Influence of complexation of thiosemicarbazone derivatives with Cu (II) ions on their antitumor activity against melanoma cells

Article abstract of DOI:10.3390/ijms22063104

A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The conformational preferences of the thiosemicarbazone system were characterized using theoretical calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions. The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes, with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites presence and the expression of oxidative stress and DNA damage response genes were evaluated. The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant enzymes expression.

Full text of DOI:10.3390/ijms22063104

International Journal of
Molecular Sciences
Article
Influence of Complexation of Thiosemicarbazone Derivatives
with Cu (II) Ions on Their Antitumor Activity against
Melanoma Cells
1
,
2
3
, Bartłomiej Rogalewicz 3
Monika Pitucha * , Agnieszka Korga-Plewko
, Agnieszka Czylkowska
,
1
4
2
2
2
Monika Drozd , Magdalena Iwan , Joanna Kubik , Ewelina Humeniuk , Grzegorz Adamczuk ,
Zbigniew Karczmarzyk 5 , Emilia Fornal 6 , Waldemar Wysocki 5 and Paulina Bartnik 5
1
Independent Radiopharmacy Unit, Faculty of Pharmacy, Medical University of Lublin,
PL-20093 Lublin, Poland; monika.drozd@umlub.pl
Independent Medical Biology Unit, Faculty of Pharmacy, Medical University of Lublin,
2
PL-20093 Lublin, Poland; agnieszka.korga@umlub.pl (A.K.-P.); joanna.kubik@umlub.pl (J.K.);
ewelinahumeniuk@umlub.pl (E.H.); grzegorzadamczuk@umlub.pl (G.A.)
3
Institute of General and Ecological Chemistry, Faculty of Chemistry, Lodz University of Technology,
Zeromskiego 116, 90-924 Lodz, Poland; agnieszka.czylkowska@p.lodz.pl (A.C.); 211150@edu.p.lodz.pl (B.R.)
Chair and Department of Toxicology, Faculty of Pharmacy, Medical University of Lublin,
PL-20093 Lublin, Poland; magda.iwan@umlub.pl
Faculty of Science, Siedlce University of Natural Sciences and Humanities, 3 Maja 54, 08-110 Siedlce, Poland;
zbigniew.karczmarzyk@uph.edu.pl (Z.K.); waldemar.wysocki@uph.edu.pl (W.W.);
paulina.bartnik95@gmail.com (P.B.)
4
5
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Pitucha, M.; Korga-Plewko,
A.; Czylkowska, A.; Rogalewicz, B.;
Drozd, M.; Iwan, M.; Kubik, J.;
6
Chair and Department of Pathophysiology, Faculty of Medicine, Medical University of Lublin,
PL-20090 Lublin, Poland; emilia.fornal@umlub.pl
Humeniuk, E.; Adamczuk, G.;
*
Correspondence: monika.pitucha@umlub.pl; Tel.: +48-81-448-7240
Karczmarzyk, Z.; et al. Influence of
Complexation of Thiosemicarbazone
Derivatives with Cu (II) Ions on Their
Antitumor Activity against
Abstract: A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity
in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed
the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The
conformational preferences of the thiosemicarbazone system were characterized using theoretical
calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions.
The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes,
with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical
and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination
compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and
SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested
compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most
promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle
analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites
presence and the expression of oxidative stress and DNA damage response genes were evaluated.
The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions
improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated
with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant
enzymes expression.
Melanoma Cells. Int. J. Mol. Sci. 2021,
2
2, 3104. https://doi.org/10.3390/
ijms22063104
Academic Editors:
Stéphane Bellemin-Laponnaz and
Diego Tesauro
Received: 28 January 2021
Accepted: 12 March 2021
Published: 18 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: synthesis; thiosemicarbazone; X-ray investigation; copper(II) complex; TG-DTG tech-
niques; melanoma; anticancer activity
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1. Introduction
Thiosemicarbazones are a group of sulfur derivatives of semicarbazones that are
obtained as a result of the condensation of appropriate aldehydes or ketones and thiosemi-
carbazides in an acidic environment. The structure of thiosemicarbazones has a significant
4
.0/).
Int. J. Mol. Sci. 2021, 22, 3104. https://doi.org/10.3390/ijms22063104
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 3104
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influence on biological activity. Their antiviral [
terial [3] activity is known.
1
], antidiabetic [2], antifungal, and antibac-
There are also reports in the literature on the antitumor activity of thiosemicarbazone
derivatives. Many substances are currently in clinical trials, for example: Triapine is a pow-
erful anti-cancer drug in Phase 3 clinical trials. It is an inhibitor of ribonucleotide reductase
with chelating properties [
ions easily [ ]. It has also been shown that the same compound, either in combination
with cisplatin or gemcitabine, effectively inhibits the growth of cell proliferation of various
types of cancer [ ]. According to the literature, triapine shows great potential for use in the
treatment of lung and ovarian cancers and some types of leukemia [ ]. On the other hand,
4]. It has been shown to form complex compounds with iron
5
6
7
the compound labeled Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone)
showed stronger antitumor effect than triapine and doxorubicin in in vitro studies, and
was effective in the exposure of chemo- and radio-resistant cancer cell lines [
tential of this molecule was to be used in the treatment of neuroblastoma, osteosarcoma
and prostate cancer [ ]. However, the compound showed strong cardiotoxic properties,
8]. The po-
9
therefore the search for safer analogues was started [10]. This led to the discovery of
di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone, which was found to be
effective in the treatment of neuroblastoma [11]. Recently, there have been reports on the
biological activity of thiosemicarbazone derivative complexes [12–15].
In our work, we have synthesized a new series of thiosemicarbazone derivatives. We
subjected selected compounds to complexation reaction with Cu (II) ions. We investigated
the effect of complexing on anti-cancer activity of new compounds.
2. Results and Discussion
2.1. Chemistry
Thiosemicarbazones can be classified as imine derivatives obtained by condensation reac-
tion of thiosemicarbazide derivatives with aldehydes or ketones. The title compounds T1–T19
were obtained by the classic method using thiosemicarbazides: 4-methylthiosemicarbazide, 4-
phenylthiosemicarbazide, 2-methylphenylthiosemicarbazide, 2-chlorophenylthiosemicarbazide,
3
-chlorophenylthiosemicarbazide, 3-chlorophenylthiosemicarbazide, as substrates. The
thiosemicarbazide was heated with the appropriate aldehyde or ketone (4-bromobenzaldehyde,
,4-dimethoxybenzaldehyde, 3,4-dichlorobenzaldehyde, 4-aminoacetophenone, acetophe-
3
none) for 2.5h in ethanol. The reaction was carried out according to Scheme 1. The
newly obtained compounds were identified by modern spectroscopic methods. For the
newly obtained compounds, additional mass measurements were performed by high-
performance liquid chromatography (Table S1). Compounds T1, T2, T14, T16 have been
described earlier [16–19]. Compounds T15, T18, and T19 were obtained in the reactions of
the corresponding hydrazone with an isothiocyanate [20,21].
In order to confirm assumed synthesis pathway and molecular structures of the
synthesized compounds the X-ray analysis for T2, T3, and T5 were performed. A view of
these molecules in conformation observed in the crystal is shown in Figure 1.

Int. J. Mol. Sci. 2021, 22, 3104
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Scheme 1. The route of synthesis of thiosemicarbazone derivatives (T1–T19) (
a
) and list of sub-
stituents (b).
Figure 1. The molecular structures of T2, T3, and T5 with atom label ligand displacement ellipsoids
(30% probability). The intramolecular hydrogen bond is shown as dashed line.
The C2=S3, N4–N5, and N5=C6 bond lengths of 1.686(4), 1.373(4), and 1.266(5) Å in
T2, 1.6896(13), 1.3809(14), and 1.2766(17) Å in T3 and 1.693(3), 1.375(3), and 1.272(3) Å in
T5, respectively, are characteristic for those observed in other organic molecules containing

Int. J. Mol. Sci. 2021, 22, 3104
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thiosemicarbazone system [22
,
23] and confirming that the thione tautomeric form is ob-
served in the crystalline state. In all analyzed molecules the thiosemicarbazone skeleton
N1/C2/S3/N4/N5/C6 is slightly distorted from planarity with the largest deviation from
the mean plane of 0.025(3) Å for N5 in T2, 0.0563(12) Å for N4 in T3 and 0.015(2) Å for
N4 in T5. The torsion angles C7–N1–C2–N4, N1–C2–N4–N5, C2–N4–N5–C6, N4–N5–C6–
◦
C21 and N6–C6–C21–C22 of
−
179.5(4),
−
3.7(5), 179.2 (3),
−
178.7(3) and
−
7.3(6) in T2,
◦
−
177.01(14), 5.86(19), 178.25(13),
−
177.95(12) and 2.4(2) in T3 and
−
174.5(3),
−
1.1(4),
◦
−
179.8(2),
−
179.3(2) and
−
3.6(4) in T5, respectively, show the trans-cis-trans-trans-cis
conformation of the tiosemicarbazone chain in all molecules. The cis conformation on
the C2–N4 bond favours the formation of N1–H1 . . . N5 intramolecular hydrogen bond
that stabilizes the conformation of the molecules observed in the crystal. The methoxy
groups in T3 are located almost coplanar with the plane of the benzene ring and they
have a trans conformation with respect to C23–C24 bond confirmed by the torsion angles
◦
C24–C23–O27–C28 and C23–C24–O28–C29 of −172.08(14) and −174.28(12) , respectively.
In all investigated molecules the benzene rings are planar within 0.005(4) Å in T2, 0.0121(14)
Å in T3 and 0.010(3) Å in T5 and the planes of these rings are inclined with respect to the
◦
mean planes of thiosemicarbazone functions at an angle of 9.65(11), 8.08(4) and 4.03(7) for
T2, T3, and T5, respectively, which shows that all molecules as a whole are almost flat.
The intermolecular hydrogen bond N4–H4 . . . S3 observed in the crystals of T2, T3,
and T5 (Table 1), linking the molecules in molecular dimers, stabilized the tautomeric form
occurring in the crystalline state.
◦
Table 1. Hydrogen-bond geometry (Å, ).
Compound
T2
D–H . . . A
D–H
H . . . A
D . . . A
D–H . . . A
N1–H1 . . . N5
N4–H4 . . . S3
0.78(5)
0.80(4)
2.26(6)
2.63(4)
2.646(5)
3.416(3)
111(5)
167(4)
i
i = 1 − x, −y, 1 − z;
T3
T5
N1–H1 . . . N5
0.812(18)
0.812(18)
0.855(18)
2.316(19)
2.521(18)
2.569(18)
2.6743(17)
3.1757(17)
3.4098(12)
107.5(16)
138.7(14)
168.0(15)
i
N1–H1 . . . O27
ii
N4–H4 . . . S3
i = 1 − x, −1/2 + y, 1/2 − z; ii = 2 − x, −y, −z;
N1–H1 . . . N5
0.76(4)
0.76(4)
0.78(4)
2.34(4)
2.86(4)
2.70(4)
2.649(3)
3.522(2)
3.447(2)
106(3)
146(3)
160(4)
i
N1–H1 . . . S3
ii
N4–H4 . . . S3
i = 1/2−x, −1/2 + y, 3/2−z; ii = −x, y, 2 − z;
It is worth noting that the pairs of these N4–H4 . . . S3 hydrogen bonds in the crys-
tal structure of T5 join the molecular chains parallel to Y crystallographic axis formed
by molecules related by 2 screw axis via N1–H1 . . . S3 intermolecular hydrogen bonds.
1
Similar molecular chains are observed in the crystal of T3, where molecules are linked via
N1–H1 . . . O27 hydrogen bonds. Moreover, in the crystal structure of T5, the benzene rings
belonging to the inversion related molecules overlap each other with centroid-to-centroid
separation of 3.8327(17) Å and the π . . . π distance of 3.6374(11) Å typical for the overlap-
ping π-aromatic ring systems. The structural motifs formed by intermolecular hydrogen
bonds in the crystal of T1, T2, and T3 are presented in Figure S1 (Supplementary Materials).
It can be assumed that the conformation of the thiosemicarbazone part in anal-
ysed molecules is one of the key factors related to the ability to coordinate the metal
in the metalloorganic complexes. A search of the Cambridge Structural Database (CSD;
ver. 2020.2.0,) [24
thiosemicarbazone system with N1-methyl and C6-phenyl substituents. The distribution of
the torsion angle = N1–C2–N4–N5 calculated for all molecules is shown in Figure 2a. One
,25] revealed 50 organic crystal structures and 59 molecules containing
ϕ

Int. J. Mol. Sci. 2021, 22, 3104
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can see that the cis conformation is observed in all found organic structures with the torsion
◦
angle
ϕ
in the range from
−
15.7 to +16.1 . Review of CSD exhibited also 7 metalloorganic
crystal structures and 12 molecules of Cu complexes with a model thiosemicarbazone
system as the ligands in these complexes. A certain tendency in torsion angle distribu-
ϕ
tion (Figure 2b) can be seen. For the S-monodentate ligands (9 hits) conformation cis is
preserved, while for S,N-bidentate ligands (3 hits) the trans conformation is observed.
Figure 2. Histogram of torsion angle
ϕ = N1–C2–N4–N5 in thiosemicarbazide system for (a) organic
molecules and (b) ligands in Cu complexes deposited in CSD.
The conformational preferences of the T2, T3 and T4 were investigated by calculation
of the energy effect of the free-rotation at the C2–N4 bond using the AM1 method. The
energies of conformations were minimized, and all geometrical parameters optimized
◦
◦
for each rotation with a 10 increment from
−
180 to 180 of torsion angle ϕ (Figure 3).
As can be seen in Figure 3 two minima of energy are observed but the minimum energy
corresponding to cis conformation (
◦
ϕ
= 0 ) is about 10 kcal/mol deeper than that of trans
◦
conformation (ϕ = ± 160 ). This energy difference is due to the possibility of the N–H . . .
N intramolecular hydrogen bond formation stabilizing the cis conformation. Changing
from cis to trans conformation requires an energy input of about 11 kcal/mol, which may
confirm the preference of thiosemicarbazone system to act as S-monodentate and does
not exclude N,S-bidentate ligands in Cu complexes. It should be emphasized that the
presented results of conformational analysis do not depend on the nature of substituents in
benzene ring and are consistent with the data obtained from X-ray investigations.
Figure 3. The energy effect of the free rotation for the torsion angle
T2, T3, and T5 using the semiempirical AM1 method.
ϕ = N1–C2–N4–N5 calculated for
Then, selected ligands from the group of thiosemicarbazone derivatives were sub-
jected to complexation reaction with copper(II) ions. In order to identify the obtained
compounds, FTIR spectra of the organic ligands and their copper(II) coordination com-
pounds were recorded Figures S2–S6 (Supplementary Materials). The
visible in the spectra of ligands in the range 3334–3246 cm . However, they do not show a
ν
(NH) bands are
−
1

Int. J. Mol. Sci. 2021, 22, 3104
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(S-H) band at ~ 2570 cm 1, but only strong bands for
−
ν
(NH), indicating that in the solid
ν
state Schiff bases are mainly in the tautomeric thione form. The
δ(NH) modes are also
−
1
present in the spectrum of all compounds. They are in the range: 1555–1492 cm for free
−
1
ligands and 1552–1499 cm for coordination compounds. The bands of ν(N-N) appear
−
1
in the range 1031–1010 cm for Schiff bases and they do not move in the case of com-
plexes. For uncoordinated ligands, the CH stretching vibrations are observed in the range
−
1
−1
γ(CH) in the ranges: 1277–1114 cm
3
145–2827 cm and also the modes of
β
(CH) and
−
1
and 799–703 cm , respectively. In the spectra of complexes, they are visible as follows:
(CH) 3167–2828 cm
of organic ligands there are several bands ascribed to
−
1
−1
−1
ν
,
β
(CH) 1278–1128 cm , and
γ
(CH) 774–718 cm . In all spectra
(C=S). Due to the coordination of
ν
the sulphur atom with the metal ion, the spectra in this range become less vibrating. The
disappearance of most of these bands in all complexes indicates formation of metal-sulphur
bonds. For complexes Cu(T10) Cl and Cu(T12) Cl the bands of
1
ν
(CN) are in the range
595–1579 cm and they are in the similar range as for uncoordinated ligands. For these
compounds, the organic ligands adopt only thione form. In the case of T1 and T16 two
strong absorption bands of (CN) become one in the complexes. However, for Cu(T13)Cl
one (CN) mode is shifted to higher wavenumbers (11 cm ) as a result of coordination.
Based on these data, it is likely that the Schiff bases (T1, T13 and T16) in these complexes
coordinate via both the thiolate sulphur and azomethine nitrogen atoms [26 27]. Thus, it
2
2
2
2
−
1
ν
2
−
1
ν
,
is clear from FTIR data that the Schiff bases bond with metal ions in different ways: as
monodentate (T10, T12) and chelating (T1, T13, T16) ligands.
2.2. Thermal Decomposition
The thermal decompositions of described complexes have been studied in air by TG-
DTG method. Pyrolysis of analyzed complexes in air is a multistage, overlapping process
and complicated to interpret. Figures S7–S11 (Supplementary Materials) show thermal
profiles of investigated complexes. Table 2 presents thermal decomposition results.
Table 2. TG-DTG analysis data for decomposition processes of copper(II) complexes.
Mass Loss (%)
Range of
Decomposition (°C)
Intermediate Solid Product or
Compound
Found
Calc.
33.45
42.83
Residue
Cu(T1)Cl2
120–350
34.0
350–680
43.0
CuO
Cu(T10)2Cl2
120–360
45.0
43.0
44.51
42.67
360–720
CuO

Int. J. Mol. Sci. 2021, 22, 3104
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Table 2. Cont.
Range of
Mass Loss (%)
Intermediate Solid Product or
Compound
Decomposition (°C)
Residue
Found
Calc.
Cu(T12)2Cl2
150–260
37.5
37.33
2
60–720
56.0
33.5
55.20
33.59
CuO
Cu(T13)Cl2
120–520
CuO + volatile copper halide
derivatives
520–800
61.5
50.60
Cu(T16)Cl2
120–360
32.0
47.5
31.01
46.90
360–780
CuO
◦
Cu(T1)Cl complex is stable up to 120 C. The first step of decomposition takes place
2
◦
in the temperature range of 120–350 C and is associated with partial destruction of an
organic ligand (mass loss: found. 34.0%, calc. 33.45%). From this temperature up to 680 C
◦
a total decomposition of complex (mass loss: found. 43.0%, calc. 42.83%) takes place. Final
solid product of decomposition is copper(II) oxide.
◦
◦
Decomposition of Cu(T10) Cl complex begins at 120 C. In the range 120–360 C the
2
2
first step of pyrolysis of an organic ligand (mass loss: found. 45.0%, calc. 44.51%) occurs.
◦
In the temperature range of 360–720 C there is a total destruction of complex (mass loss:
◦
found. 43.0%, calc. 42.67%). Final solid product appears above 720 C.
◦
Cu(T12) Cl starts to decompose at 150 C. Above this temperature decomposition of
2
2
an organic ligand begins (mass loss: found. 37.5%, calc. 37.33%). Total destruction takes
◦
place in the temperature range of 260–720 C with mass loss found 56.0% and calculated
◦
5
5.20. Final solid product of thermolysis, copper(II) oxide, is created above 720 C.
◦
◦
Decomposition of Cu(T13)Cl complex begins at 120 C. Between 120 and 520 C there
2
is first mass loss on TG curve (mass loss: found. 33.5%, calc. 33.59%) which is connected
◦
with partial destruction of organic ligand. When the temperature rises (520–800 C) further

Int. J. Mol. Sci. 2021, 22, 3104
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pyrolysis takes place (mass loss: found. 61.5%, calc. 50.60%). The process stops above
◦
8
00 C.
◦
◦
Cu(T16)Cl complex is stable up to 120 C. In the temperature ranges: 120–360 C and
60–780 C partial and total degradation occurs. The mass losses connected with these
2
◦
3
processes are: found. 32.0%, calc. 31.01% and found. 47.5%, calc. 46.90%, respectively. A
◦
constant mass level appears above 780 C.
According to the literature data, thiosemicarbazones are N, N, and S-donor ligands,
therefore they show a high ability to form coordinate bonds with metal ions [28]. There is a
theory that the antiproliferative activity of the group of thiosemicarbazones is due to this
ability, while the strength of the activity of the coordination compounds formed is higher
than that of the ligands [29–32].
2.3. Biological Assays
Cytotoxicity Analyses
Newly synthetized compounds were screened for anti cancer activity using in vitro
methods. Three melanoma cel lines and normal human fibroblast were treated with
thiosemicarbazone derivatives T1–T19 and Cu (II) complex of thiosemicarbazone deriva-
tives CuT1, CuT10, CuT12, CuT13, and CuT16. For comparison, cells were treated with
dacarbazine (DTIC)—a drug that is used in melanoma standard treatment.
Compounds T1–T19 revealed weak cytotoxic activity towards melanoma cell lines
or the compounds were toxic at the same level to melanoma cells and normal fibroblasts.
However in case of Cu (II) complexes of obtain derivatives, analysis of IC50 values revealed
that CuT1, CuT10, and CuT16 were toxic for A375 cells and at the same time less toxic for
normal cells. CuT12 and CuT13 showed similar activity towards malignant and normal cell
(
IC50 values for selected derivatives and their corresponding Cu (II) complexes were sum-
marized in Table 3). On the basis of the data received, CuT1, CuT10, and CuT16 have been
choosen for further analysis using A375 cell precise cytotoxicity curves weres presented on
Figure 4). It is worth noting that IC50 values obtained for selected compounds were lower
than IC values for drug that is used in melanoma treatment—dacarbazine (DTIC).
Table 3. IC50 values obtained for tested compounds and dacarbazine (DTIC) based on 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT test) results after 24 h treatment of
melanoma and normal fibroblast cell lines.
IC50 [µM]
BJ
G361
A375
SK-MEL-28
T1
CuT1
T10
CuT10
T12
CuT12
T13
357.64 ± 7.85
257.12 ± 4.45
402.17 ± 12.98
202.88 ± 8.67
128.87 ± 7.09
42.87 ± 1.17
412.75 ± 3.23
130.18 ± 3.45
>500
488.07 ± 1.66
135.64 ± 7.01
370.17 ± 9.34
251.19 ± 11.08
177.35 ± 4.09
157.17 ± 2.75
395.75 ± 6.74
148.07 ± 9.77
>500
320.99 ± 2.85
69.92 ± 1.87
201.77 ± 4.87
26.05 ± 1.75
111.76 ± 12.75
40.49 ± 0.98
388.64 ± 7.06
109.78 ± 2.95
>500
>500
129.51 ± 3.98
<500
132.98 ± 8.34
99.64 ± 5.76
46.13 ± 2.74
398.86 ± 4.64
118.60 ± 9.75
>500
CuT13
T16
CuT16
DTIC
450.09 ± 11.74
>500
425.98 ± 4.74
144.60 ± 19.85
478.31 ± 11.02
>500
412.77 ± 7.08
370.12 ± 9.46

Int. J. Mol. Sci. 2021, 22, 3104
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Figure 4. Viability of A375 cells, treated with CuT1, CuT10, and CuT16 compound or DMSO as
vehicle in control culture for 24 h (determined by MTT test). Data presented as % of control culture
viability (mean ± SD).
The MTT test results for selected compounds were verified by microscopic observa-
tions of cell morphology. The vehicle treated control cells revealed normal, epithelial-like
morphology and were closely arranged and well adherent. Cells treated with CuT1, CuT10,
and CuT16 in concentrations corresponding to IC50 values for 24 h became round and had
poor adherence. A large part of the cells floated in the culture medium (Figure 5).
Figure 5. Morphology of A375 cells treated with CuT1, CuT10, and CuT16 compound in concentration corresponding to
appropriate IC50 value (69.92, 26.05, and 144.60 µM, respectively) or DMSO as vehicle in control culture.

Int. J. Mol. Sci. 2021, 22, 3104
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Apoptosis/necrosis analysis with image cytometry revealed that A375 cell treated
with tested compounds were dying exclusively by apoptosis. The largest number of cells
in the early and late stage of apoptosis after 24 h incubation was observed in case of CuT1
treatment. The fewest apoptotic cells were observed after treatment with CuT10, most of
which were in the early phase (Figure 6).
Figure 6. Apoptosis/necrosis detection by image cytometry. A375 cells were treated with CuT1, Cu10, CuT16 in concen-
trations corresponding to IC50 values (69.92, 26.05, and 144.60 M respectively) or DMSO as vehicle in control cultures.
µ
The results show one representative experiment of three independently performed. Q1II—live, Q1Ir—early apoptotic,
Q1ur—late apoptotic, and Q1uI—necrotic cells.
Cytotoxicity studies have shown that A375 melanoma cell line was the most sensi-
tive to the tested compounds. The identification of a specific feature that differs this cell
line from the others might have contributed to the identification of the potential mech-
anism of action of the tested compounds. A375 and G361 have been described as low
metastatic melanoma and SK-MEL-28 as highly metastatic melanoma cell line [33,34]. In
accordance with Kim et al. results [35] A375 has intermediate migratory and invasive
potential compared to G361 and SK-HEP-28. All three cell lines express mutant B-Raf
gene. Analysis of published data revealed that A375 cells displaying low NAD(P)H:
quinone oxidoreductase (NQO1) expression levels are sensitive to prooxidant agent (2,6-
dichlorophenolindophenol) in contrast to G361 cells with high expression of NQO1 [36].
According to Sauviago et al. [37], who studied DNA repair signatures of the different

Int. J. Mol. Sci. 2021, 22, 3104
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melanoma cell lines, A375 is characterized by low DNA repair capacities in contrast to
SK-MEL-28 cell line that has high DNA repair capacities.
Greater sensitivity of A375 to oxidative stress and DNA damage was confirmed by
the unpublished results of our team’s research, in which cells of three melanoma lines were
exposed to X-rays at a dose of 10 Gy. Cell cycle analysis of irradiated cells revealed that
after 48 h only A375 cells were arrested in G2/M phase of the cell cycle (Figure 7).
Figure 7. Representative histograms of cell cycle analysis obtained for G361, A375, and SK-MEL-28
melanoma cells 48 h after X-ray exposure (10 Gy, M1: subG1, M2: G1, M3: S, M4: G2/M phase).
The absorption of ionizing radiation by living cells can directly damage the molecules,
causing chemical and biological changes or indirectly through water radiolysis products—
free radicals that can damage nucleic acids, proteins, and lipids [38]. The majority of DNA
damage after IR exposure is caused by IR-generated reactive oxygen species (ROS) [39].
Cell cycle analysis of A375 cells treated with tested compounds similarly revealed
G2/M phase arrest (Figure 8).

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Figure 8.
4 h with CuT1, CuT10, and CuT16 compounds in concentration corresponding to appropriate IC50 value (69.92, 26.05,
and 144.60 M respectively) or DMSO as vehicle in control cultures. Data presented as mean SD derived from three
independent experiments. (B) Representative histograms (M1: subG1, M2: G1, M3: S, M4: G2/M phase).
(A) Cell cycle analysis based on DAPI staining and image cytometry measurement. A375 cells were treated for
2
µ
±
G2/M phase arrest is connected to DNA repair attempt of damaged cells before mito-
sis phase. DNA damage agents (including prooxidative agents) can trigger a G2 checkpoint
that results in a delay in the activation of cyclin B/Cdc2 kinase activity at the G2/M bor-
der [40]. The numbers of basic sites (AP sites) were measurement as a marker of DNA
damage. AP sites can be formed by spontaneous depurination, but also occur as a result of
oxidative damage. Hydroxyl radicals can attack the deoxyribose moiety and lead to the
release of free bases, directly creating an AP site [41]. Moreover, these lesions are interme-
diates in base excision repair pathway where they are formed by DNA glycosylases [42].
In the DNA of cells treated with all three compounds, AP sites number was significantly
elevated. However, in case of CuT10 it was only 2.043 ± 0.02 AP/100 kbp and for CuT1
and CuT16—20.24
DNA of control cells (Figure 9).
±
0.19 and 14.09
± 0.44, respectively, vs. 0.67 ± 0.08 AP/100 kbp in

Int. J. Mol. Sci. 2021, 22, 3104
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Figure 9. The amount of AP sites/100 kbp in DNA of A375 cells treated for 24 h with CuT1, CuT10,
and CuT16 compound in concentration corresponding to appropriate IC50 value (69.92, 26.05, and
1
44.60
µ
M, respectively) or DMSO as vehicle in control cultures. Data presented as mean
±
SD
derived from three independent experiments.
In the next step expression analysis of DNA damage and oxidative stress response
genes was evaluated. Despite similar histograms of cell cycle analysis, there were not
similar pattern of tested genes expression (Table 4). Only ATM transcript level was elevated
in samples from cells treated with all three compounds. ATM is a key sensor of double
strand breaks in DNA (DSBs) in G1 and G2 phase of cell cycle that signalizes this lesion
presence to downstream effectors. ATM has been also hypothesized to play a vital role
in the response to oxidative stress [40
,43]. Thus, obtained results suggest that tested
compounds may induce DNA damage probably resulted from oxidative stress.
Table 4. The results of mRNA gene expression. Significantly changed mean RQ levels are marked with a color scale.
Control CuT1 CuT10 CuT16
Mean Mean Mean Mean
Gene symbol
Scale (RQ)
SD
SD
SD
SD
RQ
RQ
RQ
RQ
DNA damage response genes
ERCC
MGMT
OGG1
MLH1
PARP1
ATM
1.001
1.007
1.011
1.002
1.011
1.005
0.951
1.003
0.066
0.147
0.185
0.078
0.193
0.127
0.044
0.094
0.401
0.715
0.902
1.001
0.908
1.452
1.038
0.973
0.010
0.088
0.022
0.163
0.056
0.102
0.087
0.100
1.215
0.448
0.264
0.337
0.342
2.038
1.456
1.337
0.155
0.024
0.071
0.057
0.108
0.166
0.112
0.436
0.267
0.361
0.952
0.929
0.640
2.724
1.438
0.936
0.036
0.053
0.160
0.068
0.069
0.295
0.244
0.093
>2.00
1.51–1.99
1.11–1.50
0.91–1.10
0.51–0.90
0.31–0.5
<0.31
XPC
MSH2
oxidative stress response genes
CAT
GPX2
SOD2
1.003
1.010
1.009
0.101
0.184
0.173
0.773
0.000
1.510
0.044
0.000
0.114
0.801
0.022
1.430
0.065
0.000
0.082
0.387
0.022
1.393
0.056
0.002
0.151
The lower AP sites level in cells treated with CuT10 can possibly be connected with
decreased expression of 8-oxoguanine-DNA glycosylase 1 (OGG1, RQ = 0.264 0.071).
±
OGG1, is the major enzyme for repairing 8-oxoguanine (8-oxoG), a critical mutagenic DNA
lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from
an 8-oxoG, leaving AP sites as the major product [44].
Analysis of oxidative stress response genes revealed that in all samples superoxide
dismutase 2 (SOD2) expression was elevated and catalase (CAT) and glutathione peroxidase
1
(GPX1) was downregulated, where the expression of GPX has decreased to an almost

Int. J. Mol. Sci. 2021, 22, 3104
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indeterminate level (Table 3). It is difficult to determine how the studied complexes of
thiosemicarbazone derivatives affect the expression of these genes but regardless of the
mechanism of this phenomenon, that expression profile may suggest that SOD is activated
in response to oxidative stress (superoxide anion presence), but its product, hydrogen
peroxide, cannot be effectively deactivated due to the absence of GPX (unable GSH–GSSG
cycling) and lower level of catalase that mediates the breakdown of hydrogen peroxide to
water and oxygen.
It was revealed that copper compounds can act by different mechanisms. One of them
is generation of ROS by redox-cycling, that lead to an oxidative cell damage [45]. Obtained
results suggest that anticancer activities of tested compounds result from oxidative damages
of DNA connected with reduced ability to ROS neutralization.
3. Materials and Methods
3.1. Chemistry
All chemicals used for the synthesis were purchased from the companies Sigma-
Aldrich, AlfaAesar, POCH and used without further purification. Melting points of ob-
tained compounds were determined using Fisher-Johns block and presented without
1
l3
corrections. The H and C NMR spectra were recorded on a Bruker Avance 600 spectrom-
eter (Bruker BioSpin GmbH, Rheinstetten, Germany) in DMSO-d6. The chemical shifts are
given in δ (ppm) scale using TMS as the standard reference. The attenuated total reflectance
−
1
ATR-IR spectra were recorded over the range 4000–400 cm on the Thermo Scientific Nico-
let 6700 FTIR spectrophotometer. LC-QTOF HRMS measurements were performed using
1290 HPLC coupled to 6550 ifunnel QTOF LC/MS (Agilent Technologies, Santa Clara, CA,
USA). MS data and MS spectrograms are presented in Supplementary Material (Table S1,
Figures S2 and S3). The content of Cu (II) in solid complex was determined by the F-AAS
spectrometer with a continuum source of light and using air/acetylene flame (Analityk
Jena, contraAA 300, Jena, Germany). Absorbance was measured at analytical spectral line
3
24.7 nm. Limit of quantification was 0.04 mg/L. Solid sample was decomposed using
the Anton Paar Multiwave 3000 closed system instrument. Mineralization was carried
◦
out for 45 min at 240 C under pressure 60 bar. The contents of carbon, hydrogen and
nitrogen were determined by a Vario micro company Elementar Analysensysteme GmbH.
−
1
FTIR spectra were recorded with an IRTracer-100 Schimadzu Spectrometer (4000–600 cm
)
−
1
with accuracy of recording 1 cm using KBr pellets. The thermolysis of compounds in air
atmosphere were studied by TG-DTG techniques in the range of temperature 25–800 C at
◦
◦
−1
a heating rate of 10 C
·
min ; TG and DTG curves were recorded on Netzsch TG 209 appa-
−
1
ratus under air atmosphere v = 20 mL
material ceramic crucibles were used.
·
min using ceramic crucibles, and as a reference
3.1.1. General Procedure for the Synthesis of Thiosemicarbazone Derivatives (T1–T19)
Thiosemicarbazone derivatives (T1–T19) were synthesized according to the general
procedure by condensation of a thiosemicarbazide (0.01 mol) with an appropriate aldehyde
or ketone (0.01 mol) in ethanol. Thiosemicarbazide was dissolved in ethanol while warm
and the carbonyl compound dissolved in ethanol was added to the warm solution. The
resulting mixture was heated to reflux for 1–2 h. Then, the reaction mixture was cooled
to room temperature, the resulting precipitate was filtered off and, after drying, it was
crystallized from ethanol.
3
.1.2. T1. 2-[(3,4-Dimethoxyphenyl)methylidene-N-phenyl- hydrazine-1-carbothioamide
◦
1
C H N O S (315.39 g/mol), yield: 86%, m.p. 161–163 C. H NMR (DMSO-d ) δ:
16
17
3
2
6
3
.80 (s, 3H, CH ), 3.83 (s, 3H, CH ), 6.99–7.58 (m, 8H, CHaromat), 8.09 (s, 1H, CH), 10.03 (s,
3
3
13
1H, NH), 11.74 (s, 1H, NH). C NMR (DMSO-d6) δ: 56, 56, 109, 111, 122, 125, 126, 127, 128,
−
1
1
ν
39, 143, 149, 151, 176 [16]. FTIR spectra (KBr, cm ):
(CN) 1598, 1573; (NH) 1540, 1507; (CH) 1266, 1238, 1194, 1160, 1136;
76–862; γ(CH) 790, 755, 732.
ν
(NH) 3334, 3311;
ν
(CH) 3145–2834;
δ
β
ν
(NN) 1021; (CS)
ν
9

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3
.1.3. T2. 2-[(4-Bromophenyl)methylidene-N-methyl- hydrazine-1-carbothioamide
◦
1
C H N SBr (272.16 g/mol), yield: 81%, m.p. 181–183 C. H NMR (DMSO-d ) δ:
9
10
3
6
3
8
.02–3.03 (d, J = 4.8 Hz, 3H, CH ), 7.59–7.61 (m, 2H, CHaromat), 7.75–7.77 (m, 2H, CHaromat),
3
13
.01 (s, 1H, CH), 8.57–8.58 (m, 1H, NH), 11.55 (s, 1H, NH). C NMR (DMSO-d6) δ: 31, 123,
−
1
129, 132, 134, 140, 178. IR cm : 3152, 2952, 1602 [17].
3
.1.4. T3. 2-[(3,4-Dimethoxyphenyl)methylidene-N-methyl- hydrazine-1-carbothioamide
◦
1
C H N O S (253.32 g/mol), yield: 89%, m.p. 171–173 C. H NMR (DMSO-d )
11
15
3
2
6
δ
: 3.04–3.05 (d, J = 4.8 Hz, 3H, CH ), 3.79 (s, 3H, CH ), 3.83 (s, 3H, CH ), 6.96–6.98 (d,
3 3 3
J = 8.4 Hz, 1H, CHaromat), 7.19–7.20 (d, J = 1.8 Hz, 1H, CHaromat), 7.46–7.47 (d, J = 1.8 Hz, 1H,
1
3
CHaromat), 7.98 (s, 1H, CH), 8.42–8.43 (q, J = 4.2 Hz, 1H, NH), 11.39 (s, 1H, NH). C NMR
−
1
(
DMSO-d6) δ: 31, 56, 109, 111, 122, 127, 142, 149, 151, 177. IR cm : 3351, 2990, 1601 [46].
LC-QTOF HRMS (m/z): calculated monoisotopic mass: 253.0885, measured monoisotopic
mass: 253.0889.
3
.1.5. T4. 2-[1-(4-Aminophenyl)ethylidene]-N-methyl-hydrazine-1-carbothioamide
◦
1
C H N S (222.31 g/mol), yield: 87%, m.p. 170–172 C. H NMR (DMSO-d )
δ: 2.18
10
14
4
6
(
s, 3H, CH ). 3.00 (s, 3H, CH ), 3.83 (s, 3H, CH ), 5.46 (s, 2H, CH ), 6.54–6.55 (q, J = 8.4 Hz,
3
3
3
2
2H, CHaromat), 7.63–7.64 (d, J = 8.4 Hz, 2H, CHaromat), 8.26–8.27 (d, J = 4.2 Hz, 1H, NH), 9.96
(s, 1H, NH). LC-QTOF HRMS (m/z): calculated monoisotopic mass: 222.0939, measured
monoisotopic mass: 222.0944.
3
.1.6. T5. 2-[(3,4-Dichlorophenyl)methylidene]-N-methyl- hydrazine-1-carbothioamide
◦
1
C H N SCl (262.26 g/mol), yield: 80%, m.p. 192–194 C. H NMR (DMSO-d ) δ:
9
9
3
2
6
3
.02 (s, 3H, CH ), 7.66–7.99 (m, 3H, CHaromat), 8.21 (s, 1H, CH), 8.69 (q, J = 4.2 Hz, 1H,
3
13
−1
:
NH), 11.64 (s, 1H, NH). C NMR (DMSO-d6)
150, 2930, 1587 [18]. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 260.9894,
measured monoisotopic mass: 260.9896.
δ
: 31, 128, 131, 132, 135, 139, 178. IR cm
3
3
.1.7. T6. 2-[(4-Bromophenyl)methylidene]-N-(2-methylphenyl)hydrazine-
1
-carbothioamide
◦
1
C H N SBr (348,26 g/mol), yield: 85%, m.p. 178–180 C. H NMR (DMSO-d ) δ:
15
14
3
6
2
.23 (s, 3H, CH ), 7.21–7.28 (m, 4H, CHaromat), 7.61–7.88 (m, 4H, CHaromat), 8.10 (s, 1H, CH),
3
−
1
1
0.05 (s, 1H, NH), 11.58 (s, 1H, NH). IR cm : 3150, 2930, 1587. LC-QTOF HRMS (m/z):
calculated monoisotopic mass: 347.0092, measured monoisotopic mass: 347.0096.
3
.1.8. T7. 2-[(3,4-Dimethoxyphenyl)methylidene]-N-(2-methylphenyl)hydrazine-
1
-carbothioamide
◦
1
C H N O S (329.41 g/mol), yield: 85%, m.p. 205–206 C. H NMR (DMSO-d ) δ:
17
19
3
2
6
2
.25 (s, 3H, CH ), 3.80 (s, 3H, CH ), 3.82 (s, 3H, CH ), 6.98–6.99 (m, 1H, CHaromat), 7.21–7.24
3
3
3
(
1
m, 4H, CHaromat), 7.28–7.29 (m, 1H, CHaromat), 7.35–7.37 (m, 1H, CHaromat), 7.59–7.60 (m,
H, CHaromat), 8.08 (s, 1H, CH), 9.87 (s, 1H, NH), 11.70 (s, 1H, NH). LC-QTOF HRMS (m/z):
calculated monoisotopic mass: 329.1198, measured monoisotopic mass: 329.1202.
3
.1.9. T8. 2-[(3,4-Dichlorophenyl)methylidene]-N-(2-methylphenyl)hydrazine-
1
-carbothioamide
◦
1
C H N SCl (338,25 g/mol), yield: 84%, m.p. 200–202 C. H NMR (DMSO-d )
15
13
3
2
6
δ
: 2.24 (s, 3H, CH ), 7.22–7.30 (m, 4H, CHaromat), 7.65–7.67 (m, 1H, CHaromat), 7.77–7.78
3
(
(
m, 1H, CHaromat), 7.79–8.10 (m, 1H, CHaromat), 8.37 (s, 1H, CH), 10.15 (s, 1H, NH), 11.94
s, 1H, NH). LC-QTOF HRMS (m/z): calculated monoisotopic mass: 337.0207, measured
monoisotopic mass: 337.0211.

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3
.1.10. T9. 2-[(4-Bromophenyl)methylidene]-N-(2-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N SBrCl (368,68 g/mol), yield: 86%, m.p. 209–211 C. H NMR (DMSO-d )
14
11
3
6
1
3
δ
: 7.31–7.86 (m, 8H, CHaromat), 8.13 (s, 1H, CH), 10.15 (s, 1H, NH), 12.04 (s, 1H, NH).
C
−
1
NMR (DMSO-d6)
δ
: 123, 127, 128, 129, 130, 131, 132, 133, 137, 142, 177. IR cm : 3137,
2
970, 1738. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 366.9546, measured
monoisotopic mass: 366.9545.
3
.1.11. T10. 2-[(3,4-Dimethoxyphenyl)methylidene-N-(2-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N O SCl (349.84 g/mol), yield: 78%, m.p. 179–180 C. H NMR (DMSO-d ) δ:
16
16
3
2
6
3
.80 (s, 3H, CH ), 3.82 (s, 3H, CH ), 7.00–7.86 (m, 7H, CHaromat), 8.10 (s, 1H, CH), 10.03
3
3
13
(s, 1H, NH), 11.94 (s, 1H, NH). C NMR (DMSO-d6) δ: 56, 109, 111, 123, 126, 127, 128,
−
1
1
ν
29, 130, 136, 143, 149, 151, 176. FTIR spectra (KBr, cm ):
(CN) 1595, 1579; (NH) 1552, 1507; (CH) 1267, 1238, 1194, 1162, 1134;
78–858; (CH) 799, 753, 723. LC-QTOF HRMS (m/z): calculated monoisotopic mass:
ν
(NH) 3246;
ν
(CH) 3144–2827;
δ
β
ν
(NN) 1023; (CS)
ν
9
3
γ
49.0652, measured monoisotopic mass: 349.0653.
3
.1.12. T11. N-(2-Chlorophenyl)-2-(1-phenylethylidene)hydrazine-1-carbothioamide
◦
1
C H N SCl (303.80 g/mol), yield: 78%, m.p. 115–117 C. H NMR (DMSO-d )
δ
: 2.41
15
14
3
6
1
3
(
(
(
s, 3H, CH ), 7.28–8.01 (m, 8H, CHaromat), 10.12 (s, 1H, NH), 10.89 (s, 1H, NH). C NMR
DMSO-d6)
m/z): calculated monoisotopic mass: 303.0597, measured monoisotopic mass: 303.0601.
3
−
1
δ: 125, 126, 127, 129, 136, 179. IR cm : 3599, 2970, 1594. LC-QTOF HRMS
3
.1.13. T12. 2-[(3,4-Dichlorophenyl)methylidene]-N-(2-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N SCl (358.67 g/mol), yield: 81%, m.p. 210–211 C. H NMR (DMSO-d ) δ:
14
10
3
3
6
1
3
7
.33–8.11 (m, 7H, CHaromat), 8.34 (s, 1H, CH), 10.24 (s, 1H, NH), 12.11 (s, 1H, NH).
C
−
1
NMR (DMSO-d6)
(NH) 3315, 3258;
114; (NN) 1031;
δ
: 127, 128, 129, 131, 132, 135, 137, 140, 177. FTIR spectra (KBr, cm ):
(CN) 1591; (NH) 1545, 1512; (CH) 1265, 1204, 1197,
(CH) 787, 736, 723. LC-QTOF HRMS (m/z): calculated
ν
ν
(CH) 3140–2983;
ν
δ
β
1
ν
ν
(CS) 960–825;
γ
monoisotopic mass: 356.9661, measured monoisotopic mass: 356.9654.
3
.1.14. T13. 2-[(4-Bromophenyl)methylidene]-N-(3-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N SBrCl (368.68 g/mol), yield: 86%, m.p. 162–164 C. H NMR (DMSO-d )
14
11
3
6
1
3
δ
: 7.26–7.89 (m, 8H, CHaromat), 8.14 (s, 1H, CH), 10.23 (s, 1H, NH), 12.02 (s, 1H, NH).
C
−
1
NMR (DMSO-d6)
(NH) 3330; (CH) 3128–2972;
129; (NN) 1009; (CS) 964–867;
monoisotopic mass: 366.9546, measured monoisotopic mass: 366.9551.
δ
: 123, 124, 125, 130, 132, 132, 133, 141, 142, 176. FTIR spectra (KBr, cm ):
(CN) 1578; (NH) 1536, 1499; (CH) 1277, 1253, 1204,
(CH) 780, 746, 703. LC-QTOF HRMS (m/z): calculated
ν
ν
ν
δ
β
1
ν
ν
γ
3
.1.15. T14. 2-[(4,5-Dimethoxyphenyl)methylidene-N-(3-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N O SCl (349.84 g/mol), yield: 80%, m.p. 180–182 C. H NMR (DMSO-d ) δ:
16
16
3
2
6
3
.81 (s, 3H, CH ), 3.84 (s, 3H, CH ), 6.99–7.79 (m, 7H, CHaromat), 8.11 (s, 1H, CH), 10.10 (s,
3
3
13
1
H, NH), 11.88 (s, 1H, NH). C NMR (DMSO-d )
δ:. 56, 109, 111, 122, 124, 125, 126, 130,
6
−
1
132, 141, 144, 149, 151, 175. IR cm : 3140, 2834, 1597 [18].
3
.1.16. T15. N-(3-Chlorophenyl)-2-[(3,4-dichlorophenyl)methylidene]hydrazine-
1
-carbothioamide
◦
1
C H N SCl (358.67 g/mol), yield: 77%, m.p. 210–212 C. H NMR (DMSO-d ) δ:
14
10
3
2
6
7.28–8.12 (m, 7H, CHaromat), 8.33 (s, 1H, CH), 10.30 (s, 1H, NH), 12.09 (s, 1H, NH) [20].

Int. J. Mol. Sci. 2021, 22, 3104
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3
.1.17. T16. 2-[(4,5-Dimethoxyphenyl)methylidene-N-(4-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N O SCl (349.84 g/mol), yield: 76%, m.p. 200–202 C. H NMR (DMSO-d ) δ:
16
16
3
2
6
3
.80 (s, 3H, CH ), 3.83 (s, 3H, CH ), 6.99–7.64 (m, 7H, CHaromat), 8.10 (s, 1H, CH), 10.07 (s,
3
3
13
1
1
1
H, NH), 11.83 (s, 1H, NH). C NMR (DMSO-d )
49, 151, 176 [19]. FTIR spectra (KBr, cm ):
599, 1574; (NH) 1545, 1505;
δ
: 56, 109, 111, 122, 126, 128, 129, 138, 144,
(NH) 3331, 3326; (CH) 3142–2828; (CN)
(CH) 1265, 1235, 1210, 1140; (NN) 1019; (CS) 976–832;
6
−
1
ν
ν
ν
δ
β
ν
ν
γ(CH) 795, 768, 746, 716.
3
.1.18. T17. N-(4-Chlorophenyl)-2-[(3,4-dichlorophenyl)methylidene]hydrazine-
1
-carbothioamide
◦
1
C H N SCl (358.67 g/mol), yield: 83%, m.p. 202–204 C, H NMR (DMSO-d ) δ:
14
10
3
3
6
7
.43–8.12 (m, 7H, CHaromat), 8.34 (s, 1H, CH), 10.29 (s, 1H, NH), 12.05 (s, 1H, NH). 13C
−
1
NMR (DMSO-d6)
δ
: 128, 129, 130, 131, 132, 135, 138, 141, 177. IR cm : 3122, 2970, 1589.
LC-QTOF HRMS (m/z): calculated monoisotopic mass: 356.9661, measured monoisotopic
mass: 356.9664.
3
.1.19. T18. 2-[(4-Bromophenyl)methylidene]-N-(4-chlorophenyl)hydrazine-
1
-carbothioamide
◦
1
C H N SBrCl (368.68 g/mol), yield: 89%, m.p. 182–184 C. H NMR (DMSO-d ) δ:
14
11
3
6
1
3
7
(
.42–7.89 (m, 8H, CHaromat), 8.13 (s, 1H, CH), 10.21 (s, 1H, NH), 11.98 (s, 1H, NH). C NMR
−
DMSO-d ) δ: 123, 128, 129, 130, 132, 133, 138, 142, 176, IR cm : 3128, 2971, 1589 [20].
6
1
3
.1.20. T19. N-(3-Chlorophenyl)-2-(1-phenylethylidene) hydrazine-1-carbothioamide
◦
1
C H N SCl (303,81 g/mol), yield: 80%, m.p. 162–165 C. H NMR (DMSO-d ) δ:
15
14
3
6
2
.39 (s, 3H, CH ), 7.26–7.69, 8,02 (m, 9H, CHaromat), 10.11 (s, 1H, NH), 10.77 (s, 1H, NH).
3
1
3
−1
C NMR (DMSO-d ) δ: 122, 123, 124, 130, 132, 141, 176. IR cm : 3138, 2970, 1586 [21].
6
3.2. X-ray Structure Determination
X-ray data of T2, T3 and T5 were collected on the KUMA Diffraction KM-4 CCD
diffractometer; MoK
.50 × 0.50 × 0.10 mm for T2, 0.50
for T5, absorption correction: multi-scan CrysAlisPro [47], Tmin/Tmax of 0.0054/1.0000,
.7525/1.0000 and 0.2426/1.0000 for T2, T3, and T5, respectively. The structures were
α
(
λ
= 0.71073 Å) radiation,
ω
scans, T = 296(2) K; crystal sizes
0
×
0.50 0.30 mm for T3 and 0.50
×
×
0.40 0.30 mm
×
0
solved by direct methods using SHELXS97 [48] and refined by full-matrix least-squares
with SHELXL-2014/7 [48]. The N-bound H atoms were located by difference Fourier
synthesis and refined freely. The remaining H atoms were positioned geometrically and
treated as riding on their parent C atoms with C-H distances of 0.93 Å (aromatic) and 0.96 Å
(
CH ). All H atoms were refined with isotropic displacement parameters taken as 1.5 times
3
those of the respective parent atoms. All calculations were performed using WINGX
version 1.64.05 package [49]. CCDC-2058475 for T2, 2058476 for T3, and 2058477 for T5
contain the supplementary crystallographic data for this paper. These data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html (accessed on 15 Mar 2021) [or
from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge
CB2 1EZ, UK; fax: +44(0)-1223-336-033; email: deposit@ccdc.cam.ac.uk].
Crystal data of T2: C H N SBr, M = 272.17, orthorhombic, space group Pbca,
9
10
3
3
a = 13.580(15), b = 8.5494(8), c = 19.0926(18) Å, V = 2216.7(4) Å , Z = 8, d
) = 3.862 mm , T = 296K, 12,588 measured reflections (
.13–28.10 ), 2431 unique reflections, final R = 0.049, wR= 0.129, S = 1.009 for 1713 reflections
= 1.631 Mg
calc
−
3, F
−1
m
2
(000) = 1088,
µ
(MoK
α
θ
range
◦
with I > 2σ(I).
Crystal data of T3: C H N3O2S, M = 253.32, monoclinic, space group P2 /c,
1
1
15
1
◦
−1
3
a = 8.5409(7), b = 8.1329(5), c = 18.8922(12) Å,
β
= 102.099(7) , V = 1283.15(16) Å ,
) = 0.247 mm , T = 296K, 8182 mea-
−
3, F
Z = 4, dcalc = 1.311Mg m
sured reflections (
(000) = 536,
µ
(Mo K
α
◦
θ
range 2.20–27.78 ), 2666 unique reflections, final R = 0.031, wR= 0.085,
S = 1.045 for 2366 reflections with I > 2σ(I).

Int. J. Mol. Sci. 2021, 22, 3104
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Crystal data of T5:C H N SCl , M = 262.15, monoclinic, space group C2/c,
9
9
3
2
3
a = 12.8992(10), b = 10.0640(8), c = 18.4826(13) Å,
β
= 96.754(7), V = 2382.7(3) Å ,
−
3, F
−1
Z = 8, dcalc = 1.462 Mg m
sured reflections (
(000) = 1072,
µ
(MoK
α
) = 0.690 mm , T = 296K, 6897 mea-
◦
θ
range 2.22–28.06 ), 2493 unique reflections, final R = 0.044, wR = 0.127,
S = 1.066 for 1981 reflections with I > 2σ(I).
3.3. Theoretical Calculations
The conformational analysis for T2, T3 and T5 was performed using semiempirical
AM1 method implemented in GAUSSIAN 03 [50]. The structures were fully optimized and
the initial geometries were obtained from their crystallographic data.
3.4. General Procedure for the Synthesis of Cu (II) Complex of Thiosemicarbazone Derivatives
Cu(T1)Cl , Cu(T10) Cl , Cu(T12) Cl , Cu(T13)Cl , Cu(T16)Cl ) Synthesis of com-
2
2
2
2
2
2
2
plexes was carried out in MeOH/EtOH (v/v = 1/1) mixture. In all cases molar ratio of
organic ligand and copper(II) chloride dihydrate was 1:1. Total volume of reaction mixtures
did not exceed 60 mL. After 6 h, green precipitates of Cu(T1)Cl , Cu(T10) Cl , Cu(T13)Cl ,
2
2
2
2
Cu(T16)Cl , and yellow precipitate of Cu(T12) Cl were filtered and washed three times
2
2
2
with small amounts of MeOH/EtOH. Solid compounds were then dried in open air for
several days and weighted.
Cu(T1)Cl (C H N SO CuCl ) (449.87 g/mol), yield (47%), anal. calculated (%):
2
16 17
3
2
2
Cu, 14.12; C, 42.72; H, 3.81; N, 9.34. Found (%): Cu, 14.39; C, 42.50; H, 3.79; N, 8.85. FTIR
−
1
spectra (KBr, cm ): ν(NH) 3442, 3343; ν(CH) 3056–2833; ν(CN) 1590; δ(NH) 1500; β(CH)
266, 1168, 1139; ν(NN) 1021; ν(CS) 853, 808; γ(CH) 764, 728.
Cu(T10) Cl (C H N S O CuCl ) (834.18 g/mol), yield (75%), anal. calculated (%):
1
2
2
32 32
6
2
4
4
Cu, 7.62; C, 46.07; H, 3.87; N, 10.08. Found (%): Cu, 8.08; C, 46.25; H, 3.83; N, 9.66. FTIR
−
1
spectra (KBr, cm ):
β(CH) 1267, 1240; ν(NN) 1021; ν(CS) 939, 851; γ(CH) 738, 722.
Cu(T12) Cl (C H N S CuCl ) (851.82 g/mol), yield (69%), anal. calculated (%):
ν(NH) 3251; ν(CH) 3116–2828; ν(CN) 1597, 1576; δ(NH) 1555, 1508;
2
2
28 20
6
2
8
Cu, 7.46; C, 39.48; H, 2.37; N, 9.87. Found (%): Cu, 6.86; C, 39.69; H, 2.40; N, 9.71. FTIR
−
1
spectra (KBr, cm ):
β(CH) 1278, 1205, 1197, 1128; ν(NN) 1031; ν(CS) 941, 900, 877; γ(CH) 745, 731.
Cu(T13)Cl (C H N SCuCl Br) (503.14 g/mol), yield (62%), anal. calculated (%):
ν(NH) 3305, 3272; ν(CH) 3092, 2923; ν(CN) 1592; δ(NH) 1549, 1520;
2
14 11
3
3
Cu, 12.63; C, 33.42; H, 2.20; N, 8.35. Found (%): Cu, 12.35; C, 33.26; H, 2.16; N, 8.23. FTIR
−
1
spectra (KBr, cm ):
β(CH) 1278, 1253, 1205, 1128; ν(NN) 1010; ν(CS) 959, 912, 871; γ(CH) 774, 745.
Cu(T16)Cl (C H N SO CuCl ) (484.31 g/mol), yield (35%), anal. calculated (%):
ν(NH) 3382, 3279; ν(CH) 3167–3067; ν(CN) 1589; δ(NH) 1537, 1505;
2
16 16
3
2
3
Cu, 13.12; C, 39.68; H, 3.34; N, 8.68. Found (%): Cu, 13.15; C, 39.93; H, 3.30; N, 8.21. FTIR
−
1
spectra (KBr, cm ):
ν(NH) 3424, 3265; ν(CH) 3096–2834; ν(CN) 1590; δ(NH) 1528, 1492;
β(CH) 1268, 1177; ν(NN) 1020; ν(CS) 939, 858, 828; γ(CH) 768, 746, 718.
3.5. Biological Assays
3.5.1. Cell Culturing
The evaluation of cytotoxicity of tested compounds was carried out on the G361, A375,
and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells. The cell lines
were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The
◦
cells were cultured at 37 C in the presence of 5% CO in atmosphere. The culture medium
2
(
Dulbecco’s Modified Eagle’s Medium for G361 and A375 and Eagle’s Minimum Essential
Medium for SK-MEL-28 and BJ) was supplemented with 10% foetal bovine serum. The
cells morphology was examined under inverted phase contrast microscope Nikon Eclipse
Ti (Nikon, Tokyo, Japan). The authenticity of tested cell lines was verified by short tandem
repeat (STR) genotyping in the Department of Forensic Medicine (Medical University of
Lublin, Lublin, Poland).

Int. J. Mol. Sci. 2021, 22, 3104
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3
.5.2. Cell Viability Assay
The cells were seeded into 96-well plates (in concentration: BJ, A375, and SK-MEL-
5
5
2
8—1
×
10 cells/mL, G361—1.5
×
10 cells/mL) and cultured until they reach 70–80%
confluency. Then the cells were incubated with tested compounds and dacarbazine in
concentrations ranging from 10 to 500 M or DMSO as vehicle in control cultures for next
4 h (max. DMSO concentration <0.5%). The compounds cytotoxicity was evaluated with
µ
2
the MTT colorimetric method based on the ability of viable cells to the transformation of
tetrazolium salts (3-[4,5-dimethylthiazol-2-yl]-2,5-) diphenyltetrazolium bromide, MTT)
to purple formazan, by cellular dehydrogenases. After 24-h-incubation cell cultures were
supplemented with 20
in PBS (Corning, NY, USA), and the incubation was continued for 4 h at 37 C. Next,
µ
L of 5 mg/mL MTT (Thermofisher, Waltham, MA, USA) stock
◦
the medium with MTT was removed, and the formed crystals were dissolved in 200
of DMSO (dimethyl sulfoxide, Avantor, Gliwice, Poland). The solution absorbency was
measured at 570 nm, using a PowerWave xs microplate spectrophotometer (BioTek
µL
™
Instruments, Winooski, VT, USA). The experiment was performed three times with three
replicates for each concentration of tested compounds. IC50 values were determined using
the AAT Bioquest IC50 calculator [51]. On the basis of obtained IC50 values for normal
and malignant cells, A375 cell line and three tested compounds (CuT1, Cu10, CuT16) were
chosen for further investigation.
3.5.3. Cell Cycle Assay
Cell-cycle assay was performed using NC 3000 system (ChemoMetec, Lillerød, Den-
mark) according to manufacturer’s protocol for the two-step cell cycle analysis. The A375
5
cells were seeded in 12-well plates at density of 1
×
10 cells/mL and after 70–80% con-
fluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations
corresponding to IC50 values or DMSO as vehicle in control cultures and analysed after
2
4h incubation. The cells were washed with PBS (Corning, New York, NY, USA), incubated
◦
with lysis buffer (Solution 10) supplemented with 10
µg/mL of DAPI for 5 min at 37 C.
After addition of stabilization buffer (Solution 11) the samples were analysed. Fluorescence
®
signal was quantified using a NucleoCounter NC-3000
™ image cytometer (ChemoMetec
USA Inc., Lillerød, Denmark). Data were derived from three independent experiments
with three replicates for each concentration of tested compound.
3.5.4. Cell Apoptosis Assay
Cell apoptosis assay was performed using NC 3000 system (ChemoMetec USA Inc.,
Lillerød, Denmark) according to the manufacturer’s protocol for Annexin V Assay. The
5
A375 cells were seeded in 6-well culture plates at a density 1
7
×
10 cells/mL and after
0–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concen-
trations corresponding to IC50 values or DMSO as vehicle in control cultures and analysed
after 24h incubation. The cells were dissociated into single-cell suspensions in PBS (Corn-
5
ing, New York, NY, USA) and app. 4
×
10 cells were resuspended in 100
µ
L of Annexin V
binding buffer and incubated with Annexin V—CF488A conjugate and Hoechst 33,342 for
5 min at 37 C. Next, the cells were washed with Annexin V binding buffer and the cell
◦
1
pellets were resuspended in 100 mL of Annexin V binding buffer supplemented with
1
0
µ
g/mL of propidium iodide (PI) and Annexin V Assay was performed immediately.
®
Cellular fluorescence was quantified using a NucleoCounter NC-3000
™ image cytometer
(ChemoMetec USA Inc., Lillerød, Denmark). Data were derived from three independent
experiments with three replicates for each concentration of tested compounds.
3.5.5. DNA Damage Evaluation
DNA damage was evaluated on the basis of a basic (AP) sites presence, that arose
in response to oxidative damage or as an intermediate in base excision repair pathway.
5
The A375 cells were seeded in 6-well culture plates at a density 1
×
10 cells/mL and
after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16

Int. J. Mol. Sci. 2021, 22, 3104
20 of 23
in concentrations corresponding to IC50 values or DMSO as vehicle in control cultures.
After 24 h of incubation genomic DNA was isolated using Syngen DNA Mini Kit (Syngen
Biotech, Wroclaw, Poland) according to the manufacturer’s instruction. The concentrations
of isolated DNA for all samples were equalized to 100 µg/mL. The measurements of the
number of AP sites were evaluated with DNA Damage Quantification Kit (Dojindo, Japan)
according to the manufacturer’s instruction. The DNA was treated with Aldehyde Reactive
Probe (ARP) to tag all of the AP sites with a biotin residue. Then all of the AP sites were
quantified using avidin—biotin assay. The coloured product of horseradish peroxidase
conjugated to the avidin was detected at 650 nm using a PowerWave
™ xs microplate
spectrophotometer (BioTek Instruments, Winooski, VT, USA).
3.5.6. Gene Expression Evaluation
The expression level of genes connected with DNA repair and oxidative stress re-
sponse was evaluated with quantitative real-time PCR (qRT-PCR) method. The A375
5
cells were seeded in 6-well culture plates at a density 1
×
10 cells/mL and after 70–80%
confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations
corresponding to IC50 values or DMSO as vehicle in control cultures. After 24h of incuba-
tion total RNA was isolated using the Syngen Blood/Cell RNA Mini Kit (Syngen Biotech,
Wroclaw, Poland). All samples were transcribed using NG dART RT-PCR reagents (EURx,
Gdansk, Poland) according to the manufacturer’s instructions. The relative mRNA expres-
sion level was determined by relative quantification method (∆∆Ct) using 18S ribosomal
N5 (RNA18SN5) and beta-actin (ACTB) as endogenous controls. The reference genes were
selected on the basis of our preliminary studies, where RNA18SN5 and ACTB remained
unaffected by the experimental conditions. The PCR was conducted in triplicate using
®
Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City,
CA, USA) and SG qPCR Master Mix (2 ) (EURx, Gdansk, Poland) in accordance with the
×
−
∆∆Ct
manufacturer’s protocol. Data was presented and analysed as RQ values (RQ = 2
The genes and sequences of used primers were listed in Table 5.
).
Table 5. Primers used in gene expression evaluation.
0
0
0
0
Gene Symbol
Protein Name
Forward Sequence (5 →3 )
Reverse Sequence (5 →3 )
ERCC
ERCC Excision Repair 1
CTCGGAGTTTTGTGGGGGAC
CACTGGCGTCTACGTTCTCA
TGCTCACAACCAGACAGCTC
O-6-methylguanine-DNA
methyltransferase
MGMT
ACCGTTTGCGACTTGGTACT
OGG1
MLH1
PARP1
8-oxoguanine DNA glycosylase
mutL homolog 1
CCTGTGGGGACCTTATGCTG
GCACCGGGATCAGGAAAGAA
CCCCACGACTTTGGGATGAA
TGTGAATCCCCTCTCCCGAT
GCCTCACCTCGAAAGCCATA
AGACTGTAGGCCACCTCGAT
poly(ADP-ribose) polymerase 1
ataxia telangiectasia mutated
protein kinase
ATM
XPC
GCCGCGGTTGATACTACTTTG
GCGAAGTGGAATTTGCCCAG
GCAGCAGGGTGACAATAAACA
TTGGCCTTGGATTTCTGGCT
xeroderma pigmentosum group
C-complementing protein
MSH2
SOD2
MutS homolog 2
superoxide dismutase 2
glutathione peroxidase 1
catalase
CAGGAGGTGAGGAGGTTTCG
CTTCAGGGTGGTATGGCTGT
TTGACATCGAGCCTGACATC
AGCTTAGCGTTCATCCGTGT
GAAACTGCGAATGGCTCATTAAA
AGAGCTACGAGCTGCCTGAC
CCGTGCGCCGTATAGAAGTC
TGGCCAGACCTTAATGTTCC
ACTGGGATCAACAGGACCAG
TCCAATCATCCGTCAAAACA
CACAGTTATCCAAGTGGGAGAGG
AGCACTGTGTTGGCGTACAG
GPX1
CAT
RNA18SN5
ACTB
18S ribosomal N5
beta-actin
3.5.7. X-ray Radiation
G361, A375 and SK-MEL-28 were seeded in 6-well plates at following density: A375
5
5
and SK-MEL-28—1 × 10 cells/mL, G361—1.5 × 10 cells/mL and were irradiated using
the RS-2000 Biological Research Irradiator (Rad Source Technologies, Buford, GE, USA). In

Int. J. Mol. Sci. 2021, 22, 3104
21 of 23
the studies radiation at the dose of 10 Gy was used. X-ray treated cells were incubated for
further 48 h under standard culturing conditions (characterised in Cells culturing section).
Control cells that had not been exposed to X-rays were also cultured for 48 h. To compare
all three cell lines responses, cell cycle analysis was conducted using NC 3000 system as
described above.
3.5.8. Statistical Analysis
Statistical analysis was performer using STATISTICA 13 software (StatSoft, Krakow,
Poland). Comparison of values was performed by one-way analysis of variance (ANOVA)
and post hoc multiple comparisons with Tukey’s honest significant difference test (Tukey’s
HSD test). The data were calculated as mean
results were considered statistically significant.
±
SD. If the p-value was under 0.05, the
4. Conclusions
A number of thiosemicarbazone derivatives have been prepared as potential anti-
cancer agents. X-ray examinations carried out for T2, T3 and T5 confirmed the synthesis
pathway, the assumed molecular structures and showed that the tautomeric form of
thione is the dominant crystal. All the molecules as a whole are almost flat and their
conformation is strongly dependent on the intramolecular N–H . . . N hydrogen bond in
the thiosemicarbazone part. In the next step, five Cu (II) thiosemicarbazone complexes
have been successfully synthesized and described. The way of complexation has been
established using adequate analytical techniques to confirm their formulae.
In vitro studies of the anti-tumor activity of ligands and complexes showed that the
complexation of thiosemicarbazone derivatives with Cu (II) ions improves their anti-tumor
activity against melanoma cells. The observed cytotoxic effect is related to DNA damage
and the G2/M phase of cell cycle arrest and disturbed expression of antioxidant enzymes.
Presented results are outstanding and highlight the potential of complexation of biologically
active compounds. Further studies on this topic have to be performed, as complexation is
a powerful tool in the search for more efficient and reliable drugs.
Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/1422
-
0067/22/6/3104/s1.
Author Contributions: Conceptualization, M.P. and A.C.; methodology, M.D., M.I.; software, Z.K.,
W.W., J.K.; validation, E.F., G.A.; formal analysis, E.F., A.C.; investigation, Z.K., W.W., B.R., P.B.,
J.K., G.A., E.H.; data curation, J.K.; writing—original draft preparation, M.P., A.C., A.K.-P.; writing—
review and editing M.P., A.C., M.I.; visualization, A.C., A.K.-P.; supervision, M.P. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
MDPI Multidisciplinary Digital Publishing Institute
DOAJ Directory of open access journals
TLA
LD
Three letter acronym
linear dichroism

Int. J. Mol. Sci. 2021, 22, 3104
22 of 23
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