Discovery of novel pyrazoline derivatives containing methyl-1H-indole moiety as potential inhibitors for blocking APC-Asef interactions

Article abstract of DOI:10.1016/j.bioorg.2020.103838

A series of novel pyrazoline derivatives containing methyl-1H-indole moiety were discovered as potential inhibitors for blocking APC-Asef interactions. The top hit Q19 suggested potency of inhibiting APC-Asef interactions and attractive preference for human-sourced colorectal cells. It was already comparable with the previous representative and the positive control Regorafenib before further pharmacokinetic optimization. The introduction of methyl-1H-indole moiety realized the Mitochondrial affection thus might connect the impact on the protein-interaction level with the apoptosis events. The molecular docking simulation inferred that bringing trifluoromethyl groups seemed a promising approach for causing more key interactions such as H-bonds. This work raised referable information for further discovery of inhibitors for blocking APC-Asef interactions.

Full text of DOI:10.1016/j.bioorg.2020.103838

Journal Pre-proofs
Discovery of novel pyrazoline derivatives containing methyl-1H-indole moi-
ety as potential inhibitors for blocking APC-Asef interactions
Peng-Fei Qi, Li Fang, Hua Li, Shu-Kai Li, Yu-Shun Yang, Jin-Liang Qi,
Chen Xu, Hai-Liang Zhu
PII:
S0045-2068(19)31867-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.103838
YBIOO 103838
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Bioorganic Chemistry
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1 March 2020
6 April 2020
Please cite this article as: P-F. Qi, L. Fang, H. Li, S-K. Li, Y-S. Yang, J-L. Qi, C. Xu, H-L. Zhu, Discovery of
novel pyrazoline derivatives containing methyl-1H-indole moiety as potential inhibitors for blocking APC-Asef
interactions, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103838
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Discovery of novel pyrazoline derivatives containing methyl-1H-indole moiety as
potential inhibitors for blocking APC-Asef interactions
Peng-Fei Qi,† Li Fang,† Hua Li, Shu-Kai Li, Yu-Shun Yang, Jin-Liang Qi,* Chen Xu,* and
Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023,
China
†Both authors contributed equally to the work
*Corresponding author. Tel.
&
Fax: +86-25-89682672; E-mail: zhuhl@nju.edu.cn;
xuchn@nju.edu.cn; QiJL@nju.edu.cn.
Abstract: A series of novel pyrazoline derivatives containing methyl-1H-indole
moiety were discovered as potential inhibitors for blocking APC-Asef interactions.
The top hit Q19 suggested potency of inhibiting APC-Asef interactions and attractive
preference for human-sourced colorectal cells. It was already comparable with the
previous representative and the positive control Regorafenib before further
pharmacokinetic optimization. The introduction of methyl-1H-indole moiety realized
the Mitochondrial affection thus might connect the impact on the protein-interaction
level with the apoptosis events. The molecular docking simulation inferred that
bringing trifluoromethyl groups seemed a promising approach for causing more key
interactions such as H-bonds. This work raised referable information for further
discovery of inhibitors for blocking APC-Asef interactions.
Keywords: anti-cancer; pyrazoline derivatives; methyl-1H-indole moiety; APC-Asef
interactions; molecular docking.
1

1. Introduction
Focusing on the topic of anti-cancer agents and therapies, researchers have been
attracted by Protein-protein interactions (PPIs) due to their regulation of proliferation
and migration [1-4]. Considering the tumor classification, when we keep down-to
earth and study the colon carcinoma, we found that recent in-depth therapeutic study
revealed the central role of one specific protein-protein interaction in this kind of
cancer cases [5,6]. Originated from colonic epithelial cells, colon cancer would
transformed into adenomatous polyps pathogenically and become infectious [7-10].
Among the corresponding pathogenical mutation processes of adenomatous polypoids,
adenomatous polyposis coli (APC) is a significant suppressor protein. It consists of
the N-terminal Armadillo (ARM) domain, the middle β-catenin binding domain, and
C-terminal binding domain with DLG tumor-inhibiting factor [11-14]. According to
clinical studies, truncated APC protein was associated with over 85% of congenital
colon cancer patients and 80% of the acquired cases [15,16]. Pathological APC
signaling stimulation blocked the negative regulation of Asef (the receptor of APC),
leading to proliferation and migration in human colorectal cancer [17-20]. Knocking
down either APC or Asef could prevent their interaction thus decrease the
deterioration of colorectal cancer [21-24]. More therapeutically, investigators
achieved in expressing exogenous wild-type APC gene by transfecting into
pathological cell and restoring full-length APC to disrupt the Asef binding with
mutant APC [25]. However, knock-down, exogenous-expression and genetic
induction are difficult to control for in vivo approaches [26-28]. Therefore, blocking
the APC-Asef interaction by small functional molecules, referring to the similar
binding mechanism of drugs, has drawn the attention of researchers [29-33].
In our previous work, we have exploited some novel morpholine-containing 2H
pyrazole derivatives for inhibiting APC-Asef interaction [34]. The top hit indicated
attractive potency (IC50 = 0.18±0.01 μM). However, according to the molecular
docking patterns, the introduced morpholine ring did not bring strong interactions
such as H-bonds or π interactions. Moreover, since the results inferred the connection
2

between the bioactivity and apoptosis, here we wonder if a Mitochondria-linked
moiety could provide improved potency or at least valuable information. According to
virtual screening, we selected 1-methyl-1H-indole to replace naphthalene. With
similar steric effect, this choice might improve the druggability by offering better
parameters (LogP and Polar Surface Area), or at least maintain good druggability
when introducing the same pharmacokinetic group. Accompanying structural changes
led to a new series of candidates, among whom inhibitory hits for blocking APC-Asef
interaction were picked after stepwise evaluation.
Figure 1. The designing concept of the pyrazoline derivatives containing methyl-1H-indole moiety
as potential inhibitors for blocking APC-Asef interactions
2. Results and discussion
2.1. Synthesis of the compounds
The general synthesis route of the pyrazoline derivatives Q1-Q20 was outlined
in Scheme 1. They were prepared in four steps. First, the methylation of
1H-indole-5-carbaldehyde was conducted by using alkyliodide. Second, the
methylated aldehyde was treated with the corresponding acetophenones, respectively,
yielding certain chalcone analogues. Third, the cyclization reaction was conducted by
3

using 4-hydrazinobenzoic acid. Finally, the aniline derivatives were added to achieve
the target compounds Q1-Q20. All of them were reported for the first time with
satisfactory analytical and spectroscopic data.
Compounds
Q1
R1
H
R2
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
H
R3
H
R4
H
R5
H
R6
H
H
CH3O
H
H
H
H
Q2
H
Br
H
H
H
Q3
H
H
Cl
F
H
Q4
H
H
H
H
Q5
H
CH3
H
H
H
H
Q6
H
Cl
NO2
CF3
F
Cl
NO2
H
H
Q7
H
H
H
Q8
H
H
CF3
H
Q9
H
H
F
Q10
Q11
Q12
Q14
Q15
Q16
Q17
Q18
Q19
Q20
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
H
H
H
H
H
CH3O
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
CH3
H
H
H
H
H
Cl
H
Cl
NO2
H
H
H
H
H
H
H
CF3
F
CF3
H
H
H
F
Scheme 1. General synthesis of target compounds Q1-Q20. Reagents and conditions: i)
4

alkyliodide, DMF, NaH, 0 ℃, 30 min, r. t., 12 h; ii) acetophenone derivatives, NaOH, ethanol, r. t.,
4 h; iii) CH3COOH, methanol, 4-hydrazinobenzoic acid, reflux, 8 h; iv) aniline derivatives, EDC,
HOBt, r. t., 5 h.
2.2. Biological evaluation
2.2.1. Antiproliferation assay
Initially, all the synthesized compounds Q1-Q20 were evaluated for their
antiproliferation activities against five cancer cell lines, HCT116, SW480, HT29
(human colorectal cell lines), CT26 (mice colorectal cell lines), HeLa (human cervix
cell line), and one noncancer cell line, 293T (human embryonic kidney cell line).
Among the human colorectal cell lines, APC is the wild type in HCT116, but the
mutant ones in SW480 and HT29. According to the results of MTT assay shown in
Table 1, the tested compounds seemed more potent against human-sourced colorectal
cells (GI50 < 10 μM: 12/20 for HCT116, 3/20 for SW480, 9/20 for HT29; GI50 > 30
μM: 3/20 for HCT116, 8/20 for SW480, 6/20 for HT29) than either other-sourced
colorectal cells (GI50 < 10 μM: 5/20 for CT26; GI50 > 30 μM: 9/20 for CT26) or
human-sourced other cells (GI50 < 10 μM: 6/20 for HeLa; GI50 > 30 μM: 9/20 for
HeLa). The safety could be basically ensured because the toxicity upon 293T cells
was low. Among the tested cell lines, HCT116 seemed the most sensitive one to our
series, which agreed with our previous study. The top hits, Q7 (GI50 = 1.95 μM), Q8
(GI50 = 1.57 μM), Q19 (GI50 = 1.37 μM) and Q20 (GI50 = 1.98 μM), all exhibited
comparable potency with the positive control Regorafenib (GI50 = 4.36 μM).
5

Although this experiment was affected by the status of cells, we could still infer that
this series was potential for blocking the growth of human colorectal cells. Meanwhile,
the compounds did not show obvious preference for either the wild or the mutant type
because their potency upon HT29 was also high.
Table1. In vitro antiproliferation inhibitory activity and cytotoxcity of compounds Q1-Q20.
Compounds
GI50 (μM)
HT29
CC50 (μM)
293T
HCT116
17.8±1.19
20.8±1.86
34.2±0.96
3.17±0.61
6.06±0.73
11.3±1.35
1.95±0.48
1.57±0.39
3.53±0.45
2.41±0.94
13.7±1.29
33.9±1.23
31.4±1.67
9.28±1.01
2.49±0.36
19.3±1.96
2.95±0.65
8.47±0.89
1.37±0.53
1.98±0.57
4.36±0.32
SW480
CT26
HeLa
52.4±1.21
43.7±1.72
38.6±1.44
8.83±0.70
24.2±0.93
38.9±1.66
6.76±0.17
29.5±0.97
4.72±0.94
20.7±1.16
21.3±1.26
29.8±1.38
36.1±1.85
29.2±1.34
36.9±1.45
42.1±1.64
25.7±1.49
21.5±1.36
10.9±0.87
30.3±1.94
10.7±0.96
38.8±1.92
30.9±1.42
28.8±1.16
2.33±0.46
40.2±1.17
68.4±1.88
1.82±0.49
3.55±0.61
2.12±0.28
9.75±0.74
27.3±1.07
42.4±1.49
24.3±0.86
2.67±0.19
13.5±0.29
59.4±1.72
1.59±0.32
2.03±0.16
1.22±0.33
23.5±1.48
0.073±0.02
38.3±1.15
19.9±0.51
26.8±0.73
2.80±0.21
32.6±1.41
71.8±1.82
6.36±0.36
9.78±0.98
7.08±0.64
13.0±0.87
42.9±1.20
56.9±1.46
27.1±0.75
8.28±0.83
14.9±0.56
> 200
44.3±1.69
27.8±1.25
41.8±1.36
6.61±0.56
37.8±0.97
53.4±1.60
7.59±0.83
4.35±0.20
3.84±0.43
26.8±0.90
18.4±0.63
61.9±1.72
43.4±1.43
9.20±0.41
12.8±0.13
59.5±1.95
4.61±0.38
76.3±1.76
19.7±0.81
30.2±1.15
8.43±0.61
265±7.66
130±4.14
99.3±3.77
91.2±6.35
150±12.82
144±9.66
104±8.72
92.9±7.13
178±9.60
117±7.83
103±7.99
147±6.39
89.4±5.08
137±8.25
86.8±5.06
190±15.93
142±8.93
137±7.10
119±8.30
148±5.23
139±9.69
Q1
Q2
Q3
Q4
Q5
Q6
Q7
Q8
Q9
Q10
Q11
Q12
Q13
Q14
Q15
Q16
Q17
Q18
Q19
Q20
Regorafenib
15.4±0.72
78.76±2.24
46.3±1.47
47.6±1.31
33.9±1.04
2.2.2. Blocking APC-Asef interactions
Consulting the established fluorescence polarization immunoassay to evaluate
the inhibitory activity upon APC-Asef interactions [34,35], we checked the
synthesized compounds in this series. The concentrations causing 50% inhibition
6

(IC50 values) described in Table 2 indicated that some of the candidates were potential.
The majority of them showed good potency with the range of IC50 values varying
from 1.02 to 30.5 μM. Not surprisingly, the top hits against human colorectal cells
also indicated sound potency in blocking APC-Asef interactions. Therefore we could
preliminarily connect the antiproliferation performance to the effect of interfering
APC-Asef interactions according to their linear correlation analysis (Correlation
coefficient R2 = 0.8422). Generally, the difference between setting the methoxy group
on R1 or R2 was not obvious. Whereas the substituent of aniline moiety suggested the
preference of electron-withdrawing groups than electron-donating ones. After this
stage, the most attractive hit Q19 was selected for the following evaluation.
Table 2. Inhibitory activities of compounds Q1-Q20 against the APC-Asef interactions
Compounds
Q1
IC50 (μM)
14.7±1.05
25.5±1.51
28.1±1.78
6.24±0.41
8.56±0.58
12.2±0.94
4.42±0.28
4.96±0.32
5.32±0.36
1.38±0.11
Compounds
Q11
IC50 (μM)
9.05±0.73
22.8±1.62
30.5±1.95
17.2±1.04
7.11±0.47
19.5±1.23
10.4±0.86
6.75±0.47
1.02±0.10
3.08±0.26
Q2
Q12
Q3
Q13
Q4
Q14
Q5
Q15
Q6
Q16
Q7
Q17
Q8
Q18
Q9
Q19
Q10
Q20
2.2.3. Apoptosis and Mitochondrial affection
7

Although the antiproliferation and APC-Asef interaction blocking suggested a
positive correlation, there was still a long story between these two nodes. The
checkpoint of apoptosis-related parameters was examined here to hint the mechanism.
HCT116 cells treated with Q19 were analyzed by flow-cytometry with Annexin
V-FITC/PI system. As shown in Figure2A, when the incubation time was set as 24 h,
along with the increasing Q19 concentrations (0, 0.20, 0.50, 1.0, 2.0, 5.0 μM), the
percentage of apoptotic cells was significantly elevated from 1.08% to 89.8%.
Therefore, in dose-dependent manners, the top hit Q19 could induce the apoptosis of
human colorectal cell HCT116. Moreover, since we replaced the naphthalene with
1-methyl-1H-indole moiety, the Mitochondrial affection was checked here by
measuring the variation of Mitochondrial membrane potential using the JC-1 assay.
As shown in Figure 2B, with the dose increasing, the mitochondrial aggregate form of
JC-1 gradually transferred into the monomeric form, suggesting the mitochondria
depolarization of HCT116 cells. This result indicated that the introduction of
1-methyl-1H-indole was beneficial in the induction procedure of apoptosis between
the APC-Asef interaction blocking and the antiproliferation, bringing the
Mitochondrial affection as well. In Figure 2C, we used the APC-mutant HT29 cells to
conduct the apoptosis assay again. Q19 could also induce cell apoptosis in HCT29
cells in a dose-dependent manner. However, with the same time condition, the
proportion of early and late apoptosis was quite different from that in HCT116 cells.
This point might be further investigated when we explore the pharmacology between
wt/mutant APC in future.
8

Figure 2. (A) Q19 induced cell apoptosis in HCT116 cells in a dose-dependent manner. (B) The
mitochondrial membrane potential were reduced by Q19. (C) Q19 could also induce cell apoptosis
in HT29 cells in a dose-dependent manner.
2.3. In silico study
For in silico study, the ADMET simulation was conducted initially to evaluate
the druggability. The parameters of AlogP (the partition coefficient of drug in
octanol/aqueous solution calculated by ACD/PhysChem Suite Software) and PSA_2D
(the fast calculated polar surface area from the 2D structure) were used to predict the
Absorption (human intestinal absorption) and BBB (blood-brain barrier penetration)
including 95% and 99% confidence ellipses as referenced [36]. There was a major
concern of this series. When we disassembled the morpholine moiety to form the
9

compounds in this work, whether the ADMET properties were still acceptable before
regaining a pharmacokinetic group. We set three series to conduct the comparison.
They were the previous series (purple) [35], the previous series with the
pharmacokinetic group morpholine replaced by a simple benzene (blue), and the
series in this work (reddish). In Figure 3, we could infer that although the druggability
was impacted by removing the morpholine, a latter introduction of similar moieties
(orange) could recover this temporary disadvantage. This kind of introduction might
be conducted by rational extension on the indole ring or the methylene of pyrazoline.
Figure 3. ADMET properties predicted for previous series and series in this work. "PG" meant
"pharmacokinetic group". Plots located inside the innermost oval are better for this parameter. The
percentages in the figure legend meant the 95% and 99% confidence ellipses.
10

The molecular docking models gave some hints for revealing the binding
patterns. Since we used CDOCKER module to perform the docking simulation, the
minus value of CDOCKER Interaction Energy was a crucial parameter to check the
binding capability of the compounds into the binding site. In this work, the values
varied within the range of 40.87-54.37 kcal/mol, which was comparable with that of
the previous series ranging within 35.46-56.45 kcal/mol. The linear correlation
analysis between this parameter and the APC-Asef interactions-blocking effect
inferred their positive correlation (Correlation coefficient R2 = 0.8512).
When we looked into the binding pattern, the result hinted possible interactions.
The binding situations generally agreed with the structure-activity relationship. As the
representative of this work, Q19 indicated typical and unique interactions with several
key residues on two of the three chains of APC (PDB code: 3NMX). As shown in
Figure 4, two conventional hydrogen bonds (with B: Arg499 and C: Lys453) and two
halogen interactions (with B: Asp539 and B: Gln542) suggested that the
trifluoromethyl groups here could bring several strong binding points. These
interactions could hardly be retained when trifluoromethyl groups were replaced by
other options including nitro- or fluoro- groups. Among all the other interactions
including π-sulfur with B: Met503, two pairs introduced by the 1-methyl-1H-indole
were reported for the first time. The key residue, B: Lys455, participated in both the
π-alkyl with and the carbon hydrogen bond. Since almost all modified moieties
indicated attractive interactions, the designing strategy in this work seemed success.
11

The 3D and the receptor surface models also agreed with the viewpoint of raising a
good binding situation.
Figure 4. (A) The 2D docking pattern of representative compounds Q19 into APC. (B) The 3D
conformation of Q19, key residues and corresponding interactions. (C) The binding position in the
whole APC protein. (D) The receptor surface model with Q19 in APC.
3. Conclusion
In summary, this work offered the novel pyrazoline derivatives containing
methyl-1H-indole moiety as potential inhibitors for blocking APC-Asef interactions.
With the introduction of the Mitochondria-linked moiety, this series realized the
Mitochondrial affection, which might connect the impact on the protein-interaction
12

level with the apoptosis events. The top hit Q19 suggested potency of inhibiting
APC-Asef interactions (IC50 = 1.02 μM) and attractive preference for human-sourced
colorectal cells (GI50 = 1.37 μM for HCT116; GI50 = 1.22 μM for HT29). Before a
further optimization with pharmacokinetic group, Q19 was already comparable with
the previous representative and the positive control Regorafenib. After bringing more
possibilities with another substituent site, introducing trifluoromethyl groups seemed
a promising approach for causing more key interactions such as H-bonds. With the
information in this work, further researches will achieve advanced molecules with
more attractive pre-clinical and even clinical potential.
4. Experimental
4.1. Materials and measurements
All chemicals (reagent grade) were purchased and used without further
purification. All the NMR spectra were recorded on Bruker DPX400 or Bruker
DPX600 model spectrometer in DMSO, with the chemical shifts reported as parts per
million. Melting points were determined on a X4 MP apparatus (Jingsong Corp,
Shanghai, China). Thin layer chromatography (TLC) was performed on the glass
backed silica gel sheets (Silica Gel 60 GF254) and visualized in UV light (254 nm
and 365 nm). Recombinant APC protein (HZ-APC-8) was purchased from Shanghai
Huzhen Industrial Co. LTD. (Shanghai, China). APC antibody (sc-15803) and Asef
anti-body (sc-13278) were purchased from Shanghai Haoran Biotechnology Co. LTD.
Annexcin V-FITC cell apoptosis assay kit (catalog No. BA11100) was purchased
13

from BIO-BOX (Nanjing, China). Matrixgel basement membrane transwell chambers
were purchased from Guangzhou Hehua technology co. LTD. (Guangzhou, China).
4.2. Synthesis
Indole-5-carboxaldehyde (1.0 mmol) was dissolved in anhydrous DMF under the
temperature of 0 ℃. To this solution, 1.5 mmol of NaH was added portionwise and the
mixture was allowed to react for 30 min. Then, 1.5 mmol of iodomethane in DMF
was added dropwise and the reaction was warmed to room temperature and stirring
for further 12 h. Then, reaction was quenched by 1 N HCl and washed with brine.
Organic layer was separated, dried over anhydrous Na2SO4, filtered, and evaporated
in vacuo. Crude products were purified by column chromatography using
n-hexane/ethyl acetate (4:1 v:v) as mobile phase, to obtain intermediate compounds 2
with 89% of yields.
To a solution of compound 2 (6 mmol) in 10 mL absolute ethyl alcohol was
added acetophenone derivatives (6 mmol) and 40% sodium hydroxide solution (9
mmol). The reaction was stirred at room temperature for 4 h and filtered to afford
compounds 5-6, which were used without further purification.
4-Hydrazinobenzoic acid (4.0 mmol), and compounds 5-6 (2.0 mmol) were
dissolved in methanol, then glacial acetic acid (0.2 mmol) was added in the solution
stirred at room temperature for 30 min. Then, the mixture was heated to reflux
temperature and stirred overnight. The course of the reaction was checked by TLC.
The crude products were purified by column chromatography using n-hexane/ethyl
acetate (2:1 v:v) as eluent leading to the corresponding compounds 7-8 with 70, 68%
14

of yields, respectively.
To a mixture of compounds 7-8 (1 mmol) in the dichloromethane (30 mL) was
added EDC (1.6 mmol), and HOBt (0.6 mmol), then the mixture was stirred at 0 ℃for
30 min. Aniline derivatives (0.6 mmol) was added subsequently, and the reaction was
transferred at room temperature and kept overnight. Reaction was quenched by 10%
aqueous solution of sodium bicarbonate and extracted with dichloromethane. The
organic layer was washed with 10% aqueous solution of sodium bicarbonate for three
times, dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. Crude
products were purified by column chromatography using n-hexane/ethyl acetate (6:1
v:v) as eluent, to obtain the target compounds Q1-Q20.
4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-
phenylbenzamide (Q1)
Yellow crystal, yield: 80.6%, m.p. 204-212 ℃. 1H NMR (400 MHz, DMSO-d6) δ
9.82 (s, 1H), 7.76 (s, 1H), 7.74 (s, 1H), 7.71 (s, 2H), 7.69 (s, 2H), 7.46 (s, 1H), 7.39 (d,
J = 8.4 Hz, 1H), 7.31 – 7.25 (m, 5H), 7.11 (s, 1H), 7.09 (s, 1H), 7.08-7.00 (m, 2H),
6.37 (d, J = 3.0 Hz, 1H), 5.67 (dd, J = 12.1, 5.3 Hz, 1H), 3.98 (dd, J = 17.7, 12.1 Hz,
13
1H), 3.74 (s, 3H), 3.19 (dd, J = 17.6, 5.4 Hz, 1H), 2.35 (s, 3H). C NMR (101 MHz,
DMSO-d6) δ 149.71, 140.04, 139.24, 133.08, 130.73, 129.85, 129.77, 129.35, 128.92,
128.64, 126.42, 124.15, 123.50, 120.53, 119.37, 117.92, 112.35, 110.95, 100.84,
63.51, 43.99, 32.96, 21.47. HRMS (ESI-TOF) Calcd. for C32H28N4O2 [M+H]+
501.2265; Found 501.2267.
N-(4-methoxyphenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihyd
15

ro-1H-pyrazol-1-yl)benzamide (Q2)
1
White solid, yield: 86.3%, m.p. 112-117 ℃. H NMR (600 MHz, DMSO-d6) δ
9.00 (s, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.82-7.78 (m, 1H),
7.75 (t, J = 9.1 Hz, 2H), 7.70 (d, J = 7.9 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.51 (t, J =
7.7 Hz, 1H), 7.45 (s, 1H), 7.41 (dd, J = 16.7, 8.5 Hz, 1H), 7.34 – 7.29 (m, 2H), 7.27 (d,
J = 8.0 Hz, 1H), 7.10 (dd, J = 8.1, 5.9 Hz, 1H), 7.06 (ddt, J = 8.4, 4.0, 1.7 Hz, 1H),
6.95-6.90 (m, 1H), 6.38 (dd, J = 10.0, 3.1 Hz, 1H), 5.81 (dd, J = 11.8, 4.6 Hz, 1H),
4.01 (ddd, J = 45.2, 17.7, 12.0 Hz, 1H), 3.80 (s, 3H), 3.75 (d, J = 5.6 Hz, 3H), 3.22
(ddd, J = 53.3, 17.7, 5.0 Hz, 1H), 2.36 (d, J = 8.5 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6) δ 150.16, 147.61, 146.83, 136.25, 133.05, 131.18, 130.76, 129.78, 129.12,
128.63, 126.48, 123.86, 123.67, 120.62, 119.85, 119.29, 117.82, 112.45, 111.59,
111.00, 100.83, 63.33, 56.12, 44.06, 32.96, 21.47. HRMS (ESI-TOF) Calcd. for
C33H30N4O3 [M+H]+ 531.2373; Found 531.2371.
N-(3-bromophenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzamide (Q3)
1
White solid, yield: 76.5%, m.p. 110-115 ℃. H NMR (600 MHz, DMSO-d6) δ
9.98 (s, 1H), 8.04 (s, 1H), 7.94 (d, J = 8.6 Hz, 0.5H), 7.86 (d, J = 7.9 Hz, 0.5H), 7.73
(dd, J = 19.4, 8.3 Hz, 3H), 7.68 (d, J = 8.4 Hz, 1H), 7.48-7.44 (m, 1H), 7.40 (d, J =
8.5 Hz, 1H), 7.33-7.20 (m, 5H), 7.12-7.08 (m, 2H), 7.06 (dd, J = 8.5, 1.4 Hz, 1H),
6.37 (d, J = 3.0 Hz, 1H), 5.69 (dd, J = 12.1, 5.3 Hz, 1H), 3.98 (dd, J = 17.5, 12.1 Hz,
1H), 3.81 (s, 1H), 3.74 (s, 2H), 3.19 (dd, J = 17.5, 5.3 Hz, 1H), 2.36 (d, J = 5.3 Hz,
3H). 13C NMR (151 MHz, DMSO-d6) δ 165.77, 149.92, 146.89, 141.71, 139.31,
16

136.25, 133.02, 130.97, 130.76, 129.79, 129.48, 128.61, 126.46, 126.03, 123.54,
122.62, 121.80, 119.35, 119.10, 117.91, 112.36, 110.99, 100.83, 63.40, 43.95, 32.97,
21.48. HRMS (ESI-TOF) Calcd. for C32H27BrN4O2 [M+H]+ 579.1379; Found
579.1377.
N-(4-chlorophenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzamide (Q4)
1
Yellow solid, yield: 60%, m.p. 142-149 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.6 Hz, 1H), 8.00 (dd, J = 8.4, 6.4 Hz, 2H), 7.80 (d, J = 8.3 Hz, 1H), 7.76
(d, J = 8.2 Hz, 2H), 7.64 – 7.58 (m, 1H), 7.54-7.48 (m, 1H), 7.46 (d, J = 1.7 Hz, 1H),
7.41 (dd, J = 14.4, 8.5 Hz, 2H), 7.33 (d, J = 3.1 Hz, 1H), 7.30 (s, 1H), 7.29 (s, 1H),
7.23 (s, 1H), 7.07 (dd, J = 8.5, 1.8 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 5.81 (dd, J =
11.9, 4.6 Hz, 1H), 4.04 (dd, J = 17.8, 11.9 Hz, 1H), 3.75 (s, 3H), 3.26 (dd, J = 17.8,
13
4.6 Hz, 1H), 2.36 (s, 3H). C NMR (151 MHz, DMSO-d6) δ 162.87, 152.65, 149.28,
143.23, 140.08, 136.34, 132.84, 132.34, 130.89, 129.87, 129.53, 129.27, 128.68,
126.86, 125.66, 120.27, 119.15, 117.73, 113.01, 111.41, 111.13, 109.77, 100.90,
63.09, 44.13, 32.98, 21.51. HRMS (ESI-TOF) Calcd. for C32H27ClN4O2 [M+H]+
535.1813; Found 535.1812.
N-(4-fluorophenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzamide (Q5)
1
White solid, yield: 83.3%, m.p. 128-135 ℃. H NMR (600 MHz, DMSO-d6) δ
9.68 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.6 Hz,
1H), 7.71 (d, J = 8.0 Hz, 1H), 7.62-7.57 (m, 1H), 7.54 (t, J = 7.9, 1H), 7.47 (dd, J =
17

8.6, 2.9 Hz, 1H), 7.45 (d, J = 1.6 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 3.2 Hz,
1H), 7.27 (t, J = 6.4 Hz, 2H), 7.23-7.20 (m, 1H), 7.17 (t, J = 7.5, 1.7 Hz, 1H), 7.09 (d,
J = 10.5 Hz, 1H), 7.05 (t, J = 9.3 Hz, 1H), 6.37 (d, J = 3.0 Hz, 1H), 5.69 (dd, J = 12.1,
5.2 Hz, 1H), 3.98 (dd, J = 17.6, 12.1 Hz, 1H), 3.77 (d, J = 39.8 Hz, 3H), 3.19 (dd, J =
17.6, 5.2 Hz, 1H), 2.36 (d, J = 6.8 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.33,
161.80, 151.58, 148.74, 145.84, 142.16, 139.01, 135.18, 131.27, 129.81, 128.46,
127.85, 126.30, 125.55, 124.81, 124.58, 123.68, 122.50, 122.04, 119.20, 118.24,
116.79, 111.29, 110.05, 108.70, 99.75, 62.34, 43.05, 31.88, 20.40. HRMS (ESI-TOF)
Calcd. for C32H27FN4O2 [M+H]+ 519.2217; Found 519.2216.
4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-
(o-tolyl)benzamide (Q6)
1
White solid, yield: 76.9%, m.p. 112-115 ℃. H NMR (600 MHz, DMSO-d6) δ
10.13 (s, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 9.1 Hz, 2H), 8.00 (d, J = 9.4 Hz,
1H), 7.87 (d, J = 8.4 Hz, 1H), 7.83-7.80 (m, 3H), 7.67 (ddd, J = 8.1, 6.9, 0.9 Hz, 1H),
7.54 (ddd, J = 8.2, 6.9, 1.0 Hz, 1H), 7.46 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.07 (dd, J
= 8.5, 1.8 Hz, 1H), 7.05-7.03 (m, 1H), 7.00 (d, J = 2.2 Hz, 1H), 6.99 (d, J = 2.1 Hz,
1H), 6.39 (d, J = 3.1 Hz, 1H), 5.80 (td, J = 11.6, 4.5 Hz, 1H), 4.05 (ddd, J = 17.6, 11.8,
5.2 Hz, 1H), 3.82 (s, 1.5H), 3.80 (s, 3H), 3.76 (s, 1.5H), 3.26 (dd, J = 17.8, 4.6 Hz,
1H), 2.34 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.67, 165.15, 159.91, 151.76,
149.61, 146.69, 139.03, 136.62, 134.52, 133.35, 132.96, 131.32, 130.76, 129.44,
128.85, 127.80, 127.06, 124.86, 122.30, 121.95, 119.35, 118.37, 115.50, 114.34,
112.50, 110.52, 106.60, 101.40, 100.84, 63.57, 55.65, 43.91, 32.96. HRMS (ESI-TOF)
18

Calcd. for C33H30N4O2 [M+H]+ 515.2480; Found 515.2478.
N-(3,4-dichlorophenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihy
dro-1H-pyrazol-1-yl)benzamide (Q7)
1
Yellow solid, yield: 59.8%, m.p. 114-119 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 9.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.76 (d, J
= 8.2 Hz, 2H), 7.62 (ddd, J = 8.1, 6.9, 0.9 Hz, 1H), 7.51 (ddd, J = 8.2, 7.0, 1.0 Hz,
1H), 7.46 (d, J = 1.7 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 3.1 Hz, 1H), 7.30
(d, J = 8.1 Hz, 2H), 7.23 (d, J = 9.8 Hz, 1H), 7.06 (dt, J = 9.2, 2.8 Hz, 1H), 6.39 (dd, J
= 3.1, 0.8 Hz, 1H), 5.81 (dd, J = 11.8, 4.6 Hz, 1H), 4.05 (dd, J = 17.8, 11.8 Hz, 1H),
13
3.75 (d, J = 9.6 Hz, 3H), 3.26 (dd, J = 17.8, 4.6 Hz, 1H), 2.36 (s, 3H). C NMR (151
MHz, DMSO-d6) δ 167.71, 150.15, 147.61, 139.38, 136.25, 132.98, 131.18, 130.76,
129.78, 128.62, 128.26, 127.92, 126.48, 119.85, 119.22, 117.72, 112.34, 110.99,
109.99, 100.83, 63.33, 44.06, 32.96, 21.46. HRMS (ESI-TOF) Calcd. for
C32H26Cl2N4O2 [M+H]+ 569.1464; Found 569.1463.
N-(3,4-dinitrophenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydr
o-1H-pyrazol-1-yl)benzamide (Q8)
1
White solid, yield: 85.5%, m.p. 135-142 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.5 Hz, 1H), 8.01 (s, 1H), 7.99 (s, 1H), 7.83-7.78 (m, 3H), 7.61 (t, J = 7.6
Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H),
7.33 (d, J = 3.1 Hz, 1H), 7.21 (s, 1H), 7.07 (dd, J = 8.5, 1.8 Hz, 1H), 7.05 (s, 1H),
7.03 (s, 1H), 6.39 (dd, J = 3.1, 0.8 Hz, 1H), 5.79 (dd, J = 11.8, 4.5 Hz, 1H), 4.04 (dd,
J = 17.8, 11.8 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.26 (dd, J = 17.8, 4.5 Hz, 1H). 13C
19

NMR (151 MHz, DMSO-d6) δ 167.73, 160.65, 150.08, 147.70, 133.04, 131.18,
130.75, 128.62, 128.11, 125.02, 124.92, 119.76, 119.60, 119.23, 117.71, 114.66,
112.20, 110.98, 110.00, 100.83, 63.27, 55.75, 44.20, 32.96. HRMS (ESI-TOF) Calcd.
for C32H26N6O6 [M+H]+ 591.1967; Found 591.1866.
N-(3,5-bis(trifluoromethyl)phenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-
yl)-4,5-dihydro-1H-pyrazol-1-yl)benzamide (Q9)
1
White solid, yield: 90.6%, m.p. 128-140 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 9.3 Hz, 2H), 7.80 (dd, J = 8.5, 6.6 Hz, 3H), 7.61
(ddd, J = 8.1, 6.9, 0.9 Hz, 1H), 7.51 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H), 7.46 (s, 1H), 7.42
(d, J = 8.4 Hz, 1H), 7.33 (d, J = 3.1 Hz, 1H), 7.21 (s, 1H), 7.07 (dd, J = 8.5, 1.7 Hz,
1H), 7.05 (s, 1H), 7.03 (s, 1H), 6.40-6.35 (d, J = 3.0 Hz , 1H), 5.78 (dd, J = 11.8, 4.5
Hz, 1H), 4.03 (dd, J = 17.8, 11.8 Hz, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.26 (dd, J =
17.8, 4.5 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 167.74, 162.88, 161.09, 160.64,
150.07, 149.31, 147.71, 136.25, 133.04, 132.83, 131.19, 130.88, 130.74, 129.53,
128.55, 128.27, 128.10, 125.65, 125.02, 120.27, 119.61, 119.23, 117.71, 114.74,
114.65, 112.87, 110.98, 109.77, 100.83, 63.29, 55.74, 44.20, 32.95. HRMS (ESI-TOF)
Calcd. for C34H26F6N4O2 [M+H]+ 637.2044; Found 637.2043.
N-(3,4-difluorophenyl)-4-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihy
dro-1H-pyrazol-1-yl)benzamide (Q10)
1
Yellow solid, yield: 78.2%, m.p. 130-138 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.80 (dd, J = 8.4, 5.8 Hz, 3H), 7.61
(t, J = 7.6 Hz, 1H), 7.51 (t, J = 8.6Hz, 1H), 7.46 (s, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.33
20

(d, J = 3.1 Hz, 1H), 7.21 (s, 1H), 7.05 (dd, J = 15.5, 8.7 Hz, 3H), 6.39 (d, J = 3.1 Hz,
1H), 5.79 (dd, J = 11.8, 4.5 Hz, 1H), 4.04 (dd, J = 17.8, 11.8 Hz, 1H), 3.82 (s, 3H),
3.75 (s, 3H), 3.26 (dd, J = 17.8, 4.5 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ
167.73, 160.64, 152.58, 150.07, 149.31, 147.71, 136.33, 136.25, 133.04, 132.83,
131.19, 130.74, 128.63, 128.56, 128.11, 125.02, 124.53, 120.27, 119.61, 119.23,
117.71, 114.65, 112.21, 111.11, 110.98, 109.77, 100.83, 63.28, 55.75, 44.20, 32.96.
HRMS (ESI-TOF) Calcd. for C32H26F2N4O2 [M+H]+ 536.2052; Found 536.2050.
4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-
phenylbenzamide (Q11)
1
White solid, yield: 85.2%, m.p. 145-150 ℃. H NMR (600 MHz, DMSO-d6) δ
9.85 (s, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.46 (s, 1H), 7.39 (dd,
J = 16.3, 9.4 Hz, 3H), 7.34 (s, 1H), 7.32-7.27 (m, 3H), 7.12 (d, J = 8.5 Hz, 2H), 7.07
(d, J = 8.6 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.37 (d, J = 3.0
Hz, 1H), 5.71 (dd, J = 12.2, 5.4 Hz, 1H), 3.99 (dd, J = 17.5, 12.1 Hz, 1H), 3.83 (s,
3H), 3.74 (s, 3H), 3.22 (dd, J = 17.6, 5.4 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ
165.58, 159.91, 149.50, 146.58, 140.01, 136.26, 133.92, 133.00, 130.76, 130.29,
129.37, 128.94, 128.62, 124.37, 123.53, 120.50, 119.36, 118.97, 117.92, 115.49,
112.48, 111.30, 110.98, 100.83, 63.58, 55.65, 43.91, 39.54, 32.97. HRMS (ESI-TOF)
Calcd. for C32H28N4O2 [M+H]+ 501.2325; Found 501.2325.
N-(2-methoxyphenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihyd
ro-1H-pyrazol-1-yl)benzamide (Q12)
1
White solid, yield: 93.6%, m.p. 158-163 ℃. H NMR (600 MHz, DMSO-d6) δ
21

9.01 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.46 (s, 1H), 7.42-7.34
(m, 4H), 7.31 (d, J = 3.1 Hz, 1H), 7.11 (dd, J = 10.4, 7.7 Hz, 3H), 7.05 (dd, J = 13.9,
8.4 Hz, 2H), 6.98 (d, J = 7.4 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.37 (d, J = 3.1 Hz,
1H), 5.69 (dd, J = 12.1, 5.4 Hz, 1H), 3.98 (dd, J = 17.6, 12.2 Hz, 1H), 3.82 (s, 3H),
3.80 (s, 3H), 3.74 (s, 3H), 3.21 (dd, J = 17.6, 5.4 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 164.87, 159.91, 151.28, 149.54, 146.69, 136.28, 132.98, 130.77, 130.29,
129.12, 128.65, 127.74, 125.37, 123.93, 123.84, 120.62, 119.29, 118.99, 117.84,
115.57, 112.61, 111.58, 111.23, 111.00, 100.84, 63.60, 56.13, 55.64, 43.98, 32.97.
HRMS (ESI-TOF) Calcd. for C33H30N4O3 [M+H]+ 530.2451; Found 530.2450.
N-(3-bromophenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzamide (Q13)
Yellow solid, yield: 79.2%, m.p. 135-142 ℃. 1H NMR (600 MHz, DMSO-d6) δ
10.00 (s, 1H), 8.11 (d, J = 55.5 Hz, 1H), 8.02 (d, J = 9.4 Hz, 1H), 7.76 (d, J = 8.8 Hz,
1H), 7.62 (t, J = 8.0 Hz, 1H), 7.53-7.48 (m, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.39 (t, J =
7.7 Hz, 2H), 7.37-7.21 (m, 4H), 7.13 (d, J = 8.7 Hz, 1H), 7.07 (ddd, J = 8.6, 3.8, 1.8
Hz, 1H), 7.05-6.95 (m, 1H), 6.38 (ddd, J = 11.0, 3.1, 0.8 Hz, 1H), 5.77 (ddd, J = 72.1,
12.1, 5.0 Hz, 1H), 4.02 (ddd, J = 41.7, 17.8, 12.1 Hz, 1H), 3.83 (d, J = 5.2 Hz, 3H),
3.75 (d, J = 7.8 Hz, 3H), 3.27 (ddd, J = 43.9, 17.7, 5.0 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 165.77, 159.95, 152.48, 149.23, 143.22, 136.35, 133.88, 132.85, 130.97,
129.49, 129.06, 128.68, 125.68, 124.88, 123.01, 122.63, 120.28, 119.17, 117.73,
116.25, 115.54, 113.18, 112.51, 111.72, 111.00, 109.80, 100.90, 63.21, 55.72, 44.12,
32.97. HRMS (ESI-TOF) Calcd. for C32H27BrN4O2 [M+H]+ 578.1347; Found
22

578.1346.
N-(4-chlorophenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzamide (Q14)
1
Yellow solid, yield: 74.3%, m.p. 135-142 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 9.3 Hz, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.62 (t, J =
7.6 Hz, 1H), 7.51 (ddd, J = 8.2, 7.0, 1.0 Hz, 1H), 7.46 (d, J = 1.7 Hz, 1H), 7.45-7.42
(m, 2H), 7.43-7.37 (m, 3H), 7.33 (d, J = 3.1 Hz, 1H), 7.26 (s, 1H), 7.07 (dd, J = 8.5,
1.8 Hz, 1H), 7.04 (ddd, J = 8.1, 2.6, 1.1 Hz, 1H), 6.39 (dd, J = 3.1, 0.8 Hz, 1H), 5.84
(dd, J = 11.9, 4.6 Hz, 1H), 4.06 (dd, J = 17.9, 11.9 Hz, 1H), 3.83 (d, J = 9.5 Hz, 3H),
3.75 (d, J = 9.8 Hz, 3H), 3.30 (dd, J = 17.9, 4.6 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 167.69, 159.90, 149.95, 147.48, 133.81, 132.91, 131.18, 130.78, 130.29,
128.63, 120.11, 119.22, 119.04, 117.72, 115.68, 112.50, 111.26, 111.00, 100.84,
63.46, 55.65, 44.03, 32.96. HRMS (ESI-TOF) Calcd. for C32H27ClN4O2 [M+H]+
534.1894; Found 534.1892.
N-(4-fluorophenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzamide (Q15)
1
White solid, yield: 80.2%, m.p. 129-137 ℃. H NMR (600 MHz, DMSO-d6) δ
9.71 (s, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.02 (s, 1H), 8.01 (s, 1H), 7.79 (dd, J = 17.5,
8.7 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.50 (dd, J = 16.3, 8.2 Hz, 2H), 7.47-7.35 (m,
5H), 7.33 (d, J = 3.1 Hz, 1H), 7.26 (s, 1H), 7.07 (dd, J = 8.5, 1.7 Hz, 1H), 7.04 (dd, J
= 7.6, 2.2 Hz, 1H), 6.39 (d, J = 3.0 Hz, 1H), 5.83 (dd, J = 11.9, 4.6 Hz, 1H), 4.06 (dd,
J = 17.9, 12.0 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.30 (dd, J = 17.9, 4.6 Hz, 1H). 13C
23

NMR (151 MHz, DMSO-d6) δ 158.87, 151.38, 148.15, 142.15, 135.28, 132.28,
131.76, 129.83, 129.32, 128.47, 127.93, 124.59, 119.20, 118.31, 118.09, 116.67,
115.16, 112.10, 111.44, 110.65, 110.05, 108.71, 99.83, 62.15, 54.63, 43.04, 31.91.
HRMS (ESI-TOF) Calcd. for C32H27FN4O2 [M+H]+ 518.2189; Found 518.2188.
4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-
(o-tolyl)benzamide (Q16)
1
Yellow solid, yield: 70.5%, m.p. 130-144 ℃. H NMR (600 MHz, DMSO-d6) δ
10.13 (s, 1H), 8.14 (dd, J = 8.8, 5.7 Hz, 2H), 8.00 (d, J = 9.4 Hz, 2H), 7.81 (dd, J =
8.8, 3.3 Hz, 3H), 7.61 (t, J = 7.7 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 7.46-7.42 (m, 3H),
7.33 (d, J = 3.1 Hz, 1H), 7.07 (dd, J = 8.5, 1.8 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 7.03
(d, J = 2.6 Hz, 1H), 6.39 (dd, J = 3.0, 0.8 Hz, 1H), 5.79 (dd, J = 11.8, 4.5 Hz, 1H),
4.04 (dd, J = 17.8, 11.8 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.76 (s, 3H), 3.26 (dd, J =
17.8, 4.5 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 161.79, 158.82, 151.39, 148.14,
145.48, 142.14, 135.18, 132.83, 131.21, 129.70, 128.47, 127.92, 125.77, 124.90,
123.79, 119.19, 118.08, 117.88, 116.65, 115.16, 114.41, 112.09, 111.44, 110.64,
108.71, 99.82, 99.75, 62.49, 54.57, 31.89, 17.33. HRMS (ESI-TOF) Calcd. for
C33H30N4O2 [M+H]+ 514.2495; Found 514.2495.
N-(3,4-dichlorophenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihy
dro-1H-pyrazol-1-yl)benzamide (Q17)
1
White solid, yield: 93.3%, m.p. 100-112 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.9 Hz, 2H), 7.81 (d, J = 8.3 Hz, 1H), 7.62 (t, J =
7.4 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.46 (s, 1H), 7.44 (t, J = 7.6 Hz, 2H), 7.41-7.38
24

(m, 2H), 7.33 (d, J = 3.1 Hz, 1H), 7.26 (s, 1H), 7.07 (dd, J = 8.5, 1.7 Hz, 1H), 7.04
(dd, J = 8.0, 2.5 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 5.84 (dd, J = 11.9, 4.6 Hz, 1H),
4.06 (dd, J = 17.9, 11.9 Hz, 1H), 3.84 (s, 3H),3.76 (s, 3H), 3.30 (dd, J = 17.9, 4.6 Hz,
1H). 13C NMR (151 MHz, DMSO-d6) δ 158.86, 151.39, 148.14, 142.14, 135.27,
132.28, 131.76, 129.83, 129.32, 128.47, 127.93, 127.60, 124.59, 119.19, 118.31,
118.09, 116.65, 115.17, 112.09, 110.64, 110.60, 110.05, 108.71, 99.82, 62.14, 54.63,
43.04, 31.91. HRMS (ESI-TOF) Calcd. for C32H26Cl2N4O2 [M+H]+ 568.1433; Found
568.1431.
4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-
(4-nitrophenyl)benzamide (Q18)
1
White solid, yield: 75.5%, m.p. 105-110 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 9.3 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.62 (t, J =
7.6 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.46 (s, 1H), 7.44 (t, J = 7.0 Hz, 1H), 7.42-7.38
(m, 3H), 7.33 (d, J = 3.1 Hz, 1H), 7.26 (s, 1H), 7.07 (dd, J = 8.5, 1.7 Hz, 1H), 7.04
(dd, J = 8.1, 2.7 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 5.84 (dd, J = 12.0, 4.6 Hz, 1H),
4.06 (dd, J = 17.9, 11.9 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.30 (dd, J = 17.9, 4.6 Hz,
1H). 13C NMR (151 MHz, DMSO-d6) δ 159.94, 152.48, 149.22, 143.22, 136.35,
133.36, 132.84, 132.29, 130.91, 130.40, 129.56, 128.68, 125.68, 120.27, 119.39,
119.17, 117.73, 116.25, 113.17, 111.72, 111.68, 111.13, 109.80, 100.90, 63.21, 55.72,
44.12, 33.00. HRMS (ESI-TOF) Calcd. for C32H27N5O4 [M+H]+ 545.2179; Found
545.2178.
N-(3,5-bis(trifluoromethyl)phenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-
25

yl)-4,5-dihydro-1H-pyrazol-1-yl)benzamide (Q19)
1
White solid, yield: 80.4%, m.p. 138-147 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 8.3 Hz, 1H), 7.62 (t, J =
7.7 Hz, 1H), 7.51 (t, J = 7.0 Hz, 1H), 7.46 (s, 1H), 7.44 (t, J = 7.1 Hz, 2H), 7.41-7.38
(m, 2H), 7.33 (d, J = 3.0 Hz, 1H), 7.26 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 7.04 (dd, J =
8.1, 2.3 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 5.84 (dd, J = 11.9, 4.6 Hz, 1H), 4.06 (dd, J
13
= 17.9, 11.9 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.30 (dd, J = 17.9, 4.6 Hz, 1H). C
NMR (151 MHz, DMSO-d6) δ 131.10, 117.43, 116.00, 111.44, 109.40, 108.05, 99.17,
61.49, 53.97, 42.38, 38.76, 38.64, 38.62, 38.50, 38.37, 38.23, 38.09, 37.95, 37.81,
31.25. HRMS (ESI-TOF) Calcd. for C34H26F6N4O2 [M+H]+ 636.2058; Found
636.2056.
N-(3,4-difluorophenyl)-4-(3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-5-yl)-4,5-dihy
dro-1H-pyrazol-1-yl)benzamide (Q20)
1
White solid, yield: 85.7%, m.p. 125-140 ℃. H NMR (600 MHz, DMSO-d6) δ
8.14 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 9.2 Hz, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.62 (t, J =
7.8 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.47 (s, 1H), 7.44 (t, J = 7.3 Hz, 2H), 7.41-7.38
(m, 2H), 7.33 (d, J = 3.1 Hz, 1H), 7.26 (s, 1H), 7.07 (d, J = 7.0 Hz, 1H), 7.04 (dd, J =
7.8, 2.1 Hz, 1H), 6.39 (d, J = 2.9 Hz, 1H), 5.83 (dd, J = 11.9, 4.6 Hz, 1H), 4.06 (dd, J
= 17.9, 11.9 Hz, 1H), 3.83 (d, J = 9.4 Hz, 3H), 3.75 (d, J = 9.8 Hz, 3H), 3.30 (dd, J =
17.9, 4.6 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 157.47, 149.96, 146.75, 140.77,
130.89, 130.34, 128.43, 127.91, 127.05, 123.18, 117.80, 116.91, 116.69, 115.29,
113.74, 110.70, 109.26, 109.22, 108.65, 107.29, 98.44, 60.78, 53.22, 41.64, 30.51.
26

HRMS (ESI-TOF) Calcd. for C32H26F2N4O2 [M+H]+ 536.2082; Found 536.2081.
4.3. Anti-proliferative assay
Five cancer cell lines, HCT116, SW480, HT29 (human colorectal cell lines),
CT26 (mice colorectal cell lines), HeLa (human cervix cell line), and one noncancer
cell line, 293T (human embryonic kidney cell line) were used in the evaluation of
antiproliferation activities with a standard MTT-based colorimetric assay. The cells
were seeded at a density of 5 * 104 /well into 96-well plates and incubated for 12 h at
37 ℃, 5% CO2 atmosphere. Experimental groups were treated with concentrations of
synthesized compounds and Regorafenib was used as the positive control. MTT was
applied to check the status of the cells after 48 h. The absorbance (OD 570 nm) was
read on an ELISA reader (ELx800, BioTek, USA) with reference wavelength of 630
nm. Then the GI50 values were calculated.
4.4. Inhibitory activity upon APC-Asef interactions
We used the previously established fluorescence polarization immunoassay to
evaluate the inhibitory activity upon APC-Asef interactions [34,35]. The proteins and
corresponding anti-bodies were purchased as mentioned in the materials. The
concentrations of APC and Asef were set as 1 mM when the stock solution was
prepared. Along with the diluting of Asef solution, the concentrations of Corning
NBS 3995 were set in the gradient of 1, 0.1, 0.01 and 0.001 mM. After adding APC,
the mixture was incubated at room temperature for 2 h. Then tested compounds were
added at various concentrations. After a further incubation for 2 h, the fluorescent
signals at 525 nm were read with the excitation wavelength of 480 nm. Then, the IC50
27

values were calculated accordingly.
4.5. Cell apoptosis assay
HCT116 cells were seeded in six-well plates at the density of 2 * 105 /well, and
treated with compound Q19 at final concentrations of 0, 0.2, 0.5, 1.0, 2.0 and 5.0 μM
for 24 h to induce cell apoptosis. Then the cells were harvested and washed twice with
ice-cold PBS. After being resuspended by binding buffer, the cells were incubated
with Annexin-V/FITC (5 mL) and PI (5 mL) for a further 10 min in the dark. The
same protocol was also used for HT29. We used a flow-cytometer (Becton Dickinson,
USA) to analyze the result and conducted the statistical analysis with Flowjo 7.6.1
software.
4.6. Mitochondrial membrane potential assay
Mitochondrial transmembrane potential was detected with a JC-1 mitochondrial
membrane potential assay kit (Beyotime Biotech). After the incubation with 5 μg/mL
JC-1 at 37 ℃ for 20 min, the cells were washed twice with PBS and placed in fresh
medium without serum. The samples were analyzed using a FACSCalibur cytometer
(Becton Dickinson) at 488 nm.
4.7. Molecular docking simulation
The crystal structures of APC protein (PDB Code: 3NMX) was retrieved from
the RCSB Protein Data Bank (http://www.rcsb.org). After a procedure of eliminating
water and adding polar hydrogen, the protein was well prepared. Molecular docking
of all compounds was then carried out using the Discovery Studio (version 3.5) as
implemented through the graphical user interface CDOCKER protocol, which was an
28

implementation of a CHARMm based molecular docking tool using a half-flexible
receptor.
Supporting Information
NMR spectra were detailed in the Supporting Information. Supporting
Information associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.XXX.2019.XX.XXX.
Acknowledgments
This work is supported by the National Undergraduate Innovation Program and
the National Science Foundation of China (No. S201910284029).
Declaration of Competing Interest
The authors have no conflict of interests to declare.
References
[1] L. Vanaelst, M. Barr, S. Marcus, A. Polverino, M. Wigler, Complex-formation
between Ras and Raf and other protein-kinases, Proc. Natl. Acad. Sci. U.S.A. 90
(1993) 6213-6217.
[2] H. Chong, H. G. Vikis, K. L. Guan, Mechanisms of regulating the Raf kinase
family, Cell. Signal. 15 (2003) 463-469.
[3] P. Chene, Inhibiting the p53-MDM2 interaction: An important target for cancer
therapy, Nat. Rev. Cancer 3 (2003) 102-109.
[4] O. Vanunu, O. Magger, E. Ruppin, T. Shlomi, R. Sharan, Associating genes and
29