Novel thiosemicarbazide derivatives with 4-nitrophenyl group as multi-target drugs: α-glucosidase inhibitors with antibacterial and antiproliferative activity

Article abstract of DOI:10.1016/j.biopha.2017.07.049

A series of thiosemicarbazides with 4-nitrophenyl group was obtained in the reaction of carboxylic acid hydrazides with isothiocyanates. All compounds were checked for their antibacterial and antiproliferative activity. Our results have shown that derivatives 6-8 possessed antibacterial activity against S. aureus, S. epidermidis, S. mutans and S. sanguinis, moderate cytotoxicity and good therapeutic safety in vitro. Additionally, compounds 1 and 4 significantly inhibited A549, HepG2 and MCF-7 cell division. Moreover, PASS software indicated that newly obtained compounds are potential α-glucosidase inhibitors. This was confirmed by in vitro studies. To investigate the mode of interaction with the molecular target compounds were docked to glucose binding site of the enzyme and exhibited a similar binding mode as glucose.

Full text of DOI:10.1016/j.biopha.2017.07.049

Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
Available online at
ScienceDirect
www.sciencedirect.com
Novel thiosemicarbazide derivatives with 4-nitrophenyl group as
multi-target drugs:
a-glucosidase inhibitors with antibacterial and
antiproliferative activity
Maciej Wosa, Małgorzata Miazga-Karskab, Agnieszka A. Kaczorc,d, Katarzyna Klimekb,
Zbigniew Karczmarzyke, Dorota Kowalczukf, Waldemar Wysockie, Grazyna Ginalskab,
Zofia Urbanczyk-Lipkowskag, Maja Morawiakg, Monika Pituchaa,
*
a
Department of Organic Chemistry, Medical University, 4A Chodzki St., PL-20093, Lublin, Poland
Department of Biochemistry and Biotechnology, Medical University,1 Chodzki St., PL-20093, Lublin, Poland
b
c
Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Laboratory, Medical University,1 Chodzki St., PL-
20093, Lublin, Poland
d
University of Eastern Finland, School of Pharmacy, Department of Pharmaceutical Chemistry, Yliopistonranta 1, P.O. Box 1627, FI-70211, Kuopio, Finland
Department of Chemistry, Siedlce University of Natural Sciences and Humanities, 3 Maja 54 St., PL-08110, Siedlce, Poland
e
f
Department of Medicinal Chemistry, Medical University of Lublin, Jaczewskiego 4 St., PL-20090, Lublin, Poland
g
Institute of Organic Chemistry, Polish Academy of Sciences,Kasprzaka 44/52 St., PL-01224, Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of thiosemicarbazides with 4-nitrophenyl group was obtained in the reaction of carboxylic acid
hydrazides with isothiocyanates. All compounds were checked for their antibacterial and antiprolifer-
ative activity. Our results have shown that derivatives 6-8 possessed antibacterial activity against S.
aureus, S. epidermidis, S. mutans and S. sanguinis, moderate cytotoxicity and good therapeutic safety in
vitro. Additionally, compounds 1 and 4 significantly inhibited A549, HepG2 and MCF-7 cell division.
Received 6 April 2017
Received in revised form 12 June 2017
Accepted 11 July 2017
Keywords:
Antibacterial activity
Antiproliferative activity
Moreover, PASS software indicated that newly obtained compounds are potential
a-glucosidase
a-glucosidase inhibitor
inhibitors. This was confirmed by in vitro studies. To investigate the mode of interaction with the
molecular target compounds were docked to glucose binding site of the enzyme and exhibited a similar
binding mode as glucose.
Molecular docking
Thiosemicarbazide
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
On the other hand, nitro group is a structural moiety that is
often found in biologically active compounds. Nitroheterocyclic
In recent years the organo-nitrogen compounds are commonly
designed and synthesized as biologically active compounds [1–3].
Small heterocyclic molecules, such as derivatives of pyrazole,
pyridine, triazole, thiazole derivatives have interesting bioactivity
profiles [4–6]. Moreover, linear derivatives of these compounds [7–
9], including thiosemicarbazide derivatives can result in ground
breaking discovery of new class of therapeutic agents [10].
Conjugated N-N-S tridentate ligand system of thiosemicarbazide
(NH2-C(S)-NH-NH2) is very important for antibacterial, antituber-
cular, anticonvulsant, antifungal, antiproliferative and anticancer
activity [11–15].
derivatives have a wide spectrum of activity including antibacte-
rial, antifungal or anticancer properties [16,17]. A significant
number of compounds have been found to be adequate in drug
discovery process and pharmaceutical research exhibiting anti-
inflammatory, anti-HIV, anti-tubercular, antiprotozoal, antibacte-
rial, antifungal, antiproliferative and anticancer activity [18–24].
The nitro group affects biological properties of chemical com-
pounds, usually by increasing their bactericidal ability and toxicity.
In the light of above, the aim of this study was to design,
synthesize and study multi-target compounds containing thio-
semicarbazide and nitro moieties with antibacterial and anti-
proliferative activity. Due to the limited amount of information on
the activity of these compounds as a a-glucosidase inhibitors [25–
30], we decided to test this mode of activity. The rationale of this
study can be summarized as follows: (1) the possibility of
* Corresponding author.
E-mail address: monika.pitucha@umlub.pl (M. Pitucha).
http://dx.doi.org/10.1016/j.biopha.2017.07.049
0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

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M. Wos et al. / Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
Fig. 1. The molecular structure of
4 compound, with atom labelling and
displacement ellipsoids drawing at the 30% probability level.
precipitated compound was filtered, dried and crystallized from
methanol-acetonitrile (1:1). (Supplementary material includes
experimental data of all compounds).
2.3. X-ray analysis
X-ray data of compound 4 were collected on the Bruker SMART
APEX II CCD diffractometer; crystal sizes 0.60 ꢂ 0.44 ꢂ 0.33 mm,
CuKa (l= 1.54178 Å) radiation, v and ’ scans, T = 293 K, absorption
correction: multi-scan SADABS [36], Tmin/Tmax = 0.265/0.580. The
structure was solved by direct methods using SHELXS97 [37]. All
calculations were performed using WINGX version 1.64.05
package [38]. (Supplementary material includes experimental
data of compound 4).
Scheme 1. Synthesis of the investigated compounds.
combination of two important bioactive groups, i.e. thiosemicar-
bazide and nitro moieties in one molecule; (2) the ability to design
a group of multi-target compounds which is a hot topic in current
medicinal chemistry and (3) the striking epidemiologic data about
cancer, diabetes and drug-resistant bacterial infections.
2.4. Biological assays
2.4.1. Antibacterial assays
All synthesized compounds were screened for their in vitro
antibacterial activity against aerobic Gram-positive Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228 and
microaerobic Gram-positive Streptococcus mutans PCM 2502,
Streptococcus sanguinis PCM 2335 bacterial strains. Pilot disc
diffusion test and MIC, MBC assays were performed as described
previously [39] and obtained data were compared to reference
Cefepime (Maxipime).
2. Material end methods
2.1. Chemistry
The chemicals used for synthesis and analysis were purchased
from Merck Co. or Sigma-Aldrich and used without further
purification. Melting points were determined on a Fisher-Johns
block and presented without any corrections. The NMR spectra
were acquired using Bruker Avance 300 MHz spectrometer with
TMS as an internal standard. The IR spectra were recorded on the
2.4.2. Cytotoxicity assay and therapeutic index
Thermo Nicolet 6700 ATR device in the range of 500–3500 cmꢀ1
.
The cytotoxicity assay and therapeutic index determination of
synthesized compounds were performed using BJ cell line as
described previously [40].
The mass spectra were obtained by Bruker microToF apparatus (CI-
MS). The elementary analysis was performed with the application
of Perkin-Elmer analyzer (940 Winter St., Waltham, MA, USA). The
obtained results were within ꢁ0.4% of the theoretical value. The
purity of obtained compounds was checked by TLC on Merck Co.
plates (aluminium oxide 60 F254 in a CHCl3/C2H5OH (10:1 or 10:2)
solvent system with UV or in iodide visualization.
2.4.3. Cell proliferation assay and selectivity index
The cell proliferation assay was carried out using A549, HepG2,
MCF-7 and BJ cell lines as described previously [36]. After 96-h
The carboxylic acid hydrazides were obtained by the general
procedure described earlier [31]. Some of the tested compounds
have been reported in literature [32–35].
Table 1
Bacterial growth inhibition zones caused by new nitro derivatives 1-9. *Compounds
2 and 5 showed no inhibition zone.
Comp.*
Bacterial growth inhibition zones [mm]
S. aureus
S. epidermidis
S. mutans
S. sanguinis
2.2. General procedure for the preparation of 1,4-disubstituted
thiosemicarbazide 1–9
1
3
17
5
18
5
0
5
0
7
4
6
7
8
9
13
17
15
21
12
26
0
0
10 mmole of the 2–,3-,4-pyridine carboxylic acid hydrazide or
4-nitrophenyl carboxylic acid hydrazide, cyclohexyl, n-butyl, 4-
nitrophenyl, 2,4-dichlorophenyl or benzyl isothiocyanate (10
mmole) and methanol (10 mL) were placed in a round bottom
flask and heated at reflux for 30 min. After this time the
17
16
21
5
15
22
23
0
13
20
21
0
9
Cefepime
24
29
24

M. Wos et al. / Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
1271
Table 2
Antibacterial and cytotoxic effects of new nitro derivatives: MIC, MBC/MIC ratio, CC50 and therapeutic index (TI). CC50: the concentration that caused a 50% reduction of
normal fibroblast viability; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; TI (therapeutic index): the ratio between CC50 and MIC
values; NA– no activity in screening test; NT ꢀ not tested.
no
CC50 ꢁ SD [
m
g/ml]
S. aureus
S. epidermidis
S. sanguinis
S. mutans
MIC [mg/ml]
TI
MBC
MIC
MIC [m
g/ml]
TI
MBC
MIC
MIC [m
g/ml]
TI
MBC
MIC
MIC [m
g/ml]
TI
MBC
MIC
1
4
6
7
25.76 ꢁ 1.15
24.21 ꢁ 1.45
62.5
1000
125
500
31.25
0.488
0.41
0.02
1.97
0.19
2.18
nc
16
NT
2
2
2
31.25
62.5
62.5
250
31.25
0.488
0.82
0.39
3.95
0.38
2.18
nc
8
16
8
4
8
NA
NA
31.25
31.25
15.62
0.122
NA
NA
7.86
3.05
4.36
nc
NA
NA
4
4
2
NA
NA
15.62
31.25
7.81
0.122
NA
NA
15.79
3.06
8.74
nc
NA
NA
2
2
2
246.60 ꢁ 1.03
95.49 ꢁ 1.34
68.23 ꢁ 1.03
>1000
8
Cefepime
1
2
2
2
nc ꢀ non cytotoxic in tested concentrations.
incubation, the inhibitory effect of tested compounds and Cisplatin
was evaluated using SRB assay according to manufacturer protocol
(Sigma Aldrich). Due to the fact that compounds which possess
anticancer activity are often very toxic towards normal cells, the
selectivity index (SI) was determined. It was defined as the ratio of
IC50 of compounds in normal cells versus cancer cells [41–43].
Cisplatin was used as a reference drug [44].
Aldrich) was used as a positive drug control. IC50 values were
calculated by the graphic method
2.5. Statistical analysis
All in vitro experiments were performed in three independent
measurements. The results were presented as mean values ꢁ
standard deviation (SD) or as mean values ꢁ standard error of the
mean (S.E.M). The data were analyzed using unpaired Students t-
test. Differences were considered as significant with P < 0.05
(GraphPad Prism 5, Version 5.04 Software). The values of CC50 and
IC50 were calculated via 4-parameter nonlinear regression analyses
using GraphPad Prism 5, version 5.04.
2.4.4.
The
to Elya et al. [45] with some modifications, by measuring the
release of p-nitrophenol from substrate p-nitrophenyl
glucopyranoside (p-NPGP) (Sigma Aldrich). Acarbose (Sigma
a-glucosidase inhibition
a
-glucosidase inhibitory activity was examined according
a-D-
Fig. 2. Antiproliferative activity of derivatives 1, 4 and Cisplatin on cancer cells: human lung adenocarcinoma (A), human hepatocellular carcinoma (B), human breast
adenocarcinoma (C) and normal cells: normal human skin fibroblasts (D) after 96-h incubation. The results were presented as mean values ꢁ S.E.M. from three separate
experiments. *Statistically significance obtained at P < 0.05 compared to the control (unpaired Students t-Test).

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M. Wos et al. / Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
Table 3
The IC50 and SI of 1, 4 derivatives and Cisplatin on cancer cell lines (A549, HepG2, MCF-7) and normal cell line (BJ) after 96-h incubation. The IC50 values were expressed as
mean values ꢁ SD from three separate experiments. IC50 ꢀ inhibitory concentration that caused 50% inhibition of cell proliferation; SI ꢀ the ratio between IC50 on normal cell
line and IC50 on cancer cell line.
Comp.
IC50 ꢁ SD [
m
g/mL]
SI
BJ
A549
HepG2
MCF-7
BJ/
BJ/
BJ/
A549
HepG2
MCF-7
1
4
2.87 ꢁ 1.10
3.09 ꢁ 1.19
1.19 ꢁ 0.56
4.96 ꢁ 1.96
6.80 ꢁ 1.25
2.38 ꢁ 1.16
3.64 ꢁ 1.18
11.88ꢁ 3.91
2.82 ꢁ 1.79
2.53 ꢁ 1.21
3.62 ꢁ 1.07
2.97 ꢁ 1.33
0.58
0.45
0.5
0.79
0.26
0.42
1.13
0.85
0.40
Cisplatin
2.6. Molecular modeling
2.6.1. Homology modeling
compounds (1–9) were prepared in reaction of carboxylic acid
hydrazide with isothiocyanate using methanol as solvent
a
(Scheme 1). All the synthesized compounds were characterized
by spectroscopic methods. 1H NMR spectra of thiosemicarbazide
derivatives exhibited proton signals typical of the NH group in the
range of 7.98–11.93 ppm. Also, in the IR spectra the signals from the
Homology modeling was applied to construct molecular model
a-glucosidase from Saccharomyces cerevisiae (UniProt entry
of
P53341) in complex with glucose. Sequence alignment was
performed using Muscle software [46]. Homology modeling was
performed using Modeller v. 9.17 [47]. X-ray structure of
group C .
O and C = S are observed in the range 1600–1800 cmꢀ1
¼
The above observations suggest that the compounds exist mainly
in the thione form. This is consistent with the literature data [9,11].
a
-glucosidase from Geobacillus sp. HTA-462 (PDB ID: 2ZE0 [48])
was used as a template (sequence identity 40%). The glucose
moiety was transferred to the enzyme catalytic site from the
sucrose mutase-sucrose complex (PDB ID: 2PWE [49]). A popula-
tion of 100 homology models was generated and subsequently
assessed by the Modeller objective function and Discrete Opti-
mized Protein Energy profiles [50]. The best model was subjected
to quality assessments using the Schrödinger software module for
Ramachandran plots.
For derivative 4 as a model compound X-ray structure
determination was carried out It was performed in order to
confirm the synthesis pathway, assumed molecular structure of 4
and identification of its tautomeric form in the crystalline phase.
This compound crystallizes with the methanol solvent molecule
lying on special position with twofold rotation symmetry. The
structure and conformation of the molecule 4 in the crystal is
shown in Fig. 1. In the crystalline state, the molecule exists in N1-
amino/N3-amino/N4-amino/S2-thione/O5-keto tautomeric form,
as evidenced by the C2–S2 and C5–O5 bond length of 1.213(3) and
1.684(2) Å, respectively, typical for the carbonyl and thione group
[55]. Moreover, the positions of the amino-H atom are localized at
the difference electron-density map in the vicinity of N1, N3 and
N4 atoms. The carbonylthiosemicarbazide part of the molecule
adopt trans-cis-gauche-trans conformation with the C11–N1–C2–
N3, N1–C2–N3–N4, C2–N3–N4–C5 and N3–N4–C5–C51 of 174.2(2),
ꢀ8.6(3), 103.9(2) and 171.73(18)ꢃ, respectively. The torsion angle
O5–C5–C51–C56 of ꢀ20.6(4)ꢃ shows that the carbonyl group is
slightly distorted from coplanarity with the benzene ring, similarly
as disordered nitro group with the torsion angles C55–C54–N6–
O61 of 6.0(5)ꢃ, C55–C54–N6–O62A of ꢀ152.8(4)ꢃ and C55–C54–
N6–O62 B of 152.9(7)ꢃ. The thione C2 = S2 group has trans and cis
conformation with respect to N3–N4 and N1–C11 bonds,
respectively; the respective torsion angles are 172.38(16) and
2.6.2. Compound preparation
The investigated compounds were modeled using the LigPrep
protocol from the Schrödinger Suite [51]. In order to sample
different protonation states of ligands in physiological pH, Epik
module was used with default settings [52].
2.6.3. Molecular docking
Molecular docking was performed using Glide from the
Schrödinger suite of software [53]. The grid file was generated
indicating glucose as a reference ligand. Molecular docking was
performed using the SP (standard precision) protocol of Glide. 100
poses were generated for each ligand. PyMol v. 0.99 [54] was used
for visualization of results.
3. Results and discussion
3.1. Chemistry
The starting reagents for the synthesis of thiosemicarbazides 1–
9 were 2-, 3-, 4-pyridyl carboxylic acid hydrazide or 4-nitrophenyl
carboxylic acid hydrazide. These compounds were obtained by the
reaction of appropriate ester with hydrazine hydrate in anhydrous
ethanol by the procedure described earlier [31]. The title
Table 4
Probability to inhibit (Pa) and not to inhibit (Pi)
software PASS.
a-glucosidase calculated using
Compound
Pa
Pi
1
2
3
4
5
6
7
8
9
0.919
0.901
0.915
0.886
0.777
0.927
0.926
0.915
0.900
0.009
0.012
0.010
0.014
0.034
0.009
0.009
0.010
0.012
Fig. 3. The
a-glucosidase inhibitory activity and estimation of IC50 of nitro
derivatives and reference compound acarbose. The results were presented as mean
values ꢁ SD from three separate experiments. *Statistically significance obtained at
P < 0.05 compared to the control (unpaired Students t-Test).

M. Wos et al. / Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
1273
Fig. 4. Ramachandran plot for homology model of yeast
a
-glucosidase. Triangles denote glycine residues, squares ꢀ proline residues and circles ꢀ all other residues.
ꢀ6.9(4)ꢃ. The cyclohexyl ring adopts a chair conformation with
2-yl)carbonylthiosemicarbazide (1), 4-(2-chlorophenyl)-1-(4-
nitrophenyl)carbonylthiosemicarbazide (6) and 4-(2,4-dichloro-
phenyl)-1-(4-nitrophenyl)carbonylthiosemicarbazide (8) with
puckering parameters of Q = 0.554(4) Å and
u
= 178.3(4)ꢃ [56].
Other details can be found in Supplementary material.
MIC values ranging from 15.79 mg/mL to 125 mg/mL. It is known
3.2. Antibacterial activity
that an antibacterial agent is considered bacteriostatic when the
MBC/MIC ratio is ꢄ 4 while being bactericidal when the MBC/MIC
ratio is > 4 [57]. According to the data summarized in Table 2 we
found that thiosemicarbazides 6-8 showed bactericidal activity
against S. aureus, S. mutans and S. sanguinis. The MBC values against
S. epidermidis for compounds 6-8 and also 1 (R1 = 2-pyridine and
R2 = 4-NO2C6H5) were determined to be at least 4-fold higher than
the MIC values, what confirms that compounds were bacteriostat-
ic. However, cefepime remains the most active antimicrobial drug
All newly synthesized compounds were tested for their
antibacterial activity by a modified disc diffusion method. The
main inhibition zones of both aerobic and microaerobic Gram-
positive bacteria caused by thiosemicarbazides were found in a
range of 13 mm to 23 mm (Table 1). Among tested substances,
derivatives 6–8 had good activity against aerobic and microaerobic
Gram-positive bacteria. Thus, the most active compounds were the
thiosemicarbazides with
4-nitrophenyl group substituted in the position 1 and possess-
ing in position 4 the following substituents: 2-chlorophenyl (6), 4-
nitrophenyl (7) or 2,4-dichlorophenyl (8). This activity increases in
a series 2-ClC6H4 < 4-NO2C6H5 < 2,4-Cl2C6H3 for S. mutans and S.
sanguinis. For S. aureus and S. epidermidis the increase in activity is
as follows: 4-NO2C6H5 < 2-ClC6H4 < 2,4-Cl2C6H3.
The minimum inhibitory concentration (MIC) was determined
for each derivatives which exhibited the bacterial growth
inhibition zones. Table 2 presents MIC values of the most potent
molecules with good antibacterial activity. The MIC assay clearly
confirmed results obtained during modified disc diffusion test and
showed that compounds 6-8 (R1 = 4-NO2C6H5 and R2 = 2-ClC6H4, 4-
NO2C6H5 or 2,4-Cl2C6H3) had moderate to good activity against
most of the tested bacteria. Compounds 6-8 display good
inhibitory effect against tested microaerobic Gram-positive strains
(MIC 0.122
m
g/mL ꢀ 0.976
mg/mL) with high IB (1.08 to 34.6)
3.3. Cytotoxic activity
The compounds which exhibited antibacterial activity (1, 4, 6, 7,
8) were assessed for their cytotoxic activity against normal human
skin fibroblast (BJ cell line) using MTT assay. The 1,4-disubstituted
thiosemicarbazide derivatives showed different cytotoxic activi-
ties with CC50 values ranging from 24.21 to 246.60 mg/mL (Table 2).
Among all tested compounds, the 4-cyclohexyl-1-(4-nitrophenyl)
carbonylthiosemicarbazide (4) exhibited the highest cytotoxic
activity with a CC50 value of 24.21 mg/mL. On the other hand, the 4-
(2-chlorophenyl)-1-(4-nitrophenyl)-carbonylthiosemicarbazide
(6) showed the lowest cytotoxic activity in comparison with the
tested compounds. It is worth noting that compounds 4 and 6
possessed 4-NO2C6H4 group at position 1 and C6H11 (4) group and
2-ClC6H4 (6) group at position 4, respectively. The results suggest
that the presence of 2-ClC6H4 substituted in the 4 position of
with MICs of 7.81–31.25
pathogens were well inhibited by 1 4-(4-nitrophenyl)-1-(pyridine-
mg/mL. In turn, aerobic Gram-positive

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M. Wos et al. / Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
Fig. 5. Interactions of glucose and compounds 1, 3 and 8 with yeast a-glucosidase. Protein shown with grey carbon atoms in wire representation while the residues forming
hydrogen bonds (denoted as red dashed lines) are shown as sticks. Ligands shown as sticks with magenta carbon atoms. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
thiosemikarbazide greatly increases cytotoxic activity of this
derivatives. In order to estimate the in vitro therapeutic safety
and efficacy of synthesized compounds, the therapeutic index (TI)
was determined and summarized in Table 2. The high value of
therapeutic index (TI) indicates that the tested compound is
effective against bacterial strain and safe for eukaryotic cells Thus,
the TI value below 1 determines a lack of therapeutic safety
whereas TI value higher than 10 allows to perform in vivo
evaluation [40,58,59]. Among the tested compounds, three of them
(6, 7, 8) showed the TI values higher than 1. Nevertheless,
compound 6 had the highest TI values against S. epidermidis (3.95),
S. sanguinis (7.86) and S. mutans (15.79). Thus, derivative 6 is a
promising candidate for treatment of S. mutans infections and due
to excellent TI value can be administered to perform in vivo
evaluation. Also in the case of cytotoxicity tests, the results
obtained for cefepime were most favorable among all tested
substances. Cefepime was non cytotoxic in tested concentration
cancer cells versus normal cells, the IC50 and SI values were
calculated and summarized in Table 3. The SI value above 1
indicates that compound exhibited greater inhibition of cancer
cells compared to normal cells. It was observed only for MCF-7 cells
after incubation with derivative 1 (R1 = 2-pyridyl and R2 = 4-
NO2C6H5). Moreover, it should be noted that derivative 1 possessed
antiproliferative selectivity greater than Cisplatin against all tested
anticancer cells. The results indicate that thiosemicarbazide chain
substituted in position 1 by pyridin–2 yl group and in position 4 by
4-NO2C6H4 group cause that compounds possess high antiproli-
ferative activity, which is more selective to cancer cells than to
normal cells.
3.5. Inhibition of
a-glucosidase activity in vitro and in silico
In order to check if the studied compounds may exhibit
-glucosidase inhibitory activity, PASS software was used [63]. The
a
range (1000
m
g/mL).
obtained results are shown in Table 4. It can be seen that all
compounds except for compounds
(R1 = 4-NO2C6H5 and
R2 = C6H11 (R1 = 4-NO2C6H5 and R2 = C4H9) have the
and
probability to inhibit -glucosidase equal or above 0.9. Due to
results obtained using PASS software, it was decided to check
inhibition of -glucosidase by the studied compounds in vitro. The
-glucosidase from S. cerevisiae was used to demonstrate the
inhibitory activity of new nitro compounds as previously reported
[17]. The inhibitory activity of the compounds against -glucosi-
4
3.4. Antiproliferative activity
)
5
a
Among the synthesized compounds, 4-(4-nitrophenyl)-1-(pyr-
idine-2-yl)carbonylthiosemi carbazide (1) and 4-cyclohexyl-1-(4-
nitrophenyl)carbonylthiosemicarbazide (4) exhibited antiprolifer-
ative potency against cancer cells in a dose-dependent manner
(Fig. 2A–C). Since the normal skin fibroblasts are widely used as a
standard model of normal cells [60–62] in our study the BJ cells
were used as a control of normal cells (Fig. 2D). In order to evaluate
the antiproliferative selectivity of synthesized compounds towards
a
a
a
dase was tested using p-NPGP as a substrate and this was
compared with a reference drug ꢀ acarbose. To determine the
a
-glucosidase inhibition ability in vitro IC50 values were

M. Wos et al. / Biomedicine & Pharmacotherapy 93 (2017) 1269–1276
1275
calculated. As can be observed in Fig. 3, the in vitro assay of
-glucosidase confirmed the results predicted using PASS soft-
ware. The IC50 values of tested derivatives ranged from 283.24 g/
mL to 1930.55 g/mL. Compound 8 was the most active (IC50 of
283,24 g/mL), followed by 7 (with IC50 of 339.57 g/mL), 3 (with
IC50 of 368,46 g/mL) and 1 (with IC50 of 434.44 g/mL). There
POPW.01.03.00–06-010/09–00 Operational Program Development
of Eastern Poland 2007–2013, Priority Axis I, Modern Economy,
Operations 1.3. Innovations Promotion. Part of the calculations was
performed under a computational grant by Interdisciplinary
Center for Mathematical and Computational Modeling (ICM),
grant number Warsaw, Poland, grant number G30-18 and under
resources and licenses by CSC, Finland.
a
m
m
m
m
m
m
were no compounds which had IC50 lower than acarbose, but
compounds 8, 1 and 3 possess similar inhibitory activity to a
reference compound acarbose on
In order to study the molecular interactions of most active
compounds 1, 3 and 8 with yeast -glucosidase, we performed
molecular docking. First homology model of yeast -glucosidase
was constructed using X-ray structure of -glucosidase form
a-glucosidase in in vitro test.
Appendix A. Supplementary data
a
Supplementary data associated with this article can be found, in
a
the
online
version,
at
http://dx.doi.org/10.1016/j.
a
biopha.2017.07.049.
Geobacillus sp. HTA-462 (PDB ID: 2ZE0 [45]) as a template. The
quality of the homology model was confirmed by means of
Ramachandran plot presented in Fig. 4. The interactions of yeast
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presented in Fig. 5. The mode of interaction of glucose with yeast
-glucosidase resembles interactions reported for the form
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