Identification of nitroimidazole-oxime derivatives targeting the polo-box domain of polo-like kinase 1

Article abstract of DOI:10.1016/j.bmc.2017.10.035

Recent progress in the development of small molecular skeleton-derived polo-like kinase (PLK1) catalytic domain (K_D) inhibitors has led to the synthesis of multiple ligands with high binding affinity. However, few systematic analyses have been

Full text of DOI:10.1016/j.bmc.2017.10.035

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of nitroimidazole-oxime derivatives targeting
the polo-box domain of polo-like kinase 1
a
c
b,
Juan Sun a,b, , Han-Yu Liu , Ruo-Fei Xu , Hai-Liang Zhu
⇑
⇑
a School of Life Sciences, Shandong University of Technology, Zibo 255049, PR China
b Elion Nature Biological Technology Co., Ltd, Nanjing 210046, PR China
c Shandong Experimental High School, Jinan 250001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent progress in the development of small molecular skeleton-derived polo-like kinase (PLK1) catalytic
domain (KD) inhibitors has led to the synthesis of multiple ligands with high binding affinity. However,
few systematic analyses have been conducted to identify key PLK1-PBD domain and characterize their
interactions with potent PLK1 inhibitors. Therefore, we designed a series of PLK1-PBD inhibitors with
an in silico scaffold modification strategy. A docking simulation combined with a primary screen
in vitro were performed to filter for the lead compound, which was then substituted, synthesized and
evaluated by a variety of bioassays. The biological profile of 4v suggests that this compound may be
developed as a potential anticancer agent.
Received 18 July 2017
Revised 15 October 2017
Accepted 25 October 2017
Available online xxxx
Keywords:
Nitroimidazole-oxime
PLK1-PBD
Ó 2017 Elsevier Ltd. All rights reserved.
Antitumor
Molecular docking
1. Introduction
nately, the limited number of PLK1-PBD inhibitors and their mod-
est selectivity greatly limit the design based on the structure.
Polo-like kinase 1 (PLK1), a key player in mitosis,1,2 modulates
the transition through the G2/M checkpoint, is crucial in cell pro-
liferation and has been considered as a target for tumor therapy.3,4
Until now, two distinct drug targets have been identified in PLK1: a
N-terminal catalytic domain (KD) and a C-terminal domain having
2 highly homologous sequences, termed polo-box domain (PBD).
For a long period, discovery of PLK1 inhibitors has been focused
on targeting the N-terminal catalytic domain.5,6 However, protein
kinases have high similarities in the ATP-binding pockets,7 and
these efforts suffered from a lack of specificity. An alternative
approach with potential for identifying potent and highly selective
kinase inhibitors is to target the interfaces of protein-protein com-
plexes of interest.8 Therefore, the special structure of the PBD has
become an highly selective target.9,10
Several series of peptides were designed and synthesized tar-
geting PLK1-PBD with high affinity and specificity.11–16 However,
inadequate proteolytic resistance and cell permeability of the pep-
tides hinder the development of these peptide-based inhibitors
into novel therapeutic compounds. In order to overcome the short-
comings of peptide-based inhibitors, some small molecules have
been designed and synthesized targeting PLK1-PBD.17–22 Unfortu-
Therefore, clarifying the binding mode between PBD and small
molecules, as well as improving their specificity recognition, is
the key issues to be resolved.
To continue our efforts aimed at discovering and developing
PLK1-PBD inhibitors23, in this study, we used a structure-based
approach to design a novel and potent small molecule named 4v,
which exhibits a high binding affinity for PLK1-PBD.
2. Results and discussion
2.1. Structure-based design of small molecules
To identify the minimal set of key residues required for a high
binding affinity, we examined the previously reported cocrystal
structures of PLK1-PBD24 (PDB ID: 4HCO) in complex with a ligand.
Then we have found that three binding pockets are critical to bind
PLK1-PBD with high affinity, combined with our previous
research23: (1) Electrostatic-binding region (EBR) in which the
Lys540-His538 pincer clinches phosphopeptides by the phosphate.
(2) Hydrogen-bonding region (HBR) is surrounded by Trp414,
Phe535 and Arg516 residues through participating in a hydrogen
bonding interactions. (3) Hydrophobic motif (HM) formed by
Val415, Tyr417, Tyr485 and Leu490 through hydrophobic interac-
tions with the above residues (Fig. 1). These observations suggest
⇑
Corresponding authors at: School of Life Sciences, Shandong University of
Technology, Zibo 255049, PR China (J. Sun).
E-mail address: sunreiyi86@163.com (J. Sun).
https://doi.org/10.1016/j.bmc.2017.10.035
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

2
J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
In vitro anticancer activities against four human tumor cell lines.
Compounds
IC50 SD (
MGC-803
>20
lg/mL)
HepG-2
MCF-7
Hela
3
17.39 0.12
1.22 0.08
0.96 0.02
1.34 0.05
1.25 0.07
1.43 0.09
2.56 0.04
0.97 0.02
1.15 0.03
0.86 0.05
4.38 0.01
6.59 0.5
10.65 0.14
1.33 0.05
2.37 0.02
13.96 0.28
>20
0.76 0.03
0.71 0.02
0.92 0.03
0.73 0.01
0.81 0.02
0.75 0.01
2.24 0.07
>20
>20
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
1.24 0.05
0.81 0.03
0.45 0.02
0.34 0.04
0.37 0.02
1.48 0.06
0.21 0.03
0.18 0.02
0.04 0.01
2.63 0.87
2.54 0.04
3.25 0.02
0.16 0.07
0.84 0.06
3.66 0.05
3.53 0.04
0.73 0.05
0.82 0.03
0.03 0.01
0.01 0.01
0.02 0.01
2.12 0.03
1.54 0.05
2.79 0.07
3.15 0.02
0.86 0.03
2.56 0.05
1.87 0.03
0.84 0.06
1.37 0.02
3.79 0.09
7.13 0.04
2.82 0.03
1.27 0.08
2.15 0.06
>20
3.13 0.07
0.76 0.04
0.79 0.02
0.89 0.02
0.78 0.03
0.87 0.01
0.72 0.02
2.19 0.03
7.24 0.06
0.96 0.02
8.79 0.03
6.54 0.04
12.41 0.19
5.67 0.06
4.55 0.04
4.09 0.04
2.57 0.08
>20
11.35 0.07
12.35 0.09
8.53 0.06
9.76 0.05
>20
(A)
(Eur. J. Med. Chem. 2014, 81: 420-426)
(B)
(C)
Fig. 1. Three binding pockets are critical to bind PLK1-PBD with high affinity: (A)
2D figure. (B) 3D figure. (C) 3D figure.
>20
1.28 0.01
0.96 0.02
2.35 0.04
1.51 0.05
1.63 0.02
0.86 0.03
3.82 0.08
4s
4t
4u
4v
5-FU
that appropriate design of small molecules would provided drug-
like small molecule targeting PLK1-PBD with high binding affinity.
According to our previous research and the three binding pock-
ets, we have selected benzene ring and pyridine ring as the basic
scaffold. At the same time, a variety of substituents have been
introduced, aim to investigate the position effect, space effect, elec-
tron effect, hydrophilicity and hydrophobicity against to the bio-
logical activity of the target compounds. Meanwhile,
nitroimidazole-oxime skeleton has a good affinity with Lys540-
His538 pincer clinches in the EBR region according to the Docking
simulations. Therefore, in this research, we presented a nitroimida-
zole-oxime as our scaffold which provides rigidity around the
central unit.
0.005 0.003
1.8 0.02
2.3. MTT assay for cell proliferation
To test the antiproliferative activities of the synthesized com-
pounds, the target compounds were evaluated in vitro antiprolifer-
ation assays against four human cancer cell lines (MCF7, HeLa,
MGC803 and HepG2). The results were summarized in Table 1.
With few exception, the active analogs showed a remarkable
potential antiproliferative activity against MGC803 (Fig. 2), sug-
gesting that nitroimidazole-oxime derivatives could significantly
enhance antiproliferative potency. For the given compounds, it
was observed that compound 4v showed the most potent antipro-
2.2. Chemistry
liferative activity (IC50 = 0.002 lg/mL).
Twenty-two nitroimidazole-oxime analogs were synthesized
and all of them were synthesized for the first time. The synthesis
of compounds has been followed the general pathway outlined in
Scheme 1. All of the synthetic compounds 4a–4v are being
reported for the first time and gave satisfactory analytical and
spectroscopic data.
Structure–activity relationship (SAR) analysis indicated that dif-
ferent acids substitutes led to different antitumor activity, and the
potency order was niacin > benzoic acid. compounds 4q and 4r
with substituted Cl group on niacin ring showed moderate antitu-
mor activity with IC50 of 0.71–1.28 lg/mL against four human can-
cer cell lines. Based on the data obtained, compounds 4t and 4u
with niacin and Isonicotinic ring were found to be better activity
toward BGC823 cancer cell line with IC50 values, 0.01 and 0.02
lg/mL respectively.
2.4. Cytotoxicity test
Generally speaking, the inhibitory activity of the compounds is
due to cell apoptosis or toxic effect, so we performed cytotoxicity
test before detecting kinase activity. All of the compounds were
detected for their cytotoxicity on Human Kidney epithelia cells
(293T). The pharmacological results of these compounds were
summarized in Table 2. What we can see from the data is that most
of the compounds were low toxic.
2.5. PLK1-PBD inhibitory assay
To validate whether the above anti-proliferative effect was pro-
duced by interaction of PLK1 protein and the synthesized com-
pounds, the selected compounds were evaluated for their
abilities to inhibit the activity of PLK1 protein kinases relevant to
cancer. As expected, all compounds displayed the best inhibitory
Scheme 1. General synthesis of compounds 4a–4v. Reagents and conditions: (i)
2,40-dibromo-acetophenone, K2CO3, TBAB, ethanol, reflux, 0.5–1 h. (ii) Hydroxy-
lamine hydrochloride, K2CO3, ethanol, reflux, 2–4 h. (iii) Substituted benzoic acid,
niacin or isonicotinic acid, EDCꢀHCl, HoBt, rt, 8–10 h.
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Fig. 2. The inhibition rates of synthesized compounds on MGC803.
Table 2
PBD domain of PLK1 by three hydrogen bonds with Lys540 and Arg
557, one Pi-cation bond with Lys540, and one Pi-Pi bond with Trp
414. Nitroimidazole group has a good affinity with Lys540-His538
pincer clinches in the EBR region, and 6-methoxypyridine group is
perfectly fit in the HM region. Furthermore, other weak interac-
tions, such as Vander Waals and carbon-hydrogen bonds, also con-
tributed to the binding affinity of 4v with PLK1. In Fig. 5(B), 3D
models are depicted. The molecular docking suggests that com-
pound 4v maybe a potential PLK1-PBD ligand.
Cell proliferation assay on 293T and kinase inhibitory activities (mg/ml).
Compounds
CC50
PLK1-PBD
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
2.3 ꢁ 104
2.7 ꢁ 102
2.3 ꢁ 102
1.6 ꢁ 103
1.6 ꢁ 102
3.3 ꢁ 102
5.3 ꢁ 102
7.0 ꢁ 102
1.8 ꢁ 102
1.5 ꢁ 102
2.3 ꢁ 102
1.3 ꢁ 103
5.3 ꢁ 102
3.8 ꢁ 103
3.2 ꢁ 104
5.0 ꢁ 102
1.2 ꢁ 104
2.5 ꢁ 102
9.8 ꢁ 102
0.5 ꢁ 102
0.8 ꢁ 102
8.3 ꢁ 103
1.4 ꢁ 102
0.69 0.12
0.23 0.03
0.17 0.11
0.22 0.02
0.1 0.02
0.88 0.31
0.11 0.01
0.13 0.02
0.18 0.02
1.66 0.22
1.95 0.31
2.88 0.01
0.33 0.07
0.25 0.01
3.15 0.40
2.93 0.22
0.25 0.09
0.28 0.03
0.05 0.02
0.03 0.01
0.06 0.02
0.002 0.02
2.19 0.03
2.8. Single crystal X-ray diffraction
Crystals of compound 4 t (CCDC 1487468) were obtained from
methanol solution. Fig. 6A shows a perspective view of the mono-
meric unit with the atomic numbering scheme, and Fig. 6B depicts
the intramolecular and intermolecular hydrogen bonds. Crystallo-
graphic data, details of data collection and structure refinement
parameters are listed in Table 3. The hydrogen bond lengths and
bond angles are given in Table 4.
4q
4r
4s
4t
4u
4v
3. Conclusions
Thymoquinone
In this study, a series of nitroimidazole-oxime derivatives were
designed as new PLK1-PBD inhibitors based on the computer-aided
drug design. These compounds were tested in a series of experi-
ments and the results indicated that some compounds may be
promising in drug development. Compound 4v is the most impres-
sive, with a notable biological profile. Against PLK1-PBD and the
MGC803 cancer cell line, 4v demonstrated better inhibitory
potency than poloxin. Assisted by apoptosis of MGC803 cell, 4v
could induce apoptosis in a dose-dependent manner. We hope that
this study will benefit the study of cancer treatment through PLK1-
PBD inhibition.
activity for PLK1 and the results have been showed in Table 2.
Besides, a further study between the anti-proliferative activity
against MGC803 cell line and the PLK1 inhibitory activity of these
compounds was analyzed and the result indicated that there was a
moderate correlation between PLK1 inhibition and inhibition of
cancer cellular proliferation, as evidenced in Fig. 3. The correlation
coefficient r2 was found to be 0.691.
2.6. Annexin V-FITC/PI apoptosis detection
Besides, we detected the mechanism of compound 4v inhibition
activity by flow cytometry (FCM) (Fig. 4) and found that the com-
pound could induce the apoptosis of activated MGC803 cells in a
dose-dependent manner. As shown in Fig. 4, MGC803 cells were
4. Experimental results
4.1. Chemistry
treated with 0.01, 0.06 and 0.12 lM of compound 4v for 24 h.
The compound increased the percentage of apoptosis by Annexin
V-FITC/PI staining in a dose-dependent.
All chemicals (reagent grade) used were purchased from Alad-
din (Shanghai, China). All the 1H NMR spectra were recorded on
a Bruker DPX 400 model spectrometer in DMSO d6, and chemical
shifts (d) are reported as parts per million (ppm). ESI-MS spectra
were recorded by a Mariner System 5304 Mass spectrometer.
Melting points were determined on a Digital Melting Point appara-
tus (Shenguang., Shanghai, China). Thin layer chromatography
(TLC) was performed on silica gel plates (Silica Gel 60 GF254)
and visualized in UV light (254 nm and 365 nm). The PLK1 Kinase
Assay Kit was purchased from Bio-Swamp.
2.7. Docking simulations
Docking study was performed to fit compound 4v into the
active center of the polo-box domain of polo-like kinase 1 (PDB
code: 4HCO). The obtained results were presented in Fig. 5(A). In
the binding model, compound 4v is nicely bound to the active site
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

4
J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 3. Correlation between the anti-proliferative activity against MGC-803 and the PLK1-PBD inhibitory activity, which indicated that there was a moderate correlation
between PLK1-PBD inhibition and inhibition of cellular proliferation.
Fig. 4. MGC803 cells were cultured with anticancer and various concentrations of 4v for 24 h. Cells were stained by Annexin VeFITC/PI and apoptosis was analyzed by flow
cytometry. (A) Control. (B) 0.01 lM. (C) 0.06 lM. (D) 0.12 lM.
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 5. (A) 2D molecular docking model of compound 4v with 4HCO. (B) 3D interaction map between compound 4v and 4HCO binding site.
Fig. 6. Crystal structure of compound 4t. (A) Molecular structure of compound 4t with atomic numbering scheme. (B) Crystal packing of the compound 4t.
4.2. General procedure for the synthesis of compounds 4a–4v
4.2.3. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(4-chlorobenzoyl)oxime(4c)
Commercially available 2-methyl-4-nitroimidazole, potassium
carbonate and tetrabutylammonium bromide were reacted with
2,40-dibromo-acetophenone in ethanol to give compound 2. Com-
pound 2 was reacted with hydroxylamine hydrochloride in ethanol
to give compound 3. Then target compound 4a–4v were obtained
with proper substituted benzoic acid or niacin under the condition
of EDCI and HoBt in CH2Cl2.
White crystal, yield 87%, m.p. 170.4 °C. 1H NMR (400 MHz,
DMSO d6) d (ppm): 8.374 (s, 1H), 8.289–8.253 (m, 2H), 7.947–
7.918 (m, 1H), 7.620–7.537 (m, 4H), 7.254–7.183 (m, 1H), 5.638
Table 3
Crystallographic data, details of data collection and structure refinement parameters.
compound
4t
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C
18H14BrN5O4
4.2.1. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(4-fluorobenzoyl)oxime (4a)
444.24
Orthorhombic
Pbca (No. 61)
10.6734(8)
15.7179(13)
22.2989(16)
90
90
90
3740.9(5)
8
White crystal, yield 92%, m.p. 171.4–172.2 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.194 (s, 1H), 7.712–7.690 (m, 1H),
7.649–7.558 (m, 5H), 7.259–7.101 (m, 1H), 6.393–6.372 (m, 1H),
5.417 (s, 2H), 2.510–2.502 (m, 3H). MS (ESI): 463.02 (C19H15BrFN4-
O4, [M+H]+). Anal. Calcd: C, 49.48; H, 3.06; N, 12.15%; Found: C,
49.42; H, 3.11; N, 12.05%.
b (Å)
c (Å)
a
(°)
b(°)
c
(°)
V (Å3)
Z
F(0 0 0)
1792
1.578
2.233
1.07
4.2.2. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(4-methylbenzoyl)oxime (4b)
Dx(g/cm3)
Mu(MoKa) [/mm]
GOOF
White crystal, yield 84%, m.p. 171.4–171.5 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 7.419 (s, 1H), 7.276 (s, 5H), 7.243–7.146
(m, 3H), 5.163 (s, 2H), 2.764–2.735 (m, 2H), 2.578–2.565 (m, 1H),
2.329–2.247 (m, 3H). MS (ESI): 457.04 (C20H18BrN4O4, [M+H]+).
Anal. Calcd: C, 52.53; H, 3.75; N, 12.25%; Found: C, 52.19; H,
3.14; N, 12.09%.
Rint
0.0596
R1; wR2 [I > 2
r(I)]
0.0833, 0.2321
0.1313, 0.2633
2.5–25.1
273
R1; wR2 (all data)
h range (°)
T (K)
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

6
J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 4
Hydrogen bond lengths (Å) and bond angles (°) of compound 4t.
H-bond type
Symmetry code
D–H. . .A
d(D–H)
d(H. . .A)
d(D. . .A)
\DHA
Inter
1 ꢂ x, 1 ꢂ y, 2 ꢂ z
1/2 ꢂ x, ꢂ1/2 + y, z
1 + x, y, z
1/2 + x, 1/2 ꢂ y, 2 ꢂ z
C4–H4C. . .O4
C5–H5A. . .O4
C5–H5B. . .O2
C12–H12. . .N2
0.96
0.97
0.97
0.93
2.60
2.57
2.37
2.58
3.469(8)
3.431(9)
3.176(8)
3.337(8)
151
148
140
138
Intra
–
C5–H5B. . .O3
0.97
2.23
2.599(7)
101
(s, 2H), 2.449–2.351 (s, 3H). MS (ESI): 478.99 (C19H15BrClN4O4, [M
+H]+). Anal. Calcd: C, 47.77; H, 2.95; N, 11.73%; Found: C, 47.18; H,
3.06; N, 11.68%.
2.502 (m, 3H). MS (ESI): 522.94 (C19H15Br2N4O4, [M+H]+). Anal.
Calcd: C, 43.70; H, 2.70; N, 10.73%; Found: C, 43.59; H, 2.79; N,
10.65%.
4.2.4. 1-(4-bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(4-methoxy-benzoyl)oxime (4d)
4.2.10. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(2-bromo-benzoyl)oxime(4j)
White crystal, yield 85%, m.p. 172.0–172.7 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.357–8.302 (m, 1H), 8.117–8.088 (d, J
= 11.6 Hz, 1H), 7.797–7.590 (m, 5H), 7.124–7.017 (m, 2H), 5.824
(s, 2H), 3.880–3.814 (m, 3H), 2.412–2.249 (m, 3H). MS (ESI):
475.04 (C20H18BrN4O5, [M+H]+). Anal. Calcd: C, 50.76; H, 3.62; N,
11.84%; Found: C, 50.69; H, 3.57; N, 11.59%.
White crystal, yield 92%, m.p. 170.7 °C. 1H NMR (400 MHz,
DMSO d6) d (ppm): 8.289 (s, 1H), 7.936–7.905 (m, 1H), 7.853–
7.822 (m, 1H), 7.717–7.688 (m, 2H), 7.635–7.558 (m, 4H), 5.781
(s, 2H), 2.251–2.210 (m, 3H). MS (ESI): 522.94 (C19H15Br2N4O4,
[M+H]+). Anal. Calcd: C, 43.70; H, 2.70; N, 10.73%; Found: C,
43.59; H, 2.61; N, 10.84%.
4.2.5. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(4-nitro-benzoyl)oxime (4e)
4.2.11. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(2-nitro-benzoyl)oxime(4k)
Yellow crystal, yield 94%, m.p. 171.1–171.6 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.410 (s, 1H), 8.374–8.356 (m, 1H),
8.274–8.231 (m, 2H), 7.711–7.431 (m, 5H), 5.871–5.844 (m, 2H),
2.511–2.502 (m, 3H). MS (ESI): 488.01 (C19H15BrN5O6, [M+H]+).
Anal. Calcd: C, 46.74; H, 2.89; N, 14.34%; Found: C, 46.82; H,
2.83; N, 14.29%.
Yellow crystal, yield 93%, m.p. 158.5–158.6 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.763 (s, 1H), 8.572–8.534 (m, 1H),
8.375–8.341 (m, 3H), 7.951–7.894 (m, 4H), 5.843–5.823 (m, 2H),
2.591–2.578 (m, 3H). MS (ESI): 488.01 (C19H15BrN5O6, [M+H]+).
Anal. Calcd: C, 46.74; H, 2.89; N, 14.34%; Found: C, 46.59; H,
3.01; N, 14.42%.
4.2.6. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(4-bromo-benzoyl)oxime (4f)
4.2.12. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(3-methylbenzoyl)oxime(4l)
White crystal, yield 92%, m.p. 171.0–171.3 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.137–8.105 (m, 2H), 7.976–7.939 (m,
1H), 7.759–7.743 (m, 2H), 7.626–7.558 (m, 4H), 5.833 (s, 2H),
2.451–2.419 (m, 3H). MS (ESI): 522.94 (C19H15Br2N4O4, [M+H]+).
Anal. Calcd: C, 43.70; H, 2.70; N, 10.73%; Found: C, 43.61; H,
2.81; N, 10.65%.
White crystal, yield 90%, m.p. 159.5–159.6 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.580–8.558 (m, 1H), 8.423–8.407 (m,
3H), 8.098–8.078 (m, 3H), 7.634–7.612 (m, 2H), 5.896 (s, 2H),
3.353–3.330 (m, 3H), 2.260 (s, 3H). MS (ESI): 457.04 (C20H18BrN4-
O4, [M+H]+). Anal. Calcd: C, 52.53; H, 3.75; N, 12.25%; Found: C,
52.62; H, 3.65; N, 12.14%.
4.2.7. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(3-fluoro-benzoyl)oxime (4g)
4.2.13. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(2-chloro-benzoyl)oxime(4m)
White crystal, yield 91%, m.p. 171.7–172.1 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.584 (s, 1H), 8.359 (s, 1H), 8.213–7.992
(m, 1H), 7.719–7.658 (m, 4H), 7.396–7.349 (m, 2H), 5.687–5.647
(m, 2H), 2.519–2.428 (m, 3H). MS (ESI): 463.02 (C19H15BrFN4O4,
[M+H]+). Anal. Calcd: C, 49.48; H, 3.06; N, 12.15%; Found: C,
49.56; H, 3.11; N, 12.01%.
White crystal, yield 91%, m.p. 170.7–170.8 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.543 (s, 1H), 8.173–8.157 (m, 1H),
8.134–8.121 (m, 1H), 7.839–7.812 (m, 3H), 7.356–7.235 (m, 3H),
6.247 (s, 2H), 2.168–2.139 (m, 3H). MS (ESI): 478.99 (C19H15
-
BrClN4O4, [M+H]+). Anal. Calcd: C, 47.77; H, 2.95; N, 11.73%; Found:
C, 47.91; H, 3.08; N, 11.62%.
4.2.8. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(3-chloro-benzoyl)oxime(4h)
4.2.14. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(2-methylbenzoyl)oxime (4n)
White crystal, yield 95%, m.p. 170.7–170.8 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.381 (s, 1H), 8.132–8.093 (m, 2H),
7.849–7.819 (m, 1H), 7.710–7.667 (m, 2H), 7.641–7.589 (m, 3H),
5.871 (s, 2H), 2.247 (s, 3H). MS (ESI): 478.99 (C19H15BrClN4O4, [M
+H]+). Anal. Calcd: C, 47.77; H, 2.95; N, 11.73%; Found: C, 47.68;
H, 3.08; N, 11.59%.
White crystal, yield 92%, m.p. 171.0–171.5 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.359 (s, 1H), 7.713–7.692 (m, 2H),
7.613–7.567 (m, 4H), 7.439–7.383 (m, 2H), 5.763 (s, 2H), 2.569–
2.506 (m, 3H), 2.245–2.222 (m, 3H). MS (ESI): 457.04 (C20H18BrN4-
O4, [M+H]+). Anal. Calcd: C, 52.53; H, 3.75; N, 12.25%; Found: C,
52.65; H, 3.62; N, 12.31%.
4.2.9. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(3-bromo-benzoyl)oxime(4i)
4.2.15. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-benzoyloxime(4o)
White crystal, yield 92%, m.p. 172.7–172.8 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.412 (s, 1H), 8.272–8.264 (m, 1H),
8.272–8.152 (d, J = 8.0 Hz, 1H), 7.987–7.967 (d, J = 8.0 Hz, 1H),
7.712–7.690 (m, 2H), 7.612–7.559 (m, 3H), 5.885 (s, 2H), 2.511–
White crystal, yield 89%, m.p. 183.5–183.6 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.410 (s, 1H), 8.174–8.153 (m, 2H),
7.787–7.749 (m, 1H), 7.713–7.691 (m, 2H), 7.630–7.595 (m, 4H),
5.866 (s, 2H), 2.253 (s, 3H). MS (ESI): 445.03 (C19H16BrN4O4,
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
[M+H]+). Anal. Calcd: C, 51.48; H, 3.41; N, 12.64%; Found: C, 51.56;
H, 3.32; N, 12.72%.
Calcd: C, 49.80; H, 3.52; N, 15.28%; Found: C, 49.92; H, 3.47; N,
15.17%.
4.2.16. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(2-methoxy-benzoyl)oxime(4p)
4.3. Cell culture
White crystal, yield 85%, m.p. 156.9–157.2 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.319 (m, 2H), 8.194 (s, 1H), 7.714–
7.693 (m, 1H), 7.636–7.557 (m, 5H), 5.417 (s, 2H), 2.511–2.502
(m, 3H), 2.340 (s, 3H). MS (ESI): 475.04 (C20H18BrN4O5, [M+H]+).
Anal. Calcd: C, 50.76; H, 3.62; N, 11.84%; Found: C, 50.84; H,
3.51; N, 11.96%.
A human hepatoma cell line (HepG2), human gastric cell line
(MGC-803), carcinoma of cervix cell line (HeLa), human breast cell
line (MCF-7) and human kidney epithelial cell (293T) were pur-
chased from Nanjing Keygen Technology (Nanjing, China). Cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Hyclone) (High Glucose) with L-glutamine supplemented with
10% foetal bovine serum (FBS, BI), 100 U/mL penicillin and 100
mg/mL streptomycin (Hyclone), and incubated at 37 °C in a humid-
ified atmosphere containing 5% CO2.
4.2.17. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(2-chloro-nicotinoyl)oxime (4q)
White crystal, yield 87%, m.p. 185.5–185.6 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.761–8.732 (m, 1H), 8.166–8.153 (m,
2H), 7.839 (s, 1H), 7.636–7.625 (m, 2H), 7.589–7.576 (m, 2H),
4.4. Anti-proliferation assay
5.034–5.017 (m, 2H), 2.342–2.339 (m, 3H). MS (ESI): 479.98 (C18
H
Found: C, 45.28; H, 2.65; N, 14.72%.
-
14BrClN5O4, [M+H]+). Anal. Calcd: C, 45.16; H, 2.74; N, 14.63%;
The anticancer activities of the prepared compounds in vitro
have been evaluated against MGC-803, MCF-7, HeLa and HepG2
cell lines. Target tumor cells were cultured in DMEM medium sup-
plemented with 10% fetal bovine serum. After reaching a dilution
4.2.18. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(6-chloro-nicotinoyl)oxime (4r)
of 1 ꢁ 105 cells mLꢂ1 with the medium, 100
lL of the obtained cell
White crystal, yield 91%, m.p. 180.4–180.5 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 9.169–9.162 (m, 1H), 8.566–8.539 (m,
1H), 8.410 (s, 1H), 7.812–7.790 (m, 1H), 7.718–7.697 (m, 2H),
7.621–7.600 (m, 2H), 5.896 (s, 2H), 2.249 (s, 3H). MS (ESI):
479.98 (C18H14BrClN5O4, [M+H]+). Anal. Calcd: C, 45.16; H, 2.74;
N, 14.63%; Found: C, 45.30; H, 2.62; N, 14.74%.
suspension was added to each well of 96-well culture plates. Sub-
sequently, incubation was performed at 37 °C in 5% CO2 atmo-
sphere for 48 h before the cytotoxicity assessment. Tested
samples at preset concentrations were added to 6 wells with Erlo-
tinib being employed as a positive reference. After 72 h exposure
period, 25
l
L of PBS containing 2.5 mg mLꢂ1 of MTT was added
L DMSO
to each well. After 4 h, the medium was replaced by 150
l
to dissolve the purple formazan crystals produced. The absorbance
at 570 nm of each well was measured with an ELISA plate reader.
The data represented the mean of three independent experiments
in triplicate and were expressed as means SD. The IC50 value was
defined as the concentration at which 50% of the cells could
survive.
4.2.19. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(6-methoxy-nicotinoyl)oxime (4s)
White crystal, yield 92%, m.p. 176.7–176.9 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 9.030–9.025 (m, 1H), 8.399–8.365 (m,
1H), 7.707–7.685 (m, 2H), 7.616–7.579 (m, 3H), 7.013–7.014 (m,
1H), 5.875 (s, 1H), 5.417 (s, 1H), 2.510–2.502 (m, 3H), 2.251–
2.244 (m, 3H). MS (ESI): 474.03 (C19H17BrN5O5, [M+H]+). Anal.
Calcd: C, 48.12; H, 3.40; N, 14.77%; Found: C, 48.12; H, 3.40; N,
14.77%.
4.5. PLK1-PBD inhibitor screening assay
The ability of compounds 4a–4v to inhibitor PLK1-PBD was
determined using fluorescence polarization screening assay.25
4.2.20. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-nicotinoyl oxime (4t)
White crystal, yield 92%, m.p. 179.3–180.2 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.337–8.330 (m, 1H), 8.287–8.279 (m,
1H), 8.136 (m, 2H), 7.684–7.628 (m, 2H), 7.623–7.576 (m, 3H),
5.784 (s, 2H), 2.393–2.369 (m, 3H). MS (ESI): 444.02 (C18H15BrN5-
O4, [M+H]+). Anal. Calcd: C, 48.67; H, 3.18; N, 15.76%; Found: C,
48.67; H, 3.18; N, 15.76%.
4.6. Cell apoptosis assay
Approximately 105 cells/well were plated in a 24-well plate and
allowed to adhere. Subsequently, the medium was replaced with
fresh culture medium containing compound 4v at final concentra-
tions of 0, 0.01, 0.06 and 0.12 lM. Non-treated wells received an
equivalent volume of ethanol (<0.1%). After 24 h, cells were trypsi-
nized, washed in PBS and centrifuged at 2000 rpm for 5 min. The
4.2.21. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-isonicotinoyl oxime (4u)
pellet was resuspended in 500
lL staining solution (containing 5
White crystal, yield 95%, m.p. 166.3–166.6 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.295–8.272 (m, 1H), 8.046–8.023 (m,
3H), 7.932–7.729 (m, 1H), 7.715–7.698 (m, 3H), 7.663–7.657 (m,
1H), 5.349 (s, 2H), 2.385–2.369 (m, 1H), 2.147 (s, 2 H). MS (ESI):
444.02 (C18H15BrN5O4, [M+H]+). Anal. Calcd: C, 48.67; H, 3.18; N,
15.76%; Found: C, 48.74; H, 3.05; N, 15.63%.
l
L AnnexinV-FITC and 5 L PI in Binding Buffer), mixed gently
l
and incubated for 15 min at room temperature in dark. The sam-
ples were then analyzed by a FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA, USA).
4.7. Scaffold modification
4.2.22. 1-(4-Bromophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
ethanone-(6-methyl-nicotinoyl)oxime (4v)
Scaffold modification was achieved with the module Grow Scaf-
fold in Discovery Studio (version 3.5). Following the protocol, the
scaffold was docked into PLK1 (PDB code: 4HCO) binding site
beforehand. The nitroimidazole-oxime scaffold were then marked
as sites to be substituted. After calculation, modified molecules
were produced to generate a library of small molecules.
White crystal, yield 85%, m.p. 180.9–181.0 °C. 1H NMR (400
MHz, DMSO d6) d (ppm): 8.518 (s, 1H), 8.411–8.385 (m, 1H),
8.195 (s, 1H), 7.712–7.691 (m, 1H), 7.621–7.502 (m, 4H), 5.882
(s, 1H), 5.417 (s, 1H), 2.607 (s, 1H), 2.510–2.502 (m, 2H), 2.251–
2.245 (m, 3H). MS (ESI): 458.04 (C19H17BrN5O4, [M+H]+). Anal.
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035

8
J. Sun et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.8. Docking simulation
References
1. Archambault V, Lepine G, Kachaner D. Understanding the Polo kinase machine.
Oncogene. 2015;34:4799–4807.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 11.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2009)], then they were energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The Gasteiger-Hückel charges of ligands were assigned. Molecular
docking of the compounds binding the three-dimensional X-ray
structure of PLK1 (PDB code: 4HCO) was carried out using Discov-
ery Studio (version 3.5) as implemented through the graphical user
interface DS-CDOCKER protocol. For enzyme preparation, the
hydrogen atoms were added with the pH of the protein in the
range of 6.5–8.5. CDOCKER is an implementation of a CHAR Mm
based molecular docking tool using a rigid receptor. It includes
the following steps:
2. Schuldt A. Cell division:
2013;14:194–195.
a timely exit for PLK1. Nat Rev Mol Cell Biol.
3. McInnes C, Wyatt MD. PLK1 as an oncology target: current status and future
potential. Drug Discovery Today. 2011;16:619–625.
4. Murugan RN, Park JE, Kim EH, et al. Plk1-targeted small molecule inhibitors:
molecular basis for their potency and specificity. Mol Cells. 2011;32:209–220.
5. Garuti L, Roberti M, Bottegoni G. Polo-like kinases inhibitors. Curr Med Chem.
2012;19:3937–3948.
6. Li S, Zhang Y, Xu W. Developments of polo-like kinase 1 (Plk1) inhibitors as
anti-cancer agents. Mini-Rev Med Chem. 2013;13:2014–2025.
7. Noble ME, Endicott JA, Johnson LN. Protein kinase inhibitors: insights into drug
design from structure. Science. 2004;303:1800–1805.
8. DeLano WL, Ultsch MH, deVos AM, Wells JA. Convergent solutions to binding at
a protein-protein interface. Science. 2000;287:1279–1283.
9. Lee KS, Burke TR, Park JE, et al. Recent advances and new strategies in targeting
Plk1 for anticancer therapy. Trends Pharmacol Sci. 2015;36:858–877.
10. Xu J, Shen C, Wang T, et al. Structural basis for the inhibition of Polo-like kinase
1. Nat Struct Mol Biol. 2013;20:1047–1053.
(1) A series of ligand conformations are generated using high
temperature molecular dynamics with different random
seeds.
11. Elia AE, Cantley LC, Yaffe MB. Proteomic screen finds pSer/pThr-binding domain
localizing Plk1 to mitotic substrates. Science. 2003;299:1228–1231.
12. Yun SM, Moulaei T, Lim D, et al. Structural and functional analyses of minimal
phosphopeptides targeting the polo-box domain of polo-like kinase 1. Nat
Struct Mol Biol. 2009;16:876–882.
13. Liu F, Park JE, Qian WJ, et al. Serendipitous alkylation of a Plk1 ligand uncovers a
new binding channel. Nat Chem Biol. 2011;7:595–601.
14. Murugan RN, Park JE, Lim D, et al. Development of cyclic peptomer inhibitors
targeting the polo-box domain of polo-like kinase 1. Bioorg Med Chem.
2013;21:2623–2634.
15. Kim SM, Chae MK, Lee C, et al. Enhanced cellular uptake of a TAT-conjugated
peptide inhibitor targeting the polo-box domain of polo-like kinase 1. Amino
Acids. 2014;46:2595–2603.
16. Ahn M, Han YH, Park JE, et al. A new class of peptidomimetics targeting the
polo-box domain of polo-like kinase 1. J Med Chem. 2014;58:294–304.
17. Reindl W, Yuan J, Krämer A, et al. Inhibition of polo-like kinase 1 by blocking
polo-box domain-dependent protein-protein interactions. Chem Biol.
2008;15:459–466.
(2) Random orientations of the conformations are generated by
translating the center of the ligand to a specified position
within the receptor active site, and making a series of ran-
dom rotations. A softened energy is calculated and the orien-
tation is kept when it is less than a specified limit. This
process repeats until either the desired number of low-
energy orientations is obtained, or the test times of bad ori-
entations reached the maximum number.
(3) Each orientation is subjected to simulated annealing molec-
ular dynamics. The temperature is heated up to a high tem-
perature then cooled to the target temperature. A final
energy minimization of the ligand in the rigid receptor using
non-softened potential is performed.
18. Reindl W, Yuan J, Krämer A, et al. A pan-specific inhibitor of the polo-box
domains of polo-like kinases arrests cancer cells in mitosis. ChemBioChem.
2009;10:1145–1148.
(4) For each of the final pose, the CHARMm energy (interaction
energy plus ligand strain) and the interaction energy alone
are figured out. The poses are sorted according to CHARMm
energy and the top scoring (most negative, thus favorable to
binding) poses are retained. The whole PLK1-PBD domain
defined as a receptor. CHARMm was selected as the force
field. The molecular docking was performed with a simu-
lated annealing method. The heating steps were 2000 with
700 of heating target temperature. The cooling steps were
5000 with 300 cooling target temperature. Ten molecular
docking poses saved for each ligand were ranked according
to their dock score function. The pose with the highest -
CDOCKER energy was chosen as the most suitable pose.
19. Li L, Wang X, Chen J, et al. The natural product Aristolactam AIIIa as a new
ligand targeting the polo-box domain of polo-like kinase 1 potently inhibits
cancer cell proliferation. Acta Pharm Sin. 2009;30:1443–1453.
20. Shan HM, Shi Y, Quan J. Identification of green tea catechins as potent inhibitors
of the polo-box domain of polo-like kinase 1. ChemMedChem.
2015;10:158–163.
21. Srinivasrao G, Park JE, Kim S, et al. Design and synthesis of a cell-permeable,
drug-like small molecule inhibitor targeting the polo-box domain of polo-like
kinase 1. PLoS One. 2014;9:e107432.
22. Chen DX, Huang J, Liu M, Xu YG, Jiang C. Design, synthesis, and evaluation of
non-ATP-competitive small-molecule polo-like kinase 1 (PLK1) inhibitors. Arch
Pharm Chem Life Sci. 2015;348:2–9.
23. Sun J, Lv PC, Guo FJ, et al. Aromatic diacylhydrazine derivatives as a new class of
polo-like kinase 1 (PLK1) inhibitors. Eur J Med Chem. 2014;81:420–426.
24. Yin Z, Song YL, Rehse PH. Thymoquinone blocks pSer/pThr recognition by Plk1
Polo-box domain as a phosphate mimic. ACS Chem Biol. 2013;8:303–308.
25. Chen YY, Zhang J, Li DS, Jiang JD, Wang YC, Si SY. Identification of a novel Polo-
like kinase 1 inhibitor that specifically blocks the functions of Polo-Box domain.
Oncotarget. 2017;8:1234–1246.
Acknowledgments
This work is granted by Natural Science Foundation of Jiangsu
Province (BK20160570), and Jiangsu Planned Projects for Postdoc-
toral Research Funds (1601041A).
Please cite this article in press as: Sun J., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.035