Development of a Polo-like Kinase-1 Polo-Box Domain Inhibitor as a Tumor Growth Suppressor in Mice Models

Article abstract of DOI:10.1021/acs.jmedchem.0c01451

Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.

Full text of DOI:10.1021/acs.jmedchem.0c01451

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Development of a Polo-like Kinase‑1 Polo-Box Domain Inhibitor as a
Tumor Growth Suppressor in Mice Models
Pethaiah Gunasekaran,†† Min Su Yim,†† Mija Ahn, Nak-Kyun Soung, Jung-Eun Park, Jaehi Kim,
Geul Bang, Sang Chul Shin, Joonhyeok Choi, Minkyoung Kim, Hak Nam Kim, Young-Ho Lee,
Young-Ho Chung, Kyeong Lee, Eunice EunKyeong Kim, Young-Ho Jeon, Min Ju Kim,
Kyeong-Ryoon Lee, Bo-Yeon Kim, Kyung S. Lee,* Eun Kyoung Ryu,* and Jeong Kyu Bang*
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ABSTRACT: Polo-like kinase-1 (Plk1) plays a key role in mitosis
and has been identified as an attractive anticancer drug target. Plk1
consists of two drug-targeting sites, namely, N-terminal kinase
domain (KD) and C-terminal polo-box domain (PBD). As KD-
targeting inhibitors are associated with severe side effects, here we
report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which
showed a remarkable in vitro anticancer effect by inducing Plk1
delocalization, mitotic arrest, and apoptosis in HeLa cells. Further,
in vivo optical imaging analysis and antitumorigenic activities in
mouse xenograft models demonstrate that KBJK557 preferentially
accumulates in cancer cells and selectively inhibits cancer cell
proliferation. Pharmacokinetic profiles and partition coefficients
suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73
h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that
targets the Plk1 PBD.
been widely tested under various preclinical and clinical settings,
their usage has been limited because of their dose-limiting
toxicities10 that may stem from cross-reactivity with closely
related kinases, including Plk2 and Plk3.11−13 As Plk2 and Plk3
act as tumor suppressors,14 selective inhibition of Plk1 is an
essential requirement to be an efficient anticancer agent.
Unlike the kinase domain (KD), which imposes a grave
disadvantage in selectivity because of the existence of a high
degree of sequence similarity among related kinases, the C-
terminal PBD, a unique protein−protein interaction domain of
the Plk subfamily, offers an inherent advantage. Notably, only
three PBDs from Plk1, Plk2, and Plk3 appear to show a
considerable level of homology, whereas those from Plk4 and
Plk5 are considerably different in structure and primary
sequence. It is well documented that PBD-dependent
subcellular location is critical for the mitotic functions of
Plk1.15 Thus, specific inhibition of the function of Plk1 PBD is
INTRODUCTION
■
Understanding the protein−protein interaction and their
binding sites are considered as an important tool for developing
novel therapeutics. Polo-like kinases (Plks) are a class of serine/
threonine protein kinases (classified into Plk1−5) that mediate
nonoverlapping roles in various cellular processes critical for cell
proliferation.1−5 Among them, Plk1 plays essential roles during
the multiple stages of M-phase progression, including mitotic
initiation, centrosome maturation, bipolar spindle formation,
chromosome segregation, and cytokinesis.6 Remarkably, over-
expression of Plk1 appears to be required for the viability of
cancer cells bearing oncogenic Ras and inactivated p53
mutation(s) but not their isogenic wild-type (WT) cells.7
Moreover, interfering with the Plk1 function induces mitosis
arrest and subsequent apoptosis and retards tumor growth.7−9
Therefore, specific inhibition of Plk1 has been considered an
attractive strategy for anticancer therapy.
Plk1 comprises two drug target sites, namely, N-terminal
catalytic domain, especially the ATP-binding site, and the C-
terminal polo-box domain (PBD). For the past few decades, a
significant amount of work has been carried out to develop
various Plk1 inhibitors, primarily targeting the N-terminal ATP-
binding site. For instance, a dihydropteridinone-derived
inhibitor, BI-2536, and its derivative, volasertib (BI 6727),
were developed by Boehringer Ingelheim. Although they have
Received: August 20, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01451
© XXXX American Chemical Society
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Figure 1. Data obtained with a Plk1 PBD-specific FP-based assay.
Scheme 1. Synthesis and Structures of 4(a−o)
considered as an attractive therapeutic strategy for treating
cancer, as it would trigger the G2/M cell cycle arrest and
subsequent apoptosis in tumor cells.16−18
Although various studies reported the minimal peptide,
PLHSpT, and its derivatives as novel peptide-based inhib-
itors,19−21 they have severe drawbacks, such as proteolytic
instability and poor cell permeability. To overcome the
disadvantages of peptide inhibitors, small molecular inhibitors
such as poloxin,22,23 poloxin-2,9 poloxipan, purpurogallin
(PPG),24 thymoquinone (TQ),22 green tea catechins,25 and
others26,27 were identified. Even though TQ,26 poloxin,23 and
T52127 were tested for the inhibition of tumor growth in vivo,
B
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Scheme 2. Synthesis of Second-Phase Derivatives
the cancer-targeting ability of these inhibitors using imaging
studies in a mouse xenograft model was not investigated yet.
Moreover, these inhibitors were not identified through an
extensive structure−activity relationship (SAR) study.
phospho binding pocket, which possesses His538 and Lys540
residues that undergo electrostatic interaction with Plk1 PBD
inhibitors.
First-Phase Synthesis. Based on the aforementioned
preliminary structural prerequisites, we envisioned 1,3,4-
trisubstituted pyrazole core residue 4, which offers the
possibility to derivatize various compounds through anchoring
alkyl/aryl chains at the pyrazole N1 position and functionalizing
the NH of the barbituric acid arylidene tethered at the 4-position
of pyrazole. To synthesize 4, as delineated in Scheme 1, we
began with the N-alkylation of 3-phenyl-4-pyrazole carbox-
aldehyde (1) using various aryl/alkyl bromides in the presence
of K2CO3 in DMF to result in the formation of 2(b−j). Further,
condensation of various barbituric acids (3) with 2 resulted in
the formation of 4(a−o). To derive substituted barbituric acids
3(a,b), different amines were treated with 5 in methanol to yield
6, which was under treatment with malonic acid in the presence
of acetic anhydride and acetic acid, resulting in the formation of
3(a,b). After the synthesis, the hit compound 4a was tested for
its ability to inhibit Plk1 PBD using a fluorescence polarization
(FP) assay.20 To accurately assess the level of its inhibitory
activity, a previously characterized Plk1 PBD-binding peptide,
PLHSpT, and its nonbinding control, PLHST, and a potent
PLHSpT-containing p-9mer peptide (Ac-PLHSpTAIYA-
NH2)19 were included. A small-molecule Plk1 PBD inhibitor,
PPG, known to exhibit a moderate level of anti-Plk1 activity24
was also included for comparative analysis (Figure S1).
Surprisingly, the hit compound 4a was found to be more
potent than the small-molecule Plk1 PBD inhibitor, PPG
(Figure S1), which prompted us to perform a detailed SAR
study. Thus, to examine whether the interactions of NH at
barbituric acid in 4a make a significant contribution in Plk1 PBD
activity, we introduced methyl, propyl, and ethylphenyl residues
at the NH position to generate 4b, 4c, and 4d respectively.
Unfortunately, these compounds did not display any Plk1 PBD
inhibition (Figure S1). To evaluate the influence of alkyl chain
length at the NH position of pyrazole, we introduced benzyl
derivatives, including 4e, 4f (4-phenyl-S-methyl), 4g (4-
phenoxytrifluoromethyl), and 4h (3-phenoxytrifluoromethyl).
The present study, which identified a pyrazolopyrimidine-
fused small molecule as a new chemical scaffold against Plk1
PBD, makes several significant advances over other reported
Plk1 PBD inhibitors. First, the most active compound,
KBJK557, obtained through extensive SAR studies, displayed
an appreciable level of Plk1 PBD selectivity and cell
permeability. Second, KBJK557 effectively delocalized Plk1
and induced cell cycle arrest and apoptosis in cancer cells. Third,
KBJK557 conjugated with cyanine-5 showed cancer-targeting
ability in an in vivo mouse xenograft model. Fourth, intra-
venously injected KBJK557 displayed a significant effect in
inhibiting cancer growth in a mouse xenograft model with a low
dosage level. To the best of my knowledge, this is the first study
reporting a small-molecule scaffold against Plk1 PBD that
exhibits antitumor activities in a mouse xenograft model with
significant pharmacokinetic profiles.
RESULTS
■
Design, Virtual Screening, and Synthesis. To identify a
core moiety for developing the Plk1 PBD inhibitor, we
performed virtual screening with a library of compounds as
described in methods and found that 1,3,4-trisubstituted
pyrazole core skeleton 4a was a most potent hit compound
(Figure 1). To derivatize the core residue, we carefully
investigated the crystal structure of Plk1 PBD in complex with
the most potent minimal peptide 4J (PDB ID 3RQ7).20 It is
inferred from the crystal structure that interactions at three
major binding pockets (broad pyrrolidine binding, deep and
narrow tyrosine-rich channel, and phosphate-binding pocket)
determine the binding affinity and selectivity. In particular, the
broad pyrrolidine binding pocket, surrounded by Trp414,
Phe535, and Arg516, is instrumental in achieving the Plk1
selectivity from two other related Plk2 and Plk3. A deep and
narrow tyrosine-rich channel, enclosed by Tyr417, Tyr481, and
Tyr485, delivers a high binding affinity >1000-fold; in addition, a
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Figure 2. Second-phase derivatization: (a) inhibitory effects of second-phase derivatives against Plk1 PBD using an FP-based assay; (b) comparison of
FP-based assay results of hit compound 4a and lead compound KBJK557 with references; (c) evaluation of KBJK557 selectivity against PBD of Plk1,
Plk2, and Plk3 using FP-based assays. A Plk1 PBD-binding PLHSpT and its nonphospho parental PLHST19 were used as positive and negative
controls, respectively. KBJK557 showed IC50 = 3.05 μM (PBD1) and 7.52 μM (PBD2).
These compounds did not show considerable Plk1 PBD
inhibition (Figure 1). Further increment in the N-alkyl chain
length resulted in ethylphenyl (4i), N-ethylpiperidine (4j), and
N-ethylmorpholine (4k). Among them, 4k displayed Plk1 PBD
inhibition (IC50 = 71.03 μM); however, it was less than that of
the hit compound 4a (IC50 = 32.17 μM). This binding affinity
may be attributed to the presence of heteroatoms, which might
have undergone polar interactions with PBD-binding sites.
Further increase in chain lengths using four- and eight-carbon
linkers yielded 4l, 4m, 4n, and 4o, respectively. Surprisingly, 4l
showed appreciable Plk1 PBD inhibition (Figure S1).
Second-Phase Derivatization. From the above results,
especially from 4k and 4l, we speculated that a four-carbon
intervening chain attached at pyrazole nitrogen and the presence
of heteroatoms may yield an inhibitor with a significant Plk1
PBD-binding affinity. Furthermore, the amide linker could be an
optimal choice for synthesizing the derivatives, as shown in
Scheme 2. Initially, to perform the N-alkylation on pyrazole
aldehyde (1), N-Boc-bromoethylamine was treated in the
presence of K2CO3 in DMF to yield 7, further treatment of
which with 2 M HCl in diethyl ether resulted in the formation of
amine hydrochloride, 8. To derive amide derivatives 9(a−d),
various acids were treated with amine hydrochloride (8) in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI), 1-hydroxybenzotriazole (HOBt), and N,N-diisopro-
pylethylamine (DIEA). Finally, condensation of barbituric acid
with 9(a−d) in methanol under reflux conditions yielded 10(a−
d).
Initially, we synthesized 10a, in which pyrazole nitrogen and a
cyclohexyl are separated by four intervening atoms, including an
amide linkage. Similarly, to examine the influence of aromatic
substituents, we synthesized 10b by replacing the cyclohexyl
group with a phenyl moiety in 10a. Unfortunately, as shown in
Figure 2a, the assay results of both compounds did not reveal
significant binding affinity. Further, to validate the effects of
polar substituents in 10b, we introduced methane sulfonyl and
N-methyl substituents at the para position of phenyl in 10b,
yielding 10c and 10d (KBJK557), respectively. Unfortunately,
10c did not show any binding affinity; in contrast, KBJK557 was
found to be the most potent Plk1 PBD inhibitors among the
derived compounds with a binding affinity of IC50 = 16.35 μM,
which was more potent than that of the Plk1 PBD inhibitor
minimal peptide, PLHSpT (IC50 = 29.02 μM) (Figure 2b), and
the hit compound, 4a (IC50 = 32.17 μM).
Collectively, through this SAR study, we identified that (i)
pyrazole holding N-alkyl phenyl derivatives and barbituric acid
arylidene with free NH are important for the Plk1 PBD activity;
(ii) even though small alkyl chains attached at the NH position
of pyrazole showed considerable activity, manipulation of the
alkyl chain with four intervening atoms resulted in improvement
in the Plk1 PBD affinity; in particular, amide linked with a p-
aminomethylphenyl group was found to be instrumental in
obtaining enhanced Plk1 PBD affinity.
Impact of KBJK557 on Selectivity. To examine the
binding specificity of KBJK557 against PBDs of closely related
Plk1, 2, and 3, we performed a selectivity test in comparison with
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Figure 3. Plk1 delocalization at kinetochore and cell cycle arrest. (a) Plk1 signal (red) was observed at the kinetochore (green), which was represented
by the anti-crest antibody. Among the mitotic cells, prometaphase cells were captured and analyzed. The cell phase was judged by the DNA (blue)
shape. Cells were treated with DMSO, nocodazole, BI2536, and KBJK557 for 18 h. (b) Relative Plk1 intensity in kinetochore was measured and
visualized in a graph. At least 10 kinetochores were measured per cell. The value was shown based on the DMSO sample. The “n” denotes cell number.
***p < 0.001. (c) Fluorescence-activated cell sorting analysis of cell cycle arrest in HeLa cancer cells after incubation with BI2536 and KBJK557. Cells
were cultured for 24 h with BI2536 and KBJK557. Cell cycle arrest was measured by flow cytometry with PI staining.
that of Plk1 PBD-specific PLHSpT.19,20 As shown in Figure 2c,
KBJK557 recognized the Plk1 selectively over Plk3 because it
did not bind with Plk3. In the case of the KD targeting inhibitor,
BI2536, Plk1 selectivity over Plk3 was approximately 10-fold.28
Besides, KBJK557 distinguished Plk1 over Plk2 although it was
showing a narrow window of difference by twofold. Similarly,
this selectivity in the case of BI2536 was threefold.28 As the
interface between two interacting proteins is often large and the
binding site is shallow, achieving both high binding affinity and
selectivity by a small molecule is a challenging task. However,
KBJK557 showed great selectivity for Plk1 PBD over PBD3
(Figure 2c) and Plk2 is expressed only in the G1 stage of the cell
cycle.29 Thus, the effect of KBJK557 on the cell cycle and
proliferation (below) is likely caused by inhibiting Plk1 PBD.
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Disruption of Plk1 Localization by KBJK557. To
determine the ability of KBJK557 to interfere with the function
of Plk1 PBD, we examined the intracellular localization capacity
of Plk1 after treating the cells with KBJK557. BI2536, a well-
characterized ATP inhibitor against Plk1, was included for
comparison. To this end, asynchronously growing HeLa cells
were treated with either control DMSO or KBJK557 for 18 h,
immunostained, and subjected to confocal imaging analysis
(Figure 3a). Quantification of Plk1 intensities at kinetochores
showed that the Plk1 signal (red) was strong in both DMSO and
nocodazole-treated samples. In contrast, the Plk1 signal was
effectively (∼60.95%) decreased in cells treated with KBJK557.
Cells treated with BI2536 also substantially (∼68.21%) reduced
the level of Plk1 localized to the mitotic kinetochore (Figure
3b). This is not surprising because Plk1 catalytic activity is
required to localize to kinetochores through a mechanism called
self-priming and binding.1 Collectively, these results strongly
suggest that KBJK557 delocalizes Plk1 from its endogenous
localization sites by competitively blocking Plk1 PBD from
binding to its targets at the kinetochore.
Effect of KBJK557 on the Cell Cycle. Next, we analyzed
the effect of KBJK557 on cell cycle progression in HeLa cells
using flow cytometry analysis. HeLa cells were treated with
KBJK557 and BI2536 for 24 h. Interestingly, cells treated with
KBJK557 induced a partial but significant G2/M arrest
(compare 27% with 18% observed in control cells) at a level
similar to that of BI2536 (Figure 3c). Notably, KBJK557 also
significantly delayed S-phase progression, as indicated by ∼11%
increase of the cell population at this stage.
Effect of KBJK557 on the Plk1 PBD−Cdc25C Inter-
action. Plk1 PBD plays a key role in promoting mitotic entry at
the G2/M phase of the cell cycle by phosphorylating and
activating Cdc25C through its PBD-dependent interaction.30
Therefore, we directly examined whether KBJK557 can inhibit
the interaction between Plk1 PBD and its phosphorylated
binding target, p-Cdc25C. The GST-PBD H538A K540M
(AM) mutant defective in recognizing its phosphorylated
targets31 was used as a control. As shown in Figure S2, WT
PBD effectively interacted with phosphorylated Cdc25C,
whereas its respective AM mutant did not. Under these
conditions, KBJK557 substantially diminished the level of
phosphorylated Cdc25C coprecipitated with GST-Plk1 PBD.
These results confirm that KBJK557 has a capacity to
competitively inhibit Plk1 PBD-dependent interaction against
its binding targets.
Induction of Apoptotic Cell Death in Cancer Cells. The
ability of KBJK557 to specifically inhibit PBD-dependent
interaction and induce a partial G2/M arrest prompted us to
investigate whether it can induce apoptosis in HeLa cells. In a
fluorescence apoptosis assay that shows live cells in green and
dead cells in red color (Figure 4a), we observed that treatment of
HeLa cells with KBJK557 resulted in a significant level of
apoptotic cell death (24.6% at 24 h and 71.1% at 48 h). Under
these conditions, BI2536 induced slightly more apoptotic cell
death than KBJK557 (31.1% at 24 h and 91.5% at 48 h) (Figure
4b).
Figure 4. Induction of apoptosis in HeLa cells by KBJK557. (a) Cells
were treated with BI2536 and KBJK557 for 24 h. Green color indicates
live cells and red color indicates dead cells. (b) Number of live or dead
cells is shown by a bar graph. All images were observed using a
fluorescence microscope (100× magnification). **p < 0.01.
reached the nucleus. These results confirm that our compound
can penetrate the cell membrane in vitro, which paves the way for
the possibility to perform the in vivo tumor-targeting studies.
Optical Imaging Probe for Tumor Diagnosis. In order to
analyze the in vivo tumor-targeting efficacy of KBJK557, tumor-
bearing mice were injected with the Cy5-conjugated KBJK557
(Scheme S2) via a tail vein and subjected to optical imaging
analysis. Fluorescence imaging was carried out at 6, 24, 30, and
48 h after injecting the compound. Strikingly, we observed that
tumors emerging from the HeLa cell xenografts exhibited a
significant fluorescent signal along with organs, including liver,
kidney, heart, and spleen. This is not surprising because drugs
are easily distributed in these highly perfused organs. In
addition, a higher level of uptake in the liver and kidney can
also be attributable to the fact that drug elimination occurs
through these organs. However, the signal intensities of most of
the organs were found to decrease gradually in a time-dependent
manner (Figure 5a). The quantitative evaluation of tumor and
muscle fluorescence intensity showed that the fluorescence
intensity of tumor (1.75 0.12 × 109 p/s/cm2/sr) was ∼4.2-
fold higher than that of muscle (4.18 0.24 × 108 p/s/cm2/sr)
at 48 h (Figure 5b). The finding that KBJK557 was significantly
accumulated in tumors but not in the surrounding normal
Cellular Uptake of the Fluorescence-Conjugated
Inhibitor. To assess the cellular uptake of KBJK557 in vitro,
we performed fluorescence imaging analysis using Cy5-
conjugated KBJK557 (Scheme S2). As shown in the confocal
microscopic scanning image (Figure S3), Cy5-conjugated
KBJK557 (red) colocalized with DAPI-stained chromosomes
(arrows), suggesting that KBJK557 penetrated the cytosol and
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Figure 5. Fluorescence images of Cy5-conjugated KBJK557 in HeLa tumor-bearing mice. (a) Fluorescence images were obtained at 6, 24, 30, and 48 h
after injection of Cy5-conjugated KBJK557 (5 mg/kg body weight/200 μL). Red arrows indicated tumors of mice. (b) ROI analysis of fluorescence
intensity in tumors and muscles from fluorescence images. Mice tumors were measured in at least five points. The “n” means mice number. (c) After in
vivo fluorescence images, mice were sacrificed and ex vivo fluorescence images were obtained (24 and 48 h). (d) Fluorescence intensity in organs and
the tumor were measured at 24 and 48 h. Values represent mean average radiance and the error bars represent SDs. Significant differences at **p < 0.01
(unpaired two-tailed t-test).
Figure 6. In vivo anticancer effect of KBJK557 in a xenograft mice model. (a) Tumor volume changes after the treatment of BI2536 and KBJK557 (5
mg/kg body weight) for 29 days. Tumor size was monitored and measured at 3−4-day intervals. **p < 0.01 (unpaired two-tailed t-test). Values
represent the average SEM (n = 3 per group). Tumor volume = Vt (measurement of the tumor volume) − V0 (initial tumor volume). (b) Following
the injection of BI2536 and KBJK557 for 29 days, tumors were harvested. (c) Bodyweight changes (%). The average body weight changes were
determined in the control, BI2536-, and KBJK557-treated groups in the cancer xenograft mice model during 29 days.
tissues and slowly perfused tissues, such as muscles (Figure 5a),
suggests that KBJK557 has a level of tendency to accumulate in
tumor tissues. Consistent with this notion, the ratios of a tumor
to muscle were found to be 2.1-, 3.9-, and 4.11-fold at 6, 24, and
30 h, respectively (Figure 5b). In preinjection fluorescence
images, the fluorescence signals that appeared in the peritoneum
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of mice were due to the feed, which was found to decrease slowly
over time because of the fasting after the injection.
To further investigate the biodistribution of Cy5-conjugated
KBJK557, the mice were sacrificed and samples were collected at
24 and 48 h after the injection (Figure 5c). The biodistribution
studies revealed that the fluorescence intensity of the liver,
kidney, spleen, and tumor was manifest. In addition, the region
of interest (ROI analysis) of tumors were 1.78 0.09 × 109 p/s/
cm2/sr at 24 h and 1.31 0.11 × 109 p/s/cm2/sr at 48 h,
respectively (Figure 5d). These results further support our view
that Cy5-conjugated KBJK557 exhibits a tendency to accumu-
late in tumor cells and tissues. Moreover, significant
accumulation of KBJK557 in tumor cells suggests that Cy5-
conjugated KBJK557 has a potential to be further developed as a
tumor diagnostic agent.
Figure 7. Concentration−time profile of KBJK557 in the mouse plasma
after an intravenous administration of KBJK557 (mean SD, n = 5).
Anticancer Effect of KBJK557 in Tumor-Bearing Mice.
Inspired by the tumor-targeting ability, KBJK557 was examined
for its chemotherapeutic ability in mice bearing HeLa cell
xenograft tumors, which were treated with PBS, BI2536, and
KBJK557 for 29 days (n = 3). The tumor growth of the BI2536-
and KBJK557-treated group was remarkably reduced compared
to that of the PBS-treated group (Figure 6a,b). The tumor sizes
in the PBS-treated group were increased to 68.36 2.97, 106.05
Table 1. Pharmacokinetic Profiles of KBJK557 (Mean SD)
parameters
I.V. (5 mg/kg)
AUClast (h·ng/mL)
AUC∞ (h·ng/mL)
11,504.31 369.10
11,535.29 371.69
7.73 0.46
t
1/2 (h)
CL (L/h/kg)
ss (L/kg)
MRT (h)
0.43 0.01
0.31 0.04
0.72 0.10
3.08, and 153.57
5.64 mm3 after 15, 22, and 29 days,
V
respectively. In contrast, both BI2536- and KBJK557-treated
groups showed tumor sizes substantially smaller than those of
the control PBS-treated group. The highest antitumor effect of
KBJK557 (60.78%) was observed on the 25th day of treatment,
compared with the control group. To further verify that the
reduction of tumor size is not due to the stress and external
factors, we examined the body weight in all three groups and
found no dramatic changes (Figure 6c). Taken together, these
results suggest that KBJK557 exhibits an antitumorigenic
activity, which could be attributable to its selective tumor-
targeting ability but not surrounding normal tissues (Figure 5).
Its ability to antagonize Plk1 localization and function and its
antitumorigenic effect in a mouse xenograft model demonstrate
that KBJK557 has a great potential to become a model
compound for developing anti-Plk1 PBD agents.
arylidene, categorized as a substructure of pan assay interference
compounds (PAINS),32 we analyzed the PAINS using http://
zinc15.docking.org/. As expected, the alkene was found to show
PAINS during the evaluation. Thus to verify this aspect, we
evaluated KBJK557’s anti-Plk1 PBD activity in the presence of
L-Cys (Figure 8), whose thiols would react with activated
Pharmacokinetics. To evaluate the relative safety of
employing KBJK557 in vivo, we performed pharmacokinetic
studies using rodents and the pharmacokinetic profile of the
compound was examined. Male ICR mice were used and the
blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and
24 h after intravenous administration at a dose of 5 mg/kg. The
blood samples were centrifuged immediately after collection and
the concentrations of the plasma fractions were determined
using an Agilent LC−MS/MS system (Figure 7). The
pharmacokinetic profiles of KBJK557 were obtained by
noncompartmental analysis (Table 1). The organ tissues (i.e.,
liver, spleen, and kidney) were isolated at 2, 6, and 24 h after
intravenous administration at a dose of 5 mg/kg. Intravenous
injection of KBJK557 resulted in the intermediate level of
clearance (CL, 0.43 L/h/kg), half-life (t1/2, 7.73 h), and
sufficient systemic exposure (AUC, 11.5 μg·h/mL) in the body.
The partition coefficients were evaluated as 0.166, 0.045, and
0.182 in liver, spleen and kidney, respectively, which was
calculated by the ratio of AUClast for each tissue and plasma.
These data suggest that KBJK557 possesses significant
pharmacokinetic properties that can be further developed into
anticancer therapeutics.
Figure 8. FP assays with L-cysteine. millipolarization (mP) values were
determined 30 min after incubation. Data were plotted using GraphPad
Prism software.
Michael acceptors. The result showed, however, that L-Cys
failed to diminish KBJK557’s anti-Plk1 PBD activity, even when
it was provided at 30-fold higher concentration than PBD
(Figure 8). This result suggests that KBJK557 does not behave
like PAINS molecules; instead, it inhibits Plk1 PBD through
specific interaction.
Molecular Modeling. After virtual screening, in view of
understanding the molecular interactions responsible for the
Plk1 PBD inhibitory effect, we performed molecular docking
studies with the initial hit compound, 4a, using the UCSF
DOCK6.9 program (Figure 9). The results revealed that
trioxotetrahydropyrimidin (barbituric acid arylidene) in 4a
could undergo tight electrostatic interactions with the His538,
Lys540, and Asn533 residues present in the PBD lattice. Further,
pyrazole in 4a involved in π−π stacking interactions with
Pan Assay Interference Compounds. As our lead
compound consists of a Michael acceptor, barbituric acid
H
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Figure 9. Modeling structures of Plk1 PBD with the (a) 3RQ7 ligand (PDB ID 3RQ7),20 (b) hit compound 4a, (c) KBJK557, (d) overlaid structures
of the 3RQ7 ligand, 4a, and KBJK557, (e) interacting sites of 4a in Plk1 PBD, and (f) interacting sites of KBK557 in Plk1 PBD. Modeling was done
using the DOCK6.9 program.
Trp414. In the subsequent docking analysis with the most
potent molecule KBJK557, we also found that trioxotetrahy-
dropyrimidin (barbituric acid arylidene) and pyrazole in
KBJK557 engage in electrostatic interactions with His538,
Lys540, and Asn533 and π−π stacking interactions with Trp414,
respectively. Additionally, the alkyl tail attached to the N1 of
pyrazole projected toward the tyrosine-rich channel. The phenyl
group in the alkyl tail engaged in a π−π stacking interaction with
Tyr417, which might have contributed to the enhanced anti-
Plk1 PBD activity. These three interaction patterns are similar to
those observed with the 3RQ7 ligand in Plk1 PBD.
T521 was reported to show antitumor activity by inhibiting Plk1
PBD selectively by covalent binding. Consequently, it induced
defects in centrosome integrity, chromosome alignment, and
spindle assembly and subsequent apoptosis in HeLa cells.
However, compared to KBJK557, the cancer-targeting ability of
T521 is not clear because antitumorigenic activity in a mouse
xenograft tumor model was demonstrated by an intratumoral
injection method.27 Moreover, the cancer-targeting ability of the
abovementioned Plk1 PBD inhibitors using imaging studies in a
mouse xenograft model has not yet been investigated.
The data provided here suggest that a pyrazolopyrimidine-
fused small-molecule scaffold offers promising structural and
chemical features that can be further exploited for the discovery
of a new class of Plk1 PBD inhibitor. At the cellular level,
KBJK557 is capable of effectively delocalizing Plk1 from its
subcellular localization sites and consequently inducing mitotic
arrest and apoptotic cell death in cancer cells. These
observations demonstrate that KBJK557 can overcome poor
cell permeability commonly associated with peptide-derived
Plk1 PBD inhibitors and provide proof of principle that
specifically inhibiting PBD-dependent interaction is sufficient
to disrupt Plk1 function.
DISCUSSION AND CONCLUSIONS
■
Overexpression of Plk1 is predominant in many cancer cells;
thus Plk1 is considered a valid target for developing anticancer
therapeutics. As Plk1 KD inhibitors developed so far largely
exhibit a high level of side effects because of their cross-
reactivities, PBD has drawn a lot of attention as an alternative
target for anti-Plk1 drug discovery. Even though peptide-based
inhibitors targeting Plk1 PBD showed considerable potency in
vitro, achieving high in vivo efficacy remains challenging because
of their poor cell permeability and stability. To date, a very few
small-molecule Plk1 PBD inhibitors, including T521,27 TQ,29
and poloxin123 have been investigated in vivo. However, they
required high dosage levels to display appreciable effects in
cancer treatment. In particular, the sulfonyl−oxazole inhibitor
Most of the conventional chemotherapeutic drugs possess
severe setbacks in distinguishing the cancer cells from normal
cells. Thus, identifying a cancer drug with considerable
selectivity toward the cancer cells remains the greatest challenge
I
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in the field of cancer therapy.33 Here, we show that, in an optical
imaging study, Cy5-conjugated KBJK557 significantly accumu-
lated in tumors but not their surrounding normal tissues. The
liver, kidney, heart, and spleen also exhibited a significant level of
Cy5-KBJK557 uptake. However, the fluorescence intensity
gradually decreased in a time-dependent manner. The high-level
uptake in the liver and kidney is likely due to the systemic
circulation of body metabolite and subsequent elimination
through these organs.34 Spleen tissue accumulation is closely
related to the reticuloendothelial system organs.35,36 In addition,
to monitor the effects of KBJK557 in vivo, we performed the
pharmacokinetics study (Table 1). The pharmacokinetic
profiles and partition coefficients suggested the systemic
exposure of KBJK557 in the blood, thereby distributing it to
the highly perfused organs such as liver, kidney, heart, and spleen
after intravenous administration in mouse. The accumulation of
KBJK557 in the foreleg xenograft tumors hints that KBJK557
may have a capacity to target tumor sites with a level of
selectivity. Consistent with these observations, KBJK557
exhibited a significant level of antitumorigenic activities in a
mouse xenograft tumor model, comparable to the well-
characterized Plk1 KD inhibitor, BI2536.28
2H), 7.55−7.45 (m, 3H), 7.44−7.38 (m, 3H), 7.38−7.33 (m, 2H), 5.39
(s, 2H). 13C NMR (101 MHz, CDCl3): δ 185.1, 154.2, 134.6, 133.6,
131.6, 129.1, 129.0, 128.9, 128.8, 128.7, 128.4, 121.3, 56.7. MALDI-
TOF m/z: calcd for C17H14N2O, 262.11; found, 262.35.
1-(4-(Methylthio)benzyl)-3-phenyl-1H-pyrazole-4-carbaldehyde
(2c). Compound 2c was synthesized from 3-phenyl-1H-pyrazole-4-
carbaldehyde (1), 4-(methylthio)benzyl bromide (c) and potassium
1
carbonate by following general procedure A (white solid, 76%). H
NMR (400 MHz, CDCl3): δ 9.92 (s, 1H), 7.93 (s, 1H), 7.72 (dd, J =
7.9, 1.5 Hz, 2H), 7.56−7.36 (m, 3H), 7.35−7.18 (m, 4H), 5.31 (s, 2H),
2.49 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 185.0, 154.2, 139.7,
133.6, 131.5, 131.1, 129.0, 128.9, 128.9, 128.7, 126.8, 121.3, 56.2, 15.5.
MALDI-TOF m/z: calcd for C18H16N2OS, 308.09; found, 308.24.
3-Phenyl-1-(4-(trifluoromethoxy)benzyl)-1H-pyrazole-4-carbal-
dehyde (2d). Compound 2d was synthesized from 3-phenyl-1H-
pyrazole-4-carbaldehyde (1), 4-(trifluoromethoxy)benzyl bromide (d)
and potassium carbonate by following general procedure A (white solid,
90%). 1H NMR (400 MHz, CDCl3): δ 9.96 (s, 1H), 8.03 (s, 1H), 7.75
(d, J = 7.1 Hz, 2H), 7.56−7.43 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.26
(d, J = 8.2 Hz, 2H), 5.38 (s, 2H). 13C NMR (101 MHz, CDCl3): δ
185.1, 154.4, 149.4, 133.6, 133.5, 131.4, 129.7, 129.2, 128.9, 128.8,
121.5, 121.4, 55.8. MALDI-TOF m/z: calcd for C18H13F3N2O2, 346.09;
found, 346.18.
3-Phenyl-1-(3-(trifluoromethoxy)benzyl)-1H-pyrazole-4-carbal-
dehyde (2e). Compound 2e was synthesized from 3-phenyl-1H-
pyrazole-4-carbaldehyde (1), 3-(trifluoromethoxy)benzyl bromide (e)
and potassium carbonate by following general procedure A (white semi
solid, 91%). 1H NMR (400 MHz, CDCl3): δ 9.95 (s, 1H), 8.01 (s, 1H),
7.73 (dd, J = 7.9, 1.6 Hz, 2H), 7.62−7.32 (m, 4H), 7.25−7.16 (m, 3H),
5.38 (s, 2H), 1.62 (s, 1H). 13C NMR (101 MHz, CDCl3): δ 185.1,
154.5, 149.7, 137.1, 133.9, 131.4, 130.6, 129.2, 128.9, 128.8, 126.3,
121.6, 121.0, 120.6, 56.0. MALDI-TOF m/z: calcd for C18H13F3N2O2,
346.09; found, 346.17.
In summary, our data suggest that KBJK557 is the first small-
molecule Plk1 PBD inhibitor that selectively disrupts PBD-
dependent interaction in vitro and exhibits a significant level of in
vivo tumor-targeting and treatment effect. KBJK557 appears to
have a great potential to be a model for designing anticancer
drugs that show selective cancer-targeting ability by targeting
Plk1 PBD.
1-Phenethyl-3-phenyl-1H-pyrazole-4-carbaldehyde (2f). Com-
pound 2f was synthesized from 3-phenyl-1H-pyrazole-4-carbaldehyde
(1), 2-phenylethyl bromide (f), and potassium carbonate by following
general procedure A (colorless oil, 91%). 1H NMR (400 MHz, CDCl3):
δ 9.88 (s, 1H), 8.03−7.65 (m, 3H), 7.58−7.37 (m, 3H), 7.37−7.17 (m,
3H), 7.10 (d, J = 6.7 Hz, 2H), 4.40 (t, J = 7.2 Hz, 2H), 3.24 (t, J = 7.2
Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 185.0, 154.2, 137.2, 134.1,
131.7, 129.0, 128.9, 128.8, 128.7, 128.6, 127.0, 120.6, 54.3, 36.4.
MALDI-TOF m/z: calcd for C18H16N2O, 276.13; found, 346.34.
3-Phenyl-1-(2-(piperidin-1-yl)ethyl)-1H-pyrazole-4-carbaldehyde
(2g). Compound 2g was synthesized from 3-phenyl-1H-pyrazole-4-
carbaldehyde (1), 1-(2-chloroethyl)piperidine hydrochloride (g), and
potassium carbonate by following general procedure A (colorless oil,
46%). 1H NMR (400 MHz, CDCl3): δ 9.94 (s, 1H), 8.14 (s, 1H), 7.72
(dd, J = 8.0, 1.4 Hz, 2H), 7.58−7.33 (m, 3H), 4.28 (t, J = 6.5 Hz, 2H),
2.81 (t, J = 6.5 Hz, 2H), 2.65−2.33 (m, 4H), 1.58 (dt, J = 11.0, 5.6 Hz,
4H), 1.50−1.33 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 185.1,
153.8, 134.4, 131.7, 129.0, 128.9, 128.7, 120.8, 58.0, 54.6, 50.5, 26.0,
24.2. MALDI-TOF m/z: calcd for C17H21N3O, 283.16; found, 283.38.
1-(2-Morpholinoethyl)-3-phenyl-1H-pyrazole-4-carbaldehyde
(2h). Compound 2h was synthesized from 3-phenyl-1H-pyrazole-4-
carbaldehyde (1), 4-(2-chloroethyl)morpholine hydrochloride (h) and
potassium carbonate by following general procedure A (pale yellow oil,
42%). 1H NMR (400 MHz, CDCl3): δ 9.95 (s, 1H), 8.13 (s, 1H), 7.72
(dd, J = 7.9, 1.5 Hz, 2H), 7.53−7.42 (m, 3H), 4.30 (t, J = 6.3 Hz, 2H),
3.77−3.65 (m, 4H), 2.88 (t, J = 6.3 Hz, 2H), 2.59−2.43 (m, 4H). 13C
NMR (101 MHz, CDCl3): δ 185.0, 153.8, 134.4, 131.6, 129.0, 128.8,
128.6, 120.9, 66.8, 57.6, 53.5, 50.0. MALDI-TOF m/z: calcd for
C16H19N3O2, 285.14; found, 285.37.
EXPERIMENTAL SECTION
■
General Chemistry Methods. All reagents and starting materials
were purchased from commercial chemical suppliers (Sigma-Aldrich,
TCI and Across Organics) and used as received. All the anhydrous
organic solvents of purity greater than 99.9% were purchased from
Aldrich and used directly. Thin-layer chromatography (TLC) was
performed on Merck aluminum sheets with silica gel 60 F254 and they
were visualized by ultraviolet light and staining with KMnO4, PMA
stain (phosphomolybdic acid), and ninhydrin. For the purification of
compounds, column chromatography was performed on Merck silica
gel 60 (70−230 mesh or 230−400 mesh). NMR spectra including 1H
and 13C NMR spectra were recorded on a Bruker DRX-400 and DRX-
500 spectrometer. Chemical shifts (δ) are reported in parts per million
(ppm) measured relative to an internal standard and coupling constants
(J) are expressed in hertz (Hz). Mass spectra were recorded using a
Shimadzu (MALDI-TOF) mass spectrometer. The cyanine-conjugated
compound was purified by preparative reversed-phase high-perform-
ance liquid chromatography (RP-HPLC, YL9100, Younglin, Korea)
and determined to be >95% pure by analytical HPLC [C18 column (4.6
× 250 mm)]. Two different linear gradients of 0.05% aqueous TFA
(eluent A) and 0.05% TFA in CH3CN (10−90 over 30 min, eluent B)
were used at a flow rate of 1.5 mL per min at 25 °C.
General Procedure A for the Synthesis of 2(b−j). 1-Benzyl-3-
phenyl-1H-pyrazole-4-carbaldehyde (2b). 3-Phenyl-1H-pyrazole-4-
carbaldehyde (1) (0.67 g, 0.003912 mol) in anhydrous DMF (5 mL)
was added slowly to a stirred solution of benzyl bromide (b) (0.604 g,
0.00355 mol) and potassium carbonate (1.47 g, 0.01065 mol) in
anhydrous DMF (20 mL). After completion of the addition, the
temperature was slowly raised to 60 °C and the solution was stirred for
16 h. Then, the reaction mixture was quenched by the addition of water
(30 mL) and extracted with ethyl acetate (30 mL × 3). The combined
organic extracts were washed with brine (40 mL), dried over Na2SO4,
and evaporated. The crude product was purified by silica gel column
chromatography using a hexane/dichloromethane/ethyl acetate
3-Phenyl-1-(4-phenylbutyl)-1H-pyrazole-4-carbaldehyde (2i).
Compound 2i was synthesized from 3-phenyl-1H-pyrazole-4-carbalde-
hyde (1), 4-phenylbutyl bromide (i), and potassium carbonate by
1
following general procedure A (colorless oil, 86%). H NMR (400
MHz, CDCl3): δ 9.96 (s, 1H), 8.00 (s, 1H), 7.73 (dd, J = 7.7, 1.8 Hz,
2H), 7.53−7.40 (m, 3H), 7.34−7.27 (m, 3H), 7.25−7.15 (m, 3H), 4.21
(t, J = 7.1 Hz, 2H), 2.69 (t, J = 7.5 Hz, 2H), 1.99 (p, J = 7.4 Hz, 2H),
1.78−1.63 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 185.1, 154.0,
1
(1:1:0.2) mixture to afford 2b as a white solid (0.67 g, 73%). H
NMR (400 MHz, CDCl3): δ 9.95 (s, 1H), 7.96 (s, 1H), 7.84−7.69 (m,
J
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141.5, 133.4, 131.7, 129.0, 128.9, 128.7, 128.4, 128.4, 126.0, 120.8, 52.8,
35.2, 29.4, 28.2. MALDI-TOF m/z: calcd for C20H20N2O, 304.15;
found, 304.32.
5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-1,3-diphenethyl-
pyrimidine-2,4,6(1H,3H,5H)-trione (4d). Compound 4d was synthe-
sized from 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde and 3b using
piperidine by following general procedure C (pale yellow solid, 81%).
1H NMR (400 MHz, CDCl3): δ 9.84 (s, 1H), 8.61 (s, 1H), 7.95 (d, J =
7.6 Hz, 2H), 7.68 (dd, J = 7.9, 1.6 Hz, 2H), 7.63−7.53 (m, 5H), 7.45 (t,
J = 7.4 Hz, 1H), 7.38−7.29 (m, 8H), 7.27−7.20 (m, 2H), 4.34−4.12
(m, 4H), 3.05−2.83 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ
162.4, 161.3, 158.7, 150.7, 145.4, 139.0, 138.9, 134.9, 131.1, 130.4,
130.0, 129.5, 129.2, 129.1, 128.9, 128.9, 128.7, 126.8, 120.3, 115.7,
114.4, 43.3, 42.6, 33.9. MALDI-TOF m/z: calcd for C36H30N4O3,
566.23; found, 566.80.
5-((1-Benzyl-3-phenyl-1H-pyrazol-4-yl)methylene)pyrimidine-
2,4,6(1H,3H,5H)-trione (4e). Compound 4e was synthesized from 2b
and barbituric acid using piperidine by following general procedure C
(pale yellow solid, 88%). 1H NMR (400 MHz, DMSO-d6): δ 11.20 (s,
2H), 9.41 (s, 1H), 8.14 (s, 1H), 7.68−7.47 (m, 3H), 7.43−7.29 (m,
2H), 5.56 (s, 1H). 13C NMR (101 MHz, DMSO-d6): δ 164.3, 163.2,
157.9, 150.6, 144.7, 138.6, 136.7, 131.6, 130.0, 129.5, 129.3, 129.2,
128.6, 128.5, 114.0, 113.3, 55.6. MALDI-TOF m/z: calcd for
C21H16N4O3, 372.12; found, 372.16.
5-((1-(4-(Methylthio)benzyl)-3-phenyl-1H-pyrazol-4-yl)-
methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4f). Compound 4f
was synthesized from 2c and barbituric acid using piperidine by
following general procedure C (pale yellow solid, 85%). 1H NMR (400
MHz, DMSO-d6): δ 11.23 (m, 1H), 11.19 (m, 1H), 9.40 (s, 1H), 8.13
(s, 1H), 7.61−7.43 (m, 5H), 7.36 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3
Hz, 2H), 5.51 (s, 2H), 2.46 (s, 3H). 13C NMR (101 MHz, DMSO-d6):
δ 164.3, 163.2, 157.9, 150.7, 144.8, 138.8, 138.5, 133.0, 132.6, 130.0,
129.5, 129.4, 129.3, 126.5, 114.0, 113.3, 55.5, 15.0. MALDI-TOF m/z:
calcd for C22H18N4O3S, 418.11; found, 440.69 (M + Na)+.
3-Phenyl-1-(8-phenyloctyl)-1H-pyrazole-4-carbaldehyde (2j).
Compound 2j was synthesized from 3-phenyl-1H-pyrazole-4-carbalde-
hyde (1), 8-phenyloctyl bromide (j), and potassium carbonate by
1
following general procedure A (colorless oil, 87%). H NMR (400
MHz, CDCl3): δ 9.97 (s, 1H), 8.03 (s, 1H), 7.84−7.69 (m, 2H), 7.55−
7.42 (m, 3H), 7.38−7.23 (m, 3H), 7.19 (d, J = 7.4 Hz, 3H), 4.19 (t, J =
7.2 Hz, 2H), 2.61 (t, J = 7.7 Hz, 2H), 2.11−1.85 (m, 2H), 1.83−1.45
(m, 3H), 1.45−1.14 (m, 9H). 13C NMR (101 MHz, CDCl3): δ 185.2,
154.1, 142.8, 133.5, 131.8, 129.0, 128.9, 128.7, 128.4, 128.3, 125.6,
120.8, 53.0, 36.0, 31.4, 29.9, 29.3, 29.2, 29.0, 26.5. MALDI-TOF m/z:
calcd for C24H28N2O, 360.22; found, 360.27.
General Procedure B for the Synthesis of 3(a,b). 1,3-
Dipropylpyrimidine-2,4,6(1H,3H,5H)-trione (3a). 6a (220 mg, 1.5
mmol) was added to a stirred solution of malonic acid (150 mg, 1.5
mmol) in acetic acid (5 mL) and acetic anhydride (3 mL). The
resultant solution was heated to 90 °C for 4 h and cooled to room
temperature. Then, the reaction mixture was treated with water (20
mL) and extracted with ethyl acetate (3 × 10 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, and
evaporated. The resultant residue was purified by silica gel column
chromatography (hexane−ethyl acetate, 3:1) to afford 3a as a white
solid (160 mg, 51%). 1H NMR (400 MHz, CDCl3): δ 4.02−3.75 (m,
4H), 3.67 (s, 2H), 1.75−1.51 (m, 4H), 0.95 (t, J = 7.5 Hz, 6H). 13C
NMR (101 MHz, CDCl3): δ 164.6, 151.4, 43.5, 39.7, 21.3, 11.2.
MALDI-TOF m/z: calcd for C10H16N2O3, 212.11; found, 211.37.
1,3-Diphenethylpyrimidine-2,4,6(1H,3H,5H)-trione (3b). Com-
pound 3b was synthesized from 6b and malonic acid using acetic
acid and acetic anhydride by following general procedure B (white
solid, 57%). 1H NMR (400 MHz, CDCl3): δ 7.44−7.12 (m, 10H), 4.11
(t, J = 7 Hz, 4H), 3.58 (s, 2H), 2.89 (t, J = 7.4 Hz, 1H). 13C NMR (101
MHz, CDCl3): δ 164.3, 152.0, 137.8, 129.0, 128.6, 126.8, 43.0, 39.5,
34.0.
General Procedure C for the Synthesis of 4(a−o). 5-((1,3-
Diphenyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-
trione (4a). Piperidine 10 μL was added to a solution of 1,3-diphenyl-
1H-pyrazole-4-carboxaldehyde (0.36 g, 1.46 mmol) and barbituric acid
(0.170 g, 1.32 mmol) in methanol (10 mL) and stirred for 16 h. The
resultant yellow solid was filtered and washed with cold methanol (2 ×
5 mL) and dried to yield a pure compound 4a as a yellow solid (433 mg,
91%). 1H NMR (400 MHz, DMSO-d6): δ 11.35 (s, 1H), 11.33 (s, 1H),
9.81 (s, 1H), 8.19 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.70−7.54 (m, 7H),
7.48 (t, J = 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): δ 163.6,
162.7, 157.9, 150.3, 143.5, 138.6, 130.8, 129.9, 129.5, 129.4, 128.9,
128.1, 119.7, 115.2, 114.5. MALDI-TOF m/z: calcd for C20H14N4O3,
358.10; found, 358.71.
5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-1,3-dimethylpyri-
midine-2,4,6(1H,3H,5H)-trione (4b). Compound 4b was synthesized
from 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde and 1,3-dimethyl-
barbituric acid using piperidine by following general procedure C
(yellow solid, 74%). 1H NMR (400 MHz, CDCl3): δ 9.91 (s, 1H), 8.63
(s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 7.0 Hz, 2H), 7.61−7.49
(m, 5H), 7.43 (t, J = 7.3 Hz, 1H), 3.46 (s, 3H), 3.42 (s, 3H). 13C NMR
(101 MHz, CDCl3): δ 162.9, 161.7, 159.6, 151.5, 148.0, 139.0, 135.1,
130.9, 129.8, 129.6, 129.5, 129.0, 128.1, 120.1, 116.1, 112.6, 28.9, 28.3.
MALDI-TOF m/z: calcd for C22H18N4O3, 386.13; found, 386.14.
5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-1,3-dipropylpyri-
midine-2,4,6(1H,3H,5H)-trione (4c). Compound 4c was synthesized
from 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde and 1,3-dipropylbar-
bituric acid (3a) using piperidine by following general procedure C
(pale yellow solid, 71%). 1H NMR (400 MHz, CDCl3): δ 9.91 (s, 1H),
8.63 (s, 1H), 8.01−7.84 (m, 2H), 7.67 (dd, J = 7.9, 1.5 Hz, 2H), 7.61−
7.51 (m, 5H), 7.43 (t, J = 7.4 Hz, 1H), 7.28 (s, 1H), 4.12−3.84 (m, 4H),
1.90−1.59 (m, 4H), 1.13−0.83 (m, 6H). 13C NMR (101 MHz, DMSO-
d6): δ 162.5, 161.5, 158.7, 151.1, 145.2, 139.0, 134.9, 131.2, 130.4,
130.0, 129.9, 129.4, 128.7, 120.3, 115.8, 114.5, 43.6, 43.0, 21.2, 11.7,
11.6. MALDI-TOF m/z: calcd for C26H26N4O3, 442.20; found, 442.11.
5-((3-Phenyl-1-(4-(trifluoromethoxy)benzyl)-1H-pyrazol-4-yl)-
methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4g). Compound 4g
was synthesized from 2d and barbituric acid using piperidine by
following general procedure C (pale yellow solid, 40%). 1H NMR (400
MHz, DMSO-d6): δ 11.23 (s, 2H), 9.47 (s, 1H), 8.15 (s, 1H), 7.64−
7.46 (m, 7H), 7.40 (d, J = 8.1 Hz, 2H), 5.62 (s, 2H). 13C NMR (101
MHz, DMSO-d6): δ 164.3, 163.2, 158.1, 150.7, 148.5, 148.4, 144.7,
138.9, 136.2, 131.6, 130.6, 130.0, 129.6, 129.3, 121.8, 114.1, 113.5, 55.0.
MALDI-TOF m/z: calcd for C22H15F3N4O4, 456.10; found, 455.99.
5-((3-Phenyl-1-(3-(trifluoromethoxy)benzyl)-1H-pyrazol-4-yl)-
methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4h). Compound 4h
was synthesized from 2e and barbituric acid using piperidine by
following general procedure C (pale yellow solid, 47%). 1H NMR (400
MHz, DMSO-d6): δ 11.23 (s, 2H), 9.48 (s, 1H), 8.14 (s, 1H), 7.60−
7.47 (m, 6H), 7.45−7.32 (m, 3H), 5.65 (s, 2H). 13C NMR (101 MHz,
DMSO-d6): δ 164.3, 163.2, 158.1, 150.7, 148.9, 144.6, 139.4, 139.0,
131.5, 131.3, 130.0, 129.6, 129.3, 127.6, 121.0, 114.0, 113.6, 55.0.
MALDI-TOF m/z: calcd for C22H15F3N4O4, 456.10; found, 456.67.
5-((1-Phenethyl-3-phenyl-1H-pyrazol-4-yl)methylene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (4i). Compound 4i was synthe-
sized from 2f and barbituric acid using piperidine by following general
1
procedure C (pale yellow solid, 57%). H NMR (400 MHz, DMSO-
d6): δ 11.18 (s, 2H), 9.24 (s, 1H), 8.12 (s, 1H), 7.65−7.44 (m, 5H),
7.38−7.15 (m, 5H), 4.57 (t, J = 7.3 Hz, 2H), 3.21 (t, J = 7.2 Hz, 2H).
13C NMR (101 MHz, DMSO-d6): δ 164.3, 163.1, 157.8, 150.7, 144.9,
138.7, 138.3, 131.7, 130.0, 129.5, 129.3, 129.2, 128.9, 127.0, 113.6,
112.9, 53.6, 36.0. MALDI-TOF m/z: calcd for C22H18N4O3, 386.13;
found, 386.75.
5-((3-Phenyl-1-(2-(piperidin-1-yl)ethyl)-1H-pyrazol-4-yl)-
methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4j). Compound 4j
was synthesized from 2g and barbituric acid using piperidine by
following general procedure C (pale yellow solid, 66%). 1H NMR (400
MHz, DMSO-d6): δ 11.19 (s, 2H), 9.38 (s, 1H), 8.16 (s, 1H), 7.64−
7.44 (m, 5H), 4.40 (t, J = 6.2 Hz, 2H), 2.74 (t, J = 6.3 Hz, 2H), 2.46−
2.25 (m, 4H), 1.56−1.42 (m, 4H), 1.44−1.25 (m, 2H). 13C NMR (100
MHz, DMSO-d6): δ 164.4, 163.2, 157.5, 150.7, 145.0, 139.1, 131.8,
130.0, 129.4, 129.3, 113.7, 112.8, 58.0, 54.3, 50.2, 26.0, 24.2. MALDI-
TOF m/z: calcd for C21H23N5O3, 393.18; found, 391.78.
K
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5-((1-(2-Morpholinoethyl)-3-phenyl-1H-pyrazol-4-yl)methylene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (4k). Compound 4k was synthe-
sized from 2h and barbituric acid using piperidine by following general
procedure C (yellow solid, 69%). 1H NMR (400 MHz, DMSO-d6): δ
11.28−11.07 (m, 2H), 9.41 (s, 1H), 8.16 (s, 1H), 7.64−7.45 (m, 5H),
4.44 (t, J = 6.1 Hz, 2H), 3.63−3.49 (m, 4H), 2.79 (t, J = 5.9 Hz, 2H),
2.49−2.41 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 164.4, 163.2,
157.4, 150.7, 145.0, 139.2, 131.7, 130.0, 129.5, 129.3, 113.7, 112.9, 66.6,
57.5, 53.5, 49.7. MALDI-TOF m/z: calcd for C20H21N5O4, 395.15;
found, 395.77.
5-((3-Phenyl-1-(4-phenylbutyl)-1H-pyrazol-4-yl)methylene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (4l). Compound 4l was synthe-
sized from 2i and barbituric acid using piperidine by following general
procedure C (pale yellow solid, 80%). H NMR (300 MHz, DMSO-d6):
δ 11.18 (s, 2H), 9.33 (s, 1H), 8.15 (s, 1H), 7.62−7.38 (m, 5H), 7.32−
7.22 (m, 2H), 7.22−7.12 (m, 3H), 4.34 (t, J = 6.9 Hz, 2H), 2.63 (t, J =
7.6 Hz, 2H), 1.99−1.75 (m, 2H), 1.69−1.46 (m, 2H). 13C NMR (100
MHz, DMSO-d6): δ 164.3, 163.2, 157.7, 150.8, 145.0, 142.2, 138.5,
131.8, 129.9, 129.5, 129.3, 128.8, 128.7, 126.2, 113.6, 113.0, 52.3, 34.9,
29.5, 28.2. MALDI-TOF m/z: calcd for C24H22N4O3, 414.16; found,
414.77.
128.5, 126.4, 41.6, 36.5. MALDI-TOF m/z: calcd for C17H20N2O,
268.15; found, 268.38.
tert-Butyl(2-(4-formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)-
carbamate (7). Compound 7 was synthesized from 3-phenyl-1H-
pyrazole-4-carbaldehyde (1), 2-(Boc-amino)ethyl bromide, and
potassium carbonate by following general procedure A (white solid,
70%). 1H NMR (500 MHz, CDCl3): δ 9.98 (s, 1H), 8.04 (s, 1H), 7.75
(d, J = 6.8 Hz, 2H), 7.56−7.41 (m, 3H), 4.87 (s, 1H), 4.34 (s, 2H),
3.75−3.58 (m, 2H), 1.47 (s, 9H). 13C NMR (101 MHz, CDCl3): δ
185.2, 154.6, 150.3, 138.4, 134.8, 131.4, 130.1, 129.1, 128.8, 120.9,
120.3, 80.2, 52.4, 40.4, 28.3.
1-(2-Aminoethyl)-3-phenyl-1H-pyrazole-4-carbaldehyde Hydro-
chloride (8). 2 M HCl (11 mL, 22 mmol) in diethyl ether was added to
the stirred solution of 7 (700 mg, 2.22 mmol) in 10 mL of
dichloromethane. The resultant solution was stirred for 16 h at room
temperature, and the solid formed was filtered and washed with
anhydrous diethyl ether to yield 8 as a pale yellow solid (537 mg, 96%).
1H NMR (400 MHz, DMSO-d6): δ 9.89 (s, 1H), 8.66 (s, 1H), 8.64−
8.36 (m, 3H), 7.90−7.79 (m, 2H), 7.51−7.34 (m, 3H), 4.58 (d, J = 6.1
Hz, 2H), 3.34 (q, J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ
185.0, 152.6, 138.7, 132.1, 129.3, 129.1, 128.9, 121.0, 49.5, 38.7.
MALDI-TOF m/z: calcd for C12H13N3O, 215.10; found, 215.44.
General Procedure E for the Synthesis of 9(a−d). N-(2-(4-
Formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)cyclohexanecarboxamide
(9a). To a stirred solution of EDCI (607 mg, 3.18 mmol), HOBt (428
mg, 3.18 mmol) in DMF (10 mL), cyclohexanecarboxylic acid (406 mg,
3.18 mmol), and DIEA (2.76 mL, 15.86 mmol) were added and stirred
for 30 min. To the resultant solution, 8 (957 mg, 3.82 mmol) in DMF
(5 mL) was added and it was stirred at room temperature for 18 h. The
reaction mixture was treated with 5% NaHCO3 solution (20 mL) and
extracted with ethyl acetate (2 × 15 mL); the combined organic layer
was washed with H2O and brine, dried over Na2SO4, and evaporated
under vacuum. The crude residue was purified by flash column
chromatography [CH2Cl2/ethyl acetate (3:1)] to provide 9a (0.34 g,
43%) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 9.95 (s, 1H),
7.98 (s, 1H), 7.74 (dd, J = 7.8, 1.6 Hz, 2H), 7.56−7.39 (m, 3H), 6.00 (s,
1H), 4.41−4.21 (m, 2H), 3.75 (q, J = 5.7 Hz, 2H), 2.15−1.98 (m, 1H),
1.90−1.70 (m, 4H), 1.69−1.58 (m, 1H), 1.51−1.32 (m, 2H), 1.33−
1.08 (m, 3H). 13C NMR (101 MHz, CDCl3): δ 184.8, 176.7, 154.5,
134.9, 131.7, 129.2, 128.8, 128.7, 120.9, 51.9, 45.3, 39.3, 29.6, 25.7,
25.6. MALDI-TOF m/z: calcd for C19H23N3O2, 325.17; found, 325.81.
N-(2-(4-Formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)benzamide (9b).
Compound 9b was synthesized by following general procedure E
using benzoic acid and amine salt 8 in the presence of EDCI, HOBt, and
DIEA (white solid, 58%). 1H NMR (400 MHz, CDCl3): δ 9.86 (s, 1H),
7.95 (s, 1H), 7.79−7.73 (m, 2H), 7.70 (dd, J = 6.5, 3.1 Hz, 2H), 7.52−
7.42 (m, 4H), 7.41−7.33 (m, 2H), 7.33−7.24 (m, 1H), 4.46−4.32 (m,
2H), 3.92 (q, J = 5.6 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 184.8,
167.8, 154.5, 135.1, 133.9, 131.8, 131.4, 129.2, 128.8, 128.7, 128.6,
127.0, 120.9, 51.8, 40.0. MALDI-TOF m/z: calcd for C19H17N3O2,
319.13; found, 319.78.
5-((3-Phenyl-1-(8-phenyloctyl)-1H-pyrazol-4-yl)methylene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (4m). Compound 4m was synthe-
sized from 2j and barbituric acid using piperidine by following general
1
procedure C (white solid, 80%). H NMR (300 MHz, DMSO-d6): δ
11.20 (s, 2H), 9.32 (s, 1H), 8.15 (s, 1H), 7.60−7.41 (m, 5H), 7.29−
7.21 (m, 2H), 7.20−7.08 (m, 3H), 4.30 (t, J = 6.9 Hz, 2H), 2.65−2.52
(m, 2H), 1.97−1.70 (m, 2H), 1.66−1.41 (m, 2H), 1.39−1.10 (m, 8H).
13C NMR (101 MHz, DMSO-d6): δ 164.4, 163.2, 157.6, 150.8, 144.9,
142.7, 138.4, 131.8, 129.9, 129.4, 129.2, 128.9, 128.6, 126.0, 113.7,
112.9, 52.5, 35.6, 31.4, 29.8, 29.1, 29.0, 28.8, 26.3. MALDI-TOF m/z:
calcd for C28H30N4O3, 470.23; found, 470.79.
1,3-Dimethyl-5-((3-phenyl-1-(4-phenylbutyl)-1H-pyrazol-4-yl)-
methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4n). Compound 4n
was synthesized from 2i and 1,3-dimethylbarbituric acid using
1
piperidine by following general procedure C (white solid, 87%). H
NMR (400 MHz, CDCl3): δ 9.36 (s, 1H), 8.58 (s, 1H), 7.62−7.44 (m,
5H), 7.39−7.26 (m, 2H), 7.21 (t, J = 8.7 Hz, 3H), 4.28 (t, J = 7.2 Hz,
2H), 3.44 (s, 3H), 3.40 (s, 3H), 2.71 (t, J = 7.6 Hz, 2H), 2.16−1.98 (m,
2H), 1.82−1.64 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 163.1,
161.8, 159.1, 151.5, 148.7, 141.6, 138.2, 131.1, 129.7, 129.3, 128.9,
128.4, 128.4, 126.0, 114.4, 111.4, 53.0, 35.3, 29.6, 28.9, 28.3, 28.2.
MALDI-TOF m/z: calcd for C26H26N4O3, 442.20; found, 442.78.
1,3-Dimethyl-5-((3-phenyl-1-(8-phenyloctyl)-1H-pyrazol-4-yl)-
methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4o). Compound 4o
was synthesized from 2j and 1,3-dimethylbarbituric acid using
1
piperidine by following general procedure C (white solid, 89%). H
NMR (400 MHz, CDCl3): δ 9.26 (s, 1H), 8.49 (s, 1H), 7.55−7.33 (m,
5H), 7.18 (t, J = 7.5 Hz, 2H), 7.08 (d, J = 7.6 Hz, 3H), 4.22−4.09 (m,
2H), 3.33 (s, 3H), 3.29 (s, 3H), 2.51 (t, J = 7.7 Hz, 2H), 2.02−1.78 (m,
2H), 1.61−1.43 (m, 2H), 1.35−1.20 (m, 8H). 13C NMR (101 MHz,
CDCl3): δ 163.1, 161.6, 159.1, 151.5, 148.8, 142.8, 138.1, 131.2, 129.7,
129.2, 128.9, 128.4, 128.3, 125.6, 114.4, 111.3, 53.2, 35.9, 31.4, 30.0,
29.3, 29.2, 29.0, 28.8, 28.2, 26.6. MALDI-TOF m/z: calcd for
C30H34N4O3, 498.26; found, 498.81.
General Procedure D for the Synthesis of 6(a,b). 1,3-
Dipropylurea (6a). To a stirred solution of S,S′-dimethyl dithiocar-
bonate (1.17 g, 5 mmol) in methanol (10 mL), propylamine (0.719 g
10.5 mmol) was added and heated to 60 °C for 24 h. The reaction
mixture was evaporated under vacuum, and the resultant crude mixture
was treated with diethyl ether and hexane to obtain the product 6a (308
mg, 43%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 4.48 (s, 2H),
3.14 (q, J = 7.4 Hz, 4H), 1.53 (p, J = 7.4 Hz, 4H), 0.94 (t, J = 7.4 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 158.9, 42.2, 23.6, 11.4.
1,3-Diphenethylurea (6b). Compound 6b was synthesized by
following general procedure D using S,S-dimethyl dithiocarbonate and
phenylethylamine (white solid, 69%). 1H NMR (400 MHz, CDCl3): δ
7.40−7.10 (m, 10H), 4.19 (s, 2H), 3.42 (q, J = 6.7 Hz, 4H), 2.79 (t, J =
6.8 Hz, 4H). 13C NMR (101 MHz, CDCl3): δ 158.2, 139.2, 128.8,
N-(2-(4-Formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)-4-
(methylsulfonyl)benzamide (9c). Compound 9c was synthesized by
following general procedure E using 4-(methylsulfonyl)benzoic acid
and amine salt 8 in the presence of EDCI, HOBt, and DIEA (white
solid, 65%). 1H NMR (400 MHz, CDCl3): δ 10.00−9.86 (m, 1H), 8.04
(s, 1H), 8.01−7.84 (m, 4H), 7.71 (dd, J = 6.5, 3.1 Hz, 2H), 7.55−7.43
(m, 3H), 7.37 (t, J = 5.1 Hz, 1H), 4.53−4.38 (m, 2H), 3.99 (q, J = 5.5
Hz, 2H), 3.05 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 185.0,
165.9, 152.2, 143.5, 139.1, 138.2, 132.2, 129.2, 128.9, 128.9, 128.7,
127.5, 120.8, 51.6, 43.7. MALDI-TOF m/z: calcd for C20H19N3O4S,
397.10; found, 397.74.
N-(2-(4-Formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)-4-
(methylamino)benzamide (9d). Compound 9d was synthesized by
following general procedure E using 4-(methylamino)benzoic acid and
amine salt 8 in the presence of EDCI, HOBt, and DIEA (semisolid,
55%). 1H NMR (400 MHz, CDCl3): δ 9.89 (s, 1H), 7.96 (s, 1H), 7.74
(dd, J = 7.6, 1.8 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 7.52−7.39 (m, 3H),
6.80 (t, J = 5.2 Hz, 1H), 6.52 (d, J = 8.7 Hz, 2H), 4.47−4.33 (m, 2H),
4.17 (s, 1H), 3.90 (q, J = 5.6 Hz, 2H), 2.85 (s, 3H). 13C NMR (101
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MHz, CDCl3): δ 184.8, 167.7, 154.3, 152.1, 135.2, 131.5, 129.1, 128.8,
128.7, 121.7, 120.9, 111.4, 52.1, 39.9, 30.2 (traces of dichloromethane
are present). MALDI-TOF m/z: calcd for C20H20N4O2, 348.15; found,
348.77, 360.76 (M + Na)+.
144.9, 144.3, 143.6, 141.2, 139.1, 133.3, 131.7, 130.1, 129.5, 129.3,
128.1, 127.7, 127.5, 127.2, 125.6, 120.6, 113.9, 113.0, 65.8, 51.9, 47.3,
44.0. MALDI-TOF m/z: calcd for C39H32N6O6, 680.23; found, 703.2
(M + Na)+.
General Procedure F for the Synthesis of 10(a−d). N-(2-(3-
Phenyl-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
1H-pyrazol-1-yl)ethyl)cyclohexanecarboxamide (10a). Barbituric
acid (0.269 g, 2.1 mmol) was added to a stirred solution of 9a (760
mg, 2.3 mmol) in dry methanol (15 mL) and heated to reflux for 16 h.
The resultant yellow solid was filtered and washed with cold methanol
(3 × 5 mL) and dried to yield pure compound 10a as a yellow solid
(776 mg, 84%). 1H NMR (400 MHz, DMSO-d6): δ 11.21 (s, 2H), 9.27
(s, 1H), 8.16 (s, 1H), 7.86 (t, J = 5.6 Hz, 1H), 7.64−7.43 (m, 5H), 4.34
(t, J = 5.6 Hz, 2H), 3.50 (d, J = 5.6 Hz, 2H), 2.10−1.93 (m, 1H), 1.73−
1.51 (m, 5H), 1.37−0.99 (m, 5H). 13C NMR (101 MHz, DMSO-d6): δ
176.0, 164.3, 163.2, 157.9, 150.7, 145.0, 139.1, 131.8, 130.0, 129.5,
129.3, 113.8, 112.8, 52.0, 44.3, 39.0, 29.6, 25.9, 25.7. MALDI-TOF m/
z: calcd for C23H25N5O4, 435.19; found, 435.76, 457.75 (M + Na)+.
N-(2-(3-Phenyl-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)-1H-pyrazol-1-yl)ethyl)benzamide (10b). Com-
pound 10b was synthesized from 9b and barbituric acid by following
Synthesis of 12. 4-(Aminomethyl)-N-(2-(3-phenyl-4-((2,4,6-tri-
oxotetrahydropyrimidin-5(2H)-ylidene)methyl)-1H-pyrazol-1-yl)-
ethyl)benzamide (12). To the stirred solution of 11 (150 mg, 0.222
mmol) in DMF (1.4 mL), piperidine (0.6 mL) was added dropwise and
stirred for 3 h. The solid formed was filtered; the filtrate was treated with
diethylether (30 mL) and the precipitated solids were filtered and dried
1
to yield 12 as a white solid (101 mg, 53%). H NMR (500 MHz,
DMSO-d6): δ 8.52 (t, J = 4.8 Hz, 1H), 8.46 (s, 1H), 8.05 (s, 1H), 7.75
(d, J = 7.5 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.49 (t, J = 7.3 Hz, 2H),
7.42 (t, J = 7.2 Hz, 1H), 7.35 (d, J = 7.7 Hz, 2H), 4.68 (s, 2H), 4.42 (s,
2H), 3.70 (s, 2H) (traces of diethyl ether is present). 13C NMR (101
MHz, DMSO-d6): δ 166.9, 154.9, 151.4, 143.9, 133.2, 133.1, 132.1,
129.1, 128.9, 128.8, 128.5, 128.3, 127.7, 117.3, 64.6, 51.7. MALDI-TOF
m/z: calcd for C24H23N6O4, 458.17; found, 459.13, 481.11 (M + Na)+.
3,3-Dimethyl-1-(6-oxo-6-((4-((2-(3-phenyl-4-((2,4,6-trioxotetra-
hydropyrimidin-5(2H)-ylidene)methyl)-1H-pyrazol-1-yl)ethyl)-
carbamoyl)benzyl)amino)hexyl)-2-((1E,3E,5E)-5-(1,3,3-trimethylin-
dolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium Chloride (14).
To the stirred solution of 12 (5.2 mg, 0.0109 mmol) in 1.5 mL of DMF,
cyanine-5 NH ester (0.008116 mmol) was added and stirred for 16 h
under dark reaction conditions at room temperature. Purification of the
crude compound was carried out on the preparative Vydac C18 column
using a 10−90% water/acetonitrile gradient in the presence of 0.05%
TFA for 30 min (A: water buffer, B: acetonitrile buffer). The purified
compound (>95%) was assessed by RP HPLC on an analytical Vydac
C18 column. MALDI-TOF m/z: calcd for C56H59N8O5, 923.42; found,
923.52.
Structure-Based High-Throughput Virtual Screening. Struc-
ture-based virtual screening was carried out using the DOCK6.9
program (http://dock.compbio.ucsf.edu/DOCK_6/index.htm).37 In
this analysis, we confirmed that relocation of ligands is possible using an
X-ray crystal structure (3RQ7) containing a peptide-based inhibitor.20
Among the two algorithms, namely, anchor and grow (flexible) and
rigid of DOCK6.9, a rigid algorithm was confirmed as a suitable one for
further analyses. The docking was performed using the MLSMR
database. Structurally analogous compounds were searched using the
ZINC database,38 and approximately 20,000 compounds were docked.
Finally, we identified 4a as a hit compound.
PBD FP Binding Assays for Plk1, Plk2, and Plk3. FP assays were
carried out essentially as described previously.39 Briefly, an appropriate
5-carboxyfluorescein-labeled PBD-binding peptide for Plk1, Plk2, or
Plk3 was incubated at a final concentration of 2 nM with various
concentrations of the bacterially expressed and purified PBD of Plk1,
Plk2, or Plk3, respectively, in a binding buffer containing 10 mM Tris
(pH 8.0), 1 mM EDTA, 50 mM NaCl, and 0.01% Nonidet P-40. FP was
analyzed 10 min after mixing of all components in a 384-well format
using a Molecular Devices SpectraMax Paradigm Multi-Mode Micro-
plate Detection Platform. All experiments were performed in triplicate.
Obtained data were plotted using GraphPad Prism software version 6.
Pan Assay Interference Compounds. As described above, FP
assays were carried out with 65 nM of bacterially expressed Plk1 PBD
and 5 nM of an FITC-labeled PBD-binding peptide in the presence of
increasing concentrations of KBJK557 and L-cysteine at various
concentrations (0, 0.78, and 1.95 μM, respectively). mP values were
determined 30 min after incubation. Data were plotted using GraphPad
Prism software. This experiment was performed in triplicate.
Immunofluorescence Staining. For immunofluorescence obser-
vation, HeLa cells were seeded on 24-well microplates at 2 or 4 × 104
cells/mL and cultured for 24 h. The cells were further cultured with
DMSO, nocodazole (200 nM), BI2536 (200 nM), and KBJK557 (200
μM) for 18 h. The medium in the microplate wells was then removed
and cells were fixed with 4% formaldehyde in PBS for 15 min and then
permeabilized for 5 min with 0.1% NP-40 in PBS. The cells were
washed with PBS containing 1% bovine serum albumin (PBS-1% BSA)
and then treated with 5% bovine serum albumin for 20 min. The cells in
each well were treated with anti-Plk1 (mouse) and anti-CREST
1
general procedure F (pale yellow solid, 68%). H NMR (400 MHz,
DMSO-d6): δ 11.20 (s, 2H), 9.35 (s, 1H), 8.66 (t, J = 5.4 Hz, 1H), 8.15
(s, 1H), 7.80 (d, J = 7.2 Hz, 2H), 7.65−7.36 (m, 8H), 4.51 (t, J = 5.6 Hz,
2H), 3.75 (q, J = 5.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ
167.3, 164.3, 163.2, 157.8, 150.7, 144.9, 139.1, 134.8, 131.7, 131.7,
129.9, 129.5, 129.2, 128.8, 127.7, 113.8, 112.9, 51.9, 39.9. MALDI-TOF
m/z: calcd for C23H19N5O4, 429.14; found, 451.68 (M + Na)+.
4-(Methylsulfonyl)-N-(2-(3-phenyl-4-((2,4,6-trioxotetrahydropyri-
midin-5(2H)-ylidene)methyl)-1H-pyrazol-1-yl)ethyl)benzamide
(10c). Compound 10c was synthesized from 9c and barbituric acid by
1
following general procedure F (yellow solid, 81%). H NMR (400
MHz, DMSO-d6): δ 11.30−11.08 (m, 2H), 9.36 (s, 1H), 8.93 (t, J = 5.4
Hz, 1H), 8.14 (s, 1H), 8.09−7.94 (m, 4H), 7.61−7.38 (m, 5H), 4.53 (t,
J = 5.5 Hz, 2H), 3.77 (q, J = 5.3 Hz, 2H), 3.26 (s, 3H). 13C NMR (101
MHz, DMSO-d6): δ 166.1, 164.3, 163.2, 157.8, 150.6, 144.9, 143.4,
139.3, 139.1, 131.7, 129.9, 129.5, 129.3, 128.7, 127.5, 113.9, 113.0, 51.8,
43.8. MALDI-TOF m/z: calcd for C24H21N5O6S, 507.12; found,
507.68.
4-(Methylamino)-N-(2-(3-phenyl-4-((2,4,6-trioxotetrahydropyri-
midin-5(2H)-ylidene)methyl)-1H-pyrazol-1-yl)ethyl)benzamide or
KBJK557 (10d). Compound 10d was synthesized from 9d and
barbituric acid by following general procedure F (yellow solid, 83%).
1H NMR (400 MHz, DMSO-d6): δ 11.19 (s, 2H), 9.32 (s, 1H), 8.23 (t,
J = 5.4 Hz, 1H), 8.15 (s, 1H), 7.70−7.40 (m, 7H), 6.51 (d, J = 8.7 Hz,
2H), 6.19 (q, J = 4.7 Hz, 1H), 4.47 (t, J = 5.8 Hz, 2H), 3.70 (q, J = 5.8
Hz, 2H), 2.71 (d, J = 4.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ
167.2, 164.3, 163.2, 157.8, 152.7, 150.7, 144.9, 139.0, 131.7, 130.0,
129.5, 129.2, 129.2, 121.1, 113.8, 112.9, 110.8, 52.2, 39.9, 29.8 MALDI-
TOF m/z: calcd for C24H22N6O4, 458.17; found, 458.74.
(9H-Fluoren-9-yl)methyl4-((2-(3-phenyl-4-((2,4,6-trioxotetrahy-
dropyrimidin-5(2H)-ylidene)methyl)-1H-pyrazol-1-yl)ethyl)-
carbamoyl)benzylcarbamate (11). To a stirred solution of EDCI (607
mg, 3.18 mmol), HOBt (428 mg, 3.18 mmol) in DMF (10 mL), 4-
(Fmoc-aminomethyl)benzoic acid (1.1 g, 3.18 mmol), and DIEA (2.76
mL, 15.86 mmol) were added and stirred for 30 min. To the resultant
solution, 8 (957 mg, 3.82 mmol) in DMF (5 mL) was added and it was
stirred at room temperature for 18 h. The reaction mixture was treated
with 5% NaHCO3 solution (25 mL) and extracted with ethyl acetate (3
× 15 mL); the combined organic layer was washed with H2O (3 × 20
mL) and brine, dried over Na2SO4, and evaporated under vacuum. To
the crude product in methanol (15 mL), barbituric acid (0.269 g, 3.10
mmol) was added and heated to reflux for 16 h. The resultant yellow
solid was filtered and washed with cold methanol (3 × 5 mL) and dried
to yield pure compound 11 as a yellow solid (1.08 g, 50%). 1H NMR
(400 MHz, DMSO-d6): δ 11.24 (s, 1H), 11.21 (s, 1H), 9.35 (s, 1H),
8.65 (s, 1H), 8.16 (s, 1H), 7.89 (d, J = 5.6 Hz, 3H), 7.76 (d, J = 6.6 Hz,
2H), 7.71 (d, J = 6.4 Hz, 2H), 7.59−7.47 (m, 5H), 7.47−7.37 (m, 2H),
7.37−7.31 (m, 2H), 7.28 (d, J = 6.7 Hz, 2H), 4.58−4.45 (m, 2H),
4.44−4.32 (m, 2H), 4.30−4.16 (m, 3H), 3.85−3.67 (m, 2H). 13C NMR
(101 MHz, DMSO-d6): δ 167.0, 164.3, 163.2, 157.7, 156.9, 150.7,
M
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(human) antibody in PBS-0.1% BSA and then placed in a humidified
atmosphere at room temperature and incubated for 1.5 h. After being
washed with PBS-0.1% BSA, treated wells were incubated with Alexa
Fluor 488-conjugated human anti-IgG antibody and Texas Red-
conjugated mouse anti-IgG antibody in PBS-0.1% BSA and then
incubated at room temperature for 60 min. After being washed with
PBS-0.1% BSA, the dish wells were overlaid with DAPI for 10 min at
room temperature and then washed with PBS-0.1% BSA. Fluorescence
was photographed with an inverted Zeiss 710 confocal microscope
(Carl Zeiss, Oberkochen, Germany). The signal intensity was measured
by the ZEN2010 program (Carl Zeiss, Oberkochen, Germany).
Cell Cycle Arrest Study by a Fluorescence-Activated Cell
Sorter. HeLa cells were seeded into 4 × 105 cells in six-well plates and
treated with BI2536 (100 nM) and KBJK557 (200 μM) for 24 h. Cells
were washed with PBS three times and fixed using 70% cold ethanol
during vortexing. The fixed cells were stained with propidium iodide
(PI) (1 μg/mL, Sigma-Aldrich, USA) and treated with RNase (1 μg/
mL) (Thermo Scientific, USA) in PBS for 30 min at 37 °C in an
incubator. Flow cytometric analysis was performed using a
fluorescence-activated cell sorter (Beckman Coulter, CytoFLEX, USA).
Apoptosis. HeLa cells (1 × 104 cells) were cultured in 96-well
microplates for 24 h at 37 °C. Cells were treated with BI2536 (100 nM)
and KBJK557 (200 μM) at 24 and 48 h. Then, 2.5 μL of mixtures of kit
(LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen, USA) was added
to 1 mL of PBS, and 100 μL of this solution was added to each well.
After 30 min of incubation at room temperature, images of the cells
were captured by a fluorescence microscope (Nikon ECLIPSE TS100,
Japan). The cell count was measured using ImageJ software.
Cellular Uptake of a Fluorescence-Conjugated Inhibitor. Cy5
NHS ester was purchased from Lumiprobe, USA. Cells were cultured in
24-well microplates at 4 × 104 cells with plastic coverslips for 24 h. To
the wells, 200 μM of Cy5-conjugated KBJK557 was added and
incubated for 18 h. Coverslips were washed three times with PBS and
stained with DAPI for 10 min at room temperature. Subsequently, the
coverslips were washed five times again with PBS that was covered with
mount solution. Fluorescence images were captured with an inverted
Zeiss 710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Animals. Male BALB/c nude mice were purchased from the Nara
Bio animal center (NARA Biotech, Seoul, Korea) and housed under
specific pathogen-free conditions. The mice were housed in groups of
three in transparent plastic cages bedded with aspen chips and were
provided with standard mouse chow diet and tap water ad libitum when
not being treated. The environment of the animal room was carefully
controlled, with a 12 h dark−light cycle. The temperature was
maintained at around 20−21 °C with a relative humidity of 40−45%.
The animal experiments were carried out according to a protocol
approved by the KBSI Committee (KBSI-AEC-1816) and all
experiments were performed in accordance with relevant guidelines
and regulations.
in PBS into the right thigh region of 5-week-old male BALB/c nu/nu
mice. When the tumor volume (length × width × height, mm3) reached
approximately 30−40 mm3, a tail vein injection of PBS, BI2536, and
KBJK557 (5 mg/kg) was carried out. BI2536 and KBJK557 were
dissolved in Cremophor EL (Kolliphor EL, Sigma-Aldrich, USA), N,N-
dimethylacetamide (Sigma-Aldrich, USA), and 2-(hydroxypropyl)-β-
cyclodextrin (Sigma-Aldrich, USA) (10:10:80, v/v/v, 100 μL). We
injected PBS, BI2536, and KBJK557 at 3−4-day intervals for 29 days
and the tumor size was measured using calipers.
Pharmacokinetic Profiles of KBJK557. Male ICR mice were
purchased from Koatech, Kyeonggi-do, Korea and were used for the
pharmacokinetic study of KBJK557. The blood samples were collected
at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after intravenous administration
at a dose of 5 mg/kg in mouse. The blood samples were centrifuged
immediately after collection, and the concentrations of the plasma
fractions were determined using an Agilent LC−MS/MS system
(Agilent, Santa Clara, CA) consisting of an Agilent 1200 series HPLC
system connected to an Agilent 6430 mass spectrometer equipped with
a turbo electrospray interface. The organ tissues (i.e., liver, spleen, and
kidney) were isolated at 2, 6, and 24 h after intravenous administration
at a dose of 5 mg/kg into the mouse. After homogenizing the tissues, the
concentrations of the tissue were determined as well. The elution was
carried out using a linear gradient of 0.1% formic acid in water and 0.1%
formic acid acetonitrile (95:5% → 5:95%, v/v) at a flow rate of 0.4 mL/
min with a reversed-phase high-performance liquid chromatography
column (Xterra MS C18, particle size 3.5 μm, length and the internal
diameter of column 50 mm × 2.1 mm; Waters, Milford, MA). Multiple
reaction monitoring data were collected in positive ionization mode.
The samples were monitored at the following Q1/Q3 transitions (m/
z): 459.2 → 134 for KBJK557 and 237.0 → 194.0 for carbamazepine
(internal standard). The Agilent software (Data Acquisition and
Quantitative Analysis) was used for the operation of the instrument and
data collection of KBJK557 and carbamazepine. A noncompartmental
analysis was performed to calculate the pharmacokinetic parameters for
KBJK557 using the Kinetica software. The area under the plasma
concentration−time curve (AUClast) was obtained by applying the
linear trapezoidal rule. The area under the plasma concentration−time
curve from time zero to infinity (AUC∞) was obtained by applying the
linear trapezoidal rule with the method of standard area extrapolation.
The terminal half-life (t1/2) of the compound was calculated by dividing
0.693 with λ, where λ represents the terminal log-linear slope of the
KBJK557 concentration−time profile. The total clearance (CL) for
KBJK557 was calculated using dose/AUC∞, and the steady-state
volume of distribution (Vss) was then calculated by MRT × CL.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01451.
■
sı
Optical Imaging Probe for Tumor Diagnosis. The xenograft
tumor models were prepared by subcutaneous injection of 4 × 106
HeLa cancer cells in PBS into the right foreleg region of 5-week-old,
male BALB/c nu/nu mice. When the tumor volume reached
1H and 13C NMR spectra, LC chromatogram, and
additional results and schemes (PDF)
Molecular formula strings (CSV)
approximately 120
20 mm3 at 8 weeks after inoculation, Cy5-
conjugated KBJK557 (5 mg/kg body weight/200 μL) was injected via
the tail vein. Then, to verify that Cy5-conjugated KBJK557
accumulated in the tumor, we carried out in vivo fluorescence imaging
using the in vivo imaging system (IVIS) spectrum (PerkinElmer, USA)
at 6, 24, 30, and 48 h with a 640 nm excitation (680, 700, and 720 nm for
spectral unmixing) and 680 nm emission filters and a binning factor of
8. Fluorescence was expressed as the average radiance (photons/s/
cm2/steradian). After in vivo scans, the mice were sacrificed by cervical
dislocation and tissues of interest (brain, heart, spleen, liver, lung,
kidneys, and tumor tissues) were removed immediately. The tissues
were scanned using the IVIS Spectrum in the same conditions as in vivo
scan conditions. ROIs were drawn, such as tumors and other tissues of
both in vivo imaging and ex vivo imaging, and then, the average radiant
efficiency of ROIs was measured.
AUTHOR INFORMATION
Corresponding Authors
■
Kyung S. Lee − Laboratory of Metabolism, Center for Cancer
Research, National Cancer Institute, Bethesda, Maryland 20892,
United States; orcid.org/0000-0001-8326-7162;
Email: kyunglee@mail.nih.gov
Eun Kyoung Ryu − Division of Magnetic Resonance, Korea Basic
Science Institute (KBSI), Ochang, Chung Buk 28119, Republic of
Korea; Department of Bio-analytical Science, University of Science
& Technology, Daejeon 34113, Republic of Korea; orcid.org/
0000-0002-8915-2144; Email: ekryu@kbsi.re.kr
Jeong Kyu Bang − Division of Magnetic Resonance, Korea Basic
Science Institute (KBSI), Ochang, Chung Buk 28119, Republic of
Anticancer Effect in Tumor-Bearing Mice. We induced mouse
xenograft tumor models by subcutaneously injecting 2 × 105 HeLa cells
N
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Article
Author Contributions
Korea; Department of Bio-analytical Science, University of Science
& Technology, Daejeon 34113, Republic of Korea; orcid.org/
0000-0001-5916-0912; Phone: +82-43-240-5023;
Email: bangjk@kbsi.re.kr; Fax: +82-43-240-5059
††P.G. and M.S.Y. contributed equally to this work.
Notes
The authors declare no competing financial interest.
Authors
ACKNOWLEDGMENTS
■
Pethaiah Gunasekaran − Division of Magnetic Resonance, Korea
Basic Science Institute (KBSI), Ochang, Chung Buk 28119,
Republic of Korea; orcid.org/0000-0001-8436-4183
Min Su Yim − Division of Magnetic Resonance, Korea Basic
Science Institute (KBSI), Ochang, Chung Buk 28119, Republic of
Korea; Department of Bio-analytical Science, University of Science
& Technology, Daejeon 34113, Republic of Korea
Mija Ahn − Division of Magnetic Resonance, Korea Basic Science
Institute (KBSI), Ochang, Chung Buk 28119, Republic of Korea
Nak-Kyun Soung − Anticancer Agent Research Center, Korea
Research Institute of Bioscience and Biotechnology, Ochang,
Chungbuk 28116, Republic of Korea
This work was supported by the R&D Convergence Program
(CAP-16-03-KRIBB, J.K.B.), the KRIBB Research Initiative
Program, and the National Research Foundation of Korea
(NRF) grant funded by the Korean government (MSIT)
(2020M 3E5E2039159 to Y.H.J., 2020R1A2C1009289 to
J.K.B.).
ABBREVIATIONS
■
Plk1, polo-like kinase-1; KD, kinase domain; PBD, polo-box
domain; SAR, structure−activity relationship; PPG, purpur-
ogallin; DMSO, dimethylsulfoxide; DAPI, 4′,6-diamidino-2-
phenylindole; WT, wild type; ROI, region of interest; PBS,
phosphate-buffered saline; RES, reticulo-endothelial system;
Jung-Eun Park − Laboratory of Metabolism, Center for Cancer
Research, National Cancer Institute, Bethesda, Maryland 20892,
United States
T
1/2, half-life; CL, clearance rate; AUC, area under the curve;
SD, standard deviation; Vss, apparent volume of distribution
calculated through the steady state; MRT, mean residence time;
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
HOBt, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethyl-
amine; DCM, dichloromethane; MeOH, methanol; TEA,
triethylamine; TFA, trifluoroacetic acid; DMF, N,N-dimethyl-
formamide; RP HPLC, reversed-phase high-performance liquid
chromatography
Jaehi Kim − Division of Magnetic Resonance, Korea Basic Science
Institute (KBSI), Ochang, Chung Buk 28119, Republic of Korea
Geul Bang − Biomedical Omics Group, Korea Basic Science
Institute, Ochang, Chung-Buk 363-883, Republic of Korea
Sang Chul Shin − Biomedical Research Institute, Korea Institute
of Science and Technology, Seoul 02792, Republic of Korea
Joonhyeok Choi − Division of Magnetic Resonance, Korea Basic
Science Institute (KBSI), Ochang, Chung Buk 28119, Republic of
Korea
Minkyoung Kim − College of Pharmacy, Dongguk University,
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