Aromatic diacylhydrazine derivatives as a new class of polo-like kinase 1 (PLK1) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.05.026

A novel class of aromatic diacylhydrazine derivatives was designed as PLK1 inhibitors. All the 19 new synthesized compounds were assayed for antitumor activity against the respective cervical cancer cells. In which, nine compounds with better antitumor activities were further tested for their PLK1 inhibitory activity. Last, we have successfully found that compound 7k showed both the promising antitumor activity with IC₅₀ of 0.17 μM against the cervical cancer cells, and also processed the most potent PLK1 inhibitory activity with IC₅₀ of 0.03 μM. In addition, docking simulation also carried out in this study to give a potent prediction binding mode between the small molecule and PKL1 (PDB code: 1umw) protein.

Full text of DOI:10.1016/j.ejmech.2014.05.026

Accepted Manuscript
Aromatic diacylhydrazine derivatives as a new class of Polo-like kinase 1 (PLK1)
inhibitors
Juan Sun, Feng-Jiao Guo, Xin-Yi Wang, Xiao- Han, Yang Zhang, Gui-Hua Sheng,
Shao-Song Qian, Hai-Liang Zhu
PII:
S0223-5234(14)00440-1
10.1016/j.ejmech.2014.05.026
EJMECH 6982
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2013
Revised Date: 8 April 2014
Accepted Date: 6 May 2014
Please cite this article as: J. Sun, F.-J. Guo, X.-Y. Wang, X.- Han, Y. Zhang, G.-H. Sheng, S.-S. Qian,
H.-L. Zhu, Aromatic diacylhydrazine derivatives as a new class of Polo-like kinase 1 (PLK1) inhibitors,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.026.
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ACCEPTED MANUSCRIPT
Aromatic diacylhydrazine derivatives as a new class of Polo-like kinase
1 (PLK1) inhibitors
Juan Suna, Feng-Jiao Guob, Xin-Yi Wangb, Xiao- Hanb, Yang Zhangb, Gui-Hua Shenga,
Shao-Song Qianb, Hai-Liang Zhu*a,b
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, PR China
b School of Life Sciences, Shandong University of Technology,
Shandong 255049, People’s Republic of China
New diacylhydrazine derivatives have been designed and evaluated for their PLK1
inhibitory activity and anti-cervical cancer cell activity. The most active compound is 7k.

ACCEPTED MANUSCRIPT
1. The synthesis and anticancer activities of these compounds have not been reported so far.
2. Our current findings are completely new.
3. Their biological activities are also evaluated for PLK1 inhibitory activity.

ACCEPTED MANUSCRIPT
Aromatic diacylhydrazine derivatives as a new class of Polo-like
kinase 1 (PLK1) inhibitors
Juan Suna, Feng-Jiao Guob, Xin-Yi Wangb, Xiao- Hanb, Yang Zhangb, Gui-Hua Shenga,
Shao-Song Qianb, Hai-Liang Zhu*a,b
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, PR China
b School of Life Sciences, Shandong University of Technology,
Shandong 255049, People’s Republic of China
(Phone: +86-25-8359 2672; Fax: +86-25-8359 2672; E-mail: zhuhl@nju.edu.cn)
Abstract: A novel class of aromatic diacylhydrazine derivatives was designed as
PLK1 inhibitors. All the 19 new synthesized compounds were assayed for antitumor
activity against the respective cervical cancer cells. In which, nine compounds with
better antitumor activities were further tested for their PLK1 inhibitory activity. Last,
we have successfully found that compound 7k showed both the promising antitumor
activity with IC50 of 0.17 ꢀM against the cervical cancer cells, and also processed the
most potent PLK1 inhibitory activity with IC50 of 0.03 ꢀM. In addition, docking
simulation also carried out in this study to give a potent prediction binding mode
between the small molecule and PKL1 (PDB code: 1umw) protein.
Keywords: Polo-like kinase 1; Aromatic diacylhydrazine; Computer simulation
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1. Introduction
Polo-like kinases (PLKs), a type of Ser/Thr protein kinases, have gathered much
attention as important elements that regulate cell cycle progression, particularly
mitosis [1]. Human Polo-like kinase subfamily includes four closely related members,
that is, PLK1, PLK2, PLK3, and PLK4. Recently, PLK5 has been identified; however,
it lacks a kinase domain and does not seem to function in cell cycle regulation [2].
Among them, PLK1 participates in multiple steps in mitosis by phosphorylating
various substrates, but not in normal diploid cells or nondividing cells [3-6].
Inhibition of PLK1 activity in cancer cell causes mitotic arrest and finally induces
strong cell-killing effect [7-12]. It is also reported that PLK1 is overexpressed in
many clinical cancer samples, such as lung, colon, prostate, ovary, breast, head, neck
squamous cell carcinoma, melanoma and others [13-16]. All of these indicate its
involvement in carcinogenesis and its potential as a therapeutic target.
PLK1 offers two functionally crucial target sites within one molecule-the
N-terminal kinase domain and the C-terminal polo-box domain. Several crystal
structures of PLK1 kinase domain have been determined and they are complex with
an ATP pocket-binding ligand such as a nonhydrolysable ATP analogue
adenylylimidodiphosphate [17], a pyrrolo-pyrazole inhibitor (PHA-680626) [18], a
purine mimetic inhibitor [19], a quinazoline mimetic inhibitor [20], a
pyrazolopyridine inhibitor [21] and so on. Besides, polo-box domain is essential for
the subcellular localization and mitotic functions of PLK1, it is ideally suited for the
development of anti-PLK1 inhibitors that interfere with polo-box domain-dependent
protein-protein interactions [22-24]. These provide a good basis to elucidate the
structure–activity relationship of these diverse compounds and further design novel
PLK1 inhibitors. For this purpose, diacylhydrazine derivatives disclosed by Lu et al
[25] were selected as a starting point for our efforts because of its unique structure
and strong enzyme potency (Figure 1). Besides, a number of 1,4-benzodioxan
template-containing compounds have ability as potential anticancer drugs with
excellent bioavailability and low cytotoxicity [26-28]. Antitumor activities of various
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cinnamic acid derivatives were also explored by many research groups. Particularly,
cinnamic acid ester derivatives have shown the potential antitumor activity [29-30].
Therefore, we endeavor to explore a new structure class based on known PLK1
inhibitors by chemical modifications according to bioisosteric replacement. Here we
report the design and SAR study of novel diacylhydrazine PLK1 inhibitors.
2. Results and discussion
2.1. Chemistry
Methyl 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylate 3 was prepared in two
steps as shown in Scheme 1. It was prepared in two steps. Firstly,
3,4-dihydroxybenzoic acid 1 gave methyl 3,4-dihydroxybenzoate 2, catalyzed by
concentrated sulfuric acid in methanol. Secondly, compound 3 was prepared by
treatment of 2 with dibromoethane in acetone. The synthetic route to target
compounds
(7a-7s)
is
shown
in
Scheme
1.
Firstly,
2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide was prepared by treatment of 3
with hydrazine hydrate (85%) in ethanol. Then, compound 6 gave diacylhydrazine
derivatives by using carbodiimide hydrochloride and N-hydroxybenzotriazole in
anhydrous CH2Cl2 with different substituted cinnamic acids.
All of the synthetic compounds gave satisfactory analytical and spectroscopic
data, which were full accordance with their depicted structures.
2.2 Pharmacology
Generally speaking, the inhibitory activity of the compounds is due to cell
apoptosis or toxic effect, so we firstly performed cytotoxicity test before detecting
biological activity. The diacylhydrazine compounds were detected for their
cytotoxicity on macrophages cells. The pharmacological results of these compounds
were summarized in Table 1. What we can see from the data is that most of the
compounds were low toxic.
2.2.1 Cell proliferation assay (MTT method)
All the synthesized derivatives (7a-7q) were tested in vitro for the inhibition
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activity on cervical cancer cells. The results were summarized in Table 1. The table
showed that most of the synthesized compounds exhibited potent inhibitory activity
with the IC50 value at low micromolar.
Based on the data obtained, we surveyed a variety of substituents at different
positions on the phenyl ring of these diacylhydrazine derivatives, and found that
various substituents of such as halogen, methyl, methoxyl and nitro group led to
distinct anticancer activities of these target compounds. Compound 7k with a
methoxyl group on the 3-position of phenyl ring manifested the best antitumor
activity with IC50 value of 0.17 ꢀM than other compounds, while the simplest
compound 7e of this series with a methoxyl group on the 2-position of phenyl ring
displayed the worst anticancer activity. Substitutions with electron-withdrawing group
on the 2-position of phenyl ring did not improve the activity markedly, nor did
halogen substitutions on the phenyl ring, except compound 7c and 7f with IC50 value
of 2.11 and 0.89 ꢀM, respectively. Compound 7r with a nitro group on the 4-position
of phenyl ring displayed higher antibacterial activity with IC50 value of 0.61 ꢀM than
compounds with a nitro group on the 2-position (0.89 ꢀM) and 3-position (1.56 ꢀM)
of phenyl ring, while the activity gradient of substituent group on the phenyl ring in
m-position was -OCH3 > -NO2 > -CH3 > -Br > -Cl > -F. Together, these results
illustrate that diacylhydrazine derivatives bearing 4-position substitutions of phenyl
ring have potent antitumor activities.
From the above-mentioned analysis, it could be concluded that the compounds
with substitutions on the 4-position of phenyl ring and nitro substituted benzene ring
were found to be the most favorable for the antitumor activity. More importantly,
most of the analogues possess low cytotoxicity. Perhaps the electron withdrawing
groups could reduce the electron density of the benzene ring and make it difficult to
form π-π bonds with amino acids containing aromatic groups. This makes the steric
hindrance of the active pocket smaller for benzene ring part to get in. Therefore, these
compounds are easy access to the active site.
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2.2.2 PLK1 inhibitory assay
The PLK1 inhibitory potency of the diacylhydrazine derivatives was examined
and the results were summarized in Table 2. Most of the tested compounds displayed
potent PLK1 inhibiting activity. Among them, compound 7k showed the most potent
inhibitory with IC50 of 0.03 ꢀM, prior to the positive control poloxin. The results of
PLK1 inhibitory activity of the tested compounds were in agreement to the structure
relationships (SAR) of their antitumor activities. This agreement suggested that
antitumor activities of the synthesized compounds would derive from the inhibition of
PLK1 enzymatic activities.
2.3 Binding model of compounds into PLK1 structure
In an effort to elucidate the possible mechanism by which the title compounds
can induce anticancer activity and guide further SAR studies, molecular docking of
the potent inhibitors into N-terminal kinase and C-terminal polo-box domain were
performed on the binding model based on the PLK1 (1umw.pdb) separately. And
preprocessed using the Schrodinger Protein Preparation Guide [31]. In the N-terminal
kinase domain binding model, all of the compounds couldn't enter the active pocket
except compound 7s with docking calculation score of 8.0265, much lower than the
polo-box domain docking scores. Therefore, we suspect that our compounds are small
molecule inhibitors that may bind to the PLK1 PBD. The binding models of
compound 7k and PLK1 PBD were depicted in Figure 2 (A) and Figure 3. The amino
acid residues which had interaction with PLK1 as well as bond length were labeled.
The docking calculations of the other compounds were also depicted in Table 3.
In the binding model, the carbonyl and imino of the diacylhydrazine skeleton
forms a hydrogen bond with the backbone NH and carbonyl of Trp414, respectively.
And the oxygen atom of the benzodioxan forms a hydrogen bond with the backbone
NH of Asp416. In addition, compound 7k was also nicely bound to PLK1 via one
charge interaction (Lys540), one π-sigma interaction (Leu491) and one π-π interaction
(His538). Figure 2B displayed 2D interactional maps between the small molecule
ligand poloxin and 1umw protein crystal structure. Insight into this picture, we can
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see that amino acid residues Trp414, Lys540 located in the binding
pocket also seemed very important for the active conformation of compound
poloxin. These results could provide a molecular level foundation to illustrate
compound 7k can bind well at the active site of PLK1 kinase and was a potential
inhibitor of PLK1.
3. Conclusion
In conclusion, a series of diacylhydrazine derivatives have been synthesized and
evaluated for their antitumor activities. Compound 7k demonstrated the most potent
inhibitory activity that inhibited the growth of cervical cancer cells with IC50 of 0.17
ꢀM and inhibited the activity of PLK1 with IC50 of 0.03 ꢀM. Docking simulation was
performed to position compound 7k into the PLK1 active site to determine the
probable binding conformation and the result indicated that compound 7k was a
potent inhibitor of PLK1. Given the unforeseen structural differences within the active
site of some pathogenic enzymes, the key to discovering inhibitors with antitumor
activity lies in a detailed understanding of the PLK1 active sites. Further studies on
the PLK1 inhibition ability of this compound, new structural data were guiding
further modifications of the current series with the hopes of improving both enzymatic
inhibition and physical properties.
4. Experimental results
4.1. Chemistry
All chemicals and reagents used in the current study were of analytical grade. The
reactions were monitored by thin layer chromatography (TLC) on Merck pre-coated
silica GF254 plates. Melting points (uncorrected) were determined on a XT4MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained on a Mariner
1
System 5304 mass spectrometer, and H NMR spectra were collected on a Bruker
DPX300 spectrometer at room temperature with TMS and solvent signals allotted as
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internal standards. Chemical shifts are reported in ppm (δ). Elemental analyses were
performed on a CHN-O-Rapid instrument, and were within ±0.4% of the theoretical
values.
4.1.1. General procedure for the preparation of compound 2.
Protocatechuic acid (1 mmol) in methanol (30 mL) was treated with concentrated
sulfuric acid (0.5 mL) under 90ꢀC overnight. The sovlent was removed leaving oil
which was dissovled in ethyl acetate (20 mL) and extracted with water (40 mL). After
drying the organic layer with anhydrous Na2SO4 and evaporating the solvent under
reduced pressure a solid appeared. The solid was recyrstallized from ethanol to obtain
the compound 2.
4.1.2 General procedure for the preparation of compounds 3.
In the three-necked flask under a nitrogen atmosphere, compound 2(1 mmol) was
dissolved in dry acetone (10 mL), and then added anhydrous potassium carbonate (2
mmol). After that, the acetone solution containing dibromomethane (1 mmol) was
added dropwise then refluxed for 24 h. The reaction solution was evaporated reduced
pressure distillation. The appropriate amount of water was added in the residue, and
extracted with ethyl acetate (3×40 mL). Combined organic layer and dried with
anhydrous magnesium sulfate. Then the solvent was removed by reduced pressure
steam to give the crude product, the crude product is purified by column
chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 4: 1] to give white
solid (compounds 3).
4.1.3 General procedure for the preparation of cinnamic acids (compounds
5a-5o).
A mixture of aromatic aldehydes (3.2 mmol), malonic acid (3.87 mmol), piperidine
(0.387 mmol) was dissolved in pyridine and stirred on 85ꢁ for 24 h. The pyridine
was removed at the vacuum. The reaction mixture was poured into water and washed
with HCl. And the precipitate was filtered and washed with hexane for three times,
and dried under vacuum to afford the cinnamic acids.
4.1.4 General procedure for the preparation of compounds 6.
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A stirred solution of compound 3 (0.1 mol) in ethanol (50 mL) was treated with the
hydrazine hydrate (85%), under 90oC for 5 d. The sovlent was removed leaving oil
which was dissovled in ethyl acetate (20 mL) and extracted with water (40 mL). After
drying the organic layer with anhydrous Na2SO4 and evaporating the solvent under
reduced pressure a solid appeared. The solid was recyrstallized from ethanol to obtain
the compound 6.
4.1.5 General procedure for the preparation of target compounds 7a-7s.
An equimolar compound 6 (0.001 mol) and substituted cinnamic acid in anhydrous
CH2Cl2 was stirred for 8 h with carbodiimide hydrochloride (0.0015 mmol) and
N-hydroxybenzotriazole (0.0005 mmol). Then reaction mixture was extracted twice
using ethyl acetate and water. The organic layer was evaporated and recrystallized
from ethanol to give the title compounds.
4.1.5.1
(E)-N'-(3-(2-fluorophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7a)
White powder, mp: 186-188oC. 1H NMR (300 MHz, CDCl3): 4.323-4.375 (m, 4H),
6.890-6.912 (d, J = 6.6 Hz, 1H), 6.993 (s, 1H), 7.101-7.117 (m, 2H), 7.273-7.285 (m,
2H), 7.357-7.363 (m, 1H), 7.506 (s, 1H), 7.737 (s, 1H). 9.231 (s, 1H), 9.312 (s, 1H).
13C NMR (400 MHz, CDCl3) δ: 60.38, 61.59, 109.77, 112.58, 115.37, 119.77, 120.55,
122.50, 123. 81, 124.11, 126.88, 128.03, 134.21, 149.32, 151.55, 158.05, 165.77,
166.92. MS (ESI): 343 (C18H16FN2O4, [M+H]+). Anal. Calcd for C18H15FN2O4: C,
63.15; H, 4.42; N, 8.18%. Found: C, 63.32; H, 4.35; N, 8.07%.
4.1.5.2
(E)-N'-(3-(2-chlorophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7b)
White powder, mp: 229-230oC. 1H NMR (300 MHz, CDCl3): 4.223-4.256 (m, 4H),
6.891 (s, 1H), 7.072-7.226 (m, 2H), 7.356-7.368 (d, J = 3.6 Hz, 1H), 7.502-7.711 (m,
2H), 7.805-7.876 (m, 1H), 7.906-7.998 (m, 2H), 9.135 (s, 1H), 9.235(s, 1H). 13C
NMR (400 MHz, CDCl3) δ: 61.23, 62.18, 108.90, 113.06, 118.33, 122.19, 125.05,
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126.74, 128.11, 129.23, 130.24, 132.02, 132.88, 135.71, 148.97, 150.04, 163.54,
166.02. MS (ESI): 359 (C18H16ClN2O4, [M+H]+). Anal. Calcd for C18H15ClN2O4: C,
60.26; H, 4.21; N, 7.81%. Found: C, 60.19; H, 4.33; N, 7.76%.
4.1.5.3
(E)-N'-(3-(2-bromophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7c)
White powder, mp: 251-252oC. 1H NMR (300 MHz, CDCl3): 4.481-4.553 (m, 4H),
6.669-6.721 (m, 3H), 6.910-7.111 (m, 3H), 7.275-7.288 (m, 1H), 7.505 (s, 1H),
13
7.736-7.812 (m, 1H), 9.013 (s, 1H), 9.123 (s, 1H). C NMR (400 MHz, CDCl3) δ:
60.24, 60.87, 110.06, 111.41, 119.33, 121.02, 123.71, 124.93, 127.26, 127.39, 129.22,
131.44, 138.07, 141.62, 150.08, 151.66, 165.08, 167.11. MS (ESI): 403
(C18H16BrN2O4, [M+H]+). Anal. Calcd for C18H15BrN2O4: C, 53.62; H, 3.75; N,
6.95%. Found: C, 53.81; H, 3.66; N, 7.15%.
4.1.5.4
(E)-N'-(3-(o-tolyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide
(7d)
1
White powder, mp: 250-251oC. H NMR (300 MHz, CDCl3): 1.556 (s, 3H),
4.532-4.637 (m, 4H), 6.590-6.650 (d, J = 18.0 Hz, 1H), 6.894 (s, 1H), 7.101-7.211 (m,
4H), 7.256-7.278 (m, 2H), 7.359-7.506 (m, 1H), 9.250 (s, 1H), 9.733 (s, 1H). 13C
NMR (400 MHz, CDCl3) δ: 20.18, 59.98, 60.47, 109.36, 111.46, 118.34, 120.42,
125.47, 126.51, 127.21, 128.28, 130.72, 133.11, 135.74, 140.38, 149.29, 151.63,
164.54, 166.83. MS (ESI): 339 (C19H19N2O4, [M+H]+). Anal. Calcd for C19H18N2O4:
C, 67.44; H, 5.36; N, 8.28%. Found: C, 67.31; H, 5.50; N, 8.36%.
4.1.5.5
(E)-N'-(3-(2-methoxyphenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboh
ydrazide (7e)
1
White powder, mp: 247-248oC. H NMR (300 MHz, CDCl3): 1.557 (s, 3H),
4.612-4.678 (m, 4H), 6.99 (s, 1H), 7.10-7.11 (m, 2H), 7.267-7.328 (m, 2H),
7.589-7.695 (d, J = 31.8 Hz, 1H), 7.855-7.936 (m, 3H), 9.005 (s, 1H), 9.336 (s, 1H).
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13C NMR (400 MHz, CDCl3) δ: 54.82, 61.28, 62.17, 109.06, 112.36, 113.51, 116.38,
121.02, 122.38, 124.60, 125.27, 128.28, 131.55, 140.03, 150.08, 151.63, 158.67,
163.54, 165.82. MS (ESI): 355 (C19H19N2O5, [M+H]+). Anal. Calcd for C19H18N2O5:
C, 64.40; H, 5.12; N, 7.91%. Found: C, 64.32; H, 5.21; N, 8.03%.
4.1.5.6
(E)-N'-(3-(2-nitrophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydr
azide (7f)
1
White powder, mp: 204-206oC. H NMR (300 MHz, CDCl3): 4.32-4.37 (m, 4H),
6.589-6.690 (d, J = 30.3 Hz, 1H), 6.709-6.781 (d, J = 21.6 Hz, 1H), 6.991-7.012 (m,
2H), 7.150-7.179 (m, 2H), 7.257-7.322 (m, 2H), 7.605-7.830 (m, 1H), 9.250 (s, 1H),
13
9.373 (s, 1H). C NMR (400 MHz, CDCl3) δ: 61.08, 62.83, 108.96, 112.61, 117.59,
122.04, 127.02, 128.11, 129.44, 131.03, 132.17, 136.08, 140.15, 147.95, 150.48,
151.66, 164.81, 165.91. MS (ESI): 370 (C18H16N3O6, [M+H]+). Anal. Calcd for
C18H15N3O6: C, 58.54; H, 4.09; N, 11.38%. Found: C, 58.49; H, 4.15; N, 11.43%.
4.1.5.7
(E)-N'-(3-(3-fluorophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7g)
White powder, mp: 229-230oC. 1H NMR (300 MHz, CDCl3): 4.133-4.178 (m, 4H),
6.675-6.697 (m, 1H), 6.897-6.921 (m, 2H), 6.989-7.002 (d, J = 3.9 Hz, 1H), 7.159 (s,
1H), 7.352 (s, 2H), 7.527-7.612 (m, 2H), 9.203 (s, 1H), 9.510 (s, 1H). 13C NMR (400
MHz, CDCl3) δ: 60.02, 60.96, 109.06, 112.14, 113.26, 115.70, 117.04ꢂ121.02, 125.90,
128.24, 130.15, 132.49ꢂ136.56, 151.43, 152.08, 159.52,163.05, 167.12. MS (ESI):
343 (C18H16FN2O4, [M+H]+). Anal. Calcd for C18H15FN2O4: C, 63.15; H, 4.42; N,
8.18%. Found: C, 63.32; H, 4.20; N, 8.36%.
4.1.5.8
(E)-N'-(3-(3-chlorophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7h)
White powder, mp: 221-223oC. 1H NMR (300 MHz, CDCl3): 4.298-4.311 (m, 4H),
6.893-6.906 (d, J = 3.9 Hz, 1H), 7.003 (s, 1H), 7.106-7.118 (m, 2H), 7.279-7.311 (m,
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2H), 7.553-7.596 (m, 1H), 7.670-7.698 (m, 2H), 9.731 (s, 1H), 9.838 (s, 1H). 13C
NMR (400 MHz, CDCl3) δ: 61.11, 62.37, 107.66, 111.46, 118.04, 122.74, 124.43,
125.16, 126.75, 127.23, 131.60, 134.44, 136.19, 138.82, 149.42, 150.61, 165.89,
167.08. MS (ESI): 359 (C18H16ClN2O4, [M+H]+). Anal. Calcd for C18H15ClN2O4: C,
60.26; H, 4.21; N, 7.81%. Found: C, 60.33; H, 4.35; N, 7.69%.
4.1.5.9
(E)-N'-(3-(3-bromophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7i)
White powder, mp: 228-230oC. 1H NMR (300 MHz, CDCl3): 4.232-4.267 (m, 4H),
6.710-6.781 (m, 2H), 6.899 (s, 1H), 7.176-7.215 (m, 2H), 7.356-7.379 (m, 1H),
7.507-7.587 (m, 1H), 7.733-7.789 (m, 2H). 13C NMR (400 MHz, CDCl3) δ: 60.88,
61.25, 108.83, 112.49, 117.14, 122.76, 125.24, 128.61, 128.98, 130.07, 131.13,
132.61, 135.19, 136.82, 150.41, 151.66, 164.53, 166.83. MS (ESI): 403
(C18H16BrN2O4, [M+H]+). Anal. Calcd for C18H15BrN2O4: C, 53.62; H, 3.75; N,
6.95%. Found: C, 53.81; H, 3.66; N, 7.03%.
4.1.5.10
(E)-N'-(3-(m-tolyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide
(7j)
1
White powder, mp: 251-253oC. H NMR (300 MHz, CDCl3): 1.789 (s, 3H),
4.322-4.373 (m, 4H), 6.659 (s, 1H), 6.872-6.890 (d, J = 5.4 Hz, 1H), 7.005 (s, 1H),
7.230-7.261 (m, 2H), 7.572-7.628 (m, 3H), 7.775-7.798 (m, 1H), 9.311 (s, 1H), 9.556
(s, 1H). 13C NMR (400 MHz, CDCl3) δ: 21.15, 60.26, 61.94, 110.34, 112.37, 116.38,
120.39, 121.93, 124.21, 126.83, 128.27, 129.46, 135.20, 136.74, 139.49, 151.44,
152.01, 163.75, 165.96. MS (ESI): 339 (C19H19N2O4, [M+H]+). Anal. Calcd for
C19H18N2O4: C, 67.44; H, 5.36; N, 8.28%. Found: C, 67.58; H, 5.43; N, 8.19%.
4.1.5.11
(E)-N'-(3-(3-methoxyphenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboh
ydrazide (7k)
White powder, mp: 171-172oC. 1H NMR (300 MHz, CDCl3): 1.275-1.384(m, 3H),
11

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4.277-4.296 (m, 4H), 6.679 (s, 1H), 6.731 (s, 1H), 6.866-6.916 (m, 3H), 7.211-7.238
(m, 2H), 7.637 (s, 1H), 7.741 (s, 1H), 9.158 (s, 1H), 9.613 (s, 1H). 13C NMR (400
MHz, CDCl3) δ: 54.21, 61.36, 62.22, 108.06, 110.43, 113.13, 115.28, 117.08, 120.21,
122.82, 128.21, 131.33, 136.73, 138.47, 150.41, 151.37, 159.38, 163.51, 165.93. MS
(ESI): 355 (C19H19N2O5, [M+H]+). Anal. Calcd for C19H18N2O5: C, 64.40; H, 5.12; N,
7.91%. Found: C, 64.56; H, 5.09; N, 8.02%.
4.1.5.12
(E)-N'-(3-(3-nitrophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydr
azide (7l)
White powder, mp: 155-156oC. 1H NMR (300 MHz, CDCl3): 4.132-4.157 (m, 4H),
6.829-6.890 (d, J = 18.3 Hz, 1H), 6.910-6.992 (m, 2H), 6.999 (s, 1H), 7.310-7.351 (m,
3H), 7.662-7.696 (m, 2H), 9.567 (s, 1H), 9.738 (s, 1H). 13C NMR (400 MHz, CDCl3)
δ: 61.20, 62.75, 109.56, 112.44, 117.85, 120.61, 121.72, 123.83, 129.05, 131.04,
134.47, 138.93, 139.51, 151.06, 151.49, 151.88, 162.04, 165.09. MS (ESI): 370
(C18H16N3O6, [M+H]+). Anal. Calcd for C18H15N3O6: C, 58.54; H, 4.09; N, 11.38%.
Found: C, 58.67; H, 3.98; N, 11.43%.
4.1.5.13
(E)-N'-(3-(4-fluorophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7m)
White powder, mp: 202-203oC. 1H NMR (300 MHz, CDCl3): 4.032-4.102 (m, 4H),
6.992-7.021 (m, 1H), 7.275-7.338 (m, 2H), 7.635-7.716 (m, 3H), 7.750-7.798 (m, 3H),
9.223 (s, 1H), 9.351 (s, 1H). 13C NMR (400 MHz, CDCl3) δ: MS (ESI): 343
(C18H16FN2O4, [M+H]+). Anal. Calcd for C18H15FN2O4: C, 63.15; H, 4.42; N, 8.18%.
Found: C, 63.08; H, 4.55; N, 8.20%.
4.1.5.14
(E)-N'-(3-(4-chlorophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7n)
White powder, mp: 251-252oC. 1H NMR (300 MHz, CDCl3): 4.321-4.371 (m, 4H),
6.892-6.910 (d, J = 5.4 Hz, 1H), 7.103-7.125 (m, 3H), 7.335 (s, 1H), 7.510-7.571 (m,
12

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2H), 7.627-7.688 (m, 2H), 9.350 (s, 1H), 9.673 (s, 1H). 13C NMR (400 MHz, CDCl3)
δ: 61.83, 62.35, 107.92, 112.44, 118.18, 120.83, 126.34, 127.06, 129.08, 130.71,
130.94, 133.73, 134.62, 137.56, 151.08, 151.92, 164.83, 167.37. MS (ESI): 359
(C18H16ClN2O4, [M+H]+). Anal. Calcd for C18H15ClN2O4: C, 60.26; H, 4.21; N,
7.81%. Found: C, 60.33; H, 4.29; N, 7.66%.
4.1.5.15
(E)-N'-(3-(4-bromophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohyd
razide (7o)
White powder, mp: 237-238oC. 1H NMR (300 MHz, CDCl3): 4.155-4.172 (m, 4H),
6.679 (s, 1H), 6.870-6.911 (m, 2H), 7.094-7.150 (d, J = 16.8 Hz, 1H), 7.274-7.281 (m,
2H), 7.456-7.493 (m, 2H), 7.589-7.630 (d, J = 12.3 Hz, 1H), 9.56 (s, 1H), 9.73 (s, 1H).
13C NMR (400 MHz, CDCl3) δ: 61.58, 62.43, 108.06, 110.67, 118.32, 122.33, 124.65,
129.21 129.54, 129.95, 132.62, 133.78, 134.53, 138.48, 151.73, 151.92, 165.04,
165.92. MS (ESI): 403 (C18H16BrN2O4, [M+H]+). Anal. Calcd for C18H15BrN2O4: C,
53.62; H, 3.75; N, 6.95%. Found: C, 53.55; H, 3.67; N, 7.11%.
4.1.5.16
(E)-N'-(3-(p-tolyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide
(7p)
White powder, mp: 253-255oC. 1H NMR (300 MHz, CDCl3): 1.660 (s, 3H), 3.996 (s,
4H), 6.448-6.501 (d, J = 15.9 Hz, 1H), 6.567-6.595 (d, J = 8.4 Hz, 1H), 6.984-7.013
(m, 2H), 7.090-7.144 (m, 1H), 7.144-7.172 (d, J = 8.4 Hz, 1H), 7.208 (s, 1H),
7.330-7.354 (m, 1H), 7.678-7.731 (d, J = 15.9 Hz, 1H), 9.763 (s, 1H), 9.964 (s, 1H).
13C NMR (400 MHz, CDCl3) δ: 22.03, 62.28, 62.83, 107.06, 111.43, 118.14, 122.54,
127.50, 128.06, 128.86, 129.03, 130.11, 133.72, 136.17, 137.92, 149.28, 151.08,
163.51, 167.55. MS (ESI): 339 (C19H19N2O4, [M+H]+). Anal. Calcd for C19H18N2O4:
C, 67.44; H, 5.36; N, 8.28%. Found: C, 67.56; H, 5.28; N, 8.19%.
4.1.5.17
(E)-N'-(3-(4-methoxyphenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboh
ydrazide (7q)
13

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1
White powder, mp: 223-225oC. H NMR (300 MHz, CDCl3): 1.275 (s, 3H),
4.312-4.324 (m, 4H), 6.693-6.747 (d, J = 16.2 Hz, 1H), 6.929-6.957 (m, 1H),
7.090-7.197 (m, 2H), 7.380-7.410 (m, 2H), 7.440 (s, 1H), 7.551-7.560 (m, 1H),
13
7.624-7.649 (m, 1H), 9.245 (s, 1H), 9.387 (s, 1H). C NMR (400 MHz, CDCl3) δ:
55.25, 61.28, 61.28, 108.96, 111.46, 114.15, 114.15, 127.48, 117.04, 121.02, 126.73,
127.48, 128.13, 136.15, 150.07, 150.99, 161.67, 163.17, 165.93. MS (ESI): 355
(C19H19N2O5, [M+H]+). Anal. Calcd for C19H18N2O5: C, 64.40; H, 5.12; N, 7.91%.
Found: C, 64.29; H, 5.32; N, 7.81%.
4.1.5.18
(E)-N'-(3-(4-nitrophenyl)acryloyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydr
azide (7r)
White powder, mp: 199-200oC. 1H NMR (300 MHz, CDCl3): 4.342-4.387 (m, 4H),
6.894-6.912 (d, J = 5.4 Hz, 1H), 6.993 (s, 1H), 7.150-7.181 (m, 2H), 7.277-7.289 (m,
2H), 7.385-7.416 (m, 1H), 7.672-7.726 (m, 2H), 9.254 (s, 1H), 9.478 (s, 1H). 13C
NMR (400 MHz, CDCl3) δ: 62.11, 62.76, 110.06, 112.33, 116.94, 122.92, 126.05,
126.83, 127.11, 127.85, 129.62, 138.15, 140.58, 148.73, 150.18, 151.09, 164.77,
167.32. MS (ESI): 370 (C18H16N3O6, [M+H]+). Anal. Calcd for C18H15N3O6: C, 58.54;
H, 4.09; N, 11.38%. Found: C, 58.72; H, 4.15; N, 11.19%.
4.1.5.19
(E)-N'-cinnamoyl-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (7s)
1
White powder, mp: 220-221oC. H NMR (300 MHz, CDCl3): 2.660 (s, 3H),
6.538-6.589 (d, J = 18.3 Hz, 1H), 6.939-6.967 (m, 1H), 7.403-7.443 (m, 4H), 7.538 (s,
1H), 7.541-7.626 (m, 2H), 7.741-7.793 (d, J = 15.6 Hz, 1H), 9.130 (m, 2H). 13C NMR
(400 MHz, CDCl3) δ: 60.15, 61.22, 107.36, 112.14, 118.35, 122.73, 126.58, 127.08,
127.22, 128.03, 129.55, 130.24, 136.18, 139.06, 150.01, 151.66, 163.96, 165.92. MS
(ESI): 325 (C18H17N2O4, [M+H]+). Anal. Calcd for C18H16N2O4: C, 66.66; H, 4.97; N,
8.64%. Found: C, 66.81; H, 4.73; N, 8.52%.
4.2 Cytotoxicity test
The cytotoxic activity in vitro was measured using the MTT assay. Cells were
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cultured in a 96-well plate at a density of 5
*
105 cells and different concentrations of
compounds were respectively added to each well. The incubation was permitted at 37
oC, 5% CO2 at mosphere for 24 h before the cytotoxicity assessments. 20 ꢀL MTT
reagent (4 mg/mL) was added per well 4h before the end of the incubation. Four hours
later, the plate was centrifugaled at 1200 rcf for 5 min and the supernatants were
removed, each wellwas added with 200 ꢀL DMSO. The absorbance was measured at a
wave length of 570 nm (OD570 nm) on an ELISA microplate reader. Three replicate
wells were used for each concentration and each assay was measured three times,
after which the average of CC50 and SD were calculated. The cytotoxicity of each
compound was expressed as the concentration of compound that reduced cell viability
to 50% (CC50). The results were summarized in Table 1.
4.3 Cell proliferation assay
The antitumor activities of compounds 7a-7q were determined using a standard
(MTT)-based colorimetric assay (Sigma). Seed 104 cells per well into 96-well plates,
incubate at 37ꢁ, 5% CO2 for 24 hours. Then add 100 ꢀL a series concentration of
drug-containing medium into wells to maintain the final concentration of drug as 40,
20, 6.67, 2.22, 0.74, 0.25 and 0.082 ꢀg/mL. One concentration should be triplicated.
After 48 h, cell survival was determined by the addition of an MTT solution (25 ꢀL of
5 mg/mL MTT in PBS). After 4h, discard the medium and add 100 ꢀL DMSO; the
plates were votexed for 10 minutes to make completely dissolution. Optical
absorbance was measured at 570 nm.
4.4 PLK1 enzyme assay
Purified PLK1 protein was incubated with 150 µM of a substrate peptide
(RRRDELMEASFADQEAKV), 10 µM ATP, and 2.0 µCi [γ-33P]-ATP (GE
Healthcare) in reaction buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EGTA,
0.5 mM DTT) at 25°C for 20 min in 96 well plates containing serially diluted
compound-DMSO solutions. As the reaction was terminated by the addition of 15 µL
of 41.7-fold diluted H3PO4 solution, the ASFA peptide (DELMEASFADQEAKV) was
trapped on 96 well MultiScreen-PH plate (Millipore) and each well was washed with
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133-fold diluted H3PO4 solution. The radioactivity of each well was monitored with a
liquid scintillation counter (TOPCOUNT HTS, PerkinElmer (Packard)). The IC50
values were calculated using Microsoft Excel. The IC50 values are the means of at
least 2 or more assays.
4.5 Experimental protocol of docking study.
The pdb file about the crystal structure of the PLK1 (1umw.pdb) was obtained from
the RCSB protein data bank (http://www.pdb.org). The molecular docking procedure
was performed by using CDOCKER protocol for receptor-ligand interactions section
of Discovery Studio 3.1 (Accelrys Software Inc, San Diego, CA). Initially both the
ligands and the receptor were pretreated. For ligand preparation, the 3D structures of
all our synthesized compounds were generated with ChemBioOffice 2010 and
optimized with MMFF94 method. For enzyme preparation, the hydrogen atoms were
added with the pH of the protein in the range of 7.25-7.65.
Acknowledgment
The work was financed by a grant (No. J1103512) from National Natural Science
Foundation of China and a grant (No. CXLX13_038) from Jiangsu Ordinary
University Graduate Students Scientific Research Innovation Plan of China.
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Figure Captions
Table 1. Cytotoxicity assay of the compounds on macrophages cells and cell
proliferation assay on cervical cancer cells.
Table 2. PLK1 inhibitory activity of the selected compounds.
Table 3. The docking calculation of the synthesized compounds (7a-7s).
Figure 1. The general formula of Chinese patent and our design solution of aromatic
diacylhydrazine derivatives.
Figure 2. 2D Ligand interaction diagram of compound 7k with PLK1. For clarity,
only interacting residues are displayed.
Figure 3. 3D model of the interaction between compound 7k and PLK1 binding site.
Scheme 1. Synthesis of compounds 3, 5a-5q and 7a-7s.
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Table 1. Cytotoxicity assay of the compounds on macrophages cells and cell
proliferation assay on cervical cancer cells
compound
7a
R
CC50 ±SD (ꢀM)
302.00±20.12
285.27±18.00
315.17±31.11
267.82±18.11
312.00±21.12
257.45±11.95
221.25±12.11
391.23±19.76
351.22±11.09
280.98±13.02
398.23±22.01
323.15±21.23
319.13±12.45
308.78±21.20
259.22±18.36
332.18±24.12
319.22±12.36
295.26±21.08
312.00±21.12
IC50 (ꢀM)
4.51
4.88
2.11
7.71
8.06
0.89
4.11
3.56
3.33
2.89
0.17
1.56
0.99
1.13
2.33
1.05
0.93
0.61
0.55
2-F
2-Cl
2-Br
7b
7c
2-CH3
2-CH3O
2-NO2
3-F
7d
7e
7f
7g
3-Cl
7h
7i
3-Br
3-CH3
3-CH3O
3-NO2
4-F
7j
7k
7l
7m
7n
7o
4-Cl
4-Br
4-CH3
4-CH3O
4-NO2
_
7p
7q
7r
7s
20

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Table 2. PLK1 inhibitory activity of the selected compounds.
Compound
PLK1 (IC50, ꢀM)
0.19
0.03
1.55
0.59
1.55
1.05
0.19
0.12
0.09
2.31
7f
7k
7l
7m
7n
7p
7q
7r
7s
poloxin
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Table 3. The docking calculation of the synthesized compounds (7a-7s).
Compound
-CDOCKER_ENERGY
30.4561
29.5392
34.6617
28.6713
25.8179
37.1536
31.8337
31.9411
7a
7b
7c
7d
7e
7f
7g
7h
7i
33.1001
33.6739
37.7233
35.0481
36.2040
35.1197
7j
7k
7l
7m
7n
7o
3p
7q
7r
7s
34.1645
35.7668
36.1167
37.1974
37.3372
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Figure 1. The general formula of Chinese patent and our design solution of aromatic
diacylhydrazine derivatives (R means substituted benzene group).
23

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Figure 2. 2D Ligand interaction diagram of compound 7k (A) and poloxin (B) with
PLK1. For clarity, only interacting residues are displayed.
(A)
24

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(B)
25

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Figure 3. 3D model of the interaction between compound 7k and PLK1 binding site.
26

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Scheme 1. Synthesis of compounds 3, 5a-5q and 7a-7s. Reagents and conditions. (a)
methanol, concentrated sulfuric acid, 90ꢁ, 8 h; (b) dibromoethane, potassium
carbonate, acetone, 70ꢁ, 12 h; (d) pyridine, piperidine, 85ꢁ, 24 h; (f) hydrazine
hydrate, ethanol, 90ꢁ, 4 h; (g) EDCꢀHCl, HoBt, rt, 8-10 h.
O
O
OH
HO
HO
O
O
a
O
b
O
HOOC
OH
R2
2
1
3
R3
R1
CHO
d
COOH
R
5a-5q
4
O
O
O
O
NH2
O
O
O
N
H
f
6
3
R3
R2
O
R
H
N
O
O
N
COOH
H
g
O
R1
7a-7s
27