Thiazole-based nitrogen mustards: Design, synthesis, spectroscopic studies, DFT calculation, molecular docking, and antiproliferative activity against selected human cancer cell lines

Article abstract of DOI:10.1016/j.molstruc.2016.04.058

Synthesis, characterization and investigation of antiproliferative activity of ten thiazole-based nitrogen mustard against human cancer cells lines (MV4-11, A549, MCF-7 and HCT116) and normal mouse fibroblast (BALB/3T3) is presented. The structures of novel compounds were determined using ¹H and ¹³C NMR, FAB(+)-MS, and elemental analyses. Among the derivatives, 5b, 5c, 5e, 5f and 5i were found to exhibit high activity against human leukaemia MV4-11 cells with IC₅₀ values of 2.17-4.26 μg/ml. The cytotoxic activity of compound 5c and 5f against BALB/3T3 cells is up to 20 times lower than against cancer cell lines. Our results also show that compounds 5e and 5i have very strong activity against MCF-7 and HCT116 with IC₅₀ values of 3.02-4.13 μg/ml. Moreover, spectroscopic characterization and cellular localization for selected compound were performed. In order to identify potential drug targets we perform computer simulations with DNA-binding site of hTopoI and hTopoII and quantum chemical calculation of interaction and binding energies in complexes of the five most active compounds with guanine.

Full text of DOI:10.1016/j.molstruc.2016.04.058

Journal of Molecular Structure 1119 (2016) 139e150
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Thiazole-based nitrogen mustards: Design, synthesis, spectroscopic
studies, DFT calculation, molecular docking, and antiproliferative
activity against selected human cancer cell lines
,
*
b
c
Krzysztof Z. Ła˛czkowski a , Marta Switalska , Angelika Baranowska-Ła˛czkowska ,
ꢀ
Tomasz Plech d, Agata Paneth d, Konrad Misiura a, Joanna Wietrzyk b,
Barbara Czaplinska , Anna Mrozek-Wilczkiewicz g, Katarzyna Malarz e, Robert Musioł e,
e
f,
ꢀ
Izabela Grela h
a Department of Chemical Technology and Pharmaceuticals, Faculty of Pharmacy, Collegium Medicum, Nicolaus Copernicus University, Jurasza 2, 85-089,
Bydgoszcz, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wrocław, Poland
c Institute of Physics, Kazimierz Wielki University, Plac Weyssenhoffa 11, 85-072, Bydgoszcz, Poland
d
ꢀ
Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Chodzki 4a, 20-093, Lublin, Poland
e Institute of Chemistry, University of Silesia, Szkolna 9, 40-006, Katowice, Poland
f A. Chełkowski Institute of Physics, University of Silesia, Uniwersytecka 4, Katowice, 40-007, Poland
g
ꢀ
Silesian Center for Education and Interdisciplinary Research, 75 Pulku Piechoty 1A, Chorzow, 41-500, Poland
h Faculty of Chemical Technology and Engineering, University of Technology and Life Sciences, Seminaryjna 3, 85-326, Bydgoszcz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, characterization and investigation of antiproliferative activity of ten thiazole-based nitrogen
mustard against human cancer cells lines (MV4-11, A549, MCF-7 and HCT116) and normal mouse
Received 12 March 2016
Received in revised form
18 April 2016
Accepted 19 April 2016
Available online 27 April 2016
fibroblast (BALB/3T3) is presented. The structures of novel compounds were determined using 1H and 13
C
NMR, FAB(þ)-MS, and elemental analyses. Among the derivatives, 5b, 5c, 5e, 5f and 5i were found to
exhibit high activity against human leukaemia MV4-11 cells with IC50 values of 2.17e4.26 g/ml. The
m
cytotoxic activity of compound 5c and 5f against BALB/3T3 cells is up to 20 times lower than against
cancer cell lines. Our results also show that compounds 5e and 5i have very strong activity against MCF-7
and HCT116 with IC50 values of 3.02e4.13 mg/ml. Moreover, spectroscopic characterization and cellular
localization for selected compound were performed. In order to identify potential drug targets we
perform computer simulations with DNA-binding site of hTopoI and hTopoII and quantum chemical
calculation of interaction and binding energies in complexes of the five most active compounds with
guanine.
Keywords:
Antiproliferative activity
Thiazole
Nitrogen mustard
DFT calculations
Nucleobases
Topoisomerase
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
further increase to 19.3 million by 2025 [2].
DNA alkylating agents with the nitrogen mustards remain an
Cancer is one of the most significant health problems of modern
civilization [1]. It is the major cause of death, and according to the
WHO report it is estimated that 14.1 million new cancer cases and
8.2 million cancer-related deaths occurred in 2012. The highest
incidence of cancer is observed in rapidly developing countries and
it is estimated that the number of new cancer cases per year will
important group in the modern anti-cancer drug development and
therapy [3]. The preferential site in DNA alkylation depends on the
nature of alkylating agents and occurs mostly on position N7 and
O6 in guanine, N1 and N3 in adenine, and N3 in cytosine [3]. Ni-
trogen mustards are characterized by high reactivity combined
with the lack of sequence-specific binding of DNA. They rapidly lose
their activity by non-specific interaction with a number of nucle-
ophilic groups on cellular biomolecules other than DNA, which
results in side effect, mutagenicity, teratogenicity, carcinogenicity
and to promoting secondary malignancies [4e8]. However, the
* Corresponding author.
E-mail address: krzysztof.laczkowski@cm.umk.pl (K.Z. Ła˛czkowski).
http://dx.doi.org/10.1016/j.molstruc.2016.04.058
0022-2860/© 2016 Elsevier B.V. All rights reserved.

140
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
search for new substances that selectively inhibit the growth of
cancer cells without damaging healthy cells is a very difficult
problem.
of thiazole-based nitrogen mustard derivatives 5a-j were synthe-
sized by Hantzsch condensation reaction between different
substituted benzenesulfonamide chloroacetophenones 2a-j and
the 2-(4-(bis(2-chloroethyl)amino)benzylidene)hydrazinecarbo-
thioamide (4) [13,18] in refluxing absolute ethyl alcohol, with high
yield (48e97%) in order to explore the SARs of these derivatives and
to obtain potential leading compounds. All of the synthesized de-
rivatives were purified and their structures were characterized by
spectroscopic methods 1H NMR (700 and 400 MHz) and 13C NMR
(100 MHz), FAB(þ)-MS and elemental analyses. 1H NMR spectrum
In attempts to improve the selectivity and therapeutic efficacy
researchers create hybrid molecules using nitrogen mustard de-
rivatives and DNA binding pharmacophores, such as polyamides,
acridines and 9-anilinoquinolines [9e11]. One of the most impor-
tant results of this method is the development of Tallimustine-drug
having an enhanced sequence-selectivity [12]. In our earlier study it
has been observed that combining of thiazol-2-yl-hydrazine phar-
macophore with the active anticancer nitrogen mustard moiety
resulted in compounds with high antiproliferative activity against
different human cancer cells. Additionally these hybrids were
characterized by a low cytotoxicity against normal mouse fibroblast
BALB/3T3 [13].
These results encouraged us to continue our investigation on the
synthesis and molecular interaction of biologically active 2,4-
disubstituted thiazoles [14e17]. Our research began with the
design and synthesis of ten novel thiazole-based nitrogen mustards
containing benzenesulfonamide pharmacophore, and evaluation of
their antiproliferative activity against human cancer cells lines
(biphenotypic B myelomonocytic leukemia MV4-11, human lung
carcinoma A549, human breast carcinoma MCF-7, human colon
carcinoma HCT116) and normal mouse fibroblast (BALB/3T3) using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide
(MTT) or sulforhodamine B (SRB) assays. Moreover, spectroscopic
characterization and cellular localization for selected compound
were performed. Additionally, continuing our research on the
interaction between the novel mustard drugs and nucleobases, we
perform quantum chemical calculation of interaction and binding
energies in complexes of the most active drugs with guanine.
Finally, computer simulations were performed with DNA-binding
site of hTopoI and hTopoII.
of thiazoles showed singlet at
d (7.11e7.88) due to thiazole-5H
proton, which confirms the conversion of substrates in to the ex-
pected products. The characteristic proton of CH]N group occurs
in the spectra around 7.70e8.23 ppm. The mass spectra of all
compounds are fully consistent with the assigned structures. The
elemental analyses values are in good agreement with the calcu-
lated values confirming the formation of analytically pure products.
All reactions were repeated at least two times and are fully
reproducible.
2.2. Biological evaluation
Results of the in vitro studies on antiproliferative activity of
compounds 5a-j against four human cancer cell lines (MV4-11,
A549, MCF-7 and HCT116) and normal mouse fibroblast (BALB/3T3)
using cis-platin as positive control are summarized in Table 1. In our
study, compounds with IC50 below 4 mg/ml are consider as potential
drugs [19]. We started our research from testing all compounds
against biphenotypic B myelomonocytic leukemia MV4-11 cells.
Selected five highly active compounds 5b, 5c, 5e, 5f and 5i with IC50
values between 2.17 and 4.26 mg/ml were next tested against cancer
cell lines A549, MCF-7, HCT116 and normal cell line BALB/3T3.
According to our results, compound 5e has very strong activity
against human lung carcinoma A549, human breast carcinoma
MCF-7, and human colon carcinoma HCT116, with IC50 values from
2. Results and discussion
3.03 to 4.99 mg/ml. The cytotoxic activity of compound 5e against
2.1. Chemistry
normal mouse fibroblast BALB/3T3 cells is 5e8 times lower than
against cancer cell lines.
Synthesis of target thiazole-based nitrogen mustards containing
benzenesulfonamide moiety consists of two steps. In the first step
appropriate benzenesulfonamide chloroacetophenones 2a-j were
prepared by sulfamidation reaction of 1-(4-aminophenyl)-2-
Our data indicated also that compound 5f has very strong ac-
tivity against human lung carcinoma A549 and human breast car-
cinoma MCF-7, with IC50 4.58 mg/ml and 4.18 mg/ml, respectively.
This compound did not have cytotoxic activity against normal
mouse fibroblast BALB/3T3 cells.
chloroethanone
(1)
with
different
substituted
chlor-
obenzenesulfonamides in pyridine at room temperature, with
Also compound 5i has very strong activity against MCF-7 and
22e55% yield and high purity (Scheme 1). In the next step, a series
HCT116 cell lines, with IC50 3.42 mg/ml and 3.02 mg/ml, respectively.
Scheme 1. Thiazole-based nitrogen mustard 5a-j.

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
141
Table 1
Antiproliferative activity of thiazole-based nitrogen mustards 5a-j against cancer cell lines MV4-11, A549, MCF-7, HCT116 and against normal mice fibroblast BALB/3T3.
Nitrogen mustards
Ar
IC50
[m
g/ml]
logP
MV4-11
A549
MCF-7
HCT116
Balb/3T3
5a
5b
5c
5d
5e
5f
14.05 5.73
e
e
e
e
5.83
6.15
5.74
6.29
6.80
6.35
7.17
7.52
6.53
3.63 1.07
3.95 1.88
32.68 6.37
3.29 0.77
4.26 1.67
54.92 13.87
44.63 16.9
2.17 1.02
6.13 0.81
10.29 6.35
e
9.23 3.42
8.3 3.06
e
42.26 9.52
28.34 5.73
e
68.85 28.3
>100
e
4.99 1.53
4.58 2.18
e
3.03 0.58
4.16 1.67
e
4.13 1.73
43.38 4.16
e
24.7 7.4
>100
e
5g
5h
5i
e
e
e
e
20.52 4.27
3.42 0.27
3.02 0.86
3.2 0.6
5j
>100
e
e
e
e
7.52
cis-platin
0.31 0.07
2.03 0.31
2.46 0.63
3.01 1.11
1.4 0.55
e
Lipophilic parameter, logP, was calculated for each molecule by using ACDlogP program, http://www.acdlabs.com.
This activity is similar to the activity of the most commonly used
anticancer agent cis-platin. Cytotoxic activity of 5i against normal
For this purpose, as the first step we determined spectroscopic
properties of the compound 5j.
mouse fibroblast BALB/3T3 is similar (IC50 3.2
times smaller than cytotoxicity of cis-platin (IC50 1.4
Compounds 5b and 5c show good activity against human lung
carcinoma A549 and human breast carcinoma MCF-7, with IC50
m
g/ml), but still 2
g/ml).
Absorption spectra of synthesized compound 5j consists of
three bands in the range of 250e420 nm with local maxima at 265,
308 and 367 nm (Fig. 1a). Molar absorption coefficients for each
particular band are relatively high (over 103) what allow to classify
m
6.13e10.23
mg/ml. This compounds also did not have cytotoxic ac-
them as intense and implicates the p/p* type of transitions
tivity against normal mouse fibroblast BALB/3T3. Compounds 5a,
5d, 5g, 5h and 5j have no or very low antiproliferative activity
against MV4-11 cell line.
(Table 2) The maximum of fluorescence spectra is observed at
530 nm (Fig. 1b), what implies the emission in the range of visible
light and a quite large Stokes shift (8380 cmꢀ1, 163 nm). Such a big
difference between absorption and emission maxima is highly
desired since it is critical for the sensitivity of fluorescent methods.
In many cases this type of red-shift is connected with sol-
vatochromism and big change in dipole moments between ground
and excited states. However, in this case our studies did not reveal
any relationships between solvent polarity and emission wave-
length. For quantitative description of fluorescence phenomenon
the quantum yield has been calculated according to standard dye
and counts 0.05. This is relatively small value but still may be usable
in cellular imaging in vitro.
To gain a deeper understanding of spectroscopic properties of
newly synthesized compound 5j, theoretical calculations have been
carried out. For this task we applied Time-Dependent Density
Functional Theory with the B3LYP functional, using the 6-31 þ G
(d,p) basis set (TD-DFT/B3LYP/6-31 þ G (d,p)) with PCM model. The
results, that include main electronic transitions with relatively high
oscillator strength in every band, are presented in Table 3. The
transitions which are responsible for the corresponding experi-
mental absorption maxima are highlighted. The rest of calculated
electronic transitions were omitted since they have small oscillator
strength and constitute only a background for absorption bands. As
we can see, the calculated value of wavelengths at spectra maxima
Based on IC50 values we tried to establish a correlation between
the activity of the derivatives and their chemical structure and in
particular the nature of the functional groups. As can be easily
noticed, the highest activity is observed in the case of compounds
possessing electron-withdrawing groups (5b, 5c, 5e and 5i). Within
the tested series, compound 5f containing electron-donating
methoxy group also shows high activity. It is worth noticing that
the compounds containing electron-donating methyl (5d), iso-
propyl (5g) and tert-butyl (5h) groups exhibit only very low activity.
As a result of our research, we have identified new substituents
(NHSO2CF3 (5e) and NHSO2C6F5 (5i)) which significantly enhance
antiproliferative activity in almost all tested cancer cell lines MV4-
11, A549, MCF-7 and HCT116.
2.3. Spectroscopic properties
Permeability across biological membranes is a key factor in the
absorption and distribution of drugs [20]. Due to the presence of
the fluorescent dansyl group in compound 5j we decided to
determine the possible place of accumulation of this compound in
cancer cell using spectroscopic methods and take on trying to
explain the lack of activity of some nitrogen mustard derivatives.

142
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
located on the first phenyl and thiazole rings. The similar situation
occurs for S0 / S2 transition, which employs HOMO and LUMO
ꢀ1
orbitals located on the naphthalene ring. On the other hand,
comparison of the electron distribution of HOMO and LUMO or-
bitals indicates the strong charge-transfer (CT) character of S0 / S1
transition what explains the low oscillator strength. In the case of
second band, the most important transitions are S0 / S6, S0 / S9
and S0 / S7 which is assigned to next experimental absorption
maximum. All of them employs LUMOs orbital with higher energy
e LUMO
which is localized on the central part of molecule,
þ2
partially on the first and the second phenyl ring and LUMO
þ5
together with LUMOþ4 whose electron distribution is concentrated
mainly on the first phenyl ring and second phenyl ring respectively.
The S0 / S19 transition responsible for third absorption maximum
is due to two molecular orbital contributions -HOMO / LUMO
ꢀ6
and HOMO-4 / LUMOþ3 which embrace only naphthalene part of
the molecule. While electron distribution of orbitals involved in
S0 / S18 transition is spread on almost whole molecule, excluding
the naphthalene ring. Shapes of all presented orbitals implicate the
p
/ p*type of transitions in every case. Visualization of molecular
orbitals involved in main electronic transition, obtained at the TD-
DFT B3LYP/6-31 þ G(d,p) theory level are given in the Supporting
Information (S1).
The fluorescence emission energy for spin-allowed singlet-
singlet (Sn/S0) transition was also calculated for dye solution in
DMSO. Computed data including transition energy, oscillator
strength, and orbitals involved in emission process, together with
comparison of experimental data are given in Supporting Infor-
mation. The agreement of calculated and experimental data, how-
ever less impressive is still in on good level for this type of
calculation [21]. Noticeably HOMO and LUMO orbitals calculated
for optimized geometry in ground and first excited state have
different energy level and electron distribution, what reflects in
emission process.
Using theoretical data, the attempt of explanation of large
Stokes shift and low quantum yield was undertaken. Analysis of
optimized geometry of S0 and S1 state delivered important infor-
mation about changes in molecular framework after excitation. The
main alterations occurs in coordinates of a central part of the
molecule and are seen as a new plane created with thiazole and
second phenyl rings, see the Supporting Information (S2). As a
result of this comparison, we are able to connect large Stokes shift
with the geometry relaxation of molecule in excited state. Namely,
the optimized geometry of ground and first singlet excited state
differs significantly, what causes bigger difference between exci-
tation and emission energy and larger Stokes shift [22,23]. Addi-
tionally, non-radiative process like internal conversion (IC) occurs
resulting in loss of energy. According to Kasha's rule the emission
(fluorescence or phosphorescence) occurs only from the lowest
lying electronically excited singlet state S1 [23]. Thus in our case,
since the main transition is S0 / S3 (HOMO / LUMOþ1) the
molecule quickly relaxes to the lowest vibrational level (S1 state,
LUMO). From the change of electron distribution of LUMO and
HOMO orbitals calculated for ground and excited state, conforma-
tion transformation is easily noticeable (see Supporting Informa-
tion S3 and S4.
Fig. 1. Absorption (concentration
5
ꢁ
10ꢀ5 M) and fluorescence (concentration
6.25 ꢁ 10ꢀ7 M) spectra of 5j dye solution in DMSO.
Table 2
Spectroscopic properties of 5j solution in DMSO.
Absorption data
Emission data
lmax/nm
530
labs/nm
ε$103/Mꢀ1 cmꢀ1
Stokes shift/nm
163
ф
367
308
266
30.4
29.5
26.7
0.05
are in excellent correspondence with experimental data. The
biggest difference relates to maximum of the first absorption band
and amounts to 12 nm, what represents only 3% error. The spectra
prediction based on TD-DFT calculations also confirms correctness
of these results (Fig. 2).
Visualization of molecular orbitals involved in main electronic
transitions lets for better analysis of changes in molecule taking
place during excitation process. According to our calculations, we
can assume that the first absorption band is assigned to four
transitions S0 / S1, S0 / S2, S0 / S3 and S0 / S4. The S0 / S3
transition with the largest oscillatory strength is responsible for
maximum of this band, what arises from a good overlap of involved
2.4. Cellular staining
The good spectroscopic properties of compound 5j prompted us
to perform cellular staining and co-localization experiments to gain
more information on possible mechanism of action of the nitrogen
mustard derivatives that have been tested.
The toxicity level appeared to be relatively low with 15 mM for
HCT-116 as still safe concentration. This allows for convenient
orbitals e HOMO and LUMO
whose electron distribution is
þ1

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
143
Table 3
Electronic transition data obtained by TD-DFT/B3LYP/6-31 þ G(d,p) using a PCM model (solvent e DMSO) for 5j at the DFT optimized geometry of ground state.
Electronic transition
L (nm)
F (nm)
Molecular orbital
Percentage contribution (%)
Experimental
367
l (nm)
Band 1
Band 2
Band 3
S
S
S
S
S
S
S
0 / S1
0 / S2
0 / S3
0 / S4
0 / S6
0 / S7
0 / S9
403.48
390.85
379.50
346.80
313.64
306.90
300.57
0.0252
0.2562
1.1339
0.2568
0.0414
0.1467
0.1373
HOMO / LUMO
HOMOꢀ1 / LUMO
97
94
94
HOMO / LUMO
þ1
HOMOꢀ1 / LUMO
83
308
266
þ2
HOMO / LUMO
HOMO / LUMO
68
17
63
10
54
19
þ5
þ4
S
S
0 / S18
0 / S19
267.17
265.27
0.1132
0.1910
HOMOꢀ3 / LUMO
HOMOꢀ2 / LUMO
HOMOꢀ6 / LUMO
HOMOꢀ4 / LUMO
þ1
þ4
þ3
Fig. 2. Absorption 5j dye spectra: experimental, measured in DMSO; theoretical, predicted basing on TD-DFT calculations.
visualization of the compound in the living cell without serious
damage of its functions [24]. The tested compound 5j effectively
penetrated the membrane after 2 h incubation time. Its spectro-
scopic parameters allow to excitation with standard DAPI filter
while the emission is, due to large Stockes shift, pushed to greenish
region. Micrographs are presented in Supporting Information (S5).
Intense signal from the cells visible in the micrographs, despite
relatively low quantum yield of 5j support its effective penetration
through the membrane.
Apparently, this compound tends to accumulate primarily in
cellular membrane and some extent in lysosomes. This undoubt-
edly is the result of the structure of this compound. Accumulation
in cellular membrane and in lysosomes probably explains the
complete lack of activity of compound 5j, and also a small activity of
compounds 5g and 5h. Relatively high lipophilicity, logP 7.17e7.52,
and basic character due to nitrogen atoms are main factors affecting
its intracellular behavior. Such compounds generally may exhibit
lysosomotropism.
induction of endoplasmic reticulum stress by sulfur mustard de-
rivatives [26]. It is tempting to hypothesize about similar effect in
the tested compounds.
2.5. Interaction and binding energies in drug-guanine complexes
Nitrogen mustards are known to react with two guanine mol-
ecules, forming a bridge between two DNA strings, and mechanism
of this reaction can be found in Ref. [27]. Our earlier study [13]
focused of the interaction between thiazole moiety present in the
recently developed nitrogen mustard and the four DNA bases, since
the formation of such a drug-DNA base complex, with thiazole
being an additional center helping in the formation of the bridge
between the two DNA strings, can be important both, before and
after reaction of chlorine atoms of nitrogen mustard with guanine.
Our study revealed that out of the four DNA bases, the strongest
hydrogen bonded complexes were formed with guanine, both in
the case of model thiazoles and the real drug. As a continuation of
that investigation, we presently carried out quantum chemical
study of geometrical parameters, as well as interaction and binding
energies, in the complexes formed by the new potential drugs and
guanine.
It has been shown that large lipophilicity of compounds pro-
motes the accumulation of the drug in lysosomes [25]. We believe
that this hinders their penetration into the cell nucleus (S5c) so that
there is no alkylation of DNA located there. On the other hand we
have found at least partial accumulation in mitochondria (S5b) and
endoplasmic reticulum (S5a). These results confirm the possible
DNA interaction in mitochondria. There are also reports on
Optimization of geometrical parameters of investigated com-
plexes was carried out using Density Functional Theory with the
B3LYP functional and the 6-311G** basis set, and was followed by

144
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
the evaluation of vibrational frequencies within the same approx-
imation. To maximize the interaction between investigated drugs
and guanine, in the starting geometries e one per complex e the
two molecules were forming three hydrogen bonds. The optimized
DFT/B3LYP/6-311G** structures together with the corresponding
Cartesian coordinates geometries are reported in the Supporting
Information. In Table 4 the hydrogen bond intermolecular distances
(denoted as r, in Å) and angles (denoted as :, in deg) are reported.
2.6. Molecular modelling studies
Basic mechanism of anticancer activity of nitrogen mustard
derivatives is connected with the process of DNA alkylation [3,27].
The resultant DNA adducts become apoptosis promotors and cause
disturbing in the genetic material replication process. Nitrogen
mustard derivatives, depending on their chemical structure, may
have anticancer effect also due to activation or inactivation of other
processes inside human cells. Inhibition of the topoisomerases is
one of the effective mechanisms inhibiting the growth of cancer
cells [28]. These enzymes take part in DNA metabolism in the broad
sense, thus allowing the processes of replication, transcription,
recombination and condensation of chromosomes. In order to
check whether the biological activity of the synthesized com-
pounds may also be connected with their interactions with human
type I and II topoisomerases (hTopoI, hTopoII) computer simula-
tions were performed. In our theoretical calculations the crystal-
lographic models of DNA-binding domains of hTopoI (PDB id:
1SEU) and hTopoII (PDB id: 3QX3) were used. The total docking
scores reflecting affinity of the analysed compounds (i.e., 5b, 5c, 5e,
5f, 5i) towards hTopoI fell within the range between ꢀ 17.8 and ꢀ
24.2 kc al/mol vs. ꢀ 36.7 kc al/mol for the native ligand (Table 5).
The lack of linear dependence between the affinity and activity of
the analysed derivatives may be due to the specific nature of
respective cancer cells lines, intensified activity of topoisomerases
inside these cells and the effectiveness of DNA repair mechanisms.
Moreover, the final pharmacological effect also depends on the
difference in permeability through biological membranes of cancer
cells.
Much better correlation between in silico and in vitro test results
was obtained for hTopoII (PDB id: 3QX3). Compound 5i, showing
the highest affinity towards DNA-dependent subunit of this
enzyme (ꢀ5.7 kcal/mol), was also the strongest inhibitor of colo-
rectal cancer (HCT116) and leukaemia (MV4-11) cells growth, and
second, as regards the effect against breast cancer cells (MCF-7). As
shown in Fig. 3, the ligandeenzyme complex is stabilized by three
hydrogen bonds with residues Asp479, Gln778, Ala779, one
hydrogen bond with water molecule Hoh1461, and hydrophobic
interaction of pentafluorophenyl moiety with Gln778. Compound
5f showed docking score ꢀ4.2 kcal/mol and ranked second. It was
mediating hydrogen bond interactions with the residues Gln778,
Ala779, Glu447, Ala779 and water molecule Hoh1461. The
methoxyphenyl ring of 5f formed favorable hydrophobic contacts
with Ala779 and Gln778. Although remaining compounds 5b, 5c
and 5e exhibited low docking scores (within the range
Interaction energy of investigated complexes,
DE(AB), is calcu-
lated as the difference between the energy of the complex (AB) and
the energies of the monomers (A and B):
AB
AB
AB
AB
AB
AB
D
EðABÞ ¼ E ðABÞ ꢀ E ðAÞ ꢀ E ðBÞ;
where EGBðSÞ denotes energy of system S evaluated at the G opti-
mized geometry using basis set B. Additionally, binding energies
Ebind (AB) are calculated:
h
i
h
i
AB
AB
EbindðABÞ ¼ EAABBðABÞ ꢀ E ðAÞ ꢀ E ðBÞ þ EAABðAÞ ꢀ EAAðAÞ
AB
AB
h
i
þ EABBðBÞ ꢀ EBBðBÞ ;
to evaluate the size of geometry relaxation effects. Based on con-
clusions of our earlier work, evaluation of interaction and binding
energies is carried out within the DFT/M06-2X/6-311þþG**
approximation. Results are reported in Table 4.
Analysis of binding energies reported in Table 4 reveals that the
guanine complex with 5f is the strongest (binding energy
of ꢀ24.9 kcal/mol), followed by that with 5b (ꢀ24.7 kcal/mol), 5e
(ꢀ24.3 kcal/mol), and 5c (ꢀ24.2 kcal/mol). Much weaker interac-
tion is observed for the complex 5i-G (binding energy of
only ꢀ19.0 kcal/mol). The observed differences in binding energies
are reflected in the hydrogen bonding distances and angles. Inter-
action energies for the first four complexes reported in Table 4 are
approximately 5 kcal/mol lower than the corresponding binding
energies, and their values are practically identical for the com-
plexes 5b-G and 5f-G (ꢀ29.5 kcal/mol), and for complexes 5c-G and
5e-G (ꢀ29.2 kcal/mol). This difference is close to the one reported
in our previous study for the complex of the (E)-N-(4-(2-(2-(4-
(bis(2-chloroethyl)amino)benzylidene)hydrazinyl)thiazol-4-yl)
phenyl)methanesulfonamide with guanine, where the M06-2X/6-
311þþG** interaction energy was about 4 kcal/mol lower than
the binding energy. In the case of the complex 5i-G the interaction
energy (ꢀ26.4 kcal/mol) is approximately 7 kcal/mol lower than
the binding energy, indicating larger deformation of monomers'
structure during formation of the 5i-G complex than in the case of
other complexes investigated here and previously. The first four
complexes reported in Table 4 have larger values of interaction and
binding energies than the (E)-N-(4-(2-(2-(4-(bis(2-chloroethyl)
amino)benzylidene)hydrazinyl)thiazol-4-yl)phenyl)meth-
anesulfonamide-guanine complex reported in our previous study
[13].
Table 5
Docking scores for active nitrogen mustards.
Enzyme
Docking scores (kcal/mol)
5b
5c
5e
5f
5i
Etoposide
hTopoI
hTopoII
ꢀ24.2
ꢀ3.1
ꢀ23.9
ꢀ3.2
ꢀ21.1
ꢀ2.3
ꢀ20.9
ꢀ4.2
ꢀ17.8
ꢀ5.7
ꢀ36.7
ꢀ10.1
Table 4
Selected DFT/B3LYP/6-311G** geometrical parameters characterizing hydrogen bonds present in the investigated complexes, and predicted DFT/M06-2X/6-311þþG**
interaction and binding energies.
Complex
r(NeH/N) [Å]
:(NHN) [ꢂ]
r(NeH/O1) [Å]
:(NHO1) [ꢂ]
r(NeH/O2) [Å]
:(NHO2) [ꢂ]
D
E [kcal/mol]
Ebind [kcal/mol]
5b-G
5c-G
5e-G
5f-G
5i-G
3.018
3.008
3.013
3.039
3.114
172.8
172.1
172.4
173.5
173.6
2.824
2.831
2.827
2.810
2.781
174.9
174.6
174.8
175.2
174.9
3.162
3.165
3.166
3.188
3.414
149.5
149.8
149.6
147.5
135.6
ꢀ29.5
ꢀ29.2
ꢀ29.2
ꢀ29.5
ꢀ26.4
ꢀ24.7
ꢀ24.2
ꢀ24.3
ꢀ24.9
ꢀ19.0
Symbol O1 denotes oxygen atom in guanine molecule, and O2 e oxygen atom of the SO2 group forming the hydrogen bond.

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
145
Fig. 3. Interactions between 5i and the residues of the DNA-binding site of hTopoII.
between ꢀ2.3 and ꢀ3.2 kcal/mol vs. ꢀ10.1 kcal/mol for native
ligand, Table 5) they were still stabilized by at least two hydrogen
bonds with the residues Gln778, Ala779, Ser480, Leu502 and in the
case of 5c with water molecule Hoh1461. Moreover, the phenyl
moiety of 5b, 5c and 5e at sulfonamide group formed favorable
hydrophobic contacts with Gln778, Met782, Ala779. In conclusion,
the present results indicating the possibility of human top-
oisomerases inhibition as one of the mechanisms of anticancer
effect of the synthesized derivatives (Fig. 4).
3. Conclusion
In summary, we have developed an efficient method for the
synthesis of thiazole-based nitrogen mustard. As a result of our
research, we have identified new substituent (NHSO2CF3) which
significantly enhanced antiproliferative activity in all tested cancer
cell lines MV4-11, A549, MCF-7 and HCT116. Among the derivatives,
5b, 5c, 5e, 5f and 5i were found to exhibit high activity against
Fig. 4. Docking results with DNA-binding domain of hTopoII. Binding mode of 5i (up);
binding mode of 5i and etoposide (native ligand) (down).
human leukaemia MV4-11 cells, with IC50 values of 2.17e4.26 mg/
ml. The cytotoxic activity of compounds 5e against normal mouse
fibroblast BALB/3T3 cells is 5e8 times lower than against cancer
cell lines. Spectroscopic studies show that the model compound 5j
tends to accumulate in cellular membrane and to some extent in
lysosomes, which probably explains the complete lack of activity of
some tested compounds, while partial accumulation in mitochon-
dria and endoplasmic reticulum explains their plausible mecha-
nism of activity. The molecular modelling studies indicating the
possibility of human topoisomerases inhibition as one of the
mechanisms of anticancer effect of the synthesized derivatives.
Elemental analysis was performed on ELEMENTAR Vario MACRO
CHN. Melting points were determined in open glass capillaries and
are uncorrected. Analytical TLC was performed using Macherey-
Nagel Polygram Sil G/UV254 0.2 mm plates. 4-(Bis(2-chloroethyl)
amino)benzaldehyde, chlorobenzenesulfonamides, acetic acid,
thiosemicarbazide, pyridine were commercial materials (Aldrich).
4.1.1. N-(4-(2-Chloroacetyl)phenyl)benzenesulfonamide (2a).
Typical procedure
Benzenesulfonyl chloride (0.52 g, 2.95 mmol) was added to a
stirred solution of 1-(4-aminophenyl)-2-chloroethanone (1)
(0.50 g, 2.95 mmol) in dry pyridine (6 ml) at 0 ꢂC. The reaction
mixture was stirred under room temperature for 2.5 h under ni-
trogen atmosphere. After that, 10% hydrochloric acid solution
(40 ml) was added and the product was filtered off and subse-
quently washed with water. The separated precipitate was purified
on silica gel column chromatography (230e400 mesh) using
(dichloromethane/methanol, 90:10, Rf ¼ 0.83) to afford the desired
product: 0.50 g, 55%; mp 172e175 ꢂC. 1H NMR (DMSO-d6,
4. Experimental
4.1. Materials and methods
All experiments were carried out under air atmosphere unless
stated otherwise. Reagents were generally the best quality
commercial-grade products and were used without further purifi-
cation. 1H NMR (700 and 400 MHz) and 13C NMR (100 MHz) spectra
were recorded on a Bruker Avance III multinuclear instrument.
FAB(þ)-MS was performed by the Laboratory for Analysis of
Organic Compounds and Polymers of the Centre for Molecular and
400 MHz),
7.56e7.67 (m, 3H, 3CH); 7.83e7.88 (m, 4H, 4CH); 10.98 (s, 1H, NH).
13C NMR (DMSO-d6, 100 MHz),
(ppm): 47.84; 118.29 (2C); 127.17
d
(ppm): 5.06 (s, 2H, CH2); 7.23 (d, 2H, 2CH, J ¼ 9.0 Hz);
d
ꢀ ꢀ
Macromolecular Studies of the Polish Academy of Science in Łodz.
(2C); 129.71; 129.99 (2C); 130.56 (2C); 133.83; 139.66; 143.30;
MS spectra were recorded on a Finnigan MAT 95 spectrometer.
190.54. Anal. Calcd. for C14H12ClNO3S: C, 54.28; H, 3.90; N, 4.52.

146
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
Found: C, 54.31; H, 3.91; N, 4.54.
4.15.
4.1.2. N-(4-(2-Chloroacetyl)phenyl)-4-fluorobenzenesulfonamide
(2b)
4.1.7. N-(4-(2-Chloroacetyl)phenyl)-4-
isopropylbenzenesulfonamide (2g)
4-Fluorobenzene-1-sulfonyl chloride was reacted with 1. Yield:
0.40 g, 41%, (dichloromethane/methanol, 90:10, Rf ¼ 0.78); mp
4-Isopropylbenzene-1-sulfonyl chloride was reacted with 1.
Yield: 0.28 g, 27%, (dichloromethane/methanol, 90:10, Rf ¼ 0.76);
156e158 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d
(ppm): 5.07 (s, 2H,
mp 149e150 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d (ppm): 1.17 (d, 6H,
CH2); 7.24 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.40e7.46 (m, 2H, 2CH); 7.87 (d,
2CH3, J ¼ 7.0 Hz); 2.94 (sept., 1H, CH, J ¼ 7.0 Hz); 3.80 (s, 3H, CH3);
5.06 (s, 2H, CH2); 7.24 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.46 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.78 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.87 (d, 2H, 2CH,
J ¼ 9.0 Hz); 10.95 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
2H, 2CH, J ¼ 9.0 Hz); 7.90e7.94 (m, 2H, 2CH); 11.00 (s, 1H, NH). 13
C
NMR (DMSO-d6, 100 MHz), d (ppm): 47.84; 117.20 (2C); 118.49 (2C);
129.85; 130.33 (2C); 130.56 (2C); 136.03; 143.15; 166.24; 190.57.
Anal. Calcd. for C14H11ClFNO3S: C, 51.30; H, 3.38; N, 4.27. Found: C,
51.31; H, 3.37; N, 4.30.
d
(ppm): 23.79 (2C); 33.80; 47.81; 118.00 (2C); 127.34 (2C); 127.92
(2C); 129.53; 130.59 (2C); 137.29; 143.46; 154.62; 190.51. Anal.
Calcd. for C17H18ClNO3S: C, 58.03; H, 5.16; N, 3.98. Found: C, 58.00;
H, 5.15; N, 4.00.
4.1.3. N-(4-(2-Chloroacetyl)phenyl)-4-cyanobenzenesulfonamide
(2c)
4-Cyanobenzene-1-sulfonyl chloride was reacted with 1. Yield:
0.30 g, 30%, (dichloromethane/methanol, 90:10, Rf ¼ 0.76); mp
4.1.8. 4-tert-Butyl-N-(4-(2-chloroacetyl)phenyl)
benzenesulfonamide (2 h)
196e198 ꢂC. 1H NMR(DMSO-d6, 400 MHz),
d
(ppm): 1H NMR
4-tert-Butylbenzene-1-sulfonyl chloride was reacted with 1.
Yield: 0.33 g, 30%, (dichloromethane/methanol, 90:10, Rf ¼ 0.74);
(DMSO-d6, 400 MHz),
d (ppm): 5.07 (s, 2H, CH2); 7.24 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.88 (d, 2H, 2CH, J ¼ 9.0 Hz); 8.00 (d, 2H, 2CH,
mp 152e153 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d (ppm): 1.26 (s, 9H,
J ¼ 9.0 Hz); 8.07 (d, 2H, 2CH, J ¼ 9.0 Hz); 11.23 (s, 1H, NH). 13C NMR
3CH3); 5.06 (s, 2H, CH2); 7.25 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.61 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.79 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.87 (d, 2H, 2CH,
J ¼ 9.0 Hz); 10.97 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
(DMSO-d6, 100 MHz),
d (ppm): 47.87; 116.25; 117.93; 118.82 (2C);
127.93 (2C); 130.22; 130.63 (2C); 134.19 (2C); 142.62; 143.64;
190.60. Anal. Calcd. for C15H11ClN2O3S: C, 53.82; H, 3.31; N, 8.37.
Found: C, 53.82; H, 3.30; N, 8.40.
d
(ppm): 31.14 (3C); 35.37; 47.79; 117.94 (2C); 126.85 (2C); 127.07
(2C); 129.51; 130.61 (2C); 137.05; 143.48; 156.82; 190.50. Anal.
Calcd. for C18H20ClNO3S: C, 59.09; H, 5.51; N, 3.83. Found: C, 59.11;
H, 5.49; N, 3.84.
4.1.4. N-(4-(2-Chloroacetyl)phenyl)-4-methylbenzenesulfonamide
(2d)
4-Methylbenzene-1-sulfonyl chloride was reacted with 1. Yield:
0.26 g, 27%, (dichloromethane/methanol, 90:10, Rf ¼ 0.77); mp
4.1.9. N-(4-(2-Chloroacetyl)phenyl)-2,3,4,5,6-
pentafluorobenzenesulfonamide (2i)
182e184 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d
(ppm): 2.33 (s, 3H,
2,3,4,5,6-Pentafluorobenzene-1-sulfonyl chloride was reacted
with 1. Yield: 0.26 g, 22%, (dichloromethane/methanol, 90:10,
CH3); 5.05 (s, 2H, CH2); 7.22 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.37 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.74 (d, 2H, 2CH, J ¼ 9.0 Hz); 8.85 (d, 2H, 2CH,
J ¼ 9.0 Hz); 10.90 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
Rf ¼ 0.84); mp 165e168 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d (ppm):
5.10 (s, 2H, CH2); 7.28 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.95 (d, 2H, 2CH,
d
(ppm): 21.43; 47.81; 118.17 (2C); 127.23 (2C); 129.59; 130.39 (2C);
J ¼ 9.0 Hz); 12.00 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
130.54 (2C); 136.81; 143.43; 144.29; 190.53. Anal. Calcd. for
15H14ClNO3S: C, 55.64; H, 4.36; N, 4.33. Found: C, 55.62; H, 4.34; N,
d (ppm): 47.86; 113.06; 115.15 (m); 118.83 (2C); 130.73 (2C); 131.41;
C
4.36.
136.86 (m); 139.39 (m); 141.73; 143.26 (m); 145.80 (m); 190.71.
Anal. Calcd. for C14H7ClF5NO3S: C, 42.07; H, 1.77; N, 3.50. Found: C,
42.06; H, 1.74; N, 3.51.
4.1.5. N-(4-(2-Chloroacetyl)phenyl)-4-(trifluoromethyl)
benzenesulfonamide (2e)
4.1.10. N-(4-(2-Chloroacetyl)phenyl)-5-(dimethylamino)
naphthalene-1-sulfonamide (2j)
4-(Trifluoromethyl)benzene-1-sulfonyl chloride was reacted
with 1. Yield: 0.35 g, 32%, (dichloromethane/methanol, 90:10,
5-(Dimethylamino)naphthalene-1-sulfonyl chloride was reac-
ted with 1. Yield: 0.61 g, 51%, (dichloromethane/methanol, 90:10,
Rf ¼ 0.86); mp 153e155 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d (ppm):
5.07 (s, 2H, CH2); 7.25 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.88 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.99 (d, 2H, 2CH, J ¼ 9.0 Hz); 8.06 (d, 2H, 2CH,
J ¼ 9.0 Hz); 11.20 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
Rf ¼ 0.76); mp 89e91 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d (ppm):
2.81 (s, 6H, 2CH3); 5.00 (s, 2H, CH2); 7.17 (d, 2H, 2CH, J ¼ 9.0 Hz);
7.28 (d,1H, CH, J ¼ 8.0 Hz); 7.62e7.69 (m, 2H, 2CH); 7.79 (d, 2H, 2CH,
J ¼ 9.0 Hz); 8.32e8.38 (m, 2H, 2CH); 8.48 (d, 1H, CH, J ¼ 8.0 Hz);
d
(ppm): 48.79; 119.78 (2C); 123.98; 125.48; 128.28 (q, JC-
¼ 4.0 Hz); 129.19 (2C); 131.16; 131.63 (2C); 134.29 (q, JC-
11.35 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 45.50
F
¼
32.0 Hz); 143.74; 144.60; 191.58. Anal. Calcd. for
(2C); 47.72; 115.91; 117.31 (2C); 118.74; 124.07; 128.96; 129.20;
129.29; 129.47; 130.53 (2C); 130.67; 131.08; 134.63; 143.22;
152.03; 190.41. Anal. Calcd. for C20H19ClN2O3S: C, 59.62; H, 4.75; N,
6.95. Found: C, 59.60; H, 4.72; N, 6.94.
F
C
15H11ClF3NO3S: C, 47.69; H, 2.93; N, 3.71. Found: C, 47.70; H, 3.95;
N, 3.73.
4.1.6. N-(4-(2-Chloroacetyl)phenyl)-4-
methoxybenzenesulfonamide (2f)
4.1.11. (E)-2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinecarbothioamide (4)
4-Methoxybenzene-1-sulfonyl chloride was reacted with 1.
Yield: 0.38 g, 38%, (dichloromethane/methanol, 90:10, Rf ¼ 0.84);
4-(Bis-(2-chloroethyl)amino)benzaldehyde
(3)
(2.00
g,
mp 139e142 ꢂC. 1H NMR (DMSO-d6, 400 MHz),
d
(ppm): 3.80 (s, 3H,
8.12 mmol) in ethyl alcohol (20 ml) was added to a stirred hot so-
lution of thiosemicarbazide (0.74 g, 8.12 mmol) in water (24 ml) and
then (1.6 ml) glacial acetic acid was added. The reaction mixture
was stirred under reflux for 1 h, cooled to room temperature and
separate yellow precipitate was collected by filtration. Yield: 2.38 g,
CH3); 5.06 (s, 2H, CH2); 7.09 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.22 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.78 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.85 (d, 2H, 2CH,
J ¼ 9.0 Hz); 10.84 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d
(ppm): 47.82; 56.14; 115.08 (2C); 118.06 (2C); 129.48 (2C); 130.54
(2C); 131.18; 131.44; 143.54; 163.20; 190.52. Anal. Calcd. for
15H14ClNO4S: C, 53.02; H, 4.15; N, 4.12. Found: C, 53.00; H, 4.14; N,
92%; mp 192e193 ꢂC [13,18]. 1H NMR (400 MHz, DMSO-d6)
d
(ppm):
C
3.77 (m, 8H, 4CH2); 6.77 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.60 (d, 2H, 2CH,

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
147
J ¼ 9.0 Hz); 7.77 (bs, 1H, NH); 7.93 (bs, 1H, NH); 8.02 (bs, 1H, NH);
11.21 (s, 1H, CH). 13C NMR (DMSO-d6, 100 MHz)
(ppm): 41.54 (2C);
52.33 (2C); 112.20 (2C); 123.05; 129.42 (2C); 143.36; 148.34;
177.68.
4.1.15. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
d
hydrazinyl)thiazol-4-yl)phenyl)-4-methylbenzene-sulfonamide (5d)
N-(4-(2-Chloroacetyl)phenyl)-4-methylbenzenesulfonamide
(2d) was reacted with 4. Yield: 0.22 g, 61%, (dichloromethane/
methanol, 90:10, Rf ¼ 0.82); mp 168-179 ꢂC with decomp. 1H NMR
(DMSO-d6, 700 MHz), d (ppm): 2.32 (s, 3H, CH3); 3.68e3.80 (m, 8H,
4.1.12. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)benzenesulfonamide (5a). Typical
procedure
4CH2); 6.79 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.05 (d, 2H, 2CH, J ¼ 9.0 Hz);
7.11 (s, 1H, CH); 7.33 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.48 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.64 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.67 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.70 (s, 1H, CH); 10.30 (bs, 1H, NH); 11.90 (bs, 1H, NH).
Thiosemicarbazone 4 (0.26 g, 0.81 mmol) was added to a stirred
solution of N-(4-(2-chloroacetyl)phenyl)benzene-sulfonamide (2a)
(0.25 g, 0.81 mmol) in absolute ethyl alcohol (15 ml). The reaction
mixture was under reflux for 1.5 h, cooled to room temperature and
separate precipitate was collected by filtration. The crude product
was purified on silica gel column chromatography (230e400 mesh)
using (dichloromethane/methanol, 90:10, Rf ¼ 0.79) to afford the
desired product: 0.24 g, 52%; mp 187e190 ꢂC with decomp. 1H NMR
13C NMR (DMSO-d6, 100 MHz),
d (ppm): 21.42; 41.45 (2C); 52.35
(2C); 103.00; 112.36 (2C); 119.59; 120.31 (2C); 123.06; 126.98;
127.20 (2C); 128.64; 130.17 (2C); 137.07; 137.86; 143.78; 143.92;
148.10; 148.28; 168.79. FAB(þ)-MS (m/z, %): 588.2 [(Mþþ1), 100],
345.1 (16), 247.1 (28), 245.2 (44), 195.1 (20), 190.1 (28), 188.0 (40).
Anal. Calcd. for C27H27Cl2N5O2S2: C, 55.10; H, 4.62; N, 11.90. Found:
C, 55.08; H, 4.64; N, 11.91.
(DMSO-d6, 700 MHz),
d (ppm): 3.69e3.80 (m, 8H, 4CH2); 6.80 (d,
2H, 2CH, J ¼ 9.0 Hz); 7.11e7.14 (m, 3H, 3CH); 7.49 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.51e7.56 (m, 2H, 2CH); 7.58e7.61 (m, 1H, CH); 7.68 (d,
2H, 2CH, J ¼ 9.0 Hz); 7.75e7.79 (m, 2H, 2CH); 7.95 (s, 1H, CH); 10.43
(bs, 1H, NH); 12.00 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
4.1.16. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-4-(trifluoromethyl)-
benzenesulfonamide (5e)
N-(4-(2-Chloroacetyl)phenyl)-4-(trifluoromethyl)benzenesulfo-
namide (2e) was reacted with 4. Yield: 0.20 g, 48%, (dichloro-
methane/methanol, 90:10, Rf ¼ 0.81); mp 205-207 ꢂC. 1H NMR
d
(ppm): 41.48 (2C); 52.35 (2C); 103.18; 112.35 (2C); 119.73; 120.42
(2C); 122.90; 127.10; 127.15 (2C); 128.77; 129.45; 129.75 (2C);
133.43; 137.90; 139.91; 144.65; 146.21; 147.52; 148.22; 168.77.
FAB(þ)-MS (m/z, %): 574.2 [(Mþþ1), 100], 331.2 (28), 247.1 (60),
245.1 (100), 230.1 (24), 195.1 (64), 190.0 (80), 188.0 (100), 167.0 (36),
133.0 (28). Anal. Calcd. for C26H25Cl2N5O2S2: C, 54.35; H, 4.39; N,
12.19. Found: C, 54.36; H, 4.42; N, 12.22.
(DMSO-d6, 700 MHz),
d (ppm): 3.72e3.79 (m, 8H, 4CH2); 6.82 (d,
2H, 2CH, J ¼ 9.0 Hz); 7.17 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.18 (s, 1H, CH);
7.51 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.74 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.96 (s, 1H,
CH); 7.99 (m, 4H, 4CH); 10.67 (bs, 1H, NH); 11.99 (bs, 1H, NH). 13
C
NMR (DMSO-d6, 100 MHz), d (ppm): 42.49 (2C); 53.38 (2C); 104.36;
113.39 (2C); 121.99 (2C); 124.05; 125.60; 127.14; 128.04 (2C); 128.14
(2C); 129.16 (2C); 129.68 (2C); 131.35; 134.14 (q, JC-F ¼ 32.0 Hz);
138.19; 141.81; 145.19; 149.18; 169.83. FAB(þ)-MS (m/z, %): 642.1
[(Mþþ1), 100], 247.1 (40), 245.2 (68), 195.1 (40), 188.1 (60), 167.1
(12). Anal. Calcd. for C27H24Cl2F3N5O2S2: C, 50.47; H, 3.76; N, 10.90.
Found: C, 50.44; H, 3.78; N, 10.92.
4.1.13. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-4-fluorobenzene-sulfonamide (5b)
N-(4-(2-Chloroacetyl)phenyl)-4-fluorobenzenesulfonamide
(2b) was reacted with 4. Yield: 0.21 g, 58%, (dichloromethane/
methanol, 90:10, Rf ¼ 0.70); mp 195e198 ꢂC with decomp. 1H NMR
(DMSO-d6, 700 MHz),
d (ppm): 3.69e3.80 (m, 8H, 4CH2); 6.80 (d,
2H, 2CH, J ¼ 9.0 Hz); 7.12 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.14 (s, 1H, CH);
7.37e7.41 (m, 2H, 2CH); 7.48 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.69 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.80e7.84 (m, 2H, 2CH); 7.93 (s, 1H, CH); 10.42 (bs,
4.1.17. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-4-methoxybenzene-sulfonamide
(5f)
1H, NH); 11.90 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm):
N-(4-(2-Chloroacetyl)phenyl)-4-methoxybenzenesulfonamide
(2f) was reacted with 4. Yield: 0.35 g, 80%, (dichloromethane/
methanol, 90:10, Rf ¼ 0.73); mp 198-200 ꢂC with decomp. 1H NMR
41.49 (2C); 52.36 (2C); 103.21; 112.36 (2C); 116.96 (2C); 119.94;
120.74 (2C); 123.03; 127.06 (2C); 128.67 (2C); 130.23 (2C); 136.24;
137.56; 144.12; 148.15; 163.54; 166.04; 168.80. FAB(þ)-MS (m/z, %):
592.1 [(Mþþ1), 100], 349.1 (24), 247.1 (44), 245.1 (76), 230.1 (20),
207.1 (32), 195.1 (44), 190.0 (52), 188.1 (64), 181,.1 (40), 167.0 (24),
131.0 (20). Anal. Calcd. for C26H24Cl2FN5O2S2: C, 52.70; H, 4.08; N,
11.82. Found: C, 52.68; H, 4.10; N, 11.84.
(DMSO-d6, 700 MHz), d (ppm): 3.77 (s, 3H, CH3); 3.72e3.78 (m, 8H,
4CH2); 6.79 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.05 (d, 2H, 2CH, J ¼ 9.0 Hz);
7.10 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.12 (s, 1H, CH); 7.48 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.67 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.69 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.91 (s, 1H, CH); 10.23 (bs, 1H, NH); 11.94 (bs, 1H, NH).
13C NMR (DMSO-d6, 100 MHz),
d (ppm): 41.49 (2C); 52.36 (2C);
4.1.14. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-4-cyanobenzene-sulfonamide (5c)
N-(4-(2-Chloroacetyl)phenyl)-4-cyanobenzenesulfonamide (2c)
was reacted with 4. Yield: 0.18 g, 56%, (dichloromethane/methanol,
90:10, Rf ¼ 0.73); mp 160 ꢂC with decomp. 1H NMR (DMSO-d6,
56.09; 102.95; 112.26 (2C); 114.86 (2C); 120.24 (2C); 123.00; 127.02
(2C); 128.72 (2C); 129.38 (2C); 129.49; 131.56; 138.10; 144.37;
147.96; 148.19; 162.86; 168.64. FAB(þ)-MS (m/z, %): 604.3 [(Mþþ1),
100], 434.1 (12), 361.0 .920), 247.1 (28), 245.1 (48), 195.1 (24), 190.1
(32), 188.1 (40). Anal. Calcd. for C27H27Cl2N5O3S2: C, 53.64; H, 4.50;
N, 11.58. Found: C, 53.61; H, 4.53; N, 12.01.
700 MHz),
d (ppm): 3.72e3.80 (m, 8H, 4CH2); 6.79 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.10 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.15 (s, 1H, CH); 7.47 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.70 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.89 (s, 1H, CH); 7.90
(d, 2H, 2CH, J ¼ 9.0 Hz); 8.04 (d, 2H, 2CH, J ¼ 9.0 Hz); 10.59 (bs, 1H,
4.1.18. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-4-isopropylbenzene-sulfonamide
(5g)
NH); 11.85 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm):
41.49 (2C); 52.36 (2C); 103.36; 112.36 (2C); 115.86; 118.00; 120.29;
121.11 (2C); 122.70; 123.09; 127.09; 127.89; 127.92 (2C); 128.60;
133.90; 133.96 (2C); 136.91; 143.72; 143.90; 148.07; 168.84.
FAB(þ)-MS (m/z, %): 599.2 [(Mþþ1), 56], 356.2 (28), 247.1 (32),
245.1 (52), 195.1 (60), 173.1 (100), 167.0 (50), 149.0 (60), 136.0 (64).
Anal. Calcd. for C27H24Cl2N6O2S2: C, 54.09; H, 4.03; N, 14.02. Found:
C, 54.07; H, 4.03; N, 14.05.
N-(4-(2-Chloroacetyl)phenyl)-4-isopropylbenzenesulfonamide
(2g) was reacted with 4. Yield: 0.26 g, 74%, (dichloromethane/
methanol, 90:10, Rf ¼ 0.73); mp 199-202 ꢂC with decomp. 1H NMR
(DMSO-d6, 700 MHz),
d
(ppm): 1.17 (d, 6H, 2CH3, J ¼ 7.0 Hz); 2.92
(sept, 1H, CH, J ¼ 7.0 Hz); 3.70e3.78 (m, 8H, 4CH2); 6.80 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.12 (s, 1H, CH); 7.13 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.42 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.48 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.68 (d, 2H, 2CH,

148
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
J ¼ 9.0 Hz); 7.70 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.90 (s, 1H, CH); 10.34 (bs,
1H, NH); 11.88 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
(ppm):
170.1 (64), 168.0 (48), 149.1 (32), 117.0 (20). Anal. Calcd. for
C32H32Cl2N6O2S2: C, 57.56; H, 4.83; N, 12.59. Found: C, 57.59; H,
d
23.83 (2C); 33.81; 41.53 (2C); 52.39 (2C); 103.03; 112.37 (2C);
119.43; 120.01 (2C); 122.97; 127.07; 127.30 (2C); 127.69 (2C);
128.71; 137.51; 137.57; 138.01; 144.34; 146.19; 147.88; 148.17;
154.11; 168.81. FAB(þ)-MS (m/z, %): 616.2 [(Mþþ1),100], 373.3 (24),
245.1 (60), 230.1 (16), 209.1 (28), 195.1 (32), 190.1 (52), 188.0 (60),
181.1 (32), 167.0 .916). Anal. Calcd. for C29H31Cl2N5O2S2: C, 56.49; H,
5.07; N, 11.36. Found: C, 56.51; H, 5.04; N, 11.39.
4.84; N, 12.60.
4.2. Biological activity
4.2.1. Cells
Biphenotypic B myelomonocytic leukemia MV4-11, human lung
carcinoma A549, human breast carcinoma MCF-7, human colon
carcinoma HCT116 and normal mouse fibroblast BALB/3T3 cells
were obtained from American Type Culture Collection (Rockville,
Maryland, USA). All the cell lines are being maintained at the
Institute of Immunology and Experimental Therapy, Wroclaw,
Poland.
MV4-11 cells were cultured in RPMI 1640 medium (Gibco, UK)
with 2 mM L-glutamine adjusted to contain 1.0 mM sodium py-
ruvate, and 10% fetal bovine serum (FBS) (all from SigmaeAldrich,
Germany). A549 and HCT116 cells were cultured in RPMI
1640þOpti-MEM (1:1) (both from Gibco, UK), MCF-7 cells in Eagle
medium (IIET, Wroclaw, Poland), BALB/3T3 in Dulbecco medium
(IIET, Poland) supplemented with 2 mM L-glutamine and 1.0 mM
sodium pyruvate, 10% fetal bovine serum (all from Sigma-Aldrich,
Germany).
The MCF-7 cell culture was supplemented with 0.8 mg/L of in-
sulin (Sigma-Aldrich, Germany). All culture media were supple-
mented with 100 units/ml penicillin, and 100 mg/ml streptomycin
4.1.19. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-4-tert-butylbenzene-sulfonamide
(5 h)
4-tert-Butyl-N-(4-(2-chloroacetyl)phenyl)benzenesulfonamide
(2h) was reacted with 4. Yield: 0.33 g, 97%, (dichloromethane/
methanol, 90:10, Rf ¼ 0.74); mp 210-212 ꢂC with decomp. 1H NMR
(DMSO-d6, 700 MHz),
d (ppm): 1.24 (s, 9H, 3CH3); 3.72e3.78 (m,
8H, 4CH2); 6.80 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.12 (s, 1H, CH); 7.14 (d, 2H,
2CH, J ¼ 9.0 Hz); 7.48 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.57 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.68 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.71 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.90 (s, 1H, CH); 10.37 (bs, 1H, NH); 11.89 (bs, 1H, NH).
13C NMR (DMSO-d6, 100 MHz),
d
(ppm): 31.18 (3C); 35.32; 41.48
(2C); 52.36 (2C); 103.01; 112.35 (2C); 119.38; 119.92 (2C); 122.98;
126.63 (2C); 127.03 (2C); 127.07 (2C); 128.71; 137.23; 137.31;
138.02; 138.47; 144.30; 148.16; 156.45; 168.75. FAB(þ)-MS (m/z,
%): 630.3 [(Mþþ1), 100], 387.3 (28), 245.1 (80), 195.1 (40), 190.1
(64), 188.0 (88), 181.1 (20), 167.0 (16), 133.1 (16). Anal. Calcd. for
(both from Polfa Tarchomin S.A., Poland). All cell lines were grown
C
30H33Cl2N5O2S2: C, 57.13; H, 5.27; N, 11.10. Found: C, 57.15; H, 5.25;
at 37 ꢂC with 5% CO2 humidified atmosphere.
N, 11.12.
4.2.2. Compounds
4.1.20. ((E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-2,3,4,5,6-pentafluoro-
benzenesulfonamide (5i)
Prior to usage, the compounds were dissolved in DMSO and
culture medium (1: 9) to the concentration of 1 mg/ml, and sub-
sequently diluted in culture medium to reach the required con-
N-(4-(2-Chloroacetyl)phenyl)-2,3,4,5,6-
centrations (0.1, 1, 10 and 100 mg/ml).
pentafluorobenzenesulfonamide (2i) was reacted with 4. Yield:
0.24 g, 73%, (dichloromethane/methanol, 90:10, Rf
¼
0.75);
4.2.3. In vitro antiproliferative assay
mp > 260 ꢂC. 1H NMR (DMSO-d6, 700 MHz),
d
(ppm): 3.72e3.80 (m,
Twenty four hours prior to the addition of the tested com-
pounds, the cells counted using Burker hemocytometer were
plated in 96-well plates (Sarstedt, Germany) at a density of
1 ꢁ 104 cells per well or 0.5 ꢁ 104 of the HCT116 cells. The assay was
performed after 72 h of exposure to varying concentrations of the
tested agents [29,30]. The in vitro cytotoxic effect of all agents was
examined using the SRB assay for adherent cells (A549, MCF-7,
HCT116, BALB/3T3) or MTT assay for leukaemia cells (MV4-11) as
described previously. The results were calculated as an IC50
(inhibitory concentration 50) e the concentration of tested agent
which inhibits proliferation of 50% of the cancer cell population. IC
values were calculated for each experiment separately and mean
values SD are presented in the Table 1. Each compound in each
concentration was tested in triplicate in a single experiment, which
was repeated 3-7 times.
8H, 4CH2); 6.80 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.19 (d, 2H, 2CH, J ¼ 9.0 Hz);
7.20 (s, 1H, CH); 7.48 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.78 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.90 (s, 1H, CH); 11.36 (bs, 1H, NH); 11.89 (bs, 1H, NH).
13C NMR (DMSO-d6, 100 MHz),
d (ppm): 41.48 (2C); 52.35 (2C);
103.61; 112.35 (2C); 115.28 (m); 121.06 (2C); 123.11; 127.21 (2C);
128.59 (2C); 131.38; 135.81; 136.77 (m); 139.28 (m); 142.88 (m);
143.24 (m); 143.66; 145.75 (m); 148.06; 148.56; 168.85. FAB(þ)-MS
(m/z, %): 664.1 [(Mþþ1), 100], 421.1 (28), 245.1 (80), 195.1 (64),
181.0 (68),167.0 (32),145.0 (28). Anal. Calcd. for C26H20Cl2F5N5O2S2:
C, 46.99; H, 3.03; N, 10.54. Found: C, 47.01; H, 3.00; N, 10.57.
4.1.21. (E)-N-(4-(2-(2-(4-(Bis(2-chloroethyl)amino)benzylidene)
hydrazinyl)thiazol-4-yl)phenyl)-5-(dimethylamino)-naphthalene-
1-sulfonamide (5j)
N-(4-(2-Chloroacetyl)phenyl)-5-(dimethylamino)naphthalene-
1-sulfonamide (2j) was reacted with 4. Yield: 0.21 g, 64%,
(dichloromethane/methanol, 90:10, Rf ¼ 0.79); mp 180 ꢂC with
4.2.4. SRB cytotoxic test
Cells were attached to the bottom of plastic wells by fixing them
with cold 50% TCA (trichloroacetic acid, POCH, Gliwice, Poland) on
top of the culture medium in each well. The plates were incubated
at 4 ꢂC for 1 h and then washed five times with tap water. The
cellular material fixed with TCA was stained with 0.14% sulfo-
rhodamine B (SRB, Sigma-Aldrich, Germany) and dissolved in 1%
acetic acid (POCH, Gliwice, Poland) for 30 min. Unbound dye was
removed by rinsing (4X) in 1% acetic acid. The protein-bound dye
was extracted with 10 mM unbuffered Tris base (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) for determination of the op-
decomp. 1H NMR (DMSO-d6, 700 MHz),
d (ppm): 2.79e2.87 (s, 6H,
2CH3); 3.72e3.78 (m, 8H, 4CH2); 6.79 (d, 2H, 2CH, J ¼ 9.0 Hz);
7.04e7.07 (m, 3H, 3CH); 7.26e7.36 (m, 1H, CH); 7.46 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.60 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.60e7.66 (m, 2H, 2CH);
7.88 (s, 1H, CH); 8.23 (s, 1H, CH); 8.38e8.50 (m, 2H, 2CH); 10.78 (bs,
1H, NH); 11.84 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm):
41.49 (2C); 46.11 (2C); 52.36 (2C); 102.83; 112.35 (2C); 117.88;
118.68; 119.26 (2C); 122.20; 123.10; 125.06; 126.98 (2C); 128.17;
128.59 (2C); 129.25; 129.73; 129.84; 130.69; 135.57; 137.46;
143.65; 148.05; 168.76. FAB(þ)-MS (m/z, %): 667.3 [(Mþþ1), 100],
247.1 (32), 245.1 (56), 241.2 (32), 195.1 (32), 190.0 (52), 185.1 (64),
tical density (
l
¼ 540 nm) in a computer-interfaced, 96-well Syn-
ergy H4 (BioTek Instruments USA) photometer microtiter plate

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
149
reader [31].
[32] with the B3LYP exchange-correlation functional [33] and 6-
31 þ G(d,p) basis set. The effects of the solvent were evaluated
using the PCM model [33e35] in which a cavity is created via a
series of overlapping spheres [36] with standard dielectric con-
stants (ε) of 46.826 for DMSO. Electronic absorption transition
energies for 50 spin-allowed singletesinglet and fluorescence
emission spectra made of the 4 spin-allowed transitions were
considered using the external iteration (EI) approach. The molec-
ular electron densities for each derivative were determined from
the wave functions using the CUBE option implemented in
Gaussian 09 and visualized using GaussView 5.0. The molecular
energy levels were analyzed using Chemissian software [32].
Geometrical parameters of the investigated complexes were
optimized using the DFT with the B3LYP functional and the 6-
311G** basis set. Vibrational frequencies were evaluated within the
same approximation to confirm that the resulting structures
correspond to real minima on potential energy surface. Next,
interaction and binding energies were calculated using the super-
molecular approach and counterpoise-correcting the results. On
the basis of the conclusions of our recent work [13] the DFT/M06-
2X approximation and the 6-311þþG** basis set were chosen for
that purpose. All calculations were carried out using the Gaussian
09 program [32].
4.2.5. MTT cytotoxic test
Twenty microliter of MTT solution (MTT: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, stock so-
lution: 5 mg/ml) was added to each well and incubated for 4 h.
After the incubation time was complete, 80 ml of the lysis mixture
was added to each well (lysis mixture: 225 ml dimethylformamide,
67.5 g sodium dodecyl sulfate and 275 ml of distilled water). The
optical densities of the samples were read after 24 h on a Synergy
H4 (BioTek Instruments USA) photometer microtiter plate reader at
570 nm. All of chemicals were obtained from Sigma-Aldrich, Ger-
many [31].
4.2.6. Cellular staining and colocalization
To visualize the accumulation of the compound 5j within the
cells, 10 ꢁ 103 HCT116 cells in a 300
mL growth medium were plated
into an 8-well LabTek chambered cover glass (Nunc) and incubated
under standard conditions at 37 ꢂC in a humidified atmosphere at
5% CO2 for 24 h. After this, the cells were treated with the com-
pound 5j being tested at a concentration of 16 mg/ml and incubated
for a further 2 h. After incubation the cells were treated with
cellular Trackers: ER-Tracker™ (Molecular Probes) concentration
100 nM for 1 h, MitoTracker® Orange (Molecular Probes) concen-
4.5. Automated docking setup
tration 1
concentration 6.5
dyes the cells were washed three times with PBS and then 300
m
M for 0.5 h, and Hoechst (Thermo Fisher Scientific)
M for 1 h. After incubation with the appropriate
L of
m
Flexible docking was performed by means of the FlexX program
[37] as implemented in LeadIT software package [38]. Model of the
Topo I binding site on the structure deposited in the Protein Data
Bank [39] under the PDB ID 1SEU [40] and model of the Topo II
binding site under the PDB ID 3QX3 [41] were employed. The native
ligands within the active sites, the camptothecin and the etoposide
(anticancer drug), respectively, were removed. In the case of
docking simulation within Topo II binding site, two water mole-
cules (HOH-1376-A and HOH-1461-A) were allowed. The active
sites were defined to include all atoms within 6.5 Å radius of the
native ligands. The first 100 top ranked docking poses were saved
for each docking run. For all compounds their protonated forms
were considered, as recommended by FlexX program.
m
PBS was added. Observation of the cells was carried out using an
Olympus Fluoview FV1000 confocal laser scanning system equip-
ped with the Olympus IX81 inverted microscope (Olympus, Poland)
immediately after staining. Images acquisition was performed us-
ing a 60x oil immersion objective lens. Analysis and processing
images were performed using an ImageJ 1.41 (Wayne Rasband,
National Institutes of Health, USA).
4.3. Spectroscopy
The absorption and fluorescence spectra were measured at
room temperature in a 10 mm quartz cell with a U-2900 spectro-
photometer (Hitachi) and an F-7000 spectrofluorimeter (Hitachi),
respectively. The stock solutions (10 mM) that had been prepared in
DMSO were further diluted to working concentrations starting
from 0.1 mM. The fluorescence quantum yields were measured
using the comparative method with anthracene in cyclohexane as a
reference (FRef ¼ 0.34). We prepared standard and test solutions of
decreasing concentrations in order to provide absorbance in the
range of 0.1 to 0.01 at the excitation wavelength. The fluorescence
spectra of all of the solutions were measured at room temperature
in a 10 mmcell using an F-7000 spectrofluorimeter (Hitachi). The
fluorescence quantum yields of the dyes that were tested were
calculated as follows:
Conflict of interest
The authors confirm that this article content has no conflicts of
interest.
Acknowledgements
This study was supported by the Nicolaus Copernicus University
ꢀ
(grant No. 01/CM/2014). We thank Centro de Supercomputacion de
Galicia (CESGA) and Wrocław Centre for Networking and Super-
computing, WCSS, Wrocław, Poland, http://www.wcss.wroc.pl,
Grant no. 18. for computational resources.
ꢀ
.
ꢁꢀ
.
ꢁ
f ¼ fRef aDye aRef h2Dye hR2ef
Appendix A. Supplementary data
where subscripts Dye and Ref indicate test and standard samples,
respectively, is the fluorescence quantum yield, is the gradient
obtained from the plot integrated fluorescence intensity versus
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2016.04.058.
F
a
absorbance and
applied.
h is the refractive index of solvents that were
References
[1] P. Boyle, B. Levin, World cancer Report, International Agency for Research on
Cancer, Lyon, 2008. Reference available from, http://www.iarc.fr/en/
publications/pdfs.online/wcr/2008/wcr_2008.pdf.
4.4. Quantum mechanical calculations
[2] J.S.I. Ferlay, M. Ervik, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin,
D. Forman, F. Bray, Cancer Incidence and Mortality Worldwide: IARC Can-
cerBase No. 11 [Internet]; GLOBOCAN 2012 v1.0 2013, International Agency
Spectroscopic properties have been calculated within DFT and
TD-DFT approximations using the Gaussian 09 software package

150
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1119 (2016) 139e150
for Research on Cancer, Lyon, France, 2013.
Verlag, Weinheim, 2001.
[3] S. Puyo, D. Montaudon, P. Pourquier, From old alkylating agents to new minor
groove binders, Crit. Rev. Oncol. Hematol. 89 (2014) 43e61.
[24] M. Rams-Baron, M. Dulski, A. Mrozek-Wilczkiewicz, M. Korzec, W. Cieslik,
ꢀ
E. Spaczynska, P. Bartczak, A. Ratuszna, J. Polanski, R. Musioł, Synthesis of new
[4] K. Suzukake, B.P. Vistica, D.T. Vistica, Dechlorination of L-phenylalanine
mustard by sensitive and resistant tumor cells and its relationship to intra-
cellular glutathione content, Biochem. Pharmacol. 32 (1983) 165e167.
[5] J.M. Kaldor, N.E. Day, K. Hemminki, Quantifying the carcinogenicity of anti-
neoplastic drugs, Eur. J. Cancer Clin. Oncol. 24 (1988) 703e711.
[6] M.R. Osborne, P.D. Lawley, C. Crofton-Sleigh, W. Warren, Products from
alkylation of DNA in cells by melphalan: human soft tissue sarcoma cell line
RD and Escherichia coli WP2, Chem. Biol. Interact. 97 (1995) 287e296.
[7] R. Saffhill, G.P. Margison, P.J. O'Connor, Mechanisms of carcinogenesis induced
by alkylating agents, Biochim. Biophys. Acta. 823 (1985) 111e145.
[8] B.J. Sanderson, A.J. Shield, Mutagenic damage to mammalian cells by thera-
peutic alkylating agents, Mutat. Res. 355 (1996) 41e57.
[9] Y.-D. Wang, J. Dziegielewski, N.R. Wurtz, B. Dziegielewska, P.B. Dervan,
T.A. Beerman, DNA crosslinking and biological activity of a hairpin polyamide-
chlorambucil conjugate, Nucleic Acids Res. 31 (2003) 1208e1215.
[10] T.A. Gourdie, K.K. Valu, G.L. Gravatt, T.J. Boritzki, B.C. Baguley, L.P. Wakelin,
W.R. Wilson, P.D. Woodgate, W.A. Denny, DNA-directed alkylating agents. 1.
Structure-activity relationships for acridine-linked aniline mustards: conse-
quences of varying the reactivity of the mustard, J. Med. Chem. 33 (1990)
1177e1186.
styrylquinoline cellular dyes, fluorescent properties, cellular localization and
cytotoxic behavior, PLoS One 10 (2015) e0131210.
€
[25] J. Kornhuber, A.W. Henkel, T.W. Groemer, S. Stadtler, O. Welzel, P. Tripal,
A. Rotter, S. Bleich, S. Trapp, Lipophilic cationic drugs increase the perme-
ability of lysosomal membranes in a cell culture system, J. Cell Physiol. 224
(2010) 152e164.
[26] Y.-C. Chang, J.D. Wang, K.K. Svoboda, R.P. Casillas, J.D. Laskin, M.K. Gordon,
D.R. Gerecke, Sulfur mustard induces an endoplasmic reticulum stress
response in the mouse ear vesicant model, Toxicol. Appl. Pharmacol. 268
(2013) 178e187.
[27] A. Polavarapu, J.A. Stillabower, S.G.W. Stubblefield, W.M. Taylor, M.-U. Baik,
The mechanism of guanine alkylation by nitrogen mustards: a computational
study, J. Org. Chem. 77 (2012) 5914e5921.
[28] J.L. Nitiss, Targeting DNA topoisomerase II in cancer chemotherapy, Nat. Rev.
Cancer 9 (2009) 338e350.
[29] J. Bramson, A. McQuillan, R. Aubin, M. Alaoui-Jamali, G. Batist,
G. Christodoulopoulos, L.C. Panasci, Nitrogen mustard drug resistant B-cell
chronic lymphocytic leukemia as an in vivo model for crosslinking agent
resistance, Mut. Res. 336 (1995) 269e278.
ꢀ
[30] K. Sidoryk, M. Switalska, J. Wietrzyk, A. Jaromin, M. Pie˛tka-Ottlik, P. Cmoch,
ꢀ
[11] S. McClean, C. Costelloe, W.A. Denny, M. Searcey, L.P. Wakelin, Sequence
selectivity, cross-linking efficiency and cytotoxicity of DNA-targeted 4-
anilinoquinoline aniline mustards, Anticancer Drug Des. 14 (1999) 187e204.
[12] G. Pezzoni, M. Grandi, G. Biasoli, L. Capolongo, D. Ballinari, F.C. Giuliani,
B. Barbieri, A. Pastori, E. Pesenti, N. Mongelli, Biological profile of FCE 24517, a
novel benzoyl mustard analogue of distamycin A, Br. J. Cancer 64 (1991)
1047e1050.
J. Zagrodzka, W. Szczepek, L. Kaczmarek, W. Peczynska-Czoch, Synthesis and
biological evaluation of new amino acid and dipeptide derivatives of neo-
cryptolepine as anticancer agents, J. Med. Chem. 55 (2012) 5077e5087.
[31] L.V. Rubinstein, R.H. Shoemaker, K.D. Paul, R.M. Simon, S. Tosini, P. Skehan,
D.A. Sudiero, A. Monks, M.R. Boyd, Comparison of in vitro anticancer-drug-
screening data generated with a tetrazolium assay versus a protein assay
against a diverse panel of human tumor cell lines, J. Nat. Cancer Inst. 82 (1990)
1113e1118.
ꢀ
[13] K.Z. Ła˛czkowski, K. Misiura, M. Switalska, J. Wietrzyk, A. Baranowska-
ꢀ
Ła˛czkowska, B. Fernandez, A. Paneth, T. Plech, Synthesis and in vitro anti-
[32] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson,
H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino,
G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven,
J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers,
K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari,
A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, N.J. Millam,
M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski,
R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador,
proliferative activity of thiazole-based nitrogen mustards. The hydrogen
bonding interaction between model systems and nucleobases, Anti Cancer
Agents Med. Chem. 14 (2014) 1271e1281.
[14] K.Z. Ła˛czkowski, K. Motylewska, A. Baranowska-Ła˛czkowska, A. Biernasiuk,
ꢀ
K. Misiura, A. Malm, B. Fernandez, Synthesis, antimicrobial evaluation and
theoretical prediction of NMR chemical shifts of thiazole and selenazole de-
rivatives with high antifungal activity against Candida spp, J. Mol. Struct. 1108
(2016) 427e437.
[15] K.Z. Ła˛czkowski, K. Misiura, A. Biernasiuk, A. Malm, Discovery and evaluation
of efficient selenazoles with high antifungal activity against Candida spp, Med.
Chem. 11 (2015) 118e127.
€
J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz,
[16] K.Z. Ła˛czkowski, K. Misiura, A. Biernasiuk, A. Malm, A. Siwek, T. Plech, E. Ciok-
Pater, K. Skowron, E. Gospodarek, Synthesis, in vitro biological screening and
molecular docking studies of novel camphor-based thiazoles, Med. Chem. 10
(2014) 600e608.
[17] K.Z. Ła˛czkowski, K. Misiura, A. Biernasiuk, A. Malm, A. Siwek, T. Plech, Syn-
thesis, antimicrobial activities and molecular docking studies of novel 6-
hydroxybenzofuran-3(2H)-one based 2,4-disubstituted 1,3-thiazoles, Lett.
Drug Des. Discov. 10 (2013) 798e807.
[18] J. Karthikeyan, P. Parameshwara, A. Nityananda Shetty, Analytical properties
of p-[N,N-bis(2-chloroethyl)amino]-benzaldehydethiosemicarbazone: spec-
trophotometric determination of palladium(II) in alloys, catalysts, and com-
plexes, Environ. Monit. Assess. 173 (2011) 569e577.
J. Cioslowski, D.J. Fox, Gaussian 09, Revision C.01, A.02, Gaussian, Inc., Wall-
ingford, 2009.
[33] M. Cossi, V. Barone, R. Cammi, J. Tomasi, Ab initio study of solvated molecules:
a new implementation of the polarizable continuum model, Chem. Phys. Lett.
255 (1996) 327e335.
[34] J.B. Foresman, T.A. Keith, K.B. Wiberg, J. Snoonian, M.J. Frisch, Solvent effects.
5. Influence of cavity shape, truncation of electrostatics and electron corre-
lation on ab initio reaction field calculations, J. Phys. Chem. 100 (1996)
16098e16104.
[35] J. Tomasi, B. Mennucci, R. Cammi, Quantum mechanical continuum solvation
models, Chem. Rev. 105 (2005) 2999e3093.
ꢁ
[36] J. Tomasi, B. Mennucci, E. Cances, The IEF version of the PCM solvation
[19] R.I. Geran, N.H. Greenberg, M.M. MacDonald, A.M. Schumacher, B.D. Abbott,
Protocols for screening chemical agents and natural products against animal
tumors and other biological systems, Cancer Chemother. Rep. Part 3 3 (1972)
59e61.
[20] K. Sugano, M. Kansy, P. Artursson, A. Avdeef, S. Bendels, L. Di, G.F. Ecker,
B. Faller, H. Fischer, G. Gerebtzoff, H. Lennernaes, F. Senner, Nat. Rev. Drug
Discov. 9 (2010) 597e614.
method: an overview of a new method addressed to study molecular solutes
at the QM ab initio level, J. Mol. Struct. Theochem. 464 (1999) 211e226.
[37] B. Kramer, M. Rarey, T. Lengauer, Evaluation of the FlexX incremental con-
struction algorithm for protein-ligand docking, Proteins 37 (1999) 228e241.
[38] LeadIT 2.1.0, BioSolveIT, GmbH, St. Augustin, Germany, 2012.
[39] G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, The protein
data bank, Nucleic Acids Res. 28 (2000) 235e242.
[21] C. Adamo, D. Jacquemin, The calculations of excited-state properties with
time-dependent density functional theory, Chem. Soc. Rev. 42 (2013)
845e856.
[22] Y. Chen, J. Zhao, H. Guo, L. Xie, Geometry relaxation-induced large stokes shift
in red-emitting borondipyrro-methenes (BODIPY) and applications in fluo-
rescent thiol probes, J. Org. Chem. 77 (2012) 2192e2206.
[40] B.L. Staker, M.D. Feese, M. Cushman, Y. Pommier, D. Zembower, L. Stewart,
A.B. Burgin, Structures of three classes of anticancer agents bound to the
human topoisomerase I-DNA covalent complex, J. Med. Chem. 48 (2005)
2336e2345.
[41] C.C. Wu, T.K. Li, L. Farh, L.Y. Lin, T.S. Lin, Y.J. Yu, T.J. Yen, C.W. Chiang, N.L. Chan,
Structural basis of type II topoisomerase inhibition by the anticancer drug
etoposide, Science 333 (2011) 459e462.
[23] B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley-VCH