Design, synthesis and biological evaluation of novel indole-based oxalamide and aminoacetamide derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126816

A series of novel indole-based oxalamide and aminoacetamide derivatives were designed, synthesized, and evaluated for antiproliferative activities. Preliminary results revealed that compound 8g exhibited significant antiproliferative effect against PC-3,

Full text of DOI:10.1016/j.bmcl.2019.126816

Journal Pre-proofs
Design, synthesisand biological evaluation of novel indole-based oxalamide and
aminoacetamide derivatives as tubulin polymerization inhibitors
Peng-Cheng Diao, Xie-Er Jian, Peng Chen, Chuan Huang, Jie Yin, Jie Chun
Huang, Jun-Sheng Li, Pei-Liang Zhao
PII:
S0960-894X(19)30785-1
DOI:
https://doi.org/10.1016/j.bmcl.2019.126816
BMCL 126816
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
3 September 2019
14 October 2019
8 November 2019
Please cite this article as: Diao, P-C., Jian, X-E., Chen, P., Huang, C., Yin, J., Chun Huang, J., Li, J-S., Zhao, P-L.,
Design, synthesisand biological evaluation of novel indole-based oxalamide and aminoacetamide derivatives as
tubulin polymerization inhibitors, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/
j.bmcl.2019.126816
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Design, synthesis and biological evaluation of novel indole-based oxalamide
and aminoacetamide derivatives as tubulin polymerization inhibitors
Peng-Cheng Diao#, Xie-Er Jian#, Peng Chen, Chuan Huang, Jie Yin, Jie Chun Huang, Jun-Sheng Li,
Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science,
Southern Medical University, Guangzhou 510515, P.R.China
ABSTRACT: A series of novel indole-based oxalamide and aminoacetamide derivatives were
designed, synthesized, and evaluated for antiproliferative activities. Preliminary results revealed
that compound 8g exhibited significant antiproliferative effect against PC-3, HeLa and HCT-116
cell lines. Flow cytometric analysis of the cell cycle demonstrated the compound 8g induced the cell
cycle arrest at G2/M phase in HeLa cell lines. Immunocytochemistry revealed loss of intact
microtubule structure in cells treated with 8g and inhibition of tubulin polymerization. Additionally,
molecular docking analysis suggested that 8g formed stable interactions in the colchicine-binding
site of tubulin. These preliminary results demonstrated that a new class of novel indole-based
oxalamide and aminoacetamide derivatives described in the investigation could be developed as
potential scaffolds to new anticancer agents.
Keywords: Indoles; Synthesis; Tubulin polymerization; Antiproliferative activity.
——————————
 Corresponding author. E-mail: plzhao@smu.edu.cn
# These authors contributed equally to this work.
1

Tubulin, the basic protein component of microtubules, has been identified as a highly attractive
molecular target of numerous anticancer agents. 1-3 Inhibition of tubulin polymerization or blocking
microtubule disassembly disrupts essential cellular functions, such as maintenance of cell shape,
cell signaling, cell division, and even transportation of vesicles and mitochondria. 4-8 In the former
family of agents, colchicine, one of the earliest tubulin-targeting inhibitors, played a crucial role in
clarifying the properties and functions of tubulin and microtubules, and its binding site on tubulin
has attracted great attention. 9-12 However, the clinical development of colchicine as an anticancer
agent did not succeed due to its serious side effects. 13 Therefore, it is an urgent need to develop new
small moleculars as potent tubulin inhibitors with better therapeutic properties for clinical.
N
N
N
N
N
NO2
NH
NO2
NH
N
N
O
HN
N
O
O
HN
O
O
O
O
O
N
N
H
S
N
Cl
Cl
H
N
O
Ph
1 (Indibulin)
3
4
2
μ
μ
μ
IC50 = 4.04 M(HeLa)
IC50 = 0.115 M (HeLa)
M(MDA-MB-231)
IC50 = 5.01
μ
IC50 = 0.059
M
(FaDu)
I
O
O
X
O
R3
O
NH
O
HN
O
HN
O
NH
O
O
O
N
NH
NH
H
O
N
Cl
R2
R2
H
R1
O
O
R1
N
H
7a-t
N
H
Cl
8a-h
5
6
7-8 (designed compounds)
μ
IC50 = 0.04 M (T47D)
μ
IC50 = 0.50
M
(K562)
Fig. 1. Structures of representative 3-substituted indole derivatives 1–6 with antitumor activity, and newly
designed compounds 7–8.
Over the past few decades, a large number of structurally diverse anticancer agents have been
14-16
identified as potent tubulin polymerization inhibitors.
Of which, indole core is the common
structural feature of a great number of inhibitors, especially indolyl-3-glyoxamide derivatives
exemplified by compound 1 (indibulin) possessing excellent antitumor activity with a minimal
neurotoxicity in Phase I clinical trials for cancer chemotherapy, 17-18 and another recently reported
analogue 2 (Fig. 1) also displaying potent antiproliferative activities. 19 In our previous work, 20-22
a
series of tubulin polymerization inhibitors were obtained by employing 3-aminoindole as the core
structure, such as compounds 3–5 and another recently reported analogue 6 (Fig. 1), 23 all of which
2

demonstrated remarkable antiproliferative activities against a panel of cancer cell lines. Encouraged
by the therapeutic significance of indolyl-3-glyoxylamides and structural features of the
3-aminoindole scaffold, we are very interested in the indole-based oxalamide and aminoacetamide
derivatives 7–8, as shown in Fig. 1, by incorporating glyoxylamide and acetamide moiety into the
3-aminoindole nucleus. Herein we report the detailed synthesis, antiproliferative evaluation and
inhibition of tubulin polymerization of some promising analogues.
H
N
Cl
R3
HN
HN
O
O
d
c
R2
R2
R1
R1
N
H
N
H
NH2
R2
CN
8a-h
O
12
CN
a
b
O
R1
NH2
R1
NHCOOEt
O
H
N
R1
N
H
HN
9
O
10
HN
N
11
f
O
e
R2
R2
R1
N
H
R1
N
H
13
g
7a-n
H
O
O
N
OH
HN
HN
O
O
R2
h
R2
R1
N
H
R1
N
H
7o-t
14
Scheme 1. Synthesis of the target compounds 7–8. Reagents and conditions: (a) ClCO2Et, reflux; (b) i) K2CO3/
DMF/EtOH, 80°C, 1 h; ii) 12% NaOH, 80°C, 15-30 min (c) Chloroacetyl chloride, K2CO3, THF, rt, 6 h; (d)
R3-NH2, DIPEA, 2-methoxyethanol, 120°C, 20 h; (e) Methyl chlorooxoacetate, K2CO3, THF, rt, 6 h; (f)
4-aminopyridine, TBD (30 mol%), DMF, N2, 85°C, overnight; (g) i) THF, 1% NaOH (aq), rt, 1 h ii) HCl, rt, 10
min; (h) i) CDI, THF, rt, 1h; ii) phenylamine, rt, overnight.
The synthetic sequence employed for the designed compounds 7–8 was illustrated in Scheme
1. The required diverse intermediates 2,6-disubstituted-3-aminoindoles 11 were synthesized via a
highly efficient one-pot method starting from ethyl 2-cyanophenylcarbamate 10 and
α-bromoketones, which had been demonstrated in detail in our previously reported approach. 24 The
3-aminoindoles were then treated with methyl chlorooxoacetate in the presence of
potassium carbonate (K2CO3) as base to give corresponding methyloxamates 13, followed by
aminolysis reaction with 4-aminopyridine to provide the target indole-based oxalamide derivatives
7a–n in moderate to good isolated yields. Some phenyl-substituted oxalamide analogues 7o–t not
3

accessible through direct aminolysis were also generated using a straightforward two-step
procedure including alkaline hydrolysis and nucleophilic substitution reaction. Similarly,
aminoacetamides 8a–h derived from 3-amine indoles were successfully obtained by a nucleophilic
substitution reaction of suitable phenylamine with 2-chloroacetamide derivatives 12 which was
prepared by acylation of substituted 3-amine indoles 11 with chloroacetyl chloride. The structures
of the newly prepared indole-based oxalamide 7a–t and aminoacetamide derivatives 8a–h were
1
13
characterized by H NMR, C NMR, IR and HRMS spectroscopic techniques, and the results are
shown in the supplementary section.
All the synthesized target compounds were initially evaluated for in vitro antiproliferative
activities against the human cervical cancer cells (HeLa) through MTT screening assay. Moreover,
some potent analogues were further tested against HCT116 and PC-3 cells, which originated from
different tissues of human. For comparison, a well-known tubulin-binding anticancer agent
Colchicine was employed as a positive control, and the results expressed as IC50 (μM) were
summarized in Table 1 and 2. Here, the IC50 value represents the concentration of a compound
resulting in 50% inhibition of cell growth after 48 h incubation with the compound, and is the
average of three independent experiments.
Table 1 Antiproliferative activities of compounds 7–8 against HeLa cells.
O
O
H
H
N
HN
N
HN
R1
R1
O
X
R2
8a-h
R2
R3
N
N
H
H
7a-t
IC50 (μM) a
HeLa
Comp.
X
R1
R2
R3
O
7a
7b
7c
7d
7e
7f
N
N
N
N
N
N
H
H
\
\
\
\
\
\
85.26±5.88
56.79±4.21
>100
O
O
O
H
O
H
62.41±5.09
>100
O
Cl
Cl
O
21.80±2.12
O
4

O
7g
7h
7i
N
N
Cl
Cl
\
80.65±4.02
69.57±4.56
75.13±3.92
56.87±3.48
>100
O
\
O
N
CH3
CH3
CH3
CH3
H
\
O
7j
N
\
O
O
7k
7l
N
\
O
N
\
>100
7m
7n
7o
7p
7q
7r
N
H
\
>100
O
CH
CH
H
\
62.92±4.39
>100
O
O
H
\
CH Cl
CH Cl
CH CH3
CH CH3
\
29.16±3.39
>100
O
O
\
\
\
55.20±2.25
82.33±6.31
76.71±4.49
31.09±3.53
16.20±0.63
27.88±4.13
18.30±1.30
16.74±2.45
＞100
O
7s
7t
CH
H
H
H
\
O
2-CH3
4-CH3O
2-CH3
4-CH3
8a
8b
8c
8d
8e
8f
\
\
\
\
\
\
\
\
O
O
O
O
O
H
O
O
O
O
O
H
Cl
Cl
Cl
Cl
Cl
4-F
3,4,5-(CH3O)3
O
O
8g
8h
H
15.41±1.80
＞100
3,4,5-(CH3O)3
Colchicine
0.03±0.004
a IC50 values are presented as mean values of three independent experiments done in quadruplicates.
5

In general, antiproliferative activities obviously vary with respect to substitution on the
position-3 of indole ring. Almost all of the indole derivatives bearing oxalamide group 7a–t
displayed inactive (IC50 >100 μM) or poor potency against the tested cell lines, while analogues 8b,
8d, 8e, and 8g, which incorporate aminoacetamide moiety at the position-3 of indole ring, exhibited
strong antiproliferative activities. The observed results obviously demonstrate that aminoacetamide
derivatives 8a–h manifest greater antitumor activities against tested cell lines than oxalamide
analogues 7a–t. Within the series of oxalamide derivatives 7a–t, replacement of phenyl ring with
pyridine nucleus leads to slight increase of antiproliferative activities (7a vs 7n, 7e vs 7p, 7i vs 7r,
7k vs 7s, 7m vs 7t). Meanwhile in the series of aminoacetamide analogues 8a–h, antiproliferative
activities were obviously dependent on the substitution pattern of the phenyl ring in
aminoacetamide part. It is interesting that introduction of a 3,4,5-trimethoxy group in phenyl ring
(8f, 8h), a well-defined pharmacophore for the inhibitors of tubulin found in colchicine and
combretastatin A-4, dramatically decreased growth inhibitory properties.
To further explore the antiproliferative potential, the effect of highly active analogues 8b, 8d,
8e, and 8g against a panel of two tumor cell lines derived from different tissues of human tumors
was screened. All compounds showed good to excellent antiproliferative activities against HCT-116
and PC-3 cell lines (Table 2). To confirm the growth inhibitory effect of the novel compounds was
related to an interaction with the tubulin system, we investigated them direct inhibitory effects on
tubulin polymerization in vitro at 10 μM concentration. The results obtained with the test agents are
summarized in Table 2. Among them, compound 8g, exerted the most excellent antiproliferative
activities, was aslo found to display the best inhibition activity (46%), while Colchicine exhibited a
77% inhibition effect at the tested concentration.
Table 2 Antiproliferative activities and tubulin polymerization inhibitory activities of
representative selected compounds 8b, 8e, 8g, and 8h.
Tubulin
IC50(μM)
polymerization
Comp.
R1
R2
R3
% inhibition a
HCT116
PC-3
O
O
O
4-CH3O
4-CH3
8b
8d
8e
H
Cl
Cl
46.89±2.14
43.71±2.88
7
O
O
O
20.42±3.75
18.24±3.70
15.82±1.93
21.78±2.49
29
43
4-F
6

O
8g
Cl
H
11.99±1.62
0.25±0.11
14.43±2.12
0.04±0.02
46
79
Colchicine
a Compounds were tested at a final concentration of 10μM.
In order to confirm the mode of action of these analogues on cancer cells, the influence of
different concentrations of the highly active analogue 8g on cell cycle progression was studied with
HeLa cells which were treated with compound 8g at the concentrations of 7.5, 15, and 30 μM for
24h. As illustrated in Fig. 2, there was an accumulation of 14.20% (7.5 μM), 20.70% (15.0 μM),
and 25.20% (30 μM) of cells in G2/M phase of the cell cycle observed in contrast to that of
untreated control (11.76%). These observations indicated that compound 8g induced a significant
block in the G2/M phase via a concentration-dependent manner.
A
B
C
D
Fig. 2. Effect of compound 8g on cell cycle in HeLa cells. Flow cytometry analysis of HeLa cells stained with
propidium iodide and treated with 8g for 24 h. (A) Control; (B) 8g, 7.5 μM; (C) 8g, 15 μM; (D) 8g, 30 μM.
7

Since the tubulin−microtubule system plays a key role in maintenance of the cellular
5,6
morphology,
to investigate whether the most active analogue 8g could affect microtubule
dynamics in living cells, we further explored the in situ effect of compound 8g on tubulin
organization in intact cells, and the microtubule structure of cells was visualized via
immunocytochemistry. As shown in Fig. 3, the control group demonstrated an intact network
formed by α- and β-tubulin microtubules in the untreated HeLa cells, which were described with
regularly assembled, and normal filiform microtubules wrapped around the uncondensed cell
nucleus. Whereas cells treated with 15 and 30 μM of 8g for 12 h caused obvious disruption of both
tubulin subtypes, the microtubule spindles shrank significantly around the center of the cells, and
formation of cell membrane rounding, thus exhibiting the inhibition of tubulin polymerization.
Fig.3. Effect of compound 8g on tubulin expresion in HeLa cell. Cells were seeded on glass coverslips, incubated
with compound 8g (15 μM, 30 μM) for 12 h (Scale bar = 50 μm), then fixed and processed for confocal
microscopy.
The possible binding mode for the most active derivative 8g was carried out into the colchicine
binding site of the tubulin crystal structure (PDB: 1SA0) using molecular docking studies. As
depicted in Fig. 4, indole ring and benzoyl of the analogue were located deeply into the β-subunit of
tubulin, while phenylamine group extended toward the α/β-tubulin interface and analogue 8g forms
three important hydrogen bond interactions with amino acids of tubulin. An amide group of
derivative 8g established one hydrogen binding interaction in which carbonyl oxygen interacts with
8

βCys241 (2.2 Å). Furthermore, in the α/β-tubulin interface, the residues of αLys352 and βAla317
form two hydrogen bonds with the nitrogen of the phenylamine group, respectively. These
molecular docking results demonstrated that compound 8g could interact with the colchicine
binding site betweenα and β subunits of tubulin.
Lys352
2.1Å
Cys241
2.2Å
2.7Å
Ala317
Fig.4. Proposed binding mode of compound 8g in colchicine binding site of tubulin. The main interacting residues
are shown and labeled. The black dashed lines are the potential H-bond between Cys241 (2.2 Å), Ala317 (2.7 Å),
Lys352 (2.1 Å).
In conclusion, a new class of novel indole-based oxalamide and aminoacetamide derivatives
were synthesized and evaluated for their antiproliferative activities. Among them, analogue 8g
displayed significant antiproliferative activities against PC-3, HeLa and HCT-116 cell lines. The
flow cytometric analysis revealed that the compound caused cell cycle arrest at G2/M phase.
Notably, the compound 8g exhibited potent antitubulin activity and effectively inhibit microtubule
assembly and disrupt the microtubule organization in the HeLa cells. Furthermore, docking studies
revealed that 8g, as a typical potent tubulin polymerization inhibitor, formed stable interactions in
the colchicine binding site in the α/β-tubulin interface. These preliminary results encourage further
investigation on these analogues as potential scaffolds to develop new anticancer agents.
Acknowledgments
This work was supported by the Natural Science Foundation of Guangdong Province, China
(Grant No. 2018B030311067), National Natural Science Foundation of China (No. 21372113), and
the Science and Technology Program of Guangzhou City, China (Grant No. 201707010198).
9

A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/10.1016/j.bmcl.2019.xx.xxx.
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Design, synthesis and biological evaluation of novel indole-based oxalamide
and aminoacetamide derivatives as tubulin polymerization inhibitors
Peng-Cheng, Diao#, Xie-Er Jian#, Peng Chen, Chuan Huang, Jie Yin, Jie Chun Huang, Jun-Sheng
Li, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science,
Southern Medical University, Guangzhou 510515, P.R.China
12

O
HN
O
HN
N
H
Cl
compound 8g
Molecular modeling
Cell cycle arrest
Immunocytochemistry
25.
Highlights
► A series of novel indole-based oxalamide and aminoacetamide derivatives were synthesized. ►
8g exhibited significant antiproliferative effect against PC-3, HeLa and HCT-116 cell lines. ►
Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 8g.
26.
Declaration of interests
 The authors declare that they have no known competing financialinterestsor personal relationships that could
have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
13

27.
14