Search for human DNA topoisomerase II poisons in the group of 2,5-disubstituted-1,3,4-thiadiazoles

Article abstract of DOI:10.3109/14756366.2014.995179

A series of six 2,5-disubstituted 1,3,4-thiadiazole derivatives was synthesized and examined for cytotoxic activity in MCF-7 and MDA-MB-231 breast cancer cells. MTT assay confirmed that 2-(3-fluorophenylamino)-5-(3-hydroxyphenyl)-1,3,4-thiadiazole (2), 2-

Full text of DOI:10.3109/14756366.2014.995179

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J Enzyme Inhib Med Chem, Early Online: 1–6
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SHORT COMMUNICATION
Search for human DNA topoisomerase II poisons in the group of
,5-disubstituted-1,3,4-thiadiazoles
2
1
1
1
1
2
3
Tomasz Plech , Barbara Kapro n´ , Agata Paneth , Monika Wujec , Robert Czarnomysy , Anna Bielawska ,
2
1
1
4
Krzysztof Bielawski , Nazar Trotsko , Edyta Ku s´ mierz , and Piotr Paneth
1
2
Department of Organic Chemistry, Medical University of Lublin, Chodzki, Lublin, Poland, Department of Synthesis and Technology of Drugs,
3
Medical University of Białystok, Kili n´ skiego, Białystok, Poland, Department of Biotechnology, Medical University of Białystok, Kili n´ skiego,
Białystok, Poland, and Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego, Lodz, Poland
4
_
Abstract
Keywords
A series of six 2,5-disubstituted 1,3,4-thiadiazole derivatives was synthesized and examined
for cytotoxic activity in MCF-7 and MDA-MB-231 breast cancer cells. MTT assay confirmed that
1
,3,4-Thiadiazoles, breast cancer, cytotoxicity,
MTT assay, topoisomerase inhibitors
2
(
-(3-fluorophenylamino)-5-(3-hydroxyphenyl)-1,3,4-thiadiazole (2), 2-(4-bromophenylamino)-5-
2,4-dichlorophenyl)-1,3,4-thiadiazole (3), 2-(4-fluorophenylamino)-5-(2,4-dichlorophenyl)-1,3,4-
History
thiadiazole (4), had ability to inhibit MCF-7 and MDA-MB-231 cells proliferation. The IC50 values
for the mentioned compounds ranged between 120 and 160 mM (with respect to MCF-7 cells)
and from 70 to 170 mM (with respect to MDA-MB-231 cells). It turned out, moreover, that
compound 2 is a human topoisomerase II (topoII) catalytic inhibitor whereas the two other
compounds (i.e. 3 and 4) are capable of stabilizing DNA-topoII cleavage complex and thus are
topoII poisons.
Received 17 September 2014
Revised 7 November 2014
Accepted 27 November 2014
Published online 20 March 2015
Introduction
However, it was demonstrated that certain 1,3,4-thiadiazoles
0
1
inhibit DNA biosynthesis , which, in turn, may be associated
with their potential anti-topoisomerase activity.
Cancer is one of the greatest challenges of contemporary
medicine. It affects people living in all regions of the world and
belonging to all social groups. Currently, the mortality resulting
from cancer exceeds the number of deaths caused by HIV/AIDS,
tuberculosis and malaria altogether. It is estimated that in 2012,
approximately 14 million new cases of cancer were diagnosed and
approximately 8 million deaths resulting from this disease were
recorded worldwide. Due to increasing Earth’s population and a
longer life expectancy, it is predicted that as soon as in 2030 there
will be 22 million new cases of cancer and 13 million deaths .
Attempts to counteract this alarming tendency involve, among
other things, designing new methods that enable early diagnosis
2
and introducing new anticancer drugs to clinical practice .
Topoisomerases belong to the family of enzymes that catalyze
changes in the spatial structure of DNA. They enable conform-
ational changes in DNA topology necessary for transcription,
11
replication and recombination of genetic material . Thanks to
them, amplification of genetic material is possible, which is
subsequently delivered to daughter cells formed by cell division.
Due to inhibiting topoisomerases activity, DNA synthesis in cells
is limited and consequently cell division is reduced. Substances
targeting topoisomerases fall into two categories – topoisomerase
1
12
poisons and topoisomerase catalytic inhibitors . The term
‘‘topoisomerase poisons’’ reflects the fact that these compounds
convert this enzyme into a cell poison, which causes a range of
1
,3,4-Thiadiazoles are a group of compounds known to have
,4
13
irreversible damage to genetic material . The effect of topo-
3
potential effect on cancer cells . Their anticancer activity may
be achieved via different molecular mechanisms, including
isomerase poisons depends on the concentration of this enzyme in
cells. The level of topoisomerases is the highest in rapidly
14
proliferating cells, which include cancerous cells . Therefore,
5
cyclin-dependent kinase (CDK) inhibition , Abl tyrosine-kinase
6
inactivation , down-regulation of Bcl-X expression and inhib-
L
7
ition of Akt/PKB , inhibition of the so-called tumor-associated
topoisomerase poisons, and especially topoisomerase II (topoII)
poisons are known from their anticancer properties. It is also
known that topoisomerase poisons exhibit considerably higher
8
carbonic anhydrase isozymes I, II, IX . To date, a little is known
about the ability of 1,3,4-thiadiazole derivatives to act against
13
9
cancer cells through the topoisomerase-associated mechanism .
cytotoxicity than topoisomerase catalytic inhibitors . This is why
our current research focuses on searching for compounds
that inhibit human DNA topoII activity by stabilizing the
cleavable complex. We assumed that the probability of finding
compounds with desirable anticancer properties is high in the
group of 1,3,4-thiadiazole derivatives. Particularly 2,5-disubsti-
tuted 1,3,4-thiadiazoles demonstrated the ability to inhibit cancer
Address for correspondence: Tomasz Plech, Department of Organic
Chemistry, Faculty of Pharmacy, Medical University, Chodzki 4A, 20-
093 Lublin, Poland. Tel: +48 081 532 05 19. Fax: +48 081 532 45 46.
E-mail: tomasz.plech@umlub.pl

2
T. Plech et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
cell proliferation and exhibited weaker influence on the viability 118.08, 118.41, 128.66, 130.95, 131.70, 132.81, 142.56, 154.26,
1
5–17
of normal cells
and synthesized a group of 1,3,4-thiadiazole derivatives differing C H FN OS (287.31): C 58.53, H 3.51, N 14.63. Found: C
. Inspired by these observations, we designed 158.36, 158.83, 161.49, 164.00, 164.61. Anal. Calc. for
1
4
10
3
in the structure of the substituents at C2 and C5 position. Mono- 58.40, H 3.69, N 14.45.
and disubstituted aryl rings, containing electron-donating and
electron-withdrawing groups, were chosen to test the influence of 2-(4-Bromophenylamino)-5-(2,4-dichlorophenyl)-1,3,4-thiadiazole
ꢀ
1
the substitution pattern and electronic properties of the substitu- (3). Yield 80ꢀ, m.p. 250–252 C. H-NMR (300 MHz)
ents on anticancer activity of 1,3,4-thiadiazole derivatives. The (DMSO-d ) ꢀ (ppm): 7.53 (d, 2H, ArH, J ¼ 8.8 Hz), 7.60 (dd,
6
obtained compounds subsequently underwent biological tests with 1H, ArH, J ¼ 2.1 Hz, 8.6 Hz), 7.65 (d, 2H, ArH, J ¼ 8.8 Hz), 7.84
the use of MCF-7, MDA-MB-231 breast cancer cells and human (d, 1H, ArH, J ¼ 2.0 Hz), 8.10 (d, 1H, ArH, J ¼ 8.3 Hz), 10.74
1
3
normal skin fibroblasts. The mentioned cancer cell lines were (s, 1H, NH, exchangeable with D O). C-NMR (75 MHz)
2
chosen for our studies as it had been demonstrated that (DMSO-d ) ꢀ (ppm): 114.00, 120.04, 128.41, 128.61, 130.50,
6
compounds inhibiting topoII were frequently effective against 132.07, 132.15, 132.35, 135.82, 140.09, 152.90, 165.92. Anal.
1
8,19
these breast cancer cells
. Finally, the most potent compounds Calc. for C H BrCl N S (401.11): C 41.92, H 2.01, N 10.48.
14 8 2 3
were also investigated for their ability to act as poisons or Found: C 41.76, H 1.90, N 10.55.
catalytic inhibitors of human DNA topoII.
2
-(4-Fluorophenylamino)-5-(2,4-dichlorophenyl)-1,3,4-thiadiazole
1
ꢀ
Materials and methods
(4). Yield 76ꢀ, m.p. 228–230 C. H-NMR (300 MHz)
DMSO-d ) ꢀ (ppm): 7.10–7.26 (m, 2H, ArH), 7.60 (dd, 1H,
(
6
Chemistry
ArH, J ¼ 2.2 Hz, 8.6 Hz), 7.65–7.73 (m, 2H, ArH), 7.84
All reagents were purchased from Alfa-Aesar and Sigma-Aldrich, (d, 1H, ArH, J ¼ 2.1 Hz), 8.11 (d, 1H, ArH, J ¼ 8.5 Hz), 10.63
1
3
and used without further purification. Melting points were (s, 1H, NH, exchangeable with D
determined by using Fischer-Johns apparatus (Sanyo, Osaka, (DMSO-d ) ꢀ (ppm): 116.04, 116.34, 119.82, 119.94, 128.49,
Japan) and are uncorrected. The H- and C-NMR spectra were 128.59, 130.46, 132.03, 132.08, 135.72, 137.30, 152.41, 156.33,
recorded on a Bruker Avance instrument using DMSO-d as a 159.50, 166.43. Anal. Calc. for C14 Cl FN S (340.20): C 49.43,
O). C-NMR (75 MHz)
2
6
1
13
H
6
8
2
3
solvent and TMS as an internal standard. Chemical shifts are H 2.37, N 12.35. Found: C 49.65, H 2.23, N 12.38.
expressed as ꢀ (ppm). The purity of the compounds was checked
by TLC on plates precoated with silica gel Si 60 F254, produced 2-(4-Bromophenylamino)-5-[(2,4-dichlorophenoxy)methyl]-1,3,4-
ꢀ
1
by Merck Co. (Darmstadt, Germany). The spots were detected by thiadiazole (5). Yield 84ꢀ, m.p. 170–172 C. H-NMR
exposure to UV-lamp at ꢁ ¼ 254 nm. Elemental analyses were (300 MHz) (DMSO-d ) ꢀ (ppm): 5.53 (s, 2H, CH ), 7.36 (d, 1H,
6
2
performed on AMZ 851 CHX analyzer and the results were within ArH, J ¼ 9.0 Hz), 7.42 (dd, 1H, ArH, J ¼ 2.4 Hz, 8.9 Hz), 7.51
±
0.4ꢀ of the theoretical value.
(d, 2H, ArH, J ¼ 8.9 Hz), 7.57–7.64 (m, 3H, ArH), 10.52 (s, 1H,
2 6
1
NH, exchangeable with D O). C-NMR (75 MHz) (DMSO-d ) ꢀ
3
Synthesis of 1,3,4-thiadiazole derivatives (1–6)
(ppm): 65.90, 113.82, 116.58, 119.90, 123.23, 126.11, 128.67,
29.95, 132.27, 140.15, 152.40, 155.88, 165.69. Anal. Calc. for
C H BrCl N OS (431.13): C 41.79, H 2.34, N 9.75. Found:
1
A solution of 0.01 mol of respective aryl hydrazide (3-chloro-
benzhydrazide for 1, 3-hydroxybenzhydrazide for 2, 2,4-dichlor-
obenzhydrazide for 3 and 4, 2,4-dichlorophenoxyacetic acid
hydrazide for 5 and 6) and equimolar amounts of appropriate
aryl isothiocyanate (3-fluorophenyl isothiocyanate for 1 and 2,
1
5
10
2 3
C 41.67, H 2.18, N 9.85.
2
thiadiazole (6). Yield 80ꢀ, m.p. 184-185 C. H-NMR
-(4-Fluorophenylamino)-5-[(2,4-dichlorophenoxy)methyl]-1,3,4-
1
ꢀ
4
-bromophenyl isothiocyanate for 3 and 5, 4-fluorophenyl isothio-
(300 MHz) (DMSO-d ) ꢀ (ppm):5.52 (s, 2H, CH ), 7.15–7.23
6 2
cyanate for 4 and 6) in 25 ml of anhydrous EtOH was heated under
reflux for 5 min. Next, the solution was cooled and the solid formed
was filtered off, washed with diethyl ether and crystallized from
EtOH. Subsequently, thus obtained thiosemicarbazide derivatives
were dissolved in concentrated sulfuric acid (15 ml) and stirred at
room temperature for 2 h. Then, the reaction mixture was poured
onto crushed ice and the solid precipitated was filtered off, washed
several times with water, dried and crystallized from EtOH to give
products 1–6.
(
1
m, 2H, ArH), 7.34–7.44 (m, 2H, ArH), 7.60–7.68 (m, 3H, ArH),
1
3
0.49 (s, 1H, NH, exchangeable with D O). C-NMR (75 MHz)
2
(DMSO-d ) ꢀ (ppm): 65.84, 115.97, 116.27, 116.59, 119.66,
6
1
1
19.77, 123.27, 126.10, 128.64, 129.94, 137.38, 152.42, 155.39,
66.15. Anal. Calc. for C H Cl FN OS (370.23): C 48.66,
1
5
10
2
3
H 2.72, N 11.35. Found: C 48.68, H 2.60, N 11.23.
Computational studies
Automated docking setup
2-(3-Fluorophenylamino)-5-(3-chlorophenyl)-1,3,4-thiadiazole (1).
Yield 75ꢀ, m.p. 210–212 C. H-NMR (300 MHz) (DMSO-d ) ꢀ
(
ꢀ
1
6
Docking studies were conducted by means of the FlexX
0
2
ppm): 6.88 (td, 1H, ArH, J ¼ 2.5 Hz, 8.5 Hz), 7.25 (dd, 2H, ArH, program as implemented in LeadIT package (BioSolveIT,
21
J ¼ 2.0 Hz, 8.5 Hz), 7.51–7.95 (m, 4H, ArH), 8.11 (dd, 1H, ArH, Sankt Augustin, Germany) using a model of human DNA
22
J ¼ 2.5 Hz, 11.8 Hz), 10.87 (s, 1H, NH, exchangeable with D O).
2
topoII binding site complexed with etoposide (PDB id: 3QX3 )
13
C-NMR (75 MHz) (DMSO-d ) ꢀ (ppm): 112.20, 126.15, 126.54, as a native ligand. The ligand within the active site and all water
6
1
1
5
29.30, 130.05, 130.64, 131.13, 131.73, 132.30, 132.51, 134.43, molecules were removed, while magnesium ion was allowed to
42.66, 157.32, 164.63. Anal. Calc. for C H ClFN S (305.76): C
4.99, H 2.97, N 13.74. Found: C 55.08, H 3.10, N 13.70.
1
4
9
3
remain with the charge of +2. The active site was defined to
include all atoms within 6.5 A˚ radius of the native ligand. The first
1
-(3-Fluorophenylamino)-5-(3-hydroxyphenyl)-1,3,4-thiadiazole (2). validate the molecular docking protocol, etoposide was initially
00 top ranked docking poses were saved for each docking run. To
2
Yield 69ꢀ, m.p. 136–138 C. H-NMR (300 MHz) (DMSO-d ) ꢀ docked into the crystal structure of the enzyme. The docked
ꢀ
1
6
(
ppm): 6.75–8.18 (m, 8H, ArH), 10.77 (s, 1H, NH, exchangeable ligand was found to have similar binding pose to the co-
1
3
with D O), 11.25 (s, 1H, OH, exchangeable with D O). C-NMR crystallized molecule. Subsequently, compounds 1–6 were docked
2
2
(75 MHz) (DMSO-d ) ꢀ (ppm): 113.47, 113.89, 114.82, 117.80, using same docking parameters.
6

DOI: 10.3109/14756366.2014.995179
Search for human DNA topoisomerase II poisons
3
Semiempirical and DFT calculations
presence of compounds 1–6 was calculated as percentage of
control cells.
Conformational search was calculated using HyperChem 8.0.1
2
3
(
Hypercube, Gainesville, FL) . Structures were optimized to the
closest local minimum at the semiempirical level using RM1
parametrization. Convergence criterion was set to To examine the effect of the studied compounds on cells
. Electrostatic potentials were calculated proliferation, MCF-7 and MDA-MB-231 cells, as well as
3
[
H]thymidine incorporation assay
ꢁ
1
ꢁ1
˚
0
.01 kcal mol
A
2
4,25
using the B3LYP functional
6–28
expressed in the basis set of 6– normal human skin fibroblasts were seeded in six-well tissue
5
2
3
1 G(d)
as implemented in Gaussian 09 (Gaussian Inc., culture dishes at 1 ꢂ 10 cells/well with 1 ml of growth medium.
2
9
5
Wallingford, CT) . Natural population analysis (NPA) phase of After 48 h (1.8 ± 0.1 ꢂ 10 cells/well), plates were incubated with
3
0
NBO was used . The highest occupied (HOMO) and lowest varying concentrations of compounds 2–4, etoposide and 0.5 mCi
unoccupied (LUMO) molecular orbitals, as well as electrostatic of [ H]thymidine for 24 h at 37 C. Cells were rinsed three times
3
ꢀ
potentials maps were illustrated using the GaussView 5.0 program with PBS, solubilized with 1 ml of 0.1 M sodium hydroxide
(
3
Gaussian Inc., Wallingford, CT) .
1
containing 1ꢀ SDS, scintillation fluid (9 ml) was added and
radioactivity was determined by liquid scintillation counting.
3
[ H]thymidine uptake was expressed as dpm/well.
Biological assays
Materials
Relaxation assay of topoII
Stock cultures of human MCF-7 and MDA-MB-231 breast cancer
cells as well as normal human skin fibroblasts were purchased
from the American Type Culture Collection (Manassas, VA).
Dulbecco’s minimal essential medium (DMEM) and fetal bovine
serum (FBS) used in a cell culture were products of Gibco
Supercoiled pRYG DNA (0.5 mg) was incubated with 4U of
human topoII in the cleavage buffer [30 mM Tris–HCl (pH 7.8),
5
nol], in the presence of varying concentrations of the studied
0 mM KCl, 10 mM MgCl , 3 mM ATP, 15 mM mercaptoetha-
2
ꢀ
compounds. Reactions were carried out at 37 C for 1 h and then
(Waltham, MA). Glutamine, penicillin and streptomycin were
obtained from Quality Biologicals, Inc. (Gaithersburg, MD).
terminated by the addition of 2 ml, 10ꢀ SDS and 2 ml, 50 mg/ml
proteinase K. The reaction mixture was subjected to electrophor-
esis through a 0.8ꢀ agarose gel containing 0.5 mg/ml ethidium
bromide in TBE buffer (90 mM Tris–borate and 2 mM EDTA).
The gels were stained with ethidium bromide and photographed
under UV light.
3
[
H]thymidine (6.7 Ci/mmol) was purchased from NEN (Boston,
MA) and Scintillation Cocktail ‘‘Ultima Gold XR’’ from Packard
Downers Grove, IL). Sodium dodecyl sulfate (SDS) was received
(
from Bio-Rad Laboratories (Hercules, CA). Etoposide and topoII
drug screening kit were purchased from TopoGEN (Columbus,
OH). MTT and serum replacement I (CPSR1) were purchased
from Sigma Chemical Co. (St. Louis, MO).
Results
In the process of designing potential DNA topoII poisons, a
molecular docking method was used to explore interactions
between 1,3,4-thiadiazole derivatives and DNA-binding pocket of
human DNA topoII. Since the ability of simultaneous interaction
with a DNA strand and enzyme is an inherent feature of
compounds stabilizing the topoII-DNA cleavable complex, the
action of topoII poisons must occur within the DNA-dependent
subunit of topoII. The model of this subunit, obtained from
Protein Data Bank (id: 3QX3), was used in docking simulations. It
was found that compounds 1–6 show a strong affinity to DNA-
dependent subunit of topoII and interact both with the enzyme and
nucleobases of the DNA (Figure S1).
Cell culture
Human MDA-MB-231 and MCF-7 breast cancer cells maintained
in DMEM supplemented with 10ꢀ FBS, 50 U/ml penicillin,
50 mg/ml streptomycin at 37 C. Cells were cultured in Costar
flasks and subconfluent cells were detached with 0.05ꢀ trypsin
ꢀ
and 0.02ꢀ EDTA in calcium-free phosphate buffered saline
5
(
PBS), counted in hemocytometers and plated at 5 ꢂ 10 cells/well
of six-well plates (Nunc) in 2 ml of growth medium (DMEM
without phenol red with 10ꢀ CPSR1). Cells reached about 80ꢀ of
confluency at day 3 and in most cases such cells were used for the
assays.
In the majority of compounds, the docking scores were
comparable or even superior to etoposide – an anticancer drug
which is a well-known topoII poison (Table 1). Therefore, the
Cell viability assay
The assay was performed according to the method followed by designed 2,5-disubstituted 1,3,4-thiadiazole derivatives were
3
2
Carmichael et al. , using MTT. Confluent cells, cultured for 24 h synthesized in a two-stage reaction (Scheme 1), and subsequently
with various concentrations of the studied compounds in six-well subjected for biological testing on normal and cancer cell lines.
plates were washed three times with PBS and then incubated for As had been demonstrated before, compounds inhibiting topoII
ꢀ
18,19
4
h in 1 ml of MTT solution (0.5 mg/ml of PBS) at 37 C in 5ꢀ were frequently effective against breast cancer cells
CO2 in an incubator. The medium was removed and 1 ml of this is mind, the effect of compounds 1–6 on the viability of
.1 mol/l HCl in absolute isopropanol was added to the cells estrogen receptor-positive (MCF-7) and estrogen receptor-nega-
. Bearing
0
attached. Absorbance of converted dye in living cells was tive (MDA-MB-231) breast cancer cells was evaluated. Moreover,
measured at a wavelength of 570 nm. Cell viability of breast etoposide was used as a reference drug (IC50 ¼ 125 ± 2mM in
cancer cells and normal human skin fibroblasts cultured in the MCF-7 and 98 ± 2mM in MDA-MB-231 cells). Viability of breast
Table 1. Docking scores of compounds 1–6 and etoposide obtained for DNA-dependent subunit of human topoII.
Compounds
1
2
3
4
5
6
Etoposide
DNA-binding site (PDB: 3QX3)
Energy units are in kcal/mol.
ꢁ20.59
ꢁ23.69
ꢁ19.42
ꢁ20.74
ꢁ15.40
ꢁ20.35
ꢁ20.61

4
T. Plech et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
O
N
N
NCS
R1
O
R1
NH
NH NH
S
H SO (conc.)
S
2
4
EtOH/reflux
+
R1
NH
NH NH2
R2
(
1 - 6)
R2
R2
O
1
. R1
=
=
R2 = 3-F
3. R1
=
R2 = 4-Br
5.
R
=
R2 = 4-Br
1
Cl
Cl
Cl
Cl
Cl
O
4. R1
=
R2 = 4-F
6.
R
1
=
R2 = 4-F
R2 = 3-F
2
. R1
Cl
Cl
Cl
Cl
OH
Scheme 1. Synthetic route to compounds 1–6.
Table 2. IC50 values (mM) of compounds 1–6 and etoposide in breast cancer cells (MCF-7 and MDA-MB-231) and normal human
skin fibroblasts using MTT assay.
Compounds
Cells
1
2
3
4
5
6
Etoposide
MCF-7
MDA-MB-231
Fibroblasts
4200
4200
4200
140
170
4200
120
70
4200
160
130
170
4200
4200
200
4200
4200
4200
125
98
55
The error for compounds 1–6 and etoposide is ±2 mM. Mean values ± SD from three independent experiments done in duplicate
are presented.
3
2
cancer cells was measured by the method of Carmichael et al.
using tetrazolium salt (MTT).
Table 3. Cytotoxic effects of compounds 2–4 and etoposide on the
cultured breast cancer cells (MCF-7 and MDA-MB-231) and normal
3
human skin fibroblasts as measured by inhibition of [ H]thymidine
incorporation into DNA.
The MTT assay showed that compounds 2, 3 and 4 were
endowed with an ability to inhibit MCF-7 and MDA-MB-231
cells proliferation (Table 2). The IC50 values for compounds 2–4
ranged between 120 and 160 mM (with respect to MCF-7 cells)
Compounds
and from 70 to 170 mM (with respect to MDA-MB-231 cells). Cells
Among the active derivatives, 2-(4-bromophenylamino)-5-(2,4-
2
3
4
Etoposide
MCF-7
MDA-MB-231
Fibroblasts
80
170
4200
95
90
4200
135
140
160
75
72
95
dichlorophenyl)-1,3,4-thiadiazole (3) proved to be more potent
than etoposide with respect to both types of cancer cells (with
IC50 values in MCF-7 of 120 ± 2 mM, and in MDA-MB-231 of
The error for compounds 2–4 and etoposide is ±2 mM. Mean values ± SD
from three independent experiments done in duplicate are presented.
7
0 ± 2 mM). The assessment of viability of human normal skin
fibroblasts exposed to the active compounds (i.e. derivatives 2–4)
demonstrated that they are characterized with a considerably
lower toxicity than etoposide (IC 4200 mM for 2 and 3 versus
5
0
5
5 ± 2 mM for etoposide) and exhibit greater specificity to breast was concerned (e.g. IC50 for 2 was 80 ± 2 mM and4200 mM, and
cancer cells (MCF-7 and MDA-MB-231). In order to check for etoposide 75 ± 2 mM and 95 ± 2 mM towards MCF-7 and
whether the loss of viability of breast cancer cells (MCF-7 and fibroblasts, respectively). One of the causes of inhibiting DNA
MDA-MB-231) results from reduced proliferation, we analyzed biosynthesis by compounds 2–4 may be their influence on
the influence of compounds 2–4 on the process of DNA enzymes that take part in the process of genetic material
biosynthesis (Table 3).
replication. One of the most essential enzymes required for such
All the three 1,3,4-thiadiazole derivatives inhibited intracellu- a process are topoisomerases. The effect of the active compounds
lar DNA synthesis, but in MCF-7 cells compound 2 turned out to (2–4) on the activity of human DNA topoII was tested with the
be the most potent (IC50 ¼ 80 ± 2 mM), and in MDA-MB-231 use of relaxation assay kit for topoII. This type of kit, i.e. plasmid-
cells – compound 3 (IC50 ¼ 90 ± 2 mM). The derivatives of 1,3,4- based screening kit, enable not only identification of compounds
thiadiazole, in contrast to etoposide, demonstrated higher select- inhibiting topoII, but also distinguishes topoII catalytic inhibitors
ivity as far as DNA biosynthesis in cancer cells and fibroblasts from topoII poisons. Intensity of the electrophoretic band

DOI: 10.3109/14756366.2014.995179
Search for human DNA topoisomerase II poisons
5
corresponding to linear DNA (pHOT1) is increased when the
In order to give further insight into the correlations between
tested compound acts as a topoII poison. On the other hand, in anticancer activity of the studied compounds and their electronic
case of topoII catalytic inhibitors there is a clearly visible loss of properties, the frontier orbitals’ distribution and electrostatic
3
decatenated kDNA products .
3
potential maps were calculated using DFT level of theory with
The previously conducted studies showed that the concentra- B3LYP functional expressed on the basis set of 6-31G(d). The
tion of etoposide required for 50ꢀ reduction in topoII activity was highest occupied (HOMO) and lowest unoccupied (LUMO)
1
9
found to be 100 mM . The current tests reveal that a total molecular orbitals, as well as the electrostatic potential maps
inhibition of topoII enzymatic activity occurs at a concentration were illustrated using the GaussView 5.0 program (Gaussian Inc.,
of 200 mM. Worth mentioning is the fact that 1,3,4-thiadiazole Wallingford, CT) (Figure S2). Contrary to what we expected, the
derivatives 2–4 completely inhibited the activity of this enzyme at replacement of chlorine atom in 1 with a hydroxyl group in
a concentration lower by a half (i.e. at 100 mM). Furthermore, it compound 2 did not cause any significant changes in the
turned out that compound 2 is a topoII catalytic inhibitor whereas electrostatic potential of the molecule. Similarly, the HOMO and
the two other compounds (i.e. 3 and 4) are capable of stabilizing LUMO distributions along the molecules were similar in the cases
DNA–topoII cleavage complex and thus are topoII poisons.
of compounds 1 and 2. Thus, one can conclude that it is not
electrostatics of the molecules but the particular properties of the
hydroxyl group (e.g. the potential of forming hydrogen bonds),
which is the source of such large differences in anticancer activity
Discussion
Of the 1,3,4-thiadiazoles tested, two compounds (i.e. 3 and 4) were of the above-mentioned pair of compounds. In turn, a total lack of
found to be able to stabilize the DNA-topoII cleavable complex, i.e. anticancer activity of compounds 5 and, 6, containing –O–CH2–
to act as topoII poisons. However, the third of the active linker, can be associated with a completely different geometry of
compounds (2) owes its anticancer effects to catalytic inhibition the molecules and differences in HOMO/LUMO distributions. In
of topoII. From the pharmacological point of view, 2-(4- the cases of the inactive derivatives (5, 6) the HOMO orbitals are
bromophenylamino)-5-(2,4-dichlorophenyl)-1,3,4-thiadiazole (3) located at the 1,3,4-thiadiazole ring and the aromatic substituent in
seems to be the most promising. Not only did it influence the position 2 of this heterocyclic system, while for all active
viability of both types of cancer cells in a more potent way than compounds the electron density of HOMO is virtually uniform
etoposide, but also had no toxic effects on normal cells (fibroblasts) around whole molecule. Again, the analysis of molecular orbitals
within concentration ranges tested. The second topoII poison (4) distribution revealed that all active derivatives differ from the
recognized in the group of 1,3,4-thiadiazoles had a less selective inactive ones (5, 6) in low distribution of LUMO around the
effect on cancer cells. Compound 4 decreased the cells’ viability aromatic group in position 2 of the 1,3,4-thiadiazole ring. These
and DNA synthesis in both cancer and normal cells. Having observations clearly show that spatial distribution of the frontier
analyzed the results obtained, we managed to formulate some molecular orbitals as well as geometry of the molecules are of
structure-activity relationships that might be helpful in designing greater importance for this class of compounds to exhibit
novel topoII inhibitors/poisons based on 1,3,4-thiadiazole core. It anticancer activity.
is clear that the direct connection of the phenyl ring with 1,3,4-
Finally, there is one more fact that should be noted in relation
thiadiazole core at position 5 is essential for anticancer activity. to the anticancer activity of the studied compounds. Interestingly,
The derivatives in which both aromatic systems were separated the effect of compounds 3 and 4 on the viability of MCF-7 and
with –O–CH – linker (i.e. compounds 5 and 6) were devoid of any MDA-MB-231 cells is different despite the fact that they cause
2
activity and additionally inhibited DNA synthesis in normal cells. nearly equivalent inhibition of DNA biosynthesis in both types of
Nevertheless, the sole connection of both aforementioned aromatic cancer cells. This might suggest that a total anticancer effect is
rings (i.e. phenyl and 1,3,4-thiadiazole) is not a sufficient condition also determined by other molecular mechanisms, apart from
allowing anticancer effects to be evoked. The substitution pattern inhibition of intracellular DNA biosynthesis. This might also
of a phenyl ring and the electronic character of substituents are also explain the absence of a linear relationship between the anticancer
important. The replacement of chlorine atom (compound 1) with a activity of compounds 1–6 and their docking scores obtained from
hydroxyl group (compound 2) resulted in obtaining biologically preliminary docking studies.
active derivative. In the aforementioned case, it was evident that
the effect of compound 2 on the viability of cancer cells resulted
Conclusions
directly from the character of the hydroxyl group. This substituent
is not only capable of forming strong hydrogen bonds with the To sum up, we have synthesized a series of 2,5-disubstituted
active site of the enzyme, but also, as a potent electron-donating 1,3,4-thiadiazole derivatives and evaluated them for anticancer
group, it should significantly change the electrostatic potential of a and anti-topoisomerase activities. Interestingly, the active deriva-
molecule. In spite of a low number of derivatives tested, it was tives (2–4) present a better pharmacological profile than reference
possible to put forward a hypothesis that electrostatic potential of drug – etoposide. The latter inhibits the viability of fibroblasts by
this fragment of the molecule affected the character of its 50ꢀ at a concentration of 55 ± 2 mM whereas an analogous loss of
interaction with human DNA topoII. Derivatives 3 and 4, MCF-7 and MDA-MB-231 cells viability was observed at the
containing two electron-withdrawing substituents connected to concentration 125 ± 2 mM and 98 ± 2 mM, respectively. On the
the phenyl ring at position 5 of the 1,3,4-thiadiazole core, acted as other hand, we have observed no influence of compounds 2 and 3
topoII poisons whereas the presence of a potent electron-donating on the viability of human normal skin fibroblasts in the
group (i.e. –OH) favored catalytic inhibition of topoII. concentration range tested with simultaneous reduction of breast
Confirmation or refutation of this hypothesis requires further cancer cells growth. Moreover, compounds 2–4 completely
research involving a greater number of derivatives, which is inhibited the activity of human DNA topoII in the concentrations
currently being conducted. On the other hand, when considering twice as low as in the case of etoposide. Of the three active
the influence of a phenylamine group at position 2 of the 1,3,4- derivatives (2–4), 2-(4-bromophenylamino)-5-(2,4-dichlorophe-
thiadiazole skeleton, it must be remembered that replacement of nyl)-1,3,4-thiadiazole (3) was found to exhibit the most promising
fluorine atom by bromine substituent improves anticancer activity. biological effect. Its activity was even higher than that of the
Thus, it might prove that substituents with higher volume and/or reference drug. It is worth pointing out that despite structural
lower electronegativity are better tolerated in para position.
similarity of all three active 1,3,4-thiadiazole derivatives, the

6
T. Plech et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
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derivatives acted as topoII poisons.
1
1
1
Declaration of interest
The authors declare no conflict of interest.
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Supplementary material available online
Supplementary Figures S1 and S2.