1,2,4-Triazolin-5-thione derivatives with anticancer activity as CK1γ kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103806

The optimization and synthesis of new CK2 and CK1 inhibitors are the basis for the development of new therapeutic strategies for the treatment of cancer and neurodegenerative disorders associated with overexpression and abnormal functioning of these enzym

Full text of DOI:10.1016/j.bioorg.2020.103806

BioorganicChemistry99(2020)103806
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Bioorganic Chemistry
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1,2,4-Triazolin-5-thione derivatives with anticancer activity as CK1γ kinase
inhibitors
⁎
Monika Pituchaa, , Monika Janeczkob, Katarzyna Klimekc, Emilia Fornald, Maciej Wosa,
Anna Pachuta-Steca, Grazyna Ginalskac, Agnieszka A. Kaczore,f
a Independent Radiopharmacy Unit, Faculty of Pharmacy, Medical University of Lublin, Poland
b Department of Molecular Biology, Faculty of Biotechnology and Environmental Sciences, John Paul II Catholic University of Lublin, Poland
c Department of Biochemistry and Biotechnology, Faculty of Pharmacy, Medical University of Lublin, Poland
d Department of Pathophysiology, Medical University of Lublin, Poland
e Department of Synthesis and Chemical Technology of Pharmaceutical Substances, Faculty of Pharmacy, Medical University of Lublin, Lublin, Poland
f School of Pharmacy, University of Eastern Finland, Kuopio, Finland
A R T I C L E I N F O
A B S T R A C T
Keywords:
The optimization and synthesis of new CK2 and CK1 inhibitors are the basis for the development of new ther-
apeutic strategies for the treatment of cancer and neurodegenerative disorders associated with overexpression
and abnormal functioning of these enzymes. Triazole derivatives appear to be especially interesting as potential
kinase inhibitors. In this context we synthesized a series of 1,2,4-triazolin-5-thione derivatives as CK1γ kinase
inhibitors. The antiproliferative activity of synthesized compounds was assessed against cancer cells: human
lung adenocarcinoma (A549), human hepatoma (HepG2), and human breast adenocarcinoma (MCF-7).
Compound 1 exhibited antiproliferative potency against A549 cancer cells and was characterized by a selective
antiproliferative effect. Additionally, this compound has high apoptotic activity against A549, HepG2, MCF-7
cells and induced only slight amount of necrotic cells in these cell lines. In order to decipher the mechanism of
anticancer activity of the studied compounds PASS software was used and these compounds were assayed for the
inhibition of CK1γ and CK2α kinases. The reported series of 1,2,4-triazolin-5-thiones inhibits CK1γ and CK2α
kinases in micromolar range. The most active compound shows activity against isoform γ3 which at con-
centration of 50 μM reduced the kinase activity by 69% while at 100 μM by 80%. CK2α was found to be less
susceptible to the effects of the triazoles tested, as the reduction in kinase activity by 29% was observed for
compound 15, and by 27% for compound 1 only at the concentration of 100 μM. The inhibition of CK1γ and
CK2α kinases was rationalized using molecular docking.
1,2,-Triazole
Anticancer activity
Kinase inhibitor
Molecular modeling
1. Introduction
new mechanisms of molecular action for triazoles are investigated.
Triazole derivatives appear to be especially interesting as potential ki-
Triazoles constitute an important class of heterocycles because of
their varied biological activities and have been reported as anticancer,
antifungal, anti-inflammatory, antibacterial, anti-HIV, and anti-
tubercular agents. In particular, triazoles display anticancer activity
against human hepatoma, prostate cancer, lung cancer, breast cancer,
gastric cancer, colon carcinoma, cervical cancer, ovary cancer and
leukemia [1–10]. Although 1,2,4-triazole derivatives are known and
well described, they are still of interest for researchers. In numerous
studies triazole compounds have been reported as chemical scaffold
with a broad spectrum of activity, low toxicity and excellent pharma-
cokinetic property. Additionally, more and more triazole-based com-
pounds are currently developed drug candidates [11,12]. Moreover,
nase inhibitors. In particular, 1,2,4-triazolin-5-thiones are not ex-
tensively exploited and this group of compounds has been a subject of
our studies for many years. 1,2,4-Triazolin-5-thiones synthesized in our
group have never been tested against any kinases.
Protein kinases represent a large family comprising more than 500
members within the human genome, play universal roles in the reg-
ulation of many biological processes and have been implicated in a
large number of human diseases including many forms of malignancy
[13,14]. The recent analysis of available genomics data revealed nearly
one thousand kinase mutations in about seventy single-gene disorders,
fifty of which are associated with cancer [15]. Hence the growing in-
terest in the possibility of regulation of their activity through the design
⁎ Corresponding author.
E-mail address: monika.pitucha@umlub.pl (M. Pitucha).
https://doi.org/10.1016/j.bioorg.2020.103806
Received 18 December 2019; Received in revised form 27 March 2020; Accepted 28 March 2020
Availableonline02April2020
0045-2068/©2020TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
and synthesis of specific and strong inhibitors, which in the future may
result in the acquisition of specific drugs. Kinases are “druggable” and
success of Gleevec/imatinib, the first protein tyrosine kinase inhibitor
drug for the treatment of Chronic Myelogenous Leukemia (CML) was a
seminal advance demonstrating the utility of kinase inhibitors as ther-
apeutic agents [13,14]. One of the core elements of regulatory and
signaling networks based on protein phosphorylation in the eukaryotic
cell are casein kinases. The conventional term ‘casein kinase’ (CK) de-
notes three classes of kinases - CK1, CK2 and Golgi-CK (G-CK)/Fam20C
(family with sequence similarity 20, member C). They have the ability
to phoshorylate casein in vitro, but otherwise are unrelated to each
other. All CKs have been reported to be implicated in human diseases.
Numerous reviews dealing with the druggability of CK1 and CK2 are
available [16]. CK1 is involved in diverse biological functions [17],
such as membrane transport [18], regulation of DNA repair [19], cel-
lular morphology [20], modulation of the Wnt/β-catenin and Hedgehog
pathway during development [21], and regulation of circadian rhythms
[22,23]. Protein kinase CK2 is highly conserved and multifaceted
serine/threonine enzyme, found in all eukaryotic cells investigated to
date. The CK2 holoenzyme forms a heterotetrameric complex composed
of two catalytic (CK2α and CK2α’) and two regulatory (CK2β) subunits.
The assembly of CK2 complexes containing the catalytic subunits re-
quires the earlier formation of dimers of the regulatory CK2β subunit,
which can take place in the absence of the catalytic subunits [24]. The
catalytic subunit (CK2α) is distantly related to the CMGC subfamily of
kinases, such as the CDK kinases, and alone is enzymatically active. The
CK2β subunit is able to stimulate the activity of CK2 in the tetramer.
Protein kinase CK2 is constitutively active enzyme and can use ATP and
GTP for phosphorylation. It is elevated in most tumors investigated and
in rapidly proliferating tissues [25]. More than 300 substrates of CK2
have been reported to date [26]. For this reason it has been linked to
several cellular processes, being implicated in cell cycle, transcriptional
control, neuronal function and response to cellular stress [16,27]. The
role of CK2 in many pathologies is well known; in particular it is im-
plicated in cardiovascular pathology, atherosclerosis, cardiomyocyte
hypertrophy, neurodegeneration (Parkinson’s and Alzheimer’s dis-
eases), inflammation, muscle diseases as well as virus and parasite in-
fections [28,29]. The pathology where the role of CK2 is best docu-
mented and studied is cancer, where this kinase is almost invariably
upregulated [30]; recent studies have clearly demonstrated that ab-
normally elevated CK2 level is required for tumor progression, due to
its role in the regulation of almost all the processes essential for cancer
development with special reference to the suppression of apoptosis.
High CK2 activity promotes tumor cell growth in a number of respects:
it improves transformation potential of oncogenes, stabilizes onco-
kinom by activation of cochaperone CDC37, which is crucial for the
maintenance of the active conformation of kinases with oncogenic po-
tential, counteracts the effectiveness of anti-neoplastic drugs, especially
imatinib and melphalan, promotes neovascularization, and most im-
portantly generates a broad spectrum of pro-life cell signals [31].
Here we present a series of 1,2,4-triazolin-5-thione derivatives
which were tested for their anticancer activity. To determine the me-
chanism of action we used the PASS program, which indicated CK1γ3
kinase as a potential molecular target which was confirmed by in vitro
studies. The rationale of our work can be summarized as follows: (1) the
urgent need to elaborate more effective and safer therapeutics for the
treatment of cancer; (2) the privileged scaffold of 1,2,4-triazoles and (3)
the ability to decipher the molecular mechanism of the studied com-
pounds using in vitro and in silico approaches.
Scheme 1. The scheme of synthesis of compounds 1–17.
thiosemicarbazides in alkaline medium (Scheme 1). Structures of all the
obtained compounds were confirmed by spectral analysis.
2.2. Antiproliferative activity
After 96 h incubation, SRB (sulforhodamine B) assay indicated that
only two derivatives (1 and 17) possessed antiproliferative activity
against A549, HepG2, and MCF-7 cells (Table 1). Nevertheless, the cell
proliferation inhibition of derivative 1 (R1 = 2-pyridine; R2 = 4-
NO2C6H4) towards all tested cancer cells was much better than those of
derivative 17 (R1 = 2-pyridine; R2 = 3,4-Cl2C6H3). Moreover, deri-
vative 1 may be safer (exhibited in vitro higher inhibition activity to-
wards cancer than normal cells) with SI (selective index) values ranged
from 0.57 to 2.05 in comparison with derivative 17 (SI values ranged
from 0.28 to 0.61). Taking into account the potential therapeutic po-
tency of derivative 1, it was worth noting that its selectivity against
MCF-7 cells is high (SI > 2) and also greater than those of Cisplatin,
indicating that it should be qualified for in vivo evaluation [32,33].
IC50 - inhibitory concentration that caused 50% inhibition of cell
proliferation, SI - the ratio between IC50 for normal cells and IC50 for
cancer cells, * – results adopted from earlier publication [33].
Based on the results obtained, we conducted the structure-activity
relationship analysis (SAR). The variation of substituents is one strategy
to improve the interaction of a potential drug with a molecular target.
According to the literature, a slight change in the type of substituent or
its location may be of key importance for antitumor activity [34,35]. In
our work, we have modified the 1,2,4-triazoline-5-thion system by
changing substituents in positions 3 and 4. We used substituents that
can affect the physical properties and size of the designed compounds,
e.g. alkyl, mono and disubstituted aryl groups. Our research shows that
although an ortho-substituted pyridine ring is the key to anti-
proliferative activity, the type of substituent in position 4 of the 1,2,4-
triazoline-thione system is not insignificant. The presence of an alkyl
(4), 2-fluorophenyl (7), 2-chlorophenyl (8) and 2,4-dichlorophenyl (16)
substituent is not favorable for antiproliferative activity. The 3,4-di-
chlorophenyl group (17) slightly improves the IC50 value in the range of
56.15–121 µg/mL. Only an electron withdrawing nitro group at para
position of the phenyl ring substituted as R2 accompanied by pyridin-2-
yl group as R1 (1) ensures the best IC50 values of 6.88 (for A549), 3.47
(for HepG2) and 1.92 (for MCF-7). The results obtained are in line with
our previous studies [33].
2. Results and discussion
2.1. Chemistry
In this work seventeen 1,2,4-trazolin-5-thione derivatives were ob-
tained
by the heterocyclization reaction of appropriate
2

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
Table 1
Antiproliferative activity (expressed as IC50 value) and selectivity index (SI) of tested compounds.
Comp.
IC50
SD [µg/mL]
SI
BJ
A549
HepG2
MCF-7
BJ/A549
BJ/HepG2
BJ/MCF-7
1
3.94
1.25
6.88
2.46
3.47
> 400
121
1.53
1.92
1.13
0.57
–
1.14
–
2.05
–
2–16
> 200
34.22
1.19
> 400
75.89
2.38
> 400
56.15
2.97
17
5.35
0.56
3.15
1.16
3.77
1.79
2.55
1.33
0.45
0.5
0.28
0.42
0.61
0.40
Cisplatin*
2.82
2.3. Apoptotic activity
compound 4 can be attributed to its structural difference in comparison
with other tested compounds. This is the only compound with an alkyl
substituent in R1 position and it might be that it was not similar enough
to CK1γ3 inhibitors in PASS database.
In physiological conditions apoptosis, also known as “programmed
cell death” plays a key role in maintaining homeostasis of the organism.
In pathological conditions, a disorder in cell death regulation may lead
to very serious diseases. One of them is cancer, when the cells pro-
liferate in an unregulated manner, hence invading normal tissues and
organs and sometimes spreading throughout the body [36]. To date, the
mechanism of action of the majority of toxic anticancer drugs has been
associated with induction of mutations in cancer cells which trigger cell
death via apoptosis [37–40]. Thus, to establish whether anti-
proliferative activity of derivative 1 (exhibiting the highest SI values)
was associated with induction of apoptosis, the morphological changes
(i.a. nuclear shrinkage and chromatin condensation) in A549, HepG2,
and MCF-7 cells were assessed via AO/EB (acridine orange/ethidium
bromide) staining and presented in Fig. 1.
The use of appropriate in silico methods helps in designing an ex-
periment characterized by high efficiency and simultaneously reduced
implementation time and costs. The presented results indicate that
there is no clear correlation between the PASS prognosis and the ex-
perimental IC50 values. It should be stressed that the PASS software
prediction is based on the molecular similarity and as such it suffers
limitations. Thus, it should be used as a general indication about the
possible activity of the series of compounds, not as a source of activity
probability values which may be correlated with experimentally de-
termined activity.
2.4. Kinase inhibition
In turn, percentage of viable, early/late apoptotic as well as necrotic
cells were summarized in Fig. 2a–c. In the case of A549 cells (Fig. 2a), a
The studies on the inhibitory activity of triazoles against protein
kinases have shown that compounds 1–15 and 17 inhibit CK1 activity
at concentrations 50 µM and 100 µM. The most active compound shows
activity against isoform γ3 which at concentration of 50 μM reduced the
kinase activity by 69% while at 100 μM by 80%. At lower concentra-
tions of this inhibitor (1 µM and 10 µM) kinase activity were reduced to
50% and 62% respectively. Other compounds reduce kinase activity in
the range 60–90% depending on the concentration, while one com-
pound does not show any activity against the kinase under study in the
test range. On the other hand, CK2α was found to be less susceptible to
the effects of the triazoles tested, as the reduction in kinase activity by
29% was observed for compound 15, and by 27% for compound 1 only
at the concentration of 100 μM.
percentage of EA (early apoptotic) cells (13.07
2.78%) and LA (late
apoptotic) cells (8.54
1.18%) after treatment with derivative 1 were
significantly higher (P
<
0.05) compared to the untreated cells
0.19%, respectively). Cisplatin also in-
(2.94
1.07% and 0.73
duced apoptosis in A549 cell but more potently than derivative 1. The
EA cells and LA cells represented 33.51
2.78% and 11.12
1.37%
of all cells, respectively. The necrotic cells were not observed both in
population of untreated cells as well as of cells treated with derivative 1
or Cisplatin. Similarly to untreated A549 cells, in population of un-
treated HepG2 cells, spontaneous early and late apoptosis, and a lack of
necrosis were observed (Fig. 2b). Nevertheless, after incubation with
Cisplatin a small percentage of necrotic cells was found. Interestingly,
both compound 1 and Cisplatin exhibited a high percentage of apop-
The majority of the inhibitors do not behave more similarly against
CK1 and CK2 with a significant advantage of the former one. Our results
show that the protein kinase CK1γ3 is susceptible to most tested triazole
inhibitors (Table 3). With the exception of 16, the tested triazoles
suppress the phosphorylation of the kinase with IC50 values in the range
from 35 μM to 279 μM. Screened derivatives of triazoles are not as
efficient CK2α inhibitors. In this case, only five compounds showed
inhibitory activity against this enzyme with IC50 values in the ranges
72–283 μM. The most potent activity against both kinases was 1, which
inhibited of CK1γ3 twofold stronger than that of CK2α, with IC50 35 μM
and 72 μM, respectively. The inhibition of CK2α by 4 was one and a half
times stronger than that of CK1γ3. In turn, 3 induced threefold stronger
inhibition of CK1γ3 than that of CK2α, and in the case of 11, the in-
hibition of CK2α was twofold stronger than that of CK1γ. In turn, 2
induced threefold stronger inhibition of CK1γ3 than that of CK2α, and
in the case of 11, the inhibition of CK1γ3 was twofold stronger than that
of CK2α. Among active triazoles, 15 displays the least efficacy towards
the two tested kinases while compound 16 is not active at all. Thus,
after chemical modification, derivatives of triazoles may constitute a
novel class of inhibitors of protein kinase CK1γ3.
tosis with predominant LA cells (23.87
3.49% for derivative 1 and
44.03
3.23% for Cisplatin). The MCF-7 cells proved to be the most
sensitive to derivative 1 or Cisplatin treatment. Both compounds caused
a significant reduction of cell number and induced increased apoptosis
(Fig. 2c). After treatment with derivative 1, more than 50% of apoptotic
cells and a slight amount of necrotic cells (1.50
served.
1.17%) were ob-
In the case of Cisplatin the percentage of EA cells and LA cells was
comparable (approx. 20%), while the necrotic cells constituted
10
1.70% of all cells. Thus, the AO/EB staining confirmed the an-
tiproliferative activity assessed by SRB test (see Section 2.2. Anti-
proliferative activity). Derivative 1 had high apoptotic activity against
A549, HepG2 and MCF-7 cells and induced only a slight amount of
necrotic cells in these cell lines. This is a particularly important feature
of chemotherapeutic agents, due to the fact that in contrast to necrosis,
elimination of cancer cells via apoptosis does not induce inflammation
[41].
In order to decipher the mechanism of anticancer activity of com-
pounds 1–17 PASS and Pharma Expert software were used. In general
these in silico approaches indicated various kinases as possible mole-
cular targets for the investigated compounds. Table 2 presents prob-
ability that compounds 1–17 inhibit CK1γ3 kinase. It can be concluded
that all the compounds except for compound 4 are predicted to mod-
erately inhibit CK1γ3 kinase. The prediction of lack of activity of
In the light of the cellular effect caused by triazoles and, con-
currently, the moderate results of the inhibitory effects on the activity
of CK1 and CK2 kinases, it can be concluded that the mechanism of this
activity may be not only associated with the analyzed protein kinases.
Alternatively, this activity may have a very broad range and apply to
3

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
Fig. 1. Confocal microscope images showing morphological changes in cell nuclei after 96-h treatment with derivative 1 or Cisplatin (untreated cells were served as a
control); VE – viable cells, EA – early apoptotic cells, LA – late apoptotic cells, NC – necrotic cells. Magn. 400×.
other protein kinases or even other enzymatic or receptor proteins.
Given the multitude of protein kinases present in eukaryotic (also
human) cells and their conservative nature, it is very difficult to obtain
highly specific inhibitors of specific protein kinases. Therefore, it is
highly probable that the cellular effect caused by triazoles is a complex
mechanism requiring comprehensive investigations and subsequent
experiments confirming the hypotheses.
pocket by the network of hydrogen bonds mediated by water molecule.
The most potent ligand 1 interacts with CK1γ3 kinase via a salt bridge
between its nitro group and the side chain of Asp 125 and through a
hydrogen bond between its triazole nitrogen hydrogen atom and the
side chain of Leu 119 (Fig. 3A and B). In case of compound 4 its lower
activity against the studied enzyme can be attributed to the lack of salt
bridge and forming only a hydrogen bond with the kinase binding
pocket. Compound 4 forms a hydrogen bond between its triazole ni-
trogen atom and the backbone atom of Leu 119 (Fig. 3C and D). These
two compounds are slightly rotated one to another in the binding
pocket of CK1γ3 kinase. Interactions with Leu 119 were found for all
other potent compounds, including compounds 2, 6, 7, 8, 10 and 14.
Some of these compounds may form additional salt bridge with Lys 72
or Asp 125 using their nitro groups as the anchoring points. In case of
the least active compounds 13 and 15 and the inactive compound 16
the low activity or lack of activity can be attributed to the lack of
possibility to form a hydrogen bond with Leu 119 or probably un-
favorable placement of a molecule in a binding pocket with a hydrogen
bond between Leu 119 and a pyridine nitrogen atom of the ligand.
Regarding CK2α kinase, one of the crucial interaction of co-crys-
tallized inhibitor ellagic acid (PDB ID: 2ZJW) is a water-mediated
2.5. Molecular modeling
In order to study the interactions of compounds 1–17 with CK1
isoform γ3 and CK2α kinases at the molecular level, molecular docking
to respective kinases X-ray structures was performed. Results of mole-
cular docking of most potent compounds 1 and 4 are shown in Fig. 3 for
CK1γ3 kinase and in Fig. 4 for CK2α kinase.
The mode of interactions of the most potent compounds with CK1γ3
kinase resembles the mode of interactions of co-crystallized inhibitor
with this enzyme. In case of co-crystallized inhibitor {(2Z)-4-amino-2-
[(4-methoxyphenyl)imino]-2,3-dihydro-1,3-thiazol-5-yl}(4-methox-
yphenyl)methanone (PDB ID: 2IZR), the ligand forms hydrogen bonds
with backbone atom of Glu 117 and Leu 119. It is also kept in the
4

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
Table 2
PASS and Pharma Expert prediction of probability of CK1γ3 kinase inhibition
by compounds 1–17. Pa – probability that the compound is active; Pi
–
probability that the compound is inactive.
Compound
Pa
Pi
1
0.533
0.593
0.653
0.000
0.656
0.568
0.500
0.605
0.527
0.594
0.520
0.520
0.514
0.559
0.646
0.569
0.569
0.004
0.004
0.003
0.000
0.003
0.004
0.005
0.004
0.005
0.004
0.004
0.005
0.005
0.004
0.003
0.004
0.004
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Table 3
IC50 determinations for 1,2,4-triazole derivatives against protein kinases CK1γ3
and CK2α.
No
IC50 [μM]
CK1γ3
CK2α
1
35
3
4
72
6
2
51
154
14
3
176
98
16
10
No activity
4
8
65
6
5
112
55
No activity
No activity
No activity
No activity
No activity
No activity
6
4
4
3
7
43
8
41
9
123
46
10
10
11
12
13
14
15
16
17
4
132
153
279
66
11
14
25
283
25
No activity
No activity
No activity
6
257
18
167
14
No activity
No activity
No activity
83
7
presence of hydrogen bonds only: between the ligand nitro group and a
backbone atom of Arg 47 as well as between its triazole nitrogen hy-
drogen atom and a backbone atom of Val 116 (Fig. 4A and B). The
similar mode of interactions is displayed by other active compounds.
The lack of activity of most tested compounds against this kinase can be
explained by the lack of possibility to form a hydrogen bond with Val
116 and/or unfavorable rotation of a compound in a binding pocket
resulting in lower Glide docking scores. The orientation of the in-
hibitors in both kinases is very similar (Figs. 3 and 4).
Fig. 2. The percentage of viable (VE), early apoptotic (EA), late apoptotic (LA),
and necrotic (NC) cells after 96-h treatment with derivative 1 or Cisplatin
(untreated cells were served as a control); * Significantly different amount of
treated cells compared to the amount of untreated cells (control).
#
Significantly different amount of cells treated with Cisplatin compared to the
amount of cells treated with derivative 1. Statistically significance obtained at
P < 0.05 according to one-way ANOVA (analysis of variance) test followed by
Tukey’s multiple comparison test.
It can be noticed that some of active compounds, i.e. 1, 7, 8 and 17
possess 2-pyridyl group. However, inactive compound 16 possesses it
too. Furthermore, some other active compounds, such as 2, 4, 10 and
14 do not have this structural moiety. It cannot be thus concluded that
2-pyridyl moiety ensures high activity and is essential for high activity
againt the tested kinases. Good activity of compounds 1, 7, 8 and 17
bearing 2-pyridyl group can be attributed to a favorable pattern of
hydrophobic interactions as no polar or π-π stacking interactions were
found for these compounds.
hydrogen bond with a backbone atom of Val 116. Moreover, some other
water-mediated hydrogen bonds stabilize this inhibitor in the enzyme
binding pocket. In case of most potent compound 4, its good activity
against CK2α kinase can be explained by salt bridge interactions be-
tween its nitro group and the side chain of Lys 68 as well as by for-
mation of hydrogen bonds between its nitro group and a backbone atom
of Asp 175 and between its triazole nitrogen hydrogen atom and a
backbone atom of Val 116 (Fig. 4C and D). Lower activity of compound
1 against this kinase can be explained by the lack of salt bridge and the
3. Conclusion
The reported series of 1,2,4-triazolin-5-thiones inhibits CK1γ and
5

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
Fig. 3. Molecular interactions of compounds 1 (A, B) and 4 (C, D) with CK1γ3 kinase obtained using molecular docking approach. A, C − 3D view of the binding site.
Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with grey carbon atoms, main interacting residues shown as sticks. Hydrogen
bonds shown as red dashed lines. Salt bridges shown as solid violet lines. Non-polar hydrogen atoms not shown for clarity. B, D − 2D view of the binding site.
CK2α kinases in micromolar range. The optimization and synthesis of
new CK2 and CK1 inhibitors are the basis for the development of new
therapeutic strategies for the treatment of cancer and neurodegenera-
tive disorders associated with overexpression and abnormal functioning
of these enzymes. The results show that modifications of compounds
are necessary to achieve greater inhibitory activity against these two
enzymes. On the other hand, it should also be considered that the
studied compounds are multi-target in their nature and searching for
additional targets underlying their anticancer activity will be a subject
of our future work.
companies Sigma-Aldrich, AlfaAesar, POCH and used without further
purification. Thiosemicarbazide derivatives used for the reaction were
obtained in our laboratory according to the method described earlier
[42,43]. Compounds 1, 3 and 14 have been already reported [32,44]
but we obtained them using different experimental approach.
Melting points of obtained compounds were determined using
Fisher-Johns block and presented without corrections. The 1H and l3C
NMR spectra were recorded on a Bruker Avance 600 spectrometer
(Bruker BioSpin GmbH, Rheinstetten, Germany) in DMSO‑d6. The
chemical shifts are given in δ (ppm) scale using TMS as the standard
reference. The attenuated total reflectance ATR-IR spectra were re-
corded over the range 4000–400 cm−1 on the Thermo Scientific Nicolet
6700 FTIR spectrophotometer. LC-QTOF HRMS measurements were
performed using 1290 HPLC coupled to 6550 ifunnel QTOF LC/MS
(Agilent Technologies, Santa Clara, CA, USA). HPLC column Zorbax
C18 Eclipse Plus C18 50×2.1 mm, 1.7 µm (Agilent Technologies, Santa
4. Experimental
4.1. Chemistry
All chemicals used for the synthesis were purchased from the
6

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
Fig. 4. Molecular interactions of compounds 1 (A, B) and 4 (C, D) with CK2α kinase obtained using molecular docking approach. A, C − 3D view of the binding site.
Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with grey carbon atoms, main interacting residues shown as sticks. Hydrogen
bonds shown as red dashed lines. Salt bridges shown as solid violet lines. Non-polar hydrogen atoms not shown for clarity. B, D − 2D view of the binding site.
Clara, CA, USA) was used. Mobile phase consisted of water (A) and
acetonitrile (B), both with the addition of 0.1% formic acid. The linear
gradient 5 to 95B in 6 min was applied, flow 0.4 mL/min. Spectra were
acquired at the range of 100–1000 m/z in MS scan mode, 1.5 scans/s.
Internal mass calibration was enabled, two reference ions of m/z
121.0509 and 922.0098 were used. MS data and MS spectrograms are
presented in Supplementary material (Table 1S, Fig. 1S and Fig. 2S).
(m, 1H, CHpyridyl), 8.64–8.66 (m, 2H, CHpyridyl), 9.06 (s, 1H, CHpyridyl),
11.78 (s, 1H, NH). 13C NMR (DMSOd6): 116.8; 121.9; 125.3; 126.1;
138.1; 148.9; 149.5; 151.5; 157.3; 166.5. LC-QTOF HRMS (m/z):
Formula and Calculated Monoisotopic Mass: C13H9N5O2S (299.0477),
Measured mass: 299.0482.
4.1.1.2. 3-(4-Nitrophenyl)-4-(pyridin-3-yl)-4,5-dihydro-1H-1,2,4-triazol-
5-thione (2). Yield: 63% (1.79 g) as a orange solid, m.p. 219–220 °C. IR
cm−1: 3028, 2679, 1606, 1579. 1H NMR (DMSO‑d6) δ ppm: 7.33–7.35
(m, 2H, CHpyridyl), 7.41–7.42 (m, 4H, CHphenyl), 8.64–8.67 (m, 2H,
CHpyridyl), 14.41 (s, 1H, NH). 13C NMR (DMSO‑d6): 122.4; 126.4; 129.4;
131.0; 133.6; 141.0; 148.9; 150.5; 169.7. LC-QTOF HRMS (m/z):
Formula and Calculated Monoisotopic Mass: C13H9N5O2S (299.0477),
Measured mass: 299.0480.
4.1.1. General procedure for the synthesis of 3,4-disubstituted 1,2,4-
triazoline-5-thione derivatives (1–17)
0.01 Mole of previously obtained thiosemicarbazide derivative was
dissolved in 25 mL of hot 2% NaOH solution. Next, the mixture was
heated at reflux temperature for 2 h. After cooling, the solution was
neutralized with dilute hydrochloric acid (3 M). Target substance was
filtered off and crystallized from ethanol.
4.1.1.3. 3-(4-Nitrophenyl)-4-(pyridin-4-yl)-4,5-dihydro-1H-1,2,4-triazol-
5-thione (3). Yield: 64% (1.82 g) as a orange solid, m.p. 262–264 °C. IR
cm−1: 3062, 2679, 1636, 1588. 1H NMR (DMSO‑d6) δ ppm: 7.25–7.27
(m, 2H, CHphenyl), 7.74–7.77 (m, 2H, CHphenyl), 7.83–7.86 (m, 2H,
CHpyridyl), 8.29–8.39 (m, 2H, CHpyridyl), 14.48 (s, 1H, NH). 13C NMR
(d6-DMSO): 123.0; 125.5; 131.2; 133.7; 140.4; 148.6; 149.0; 151.0;
4.1.1.1. 3-(4-Nitrophenyl)-4-(pyridin-2-yl)-4,5-dihydro-1H-1,2,4-triazol-
5-thione (1). Yield: 60% (1.71 g) as a orange solid, m.p. 218–220 °C. IR
cm−1: 3099, 2926, 1662, 1591. 1H NMR (DMSO‑d6) δ ppm: 1H NMR
(DMSO‑d6): 7.53–7.57 (m, 1H, CHphenyl), 7.95–7.96 (m, 1H, CHphenyl),
7.98–7.89 (m, 1H, CHphenyl), 8.00–8.03 (m, 1H, CHphenyl), 8.06–8.07
7

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
169.7. LC-QTOF HRMS (m/z): Formula and Calculated Monoisotopic
4.1.1.10. 4-(2-Fluorophenyl)-3-(pyridin-3-yl)-1,2,4-triazolin-5-thione
Mass: C13H9N5O2S (299.0477), Measured mass: 299.0480.
(10). Yield: 83% (2.26 g) as a creamy solid, m.p. 276–278 °C. IR cm−1
:
3028, 1605, 1552. 1H NMR (DMSO‑d6) δ ppm: 7.37–7.59 (m, 2H,
CHphenyl), 7.61–7.65 (m, 1H, CHphenyl), 7.67–7.68 (m, 1H, CHphenyl),
7.70–7.72 (m, 1H, CHpyridyl), 7.73–7.74 (m, 1H, CHpyridyl), 8.54–8.64
(m, 2H, CHpyridyl), 14.53 (s, 1H, NH). 13C NMR (DMSO‑d6) δ ppm:
119.7; 124.8; 133.6; 134.2; 146.7; 156.9; 153.0; 156.8; 159.1; 161.0;
178.0. LC-QTOF HRMS (m/z): Formula and Calculated Monoisotopic
Mass: C13H9FN4S (272.0532), Measured mass: 272.0.535.
4.1.1.4. 4-Butyl-3-(4-nitrophenyl)-4,5-dihydro-1H-1,2,4-triazol-5-thione
(4). Yield: 68% (1.9 g) as a yellow solid, m.p. 196–199 °C. IR cm−1
:
3078, 22927, 1701, 1505, 1475. 1H NMR (DMSO‑d6) δ ppm: 0.73–0.78
(m, 3H, CH3); 1.10–1.19 (m, 2H, CH2); 1.46–1.56 (m, 2H, CH2); 4.08 (t,
2H, J = 7, CH2); 7.89–8.18 (m, 2H, CHphenyl); 8.33–8.41 (m, 2H,
CHphenyl); 14.15 (s, 1H, NH). 13C NMR (DMSO‑d6): 13.7; 19.4; 30.0;
44.1; 124.6; 130.5; 132.6; 149.1; 168.7. LC-QTOF HRMS (m/z):
Formula and Calculated Monoisotopic Mass:
(278.0837), Measured mass: 278.0842.
C
12H14N4O2S
4.1.1.11. 4-(2,4-Dichlorophenyl)-3-(pyridin-3-yl)-1,2,4-triazolin-5-thione
(11). Yield: 81% (2.61 g) as a creamy solid, m.p. 248–249 °C. 1H NMR
(DMSO‑d6) δ ppm: 7.43–7.44 (m, 1H, CHphenyl), 7.66–7.71 (m, 2H,
CHphenyl), 7.73–7.74 (m, 1H, CHpyridyl), 7.87–7.90 (m, 1H, CHpyridyl),
8.56–8.57 (m, 1H, CHpyridyl), 8.63–8.65 (m, 1H, CHpyridyl), 14.44 (s, 1H,
NH). 13C NMR (DMSO‑d6) δ ppm: 122.3; 124.3; 129.4; 130.5; 131.2;
133.3; 133.7; 135.5; 136.4; 148.5; 148.6; 151.9; 169.4. LC-QTOF HRMS
(m/z): Formula and Calculated Monoisotopic Mass: C13H8Cl2N4S
(321.9847), Measured mass: 321.9847.
4.1.1.5. 3-(4-Nitrophenyl)-4-(4-nitrophenyl)-4,5-dihydro-1H-1,2,4-
triazol-5-thione (5). Yield: 68% (2.33 g) as
a yellow solid, m.p.
252–254 °C. IR cm−1: 3349, 3078, 2828, 1678, 1589. 1H NMR
(DMSO‑d6) δ ppm: 6.49–6.74 (m, 2H, CHphenyl), 6.97–6.99 (m, 1H,
CHphenyl), 7.63–7.71 (m, 2H, CHphenyl), 7.86–8.17 (m, 1H, CHphenyl),
8.22–8.44 (m, 2H, CHphenyl), 14.02 (s, 1H, NH). 13C NMR (DMSO‑d6):
112.3; 113.2; 114.0; 117.5; 124.6; 125.3; 126.3; 127.2; 130.9; 131.5;
141.3; 148.3; 151.7; 168.5. LC-QTOF HRMS (m/z): Formula and
Calculated Monoisotopic Mass: C14H9N5O4S (343.0375), Measured
mass: 343.0377.
4.1.1.12. 4-(2-Fluorophenyl)-3-(pyridin-4-yl)-1,2,4-triazolin-5-thione
(12). Yield: 88% (2.39 g) as a white solid, m.p. 222–224 °C. IR cm−1
:
3429, 1606, 1505. 1H NMR (DMSO‑d6) δ ppm: 7.28 (d, 2H, CHpyridyl),
7.39–7.60 (m, 2H, CHphenyl), 7.60–7.70 (m, 2H, CHphenyl), 8.61 (d, 2H,
CHpyridyl), 14.51 (s, 1H, NH). 13C NMR (DMSO‑d6) δ ppm: 116.2; 116.9;
117.4; 126.1; 131.7; 133.2; 148.8; 150.8; 155.5; 158.9; 169.9. LC-QTOF
HRMS (m/z): Formula and Calculated Monoisotopic Mass: C13H9FN4S
(272.0532), Measured mass: 272.0535.
4.1.1.6. 4-(2-Chlorophenyl)-3-(4-nitrofenyl)-4,5-dihydro-1H-1,2,4-
triazol-5-thione (6). Yield: 77% (2.32 g) as
a yellow solid, m.p.
228–230 °C. IR cm−1: 3079, 2908, 1668, 1546. 1H NMR (DMSO‑d6) δ
ppm: 7.57–7.59 (m, 1H, CHphenyl), 7.60–7.61 (m, 1H, CHphenyl),
7.62–7.69 (m, 2H, CHphenyl), 7.76–7.79 (m, 2H, CHphenyl), 8.20–8.23
(m, 2H, CHphenyl), 14.48 (s, 1H, NH). 13C NMR (DMSO‑d6): 124.5;
129.2; 130.9; 131.8; 132.0; 132.1; 132.3; 132.6; 148.9; 149.1; 169.8.
LC-QTOF HRMS (m/z): Formula and Calculated Monoisotopic Mass:
C14H9ClN4O2S (332.0135), Measured mass: 332.0138.
4.1.1.13. 4-(2,4-Dichlorophenyl)-3-(pyridin-4-yl)-1,2,4-triazolin-5-thione
(13). Yield: 79% (2.55 g) as a creamy solid, m.p. 264–266 °C. IR cm−1
:
3066, 1608, 1512. 1H NMR (DMSO‑d6) δ ppm: 7.29–7.31 (m, 2H,
CHpyridyl), 7.69–7.70 (m, 1H, CHphenyl), 7.72–7.73 (m, 1H, CHphenyl),
7.81–7.94 (m, 1H, CHphenyl), 8.63–8.65 (m, 2H, CHpyridyl), 14.54 (s, 1H,
NH). 13C NMR (DMSO‑d6) δ ppm: 121.4; 122.2; 127.8; 129.3; 132.9;
133.2; 136.5; 139.9; 140.6; 150.6; 164.3; 165.0; 182.4. LC-QTOF HRMS
4.1.1.7. 4-(2-Fluorophenyl)-3-(pyridin-2-yl)-4,5-dihydro-1H-1,2,4-
triazol-5-thione (7). Yield: 87% (2.36 g) as
a white solid, m.p.
206–208 °C. 1H NMR (DMSO‑d6) δ ppm: 7.26–7.34 (m, 1H, CHphenyl),
7.36–7.43 (m, 1H, CHphenyl), 7.45–7.52 (m, 1H, CHphenyl), 7.53–7.56
(m, 1H, CHphenyl), 7.90–7.97 (m, 1H, CHpyridyl), 8.26–8.27 (m, 2H,
CHpyridyl), 8.28–8.30 (m, 1H, CHpyridyl), 14.38 (s, 1H, NH). 13C NMR
(d6-DMSO): 116.3, 116.5, 123.5, 124.0, 125.0, 125.7, 131.8, 138.1,
145.2, 149.6, 156.1, 159.4, 170.1. LC-QTOF HRMS (m/z): Formula and
Calculated Monoisotopic Mass: C13H9FN4S (272.0532), Measured mass:
272.0536.
(m/z): Formula and Calculated Monoisotopic Mass: C13H8Cl2N4S
(321.9847), Measured mass: 321.9851.
4.1.1.14. 4-(2-Chlorophenyl)-3-(pyridin-4-yl)-1,2,4-triazolin-5-thione
(14). Yield: 81% (2.33 g) as a yellow solid, m.p. 236–238 °C. 1H NMR
(DMSO‑d6) δ: 7.25–7.27 (m, 2H, CHpyridyl), 7.59–7.61 (m, 1H, CHphenyl),
7.68–7.70 (m, 1H, CHphenyl), 7.74–7.77 (m, 2H, CHphenyl), 8.59–8.61
(m, 2H, CHpyridyl), 14.49 (s, 1H, NH). 13C NMR (DMSO‑d6) δ ppm:
121.8; 128.4; 129.6; 130.5; 131.3; 132.4; 132.5; 132.6; 133.0; 133.7;
148.9; 151.1; 170.2. LC-QTOF HRMS (m/z): Formula and Calculated
4.1.1.8. 4-(2-Chlorophenyl)-3-(pyridin-2-yl)-1,2,4-triazolin-5-thione
(8). Yield 85% (2.45 g) as a white solid, m.p. 258–260 °C. 1H NMR
(DMSO‑d6) δ ppm: 7.36–7.41 (m, 1H, CHphenyl), 7.43–7.50 (m, 1H,
CHphenyl), 7.52–7.58 (m, 1H, CHphenyl), 7.60–7.61 (m, 1H,
CHphenyl),7.88–7.89 (m, 1H, CHpyridyl), 7.91–7.96 (m, 2H, CHpyridyl),
8.23–8.25 (m, 1H, CHpyridyl), 14.33 (s, 1H, NH). 13C NMR (d6-DMSO):
123.3; 125.6; 128.3; 130.0; 131.3, 131.6; 132.1; 133.9; 137.9; 145.3;
149.3; 149.5, 169.8. LC-QTOF HRMS (m/z): Formula and Calculated
Monoisotopic Mass:
288.0239.
C13H9ClN4S (288.0236), Measured mass:
4.1.1.15. 4-(3,4-Dichlorophenyl)-3-(pyridin-3-yl)-1,2,4-triazolin-5-thione
(15). Yield: 84% (2.71 g) as white solid, m.p. 184–185 °C. 1H NMR
(DMSO‑d6) δ: 7.41–7.44 (m, 1H, CHpyridyl), 7.45–7.53 (m, 1H,
CHpyridyl), 7.68–7.78 (m, 1H CHphenyl), 7.80–7.89 (m, 1H, CHphenyl),
8.09–8.26 (m, 1H, CHphenyl), 8.57–8.75 (m, 2H, CHpyridyl), 14.35 (s, 1H,
NH). 13C NMR (DMSO‑d6) δ: 122.8; 124.5; 130.2; 131.9; 132.1; 132.4;
133.4; 134.7; 137.0; 149.1; 149.6; 152.0; 169.5. LC-QTOF HRMS (m/z):
Formula and Calculated Monoisotopic Mass: C13H8Cl2N4S (321.9847),
Measured mass: 321.9850.
Monoisotopic Mass:
288.0238.
C13H9ClN4S (288.0236), Measured mass:
4.1.1.9. 4-(2-Chlorophenyl)-3-(pyridin-3-yl)-4,5-dihydro-1H-1,2,4-
triazol-5-thione (9). Yield: 86% (2.48 g) as
a white solid, m.p.
258–260 °C. 1H NMR (DMSO‑d6) δ ppm: 7.39–7.43 (m, 1H, CHphenyl),
7.55–7.61 (m, 1H, CHphenyl), 7.62–7.71 (m, 1H, CHphenyl), 7.72–7.75
(m, 1H, CHphenyl), 8.50–8.51 (m, 2H, CHpyridyl), 8.59–8.60 (m, 1H,
CHpyridyl), 8.61–8.62 (m, 1H, CHpyridyl), 14.40 (s, 1H, NH). 13C NMR
(DMSO‑d6) δ ppm: 122.5; 124.2; 129.1; 130.8; 132.1; 132.4; 132.6;
135.6; 148.4; 148.7; 151.9; 169.4. LC-QTOF HRMS (m/z): Formula and
4.1.1.16. 4-(2,4-Dichlorophenyl)-3-(pyridin-2-yl)-1,2,4-triazolin-5-thione
(16). Yield: 79% (2.55 g) as a yellow solid, m.p. 236–237 °C. IR cm−1
:
3079, 2908, 1546, 1551.1H NMR (DMSO‑d6) δ: 7.38–7.42 (m, 1H,
CHphenyl), 7.57–7.58 (m, 2H, CHphenyl), 7.83 (s, 1H, CHpyridyl),
7.90–8.01 (m, 2H, CHpyridyl), 8.27–8.29 (m, 1H, CHpyridyl), 14.40 (s,
1H, NH). 13C NMR (DMSO‑d6) δ: 124.1;125.5; 129.6; 131.0; 131.2;
131.3; 132.0; 136.0; 138.0; 145.2; 149.3; 149.4; 169.6. LC-QTOF HRMS
Calculated Monoisotopic Mass:
mass: 288.0238.
C13H9ClN4S (288.0236), Measured
8

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
(m/z): Formula and Calculated Monoisotopic Mass: C13H8Cl2N4S
a. Viable cells (VE). The nuclei of VE cells emit uniform bright green
(321.9847), Measured mass: 321.9850.
fluorescence and possess organized structure.
b. Early apoptotic cells (EA). The nuclei of EA cells emit green fluor-
escence but possess bright green dots as a consequence of chromatin
condensation.
4.1.1.17. 4-(3,4-Dichlorophenyl)-3-(pyridin-2-yl)-1,2,4-triazolin-5-thione
17. Yield: 72% (2.32 g) as a creamy solid, m.p. 231–233 °C. 1H NMR
(DMSO‑d6) δ: 7.03–7.06 (m, 1H, CHphenyl), 7.18–7.22 (m, 1H, CHphenyl),
7.46–7.57 (m, 1H, CHphenyl), 7.76–7.77 (m, 1H, CHpyridyl), 7.79–7.80
(m, 2H, CHpyridyl), 8.22–8.23 (m, 1H, CHpyridyl). 13C NMR (DMSO‑d6) δ:
123.0; 123.6; 129.7; 130.0; 130.5; 130.7, 131.2; 137.6; 139.5; 148.5;
149.3; 150.3; 170.9. LC-QTOF HRMS (m/z): Formula and Calculated
c. Late apoptotic cells (LA). The nuclei of LA cells emit orange to red
fluorescence and possess condensed or fragmented chromatin.
d. Necrotic cells (NC). The nuclei of NC cells emit red fluorescence and
possess organized structure.
4.5. Statistical analysis
Monoisotopic Mass:
321.9851.
C13H8Cl2N4S (321.9847), Measured mass:
The results of the in vitro cell culture experiments were presented as
mean values
standard deviation (SD). Differences were considered
4.2. Evaluation of anticancer acivity
significant with P < 0.05 in accordance with one-way ANOVA (ana-
lysis of variance) test followed by Tukey’s multiple comparison test
(GraphPad Prism 5, Version 5.04 Software). The IC50 values were cal-
culated via 4-parameter nonlinear regression analyses using GraphPad
Prism 5, version 5.04.
4.2.1. Cell lines
For in vitro experiments three cancer lines, i.e. A549 (human lung
adenocarcinoma), HepG2 (human hepatocellular carcinoma), and MCF-
7 (human breast adenocarcinoma) as well as one normal cell line BJ
(human fibroblasts) were used and cultured as described in details
earlier [42].
4.6. Kinase inhibitor assay
4.6.1. Source of enzymes
4.3. Evaluation of antiproliferative activity
In the enzymatic assays we used commercial human recombinant
kinase CK1γ3 (Sigma Aldrich, No. cat. SRP5241) with specific activity
in the range 17–23 nmol/min/mg and human recombinant CK2α with
specific activity 1 mmol/min/mg, that was produced and purified ac-
cording to the method described previously [47]. Briefly, CK2α was
overexpressed in E. coli BL21(DE3) cells harboring the plasmid pGEX-
3X::hsCK2α (kind gift from D. Litchfield) and purified by affinity
chromatography using glutathione-sepharose (Sigma Aldrich).
The specific activities of the kinases used in the experiments vary
substantially. This is related to the different sources of these enzymes.
The CK1y3 kinase purchased from Sigma Aldrich is characterized by a
very high degree of purity and high specific activity and obtained with
highly efficient recombination and overexpression methods (informa-
tion provided by the manufacturer). In turn, CK2α is an enzyme pro-
duced in laboratories with limited technical and methodological cap-
abilities; hence, their effectiveness cannot always be comparable to that
of biotechnological companies. The experimental conditions were se-
lected and optimized appropriately to compensate for these large dif-
ferences in specific enzyme activities.
In the first step, suspensions of cells in a complete growth mediums
at concentration of 3 × 104 cells/mL (A549), 1.5 × 105 cells/mL
(HepG2), 2.5 × 105 cells/mL (MCF-7) and 6 × 104 cells/mL (BJ) were
seeded in 96-well plates (100 μL/well) and incubated for 24 h at 37 °C
in a humidified atmosphere of 5% CO2. After incubation, culture
mediums were replaced with serial dilutions of tested compounds or
anticancer standard - Cisplatin. The antiproliferative activity was as-
sessed after 96 h incubation at 37 °C in a humidified atmosphere of 5%
CO2 using SRB assay in accordance with manufacturer protocol (Sigma-
Aldrich). The antiproliferative activity of compounds and Cisplatin was
evaluated in three separate experiments in octuplicate. The half max-
imal inhibitory concentration (IC50) was defined as the compound
concentration (µg/mL) required to inhibit cell proliferation to 50%.
Moreover, selectivity index (SI) which indicates the ratio between
proliferation inhibitory potential of a compound towards normal cells
and cancer cells was calculated using the following equation:
IC50 for normal cells
IC50for cancer cells
SI =
4.6.2. Substrates
4.4. Determination of apoptosis
Yeast ribosomal acidic protein P2B was overexpressed in E. coli
BL21(DE3) cells containing the plasmid pT7-7::6xHis-yP2B (kind gift
from J.P. Ballesta) and purified by affinity chromatography using Ni-
Affinity Gel (Sigma Aldrich). The dephosphorylated casein was pur-
chased from Sigma Aldrich.
For determination of apoptosis, suspensions of cancer cells in a
complete growth mediums at a concentration of 2.4 × 104 cells/mL
(A549), 8 × 104 cells/mL (HepG2) and 2 × 105 cells/mL (MCF-7) were
seeded in 24-well plates (500 μL/well) and incubated for 24 h at 37 °C
in a humidified atmosphere of 5% CO2. Then, culture medium was
replaced with 500 μL of 1 μg/mL of a selected derivative (the compound
which exhibited the highest antiproliferative activity) or 1 μg/mL of
Cisplatin and the cells were incubated for 96 h at 37 °C in a humidified
atmosphere of 5% CO2. In order to evaluate cell death, the acridine
orange/ethidium bromide (AO/EB) double staining was used. AO/EB
staining was considered as a suitable method to evaluate the morpho-
logical changes in cell nuclei [45,46,16,27]. Acridine orange stains both
live and dead cells and emits green fluorescence, while ethidium bro-
mide stains only nonviable cells and emits red fluorescence. Thus after
incubation, the cells were stained with 20 μL of dye mixture (100 µg/
mL AO and 100 µg/mL EB in distilled water) and immediately observed
under confocal microscope (Olympus Fluoview equipped with
FV1000). The apoptotic activity was assessed in two separate experi-
ments. In every experiment a minimum of 200 cells was counted in each
sample and distinguished for four cell populations as described below:
4.6.3. Phosphorylation assays
To estimate the inhibitory effect of newly synthesized triazole
analogs two different assays were employed. CK1γ3 activity was mea-
sured by incubation for 15 min at 30 °C in 25 µL samples containing
50 mM Tris/HCl pH 7.5, 6 mM Mg2+, 1 mM EDTA, 0.4 mM EGTA,
1 mM 2-mercaptoethanol, 240 ng/µl CK1γ3, 50 mM [γ-32P]ATP
(Hartmann Analytics GmbH) and as a substrate 1 mg/mL casein, and an
appropriate concentration of the tested compounds at concentration of
1, 5, 10, 20, 50 and 100 µM (Table 2S). In the case of CK2, the phos-
phorylation reactions were conducted at 30 °C for 15 min in 25 µL
samples each containing 1 pM of human recombinant CK2α, 10 µg of
ribosomal P2B protein as a substrate, and an appropriate concentration
of the tested triazole (1, 10, and 100 µM) (Table 3S). The reaction
buffer contained 20 mM [γ-32P]ATP (specific radioactivity
300–1000 cpm/pMl), 15 mM Mg2+, 20 mM Tris–HCl pH 7.5, and 6 mM
2-mercaptoethanol.
9

M. Pitucha, et al.
BioorganicChemistry99(2020)103806
The phosphorylation was terminated by addition of SDS/PAGE
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by Coomassie Brilliant Blue staining. The gels were dried and 32P-la-
belled bands of P2B or casein were excised from the gel, and radio-
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(standard deviation) of three independent experiments.
SD
4.7. Molecular modeling
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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Calculations were partially performed under a computational grant
by Interdisciplinary Center for Mathematical and Computational
Modeling (ICM), Warsaw, Poland, grant number G30-18 and under
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supported by DS2 project of Medical University of Lublin, Poland.
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