Synthesis, characterization and anticancer activity of new 2-acetyl-5-methyl thiophene and cinnamaldehyde thiosemicarbazones and their palladium(II) complexes

Article abstract of DOI:10.1016/j.ica.2020.120036

New thiosemicarbazone (TSC) ligands, AMT-C[dbnd]N-TSCH (L1), AMT-C[dbnd]N-TSC(CH₃) (L2), CIN-C[dbnd]N-TSCH (L3) and CIN-C[dbnd]N-TSC(CH₃) (L4) (AMT = 2-acetyl-5-methylthiophene, TSCH = thiosemicarbazide, TSC(CH₃) = 4-methy

Full text of DOI:10.1016/j.ica.2020.120036

InorganicaChimicaActa515(2021)120036
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Inorganica Chimica Acta
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Research paper
Synthesis, characterization and anticancer activity of new 2-acetyl-5-methyl
thiophene and cinnamaldehyde thiosemicarbazones and their palladium(II)
complexes
⁎
⁎
Eunice A. Nyawadea,b, , Nicole R.S. Sibuyic, Mervin Meyerc, Roger Lalancetted, Martin O. Onania,
a Organometallics and Nanomaterials, Department of Chemistry, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
b School of Physical Sciences, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62, 000-00200 Nairobi, Kenya
c Department of Science and Innovation/Mintek Nanotechnology Innovation Centre (DSI/Mintek NIC), Biolabels Node, Department of Biotechnology, University of the
Western Cape, Private Bag X17, Bellville 7535, South Africa
d Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
New thiosemicarbazone (TSC) ligands, AMT-C]N-TSCH (L1), AMT-C]N-TSC(CH3) (L2), CIN-C]N-TSCH (L3)
and CIN-C]N-TSC(CH3) (L4) (AMT = 2-acetyl-5-methylthiophene, TSCH = thiosemicarbazide, TSC(CH3) = 4-
methyl-3-thiosemicarbazide, CIN = cinnamaldehyde) were synthesized by condensation reaction. The reaction
of [PdCODCl2] with the ligands L1, L2 and L4in the ratio 1:1 yielded complexes [Pd(L1)Cl] (C1), [Pd(L2)Cl2]
(C2), and [Pd(L4)2Cl2] (C4) respectively (COD = 1,5-cyclooctadiene). The reaction of [Pd(COD)Cl2] with CIN-
C]N-TSCH (L3) in 1:2 metal to ligand ratio yielded the complex [Pd(L3)2] (C3). The ligands and the complexes
were characterized by UV–Vis, FT-IR, NMR, elemental analysis and conductivity measurements. The ligand L1
coordinated to the metal in a tridentate fashion while L2, L3 and L4 coordinated in a bidentate fashion. The
ligands L1 and L3 coordinated to the metal as thiol anions while ligand L4 coordinated via the thione sulphur.
Conductivity measurements of the metal complexes in dimethyl sulfoxide showed that the chloride ions com-
plexes C1, C2 and C4 are within the coordination spheres. The crystal structures of ligands L2 and L4 were
Thiosemicarbazone
Thiosemicarbazide
Palladium(II) complexes
Cinnamaldehyde
Cytotoxicity
Anticancer activity
obtained from
a Bruker Smart KAPPA APEXII DUO diffractometer with graphite monochromated Mo
Kαradiation (k = 0.71073 Å). Cytotoxic activity of the compounds was investigated against the human cancer
cell lines; Caco-2 (colon), HeLa (cervical), Hep-G2 (hepatocellular), MCF-7 (breast), PC-3 (prostate) and MCF-
12A (non-cancer breast) cells. The complex C2 exhibited excellent broad spectrum growth inhibition activity
against all the human cell lines tested except for the non-cancer breast cells. All the metal complexes synthesized
inhibited the proliferation of human prostate cancer cell.
1. Introduction
to the metal center either in the neutral form or, an anionic form
generated after loss of a proton from the thioamide nitrogen (NH) or
The chemistry of transition metal complexes of thiosemicarbazone
(TSC) ligands has attracted a lot of research interests due to the nu-
merous pharmacological properties portrayed by both the ligands and
the complexes [1]. The excellent chelating property of TSCs has been an
attractive feature in coordination chemistry hence the vast interest in
their chemistry for a span of six decades [2–4]. The TSCs are a class of
N, S bidentate Schiff base ligands which are generally synthesized by
the condensation of a ketone or an aldehyde with a thiosemicarbazide
under suitable reaction conditions.
thiol sulphur (SH). The TSC ligand coordinate to various metals in a
bidentate manner via the N and S atoms or in a tridentate fashion if
there is an extra donor group (D) such as O, N or S in the molecule to
give metal complexes of different geometries [5,6]. Various coordina-
tion modes exhibited by the TSC ligands have been documented [7]. Pd
(II) and Pt(II) complexes with TSCs derived from 2-thiophenecarbox-
aldehyde, 5-methyl-2-thiophene carboxaldehyde and other aldehydes
and ketones exhibit various coordination modes and have been re-
ported to be biologically active [8–12]. The presence of nitrogen and
sulfur donor atoms in the ligand may be responsible for their potential
Since TSCs can exist as thione-thiol tautomers, they can coordinate
⁎ Corresponding authors at: Organometallics and Nanomaterials, Department of Chemistry, University of the Western Cape, Private Bag X17, Bellville 7535, South
Africa.
E-mail addresses: enyawade@jkuat.ac.ke (E.A. Nyawade), monani@uwc.ac.za (M.O. Onani).
https://doi.org/10.1016/j.ica.2020.120036
Received 12 June 2020; Received in revised form 18 September 2020; Accepted 20 September 2020
Availableonline24September2020
0020-1693/©2020ElsevierB.V.Allrightsreserved.

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
biological activity.
2.2. Syntheses
The biological activity of TSC derivatives and their metal complexes
depend on the parent aldehyde and ketone, and the central metal ion of
the complexes. Heterocyclic TSCs and their Pd(II) and Pt(II) complexes
exhibit a great variety of biological activities against various tumors
[13,14] viruses [15,16], fungi [17], 18bacteria [15,18], protozoa
[12,19,20], malaria [21], and convulsions [22].
2.2.1. Synthesis of 2-acetyl-5-methylthiophene TSCs ligands L1 and L2
(2E)-2-[1-(5-Methyl-2-thienyl)ethylidene]hydrazinecarbothioamide
(AMTeC]NeTSCH), L1
TSC-H, (1.0 mmol; 91.13 mg) was dissolved in 10 mL methanol in a
Schlenk tube and stirred under reflux for 30 min. To the methanolic
solution of TSC-H, a solution of AMT (0.0 mmol; 140.2 mg) in prepared
in 10 mL methanol was added drop wise while stirring continuously.
Two drops of H2SO4 was added to the mixture and stirred under reflux
for 2 hr. The solution turned pale yellow. A whitish cream precipitate
appeared on cooling. The mixture was filtered by use of a cannula and
the residue was washed with cold methanol. The residue was dried
under reduced pressure for 3 h and characterized by FTIR, NMR, UV–vis
and elemental analysis. The yield was 67.0% (142.92 mg) and melting
point was found to be 172–174 °C.
The cinnamaldehyde (CIN), a naturally occurring phytochemical
compound, possesses highly significant properties such as anti-in-
flammatory, oxidative and anti-oxidative making it a suitable candidate
for anticancer drugs. It has been shown to exert chemopreventive ac-
tivity against several types of human cancer cells by inducing apoptosis
and thus regulating cancer cell invasion and metastasis [23]. CIN has
found application in treating various types of cancer, including breast,
prostate, colon, leukemia, liver, and oral cancers [24,25]. CIN has also
been shown to reduce the spontaneous mutant frequency in Salmonella
typhimurium strain TA104 and Escherichia coli by 50% [26,27]. Owing to
the potential of CIN and derivatives as therapeutic agents, researches on
chemical syntheses and modifications have been carried out to gain
chemical entities with more potent bioactivity and favorable drugg-
ability [28,29].
Analysis:
Molecular formula: C8H11N3S2. Elemental analysis: Calculated: C;
45.04, H; 5.20, N; 19.70, S; 30.06%. Found: C; 45.30, H; 5.30, N; 20.16,
S; 30.32%. 1H NMR (400 MHz, DMSO‑d6): δ 2.42 (s, 3H), δ 2.27 (s, 3H),
δ 6.78 (m, J = 1.48 Hz,1H(thiophene)), δ 7.31 (d, J = 3.62 Hz, 1H
(thiophene)), δ 8.28 (s, 1H (eNH)). δ 10.39(s, 1H (NNH)). 13C NMR
(400 MHz, DMSO‑d6): δ 178.9 (C]S), δ 145.5 (C]N), δ 142.9 (CeS), δ
140.9 (CeS), δ 128.8 (C]C), δ 126.8 (C]C), δ 15.8 (CH3), δ 14.8
(CH3). IR (KBr, solid state; cm−1): ν(HeNeH) 3366, 3263, ν(NNeH)
3119, ν(-C]N) 1629, 1521, ν(C]S) 1286, ν(CeS)thiophene 796
ν(eNeN) 993. UV–Vis (DMSO), λmax, nm (ε, M−1cm−1) 341 (1030).
(2E)-N-Methyl-2-[1-(5-Methyl-2-thienyl)ethylidene]hy-
It is worth noting that coordination of ligands to metals give them a
three dimensional orientation which aid their specific molecule re-
cognition thus improving their biological activity [30,31].
The fact that TSCs are good chelating ligands with great pharma-
cological activities and that their coordination to the Pd metal center
may trigger or enhance their biological activity, greatly inspired this
study. New TSCs based thiophene derivative (a heterocyclic ketone)
and CIN, coordinated to Pd(II) ion, were prepared and their mode of
coordination and anticancer activities determined. We therefore herein
report for the first time the synthesis and cytotoxicity studies of Pd(II)
complexes of 2-acetyl-5-methyl thiophene TSCs and CIN TSCs.
drazinecarbothioamide (AMTeC]NeTSC(CH3)), L2
The same procedure described for the synthesis of ligand L1 was
followed in the preparation of ligand L2. Solutions of TSC-CH3
(1.0 mmol, 105.2 mg) and AMT (1.0 mmol, 140.2 mg) in 10 mL me-
thanol were used. A whitish cream solid obtained in 71% yield was
characterized by elemental analysis, FTIR, NMR and UV–vis. It melted
at a temperature of 176 °C. Crystals of ligand L2 were grown by dis-
solving the whitish cream solid in ethanol followed by addition of a
layer of diethyl ether onto the solution. The solution was left un-
disturbed for 48 h. Whitish cream needle shaped crystals of ligand L2
were obtained.
2. Materials and methods
2.1. General methods and materials
All reactions herein were performed under inert conditions using
Schlenk techniques and monitored by thin layer chromatography using
Merck silica gel 60 F254 plates. 2-Acetyl-5-methylthiophene (AMT),
CIN, TSCeH and 4-methyl-3-thiosemicarbazide (TSCeCH3) were pur-
chased from Sigma Aldrich and used without further purification. The
organic solvents; ethanol, methanol, methylene chloride, diethyl ether
were obtained from Sigma Aldrich and purified by standard techniques.
Melting points were determined using a Stuart Scientific SMP 10 ap-
paratus. Solid state Fourier Transform infrared (FT-IR) on potassium
bromide (KBr) were recorded on a Perkin Elmer Spectrum Two model
FT-IR Spectrophotometer (4000–400 cm−1). 1H and 13C NMR of the
compounds in deuterated dimethylsulfoxide (DMSO‑d6) using trimethyl
silane as an internal standard was recorded at 25 °C on a Bruker Avance
III HD Nanobay 400 MHz NMR spectrometer equipped with a 5 mm
BBO probe. Electronic spectra were recorded on a Thermo Scientific
Nicolet Evolution 100 Ultraviolet visible (UV–Vis) Spectrophotometer.
Elemental analyses were performed on a LECO CNHS-932 micro-ana-
lyzer at the Central Analytical Facilities, Stellenbosch University, South
Africa. Conductivity measurements were performed on a HANNA EC
215 conductivity meter. The single crystal structures of L2 and L4 were
obtained from a Bruker Smart KAPPA APEXII DUO diffractometer with
graphite monochromated Mo Kαradiation (k = 0.71073 Å) at Rutgers,
the State University of New Jersey, Newark, USA. The metal precursor
[Pd(COD)Cl2] (COD = 1,5-cyclooctadiene) was prepared following
previously reported methods [32,33].
Analysis:
Molecular formula: C9H13N3S2. Elemental analysis: Calculated: C;
47.55, H; 5.76, N; 18.48, S; 28.21%. Found: C; 47.70, H; 5.70, N; 18.01,
S; 27.88%.1H NMR (400 MHz, DMSO‑d6): δ 2.26 (s, 3H, CH3), δ 2.43 (s,
3H, CH3), δ 3.02 (s, J = 4.62,3H, NCH3), δ 6.78(q, J = 1.50 Hz, 1H
(thiophene)), δ 7.30 (d, J = 3.61 Hz, 1H (thiophene)), δ 7.97 (d,
J = 4.35 Hz, 1H (eNH)). δ 10.25(s, 1H (NNH)). 13C NMR (400 MHz,
DMSO‑d6): δ 178.8 (C]S), δ 145.3 (C]N), δ 142.7 (CeS), δ 140.9
(CeS), δ 128.5 (C]C), δ 126.5 (C]C), δ 31.5 (CH3), δ 15.7 (CH3), δ
14.8 (CH3). IR (KBr, solid state; cm−1): ν(eNeH) 3331, ν(NNeH)3246,
ν(H2CeH) 2924, ν(eC]N) 1541, 1506, ν(C]S) 1236, ν(eNeN)
1042,ν(CeS)thiophene658. UV–Vis (DMSO), λmax, nm (ε, M−1cm−1
)
342 (1000).
2.2.2. Synthesis of CIN TSCs, L3 and L4
(2E)-2-[(2E)-3-Phenyl-2-propen-1-ylidene]hy-
drazinecarbothioamide (CINeC]NeTSCH), L3
TSC-H (1.0 mmol, 91.13 mg) in 10 mL dry methanol was added in a
clean Schlenk tube and stirred under reflux for 30 min. 10 mL metha-
nolic solution of CIN (1.0 mmol, 0.13 mL) was slowly added to the
solution followed by a drop of conc. H2SO4. The mixture was stirred
under reflux for 2 h after which it was allowed to cool to room tem-
perature. A pale yellow precipitate formed, mixture was filtered via a
canula, washed with cold methanol and dried under reduced pressure
for 3 h. The solid obtained in 70% yield melted at a temperature of
121.0 °C.
Analysis:
2

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Molecular formula: C10H11N3S. Elemental analysis: Calculated: C;
58.51, H;5.40, N; 20.47, S; 15.62%. Found: C; 58.40, H; 5.81, N; 19.98,
S; 15.27%. 1H NMR (400 MHz, DMSO, d6): δ 11.4 (s, 1H (NNH)), δ 8.19
(s, 1H (eNH)), δ 7.90 (d, J = 9.21 Hz, 1H (N = CH)), δ 7.62 (s, 1H
(–NH)), δ 7.56 (d, J = 7.28 Hz, 2H(phenyl)), δ 7.37, (m, J = 8.50 Hz,
3H(phenyl)),δ 7.0 (d, J = 16.14 Hz, 1H(allyl)), δ 6.88,(dd, J = 8.06 Hz,
1H(allyl)). 13C NMR (400 MHz, CD3OD): δ 178.1 (C]S), δ 145.2 (C]
NeTSCeCH3 (0.1 mmol, 22.73 mg) and an equimolar amount of the
precursor were used as per the procedure described in Section 2.2.3.1 to
give an orange solid that turns black and melts at 217 °C in 89% yield.
Molecular
formula:
C9H12Cl2N3PdS2.
Elemental
analysis:
Calculated: C; 26.71, H; 3.24, N; 10.38, S; 15.85%. Found: C; 26.65, H;
3.48, N; 10.36, S; 15.81%. 1H NMR (400 MHz, DMSO‑d6): δ 2.46 (s,
3H), δ 2.58 (s, 3H (thiophene)), δ 2.94 (s, 3H (NCH3)), δ 6.8762 (d,
J = 3.36 Hz, 1H(thiophene)), δ 7.49 (d, J = 3.91 Hz, 1H (thiophene)),
δ 8.72 (s, 1H (eNH)), δ 10.26 (s, 1H (NNH)). IR (KBr, solid state;
cm−1): ν(eNeH) 3273, ν(NNeH) 3181, ν(H2CeH) 3004, 2932, ν(eC]
N) 1565, 1514, ν(eC]Ce) 1441, 1397, ν(C]S) 1229, ν(eNeN) 1048,
ν(CeS)thiophene 536, ν(PdeN) 530, ν(PdeS) 450, 497. UV–Vis
(DMSO), λmax nm, (ε, M−1cm−1), 340 (1472), 393sh (417), 486 (87.2).
N),
δ 136.3, 129.4, 129.3, 127.4, (C(Phenyl)), 139.3, 125.5
(C]C(allyl)). IR (KBr, solid state; cm−1): ν(HeNeH) 3415, 3254,
ν(NNeH),3147,ν(phenyleCeH) 3000, ν(eC]N) 1588,1521, ν(C]S)
1277,ν(eNeN) 1085. UV–Vis (DMSO), λmax, nm (ε, M−1cm−1) 345
(1470), 258sh (1248).
(2E)-N-Methyl-2-[(2E)-3-Phenyl-2-propen-1-ylidene]hy-
drazinecarbothioamide CINeC]NeTSC(CH3)), L4
The same procedure described for the ligand L3 was followed in the
preparation of ligand L4. Solutions of AMT (1.0 mmol, 105.2 mg) and
CIN (1.0 mmol, 0.13 mL) in 10 mL methanol were used. A yellow
powder obtained in 68% yield was characterized by elemental analysis,
FTIR, NMR and UV–vis. It melted at a temperature of 168–170 °C.
Crystals suitable for single crystal x-ray diffraction was obtained by
following the procedure described for crystal growth in Section 2.2.1
Pale yellow needle-shaped crystals were obtained.
2.2.3.3. Synthesis of complex C3, Pd(L3)2. To a methanolic solution of
ligand L3 (1.0 mmol, 205.28 mg) in a Schlenk tube, a solution of Pd
(COD)Cl2 (0.5 mmol, 142.76 mg) in methylene chloride was added and
the rest of the procedure followed as described in Section 2.2.3. An
orange solid was obtained in 87% yield. The solid decomposes at a
temperature above 281 °C.
Analysis:
Molecular formula: C20H20N6PdS2. Elemental analysis: Calculated:
C; 46.65, H; 3.91, N; 16.32, S; 12.45%. Found: C; 46.39, H; 4.09, N;
16.11, S; 12.29%. 1H NMR (400 MHz, DMSO‑d6): δ 9.14 (2H, NH2), δ
8.08 (m, J = 5.42 Hz, 1H (CH, allyl)), 7.62 (s, 1H (eCH, allyl)), δ 7.73
(m, J = 16.10 Hz, 5H(phenyl)). IR (KBr, solid state; cm−1): ν(HeNeH)
3421, 3259, ν(phenyl-CeH) 2975, 2828, ν(eC]N) 1583, 1513,
ν(aromatic eC]C) 1481, ν(eNeN) 977, ν(PdeN) 508, ν(PdeS) 475.
UV–Vis (DMSO), λmax nm, (ε, M−1cm−1), 303 (1770), 362 (1920), 417
(1412), 486 (97).
Analysis:
Molecular formula: C11H13N3S. Elemental analysis: Calculated: C;
60.24, H; 5.97, N; 19.16, S; 14.64%. Found: C; 60.41, H; 5.97, N; 19.01,
S; 14.69%.1H NMR (400 MHz, DMSO‑d6): δ 11.46 (s, 1H (NNH)), δ 8.30
(d, J = 4.49 Hz, 1H (eNH)), δ 7.91 (d, J = 9.20, 1H (N = CH)), δ
7.55(d, J = 7.33 Hz, 2H(phenyl)), δ 7.37, (m, J = 35 Hz, 3H(phenyl)),δ
7.0 (d, J = 16.13 Hz, 1H(allyl)), δ 6.87 (dd, J = 8.44 Hz, 1H(allyl)), δ
2.98 (d, J = 4.55 Hz, 3H (CH3)). 13C NMR (400 MHz, DMSO –d6): δ
177.9 (C]S),
δ 144.7 (C]N), δ 136.6, 129.4, 129.3, 127.3,
(C(Phenyl)), 139.1, 125.5 (C]C(allyl)), 31.3 (NeCH3) IR (KBr, solid
state; cm−1): ν(eNeH)3353, ν(NNeH) 3164, ν(phenyl-CeH) 2991,
ν(eC]N) 1532, ν(C]S) 1256, 748, ν(eNeN) 1084. UV–Vis (DMSO),
2.2.3.4. Synthesis of complex C4, Pd(L4)Cl2. The ligand L4 (1.0 mmol,
219.31 mg) and Pd(COD)Cl2(1.0 mmol, 285.51 mg) were used and the
procedure described in Section 2.2.3.3 followed to give an orange solid
in 81% yield. Solid turns black and decomposes above 235 °C.
Analysis:
λ
max, nm (ε, M−1cm−1) 344 (1487), 361sh (1163).
2.2.3. Synthesis of the Pd(II) complexes
Molecular
formula:
C
11H13Cl2N3PdS.
Elemental
analysis:
The TSC ligand was generally dissolved in methanol in a Schlenk
tube and stirred for 10 min at room temperature. A solution of Pd
(COD)Cl2 in methylene chloride was added to the contents of the
Schlenk tube drop wise as the mixture was continuously stirred. An
orange colored precipitate appeared almost immediately. The mixture
was stirred at room temperature for 4 h and the solid was allowed to
settle at the bottom of the Schlenk tube. The mixture was filtered; the
residue was washed with cold methanol, rinsed with diethyl ether and
dried under reduced pressure. The products were characterized by
FTIR, UV–vis, 1H NMR, melting point and elemental analysis.
Calculated: C; 33.31, H; 3.30, N; 10.59, S; 8.08%. Found: C; 33.21, H;
3.44, N; 10.52, S; 8.07%.1H NMR (400 MHz, DMSO, d6): δ 11.10 (1H
(NNH)), δ 8.08 (d, J = 9.61 Hz, 1H (N]CH)), δ 7.62 (m, J = 4.88 Hz,
1H (–NH)), δ 7.63–7.39 (m, 5H (phenyl)),δ 7.32 (d, J = 7.53 Hz, 1H
(allyl)), δ 7.28 (dd, J = 8.44 Hz, 1H(allyl)), δ 2.83 (d, J = 8.78 Hz, 3H
(CH3). IR (KBr, solid state; cm−1): ν(eNeH) 3246, ν(phenyleCeH)
3010, 2932, ν(eC]N) 1607, 1527 ν(aromatic eC]C) 1445, ν(C]S)
1165, ν(eNeN) [1018],ν(PdeN) 501, ν(PdeS) 458. UV–Vis (DMSO),
λmax nm, (ε, M−1cm−1), 297 (1271), 370 (1690), 388 (1681), 470
(96.6).
2.2.3.1. Synthesis of complex C1, Pd(L1)Cl. The ligand L1 (0.5 mmol,
106.66 mg) was dissolved in methanol in a Schlenk tube at room
temperature and stirred. An equimolar amount of Pd(COD)Cl2 solution
in methylene chloride was added to the Schlenk tube. The rest of the
procedure as described in Section 2.2.3 was followed. A dark orange
precipitate was formed in 83% yield. The solid decomposes at 248 °C.
Analysis:
2.3. Crystal data determination and structure refinement ligands L2 and L4
Crystals fit for single crystal X-ray diffraction studies were grown by
layering solutions of the compounds in dry ethanol with a three times
excess volume of diethyl ether and allowing it to stand for 48 h. L2 and
L4 crystals were selected, glued onto the tip of glass fibers, mounted in
a stream of cold nitrogen at 100(1) K and centered in the X-ray beam by
using a video camera. Reflections were successfully indexed by an au-
tomated indexing routine built in the APEXII program suite [34]. The
structures were solved by direct methods using SHELXS [35] and re-
fined [34]. All structures were checked for solvent-accessible cavities
using PLATON [36] and the graphics were performed with the DIA-
MOND [37] visual crystal structure information system software. The
absorption correction was based on fitting a function to the empirical
transmission surface as sampled by multiple equivalent measurements
[34]. The CIF files for the crystals can be obtained from the Cambridge
Crystallographic Data Centre (Supplementary Material file). The crystal
data and structure refinement for the ligands L2 and L4 are summarized
Molecular formula: C8H10ClN3PdS2. Elemental analysis: Calculated:
C; 27.13, H; 2.85, N; 11.86, S; 18.10%. Found: C; 27.02, H; 2.50, N;
11.69, S; 18.01%. 1H NMR (400 MHz, DMSO‑d6): δ 2.36 (s, 3H (CH3)), δ
2.45 (s, 3H, (CH3 thiophene)), δ 6.87 (d, J = 3.92 Hz, 1H(thiophene)),
δ 7.4554 (d, J = 3.96 Hz, 1H (thiophene)), δ 9.23 (s, 2H (NH2). IR (KBr,
solid state; cm−1): ν(HeNeH) 3342, ν(eC]N) 1624,1530, (NeN)
1046, ν(CeS)thiophene 646, ν(PdeN) 551, ν(PdeS) 406, 478. UV–Vis
(DMSO), λmax nm, (ε, M−1cm−1) 291 (1589), 342 (1166), 393sh
(433), 480sh (218).
2.2.3.2. Synthesis of complex C2, Pd(L2)Cl2. The ligand AMTeC]
3

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Table 1
Crystal data and structure refinement for Ligands L2 and L4.
L2
L4
Empirical formula
Formula weight
Temperature
C9H13N3S2
227.3
C11H13N3S
219.30
100 K
100 K
Wavelength
1.54178 Å
Monoclinic
P 21/c
1.54178 Å
Monoclinic
P 21/n
Crystal system
Space group
Unit cell dimensions (Å, °)
a = 5.5173(4)
b = 11.1578(8)
c = 18.0817(3)
1106.98(14) Å3
4
α = 90
a = 5.4418(2)
b = 9.2819(3)
c = 22.4502(7)
1133.27(7) Å3
4
α = 90
β = 96.00(2)
γ = 90
β = 92.056(2)
γ = 90
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1.364 Mg/m3
4.075 mm−1
483.62
1.285 Mg/m3
2.290 mm−1
466.41
h,klmax
6, 13, 21
6, 11, 22
Nref (reported)
Data completeness
Theta max
1954
2004
0.970
0.960
68.116
68.545
Absorption correction
Max. and min. transmission
R(reflections)
wR2(reflections)
S
Numerical
0.799 and 0.544
0.0267 (1809)
0.0691 (1954)
1.045
Numerical
0.843 and 0.396
0.0331 (1892)
0.0814 (2004)
1.099
Npar
136
138
in Table 1.
DMSO. The reduction of MTT was read at 570 nm using POLARStar
Omega plate reader. Cell viability was calculated using this formula:
2.4. Anticancer activity
Average absorbances of treated cells
Average absorbances of control
\% Cell Viability =
× 100 \%
Cytotoxicity of the compounds was tested against human cell lines
obtained from American Type Culture Collection (Manassas, United
States of America): Caco-2 (colon), HeLa (cervical), Hep-G2 (hepato-
cellular), MCF-7 (breast), PC-3 (prostate) cancers and MCF-12A (non-
cancer breast) cells. The cells were cultured in their respective media
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-
streptomycin cocktail (100 μg/mL penicillin and 100 μg/mL strepto-
mycin). Caco-2, HeLa, HepG2, and MCF-7 cells were grown in
Dulbecco's Modified Eagle Medium (DMEM); MCF-12A in DMEM-F12
containing insulin, EGF and hydrocortisone; while PC-3 cells were
cultured in RPMI-1640 media. The cells were grown at 37 °C in a 5%
CO2 humidified incubator (Labotech, South Africa). The cells were
seeded at 1 × 105 cells/mL density in a 96-well plate and incubated at
37 °C for 24 h. The cells were treated with varying concentrations of
compounds (0–100 µg/mL) and incubated at 37 °C for 24 h.
Cytotoxicity of the compounds was assessed by MTTassay, a colori-
metric assay that measures the reduction of yellow 3-(4,5-di-
methythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)by mi-
tochondrial succinate dehydrogenase, following manufacturer’s
instructions. After treatment, 10 μL of 5 mg/mL MTT (Sigma, USA) was
added to each well (100 μL/well) and incubated at 37 °C for 3 h. The
insoluble formazan crystals were solubilized by adding 100 μL of
The concentration of the compounds that inhibited 50% cell pro-
liferation (IC50) on each cell line was estimated using GraphPad Prism
software version 5 (GraphPad Software, California, USA).
3. Results and discussion
3.1. Synthesis and characterization of the ligands
The reactions of 2-acetyl-5-methylthiophene with thiosemicarba-
zide and 4-methyl-3-thiosemicarbazide in methanol under reflux
formed the 2-acetyl-5-methylthiophene TSC ligands L1 and L2 as
whitish cream solids, respectively. The CIN TSC ligands L3 and L4 were
obtained by the reaction of cinnamaldehyde with thiosemicarbazide
and 4-methyl-3-thiosemicarbazide, respectively, in methanol as a sol-
vent under reflux respectively (Fig. 1). The TSC ligands were char-
acterized by FTIR, UV–Vis, 1H and 13C NMR and elemental analysis.
3.1.1. 1H NMR and FTIR analysis
The IR spectra of the ligands L1 to L4 displayed stretching fre-
quency bands for the ν(C]N) and ν(C]S) in the regions 1584–1521
and 1256–1225 cm−1, respectively. The absence of bands in the
Fig. 1. General reaction scheme for the synthesis of ligands L1, L2, L3 and L4.
4

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Fig. 4. Crystal structure of L4 with displacement ellipsoids drawn at the 50%
probability level.
carbon signals observed in the range 177.9–178.9 ppm and
44.5–142.5 ppm attributed to the thione C]S and imine C]N carbon
atoms respectively further confirms the formation and existence of the
TSCs in the thione tautomer. The NMR and FT–IR results therefore
suggest that the thiosemicarbazone ligands exist as E form of the thione.
It is worth noting that the crystal structure of the ligands reveal the
existence of the ligands in the E-isomers (Figs. 2 and 4).
Fig. 2. Ligand L2 in asymmetric unit, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
carbonyl stretching frequency ν(C]O) in the region 1690–1650 cm−1
and appearance of the imine stretching frequencies in the IR spectra of
the ligands indicated that the imine bond was actually formed by
condensation. The 1H NMR spectra of TSCs L3 and L4 exhibited a signal
associated with the imine proton C]NH at 7.9 ppm, approximately
1.77 ppm upfield compared to the absent carbonyl proton HC]O signal
of the cinnamaldehyde (9.69 ppm), a confirmation that the imine bond
was actually formed. The elemental analyses data further confirmed the
chemical composition of the TSC. Since thiosemicarbazones have the
thioamine functional group eNHeC(S), they are capable of exhibiting
the thione-thiol tautomeric forms. They can therefore exist as thiols or
thiones or a mixture of both [38]. The absence of absorption bands
associated with the eSeH vibration in the region 2600–2500 cm−1
confirmed that the thione form of the synthesized TSCs is predominant
in the solid state [39,40]. This is further confirmed by the single crystal
XRD structures of L2 and L4 (Figs. 2 and 3). The 1H NMR in DMSO-d6of
TSCs L1 and L2 exhibited a proton signal at 10.32 and 10.24 ppm re-
spectively while L3 and L4 exhibited a proton signal at 11.40 and
11.42 ppm respectively; the proton signals are attributed to the N–NH
proton. A proton signal at 4.00 ppm attributable to the eSH proton was
absent [8], a clear indication that the TSCs existed as the thione tau-
tomer even in the presence of a solvent as polar as DMSO [8]. TSCs can
also exist in Z and E-isomeric forms or as a mixture of both isomers in
solution. The 1H NMR signals observed in the 10.24–11.42 ppm range
are attributed to the hydrazine NeNH protons falls within the literature
range of 9.00–12.00 ppm reported for the E-isomer [8]. The absence of
proton signals in the range of 14.00–15.00 ppm, normally associated
with the hydrazine N-NH protons in the Z-isomer, further confirmed
3.1.2. Crystal structures of ligands L2 and L4
A few selected geometric parameters for the crystal structures of L2
and L4 are summarized in Table 2.
The ligand L2 crystallizes as whitish cream needle shaped crystals
with four molecules in the asymmetric unit. The crystal structure of the
ligand L2 is represented in Fig. 2. The crystal belongs to monoclinic
crystal class and space group P 21/c. The crystal data is given in
Table 1. The bond lengths and bond angles (Table 2) of the four mo-
lecules in a unit cell are equal and are in agreement with values re-
ported for analogous molecules, 1-(Thiophen-2-yl)ethanone TSC
[41–43]. The TSC fragment is close to being planar with the maximum
deviation from planarity being 0.0435 Å for atom C(1). The maximum
deviation from planarity for the thiophene fragment is 0.045 Å at atom
C(5).
The
thiophene
fragment’s
torsion
angles
are
S
(2) C(6) C(5) C(4) = 0.9(2)° and S(2)–C(3) –C(4)–C(5) = −0.0(2)°
further confirms planarity of the thiophene ring. The torsion angles
177.3(1)° and 168.8(1)° for N(2)–N(1)–C(2)–C(3) and S(1)–C(1)–N
(2)–N(1) respectively, suggests that the TSC fragment has an extended
conformation which may allow for intramolecular N(3)–H(3) N(1) hy-
drogen bond formation, and generation of a 5-member ring motif.
However the crystal packing does not display the anticipated N(3)–H(3)
N(1) intramolecular hydrogen bonds. The molecules are held together
by a network of interactions which include hydrogen bonds S(1) H
(2)–N(2),S(1) H(8A)–C(8), S(1) H(3)–N(3) and N(3) H(8A)–C(8);
that the TSC ligands existed as E-isomers in the DMSO solution [8]. 13
C
Fig. 3. Crystal packing diagram of L2 as viewed along the crystallographic a-axis. Intermolecular interactions are indicated by dashed lines.
5

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Table 2
Geometric parameters of ligands L2 and L4.
Bond distance in Angstroms (Å)
L2
L4
C(2) N(1)
N(1) N(2)
N(2) C(1)
C(1) S(1)
C(1) N(3)
N(3) C(9)
1.292(2)
1.376(2)
1.361(2)
1.686(2)
1.330(2)
1.443(2)
C(3) N(3)
N(3) N(2)
N(2) C(1)
C(1) S(1)
C(1) N(1)
N(1) C(2)
1.290(2)
1.376(2)
1.355(2)
1.695(2)
1.324(2)
1.453(2)
Bond angles in degrees (°)
L2
L4
C(2) N(1) N(2)
N(1) N(2) C(1)
C(1) N(3) C(9)
N(2) C(1) N(3)
N(2) C(1) S(1)
N(3) C(1) S(1)
118.3(1)
118.2(1)
124.5(1)
115.8(1)
120.2(1)
123.9(1)
C(3) N(3) N2
N(3) N(2) C(1)
C(1) N(1) C(2)
N(2) C(1) N(1)
N(1) C(1) S(1)
N(2) C(1) S(1)
115.8(1)
118.8(1)
124.7(1)
116.4(1)
124.2(1)
119.5(1)
Fig. 5. Crystal packing diagram of L4 as viewed along the crystallographic a-
Torsion angles in degrees(°)
Ligand L2
L4
axis. Intermolecular interactions are indicated by dashed lines.
C(2) N(1) N(2) C(1)
N(1) N(2) C(1) S(1)
N(1) N(2) C(1) N(3)
N(2) C(1) N(3) C(9)
S(2) C(6) C(5) C(4)
S(2) C(3) C(4) C(5)
N(2) N(1) C(2) C(3)
179.9(1)
168.8(1)
10.1(2)
1.3(2)
C(3) N(3) N(2) C(1)
N(3) N(2) C(1) S(1)
N(3) N(2) C(1) N(1)
N(2) C(1) N(1) C(2)
177.7(1)
176.8(1)
4.6(2)
associated with the thioamide vibrations and imine vibrations are
generally expected to shift to lower frequencies signaling coordination
of the ligand to the metal via the sulphur atom and the imine nitrogen
atoms respectively. The shift of spectral bands on chelation of the li-
gands to the metal through the imine and thioamide linkages reduces
the electron density on the pi-bonds, decreases the pi-character and the
bond is weakened. In this study, it was observed that the bands in the
region 1680–1500 cm−1 associated with imine vibrations are generally
split and could be overlapping with the ν(NeH) asymmetric stretches,
for complexes C1 and C3, and, the thiophene/benzene aromatic
ν(C]C) stretch. These overlaps camouflaged the expected shift to lower
energy of the band due to the imine bond ν(C]N) vibration and hence
may not be very obvious in the spectra. Despite this observation, the
bands associated with imine vibrations are generally shifted to lower
frequencies in the spectra of the complexes as compared to the free
ligand (Table 3). The thioamide ν(C]S) bond vibration in complex C4
observed at 1164 cm−1, a lower frequency than that observed for the
free ligand, an indication of coordination via the thioamide sulphur
atom. The ν(C]S) vibrations were absent in complexes C1 and C3, a
sign that the ligands coordinated to the metal in their anionic form
through the sulphide, S−1. A very slight shift (7 cm−1) was observed in
the ν(C]S) vibrations for complex C2 as compared to ligand L2,
probably due to non-involvement of the C]S bond in coordination. The
coordination of the TSC ligands through the sulphur and nitrogen atoms
was further confirmed by the presence of two new bands at
406–458 cm−1 and 478–508 cm−1 regions due to ν(Pd–N) and ν(Pd–S)
vibrations respectively. It’s worth noting that the ν(N–NH) bands in
complexes C1 and C3 shifted to higher frequencies. This shift is asso-
ciated with the electronic delocalization and may have occurred as a
consequence of the coordination through the imine nitrogen and de-
protonation of TSCs [45]. On the contrary, the ν(N–NH) vibrations in
complexes C2 and C4 did not shift significantly due to none deproto-
nation of the TSCs and hence absence of resonance. The presence of the
a hydrazinic band ν(NN–H) in the region 3180–3116 cm−1 for com-
plexes C2 and C4 suggests coordination of the TSC ligands in a neutral
manner while its absence in complex C1 and C3 suggest deprotonation
of the hydrazine nitrogen. The TSC ligands exist in the neutral thione
form in the complexes C2 and C4 while in complexes C1 and C3, it
probably exists as the anionic thiol form. Despite the expected shift to
higher frequency on coordination as a neutral ligand and shift to
slightly lower frequency on coordination as a thiolate anion[7], the
amine bands ν(–NeH) slightly shifted to lower frequency in the com-
plexes compared to the free ligands. The 2-acetyl-5-methyl TSC Pd(II)
complexes, C1 and C2 exhibited a shift to lower frequency of the bands
associated with thiophene ν(CeS) 646 and 536 cm−1 respectively
180.0(2)
0.9(2)
0.0(2)
177.3(1)
π hydrogen interactions C(5) H(9A)–C(9), and C(1) H(5A)–C(5); and
Van de Waals forces C(8) S(1), C(9) (9) thereby forming a three di-
mensional lattice structure (Fig. 3). Two adjacent molecules in the
crystal lattice are joined together by S(1) H(2) N(2) hydrogen bonds
forming inversion dimmers. The C(1) N(2) bond distance, 1.361 Å, is
longer than the C(2) N(1) bond distance, 1.282 Å; a clear indication
that an imine bond exists in ligand L2.
The ligand L4 formed needle shaped monoclinic crystals in P 21/n
space group with four molecules in a unit cell. Fig. 4 gives an illus-
tration of the crystal structure.
The benzene ring is planar with a slight variation in the torsion
angles ranging from −0.14° to −0.84°. The TSC fragment is fairly
planar with torsion angles of 177.7(1)° and −4.6(2)° for C(3)–N(3)–N
(2)–C(1) and N(3)–N(2)–C(1)– N(1) respectively, The benzene ring is
trans-related to the thiosemicarbarzone fragment about C(3) = C(4)
double bond. The bond distances and bond angles are generally the
same as those reported in literature [44]. Bond angles, torsion angles
and bond distances for ligands L2 and L4 are summarized in Table 2.
Intermolecular interactions S(1) N(2) and S(1) H(2)–N(2) between two
adjacent molecules related to each other by inversion lead to formation
of centrosymmetric dimmers. The crystal lattice is held together by a
network of interactions shown in Fig. 5.
3.2. Synthesis and characterization of the Pd(II) complexes
The Pd(II) complexes were synthesized by the reaction of the TSC
ligands with [Pd(COD)Cl2] in the ratio of 1:1 for complexes C1, C2 and
C4, and a ratio of 2:1 for complex C3 at room temperature (Scheme 1).
The complexes were obtained as orange, air and moisture stable solids
generally soluble in DMSO and DMF but insoluble in ethanol, methanol
and water. Complexes C1 and C2 are soluble in methylene chloride and
chloroform while complexes C3 and C4 are not. The new thiosemo-
carbazone Pd(II) complexes were characterized by elemental analysis,
FTIR, UV–Vis and 1H NMR spectroscopy.
3.2.1. IR spectroscopic studies
The infrared spectroscopic data for the imine ν(C]N) and thioa-
mide ν(C]S) vibrations provides very crucial information in de-
termining the coordination mode of the ligand (Table 3). The bands
6

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Scheme 1. Synthetic routes for the complexes C1, C2, C3 and C4.
compared to the free ligand bands observed at 796 and 657 cm−1 re-
spectively. This evidence of probable coordination of ligands L1 and L2
to the metal via the thiophene sulphur is further supported by the ob-
servation of two new Pd–S bands; one for coordination via the thio-
phene sulphur and the other for coordination via the thioamide sul-
phur. The spectral studies suggests that the TSC ligand L1 is
coordinated to the palladium metal in a tridentate manner forming a
complex of general formula [Pd(L)Cl] (Scheme 1) while ligands L2, L3
and L4 are coordinated in a bidentate manner forming complexes of
general formulae [PdL2] for complex C3 and [PdLCl2] for complexes C2
and C4 (Scheme 1).
absent the spectra of complexes C1 and C3, affirming the proposal
made from IR data that the ligands L1 and L3 exist in the deprotonated
form in the complexes (Figs. S2 and S3). The 1H NMR spectra of com-
plexes showed a downfield shift for the protons of the methyl group
attached to the azomethine carbon atom, (CH3)C]N in complexes C1
and C2, and the azomethine protons HC]N on complexes C3 and C4
further confirming coordination of the ligand to the metal centers via
the azomethine nitrogen. A downfield shift was also observed for the
two thiophene ring protons in the complexes C1 and C2, an indication
that the ligand may have coordinated to the metal via the thiophene
sulphur atom (Table 5).
3.2.2. 1H NMR analysis
3.2.3. UV–Vis electronic spectra analysis
The 1H NMR data of the complexes and ligands in DMSO‑d6 are
summarized in Table 4. The assignment of the signals in the spectra was
based on integrations and assignments previously made for related
compounds [7–9]. The 1H NMR data show that the spectra of complexes
C2 and C4 contain the proton on nitrogen atom of NN-H group signal
observed in the free ligand, further proof that the ligand is coordinated
to the central metal in the neutral thione form. On the other hand, the
NN–H group proton signal present in NMR spectra of the free ligand is
The electronic spectra of the ligands and complexes in the ultra
violet and visible (UV–Vis) region were obtained in dimethyl sulph-
oxide (DMSO) solvent. The spectra of the ligands exhibited two intra-
ligand bands in the 252–267 nm and 337–343 nm regions attributed to
π–π* and n–π* transitions, respectively. The intense bands are generally
associated with the C]S group, C]N group and thiophene ring, which
are overlapped in ligands L1 and L2 (Fig. S4). Ligands L3 and L4 ex-
hibited intense broad band with shoulders in the region 325–359 nm
Table 3
Selected IR vibration frequencies (cm−1) for selected bonds in the ligands and complexes.
Compound
ν(eC]N)
ν(eC]S)
ν(eNeN)
ν(eCeS) (Thiophene)
ν(Pd–S)
ν(Pe–N)
NNeH
eNeH
Ligand L1
1629, 1521
1624, 1530
1541, 1506
1565, 1514
1588, 1521
1583, 1513
1532
1286
–
993
796
646
657
536
–
–
–
3119
–
3366, 3263
3342, 3271
3331
Complex C1
Ligand L2
1046
1042
1048
1085
977
406, 478
551
–
1236
1229
1277
–
–
3242
3181
3147
–
Complex C2
Ligand L3
450, 497
530
–
3272
–
3415, 3254
3420, 3259
3353
Complex C3
Ligand L4
–
475
–
508
–
1256
1164
1084
1018
–
3164
3041
Complex C4
1607
–
458
501
3246
The IR spectra of the complexes can be found in the supplementary material file (Fig. S1).
7

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Table 4
1H NMR data (ppm) for the ligands and complexes.
Compound
δ(NNH)
δ(NH2)/(NH)
δ(HC]N)
δ(C4H2S) (thiophene)
δ(CH3) (thiophene)
δ(CH3)-C]N
δ(CH3) N(CH3)
L1
C1
L2
C2
L3
C3
L4
C4
10.32
–
7.34
8.25
7.98
–
–
7.31, 6.78
7.45, 6.85
7.30, 6.72
7.49, 6.87
–
2.28
2.36
2.26
2.47
–
2.42
2.45
2.43
2.58
–
–
–
–
10.25
–
–
3.02
2.90
–
–
11.4
–
8.19, 7.62
7.62
8.30
7.62
7.9
8.08
7.91
8.08
–
–
–
–
11.46
11.10
2.94
2.83
–
–
–
Table 5
C1, C2 and C4 are within the coordination sphere. This is an indication
that the complexes are neutral non-electrolytes with coordinated
chloride.
Electronic data for the ligands and Pd(II) complexes.
Compounds λmax, nm (ε, M−1cm−1
)
The spectroscopic analyses, elemental analysis data and con-
ductivity measurements suggest that the palladium complexes the
complexes exhibit coordination modes and structures illustrated in
Scheme 1.
*L1
341 (1030)
C1
291 (1589) 342 (1166) 393 (433)
342 (1000)
480 (218)
486 82.7)
L2
It is worth noting that attempts to grow crystals for single crystal X-
ray crystallography was not successful.
C2
340 (1472) 393 (417)
**L3
345 (1470) 358sh
(11248)
*C3
L4
303 (1770) 362 (1920) 417 (1430) 486 (97)
344 (1487) 361 (1163)
3.3. Biological studies
C4
297 (1271)
370 (1690) 388 (1681) 470 (46.2)
3.3.1. Cytotoxicity studies
Note: 10−3M solution of each compound in DMSO was scanned at 200–800 nm
range; * 5 × 10−4 M solution used; **1.5 × 10−3M.
The cytotoxicity of the ligands and their corresponding Pd(II)
complexes were screened against the human cancer (Caco-2, HeLa,
HepG2, MCF-7, PC-3) and non-cancer (MCF-12A) cells. The cells were
exposed to 100 μM of each compound for 24 h, the cell viability in
response to treatments was assessed following MTT assay and expressed
as % cell viability. As shown in Fig. 6, the ligands demonstrated varying
degrees of toxicity against the test cells. L1 and L3 were the most active
and exhibited < 40% cell viability in two or three cell lines i.e. PC-3,
HepG2 and Caco-2 cells. The PC-3 and HepG2 cells showed higher
susceptibility to three (L1, L3 and L4) and four ligands, respectively.
The cytotoxic effect of the ligands on the cells was enhanced after
coordination to the Pd metal, thus the cytotoxic effects of the Pd(II)
complexes were significantly higher than the ligands. Treatment with
100 μM of the Pd(II) complexes resulted in non-selective reduction in
proliferation of both cancer and non-cancer cells. In essence, the com-
plexes were more lethal to the cancer cells compared to their respective
ligands. A few exceptions where the ligands were more potent than the
Pd(II) complexes were observed, for instance, C3 versus L3 against
Caco-2 (29.4% vs 35.5%) and C1 versus L1 against PC-3 (7.7% vs 10%)
cells.
associated with the overlap of the eC]Ce group of the benzene ring.
These bands associated with the C]S and C]N groups exhibited a
red shift of between 20 and 61 nm after coordination of the ligands L2
and L4 to the metal center through the imine nitrogen and thiol sulphur
respectively. This observation is in agreement with others earlier re-
ported for palladium TSC complexes [9,46]. The spectra of complexes
exhibited new strong bands resulting from ligand to metal charge
transfer (LMCT) in the region 338–478 nm. These bands may be as-
signed to S–Pd charge transfer further supporting coordination of the
ligand to the metal via the sulphur atom of the C]S group in the
neutral thiol form in C2 and C4 and anionic form in C1 and C3. The
spectra of complexes C1 and C2 each had a new band at 483 nm, a
further indication that the thiophene sulphur is also involved in co-
ordination as suggested by the FT-IR data. On the contrary, the UV–Vis
spectra of complexes C3 and C4, which do not bear the thiophene
group, did not show any bands at 483 nm. The Pd(II) ion has d8 con-
figuration and is expected to show three bands in the electronic spec-
trum since palladium complexes have a square planar geometry cor-
1
responding to the A1g
→
1A2g,1A1g
→
1B1g, and1A1g
→
1Eg transitions
The concentration of the compounds that inhibited 50% cell pro-
liferation (IC50) on each cell line was estimated using GraphPad Prism
software version 5 (Table 7). The IC50 values revealed that for the li-
gands to inhibit 50% cell proliferation, IC50 values of more 100 μM was
required, with the exception of L1 and L2 on HepG2 and MCF-7 cells.
L1 had an IC50 value of 45.3 μM against the human breast cancer cells
(MCF-7) and 85 μM on HepG2; and 54 μM for L2 on HepG2 cells. The
Pd(II) complexes were observed to be more active in preventing cell
proliferation than the free ligands. Some of the Pd(II) complexes were
more potent against specific cell lines with C2, C3 and C4 exhibiting a
broad spectrum potency against all the human cancer cell lines tested.
The activity of C2 against the proliferation of the cells tested increased
in the order Caco-2 < HepG2 < MCF-7 < HeLa < PC3. All the Pd
(II) complexes (C1, C2, C3 and C4) exhibited very good activity against
the human prostate cancer cell (PC-3) in the order C3 > C2 > C1 >
C4, with the IC50 values ranging between 2.0 µM (C3) and 9.6 µM (C4).
The IC50 values previously reported for the Pd(II) thiosemicazone
complexes against PC-3 cells were much higher (48–58 μM) [4] when
compared to the ones reported herein (2.0–9.0 μM). C2 was an ex-
cellent candidate for breast cancer (MCF-7) cells because the
[8]. The presence of the S and N atoms makes the spectra of Pd(II)
complexes complicated since the tails of the S–Pd band may extend up
to the visible region of the spectrum thus obscuring the expected d–d
transitions. The elemental analysis data obtained for the complexes and
the ligands further confirmed the composition and reaction ratio of the
ligands and metal ions and hence the proposed molecular structures
and coordination modes.
The conductivity measurements of solutions the metal complexes in
DMSO indicated that the Pd(II) complexes are non-electrolytes since the
values obtained for the compounds were the same as that observed for
pure DMSO (Table 6). This clearly shows that the chloride in complexes
Table 6
Conductivity values for the Pd(II) complexes in DMSO.
Metal complexes
[Pd(L1)Cl] [Pd(L2)Cl2] [Pd(L3)2] [Pd(L4)Cl2] DMSO
Conductivity mS/
cm
0.24 0.23 0.24 0.23 0.25
8

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Fig. 6. Effect of the ligands and their Pd (II) complexes against cancer and non-cancer cell lines. The cells were exposed to 100 μM of L1 vs C1 (A), L2 vs C2 (B), L3 vs
C3 (C), and L4 vs C4 (D).*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
concentration needed to inhibit 50% cell proliferation was 7.6 µM
compared to the IC50 for the non-cancer breast (MCF-12A) cells. The
IC50 value for C2 against the MCF-7 cells is comparable to the IC50
values (4.41 µM and 48.0 µM) reported for Pd complexes on the same
cell line [10]. Of all the compounds tested, C2 was the most potent
against the cancer cells. The IC50 values observed in this study are
comparable to those reported for Pd(II) complexed with indole-3-car-
baldehyde thiosemicarzone in literature [47]. It is worth noting, from
the data obtained, that the presence or absence of coordinated chloride
ion may or may not influence the inhibition of cancer cell proliferation.
For instance, C2 and C4 each had two coordinated chloride ions but C2
exhibited higher cytotoxicity mostly on cancer cells while C4 was toxic
to all cells (Table 7). The difference in activity between the two com-
plexes can probably be associated with the presence of the two sulphur
atoms in C2 and only one sulphur atom in C4.
hepatocellular, breast and prostate) and non-cancer breast cells were
determined. The thiosemicarbazone L2, L3 and L4 coordinated to the
metal via the nitrogen and sulphur (N,S fashion) of the of the thiose-
micarbazide fragment in a bidentate manner while L1 coordinated to
the metal in a tridentate manner via the nitrogen and Sulphur of the
thiosemicarbazide fragment, and Sulphur of the thiophene fragment
(S,N,S fashion). Conductivity measurements done on the complexes
showed that the complexes are non-electrolytes an indication that the
chloride ions are within the coordination sphere. This also confirms that
the thiosemicarbazones in complexes C1, C2 and C4 are coordinated
anions of the thiol tautomer. Except for L1 which gave an IC50 value of
45.3 μM against HepG2 cancer cells, the ligands generally did not show
growth inhibition against any of the cells tested at IC50 values below
50 μM. The Pd(II) complexes exhibited excellent growth inhibition
against all the human cancer cells tested, which was reduced in the non-
cancer breast cells. C3 had an IC50 value of 2.0 μM against the PC-3
cancer cells and can be a suitable candidate for prostate cancer. Further
studies are underway to investigate the selectivity of the complexes and
their mechanism of action.
4. Conclusion
The crystal structures confirm the formation of L2 and L4. The new
Pd(II) complexes of 2-acetyl-5-methyl thiophene and CIN thiosemi-
carbazones were successfully synthesized and their in vitro anticancer
activities against five human cancer cell lines (colon, cervix,
Table 7
IC50 of ligands and Pd complexes.
Samples
IC50 (µM)
Caco-2
MCF-12A
HeLa
PC-3
HepG2
MCF-7
L1
C1
L2
C2
L3
C3
L4
C4
> 100
74.14
> 100
15.92
> 100
27.87
> 100
76.24
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
7.92
> 100
5.53
84.85
16.26
54.03
11.68
> 100
> 100
> 100
9.61
2.01
1.08
3.54
2.79
45.25
72.65
> 100
9.53
2.57
1.96
3.16
2.01
0.50
3.15
0.49
> 100
5.3
0.07
0.03
2.90
> 100
> 100
> 100
52.44
> 100
1.99
> 100
10.06
> 100
48.76
5.15
90.6
21.92
0. 52
5.52
0.05
0.21
3.31
4.55
> 100
9.6
0.63
2.32
0.58
aData represents the mean values of three independent experiments.
9

E.A. Nyawade, et al.
InorganicaChimicaActa515(2021)120036
Declaration of Competing Interest
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Acknowledgements
We would like to thank Jomo Kenyatta University of Agriculture
and Technology in Kenya for granting Dr. Eunice Nyawade study leave
to undertake this study at the University of the Western Cape, South
Africa. We would also like to thank the following research groups:
“Organometallics and Nanomaterials” and “DSI/Mintek NIC Biolabels
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Funding
[23] T. Nitoda, M.D. Fan, I. Kubo, Phytother. Res. 22 (2008) 809–813.
[24] L. Liang-Tzung, T. Chen-Jei, C. Shun-Pang, C. Jin-Liang, W. Shu-Jing, L. Chun-
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This work was supported by the National Research Foundation,
South Africa [grant numbers UD: 116692.
[26] D.T. Shaughnessy, R.W. Setzer, D.M. DeMarini, Mutat. Res./Fundam. Mol. Mech.
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Mech. Mutag. 107 (1983) 219–227.
CCDC2009031 and 2009034 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of charge
via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: +44 1223 336033). Supplementary data to this ar-
ticle can be found online at https://doi.org/10.1016/j.ica.2020.
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