Novel nicotinoyl pyrazoline derivates bearing N-methyl indole moiety as antitumor agents: Design, synthesis and evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.07.044

In the present work, twenty-five nicotinoyl pyrazoline derivates bearing N-methyl indole moiety have been designed and synthesized. The biological evaluation of these compounds as tubulin assembly inhibitors revealed that most of them were potential antitumor agents. Among them, compound 28 exhibited most potency against cancer cell line panels (GI₅₀ = 29–90 nM for HeLa, HepG2 and MCF-7 cells) without toxicity to non-tumor cells (CC₅₀ > 300 μM for 293 T cell), bound to the colchicine site of tubulin and displayed excellent inhibitory activity in tubulin assembly assay (IC₅₀ = 1.6 μM, better than CA-4). Molecular dynamics simulation was carried out to validate the docking pose of compound 28 with tubulin crystalline. Further investigation on HepG2 and HeLa cells demonstrated that compound 28 could cause mitosis arrest to G2/M phase, and subsequently induced cell apoptosis. The efficiency in vivo of compound 28 was also evaluated on HeLa-Xenograft nude mice, and the relative tumor inhibition ration was up to 61.52% without noticeable weight loss and tissue damage (examined by H&E staining), which was comparable to CA-4 (inhibited 59.92%). In brief, compound 28 is a promising candidate for tumor therapy as tubulin assembly inhibitor.

Full text of DOI:10.1016/j.ejmech.2018.07.044

Accepted Manuscript
Novel nicotinoyl pyrazoline derivates bearing N-methyl indole moiety as antitumor
agents: Design, synthesis and evaluation
Kun Chen, Ya-Liang Zhang, Jing Fan, Xiang Ma, Ya-Juan Qin, Hai-Liang Zhu
PII:
S0223-5234(18)30600-7
10.1016/j.ejmech.2018.07.044
EJMECH 10579
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 May 2018
Revised Date: 12 July 2018
Accepted Date: 16 July 2018
Please cite this article as: K. Chen, Y.-L. Zhang, J. Fan, X. Ma, Y.-J. Qin, H.-L. Zhu, Novel nicotinoyl
pyrazoline derivates bearing N-methyl indole moiety as antitumor agents: Design, synthesis and
evaluation, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.044.
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Novel nicotinoyl pyrazoline derivates bearing N-methyl indole moiety as antitumor agents:
design, synthesis and evaluation
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Kun Chen * , Ya-Liang Zhang , Jing Fan , Xiang Ma , Ya-Juan Qin* , Hai-Liang
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Zhu*
The Joint Research Center of Guangzhou University and Keele Univeristy for Gene
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Interference and Application, School of Life Science, Guangzhou University, Guangzhou
510006, People’s Republic of China.
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State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210023, People’s Republic of China.
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Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University,
Nanjing 211166, P. R. China. Email: yjqin@njmu.edu.cn.
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*Corresponding author:
Hai-Liang Zhu, Tel. & fax: +86-25-89682572; E-mail: zhuhl@nju.edu.cn
Kun Chen, Tel. & fax: +86-20-39366915; E-mail: Kchennju@hotmail.com
†
These two authors contributed equally to this paper.

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Abstract
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In the present work, twenty-five nicotinoyl pyrazoline derivates bearing N-methyl indole
moiety have been designed and synthesized. The biological evaluation of these
compounds as tubulin assembly inhibitors revealed that most of them were potential
antitumor agents. Among them, compound 28 exhibited most potency against cancer cell
line panels (GI = 29 ~ 90 nM for HeLa, HepG2 and MCF-7 cells) without toxicity to
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non-tumor cells (CC > 300 µM for 293T cell), bound to the colchicine site of tubulin
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and displayed excellent inhibitory activity in tubulin assembly assay (IC50 = 1.6 µM,
better than CA-4). Molecular dynamics simulation was carried out to validate the docking
pose of compound 28 with tubulin crystalline. Further investigation on HepG2 and HeLa
cells demonstrated that compound 28 could cause mitosis arrest to G2/M phase, and
subsequently induced cell apoptosis. The efficiency in vivo of compound 28 was also
evaluated on HeLa-Xenograft nude mice, and the relative tumor inhibition ration was up
to 61.52% without noticeable weight loss and tissue damage (examined by H&E
staining), which was comparable to CA-4 (inhibited 59.92%). In brief, compound 28 is a
promising candidate for tumor therapy as tubulin assembly inhibitor.
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Keywords
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Nicotinoyl pyrazoline; tubulin assembly inhibition; molecular dynamics; cell cycle arrest;
Xenograft model
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1. Introduction
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Despite the rapid development of anticancer therapies, cancers remain a major public
health problem and a leading cause of death all over the world [1,2]. Whether it is the
developed or developing countries, the burden led by cancers is growing worldwide
because of the growth and aging of the population [3,4]. While emerging therapies, such
as hyperthermia, photodynamic therapy and immunotherapy, were booming,
chemotherapy is still the most commonly used method to prolong the life of patients
[5,6]. In the clinic, tubulin targeting agents cover a large part of chemotherapy drugs,
such as paclitaxel and vinblastine [7].
Microtubules (MTs), a slender and fibrous structure, are a crucial component of
cytoskeleton. MTs are the highly dynamic polymers mainly formed by α, β-tubulin
heterodimers which turn over continuously [8]. MTs have a vital effect in a number of
cellular processes. In all eukaryotic cells, the dynamic instability and treadmilling of
microtubules highly take various fundamental cellular processes, including cell shape
maintenance, cell division, cell signaling and even the transport of vesicles throughout
cells [9,10]. In dividing mammalian cells, microtubules forming the mitotic spindle are
highly dynamic and incisively sensitive to destabilizing agents [11], such as colchicine
and vinblastine. Cancer cells proliferate rapidly and are more prone to be affected by
mitosis stress. Interrupting microtubule dynamics proves to be an efficient strategy in the
clinical therapy [12,13].
Most of tubulin-binding agents currently are natural products that originated from plants.
Based on the mechanism of action, the tubulin targeting agents could be divided into
microtubule stabilizing and destabilizing agents, which promote and inhibit microtubule

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polymerization, respectively. The most well-known anticancer drug as microtubule
stabilizing agent is paclitaxel, which is isolated from the bark of the yew tree and used to
treat a number of cancers [14,15]. Microtubule destabilizing drugs could bind to different
sites of tubulin heterodimers, in which the most mentioned are colchicine binding site and
vinca alkaloids binding site. Colchicine (compound a, Fig. 1), one of well-known
microtubule destabilizing drugs, could effectively prevent microtubule from
polymerization. Its binding site on tubulin has been characterized already [16]. However,
the clinical development of colchicine for cancer therapy had to be terminated because of
severe side effects [13]. Another colchicine domain inhibitor is combretastatin A4 (CA-4,
compound b, Fig. 1), which was isolated from the bark of the South African tree,
Combretum caffrum. It can potently bind to the colchicine domain, leading to severe
cytotoxicity against multiple human cancer cell lines [17]. However, CA-4 easily
undergoes cis-trans isomerization under natural light, causing a sharp reduction in the
anti-tubulin activity, which limits its further applications [18]. Its diphosphate (CA-4P) is
in clinic Phase II trial [19–21]. In spite of long historical research, tubulin inhibitors
targeting colchicine binding site still need more work to meet clinic therapy as
commercial anticancer drugs. In this way, further studies are mandatory [22–27].
In the past decades, there is a continuous interest in tubulin inhibitors of colchicine
domain, mainly focusing on structural simplified synthesized compounds, such as
compound c (SAMRT) [28], d [29], e (A-204197) [30], f (indibulin) [31] and g [32] (Fig.
1). After analysis of reported inhibitors, we extracted from compound c-e and employed
3,5-diaryl pyrazoline as the scaffold of compounds, considering the atomic arrangement
and space angle of five membered heterocyclic ring. Three of them bore heterocyclic ring

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as central contact bridge, and the five atoms in same plane caused cytotoxicity lose,
comparing the IC of d and e. And the 3,4,5-trimethoxyl motif also was obtained as its
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significance for tubulin targeting inhibitors [32]. As reported, the indole nucleus was
widely presented in several tubulin binding inhibitors, whether natural products or
synthetic small molecules [33]. Furthermore, the methylation of the nitrogen atom of
indole greatly improve the potency of tubulin polymerization inhibition and cytotoxicity
[34]. By adding nicotinic acid to pyrazoline, the molecular stability will be improved,
avoiding the oxidizable property of pyrazoline. A class of novel 1-methyl-1H-indole-
pyrazoline hybrids were reported as potential tubulin polymerization inhibitors [35]. We
hypothesized that the integration of these groups would help to increase tubulin inhibitory
potency (compound 5-29, Fig. 1) based on the previous researches. Herein, we describe
the synthetic method and the bioactivities of this series of compounds with novel
scaffold. Docking and molecular dynamics simulations are also performed to explore the
binding modes of these compounds at the active site of tubulin. The most potent
compound in vitro was further evaluated on the Xenograft model in vivo.
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2.1. Chemistry
The synthetic route of compounds 5-29 was outlined in Scheme 1. The substituted N-
methyl-indole-3-carboxaldehyde 3a-c and acetophenone went on aldol condensation in
alkaline environment, forming condensation products chalcones 4a-q. Then the
intermediates pyrazolines were synthesized from cyclization between chalcones 4a-q and
hydrazine hydrate. Because of the oxidable property, the pyrazolines were put into next

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steps immediately without any purification and reacted with nicotinic acid under inert
environment to afford the target materials 5-29. The starting materials 3a-c were
synthesized from indoles 1a-c undergoing Vilsmeier-Haack reaction and methylation
[36].
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2.2. Crystal structure of compound 26
The single crystal of compound 26 was obtained accidentally when recrystallized. Its
spatial structure was determined by X-ray diffraction analysis. The crystal data presented
in Table 1 and Fig. 2 gave perspective views with the atomic labeling system. The
crystallographic data has been deposited at the Cambridge Crystallographic Data Centre
(CCDC) and numbered 1836503.
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2.3. Antiproliferative and toxic activity in vitro
The synthesized compounds were evaluated by MTT method for antiproliferative activity
against four different human cancer cell lines including MCF-7 (human breast
adenocarcinoma), A549 (human non-small cell lung carcinoma), HepG2 (human liver
hepatocellular carcinoma) and HeLa (human cervix carcinoma). Data are expressed as the
50% growth inhibitory concentration (GI ), defined as the concentration at which the
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compounds reduced cancer cell proliferation by 50%. The results were summarized in
Table 2. As a whole, these compounds mostly showed activity to inhibit the grow of
cancer cells with smaller GI value than 10 µM. But when 3,4,5-trimethoxyl occupied
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R , such as compound 18 and 26, the inhibitory was dramatically weaken and GI was
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above 10 µM. However, compound 11 exhibited relatively good inhibitory activity to four

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cancer cell lines and its GI50 values were 0.18 µM, 0.23 µM, 0.31 µM and 0.21 µM for
MCF-7 , A549, HepG2 and HeLa, respectively. To compare the different substituted
groups on indole ring, it could be conducted the priority sequence was methoxyl, bromine
and hydrogen. For instance, compounds inhibited cells as following 21 > 12 > 7. When
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R was methoxyl and R was 6-methoxyl, different R contributed variously to the
antiproliferative and showed the order as 3-OCH > 4-OCH > 4-F > 4-Cl > 4-Br. The
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compound with 4-F, 22, displayed high inhibitory activity. When R was methoxyl and R
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was 3-methoxyl, there was no obvious difference between methyl and methoxyl on R ,
but both was better than none substituted.
The cytotoxicity of these nicotinoyl pyrazoline derivates was also evaluated on human
embryonic kidney cell line 293T (Table 2). Data were expressed as the 50% cytotoxic
concentration (CC ), defined as the concentration at which the compounds reduced non-
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tumor cell proliferation by 50%. The results of CC indicated that nearly all of them
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were less toxic to non-tumor cell. Especially for compound 28, the ratio of efficacy and
toxicity (CC50/GI50) value was at 3-10 thousand level, implying that compound 28
displayed different actions in tumor and non-tumor cells. The molecular mechanism of
this difference is still unknown, and it needs further investigation.
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2.4. Tubulin assembly inhibition
The ability of compounds 5-29 to inhibit tubulin assembly with OD340 method was
evaluated in vitro to investigate whether the antiproliferative activity of these agents was
resulted from an interaction with tubulin (Table 3). The IC values of these compounds
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Among the series of compounds, 11, 21, 22, 25, 27, 28 and 29 strongly inhibited tubulin
assembly in vitro, whose IC value against tubulin was lower than 3.0 µM. Compound
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28 showed the most potency of anti-tubulin assembly with IC value of 1.6 µM, better
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than reference drug CA-4 (IC =2.1 µM). The inhibition curve of compound 28 was
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shown in Fig. 3A Therefore, it was concluded that most of the synthesized compounds
could inhibit tubulin assembly. These results correlated positively with the
antiproliferative activity, revealing that these compounds inhibited cells growth through
preventing from microtubule assembly.
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2.5. Colchicine competition assay
To investigate the exact binding site of compound 28 on tubulin, the modified colchicine
competition assay was employed. Preincubate tested compounds with tubulin to let them
occupy the binding site of tubulin. Then add colchicine to compete with tested
compounds bound to tubulin, and unbound colchicine was tested by ultra-performance
liquid chromatography - tandem mass spectrometer (UPLC-MS/MS). The results were
shown in Fig. 3B. It indicated that compound 28 bound strongly to the colchicine binding
site of tubulin, as well as CA-4, competing with colchicine.
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2.6. Molecular simulation
As mentioned above, compound 28 bound to the colchicine binding site and exhibited
most activity against tubulin assembly, driving us to investigate the interaction mode.
Employing the crystalline complex of DAMA-colchicine binding to tubulin (PDB
ID:1SA0 [16]) and Discovery Studio (Accelrys, Co. Ltd, vision 3.5 [37]), the interaction

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mode of compound 28 with tubulin was analyzed, while the crystallized ligand DAMA-
colchicine was set as reference structure. As shown in Fig. 4A, compound 28 was soaked
in deep inside of β-tubulin between α, β-heterodimer. Compound 28 occupied the space
of colchicine binding site (CBS), similarly maintaining the orientation and shape of
DAMA-colchicine. To analysis the interaction of original ligand, it was found that the
3,4,5-trimethoxy phenyl connected CYS241 from β-subunit by hydrogen bond (H-bond,
angle S-H…O 118.56°, distance 2.01 Å). In the same way, m-methoxy phenyl from
compound 28 maintained the H-bond interaction with CYSβ241 (angle S-H…O 133.49°,
distance 2.14 Å) and was surrounded by hydrophobic residues such as LEUβ248,
LEUβ255, ALAβ316, ALAβ317, VALβ318, ALAβ354, ILEβ378 (green balls in Fig. 4D).
N-methyl indole moiety orientated against β-subunit and placed in the gap between
subunits (Fig. 4C). In addition, another H-bond (between carbonyl and ASNβ258, angle
N-H…O 107.363°, distance 2.42 Å) may contribute to stronger binding interaction for 28
compared with colchicine.
To validate the docking results, a 12 ns MD simulation was carried out for 28-tubulin
complex starting from the docking pose above already mentioned. When the simulation
accomplished, the initial and final poses of complex were aligned (Fig. 4E). Compound
28 hold original orientation and conformation of interaction with tubulin, despite of only
a short shift. The evolution in time of RMSD calculated on the backbone atoms of the
protein to the initial pose is represented in Fig. 4F. The 28-tubulin complex settled into
equilibrium soon with fluctuations wobbling in narrow range. Thus, it suggested the
virtual simulations were convincible to demonstrate the binding model of 28 with tubulin.

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2.7. Compound 28 interrupted microtubule network in HepG2 and HeLa cells
The functional feature of compound 28 affecting cellular microtubules was analyzed by
immunofluorescence staining. HepG2 and HeLa cells were treated with colchicine (0.04
µM), paclitaxel (0.04 µM) and compound 28 (0.04 µM and 0.1 µM) for 12 h. The
microtubules and nuclei were labeled by FITC-antibody (green) and DAPI (blue),
respectively. As shown in Fig. 5, the microtubule network of control cells was spindly,
fibrous and clearly visible, with typical normal spindle shape. Besides causing the cell
shape alternation, colchicine and paclitaxel mainly led to abnormal microtubule network.
Colchicine, typical tubulin assembly inhibitor, break down the microtubule and made it
short and dispersive in the cytoplasm. In contrast, paclitaxel dramatically enhanced
tubulin polymerization to form long thick microtubule fibers. After treatment of
compounds 28, cells altered normal shape and became round. To observe the
microtubules, compound 28 resulted in colchicine-like effect on HepG2 and HeLa cells
but differed from paclitaxel. With the concentration increasing of compound 28, the
percentage of shrunken or death cells raised up. Meanwhile, especially in HepG2 cells,
multinucleate cell and abnormal mitotic spindle, the typical characters of mitotic
catastrophe, were apparently observed. It indicated that compound 28 interrupted the
microtubule network in HepG2 and HeLa cells.
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2.8. Compound 28 arrest cell cycle at G2/M phase in cells
Tubulin assembly inhibitors typically interfere with the process of mitosis, causing the
cell cycle arrest to G2/M phase and failure of DNA division [38]. To test whether
compound 28 caused G2/M arrest, DNA content analysis with PI staining method was

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performed by flow cytometry. HepG2 and HeLa cells were treated with various
concentrations of 28 (0, 0.020, 0.040, 0.060, 0.080 and 0.10 µM) for 6 h, 12 h and 24 h.
The test results were collected in Fig. 6. Compound 28 significantly caused mitosis arrest
at G2/M phase in dose-dependent manner with corresponding decrease of cells in other
phases. After treatment of compound 28 to HepG2 cells for 6 h, the percent of G2/M cells
raised and reached plateau quickly despite of increasing concentration of 28 (Fig. 6A). To
prolong the time of administration, the G2/M cells kept increasing up and the maximum
was beyond 60%. However, after 24 h, the percent of cells in G2/M phase decreased
compared with shorter treatment. It may be because long treatment has led to cells
apoptosis and S phase cells increase somehow, which could be found in Fig. 6B. The
effect of compound 28 to HeLa was alike that to HepG2 (Fig. 6C). The percent of
arrested cells increased accompanied by time and dose of treatment and the maximum
was 66.08% (0.080 µM for 12 h). What differed from HepG2, when treated for 24 h, the
curve of G2/M cells presented standard s-shape. The maximum effect for 24 h was equal
to that for 12 h. Sub-G1 cells, implying cell apoptosis, also were observed (Fig. 6D).
Thus, it was established that compound 28 could induce cell cycle arrest in mitosis.
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2.9. Compound 28 caused cells apoptosis
To investigate whether compound 28 induced apoptosis, a double staining (Annexin V-
FITC/PI) assay was carried on by flow cytometry. HepG2 and HeLa cells were treated
with various concentrations (0, 0.02, 0.040, 0.060, 0.080 and 0.10 µM) of compound 28
for 6 h, 12 h and 24 h. The apoptotic rate was calculated and analyzed as percentage of
FITC positive cells, which included both early and late apoptotic cells. As presented in

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Fig. 7, apoptotic HepG2 cells increased with the escalating concentrations of compound
28. Given longer treatment, apoptotic cells increased up to maximum (20-30%). For
instance, the percentages of apoptosis 5.52%, 6.49%, 13.73%, 12.93%, 16.17% and
27.67% correspond to the concentration 0, 0.020, 0.040, 0.060, 0.080 and 0.10 µM for 24
h, respectively. However, there was no significant difference between treatment for 12 h
and 24 h. Compared with HepG2 cells, HeLa apoptosis seemed be prone to underlie with
the treat of compound 28. Alike results of HepG2, there was no or slight sign of apoptosis
for 6 h and 12 h. When treated with 0.10 µM for 24 h, the apoptotic rate of HeLa cells
increased nearly 4-fold. The percentages of apoptosis 12.27%, 11.44%, 14.93%, 31.64%,
37.90% and 40.89% correspond to the concentration 0, 0.020, 0.040, 0.060, 0.080 and
0.10 µM for 24 h, respectively. Compound 28 induced cell apoptosis in HeLa cells in
both dose- and time-dependent fashion. In addition, it was found that short treatment to
two cell lines did not cause significantly apoptosis, which indicated that mitosis arrest
maybe led to cell apoptosis (at least, mitosis arrest preceded apoptosis). Thus, it could be
concluded that compound 28 could induce apoptosis, just like other tubulin-binding
agents.
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2.10. Compound 28 suppressed tumor growth on HeLa-Xenograft model
To test the potent activity of compound 28 in vivo, HeLa cells Xenograft nude mice
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model was established. When the tumor size approximated to 100 mm after HeLa cells
injection, twenty-eight tumor-bearing mice were randomly decided into four groups and
injected compounds intraperitoneally: vehicle, CA-4 (10 mg/kg), 28 (10 mg/kg) and 28
(20 mg/kg). As shown in Fig. 8A, compared with rapid tumor growth of vehicle group,

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tumor growth of treated groups was greatly suppressed, whereas compound 28 treated
groups did not significantly differ from reference drug-treated group. After continuous
treatment of 14 days, CA-4 (10 mg/kg), 28 (10 mg/kg) and 28 (20 mg/kg) groups gave
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918.61, 1213.02, and 881.93 mm of final tumor volume, respectively, comparing the
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2292.17 mm of vehicle group. Further data analysis indicated that relative inhibition
ration (RIR) of 28 (20 mg/kg) group was up to 61.52%, slightly better than 59.92% of
vehicle group, while that of 28 (10 mg/kg) group also reached by 47.08% (Fig. 8B). The
executed tumor also was weighted and recorded. The average tumor weight of CA-4 (10
mg/kg), 28 (10 mg/kg) and 28 (20 mg/kg) groups was 0.59, 0.91 and 0.53 g, respectively,
compared to 1.54 g of vehicle group. No obvious ulceration was observed in treated
groups, while slight ulceration was found in control group throughout the observation
period. Furthermore, in comparison with vehicle group, compound 28 caused neither an
obvious loss of body weight (Fig. 8C) nor the weight change of main organs (Fig. 8D),
which indicated that compound 28 displayed no signs of toxicity in these mice, especially
in high dose.
The pathological change in tumors by H&E staining also was studied. In general, the
tumor tissue arranges disorderly and giant colorable nuclei occupy most of space in cells.
As Fig. 9, untreated tumor tissues displayed in violet as normal morphological characters.
After treatment of CA-4 or compound 28, the tumor cells arranged loosely and
irregularly, and extensive necrosis in light pink without colorable nuclei was observed in
different degrees. In high group of 28, more interspace even formed because of severe
necrosis, representing the strongest antitumor ability. Meanwhile in Fig. 9, there was no
obvious cellular inflammatory infiltration, edema or necrosis in the organ tissues. The

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cells of heart, liver, kidney and spleen in drug treated groups kept a complete
morphology, which implied no noticeable toxicity to organs. The results of tumor growth
inhibition, body weight changes and H&E staining indicated that compound 28 exhibited
obvious antitumor efficacy in HeLa xenografts via the suppression of proliferation
without local and systemic toxicity.
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In this study, a series of nicotinoyl pyrazoline derivates bearing N-methyl indole moiety
(5-29) were designed, synthesized and evaluated as potential tubulin assembly inhibitors.
Most of them showed potent anti-proliferative activity, and compound 28 displayed the
most potent activity against MCF-7, A549, HepG2 and HeLa cells with GI values of
5
0
0.09 µM, 0.59 µM, 0.029 µM and 0.034 µM, respectively. Also, compound 28 exhibited
great ability to inhibit tubulin assembly, whose IC50 was 1.6 µM and better than CA-4
(IC50 2.1 µM). The colchicine competition assay proved that the compound 28 could bind
to the colchicine domain of tubulin. Besides, molecular docking and molecular dynamics
studies were performed to predict the inhibitor-tubulin interactions. According to the
analysis of the binding modes between compound 28 and tubulin, several interactions
were observed with the protein residues in the colchicine binding site, which may
contribute to its anti-tubulin polymerization and antiproliferative activity. Further
mechanism of action studies proved that compound 28 induced the colchicine-like effect
on HepG2 and HeLa cells. It could cause mitosis block in G2/M phase, as well as severe
disruption of the microtubule system. Compound 28 also was a potent inducer of
apoptosis on HepG2 and HeLa cells. The efficiency in vivo of compound 28 was also

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evaluated on HeLa-Xenograft nude mice, and the relative tumor ration was up to 61.52%
without noticeable weight loss and tissue damage, which was comparable to CA-4. In the
light of current data, compound 28 was a promising candidate for tumor therapy as
tubulin assembly inhibitor.
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25 4. Experimental section
26
4.1. Chemistry
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4.1.1. General
All chemical solvents and reagents used in current study were analytical grade and
purchased at Nanjing Chemical Reagent CO., LTD (Nanjing, China) or Aladdin
(Shanghai, China). The reactions were monitored by thin layer chromatography (TLC),
which was performed on the glass-backed silica gel sheets (silica gel 60 GF254) and
detected using UV light (254 nm or 365 nm). Melting points were determined on an XT4
1
13
MP apparatus (Taike Corp., Beijing, China). H NMR and C NMR spectra were
collected on a Bruker DPX 400 model spectrometer in DMSO-d or CDCl . ESI-MS
6
3
spectra were recorded on a Mariner System 5304 Mass spectrometer. Elemental analyses
were performed on a CHN-O-Rapid instrument. All compounds gave satisfactory
chemical analyses (±0.4%).
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4.1.2. General procedure for synthesis of 1H-indole-3-carboxaldehyde (2a-c)
The relevant indole derivate (1a-c, 40 mmol) and phosphorus oxychloride (10 mL) were
dissolved into DMF (20 mL), respectively. The solution of indole was added dropwise to
3
POCl at 0 °C and stirred for 2 h. Then the reaction mixture was diluted with ice water

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(300 mL), neutralized with NaOH to pH 9.0, and extracted with ethyl acetate (50 mL ×
3). The organic layer was washed with brine, dried over anhydrous sodium sulfate,
filtered and concentrated in vacuum. Recrystallization afforded compound 2a-c with
yields of 95.3-98.1%.
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4.1.3. General procedure for synthesis of 1-methyl-1H-indole-3-carboxaldehyde (3a-c)
The relevant indole-3-carboxaldehyde (2a-c, 10 mmol) in THF (25 mL) was added
dropwise to a stirred solution of NaH (25 mmol) in THF (25 mL) at 0 °C and CH I (13.2
3
mmol) was added after 15 min stirring. The reaction mixture was moved to room
temperature and stirred for further 24 h. Then the solvent was removed in vacuum and the
residue was extracted by ethyl acetate. The organic layer was washed with brine, dried
over anhydrous sodium sulfate, filtered and concentrated in vacuum. Recrystallization
afforded compound 3a-c with yields of 93.4-97.5%.
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4.1.4. General procedure for synthesis of chalcones (4a-q)
The relevant N-methyl intermediate (3a-c, 2 mmol), acetophenone derivate (2 mmol) and
potassium hydroxide (6 mmol) was dissolved in ethanol (20 mL) and stirred for 24 h.
Then the reaction mixture was filtered and washed with water and cold ethanol. The
crude product was purified by recrystallization from ethanol and dichloromethane to give
pure chalcone (4a-q) with yields of 34.2-93.1%.

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4.1.5. General procedure for synthesis of Nicotinoyl (5-(1-Methyl-1H-indol-3-yl)-3-
phenyl-pyrazoline derivates (5-29)
A mixture of chalcone (4a-q, 1 mmol) and hydrazine hydrate (2 mmol) was dissolved in
ethanol (5 mL) and refluxed for 2h. The reaction mixture was cooled and kept at -20 °C
overnight. Then the precipitate in the mixture was filtered off, washed with petroleum
ether and taken to next step immediately.
To a stirred solution of relevant nicotinic acid (2 mmol), EDC·HCl (3 mmol) and HOBt
(1.2 mmol) in acetonitrile or dichloromethane (5 mL), the precipitate (1 mmol) from last
step was added at room temperature with the protection of inert gas. After 24 h, the
reaction mixture was filtered, washed (cool ethanol and water) and purified by
recrystallization to obtain target compound 5-29, yield 14.7-52.1%.
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4.1.6. (5-(1-Methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-3-
yl)methanone (5)
1
Red crystal. Yield: 29.8%. m. p. 165 ~ 166 °C. H NMR (DMSO-d , 400 MHz) δ: 9.00 (s,
6
1H), 8.69 (dd, J = 4.7, 1.3 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.78 (dd, J = 6.5, 2.9 Hz,
2H), 7.59 – 7.34 (m, 7H), 7.15 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.07 (dd, J =
11.7, 4.7 Hz, 1H), 3.94 (dd, J = 18.0, 11.8 Hz, 1H), 3.75 (s, 3H), 3.35 (dd, J = 18.0, 4.8
1
3
3
Hz, 1H). C NMR (CDCl , 151 MHz) δ: 164.18, 156.17, 151.00, 150.95, 137.86, 137.60,
131.18, 130.75, 130.68, 128.87, 127.70, 126.92, 124.96, 122.81, 121.93, 119.66, 118.74,
+
113.87, 109.84, 54.94, 40.14, 32.83. MS (ESI) m/z: 381.16 (C24
H
21
N
4
O, [M+H] ). Anal.
Calcd for C H N O: C, 75.77; H, 5.30; N, 14.73. Found: C, 75.87; H, 5.31; N, 14.76.
24
20
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4.1.7. (6-Chloropyridin-3-yl)(5-(1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)methanone (6)
1
Light yellow powder. Yield: 22.1%. m. p. 129 ~ 130 °C. H NMR (DMSO-d , 400 MHz)
6
δ: 8.85 (s, 1H), 8.27 (d, J = 6.3 Hz, 1H), 7.87 – 7.72 (m, 2H), 7.65 (d, J = 8.3 Hz, 1H),
7.59 – 7.36 (m, 6H), 7.15 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.06 (dd, J = 11.7,
4.7 Hz, 1H), 3.95 (dd, J = 18.1, 11.7 Hz, 1H), 3.75 (s, 3H), 3.36 (dd, J = 18.0, 4.8 Hz,
+
1H). MS (ESI) m/z: 415.12 (C24
H
20ClN
4
O, [M+H] ). Anal. Calcd for C24
H19ClN
4
O: C,
69.48; H, 4.62; N, 13.50. Found: C, 69.44; H, 4.62; N, 13.52.
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3
3
3
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4
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4
4
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97
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01
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4.1.8. (6-Methoxypyridin-3-yl)(5-(1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)methanone (7)
1
Orange powder. Yield: 36.4%. m. p. 174 ~ 176 °C. H NMR (DMSO-d
6
, 400 MHz) δ:
8.75 (s, 1H), 8.18 (dd, J = 8.7, 2.2 Hz, 1H), 7.81 (dd, J = 6.5, 3.0 Hz, 2H), 7.58 – 7.46
(m, 3H), 7.42 (d, J = 8.5 Hz, 2H), 7.37 (s, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.99 (t, J = 7.4
Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.04 (dd, J = 11.8, 4.8 Hz, 1H), 4.07 – 3.84 (m, 4H),
+
3.75 (s, 3H), 3.33 – 3.30 (m, 1H). MS (ESI) m/z: 411.17 (C H N O , [M+H] ). Anal.
25
23
4
2
Calcd for C H N O : C, 73.15; H, 5.40; N, 13.65. Found: C, 73.34; H, 5.39; N, 13.64.
25
22
4
2
4
4
4
4
4
05
06
07
08
09
4.1.9. (5-(1-Methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(6-
methylpyridin-3-yl)methanone (8)
1
Light yellow powder. Yield: 21.2%. m. p. 158 ~ 160 °C. H NMR (DMSO-d , 400 MHz)
6
δ: 8.90 (s, 1H), 8.10 (d, J = 6.6 Hz, 1H), 7.79 (dd, J = 6.6, 2.9 Hz, 2H), 7.58 – 7.33 (m,
7H), 7.15 (t, J = 7.9 Hz, 1H), 7.01 (t, J = 7.7 Hz, 1H), 6.06 (dd, J = 11.7, 4.8 Hz, 1H),

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3.95 (dd, J = 18.0, 11.8 Hz, 1H), 3.76 (s, 3H), 3.35 (dd, J = 17.9, 4.9 Hz, 1H), 2.54 (s,
+
3H). MS (ESI) m/z: 395.18 (C H N O, [M+H] ). Anal. Calcd for C H N O: C, 76.12;
2
5
23
4
25 22
4
H, 5.62; N, 14.20. Found: C, 75.89; H, 5.60; N, 14.21.
4
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4
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4
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16
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4.1.10. (2-Chloropyridin-3-yl)(5-(1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)methanone (9)
1
Light yellow powder. Yield: 48.2%. m. p. 191 ~ 193 °C. H NMR (DMSO-d , 400 MHz)
6
δ: 8.51 (dd, J = 4.8, 1.9 Hz, 1H), 7.89 (dd, J = 7.5, 1.9 Hz, 1H), 7.65 (d, J = 6.3 Hz, 2H),
7.54 (dd, J = 7.5, 4.8 Hz, 1H), 7.51 – 7.37 (m, 6H), 7.18 (t, J = 7.3 Hz, 1H), 7.02 (t, J =
7.3 Hz, 1H), 6.04 (dd, J = 11.7, 4.7 Hz, 1H), 4.00 (dd, J = 18.2, 11.7 Hz, 1H), 3.79 (s,
1
3
3H), 3.38 (dd, J = 18.2, 4.7 Hz, 1H). C NMR (CDCl , 151 MHz) δ: 163.63, 156.52,
3
149.76, 148.04, 138.03, 137.66, 132.95, 131.02, 130.72, 128.78, 127.74, 126.93, 124.92,
122.03, 121.96, 119.55, 118.87, 113.48, 109.84, 54.48, 41.00, 32.88. MS (ESI) m/z:
+
415.12 (C H ClN O, [M+H] ). Anal. Calcd for C H ClN O: C, 69.48; H, 4.62; N,
24
20
4
24 19
4
13.50. Found: C, 69.67; H, 4.63; N, 13.49.
4
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4
4
4
4
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28
29
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31
32
4.1.11. (5-(1-Mmethyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-
yl)methanone (10)
1
Light purple powder. Yield: 23.0%. m. p. 175 ~ 176 °C. H NMR (DMSO-d , 400 MHz)
6
δ: 8.72 (d, J = 5.6 Hz, 2H), 7.86 – 7.76 (m, 2H), 7.70 (d, J = 5.6 Hz, 2H), 7.59 – 7.36 (m,
6H), 7.15 (t, J = 7.5 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.05 (dd, J = 11.7, 4.6 Hz, 1H),
1
3
3.95 (dd, J = 18.1, 11.8 Hz, 1H), 3.76 (s, 3H), 3.44 – 3.31 (m, 1H). C NMR (CDCl ,
3
151 MHz) δ: 159.71, 151.64, 144.89, 137.40, 132.86, 126.39, 126.01, 124.15, 123.04,

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122.16, 120.18, 119.00, 117.21, 114.92, 113.95, 108.93, 105.13, 50.21, 35.38, 28.09. MS
+
(ESI) m/z: 381.16(C H N O, [M+H] ). Anal. Calcd for C H N O: C, 75.77; H, 5.30;
24
21
4
24 20
4
N, 14.73. Found: C, 75.85; H, 5.28; N, 14.74.
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4.1.12. (5-(1-Methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-
1-yl)(6-methylpyridin-3-yl)methanone (11)
1
Brown crystal. Yield: 52.1%. m. p. 217 ~ 219 °C. H NMR (DMSO-d , 400 MHz) δ: 8.91
6
(s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.53 – 7.30 (m, 4H), 7.15 (t, J = 7.6 Hz, 1H), 7.06 (s,
2H), 7.02 (t, J = 7.5 Hz, 1H), 6.06 (dd, J = 11.7, 4.6 Hz, 1H), 3.90 (dd, J = 18.1, 11.7 Hz,
1H), 3.81 (s, 6H), 3.75 (s, 3H), 3.71 (s, 3H), 3.40 (dd, J = 18.1, 4.7 Hz, 1H), 2.53 (s, 3H).
1
3
C NMR (CDCl , 151 MHz) δ:164.19, 160.46, 155.72, 153.48, 150.56, 140.47, 138.28,
3
137.60, 127.82, 127.55, 126.76, 124.95, 122.32, 121.92, 119.61, 118.76, 114.03, 109.83,
+
104.20. MS (ESI) m/z: 485.21 (C28
H
29
N
4
O
4
, [M+H] ). Anal. Calcd for C28
H
28
N
4
4
O : C,
69.41; H, 5.82; N, 11.56. Found: C, 69.19; H, 5.83; N, 11.53.
4
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4
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4
4
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4.1.13. (5-(5-Bbromo-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)(pyridin-3-yl)methanone (12)
1
Orange crystal. Yield: 40.1%. m. p. 185 ~ 186 °C. H NMR (DMSO-d , 400 MHz) δ:
6
9.00 (s, 1H), 8.70 (dd, J = 4.8, 1.5 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.87 – 7.73 (m, 2H),
7.71 (s, 1H), 7.64 – 7.38 (m, 6H), 7.28 (dd, J = 8.7, 1.8 Hz, 1H), 6.07 (dd, J = 11.6, 4.6
Hz, 1H), 3.95 (dd, J = 18.0, 11.6 Hz, 1H), 3.75 (s, 3H), 3.40 – 3.30 (m, 1H). MS (ESI)
+
m/z: 459.07 (C H BrN O, [M+H] ). Anal. Calcd for C H BrN O: C, 62.76; H, 4.17;
24
20
4
24 19
4
N, 12.20. Found: C, 62.88; H, 4.18; N, 12.21.

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4.1.14. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-
1-yl)(pyridin-3-yl)methanone (13)
1
Light yellow powder. Yield: 19.9%. m. p. 166 ~ 168 °C. H NMR (DMSO-d
6
, 400 MHz)
δ: 8.99 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.20 (d, J = 7.9 Hz, 1H), 7.83 (dd, J = 8.8,
5.5 Hz, 2H), 7.71 (s, 1H), 7.53 (dd, J = 7.8, 4.9 Hz, 1H), 7.49 – 7.39 (m, 2H), 7.39 – 7.24
(m, 3H), 6.07 (dd, J = 11.6, 4.6 Hz, 1H), 3.94 (dd, J = 18.1, 11.6 Hz, 1H), 3.75 (s, 3H),
+
3.44 – 3.31 (m, 1H). MS (ESI) m/z: 477.06 (C H BrFN O, [M+H] ). Anal. Calcd for
24
19
4
C H BrFN O: C, 60.39; H, 3.80; N, 11.74. Found: C, 60.50; H, 3.79; N, 11.74.
24
18
4
4
4
4
4
4
4
4
4
4
4
4
4
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69
70
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4.1.15. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-chlorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(pyridin-3-yl)methanone (14)
1
White powder. Yield: 21.4%. m. p. 202 ~ 203 °C. H NMR (DMSO-d , 400 MHz) δ: 9.00
6
(s, 1H), 8.70 (d, J = 3.5 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.71
(s, 1H), 7.55 (d, J = 8.6 Hz, 3H), 7.46 (s, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.28 (dd, J = 8.7,
1.7 Hz, 1H), 6.08 (dd, J = 11.6, 4.6 Hz, 1H), 3.94 (dd, J = 18.1, 11.7 Hz, 1H), 3.74 (s,
1
3
3
3H), 3.35 (dd, J = 18.1, 4.7 Hz, 1H). C NMR (CDCl , 151 MHz) δ: 159.67, 150.34,
146.66, 146.33, 132.85, 132.04, 131.46, 125.61, 124.76, 124.43, 123.61, 123.37, 121.87,
120.17, 118.09, 116.52, 108.82, 108.35, 106.61, 50.01, 35.46, 28.28. MS (ESI) m/z:
+
493.04 (C H BrClN O, [M+H] ). Anal. Calcd for C H BrClN O: C, 58.38; H, 3.67;
24
19
4
24 18
4
N, 11.35. Found: C, 58.46; H, 3.66; N, 11.37.

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4.1.16. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(pyridin-3-yl)methanone (15)
1
White powder. Yield: 26.5%. m. p. 213 ~ 214 °C. H NMR (DMSO-d , 400 MHz) δ: 8.99
6
(s, 1H), 8.70 (dd, J = 4.7, 1.2 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.89 – 7.64 (m, 5H), 7.61
– 7.52 (m, 1H), 7.45 (dd, J = 26.9, 16.2 Hz, 2H), 7.28 (dd, J = 8.7, 1.8 Hz, 1H), 6.08 (dd,
J = 11.6, 4.6 Hz, 1H), 3.94 (dd, J = 18.1, 11.7 Hz, 1H), 3.74 (s, 3H), 3.36 – 3.28 (m, 1H).
1
3
3
C NMR (CDCl , 151 MHz) δ 159.68, 150.41, 146.69, 146.34, 132.83, 131.45, 127.38,
125.58, 125.20, 123.61, 123.56, 121.87, 120.42, 120.17, 118.08, 116.51, 108.82, 108.36,
+
106.62, 50.02, 35.41, 28.28. MS (ESI) m/z: 536.98 (C24
H19Br
2
N
4
O, [M+H] ). Anal.
Calcd for C H Br N O: C, 53.56; H, 3.37; N, 10.41. Found: C, 53.44; H, 3.38; N,
24
18
2
4
10.43.
4
4
4
4
4
4
4
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4
4
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91
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4.1.17. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(pyridin-3-yl)methanone (16)
1
White crystal. Yield: 25.4%. m. p. 180 ~ 181 °C. H NMR (DMSO-d , 400 MHz) δ: 9.00
6
(s, 1H), 8.69 (dd, J = 4.8, 1.5 Hz, 1H), 8.20 (d, J = 7.9 Hz, 1H), 7.79 – 7.64 (m, 3H), 7.53
(dd, J = 7.8, 4.9 Hz, 1H), 7.48 – 7.38 (m, 2H), 7.27 (dd, J = 8.7, 1.8 Hz, 1H), 7.03 (d, J =
8.9 Hz, 2H), 6.04 (dd, J = 11.5, 4.4 Hz, 1H), 3.91 (dd, J = 18.0, 11.6 Hz, 1H), 3.81 (s,
+
3H), 3.74 (s, 3H), 3.34 – 3.29 (m, 1H). MS (ESI) m/z: 489.08 (C25
H22BrN
4
O
2
, [M+H] ).
Anal. Calcd for C25
11.43.
4 2
H21BrN O : C, 61.36; H, 4.33; N, 11.45. Found: C, 61.49; H, 4.32; N,

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4.1.18. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(pyridin-3-yl)methanone (17)
1
Pink powder. Yield: 32.3%. m. p. 112 ~ 114 °C. H NMR (DMSO-d , 400 MHz) δ: 9.02
6
(s, 1H), 8.69 (d, J = 4.1 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.53 (dd, J = 7.7,
5.0 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.39 – 7.24 (m, 3H), 7.05 (d, J = 8.4 Hz, 1H), 6.05
(dd, J = 11.4, 4.2 Hz, 1H), 3.91 (dd, J = 18.0, 11.5 Hz, 1H), 3.81 (s, 3H), 3.78 (s, 3H),
+
3.75 (s, 3H), 3.39 – 3.31 (m, 1H). MS (ESI) m/z: 519.10 (C26
H24BrN
4
O
3
, [M+H] ). Anal.
Calcd for C26
10.78.
4 3
H23BrN O : C, 60.12; H, 4.46; N, 10.79. Found: C, 59.99; H, 4.47; N,
5
5
5
5
5
5
5
5
5
5
5
5
10
11
12
13
14
15
16
17
18
19
20
21
4.1.19. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)(pyridin-3-yl)methanone (18)
1
White powder. Yield: 19.2%. m. p. 194 ~ 195 °C. H NMR (DMSO-d
6
, 400 MHz) δ: 9.03
(s, 1H), 8.70 (dd, J = 4.7, 1.3 Hz, 1H), 8.24 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.53 (dd, J
= 7.7, 5.0 Hz, 1H), 7.45 – 7.38 (m, 2H), 7.29 (dd, J = 8.7, 1.8 Hz, 1H), 7.05 (s, 2H), 6.09
(dd, J = 11.4, 4.2 Hz, 1H), 3.92 (dd, J = 18.0, 11.5 Hz, 1H), 3.81 (s, 6H), 3.73 (d, J = 12.5
1
3
Hz, 6H), 3.41 (dd, J = 18.0, 4.4 Hz, 1H). C NMR (CDCl , 151 MHz) δ: 159.21, 151.52,
3
148.75, 146.07, 145.93, 135.90, 133.36, 131.48, 125.92, 123.48, 121.89, 121.66, 120.17,
118.18, 116.62, 108.93, 108.33, 106.59, 99.50, 56.28, 51.61, 50.02, 35.80, 28.28. MS
+
(ESI) m/z: 549.11 (C27
H26BrN
4
O
4
, [M+H] ). Anal. Calcd for C27
H25BrN
4
O
4
: C, 59.02; H,
4.59; N, 10.20. Found: C, 59.19; H, 4.58; N, 10.21.

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5
5
5
5
5
22
23
24
25
26
27
28
29
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31
32
33
4.1.20. (5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(pyridin-4-yl)methanone (19)
1
Gray powder. Yield: 18.9%. m. p. 168 ~ 169 °C. H NMR (DMSO-d , 400 MHz) δ: 8.74
6
(d, J = 5.7 Hz, 2H), 7.75 (d, J = 5.8 Hz, 2H), 7.71 (s, 1H), 7.42 (d, J = 7.3 Hz, 2H), 7.40 –
7.23 (m, 3H), 7.04 (d, J = 8.5 Hz, 1H), 6.05 (dd, J = 11.4, 4.2 Hz, 1H), 3.90 (dd, J = 18.0,
1
3
11.5 Hz, 1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.75 (s, 3H), 3.40 – 3.32 (m, 1H). C NMR
(CDCl , 151 MHz) δ: 164.08, 156.53, 151.61, 149.29, 149.24, 142.47, 136.22, 128.43,
3
126.63, 124.89, 123.90, 123.71, 121.38, 120.90, 113.53, 113.08, 111.34, 110.83, 108.91,
+
56.03, 55.99, 54.63, 40.39, 33.03. MS (ESI) m/z: 519.10 (C26
H24BrN
4
O
3
, [M+H] ). Anal.
Calcd for C H BrN O : C, 60.12; H, 4.46; N, 10.79. Found: C, 60.02; H, 4.47; N,
26
23
4
3
10.81.
5
5
5
5
5
5
5
5
5
5
34
35
36
37
38
39
40
41
42
43
4.1.21. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)(pyridin-3-yl)methanone (20)
1
Pink powder. Yield: 24.3%. m. p. 134 ~ 136 °C. H NMR (DMSO-d , 400 MHz) δ: 8.99
6
(s, 1H), 8.68 (dd, J = 4.8, 1.6 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.80 (dd, J = 6.5, 3.0 Hz,
2H), 7.63 – 7.44 (m, 4H), 7.44 – 7.29 (m, 2H), 6.88 (d, J = 1.9 Hz, 1H), 6.80 (dd, J = 8.9,
2.4 Hz, 1H), 6.05 (dd, J = 11.6, 4.1 Hz, 1H), 3.94 (dd, J = 18.0, 11.7 Hz, 1H), 3.71 (s,
+
3H), 3.56 (s, 3H), 3.35 – 3.31 (m, 1H). MS (ESI) m/z: 411.17 (C25
23
H N
4
2
O , [M+H] ).
Anal. Calcd for C25
14.68.
22 4 2
H N O : C, 75.77; H, 5.30; N, 14.73. Found: C, 75.89; H, 5.30; N,

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46
47
48
49
50
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4.1.22. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(6-
methoxypyridin-3-yl)methanone (21)
1
White powder. Yield: 17.3%. m. p. 148 ~ 150 °C. H NMR (DMSO-d , 400 MHz) δ: 8.76
6
(s, 1H), 8.19 (dd, J = 8.7, 2.2 Hz, 1H), 7.92 – 7.77 (m, 2H), 7.59 – 7.42 (m, 3H), 7.31 (d,
J = 8.6 Hz, 2H), 6.92 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 2.3 Hz, 1H), 6.78 (dd, J = 8.8, 2.4
Hz, 1H), 6.02 (dd, J = 11.7, 4.3 Hz, 1H), 4.04 – 3.82 (m, 4H), 3.71 (s, 3H), 3.55 (s, 3H),
+
3.32 (dd, J = 18.0, 4.3 Hz, 1H). MS (ESI) m/z: 441.18(C26
Calcd for C26
H
25
N
4
O
3
, [M+H] ). Anal.
H
24
N
4
O
3
: C, 70.89; H, 5.49; N, 12.72. Found: C, 70.91; H, 5.48; N, 12.74.
5
5
5
5
5
5
5
5
5
53
54
55
56
57
58
59
60
61
4.1.23. (3-(4-Fluorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazol-1-yl)(6-methoxypyridin-3-yl)methanone (22)
1
Brown powder. Yield: 14.7%. m. p. 123 ~ 126 °C. H NMR (DMSO-d
6
, 400 MHz) δ:
8.76 (s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.89 (dd, J = 8.7, 5.5 Hz, 2H), 7.40 – 7.28 (m, 4H),
6.92 (d, J = 8.7 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.79 (dd, J = 8.8, 2.3 Hz, 1H), 6.03
(dd, J = 11.7, 4.3 Hz, 1H), 3.97 – 3.85 (m, 4H), 3.71 (s, 3H), 3.58 (s, 3H), 3.33 (dd, J =
+
18.0, 4.3 Hz, 1H). MS (ESI) m/z: 459.18 (C H FN O , [M+H] ). Anal. Calcd for
26
24
4
3
C H FN O : C, 68.11; H, 5.06; N, 12.22. Found: C, 68.29; H, 5.07; N, 12.26.
26
23
4
3
5
5
5
5
5
62
63
64
65
66
4.1.24. (3-(4-Chlorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazol-1-yl)(6-methoxypyridin-3-yl)methanone (23)
1
Yellow powder. Yield: 18.6%. m. p. 157 ~ 159 °C. H NMR (DMSO-d , 400 MHz) δ:
6
8.75 (s, 1H), 8.17 (dd, J = 8.6, 2.1 Hz, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.6 Hz,
2H), 7.38 – 7.29 (m, 2H), 6.92 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.80 (dd, J =

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68
69
70
71
8.9, 2.3 Hz, 1H), 6.03 (dd, J = 11.7, 4.4 Hz, 1H), 4.00 – 3.84 (m, 4H), 3.71 (s, 3H), 3.59
+
(s, 3H), 3.32 (dd, J = 18.1, 4.4 Hz, 1H). MS (ESI) m/z: 475.15 (C H ClN O , [M+H] ).
26
24
4
3
Anal. Calcd for C H ClN O : C, 65.75; H, 4.88; N, 11.80. Found: C, 65.81; H, 4.88; N,
26
23
4
3
11.78.
5
5
5
5
5
5
5
5
5
72
73
74
75
76
77
78
79
80
4.1.25. (3-(4-Bromophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazol-1-yl)(6-methoxypyridin-3-yl)methanone (24)
1
Orange crystal. Yield: 21.7%. m. p. 163 ~ 164 °C. H NMR (DMSO-d , 400 MHz) δ:
6
8.75 (s, 1H), 8.17 (dd, J = 8.7, 2.0 Hz, 1H), 7.74 (dd, J = 23.5, 8.6 Hz, 4H), 7.37 – 7.29
(m, 2H), 6.92 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.80 (dd, J = 8.8, 2.3 Hz, 1H),
6.03 (dd, J = 11.7, 4.4 Hz, 1H), 3.96 – 3.81 (m, 4H), 3.71 (s, 3H), 3.59 (s, 3H), 3.31 (dd,
+
J = 18.1, 4.5 Hz, 1H). MS (ESI) m/z: 519.10 (C26
H
24BrN
4
O
3
, [M+H] ). Anal. Calcd for
26 4 3
C H23BrN O : C, 60.12; H, 4.46; N, 10.79. Found: C, 60.19; H, 4.47; N, 10.78.
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5
5
5
5
5
5
5
5
81
82
83
84
85
86
87
88
89
4.1.26. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(6-methoxypyridin-3-yl)methanone (25)
1
Light yellow powder. Yield: 17.8%. m. p. 90 ~ 93 °C. H NMR (DMSO-d
6
, 400 MHz) δ:
8.76 (s, 1H), 8.23 – 8.17 (m, 1H), 7.80 – 7.73 (m, 4H), 7.32 (d, J = 8.6 Hz, 2H), 6.92 (d, J
= 8.7 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.79 (dd, J = 8.9, 2.3 Hz, 1H), 6.00 (dd, J = 11.5,
4.2 Hz, 1H), 3.92 – 3.79 (m, 7H), 3.71 (s, 3H), 3.57 (s, 3H), 3.29 (dd, J = 18.1, 4.2 Hz,
+
1H). MS (ESI) m/z: 471.20 (C H N O , [M+H] ). Anal. Calcd for C H N O : C,
27
27
4
4
27 26
4
4
68.92; H, 5.57; N, 11.91. Found: C, 69.04; H, 5.56; N, 11.94.

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4.1.27. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)(6-methoxypyridin-3-yl)methanone (26)
1
Yellow crystal. Yield: 18.9%. m. p. 175 ~ 177 °C. H NMR (DMSO-d , 400 MHz) δ: 8.71
6
(s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 7.37 – 7.23 (m, 2H), 7.09 (s, 2H), 6.93 (d, J = 9.1 Hz,
2H), 6.80 (dd, J = 8.9, 2.3 Hz, 1H), 6.03 (dd, J = 11.6, 4.0 Hz, 1H), 3.92 (s, 3H), 3.90 –
3.76 (m, 7H), 3.72 (d, J = 2.2 Hz, 6H), 3.61 (s, 3H), 3.43 – 3.38 (m, 1H). MS (ESI) m/z:
+
531.22 (C29
H
31
N
4
O
6
, [M+H] ). Anal. Calcd for C29
H
30
N
4
O
6
: C, 65.65; H, 5.70; N, 10.56.
Found: C, 65.75; H, 5.71; N, 10.58.
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6
6
6
6
6
6
6
6
6
99
00
01
02
03
04
05
06
07
08
4.1.28. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(pyridin-3-yl)methanone (27)
1
Orange powder. Yield: 21.2%. m. p. 145 ~ 148 °C. H NMR (DMSO-d
6
, 400 MHz) δ:
8.99 (s, 1H), 8.69 (dd, J = 4.8, 1.4 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.54 (dd, J = 7.7, 4.9
Hz, 1H), 7.48 – 7.26 (m, 5H), 7.22 – 7.02 (m, 1H), 6.89 (d, J = 1.5 Hz, 1H), 6.81 (dd, J =
8.9, 2.3 Hz, 1H), 6.05 (dd, J = 11.6, 4.1 Hz, 1H), 3.93 (dd, J = 18.0, 11.7 Hz, 1H), 3.79 (s,
3H), 3.72 (s, 3H), 3.59 (s, 3H), 3.47 – 3.27 (m, 1H). MS (ESI) m/z: 441.18 (C H N O ,
26
25
4
3
+
[M+H] ). Anal. Calcd for C H N O : C, 70.89; H, 5.49; N, 12.72. Found: C, 70.86; H,
26
24
4
3
5.51; N, 12.75.
6
6
6
6
09
10
11
12
4.1.29. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(6-methylpyridin-3-yl)methanone (28)
1
Yellow powder. Yield: 36.4%. m. p. 138 ~ 139 °C. H NMR (DMSO-d , 400 MHz) δ:
6
8.89 (s, 1H), 8.10 (d, J = 7.4 Hz, 1H), 7.42 – 7.27 (m, 6H), 7.08 (s, 1H), 6.88 (d, J = 1.8

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14
15
16
17
Hz, 1H), 6.79 (dd, J = 8.9, 2.3 Hz, 1H), 6.02 (dd, J = 11.6, 4.1 Hz, 1H), 3.90 (dd, J =
18.0, 11.8 Hz, 1H), 3.79 (s, 3H), 3.71 (s, 3H), 3.58 (s, 3H), 3.32 – 3.28 (m, 1H), 2.53 (s,
+
3H). MS (ESI) m/z: 455.20 (C H N O , [M+H] ). Anal. Calcd for C H N O : C,
27
27
4
3
27 26
4
3
71.35; H, 5.77; N, 12.33. Found: C, 71.44; H, 5.78; N, 12.33.
6
6
6
6
6
6
6
6
6
18
19
20
21
22
23
24
25
26
4.1.30. (5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)(6-methoxypyridin-3-yl)methanone (29)
1
Gray powder. Yield: 25.5%. m. p. 133 ~ 135 °C. H NMR (DMSO-d , 400 MHz) δ: 8.75
6
(s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 5.3 Hz, 2H), 7.39 – 7.29 (m, 3H), 7.23 –
6.97 (m, 1H), 6.93 (d, J = 8.7 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.80 (dd, J = 8.8, 2.3 Hz,
1H), 6.02 (dd, J = 11.7, 4.2 Hz, 1H), 3.96 – 3.82 (m, 4H), 3.81 (s, 3H), 3.71 (s, 3H), 3.58
+
(s, 3H), 3.33 – 3.28 (m, 1H). MS (ESI) m/z: 471.20 (C27
H
27
N
4
O
4
, [M+H] ). Anal. Calcd
26 4 4
for C27H N O : C, 68.92; H, 5.57; N, 11.91. Found: C, 69.04; H, 5.56; N, 11.93.
6
6
6
6
6
6
6
6
6
27
28
29
30
31
32
33
34
35
4.1.31. Crystal structure determination
Crystal structure of compound 26 was determined at 293 K by performing a Bruker D8
VENTURE PHOTON diffractometer equipped with graphite-monochromated Mo Kα (λ
= 0.71073 Å) radiation. The structure was solved by direct methods and refined on F2 by
full-matrix least-squares methods using SHELXTL [39,40] (Bruker, German). All the
non-hydrogen atoms were refined with anisotropic thermal parameters. All the hydrogen
atoms were placed in geometrically idealized positions and constrained to ride on the
parent atoms.