Design, synthesis, and evaluation of non-ATP-competitive small-molecule polo-like kinase 1 (Plk1) inhibitors

Article abstract of DOI:10.1002/ardp.201400294

A series of small-molecule Plk1 inhibitors targeting the substrate-binding pocket were designed through rational drug design for the first time. The designed compounds were synthesized and their activities were evaluated in vitro. Some of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 5i showed Plk1 inhibitory activity with an IC₅₀ value of 0.68 μM. Compound 5i also showed cell growth inhibitory activity on HeLa cells with an IC₅₀ value of 0.51 μM, which is about four times more potent compared to thymoquinone. The mechanism of action suggested that 5i was an ATP-independent and substrate-dependent Plk1 inhibitor. Compound 5i demonstrated excellent Plk1 inhibitory selectivity against Plk2, Plk3, and five serine/threonine and tyrosine kinases. Our discovery and structure-activity relationship study may provide useful lead compounds for further optimization of non-ATP-competitive Plk1 inhibitors.

Full text of DOI:10.1002/ardp.201400294

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
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Full Paper
Design, Synthesis, and Evaluation of Non-ATP-Competitive
Small-Molecule Polo-Like Kinase 1 (Plk1) Inhibitors
1
,2
3
1,2
1,2
1,2
Dong-Xing Chen , Jie Huang , Meng Liu , Yun-Gen Xu , and Cheng Jiang
1
Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
China
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
Gansu Institute for Food and Drug Control, Lanzhou, China
2
3
A series of small-molecule Plk1 inhibitors targeting the substrate-binding pocket were designed through
rational drug design for the first time. The designed compounds were synthesized and their activities
were evaluated in vitro. Some of the targeted compounds showed potent Plk1 inhibitory activities and
anti-proliferative characters. Particularly, 5i showed Plk1 inhibitory activity with an IC50 value of 0.68 mM.
Compound 5i also showed cell growth inhibitory activity on HeLa cells with an IC50 value of 0.51 mM,
which is about four times more potent compared to thymoquinone. The mechanism of action suggested
that 5i was an ATP-independent and substrate-dependent Plk1 inhibitor. Compound 5i demonstrated
excellent Plk1 inhibitory selectivity against Plk2, Plk3, and five serine/threonine and tyrosine kinases. Our
discovery and structure–activity relationship study may provide useful lead compounds for further
optimization of non-ATP-competitive Plk1 inhibitors.
Keywords: Non-ATP-competitive / Polo-like kinase 1 / Rational drug design / Small-molecule / Structure–
activity relationship
Received: July 28, 2014; Revised: October 11, 2014; Accepted: October 17, 2014
DOI 10.1002/ardp.201400294
characteristic substrate-binding PBD, which has been proved
Introduction
to be essential for bringing the catalytic activity of Plk1 to its
binding targets at specific subcellular locations [2–4]. Thus, the
PBD is ideally suited to the exploration of the feasibility of
inhibiting a serine/threonine kinase by interfering with its
intracellular localization, rather than targeting the conserved
ATP-binding site [5].
The serine/threonine kinase Polo-like kinase 1 (Plk1) is a member
of the Polo-like kinases family (Plk1–5 in humans; collectively
termed Plks) that act as a regulator in multiple stages of mitotic
progression. Plk1 is considered an attractive anti-cancer drug
target due to its ability to promote tumorigenesis [1]. Until
now, two distinct drug targets have been identified in Plk1:
an N-terminal catalytic domain and a C-terminal Polo-box
domain (PBD). For a long period, discovery of Plk1 inhibitors
has been focused on ATP-competitive compounds targeting
the N-terminal catalytic domain [1]. However, as the catalytic
domain of Plk1 is closely related to several members of the
superfamily of protein kinases, these efforts suffered from
a lack of target specificity. Like other Plks, Plk1 contains a
Several series of peptides were designed and synthesized
targeting Plk1 PBD with high affinity and specificity [6–12].
Some small-molecule compounds (Fig. 1) were also identified
as Plk1 PBD inhibitors [5, 13–16]. To our knowledge, all the
reported small-molecule Plk1 PBD inhibitors were discovered
by screening. Herein, we report the identification of a series of
small-molecule Plk1 PBD inhibitors through rational drug
design according to the binding pocket of Plk1 PBD.
Results
Correspondence: Prof. Yun-Gen Xu, Jiang Su Key Laboratory of
Drug Design and Optimization, Department of Medicinal
Chemistry, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, China.
Rational design
Rational design of the target compounds is depicted in Fig. 2.
Compound CJ-032 was obtained from the screening of a series
of in-house compounds. The Plk1 IC50 value of CJ-032 was
E-mail: cpujc7@126.com
Fax: þ86 25 83271351
5.04 mM. Then CJ-032 was docked in to the Plk1 PBD to do
Additional correspondence: Dr. Cheng Jiang
E-mail: jc@cpu.edu.cn
some modification.
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pocket formed by Trp410, Asn496, Arg557, and Phe559. Based
on these, a substituted or unsubstituted phenyl group was
introduced through an amide linker. The modified structure
was then validated by docking again. As shown in Fig. 2, the
introduced phenyl group linked with an amide merged into
the hydrophobic pocket formed by Trp410, Asn496, Arg557,
and Phe559. Interestingly, the carbonyl group could form an
additional H-bond with the amino group in Lys540 as shown
in Fig. 2. The benzimidazole ring could be substituted by
an indole group as an isostere. A series of benzimidazoles and
indoles were designed as shown in Table 1.
O
OH
O
N
O
O
N
O
O
Thymoquinone
Poloxin
Poloxime (PXE)
O
O
O
H
S
NH
Chemistry
O
O
H
The synthesis of designed compounds 5 and 6 is provided in
Scheme 1. Commercially available benzimidazole or indole (1)
was reacted with 1-bromo-3-chloropropane in acetone at
reflux temperature to give 2. Compound 2 was reacted with
O
S
N
O
O
HN
O
N
COOH
ON01910
2
-nitrophenol in DMF to give nitro 3. Aniline 4 was obtained
after reduction of nitro 3. Then, the designed compounds 5 or
were obtained after acylation or benzylation.
I2
6
Figure 1. Chemical structures of the reported small-molecule
Plk1 PBD inhibitors.
In vitro Plk1 and cancer cell line growth
inhibition assays
According to the docking result of CJ-032 shown in Fig. 2, the
benzimidazole residue of CJ-032 may form hydrophobic
interaction with the indole ring of Trp414. The 2-methyl
phenyl group in CJ-032 may interact with a hydrophobic
Designed compounds were synthesized and evaluated by
Plk1 inhibitory assay [15] and in vitro cell line proliferation
inhibition assay. As shown in Table 1, compounds with
benzamide substitutions (5a–m) displayed similar inhibition
Figure 2. Design of new small-molecule Plk1 inhibitors targeting PBD. (A) Modification of CJ-032. (B) Docking result of CJ-032
yellow) to Plk1 PBD. (C) Docking result of designed compound (blue) to Plk1 PBD. (D) Docking result of CJ-032 (yellow) merged with
(
designed compound (blue) to Plk1 PBD.
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Non-ATP-Competitive Small-Molecule Plk1 Inhibitors
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Table 1. In vitro Plk1 and cancer cell line growth inhibition of 5 and 6.
a)
Cell growth inhibition, IC50 ꢀ SD (mM)
Plk1 inhibition,
a)
Compound
X
R
IC50 ꢀ SD (mM)
HeLa
MCF-7
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
j
k
l
m
a
b
c
d
e
f
N
N
N
N
N
N
N
C
C
C
C
C
C
N
N
N
N
N
C
–
Benzoyl-
6.33 ꢀ 1.06
0.93 ꢀ 0.20
7.09 ꢀ 2.64
2.60 ꢀ 0.82
>10
10.03 ꢀ 3.02
>10
4-Methoxybenzoyl-
4-Methylbenzoyl-
2-Methylbenzoyl-
4-Nitrobenzoyl-
2-Nitrobenzoyl-
Acetyl-
2.18 ꢀ 1.41
5.24 ꢀ 0.86
5.81 ꢀ 0.62
>10
>10,
>10
–
–
–9 .28 ꢀ 1.25
–
–
>10
–
>10
6.83 ꢀ 0.52
Benzoyl-
>10
4-Methoxybenzoyl-
2-Methylbenzoyl-
4-Nitrobenzoyl-
2-Nitrobenzoyl-
Acetyl-
0.68 ꢀ 0.07
5.59 ꢀ 0.89
>10
0.51 ꢀ 0.18
3.73 ꢀ 1.02
2.97 ꢀ 0.74
>10
–
–
–
–
>10
4.70 ꢀ 0.48
9.48 ꢀ 0.95
3.22 ꢀ 0.22
2.05 ꢀ 0.51
4.66 ꢀ 1.01
>10
>10
>10
>10
>10
–2 .83 ꢀ 1.25
8.03 ꢀ 2.07
Benzyl-
>10
4-Chlorobenzyl-
4-Methoxybenzyl-
4-Methylbenzyl-
4-Nitrobenzyl-
Benzyl-
>10
>10
–6 .56 ꢀ 1.83
>10
7.02 ꢀ 1.41
5.14 ꢀ 1.00
Thymoquinone
–
2.18 ꢀ 0.31
2.06 ꢀ 0.27
6.19 ꢀ 0.97
a)
Values are the average of three independent experiments.
to Plk1, compared to compounds containing corresponding
benzylamine group (6a–f). Compounds with benzimidazole
core also showed similar Plk1 inhibitory activity compared to
those with indole core. N-Acetyl compounds (5g and 5m)
showed similar Plk1inhibitory activity as CJ-032. Compounds
also showed potent in vitro anti-proliferative activities. For
example, 5i showed inhibitory activity to HeLa cell line with
an IC50 value of 0.51 mM, which is about four times more
potent compared to thymoquinone.
5
a and 6a, which is N-benzoyl and N-benzyl substituted,
Plk1 inhibition characters of 5i
showed similar Plk1 inhibition to CJ-032. The substitution on
the phenyl ring had big effect to both the inhibition to plk1
and anti-proliferative activities. Compounds with electron
withdrawing groups (5e, 5f, 5k, 5l, and 6e) on the phenyl ring
displayed decreased inhibition to Plk1. Electron donating
groups gave an improvement for the Plk1 inhibition. For
example, compounds 5b and 5i showed Plk1 inhibitory activity
with an IC50 value of 0.93 and 0.68 mM, respectively, about five
and eight times more potent than CJ-032. Compounds 5b and
The effects of increasing concentrations of ATP or the
substrate Cdc25C on the inhibitory activity of the compound
were examined using the method reported by Gumireddy
et al. [15]. Ten nanograms of recombinant Plk1 was mixed
with different concentrations of 5i, followed by the addition
3
2
of a reaction mixture containing g P-ATP and varying
concentrations of ATP (20, 40, 80, and 160 mM). The values
from individual samples were analyzed and plotted as a
function of inhibitor concentration using Prism 4 Graphpad
software. The results are displayed as Fig. 3. These analyses
showed that the IC50 values for the inhibitor were similar in
the presence of increasing concentrations of ATP, suggesting
that 5i is not an ATP-competitive inhibitor (Fig. 3A). Then, the
5
i were two to three times more potent than thymoquinone, a
reported natural product as Plk1 PBD inhibitor. This result
suggests that the electronic density on the phenyl ring is
critical for Plk1 inhibitory activity. Compounds 5b and 5i
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Cl
H
NO2
O
N
a
b
N
X
N
X
X
1
2
3
1
1
a X = N
b X = C
2a X = N
2b X = C
3a X = N
3b X = C
NH2
HN R
O
O
d
or e
c
N
X
N
X
4
or
6
5
4
4
a X = N
b X = C
5a X = N, R = benzoyl-
6a X = N, R = benzyl-
5b X = N, R = 4-methoxybenzoyl-
6b X = N, R = 4-chlorobenzyl-
6c X = N, R = 4-methoxybenzyl-
6d X = N, R = 4-methylbenzyl-
6e X = N, R = 4-nitrobenzyl-
6f X = C, R = benzyl-
5
5
5
5
5
5
5
5
5
5
5
c X = N, R = 4-methylbenzoyl-
d X = N, R = 2-methylbenzoyl-
e X = N, R = 4-nitrobenzoyl-
f
X = N, R = 2-nitrobenzoyl-
g X = N, R = acetyl-
h X = C, R = benzoyl-
i
j
X = C, R = 4-methoxybenzoyl-
X = C, R = 2-methylbenzoyl-
k X = C, R = 4-nitrobenzoyl-
l
X = C, R = 2-nitrobenzoyl-
m X = C, R = acetyl-
Scheme 1. Synthetic route of compounds 5 and 6. Reagents and conditions: (a) 1-Bromo-3-chloropropane, K
2 h; (b) 2-nitrophenol, KOH, DMF, 80˚C, 12 h, 17.7–31.8% yield for two steps; (c) 10% Pb/C, MeOH, H , r.t., 4 h, 87.6–91.6%; (d) acyl
chloride, CH Cl , TEA, r.t., 2 h, 42.0–94.5%; and (e) benzyl bromide, K CO , acetone, reflux, 12 h, 36.7–62.3%.
2 3
CO , acetone, reflux,
1
2
2
2
2
3
Figure 3. Character of Plk1 inhibition by 5i. (A) IC50 curves for Plk1 with different concentrations of 5i in the presence of varying
concentrations of ATP. (B) IC50 curves for Plk1 with different concentrations of 5i in the presence of varying concentrations of Cdc25C
substrate.
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Non-ATP-Competitive Small-Molecule Plk1 Inhibitors
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Table 2. In vitro kinase inhibitory profile of 5i.
proliferative inhibition profiles made it a promising com-
pound. However, later studies showed that ON01910 exhib-
ited little activity against Plk1 in vitro. Cell-based phenotypes
do not correlate with Plk1 inhibition but are consistent with
affecting microtubule dynamics [17].
a)
IC50 ꢀ SD(mM)
5i
Protein kinase
Thymoquinone
Plk1
Plk2
Plk3
Aurora-A
CDK1
CDK2
EGFR
Src
0.68 ꢀ 0.07
>50
2.18 ꢀ 0.31
14.72 ꢀ 4.07
BI 2536 is a dihydropteridinone developed by Boehringer
Ingelheim, which exhibits a potent inhibitory activity against
Plk1 in vitro (IC50 ¼ 0.83 nM). This compound shows more than
28.05 ꢀ 5.31
>50
>50
–
–
–
–
–
21.46 ꢀ 4.78
>50
>50
1000-fold selectivity against a large panel of protein kinases,
but it affects the activities of other Plk family members,
such as Plk2 (IC50 ¼ 4 nM) and Plk3 (IC50 ¼ 9 nM) [18]. BI 6727 is
another dihydropteridinone developed by Boehringer Ingel-
heim. It strongly inhibits Plk1, Plk2, and Plk3 at IC50 values of
>50
a)
Values are the average of three independent experiments.
0
.87, 5, and 56 nM, respectively [19, 20]. BI 2536 and BI 6727
were both ATP-competitive Plk1 inhibitors. Compared to 5i, BI
536 and BI 6727 were more potent but less selective Plk1
2
effects of increasing concentrations of substrate Cdc25C
100, 200, 400, and 800 ng) on the inhibitory activity of the
compound in the presence of a constant amount of ATP
100 mM) were examined. It showed that increasing the
inhibitors. BI 2536 and BI 6727 are now in phase II clinical
trials [20, 21].
(
SBE13 is a type II Plk1 inhibitor, which stabilizes an inactive
conformation of the ATP-binding site. SBE13 had subnano-
molar inhibitory activity against Plk1 (IC50 ¼ 0.2 nM) and was
found to be selective for Plk1 over Plk2 (IC50 > 66 mM) or Plk3
(IC50 ¼ 875 nM) [22], which was more potent and selective
than 5i. However, SBE13 showed moderate proliferative
inhibition to HeLa cells (EC50 ¼ 18 mM) [22].
(
concentration of the substrate resulted in increased IC50
values of the inhibitor (Fig. 3B; IC50 curves shift right). Thus,
inhibitor 5i demonstrated the substrate-dependent and
ATP-independent nature of inhibition. This result may also
confirm indirectly that inhibitor 5i binds to the binding pocket
of Cdc25C rather than the ATP-binding pocket.
We further carried out a specificity test for 5i against
two closely related kinases Plk2 and Plk3 using the
protocol described in Section 4. Plk2 and Plk3 were
available as recombinant proteins purchased from commer-
cial sources (Abnova, Inc.). As shown in Table 2, thymoqui-
none showed excellent Plk1 inhibitory selectivity against
Plk3 (IC50 > 50 mM) and moderate inhibitory selectivity
against Plk2 (IC50 ¼ 14.72 ꢀ 4.07 mM, about sevenfold higher
than Plk1 IC50). Compound 5i showed excellent Plk1
inhibitory selectivity against Plk2 (IC50 > 50 mM) and Plk3
Conclusion
In this study, a series of benzimidazole and indole-containing
compounds were designed as new Plk1 inhibitors through
rational design according to the substrate-binding pocket of
Plk1. Two compounds showed potent Plk1 inhibitory activity
together with potent anti-proliferative activity. The SARs
revealed that the electronic density on the phenyl ring was
critical for the Plk1 inhibition. The mechanism of action of the
optimal compound 5i was also examined, which suggested
that 5i was an ATP-independent and substrate-dependent
Plk1 inhibitor. In the specificity test, 5i showed excellent Plk1
selectivity against Plk2 and 3, it also showed excellent
selectivity against a panel of serine/threonine and tyrosine
kinases. Our discovery may provide useful lead compounds for
further optimization of non-ATP-competitive Plk1 inhibitors.
(
IC50 ¼ 28.05 ꢀ 5.31 mM, about 40-fold higher than Plk1 IC50).
The inhibitory effects of 5i on five serine/threonine and
tyrosine kinases (Aurora-A, CDK1, CDK2, EGFR, and Src) were
also examined. These studies showed that at 30-fold higher
concentrations (IC50 ¼ 21.46 ꢀ 4.78 mM), 5i exhibits inhibitory
activity against one of the other kinases tested, the CDK1.
To the other kinases, IC50 value of 5i was more than 50 mM
(
Table 2). Among these different kinases, 5i was the most
active against Plk1. These results proved that the 5i was a
highly selective inhibitor of Plk1.
Experimental
In the pursuing of potent and selective Plk1 inhibitors,
several series of Plk1 inhibitors (ATP-competitive or non-ATP-
competitive) have been reported. Some of these inhibitors
show excellent characters as promising candidates.
Chemistry
All reagents were purchased from commercial sources and
were used without further purification. Melting points (m.p.)
were determined on a Mel-Temp II apparatus and are
ON01910 was initially reported to inhibit Plk1 (IC50 ¼ 9 nM)
in a substrate-dependent and an ATP-independent man-
ner [15]. ON01910 has been shown to inhibit the cell
proliferation in more than 100 cancer cell lines with IC50
values ranging from 50 to 250 nM. This candidate was more
potent and selective than 5i, and the potent cancer cell line
1
uncorrected. H NMR spectra were recorded on a Bruker
ACF-300 spectrometer (300 MHz). Chemical shifts are
expressed in ppm related to tetramethylsilane. Mass spectra
were obtained on either a Mariner Mass Spectrum or a
MAT-212 mass spectrometer.
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1-(3-(2-Nitrophenoxyl)propyl)-1H-benzo[d]imidazole (3a)
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
Benzimidazole (3.0 g, 25.4 mmol), 1-bromo-3-chloropropane
methoxybenzamide (5b)
Yellow solid, yield 90.6%, m.p. 88–90˚C. H NMR (300 MHz,
1
(
6.0 g, 38.2 mmol), K
2
CO
3
(4.2 g, 30.4 mmol) and acetone
(
30 mL) were mixed and heated to reflux for 12 h. Then, the
CDCl
3
): d 8.46 (s, 1H), 8.25 (m, 1H), 7.91–6.92 (m, 12H), 4.12 (t,
insoluble was filtered off, the solvent was removed, and the
residue was used without further purification. The residue
was mixed with 2-nitrophenol (5.22 g, 37.5mmol), KOH (2.1 g,
J ¼ 6.9 Hz, 2H), 3.85 (s, 3H), 3.71 (t, J ¼ 5.7 Hz, 2H), 2.61 (m, 2H).
1
3
3
C NMR (75 MHz, CDCl ): d 164.6, 161.8, 151.0, 142.2, 134.7,
131.7, 129.5, 127.0, 126.5, 125.8, 125.0, 124.7, 120.3, 116.2,
3
7.5 mmol), and DMF (45 mL) and the mixture was allowed to
113.7, 112.1, 112.0, 64.4, 55.4, 42.6, 28.6. EI-MS (m/z): 401 [M].
stir at 80˚C for 12 h. Then, the reaction mixture was diluted
with water (50 mL) and extracted with ethyl acetate (50 mL
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
ꢁ
3). The combined organic layer was washed with brine
methylbenzamide (5c)
White solid, yield 83.0%, m.p. 174˚C. H NMR (300 MHz,
1
(
100 mL), dried over anhydrous MgSO , filtered, and concen-
4
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/EtOAc, 2:1 v/v) to give compound 3
CDCl
3
): d 9.55 (s, 1H), 9.48 (s, 1H), 7.85–7.02 (m, 12H), 4.63
(t, J ¼ 6.57 Hz, 2H), 4.07 (t, J ¼ 5.19 Hz, 2H), 2.50 (m, 2H), 2.38
1
13
as a yellow oil (2.36 g, 31.8% yield for two steps). H NMR
3
(s, 3H). C NMR (75 MHz, CDCl ): d 164.9, 150.9, 141.5, 131.6,
(
300 MHz, CDCl
3
): d 7.96–6.92 (m, 9H), 4.55 (t, J ¼ 6.42 Hz, 2H),
131.2, 131.0, 129.0, 127.5, 126.9, 126.1, 125.9, 125.7, 125.0,
3
.95 (t, J ¼ 5.46 Hz, 2H), 2.38 (m, 2H) EI-MS (m/z): 297 [M].
120.4, 115.0, 112.8, 112.2, 64.4, 43.2, 28.4, 20.9. EI-MS (m/z):
385 [M].
1-(3-(2-Nitrophenoxy)propyl)-1H-indole (3b)
Compound 3b was prepared under similar conditions as
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-2-
1
described for 3a (yield 17.7% for two steps). H NMR
methylbenzamide (5d)
Pale pink solid, yield 87.0%, m.p. 234–236˚C. H NMR
1
(
300 MHz, CDCl
3
): d 7.81–6.43 (m, 10H), 4.35 (t, J ¼ 6.42 Hz,
2
H), 3.78 (t, J ¼ 5.55 Hz, 2H), 2.21 (m, 2H). EI-MS (m/z): 296 [M].
(300 MHz, CDCl
J ¼ 6 Hz, 2H), 4.05 (s, 2H), 2.41 (m, 5H). C NMR (75 MHz,
CDCl ): d 164.8, 150.7, 145.4, 141.4, 136.9, 135.3, 131.6, 131.0,
3
): d 9.58 (s, 2H), 7.94–7.01 (m, 12H), 4.70 (t,
1
3
1-(3-(2-Aminophenoxyl)propyl)-1H-benzo[d]imidazole
3
(
4a)
To a solution of 3 (0.85 g, 2.86 mmol) in methanol (15 mL),
0% Pd-C (0.05 g) was added. The mixture was hydrogenated
130.5, 129.6, 127.3, 126.6, 126.2, 125.9, 125.7, 125.6, 120.3,
114.9, 112.2, 64.4, 43.3, 28.4, 19.5. EI-MS (m/z): 385 [M].
1
at normal pressure for 4 h. The insoluble was filtered off and
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
the solvent was removed to gain 4 as a brown oil (0.70 g, yield
nitrobenzamide (5e)
Green solid, yield 92.8%, m.p. 80–82˚C. H NMR (300 MHz,
1
1
9
1.6%). H NMR (300 MHz, CDCl
3
): d 7.92–6.70 (m, 9H), 4.45 (t,
J ¼ 6.6 Hz, 2H), 4.00 (t, J ¼ 5.64 Hz, 2H), 3.10 (s, 2H), 2.40 (m,
H). EI-MS (m/z): 267 [M].
CDCl
3
): d 10.01 (s, 1H), 9.57 (s, 1H), 8.33 (d, J ¼ 8.67 Hz, 2H), 8.21
2
(d, J ¼ 8.73 Hz, 2H), 7.85–7.05 (m, 8H), 4.12 (t, J ¼ 6.63 Hz, 2H),
1
3
4
.09 (t, J ¼ 5.43 Hz, 2H), 2.38 (m, 2H). C NMR (75 MHz, CDCl
3
):
1-(3-(2-Aminophenoxyl)propyl)-1H-indole (4b)
d 163.6, 151.4, 149.0, 145.7, 141.3, 140.0, 130.9, 130.8, 130.6,
129.2, 126.6, 126.2, 126.1, 125.7, 123.4, 120.3, 114.8, 112.9,
112.4, 64.4, 43.4, 28.3. EI-MS (m/z): 416 [M].
Compound 4b was prepared under similar conditions as
1
described for 4a (yield 87.6%). H NMR (300 MHz, CDCl
3
): d
.62–6.48 (m, 10H), 4.37 (t, J ¼ 6.48 Hz, 2H), 3.93 (t, J ¼ 5.67 Hz,
H), 3.77 (s, 2H), 2.31 (m, 2H). EI-MS (m/z): 266 [M].
7
2
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-2-
nitrobenzamide (5f)
Yellow solid, yield 70.4%, m.p. 116–118˚C. H NMR (300 MHz,
1
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-
benzamide (5a)
CDCl
3
): d 10.05 (d, J ¼ 12.69 Hz, 1H), 9.56 (d, J ¼ 12.69 Hz, 1H),
To a solution of 4a (0.20 g, 0.75 mmol) in CH
0.11 g, 1.13 mmol) was added. Then, the solution of benzoyl
chloride (0.13 g, 0.90mmol) in CH Cl (5 mL) was added in
2 2
Cl (5 mL), TEA
8.34 (d, J ¼ 8.7 Hz, 1H), 8.21 (d, J ¼ 8.73 Hz, 1H), 7.94–7.05 (m,
1
3
(
10H), 4.65 (t, J ¼ 6.69 Hz, 2H), 4.08 (m, 2H), 2.39 (m, 2H).
C
2
2
NMR (75 MHz, CDCl ): d 165.8, 151.4, 147.7, 141.4, 131.9, 130.9,
3
drops. After the addition of benzoyl chloride, the mixture was
allowed to stir at room temperature for 2 h. Compound 5a was
obtained after silica gel column chromatography (hexane/
127.8, 127.6, 127.0, 126.9, 126.1, 125.9, 125.7, 125.0, 120.4,
114.9, 112.8, 112.2, 64.5, 43.3, 28.3. EI-MS (m/z): 416 [M].
EtOAc, 1:1 v/v) as a brown solid (0.21 g, yield 76.9%). m.p. 106–
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-
1
1
1
2
1
1
08˚C. H NMR (300 MHz, CDCl
3
): d 8.38 (m, 2H), 7.97–6.75 (m,
acetamide (5g)
Pale pink solid, yield 42.0%, m.p. 104–106˚C. H NMR
1
3H), 4.35 (t, J ¼ 6.9Hz, 2H), 4.02 (t, J ¼ 5.79 Hz, 2H), 2.36 (m,
1
3
H). C NMR (75 MHz, CDCl
3
): d 165.1, 151.1, 134.4, 131.6,
3
(300 MHz, CDCl ): d 9.03 (s, 1H), 8.23 (s, 1H), 7.81–6.91
28.4, 127.5, 126.7, 126.1, 125.1, 124.5, 120.3, 116.4, 112.2,
11.9, 64.4, 42.4, 28.6. EI-MS (m/z): 371 [M].
(m, 8H), 4.49 (t, J ¼ 6.87 Hz, 2H), 3.94 (t, J ¼ 5.88 Hz, 2H), 2.28
1
3
3
(m, 2H), 2.11 (s, 3H). C NMR (75 MHz, CDCl ): d 168.3, 149.1,
The synthetic methods for the targeted compounds 5b–m
144.0, 143.4, 133.7, 127.3, 124.5, 122.9, 122.2, 121.4, 120.2,
were similar to the procedure for the synthesis of 5a.
119.4, 111.9, 110.2, 64.9, 40.9, 28.9, 20.6. EI-MS (m/z): 309 [M].
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6

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
Non-ATP-Competitive Small-Molecule Plk1 Inhibitors
ARC HH PHARM
Archiv der Pharmazie
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)benzamide (5h)
Brown solid, yield 51.5%, m.p. 104–106˚C. H NMR (300 MHz,
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-
benzylaniline (6a)
1
CDCl
H), 4.05 (t, J ¼ 5.88 Hz, 2H), 2.39 (m, 2H).
75 MHz, CDCl ): d 164.6, 146.7, 135.3, 134.8, 131.3, 128.4,
28.1, 127.3, 127.2, 126.4, 123.5, 121.2, 121.0, 120.6, 119.7,
3
): d 8.53 (m, 2H), 7.89–6.48 (m, 14H), 4.36 (t, J ¼ 6.75 Hz,
The mixture of 4a (0.25 g, 0.9 mmol), benzyl bromide (0.19 g,
1
3
2
C NMR
2 3
1.1 mmol), K CO (0.16 g, 1.1 mmol), and acetone (10 mL) was
(
3
mixed and heated to reflux for 12 h. Then, the insoluble was
filtered off, the solvent was removed and the residue was
purified using silica gel column chromatography (hexane/
1
1
3
19.0, 110.4, 108.5, 101.2, 64.9, 42.4, 29.4. EI-MS (m/z):
92 [M].
EtOAc, 1:1 v/v) to obtain 6a as a brown oil (0.18 g, yield
1
5
3.9%). H NMR (300 MHz, CDCl
3
): d 7.89 (s, 1H), 7.80–6.63 (m,
1
3
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)-4-
13H), 4.40 (m, 4H), 4.00 (t, J ¼ 5.64 Hz, 2H), 2.37 (m, 2H).
C
methoxybenzamide (5i)
Brown solid, yield 46.5%, m.p. 122–124˚C. H NMR (300 MHz,
NMR (75 MHz, CDCl ): d 142.6, 137.7, 128.1, 127.8, 127.6, 126.8,
3
1
126.7, 126.4, 122.7, 121.9, 121.4, 119.7, 116.3, 110.3, 110.1,
109.1, 64.1, 47.5, 41.3, 29.0. EI-MS (m/z): 357 [M].
3
CDCl ): d 8.51 (m, 1H), 8.47 (s, 1H), 7.85–6.49 (m, 13H), 4.37 (t,
J ¼ 6.63 Hz, 2H), 4.05 (t, J ¼ 5.73 Hz, 2H), 3.87 (s, 3H), 2.39 (m,
The synthetic methods for the targeted compounds 6b–f
were similar to the procedure for the synthesis of 6a.
1
3
2
1
1
3
H). C NMR (75 MHz, CDCl ): d 161.9, 146.6, 128.2, 127.5,
27.2, 126.9, 123.2, 121.2, 121.0, 120.6, 119.6, 119.0, 113.6,
10.4, 108.5, 101.2, 65.0, 54.9, 42.5, 29.4. EI-MS (m/z): 422 [M].
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
chlorobenzylaniline (6b)
1
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)-2-
Brown oil, yield 40.8%. H NMR (300 MHz, CDCl
3
): d 7.95 (s,
methylbenzamide (5j)
Brown solid, yield 54.2%, m.p. 118–120˚C. H NMR (300 MHz,
1H), 7.81–6.53 (m, 12H), 4.42 (t, J ¼ 6.81 Hz, 2H), 4.34 (s, 2H),
1
13
4.01 (t, J ¼ 5.67 Hz, 2H), 2.39 (m, 2H). C NMR (75 MHz, CDCl
3
):
CDCl
3
): d 8.53 (s, 1H), 8.07 (s, 1H), 7.61–6.43 (m, 13H), 4.3 (t,
d 144.9, 143.3, 142.6, 137.7, 137.3, 133.2, 132.2, 129.0, 128.2,
128.1, 127.9, 122.6, 121.8, 121.3, 119.9, 116.5, 110.2, 110.1,
109.0, 64.2, 46.8, 41.3, 28.9. EI-MS (m/z): 391 [M].
J ¼ 6.66 Hz, 2H), 3.99 (t, J ¼ 5.88 Hz, 2H), 2.56 (s, 3H), 2.32 (m,
1
3
2
1
1
1
3
H). C NMR (75 MHz, CDCl ): d 167.2, 146.7, 136.1, 136.0,
35.3, 131.0, 129.9, 128.1, 127.2, 126.2, 125.6, 123.6, 121.2,
20.9, 120.6, 119.8, 119.0, 110.5, 108.5, 101.1, 64.9, 42.4, 29.3,
9.6. EI-MS (m/z): 406 [M].
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
methoxybenzylaniline (6c)
1
Brown oil, yield 57.3%. H NMR (300 MHz, CDCl
3
): d 7.89 (s,
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)-4-nitrobenzamide
5k)
Brown solid, yield 49.3%, m.p. 124–126˚C. H NMR (300 MHz,
CDCl
1H), 7.81–6.67 (m, 12H), 4.39 (t, J ¼ 6.81 Hz, 2H), 3.99 (t,
13
J ¼ 5.67 Hz, 2H), 3.80 (s, 2H), 3.79 (s, 3H), 2.37 (m, 2H). C NMR
(75 MHz, CDCl ): d 158.1, 142.6, 130.4, 128.9, 128.2, 122.5,
(
1
3
3
): d 8.46 (d, J ¼ 3.69 Hz, 1H), 8.35 (m, 4H), 7.95–6.45 (m,
121.7, 121.4, 119.8, 116.2, 113.6, 113.2, 110.1, 109.1, 64.1, 54.7,
1
2
1
1
0H), 4.35 (t, J ¼ 6.54 Hz, 2H), 4.09 (t, J ¼ 5.82 Hz, 2H), 2.40 (m,
47.0, 41.2, 29.0. EI-MS (m/z): 387 [M].
1
3
3
H). C NMR (75 MHz, CDCl ): d 163.6, 151.6, 149.1, 140.2,
35.5, 130.6, 129.0, 128.5, 128.0, 126.7, 126.2, 125.6, 123.6,
20.8, 120.3, 120.2, 118.8, 112.2, 109.4, 100.5, 64.8, 42.1, 29.4.
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
methylbenzylaniline (6d)
1
EI-MS (m/z): 415 [M].
Yellow oil, yield 36.7%. H NMR (300 MHz, CDCl
3
): d 7.88 (s,
1
H), 7.82–6.58 (m, 12H), 4.39 (t, J ¼ 6.8 Hz, 2H), 4.35 (s, 2H), 3.99
13
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)-2-nitrobenzamide
(t, J ¼ 5.7 Hz, 2H), 2.42–2.29 (m, 5H). C NMR (75 MHz, CDCl
3
):
(
5l)
Brown solid, yield 46.6%, m.p. 106–108˚C. H NMR (300 MHz,
CDCl
d 145.0, 143.2, 142.6, 137.7, 136.3, 136.0, 133.2, 128.9, 128.2,
128.0, 126.9, 122.6, 121.7, 121.4, 119.9, 116.2, 110.2, 110.1,
109.1, 64.0, 47.3, 41.2, 29.0, 20.6. EI-MS (m/z): 371 [M].
1
3
): d 8.48–8.46 (d, J ¼ 6.72 Hz, 1H), 8.38 (s, 1H), 8.34–6.44
(
(
m, 13H), 4.35 (t, J ¼ 6.48 Hz, 2H), 4.09 (t, J ¼ 5.94 Hz, 2H), 2.41
1
3
m, 2H). C NMR (75 MHz, CDCl
3
): d 162.4, 149.2, 146.7, 140.0,
30.5, 128.1, 127.5, 127.1, 126.5, 124.3, 123.6, 121.2, 121.0,
20.7, 119.9, 119.1, 110.5, 108.3, 101.3, 65.4, 42.7, 29.3. EI-MS
N-(2-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-4-
1
1
nitrobenzylaniline (6e)
Yellow oil, yield 55.2%. H NMR (300 MHz, CDCl
1
3
): d 8.20
(
m/z): 415 [M].
(s, 1H), 8.17 (s, 1H), 8.03 (m, 1H), 7.80–6.38 (m, 10H), 4.47
1
3
(
t, J ¼ 6.33 Hz, 4H), 4.06 (t, J ¼ 5.7Hz, 2H), 2.44 (m, 2H). C NMR
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)acetamide (5m)
Brown solid, yield 94.5%, m.p. 64–66˚C. H NMR (300 MHz,
(75 MHz, CDCl ): d 147.2, 145.0, 143.3, 142.6, 136.7, 133.2,
3
1
128.2, 127.0, 123.3, 123.1, 122.5, 121.7, 121.3, 119.9, 116.8,
CDCl
3
): d 9.03 (s, 1H), 8.23 (s, 1H), 7.81–6.91 (m, 9H), 4.49
110.2, 110.0, 109.0, 64.5, 46.7, 41.6, 28.9. EI-MS (m/z): 402 [M].
(
(
t, J ¼ 6.87 Hz, 2H), 3.94 (t, J ¼ 5.88 Hz, 2H), 2.28 (m, 2H), 2.11
1
3
s, 3H). C NMR (75 MHz, CDCl
3
): d 167.6, 146.3, 135.3,
N-(2-(3-(1H-Indol-1-yl)propoxy)phenyl)benzylaniline (6f)
1
1
1
3
28.1, 127.3, 127.1, 123.3, 121.2, 120.8, 120.7, 119.9, 119.0,
Pale yellow oil, yield 62.3%. H NMR (300 MHz, CDCl
3
): d 7.62–
10.3, 108.6, 101.1, 65.2, 42.8, 29.2, 24.3. EI-MS (m/z):
6.46 (m, 15H), 4.60 (s, 1H), 4.35(m, 4H), 3.95 (t, J ¼ 5.67 Hz, 2H),
13
2.32 (m, 2H). C NMR (75 MHz, CDCl
08 [M].
3
): d 145.4, 139.3, 137.9,
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7

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
D.-X. Chen et al.
ARC HH PHARM
Archiv der Pharmazie
1
1
35.6, 128.3, 127.6, 127.0, 126.8, 121.3, 121.2, 120.7, 119.0,
16.4, 110.3, 110.1, 108.9, 101.0, 64.6, 47.7, 42.6, 29.5. EI-MS
each concentration point and the results were reported and
presented as mean. Cell inhibitory ratio was calculated by the
following formula:
(
m/z): 402 [M].
ꢀðA
Þꢁ
control
ꢂ Atreated
Docking study
Inhibitory ratio ð%Þ ¼
ꢁ 100%
Acontrol
Glide was selected as the molecular docking tool. The
crystallized structure of Plk1-PBD in complex with thymoqui-
none (PDB ID: 4HCO) was prepared using the Protein
PreparationWizard workflow. Ligand was deleted and a
receptor grid was generated on the center of the cocrystal-
lized ligand, which was defined as the ligand-binding site
search region. The compounds to be docked were confirmed
by an enclosing box that was similar in size to the cocrystal-
lized ligand. Furthermore, the compound set was minimized
using the LigPrep module. The best conformation of each
compound was output on the basis of the Glide score and
interactions formed between the compounds and the
active site. Finally, the potential compounds were flexibly
docked into the binding site using the extra precision (XP)
docking mode. All the remaining parameters were kept as
default.
The IC50 was taken as the concentration that caused 50%
inhibition of cell proliferation.
This work was financially supported by Jiangsu Province
Natural Science Foundation (No. BK20110623) and “Qinglan
project” of Jiangsu province.
The authors have declared no conflict of interest.
References
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ARC HH PHARM
Archiv der Pharmazie
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9