(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides as tubulin polymerization inhibitors: Structure-based bioisosterism design, synthesis, biological evaluation, molecular docking and in silico ADME prediction

Article abstract of DOI:10.1016/j.bmcl.2018.09.004

A series of (E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides have been synthesized and evaluated for their anticancer activity in human hepatocellular liver carcinoma HepG2 and breast adenocarcinoma MCF-7 cell lines. Among all the test

Full text of DOI:10.1016/j.bmcl.2018.09.004

Accepted Manuscript
(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides as tu-
bulin polymerization inhibitors: Structure-based bioisosterism design, synthe-
sis, biological evaluation, molecular docking and in silico ADME prediction
Guangcheng Wang, Zhiyun Peng, Shanshan Peng, Jie Qiu, Yongjun Li, Yanyu
Lan
PII:
S0960-894X(18)30735-2
https://doi.org/10.1016/j.bmcl.2018.09.004
BMCL 26025
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 July 2018
17 August 2018
4 September 2018
Please cite this article as: Wang, G., Peng, Z., Peng, S., Qiu, J., Li, Y., Lan, Y., (E)-N-Aryl-2-oxo-2-(3,4,5-
trimethoxyphenyl)acetohydrazonoyl cyanides as tubulin polymerization inhibitors: Structure-based bioisosterism
design, synthesis, biological evaluation, molecular docking and in silico ADME prediction, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.09.004
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(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazono
yl cyanides as tubulin polymerization inhibitors:
Structure-based bioisosterism design, synthesis, biological
evaluation, molecular docking and in silico ADME
prediction
Guangcheng Wanga,c,*, Zhiyun Penga,c, Shanshan Pengc, Jie Qiuc, Yongjun Lib,*,
Yanyu Lanb
aState Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou
Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang
550004, China
bEngineering Research Center for the Development and Application of Ethnic
Medicine and TCM (Ministry of Education), Guizhou Medical University, Guiyang
550004, China
cCollege of Chemistry and Chemical Engineering, Jishou University, Jishou 416000,
PR China
*Corresponding Author
Tel.: +86 851 86908468; Fax: +86 851 86908468
E-mail address: wanggch123@163.com (G. Wang); liyongjun_gmu@163.com,
liyongjun026@126.com (Y. Li)
1

Abstract.
A series of (E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides
have been synthesized and evaluated for their anticancer activity in human
hepatocellular liver carcinoma HepG2 and breast adenocarcinoma MCF-7ꢀcell
lines. Among all the tested compounds, compound 3a, 3e and 3n displayed more
activity than lead compound with IC50 value of 0.26-0.61 μM. Meanwhile, these
compounds (3a, 3e and 3n) showed potent antiproliferative activity against a panel of
cancer cells and the HCT-8/T multidrug resistant cell line with IC50 values in the
range of 0.077 to 7.44 μM. Flow cytometric analyses revealed that compound 3n
induced cell cycle arrest in G2/M phases in a dose dependent manner. The
compound 3n also displayed potent tubulin polymerization inhibition with an
IC50 value of 0.9ꢀµM, with ten folds more active than colchicine (IC50 = 9 μM).
Molecular docking studies revealed that compound 3n efficiently interacted with the
colchicine binding site of tubulin through hydrophobic, cation-π and hydrogen bond
interaction. Furthermore, in silico pharmacokinetic prediction shown that these
compounds have a good ADME-related physicochemical parameters. These results
demonstrate that 3n exhibits potent cytotoxicity in cancer cells by targeting the
colchicine binding site of tubulin and potentially acts as a therapeutic lead compound
for the development of anticancer drugs.
Keywords: Tubulin; Bioisosterism; ADME prediction; Anticancer activity;
Acetohydrazonoyl cyanide
2

Cancer is the uncontrolled growth of abnormal cells in the body and is a constant
threat for human health. According to the World Health Organization (WHO)1, cancer
is the second leading cause of death globally and accounts for nearly 1 of 6 deaths.
Cancer was responsible for 8.8 million deaths in 2015 and the number of new cases is
expected to rise by about 70 percent over the next two decades1. Therefore, the
development and discovery of new anticancer agents is remains critically important.
Antimitotic agents are one of the important class of drugs for the treatment of cancer
and tubulin has been considered as an important target for the development of
anticancer drugs2. Microtubules, constructed from α and β tubulin, are essential
components of the cytoskeleton in all eukaryotic cells, which play an important role in
many cellular functions such as maintenance of cell integrity, cell division, cell
signaling, regulation of motility, cell secretion and intracellular transports3. Therefore,
the disruption of microtubule function can arrest the cell division at mitosis and
results in cell apoptosis. Over the last few decades, a large number of antimitotic
agents have been reported in literatures4-7. Among these inhibitors, paclitaxel,
docetaxel, epothilone, vinblastine and vincristine have been used in clinic for the
treatment of cancers (Figure 1).
3

Figure 1. The structures of some tubulin polymerization inhibitors.
Bioisosteric replacement are important strategy to modify and optimize the properties
of drug candidates in medicinal chemistry. It may help improve stability, optimize
activity, and/or remove unwanted side effects, and has been widely used on the design
and development of novel bioactive compounds8, 9. During our research on the design
and synthesis of tubulin polymerization inhibitors10-15, one years ago we reported the
synthesis of a series of (E)-3-Aryl-2-(3,4,5-trimethoxybenzoyl) acrylonitrile
derivatives13. Biological evaluation indicates that the most active compound exhibited
potent anticancer activity against HepG2, A549, MCF-7, A2780S, SKOV3, HT29 and
CHL-1 cell lines with IC50 values of 1.20, 2.09, 1.84, 1.88, 1.27, 1.28 and 1.22 µM,
respectively (Figure 2, lead compound). Molecular docking studies suggested that
this compound is a new tubulin polymerization inhibitor that target the colchicine
binding site of tubulin.
4

As replacement of –C= with –N= is a classical bioisosteric replacement16,17, to
explore new chemical structure and improve anticancer activity, we report herein the
design and synthesis a series of (E)-N-aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)
acetohydrazonoyl cyanides based on bioisosteric strategy (Figure 2).
Figure 2. Rational design of (E)-N-aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)
acetohydrazonoyl cyanides using a bioisosteric strategy.
The synthesis of (E)-N-aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl
cyanides 3a-3n was shown in Scheme 1. 3-Oxo-3-(3,4,5-trimethoxyphenyl)
propanenitrile 2 were synthesized by the Claisen-type condensation. NaH was added
by portion to a solution of methyl 3,4,5-trimethoxybenzoate 1 and CH3CN in
anhydrous toluene. The mixture was stirred at reflux for overnight and the solid
precipitate was collected by filtration. The precipitate was dissolved in ice water (100
mL) and adjust pH to 2-3 with 10% HCl. The product 2 was isolated by filtration and
recrystallization (EtOH). Aromatic diazonium compounds (diazo salts) was prepared
by reaction of aryl anilines with NaNO2 under acid condition. Condensation of 2 with
appropriate diazonium salts in ethanol in the presence of NaOAc to afford the
5

corresponding
(E)-N-aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl
cyanides 3a-3n18. To the best of our knowledge, all these target compounds have not
1
previously been reported in the literature. Their structures were characterized by H
NMR and 13C NMR spectroscopy.
Scheme 1. Reagents and conditions: (a) NaH, CH3CN, toluene, reflux, overnight; (b) i)
ArNH2, concentrated HCl, H2O, NaNO2, 0 oC, 1 h; ii) NaOAc, H2O, EtOH, r.t., 1 h.
The newly synthesized (E)-N-aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydra-
zonoyl cyanides 3a-3n were screened for their in vitro anticancer activity against
human hepatocellular liver carcinoma cell (HepG2) and human breast
adenocarcinoma cell (MCF-7) by using MTT method. The IC50 values of the tested
compounds and the lead compound are summarized in Table 1. The majority of the
screened compounds (except 3c, 3h, 3l and 3m) exhibited moderate to potent
antiproliferative activity with IC50 values among 0.26ꢀμM to 9.84ꢀμM against the two
human tumor cell lines. Among the series, compound 3n, which contained 4-methoxyl
and 3-hydroxyl group on the phenyl ring, showed the most potent antiproliferative
activity against HepG2ꢀcell and HCT-116ꢀcell, with IC50 values of 0.32 and 0.26ꢀμM,
respectively. Additionally, compounds 3a (4-methyl group) and 3e (4-methoxyl group)
also exhibited potent antiproliferative activity (IC50 < 1.0 μM).
6

The structure-activity relationship (SAR) of this series of compounds has been
summarized. Introduction of electron withdrawing groups such as chlorine and nitro
into the phenyl ring (3l and 3m), results in dramatic decrease of antiproliferative
activity. However, introduction of fluorine-containing groups such as trifluoromethyl
and fluorine into the phenyl ring, results in increase or slight decrease of
antiproliferative activity. Additional, the introduction of methyl or methoxyl group at
the para position of phenyl ring (3a, 3e and 3n) resulted in a remarkable increase of
the antiproliferative activity. However, shifting these groups to the other position
decreased the antiproliferative activity drastically. The results suggested that methyl or
methoxyl at the para positon on the phenyl ring are crucial pharmacophoric group for the
anticancer activity.
Table 1. Antiproliferative activity of (E)-N-aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)
acetohydrazonoyl cyanides 3a-3n.
Antiproliferative activity IC50 (μM)
Compound
R
HepG2
0.61±0.07
6.11±0.36
>10.0
MCF-7
0.43±0.04
5.89±0.47
>10.0
4-CH3
3-CH3
3a
3b
3c
3d
3e
3f
3,4,5-triCH3O
3-CH3O
4-CH3O
H
6.46±0.52
0.44±0.03
5.19±0.31
8.82±0.45
0.28±0.01
6.38±0.43
7

4-CH3CH2O
2-CH3CH2O
4-CF3
6.62±0.29
>10.0
7.01±0.36
>10.0
3g
3h
2.68±0.14
9.71±0.53
2.10±0.14
>10.0
3.02±0.21
9.84±0.48
1.69±0.09
>10.0
3i
4-F
3j
2-F
3k
2,4-diCl
4-NO2
3l
>10.0
>10.0
3m
3n
3-OH-4-CH3O
0.32±0.02
1.38±0.08
0.26±0.01
1.93±0.17
lead compound
Then we focused on the evaluation of the antiproliferative activity of compound 3a,
3e and 3n against various human cancer cell lines, with colchicine as the positive
controls. The selected cancer cell lines including prostate (DU145), skin (B16-F10),
breast (MDA-MB-231), colon (HCT116), cervical (Hela), and drug resistant
(HCT-8/T). The results were presented in Table 2. These compounds (3a, 3e and 3n)
showed potent antiproliferative activity with IC50 values in the range of 0.077 to 7.44
μM against all tested cancer cell lines.
Table 2. Antiproliferative activity of selected compound 3a, 3e and 3n.
Antiproliferative activity IC50 (μM)
Cell lines
3a
3e
3n
Colchicine
(nM)
Prostate
DU145
0.098±0.008 0.12±0.02 0.077±0.005
3.45±0.35
8

Skin
Breast
Colon
B16-F10
2.23±0.18 3.26±0.25 2.11±0.17
44.25±6.71
77.62±3.54
26.18±3.27
8.89±0.68
MDA-MB-231 2.24±0.28 2.46±0.36 0.72±0.09
HCT116
Hela
0.87±0.07 3.19±0.33 0.81±0.14
0.31±0.08 0.22±0.03 0.081±0.007
7.64±0.85 4.62±0.41 3.96±0.26
Cervical
Drug
HCT-8/T
227.40±95.3
resistant
Previous studies have been confirmed that tubulin polymerization inhibitors could
induce cell cycle arrest in G2/M phase. In order to investigate whether this series
compounds could arrest cell cycle in G2/M phase, the effect of compound 3n on the
cell cycle was analyzed by flow cytometry assay. Treatment of HepG2 cells with
DMSO (control) or different concentration of compound 3n (0.25 μM or 0.50 μM) for
24ꢀh, its effect on cell cycle distribution was analyzed by flow cytometry. The results
were shown in Figure 3. In the control group, about 21.09 % of HepG2ꢀcells were
distributed in G2/M phase, whereas after treatment of 24 h, the percentage of HepG2
cells in G2/M phase increased to 78.79% and 94.12%, respectively. The results
indicated that this series of compound could arrest cell cycle in G2/M phase in a
dose-dependent manner and might be used as lead compounds for development of
new antitubulin agents.
9

Figure 3. Cell cycle analysis of HepG2 after incubation with compound 3n (0.25 μM
and 0.50 μM) for 24ꢀh. DMSO diluent was used as a control.
To determine if anticancer activity of this series of compounds was related to their
capacity to inhibit tubulin polymerization, the most active compound 3n was selected
to evaluate its inhibition effect on tubulin polymerization. Colchicine was selected as
positive control. As shown in Figure 4, compound 3n inhibited tubulin assembly in a
concentration-dependent manner. As comparison to the standard colchicine, it
displayed potent tubulin polymerization inhibition with an IC50 value of 0.9ꢀµM, with
ten folds more active than colchicine (IC50 = 9 μM, The value of IC50 has been
reported in our previous study15). The results suggested that compound 3n is a new
10

tubulin polymerization inhibitor.
Figure 4. Effect of compound 3n and colchicine on the tubulin polymerization.
The theoretical binding mode between 3n and tubulin was shown in Figure 5.
Compound 3n adopted a “L-shaped” conformation in the pocket of the tubulin. The
3,4,5-trimethoxyphenyl group of 3n located at the hydrophobic pocket, surrounded by
the residues Val-238, Cys-241, Leu-242, Leu-248, Ala-250, Leu-255, Ala-316,
Ala-317 and Ile-318, forming a strong hydrophobic binding. Detailed analysis showed
that one of the phenyl group of 3n formed a cation-π interaction with the residue
Lys-352. It was shown that the residue Ser-178 (bond length: 2.4 Å) formed a
hydrogen bond interaction with 3n, which was the main interaction between 3n and
tubulin. All these interactions helped 3n to anchor in the binding site of the tubulin.
11

Figure 5. The predicted binding mode between 3n and tubulin (α-tubulin was colored
in cyan and β-tubulin was colored in green).
Unfavourable absorption, distribution, metabolism and elimination (ADME)
properties have been identified as one of the important causes of failure for
potential therapeutic agents in drug development. In order to evaluate the ADME
properties of this series of compounds, the pharmacokinetic parameters of all
synthesized compound were calculated by using Molinspiration online property
calculation toolkit (http://www.molinspiration.com/) and all the obtained parameters
are presented in Table 3. Among the series, all the synthesized compounds 3a-3n
exhibited excellent % absorption range from 61.12% to 76.93%. According to the
Lipinski’s rule of five19, a potentially biologically active substance should have
12

designed for oral route should have a molecular weight ≤ꢀ500, values of calculated
log P ≤ 5, number of hydrogen bond donor ≤ꢀ5 and number of hydrogen bond
acceptor ≤ꢀ10. The results were shown that all the synthesized compounds except 3l
were compliance with Lipinski’s rule of five which is recommended for these
compounds have a good oral bioavailability. In summary, the results of in silico
ADME prediction suggest that this series of compounds can be further developed as
drug candidates.
Table 3. ADME prediction through Molinspiration online software.
Compd %ABSa TPSAb n-rotbc MWd
MVe miLogPf n-OHNHg n-ONh Lipinski’s
(A0)
violation
Rule
<5
<5
1
<10
7
<1
0
76.93
76.93
92.96
92.96
7
7
353.38 320.16
353.38 320.16
429.43 380.23
369.38 329.14
369.38 329.14
339.35 303.60
383.40 345.94
383.40 345.94
407.35 334.89
357.34 308.53
4.25
4.23
3.43
3.84
3.86
3.80
4.24
4.19
4.70
3.97
3a
3b
3c
3d
3e
3f
1
7
0
67.37 120.66
73.74 102.19
73.74 102.19
10
8
1
10
8
0
1
0
8
1
8
0
76.93
92.96
7
1
7
0
73.74 102.19
73.74 102.19
9
1
8
0
3g
3h
3i
9
1
8
0
76.93
76.93
92.96
92.96
8
1
7
0
7
1
7
0
3j
13

76.93
76.93
92.96
92.96
7
7
8
8
357.34 308.53
408.24 330.67
384.35 326.93
3.92
5.09
3.76
3.14
1
1
1
2
7
7
0
1
0
0
3k
3l
61.12 138.78
66.77 122.42
10
9
3m
3n
385.38 337.16
a
b
c
Percentage of absorption (%ABS); Topological polar surface area (TPSA);
d
e
Number of rotatable bonds (n-rotb); Molecular weight (MW); Molecular volume
f
g
(MV); Logarithm of partition coefficient between n-octanol and water (miLogP);
h
Number of hydrogen bond donors (n-OHNH); Number of hydrogen bond acceptors
(n-ON).
In
summary,
a
series
of
tubulin
polymerization
inhibitors,
(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides have been
designed and synthesized based on the lead compound. All newly synthesized
compounds were screened for their antiproliferative activity against HepG2 and
MCF-7ꢀcell lines, and the results indicated that compound 3a, 3e and 3n displayed
more activity than lead compound with IC50 value of 0.26-0.61 μM. The selected
compounds 3a, 3e and 3n were further identified to possess potent anticancer activity
in six additional cancer cells (DU145, B16-F10, MDA-MB-231, HCT116, Hela, and
HCT-8/T). Among these compounds, 3n exhibited the most potent biological activity
and was used in further biological studies. Flow cytometry analysis demonstrated
that 3n arrests HepG2ꢀcells in G2/M phase in a dose-dependent manner. In vitro
tubulin polymerization assays revealed that 3n displayed more potent tubulin
14

polymerization inhibition than colchicine. Furthermore, molecular docking study
showed that 3n had high binding affinities for the colchicine binding site of tubulin.
Hence, this study demonstrated that compound 3n is a promising tubulin inhibitor and
can be used as lead compound for the development of anticancer drugs.
Acknowledgments
This work was supported by National Natural Science Foundation of China
(81660574).
Supplementary material
Supplementary data associated with this article can be found, in the online version, at
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Highlights
16

►(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides were
designed and synthesized.
► In vitro anticancer activities were determined.
►3n was most cytotoxic against a panel of cancer cells and multidrug resistant cells.
►3n inhibited tubulin polymerization and caused HepG2 cells arrest in G2/M phase.
►Molecular docking and ADME prediction studies were performed.
17