Discovery of novel 2-aryl-3-sulfonamido-pyridines (HoAns) as microtubule polymerization inhibitors with potent antitumor activities

Article abstract of DOI:10.1016/j.ejmech.2020.113117

Microtubules play a vital role in cell mitosis. Drugs targeting taxol or vinca binding site of tubulin have been proved an effective way to against cancer. However, drug resistance and cancer recurrence are inevitable, there is an urgent need to search for new microtubule-targeting agents (MTAs). In our study, a series of novel 2-aryl-3-sulfonamido-pyridines (HoAns) had been designed, synthesized, and evaluated for their antiproliferative activities in vitro and in vivo. Among them, compound HoAn32 exhibited the most potent activity with IC₅₀ values ranging from 0.170 to 1.193 μM in a panel of cancer cell lines. Mechanism studies indicated that compound HoAn32 bound to the colchicine site of β-tubulin, resulting in colony formation inhibition, G2/M phase cell cycle arrest, cell apoptosis as well as increased the generation of ROS in both RKO and SW620 cells. In addition, compound HoAn32 showed potent anti-vascular activity in vitro. Furthermore, compound HoAn32 also exhibited outstanding antitumor activity in SW620 xenograft tumor models without observable toxic effects, which was more potent than that of ABT-751. In conclusion, our findings suggest that compound HoAn32 may be a promising microtubule destabilizing agent and deserves for further development in cancer therapy.

Full text of DOI:10.1016/j.ejmech.2020.113117

European Journal of Medicinal Chemistry 211 (2021) 113117
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel 2-aryl-3-sulfonamido-pyridines (HoAns) as
microtubule polymerization inhibitors with potent antitumor
activities
Heping Zhu a, Shilong Ying b, Bingluo Zhou b, Xiao Liang b, Quan He a, Ping Song b,
,
, *
Xinyang Hu b, Keqiang Shi b, Mingteng Xiong a, Hongchuan Jin b **, Yuanjiang Pan a
a Department of Chemistry, Zhejiang University, Hangzhou, 310027, PR China
b Laboratory of Cancer Biology, Key Lab of Biotherapy in Zhejiang, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, 310020, PR
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Microtubules play a vital role in cell mitosis. Drugs targeting taxol or vinca binding site of tubulin have
been proved an effective way to against cancer. However, drug resistance and cancer recurrence are
inevitable, there is an urgent need to search for new microtubule-targeting agents (MTAs). In our study, a
series of novel 2-aryl-3-sulfonamido-pyridines (HoAns) had been designed, synthesized, and evaluated
for their antiproliferative activities in vitro and in vivo. Among them, compound HoAn32 exhibited the
Received 8 October 2020
Received in revised form
14 December 2020
Accepted 16 December 2020
Available online 25 December 2020
most potent activity with IC50 values ranging from 0.170 to 1.193
m
M in a panel of cancer cell lines.
-tubulin, resulting
Mechanism studies indicated that compound HoAn32 bound to the colchicine site of
b
Keywords:
in colony formation inhibition, G2/M phase cell cycle arrest, cell apoptosis as well as increased the
generation of ROS in both RKO and SW620 cells. In addition, compound HoAn32 showed potent anti-
vascular activity in vitro. Furthermore, compound HoAn32 also exhibited outstanding antitumor activ-
ity in SW620 xenograft tumor models without observable toxic effects, which was more potent than that
of ABT-751. In conclusion, our findings suggest that compound HoAn32 may be a promising microtubule
destabilizing agent and deserves for further development in cancer therapy.
2-Aryl-3-sulfonamido-pyridines
Synthesis
Tubulin polymerization
Microtubule-targeting agents
Antitumor activity
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
multiple functions, including maintaining cell morphology, medi-
ating cell signal transduction, participating cell mitosis, cyto-
Microtubules are ubiquitous in eukaryotic cells with hollow
tubular structure, which mainly consist of - and -tubulin heter-
odimers [1]. The dynamic properties of continuous polymerization
and depolymerization of the microtubule are the basis for its
plasmic transportation and organelle localization, etc. [2e5]
Comparing with normal cells, cell mitosis in tumor cells is abnor-
mally frequent, and microtubules play an important role in it [6].
Inhibiting or promoting tubulin polymerization could block tumor
cell mitosis, induce apoptosis and promote cell death eventually
[7]. Microtubule-targeting agents (MTAs) can be divided into two
categories, one is microtubule stabilizing agent targeting the taxol
binding site [8] or laulimalide binding site [9], and the other is
microtubule destabilizing agent, which specially targeting the
colchicine binding site [10] or vinca binding site [11]. Classic MTAs
such as paclitaxel, docetaxel, vincristine, vinorelbine and so forth
have been used in clinic for a long history. Although these drugs
exhibited strong antitumor effects, some limitations such as severe
side effects, drug resistance and cancer recurrence must be noted
[12,13]. Lots of efforts had been made in the process to discover
small molecular colchicine site binding agents [14], and some
excellent agents such as Combretastatin A-4 phosphate (CA4P)
a
b
Abbreviations: BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-
phenylindole; DCFH-DA, 20, 70-dichlorofluorescein diacetate; DMAP, 4-
dimethylaminopyridine; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EA, ethyl
acetate; EBI, N,N0-ethylene-bis(iodoacetamide); H&E, Hematoxylin/ eosin; MTAs,
Microtubule-targeting
agents;
MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate buff-
ered saline; Pd(PPh3)4, Terakis(triphenylphosphine)palladium(0); PE, petroleum
ether; PI, propidium iodide; ROS, reactive oxidative stress; SDS, sodium dodecyl
sulfate; TEA, triethylamine; THF, tetrahydrofuran; TLC, thin-layer chromatography;
Tris, 2-amino-2-hydroxymethyl-1,3-propandiol.
* Corresponding author.
** Corresponding author.
E-mail addresses: jinhc@zju.edu.cn (H. Jin), panyuanjiang@zju.edu.cn (Y. Pan).
https://doi.org/10.1016/j.ejmech.2020.113117
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
[15], ABT-751 [16], Plinabulin (NPI-2358) [17] and so forth had
already been successfully entering clinical trials. Despite the in-
vestigations of these agents as a single drug or in combination with
other drugs in cancer patients, there is still a long way to go to be
finally marketed. For example, ABT-751 is a classic sulfonamide
tubulin polymerization inhibitor, which has more than 10 clinical
research projects (see https://clinicaltrials.gov/) up to now, how-
ever, there are few reports of its further clinical progress, which
may be associated with its low objective response rate in cancer
patients [18e20]. Considering the far unmet needs for clinical
oncology patients, searching for new colchicine site binding agents
is of great significance.
In this work, the strategy of pharmacophore fusion was applied
in our design. On the one hand, the biaryl moieties have frequently
been observed in the naturally occurred products with potential
antitubulin activity (e.g. Honokiol [21], Fig. 1) as well as in the
synthetic antitubulin agents (e.g. compound 4v [22] and com-
pound 21 [23], Fig. 1). On the other hand, some agents containing
arylsulfonamide motif had been proved as MTAs with strong anti-
cancer activities such as T138067 [24], ABT-751 [16] as well as
ELR510444 [25] (Fig. 1), etc. We speculated that the fusion of these
two pharmacophores may produce a new structure type of anti-
tubulin agents. In view of this, we merged these two pharmaco-
phores and designed a series of novel 2-aryl-3-sulfonamido-
pyridines, which we named HoAns uniformly (Fig. 1). Herein, we
reported the discovery of these novel derivatives as potent micro-
tubule polymerization inhibitor targeting the colchicine binding
site and exploring their antitumor activities in vitro and in vivo.
when the reaction temperature turned up to 120 ꢀC and the solvent
replaced by 1,4-dioxane/H2O between intermediate 1 and boric
acid containing N heterocyclic ring. Subsequently, the final prod-
ucts HoAn25-HoAn27 were obtained via the reaction between
intermediate 2u-2w and 4-methoxybenzenesulfonyl chloride.
Starting with intermediate 1, Suzuki coupling with different
substituted phenylboronic acid could get intermediate 2a-2t. Then,
the final products HoAn1-HoAn10, HoAn14-HoAn16, HoAn18 and
HoAn20-HoAn24 obtained by intermediate 2a-2s reacted with 4-
methoxybenzenesulfonyl chloride, the reaction condition was
similar to the way to produce intermediate 2. For the synthesis of
HoAn28-HoAn31, intermediate 2d-2f or 2q reacted with 4-
methoxybenzenesulfonyl chloride in another condition, which
used TEA instead of DMAP as the catalyst and the dosage of TEA was
elevated to three times. The target products HoAn17 and HoAn19
could be obtained through reduction reaction, which via HoAn16 or
HoAn18 reacted in NH4Cl condition and catalysis with iron power
directly.
For the compounds designed in the second step, the synthetic
routes were showed in Scheme 3. The desire products HoAn32-
HoAn40 could be obtained via the reaction between intermediate
2t and various substituted benzenesulfonyl chloride or naph-
thalenesulfonyl chloride with a synthetic condition similar to the
synthesis of intermediate 2.
2.2. Biology
2.2.1. In vitro human cancer cell line growth inhibition assay
The in vitro antiproliferative efficacy of compounds HoAn1-
HoAn31 were first evaluated towards three different kinds of hu-
man cancer lines, including MCF-7 (human breast cancer cell), SK-
HEP-1 (human hepatoma cells) and RKO (human colon adenocar-
cinoma cell) by MTS assay and compared with Honokiol as well as
ABT-751. As shown in Fig. 2, among all the 31 newly designed
HoAns, four of which including HoAn15 (R1 ¼ 4-CF3), HoAn16
(R1 ¼ 4-NO2), HoAn20 (R1 ¼ 4-CN) and HoAn24 (R1 ¼ H) displayed
better inhibitory activities against these three cancer cell lines. In
detail, the cell viability of MCF-7 cells after treatment with these
2. Results and discussion
2.1. Chemistry
In order to obtain the newly designed 2-aryl-3-sulfonamido-
pyridines (HoAns), several classic organic reactions were applied.
Compounds HoAn1-HoAn31 were designed and synthesized
firstly. The synthetic routes of HoAn1 to HoAn31 were shown in
Schemes 1 and 2. Nucleophilic substitution reaction between
commercially available 2-bromopyridin-3-amine (1) and 4-
methoxybenzenesulfonyl chloride in alkaline condition with
DMAP catalysis produced intermediate 2. Then, Suzuki coupling of
intermediate 2 with phenylboronic acid substituted with hydroxyl
groups in different positions by using cheap metal catalyst
Pd(PPh3)4 under K2CO3 condition afforded the desired products
HoAn11-HoAn13. When we prepared the intermediate 2u-2w, the
Suzuki coupling reaction would take place with a moderate yield
four compounds and ABT-751 for 72 h at 10 mM was 63.4%, 58.90%,
54.95%, 65.68% and 40.00%, respectively. However, the MCF-7 cells
seem not sensitive to the other twenty-seven HoAns (HoAn1-
HoAn14, HoAn17-HoAn19, HoAn21-HoAn23 and HoAn25-
HoAn31) as well as Honokiol, the cell viabilities of which were all
exceeded 70% (Fig. 2). The similar results also happened in SK-HEP-
1 cells. As illustrated in Fig. 2, after treatment with these com-
pounds for 72 h at 10 mM, only HoAn15, HoAn16, HoAn20, HoAn24
Fig. 1. Design strategy of novel 2-aryl-3-sulfonamido-pyridines (HoAns).
2

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Scheme 1. Synthetic Route of Key Intermediates 2 and 2a-2wa.
aReagents and conditions: a). 4-methoxybenzenesulfonyl chloride (1.2 eq), DMAP (0.5 eq), pyridine, CH2Cl2, 60e70 ꢀC, reflux, 8e12 h; b). various substituted phenylboronic acids
(1 eq), Pd(PPh3)4 (0.05 eq), K2CO3 (3 eq), toluene/H2O ¼ 2/1 (v/v), N2, 80e85 ꢀC, reflux, 8e10 h; c). various pyridylboronic acids (1 eq) or 5-pyrimidinylboronic acid (1 eq), Pd(PPh3)4
(0.05 eq), K2CO3 (3 eq), 1,4-dioxane/H2O ¼ 2/1 (v/v), N2, 120 ꢀC, reflux, 12 h.
and ABT-751 showed preferable inhibitory activities, the cell via-
bilities of SK-HEP-1 were 64.00%, 40.76%, 12.44%, 68.04% and 5.97%,
respectively. For RKO cells, after incubated with these compounds
under the same condition, HoAn15, HoAn16, HoAn20, HoAn24 and
ABT-751 still showing the best antiproliferative efficacy, the cell
viabilities were all below 40% and the values were 33.08%, 21.80%,
13.44%, 35.10% and 16.30%, respectively. Meanwhile, several HoAns
such as HoAn18 (R1 ¼ 3-NO2), HoAn21 (R1 ¼ 3-CN) and HoAn25
(X ¼ Z ¼ C, Y]N) as well as Honokiol also exhibited inhibitory
efficacy, the cell viability of which was 60.62%, 51.79%, 58.57% and
49.67%, respectively (Fig. 2). On the basis of these results, we chose
HoAn15, HoAn16, HoAn20 and HoAn24 as representative com-
pounds for further antiproliferative efficacy screening.
Subsequently, the IC50 values of compounds HoAn15, HoAn16,
HoAn20 and HoAn24 were measured by MTS assay towards ten
different kinds of human cancer cell lines, including two human liver
cancer cell lines (PLC/PRF/5 and HCC-LM3), two human breast cancer
cell lines (BT474 and SK-BR-3), three human colon cancer cell lines
(RKO, SW620 and SW480), two lung cancer cell lines (A549 and
H460) and one human pancreatic cancer cell line (PANC-1) in com-
parison with ABT-751. As shown in Table 1, among these represen-
tative HoAns, compound HoAn20 showed the strongest and broad-
spectrum antiproliferative activities against these ten cancer cell
H460 cells (IC50 > 10
to HoAn16, the IC50 values of which were ranged from 1.403
7.589 M. Notably, although HoAn20 showed better antiproliferative
activities in three different human colon cancer cell lines and BT474
m
M), other seven cancer cell lines were sensitive
mM to
m
(IC50 ¼ 1.336
m
M) as well as H460 (IC50 ¼ 1.496
m
M) than ABT-751
(IC50 ¼ 8.390
mM for BT474 cells and IC50 ¼ 2.036
mM for H460 cells),
the efficacy of HoAn20 against other cancer cell lines, including PLC/
PRF/5, HCC-LM3, SK-BR-3, A549 and PANC-1 was inferior to ABT-751
under the same conditions (Table 1). Therefore, the structural
modification of HoAn20 was carried out afterwards.
Previously, Meanwell NA [26] reviewed the functions and ap-
plications of fluorine and fluorinated motifs as bioisosteres in drug
design. Inspired by his article, we introduced a fluorine atom
adjacent to the cyano group and made some modifications on the
benzenesulfonamide part, and finally designed nine new HoAns
(HoAn32-HoAn40) to compare with HoAn20. The antiproliferative
activities of these new compounds against three different cancer
cell lines, including HCC-LM3, RKO and PANC-1 were evaluated
initially. As shown in Table 2, the antiproliferative efficacy against
RKO and PANC-1 cells was both improved after incubated with
HoAn32 (R2 ¼ 400-OCH3) for 72 h, and the IC50 values of which were
0.170
mM and 0.729
mM, respectively, and the efficacy against HCC-
LM3 of HoAn32 (IC50 ¼ 0.963
mM) was not lost much. To our sur-
lines, the IC50 values of which were ranged from 0.204
2.457 M. We observed that RKO, SW620 and SW480 cells were
more sensitive to HoAn20 than other types of cancer cells, the IC50
values of which were 0.259 M, 0.607 M and 0.204 M, respec-
tively, and the efficacy was better than ABT-751 (IC50 ¼ 0.633 M,
2.787 M and 3.421 M, respectively). Compound HoAn15 showed
less potent against these cell lines, the IC50 values were all exceeded
10 M apart from colon cancer cell lines, the IC50 values were
4.048 M, 6.818 M and 9.214 M, respectively after acting on RKO,
mM to
prise, modifications on the benzenesulfonamide part were inop-
erative for improving antiproliferative efficacy, but reduced the
efficacy. The efficacy against RKO cells was about 35-fold loss for
HoAn33 (R2 ¼ 400-CH3) and 11-fold loss for HoAn38 (R2 ¼ 200-
naphthalene), and the efficacy against HCC-LM3 cells was about 12-
fold loss for HoAn38 when compared with HoAn20. For the rest
compounds, including HoAn34 (R2 ¼ 400-Cl), HoAn35 (R2 ¼ 400-Br),
HoAn36 (R2 ¼ 400-CF3), HoAn37 (R2 ¼ H), HoAn39 (R2 ¼ 400-OCF3)
and HoAn40 (R2 ¼ 300, 400-OCH3), the IC50 values of which against
m
m
m
m
m
m
m
m
m
m
m
SW620 and SW480 cells for 72 h. Likewise, compound HoAn24 was
these three cancer cell lines were all over 10 mM. Next, we tested
also exhibited antiproliferative activities only in human colon cancer
the antiproliferative efficacy of HoAn32 in other five human cancer
cell lines. The results indicated that, comparing with HoAn20,
compound HoAn32 maintained or even improved the anti-
proliferative activities, the IC50 values of which against SK-BR-3,
cell lines, the IC50 values were 4.324
RKO and SW620 cells, while the IC50 values against other cell lines
were all more than 10 M. Additionally, except BT474, A549 and
mM and 7.886 mM when against
m
3

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Scheme 2. Synthetic Route of Target Compounds HoAn1-HoAn31a.
aReagents and conditions: The synthetic conditions of routes a and b were same as the routes described in Scheme 1; d). iron powder (10 eq), NH4Cl (3 eq), ethanol/H2O ¼ 2/1 (v/
v), 95 ꢀC, reflux, 1e3 h; e). 4-methoxybenzenesulfonyl chloride (3 eq), TEA (5 eq), CH2Cl2, 60e70 ꢀC, reflux, 10e12 h.
Scheme 3. Synthetic Route of Target Compounds HoAn32-HoAn40a.
aReagents and conditions: f). various substituted benzenesulfonyl chloride (1.2 eq) or 2-naphthalenesulfonyl chloride (1.2 eq), DMAP (0.5 eq), pyridine, CH2Cl2, 60e70 ꢀC, reflux,
10e12 h.
BT474, SW620, SW480 and SGC-7901 cells were 0.431
m
M,
for another 10 days with fresh culture medium until staining with
crystal violet and photographed. The results showed that HoAn32
treatment produced a stronger inhibitory effect on both RKO
(Fig. 4A) and SW620 (Fig. 4B) cells growth in a dose-dependent
1.193 M, 0.673 M, 0.338 M and 0.617 M, respectively (Table 2).
m
m
m
m
The results also indicated that compound HoAn32 possesses better
antiproliferative activities in these cell lines except HCC-LM3 and
SK-BR-3 cell lines than ABT-751 (Table 2). The summarized SARs of
the newly synthesized compounds was illustrated in Fig. 3.
Furthermore, the long-term inhibitory effect of HoAn32 treat-
ment was performed by colony-formation assay. After treatment
with HoAn32 or ABT-751 at different concentrations for 24 h in
RKO and SW620 cells, the cell colonies were continuing to cultivate
manner (from 0.125
mM to 1
mM) when compared with the nega-
tive control. At the same concentration (1
mM), the inhibitory effect
against both cell lines of HoAn32 was better than ABT-751.
Based on the in vitro antiproliferative activity results, undoubt-
edly, we selected compound HoAn32 for further anticancer
mechanism studies.
4

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 2. In Vitro Antiproliferative Activities of Compounds HoAn1-HoAn31, Honokiol and ABT-751 against MCF-7, SK-HEP-1 and RKO cell. The cells were treated with HoAn1-
HoAn31, Honokiol and ABT-751 at 10 mM for 72 h, and the cell viability was finally determined by MTS assay.
Table 1
In Vitro Antiproliferative Activities of Representative Compounds HoAn15, HoAn16, HoAn20 and HoAn24 as well as ABT-751 against Different Kinds of Cancer Cell Linesa.
Comp.
HoAn15
4-CF3
HoAn16
4-NO2
HoAn20
4-CN
HoAn24
-H
ABT-751
R1
Cell Types
Liver cancer
Cell Lines
IC50 (m
M)b
PLC/PRF/5
HCC-LM3
BT474
SK-BR-3
RKO
SW620
SW480
A549
>10
>10
>10
>10
4.048 0.242
6.818 0.573
9.214 1.189
>10
5.296 0.151
7.598 0.186
>10
6.307 0.088
1.403 0.042
3.625 0.443
3.855 0.289
>10
0.816 0.024
0.747 0.018
1.336 0.243
0.746 0.066
0.259 0.013
0.607 0.031
0.204 0.019
2.457 0.162
1.496 0.189
1.089 0.146
>10
>10
>10
>10
4.324 0.507
7.886 0.295
>10
>10
>10
0.792 0.019
0.671 0.021
8.390 1.028
0.143 0.015
0.633 0.011
2.787 0.072
3.421 0.141
1.376 0.147
2.036 0.282
0.837 0.016
Breast cancer
Colon cancer
Lung cancer
H460
PANC-1
>10
>10
>10
7.192 0.102
Pancreatic cancer
>10
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by an MTS assay.
IC50 values are indicated as the mean SD (standard deviation) and all the experiments performed at least three times.
b
Table 2
In vitro antiproliferative activities of compounds HoAn32eHoAn40 and ABT-751 against different kinds of cancer cell Linesa.
Comp.
R2
IC50 (m
M)b
HCC-LM3
RKO
PANC-1
SK-BR-3
BT474
SW620
SW480
SGC-7901
HoAn32
HoAn33
HoAn34
HoAn35
HoAn36
HoAn37
HoAn38
400-OCH3
400-CH3
400-Cl
400-Br
400-CF3
-H
0.963 0.157
>10
>10
>10
>10
>10
8.865 0.514
0.170 0.012
9.207 0.699
>10
>10
>10
0.729 0.037
>10
>10
>10
>10
>10
10.02 0.852
0.431 0.031
1.193 0.146
0.673 0.015
0.338 0.018
0.617 0.035
N.Dc
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
>10
2.922 0.256
HoAn39
HoAn40
ABT-751
400-OCF3
300,400-OCH3
>10
>10
0.671 0.021
>10
>10
0.633 0.011
>10
>10
0.837 0.016
N.D
N.D
0.143 0.015
N.D
N.D
8.390 1.028
N.D
N.D
2.787 0.072
N.D
N.D
3.421 0.141
N.D
N.D
0.738 0.045
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by MTS assay.
IC50 values are indicated as the mean SD (standard deviation) and all the experiments performed at least three times.
N.D means not detected.
b
c
2.2.2. Compound HoAn32 inhibited microtubule assembly and
colchicine binding effects
determine whether the broad-spectrum antiproliferative activities
of HoAn32 was caused by interfering microtubule systems or not.
As shown in Fig. 5, the tubulin polymerization was almost
The in vitro tubulin polymerization assay was performed to
5

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 3. SARs summary of the newly synthesized compounds.
Fig. 4. Effect of compound HoAn32 on RKO cells (A) and SW620 cells (B) colony formation. Cells were incubated with HoAn32 (0.125, 0.25, 0.5 and 1 mM) and ABT-751 (1 mM) for
24 h and continue to grow for another 10 days with fresh culture medium. Visualized colonies were photographed.
completely inhibited after pre-incubated with HoAn32 at 10
mM
while in the higher concentration group (HoAn32, 1 mM), the cell
concentration, and the inhibition efficacy was similar with ABT-
751, while the positive control paclitaxel strongly and consistently
promoted tubulin polymerization under the same conditions. We
next observed the effect of HoAn32 on the microtubule network by
immunofluorescence via fluorescence microscopy. As illustrated in
Fig. 6, the microtubule morphology in RKO cells was incompact and
morphology changed more obvious, the shapes of which gradually
turned to round from fusiform, and the phenomenon was also
observed in ABT-751 (1
mM) administration group. Meanwhile,
paclitaxel (1 M) displayed an opposite way of disturbing the
m
microtubule network, which promoted tubulin polymerization and
made microtubule morphology more densification and aggregation
(Fig. 6).
fragmentation after treatment with HoAn32 at 0.5
mM for 24 h,
6

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 5. Effect of compound HoAn32 on microtubule polymerization in vitro. The microtubule polymerization experiments were performed with a commercial kit (#BK004P,
Cytoskeleton). In brief, tubulin (4 mg/mL) was exposed to vehicle control and 10
mM of the compounds (HoAn32, ABT-751 and paclitaxel), and then the absorbance at 340 nm was
monitored at 37 ꢀC every minute for 60 min by microplate reader.
Fig. 6. Immunofluorescence assay analysis of the effect of compound HoAn32 on the cellular microtubule network. RKO cells were treated with DMSO, HoAn32 (0.5
mM and 1 mM),
ABT-751 (1 M) and Paclitaxel (1 M) for 24 h, and then the cells were fixed and stained with the anti- -tubulin antibody (red), Alexa Fluor Plus 594 dye and nucleuses were stained
m
m
b
with DAPI (blue). Images were performed by using Olympus BX53 microscope (Tokyo, Japan). (For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
EBI (N,N0-ethylene-bis(iodoacetamide)) is an alkylating agent,
which can alkylate the colchicine site of -tubulin in Cys-239 and
Cys-354 sites to form -tubulin-EBI adducts [27]. To find out the
detailed mechanism of HoAn32 acting on tubulin polymerization,
we next carried out the EBI competition assay to determine
2.2.3. Molecular docking studies of HoAn32 with tubulin
b
Based on the above results, to further gain insight on the
possible binding mode of HoAn32 with tubulin, molecular docking
studies were performed using LeDock software. The co-crystal
structure of tubulin complexed with ABT-751 [29] (PDB ID:
3HKC) was downloaded from RCSB protein bank (http://www.rcsb.
org) and selected as the docking protein. Before the docking study
started, we measured the feasibility of the docking model by
redocking the native ligand ABT-751 to the crystal structure
(Fig. 7B), and the RMSD value of the best-docked pose was 0.540 Å,
thus the docking method using LeDock was suitable. As shown in
Fig. 7C, when focusing on the binding mode of the best docked pose
of HoAn32, there are two hydrogen bonds formed between C^N
b
whether it could bind to the colchicine site of
shown in Fig. 7A, the results revealed that HoAn32 indeed could
compete with EBI (100 M) binding to the colchicine site in
tubulin with a dose dependent manner (from 10 M to 40 M) in
SW620 cells, and the phenomenon was also occurred in ABT-751
(20 and 40 M) treatment group, which had been proved binding
with colchicine site of tubulin previously [28]. The results indicated
that HoAn32 bind to the colchicine site of -tubulin.
b-tubulin or not. As
m
b-
m
m
m
b
7

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 7. Compound HoAn32 binds in colchicine site of tubulin. (A). EBI competition assay. SW620 cells were incubated with HoAn32 (0, 10
(20 M and 40 M) for 2 h, followed by EBI (100 M) for another 2 h. Then the -tubulin and -tubulin-EBI adducts were determined by western blot; (B). The redocking result of
ABT-751 with -tubulin (PDB: 3HKC) (the orange stick represents the ligand in co-crystal structure and the blue stick represents the redocked ligand); (C). Proposed binding mode
mM, 20 mM and 40 mM) and ABT-751
m
m
m
b
b
b
of HoAn32 (shown in green stick) with tubulin (PDB: 3HKC); (D). The binding mode of ABT-751 with tubulin (shown in orange stick); (E). Superimposition of HoAn32 (green stick)
and ABT-751 (orange stick). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
group and Val-238 as well as Tyr-202 residues (3.4 Å and 3.3 Å,
respectively) in the colchicine site from the tubulin chain, and the
while for ABT-751 treatment group, the expression level of cyclin
B1 was also decreased in SW620 cells, and the effect was not
b
binding mode of which was similar to the co-crystal structure of
tubulin complexed with ABT-751, in which OH group also formed
two hydrogen bonds (3.1 Å with Val-238 and 3.2 Å with Tyr-202) in
the same place (Fig. 7D). Furthermore, when we superimposed
HoAn32 and ABT-751, the binding posture of them has a high
similarity (Fig. 7E). These molecular docking results indicated that
compound HoAn32 tightly bind in the colchicine site and exhibit a
strong polymerization inhibiting activity against tubulin, while the
binding mode of HoAn32 is different from colchicine with tubulin
[29].
obvious after incubated with ABT-751 at 1 mM for 24 h.
2.2.5. Compound HoAn32 induced apoptosis in human cancer cell
lines
The mitotic arrest of tumor cells by MTAs is generally associated
with cellular apoptosis [32]. To evaluate the capacity of compound
HoAn32 to induce apoptosis in human cancer cell lines, FITC con-
jugated Annexin V/PI assay was performed in RKO and SW620 cells.
As shown in Fig. 9A and B, after exposed to HoAn32 from 0 to 1 mM
for 24 h, the percentage of total apoptotic cells were increased from
3.39% to 38.17% in RKO cells, and from 6.07% to 14.35% in
SW620 cells, while the percentage of which were 11.47% and 12.67%
2.2.4. Compound HoAn32 induced G2/M cell cycle in human cancer
cell lines
after incubated with ABT-751 at 1 mM for 24 h, respectively.
Accumulating evidence has indicated that there exist some
pivotal proteins such as Bcl-2 family (e.g. Bax, Bad and Bcl-2) and
Caspase family (e.g. caspase-3, caspase-9) of proteins influence the
process of cell apoptosis [33,34]. As illustrated in Fig. 9C, the level of
Bax and cleaved PARP was efficiently up-regulated after treatment
As most MTAs could disrupt the cell cycle distributions of cancer
cells [30], we next detected the effect of HoAn32 on the cell cycle of
three different cancer cell lines by flow cytometer analysis. As
shown in Fig. 8A and B, RKO, SW620 and PANC-1 cells were all
arrested in the G2/M phase after treatment with HoAn32 from 0 to
1 mM for 24 h, and the percentages of RKO, SW620 and PANC-1 cells
in the G2/M phase were increased from 10.79% to 47.76%, 13.83% to
76.28% and 3.65% to 28.01%, respectively. The positive control ABT-
751 (1 mM) also showed the ability to disrupt cell cycle distribu-
with HoAn32 from 0 to 1
mM for 24 h, and the effect was also
observed in ABT-751 (1 M) administration group. The above re-
m
sults revealed that compound HoAn32 exert notable anti-
proliferative activity in vitro, display tubulin polymerization
inhibition, and induce significant cell cycle arrest and cell
apoptosis.
tions, and the percentage of RKO and PANC-1 cells in the G2/M
phase was reached 52.80% and 64.83%, respectively, while the effect
on SW620 cells was not obvious after incubated with ABT-751 at
1
m
M for 24 h, the percentages of G2/M phase of which was 17.69%.
Activation of cdc2 kinase was an important event in mitosis of
2.2.6. Compound HoAn32 induced ROS generation in human cancer
cell lines
eukaryotic cells, which was controlled by several factors such as
cyclin B1 and cdc25c, etc. [31] Thus, western blot assay was per-
formed to detect the expression levels of cdc2 and cyclin B1 pro-
teins in RKO and SW620 cells with the presence or absence of
HoAn32 to determine whether the effects on cell cycle was regu-
lated by these factors. As depicted in Fig. 8C, the expression levels of
cdc2 and cyclinB1 were gradually decreased after treatment with
Growing evidence has indicated that increased levels of reactive
oxygen species (ROS) in cancer cells play an important role in
apoptosis induction [35,36]. Thus, the intracellular ROS levels were
measured using the fluorescent probe 20, 70-dichlorofluorescein
diacetate (DCFH-DA) in the presence or absence of compound
HoAn32 or ABT-751 by flow cytometry. As shown in Fig. 10, after
incubation with HoAn32 at 0.25
mM and 0.5 mM for 24 h in
HoAn32 from 0 to 1 mM for 24 h in both RKO and SW620 cells,
SW620 cells, the ROS level elevated about 1.5 times compared with
8

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 8. Compound HoAn32 induced G2/M phase arrest in different cancer cells. (A) RKO, SW620 and PANC-1 cells were treated with DMSO, HoAn32 (0.5
mM and 1 mM) and ABT-751
(1 M) for 24 h. Cells were harvested and stained with PI, and then analyzed by flow cytometry. (B) The percentage of G2/M phase cell cycle distribution was displayed by his-
m
tograms. (C) Western blot analysis was used to detect the expression of G2/M regulatory proteins. The experiments were performed at least three times, and the representative
experiment results are shown. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group.
Fig. 9. Compound HoAn32 induced apoptosis in different cancer cells. (A) RKO and SW620 cells were treated with DMSO, HoAn32 (0.5 mM and 1 mM) and ABT-751 (1 mM) for 24 h.
The cells were collected and stained with PI/Annexin V, and then analyzed by flow cytometry. (B) The percentage of apoptotic cells was displayed by histograms. (C) Western blot
analysis was used to detect the expression of apoptosis-related proteins. The experiments were performed at least three times, and the representative experiment results are shown.
***P < 0.001 vs control group.
the control group, while which was about 1.8 times for ABT-751
(0.5 M) treatment group. Similar results also appeared in RKO cells
(Fig. 10). When cells exposure to 0.25 M and 0.5 M of HoAn32,
the ROS level was elevated about 1.5 times compared with control
group cells after treatment with HoAn32 at 0.25 M and 0.5 M for
24 h and the relative ratio was about 2.0 times for ABT-751 (0.5 M)
compound HoAn32 possessed potent vascular disrupting activity.
As shown in Fig. 11A, after treatment with HoAn32 for 24 h, HUVEC
m
m
m
migration was disturbed in a dose-dependent way from 0.5
M when comparing with DMSO treatment group, and the
wound closure percentage of which decreased from 71.8% to 20.1%,
mM to
1
m
m
m
m
while the value of which was 18.0% for ABT-751 (1 mM) treatment
treatment group when compared with the control group. These
results indicated that ROS mediated apoptosis in SW620 and RKO
cells incubated with HoAn32.
group (Fig. 11B).
In order to exclude the possibility that the anti-vascular activity
of HoAn32 was due to a cytotoxic action of HoAn32, we also
determined the antiproliferative activity of HoAn32 against
HUVECs after a 24 h treatment by an MTS assay. The IC50 values of
2.2.7. Compound HoAn32 exhibited potent vascular disrupting
activity
HoAn32 and ABT-751 against HUVECs were 8.9
9.2 1.1 M, respectively, which are both higher than the con-
centration (1 M) required for the obvious inhibition of cell
0.8 mM and
m
Most antitubulin agents possess anti-vascular activity, which
contributed to the disruption of microtubule dynamics to induce
endothelial cell shape change [37]. Considering the generation of
new blood vessels are closely related to HUVEC migration [38], so
the wound healing assay was performed to evaluate whether
m
migration. These results indicated that compound HoAn32
possessed potent anti-vascular activity.
9

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 10. Compound HoAn32 induced ROS generation in SW620 and RKO cells. (A). ROS detecting fluorescent dye DCFH-DA was used to detect the generation of ROS in RKO and
SW620 cells after incubation with HoAn32 (0.25 M and 0.5 M) and ABT-751 (0.5 M) for 24 h, and then the ROS level was determined by flow cytometry. (B). The relative ratio of
m
m
m
treatment group vs. control group was displayed by histograms. The experiments were performed at least three times, and the representative experiment results are shown.
***P < 0.001 vs control group.
Fig. 11. Effects on HUVECs migration of compound HoAn32. (A). Sterile 200
0 and 24 h after treatments with 0, 0.5 M, and 1 M of HoAn32 or 1 M of ABT-751. (B). Histograms display the percent wound closure of the scratches at 0 and 24 h after
treatments with 0, 0.5 M, and 1 M of HoAn32 or 1 M of ABT-751, ***P < 0.001 vs control group.
mL pipette tips were used to produce scratches, and images were captured using inverted microscope at
m
m
m
m
m
m
2.2.8. Physicochemical properties of Compound HoAn32
2.2.9. Compound HoAn32 inhibited the growth of SW620 xenograft
tumor in mice
Based on the excellent in vitro antiproliferative activity, com-
pound HoAn32 was selected to be evaluated in vivo antitumor ac-
tivity using a SW620 xenograft model in nude mice, which was
established by subcutaneous injection of SW620 cells into the right
In order to evaluate the drug-likeness of compound HoAn32, we
predicted the physicochemical properties of compound HoAn32 us-
ing DataWarrior [39]. As shown in Table 3, compound HoAn32 basi-
cally conformed to Lipinski’s rules of five and owned moderate water
solubility, and the parameters of which are similar with ABT-751.
10

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Table 3
indicated that there were no observable major organ-related tox-
icities after HoAn32 treatment (Fig. 13). Overall, these data sug-
gested that compound HoAn32 was efficacious in inhibiting colon
tumor growth in vivo with no observable toxicity.
Physicochemical properties of compounds HoAn32 and ABT-751.
Comp.
MWa
HBAb
HBDc
cLog Pd
tPSAe
cLog Sf
HoAn32
ABT-751
RO5g
383.40
371.41
<500
6
7
<10
1
3
<5
2.57
2.42
<5
100.46
108.93
<90
ꢁ5.03
ꢁ3.97
3. Conclusions
a
MW ¼ molecular weight.
b
c
d
e
f
HBA ¼ hydrogen-bond acceptor atoms.
HBD ¼ hydrogen-bond donor atoms.
In summary, we reported the design, synthesis and detailed
cLog P ¼ calculated logarithm of the octanolꢁwater partition coefficient.
tPSA ¼ topological polar surface area.
SARs of
a series of novel 2-aryl-3-sulfonamido-pyridines
(HoAns). Among all these new compounds, HoAn20 and HoAn32
exhibited excellent broad-spectrum antiproliferative activities
against a panel of human cancer cell lines in vitro. The IC50 values
cLog S ¼ calculated logarithm of the water solubility in mol/L, PH ¼ 7.5, 25 ꢀC,
calculated using DataWarrior (http://www.openmolecules.org/datawarrior/).
g
RO5 ¼ Lipinski’s rule of five.
of compounds HoAn20 and HoAn32 were ranging from 0.204
mM
to 2.457 M, and from 0.170 M to 1.193 M, respectively. Further
m
m
m
investigations of the optimal compound HoAn32 found that it
could induce tubulin depolymerization in vitro and bind in the
armpit of mice. Once tumors reached about 100 mm3 in volume,
the mice were randomly allocated into three groups: vehicle (p.o.,
group 1), HoAn32 (p.o., 25 mg/kg per 2 days, group 2), ABT-751
(p.o., 25 mg/kg per 2 days, group 3), with six mice per group
(Fig. 12A). As shown in Fig. 12B,D, after 20 days, the group receiving
25 mg/kg treatments of HoAn32 reduced tumor growth by 53.6%,
while the group receiving the same dose treatments of ABT-751
achieved a tumor growth inhibition of 49.3%. The one-way ANOVA
analysis followed by Dunnett’s multiple comparison test also
showed that there was a significant decrease compared to the
control for both concentrations of HoAn32-treated groups
(P < 0.001) and ABT-751-treated group (P < 0.001). Meanwhile, no
significant body weight loss of mice was observed in HoAn32 or
ABT-751 treatment group (Fig. 12C).
colchicine site of
b-tubulin, and molecular docking studies
showed that compound HoAn32 took a similar binding mode
with tubulin comparing to the reference compound ABT-751
[29]. Moreover, compound HoAn32 induced colony formation
inhibition, G2/M phase cell cycle arrest, cell apoptosis, and
elevated the generation of ROS in both RKO and SW620 cells. In
addition, compound HoAn32 possessed potent vascular dis-
rupting activity, which effectively inhibited HUVEC migration
in vitro. Furthermore, the results from in vivo experiments indi-
cated that compound HoAn32 effectively inhibited tumor growth
in mice SW620 xenograft models without observable toxicity.
Collectively, our findings provide a new structure type as po-
tential microtubule polymerization agents in cancer therapy field
and the most active compound HoAn32 deserves for further
development in future.
Additionally, H&E staining of the major organs including heart,
liver, spleen, lung and kidney collected at the end of the study also
Fig. 12. Compound HoAn32 showed antitumor activities in SW620 xenograft model. Nude mice bearing tumors formed by SW620 cells were administered with HoAn32 (p.o.,
25 mg/kg, per 2 days), ABT-751 (p.o., 25 mg/kg, per 2 days), or vehicle. (A). Images of isolated tumors from mice at the end of observation. (B). Tumor volumes changes of nude mice
(n ¼ 6). (C). Body weight changes of mice during treatment. (D). Tumor weight of the individual tumors of each group. ***P < 0.001 vs control group.
11

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
Fig. 13. H&E staining of major organs including heart, liver, spleen, lung and kidney of mice. No abnormality or apparent toxicities of these organs were observed.
4. Experimental section
The crude product was purified by silica gel column chromatog-
raphy (PE: EA ¼ 2: 1) to give a yellowish solid 2-(2-methoxyphenyl)
pyridin-3-amine (2a) (310 mg, 60%).
4.1. Chemistry
Compound 2a (100 mg, 0.499 mmol) and DMAP (61 mg,
0.499 mmol) was dissolved in CH2Cl2 (8 mL), and pyridine (1 mL)
was added subsequently. 4-methoxybenzenesulfonyl chloride
(103 mg, 0.499 mmol) was added dropwise into the mixture at
room temperature. Then, the mixture turned to 60 ꢀC and stirred
under reflux for 6 h. CH2Cl2 was removed and the mixture was
extracted with EA/H2O (3/1, v/v, 30 mL) for three times. The organic
layer was collected and dried with Na2SO4, then concentrated un-
der reduced pressure to obtain the crude product. The crude
product was purified by silica gel column chromatography (PE:
EA ¼ 3: 1) to give a white solid HoAn1 (122 mg, 66%). M.p:
130.1e130.8 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 8.51 (1H, dd,
J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-6), 8.00 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-
60), 7.36-7.26 (2H, m, H-4, H-5), 7.16-7.12 (2H, m, H-200, H-600), 6.96
(1H, d, J ¼ 8.0 Hz, H-3), 6.86 (1H, td, J1 ¼ 7.6 Hz, J2 ¼ 1.2 Hz, H-50),
6.74 (1H, dd, J1 ¼7.6 Hz, J2 ¼ 1.6 Hz, H-40), 6.62-6.58 (2H, m, H-300, H-
500), 3.85 (3H, s, 2-OCH3), 3.80 (3H, s, 400-OCH3). 13C NMR (100 MHz,
CDCl3) (ppm): 162.8, 154.9, 151.5, 147.3, 133.8, 131.9, 131.6, 130.4,
130.1, 128.5 ꢂ 2, 126.6,122.9,121.7,113.9 ꢂ 2,111.3, 56.4, 55.5. HRMS
(ESI, m/z) calcd. for C19H18N2O4S (MþH)þ 371.1060, found 371.1056.
Purity: 99% by HPLC (tR ¼ 3.500 min).
General Methods. All solvents and reagents were commercially
available and used without further purification. All reagents were
weighed and handled in the air at room temperature. The reactions
were monitored by thin-layer chromatography (TLC), which
employed glass 0.25 mm silica gel plates (GF254) and visualized
under UV light. Column chromatography was performed using
200e300 mesh silica gel (Qingdao Haiyang Chemical Co., Ltd,
China). NMR Spectra were performed at 298 K, and all NMR spectra
were recorded on Bruker AVANCE III 400 (Bruker Company, Ger-
many) for 1H NMR and 13C NMR in the indicated solvents (CDCl3 or
DMSO‑d6). The NMR chemical shift was measured in
d
(ppm) with
residual solvent peaks as internal standards relative (CDCl3,
d
d
7.26 ppm in 1H NMR,
2.50 ppm in 1H NMR,
d
77.20 ppm in 13C NMR; DMSO‑d6,
d
39.52 ppm in 13C NMR). Coupling con-
stants (J) were reported in hertz, and the splitting abbreviations
used were as follows: s, singlet; d, doublet; dd, doublet of doublets;
t, triplet; m, multiplet; td, triplet of doublets; br.s, broad singlet.
High-resolution mass spectra (HRMS) were tested on SHIMADZU
LCMS-IT-TOF, which was equipped with an electrospray ion source
(ESI) operating in positive or negative ion mode. Melting points
were recorded with SGW®X-4A micro melting point apparatus
(Shanghai INESA Physico-Optical Instrument Co., Ltd, China). High
Performance Liquid Chromatography (HPLC) analysis was per-
formed on an Agilent 1260 Infinity II LC system fitting with an
Compounds HoAn2-HoAn10, HoAn14-HoAn16, HoAn18 and
HoAn20-HoAn24 were prepared with a similar procedure which
described for the synthesis of HoAn1.
4-methoxy-N-(2-(3-methoxyphenyl)pyridin-3-yl)benzene-
sulfonamide (HoAn2). White powder. Yield: 65%. M.p:
132.3e133.1 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.73 (1H, s,-
NHSO2-), 8.48 (1H, dd, J1 ¼ 4.4 Hz, J2 ¼ 1.6 Hz, H-6), 7.54 (1H, dd,
J1 ¼ 7.2 Hz, J2 ¼ 2.0 Hz, H-60), 7.47 (2H, d, J ¼ 8.8 Hz, H-200, H-600),
7.34-7.24 (2H, m, H-2, H-5), 7.07-7.00 (3H, m, H-4, H-40, H-50), 6.96
(2H, d, J ¼ 8.8 Hz, H-300, H-500), 3.81 (3H, s, 3-OCH3), 3.76 (3H, s, 400-
OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 163.8, 162.2, 147.1,
139.3, 134.9, 131.8, 131.0, 130.3, 128.7, 128.5 ꢂ 2, 122.8, 121.3, 114.4,
114.2 ꢂ 2, 113.8, 55.5, 54.9. HRMS (ESI, m/z) calcd. for C19H18N2O4S
(MþH)þ 371.1060, found 371.1056. Purity: 99% by HPLC
(tR ¼ 5.724 min).
Ultimate™ XB-C18 column (4.6 mm ꢂ 150 mm, 5
mm particle size)
by using Acetonitrile-Water (v/v) mixture as eluent. The purities of
all synthetic compounds were determined by HPLC analysis and
was ꢃ95%.
Procedure
A
for Synthesizing of 4-methoxy-N-(2-(2-
methoxyphenyl)pyridin-3-yl)benzenesulfonamide (HoAn1). 2-
bromopyridin-3-amine (1) (500 mg, 2.89 mmol) and (2-
methoxyphenyl)boronic acid (483 mg, 3.18 mmol) was dissolved
in toluene/H2O (2/1, v/v, 15 mL). Then, K2CO3 (1.6 g, 11.56 mmol)
and Pd(PPh3)4 (167 mg, 0.145 mmol) were added, the reaction
mixture was stirred at 80 ꢀC for 8 h with N2 atmosphere. The sol-
vent was removed under reduced pressure, then the mixture was
extracted with EA/H2O (3/1, v/v, 50 mL) for three times and the EA
layer was merged. The organic layer was dried with Na2SO4 and
concentrated under reduced pressure to obtain the crude product.
4-methoxy-N-(2-(4-methoxyphenyl)pyridin-3-yl)benzene-
sulfonamide (HoAn3). Yellowish powder. Yield: 60%. M.p:
166.8e167.7 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 8.38 (1H, dd,
J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 8.01 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-
12

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
40), 7.53 (2H, d, J ¼ 8.8 Hz, H-2, H-6), 7.23 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 4.8 Hz, H-50), 7.04 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 6.91 (2H, d, J ¼
8.8 Hz, H-300, H-500), 6.86 (2H, d, J ¼ 8.8 Hz, H-3, H-5), 3.85 (3H, s, 4-
OCH3), 3.84 (3H, s, 400-OCH3). 13C NMR (100 MHz, CDCl3) (ppm):
163.4, 160.2, 150.9, 145.6, 131.5, 131.0, 130.2, 129.7 ꢂ 2, 129.2 ꢂ 2,
128.9, 122.7, 114.6 ꢂ 2, 114.3 ꢂ 2, 55.6, 55.4. HRMS (ESI, m/z) calcd.
for C19H18N2O4S (MþH)þ 371.1060, found 371.1058. Purity: 95% by
HPLC (tR ¼ 5.516 min).
7.46 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.43 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.2 Hz, H-6), 7.27 (1H, dd, J1 ¼8.0 Hz, J2 ¼ 4.4 Hz, H-50), 7.14 (1H,
dd, J1 ¼ 8.4 Hz, J2 ¼ 2.0 Hz, H-40), 7.09 (1H, s, H-2), 6.95-6.91 (3H, m,
H-5, H-300, H-500), 3.81 (3H, s, 3-OCH3), 3.80 (3H, s, 4-OCH3), 3.73
(3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.1, 154.7,
148.8, 148.0, 147.1, 135.1, 132.0, 130.4, 130.0, 128.5 ꢂ 2, 122.2, 121.7,
114.1 ꢂ 2, 112.6, 110.8, 55.5, 55.4, 55.2. HRMS (ESI, m/z) calcd. for
C
20H20N2O5S (MþH)þ 401.1166, found 401.1163. Purity: 98% by
N-(2-(2,3-dimethoxyphenyl)pyridin-3-yl)-4-
HPLC (tR ¼ 7.658 min).
methoxybenzenesulfonamide (HoAn4). White powder. Yield:
63%. M.p: 106.8e107.7 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
8.92 (1H, s, -NHSO2-), 8.42 (1H, dd, J1 ¼8.8 Hz, J2 ¼ 1.2 Hz, H-6), 7.63
(1H, dd, J1 ¼8.4 Hz, J2 ¼ 1.2 Hz, H-60), 7.42 (2H, d, J ¼ 8.8 Hz, H-200, H-
600), 7.36 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-5), 7.08 (1H, dd,
J1 ¼8.4 Hz, J2 ¼ 1.2 Hz, H-4), 6.98 (1H, t, J ¼ 8.0 Hz, H-50), 6.93 (2H, d,
J ¼ 8.8 Hz, H-300, H-500), 6.41 (1H, dd, J1 ¼ 7.8 Hz, J2 ¼ 1.2 Hz, H-40),
3.85 (3H, s, 2-OCH3), 3.80 (3H, s, 3-OCH3), 3.50 (3H, s, 400-OCH3). 13C
NMR (100 MHz, DMSO‑d6) (ppm): 162.4, 152.2, 151.5, 146.1, 145.7,
132.0, 131.9, 131.4, 131.1, 128.4 ꢂ 2, 123.6, 122.9, 122.5, 114.2 ꢂ 2,
113.0, 60.6, 55.7, 55.6. HRMS (ESI, m/z) calcd. for C20H20N2O5S
(MþH)þ 401.1166, found 401.1174. Purity: 96% by HPLC
(tR ¼ 3.717 min).
N-(2-(3,5-dimethoxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn9). White powder. Yield:
61%. M.p: 148.3e149.2 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
9.73 (1H, s, -NHSO2-), 8.47 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, H-60),
7.49 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-40), 7.46 (2H, d, J ¼ 8.8 Hz, H-
200, H-600), 7.33 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-50), 6.95 (2H, d, J ¼
8.8 Hz, H-300, H-500), 6.92 (2H, d, J ¼ 2.0 Hz, H-2, H-6), 6.49 (1H, t, J ¼
2.0 Hz, H-4), 3.81 (3H, s, 400-OCH3), 3.74 (6H, s, 3-OCH3, 5-OCH3). 13C
NMR (100 MHz, DMSO‑d6) (ppm): 162.2, 159.8 ꢂ 2, 154.7, 147.0,
139.7, 135.1, 131.9, 130.2, 128.5 ꢂ 2, 122.9, 114.1 ꢂ 2, 107.1 ꢂ 2, 100.2,
55.5, 55.1 ꢂ 2. HRMS (ESI, m/z) calcd. for C20H20N2O5S (MþH)þ
401.1166, found 401.1171. Purity: 96% by HPLC (tR ¼ 3.391 min).
4-methoxy-N-(2-(3,4,5-trimethoxyphenyl)pyridin-3-yl)ben-
zenesulfonamide (HoAn10). Yellowish powder. Yield: 58%. M.p:
143.6e144.5 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.79 (1H, s,
-NHSO2-), 8.48 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, H-60), 7.49 (1H, dd,
J1 ¼8.4 Hz, J2 ¼ 1.6 Hz, H-40), 7.46 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.33
(1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-50), 6.94 (2H, d, J ¼ 8.8 Hz, H-300,
H-500), 6.80 (2H, s, H-2, H-6), 3.80 (3H, s, 400-OCH3), 3.76 (6H, s, 3-
OCH3, 5-OCH3), 3.72 (3H, s, 4-OCH3). 13C NMR (100 MHz,
DMSO‑d6) (ppm): 162.1, 154.8, 152.2 ꢂ 2, 147.1, 137.5, 135.5, 133.2,
132.1, 130.0, 128.4 ꢂ 2, 122.7, 114.1 ꢂ 2, 106.6 ꢂ 2, 59.9, 55.7 ꢂ 2,
55.5. HRMS (ESI, m/z) calcd. for C21H22N2O6S (MþH)þ 431.1271,
found 431.1268. Purity: 95% by HPLC (tR ¼ 4.579 min).
N-(2-(2,4-dimethoxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfinamide (HoAn5). Yellowish oil. Yield: 59%.
1H NMR (400 MHz, DMSO‑d6) (ppm): 8.66 (1H, s, -NHSO2-), 8.38
(1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 1.2 Hz, H-6), 7.60 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.2 Hz, H-60), 7.40 (2H, d, J ¼ 9.2 Hz, H-200, H-600), 7.30 (1H, dd,
J1 ¼8.0 Hz, J2 ¼ 4.8 Hz, H-5), 6.92 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 6.72
(1H, d, J ¼ 8.0 Hz, H-50), 6.58 (1H, d, J ¼ 2.4 Hz, H-3), 6.45 (1H, dd,
J1 ¼ 8.4 Hz, J2 ¼ 2.4 Hz, H-40), 3.82 (3H, s, 4-OCH3), 3.81 (3H, s, 400-
OCH3), 3.72 (3H, s, 2-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm):
162.3, 160.8, 156.9, 151.7, 146.2, 131.9, 131.8, 131.7, 131.4, 128.4 ꢂ 2,
122.4, 119.3, 114.1 ꢂ 2, 105.1, 98.1, 55.6 ꢂ 2, 55.2. HRMS (ESI, m/z)
calcd. for C20H20N2O5S (MþH)þ 401.1166, found 401.1168. Purity:
99% by HPLC (tR ¼ 3.526 min).
N-(2-(4-(benzyloxy)phenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn14). Yellowish powder.
Yield: 68%. M.p: 109.9e110.8 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
(ppm): 9.71 (1H, s, -NHSO2-), 8.44 (1H, dd, J1 ¼8.4 Hz, J2 ¼ 1.2 Hz, H-
60), 7.51-7.40 (10H, m, 5H-benzyloxy, H-2, H-6, H-40, H-200, H-600),
7.25 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-50), 6.99 (2H, d, J ¼ 8.8 Hz, H-
3, H-5), 6.95 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 5.15 (2H, s, -OCH2-
phenyl), 3.80 (3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6)
(ppm): 162.2, 158.2, 154.6, 147.0, 137.0, 134.8, 131.9, 130.4 ꢂ 2, 129.4,
128.6 ꢂ 2, 128.4 ꢂ 2, 127.8, 127.7 ꢂ 2, 127.6, 122.2, 114.2 ꢂ 2,
113.9 ꢂ 2, 69.1, 55.6. HRMS (ESI, m/z) calcd. for C25H22N2O4S
(MþH)þ 447.1373, found 447.1380. Purity: 95% by HPLC
(tR ¼ 17.378 min).
N-(2-(2,5-dimethoxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn6). White powder. Yield:
54%. M.p: 110.1e110.9 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 8.54
(1H, s, -NHSO2-), 8.44 (1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 1.2 Hz, H-60), 7.66
(1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-6), 7.38 (1H, dd, J1 ¼ 8.0 Hz,
J2 ¼ 4.8 Hz, H-3), 7.29 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.01-6.92 (2H,
m, H-4, H-50), 6.86 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 6.28 (1H, d, J ¼
3.2 Hz, H-40), 3.79 (3H, s, 5-OCH3), 3.69 (3H, s, 400-OCH3), 3.64 (3H, s,
2-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.3, 152.9, 151.8,
149.5, 146.7, 133.2, 131.5, 131.0, 128.3 ꢂ 2, 127.2, 122.9, 116.0, 115.0,
114.1 ꢂ 2, 112.5, 56.2, 55.4, 55.1. HRMS (ESI, m/z) calcd. for
C
20H20N2O5S (MþH)þ 401.1166, found 401.1172. Purity: 98% by
4-methoxy-N-(2-(4-(trifluoromethyl)phenyl)pyridin-3-yl)
benzenesulfonamide (HoAn15). Yellowish powder. Yield: 56%.
M.p: 165.5e166.3 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.90
(1H, s, -NHSO2-), 8.53 (1H, dd, J1 ¼8.4 Hz, J2 ¼ 1.2 Hz, H-60), 7.71 (2H,
d, J ¼ 8.8 Hz, H-2, H-6), 7.68 (2H, d, J ¼ 8.4 Hz, H-3, H-5), 7.50 (1H,
dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-40), 7.45 (2H, d, J ¼ 8.8 Hz, H-200, H-600),
7.39 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.4 Hz, H-50), 6.95 (2H, d, J ¼ 8.8 Hz, H-
300, H-500), 3.80 (3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6)
(ppm): 162.3, 153.6, 147.5, 135.4, 134.6, 131.4, 130.6, 129.8 ꢂ 2,
128.6 ꢂ 4, 124.6, 124.5, 123.6, 114.2 ꢂ 2, 55.5. HRMS (ESI, m/z) calcd.
for C19H15F3N2O3S (MþH)þ 409.0828, found 409.0833. Purity: 98%
by HPLC (tR ¼ 2.851 min).
HPLC (tR ¼ 3.603 min).
N-(2-(2,6-dimethoxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn7). White powder. Yield:
48%. M.p: 145.0e145.8 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
8.77 (1H, s, -NHSO2-), 8.27 (1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 1.2 Hz, H-60),
7.63 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.60 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.2 Hz, H-4), 7.35 (1H, t, J ¼ 8.4 Hz, H-50), 7.22 (1H, dd,
J1 ¼ 8.4 Hz, J2 ¼ 4.8 Hz, H-40), 7.02 (2H, d, J ¼ 8.8 Hz, H-300, H-500),
6.67 (2H, d, J ¼ 8.4 Hz, H-3, H-5), 3.81 (3H, s, 400-OCH3), 3.55 (6H, s,
2-OCH3, 6-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.3,
157.8 ꢂ 2, 147.4, 144.8, 133.1, 132.0, 131.5, 130.0, 128.7 ꢂ 2, 128.2,
122.3, 114.2 ꢂ 2, 103.9 ꢂ 2, 55.6, 55.4 ꢂ 2. HRMS (ESI, m/z) calcd. for
4-methoxy-N-(2-(4-nitrophenyl)pyridin-3-yl)benzenesulfo-
namide (HoAn16). Yellowish powder. Yield: 50%. M.p:
170.2e171.1 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.94 (1H, s,
-NHSO2-), 8.56 (1H, dd, J1 ¼8.4 Hz, J2 ¼ 1.2 Hz, H-60), 8.20 (2H, d, J ¼
8.8 Hz, H-2, H-6), 7.74 (2H, d, J ¼ 8.4 Hz, H-3, H-5), 7.51 (1H, d, J ¼
7.2 Hz, H-40), 7.43-7.41 (3H, m, H-50, H-200, H-600), 6.94 (2H, d, J ¼
8.8 Hz, H-300, H-500), 3.80 (3H, s, 400-OCH3). 13C NMR (100 MHz,
C
20H20N2O5S (MþH)þ 401.1166, found 401.1164. Purity: 99% by
HPLC (tR ¼ 2.906 min).
N-(2-(3,4-dimethoxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn8). Yellowish powder. Yield:
52%. M.p: 161.8e162.6 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
9.72 (1H, s, -NHSO2-), 8.46 (1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 1.2 Hz, H-60),
13

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
DMSO‑d6) (ppm): 162.4, 153.1, 147.7, 146.9, 144.7, 135.9, 131.2, 130.8,
130.3 ꢂ 2, 128.6 ꢂ 2, 124.0, 122.8 ꢂ 2, 114.2 ꢂ 2, 55.5. HRMS (ESI, m/
z) calcd. for C18H15N3O5S (MþH)þ 386.0805, found 386.0801. Pu-
rity: 99% by HPLC (tR ¼ 6.279 min).
Purity: 98% by HPLC (tR ¼ 5.354 min).
4-methoxy-N-(2-phenylpyridin-3-yl)benzenesulfonamide
(HoAn24). White powder. Yield: 83%. M.p: 141.6e142.5 ꢀC. 1H NMR
(400 MHz, DMSO‑d6) (ppm): 9.74 (1H, s, -NHSO2-), 8.48 (1H, dd,
J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.52-7.45 (5H, m, H-2, H-4, H-6, H-200,
H-600), 7.38-7.35 (3H, m, H-3, H-5, H-40), 7.31 (1H, dd, J1 ¼ 8.0 Hz,
J2 ¼ 4.4 Hz, H-50), 6.98 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 3.81 (3H, s, 400-
OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.3, 154.8, 147.0,
138.1, 134.5, 131.8, 130.4, 129.0 ꢂ 2, 128.6 ꢂ 2, 128.0, 127.7 ꢂ 2, 122.7,
114.3 ꢂ 2, 55.6. HRMS (ESI, m/z) calcd. for C18H16N2O3S (MþH)þ
341.0954, found 341.0939. Purity: 98% by HPLC (tR ¼ 3.141 min).
Procedure B for Synthesizing of N-(2-(2-hydroxyphenyl)pyr-
4-methoxy-N-(2-(3-nitrophenyl)pyridin-3-yl)benzenesulfo-
namide (HoAn18). Yellowish powder. Yield: 52%. M.p:
185.1e185.9 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.97 (1H, s,
-NHSO2-), 8.57 (1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-6), 8.22-8.20 (2H,
m, H-2, H-4), 7.97 (1H, d, J ¼ 7.6 Hz, H-60), 7.68-7.63 (1H, m, H-5),
7.56 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-40), 7.44 (1H, dd, J1 ¼ 8.0 Hz,
J2 ¼ 4.8 Hz, H-50), 7.36 (2H, d, J ¼ 9.2 Hz, H-200, H-600), 6.89 (2H, d,
J ¼ 8.8 Hz, H-300, H-500), 3.77 (3H, s, 400-OCH3). 13C NMR (100 MHz,
DMSO‑d6) (ppm): 162.3, 153.1, 147.9, 147.3, 139.6, 136.6, 135.4, 131.1,
130.5, 129.3, 128.5 ꢂ 2, 123.9, 123.6, 122.8, 114.1 ꢂ 2, 55.5. HRMS
idin-3-yl)-4-methoxybenzenesulfonamide
(HoAn11).
2-
bromopyridin-3-amine (1) (500 mg, 2.89 mmol), pyridine (1 mL)
and DMAP (176 mg, 1.44 mmol) was dissolved in CH2Cl2, and then
4-methoxybenzenesulfonyl chloride (716 mg, 3.47 mmol) was
added slowly, the reaction mixture was stirred under reflux at 66 ꢀC
for 10 h. After that, CH2Cl2 was removed under reduced pressure
and extracted with EA/H2O (2/1, v/v, 400 mL) for three times, the EA
layer was converged and removed to give the crude product. The
crude product was purified by silica gel column chromatography
(PE: EA ¼ 6: 1) to obtain a yellowish solid N-(2-bromopyridin-3-yl)-
4-methoxybenzenesulfonamide (2) (793 mg, 80%).
(ESI, m/z) calcd. for
C
18H15N3O5S (MþH)þ 386.0805, found
386.0793. Purity: 96% by HPLC (tR ¼ 6.072 min).
N-(2-(4-cyanophenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn20). White powder. Yield:
50%. M.p: 208.0e208.8 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
9.90 (1H, s, -NHSO2-), 8.54 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.2 Hz, H-60),
7.82 (2H, d, J ¼ 8.4 Hz, H-2, H-6), 7.65 (2H, d, J ¼ 8.4 Hz, H-3, H-5),
7.49 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, H-40), 7.43 (2H, d, J ¼ 8.8 Hz, H-
200, H-600), 7.39 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.4 Hz, H-50), 6.96 (2H, d,
J ¼ 9.2 Hz, H-300, H-500), 3.82 (3H, s, 400-OCH3). 13C NMR (100 MHz,
DMSO‑d6) (ppm): 162.4, 153.4, 147.6, 142.7, 135.7, 131.7 ꢂ 2, 131.3,
130.7, 129.9 ꢂ 2, 128.6 ꢂ 2, 123.8, 118.8, 114.2 ꢂ 2, 110.6, 55.6. HRMS
Compound 2 (200 mg, 0.582 mmol) and (2-hydroxyphenyl)
boronic acid (80 mg, 0.582 mmol) was dissolved in THF/H2O (2/1, v/
v, 15 mL), and then Pd(PPh3)4 (34 mg, 0.029 mmol) and K2CO3
(121 mg, 0.874 mmol) was added. The mixture was stirred under
reflux at 70 ꢀC for 5 h. When the reaction finished, the solvent was
removed under reduced pressure and extracted with EA/H2O (1/1,
v/v, 300 mL) for three times. The organic layer was concentrated
and purified by silica gel column chromatography (PE: EA ¼ 2: 1). A
yellowish solid HoAn11 (102 mg, 49%) was obtained. M.p:
150.8e151.6 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 10.80 (1H, s,
-NHSO2-), 8.62 (1H, s, 2-OH), 8.48 (1H, d, J ¼ 3.6 Hz, H-6), 7.73 (1H,
dd, J1 ¼8.0 Hz, J2 ¼ 1.2 Hz, H-60), 7.40 (1H, dd, J1 ¼8.0 Hz, J2 ¼ 4.4 Hz,
H-50), 7.23 (1H, t, J ¼ 8.4 Hz, H-4), 7.15 (2H, d, J ¼ 8.8 Hz, H-200, H-600),
6.94 (1H, d, J ¼ 8.4 Hz, H-5), 6.83 (1H, d, J ¼ 6.8 Hz, H-3), 6.75 (2H, d,
J ¼ 8.8 Hz, H-300, H-500), 6.72 (1H, d, J ¼ 7.6 Hz, H-40), 3.77 (3H, s, 400-
OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.4, 152.8, 147.0,
146.9, 133.8, 131.7, 129.9, 128.7, 128.6, 128.0 ꢂ 2, 124.7, 122.7, 119.4,
115.6, 114.1 ꢂ 2, 55.5. HRMS (ESI, m/z) calcd. for C18H16N2O4S
(MþH)þ 357.0904, found 357.0896. Purity: 98% by HPLC
(tR ¼ 4.174 min).
(ESI, m/z) calcd. for
C
19H15N3O3S (MþH)þ 366.0907, found
366.0907. Purity: 98% by HPLC (tR ¼ 2.931 min).
N-(2-(3-cyanophenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn21). White powder. Yield:
62%. M.p: 200.2e201.1 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 8.47
(1H, d, J ¼ 4.0 Hz, H-6), 8.09 (1H, d, J ¼ 8.4 Hz, H-60), 7.67 (1H, d,
J ¼ 6.8 Hz, H-4), 7.53-7.46 (4H, m, H-5, H-40, H-200, H-600), 7.36 (1H,
dd, J1 ¼ 8.0 Hz, J2 ¼ 4.4 Hz, H-50), 7.08 (1H, s, H-2), 6.93 (2H, d,
J ¼ 8.4 Hz, H-300, H-500), 6.55 (1H, s, -NHSO2-), 3.89 (3H, s, 400-OCH3).
13C NMR (100 MHz, CDCl3) (ppm): 163.7, 149.8, 146.6, 137.9, 132.9,
132.4, 132.1, 131.8, 131.0, 129.9, 129.8, 129.0 ꢂ 2, 124.0, 117.9,
114.7 ꢂ 2, 113.2, 55.8. HRMS (ESI, m/z) calcd. for C19H15N3O3S
(MþH)þ 366.0907, found 366.0910. Purity: 95% by HPLC
(tR ¼ 5.004 min).
N-(2-(benzo[d]
[1,3]dioxol-5-yl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn22). Yellowish powder.
Yield: 67%. M.p: 170.9e171.8 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
(ppm): 9.69 (1H, s, -NHSO2-), 8.44 (1H, d, J ¼ 3.6 Hz, H-60), 7.47 (1H,
d, J ¼ 6.4 Hz, H-6), 7.45 (2H, d, J ¼ 8.4 Hz, H-200, H-600), 7.29 (1H, dd,
J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-50), 6.99-6.93 (4H, m, H-2, H-5, H-300, H-
500), 6.87 (1H, d, J ¼ 8.0 Hz, H-40), 6.05 (2H, s, -OCH2O-), 3.81 (3H, s,
400-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.3, 154.6, 147.1,
146.7, 135.4, 131.9, 131.8, 131.0, 130.0, 128.5 ꢂ 2, 123.1, 122.5,
114.1 ꢂ 2, 109.3, 107.6, 101.0, 55.5. HRMS (ESI, m/z) calcd. for
Compounds HoAn12 and HoAn13 were prepared with a similar
procedure which described for the synthesis of HoAn11.
N-(2-(3-hydroxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn12). White powder. Yield:
55%. M.p: 185.8e186.6 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
9.64 (1H, s, -NHSO2-), 9.42 (1H, s, 3-OH), 8.43 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.2 Hz, H-60), 7.54 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.43 (1H, dd,
J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz, H-6), 7.27 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-
50), 7.15 (1H, t, J ¼ 8.4 Hz, H-5), 6.99 (2H, d, J ¼ 8.8 Hz, H-300, H-500),
6.91-6.90 (2H, m, H-2, H-4), 6.77 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, H-
40), 3.81 (3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm):
162.3, 156.9, 154.4, 146.6, 139.2, 133.6, 131.8, 130.4, 128.6 ꢂ 2, 128.6,
122.6, 119.7, 116.2, 115.0, 114.2 ꢂ 2, 55.5. HRMS (ESI, m/z) calcd. for
C
19H16N2O5S (MþH)þ 385.0853, found 385.0862. Purity: 99% by
HPLC (tR ¼ 5.291 min).
N-(2-(dibenzo[b,d]furan-4-yl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn23). White powder. Yield:
61%. M.p: 162.3e163.2 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
9.67 (1H, s, -NHSO2-), 8.54 (1H, dd, J1 ¼ 4.4 Hz, J2 ¼ 1.2 Hz, H-60),
8.16 (1H, d, J ¼ 8.0 Hz, H-6), 8.14 (1H, d, J ¼ 7.6 Hz, H-4), 7.72 (1H, dd,
J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-5(benzofuran)), 7.61 (1H, d, J ¼ 8.0 Hz, H-
2(benzofuran)), 7.53-7.35 (6H, m, H-5, H-50, H-200, H-600, H-
3(benzofuran), H-4(benzofuran)), 7.24 (1H, dd, J1 ¼ 7.2 Hz,
J2 ¼ 1.2 Hz, H-40), 6.77 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 3.60 (3H, s, 400-
OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.0, 155.4, 153.0,
150.8, 146.8, 134.1, 131.7, 131.4, 128.3 ꢂ 2, 128.2, 127.4, 123.9, 123.5,
123.5, 122.9, 122.6, 122.3, 121.1, 121.0, 113.9 ꢂ 2, 111.6, 55.3. HRMS
(ESI, m/z) calcd. for C24H18N2O4S (MþH)þ 431.1060, found 431.1060.
C
18H16N2O4S (MþH)þ 357.0904, found 357.0900. Purity: 99% by
HPLC (tR ¼ 2.343 min).
N-(2-(4-hydroxyphenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn13). White powder. Yield:
57%. M.p: 149.1e150.0 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm):
9.62 (1H, s, -NHSO2-), 9.61 (1H, s, 4-OH), 8.41 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.2 Hz, H-60), 7.52 (2H, d, J ¼ 9.2 Hz, H-2, H-6), 7.40 (1H, dd,
J1 ¼8.0 Hz, J2 ¼ 1.6 Hz, H-40), 7.35 (2H, d, J ¼ 8.4 Hz, H-200, H-600), 7.22
(1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-50), 6.98 (2H, d, J ¼ 8.8 Hz, H-300,
14

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
H-500), 6.73 (2H, d, J ¼ 8.4 Hz, H-3, H-5), 3.82 (3H, s, 400-OCH3). 13C
NMR (100 MHz, DMSO‑d6) (ppm): 162.3, 157.5, 154.7, 146.8, 134.3,
132.0, 130.4 ꢂ 2, 130.0, 128.8, 128.6 ꢂ 2, 121.8, 114.5 ꢂ 2, 114.2 ꢂ 2,
55.5. HRMS (ESI, m/z) calcd. for C18H16N2O4S (MþH)þ 357.0904,
found 357.0904. Purity: 99% by HPLC (tR ¼ 2.276 min).
CDCl3) (ppm): 163.5,150.0 ꢂ 2,149.1,146.5,144.8,131.3,131.2,130.5,
129.1 ꢂ 2, 124.1, 123.3 ꢂ 2, 114.5 ꢂ 2, 55.7. HRMS (ESI, m/z) calcd. for
C
17H15N3O3S (MþH)þ 342.0907, found 342.0900. Purity: 98% by
HPLC (tR ¼ 2.042 min).
Compounds HoAn26 and HoAn27 was prepared with a similar
procedure which described for the synthesis of HoAn25.
Procedure C for Synthesizing of N-(2-(4-aminophenyl)pyr-
idin-3-yl)-4-methoxybenzenesulfonamide (HoAn17). HoAn16
(100 mg, 0.259 mmol), iron powder (43 mg, 0.778 mmol) and
NH4Cl (138 mg, 2.59 mmol) was dissolved in ethanol/H2O (3/1, v/v,
10 mL). The mixture was stirred under reflux at 100 ꢀC for 3 h. After
the reaction was finished, the iron powder was removed by filtering
with diatomite, and the filtrate was removed under reduced pres-
sure. The left mixture was extracted with EA/H2O (2/1, v/v, 200 mL)
for three times and then the organic layer was collected and
concentrated to give the crude product. Further purified by silica
gel column chromatography (PE: EA ¼ 1: 1.5) could obtain the final
yellow solid HoAn17 (74 mg, 80%). M.p: 170.0e170.9 ꢀC. 1H NMR
(400 MHz, DMSO‑d6) (ppm): 9.53 (1H, s, -NHSO2-), 8.36 (1H, d,
J ¼ 3.2 Hz, H-60), 7.55 (2H, d, J ¼ 8.8 Hz, H-2, H-6), 7.35 (1H, dd,
J1 ¼8.0 Hz, J2 ¼ 1.2 Hz, H-40), 7.28 (2H, d, J ¼ 8.4 Hz, H-200, H-600), 7.14
(1H, dd, J1 ¼8.0 Hz, J2 ¼ 4.8 Hz, H-50), 7.00 (2H, d, J ¼ 8.8 Hz, H-300, H-
500), 6.53 (2H, d, J ¼ 8.8 Hz, H-3, H-5), 5.32 (2H, s, -NH2), 3.82 (3H, s,
400-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm): 162.2, 154.9, 148.9,
146.6, 133.8, 132.1, 130.0 ꢂ 2, 129.6, 128.6 ꢂ 2, 125.2, 121.1, 114.2 ꢂ 2,
112.9 ꢂ 2, 55.6. HRMS (ESI, m/z) calcd. for C18H17N3O3S (MþH)þ
356.1063, found 356.1059. Purity: 95% by HPLC (tR ¼ 3.299 min).
Compound HoAn19 was prepared with a similar procedure
which described for the synthesis of HoAn17.
N-([2,3′-bipyridin]-3-yl)-4-methoxybenzenesulfonamide
(HoAn26). Yellow powder. Yield: 58%. M.p: 154.6e155.5 ꢀC. 1H
NMR (400 MHz, CDCl3) (ppm): 8.55 (1H, s, H-2), 8.46 (1H, d,
J ¼ 4.4 Hz, H-6), 8.39 (1H, s, H-4), 7.99 (1H, d, J ¼ 8.0 Hz, H-60), 7.55
(2H, d, J ¼ 9.2 Hz, H-200, H-600), 7.53 (1H, d, J ¼ 8.0 Hz, H-5), 7.32-7.29
(2H, m, H-40, H-50), 6.87 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 3.84 (3H, s,
400-OCH3). 13C NMR (100 MHz, CDCl3) (ppm): 163.4, 149.6, 149.1,
146.5, 136.5, 131.5, 131.3, 131.2, 130.5, 130.4, 129.1 ꢂ 2, 123.7, 123.6,
114.4 ꢂ 2, 55.7. HRMS (ESI, m/z) calcd. for C17H15N3O3S (MþH)þ
342.0907, found 342.0907. Purity: 97% by HPLC (tR ¼ 2.035 min).
4-methoxy-N-(2-(pyrimidin-5-yl)pyridin-3-yl)benzenesulfo-
namide (HoAn27). Yellowish powder. Yield: 69%. M.p:
164.7e165.6 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 10.08 (1H, s,
-NHSO2-), 9.17 (1H, s, H-4), 8.88 (2H, s, H-2, H-6), 8.59 (1H, dd,
J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.49 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-
40), 7.45-7.42 (3H, m, H-50, H-200, H-600), 6.98 (2H, d, J ¼ 9.2 Hz, H-300,
H-500), 3.81 (3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm):
162.5, 157.4, 156.2 ꢂ 2, 150.0, 148.1, 135.8, 132.1, 131.2, 130.7,
128.6 ꢂ 2, 124.2, 114.4 ꢂ 2, 55.6. HRMS (ESI, m/z) calcd. for
C
16H14N4O3S (MþH)þ 343.0859, found 343.0844. Purity: 97% by
HPLC (tR ¼ 1.879 min).
Procedure E for Synthesizing of N-(2-(2,3-dimethoxyphenyl)
pyridin-3-yl)-4-methoxy-N-((4-methoxyphenyl)sulfonyl)benze-
N-(2-(3-aminophenyl)pyridin-3-yl)-4-
methoxybenzenesulfonamide (HoAn19). Yellow powder. Yield:
63%. M.p: 115.1e115.9 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.49
(1H, s, -NHSO2-), 8.38 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.2 Hz, H-60), 7.58
(2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.45 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz,
H-40), 7.25 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-50), 7.02-6.99 (3H, m,
H-6, H-300, H-500), 6.67 (1H, s, H-2), 6.61-6.57 (2H, m, H-4, H-5), 5.08
(2H, s, -NH2), 3.82 (3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6)
(ppm): 162.3,154.5,148.3,146.2,138.5,132.5,131.8, 130.4,128.7 ꢂ 2,
128.1, 122.3, 116.6, 114.9, 114.2 ꢂ 2, 113.7, 55.6. HRMS (ESI, m/z)
calcd. for C18H17N3O3S (MþH)þ 356.1063, found 356.1063. Purity:
95% by HPLC (tR ¼ 2.406 min).
nesulfonamide
(HoAn28).
The
intermediate
2-(2,3-
dimethoxyphenyl)pyridin-3-amine (2d) was prepared with
similar procedure which described for the synthesis of the inter-
mediate 2a in Procedure A with a 58% yield.
a
Compound
2d
(100
mg,
0.434
mmol),
4-
methoxybenzenesulfonyl chloride (269 mg, 1.30 mmol) and TEA
(0.301 mL, 2.17 mmol) was dissolved in CH2Cl2, and the mixture
was stirred under reflux at 68 ꢀC for 12 h. After the reaction was
completed, the solvent was removed and extracted with EA/H2O (1/
1, v/v, 200 mL) for three times. The EA layer was concentrated and
purified by silica gel column chromatography (PE: EA ¼ 2: 1) to
obtain a yellow solid HoAn28 (132 mg, 76%). M.p: 176.3e177.2 ꢀC.
1H NMR (400 MHz, CDCl3) (ppm): 8.72 (1H, dd, J1 ¼ 4.4 Hz,
J2 ¼ 1.6 Hz, H-60), 7.73 (4H, d, J ¼ 7.6 Hz, H-2’’ ꢂ 2, H-6’’ ꢂ 2), 7.37
(1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-6), 7.29 (1H, dd, J1 ¼ 8.0 Hz,
J2 ¼ 1.6 Hz, H-4), 7.24 (1H, dd, J1 ¼8.0 Hz, J2 ¼ 4.4 Hz, H-50), 6.95 (1H,
t, J ¼ 7.6 Hz, H-5), 6.87-6.83 (5H, m, H-40, H-3’ ꢂ 20, H-5’’ ꢂ 2), 3.88
(6H, s, 400-OCH3 ꢂ 2), 3.86 (3H, s, 3-OCH3), 3.74 (3H, s, 2-OCH3). 13C
NMR (100 MHz, CDCl3) (ppm): 163.7 ꢂ 2, 158.9, 152.7, 149.7, 147.4,
139.4, 132.6, 131.5 ꢂ 2, 130.9, 130.3 ꢂ 2, 123.0, 122.4 ꢂ 4, 113.7 ꢂ 4,
112.8, 61.1, 55.6 ꢂ 2, 55.5. HRMS (ESI, m/z) calcd. for C27H26N2O8S2
(MþH)þ 571.1203, found 571.1203. Purity: 95% by HPLC
(tR ¼ 7.342 min).
Procedure D for Synthesizing of N-([2,4′-bipyridin]-3-yl)-4-
methoxybenzenesulfonamide (HoAn25). 2-bromopyridin-3-
amine (1) (500 mg, 2.89 mmol), pyridin-4-ylboronic acid
(355 mg, 2.89 mmol), Pd(PPh3)4 (167 mg, 0.144 mmol) and K2CO3
(1.20 g, 8.67 mmol) was dissolved in dioxane/H2O (2/1, v/v, 25 mL).
The mixture was stirred under reflux at 120 ꢀC for 12 h containing
N2 atmosphere. After cooling to room temperature, the solvent was
concentrated and extracted with EA/H2O (1/1, v/v, 500 mL) for three
times. The organic layer was concentrated and the crude product
was purified by silica gel column chromatography (PE: EA ¼ 1: 2) to
give a brown solid [2,40-bipyridin]-3-amine (2u) (178 mg, 36%).
Compound
2u
(100
mg,
0.584
mmol),
4-
methoxybenzenesulfonyl chloride (145 mg, 0.701 mmol), pyridine
(0.5 mL) and DMAP (35 mg, 0.292 mmol) was dissolved in CH2Cl2
and stirred under reflux at 70 ꢀC for 10 h. Then the reaction mixture
was concentrated under reduced pressure, extracted with EA/H2O
(2/1, v/v, 150 mL) for three times. The organic layer was collected
and removed, then the crude product was purified by silica gel
column chromatography (PE: EA ¼ 1: 2) to give a white solid
HoAn25 (109 mg, 55%). M.p: 176.5e177.4 ꢀC. 1H NMR (400 MHz,
CDCl3) (ppm): 8.53 (2H, d, J ¼ 6.0 Hz, H-3, H-5), 8.46 (1H, dd,
J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.97 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, H-
40), 7.57 (2H, d, J ¼ 9.2 Hz, H-200, H-600), 7.33 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 4.8 Hz, H-50), 7.11 (2H, d, J ¼ 6.0 Hz, H-2, H-6), 6.88 (2H, d,
J ¼ 9.2 Hz, H-300, H-500), 3.85 (3H, s, 400-OCH3). 13C NMR (100 MHz,
Compounds HoAn29-HoAn31 was prepared with a similar
procedure which described for the synthesis of HoAn28.
N-(2-(2,4-dimethoxyphenyl)pyridin-3-yl)-4-methoxy-N-((4-
methoxyphenyl)sulfonyl)benzenesulfonamide (HoAn29). Yellow
powder. Yield: 78%. M.p: 182.1e182.9 ꢀC. 1H NMR (400 MHz, CDCl3)
(ppm): 8.73 (1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.69-7.65 (5H, m,
H-6, H-2’’ ꢂ 2, H-6’’ ꢂ 2), 7.29 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, H-40),
7.21 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.4 Hz, H-50), 6.85 (4H, d, J ¼ 8.8 Hz, H-
3’’ ꢂ 2, H-5’’ ꢂ 2), 6.43 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 2.4 Hz, H-5), 6.31
(1H, d, J ¼ 2.0 Hz, H-3), 3.89 (6H, s, 400-OCH3 ꢂ 2), 3.83 (3H, s, 4-
OCH3), 3.62 (3H, s, 2-OCH3). 13C NMR (100 MHz, CDCl3) (ppm):
163.7 ꢂ 2, 161.2, 158.4, 149.9, 139.4, 131.3 ꢂ 5, 131.1, 130.5 ꢂ 2,
122.1 ꢂ 2,113.7 ꢂ 5,103.6, 99.0, 55.6 ꢂ 2, 55.4, 55.3. HRMS (ESI, m/z)
15

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
calcd. for C27H26N2O8S2 (MþH)þ 571.1203, found 571.1185. Purity:
99% by HPLC (tR ¼ 5.687 min).
125.0, 124.4, 117.2, 117.0, 113.4, 21.5. HRMS (ESI, m/z) calcd. for
C
HPLC (tR ¼ 3.753 min).
19H14FN3O2S (MþH)þ 368.0864, found 368.0868. Purity: 99% by
N-(2-(2,5-dimethoxyphenyl)pyridin-3-yl)-4-methoxy-N-((4-
methoxyphenyl)sulfonyl)benzenesulfonamide (HoAn30). Yellow
powder. Yield: 70%. M.p: 154.1e155.0 ꢀC. 1H NMR (400 MHz,
DMSO‑d6) (ppm): 8.70 (1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.64-
7.53 (4H, m, H-2’’ ꢂ 2, H-6’’ ꢂ 2), 7.43 (1H, dd, J1 ¼8.0 Hz, J2 ¼ 4.8 Hz,
H-50), 7.23 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-3), 7.19 (1H, d,
J ¼ 2.8 Hz, H-6), 7.04 (4H, d, J ¼ 8.8 Hz, H-3’’ ꢂ 2, H-5’’ ꢂ 2), 6.92-
6.84 (2H, m, H-4, H-40), 3.88 (6H, s, 400-OCH3 ꢂ 2), 3.68 (3H, s, 5-
OCH3), 3.53 (3H, s, 2-OCH3). 13C NMR (100 MHz, DMSO‑d6)
(ppm): 163.7 ꢂ 2, 157.9, 152.1, 151.3, 150.1, 138.9, 130.8, 130.0,
129.2 ꢂ 4, 127.0, 123.1 ꢂ 2, 115.6, 114.8, 114.2 ꢂ 4, 112.6, 55.9 ꢂ 2,
55.6, 55.4. HRMS (ESI, m/z) calcd. for C27H26N2O8S2 (MþH)þ
571.1203, found 571.1177. Purity: 95% by HPLC (tR ¼ 5.695 min).
N-(2-(benzo[d] [1,3]dioxol-5-yl)pyridin-3-yl)-4-methoxy-N-
4-chloro-N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)benze-
nesulfonamide (HoAn34). Grey-white powder. Yield: 38%. M.p:
180.9e181.8 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 8.52 (1H, d,
J ¼ 4.0 Hz, H-60), 7.94 (1H, d, J ¼ 8.0 Hz, H-6), 7.66 (1H, t, J ¼ 7.2 Hz,
H-5), 7.55 (2H, d, J ¼ 8.4 Hz, H-200, H-600), 7.44 (2H, d, J ¼ 8.4 Hz, H-300,
H-500), 7.38 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-2), 7.17 (1H, d,
J ¼ 8.0 Hz, H-50), 7.02 (1H, d, J ¼ 9.6 Hz, H-40), 6.59 (1H, s, -NHSO2-).
13C NMR (100 MHz, CDCl3) (ppm): 170.2, 153.1, 147.3, 140.6, 137.1,
133.9, 132.1, 130.4, 129.7 ꢂ 2, 128.4 ꢂ 2, 125.0, 124.9, 124.5, 117.2,
116.9, 113.2. HRMS (ESI, m/z) calcd. for C18H11ClFN3O2S (MþH)þ
388.0317, found 388.0310. Purity: 99% by HPLC (tR ¼ 3.465 min).
4-bromo-N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)benze-
nesulfonamide (HoAn35). Grey-white powder. Yield: 53%. M.p:
192.8e193.6 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 10.28 (1H, s,
-NHSO2-), 8.60 (1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.89 (1H, t,
J ¼ 7.2 Hz, H-5), 7.65 (2H, d, J ¼ 8.4 Hz, H-200, H-600), 7.57 (1H, dd,
J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz, H-2), 7.52-7.46 (3H, m, H-6, H-40, H-50), 7.38
(2H, d, J ¼ 8.4 Hz, H-300, H-500). 13C NMR (100 MHz, DMSO‑d6) (ppm):
163.1, 160.6, 152.7, 148.3, 145.5, 145.4, 138.9, 137.3, 133.3, 132.2,
130.2, 128.3, 126.8, 126.1, 124.5, 116.8, 116.6, 113.8. HRMS (ESI, m/z)
calcd. for C18H11BrFN3O2S (M-H)- 429.9667, found 429.9682. Purity:
97% by HPLC (tR ¼ 3.621 min).
((4-methoxyphenyl)sulfonyl)benzenesulfonamide
(HoAn31).
Yellow powder. Yield: 77%. M.p: 188.8e189.7 ꢀC. 1H NMR (400 MHz,
CDCl3) (ppm): 8.70 (1H, dd, J1 ¼4.4 Hz, J2 ¼ 1.6 Hz, H-60), 7.72 (4H, d,
J ¼ 9.2 Hz, H-2’’ ꢂ 2, H-6’’ ꢂ 2), 7.31-7.26 (2H, m, H-5, H-6), 7.22 (1H,
dd, J1 ¼ 8.0 Hz, J2 ¼ 4.4 Hz, H-50), 7.09 (1H, d, J ¼ 2.0 Hz, H-2), 6.88
(4H, d, J ¼ 8.8 Hz, H-3’’ ꢂ 2, H-5’’ ꢂ 2), 6.70 (1H, d, J ¼ 8.4 Hz, H-40),
5.93 (2H, s, -OCH2O-), 3.90 (6H, s, 400-OCH3 ꢂ 2). 13C NMR (100 MHz,
CDCl3) (ppm): 163.9 ꢂ 2, 159.5, 150.6, 148.0, 147.3, 139.9, 131.4 ꢂ 4,
130.5 ꢂ 2, 130.2, 124.0 ꢂ 2, 122.2, 113.8 ꢂ 4, 110.1, 107.9, 101.1,
55.7 ꢂ 2. HRMS (ESI, m/z) calcd. for C26H22N2O8S2 (MþH)þ
555.0890, found 555.0880. Purity: 98% by HPLC (tR ¼ 7.238 min).
N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)-4-(tri-
fluoromethyl)benzenesulfonamide (HoAn36). Yellowish powder.
Yield: 68%. M.p: 183.7e184.6 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm):
8.54 (1H, d, J ¼ 4.0 Hz, H-60), 7.93 (1H, d, J ¼ 8.0 Hz, H-6), 7.74 (4H,
m, H-200, H-300, H-500, H-600), 7.63 (1H, t, J ¼ 7.2 Hz, H-5), 7.39 (1H, dd,
J1 ¼8.0 Hz, J2 ¼ 4.8 Hz, H-2), 7.16 (1H, d, J ¼ 8.0 Hz, H-50), 6.99 (1H, d,
J ¼ 9.2 Hz, H-40), 6.80 (1H, s, -NHSO2-). 13C NMR (100 MHz, CDCl3)
(ppm): 164.2, 161.6, 149.6, 147.5, 143.8, 143.7, 142.2, 133.9, 132.5,
130.1, 127.5 ꢂ 2, 126.6, 126.5, 125.0, 124.5, 117.1, 116.9, 113.2. HRMS
Procedure
fluorophenyl)pyridin-3-yl)-4-methoxybenzenesulfonamide
(HoAn32). The intermediate 4-(3-aminopyridin-2-yl)-2-
F
for Synthesizing of N-(2-(4-cyano-3-
fluorobenzonitrile (2t) was prepared with a similar procedure
which described for the synthesis of the intermediate 2a in Pro-
cedure A with a 70% yield.
Compound 2t (100 mg, 0.469 mmol), DMAP (28 mg,
0.234 mmol) and pyridine (0.5 mL) was dissolved in CH2Cl2, and
then 4-methoxybenzenesulfonyl chloride (116 mg, 0.562 mmol)
was added slowly within 5 min. The reaction mixture was stirred
under reflux at 65 ꢀC for 12 h. Till the reaction finished, the mixture
was concentrated and extracted with EA/H2O (2/1, v/v, 250 mL) for
three times. EA was removed under reduced pressure and the crude
product was separated by silica gel column chromatography (PE:
EA ¼ 4: 1) to obtain a white solid HoAn32 (79 mg, 44%). M.p:
158.6e159.5 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 9.95 (1H, s,
-NHSO2-), 8.56 (1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.90 (1H, t,
J ¼ 7.2 Hz, H-6), 7.55 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-5), 7.51-7.43
(3H, m, H-2, H-40, H-50), 7.40 (2H, d, J ¼ 8.8 Hz, H-200, H-600), 6.96 (2H,
d, J ¼ 9.2 Hz, H-300, H-500), 3.82 (3H, s, 400-OCH3). 13C NMR (100 MHz,
DMSO‑d6) (ppm): 162.4, 160.6, 152.2, 147.8, 145.6, 145.5, 136.5,
133.2, 131.1, 130.7, 128.6 ꢂ 2, 126.0, 124.4, 116.8, 114.2 ꢂ 2, 113.9,
55.6. HRMS (ESI, m/z) calcd. for C19H14FN3O3S (MþH)þ 384.0813,
found 384.0816. Purity: 98% by HPLC (tR ¼ 6.219 min).
(ESI, m/z) calcd. for
C
19H11F4N3O2S (M-H)- 420.0435, found
420.0456. Purity: 95% by HPLC (tR ¼ 10.878 min).
N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)benzenesulfona-
mide (HoAn37). White powder. Yield: 60%. M.p: 188.8e189.7 ꢀC. 1H
NMR (400 MHz, DMSO‑d6) (ppm): 10.14 (1H, s, -NHSO2-), 8.56 (1H,
dd, J1 ¼ 4.8 Hz, J2 ¼ 1.2 Hz, H-60), 7.91 (1H, t, J ¼ 7.2 Hz, H-6), 7.64-
7.60 (1H, m, 400), 7.53-7.42 (8H, m, H-2, H-5, H-40, H-50, H-200, H-300,
H-500, H-600). 13C NMR (100 MHz, DMSO‑d6) (ppm): 163.1, 160.6,
152.2, 147.9, 145.6, 145.5, 136.2, 133.3, 132.8, 130.6, 129.2 ꢂ 2,
126.3 ꢂ 2, 126.0, 124.4, 116.8, 113.9. HRMS (ESI, m/z) calcd. for
C
18H12FN3O2S (MþH)þ 354.0707, found 354.0716. Purity: 96% by
HPLC (tR ¼ 3.158 min).
N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)naphthalene-2-
sulfonamide (HoAn38). White powder. Yield: 51%. M.p:
168.9e169.8 ꢀC. 1H NMR (400 MHz, DMSO‑d6) (ppm): 10.25 (1H, s,
-NHSO2-), 8.57 (1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 8.09 (1H, s, H-
200), 8.04-7.97 (3H, m, H-6, H-500, H-600), 7.73-7.63 (3H, m, H-5, H-a,
H-d), 7.59 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz, H-2), 7.52 (1H, dd,
J1 ¼ 8.8 Hz, J2 ¼ 1.6 Hz, H-b), 7.47-7.40 (3H, m, H-c, H-40, H-50). 13C
NMR (100 MHz, DMSO‑d6) (ppm): 162.9, 160.4, 152.6, 148.2, 137.3,
136.7, 134.1, 133.0, 131.4, 130.4, 129.3, 129.1, 129.0, 127.7, 127.6, 127.3,
125.9, 124.4, 121.7, 116.6, 116.4, 113.7. HRMS (ESI, m/z) calcd. for
Compounds HoAn33-HoAn40 was prepared with a similar
procedure which described for the synthesis of HoAn32. (The re-
agent 4-methoxybenzenesulfonyl chloride was replaced by
different substituted benzensulfonyl chloride.)
N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)-4-
methylbenzenesulfonamide (HoAn33). Yellowish powder. Yield:
40%. M.p: 176.6e177.5 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 8.48
(1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.6 Hz, H-60), 7.99 (1H, dd, J1 ¼ 8.0 Hz,
J2 ¼ 1.2 Hz, H-6), 7.59 (1H, t, J ¼ 7.2 Hz, H-5), 7.46 (2H, d, J ¼ 8.0 Hz,
H-200, H-600), 7.37 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 4.8 Hz, H-2), 7.24 (2H, d,
J ¼ 8.8 Hz, H-300, H-500), 7.12 (1H, dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, H-50),
6.86 (1H, dd, J1 ¼ 9.2 Hz, J2 ¼ 1.2 Hz, H-40), 6.74 (1H, s, -NHSO2-),
2.44 (3H, s, 400-CH3). 13C NMR (100 MHz, CDCl3) (ppm): 164.3, 161.7,
149.3, 146.9, 145.0, 135.6, 133.7, 132.4, 130.8, 130.0 ꢂ 2, 126.9 ꢂ 2,
C
22H14FN3O2S (M-H)- 402.0718, found 402.0744. Purity: 99% by
HPLC (tR ¼ 4.240 min).
N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)-4-(tri-
fluoromethoxy)benzenesulfonamide (HoAn39). Yellowish pow-
der. Yield: 35%. M.p: 162.6e163.5 ꢀC. 1H NMR (400 MHz, CDCl3)
(ppm): 8.53 (1H, d, J ¼ 3.6 Hz, H-60), 7.94 (1H, d, J ¼ 7.6 Hz, H-6), 7.67
(2H, d, J ¼ 8.8 Hz, H-200, H-600), 7.63 (1H, d, J ¼ 7.2 Hz, H-5), 7.38 (1H,
dd, J1 ¼ 8.0 Hz, J2 ¼ 4.8 Hz, H-2), 7.28 (2H, d, J ¼ 8.4 Hz, H-300, H-500),
7.16 (1H, d, J ¼ 7.6 Hz, H-50), 7.01 (1H, d, J ¼ 9.2 Hz, H-40), 6.77 (1H, s,
16

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
-NHSO2-). 13C NMR (100 MHz, CDCl3) (ppm): 164.3, 161.7, 152.8,
149.4, 147.4, 143.9, 143.8, 136.9, 133.9, 132.3, 130.3, 129.2 ꢂ 2, 125.0,
124.5, 121.0, 117.1, 116.9, 113.2. HRMS (ESI, m/z) calcd. for
4.2.3. MTS assay
The in vitro antiproliferative effect of compounds was deter-
mined by the MTS assay. Briefly, cells grown in the logarithmic
phase were seeded into 96-well plates (5 ꢂ 103e6 ꢂ 103 cells per
well, depending on the growth rate of the cell line) for 24 h. After
C
19H11F4N3O3S (M-H)- 436.0384, found 436.0403. Purity: 97% by
HPLC (tR ¼ 3.074 min).
N-(2-(4-cyano-3-fluorophenyl)pyridin-3-yl)-3,4-
that, fresh medium (100 ml) containing the test compound at a
dimethoxybenzenesulfonamide (HoAn40). Yellowish powder.
Yield: 66%. M.p: 141.5e142.3 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
(ppm): 9.92 (1H, s, -NHSO2-), 8.57 (1H, d, J ¼ 3.6 Hz, H-60), 7.88 (1H,
t, J ¼ 7.2 Hz, H-6), 7.60 (1H, d, J ¼ 8.0 Hz, H-5), 7.49-7.44 (3H, m, H-2,
H-200, H-600), 6.99-6.91 (3H, m, H-40, H-50, H-500), 3.82 (3H, s, 300-
OCH3), 3.67 (3H, s, 400-OCH3). 13C NMR (100 MHz, DMSO‑d6) (ppm):
163.0, 160.5, 152.3, 148.4, 147.9, 145.4, 145.3, 137.0, 133.0, 130.7,
126.0, 124.4, 120.3, 116.7, 116.5, 113.9, 110.7, 108.5, 55.7, 55.3. HRMS
(ESI, m/z) calcd. for C20H16FN3O4S (M-H)- 412.0773, found 412.0794.
Purity: 98% by HPLC (tR ¼ 2.832 min).
certain concentration was added to each well and incubated for
72 h in five replicates. Then the old medium was replaced by fresh
medium (100 ml) containing 10% MTS and the cells were incubated
in dark at 37 ꢀC for at least 30 min. The optical density value at
490 nm of each well was recorded in BioTek Synergy H4 microplate
Reader (Vermont, USA). The cytotoxic effect of test compound was
expressed as the inhibitory rate or IC50 value, which was calculated
by the nonlinear regression analysis using GraphPad Prism 7.0
(GraphPad Software, San Diego, CA).
4.2.4. In vitro tubulin polymerization assay
The tubulin polymerization assay was performed according to
the procedures provided by a commercial kit (#BK004P) purchased
from Cytoskeleton (Danvers, MA, USA) with appropriate modifi-
cation. Briefly, porcine tubulin protein (4 mg, >97% pure) was
4.2. Biology
4.2.1. Cell lines and culture
The human cancer cell lines (MCF-7, SK-HEP-1, RKO, PLC/PRF/5,
HCC-LM3, BT474, SK-BR-3, SW620, SW480, A549, H460, PANC-1
and SGC-7901) were purchased from Cell Bank of the Chinese
Academy of Science (Shanghai, China), and the HUVEC cell line was
purchased from ATCC (Washington, DC, USA). Cell lines including
MCF-7, SK-HEP-1, PLC/PRF/5, HCC-LM3, SK-BR-3, A549, PANC-1 and
HUVEC were cultivated in DMEM medium (CR-12800-S, Zhejiang
Senrui Biological Technology Co., Ltd, Huzhou, China). Cell lines
including BT474, RKO, SW620, SW480, H460 and SGC-7901 were
maintained in RPMI 1640 medium (CR-31800-S, Zhejiang Senrui
Biological Technology Co., Ltd, Huzhou, China). The medium used in
all cell lines were supplemented with 10% (v/v) fetal bovine serum
resuspended with 400
PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, pH ¼ 6.9) to be tubulin stock
solution (10 mg/mL). Then, 176 L tubulin stock solution was
diluted with 264 L ice-cold general tubulin buffer and then added
L GTP stock solution (100 mM) to be tubulin reaction mix (4 mg/
mL). Meanwhile, 5 L of the 2 mM test compound (HoAn32, ABT-
751, paclitaxel and DMSO) stock solution was diluted with 95 L of
room temperature general tubulin buffer to form a solution with a
concentration of 100 M. Half area 96-well plate was pre-warmed
in Synergy H4 microplate reader (BioTek, Winooski, Vermont, USA)
at 37 ꢀC for 30 min, and then 10
L of the test compounds (100 M)
mL ice-cold general tubulin buffer (80 mM
m
m
4
m
m
m
m
m
m
was added into the wells of the pre-warmed plate and incubated for
another 5 min at 37 ꢀC before detecting the tubulin polymerization
reaction. After that, pre-cooled tubulin reaction mix was added into
(FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin. All cell
lines were cultivated at 37 ꢀC in a humidified atmosphere with 5%
CO2.
the corresponding wells (100
mL per well) immediately and the
absorbance was detected by Synergy H4 microplate reader at
4.2.2. Antibodies and reagents
340 nm at 37 ꢀC every 1 min for 60 min.
Primary antibodies including anti-b-actin (4970, 1:1000), anti-
Cdc2 (77055, 1:1000), anti-Cyclin B1 (4138, 1:1000), anti-Bax
(2772, 1:1000) and anti-Cleaved PARP (9541, 1:1000) were pur-
chased from Cell Signaling Technology (Shanghai, China), and anti-
4.2.5. EBI competition assay
SW620 cells were seeded to 6-well plates (5 ꢂ 105 cells per
well). Firstly, the cells were incubated with DMSO, HoAn32 (10, 20
b
-tubulin antibody (ET1602-4, 1:2000) was purchased from
and 40
mM) and ABT-751 (20 and 40
mM) for 2 h and then the cells
Hangzhou HuaAn Biotechnology Co., Ltd (Zhejiang, China). Second
antibodies including the goat anti-rabbit IgG (HþL) highly cross-
adsorbed secondary antibody/Alexa Fluor Plus 594 (A32740) was
purchased from Invitrogen (Camarillo, CA, USA) and Peroxidase
AffiniPure Goat Anti-Rabbit IgG (HþL) (111-035-003, 1:5000) was
purchased from Jackson ImmunoResearch (Pennsylvania, USA). The
cell cycle staining kit (CCS012) was purchased from Multi Sciences
(Hangzhou, China) and FITC Annexin V apoptosis detection kit
(556547) was purchased from BD Biosciences (New Jersey, USA).
Reactive Oxygen Species assay kit (S0033S) was purchased from
Beyotime (Shanghai, China). A commercial kit (#BK004P) used for
the tubulin polymerization assay was purchased from Cytoskeleton
were treated with EBI (100
m
M) for 2 h. The cells were harvest and
treatment with 1 ꢂ loading buffer (50 mM Tris-HCl PH ¼ 6.8, SDS
2%, bromophenol blue 0.1%, glycerol 10%, DTT 1.5%) to prepare the
cell extracts for western blot analysis. The next processes were
similar to the steps described in chapter 4.2.9 Western Blot Assay
section, in which anti-
antibody.
b-tubulin antibody was used as primary
4.2.6. Immunofluorescence microscopy
RKO cells were seeded to 6-well plates (2.5 ꢂ 105 cells per well)
with glass coverslips and incubated overnight. The cells were
treated with DMSO, HoAn32 (0.5
mM and 1 mM), ABT-751 (1 mM)
(Danvers,
MA,
USA).
3-(4,5-dimethylthiazol-2-yl)-5-(3-
and paclitaxel (1 M) and incubated for 24 h. Then the cells were
m
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS,
G111A) was purchased from Promega (Madison, WI, USA). Honokiol
washed with PBS for three times and fixed with pre-cooled
methanol for 10 min. After permeabilization with 0.25% Triton X-
100 for 10 min, the cells were washed with PBS for three times and
blocked in 1% bovine serum albumin (BSA) at 4 ꢀC overnight. The
(CSN19405, purity
ꢃ
99.9%) and ABT-751 (CSN16987,
purity ꢃ 99.9%) were purchased from CSNpharm (Shanghai, China).
Corn oil (C116023) was obtained from aladdin (Shanghai, China).
N,N0-ethylene-bis(iodoacetamide) (EBI, E917500) was obtained
from TRC (Toronto, Canada). Stock solution (20 mM) of compounds
was prepared with dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St.
Louis, MO) and freshly diluted with cell culture medium before use.
cells were incubated with rabbit anti-b-tubulin primary antibody
containing 1% BSA (v/v, 1: 100) at room temperature for 2 h and
washed with PBS for three times. After that, the cells were incu-
bated with goat anti-rabbit IgG (HþL) highly cross-adsorbed sec-
ondary antibody/Alexa Fluor Plus 594 containing 1% BSA (v/v, 1:
17

H. Zhu, S. Ying, B. Zhou et al.
European Journal of Medicinal Chemistry 211 (2021) 113117
500) at room temperature for 1 h and washed with PBS for three
times. 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Sci-
entific) was added and keep in dark for 1 h. Finally, fluorescence
images of the cells were recorded on BX53 Upright Microscope
(Olympus, Japan).
glycerol 10%, DTT 1.5%). Then, the protein extracts were boiled for
15 min, sonicated, run in 8%e15% SDS-PAGE gels, transferred to
polyvinylidene fluoride (PVDF) membranes (Beyotime, China) and
blocked with 5% non-fat milk (dissolved in 1 ꢂ TBST). After that, the
membranes were incubated with corresponding primary anti-
bodies at 4 ꢀC overnight, washed with 1 ꢂ TBST (TBS with 0.1%
Tween-20) for three times, followed by incubating with horse-
radish peroxidase (HRP)-conjugated secondary antibody at room
temperature for 2 h and washed with 1 ꢂ TBST for three times
again. Finally, the immunoreactivity of the membranes was visu-
alized by using a LAS-4000mini Luminescent Imaging Analyzer
(Fujifilm, Japan) with an enhanced chemiluminescence kit (Beyo-
time, China) for HRP.
4.2.7. Cell cycle analysis
RKO, SW620 and PANC-1 cells were seeded into 6-well plates
(4 ꢂ 105e5 ꢂ 105 cells per well) and incubated for 24 h. Subse-
quently, the cells were treated with DMSO, HoAn32 (0.5 and 1 mM)
and ABT-751 (1 mM) for another 24 h. Then the cells were harvested
by centrifugation and washed with PBS for three times, ice-cold
75% ethanol was added to fix cells at 4 ꢀC overnight. After that,
ethanol was removed and the cells were resuspended with ice-cold
PBS, and then RNase A and the DNA staining solution propidium
iodide (PI) (Multi Sciences, China) was added and keep the cells in
the darkness at 37 ꢀC for 30 min before detection. Finally, the cell
cycle distribution was detected by a FACS Calibur flow cytometer
(Bectone Dickinson, San Jose, CA, USA).
4.2.12. Molecular modeling
The crystal structure of tubulin in complex with ABT-751 was
downloaded from the Protein Data Bank (PDB codes: 3HKC, http://
www.rcsb.org). The whole docking process took place in LeDock
software. Briefly, molecular HoAn32 was prepared in ChemBio3D
Ultra 14.0 and optimized to obtain the lowest energy conformer.
The protein structure was prepared in Lepro module. After
extracting the ligand, hydrogen atoms were added to the crystal
and then the charges were added to biopolymer by the CHARMm
force field. Finally, the docking studies of HoAn32 with the pre-
pared protein occurred in Ledock module with the default settings.
6 poses were exported for the next analysis. The structural images
were produced by PyMOL software.
4.2.8. Cell apoptosis analysis
RKO and SW620 cells were seeded into 6-well plates
(4 ꢂ 105e5 ꢂ 105 cells per well) and treatment with DMSO,
HoAn32 (0.5 and 1 mM) or ABT-751 (1 mM) for 24 h. After incuba-
tion, the cells were harvested and washed with PBS for three times.
Then, 100 L of the mixture of Annexin V/FITC (BD Biosciences,
USA) and PI in binding buffer (v/v, 5: 5: 100) was added and keep in
the darkness at 37 ꢀC for 30 min. Before detection, another 300
m
mL
binding buffer was added into the sample and then apoptosis was
analyzed by a FACS Calibur flow cytometer (Bectone Dickinson, San
Jose, CA, USA).
4.2.13. Wound healing assays
HUVEC cells were seeded in 6-well plates at a density of
1 ꢂ 106 cells per well for 24 h. After producing scratches by using
200
m
L pipette tips, the wounds were washed twice with 1 ꢂ PBS to
4.2.9. Clonogenic assay
RKO or SW620 cells were embedded at a density of 1000 cells
per well in 6-well plates overnight and then treated with DMSO、
remove nonadherent cell debris. Then, the cells were incubated
with 0, 0.5 M, and 1 M of HoAn32 or 1 M of ABT-751 for 24 h.
m
m
m
Cells which migrated across the wound area were photographed
through inverted microscopy (Primovert, ZEISS) at 0 and 24 h.
HoAn32 (0.125
mM、0.25 mM、0.5 mM and 1 mM) and ABT-
751(1 M) for another 24 h. After that, the culture medium was
m
replaced with fresh culture medium and the cells were continue to
grow for another 10 days until analysis. After this stage, the culture
medium was removed and washed with 1 ꢂ PBS for twice. Then,
the cells were fixed with methanol for 15 min, and stained with
0.1% crystal violet for another 30 min. Finally, the cells were washed
with water, dried and then photographed for visualization. More
than 50 cells per colony were counted.
4.2.14. In vivo antitumor efficacy of HoAn32 in the SW620 colon
cancer xenograft model
All the animal experimental procedures performed in accor-
dance with the animal experiment guidelines of the Animal Care
and Welfare Committee of Zhejiang University. The Five to six-
week-old male athymic nude mice were purchased from
Shanghai Laboratory Animal Center, CAS (SLACCAS) and fed under
pathogen-free condition for one week before use. A total of 5 ꢂ 106
4.2.10. Measurement of intracellular ROS generation
SW620 cells containing 125 mL PBS were subcutaneously inoculated
Peroxide-sensitive fluorescent probe DCFH-DA was used for
detecting intracellular ROS generation. In brief, cells were seeded
into 6-well plates (5 ꢂ 105 cells per well) and incubated with
into the right armpit of mice. When the tumor volumes reached
about 100 mm3, mice were randomized into vehicle or treatment
groups (n ¼ 6). HoAn32 and ABT-751 were dissolved in a 1:9 ratio
of DMSO: corn oil mixed solution to produce desired concentra-
tions. The vehicle control solution was prepared with equal parts of
DMSO、HoAn32 (0.25 mM and 0.5 mM) and ABT-751 (0.5 mM) for
24 h. Then the culture medium was replaced by 1 mL serum-free
medium containing 5 M DCFH-DA (1: 2000) and incubated for
m
DMSO and corn oil only. Then, the mice were treatment with 100 mL
another 30 min at 37 ꢀC. After that, DCFH-DA was washed with
serum-free medium for twice. Then the cells were harvested,
centrifugated and washed with PBS for twice. Finally, the samples
were analyzed by a FACS Calibur flow cytometer (Bectone Dick-
inson, San Jose, CA, USA) at an excitation wavelength of 488 nm and
an emission wavelength of 525 nm.
HoAn32 (p.o., 25 mg/kg, per 2 days), ABT-751 (p.o., 25 mg/kg, per 2
days) or vehicle solution (p.o., per 2 days) for 20 days. Tumor vol-
ume and body weights were measured with Vernier calipers every
2 days after the start of drug administration. At the end of the
experiment, the mice were sacrificed and the tumor bulks were
peeled off for weighing. The data was dealt with by the following
formula: tumor volume ¼ L (length) ꢂ W2 (width)/2; The ratio of
inhibition of tumor (%) ¼ (1 - tumor weight of treated group/tumor
weight of vehicle group) ꢂ 100%.
4.2.11. Western blot analysis
Cells were seeded into 6-well plates (4 ꢂ 105e5 ꢂ 105 cells per
well) and incubated with DMSO, HoAn32 (0.5 and 1 mM) and ABT-
751 (1
mM) for 24 h. After incubation, cells were washed with ice-
4.2.15. H&E staining
cold PBS and the protein extracts were prepared in 1 ꢂ loading
The H&E staining experiences were conducted at Hangzhou
HuaAn Biotechnology Co., Ltd (Hangzhou, China). The major organs
buffer (50 mM Tris-HCl PH ¼ 6.8, SDS 2%, bromophenol blue 0.1%,
18

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European Journal of Medicinal Chemistry 211 (2021) 113117
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Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This study was supported by the China State Key Research, Grant
No. 2016YEF0200503 and the National Natural Science Foundation
of China (No. 21532005) as well as the National Natural Science
Foundation of China (No. 81761138047).
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proliferative properties, J. Med. Chem. 63 (2020) 827e846, https://doi.org/
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[23] M.J. Lai, H.Y. Lee, H.Y. Chuang, L.H. Chang, A.C. Tsai, M.C. Chen, H.L. Huang,
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aminobiaryls as antitubulin agents and inhibitors of signal transducers and
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113117.
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