2,4-dichlorophenoxyacetic thiosemicarbazides as a new class of compounds against stomach cancer potentially intercalating with dna

Article abstract of DOI:10.3390/biom10020296

Thiosemicarbazide is a useful structural moiety that has the biological potential. Optimization of this structure can result in groundbreaking discovery of a new class of therapeutic agents. In the light of this, 1-(2,4-dichlorophenoxy)acetyl-4-(1-naphthy

Full text of DOI:10.3390/biom10020296

Article
2,4‐Dichlorophenoxyacetic Thiosemicarbazides
as a New Class of Compounds Against Stomach
Cancer Potentially Intercalating with DNA †
1,
2
3
2
Monika Pitucha *, Agnieszka Korga‐Plewko , Pawel Kozyra , Magdalena Iwan
4,5
and Agnieszka A. Kaczor
1
Independent Radiopharmacy Unit, Department of Organic Chemistry, Faculty of Pharmacy, Medical
University of Lublin, PL‐20093 Lublin, Poland
2
3
4
5
Independent Medical Biology Unit, Faculty of Pharmacy, Medical University of Lublin,
PL‐20093 Lublin, Poland; agnieszkakorga@umlub.pl
Student research group, Independent Radiopharmacy Unit, Faculty of Pharmacy,
Medical University of Lublin, PL‐20093 Lublin, Poland; pawekoz@interia.pl
Department of Synthesis and Chemical Technology of Pharmaceutical Substances,
Faculty of Pharmacy, Medical University of Lublin, PL‐20093 Lublin, Poland; agnieszka.kaczor@umlub.pl
School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, P.O. Box 1627,
FI‐70211 Kuopio, Finland
* Correspondence: monika.pitucha@umlub.pl; Tel.: 48‐81‐448‐72‐40
† This work was partially presented at the 11th Joint Meeting on Medicinal Chemistry 2019,
Prague, Czech, 27–30, June 2019, P‐70.
Received: 14 December 2019; Accepted: 11 February 2020; Published: 13 February 2020
Abstract: Thiosemicarbazide is a useful structural moiety that has the biological potential.
Optimization of this structure can result in groundbreaking discovery of a new class of therapeutic
agents. In the light of this, 1‐(2,4‐dichlorophenoxy)acetyl‐4‐(1‐naphthyl)thiosemicarbazide (1) and
1,4‐bis[(2,4‐dichlorophenoxy)acetylthiosemicarbazide]phenyl
(2)
were
synthesized
and
characterized by spectroscopic method. Cytotoxicity of obtained compounds was evaluated on
MKN74 gastric cancer cell line and human skin fibroblast BJ based on methylthiazolyldiphenyl‐
tetrazolium bromide (MTT) test. The apoptosis/necrosis and cell cycle analysis were conducted
using image cytometry. Additionally, in DNA of treated cells, abasic sites (AP) and double strands
breaks (DSB) presence were measured. Intercalating properties of active compounds were evaluated
using the UV–spectroscopic method. Among newly synthesized derivatives, compound 2 showed
toxic effects on gastric cancer cells with simultaneous lack of toxicity to normal fibroblasts. Cell cycle
analysis revealed that both compounds influence cell division mainly at the stage of replication.
Simultaneously with DNA synthesis disorders, DNA damages like AP‐sites and DSBs were
observed. Spectroscopic studies revealed possible DNA intercalating properties of tested
compounds. Obtained results indicate that the newly synthesized thiosemicarbazide derivatives are
a promising group of compounds with potential anticancer activity resulted from interactions with
DNA and cell cycle interrupt.
Keywords: thiosemicarbazide; cytotoxicity; cell cycle; DNA; DNA intercalators
1. Introduction
Cancer belongs to the main reasons for death worldwide in countries of all income levels [1].
According to WHO, cancer is the second leading cause of death globally and is estimated to account
for 9.6 million death in 2018. Globally, about 1 in 6 deaths is due to cancer. Lung, prostate, colorectal,
stomach, and liver cancer are the most common types of cancer in men, while breast, colorectal, lung,
Biomolecules 2020, 10, 296; doi:10.3390/biom10020296
www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 296
2 of 13
cervix, and thyroid cancer are the most common among women [2]. Among various types of cancer,
gastric cancer is the second most common reason for cancer‐related deaths worldwide [3].
Surgery and radiotherapy are the most efficient and valuable methods for local and non‐
metastatic cancer treatment, but they are not effective when cancer has spread throughout the
organism [4]. The application of anticancer drugs, i.e., chemotherapy, hormone, and biological
therapies, is the current method of choice for the treatment of metastatic cancers since they are
capable of reaching each part of the body via the bloodstream [4]. The currently available anticancer
drugs can be divided into cytotoxic‐based drugs and target‐based drugs. The cytotoxic compounds
are able to rapidly kill dividing cells by targeting components of the mitotic and/or DNA replication
pathways while target‐based drugs block the cancer growth by interactions with molecular targets
involved in cancer progression [5]. Novel drugs to treat cancer are still developed [6–9] to meet the
need of alarming cancer epidemiology.
Our research focuses for a long time on searching for novel compounds with anticancer
activity [10–13]. In particular, we focus on thiosemicarbazides as a privileged scaffold in medicinal
chemistry, displaying a wide range of activities, i.e., antibacterial, antifungal, antioxidant [14–18], and
anticancer activity [17–19]. Among a series of different thiosemicarbazides derivatives studied
against human gastric cancer cell line MKN74, we selected two most promising based on IC50 values.
Here we present synthesis and characterization of these two compounds, cell cycle analysis, oxidative
DNA damage, and spectrophotometry study on the interactions of the compounds with ds‐DNA as
well as molecular docking results on the interactions of the compounds with DNA.
2. Materials and Methods
All chemicals used for the synthesis were purchased from Sigma‐Aldrich (Sigma‐Aldrich, St.
Louis, Missouri, USA), Alfa Aesar (Haverhill, MA, United States), and POCH (Polish Chemical
Reagents, Gliwice, Poland) companies. Melting points were determined using Fisher–Johns block.
1
l3
The H and C NMR spectra were recorded on a Bruker Avance 600 spectrometer (Bruker BioSpin
GmbH, Rheinstetten, Germany). The attenuated total reflectance ATR‐IR spectra were recorded on
the Thermo Scientific Nicolet 6700 FTIR spectrophotometer (Thermofisher, Waltham, Massachusetts,
USA). The mass measurements were made using an Agilent Technologies liquid chromatograph 1290
coupled to an Agilent Technologies 6550 iFunnel Q‐TOF LC/MS (Agilent Technologies, Santa Clara,
CA, USA) [20]. Compound 1 was first obtained by Gupta et al. [21].
2.1. General Procedure for the Synthesis of 1,4‐Disubstituted thiosemicarbazide derivatives (1,2)
0.5 g of 2,4‐Dichlorophenoxyacetic acid hydrazide was mixed with appropriate isothiocyanate (1‐
naphthylisothiocyanate or 1,4‐phenylenediisothiocyanate) in 15 mL methanol solution. Next, the
mixture was heated at reflux temperature for 2h. After cooling, the target substance was filtered off
and crystallized from methanol.
2.1.1. 1‐(2,4‐Dichlorophenoxy)acetyl‐4‐(1‐naphtyl)thiosemicarbazide (1) [21]
−1
1
Yield: 80 %, m.p. 161‐162 °C. IR cm : 3307 (NH), 3183 (NH), 3057 (NH), 1661 (C=O). H NMR (DMSO‐
d6) δ ppm: 4.86 (s, 2H), 7.16‐7.62 (m, 7H, CHar), 7.87‐7.97 (m, 3H, CHar), 9.78 (s, 1H, NH), 9.94 (s, 1H,
13
NH), 10.41 (s, 1H, NH). C NMR (DMSO‐d6) δ ppm: 67.1; 115.9; 122.9; 124.1; 125.5; 125.9; 126.4; 126.5;
127.0; 127.5; 128.3; 129.3; 131.1; 134.2; 135.9; 153.1; 167.1; 170.6; 182.9. LC‐QTOF MS (m/z): calculated
monoisotopic mass: 419.0262, measured monoisotopic mass: 419.0266 (Figure S1).
2.1.2. 1‐[[2‐(2,4‐Dichlorophenoxy)acetyl]amino]‐3‐[4‐[[[2‐(2,4‐dichlorophenoxy)acetyl]amino]
carbamoylamino]phenyl]thiourea (2)
−1
1
Yield: 77 %, 158‐159 °C. IR cm : 3315 (NH), 3165 (NH), 1693 (C=O). H NMR (DMSO‐d6) δ ppm: 4.80
(s, 4H, 2xCH2), 7.14‐7.36 (m, 2H, CHar), 7.35‐7.43 (m, 6H, CHar), 7.55‐7.60 (m, 2H, CHar), 9.68‐9.86 (m,
13
4H, 4NH), 10.26 (s, 2H, 2NH). C NMR (DMSO‐d6): 67.0; 115.5; 115.9; 122.5; 122.8; 125.0; 125.7; 126.2;

Biomolecules 2020, 10, 296
3 of 13
126.8; 128.1; 128.3; 129.8; 133.6; 136.5; 139.0; 152.0; 153.0; 167.3; 170.7; 182.2. LC‐QTOF MS (m/z):
calculated monoisotopic mass: 659.9742, measured monoisotopic mass: 659.9736 (Figure S2).
2.2. Cell Culturing
The human gastric cancer cell line MKN74 (Japanese Cancer Research Resources Bank, JCRB,
Tokyo, Japan) and human skin fibroblast BJ (ATCC, Manassas, Wirginia, USA) were cultured in
RPMI and Eagleʹs Minimum Essential Medium (EMEM) (Corning, New York, New York, USA)
respectively, supplemented with 10 % fetal bovine serum (Corning, New York, New York, USA) and
a set of antibiotics: penicillin (100 units) and streptomycin (100 μg/mL) (Corning, New York, New
York, USA). Cells were incubated at 37 °C with 5% CO2 in air atmosphere. The cell morphology was
analyzed under a phase‐contrast microscope Nikon Eclipse Ti using NIS‐Elements Imaging Software
(Nikon, Tokyo, Japan). The authenticity of tested cell lines was verified by short tandem repeat (STR)
genotyping in the Department of Forensic Medicine (Medical University of Lublin, Lublin, Poland).
2.3. The Cytotoxicity Analysis
The cytotoxicity of tested compounds was evaluated using the MTT (methylthiazolyldiphenyl‐
tetrazolium bromide) test. The test is based on the living cells’ ability to reduce orange tetrazolium
salt to water‐insoluble purple formazan crystals. The cells were seeded into 96‐well plates: MKN74
5
5
in the concentration of 1.5×10 cells/mL and BJ in the concentration of 1x10 cells/mL. The tested
compounds (in the final concentration of 10, 125, and 250 μM) were added when 70%–80% of
confluence was achieved. The MTT ‐ 3‐(4,5‐dimethylthiazol‐2‐yl)‐ 2,5diphenyltetrazolium bromide
(Thermofisher, Waltham, Massachusetts, USA) solution (4.0 mg/mL) was added to the culture 24 h
after chemicals. After 4 h of incubation, the medium with MTT was removed, and the formed crystals
were dissolved in DMSO (dimethyl sulfoxide, 200 mL/well, POCH, Poland). The solution absorbency
was measured at 540 nm using PowerWave microplate spectrophotometer (BioTek Instruments,
USA). Each assay was conducted three times and was measured in triplicates. IC50 values were
determined using the AAT Bioquest IC50 calculator [22].
2.4. Cell Cycle Analysis
Cell cycle analysis was conducted using NucleoCounter NC‐3000 (ChemoMetec, Lillerød,
Denmark) according to manufacturer instructions. The MKN74 cells were seeded into 6‐well plates
5
at a concentration of 1.5 × 10 cells/mL and the tested compounds were added when 70%–80% of
confluence was achieved. After 24 h of incubation with tested compounds, the cells were collected
and analyzed according to the manufacturer’s protocol‐ 2‐step Cell Cycle Assay (ChemoMetec,
Lillerød, Denmark). The culture medium was removed and cells were washed with PBS and
resuspended in Solution 10 supplemented with DAPI (4′,6‐Diamidine‐2′‐phenylindole
dihydrochloride) 10 μg/mL. Then, the cells were incubated at 37 °C for 5 minutes, and Solution 11
was added. Thirty microliters of the cell suspension was loaded into NC‐Slide and read in
NucleoCounter. Each experiment was conducted three times with measurement in triplicate.
2.5. Determination of DNA Oxidative Damage
The oxidative DNA damage was evaluated by measuring the number of abasic sites (AP sites)
that are one of the major types of damage generated by reactive oxygen species (ROS). The cells were
5
seeded into 6‐well plates in a concentration of 1.5 × 10 cells/mL. The tested compounds (in a final
concentration of 10, 125, and 250 μM) were added when 70%–80% of confluence was achieved. After
24‐hour incubation, the DNA was isolated with the Syngen DNA Mini Kit (Syngen, Poland)
according to the manufacturer’s protocol. The concentration of the genomic DNA was measured
using the MaestroNano Micro‐Volume Spectrophotometer (Maestrogen Inc., Hsinchu, Taiwan) and
adjusted to 100ꢀμg/mL in the TE buffer. The number of abasic sites (AP sites) was evaluated with the
DNA Damage Quantification Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s

Biomolecules 2020, 10, 296
4 of 13
instructions. The method is based on a specific reaction of an aldehyde‐reactive probe (ARP; N′‐
aminooxymethylcarbonylhydrazin‐D‐biotin) with an aldehyde group present on the open‐ring form
of AP sites. AP sites were tagged with biotin residues and were quantified using avidin‐biotin assay
followed by colorimetric detection of peroxidize‐conjugated to the avidin at 650ꢀnm using
PowerWave™ microplate spectrophotometer (BioTek Instruments, Winooski, Vermont, USA). Each
experiment was conducted three times with measurement in triplicate.
2.6. DNA Damage – Double Strand Breaks (DSB)
5
The cells were seeded into 96‐well plates in a concentration of 1.5 × 10 cells/mL. The tested
compounds (in a final concentration of 10, 125, and 250 μM) were added when 70%–80% of
confluence was achieved. After 24‐hour incubation, the DNA damage (DSB) was determined using
the HCS DNA Damage Kit (Thermofisher, Waltham, Massachusetts, USA) according to the
manufacturer’s instruction. Phosphorylation of H2A histones is a known response to DSBs formation
specifically. The DSBs level was measured by specific antibody‐based detection of phosphorylated
H2AX (Ser139) in the nucleus. The fluorescence of secondary antibody was measured using the
SpectraMax i3 Multi‐Mode Platform (Molecular Devices, San Jose, California, USA). Each experiment
was conducted three times with measurement in triplicate.
2.7. Spectrophotometry Study on the Interaction of the Compounds with ds‐DNA
Calf thymus DNA (CT‐DNA) was supplied by Sigma‐Aldrich,( St. Louis, Missouri, USA). The
stock solution of DNA was prepared by dissolving 0.25 mg of DNA in 1 mL of phosphate buffer (pH
7.4). The absorption was titrated by keeping the concentrations of CT DNA constant with varying
concentrations of the tested compound – 50, 40, 30, 20, and 10 μM. The absorption spectra were
recorded in the wavelength range of 240–350 nm using PowerWave microplate spectrophotometer
with 1‐cm path length quartz cuvettes (BioTek Instruments, Winooski, Vermont, USA).
2.8. Statistical Analysis
The results were analyzed statistically in the STATISTICA vs. 12 application (StatSoft, Krakow,
Poland). The data were calculated as meanꢀ ± ꢀSD. To compare more than two groups, one‐way
analysis of variance (ANOVA) and post hoc multiple comparisons with Tukey’s HSD test were used.
All parameters were considered statistically significantly different if P values were less than 0.05.
2.9. Molecular Modeling
2.9.1. Compound Preparation
Compounds 1 and 2 were modeled using LigPrep module [23] of Schrödinger suite of software,
v. 2019‐4 as previously reported [24–28]. To identify the protonation state, Epik module [29] of
Schrödinger suite of software, v. 2019‐4 was applied.
2.9.2. Molecular Target
DNA fragment in complex with
a threading intercalator 5‐bromo‐9‐amino‐N‐ethy
(diaminomethyl)acridine‐4‐carboxamide (PDB ID: 367D [30]) was used for molecular docking. DNA
was prepared using respective tools of Schrödinger suite of software, v. 2019‐4.
2.9.3. Molecular Docking
Standard Precision (SP) method of Glide [31] from Schrödinger v. 2019‐4 was used for molecular
docking. The grid file was generated based on the co‐crystallized ligand. 100 poses were obtained for
each compound. The final poses were selected based on the Glide scoring function and visual
inspection. Visualization of molecular modeling results was achieved with Maestro Release 2019.4
[32] and PyMol 2.0.4 [33] software.

Biomolecules 2020, 10, 296
5 of 13
3. Results and Discussion
The title compounds were obtained by reacting 2,4‐dichlorophenoxyacetic hydrazide with 1‐
naphthylisothiocyanate (1) and 1,4‐phenylene diisothiocyanate (2). The reactions were carried out in
methanol in accordance with the procedures developed in our laboratory [12]. Structures of the
1
13
obtained compounds (Figure 1) were determined by H and C NMR, IR, and MS.
O
Cl
Cl
S
Cl
NH
NH
O
S
NH
NH
NH
NH
O
NH
S
O
NH
NH
Cl
O
Cl
Cl
O
1
2
Figure 1. Structure of tested compounds.
Cytotoxicity of tested compounds was evaluated by the MTT test. Both compounds revealed
toxicity against MKN74 cells (Figure 2). Studies on normal human fibroblasts showed lower toxicity
of the tested compounds: IC50 = 631.45 vs 137.38 μM for compound 1 and 756.85 vs. 143.54 μM for
compound 2 (Figure 3).
Figure 2. The relative MKN74 gastric cancer cells viability treated with compound 1 or 2 for 24h
determined by MTT assay. The results were calculated as % of control cultures’ viabilities which were
averaged to define the 100%. Values were presented as meanꢀ ± ꢀSD derived from three independent
experiments. *pꢀ <ꢀ 0.05, ***p < 0.001 vs. control; (ANOVA followed by Tukey’s HSD post hoc analysis).
IC50 value was determined using the AAT Bioquest IC50 calculator.
Figure 3. The relative BJ fibroblast cells viability treated with compound 1 or 2 for 24h determined by
MTT assay. The results were calculated as % of control cultures’ viabilities which were averaged to
define the 100%. Values were presented as meanꢀ ± ꢀSD derived from three independent experiments.
**pꢀ <ꢀ 0.01, ***p < 0.001 vs. control; (ANOVA followed by Tukey’s HSD post hoc analysis). IC50 value
was determined using the AAT Bioquest IC50 calculator.

Biomolecules 2020, 10, 296
6 of 13
IC50 values for tested compounds were relatively high, as clinically useful drugs work at much
lower concentrations. 5‐fluorouracyl is the first‐choice drug for the treatment of advanced gastric
cancer but its effectiveness is limited by drug resistance [34]. The 5‐fluorouracil toxicity toward
MKN74 and BJ was determined. The obtained IC50 value in the case of cancer cells was clearly lower
than the tested compounds: 37.54 vs. 631.45 or 756.85 μM (Figure 4). However, the ratio of IC50 values
for normal vs. neoplastic cells was more beneficial for the tested compounds: 85.04/37.54 μM vs.
631.45/137.38 μM or 756.85/143.54 μM for compound 1 and 2, respectively. This observation justifies
the further studies of new compounds that we obtained.
Figure 4. The relative MKN74 gastric cancer cells and BJ fibroblast viability treated with 5‐fluorouracil
for 24h determined by MTT assay. The results were calculated as % of control cultures’ viabilities
which were averaged to define the 100%. Values were presented as meanꢀ ± ꢀSD derived from three
independent experiments. **pꢀ < ꢀ0.01, ***p < 0.001 vs. control; (ANOVA followed by Tukey’s HSD post
hoc analysis). IC50 value was determined using the AAT Bioquest IC50 calculator.
The MTT test results for compounds 1 and 2 were verified by microscopic observations of cell
morphology. The cells in control cultures revealed normal, epithelial‐like morphology and were
closely arranged and well adherent. Cells treated with compounds 1 and 2 for 24 hours became round
and had poor adherence especially in case of the highest concentrations of the compounds (Figure 5).
Figure 5. MKN74 cells morphology analyzed under a phase‐contrast microscope Nikon Eclipse Ti.
The cells were treated with compounds 1 or 2 (250 μM) for 24 hours (magnification ×400).
Cytotoxicity was confirmed by apoptosis/necrosis analysis. After the use of compound 1 at a
concentration of 250 μM, both late apoptotic and necrotic cells were observed. Compound 2 in the
same concentration caused mainly apoptosis (88% of cells were in late apoptotic phase, Figure 6).

Biomolecules 2020, 10, 296
7 of 13
Figure 6. Cell apoptosis/necrosis of MKN74 cells, stained with Annexin V‐FITC and PI for image
cytometry, analyzed by NucleoCounter NC‐3000. (A) Live, (B) early apoptotic, (C) late apoptotic,
and (D) necrotic cells. The cells were treated with compounds 1 and 2 (250 μM) for 24 hours.
Cell cycle analysis by image cytometry revealed that both compounds arrested cell cycle.
Compound 2 acted clearly in S phase while the action of compound 1 was more complex ‐ an increase
in the population of cells in either S‐phase or G2 phase was observed. Due to the fact that DNA
synthesis has been inhibited and consequently cell division, a decrease in cell population in G1 phase
has been observed (Figure 7). These results indicate that both compounds influence cell division
mainly at the stage of replication, although the mechanism of their action is not identical.
A
B
Figure 7. A) Cell cycle analysis through DAPI (4′,6‐Diamidine‐2′‐phenylindole dihydrochloride)
staining and image cytometry by NucleoCounter NC‐3000. The results were calculated as % of control
values that were averaged to define the 100%. The MKN74 cells were treated with compounds 1 and
2 (250 μM) for 24 hours. Values were presented as meanꢀ ± ꢀSD derived from three independent
experiments. **pꢀ <ꢀ 0.01, ***p < 0.001 vs. control; (ANOVA followed by Tukey’s HSD post hoc
analysis). B) Representative histograms (M1‐4: cell cycle phases subG1, G1, S, G2/M, respectively).

Biomolecules 2020, 10, 296
8 of 13
DNA synthesis disorders are closely related to DNA helix damages. The analysis of oxidative
DNA damage showed a significant increase of the AP sites accumulation in the DNA isolated from
the MKN74 cells treated with compound 2 in a concentration of 250 μM (12.03 ± 1.89 AP sites/100
kbp) in comparison to the control culture (1.90 ± 0.41 AP sites/100 kbp, see Figure 8). Lesser, but
statistically significant increases were observed for compound 2 in concentration of 125 μM and
compound 1 in a concentration of 250 μM (5.40 ± 1.21 and 4.13 ± 0.32 AP sites/100 kbp, respectively).
Both tested compound treatment resulted in DNA damages in the form of DSBs, mostly compound
1 in concentration of 250 μM (283, 33 ± 35, 12% of control culture, Figure 9).
Figure 8. Abasic sites’ (AP) number per 100 kbp in DNA of MKN74 cells treated with compound 1 or
2 for 24 hours. The AP site detection was based on a specific reaction of an aldehyde‐reactive probe
(ARP) with an aldehyde group present on the open‐ring form of AP sites. AP sites were tagged with
biotin residues and were quantified using avidin‐biotin assay followed by colorimetric detection of
peroxidase conjugated to the avidin at 650ꢀnm. Values were presented as meanꢀ ± ꢀSD. *pꢀ<ꢀ0.05,
***p<0.001 vs. control; (ANOVA followed by Tukey’s HSD post hoc analysis).
Figure 9. The content of double strands breaks (DSB) in tested cell’s DNA (based on phosphorylated
H2AX level) presented as a % of control. The cells were treated with compound 1 or 2 for 24 hours.
The DSBs level was measured by specific antibody‐based detection of phosphorylated H2AX (Ser139)
in the nucleus. The fluorescence of secondary antibody was measured using the SpectraMax i3 Multi‐
Mode Platform. Values were presented as meanꢀ ± ꢀSD. *p ꢀ<ꢀ 0.05, ***p < 0.001 vs. control; (ANOVA
followed by Tukey’s HSD post hoc analysis).
Observed cell cycle arrest, as well as DNA damages, may be a result of DNA intercalation. To
examine whether the tested compounds intercalate into DNA, UV–spectroscopic studies were
evaluated. The spectrum of CT‐DNA has been changed in the presence of varying amounts of
compounds 1 and 2. The hypochromatic effect that was observed (Figure 10 and 11) generally results
from the damage in the DNA double‐helix structure and is connected to the intercalation of the
compound [35,36].

Biomolecules 2020, 10, 296
9 of 13
Figure 10. Absorption spectra of calf thymus DNA (250 μg/mL) in phosphate buffer (pH 7.4) upon
addition of compound 1 (10, 20, 30, 40, and 50 μM, respectively).
Figure 11. Absorption spectra of calf thymus DNA (250 μg/mL) in phosphate buffer (pH 7.4) upon
addition of compound 2 (10, 20, 30, 40, and 50 μM, respectively).
The new thiosemicarbazide derivatives have shown toxic effects on the highly resistant gastric
cancer cell line while not affecting normal cells. The selectivity of new cancer drugs is extremely
important because of the serious side effects of existing therapies. The main goal of cancer therapy is
to stop cell division and induce apoptosis. The tested derivatives induced apoptosis in tumor cells
probably by inducing DNA damage, especially DSBs. The second of the detected DNA damages ‐ AP
sites can block the advancement of DNA replication forks, and also block the DNA polymerases [37].
For the above reasons, cell cycle arrest was expected. The analysis showed an increase in the
population of cells in S phase and, additionally, in the case of compound 2 also in G2 phase. The DNA
biosynthesis process involves an internal S‐phase checkpoint system that limits the rate of replication
in the event of physical DNA damage, especially DSB, and blocks the possibility of initiating further
steps of the cell cycle by those cells. Any structural disorder forces a slowdown in the rate of
replication fork motion and DNA damage response (DDR). The main goal of DDR is DNA repair or
if it is ineffective, induction of apoptosis [38,39].
Based on the results obtained, it is not possible to assess the mechanism of interaction of the
tested compounds on DNA. The presence of AP‐sites may indicate the generation of oxidative stress
[40]. In turn, UV–spectroscopic studies have shown direct interactions, i.e., the formation of
complexes of tested compounds with DNA. However, the exact type of binding cannot be determined
on this basis.
As experimental results described above indicated that a possible mechanism of anticancer
activity of compounds 1 and 2 is interaction with DNA, we decided to use molecular docking to find
how the compounds can interact with DNA. We assumed that the compounds are DNA intercalators
as intercalation is one of the most common binding modes of small aromatic molecules with DNA

Biomolecules 2020, 10, 296
10 of 13
[41]. Intercalation is the insertion of a small molecule or fragment between two adjacent base pairs
(usually CG) of the DNA strand either with or without additional interactions with the grooves [33].
Figure 12 presents the docking poses of compounds 1 and 2 in a double‐stranded DNA fragment.
DNA intercalators commonly possess a planar polyaromatic system that forms π−π interactions with
the two flanking bases [41]. These interactions are the most important driving force of the
intercalation binding mode [41]. The aromatic rings of compounds 1 and 2 are located between two
guanine ring systems, which constitute the intercalation binding mode. Moreover, compound 1 forms
two hydrogen bonds between its 2,4‐dichlorophenoxy oxygen atom and one of its nitrogen/hydrogen
atom and the carbonyl group of guanine. In the case of compound 2, a hydrogen bond is formed
between one of its nitrogen/hydrogen atom and the carbonyl group of guanine, and another
hydrogen bond is formed between its oxygen atom and the amino group of guanine. The values of
Glide scoring functions were ‐6.55 for a selected pose of compound 2 and ‐6.48 for a selected pose of
compound 1. These values are comparable; however, they indicate slightly stronger interactions of
compound 2, which is in accordance with its better anticancer activity.
Figure 12. Compound 1 (A) and compound 2 (B) intercalating with DNA as obtained using molecular
docking approach of flexible ligands to rigid DNA fragment. DNA is shown in wire representation
with magenta carbon atoms. Ligands are shown in stick representation with grey carbon atoms. Non‐
polar hydrogen atoms not shown for clarity. Hydrogen bonds are shown as red dashed lines.
4. Conclusions
Compounds 1 and 2 revealed cytotoxicity against a gastric cancer cell line without significant
toxicity against normal cells. Compound 1 arrested cell cycle in S and G2 phase, compound 2 visibly
arrested cell cycle in S phase. Both compounds generated DNA damages in the form of AP‐sites and
DSBs presence. UV spectrum analysis indicates the possibility of intercalation of both compounds to
DNA.
Supplementary Materials: Supplementary Materials are available online.
Author Contributions: Supervision, M.P.; project administration, A.K‐P.; investigation, M.P., A.K‐P., P.K., M.I.,
and A.A.K.; validation and visualization, A.A.K. and A.K‐P. All authors have read and agreed to the published
version of the manuscript.
Funding: Calculations were partially performed under a computational grant by Interdisciplinary Center for
Mathematical and Computational Modeling (ICM), Warsaw, Poland, grant number G30‐18 (to A.A.K.), under
resources and licenses from CSC, Finland (to A.A.K).
Acknowledgements: The authors thank Emilia Fornal (Department of Pathophysiology, Medical University of
Lublin, Poland) for LC/QTOF data acquisition and analysis.
Conflicts of Interest: The authors declare no conflict of interest.

Biomolecules 2020, 10, 296
11 of 13
Abbreviations
AB
abasic sites
DAPI
DSB
MTT
ROS
SP
4′,6‐Diamidine‐2′‐phenylindole dihydrochloride
double strands breaks
Methylthiazolyldiphenyl‐tetrazolium bromide
Reactive Oxygen Species
Standard Precision
References
1.
Torre, L.A.; Siegel, R.L.; Ward, E.M.; Jemal, A. Global Cancer Incidence and Mortality Rates and Trends‐
An Update. Cancer Epidemiol. Biomark. Prev. 2016, 25, 16–27.
2.
3.
Available online: https://www.who.int/health‐topics/cancer (accessed on 14 December 2019).
Sitarz, R.; Skierucha, M.; Mielko, J.; Offerhaus, G.J.A.; Maciejewski, R.; Polkowski, W.P. Gastric cancer:
Epidemiology, prevention, classification, and treatment. Cancer Manag. Res. 2018, 10, 239–248.
Pérez‐Herrero, E.; Fernandez‐Medarde, A. Advanced targeted therapies in cancer: Drug nanocarriers, the
future of chemotherapy. Eur. J. Pharm. Biopharm. 2015, 93, 52–79.
Sun, J.; Wei, Q.; Zhou, Y.; Wang, J.; Liu, Q.; Xu, H. A systematic analysis of FDA‐approved anticancer drugs.
BMC Syst. Boil. 2017, 11, 87.
Wu, C.; Wu, K.‐J.; Liu, J.‐B.; Zhou, X.‐M.; Leung, C.‐H.; Ma, D.‐L. A dual‐functional molecular strategy for
in situ suppressing and visualizing of neuraminidase in aqueous solution using iridium(iii) complexes.
Chem. Commun. 2019, 55, 6353–6356.
4.
5.
6.
7.
Yang, G.‐J.; Ko, C.‐N.; Zhong, H.‐J.; Leung, C.‐H.; Ma, D.‐L. Structure‐Based Discovery of a Selective
KDM5A Inhibitor that Exhibits Anti‐Cancer Activity via Inducing Cell Cycle Arrest and Senescence in
Breast Cancer Cell Lines. Cancers 2019, 11, 92.
8.
9.
Xiao, M.; Lai, W.; Wang, F.; Li, L.; Fan, C.; Pei, H. Programming Drug Delivery Kinetics for Active Burst
Release with DNA Toehold Switches. J. Am. Chem. Soc. 2019, 141, 20354–20364.
Lee, S.H.; Park, O.K.; Kim, J.; Shin, K.; Pack, C.G.; Kim, K.; Ko, G.; Lee, N.; Kwon, S.‐H.; Hyeon, T. Deep
Tumor Penetration of Drug‐Loaded Nanoparticles by Click Reaction‐Assisted Immune Cell Targeting
Strategy. J. Am. Chem. Soc. 2019, 141, 13829–13840.
10. Pitucha, M.; Rzymowska, J.; Olender, A.; Grzybowska‐Szatkowska, L. Synthesis of 1,6‐hexanediyl‐
bis(semicarbazides) and 1,6‐hexanediyl‐bis(1,2,4‐triazol‐5‐ones) and their antiproliferative and
antimicrobial activity. J. Serb. Chem. Soc. 2012, 77, 1–8.
11. Kaczor, A.A.; Pitucha, M.; Karczmarzyk, Z.; Wysocki, W.; Rzymowska, J.; Matosiuk, D. Structural and
molecular docking studies of 4‐benzyl‐3‐[(1‐methylpyrrol‐ 2‐yl)methyl]‐4,5‐dihydro‐1H‐1,2,4‐triazol‐5‐one
with anticancer activity. Med. Chem. 2013, 9, 313–328.
12. Pitucha, M.; Woś, M.; Miazga‐Karska, M.; Klimek, K.; Mirosław, B.; Pachuta‐Stec, A.; Gładysz, A.; Ginalska,
G. Synthesis, antibacterial and antiproliferative potential of some new 1‐pyridinecarbonyl‐4‐substituted
thiosemicarbazide derivatives. Med. Chem. Res. 2016, 25, 1666–1677.
13. Wos, M.; Miazga‐Karska, M.; Kaczor, A.A.; Klimek, K.; Karczmarzyk, Z.; Kowalczuk, D.; Wysocki, W.;
Ginalska, G.; Urbanczyk‐Lipkowska, Z.; Morawiak, M.; et al. Novel thiosemicarbazide derivatives with 4‐
nitrophenyl group as multi‐target drugs: α‐glucosidase inhibitors with antibacterial and antiproliferative
activity. Biomed. Pharmacother. 2017, 93, 1269–1276.
14. Shefali, A.; Shilpi, A.; Shailey, S. Anticancer activity of thiosemicarbazides/thiosemicarbazones: A review.
Int. J. Pharm. Pharm. Sci. 2014, 6, 34–41.
15. Singhal, S.; Arora, S.; Agarwal, S.; Sharma, R.; Singhal, N. A review on potential biological activities of
thiosemicarbazides. WJPPS 2013, 2, 4661–4681.
16. Reshma,N.J.; Avinash, D.S. Antibacterial activity of thiosemicarbazide derivatives. Der Pharma Chemica
2013, 5, 45–49.
17. Zhang, X.; Lei, P.; Sun, T.; Jin, X.; Yang, X.; Ling, Y. Design, Synthesis, and Fungicidal Activity of Novel
Thiosemicarbazide Derivatives Containing Piperidine Fragments. Molecules 2017, 22, 2085.
18. Chen, R.; Huo, L.; Jaiswal, Y.; Huang, J.; Zhong, Z.; Zhong, J.; Williams, L.; Xia, X.; Liang, Y.; Yan, Z. Design,
Synthesis, Antimicrobial, and Anticancer Activities of Acridine Thiosemicarbazides Derivatives. Molecules
2019, 24, 2065.

Biomolecules 2020, 10, 296
12 of 13
19. He, J.; Wang, X.; Zhao, X.; Liang, Y.; He, H.; Fu, L. Synthesis and antitumor activity of novel quinazoline
derivatives containing thiosemicarbazide moiety. Eur. J. Med. Chem. 2012, 54, 925–930.
20. Pitucha, M.; Karczmarzyk, Z.; Swatko‐Ossor, M.; Wysocki, W.; Wos, M.; Chudzik, K.; Ginalska, G.;
Fruzinski, A. Synthesis, In Vitro Screening and Docking Studies of New Thiosemicarbazide Derivatives as
Antitubercular Agents. Molecules 2019, 24, 251.
21. Sen Gupta, A.K.; Misra, H.K. Studies on potential pesticides. Part XIII: Synthesis and evaluation of S‐(3‐
substituted phenoxymethyl‐4‐aryl/ayclohexyl‐4H‐1,2,4‐triazol‐5‐yl)‐2‐mercaptomethylbenzimidazo‐ les
for antibacterial and insecticidal activities. J. Indian. Chem. Soc. 1981, 58, 508–511.
22. AAT Bioquest, Inc. Quest Graph™ IC50 Calculator. Available online: www.aatbio.com/tools/ic50‐
calculator (accessed on 14 January 2020).
23. Release, S. Schrödinger Release 2019‐4: LigPrep; Schrödinger, LLC: New York, NY, USA, 2019.
24. Kaczor, A.A.; Silva, A.G.; Loza, M.I.; Kolb, P.; Castro, M.; Poso, A. Structure‐Based Virtual Screening for
Dopamine D 2 Receptor Ligands as Potential Antipsychotics. ChemMedChem 2016, 11, 718–729.
25. Kaczor, A.A.; Targowska‐Duda, K.M.; Budzyńska, B.; Biała, G.; Silva, A.G.; Castro, M. In vitro, molecular
modeling and behavioral studies of 3‐{[4‐(5‐methoxy‐1H‐indol‐3‐yl)‐1,2,3,6‐tetrahydropyridin‐1‐
yl]methyl}‐1,2‐dihydroquinolin‐2‐one (D2AAK1) as a potential antipsychotic. Neurochem. Int. 2016, 96, 84–
99.
26. Kaczor, A.A.; Jörg, M.; Capuano, B. The dopamine D2 receptor dimer and its interaction with homobivalent
antagonists: Homology modeling, docking and molecular dynamics. J. Mol. Model. 2016, 22, 203.
27. Kondej, M.; Wróbel, T.M.; Silva, A.G.; Stępnicki, P.; Koszła, O.; Kędzierska, E.; Bartyzel, A.; Biała, G.;
Matosiuk, D.; Loza, M.I.; et al. Synthesis, pharmacological and structural studies of 5‐substituted‐3‐(1‐
arylmethyl‐1,2,3,6‐tetrahydropyridin‐4‐yl)‐1H‐indoles as multi‐target ligands of aminergic GPCRs. Eur. J.
Med. Chem. 2019, 180, 673–689.
28. Kondej, M.; Bartyzel, A.; Pitucha, M.; Wróbel, T.M.; Silva, A.G.; Matosiuk, D.; Castro, M.; Kaczor, A.A.
Synthesis, Structural and Thermal Studies of 3‐(1‐Benzyl‐1,2,3,6‐tetrahydropyridin‐4‐yl)‐ 5‐ethoxy‐1H‐
indole (D2AAK1_3) as Dopamine D₂ Receptor Ligand. Molecules 2018, 23, 2249.
29. Release, S. Schrödinger Release 2019‐4: Epik; Schrödinger, LLC: New York, NY, USA, 2019.
30. Todd, A.K.; Adams, A.; Thorpe, J.H.; Denny, W.A.; Wakelin, L.P.G.; Cardin, C.J. Major Groove Binding and
‘DNA‐Induced’ Fit in the Intercalation of a Derivative of the Mixed Topoisomerase I/II PoisonN‐(2‐
(Dimethylamino)ethyl)acridine‐4‐ carboxamide (DACA) into DNA: X‐ray Structure Complexed to d(CG(5‐
BrU)ACG)2at 1.3‐Å Resolution. J. Med. Chem. 1999, 42, 536–540.
31. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.;
Mainz, D.T. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure
for Protein−Ligand Complexes. J. Med. Chem. 2006, 49, 6177–6196.
32. Release, S. Schrödinger Release 2019‐4: BioLuminate; Schrödinger, LLC: New York, NY, USA, 2019.
33. The PyMOL Molecular Graphics System, Version 2.0; Schrödinger, LLC: New York, NY, USA, 2019.
34. Xu, Z.‐Y.; Tang, J.‐N.; Xie, H.‐X.; Du, Y.‐A.; Huang, L.; Yu, P.‐F.; Cheng, X.‐D. 5‐Fluorouracil Chemotherapy
of Gastric Cancer Generates Residual Cells with Properties of Cancer Stem Cells. Int. J. Boil. Sci. 2015, 11,
284–294.
35. Bathaie, S.Z.; Nikfarjam, L.; Rahmanpour, R.; Moosavi‐Movahedi, A. Spectroscopic studies of the
interaction of aspirin and its important metabolite, salicylate ion, with DNA, A∙T and G∙C rich sequences.
Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 2010, 77, 1077–1083.
36. Dehghan, G.; Dolatabadi, J.E.N.; Jouyban, A.; Zeynali, K.A.; Ahmadi, S.M.; Kashanian, S. Spectroscopic
Studies on the Interaction of Quercetin–Terbium(III) Complex with Calf Thymus DNA. DNA Cell Boil. 2011,
30, 195–201.
37. Maga, G.; van Loon, B.; Crespan, E.; Villani, G.; Hübscher, U. The block of DNA polymerase delta strand
displacement activity by an abasic site can be rescued by the concerted action of DNA polymerase beta and
Flap endonuclease 1. J. Biol. Chem. 2009, 284, 14267–14275.
38. Herrick, J.; Bensimon, A. Global regulation of genome duplication in eukaryotes: An overview from the
epifluorescence microscope. Chromosoma 2008, 117, 243–260.
39. Cann, K.L.; Hicks, G.G. Regulation of the cellular DNA double‐strand break response. Biochem. Cell Boil.
2007, 85, 663–674.

Biomolecules 2020, 10, 296
13 of 13
40. Chastain, P.D.; Nakamura, J.; Swenberg, J.; Kaufman, D. Nonrandom AP site distribution in highly
proliferative cells. FASEB J. 2006, 20, 2612–2614.
41. Gilad, Y.; Senderowitz, H. Docking studies on DNA intercalators. J. Chem. Inf. Model. 2014, 54, 96–107.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).