Evaluation of 4-phenylamino-substituted naphthalene-1,2-diones as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.07.047

A series of 4-phenylamino-substituted naphthalene-1,2-dione derivatives were prepared and evaluated as effective antiproliferative agents. MTT assays showed that the compounds with a methyl group on the nitrogen linker exhibited potent antiproliferative activities against human cancer cells. The mechanistic study revealed that these compounds could induce mitochondrial depolarization, which resulted in intracellular ROS production, and they also acted as tubulin polymerization inhibitors. Moreover, the typical compound could arrest A549 cells in the G2/M phase, resulting in cellular apoptosis and induced mitotic arrest in A549 cells through disrupting microtubule dynamics.

Full text of DOI:10.1016/j.bmcl.2018.07.047

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of 4-phenylamino-substituted naphthalene-1,2-diones as tubulin
polymerization inhibitors
Honghao Yanga, Baijiao Anb, Xingshu Lib,⁎, Wei Zenga,⁎
a
Key Laboratory of Functional Molecular Engineering of Guangdong Province, School of Chemistry and Chemical Engineering, South China University of Technology,
Guangzhou 510641, China
b
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antitumor agents
Tubulin polymerization inhibitors
Mechanism
Reactive oxygen species
A series of 4-phenylamino-substituted naphthalene-1,2-dione derivatives were prepared and evaluated as ef-
fective antiproliferative agents. MTT assays showed that the compounds with a methyl group on the nitrogen
linker exhibited potent antiproliferative activities against human cancer cells. The mechanistic study revealed
that these compounds could induce mitochondrial depolarization, which resulted in intracellular ROS produc-
tion, and they also acted as tubulin polymerization inhibitors. Moreover, the typical compound could arrest
A549 cells in the G2/M phase, resulting in cellular apoptosis and induced mitotic arrest in A549 cells through
disrupting microtubule dynamics.
Malignant tumors, commonly known as cancer, are a major disease
that is the second most serious threat to human life and health after
cardiovascular disease, and cancer has become the world's leading “life
killer.” It is estimated that by 2020, the number of deaths worldwide
may double due to cancer, and approximately 84 million people will die
from cancer in the next 10 years.1,2 With the rapid increase in cancers
worldwide, the existing drugs are far from meeting the needs. There-
fore, in the past several decades, many anticancer agents aimed at
various targets have been developed.3–6 Among them, tubulin poly-
merization or depolymerization inhibitors have attracted much atten-
tion, as tubulin-microtubule systems play a critical role for a wide range
of cellular processes and represent a prominent cancer drug target. The
use of taxane and vinca alkaloids to treat a variety of human cancers in
the past decades are such an example.7 At the same time, new tubulin
inhibitors are also being developed. For example, MPC-6827, a quina-
zoline amino-substituted derivative, could effectively inhibit the poly-
merization of tubulin in vitro and disrupt the formation of microtubules
in a variety of cancer cell lines, and has reached phase II for the
treatment of recurrent glioblastoma.8–11 Since cancer is prone to drug
resistance, from the view point of both the academic and applied sci-
ence realms, the discovery and development of highly effective and low
toxicity candidate drug molecules for the treatment of cancer is still
important.
under the condition of a modest rise in the intracellular ROS. On the
other hand, apoptosis is also induced at higher levels of ROS. Small
molecules that modulate antioxidant levels and/or enhance in-
tracellular ROS could disturb the cellular oxidative environment and
induce cell death. Thus, the utilization of ROS-inducing small molecules
to target cancer has been considered as a potential strategy. The en-
hancement of intracellular ROS could be achieved by endogenous an-
tioxidant inhibition, thus modulating the functions of proteins re-
sponsible for maintaining redox homeostasis in the presence of small
molecule ROS.12 Quinones are one of the major sources of ROS in cells,
and many quinone derivatives, such as mitomycin C,13 geldanamycin,14
mitoxantrone,15 and eoxynyboquinone,16 have been used as anticancer
agents. Recently, Potter et al. reported that β-lapachones are potent,
reversible, and selective inhibitors of human liver carboxylesterase
(CEs) with Ki values in the nanomolar range, which are ubiquitous
enzymes that are responsible for the metabolism of xenobiotics in-
cluding drugs such as the anticancer drugs irinotecan and oseltamivir.17
Inspired by the structure and anticancer activity of compound MPC-
6827, we proposed that 4-phenylamino-substituted naphthalene-1,2-
diones should possess the antiproliferative activity against cancer cells.
To further promote the application of these compounds in the treatment
of cancer, and to investigate the structure-activity relationships, herein,
we reported the evaluation of a series 4-phenylamino-substituted
naphthalene-1,2-diones as antiproliferative agents as well as their me-
chanism of action.
Reactive oxygen species (ROS) are closely related to tumorigenesis
and treatment. In cancer cells, growth and proliferation are encouraged
⁎
Corresponding authors.
E-mail addresses: lixsh@mail.sysu.edu.cn (X. Li), zengwei@scut.edu.cn (W. Zeng).
https://doi.org/10.1016/j.bmcl.2018.07.047
Received 15 June 2018; Received in revised form 30 July 2018; Accepted 31 July 2018
0960-894X/©2018PublishedbyElsevierLtd.
Pleasecitethisarticleas:Yang,H.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.07.047

H. Yang et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
R1
R1
R2
b
a,
N
R2
H2N
H
R3
R3
1
2
R1
R2
R1
SO3Na
N
HN
R2
c
c
R3
O
O
O
O
O
O
1
1
:
2=R3
:
2=R3
=H
3a R =OCH
R
=H
3g R =CH
R
,
1
2=H
2=F
,
3
3
:
4a R
R
R
=OCH
,
,
3
1
1
3b R =OCH R2=F, R3=H 3h R =F, R
=H
2=R3
:
:
2=R3
1
,
3
:
4b R
=OCH
3
1
3
1
3c R =OCH R2
R =H 3i R =CN, R
=H
:
:
=Cl,
,
3
1=R2
:
1=R2
3
3
:
3d R
=F, R =H
3j R
=H, R =OCH3
2=R3=H
1=R2
:
3
1
:
3e R
=Cl R =H
3k R =SeCH
R
,
3
1
2
:
3=H
3f R =F, R
R
=Cl,
Scheme 1. The synthesis of 4-phenylamino-substituted naphthalene-1,2-diones. Reagents and conditions Reagents and conditions: (a) Na, MeOH, (CH2O)n. (b)
NaBH4, 60 °C. (c) 1 or 2, H2O, rt.
Compounds 3a, 3g, 3h and 4a were previously synthesized and
evaluated as selective inhibitors of human liver CEs.17 To study their
activity of anti-tubulin aggregation, we prepared 3a–k and 4a–b
(Scheme 1) according the method described in the same literatures.17,18
Briefly, aniline derivatives reacted with paraformaldehyde in the pre-
sence of sodium, methanol and sodium borohydride to afford N-methyl
aniline derivatives. The commercial sodium 3,4-dioxo-3,4-dihy-
dronaphthalene-1-sulfonic acid reacted with the N-methyl aniline de-
rivatives gained above or other aniline derivatives to afford target
compounds 3 or 4 in water under mild conditions.
To evaluate the antiproliferative activities of the 4-(phenylamino)
naphthalene-1,2-dione derivatives, an MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay was performed toward five
human cancer cell lines (A549, nonsmall cell lung carcinoma; HeLa,
human epithelial cervical cancer cell line; HepG2, human hepatoma
carcinoma cells; RKO and HCT-116, human colon cancer cell lines), and
the results are listed in Table 1. Among the tested compounds, 3a (4-
((4-methoxyphenyl)(methyl)amino)naphthalene-1,2-dione) and 3b ex-
hibited potent antiproliferative activities with IC50 values ranging from
0.091 to 0.471 μΜ. The substitutes on the ortho position of the methoxy
group, especially larger groups, seem unfavorable for the activity; 3c,
with Cl at the R2 position, gave relatively weak activity
(0.684–1.441 μΜ). Compounds 3d, 3e and 3f containing halogen
groups exhibited weak activities, which suggested that the methoxy at
the 4-position of the phenylamino moiety is necessary. Compound 3g,
with methyl group at the 4-position of the phenylamino moiety ex-
hibited good activity with 1.091–2.352 μΜ of the IC50 values. However,
cyano group at the 4-position provided very poor activity (3i: > 10 μΜ
of the IC50 values for five cancer cell lines). The position of methoxy at
the phenylamino moiety is also important to the activity; 3j, with
methoxy group at the 2-position, exhibited 5.924–6.853 μΜ of the IC50
values, which was much weaker than its isomer 3a. We previously re-
ported the synthesis and evaluation of selenium-containing 4-anilino-
quinazoline derivatives as novel antimitotic agents. Among them, the
optimal compound, N,2-dimethyl-N-(4-(methylselanyl)phenyl)quina-
zolin-4-amine, exhibited IC50 values of 2–9 nM against six human
cancer cell lines including A549 and Hct116.19 However, compound 3g,
with SeCH3 at the 4-position of the phenylamino moiety, provided
moderate activities, with the IC50 values ranging from 1.005 to
3.515 μΜ. To examine the effect of methyl group on the linker nitrogen,
Table 1
Antiproliferative activities (μΜ) of 3a–3k and 4a–4c against five human cancer cell lines.a,b
A549
HELA
HEPG2
RKO
HCT116
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
4a
4b
Colchicine
MPC6827
0.097
0.093
0.684
2.154
4.933
1.036
1.091
1.63
> 10
6.463
1.003
0.159
6.632
0.023
0.005
0.004
0.019
0.015
0.036
0.351
0.063
0.011
0.021
0.091
0.092
0.864
4.081
4.059
1.985
1.230
1.59
> 10
6.910
1.661
0.416
4.905
0.051
0.007
0.001
0.014
0.044
0.324
0.036
0.095
0.012
0.027
0.096
0.101
0.785
6.546
5.091
2.468
1.295
1.89
> 10
6.751
3.515
0.776
3.967
0.046
0.006
0.001
0.008
0.015
0.063
0. 358
0.117
0.012
0.025
0.116
0.332
0.784
4.641
2.208
1.447
1.371
1.713
> 10
5.924
2.572
2.490
8.407
0.043
0.005
0.010
0.022
0.059
0.158
0.321
0.021
0.015
0.031
0.471
0.106
1.441
6.229
3.851
8.4245
2.352
1. 915
> 10
6.853
2.242
2.496
8.591
0.039
0.009
0.025
0.012
0.011
0.327
0.253
0.215
0.012
0.023
0.035
0.081
0.071
0.072
0.003
0.001
0.032
0.142
0.056
0.0188
0.006
0.002
0.037
0.092
0.041
0.039
0.005
0.002
0.032
0.157
0.114
0.603
0.006
0.001
0.042
0.106
0.098
0.548
0.005
0.002
a
Cell lines were treated with the compounds for 48 h. The cell viability was measured by the MTT assay as described in the Experimental Section.
b
IC50 values are indicated as the mean
SD (standard error) of at least three independent experiments.
2

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Fig. 1. Effects of compounds 3b on microtubule dynamics. Purified tubulin protein at 10 μΜ in a reaction buffer was incubated at 37 °C in the absence (control) or
presence of 3b at the indicated concentrations (ranging from 1 to 10 μM). The experiments were performed for three times.
compounds 4a and 4b, without methyl on the nitrogen, were prepared
and evaluated. It can be seen that the two compounds provided slightly
weaker antiproliferative activities than their homologues 3a and 3b,
which shows the same trend as the literature.20
Considering the structural similarity of 4-phenylamino-substituted
naphthalene-1,2-diones with the tubulin polymerization inhibitor MPC-
6827, we proposed that these compounds should also possess the ability
to inhibit tubulin polymerization. As compounds 3a and 3b showed
better anti-proliferative activity than the others in the initial cytotoxi-
city screening, we chose 3b as the typical for further study, which may
have better stability due to the presence of the fluorine atom on the 3-
position of the aniline moiety. We incubated purified, unpolymerized,
Fig. 2. Effect of 3b on cell cycle arrest. (A) The A549 cells were treated with compound 3b at 0.05, 0.10, or 1.0 μM for 24 h or 48 h. (B) Quantitative analysis of the
percentage of cells in each cell cycle phase was carried out by EXPO32 ADC analysis software. The experiments were performed three times, and the results of
representative experiments are shown.
3

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Fig. 3. 3b disrupted the organization of the cellular microtubule network at the indicated concentrations. A549 cells were plated in confocal dishes and incubated
with 3b at 0.05, 0.10, and 1.0 μM for 24 h, followed by a direct microscopy assay. The detection of the fixed and stained cells was performed with a LSM 570 laser
confocal microscope (Carl Zeiss, Germany). The experiments were performed three times, and the results of the representative experiments are shown.
and microtubule-associated protein-rich tubulin with various con-
centrations of 3b and measured the polymerization according to the
literature method reported by Bonne et al.21,22 It can be seen from the
results in Fig. 1 that the fluorescence intensity at 410 nm of the control
increased with the extension of time, while in the presence of 3b at
concentrations of 1, 1.75, 2.5, 5 and 10 μM, respectively, the fluores-
cence intensity increases significantly more slowly than that of the
control, which indicated that tubulin polymerization was indeed in-
hibited. The inhibitory concentration that reduces polymerized tubulin
concentration of 0.1 μM, 39.88% of the G2/M phase was arrested, and
the data rose to 46.18% when the concentration was increased to 1 μM.
When the incubation time was extended to 48 h, the G2/M cell cycle
arrest effect was more obvious at 0.1 μΜ (68.79%). However, when
exposed at a concentration of 1 μΜ, the sub-G1 peak (a characteristic
hypodiploid peak) was the main peak (55.94%), which implied that
most cells underwent DNA fragmentation.
The tubulin-microtubule system is very important in the main-
tenance of cellular morphology. To determine whether compound 3b
could affect microtubule dynamics in living cells, we performed a laser
confocal microscopy assay that involved the disruption of microtubule
dynamics with A549 cells. The results in Fig. 3 clearly show the heavy
disruption of the microtubule system in A549 cells after treatment with
different concentrations of compound 3b compared with the control.
The microtubule network became disorganized with the treatment of
compound 3b and was completely disrupted as the concentration of 3b
increased. When the concentration was increased to 1.0 μM, the mi-
crotubule spindles had obviously shrunken around the center of the
cells; the cytoplasm and the chromosomes appeared more irregular
than in the cells treated with the 0.05 and 0.1 μM concentrations. In
comparison, the control cells predominantly exhibited at interphase of
the cell cycle, which was characterized by uncondensed chromosomes
and the regularly assembled, slim, and fibrous microtubules wrapped
by 50% is 4.978
substituted naphthalene-1,2-diones are effective tubulin polymerization
inhibitors.
0.13 μM, which indicated that 4-phenylamino-
It is known that tubulin-destabilizing agents block the cell cycle in
the G2/M phase due to microtubule depolymerization and cytoskeleton
disruption.23 To investigate whether the cytotoxicity induced by the 4-
(phenylamino)naphthalene-1,2-dione derivatives was due to cell cycle
arrest, we performed flow cytometry analysis using human A549 non-
small cell lung carcinoma cells to evaluate the arrest effect on the G2/M
transition. After the cells were treated with compound 3b at 0, 0.05,
0.1, or 1 μM for 24 h, the cells were harvested, and the cell cycle phases
were analyzed. As the results show in Fig. 2, 3b significantly arrested
the cell cycle at the G2/M phase in a dose-dependent manner with a
concomitant decrease in G1 phase cells. After treatment for 24 h at a
4

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Fig. 4. Compound 3b induced apoptosis and decreased the mitochondrial membrane potential in a dose- and time-dependent manner. Dot plot representation of
Annexin-V-FITC-fluorescence (x-axis) vs PI-fluorescence (y-axis) of the apoptotic A549 cells (Annexin-V positive) cells, treated with compound 3b (0.05, 0.1, and
1.0 μM) for 24 h (A) or 48 h (B). A549 cells were treated with 3b at the indicated concentration (0.05, 0.1, and 1.0 μM) or DMSO (0.01%) for 24 h, followed by
incubation with the fluorescence probe JC-1 for 30 min. Then, the cells were analyzed by fluorescence microscope (D). The experiments were performed at least three
times, and the results of the representative experiments.
around the cell nucleus.
aggregated to green-monomeric forms.
Apoptosis play an important role in tumor biology. To evaluate the
ability of compound 3b to induce the apoptosis of cancer cells, we
carried out flow cytometry assay using propidium iodide (PI) and
fluorescent immunolabeling of the protein annexin-V (V-FITC) with
A549 cells and the result was shown in Fig. 4. At the concentrations of
0.05, 0.1 and 1.0 μΜ of 3b for 24 h, the early and late apoptosis cells
were found to be 12.39%, 30.91%, 54.58% comparing with 1.04% of
the control (DMSO, 0.01%), respectively (Fig. 4A). The total apoptosis
cells increased to 55.62%, 63.52%, 64.99% when the incubation time
was extended to 48 h (Fig. 4B), respectively.
To evaluate the ability of 3b to generate ROS in cancer cells, an
assay with an oxidation-sensitive fluorescent probe (DCFH-DA) was
performed, and the results are shown in Fig. 5. The green fluorescence
reflects the level of intracellular ROS, which was hardly observed in the
control group. After compound 3b was added to the A549 cell line for
12 h, the intensity of the green fluorescence increased significantly in
contrast to the control group. Furthermore, there is also a dose-de-
pendent relationship between 3b and the ROS. It was obvious that the
intensity of the green fluorescence became progressively brighter when
the concentration of compound 3b was increased.
Mitochondrial dysfunction plays a vital role in the progression of
apoptosis,24 To investigate the possible involvement of mitochondrial
dysfunction in 3b-induced apoptosis of the cells, the quantitative mi-
tochondrial transmembrane potential (MMP) assay was performed with
JC-1 staining. The results in Fig. 4d demonstrated that when the A549
cells were exposed to the concentrations at 0.05, 0.10, and 1.0 μM for
24 h, a rapid collapse of MMP was detected in contrast to the control
(DMSO, 0.01%), which accumulate fluorescent dye from its red-
To evaluate whether the new 4-phenylamino-substituted naphtha-
lene-1,2-diones could act as carboxylesterase inhibitors, compound 3b,
which exhibited potent antiproliferative activities for human cancer cell
lines, was chosen for the carboxylesterase inhibition assay according
the method reported in literature25 using 3a and 3h as the reference
compounds. The results in Table 2 indicated that all the three com-
pounds were reasonable inhibitors of human liver carboxylesterase
with the Ki values range from 15.6 to 92.3 nM.
5

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Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 5. The A549 cells were treated with 3b at different concentrations (0.1, 0.5, and 1 μM) or DMSO (0.01%) for 12 h using an oxidation-sensitive fluorescent probe
(DCFH-DA). The green fluorescence intensity, which corresponded to the intracellular ROS level, was measured using a microplate reader with the excitation and
emission wavelengths set at 488 and 525 nm, respectively.
as the anticancer drugs irinotecan and oseltamivir, we synthesized and
evaluated a series of 4-phenylamino-substituted naphthalene-1,2-dione
derivatives as effective antiproliferative agents. The MTT assay results
indicated that some of the compounds, especially 3a and 3b, exhibited
potent antiproliferative activities against five human cancer cells with
IC50 values below the micromolar level. We have studied the anti-
proliferative mechanism of these compounds, such as inducing mi-
tochondrial depolarization, resulting in intracellular ROS production,
and acting as tubulin polymerization inhibitors. All of these results
indicated that compound 3a or 3b has the potential to act as a lead
compound for further evaluation, especially as a combination drug with
irinotecan and oseltamivir that are easily metabolized by liver car-
boxylesterase.
Table 2
Esterase Inhibition data for Compound 3a, 3b and 3h.
R1
N
O
R2
O
R1
R2
hCE1 Ki (nM
SE; o-NPA)
Appendix A. Supplementary data
Comp.
3a
3b
3h
OCH3
OCH3
CH3
H
F
H
87.5
92.3
15.6
5.1 (81.6
4.3
0.7 (11.1
4.7)a
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmcl.2018.07.047.
0.2)a
a
The literature value (brackets) in Ref. 17.
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IC50 (μM) against LO2 cell lines
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42.23
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a
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