Design, synthesis, and anticancer evaluation of benzophenone derivatives bearing naphthalene moiety as novel tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104265

A series of benzophenone derivatives bearing naphthalene moiety were designed, synthesized, characterized by ¹H NMR, ¹³C NMR, and HRMS and evaluated for their antiproliferative activity against human breast cancer cell line (MCF-7). Most of the tested derivatives showed good to moderate cytotoxicity against MCF-7 cell line. Among them, compound 4u (IC₅₀ = 1.47 ± 0.14 μM) was found to be the most active compound, which is more active than the standard drug cisplatin (IC₅₀ = 15.24 ± 1.27 μM). In vitro tubulin polymerization inhibition assay, EBI competition assay, cell cycle analysis, and cell apoptosis assay identified that compound 4u was a new tubulin polymerization inhibitor by targeting the colchicine binding site. Besides, molecular docking study showed that compound 4u has high binding affinities with the colchicine binding site of tubulin through hydrogen bond, cation-π, and hydrophobic interaction.

Full text of DOI:10.1016/j.bioorg.2020.104265

BioorganicChemistry104(2020)104265
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and anticancer evaluation of benzophenone derivatives
bearing naphthalene moiety as novel tubulin polymerization inhibitors
⁎
Guangcheng Wanga,b, , Wenjing Liud,e, Juan Tangf, Xue Mad, Zipeng Gonga, Yong Huanga,
⁎
Yongjun Lid, Zhiyun Pengc,
a State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang,
China
b College of Chemistry and Chemical Engineering, Jishou University, Jishou, China
c College of Food Science and Technology, Shanghai Ocean University, Shanghai, China
d Engineering Research Center for the Development and Application of Ethnic Medicine and TCM (Ministry of Education), Guizhou Medical University, Guiyang, China
e School of Pharmacy, Guizhou Medical University, Guiyang, China
f The Affiliated Hospital of Guizhou Medical University, Guiyang, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of benzophenone derivatives bearing naphthalene moiety were designed, synthesized, characterized by
1H NMR, 13C NMR, and HRMS and evaluated for their antiproliferative activity against human breast cancer cell
line (MCF-7). Most of the tested derivatives showed good to moderate cytotoxicity against MCF-7 cell line.
Benzophenone
Naphthalene
Tubulin polymerization inhibitors
Anticancer activity
Among them, compound 4u (IC50 = 1.47
0.14 μM) was found to be the most active compound, which is
more active than the standard drug cisplatin (IC50 = 15.24
1.27 μM). In vitro tubulin polymerization in-
hibition assay, EBI competition assay, cell cycle analysis, and cell apoptosis assay identified that compound 4u
was a new tubulin polymerization inhibitor by targeting the colchicine binding site. Besides, molecular docking
study showed that compound 4u has high binding affinities with the colchicine binding site of tubulin through
hydrogen bond, cation-π, and hydrophobic interaction.
1. Introduction
antimalarial activities [12]. The literature survey reveals that large
number of benzophenone derivatives emerging day by day with potent
Microtubules, comprising α- and β-tubulin heterodimers, are major
cytoskeletal components with important roles in many essential cellular
functions, such as intracellular transport, guideline of motility, cell
signaling, formation and maintenance of cell shape, and cell division in
eukaryotic cells [1–3]. Therefore, microtubule has become an attractive
molecular target for anticancer drug research [4–6]. A larger number of
anti-microtubule agents have been developed to date and some of them
(eg. paclitaxel, docetaxel, vinblastine, and vincristine) have been
widely used in clinical for the treatment various cancer including breast
cancer, leukemia, lymphoma, ovarian cancer, colon cancer and small
cell lung cancer [1,6].
anticancer activity in recent years. For their mode of action, inhibit
tubulin polymerization is one of the most common anticancer me-
chanisms. Belonging to this class of molecules, phenstatin (Fig. 1, I), a
potent inhibitor of tubulin polymerization, shown strongly inhibited
cancer cell growth and arrest the G2/M phase of the cell cycle by tar-
geting the colchicine binding site of microtubules [13]. Hsieh et al.
introduced 3-amino-4-methoxyphenyl moiety into the phenstatin re-
sulted in compound II (Fig. 1), that exhibited greater cytotoxic activity
than phenstatin [14]. Hayashi et al. reported a new benzophenone-di-
ketopiperazine-type potent antimicrotubule agent (Fig. 1, KPU-300,
III), which developed by modifying the structure of the clinical candi-
date plinabulin. In particularly, KPU-300 showed a sufficiently high
potent cytotoxicity (IC50 = 7.0 nM), twice the activity of plinabulin
[15]. Recently, inspired by the potent inhibition activity of phenstatin
and millepachine, Li group designed and synthesized two new class of
benzophenone series (Fig. 1, IV, and V), and some of the obtained
Benzophenones are the privileged class of pharmacologically active
molecules, which are present in many natural products and synthetic
compounds [7]. Several compounds possessing benzophenone moiety
displayed a broad spectrum of biological activities, such as anticancer
[8], anti-inflammatory [9], anti-HIV [10], antibacterial [11], and
⁎ Corresponding authors at: State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou
Medical University, Guiyang, China (G. Wang); College of Food Science and Technology, Shanghai Ocean University, Shanghai, China (Z. Peng).
E-mail addresses: wanggch123@163.com (G. Wang), pengzhiyun1986@163.com (Z. Peng).
https://doi.org/10.1016/j.bioorg.2020.104265
Received 15 July 2020; Received in revised form 30 August 2020; Accepted 31 August 2020
Availableonline03September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

G. Wang, et al.
BioorganicChemistry104(2020)104265
2.2. In vitro antiproliferative activities
All the synthesized compounds 4a-4u were evaluated for their an-
tiproliferative activity against human breast cancer cell line (MCF-7) by
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay with cisplatin and colchicine as reference drugs. The
IC50 values of the tested compounds 4a-4u are summarized in Table 1.
Most of the tested derivatives showed good to moderate cytotoxicity
against MCF-7 cell line. Among them, compound 4u
(IC50 = 1.47
0.14 μM) bearing 3-hydroxy-4-methoxyphenyl was
found to be the most active compound, which is more active than the
standard drug cisplatin (IC50 = 15.24 1.27 μM). Compound 4s
(IC50 = 1.86
0.23 μM) with 3-amino-4-methoxyphenyl moiety was
found to be the second most active member of the series.
The structure-activity relationship (SAR) of this class of compounds
has been summarized. Compound 4a with the unsubstituted phenyl
Fig. 1. The structures of some tubulin polymerization inhibitors with benzo-
phenone moiety.
ring displayed low active (IC50
> 10.0 μM). The introduction of
electron-withdrawing groups (F, Cl, NO2) resulted in a slight increase in
antiproliferative activity, such as compounds 4c, 4d, and 4o. The in-
troduction of electron-donating group methyl (4i-4k) and methoxy (4f-
4h) into the phenyl ring, results in increases antiproliferative activity.
Compared to the inhibitory activity of these compounds (4i-4k and 4f-
4h), the results were shown that the electron-donating group located at
the 4-position of the phenyl ring resulted in better activity. It was in-
teresting to find that compound 4h containing 4-methoxy group at the
phenyl ring shown potent antiproliferative activity, indicating that 4-
methoxy group plays an important role in the improvement of anti-
proliferative activity, and other compounds (4m, 4q-4u) having this
group also displayed a certain extent of antiproliferative activity.
Remarkably, compounds 4s and 4u represented the most potent anti-
proliferative activity in this series, which containing 3-amino-4-
methoxy or 3-hydroxy-4-methoxy group, respectively. Interestingly,
this pharmacophore (3-amino-4-methoxy or 3-hydroxy-4-methoxy
group) was also found in other tubulin inhibitors, such as compounds I,
II, IV and V (Fig. 1). Furthermore, the introduction of electron-donating
group tertiary butyl at the 4-position of the phenyl ring (4l) resulted in
drastically decreased the inhibitory activity, due to tert-butyl group is
bulky and has steric hindrance.
compounds exhibited better antiproliferative activity than the lead
compound millepachine [16,17].
In our previous work, we have found a novel series of chalcone
derivatives containing naphthalene moiety with potent anticancer ac-
tivity through inhibition of tubulin polymerization [18–20]. Based on
this interesting property of benzophenone analogs and in our con-
tinuing search for new molecules with anticancer activity, herein we
report the synthesis, biological evaluation, and mechanism study of a
series of benzophenone derivatives bearing naphthalene moiety as tu-
bulin polymerization inhibitors by targeting colchicine binding site
(Fig. 2).
2. Results and discussion
2.1. Chemistry
A series of benzophenone derivatives bearing naphthalene moiety
(4a-4u) was synthesized according to the pathways described in
Schemes 1. The commercially available naphthalen-1-ol 1 was reacted
with methyl iodide in the presence of K2CO3 as the base at room tem-
perature to give product 2. According to a recent literature report by Xu
et al. [21], intermediate 2 was reacted with substituted aromatic car-
boxylic acid 3 and trifluoroacetic acid anhydride (TFAA) in tri-
fluoroacetic acid (TFA) at room temperature to give the title com-
pounds 4a-4u. The chemical structures of these compounds were
confirmed by 1H NMR, 13C NMR, and HRMS. For instance, the 1H NMR
spectrum of 4f shown two singlets at δ 3.64 and 3.97 ppm was attrib-
uted to two methoxy groups in the phenyl ring. Multiple peaks observed
at δ 6.64–9.5 ppm were assigned to aromatic protons. In the 13C NMR
spectrum, the number of signals equals the number of different carbons.
The high-resolution mass spectrum of compound 4f showed a molecular
ion peak at m/z 315.0991 as [M+Na]+ which also supports the pro-
posed structure of the compound. The HRMS of compounds 4a-4u
showed a molecular ion peak equivalent to their molecular formulae.
Besides, the 1H and 13C NMR spectra data of 4a, 4e, 4f, 4g, 4k, 4p, and
4q were closely similar to the previous report [22].
2.3. Effect on tubulin polymerization
To evaluate whether this class of compounds exhibited anti-
proliferative activity by disturb the tubulin-microtubule system, the
most active compound 4u was selected to test its effect on tubulin
polymerization. The tubulin polymerization inhibitory potential was
evaluated for compound 4u at different concentrations (0.8, 1.5, 3.0,
6.0, 12.5, 25, 50, and 100 μM), with colchicine (12.5 μM) as a positive
control. The results were shown in Fig. 3, compound 4u displayed
potent tubulin polymerization inhibitory activity with IC50 value was
8.1 μM. Interestingly, compound 4u shown a similar inhibitory manner
with the reference compound colchicine, indicated that compound 4u is
a new tubulin polymerization inhibitor.
2.4. Tubulin interaction at the colchicine binding site
Based on the literature, there are four unique binding sites in
Fig. 2. Rationale design of the title compounds of
this study.
2

G. Wang, et al.
BioorganicChemistry104(2020)104265
Scheme 1. (a) CH3I, K2CO3, DMF, room temperature, 4 h; (b) TFAA, TFA, room temperature, 12 h; (c) Fe, NH4Cl, EtOH/H2O (v/v = 3:1), reflux, 3 h.
Table 1
The antiproliferative activities of all title compounds 4a-4u against the human breast cancer cell line (MCF-7).
No.
Ar
IC50 (μM)
No.
Ar
IC50 (μM)
4a
> 10.0
4m
6.69
0.47
4b
4c
> 10.0
4n
4o
7.12
6.66
0.51
0.32
8.61
0.58
4d
4e
8.04
0.69
4p
4q
> 10.0
8.33
> 10.0
0.61
0.46
4f
7.70
0.45
4r
7.82
4g
4h
4i
7.61
0.42
0.32
4s
4t
1.86
6.62
1.47
0.23
0.53
0.14
2.54
> 10.0
4u
4j
7.66
0.64
0.40
cisplain
15.24
0.083
1.27
4k
4l
6.67
colchicine
0.008
> 10.0
3

G. Wang, et al.
BioorganicChemistry104(2020)104265
Fig. 3. Effect of compound 4u and colchicine on the tubulin polymerization.
Fig. 4. EBI competition assay of compound 4u in MDA-MB-231 cell line. Competition assay of compound 4u (2, 10, and 50 µM), vincristine (5 and 25 μM) and
colchicine (5 μM) with EBI (100 µM) on MDA-MB-231 cells.
tubulin (taxanes, vinca alkaloids, laulimalide, and colchicine) [23]. To
evaluate whether compound 4u binds to the colchicine binding site of
tubulin, N, N’-ethylenebis (iodoacetamide) (EBI) competition assay was
carried out in human breast cancer cell line (MDA-MB-231) according
to the previously published references [24,25]. EBI is an alkylating
agent that has the property to specifically cross-link the Cys239 and the
Cys354 residues of β-tubulin involved in the colchicine-binding site
[24]. Tubulin polymerization inhibitors occupy the colchicine-binding
site of tubulin could inhibit the formation of the EBI:β-tubulin adduct,
which could be detected by Western blot. As shown in Fig. 4, compound
4u can inhibit the formation of EBI-β-tubulin adducts in a concentra-
tion-dependent manner, resulting in the decrease of the adduct band
comparing with DMSO and EBI treatment, which similar to positive
control colchicine. Vincristine has a different binding site compared
with colchicine, was used as a negative control. These results proved
that compound 4u can directly bind to the colchicine binding site of
tubulin and inhibit tubulin polymerization.
to 10.6%, and the percentage of early apoptotic cells from 1.89 to
11.2%, indicating that compound 4u effectively induced cell apoptosis
in MCF-7 cells.
2.7. Molecular docking study
To elucidate the binding mode of compound 4u with tubulin, the
molecular docking study of compound 4u into the colchicine binding
pocket of tubulin (PDB: 1SA0) was performed using Autodock vina
1.1.2 [26]. Colchicine was first docked into the colchicine binding site
of tubulin, its co-crystallized conformation was reproduced approxi-
mately (Fig. 6A, RMSD: 1.10 Å), suggesting that the methods are ap-
propriate for the docking study. Then, we investigate the binding mode
of compound 4u with tubulin. From the docking study (Fig. 6B), it can
be seen that compound 4u and colchicine can be well superimposed.
The docking results was shown in Fig. 7, it was observed that compound
4u adopted an “L-shaped” conformation in the colchicine-binding do-
main of the tubulin, with the estimated binding energy of
−8.4 kcal·mol−1. The naphthyl group of 4u is located at the hydro-
phobic pocket, surrounded by the residues Leu-248, Leu-255, Met-259,
Val-315, Ala-316, and Ala-354, forming a strong hydrophobic binding.
Detailed analysis showed that the phenyl and naphthyl groups of 4u
formed cation-π interactions with the residues Lys-254 and Lys-352,
respectively. It was shown that the residue Asn-249 (bond length:
2.5 Å) formed a hydrogen bond with 4u, which was the main interac-
tion between 4u and tubulin.
2.5. Cell cycle study
Due to microtubules play an important role in eukaryotic cell di-
vision, tubulin polymerization inhibitors can arrest the cell cycle in G2/
M phase and lead to cell apoptosis. Based on the above results, the
effect of most active compound 4u on cell cycle was investigated in
MCF-7 cells using flow cytometry analysis. The result was showed in
Fig. 5, the percentage of cells in the G2/M phase increased to 54.21%
from 14.10% after treatment with compound 4u at the concentration of
1.25 μM. These results verified that compound 4u can induced cell
cycle arrest in the G2/M phase.
3. Conclusion.
In conclusion, a series of benzophenone derivatives bearing naph-
thalene moiety 4a-4u were synthesized and evaluated for their anti-
proliferative activity against human breast cancer cell line (MCF-7).
Antiproliferative screening suggested that most of the tested derivatives
showed good to moderate cytotoxicity. Among the series, compound 4u
2.6. Induction of cell apoptosis
To identify whether compound 4u could induce cell apoptosis, cell
apoptosis assay was explored using Annexin V-FITC/PI assay in MCF-7
cell line. As shown in Fig. 5, after treatment with 4u at the con-
centration of 1.25 μM, the percentage of late apoptotic cells from 4.18
(IC50 = 1.47
0.14 μM) bearing 3-hydroxy-4-methoxyphenyl was
found to be the most active compound, which are more active than the
4

G. Wang, et al.
BioorganicChemistry104(2020)104265
Fig. 5. Effect of compound 4u on cell cycle arrest and cell apoptosis in MCF-7 cells. (A, B) Cell cycle analysis of MCF-7 cells after treated with DMSO (control) or
compound 4u (1.25 μM) for 24 h; (C, D) Induction of apoptosis by DMSO (control) and compound 4u (1.25 μM).
Fig. 6. (A) Redocking conformation superimposed on crystal conformation, rose red and violet are the crystal conformation and docking conformation of colchicine
ligands, respectively; (B) docked conformation of compound 4u (purple) superimposed on crystal conformation of the colchicine (rose red). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
5

G. Wang, et al.
BioorganicChemistry104(2020)104265
Fig. 7. Molecular docking of most potent compound 4u with the colchicine binding site of tubulin (PDB ID: 1SA0).
standard drug cisplatin (IC50 = 15.24
1.27 μM). The preliminary
(TFA, 1 mL) and stirred at room temperature for 12 h. After the com-
pletion of the reaction, the solvent was removed under reduced pres-
sure and the residue was purified by chromatography to give the de-
sired products 4a-4u. Spectral data of all title compounds (4a-4u) were
provided in Supplementary Material.
structure-activity relationships shown that 3-amino-4-methoxy or 3-
hydroxy-4-methoxy group in the phenyl ring play an important role in
the improvement of the antiproliferative activity. In vitro tubulin
polymerization inhibition assay, EBI competition assay, cell cycle ana-
lysis, and cell apoptosis assay identified that compound 4u was a new
tubulin polymerization inhibitor by targeting the colchicine binding
site. Furthermore, molecular docking study showed that compound 4u
has high binding affinities with the colchicine binding site of tubulin.
Hence, this study identified compound 4u can be used as a lead mo-
lecule for further research and development of new tubulin poly-
merization inhibitors.
4.2. Evaluation of in vitro antiproliferative activity
The antiproliferative activity of all synthesized compounds on the
MCF-7 cell line was evaluated using MTT assay [28]. Briefly, cells were
seeded in 96-well plates and cultured for overnight. Then the cells were
treated with various concentrations of tested compounds for 48 h. After
incubation, cell viability was determined by the MTT method.
4. Experimental section
4.3. In vitro tubulin polymerization inhibition assay
4.1. Chemistry
The purified tubulin protein was purified from fresh pig brain by
three cycles of temperature-dependent assembly/disassembly in
100 mM PIPES (pH 6.5), 1 mM MgSO4, 2 mM EGTA, 1 mM GTP and
1 mM 2-mercaptoethanol [29]. In the first cycle of polymerization,
glycerol and phenylmethylsulfonyl fluoride were added to 4 M and
0.2 mM, respectively. Homogeneous tubulin was prepared from mi-
crotubule protein by phosphocellulose (P11) chromatography. The
purified proteins were stored in aliquots at −70 °C. After purification,
tubulin protein was mixed with different concentrations of compound
4u (0.8, 1.5, 3.0, 6.0, 12.5, 25, 50 and 100 μM) or colchicine (12.5 μM)
in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA) con-
taining 1 mM GTP and 5% glycerol. Microtubule polymerization was
monitored at 37 °C by light scattering at 340 nm using a SPECTRA MAX
190 (Molecular Device) spectrophotometer. The plateau absorbance
values were used for calculations.
All starting materials and reagents were purchased from commercial
suppliers. TLC was performed on 0.20 mm Silica Gel 60 F254 plates
(Qingdao Ocean Chemical Factory, Shandong, China). Nuclear mag-
netic resonance spectra (NMR) were recorded on a Bruker spectrometer
(400 MHz) with TMS as an external reference and reported in parts per
million.
4.1.1. 1-Methoxynaphthalene (2)
A mixture of naphthalen-1-ol (1 mmol), K2CO3 (3 mmol) and CH3I
(3 mmol) in DMF (10 mL) was stirred at room temperature for 4 h. The
reaction mixture was poured into H2O (100 mL) and extracted with
EtOAc (50 mL × 3). The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to give product 2 as a light brown liquid (83%).
4.1.2. General procedure for the synthesis of 4a-4u
4.4. EBI competition assay
Trifluoroacetic acid anhydride (TFAA, 2 mmol) was added to a so-
lution of 1-methoxynaphthalene 2 (1.5 mmol) and various commercial
available aromatic carboxylic acid (1 mmol) in Trifluoroacetic acid
MDA-MD-231 cells (1 × 106) were seeded in 6-well culture plate
and allowed to adhere overnight. The cells were treated with different
6

G. Wang, et al.
BioorganicChemistry104(2020)104265
concentrations of compound 4u (2, 10, and 50 µM), colchicine (5 μM),
or vincristine (5 and 25 μM) for 4 h and afterward treated with EBI
(100 mM) for 1.5 h. The cells were harvested and analyzed by Western
blot according to the previously published references [27]. β-Actin was
employed as the loading control.
and Technology Bureau ([2017]30-29).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104265.
4.5. Cell cycle analysis
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Acknowledgments
This work was supported by National Natural Science Foundation of
China (81660574), One Thousand Talents Program of Guizhou
Province (the fifth group, [2019]4), Guizhou Provincial Natural Science
Foundation ([2019]1256), the Doctor Foundation of Guizhou Medical
University ([2018]004), Guizhou Science and Technology Department
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