Discovery of novel diarylamide n-containing heterocyclic derivatives as new tubulin polymerization inhibitors with anti-cancer activity

Article abstract of DOI:10.3390/molecules26134047

Tubulin has been regarded as an attractive and successful molecular target in cancer therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer activity. As the continuation of our research work on colchicine binding site tubulin inhibitors, we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-803, PC-3 and EC-109) with IC₅₀ values of 1.56 μM, 3.56 μM and 14.5 μM, respectively. Besides, the SARs of these compounds were preliminarily studied and summarized. The most active compound 15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results of molecular docking also suggested compound 15b could tightly bind into the colchicine binding site of β-tubulin.

Full text of DOI:10.3390/molecules26134047

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molecules
Article
Discovery of Novel Diarylamide N-Containing Heterocyclic
Derivatives as New Tubulin Polymerization Inhibitors with
Anti-Cancer Activity
Xu Liu 1,†, Xiao-Jing Pang 1,†, Yuan Liu 1, Wen-Bo Liu 1, Yin-Ru Li 2, Guang-Xi Yu 1, Yan-Bing Zhang 1, Jian Song 1,
*
and Sai-Yang Zhang 1,2,
*
1
Key Laboratory of Advanced Drug Preparation Technologies (Ministry of Education),
Institute of Drug Discovery & Development, School of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou 450001, China; lx012496@163.com (X.L.); summer_pxj@163.com (X.-J.P.);
liuyuan9720@163.com (Y.L.); 17698567571@163.com (W.-B.L.); ygx19990110@126.com (G.-X.Y.);
zhangyb@zzu.edu.cn (Y.-B.Z.)
School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, 450001, China; 17603868158@163.com
Correspondence: mumuandzz@163.com (J.S.); saiyangz@zzu.edu.cn (S.-Y.Z.)
These authors contributed equally to this work.
2
*
†
Abstract: Tubulin has been regarded as an attractive and successful molecular target in cancer
therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin
inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer
activity. As the continuation of our research work on colchicine binding site tubulin inhibitors,
we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the
combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though
proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group
exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Liu, X.; Pang, X.-J.; Liu, Y.;
Liu, W.-B.; Li, Y.-R.; Yu, G.-X.; Zhang,
Y.-B.; Song, J.; Zhang, S.-Y. Discovery
of Novel Diarylamide N-Containing
Heterocyclic Derivatives as New
Tubulin Polymerization Inhibitors
with Anti-Cancer Activity. Molecules
2021, 26, 4047. https://doi.org/
10.3390/molecules26134047
803, PC-3 and EC-109) with IC50 values of 1.56 µM, 3.56 µM and 14.5 µM, respectively. Besides, the
SARs of these compounds were preliminarily studied and summarized. The most active compound
15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused
microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as
a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results
of molecular docking also suggested compound 15b could tightly bind into the colchicine binding
site of β-tubulin.
Academic Editor: Maria Emília de
Sousa
Keywords: tubulin; CA-4; vicinal diaryl; N-containing heterocyclics; anti-proliferative activity;
colchicine binding site
Received: 8 May 2021
Accepted: 29 June 2021
Published: 2 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Tubulin is an important component of the cytoskeleton and plays a vital role in
the process of maintaining normal cell formation, cell mitosis, signal transduction and
material transportation [
molecular target in cancer therapy and drug discovery [
excellent anti-cancer activity such as paclitaxel/Taxol, vincristine and vinblastine have been
successfully used in clinical treatment [ ]. However, owing to the poor water solubility,
drug resistance or side effects of clinical used tubulin inhibitors [ ], it is necessary to
develop novel tubulin inhibitors [ ]. However, colchicine site tubulin inhibitors have
1
,
2
]. Tubulin has been regarded as an attractive and successful
3]. Tubulin-targeting agents with
4
Copyright:
© 2021 by the authors.
5–7
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
6
received extraordinary attention in the recent years, which could largely overcome the
above drawbacks and have more therapeutic advantages over other binding sites tubulin
inhibitors [8,9]. In addition, no colchicine site tubulin inhibitors have not been approved
for clinical use; therefore, it is necessary to develop novel colchicine site tubulin inhibitors.
Molecules 2021, 26, 4047. https://doi.org/10.3390/molecules26134047
https://www.mdpi.com/journal/molecules

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Molecules 2021, 26, 4047
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Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors,
which is also an important pharmacophoric point of tubulin binding and anti-cancer
activity [10–13]. Natural colchicine site tubulin inhibitor combretastatin A-4 (CA-4) is a 1,2-
diarylethylene analog, which exhibits potent inhibitory potency against many cancer cells
including multidrug resistant cancer cells and could effectively inhibit the polymerization
of tubulin [14]. However, due to the poor water solubility, low bioavailability and unstable
cis double bond of CA-4 [12,15], its clinical application is limited; therefore, clinical work
with CA-4 was carried out with the corresponding water-soluble phosphate prodrug salt
(
CA-4P) [16]. Therefore, many groups have reported novel colchicine site tubulin inhibitors
with vicinal diaryl cores base on the structural optimizations of CA-4 [17–25]. Cushman
et al. reported a novel tubulin inhibitor benzyl aniline in which the olefinic bridge of the
CA-4 was replaced by an aminomethylene hydrochloride moiety. Compound retained
1
1
significant anti-proliferative activity and inhibitory potency of tubulin polymerization [23].
Romagnoli et al. replaced the usual ethylene bridge of the led CA-4 with a five membered
heterocyclic ring to obtain 1,5-disubstituted 1,2,4-triazole
inhibitory effect on tubulin polymerization (IC50 = 2.3 M), and significantly suppressed
the growth of human cancer cells at nano-molar levels [24]. Meegan0s group reported
a class of diaryl- -lactam derivatives which contained the -lactam ring system in place of
the ethylene bridge of the led CA-4 [25]. Compound displayed sub nano-molar inhibitory
2. Compound 2 exhibited potent
µ
β
β
3
activity against breast cancer cells MCF-7 and MDA-MB-231 (IC50 = 34 and 78 nM, respec-
tively) together with significant inhibition of tubulin polymerization (Figure 1). Therefore,
tubulin inhibitors bearing the vicinal diaryl moiety may be favorable for the interactions
with tubulin.
Figure 1. Structures of CA-4 and CA-4 analogs as tubulin inhibitors.
N-containing heterocyclic skeleton is one of most attractive frameworks in bioactive
compounds with strong pharmacological significance [26], especially the anti-cancer ability.
For example, indole [27
have also been used to design novel colchicine binding site tubulin inhibitors with po-
tent anti-cancer activity. Chalcone indole derivative potently inhibited cancer cell
growth with IC50 values ranging from 0.22 to 1.80 M [30]. Compound induced
cell cycle arrest in G2/M phase and effectively inhibited the polymerization of tubu-
lin. Quinoline-indole derivative as an anti-tubulin agent targeting the colchicine binding
–32], indoline [33–35] and tetrahydroquinoline derivatives [36–38]
4
µ
4
5
site showed effective inhibition effect against tested cancer cell lines with IC50 values
ranging from 2 to 11 nM together with significant in vitro inhibition of tubulin poly-
merization (IC50 = 2.09
inhibitory effects on the growth of cancer cells with an average IC50 value of 50 nM to-
gether with significant in vitro inhibition of tubulin polymerization (IC50 = 2.52 µM) [32]
The 7-Aroyl-aminoindoline-1-sulfonamide 7 potently inhibited the tubulin polymeriza-
tion (IC50 = 1.1 M) by binding to colchicine binding site. Compound effectively sup-
µM) [31]. Benzimidazole-indole derivative 6 exhibited potent
.
µ
7
pressed the growth of KB, MKN45, H460, HT29 and TSGH cells with IC50 values rang-
ing from 8.6 nM to 10.8 nM [35]. Tetrahydroquinoline-pyrimidine [38] showed strong
anti-proliferative activity (IC50 values ranging from 5.6 to 18.3 nM) via tubulin polymer-
ization inhibition and potently inhibited tumor growth in an A375 melanoma xenograft
model (Figure 2).

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Figure 2. Structures of N-containing heterocyclic derivatives as tubulin inhibitors.
Based on the above findings and as the continuation of our group work on colchicine
binding site tubulin inhibitors, we designed and synthesized a series of diarylamide
N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-
containing heterocyclic skeleton into one hybrid though proper linkers (Figure 3). Among
of these compounds, compound 15b was identified as a tubulin inhibitor with potent
anti-cancer activity.
Figure 3. Design of diarylamide N-containing heterocyclic derivatives in this work.
2. Results and Discussion
2.1. Chemistry
As shown in Scheme 1, all the target compounds 15a
by starting from commercially available 4-methoxybenzylchloride
10. The 4-Methoxybenzylchloride reacted with the 3,4,5-trimethoxyaniline 10 in the pres-
–15h and 19a–19g were synthesized
9
and 3,4,5-trimethoxyaniline
9
ence of K2CO3 in DMF to give compound 11. Then compound 11 reacted with chloroacetyl
chloride to afford compound 13 in DMF. Substitution reactions between compound 13 with
indoles, indolines or tetrahydroquinolines 14 in the presence of K2CO3 in acetonitrile gave
compounds 15a
–h. In the synthesis of another series of compounds, compounds 14 reacted
with 3-bromopropyne 16 in the presence of K2CO3 in acetonitrile to obtain compound 17
.
Substitution reaction between compound 15 with sodium azide in the presence of K2CO3
in acetonitrile gave compound 18. Then click reactions between compound 17 with 18 in
the presence of CuSO4 and sodium ascorbate in THF/H2O (1:1) gave compounds 19a–e.
Finally, all the target compounds were fully characterized by NMR and HMRS which was
showed in the Supplementary Materials.

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Scheme 1. Synthetic routes of diarylamide N-containing heterocyclic derivatives.
2.2. Anti-proliferative Activity and Structure Activity Relationships
According to the latest cancer data in China [39] and the actual situation of our
laboratory, the in vitro anti-proliferative activity of new target compounds 15a and
19a were evaluated against MGC-803 cell line (human gastric cancer), HCT-116 cell line
–
h
–
e
(human colon cancer) and PC-3 cell line (human prostate cancer) using MTT assays with
the well characterized tubulin inhibitor colchicine was used as a positive control. The
following Tables 1 and 2 depicted the results of in vitro anti-proliferative activity.
Table 1. In vitro anti-proliferative activity of compounds 15a
–
h
against MGC-803, PC-3 and
EC-109 cells.
IC50 (µM) a
Compounds
MGC-803
PC-3
EC-109
15a
15b
15c
15d
15e
15f
15g
15h
1.88 ± 0.58
1.56 ± 0.58
6.25 ± 2.73
20.99 ± 1.32
10.83 ± 6.58
11.89 ± 6.15
9.93 ± 6.83
10.41 ± 3.60
0.18 ± 0.02
6.27 ± 0.19
3.56 ± 0.07
12.03 ± 1.04
18.09 ± 1.28
29.78 ± 2.51
19.83 ± 1.47
16.29 ± 1.01
22.56 ± 1.54
0.23 ± 0.08
19.61 ± 1.07
14.5 ± 0.68
24.61 ± 2.07
54.07 ± 2.83
47.72 ± 2.49
46.81 ± 2.89
31.87 ± 42.6
21.71 ± 1.49
0.32 ± 0.12
Colchicine
a
In vitro anti-proliferative activity was assayed by exposure for 48 h.