Anticancer and structure-activity relationship evaluation of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide analogs of chalcone

Article abstract of DOI:10.1016/j.bioorg.2016.08.003

To identify new potent chemotherapeutic agents, we synthesized compounds with 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC) skeletons and evaluated their cytotoxicities using a clonogenic long-term survival assay. Their half-maximal

Full text of DOI:10.1016/j.bioorg.2016.08.003

Accepted Manuscript
Anticancer and structure-activity relationship evaluation of 3-(naphthalen-2-
yl)-N,5-diphenyl-pyrazoline-1-carbothioamide analogs of chalcone
Youngshim Lee, Beom Soo Kim, Seunghyun Ahn, Dongsoo Koh, Young Han
Lee, Soon Young Shin, Yoongho Lim
PII:
S0045-2068(16)30089-X
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.08.003
YBIOO 1937
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 June 2016
3 August 2016
4 August 2016
Please cite this article as: Y. Lee, B.S. Kim, S. Ahn, D. Koh, Y.H. Lee, S.Y. Shin, Y. Lim, Anticancer and structure-
activity relationship evaluation of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide analogs of
chalcone, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.08.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anticancer and structure-activity relationship evaluation of 3-(naphthalen-2-yl)-N,5-
diphenyl-pyrazoline-1-carbothioamide analogs of chalcone
Youngshim Leea,+, Beom Soo Kima,+, Seunghyun Ahna, Dongsoo Kohb, Young Han Leec, Soon
Young Shinc,*, and Yoongho Lima,*
aDivision of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 143-701, Korea
bDepartment of Applied Chemistry, Dongduk Women’s University, Seoul 136-714, Korea
cDepartment of Biological Sciences, Konkuk University, Seoul 143-701, Korea
+These authors contributed equally to this work.
*Corresponding authors:
Prof. Yoongho Lim; Division of Bioscience and Biotechnology, Konkuk University
Hwayang-Dong 1, Kwangjin-Ku, Seoul, Korea 143-701
Tel.: 82-2-450-3760; Fax: 82-2-3437-6106; Email: yoongho@konkuk.ac.kr
Prof. Soon Young Shin; Department of Biological Sciences, Konkuk University
Hwayang-Dong 1, Kwangjin-Ku, Seoul, Korea 143-701
Tel.: 82-2-2030-7946; Fax: 82-2-3437-9781; Email: shinsy@konkuk.ac.kr
Abbreviations: CoMFA, comparative molecular field analysis; CoMSIA, comparative
molecular similarity index analysis; GI50, half-maximal cell growth inhibitory concentration
NDPC, 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide
1

Abstract
To identify new potent chemotherapeutic agents, we synthesized compounds with 3-(naphthalen-
2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC) skeletons and evaluated their
cytotoxicities using a clonogenic long-term survival assay. Their half-maximal cell growth
inhibitory concentrations ranged from a few hundred nanomolars to a few micromolars. Further
biological experiments including flow cytometry and western blotting analysis were performed
with the derivative showing the best cytotoxicity. To identify a target protein of the selected
compound, an in vitro kinase assay was carried out, which revealed that aurora kinases A and B
were inhibited by the test compound, and this was confirmed using western blot analysis. The
molecular binding mode between the selected compound and the kinases was elucidated using in
silico docking. The structural conditions required for good cytotoxicity were identified based on
the quantitative relationships between the physicochemical properties of the derivatives and their
cytotoxicities.
Keywords: colorectal, clonogenicity, QSAR, flow cytometry, immunofluorescence microscopy,
in silico docking
2

1. Introduction
Compounds containing pyrazoline-1-carbothioamide moiety show antidepressant and
anticonvulsant activities [1], as well as pesticide activities against the yellow fever mosquito
vector (Aedes aegypti) [2]. However, their anticancer activities have been rarely reported.
Compounds containing diphenylpyrazoline moiety show anticancer activities [3]. To identify
compounds with potential for use as chemotherapeutic agents, we combined the structures of
pyrazoline-1-carbothioamide and diphenylpyrazoline to create diphenylpyrazoline-1-
carbothioamide (Figure 1A). It has been reported diphenylpyrazoline-1-carbothioamide
derivatives show anticonvulsant and antidepressant activities [1]. Furthermore, to obtain
asymmetrical structures, one phenyl ring of the compound was switched with naphthalene. Since
the methoxylation of polyphenols can enhance cell permeability and stability [4], methoxylated
derivatives were designed (Figure 1B).
Colorectal cancer is one of the most common types of cancer in both developing and advanced
countries. Most colorectal cancers begin as a polyp in the inner lining of the large intestine and
over 90% occurred in gland cells lining the inside of the colon and rectum. The exact causes of
colorectal cancer are not known exactly, but some risk factors have been identified including a
high-fat diet, family history, genetic mutations, age, smoking, and ulcerative colitis. Several
studies have demonstrated that numerous bioactive polyphenols have chemopreventive or
therapeutic effects against colon cancer cells [5].
Because cancer cells divide faster than normal cells do, a cytotoxicity test can be a screening tool
to measure the anticancer activities of compounds. The compounds synthesized in this study
have the same basic 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC)
skeleton, and differ only in except the methoxy groups (Figure 1C). In addition, it was found that
hydroxylation of naphthalenyl group increased the inhibitory effect on aurora kinases [3], so that
1-hydroxy group was added to naphthalenyl group (Figure 1D). To distinguish the cytotoxicities
of the synthesized compounds, a clonogenic long-term survival assay was used because the result,
which is obtainable after 7 days, can clarify the distinction between similar structures [6]. Here,
36 compounds containing NDPC skeletons were synthesized, and their colonogenicities were
3

tested. In addition, further biological experiments including flow cytometry and western blotting
analysis were performed to elucidate the cytotoxicities of the compounds. Furthermore, to
identify the target protein of the compounds, an in vitro kinase assay was carried out based on
the superior half-maximal cell growth inhibitory concentration (GI50) exhibited by the selected
compound in inhibiting these kinases. The molecular binding mode between the selected
compound and the kinases was also elucidated using in silico docking. Moreover, the structural
conditions required for good cytotoxicity were determined based on the evaluation of the
quantitative relationships between the physicochemical properties of the compounds and their
cytotoxicities.
2. Materials and Methods
2.1.
Preparation of NDPC derivatives
All the compounds (1–36) containing the NDPC skeleton were synthesized using previously
reported methods [7,8]. Synthetic procedures are shown in Scheme 1 where the intermediate
chalcone III was obtained from Claisen-Schmidt condensation reaction between 1’-hydroxy-2’-
acetonaphthone I and methoxy-substituted benzaldehyde II. Chalcone III was treated with
excess amount of hydrazine to give pyrazoline IV, which was reacted with isothiocyanate in
ethanol to furnish desired compounds NDPC 1-36. After recrystallization, analytically pure
compounds of NDPC 1-36 were obtained. Their chemical identification was performed by
analyzing basic one- and two-dimensional (1D and 2D, respectively) nuclear magnetic resonance
(NMR) spectroscopy and high-resolution electron impact ionization mass spectrometry (MS)
spectra as reported previously [7,8]. All the NDPC derivatives prepared here are novel, and their
structures are listed in Table 1. Their melting points were measured using a Mel-Temp II melting
point apparatus (LabX, Midland, ON, Canada) and were uncorrected while the infrared (IR)
spectra were acquired using a Fourier transform (FT)-IR 4200 (JASCO, Easton, MD, USA) with
attenuated total reflection (ATR, PR0450-S). The column chromatography purifications were
performed on Silica gel 60 (70−230-mesh, Merck, Whitehouse Station, NJ, USA) when it was
necessary [9]. The chemical data including names, colors, melting points, yields, IR, NMR, and
4

HRMS were determined completely as provided in Supplementary materials [7,8].
2.2.
Clonogenic assay
The HCT116 human colorectal cancer cells were obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA) and were grown in Dulbecco’s modified Eagle’s
medium (DMEM, Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT, USA). For the experiment,
the HCT116 cells (3 × 103 cells/well) were seeded in 24-well tissue culture plates (BD Falcon,
Franklin Lakes, NJ, USA) in the absence or presence of different concentrations of the 36
compounds, incubated for 7 days, and were tested on four plates treated with 0, 5, 10, and 20 µM
of the samples. The colorectalies that formed were fixed with 6% glutaraldehyde and stained
with 0.1% crystal violet as described previously [6]. Since most of the compounds containing the
NDPC skeleton inhibited the cancer cell lines completely, the clonogenic long-term survival
assays were repeated using lower concentrations (0, 50, 500, and 5000 nM). The results of the
clonogenic survival assay were analyzed using densitometry (MultiGuage, Fujifilm, Japan). The
GI50 values were determined using the SigmaPlot software (SYSTAT, Chicago, IL, USA) and
their negative logarithmic scales (pGI50) were considered as the biological data of each NDPC
compound (Table 1) [10].
2.3.
Quantitative structure–activity relationship (QSAR)
The quantitative structure-activity relationship (QSAR) calculations were carried out using an
Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC with the Sybyl 7.3 software (Tripos, St. Louis,
MO, USA) using comparative molecular field analysis (CoMFA) and comparative molecular
similarity index analysis (CoMSIA) [11]. The previously unknown 3D structures of all the
NDPC derivatives needed to be determined, and the 3D structure of N,3,5-triphenyl-pyrazoline-
1-carbothioamide (Suppl. Figure 1) obtained from National Center for Biotechnology
Information (NCBI, PubChem CID: 3794935) was used as a template for the determination. The
3D structures constructed based on N,3,5-triphenyl-pyrazoline-1-carbothioamide were subjected
to energy minimization using the molecular mechanic algorithms provided by Sybyl 7.3 [3]. The
5

compounds that conformed to the lowest energy were selected for the QSAR calculations. The
conformational search to identified the most stable structure, the minimization process, and
CoMFA and CoMSIA calculations were performed according to methods reported previously [9].
2.4.
Cell cycle and cell death analysis
Flow cytometry was used to assess the cell cycle by measuring the propidium iodide (PI)-stained
DNA contents using a NucleoCounter NC-3000 image cytometer (ChemoMetec, Allerød,
Denmark) as described previously [12].
2.5.
Western blot analysis
The HCT116 cells were treated with 20 μM NDPC compound 4 (NDPC4) for different
incubation times, harvested, and then lysed in a buffer containing 20 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES, pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM
sodium chloride (NaCl), 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The
total cell lysates containing 10–20 g protein were separated using sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes,
which were probed with the primary antibodies of interest. The blots were subsequently
incubated with horseradish peroxidase-conjugated secondary antibodies and were visualized
using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech Inc.)
[13].
2.6.
Kinases assay
We used the following 17 kinases in the cell cycle analysis and in vitro kinase assays: aurora
kinase A and B (aurA and aurB, respectively), calcium/calmodulin-dependent protein kinase
type IV, dual specificity tyrosine-phosphorylation-regulated kinase 2, ephrin type-A receptor 2,
haspin Serine/threonine-protein kinase, hepatocyte growth factor receptor, insulin-like growth
factor-1 receptor kinase, nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor (IκB) kinase α, polo-like kinase 1, protein kinase B, proto-oncogene serine/threonine-
protein kinase, serine/threonine-protein kinase PAK 4, TANK-binding kinase 1, transforming
growth factor beta-activated kinase-1, tyrosine-protein kinase HCK, and vascular endothelial
6

growth factor receptor 1. The kinase assay was performed using the EMD Millipore
KinaseProfiler service assay protocol (EMD Millipore Corporation, Billerica, MA, USA) [10].
2.7.
In silico docking
The docking experiments were performed with an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC
with Sybyl 7.3 [14]. The 3D structure of aurA was adapted from 2J4Z.pdb deposited in the
protein data bank (PDB) [15] while that of aurB was adapted from 4AF3.pdb [16]. The detailed
procedures for in silico docking were performed according to previously reported methods [9].
2.8.
Statistical analysis
The statistical significance was analyzed using the Student’s t-test and a p < 0.05 was considered
statistically significant. All the experiments were performed in triplicate [13].
3. Results and Discussion
3.1.
Clonogenic long-term survival assay
As shown in Suppl. Figure 2, the clonogenic assay results obtained with the samples at 0, 5, 10,
and 20 µM could not distinguish the activity of the compounds because most of them inhibited
the cancer cells completely. Therefore, the GI50 values were obtained from the clonogenic long-
term survival assays performed at lower different concentrations (0, 50, 500, and 5000 nM,
Figure 2). They ranged from 210.55 to 4788.65 nM (NDPC compounds 4 and 3 respectively).
The GI50 values were obtained using the SIGMAPLOT software (SYSTAT, Chicago, IL), and
their negative logarithmic scales (pGI50) were used for the QSAR calculations (Table 1).
3.2.
QSAR calculations
The structures of the 36 NDPC derivatives showed slight differences in the position of the
methoxy substituents. However, the effects of the compounds on the human colorectal cancer
cells vary from 210.55 to 4788.65 nM. To explain why derivatives with similar structures
exhibited 22.7 times higher activities than the other derivative did, the QSAR calculations were
7

performed using an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC with the Sybyl 7.3 software
[11]. Furthermore, CoMFA) and CoMSIA were used for the QSAR calculations. The compounds
were divided into two groups, the training and test sets, for the validation of the QSAR model.
Seven derivatives (2, 9, 14, 19, 24, 27, and 35) were selected randomly as the test set for
evaluating the relationship between the structures and cancer cell growth inhibitory activity
while other derivatives were used to establish the QSAR model. The derivatives of the test set
were analyzed using a hierarchical clustering tree. As shown in Suppl. Figure 3, the seven
derivatives that were chosen as the test set belonged to separate structural groups. Therefore, the
test set could be used to validate the reliability of the QSAR models used in this experiment.
For the QSAR calculation using CoMFA, a partial least square (PLS) regression was used to
elucidate the linear correlation between the calculated fields such as the steric and electrostatic
fields and the biological data. The cross-validated correlation coefficient (q2) and non-cross-
validated correlation coefficient (r2) were calculated based on the overall compounds in the
training set. Among the models generated by the CoMFA, the model showing the best cross-
validated correlation coefficient (q2 = 0.598) was chosen for the further analysis. The final non-
cross-validated (r2 = 0.920) analysis was performed using the optimal number of components (n
= 6) obtained using the leave-one-out (LOO) method. In the PLS analysis, the standard error of
the estimate was 0.137 and F value was 42.443. To evaluate the CoMFA model, the activities of
36 derivatives were predicted, and their values were compared to the experimental data as shown
in Suppl. Table 1. The residuals between the experimental and predicted values for the training
set ranged from 0.03% to 10.82%. For the test set, the residuals ranged between 4.04% and
21.78%. The experimental data was plotted against the values predicted by the CoMFA model
(Suppl. Figure 4).
To visualize the relationships between the structures of the 36 compounds and their inhibitory
activities in the clonogenicity assay, CoMFA contour maps were generated in Suppl. Figure 5.
The steric and electrostatic field descriptors contributed 95.5% and 4.5%, respectively. In
addition, the only difference between the derivatives was the position of the methoxy
substitutions on rings A and B, which can contribute to the steric property considerably, and
8

therefore, a detailed analysis was conducted on the steric field. The favored steric bulky region
was 90%, and the disfavored region was 10%. The electronegative favored region was 90%, and
the electropositive region was 10%. As shown in Suppl. Figure 5, a yellow contour observed at
the C2 position of the A benzene ring indicates that the absence of a bulky group at C2 is
required for the inhibitory activity. All the derivatives that had a methoxy group at C2 showed
low inhibition activity such as derivatives 3, 6, 9, 12, 14, 17, 20, 23, and 32. On the other hand, a
green contour at C5 position was observed, suggesting that this bulky group at this region
increase activity. This was based on the observation of compounds 7, 13, 18, 24, and 30. The
remaining yellow contours, which were positioned at C2′ and C3′ of the B ring, need a
complicated explanation because we assumed that the combination of bulky group substitution is
important. For example, compounds dimethoxylated at both meta positions, C3′ and C5′, showed
good inhibitory activity supported by NDPC4, 15, 16, and 18. Compounds 31–36, which
contained tri-methoxy groups at C2′, C4′, and C6′, showed low inhibitory effects. In addition, the
derivatives that were dimethoxylated at positions C2′ and C4′ showed better activity than that
shown by the derivatives dimethoxylated at C2′ and C3′, when the same substitution was
included in the A ring. This observation was based on the comparison of compounds 2 and 11, 1
and 8, and 5 and 10.
Additional information on the QSAR model was obtained by calculating the CoMSIA. Among
the numerous CoMSIA models, the best model that showed q2 and r2 values of 0.6 and 0.893,
respectively. The PLS statistical parameter were the number of components, the standard error of
estimate, and the F values, which were 6, 0.159, and 30.543, respectively. To evaluate the
CoMSIA model, the activities of the compounds were predicted and compared with that of the
experimental data. All the residuals between the experimental and predicted activity values are
shown in Suppl. Table 1. For the test set, the residuals between the experimental and predicted
values ranged from 2.23% to 23.92%. The residuals for the derivatives in the training set ranged
from 0.4% to 11.4%. The experimental data was plotted against the values predicted using the
CoMFA model (Suppl. Figure 6) and based on these results, the selected CoMSIA model was
determined to be reliable.
9

In this study, the selected CoMSIA model provided information that the contributions of the
steric, electrostatic, hydrophobic, and H-bond acceptor field descriptors were 21.6%, 6.9%,
62.3%, and 9.2%, respectively. The contour maps are shown in Suppl. Figure 7. The steric bulky
favored region was 85% while the disfavored region was 15%. The electronegative favored
region was 90%, and the electropositive region was 10%. The hydrophobic favored region was
75%, and the disfavored region was 25%. The H-bond acceptor favored region was 8%, and the
disfavored region was 92%. Because the information on the steric and electrostatic fields
obtained from the CoMSIA maps was similar to that from the CoMFA, the contour maps of the
hydrophobic and H-bond acceptor fields were examined. As shown in Suppl. Figure 7 (C), a
yellow contour at the C2 position indicates that the hydrophobic substituents at this position
increased the inhibitory activity. For examples, the GI50 values of derivatives 1 and 19 were
better than those of derivatives 3 and 20. The C2′ position was also associated with the yellow
color, which indicates that the methoxy group at that position decreased the activity. Furthermore,
derivatives 25, 27, and 28 that possess the methoxy group at the C5′ position showed less activity
than derivatives 8, 10, and 11, did. The gray contour at position C3 suggests that hydrophilic
groups at this positions were favored. This observation is supported by the evidence that
derivatives 10, 13, 15, and 18, which have methoxy groups at this position, showed relatively
good inhibitory activity. As shown in Suppl. Figure 7 (D), a purple contour was observed at the
hydroxyl group of the naphthalene ring, which was a common structure in all 36 compounds
while the cyan contour was not observed in this map. The color of the H-bond acceptor favored
and disfavored was cyan and purple, respectively. Therefore, the importance of the H-bond
acceptor field was not discovered in this study.
3.3.
Evaluation of cellular activities of NDPC4
Next, we evaluated the biological activities of NDPC4 including its cytotoxicity against HCT116
cells using a cell counting kit-8 (CCK-8). Treatment with NDPC4 resulted in a concentration-
dependent inhibition of the cell viability with an IC50 value of ~10 μM (Figure 3A), suggesting
that the NDPC4 exhibited a growth inhibitory effect in cancer cells. To assess whether this
decrease in cell viability is associated with inhibition of the cell cycle progression, we analyzed
the cell cycle phase distribution using flow cytometry. As shown in Figure 3B, NDPC4 caused a
10

considerable decrease in G1 phase cells (from 54% to 29%) with a concomitant increase in S
phase cells (from 18% to 35%), whereas the total number of G2/M phase cells was not changed
compared with the vehicle-treated control cells. This result suggests that NDPC4 inhibited cell
cycle progression. It is well established that cell cycle progression is regulated by cell cycle
regulatory proteins such as cyclin and cyclin-dependent kinase (Cdks) complexes. Among them,
cyclin D1 triggers G1 cell cycle progression while cyclin B1 is involved in the regulation of
G2/M phase progression. To explore the mechanism underlying the NDPC4-induced cell cycle
dysregulation, we examined the expression level of cyclin D1 and cyclin B1 using a western
blotting assay. We found that treatment of HCT116 cells with 10 μM NDPC4 remarkably
decreased the levels of both cyclin D1 and cyclin B1 in a time-dependent fashion (Figure 3C),
suggesting that NDPC compound 4 inhibited the cell cycle progression by downregulating G1
and G2/M cell cycle regulators. Notably, following NDPC4 treatment at a concentration of 10
μM, the population of sub-G1 cells was increased from 1% (control) to 7% (after treatment),
suggesting an induction of apoptosis.
Therefore, we subsequently examined whether NDPC4 induces apoptosis. It is well established
that phosphatidylserine (PS), a membrane phospholipid, is located on the outer membrane of
apoptotic cells [17]. Annexin V binds to PS [18] and, therefore, we analyzed annexin V-positive
cells (AV+) after NDPC4 treatment of cells using flow cytometry. PI was used as a counterstain
to detect late apoptotic or necrotic dead cells or both. As shown in Figure 4A, treatment with
NDPC4 decreased the population of viable cells (lower left quadrant, AV and PI double-negative,
AV-PI-) from 83% to 14% while the early apoptotic cells (lower right quadrant; AV+PI-)
increased from 13% to 80%. The late apoptotic or dead cells, or both (upper right quadrant;
PI+AV+) increased from 3% to 6%. Therefore, the total dead cells were increased from 16% to
86% following NDPC4 treatment (Figure 4B). These data demonstrate that NDPC4 triggered
cell death through apoptosis of HCT116 cells.
Caspases are a family of cysteine-dependent aspartate-directed proteases that play central roles in
regulating apoptosis. Caspases are subclassified by their roles as initiator caspases (caspase-9
and -8) and effector caspases (caspase-7 and -3). Following exposure to apoptotic stimuli,
11

initiator caspases are activated by autocatalytic cleavage. The active initiator caspases then
cleave and activate effector caspases, thereby accelerating the cleavage of numerous cellular
proteins including the DNA repair enzyme poly(adenosine diphosphate [ADP]-ribose)
polymerase (PARP), to induce apoptosis [19]. To explore whether NDPC4 activates caspases,
the HCT116 cells were treated with 20 μM, which is sufficient to induce apoptosis and then the
levels of active caspase 7 were determined using a western blotting assay. We found that the
cleavage of caspase 7 and its substrate PARP, were increased in response to NDPC4 treatment
(Figure 4C). These results suggest that NDPC4 induced apoptosis by a caspase-dependent
mechanism.
3.4.
In vitro kinase assay
The in vitro kinases assays of NDPC4 were performed using 17 kinases to evaluate the effects
on the cell cycle including aurA, aurB, calcium/calmodulin-dependent protein kinase type IV,
dual specificity tyrosine-phosphorylation-regulated kinase 2, ephrin type-A receptor 2, haspin
serine/threonine-protein kinase, hepatocyte growth factor receptor, insulin-like growth factor-1
receptor kinase, IκB kinase α, polo-like kinase 1, protein kinase B, proto-oncogene
serine/threonine-protein kinase, serine/threonine-protein kinase PAK 4, TANK-binding kinase 1,
transforming growth factor beta-activated kinase-1, tyrosine-protein kinase HCK, and vascular
endothelial growth factor receptor 1. Of these kinases, aurA and aurB were inhibited by NDPC4
and to verify this effect, the phosphorylation status of both kinases was determined using western
blot analysis. Aurora kinase activity is accompanied by an increase in its phosphorylation [20].
Treatment with NDPC4 decreased the phosphorylation of aurA, aurB, and aurC on threonine-
288, -232, and -198, respectively (Figure 5A), as well as that of histone H3, a substrate of aurA
and aurB, at serine 10 (Figure 5B) in a time-dependent fashion [21]. These data suggest that
decreased phosphorylation of aurora kinases induced by NDPC4 inhibited their kinase activity.
3.5.
Molecular binding modes between NDPC4 and aurA and aurB
To evaluate the molecular binding mode between the aurora kinases and NDPC4, the in silico
docking experiments were chosen. The 3D structure of NDPC4 was generated using energy
minimization based on the 3D structure of N,3,5-triphenyl-pyrazoline-1-carbothioamide (Suppl.
12

Figure 1) provided by the NCBI (PubChem CID: 3794935). The procedure used to determine the
energy minimization was based on a previously reported method [11]. The 3D structure of aurA
was obtained from the crystallographic structure of the complex of aurA and a ligand, 4-(4-
methylpiperazin-1-yl)-N-[5-(2-thienylacetyl)-1,5-dihydropyrrolo[3,4-C]pyrazol-3-yl]benzamide
(named 626) [Suppl. Figure 8], which was deposited in the PDB as 2J4Z.pdb. More than 100 3D
structures of aurA can be found in the PDB, but 2J4Z.pdb contains most residues (126–395). The
2J4Z.pdb consists of two polypeptide chains, A and B, and chain B includes more residues than
A. Therefore, 2J4Z-B was chosen for the in silico docking experiment. First, apoprotein was
prepared using the Sybyl program. After its energy minimization, the root-mean-square deviation
between the crystallographic structure and the energy-minimized structure was 0.30 Å. Since we
used a flexible docking method, the docking radius was set to 6.5 Å. The docking process was
repeated 30 times to generate 30 complexes. The substrate binding sites were determined based
on the interpretation of the LigPlot analysis [22] [Suppl. Figure 9A]. Fifteen residues were used
included in the binding site. When the original ligand, 626 was docked into the apoprotein, the
binding energy ranged from -24.25 to -27.32 kcal/mol∙Å. Similarly, NDPC4 was docked into the
apoprotein, and 30 complexes of aurA and NDPC4 were obtained. Their binding energy ranged
between -22.24 and -24.85 kcal/mol∙Å. The first complex showed the best binding pose and
lowest binding energy and, therefore, it was further analyzed. The LigPlot analysis revealed that
10 residues participated in the hydrophobic interactions between the apoprotein and NDPC4, and
two residues formed four hydrogen bonds (H-bonds) [Suppl. Figure 9B]. Eight residues
consisting of Agr137, Leu139, Ala160, Tyr212, Ala213, Pro214, Thr217, and Leu263 were
observed in both complexes while in the aurA-626 and aurA-NDPC4 complex, two residues
each (Glu211 and Ala213, and Ala137 and Tyr212) formed two and four H-bonds, respectively.
The 3D images generated using the PyMol program (PyMOL Molecular Graphics System,
version 1.3, Schrödinger, LLC) demonstrated the presence of different H-bonds [Suppl. Figure
10]. The binding energy of the aurA-NDPC4 and aurA-626 complexes could be matched.
Furthermore, even a lower number of residues in the aurA-NDPC4 complex participated in the
binding site than participated in that of the aurA-626 complex. Therefore, the four H-bonds of
the aurA-NDPC4 complex may have created a stronger interaction than the two H-bonds of the
13

aurA-626 complex did.
Similarly, the binding mode between NDPC4 and aurB was elucidated using in silico
docking. Because only one 3D structure of aurB was deposited in the PDB as 4AF3.pdb, it was
chosen for the in silico docking. It contains a ligand, cyclopropanecarboxylic acid {4-[4-(4-
methyl-
piperazin-1-yl)-6-(5-methyl-2h-pyrazol-3-ylamino)-pyrimidin-2-ylsulfanyl]-phenyl}-
amide (named VX6) [Suppl. Figure 11]. The binding energy of VX6 docked into the apo-aurB
ranged from -24.43 to -29.87 kcal/mol∙Å. The residues participating in the binding site were
analyzed using LigPlot [Suppl. Figure 12A]. Eleven residues were found in the binding site of
VX, and nine of them showed hydrophobic interactions while two formed three H-bonds. The
docking process was applied to NDPC4, and the binding energy of the aurB-NDPC4 complex
ranged between -24.93 and -29.88 kcal/mol∙Å. Furthermore, 14 residues were observed in the
binding site analyzed using LigPlot [Suppl. Figure 12B] and one (Glu161) formed an H-bond
while eight residues consisting of Leu83, Val91, Ala104, Glu155, Tyr156, Ala157, Gly160, and
Leu207 were observed in both binding sites. The 3D images of the aurB-VX6 and aurB-NDPC4
complex were generated using PyMol [Suppl. Figure 13]. In the aurB-NDPC4 and aurB-VX6
complexes, 14 and 11 residues participated in the binding site. However, the aurB-NDPC4
complex included one H-bond while the aurB-VX6 complex had three. This phenomenon may
be responsible for the similar binding energy observed between the aurB-NDPC4 and aurB-VX6
complexes. In order to develop NDPC derivatives for clinical cancer therapy, further rigorous
studies are needed to determine the in vivo bioavailability and safety of the NDPC derivatives,
including the pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) and
toxicokinetic properties (e.g. acute toxicity and repeated-dose toxicity).
4. Conclusion
The cytotoxicities of most natural chalcones against HCT116 human colorectal cancer cells are
evident at micromolar levels [23,24]. The NDPC derivatives investigated are derived chalcones,
and their cytotoxicities ranged between 210 and 4788 nM. Therefore, a substitution of the α,β-
unsaturated carbonyl group of chalcone with a pyrazoline group improved the cellular
cytotoxicities. Furthermore, the 36 derivatives evaluated here possess the same skeleton, but the
number and position of methoxy groups vary. To elucidate the relationships between their
14

cytotoxicities and the number and position of the methoxy groups, CoMFA and CoMSIA were
used, and the analysis of their contour maps revealed that the C2 position favors hydrophobic
and less bulky group while the other positions of the A ring are less involved in the cell growth
inhibition. On the other hand, the inhibitory activity was affected by a combination of
substituents on the B ring. It is possible that altering the 3D structure of the NDPC compounds
with different substituents on B ring induced differential effects on the target protein. Therefore,
we first sought to identify the target protein for NDPC4, which exhibited the best activity in the
long-term survival clonogenic assay, using in vitro kinases assays with 17 kinases that are known
to participate in the cell cycle. Because aurA and aurB were inhibited by NDPC4, we verified if
this effect was mediated by the alteration of the phosphorylation status of the aurora kinases
using western blot analysis. The result revealed that NDPC4 decreased the phosphorylation of
the aurora kinases by inhibiting their kinase activity. To evaluate the molecular binding mode
between the aurora kinases and NDPC4, the in silico docking experiments were carried out. The
binding energy of the aurA and NDPC4 complex was matched with that of the aurA and ligand
626 contained in the aurA complex. Similarly, the binding energy between the aurB and NDPC4
complex competed with that between the aurB and ligand VX6 contained in the aurB complex.
Next, we evaluated the biological activities of NDPC4, which induced apoptosis and triggered
cell death by apoptosis in HCT116 cells by a caspase-dependent mechanism. These findings
could be used for the further development of NDPC derivatives as potential chemotherapeutic
agents.
Acknowledgments
This work was supported by the Priority Research Centers Program (NRF, 2009-0093824) and
Cooperative Research Program for Agriculture Science & Technology Development (RDA,
PJ01130302). SY Shin was supported by the KU Research Professor Program of Konkuk
University.
5. References
15

[1] N. Siddiqui, P. Alam, W. Ahsan, Arch Pharm (Weinheim). 342 (2009) 173-181.
[2] B. Kocyigit-Kaymakcioglu, N. Beyhan, N. Tabanca, A. Ali, D.E. Wedge, S.O. Duke, U.R.
Bernier, I.A. Khan, Med Chem Res. 24 (2015) 3632-3644.
[3] S.Y. Shin, H. Yoon, D. Hwang, S. Ahn, D.W. Kim, D. Koh, Y.H. Lee, Y. Lim, Bioorg Med
Chem. 21 (2013) 7018-7024.
[4] X. Wen, T. Walle, Drug Metab Dispos. 34 (2006) 1786-1792.
[5] J.R. Araujo, P. Goncalves, F. Martel, Nutr Res. 31 (2011) 77-87.
[6] N.A. Franken, H.M. Rodermond, J. Stap, J. Haveman, C. van Bree, Nat Protoc. 1 (2006)
2315-2319.
[7] H. Jung, S. Ahn, M. Park, H. Yoon, H.J. Noh, S.Y. Kim, J.S. Yoo, D. Koh, Y. Lim, Magn
Reson Chem. 53 (2015) 383-390.
[8] H. Jung, S. Ahn, Y. Jung, H.J. Noh, S.Y. Kim, D. Koh, Y. Lim, Magn Reson Chem. 53
(2015) 391-397.
[9] S.Y. Shin, H. Jung, S. Ahn, D. Hwang, H. Yoon, J. Hyun, Y. Yong, H.J. Cho, D. Koh, Y.H.
Lee, Y. Lim, Bioorg Med Chem. 22 (2014) 1809-1820.
[10] H. Yoon, T.W. Kim, S.Y. Shin, M.J. Park, Y. Yong, D.W. Kim, T. Islam, Y.H. Lee, K.Y.
Jung, Y. Lim, Bioorg Med Chem Lett. 23 (2013) 232-238.
[11] J. Hyun, S.Y. Shin, K.M. So, Y.H. Lee, Y. Lim, Bioorg Med Chem Lett. 22 (2012) 2664-
2669.
[12] S.Y. Shin, J.M. Lee, M.S. Lee, D. Koh, H. Jung, Y. Lim, Y.H. Lee, Clin Cancer Res. 20
(2014) 4302-4313.
[13] Y. Jung, S.Y. Shin, Y. Yong, H. Jung, S. Ahn, Y.H. Lee, Y. Lim, Chem Biol Drug Des. 85
(2015) 574-585.
[14] S.Y. Shin, H. Yoon, S. Ahn, D.W. Kim, S.H. Kim, D. Koh, Y.H. Lee, Y. Lim, Bioorg Med
Chem. 21 (2013) 4250-4258.
[15] D. Fancelli, J. Moll, M. Varasi, R. Bravo, R. Artico, D. Berta, S. Bindi, A. Cameron, I.
Candiani, P. Cappella, P. Carpinelli, W. Croci, B. Forte, M.L. Giorgini, J. Klapwijk, A.
Marsiglio, E. Pesenti, M. Rocchetti, F. Roletto, D. Severino, C. Soncini, P. Storici, R.
Tonani, P. Zugnoni, P. Vianello, J Med Chem. 49 (2006) 7247-7251.
16

[16] J.M. Elkins, S. Santaguida, A. Musacchio, S. Knapp, J Med Chem. 55 (2012) 7841-7848.
[17] J.J. Lum, D.E. Bauer, M. Kong, M.H. Harris, C. Li, T. Lindsten, C.B. Thompson, Cell. 120
(2005) 237-248.
[18] H.A. Andree, C.P. Reutelingsperger, R. Hauptmann, H.C. Hemker, W.T. Hermens, G.M.
Willems, J Biol Chem. 265 (1990) 4923-4928.
[19] D.R. McIlwain, T. Berger, T.W. Mak, Cold Spring Harb Perspect Biol. 5 (2013) a008656.
[20] A.O. Walter, W. Seghezzi, W. Korver, J. Sheung, E. Lees, Oncogene. 19 (2000) 4906-4916.
[21] C. Crosio, G.M. Fimia, R. Loury, M. Kimura, Y. Okano, H. Zhou, S. Sen, C.D. Allis, P.
Sassone-Corsi, Mol Cell Biol. 22 (2002) 874-885.
[22] A.C. Wallace, R.A. Laskowski, J.M. Thornton, Protein Eng. 8 (1995) 127-134.
[23] D.H. Lee, Y.J. Jung, D. Koh, Y. Lim, Y.H. Lee, S.Y. Shin, Cancer Lett. 372 (2016) 1-9.
[24] J.M. Lee, M.S. Lee, D. Koh, Y.H. Lee, Y. Lim, S.Y. Shin, Cancer Lett. 354 (2014) 348-354.
17

Figure legends
Figure 1. Structures of (A) diphenylpyrazoline-1-carbothioamide, (B) 3-naphthalen-2-yl-5-
methoxyphenyl-pyrazoline-1-carbothioamide, (C) 3-(naphthalen-2-yl)-N,5-dimethoxyphenyl-
pyrazoline-1-carbothioamide, and (D) 3-(1-hydroxynaphthalen-2-yl)-N,5-dimethoxyphenyl-
pyrazoline-1-carbothioamide.
Figure 2. Clonogenic long-term survival assays of 36 3-(naphthalen-2-yl)-N,5-diphenyl-
pyrazoline-1-carbothioamide (NDPC) derivatives at four different concentrations (0, 50, 500,
and 5000 nM).
Figure 3. Antiproliferative effect of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-
carbothioamide (NDPC)-4 in HCT116 cells. (A) Cell viability test using cell counting kit-8
(CCK-8). HCT116 cells (1 × 103 cells/sample) were treated with different concentrations of
NDPC4 (0, 5, 10, 20, and 40 μM) for 24 h. (B) Effect of NDPC4 on cell cycle distribution. Cells
were treated with 10 μM NDPC4 for 24 h, fixed with ethanol, and stained with propidium iodide
(PI). Cellular DNA content was determined using flow cytometry using a NucleoCounter NC-
3000; x-axis, cell counts; y-axis, PI-stained DNA contents; 2N, diploid; 4N, tetraploid. (C) Effect
of NDPC4 on expression of cyclin D1 and cyclin B1. Cells were treated with 10 μM NDPC4 for
0, 3, 6, 12, and 24 h. Whole-cell lysates were prepared and subjected to western blotting.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the internal control used to ensure
equal protein loading.
Figure 4. Effect of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC)-4 on
induction of apoptosis. HCT116 cells were treated with 20 μM NDPC4 for 24 h and then stained
with fluorescein isothiocyanate (FITC)-annexin-V and propidium iodide (PI). Cells were
harvested, washed, and analyzed for fluorescence intensity using flow cytometer. (A) Scatter
plots show the FITC-annexin V intensity versus that of PI. (B) Histograms show the FITC-
annexin V intensity of the cell counts (%). (C) Cells were treated with 10 μM NDPC4 for 0, 12,
and 24 h. Whole-cell lysates were prepared and subjected to western blotting using antibodies
18

against cleaved caspase-7 and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP).
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was the internal control used to
show equal protein loading.
Figure 5. Effect of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC)-4 on
phosphorylation of aurora kinases in HCT116 cells treated with 20 μM NDPC4 for various
lengths of time. Whole cell lysates were prepared, and we analyzed phosphorylated levels of (A)
aurora kinase A (aurA), aurB, and aurC on threonine-288,-232, and -198 and (B) histone H3 on
serine-10 using western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
antibody was the internal control used to show equal protein loading.
Scheme 1. The synthetic procedures of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-
carbothioamide derivatives 1-36.
Table 1. Structures and biological data of 36 synthesized 3-(naphthalen-2-yl)-N,5-diphenyl-
pyrazoline-1-carbothioamide (NDPC) derivatives.
19

Suppl. Table 1. Experimental half-maximal cell growth inhibitory concentration negative
logarithmic scale (pGI50) data and pGI50 values predicted using comparative molecular field
analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) models.
Suppl. Figure 1. Structure of N,3,5-triphenyl-pyrazoline-1-carbothioamide.
Suppl. Figure 2. Clonogenic long-term survival assays of 36 3-(naphthalen-2-yl)-N,5-diphenyl-
pyrazoline-1-carbothioamide (NDPC) derivatives at four different concentrations (0, 5, 10, and
20 μM).
Suppl. Figure 3. Hierarchical clustering tree; *indicates selective derivatives for the test set used
for the quantitative structure-activity relationship (QSAR) calculation.
Suppl. Figure 4. Plot of experimental data versus values predicted using comparative molecular
field analysis (CoMFA). Diamonds and squares denote training and test sets, respectively.
Suppl. Figure 5. Comparative molecular field analysis (CoMFA) contour maps. (A) Steric field
contours are shown in green (more bulkiness favored) and yellow (less bulkiness favored). (B)
Electrostatic field contours are shown in red (electronegative substituents favored) and blue
(electropositive substituents favored).
Suppl. Figure 6. Plot of experimental data versus values predicted using comparative molecular
similarity index analysis (CoMSIA). Diamonds and squares denote training and test sets,
respectively.
Suppl. Figure 7. Comparative molecular similarity index analysis (CoMSIA) contour maps. (A)
Steric field contours are shown in green (more bulkiness favored) and yellow (less bulkiness
favored). (B) Electrostatic field contours are shown in red (electronegative substituents favored)
and blue (electropositive substituents favored). (C) Hydrophobic field contours are shown in
orange (hydrophobic favored) and white (hydrophobic disfavored). (D) H-bond acceptor filed
20

contours are shown in magenta (H-bond acceptor favored) and cyan (H-bond acceptor
disfavored).
Suppl. Figure 8. Structure of 4-(4-methylpiperazin-1-yl)-N-[5-(2-thienylacetyl)-1,5-
dihydropyrrolo[3,4-C]pyrazol-3-yl]benzamide (named 626) contained in 2J4Z.pdb as a ligand.
Suppl. Figure 9. Residues participating in binding sites of (A) aurora kinase A (aurA)-626 and
(B) aurA-3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamid (NDPC)-4 complexes
were analyzed using LigPlot program. Residues in half circles create hydrophobic interactions
with the ligands, and dotted lines denote H-bonds.
Suppl. Figure 10. Three-dimensional (3D) images of binding sites of (A) aurora kinase A
(aurA)-626 complex and (B) aurA-3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamid
(NDPC)-4 complex were generated using PyMOL program. Ligands 626 and NDPC4 are shown
in yellow and magenta, respectively. Cyan-colored lines represent H-bonds.
Suppl. Figure 11. Structure of cyclopropanecarboxylic acid {4-[4-(4-methyl- piperazin-1-yl)-6-
(5-methyl-2h-pyrazol-3-ylamino)-pyrimidin-2-ylsulfanyl]-phenyl}-amide
(named
VX6)
contained in 4AF3.pdb as a ligand.
Suppl. Figure 12. Residues participating in binding sites of (A) aurora kinase B (aurB)-VX6
complex and (B) aurB-3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamid (NDPC)-4
complexes were analyzed using LigPlot program. Residues in half circles create hydrophobic
interactions with ligands, and dotted lines denote H-bonds.
Suppl. Figure 13. Three-dimensional (3D) images of binding sites of (A) aurora kinase B
(aurB)-VX6 and (B) aurB-3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamid
(NDPC)-4 complexes were generated using PyMOL program. Ligands VX6, and NDPC4 are
shown in yellow and magenta, respectively. Cyan-colored lines represent H-bonds.
21

Fig.1
S
(A)
NH2
S
N
NH2
NH
N
N
N
N
+
pyrazoline-1-carbothioamide
diphenylpyrazoline
diphenylpyrazoline-1-carbothioamide
(B)
S
NH2
N
N
n(H3C)

Fig.1
(CH3)n
(C)
S
NH
N
N
n(H3C)
(CH3)n
(D)
S
NH
OH
N
N
n(H3C)

Fig.2

Fig.2

Fig.3
Time (h)
3 6 12 24
A
C
0
100
80
60
40
20
0
Cyclin D1
Cyclin B1
GAPDH
0
10
20
30
[NDPC 4] (µM)
40
Vehicle
NDPC 4
B
sub-G1: 1 %
sub-G1: 7 %
G1: 54 %
S: 18 %
G2/M: 23 %
G1: 29 %
S: 35 %
G2/M: 24 %
2N 4N
2N 4N

Fig.4
Vehicle
NDPC 4
A
C
Time (h)
12
AV+P+
AV+P+
0
24
AV+P-
AV+P-
Cleaved Casp-7
Pro-PARP
Cleaved PARP
B
16%
86%
GAPDH

Fig.5
A
Time (h)
0 0.5 6 12
p-Aurora A (Thr-288)
p-Aurora B (Thr-232)
p-Aurora C (Thr-198)
GAPDH
Time (h)
B
0
3
6
12 24
p-Histone H3
(Ser-10)
GAPDH

Scheme 1
OH O
O
OH O
KOH(50%)
EtOH
H
(OCH3)x
+
(OCH3)x
I
II
III
OH
N
NH
NH2NH2·H2O
EtOH/reflux
(OCH3)x
(OCH3)y
IV
NCS
V
S
NH
(OCH3)y
OH N
N
EtOH/reflux
(OCH3)x
NDPC 1 - 36