Cytotoxic effects, microbiological analysis and inhibitory properties on carbonic anhydrase isozyme activities of 2-hydroxy-5-methoxyacetophenone thiosemicarbazone and its Cu(II), Co(II), Zn(II) and Mn(II) complexes

Article abstract of DOI:10.1007/s11164-020-04284-8

Metal complexes of thiosemicarbazones have been receiving considerable attention in biological applications such as antimicrobial and anticancer therapies. In this work, Co(II), Zn(II) and Mn(II) complexes of 2-hydroxy-5-methoxyacetophenone thiosemicarbazone (HMAT) were synthesized for the first time and characterized by EPR, FT-IR, NMR, UV–Vis spectroscopies, TG/DSC and elemental analysis. X-ray powder diffraction analysis was carried out for Zn(II) complex. HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes were tested as enzyme inhibitory agents. All compounds are effective inhibitor of cytosolic carbonic anhydrase I and II isoforms (hCA I and II) enzymes. IC₅₀ values of HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes were determined as 93.35, 324.46, 25.67, 1.06 and 22.36?μM for CA I isozyme and 99.02, 86.64, 57.76, 10.34 and 36.48?μM for CA II isozyme, respectively. The evaluation of potential cytotoxic effects of the compounds was performed against normal epithelial breast mammary gland CRL-4010, estrogen-positive low metastatic MCF-7 and triple negative highly metastatic MDA-MB-231 breast adenocarcinoma cell lines by MTT assay. The results showed that the tested metal complexes have high cytotoxic effects than their ligand molecule. In particular, the Cu(II) complex displayed preciously high cytotoxic properties different from the others. Given these facts, the Cu(II) complex could be debated as potential chemotherapeutic molecule against drug-resistant breast cancer cells. Minimum inhibitory concentrations of the compounds against the test organisms were also detected for the microbiological analysis.

Full text of DOI:10.1007/s11164-020-04284-8

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04284-8
Cytotoxic efects, microbiological analysis and inhibitory
properties on carbonic anhydrase isozyme activities
of 2‑hydroxy‑5‑methoxyacetophenone thiosemicarbazone
and its Cu(II), Co(II), Zn(II) and Mn(II) complexes
Asuman Ucar, et al. [full author details at the end of the article]
Received: 3 July 2020 / Accepted: 26 September 2020
© Springer Nature B.V. 2020
Abstract
Metal complexes of thiosemicarbazones have been receiving considerable atten-
tion in biological applications such as antimicrobial and anticancer therapies. In this
work, Co(II), Zn(II) and Mn(II) complexes of 2-hydroxy-5-methoxyacetophenone
thiosemicarbazone (HMAT) were synthesized for the frst time and characterized
by EPR, FT-IR, NMR, UV–Vis spectroscopies, TG/DSC and elemental analysis.
X-ray powder difraction analysis was carried out for Zn(II) complex. HMAT and
its Cu(II), Co(II), Zn(II) and Mn(II) complexes were tested as enzyme inhibitory
agents. All compounds are efective inhibitor of cytosolic carbonic anhydrase I and
II isoforms (hCA I and II) enzymes. IC50 values of HMAT and its Cu(II), Co(II),
Zn(II) and Mn(II) complexes were determined as 93.35, 324.46, 25.67, 1.06 and
22.36 μM for CA I isozyme and 99.02, 86.64, 57.76, 10.34 and 36.48 μM for CA
II isozyme, respectively. The evaluation of potential cytotoxic efects of the com-
pounds was performed against normal epithelial breast mammary gland CRL-4010,
estrogen-positive low metastatic MCF-7 and triple negative highly metastatic MDA-
MB-231 breast adenocarcinoma cell lines by MTT assay. The results showed that
the tested metal complexes have high cytotoxic efects than their ligand molecule.
In particular, the Cu(II) complex displayed preciously high cytotoxic properties dif-
ferent from the others. Given these facts, the Cu(II) complex could be debated as
potential chemotherapeutic molecule against drug-resistant breast cancer cells. Min-
imum inhibitory concentrations of the compounds against the test organisms were
also detected for the microbiological analysis.
Keywords Thiosemicarbazone · Complexes · Carbonic anhydrase · Antimicrobial
and anticancer activity · Spectroscopic and thermal analysis
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-020-04284-8) contains supplementary material, which is available to authorized users.
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A. Ucar et al.
Introduction
Aromatic thiosemicarbazone analogues were used in medicinal chemistry due to
their antiproliferative, antibacterial, antitumor, antifungal and antiviral activities
[1–6]. Thiosemicarbazones have antiparasitic and antibacterial efects against
many bacterial strains and this can be used in their identifcation [7]. As the
responsible moiety, the conjugated N–N–S system in the thiosemicarbazone scaf-
folds has been identifed for the designated biological properties [8]. Especially
with this system, obtaining chelate metal complexes even more increases the bio-
logical activity [9]. Copper is a crucial nutrient required for important biological
processes [10] such as in redox reactions and required as an allosteric component
for many cellular enzymes in all organisms. For example in humans, the essen-
tial enzymes that require copper ion as a cofactor for their functions include Cu/
Zn superoxide dismutase (SOD), cytochrome c oxidase, ceruloplasmin, lysyl oxi-
dase (LOX) and tyrosinase [11]. The anticancer activity of copper complexes is
dependent on generation of highly reactive oxygen species (ROS) by ionic copper
due to its redox activation in the cytosol. By this way, free copper could cause
damage to cellular biomolecules such as lipids, proteins and nucleic acids. The
tumor cells in comparison with normal cells contain raised ROS depending on
reduced glutathione mediated antioxidant capacity. Therefore, they have become
more sensitive to pro-oxidant ionophoric copper. Accordingly, the copper com-
plexes increased intracellular copper ionophores, producing toxic levels of intra-
cellular ROS in cancer cells, but not in normal cells. The potential for copper
molecules to be used as hypoxia markers has been thoroughly evaluated in both
preclinical and clinical studies [11, 12]. Recently, it has been determined that
cancer cells showed greater copper uptake than normal cells [13]. The copper
transporter-1 is known to play a role in the cellular uptake of copper ions and
assessed as a new reporter for 64Cu PET imaging in cancer [14].
The carbonic anhydrase enzyme (CA, 4.2.1.1), which contains Zn2+ ions in its
active site and is commonly found in all organisms, catalyzes the reversible reac-
−
tion between CO2 and H2O that yields H+ and HCO3 [15]. In mammals, there
are 17 isoenzymes of CA that are found in cytosol, membrane-bound, mitochon-
dria, extracellular space and saliva, with diferent catalytic activities and behav-
ior against inhibitors. Of these, CA I and II are cytosolic [16]. CAs have been
reported to participate in biological events such as respiration, acid–base and ion
homeostasis, tumorigenesis and urea formation [17]. CA I is the most abundant
protein in erythrocytes that does not contain hemoglobin and is expressed about
5–6 times more than CA II. Although it has been determined that the defciency
does not cause any abnormality, it can be said that this is due to the presence
of CA II [18]. However, Zheng and colleagues reported that CA I expression
in breast cancer is increased, induces abnormal cell calcifcation, apoptosis and
migration, and may be a potential oncogene [19]. CA II remains a target for the
treatment of diseases such as glaucoma, edema, epilepsy and attitude sickness.
In addition, CA II has important roles in thyroid hormone synthesis. CAs have
become an interesting enzyme, especially after their inhibitors have been found
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Cytotoxic efects, microbiological analysis and inhibitory…
to act diuretically [17, 20]. Today the carbonic anhydrases could be inhibited by
several compounds such as sulfonamides, sulfonates and sulfamides for cancer
and other metabolic disorder therapies. Generally, these chemical inhibitors target
the catalytic zinc ion in the enzyme cavity. In particular, indisulam and COU-
MATE-667 compounds are still under consideration as anticancer agents. Until
today, CA inhibitors were commonly used as diuretic and antiglaucoma agents,
but turn out that several of them showed signifcant antitumorigenic efects. How-
ever, most of the CA inhibitors show undesirable side efects due to the random
or the of target inhibition of CA isoforms [21]. Therefore, specifc isozyme selec-
tive new compounds with fewer side efects still need to be developed.
Herein we report the synthesis and characterization of Co(II), Zn(II) and Mn(II)
complexes of HMAT for the frst time. We had synthesized the HMAT and its Cu(II)
complex in our previous work and presented their characterizations. In other part
of our study, we investigated the antimicrobial, anticancer and carbonic anhydrase
inhibition (CA I and II inhibitory) efects of HMAT and of its Cu(II), Co(II), Zn(II)
and Mn(II) complexes. According to the results, the biological activities of the com-
pounds were evaluated.
Experimental
Chemistry
All chemicals were of analytical reagent grade. 1H, 13C NMR spectra were recorded
with a Bruker AC 400 (400 MHz) NMR spectrometer. For structural analysis of the
samples, they were measured in the range of 4000–500 cm−1 using attenuated total
refection-Fourier transformed infrared (ATR-FT-IR) spectrometer (PerkinElmer
100). UV–Vis absorption spectrum was obtained on Shimadzu UV-1800 double-
beam spectrophotometer. Thermogravimetric analysis (TGA, 25–900 °C) was per-
formed using Setaram thermal gravimetric analyzer at heating rate 10 °C/min and
nitrogen fow rate of 20 ml/min. XRD measurements were taken using Bruker axis
difractometer (Bruker D8 ADVANCE) with Cu Kα radiation, operating at 40 kV
and 30 mA with a rate of 21 ml/min. EPR spectra of Mn(II) complex in polycrystal-
line form were investigated using JEOL JesFa300 X-band spectrometer at the 300
and 123 K temperatures, respectively. Carbonic anhydrase enzyme activity was
determined by using UV–Vis spectrophotometer—Optizen POP.
Synthesis of HMAT
HMAT was prepared according to our previous article [22]. The general procedure
is illustrated in Scheme 1.
Anal. Calcd. For C10H13N3O2S: C, 50.19; H, 5.48; N, 17.56%. Found: C, 49.91;
1
H, 3.59; N, 17.40%. H NMR (400 MHz DMSO-d6): δ (ppm): 12.62 (s, 1H, –OH),
7.1–6.92 (d, 2H, HAr), 3.59–2.38 (s, 3H, –CH3). FT-IR (cm−1) ʋ:, 3388, 3185,
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A. Ucar et al.
Scheme 1 Synthetic routes for 2-hydroxy-5-methoxyacetophenone thiosemicarbazone (HMAT)
Scheme 2 Proposed structures of complexes
2359, 1646, 826, 539. 13C NMR (400 MHz, CDCl3): δ (ppm): 181.1 (C1 =S), 146.6
(C2 =N), 154.0 (C3–OH), 114.0–120.1 (CAr).
Synthesis of metal complexes
Cu(II) complex was prepared according to our previous article [23]. Co(II), Zn(II)
and Mn(II) complexes were synthesized for the frst time in this study following
this general procedure: In an amount of 1:2 metal/ligand molar ratio, CoCl2 6H2O,
ZnCl2 and Mn(NO3)2 4H2O in the minimum quantity of EtOH were added drop-
wise to the 3.0 mmol of HMAT in EtOH (60 mL) separately. After refuxing for 2 h,
the resulting solids were fltered and washed with anhydrous ether. Physical data of
HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes are given in Table 1. The
proposed structures of complexes are shown in Scheme 2 considering similar struc-
tures in the related publications [24–27].
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Cytotoxic efects, microbiological analysis and inhibitory…
Table 1 Physical data of HMAT
Compound
Color
M.p. ( °C)
Yield %
and its Cu(II), Co(II), Zn(II) and
Mn(II) complexes
HMAT
Cream
Dark green
Brown
Yellow
Dark red
186–188
187–189
190–192
189–191
191–193
52
47
43
48
41
Cu(II)
Co(II)
Zn(II)
Mn(II)
Cu(II) komplex:
Anal. Calcd. For C10H11ClCuN3O2S: C, 35.72; H, 3.30; N, 12.50%. Found: C,
35.75; H, 3.50; N, 12.78%. 1H NMR (400 MHz, DMSO-d6): δ (ppm)): 7.29–6.90 (d,
2H, HAr), 6.59 (s, 2H, –NH2), 3.57–2.58 (s, 3H, –CH3). FT-IR (cm−1) ʋ: 3487, 3261,
1523, 1262, 824, 526.
Co(II) komplex:
Anal. Calcd. C20H22N6O4S2Co: C, 45.03; H, 4.16; N, 15.75%. Found: C, 45.13; H,
1
4.21; N, 15.78%. %. H NMR (400 MHz, DMSO-d6: δ (ppm): 7.29–6.75 (d, 2H,
HAr), 6.90 (s, 2H, –NH2), 3.62–2.52 (s, 3H, –CH3). FT-IR (cm−1) ʋ: 3522, 3099,
1537, 1217, 1031, 783, 590.
Zn(II) komplex:
Anal. Calcd. C20H22N6O4S2Zn: C, 44.49; H, 4.11; N, 15.56%. Found: C, 45.03; H,
1
4.19; N, 15.68%. H NMR (400 MHz, DMSO-d6): δ (ppm): 7.24 (s, 2H, –NH2),
6.93–6.84 (d, 2H, HAr), 3.78–2.35 (s, 3H, –CH3). FT-IR (cm−1) ʋ: 3486, 3192, 1540,
1215, 1030, 783, 585.
Mn(II) komplex:
Anal. Calcd. C20H22N6O4S2Mn: C, 45.37; H, 4.19; N, 15.87%. Found: C, 45.53; H,
1
4.29; N, 15.98%. H NMR (400 MHz, DMSO-d6): δ (ppm): 7.63–7.29(d, 2H, Har),
7.15 (s, 2H, –NH2), 3.73–2.34 (s, 3H, –CH3). FT-IR (cm−1) ʋ: 3351, 3190, 1629,
1200, 1028, 783, 583.
Purifcation of CA I, II and esterase activity assay
CA I and II enzymes were purifed from human erythrocytes using the method
described earlier by Kuzu and co-workers [28]. Erythrocytes were obtained from
Agri State Hospital blood bank. Enzyme activity was measured spectrophotometri-
cally at 348 nm using 4-nitrophenyl acetate as substrate [29].
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A. Ucar et al.
Microbiological analysis
The following species of bacteria were used in the study for the microbiological
analysis: Staphylococcus aureus ATCC 25,923, S. Aureus ATCC 43,300 (Resist-
ant to methicillin), Enterococcus faecalis ATCC 29,212, Escherichia coli ATCC
25,922, Salmonella enteriditis ATCC 13,076, Pseudomonas aeuroginosa ATCC
2783, P. Aeuroginosa ATCC 2783 and Candida albicans ATCC90028 (yeast). Mini-
mum inhibitory concentrations (MICs) of HMAT and its Cu(II), Co(II), Zn(II) and
Mn(II) complexes against the test organisms were detected according to the recom-
mendations of the Clinical and Laboratory Standards Institute [30].
Cell viability assay
To evaluate the cytotoxic efects of HMAT and its Cu(II), Co(II), Zn(II) and Mn(II)
complexes on CRL-4010, MCF-7 and MDA-MB-231 cell lines, the cells were cul-
tured in complete DMEM (Dulbecco’s modifed Eagle medium) (Sigma) and seeded
into 96-well plates (Costar, Corning Inc) at a density of 1×104 cells per well. After
overnight culturing, the cells were treated with the ligand and its complexes at a fnal
concentration of 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 μM in 100 μl medium for
72 h. Control cells were treated with DMSO simultaneously. All doses of tested com-
pounds and control wells were performed in triplicate. At the end of treatment, 10 μL
thiazolyl blue tetrazolium bromide (MTT) was added to each well and incubated for
4 h. Then supernatants were discarded and DMSO was added to dissolve formazan.
The optical density values of each well were determined by spectrophotometer (Mul-
tiscan GO, Thermo Fisher Inc.) at 570 and 690 nm. The % cell viability of the sam-
ples was calculated by using the following formula: % cytotoxicity=(1—the mean
OD of experimental wells)/(the mean OD of control wells)×100%. The IC50 values
of the ligand and its metal complexes for CRL-4010, MCF-7 and MDA-MB-231
cells were estimated separately from the data of generated dose–response curve.
Statistical analysis
The IC50 values of each compound were calculated by logarithmic mean of the per-
centage of inhibition concentrations, and 95% confdence interval (CI) values and
mean standard errors (SEM) were determined by using the GraphPad Prism8
software.
Results and discussion
Characterizations of compounds
1H NMR, FT-IR and TGA analyses of HMAT and its Cu(II) complex have been
reported in our previous studies [22, 23]. Detailed characterization of the frst syn-
thesized Co(II), Zn(II) and Mn(II) complexes is given below.
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Cytotoxic efects, microbiological analysis and inhibitory…
Fig. 1 1H NMR spectra of Co(II), Zn(II) and Mn(II) complexes
1
In the H NMR spectra of Co(II), Zn(II) and Mn(II) complexes (Fig. 1), -OH
(12.62 ppm) and –NH signal (11.12 ppm) of HMAT (Fig. S1) [31, 32] were disap-
peared due to the coordination of the deprotonated phenolate component after the
complexation. The proton signal of the –NH2 of Co(II), Zn(II) and Mn(II) com-
plexes was appeared as a singlet at 6.90, 7.24, 7.15 ppm, respectively [33, 34]. The
protons in the aromatic ring have signals in the range of 6.75–7.63 ppm for the com-
plexes. A singlet was appeared at 3.62, 3.78, 3.73 ppm for the proton present in the
methoxy group [35], and a singlet appeared around 2.52, 2.35, 2.34 ppm was attrib-
uted to CH3 proton of azomethine group [36] for Co(II), Zn(II) and Mn(II) com-
plexes, respectively.
13C NMR spectrum of HMAT is shown in Fig. S2. The signals for the carbon
atom of C=S group and C–OH were appeared at 181.1 and 154.0 ppm, respectively.
The signals at the range of 114.0–120.1 ppm can be attributed to aromatic carbons
(CAR). The obtained results are compatible with the studies [37, 38].
The FT-IR spectra of the complexes and HMAT are shown in Fig. 2a and
Fig. S3, respectively. υ(O–H) and υ(N–H) vibrations, which are the character-
istic peaks of thiosemicarbazone and disappeared in the complexes, have been
found at 3388 cm−1 and 3185 cm−1 for HMAT (Fig. S3), respectively [36].
υ(–NH2) stretching frequencies have been found between 3522–3351 cm−1 in all
complexes [36]. The band observed at 826 cm−1 in HMAT which attributed to
ν(C = S) was not seen in the spectrum of the complexes. A new group appeared
at higher energy (783 cm−1) indicating coordination of sulfur atom to metal [39].
Other characteristic peak υ(C = N) was observed at 1537 cm−1 for Co(II), 1540
for Zn(II) and 1629 for Mn(II) [27]. After complexation, metal-O vibrations in
the complexes around 590 cm−1 evidence the coordination through oxygen [40].
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A. Ucar et al.
Fig. 2 FT-IR a and UV–Vis b spectra of Co(II), Zn(II) and Mn(II) complexes
In the UV–Vis spectra of HMAT (Fig. S4), the band at 305 nm was assigned
to π—π* transitions of azomethine, while the absorption at 355 nm corresponds
to a n—π* transition of thioamide [22]. The absorption bands of the complexes
(Fig. 2b) appear at 295–298 nm owing to π—π*, and the other one at 343–347 nm
corresponds to n—π* transitions. By comparing the frequency of HMAT and
the corresponding complexes, the electronic transitions of π—π* are shifted to a
lower value due to the formation of the complexes and coordination of the ligand
to the metal.
TG/DSC curve of new synthesized Co(II), Zn(II) and Mn(II) complexes is
represented in Fig. 3. The Co(II) complex is stable up to 200 °C, indicating the
absence of water. The frst weight loss at 310 °C (Cald; 10.22%, Obs; 10.17%)
can be attributed disintegrated units of ligand molecules [41]. The other weight
loss due to the residue of the product can be seen in the range of 375 °C and
450 °C (Cald; 3.77%, Obs; 3.86%). The Zn(II) complex is stable up to 180 °C,
indicating the absence of water. The frst weight loss at 225 °C (Cald; 10.11%,
Obs; 10.14%) can be attributed disintegrated units of ligand molecules. The other
weight loss due to the residue of the product can be seen in the range of 275 °C
and 375 °C (Cald; 50.77%, Obs; 50.86). The frst stage of the TG degradation of
the Mn(II) complex was from 69–118 °C, which corresponded to the lost of lat-
tice water (Cald; 6.37%, Obs; 6.87). The other weight loss due to the residue of
the product decomposition can be seen in the range of 200 °C and 290 °C (Cald;
19.74%, Obs; 19.96%). The decomposition of the complexes to CoO, ZnO and
Fig. 3 TG/DSC curve of Co(II), Zn(II) and Mn(II) complexes
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Cytotoxic efects, microbiological analysis and inhibitory…
MnO starts within the temperature ranging from 500 °C to 800 °C. As can be
seen in the DSC curve of Co(II), Zn(II) and Mn(II) complexes, a single peak
observed at 243.06 °C, 188.38 °C, 234.46 °C, respectively, indicates that com-
pounds were obtained in pure form.
EPR analysis of complexes
EPR, a nondestructive spectroscopic method, can be directly used to detect and
characterize the paramagnetic centers having unpaired electron(s) such as transition
metals [42]. Detailed EPR spectrum examination of Cu(II) complex of HMAT was
done in our previous study [23]. The EPR spectrum of Zn(II) complex of HMAT
could not be studied since Zn(II) is not paramagnetic. The EPR spectrum shown
in Fig. 4 recorded at 300 K has broadened isotropic EPR signal with the value
ΔHpp =64.13 mT. This can be interpreted as enhanced spin lattice relaxation due
to dipolar interaction and random orientation of Mn (II) ions [43]. In order to elimi-
nate unwanted efects and obtain the spectra with the highest resolution, powdered
complex was diluted in DMF and EPR spectra were recorded at 123 K. Due to the
hyperfne interaction between the unpaired electron and metal nuclear spins (55Mn,
I=5/2) of the complex, a hyperfne sextet was observed at the spectrum given in
Fig. 5. By considering the resonance condition giso =hυ/gβHr, the giso value was
measured as 2.0036 which is very close to free electron spin value suggestive of the
absence of spin orbit coupling in the ground state. The average value of hyperfne
splitting for allowed transitions corresponding to ΔmI =0 was measured as 9.5 mT.
This value is consistent with octahedral coordination because in tetrahedral sites Aiso
is 20–25% lower than octahedral sites. In addition to allowed transitions, between
each of the two main hyperfne lines a pair of low-intensity forbidden lines with an
average spacing of 2.3 mT is observed corresponding to ΔmI = 1 transitions [44].
This forbidden line is observed because of the mixture of the nuclear hyperfne lev-
els by the zero-feld splitting factor of the Hamiltonian [45]. The measured values of
main line splitting from low to high feld (8.8, 9.3, 9.5, 9.8 10.1 mT) and the doublet
Fig. 4 Powder EPR spectrum of Mn(II) complex at 30 0 K temperature (ΔHpp =64.13 mT)
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A. Ucar et al.
Fig. 5 EPR spectrum of Mn(II) complex at 123 K temperature in frozen DMF solution
Fig. 6 EPR spectrum of Co(II) complex in frozen DMF solution at 123 K temperature
splitting from low to high feld (1.9, 2.2, 2.5, 2.4, 2.5 mT) are in good agreement
with the values given in the literature [46]. The magnetic moment was determined
as 5.93 B.M using the µ=giso[S(S+1)]1/2 equation which supports the d5 high-spin
Mn(II) complex [47].
EPR spectra of Co(II) complex were recorded at 123 K in frozen DMF solu-
tion using the JEOL JesFa300 X-band spectrometer (Fig. 6). An axially symmetric
spectral pattern was obtained at this temperature which supports the low-spin com-
plex with S=1/2 since for the high-spin complexes with S=3/2 very low tempera-
tures are necessary due to the short spin-lattice relaxation time [48]. The parallel
and perpendicular components of the spectroscopic splitting value were measured
as g||=2.1684 and g⊥ =2.0535, and the order g||>g⊥ >ge indicates the presence of
2
unpaired electron in dx -y2 orbital [49].
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Cytotoxic efects, microbiological analysis and inhibitory…
Fig. 7 % Activity–[I] plots for HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes having inhibi-
tion efect
Table 2 IC50 values of HMAT
Compound
IC50 CA I (μM)
IC50 CA II (μM)
and its Cu(II), Co(II), Zn(II) and
Mn(II) complexes on CA I and
CA II isoenzymes
HMAT
Cu(II)
Co(II)
Zn(II)
Mn(II)
98.35
324.46
25.67
1.06
99.02
86.64
57.76
10.34
36.48
22.36
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A. Ucar et al.
Carbonic anhydrase inhibition
In order to determine the efects of HMAT and its Cu(II), Co(II), Zn(II) and Mn(II)
complexes on enzymatic activity, activity measurements were taken at fve difer-
ent concentrations. % Activity–[compound] graphs were drawn using the results
obtained by taking control measurement as 100% (Fig. 7). The concentration of sub-
stance causing 50% inhibition was calculated from the graph equation. Accordingly,
the Zn(II) complex showed the strongest inhibitory efect for both CA I and CA II.
The results are summarized in Table 2.
Cytosolic CA II is the most widely spread isoenzyme and is found in almost all
tissues. The main role of CA II is to promote H+ production and acid–base homeosta-
sis, pH balance and metabolic acidosis [50]. It has been reported that CA I plays an
important role in the formation of retinal and cerebral edema, and its inhibition may
be advantageous in the elimination of these conditions [51]. CA I has been shown to
be an indicator for diferentiating autoimmune hemolytic anemia from other types
of anemia [52]. In addition, it has been reported that CA I and II are associated with
gastrointestinal tumors, and CA II may be a novel biomarker for gastrointestinal stro-
mal tumors [53]. It has been stated that CA enzymes are important therapeutic targets
and that both their inhibitors and activators are already used as drugs. Approximately
25 drugs have been developed that are used clinically and targeting CAs, utilized as
diuretics, antiglaucoma, anticonvulsant and anticancer [50]. Besides, CA inhibitors
have been shown to be antifungal and bacterial agents [54]. Previous studies have
identifed natural and synthetic substances that have an in vivo or in vitro inhibitory
efect on CA, and have also reported that some metal ions inhibit the enzyme [51,
55–57]. Therefore, synthesis of new and potent CA inhibitors is important.
Microbiological analysis
HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes have bacteriostatic activ-
ity on tested organism at diferent degrees (Table 3). Although antibacterial activity
Table 3 MIC values of HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes
Test organisms
MIC (mg/ml)
HMAT Cu(II) Co(II) Zn(II) Mn(II)
C. Albicans ATCC90028
S. Enteriditis ATCC 13,076
P. Aeuroginosa ATCC 2783
Klebsiella pneumoniae ATCC 70,060
E. Coli ATCC 25,922
Yeast
Gram-negative 1.25
0.31
0.31
1.25
0.625 1.25
1.25
2.5
0.31
2.5
1.25
1.25
0.31
1.25
1.25
1.25
2.5
1.25
2.5
2.5
0.31
1.25
1.25
1.25
1.25
0.31
2.5
1.25
1.25
2.5
1.25
1.25
1.25
1.25
2.5
E. Faecalis ATCC 29,212
S. Aureus ATCC 25,923
Gram-positive 1.25
2.5
2.5
S. Aureus ATCC 43,300 (Resistant to
2.5
2.5
methicillin)
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Cytotoxic efects, microbiological analysis and inhibitory…
of HMAT on C. Albicans and gram-positive bacteria was not observed, Zn(II) and
Cu(II) complexes have antibacterial efect on these microorganisms. The inhibi-
tory efects of the Mn(II) and Co(II) complexes on C. Albicans are particularly
noteworthy.
In previous studies [58–61], the in vitro antibacterial activity of the diferent
thiosemicarbazone complexes was shown against gram-positive and gram-negative
bacteria and also fungi in diferent concentrations. Noruzi et al. (2020) [62] were
detected a considerable antibacterial activity against gram-positive and gram-neg-
ative bacteria in particular MIC=31.25 µg/mL concentration of thiosemicarbazone
complexes. Similar to our study, they reported the fungal strains were resistant
against complexes except for cobalt complex.
Evaluation of anticancer activity on breast cancer cell lines
The cytotoxic properties of HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) com-
plexes against normal breast epithelium CRL-4010, low metastatic MCF-7 and
highly metastatic MDA-MB-231 cells were assessed by MTT assay (Fig. 8). By
all accounts, the IC50 values and their 95% confdence intervals (CI) of each com-
pound were calculated by GraphPad Prism8 software (Table 4). According to the
results, metal complexes have higher toxic properties than their ligand. Also, the
tested compounds were found to have weaker cytotoxic efects on normal cells than
on MCF-7 and MDA-MB-231 breast cancer cells. Among these metal complexes,
the Cu(II) complex was found as the highest cytotoxic compound. In detail, the IC50
value of HMAT on normal cells was 234.5 μM, while the IC50 value on cancer cells
was 210.2 μM on MCF-7 and 110.7 μM on MDA-MB-231 cells. The IC50 values
of Co(II), Zn(II) and Mn(II) complexes were ranged at 115.3–228 µM on normal
cells, 46.77–103.4 µM on MCF-7 cells and 36.92–71.82 µM on MDA-MB-231 cells.
Remarkably, the IC50 values of Co(II) complex were ranging from 0.8 to 2.6 µM
at the tested cell lines. By last result, the Cu(II) complex was determined to have
30–120 times higher cytotoxic properties than the other compounds. For benchmark-
ing of these efects, the chemosensitivity (IC50 values) of the frst and widely used
metal (platinum)-containing drug, cisplatin (cis-diammineplatinum(II) dichloride), is
known to be 97.86 µM for MCF-7 and 36.20 µM for MDA-MB-231 cell line. In this
direction, it could be generalize that the tested Cu(II) complex was highly cytotoxic
113-fold to MCF-7 and 30-fold to MDA-MB-231 cells than cisplatin. In addition, the
Fig. 8 Cytotoxic properties of HMAT and its Cu(II), Co(II), Zn(II) and Mn(II) complexes against normal
breast epithelium CRL-4010, low metastatic MCF-7 and highly metastatic MDA-MB-231 cells
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A. Ucar et al.
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Cytotoxic efects, microbiological analysis and inhibitory…
another prominent complex that we regard as signifcant was Zn(II) complex, due to
exert similar cytotoxic properties to cisplatin. In brief, it could be said that the our
synthesized metal complexes have at least as much cytotoxic efects as cisplatin.
Currently, the screening for efective chemotherapeutic agents for the treatment
of drug-resistant tumor cells is ongoing. In addition, the search for novel adjuvant
molecules that will increase the efect of existing drugs continues rapidly. For this
purpose, in this study we investigated the possible anticancer efects of HMAT and
its new metal complexes. In light with our fndings, the metal complexes were more
cytotoxic than their ligand and had a higher cytotoxic efects against MCF-7 and
MDA-MB-231 breast cancer cell lines compared to normal breast epithelial cells.
With this aspect, our fndings were clearly remarkable. A notable consequence was
that the Cu(II) complex had an extremely high anticancer activity than the other
metal complexes. Copper is an essential nutrient which is important in several bio-
chemical processes and by this regard required by all living organisms. Copper is a
transition metal that could transform from the oxidized form (Cu2+) to the reduced
form (Cu+) of copper. Due to this redox activity in cytosol, copper is an essential
cofactor for many enzymatic processes and pathways. Also it could be considered
potentially toxic to the cells when it is in excess amount. This high redox activity of
copper could elevate reactive oxygen species (ROS) in the cytosol. When there is an
imbalance between the number of ROS formed and the neutralization of these by
antioxidant defenses, there is evidence that the toxicity of copper causes oxidative
stress. By this way, the intracellular oxidative stress could cause cellular damage
through the efects of ROS attack on macromolecules such as sugars, DNA, proteins
and lipids [63]. Due to these biological properties of the copper ion, it could be
extrapolated that why we fnd out higher cytotoxic properties in the copper complex.
Moreover, remarkably the Zn(II) complex showed approximately 2.8-fold higher
cytotoxic efects on MDA-MB-231 cells compared to MCF-7 cells and approxi-
mately 3.1-fold higher than normal breast epithelium cells.
Conclusion
The synthesis of thiosemicarbazone ligand containing 2-hydroxy-5-methoxyaceto-
phenone (HMAT) and its Cu(II) complex has been included in our previous studies,
while Co(II), Zn(II) and Mn(II) complexes were synthesized for the frst time. In this
study, inhibition efects of synthesized thiosemicarbazone and its metal complexes
on CA I and II enzymes which are therapeutic target in the treatment of some dis-
eases were revealed. According to our results, we found that the Zn(II) complex had
higher inhibitory efect against CA I and II enzymes among other metal complexes.
In this regard, it is thought that these synthesized substances may be promising for
the discovery of new and more potent CA inhibitors.
Copper complexes are promising new more efective drugs for the treatment of
patients with aggressive cancer and to overcome cisplatin resistance. Metallothera-
peutic drugs, such as cisplatin, cause undesirable side efects and resistance to cis-
platin hampered clinical use of these metallotherapeutic agents. Therefore, the stud-
ies are still continuing to discover new metallotherapeutic drugs such as copper
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A. Ucar et al.
complexes with strong anticancer activity for drug-resistant cancer treatment [64].
Considering cytotoxic properties of the compounds, in particular, the Cu(II) com-
plex displayed preciously strong cytotoxic. Given these facts, the Cu(II) and Zn(II)
complexes could be debated as potential chemotherapeutic molecule against drug-
resistant breast cancer cells. Microbiological fndings indicate that synthesized prod-
ucts may become useful for the development of new antibacterial agents.
Acknowledgements This study was supported by the Necmettin Erbakan University Scientifc Research
Foundation (BAP-161715001)
Compliance with ethical standards
Confict of interest The authors declare that they have no confict of interest.
References
1. J. Deng, T. Li, G. Su, Q.-P. Qin, Y. Liu, Y. Gou, J Mol Struct 1167, 33 (2018)
2. D.S. Kalinowski, P. Quach, D.R. Richardson, Med Chem 1, 1143 (2009)
3. N.S.H.N. Moorthy, N.M.F.S.A. Cerqueira, M.J. Ramos, P.A. Fernandes, Recent Pat Anticancer
Drug Discov 8, 168 (2013)
4. E. Bozgeyik, D. Tasdemir-Kahraman, K. Arman, I. Bozgeyik, A. Karakucuk-Iyidogan, E.A.
Cakmak, Gene Rep 18, 100578 (2020)
5. H. Muğlu, Res Chem Intermed 46, 2083 (2020)
6. Z. Bakherad, M. Safavi, A. Fassihi, H. Sadeghi-Aliabadi, M. Bakherad, H. Rastegar, J.B. Gha-
semi, S. Sepehri, L. Saghaie, M. Mahdavi, Res Chem Intermed 45, 2827 (2019)
7. W.M. Elhusseiny, Polycycl Aromat Compd 0, 1 (2020)
8. M. Bingul, E. Şenkuytu, M.F. Saglam, M. Boga, H. Kandemir, I.F. Sengul, Res Chem Intermed
45, 4487 (2019)
9. R.V. Singh, P. Chaudhary, S. Chauhan, M. Swami, Spectrochim Acta Part A Mol Biomol Spec-
trosc 72, 260 (2009)
10. L.N. Sharada, S.N.T. Sunitha, M. Sabha, T.L. Viveka, Y. Aparna, M. Prasad, Res Chem Intermed
46, 3341 (2020)
11. D. Denoyer, S.A.S. Clatworthy, M.A. Cater, Met Ions Life Sci 18, 469 (2018)
12. H. Karlsson, M. Fryknäs, S. Strese, J. Gullbo, G. Westman, U. Bremberg, T. Sjöblom, T. Pand-
zic, R. Larsson, P. Nygren, Oncotarget 8, 30217 (2017)
13. J.B. Foo, M.L. Low, J.H. Lim, Y.Z. Lor, R. Zainol Abidin, V. Eh Dam, N. Abdul Rahman, C.Y.
Beh, L.C. Chan, C.W. How, Y.S. Tor, L. Saiful Yazan, Biometals 31, 505 (2018)
14. K. Il Kim, S.J. Jang, J.H. Park, Y.J. Lee, T.S. Lee, K.S. Woo, H. Park, J.G. Choe, G. Il An, J.H.
Kang, J Nucl Med 55, 1692 (2014)
15. M. Kuzu, R. Demirdağ, A. Özkaya, V. Çomaklı, Z. Şahin, U. Dardağan, K. Uzun, M. Koca, J
Biol Chem 44, 409 (2016)
16. X. Liu, D. Lu, R. Bowser, J. Liu, Int J Mol Sci 17, 1820 (2016)
17. A. Alver, A. Mentes, S.C. Karahan, C. Erem, E.E. Keha, M.K. Arikan, M.S. Eminag, O. Deger,
Exp Clin Endocrinol Diabetes 115, 287 (2007)
18. W.S. Sly, P.Y. Hu, Annu Rev Biochem 64, 375 (1995)
19. Y. Zheng, B. Xu, Y. Zhao, H. Gu, C. Li, Y. Wang, X. Chang, BMC Cancer 1, 1 (2015)
20. M. Jakubowski, E. Szahidewicz-krupska, A. Doroszko, BioMed Res Int 2018 (2018)
21. S.M.I. Morsy, A.M. Badawi, A. Cecchi, C.T. Supuran, A. Cecchi, J J Enzyme Inhib Med Chem
24, 499 (2009)
22. E.G. Akgemci, A.O. Saf, H.U. Tasdemir, E. Türkkan, H. Bingol, S.O. Turan, M. Akkiprik, Spec-
trochim Acta Part A Mol Biomol Spectrosc 136, 719 (2015)
23. E. Türkkan, U. Sayin, N. Erbilen, S. Pehlivanoglu, G. Erdogan, H.U. Tasdemir, A.O. Saf, L.
Guler, E.G. Akgemci, J Organomet Chem 831, 23 (2017)
1 3

Cytotoxic efects, microbiological analysis and inhibitory…
24. A.B.M. Ibrahim, M.K. Farh, S.A. ElGyar, M.A. ELGahami, D.M. Fouad, F. Silva, I.C. Santos,
A. Paulo, Inorg Chem Commun 96, 194 (2018)
25. C.C. Gatto, M.A.S. Chagas, I.J. Lima, F. Mello Andrade, H.D. Silva, G.R. Abrantes, E.P.S. Lac-
erda, Transit Met Chem 44, 329 (2019)
26. M. Selvaganapathy, N. Raman, J Chem Biol Ther 01, 108 (2016)
27. M.S. Refat, H.K. Ibrahim, S.Z.A. Sowellim, M.H. Soliman, E.M. Saeed, J Inorg Organomet
Polym Mater 19, 521 (2009)
28. M. Kuzu, V. Çomaklı, E. Akkemik, M. Çiftci, Ö.İ. Küfrevioğlu, Fish Physiol Biochem 44, 1119
(2018)
29. J.A. Verpoorte, S. Mehta, J.T. Edsall, J Biol Chem 242, 4221 (1967)
30. J.B. Patel, R.F. Cockerill, A.P. Bradford, M.G. Eliopoulos, A.J. Hindler, G.S. Jenkins, S.J.
Lewis, B. Limbago, A.L. Miller, P.D. Nicolau, M. Pwell, M.J. Swenson, M.M. Traczewski, J.D.
Turnidge, CLSI Clin Lab Stand Inst 35, 1 (2015)
31. Ş. Güveli, I. Kılıç-Cıkla, B. Ülküseven, M. Yavuz, T. Bal-Demirci, J Mol Struct 1173, 366
(2018)
32. R.A. Bedier, T.A. Yousef, G.M. Abu El-Reash, O.A. El-Gammal, J Mol Struct 1139, 436 (2017)
33. D. Sharma, J.P. Jasinski, V.A. Smolinski, M. Kaur, K. Paul, R. Sharma, Inorganica Chim Acta
499, 119187 (2020)
34. R. Konakanchi, J. Haribabu, J. Prashanth, V.B. Nishtala, R. Mallela, S. Manchala, D. Ganda-
malla, R. Karvembu, B.V. Reddy, N.R. Yellu, L.R. Kotha, Appl Organomet Chem 32, 1 (2018)
35. M.A. Arafath, F. Adam, M.R. Razali, L.E. Ahmed Hassan, M.B.K. Ahamed, A.M.S.A. Majid, J
Mol Struct 1130, 791 (2017)
36. I. Kılıç-Cıkla, Ş. Güveli, M. Yavuz, T. Bal-Demirci, B. Ülküseven, Polyhedron 105, 104 (2016)
37. L. V Kumar, S. Sunitha, G. Rathika Nath, Mater. Today Proc. (2020 in press) https://doi.
org/10.1016/j.matpr.2020.05.376
38. A. Dehno Khalaji, E. Shahsavani, N. Feizi, M. Kučeráková, M. Dušek, R. Mazandarani, A.
Amiri, Comptes Rendus Chim 20, 125 (2017)
39. O. Abdullahi, W. Handy, M. Shazwan, S. Jamil, M. Shamsuddin, Mal J Fund Appl Sci 16, 351
(2020)
40. M.A. Afan, M.A. Salam, F.B. Ahmad, F. White, H.M. Ali, Inorganica Chim Acta 387, 219
(2012)
41. S. Paswan, M.K. Bharty, P. Bharati, P.K. Sonkar, V. Ganesan, R.J. Butcher, New J Chem 41,
15466 (2017)
42. A. Abragam, B. Bleaney, Electron paramagnetic resonance of transition ions (Clarendon Press,
UK, 1970)
43. V.L. Siji, M.R.S. Kumar, S. Suma, M.R.P. Kurup, Spectrochim Acta Part A Mol Biomol Spec-
trosc 76, 22 (2010)
44. V. Philip, V. Suni, M.R. Prathapachandra, Spectrochim Acta Part A Mol Biomol Spectrosc 64,
171 (2006)
45. B. Bleaney, R.S. Rubins, Proc Phys Soc 77, 103 (1961)
46. L. Burlamacchi, E. Tiezzi, Chem Phys Lett 4, 173 (1969)
47. C.G. Oliveira, P. Ivo, S. Maia, P.C. Souza, F.R. Pavan, C.Q.F. Leite, R.B. Viana, A.A. Batista,
O.R. Nascimento, V.M. De, J Inorg Biochem 132, 21 (2014)
48. A.C. Rizzi, N.I. Neuman, P.J. González, C.D. Brondino, Eur J Inorg Chem 2016, 192 (2016)
49. A. Sasmal, E. Garribba, V. Ugone, C. Rizzoli, S. Mitra, Polyhedron 121, 107 (2017)
50. MdI Hassan, B. Shajee, A. Waheed, F. Ahmad, S. William Sly, Bioorg Med Chem 21, 1570
(2012)
51. L. Safarova, A. Sujayev, I. Gulc, M. Abbasova, P. Taslimi, S.H. Alwasel, C.T. Supuran, V. Far-
zaliyev, J Enzyme Inhib Med Chem 32, 1174 (2017)
52. W. Kuo, S. Yang, Y. Hsieh, C. Tsai, H. Wen-Li, C. Shu-Chen, Clin Chim Acta 351, 79 (2005)
53. A. Mentese, E. Fidan, A. Alver, S. Demir, S.O. Yaman, A. Sumer, S. Fidan, H. Kavgaci, I. Turan,
Clin immunol 42, 73 (2017)
54. H.M. Becker, M. Klier, J.W. Deitmer, Carbonic anhydrase: mechanism, regulation, links to dis-
ease, industrial applications (Springer, Dordrecht, 2014), pp. 105–134
55. H. Nadaroglu, N.H. Kishali, B. Sever, M.D. Altintop, J Mol Struct 1197, 386 (2019)
56. R. Demirdag, V. Comakli, M. Kuzu, E. Yerlikaya, M. Şentürk, J Biochem Mol Toxicol 29, 123
(2015)
1 3

A. Ucar et al.
57. M. Kuzu, A. Özkaya, Z. Şahin, Ü. Dağ, V. Çomaklı, R. Demirdağ, Turkish J Phamaceutical Sci
14, 9 (2017)
58. I. Kizilcikli, Y.D. Kurt, B. Akkurt, A.Y. Genel, S. Birteksöz, G. Ötük, B. Ülküseven, Folia
Microbiol Praha 52, 15 (2007)
59. K.S.A. Melha, J Enzyme Inhib Med Chem 23, 4 (2008)
60. E. Pahontu, F. Julea, T. Rosu, V. Purcarea, Y. Chumakov, J Cell Mol Med 19, 865 (2015)
61. F. Bisceglie, C. Bacci, A. Vismarra, E. Barilli, M. Pioli, J Inorg Biochem 203, 110888 (2020)
62. E.B. Noruzi, B. Shaabani, S. Geremia, N. Hickey, P. Nitti, H.S. Kaf, Molecules 25, 370 (2020)
63. Z. Zu, R. McKendry, C.L. Chavez, Signaling in copper ion homeostasis (Elsevier BV, Amsterdam,
2000), pp. 293–300
64. F. Xie, F. Peng, J Fluoresc 28, 89 (2018)
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Afliations
Asuman Ucar1 · Mukerrem Findik2 · Muslum Kuzu3 · Suray Pehlivanoglu4 ·
Ulku Sayin5 · Zafer Sayin6 · Emine Guler Akgemci7
* Asuman Ucar
asucar340@gmail.com
1
Department of Science Education, Education Faculty, Agri Ibrahim Cecen University, Agri,
Turkey
2
Department of Chemistry Education, Research Laboratory, Ahmet Kelesoglu Education Faculty,
Necmettin Erbakan University, Konya, Turkey
3
Department of Nutrition and Dietetics, Health Sciences Faculty, Karabuk University, Karabuk,
Turkey
4
Department of Molecular Biology and Genetics, Faculty of Science, Necmettin Erbakan
University, Konya, Turkey
5
Physics Department, Science Faculty, Selcuk University, Campus, Konya, Turkey
6
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Selcuk
University, Konya, Turkey
7
Department of Chemistry Education, Ahmet Kelesoglu Education Faculty, Necmettin Erbakan
University, Konya, Turkey
1 3