Platinum(II) and palladium(II) aryl-thiosemicarbazone complexes: Synthesis, characterization, molecular modeling, cytotoxicity, and antimicrobial activity

Article abstract of DOI:10.1080/00958972.2014.900664

Platinum(II) and palladium(II) complexes [ML₂] have been isolated from reaction of K₂PtCl₄ or K ₂PdCl₄ and ligands (L) derived from thiosemicarbazones. The complexes were characterized by elemental analysis, Raman, IR, and NMR spectroscopy. In addition, quantum mechanical calculations were used to predict their structures and spectroscopic properties. For the first time, theoretical calculations using ¹⁹⁵Pt NMR data were used to support the suggested structures. The results indicate that the thionic sulfur and the azomethine nitrogen are bonded to the metal ion in a trans configuration. Antibacterial activities and cytotoxicities of the complexes to B16-F10 and CT26.WT cell lines were also investigated. Some of the complexes demonstrated superior cytotoxic activity compared to cisplatin. 2014

Full text of DOI:10.1080/00958972.2014.900664

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Platinum(II) and palladium(II) aryl-
thiosemicarbazone complexes:
synthesis, characterization,
molecular modeling, cytotoxicity, and
antimicrobial activity
T.T. Tavaresa, D. Paschoala, E.V.S. Mottab, A.G. Carpaneza, M.T.P.
Lopesc, E.S. Fontesb, H.F. Dos Santosa, H. Silvaa, R.M. Grazula &
A.P.S. Fontesa
a Departamento de Química, Universidade Federal de Juiz de
Fora, Juiz de Fora, Brazil
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b Departamento de Bioquímica, Universidade Federal de Juiz de
Fora, Juiz de Fora, Brazil
c Departamento de Farmacologia, Universidade Federal de Minas
Gerais, Belo Horizonte, Brazil
Accepted author version posted online: 05 Mar 2014.Published
online: 01 Apr 2014.
To cite this article: T.T. Tavares, D. Paschoal, E.V.S. Motta, A.G. Carpanez, M.T.P. Lopes,
E.S. Fontes, H.F. Dos Santos, H. Silva, R.M. Grazul & A.P.S. Fontes (2014) Platinum(II) and
palladium(II) aryl-thiosemicarbazone complexes: synthesis, characterization, molecular modeling,
cytotoxicity, and antimicrobial activity, Journal of Coordination Chemistry, 67:6, 956-968, DOI:
10.1080/00958972.2014.900664
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Journal of Coordination Chemistry, 2014
Vol. 67, No. 6, 956–968, http://dx.doi.org/10.1080/00958972.2014.900664
Platinum(II) and palladium(II) aryl-thiosemicarbazone
complexes: synthesis, characterization, molecular modeling,
cytotoxicity, and antimicrobial activity
T.T. TAVARES†, D. PASCHOAL†, E.V.S. MOTTA‡, A.G. CARPANEZ†, M.T.P. LOPES§,
E.S. FONTES‡, H.F. DOS SANTOS†, H. SILVA†, R.M. GRAZUL† and
A.P.S. FONTES*†
†Departamento de Química, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil
‡Departamento de Bioquímica, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil
§Departamento de Farmacologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
(Received 19 September 2013; accepted 1 February 2014)
Platinum(II) and palladium(II) complexes [ML2] have been isolated from reaction of K2PtCl4 or
K2PdCl4 and ligands (L) derived from thiosemicarbazones. The complexes were characterized by
elemental analysis, Raman, IR, and NMR spectroscopy. In addition, quantum mechanical calcula-
tions were used to predict their structures and spectroscopic properties. For the first time, theoretical
calculations using 195Pt NMR data were used to support the suggested structures. The results indi-
cate that the thionic sulfur and the azomethine nitrogen are bonded to the metal ion in a trans con-
figuration. Antibacterial activities and cytotoxicities of the complexes to B16-F10 and CT26.WT cell
lines were also investigated. Some of the complexes demonstrated superior cytotoxic activity com-
pared to cisplatin.
Keywords: Thiosemicarbazones; Pt(II) and Pd(II) complexes; Cytotoxicity; Molecular modeling
1. Introduction
The majority of potential antitumor metal complexes are structural analogs of cisplatin.
However, due to severe side effects and other undesirable properties [1, 2], there has been a
*Corresponding author. Email: ana.fontes@ufjf.edu.br
© 2014 Taylor & Francis

Platinum(II) and palladium(II) thiosemicarbazones
957
decrease in the number of compounds based on the cisplatin structure. This has motivated
the search for complexes presenting novel structures and promising activity, mainly those
with the ability to overcome cisplatin resistance [3].
Metal coordination to biologically active molecules can be used as a strategy to enhance
activity and overcome resistance. For instance, metal complexes of thiosemicarbazones are
often more active than the free ligand, or even act as a vehicle for activation of the ligand
as the cytotoxic agent [2, 3]. The coordination chemistry of thiosemicarbazones has gener-
ated interest for their variable donor ability, structural diversity, and biological applications
[4, 5]. Thiosemicarbazones are among the most potent inhibitors of ribonucleotide reductase
activity. This enzyme catalyzes the synthesis of DNA and is responsible for maintaining the
balanced supply of deoxyribonucleotides required for DNA synthesis and repair. Strong
positive correlation has been established between ribonucleotide reductase activity and anti-
tumor activity [6]. Platinum(II) and palladium(II) complexes with thiosemicarbazones are
particularly attractive because of their antitumor [7], antibacterial [8], antiviral [9], and cyto-
toxic activities [10, 11].
The combination of thiosemicarbazones with platinum(II) and palladium(II) can also pro-
duce synergistic inhibition of tumor growth and may lead to improvements in the efficacy
of cancer chemotherapy [4, 12, 13]. Palladium complexes with thiosemicarbazones show
antitumor activities and some are also active in cell lines resistant to cisplatin, presenting
decreased nephrotoxicity [14, 15].
Thiosemicarbazones may incorporate heterocyclic or aromatic rings. Due to their biologi-
cal activities, such molecules have attracted interest. They have been previously described
to present nematocidal, insecticidal, antibacterial, antifungal, antiviral, and anti-inflamma-
tory activities, especially for furan and thiophene side groups [16].
The coordination chemistry of metal complexes with thiosemicarbazones has been inten-
sively investigated. Usually they bind to a metal ion via dissociation of the hydrazine pro-
ton, as a bidentate N, S donor, forming a five-membered chelate ring [17]. The ligands may
feature more than two binding sites, depending on the precursor aldehyde, and on the tauto-
meric equilibrium of the thiosemicarbazone.
As part of our continuing investigations on metal complexes as anticancer [18, 19] and
antimicrobial [20, 21] agents, we report herein the synthesis, characterization, and cytotoxic/
antimicrobial activity of palladium(II) and platinum(II) complexes containing thiosemicarba-
zone ligands. The compounds were characterized by Raman, IR, and NMR (1H, 13C, 195Pt)
spectroscopy and elemental analysis, in addition to quantum mechanical calculations.
2. Experimental
2.1. Materials and methods
All reagents and solvents were used as obtained from commercial sources (Aldrich Sigma)
without purification. The thiosemicarbazones were prepared according to previously
reported methods [22].
Melting points were determined with a MQAPF-Microquímica hot-stage apparatus. Ele-
mental analyses were performed at the University of São Paulo, Brazil. IR spectra were
obtained on a Bomem FT IR MB-102 spectrometer as KBr pellets. Fourier-transform
Raman spectroscopy was carried out using a Bruker RFS 100 instrument equipped with an

958
T.T. Tavares et al.
Nd3+/YAG laser operating at 1064 nm in the near infrared with a CCD detector cooled with
1
liquid nitrogen with an average of 1000 scans and a spectral resolution of 4 cm−1. H NMR
(300 MHz), 13C NMR (75 MHz), and 195Pt NMR (64 MHz) spectra were recorded on a
Bruker spectrometer by dissolving the complexes in DMSO-d6. The chemical shifts are
expressed as δ (in ppm) from internal reference standards, TMS (1H and 13C NMR) and
K2PtCl6 (195Pt NMR). Mass spectra were obtained on an AXIMA MALDI-TOF-TOF Shi-
madzu Biotech equipment. A nitrogen laser (λmax = 337 nm) was employed for excitation in
a α cyano matrix. 200 scans were accumulated with 20 repetitions each.
2.2. Synthesis of ligands
The thiosemicarbazone ligands were prepared according to the literature procedure [22]
(scheme 1). To a hot solution of thiosemicarbazide (31.25 mM) in 50 mL of methanol, a
solution of the corresponding aromatic aldehyde (31.25 mM) in 30 mL of methanol was
added dropwise. The mixture was stirred and refluxed for 4 h, cooled, and then filtered. The
volume of the filtrate was reduced and kept in a refrigerator. After several hours, yellow or
white solids were obtained, isolated by filtration, washed with cold ethanol, and dried under
vacuum. (a) (C8H9N3S), white solid, yield = 62%, m.p. 158–160 °C; (b) (C9H11ON3S),
white solid, yield = 82%, m.p. 174–175 °C; (c) (C6H7N3S2), pale yellow solid, yield = 74%,
m.p. 189–190 °C; (d) (C6H7ON3S), pale yellow solid, yield = 61%, m.p. 152–153 °C;
(e) (C7H8N4S), beige solid, yield = 88%, m.p. 217–219 °C; (f) (C8H9ON3S), beige solid,
yield = 65%, m.p. 237–238 °C.
2.3. Synthesis of complexes
A suspension of potassium tetrachoroplatinate or potassium tetrachloropalladate (0.5 mM)
in methanol (10 mL) was added with stirring to a solution of the corresponding ligand
(1 mM) in methanol (10 mL) containing triethylamine (2 mM; scheme 2). The solution was
refluxed for 24 h for the palladium complexes. For the platinum complexes, reflux was
maintained between 10 and 20 days. The complete consumption of the starting material
was evidenced by thin-layer chromatography (TLC; eluent: 9:1 dichloromethane/methanol).
The precipitate thus formed was collected by filtration and dried in vacuum. Yields: (1)
74%, (2) 42%, (3) 64%, (4) 61%, (5) 67%, (6) 31%, (7) 40%, (8) 79%, (9) 70%, (10)
87%, (11) 61%, (12) 66%.
(1) IR νmax KBr (cm−1): 3420, 3274, 1598, 1586, 1025, 847; Raman (cm−1): 1578, 1556,
1180, 872, 433, 400. 1H NMR (300 MHz, DMSO-d6) δ: 7.79 m, 2H, NH2); 7.78,7.44
(m, 6H, Ph), 8.14 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6) δ: 132.38, 132.32,
130.70, 128.29, 128.10 and 11.32 (C, Ph); 143.36 (HC=N); 176.16 (C-S); 195Pt NMR
(64 MHz, DMSO-d6) δ: –3126. Anal. Calcd for C16H16N6S2Pt (%): C, 34.85; H, 2.90; N,
15.25. Found: C, 34.72; H, 3.02; N, 14.71. MS (MALDI): m/z Calcd for [C16H17N6S2Pt]
[M + H]+ Calcd (552.1) found 552.5.
(2) IR νmax KBr (cm−1): 3468, 3359, 1603, 1563, 1012, 856; Raman (cm−1): 1585,
1
1537, 1165, 869, 486, 400. H NMR (300 MHz, DMSO-d6) δ: 3.83 (s, 3H, CH3); 7.33
(s, 2H, NH2); 8.18, 8.15, 6.99, 6.96 (dd, 6H, Ph), 7.72 (s, 1H, HC=N); 13C NMR (75 MHz,
DMSO-d6) δ: 160.91, 134.59, 125.43 and 113.49 (C, Ph); 154.16 (HC=N); 175.08 (C–S);
195Pt NMR (64 MHz, DMSO-d6) δ: −3143. Anal. Calcd for C18H20N6S2O2Pt (%): C,
35.35; H, 3.27; N, 13.03. Found: C, 35.31; H, 3.50; N, 13.75. MS (MALDI): m/z Calcd for
[C18H21N6S2O2Pt] [M + H]+ Calcd (612.1) found 612.2.

Platinum(II) and palladium(II) thiosemicarbazones
959
(3) IR νmax KBr (cm−1): 3461, 3358, 1599, 1572, 1062, 813. Raman (cm−1): 1578,
1
1522, 1083, 857, 456, 388. H NMR (300 MHz, DMSO-d6) δ: 7.34 (s, 2H, NH2); 8.07,
7.94, 7.64, 7.15 (dd, 4H, Ph), 8.11 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 149.42, 141.89, 140.34, 132.44 (C, Ph); 153.01 (HC=N); 182.04 (C-S); 195Pt NMR
(64 MHz, DMSO-d6) δ: −3012. Anal. Calcd for C12H12N6S4Pt (%): C, 25.58; H, 2.13;
N, 14.92. Found: C, 25.79; H, 2.18; N, 14.15. MS (MALDI): m/z Calcd for [C12H13N6S4Pt]
[M + H]+ Calcd (564.0) found 564.5.
(4) IR νmax KBr (cm−1): 3405, 3295, 1608, 1588, 1091, 862. Raman (cm−1): 1590,
1
1503, 1146, 823, 452, 418. H NMR (300 MHz, DMSO-d6) δ: 7.56 (s, 2H, NH2); 7.77,
7.76, 7.70, 6.75 (dd, 4H, Ph), 7.90 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 146.07, 141.76, 121.54, 121.54 and 113.03 (C, Ph); 147.69 (HC=N); 175.79 (C-S);
195Pt NMR (64 MHz, DMSO-d6) δ: −2999. Anal. Calcd for C12H12N6S2O2Pt (%): C,
27.12; H, 2.26; N, 15.49. Found: C, 27.16; H, 2.42; N, 15.82. MS (MALDI): m/z Calcd for
[C12H13N6S2O2Pt] [M + H]+ Calcd (532.0) found 532.3.
(5) IR νmax KBr (cm−1): 3417, 3288, 1632, 1589, 1040, 859. Raman (cm−1): 1593, 1541,
1
1154, 850, 418, 384. H NMR (300 MHz, DMSO-d6) δ: 7.07 (s, 2H, NH2); 8.69, 8.36,
7.85, 7.68, 7.61, 7.38 (m, 5H, Ph), 8.08 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 151.96, 138.49, 125.19 (C, Ph); 150.11 (HC=N); 178.19 (C-S); 195Pt NMR (64 MHz,
DMSO-d6) δ: −2932. Anal. Calcd for C14H14N8S2Pt (%): C, 30.38; H, 2.74; N, 20.25.
Found: C, 31.31; H, 3.11; N, 22.30. MS (MALDI): m/z Calcd for [C14H15N8S2Pt] [M + H]+
Calcd (554.1) found 554.1.
(6) IR νmax KBr (cm−1): 3426, 3321, 1598, 1574, 1025, 827. Raman (cm−1): 1560,
1
1537, 1169, 869, 414, 384. H NMR (300 MHz, DMSO-d6) δ: 7.22 (s, 2H, NH2); 8.06,
8.03, 6.81, 6.78 (m, 6H, Ph), 7.65 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 160.13, 135.13, 124.32, 115.22 (C, Ph); 135.16 (HC=N); 174.87 (C-S); 195Pt NMR
(64 MHz, DMSO-d6) δ: −2931. Anal. Calcd for C16H16N6S2O2Pt (%): C, 32.93; H, 2.74;
N, 14.41. Found: C, 31.80; H, 2.55; N, 13.97. MS (MALDI): m/z Calcd for
[C16H17N6S2O2Pt] [M + H]+ Calcd (584.1) found 584.3.
(7) IR νmax KBr (cm−1): 3452, 3330, 1615, 1574, 1023, 868. Raman (cm−1): 1596,
1
1574, 1173, 874, 470, 405. H NMR (300 MHz, DMSO-d6) δ: 7.27 (m, 2H, NH2); 7.66,
7.58, 7.37, 6.79 (m, 6H, Ph), 7.37 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 132.24, 130.62, 130.03, 127.29 (C, Ph); 155.02 (HC=N); 172.81 (C–S); Anal. Calcd for
C16H16N6S2Pd (%): C, 41.52; H, 3.83; N, 18.17. Found: C, 39.92; H, 3.91; N, 17.27. MS
(MALDI): m/z Calcd for [C16H17N6S2Pd] [M + H]+ Calcd (463.1) found 463.5.
(8) IR νmax KBr (cm−1): 3458, 3336, 1609, 1566, 1018, 827. Raman (cm−1): 1593,
1
1571, 1173, 880, 422, 396. H NMR (300 MHz, DMSO-d6) δ: 3.79 (s, 3H, CH3); 6.66
(s, 2H, NH2); 7.56, 7.53, 6.88, 6.85 (dd, 6H, Ph), 7.38 (s, 1H, HC=N); 13C NMR (75 MHz,
DMSO-d6) δ: 160.84, 131.46, 124.40, 112.24 (C, Ph); 154.76 (HC=N); 171.48 (C–S).
Anal. Calcd for C18H20N6S2O2Pd.CH3OH (%): C, 41.12; H, 4.32; N, 15.15. Found: C,
40.08; H, 4.16; N, 15.12. MS (MALDI): m/z Calcd for [C18H21N6S2O2Pd] [M + H]+ Calcd
(523.0) found 523.3.
(9) IR νmax KBr (cm−1): 3443, 3281, 1598, 1572, 1057, 825. Raman (cm−1): 1578,
1
1576, 1146, 857, 418, 317. H NMR (300 MHz, DMSO-d6) δ: 7.13 (s, 2H, NH2); 7.87,
7.86, 7.11, 7.10 (dd, 4H, Ph), 7.58 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 135.58, 133.95, 132.89, 126.56 (C, Ph); 146.78 (HC=N); 173.07 (C–S). Anal. Calcd for
C12H12N6S4Pd (%): C, 30.35; H, 2.53; N, 17.71. Found: C, 30.32; H, 2.68; N, 17.29. MS
(MALDI): m/z Calcd for [C12H13N6S4Pd] [M + H]+ Calcd (474.9) found 475.1.

960
T.T. Tavares et al.
(10) IR νmax KBr (cm−1): 3478, 3303, 1609, 1592, 1082, 889. Raman (cm−1): 1585,
1
1560, 1150, 828, 425, 381. H NMR (300 MHz, DMSO-d6) δ: 7.34 (s, 2H, NH2); 7.62,
7.64, 7.18, 6.70, 6.69 (dd, 4H, Ph), 7.84 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-d6)
δ: 146.09, 140.85, 121.21, 113.32 (C, Ph); 147.17 (HC=N); 174.57 (C–S). Anal. Calcd
for C12H12N6S2O2Pd (%): C, 32.55; H, 2.71; N, 18.99. Found: C, 32.77; H, 2.91; N,
18.90. MS (MALDI): m/z Calcd for [C12H13N6S2O2Pd] [M + H]+ Calcd (443.0) found
443.3.
(11) IR νmax KBr (cm−1): 3470, 3410, 1619, 1593, 1027, 865. Raman (cm−1):
1
1593, 1574, 1199, 857, 444, 392. H NMR (300 MHz, DMSO-d6) δ: 7.25 (s, 2H, NH2);
8.65, 8.63, 8.19, 7.66, 7.63, 7.55 (m, 5H, Ph), 8.21 (s, 1H, HC=N); 13C NMR (75 MHz,
DMSO-d6) δ: 138.31, 137.87, 125.29, 125.09 (C, Ph); 147.27 (HC=N); 176.60 (C–S).
Anal. Calcd for C14H14N8S2Pd (%): C, 36.18; H, 3.01; N, 24.12. Found: C, 35.82; H,
2.86; N, 23.15. MS (MALDI): m/z Calcd for [C14H15N8S2Pd] [M + H]+ Calcd (465.0)
found 465.4.
(12) IR νmax KBr (cm−1): 3392, 3305, 1600, 1598, 1029, 816. Raman (cm−1): 1588,
1512, 1176, 805, 414, 343. 1H NMR (300 MHz, DMSO-d6) δ: 6.98 (s, 2H, NH2);
7.99, 7.97, 6.97, 6.76 (m, 6H, Ph), 7.14 (s, 1H, HC=N); 13C NMR (75 MHz, DMSO-
d6) δ: 160.08, 135.10, 123.85, 115.28 (C, Ph); 153.99 (HC=N); 173.36 (C–S). Anal.
Calcd for C16H16N6S2O2Pd (%): C, 38.83; H, 3.24; N, 16.99. Found: C, 38.06; H,
3.18; N, 16.97. MS (MALDI): m/z Calcd for [C16H17N6S2O2Pd] [M + H]+ Calcd
(495.0) found 495.3.
2.4. Cytotoxicity assay
Cytotoxic activities were investigated against the following tumor cell lines: B16-F10 –
mouse metastatic skin melanoma, CT26.WT – colon cancer cells (Ludwig Institute of Can-
cer Research – São Paulo – Brazil), and the non-tumor cell line BHK-21 – Baby Hamster
Kidney (Centro Panamericano de Febre Aftosa – Rio de Janeiro – Brazil). All cell lines
were propagated in RPMI 1640 culture medium at pH 7.4, supplemented with 10% heat-
inactivated fetal bovine serum (FBS), HEPES (4.0 mM), NaHCO3 (14.0 mM), ampicillin
(0.27 mM), and streptomycin (0.06 mM).
Cells were harvested by trypsinization and seeded in 96-well tissue culture plates
(100 μL/well) at different densities according to the cell line (0.5 × 103–2 × 103 viable cells/
well) and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 24 h. Stock
solutions of the test substances in DMSO were serially diluted in cell culture medium (<1%
DMSO). After drug exposure for 72 h at 37 °C and 5% CO2, cells were incubated with
MTT (0.01 M in water – 10 μL/well) for 4 h at 37 °C and 5% CO2. MTT is metabolized by
viable cells resulting in a violet product that, after being solubilized in 100 μL of DMSO,
can be quantified through colorimetric assay using an ELISA reader (absorbance at
570 nm).
The negative control (100% value of viability) was obtained with cells exposed to RPMI
1640 medium supplemented with 10% FBS. Positive controls (cisplatin) were also used
against these cell lines.
The raw data were normalized to the untreated control cells and set relative to the meta-
bolic activity of the viable treated cells. IC50 values were calculated by four parametric non-
linear regression using Graph Pad Prism 5.0 software.

Platinum(II) and palladium(II) thiosemicarbazones
2.5. Antimicrobial assay
961
The samples were evaluated against the bacterial strains Staphylococcus aureus (ATCC
29213), Pseudomonas aeruginosa (ATCC 27853), Shigella dysenteriae (ATCC 13313),
Salmonella enterica subsp. enterica serovar typhimurium (ATCC 13311), Escherichia coli
(ATCC 10536), and Enterobacter cloacae (ATCC 23355).
The MIC of each sample was determined using broth microdilution techniques for bacte-
ria (NCCLS, 2002). MIC values were determined in Mueller-Hinton broth (MHB). Bacterial
strains were cultured overnight at 37 °C in Brain Heart Infusion agar. Sample stock solu-
tions were twofold diluted from 200 to 1.56 μg mL−1 (final volume = 80 μL) and a final
DMSO concentration ≤ 1%. Then, MHB (100 μL) was added onto 24 microplates. Finally,
20 μL of 106 CFU/mL (values of 0.08–0.10 at 625 nm, according to McFarland turbidity
standards) of standardized bacterial suspensions were inoculated onto microplates and the
test was performed with a volume of 200 μL. Plates were incubated at 37 °C for 24 h. The
same tests were performed simultaneously for growth control (MHB + bacteria) and sterility
control (MHB + extract). The MIC values were calculated as the highest dilution showing
complete inhibition of the tested strain. Chloramphenicol (100–0.78 μg mL−1) was used as
positive control [23].
2.6. Calculations
Quantum mechanical calculations using density functional theory (DFT) were used to
predict structures and spectroscopic properties of the Pt(II) and Pd(II)-thiosemicarbazone
complexes. The geometries for the cis and trans isomers were optimized in the gas
phase at the B3LYP level [24] with the 6-31+G(d) basis set used for hydrogen and
heavy elements, except for the metal for which the LanL2DZ [25] effective core poten-
tial was set for core electrons with its balanced double-zeta basis set for valence elec-
trons [26]. This calculation scheme is abbreviated as B3LYP/LanL2DZ/6-31+G(d) and
was recently evaluated for cisplatin reactivity [27]. The same level of theory was used
for vibrational frequency calculation in order to characterize the structures as true min-
ima on the potential energy surface (all harmonic frequencies real) as well as to analyze
the vibrational normal modes. NMR predictions were also accomplished for 1H, 13C,
and 195Pt nuclei in order to assist the experimental assignments. The gage-independent
atomic orbital method implemented by Wolinski, Hilton and Pulay [28] was used for
calculation of magnetic shielding constants (σ) and the chemical shifts (δ), obtained on a
δ-scale relative to the TMS (1H and 13C) and K2PtCl6
(
195Pt). For Pd(II) complexes,
with only H and 13C nuclei active, the B3LYP/LanL2DZ/6-311++G(2df,2pd)// level of
calculation was used with solvent effect (DMSO) included through the IEFPCM contin-
uum model [29]. The double slash signifies that the geometries were those calculated at
B3LYP/LanL2DZ/6-31+G(d) level. For Pt(II), in which the 195Pt nuclei is also active, a
distinct methodology was applied. The B3LYP level was used accounting for Douglas–
Kroll–Hess (DKH) fourth-order relativistic calculations including spin-orbit terms [30].
The basis set for all atoms was the NMR-TZPP-DKH, which was specifically con-
structed for 195Pt NMR prediction [31]. This calculation scheme is abbreviated as
B3LYP-DHKSO/NMR-TZPP-DKH//. All calculations were carried out using the
Gaussian 09 suite of programs [32].
1

962
T.T. Tavares et al.
3. Results and discussion
3.1. Structures and spectroscopic characterization
The thiosemicarbazones used as ligands were prepared by the classical method that consists
of condensation of the aldehyde and thiosemicarbazide [22]. They were characterized using
1
IR, H, and 13C NMR and by comparison with the data reported in the literature [33, 34].
Structures for Pd(II) and Pt(II) complexes with benzaldehyde thiosemicarbazone have
been described by Hernández and collaborators [35]. They found that Pd forms a bis-che-
lated complex whereas the Pt complex is tetranuclear. To the best of our knowledge, the
other complexes are new, although different structures with the same ligands have been
described. For example, Lobana and coworkers [36] have described a palladium complex
with triphenylphosphine- and thiophene thiosemicarbazone-coordinated tridentate through
the N, S, and C. Antelo et al. [37] reported the study of palladium and platinum bimetallic
compounds with p-hydroxyphenyl thiosemicarbazone.
We were not able to obtain crystals for X-ray diffraction, therefore definitive solid-state
structures could not be obtained. Nonetheless, gas-phase geometries were predicted with the
aid of DFT calculations. The metal : ligand 1 : 2 stoichiometry was observed from elemental
analysis with the thiosemicarbazone in the Z form. The cis and trans forms are presented in
figure 1 (for the benzene analog) with the main structural data quoted in table S1 and given
as Supplementary materials (see online supplemental material at http://dx.doi.org/10.1080/
00958972.2014.900664). The trans isomer was more stable than the cis isomer, regardless
of the ligand derivative, with Gibbs free energy difference ~3 kcal M−1 (Pt complexes) and
~1 kcal M−1 (Pd complexes). The kinetics for formation of cis and trans isomers also favors
the trans form. Using a simpler model with R=CH3 in scheme 2, the free-energy barrier
was 4 kcal M−1 lower for the reaction leading to the trans isomer than the corresponding
process for the cis complex in methanol solution at 298.15 K. Therefore, the theoretical
analysis presented here is focused on the trans isomer with some data for the cis form given
as comparison. The trans complexes were planar with a slightly distorted square-planar
geometry around the metal. The M–N and M–S bond lengths (M=Pt or Pd) were predicted
as 2.07 and 2.37 Å, respectively, and the S–M–N angle was predicted to be 82° (see table
S1). These values are in agreement with X-ray data reported for the Pd(II) complex of
m-cyanobenzaldehyde thiosemicarbazone which were found to be 2.05, 2.30 Å, and 82.8°,
respectively [35].
Figure 1. B3LYP/LanL2DZ/6-31+G(d) optimized geometries for trans (a) and cis (b) forms of 1.

Platinum(II) and palladium(II) thiosemicarbazones
963
S
O
H
S
MeOH
H2O
R
N
+
R
+
H2N
N
H
NH2
N
H
NH2
Ligands a-f
(a)
(c)
(b)
R =
*
*
*
*
S
H3CO
(f)
*
(e)
(d)
*
N
HO
O
Scheme 1.
The experimental IR spectra of the free ligands showed broad bands at 3145 cm−1
assigned to N–H stretch, which disappear in spectra of the complexes indicating deprotona-
tion of the HN-CS. A strong band was observed at 1605 cm−1 for the free ligands due to
ν(C=N) [38]. Theoretically, two transitions were calculated at 1688 cm−1 (ν(C=N) – very
weak) and 1666 cm−1 (δ(NH2) – very strong. Theoretical frequencies in this region are
overestimated and it is common to scale those using linear factors. Herein, we preferred to
keep values without scaling, as the purpose is to assist the vibrational assignment and not
reproduce exactly the vibrational frequencies. Theoretical IR calculations also predicted an
intense transition at ~1577 cm−1 due to the H–NCS in-plane deformation, which could be
S
K2PtCl4
ou
K2PdCl4
Et3N
Complexes
1-6 e 7-12
R
N
+
N
H
NH2
(3)(9)
(2)(8)
(1)(7)
R =
*
*
*
*
S
H3CO
(6)(12)
HO
(4)(10)
(5)(11)
N
*
*
O
Scheme 2.

964
T.T. Tavares et al.
useful to follow deprotonation upon complexation. Experimentally, for the complexes, the
band at ~1605 cm−1 (free ligand) was shifted to lower frequencies (~1578 cm−1) suggesting
coordination through the imine nitrogen in a bis-chelate mode [39]. The calculated values
for ν(C=N) ranged from 1630 to 1653 cm−1, with the lowest predicted for the pyridine ana-
logs (5 and 11) and the highest frequency calculated for the furan derivatives (4 and 10).
Note that a shift to lower frequencies was observed due to metal coordination to nitrogen.
As discussed previously for the free ligand, the δip(NH2) mode, calculated at ~1665 cm−1
for the complexes (the same frequency as the ligand), is more intense than ν(C=N). A very
intense transition was calculated at 1544–1571 cm−1 assigned to ν(SC–N). Therefore, focus-
ing on the theoretical IR in the 1500–1700 cm−1 region, two intense bands were predicted
for the free ligand and complexes. For the free ligand, the bands were centered at
1577 cm−1 (δip(H-NCS)) and 1666 cm−1 (δ(NH2)); while for the complexes, they were
found at ~1560 cm−1 (ν(SC–N)) and ~1667 cm−1 (δip(NH2)). The IR assignment of the first
band is distinct for the ligand and complex due to deprotonation upon coordination. The
SC–N stretch was calculated at a fairly high frequency due to double-bond character of this
bond (see figure 1). The next set of intense IR bands was found around 1340 cm−1 and
assigned mainly to ν(C-NH2). For ligands, this same mode was calculated around
1290 cm−1. Experimentally, ν(C=S) was observed as medium bands at 1090–835 cm−1 (free
ligands) and at lower frequencies (~1025–813) on coordination of the thiocarbonyl sulfur to
platinum(II) or palladium(II). The ν(C=S) mode was calculated at ~770 cm−1 (free ligands)
and ~670 cm−1 (complexes) with very low intensity. These IR data are in agreement with
those observed for thiosemicarbazone complexes [35]. The calculated vibrational frequen-
cies and assignments are given in table S2 as Supplementary materials.
In Raman spectra, ν(C=N) was predicted to be very intense at 1634 cm−1 coupled to
ν(C=C) of the aromatic ring. A set of weak bands was observed around 1500 cm−1 due to
δip(CH-Ar) with the most intense transition within this set assigned to δip(C–H) at
~1450 cm−1. The stretching mode ν(HC–C) was also active in the Raman spectrum at
~1235 cm−1 as well as the ν(N–N) vibration at ~1183 cm−1. Metal–ligand modes were also
predicted in Raman at ~535 ν(M–N) and ~317 ν(M–S) with very low intensity. Experimen-
tally these M–L bands were observed around 440 and 380 cm−1, respectively.
The NMR spectra of the thiosemicarbazones and their platinum(II) and palladium(II)
1
complexes were recorded in DMSO-d6. In the H-NMR spectra of the ligands, signals of
the = N-NH protons were observed as a singlet at δ 11.4. This is indicative of the predomi-
1
nance of the thione form in the E configuration [40]. This signal disappeared in H NMR
spectra of the complexes due to deprotonation of = N–NH, which indicates the formation of
the complexes. The signal of the HC=N protons which appeared as a singlet at δ 8.1 in
spectra of the ligands (calculated at δ 10.4) showed an upfield shift after complexation,
which indicates coordination of the imine nitrogen to the metal [41]. The calculated values
range from δ 7.40–8.10 (Pt complexes) and 6.87–7.53 (Pd complexes), which agree satis-
factorily with the experimental data. The signals of the aromatic protons of the complexes
were observed at δ 7.1–8.3, and they did not suffer relevant changes compared to the free
ligands. The NH2 signal in the ligands appeared as doublets centered at δ 8.1 due to non-
equivalence of the amine protons, attributed to restricted rotation around the C–NH2 bond
(thiocarbonyl carbon and terminal amine nitrogen) due to its partial-double-bond character
[42]. In spectra of the complexes, this signal was observed at δ 7.5.
In 13C NMR spectra of the complexes, the signals of the HC=N group occur at δ 154.
The C=S signal, observed at δ 173, is characteristic of this group [43], while the aromatic
carbons were observed in the δ 154–114 region. The calculated values for HC=N and C=S

Platinum(II) and palladium(II) thiosemicarbazones
965
were slightly overestimated (~5%), the average chemical shifts found at 160 and 185 ppm,
respectively.
Theoretical calculations concerning the predicted 195Pt NMR spectra were used for the
first time [31]. In 195Pt NMR spectra of the complexes, a signal around δ –3000 was
observed, expected for coordination of platinum to two sulfurs and two nitrogens [44]. The
calculated values were obtained for both the cis and trans complexes in order to support the
predominance of the trans isomer. The agreement with experimental results was much bet-
ter for the trans structure, with an average absolute deviation of approximately 283 ppm
(~10%), whereas for the cis isomer, it was 622 ppm (~20%). According to our previous
study [31], where the methodology was developed and tested for 195Pt NMR prediction, the
average deviation was ~60 ppm, with maximum error for a set of 27 molecules equal to
150 ppm. The shift predicted for cis and trans signals in the present study (~300 ppm) is
higher than our confidence limit, therefore, the level of theory applied here might be satis-
factory to distinguish between isomeric forms.
3.2. Cytotoxic activity
The cytotoxicities of the complexes were evaluated in comparison with cisplatin in two
tumor cell lines: colon cancer cells (CT26.WT) and melanoma (B16-F10). The complexes
were also examined for their cytotoxic properties on one normal, i.e., non-tumor kidney cell
line (BHK-21). The tumor cells were chosen to represent two different tumor types, mela-
noma and carcinoma, and to also assess the activity in cell lines of different embryonic ori-
gin such as epithelial and fibroblast. This was done in an effort to overcome and control any
differences. The third cell line is a normal cell, which was used to evaluate the selectivity
index, allowing comparison of the cytotoxicity of the compounds in tumor and normal cells.
Table 1. Cytotoxic activities against tumor cell lines (B16-F10 and CT26.WT) and non-tumor cell lines (BHK).
IC50 (μM SD)*.
Compound
B16-F10 (μM SD)*
SI**
CT26.WT (μM SD)*
SI**
BHK-21 (μM SD)*
Ligand a
Ligand b
Ligand c
Ligand d
Ligand e
Ligand f
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
Complex 6
Complex 7
Complex 8
Complex 9
Complex 10
Complex 11
Complex 12
Cisplatin
>100
>100
>100
>100
>100
NA
NA
NA
NA
NA
NA
4.3
2.2 2.1
2.7 1.7
1.0 0.7
2.6 2.7
8.6 0.4
1.6 0.7
4.4 3.4
<0.1
9.6 0.4
0.6 0.4
2.0 1.1
0.5 0.2
2.7 0.4
<0.1
NA
19.6
66.3
NA
NA
NA
3.5
335.0
5.3
100.7
NA
NA
6.7
NA
16.5
3.11
26.8
NA
>100
53.1 4.1
66.3 1.3
>100
>100
>100
15.5 2.9
33.5 2.1
50.9 14.9
60.4 3.2
>100
>100
3.6 0.6
0.4 0.3
12.3 3.7
0.4 0.3
11.4 1.4
<0.1
1.7 0.6
<0.1
<0.1
83.7
4.1
151.0
NA
NA
10.6
NA
NA
NA
68.9
52.6
43.6
>100
18.1 5.1
16.3 2.1
6.6 1.1
2.8 0.4
96.5 6.5
>100
0.4 0.3
0.9 0.4
3.6 1.0
15.4 8.5
<0.1
<0.1
1.4 0.6
1.9 0.4
0.9 0.7
392.0
39.2 1.0
*SD (standard deviation of triplicate of two independent experiments).
**SI (selectivity index).

966
T.T. Tavares et al.
IC50 values, calculated from the concentration response curves obtained after 72 h drug
treatment employing the MTT test, are shown in table 1. Some compounds presented equal
or superior activity when compared to cisplatin.
The free ligands, in B16F10 cell line, did not show any toxicity; however, they were
toxic to the CT26.WT cells and showed a high selectivity index in comparison to the nor-
mal BHK-21 cell.
Complexes 1–12 were cytotoxic against B16F10 cell while the free ligands were not.
Especially for platinum complexes, 2, 4, and 6 were 10-fold more potent and 20-fold more
selective than the others. All of the palladium complexes showed a high selectivity index.
To the CT26.WT cell line, the complex cytotoxicities were very similar to the free ligands.
However, platinum compounds 2, 4, and 6 and palladium compounds 8, 9, and 11 showed
a high selectivity index.
The formation of the complexes significantly improved their cytotoxicity in tumor cells,
especially for the B16-F10 cell line, without showing toxicity to normal cells (BHK21),
improving the selectivity index for most of the complexes.
3.3. Antibacterial activity
The antibacterial activities for all complexes were evaluated in six different bacterial strains
and compared to chloramphenicol (table S3). Complexes 1 and 7 showed better activity in
S. aureus (45 and 14 μM, respectively) than chloramphenicol (64 μM) for this strain. The
same complexes were also active against S. dysenteriae (90 and 54 μM) compared to the
same positive control (64 μM). All other compounds did not show antibacterial activity
against the tested strains.
Complexes 1 and 7 present the same R substituent in the thiosemicarbazone, a benzyl
ring, which could be important to establish structure–activity relationships.
4. Conclusion
Potential anticancer Pt(II) and Pd(II) thiosemicarbazone complexes were prepared. The
structures of the synthesized complexes were elucidated on the basis of their spectroscopic
properties and analytical techniques in addition to quantum mechanical calculations. Theo-
retical calculations suggest the trans configuration of the complexes.
Despite previous reports that many Pd(II) and Pt(II) complexes exhibit antibacterial and
antifungal activity [45–47], even complexes with thiosemicarbazones [48, 49], the present
compounds did not show significant antibacterial activity.
Nonetheless, the complexes described herein were more cytotoxic than the corresponding
free ligands in the tested cell lines, some of them exhibiting activity superior to cisplatin.
The development of potential anticancer metal complexes is relevant considering that rela-
tively few of these are in clinical use currently. Platinum complexes are classical antitumor
agents and many of them show significant cytotoxicity [50, 51]. On the other hand,
although several palladium compounds exhibit antiproliferative properties [52–55], none are
approved for commercial distribution yet and it is important to design complexes that are
potentially active against cancer.

Platinum(II) and palladium(II) thiosemicarbazones
967
Acknowledgments
The authors wish to thank CNPq, FAPEMIG and CAPES for financial supports and fellowships.
This work is a collaborative research project with members of the Rede Mineira de Química
(RQ-MG), supported by FAPEMIG.
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