N(4)-Tolyl-2-acetylpyridine thiosemicarbazones and theirplatinum(II,IV) and gold(III) complexes: cytotoxicityagainst human glioma cells and studies on the modeof action

Karina S. O. Ferraz • Jeferson G. Da Silva •

Flavia M. Costa • Bruno M. Mendes • Bernardo L. Rodrigues •

Raquel G. dos Santos • Heloisa Beraldo

Received: 26 November 2012 / Accepted: 26 May 2013 / Published online: 9 June 2013

� Springer Science+Business Media New York 2013

Abstract Complexes [Au(2Ac4oT)Cl][AuCl2] (1),

[Au(Hpy2Ac4mT)Cl2]Cl�H2O (2), [Au(Hpy2Ac4pT)

Cl2]Cl (3), [Pt(H2Ac4oT)Cl]Cl (4), [Pt(2Ac4mT)Cl]�H2O (5), [Pt(2Ac4pT)Cl] (6) and [Pt(L)Cl2OH], L =

2Ac4mT (7), 2Ac4oT (8), 2Ac4pT (9) were prepared

with N(4)-ortho- (H2Ac4oT), N(4)-meta- (H2Ac4mT)

and N(4)-para- (H2Ac4pT) tolyl-2-acetylpyridine

thiosemicarbazone. The cytotoxic activities of all

compounds were assayed against U-87 and T-98

human malignant glioma cell lines. Upon coordination

cytotoxicity improved in 2, 5 and 8. In general, the

gold(III) complexes were more cytotoxic than those

with platinum(II,IV). Several of these compounds

proved to be more active than cisplatin and auranofin

used as controls. The gold(III) complexes probably act

by inhibiting the activity of thioredoxin reductase

enzyme whereas the mode of action of the plati-

num(II,IV) complexes involves binding to DNA. Cells

treated with the studied compounds presented mor-

phological changes such as cell shrinkage and blebs

formation, which indicate cell death by apoptosis

induction.

Keywords Thiosemicarbazones � Gold(III)

complexes � Platinum(II,IV) complexes �Cytotoxicity � Mode of action

Introduction

Platinum(II) compounds such as cisplatin, carboplatin

and oxaliplatin are widely used in the clinics against

solid tumors. However, the clinical use of the current

platinum-based drugs has been limited due to the

appearance of resistance and severe side effects (Jung

and Lippard 2007). Therefore, effort in designing new

platinum and non-platinum alternative antitumor

drugs is still quite intense.

Platinum(IV) complexes could be an interesting

alternative to overcome the drawbacks of platinum(II)

compounds (Hall et al. 2007). The literature reports

that platinum(IV) complexes must be reduced to

platinum(II) to be activated. Platinum(IV) compounds

are inert and could serve as pro-drugs acting through

the release of clinically effective levels of the plati-

num(II) counterparts (Dhar and Lippard 2009).

Thiosemicarbazones are very promising com-

pounds which present a wide range of pharmacolog-

ical applications as antimicrobial, antitumor and

antiviral agents (Beraldo and Gambino 2004; Dilovic

et al. 2008). In many cases coordination of

K. S. O. Ferraz � J. G. Da Silva � B. L. Rodrigues �H. Beraldo (&)

Departamento de Quımica, Universidade Federal de

Minas Gerais, Belo Horizonte, MG 31270-901, Brazil

e-mail: hberaldo@ufmg.br

F. M. Costa � B. M. Mendes � R. G. dos Santos

Centro de Desenvolvimento da Tecnologia Nuclear

(CDTN), Belo Horizonte, MG 31270-901, Brazil

123

Biometals (2013) 26:677–691

DOI 10.1007/s10534-013-9639-x

thiosemicarbazones to metal ions result in improved

pharmacological activities. It has been shown that

platinum(II,IV) complexes with thiosemicarbazones

are active against cisplatin-resistant human cancer cell

lines, probably because their mode of action involves

interstrand crosslinks with DNA, while intrastrand

crosslinks is the major coordination mode of cisplatin

(Roberts and Friedlos 1981; Quiroga et al. 1998).

Our group demonstrated that 2-benzoylpyridine-

derived thiosemicarbazones and their platinum(II)

complexes present cytotoxic activity against solid

tumor and leukemia cells (Ferraz et al. 2011). In many

cases cytotoxicity increased upon coordination.

Gold(III) ion is isostructural (quadratic) and iso-

electronic (5d8) with platinum(II), and the literature

reports that gold(III) compounds also exhibit antitu-

mor activity (Henderson et al. 2001; Casini et al. 2008;

Kouodom et al. 2012; Arsenijevic et al. 2012).

Gold(I) complexes have shown activity against a

range of tumor types (Kaps et al. 2012; Langner et al.

2012; Maia et al. 2012). Triethylphosphinegold(I) tet-

raacetatothioglucose (auranofin), a drug used in the

clinics to treat rheumatoid arthritis, has shown high

cytotoxic activity against different tumor cells (Cox

et al. 2008; Marzano et al. 2007), including cisplatin-

resistant lineages.

Thioredoxin reductase (TrxR) is a selenoenzyme

involved in the biosynthesis of DNA, which is

considered as one of the most critical targets for gold

complexes (Bindoli et al. 2009; Rubbiani et al. 2011).

The literature reports that complexes with different

metals inhibit the TRxR catalytic activity (Prast-

Nielsen et al. 2010). Our group demonstrated that

gold(I) complexes with thiosemicarbazones are highly

cytotoxic against human tumor cell lines and act by

inhibiting TrxR activity (Lessa et al. 2011, 2012).

Malignant gliomas are the most common and

aggressive primary brain tumors. Despite treatment

advances the outcome of patients with gliomas

remains dismal (Kiaris et al. 1999; Giannopoulou

et al. 2010). In previous works we showed that

2-acetylpyridine-derived thiosemicarbazones (Lessa

et al. 2010, Soares et al. 2012) present cytotoxic

activity against glioma cells. In the present work

gold(III) and platinum(II,IV) complexes were pre-

pared with N(4)-ortho- (H2Ac4oT), N(4)-meta-

(H2Ac4mT) and N(4)-para- (H2Ac4pT) tolyl-2-ace-

tylpyridine thiosemicarbazone (see Fig. 1). The com-

pounds were evaluated for their in vitro cytotoxic

activity against malignant U-87 (expressing p53 wild

type protein) and T-98 (expressing p53 mutant

protein) glioma cell lines. The interactions of the

studied complexes with TrxR and with plasmid-DNA

were also investigated.

Experimental section

Materials and instrumentation

Partial elemental analyses were performed on a Perkin

Elmer CHN 2400 analyzer. Infrared spectra were

recorded on a Perkin Elmer FT-IR Spectrum GX

spectrometer using KBr pellets (4000–400 cm-1) and

Nujol mulls between CsI plates (700–200 cm-1); an

YSI model 31 conductivity bridge was employed for

molar conductivity measurements; NMR spectra were

obtained at room temperature with a Brucker DRX-

200 Avance (200 MHz) spectrometer using deuterated

dimethyl sulfoxide (DMSO-d6) and methanol (MeOD-

d4) as the solvent and tetramethylsilane (TMS) as

internal reference. Splitting patterns are designated as

follows: s, singlet; d, doublet; t, triplet; q, quartet; m,

multiplet. Analyses by direct infusion ESI were

performed using a mass spectrometer (model IT-

TOF, Shimadzu, Tokyo, Japan) with two analyzers in

tandem: ion trap (IT) and time-of-flight (TOF), and

using methanol (P.A.) as solvent.

N1

N2

CH3

NH3

S NH4

CH3CH3

N1

N2

CH3

NH3

S NH4

(E ) (Z )

Fig. 1 General structure of the configurational isomers of 2-

acetylpyridine N(4)-ortho-, (H2Ac4oT), N(4)-meta- (H2Ac4mT),

and N(4)-para- (H2Ac4pT) tolyl-thiosemicarbazones

678 Biometals (2013) 26:677–691

123

Synthesis

Synthesis of N(4)-ortho-tolyl (H2Ac4oT), N(4)-meta-

tolyl (H2Ac4mT) and N(4)-para-tolyl (H2Ac4pT)

2-acetylpyridine thiosemicarbazone

H2Ac4oT, H2Ac4mT and H2Ac4pT were prepared

according to previously reported procedures (Mendes

et al. 2006, 2007).

Synthesis of gold(III) complexes with H2Ac4oT,

H2Ac4mT and H2Ac4pT

The gold(III) complexes were obtained by adding

solid portions of the desired thiosemicarbazone

(1 mmol) to a solution of HAuCl4 (1.1 mmol) in

methanol (5 mL). The reaction was kept under reflux

for 1 h. The obtained solids were filtered off, washed

with hot methanol and dried.

[Au(2Ac4oT)Cl][AuCl2] (1) Brown solid. Yield:

94 %. Anal. Calcd. for C15H15Au2Cl3N4S (%): C,

22.99; H, 1.93; N, 7.15. Found: C, 23.29; H, 1.80; N,

7.22. FW: 783.66 g mol-1. Molar conductivity

(1 9 10-3 mol L-1, DMF): 51 X-1 cm2 mol-1. IR

(KBr, CsI/Nujol, cm-1): m(C=N) 1537m, m(C=S) 773m,

q(py) 617w, m(M–N) 398, m(M–Npy) 385, m(M–S) 351,

m(M–Cl) 315. The main signals in 1H NMR (MeOD-d4,

200 MHz) [d (ppm)]: 9.03 (1H, d, H(6)), 7.99 (1H, t,

H(5)), 8.50 (1H, t, H(4)), 8.24 (1H, d, H(3)). ESI–MS

(positive): m/z 515.0357 [Au(2Ac4pT)Cl]?; ESI–MS

(negative): m/z 266.9003 [AuCl2]-.

[Au(Hpy2Ac4mT)Cl2]Cl�H2O (2) Brown solid. Yield:

70 %. Anal. Calcd. for C15H18AuCl3N4OS (%): C,

29.74; H, 3.00; N, 9.25. Found: C, 29.98; H, 2.88; N,

9.21. FW: 605.72 g mol-1. Molar conductivity (1 9

10-3 mol L-1, DMF): 60 X-1 cm2 mol-1. IR (KBr,

CsI/Nujol, cm-1): m(Npy–H) 2808s, m(C=N) 1598m,

m(C=S) 859m, q(py) 620w, m(M–N) 374, m(M–S) 351,

m(M–Cl) 328. The main signals in 1H NMR (MeOD-d4,

200 MHz) [d (ppm)]: 9.10 (1H, d, H(6)), 8.04 (1H, t,

H(5)), 8.55 (1H, t, H(4)), 8.34 (1H, d, H(3)).

[Au(Hpy2Ac4pT)Cl2]Cl (3) Brown solid. Yield:

81 %. Anal. Calcd. C15H16AuCl3N4S (%): C, 30.65; H,

2.74; N, 9.53. Found: C, 30.30; H, 2.73; N, 9.55. FW:

587.71 g mol-1. Molar conductivity (1 9 10-3 mol

L-1, DMF): 50 X-1 cm2 mol-1. IR (KBr, CsI/Nujol,

cm-1): m(Npy–H) 2841s, m(C=N) 1599m, m(C=S) 826m,

q(py) 619w,m(M–N) 398,m(M–S) 351, m(M–Cl) 315. The

main signals in 1H NMR (MeOD-d4, 200 MHz) [d(ppm)]: 9.08 (1H, d, H(6)), 8.03 (1H, t, H(5)), 8.54 (1H, t,

H(4)), 8.32 (1H, d, H(3)). ESI–MS (positive): m/z 515.15

[Au(2Ac4pT)Cl]?.

Synthesis of platinum(II) complexes with H2Ac4oT,

H2Ac4mT and H2Ac4pT

The platinum(II) complexes were prepared by mixing

the desired ligand in ethanol with an aqueous solution

of K2PtCl4 in equimolar amounts (1 mmol). The

reaction mixture was kept under reflux for 2 h. The

obtained solids were filtered off and washed with

ethanol, water and ether, and dried.

[Pt(H2Ac4oT)Cl]Cl (4) Brown solid. Yield: 81 %.

Anal. Calcd. for C15H16Cl2N4PtS (%): C, 32.73; H, 2.93;

N, 10.18. Found: C, 32.73; H, 2.63; N, 9.96. FW:

550.36 g mol-1. Molar conductivity (1 9 10-3 mol

L-1, DMF): 21 X-1 cm2 mol-1. IR (KBr, CsI/Nujol,

cm-1): m(C=N) 1587m, m(C=S) 760m, q(py) 618w, m(M–

N) 425, m(M–Npy) 295, m(M–S) 342, m(M–Cl) 338. The

main signals in 1H NMR (DMSO-d6, 200 MHz) [d(ppm)]: 8.74 (1H, d, 1J = 5.40 Hz, H(6)), 7.70 (1H, t,1J = 6.36 Hz, H(5)), 8.14 (1H, t, 1J = 8.14 Hz,2J = 1.35 Hz, H(4)), 7.70 (1H, d, 1J = 7.92 Hz, H(3)),

2.20 (3H, s, H(16)), 9.81 (1H, s, N(3)H), 9.30 (1H, s,

N(4)H). 13C NMR (DMSO-d6, 50 MHz) [d (ppm)]:

181.6 (C8=S), 156.5 (C7=N), 146.1 (C6), 126.3 (C5),

138.3 (C4), 125.9 (C3), 152.2 (C2), 13.2 (C16).

[Pt(2Ac4mT)Cl]�H2O (5) Brown solid. Yield: 74 %.

Anal. Calcd. for C15H17ClON4PtS (%): C, 33.87; H,

3.22; N, 10.53. Found: C, 34.44; H, 3.28; N, 10.54.

FW: 531.92 g mol-1. Molar conductivity (1 9

10-3 mol L-1, DMF): 14 X-1 cm2 mol-1. IR (KBr,

CsI/Nujol, cm-1): m(C=N) 1592m, m(C=S) 774m,

q(py) 637w, m(M–N) 428, m(M–Npy) 290, m(M–S)

340, m(M–Cl) 313. The main signals in 1H NMR

(DMSO-d6, 200 MHz) [d (ppm)]: 8.77 (1H, d,1J = 5.15 Hz, H(6)), 7.66 (1H, t, 1J = 5.92 Hz,

H(5)), 8.17 (1H, t, 1J = 7.54 Hz, H(4)), 7.84 (1H, d,1J = 7.64 Hz, H(3)), 2.44 (3H, s, H(16)), 10.19 (1H, s,

N(4)H), 5.46 (2H, s, H2O). 13C NMR (DMSO-d6,

50 MHz) [d (ppm)]: 159.2 (C7=N), 146.2 (C6), 127.4

(C5), 137.7 (C4), 124.0 (C3), 156.2 (C2), 13.8 (C16).

Biometals (2013) 26:677–691 679

123

[Pt(2Ac4pT)Cl] (6) Brown solid. Yield: 89 %. Anal.

Calcd. for C15H15ClN4PtS (%): C, 35.06; H, 2.94; N,

10.90. Found: C, 35.23; H, 2.91; N, 10.49. FW:

513.90 g mol-1. Molar conductivity (1 9

10-3 mol L-1, DMF): 17 X-1 cm2 mol-1. IR (KBr,

CsI/Nujol, cm-1): m(C=N) 1557m, m(C=S) 766m,

q(py) 636w, m(M–N) 427, m(M–Npy) 307, m(M–S)

382, m(M–Cl) 340. The main signals in 1H NMR

(DMSO-d6, 200 MHz) [d (ppm)]: 8.81 (1H, d,1J = 5.29 Hz, H(6)), 7.71 (1H, t, 1J = 6.48 Hz,

H(5)), 8.20 (1H, t, 1J = 7.55 Hz, H(4)), 7.85 (1H, d,1J = 7.94 Hz, H(3)), 2.43 (3H, s, H(16)), 10.22 (1H, s,

N(4)H). 13C NMR (DMSO-d6, 50 MHz) [d (ppm)]:

159.2 (C7=N), 146.2 (C6), 127.2 (C5), 137.8 (C4),

120.1 (C3), 158.3 (C2), 13.8 (C16).

Synthesis of platinum(IV) complexes with H2Ac4oT,

H2Ac4mT and H2Ac4pT

The platinum(IV) complexes were prepared by mixing

the desired ligand in ethanol with an aqueous solution

of K2PtCl6 in equimolar amounts (1 mmol). The

reaction mixture was kept at room temperature for

7 days. The solids which formed were filtered off and

washed with water and ethanol, and dried.

[Pt(2Ac4oT)Cl2OH] (7) Brown solid. Yield: 86 %.

Anal. Calcd. for C15H16Cl2N4OPtS (%): C, 31.81; H,

2.85; N, 9.89. Found: C, 32.51; H, 2.62; N, 9.79. FW:

566.36 g mol-1. Molar conductivity (1 9 10-3 mol

L-1, DMF): 22 X-1 cm2 mol-1. IR (KBr, CsI/Nujol,

cm-1): m(C=N) 1588m, m(C=S) 760m, q(py) 631w,

m(M–OH) 520, m(M–N) 419, m(M–S) 385, m(M–Cl)

337, m(M–Npy) 304. The main signals in 1H NMR

(DMSO-d6, 200 MHz) [d (ppm)]: 8.80 (1H, d,1J = 5.03 Hz, H(6)), 7.70 (1H, t, 1J = 6.20 Hz,

H(5)), 8.18 (1H, t, 1J = 7.73 Hz, H(4)), 7.77 (1H, d,1J = 8.08 Hz, H(3)), 2.24 (3H, s, H(16)), 9.86 (1H, s,

N(4)H), 3.98 (s, OH). 13C NMR (DMSO-d6, 50 MHz)

[d (ppm)]: 156.5 (C7=N), 173.1 (C8–S), 146.4 (C6),

127.9 (C5), 140.1 (C4), 124.9 (C3), 156.1 (C2); 12.8

(C16).

[Pt(2Ac4mT)Cl2OH] (8) Brown solid. Yield: 89 %.

Anal. Calcd. for C15H16Cl2N4OPtS (%): C, 31.81; H,

2.85; N, 9.89. Found: C, 31.97; H, 2.80; N, 9.53 %. FW:

566.36 g mol-1. Molar conductivity (1 9 10-3 mol

L-1 DMF): 16 X-1 cm2 mol-1. IR (KBr, CsI/Nujol,

cm-1): m(C=N) 1563m, m(C=S) 774m, q(py) 636w,

m(M–OH) 511, m(M–N) 430, m(M–S) 390, m(M–Cl) 342,

m(M–Npy) 304. The main signals in 1H NMR (DMSO-

d6, 200 MHz) [d (ppm)]: 8.81 (1H, d, 1J = 5.51 Hz,

H(6)), 7.72 (1H, t, 1J = 6.17 Hz, H(5)), 8.20 (1H, t,1J = 8.08 Hz, H(4)), 7.86 (1H, d, 1J = 7.81 Hz, H(3)),

2.44 (3H, s, H(16)), 10.22 (1H, s, N(4)H), 3.72 (s, OH).13C NMR (DMSO-d6, 200 MHz) [d (ppm)]: 159.2

(C7=N), 177.6 (C8–S), 146.5 (C6), 127.4 (C5), 137.8

(C4), 124.0 (C3), 156.7 (C2), 13.8 (C16).

[Pt(2Ac4pT)Cl2OH] (9) Brown solid. Yield: 88 %.

Anal. Calcd. for C15H16Cl2N4OPtS (%): C, 31.81; H,

2.85; N, 9.89. Found: C, 31.83; H, 2.74; N, 10.03. FW:

566.36 g mol-1. Molar conductivity (1 9 10-3 mol

L-1 DMF): 21 X-1 cm2 mol-1. IR (KBr, CsI/Nujol,

cm-1): m(C=N) 1560m, m(C=S) 768m, q(py) 634w,

m(M–OH) 513, m(M–N) 431, m(M–Npy) 304, m(M–S)

385, m(M–Cl) 343. The main signals in 1H NMR

(DMSO-d6, 200 MHz) [d (ppm)]: 8.80 (1H, d,1J = 5.34 Hz, H(6)), 7.70 (1H, t, 1J = 6.10 Hz, H(5)),

8.20 (1H, t, 1J = 7.69 Hz, H(4)), 7.85 (1H, d,1J = 7.89 Hz, H(3)), 2.42 (3H, s, H(16)), 10.22 (1H, s,

N(4)H), 4.00 (s, OH). 13C NMR (DMSO-d6, 50 MHz) [d(ppm)]: 159.2 (C7=N), 177.5 (C8–S), 146.2 (C6), 127.3

(C5), 137.8 (C4), 120.2 (C3) 158.4 (C2) 13.8 (C16).

X-ray crystallography

Crystals of [Pt(2Ac4mT)Cl] (5a), [Pt(2Ac4oT)Cl]

(7a) and [Pt(2Ac4pT)Cl] (9a) were formed and their

crystal structures were determined using single-crystal

X-ray diffractometry. A summary of crystals data,

data collection details and refinement results are listed

in Table 1. Molecular graphics and packing figures

were prepared using ORTEP (Farrugia 1997).

Cell lines and culture conditions

U87 (multiform glioblastoma wild-type) and T98

(multiform glioblastoma P53 mutant) malignant

human tumor cells and MRC5 (Human Fetal Lung

Fibroblast) cells (normal cells) were obtained from the

American Type Culture Collection (ATCC, USA).

Cell lines were grown as monolayer in Dulbecco’s

Modified Eagle’s Medium (DMEM, Gibco), supple-

mented with 10 % fetal bovine serum (Cultilab) and

antibiotics (50 U/mL penicillin/50 lM streptomycin),

in a humidified atmosphere air/CO2 (5 %/95 %) at

37 �C. Cells 80 % confluents were used in all

680 Biometals (2013) 26:677–691

123

experiments. For all experiments, cells were seeded in

96-well plates, at a density of 2000 cells/well. After

24 h incubation cells were treated.

Cytotoxic activity

To determine the IC50 values the cytotoxic effects

were quantified using the 3-(4,5-dimethyl-2-

thioazolyl)-2,5-diphenyl tetrazolium bromide (MTT)

colorimetric assay (Freshney 2000). Briefly, logarith-

mic phase glioma cells were treated with increasing

concentrations (10-12, 10-11, 10-10, 10-9, 10-8,

10-7, 10-6, 10-5, 10-4 mol L-1) of either cisplatin

(positive control) or test compounds. Drugs were

previously dissolved in dimethyl sulfoxide (DMSO)

and the final concentrations were adjusted in DMEM

Table 1 Crystal data and

structure refinement for

[Pt(2Ac4mT)Cl] (5a),

[Pt(2Ac4oT)Cl] (7a) and

[Pt(2Ac4pT)Cl] (9a)

Identification code 5a 7a 9a

Empirical formula C15H15N4ClPtS

Formula weight

(g mol-1)

513.91

Temperature (K) 150(2) 150(2) 150(2)

Wavelength, k (A) 0.71073 0.71073 0.71073

Crystal system Triclinic Monoclinic Triclinic

Theta range for data

collection (�)

2.58–32.91 2.59–29.49 2.32–29.63

Space group Pı P21/n Pı

Unit cell dimensions

a (A) 7.6653(2) 7.2028(2) 7.5196(8)

b (A) 8.1208(4) 26.4162(10) 9.5699(9)

c (A) 13.0087(5) 8.2669(3) 11.7345(14)

a (�) 100.340(3) 90 85.350(9)

b (�) 101.448(3) 92.134(3) 84.494(9)

c (�) 96.781(3) 90 66.915(9)

Volume (A3) 770.79(5) 1571.86(9) 772.36(14)

F(000) 488 976 488

Z 2 4 2

Calculated density

(mg m-3)

2.214 2.172 2.210

Absorption coefficient, l(mm-1)

9.411 9.230 9.392

Limiting indices -11 B h B 11,

-12 B k B 12,

-19 B l B 19

-9 B h B 9,

-26 B k B 35,

-7 B l B 10

-9 B h B 10,

-12 B k B 12,

-16 B l B 15

Reflections collected 49,856 7,895 6,967

Reflections unique/R(int) 5,529/0.0574 3,718/0.0402 3,656/0.0683

Completeness to theta 100 % (h = 30.50�) 100.0 %

(h = 26.37�)

100.0 %

(h = 26.37�)

Data/restraints/

parameters

5,529/0/201 3,718/0/201 3,656/0/201

Goodness-of-fit on F2 1.055 1.016 1.082

R [I [ 2r(I)] R1 = 0.0229,

wR2 = 0.0413

R1 = 0.0369,

wR2 = 0.0478

R1 = 0.0582,

wR2 = 0.1332

R (all) R1 = 0.0305,

wR2 = 0.0437

R1 = 0.0583,

wR2 = 0.0526

R1 = 0.0701,

wR2 = 0.1415

Dq min/max (e A3) 1.779/-0.929 1.136/-1.313 5.447/-2.454

Biometals (2013) 26:677–691 681

123

in such manner that final DMSO concentration was

lower than 0.5 %. Following 48 h treatment, MTT

reagent was added to each well. Following another 4 h

of incubation at 37 �C, DMSO was added to each well

to dissolve formazan precipitate and absorbance was

measured at 570 nm. Tests using DMSO (0.5 % in

DMEM) as negative control were carried out in

parallel. All tests were performed in triplicates with

full agreement between the results. Statistical analysis

was carried out by the unpaired, one-tailed Student’s

t test.

Studies of interactions with supercoiled

plasmid DNA

Studies of interactions between the compounds under

study and supercoiled plasmid DNA by agarose gel

electrophoresis were carried out. Thus, 136 ng of

purified plasmid DNA-pUC 19 from Escherichia coli

(Sigma) were incubated with compounds (HAc4mT,

2, 5, 8, HAuCl4, K2PtCl4, K2PtCl6, cisplatin and

auranofin) at 200 lM in Tris–HCl buffer (NaCl

50 mM, Tris–HCl 5 mM, pH 7.2). The mixture was

incubated at 37 �C for 24 h. Thereafter, the reactions

were quenched by adding 5 lL of the loading buffer

solution (50 mmol L-1 Tris, pH 7.2, 0.01 % bromo-

phenol blue, 50 % glycerol, and 250 mmol L-1

EDTA). The samples were analyzed by 1 % agarose

gel electrophoresis in 0.59 TBE buffer for 1 h 40 min

at 75 mV. The gel was stained after electrophoresis in

0.59 TBE buffer with 2.5 lg mL-1 ethidium bromide

for 15 min and visualized by UV light.

Inhibition of TrxR activity

Rat liver TrxR (Sigma) was used to determine TrxR

inhibition by the compounds. The assay was performed

according to the manufacturer’s instructions (Sigma

product information sheet T9698) and Ott et al. (2009)

with appropriate modifications. Initially, the TrxR rat

liver solution was diluted with 1.0 M potassium phos-

phate buffer, pH 7.0. To 20 lL aliquots of this solution

(each containing approximately 0.10 units of the

enzyme) 20 lL of a solution of HAc4mT, 2, 5, 8,

HAuCl4, K2PtCl4, K2PtCl6, cisplatin and auranofin in

5 % DMF/potassium phosphate buffer pH 7.0 at 10 lM

or vehicle without compound (control) were added, and

the resulting solutions were incubated for 1 h at 37 �C

with moderate shaking. The solutions were transferred

quantitatively to 96-well plates, and to each well 200 lL

of reaction mixture (10 mL of reaction mixture con-

sisted of 1.0 mL of 1.0 M potassium phosphate buffer,

pH 7.0, 0.20 mL of 500 mM EDTA solution pH 7.5,

0.80 mL of 63 mM dithiobisnitrobenzoic acid (DTNB)

in ethanol, 0.10 mL of 20 mg/mL bovine serum albu-

min, 0.05 mL of 48 mM nicotinamide adenine dinu-

cleotide phosphate (NADPH) and 7.85 mL of water)

were added. To correct for non-enzymatic product

formation, 40 lL of 2.5 % DMF potassium phosphate

buffer 1.0 M, pH 7.0, and 200 lL of reaction mixture

were processed simultaneously (blank). After proper

mixing the formation of 5-thionitrobenzol (TNB) was

monitored in a microplate reader (Thermo Scientific

Multiskan� Spectrum) at 412 nm in 2 s intervals for

4 min. The absorbance of the blank was subtracted from

those of the control and treated wells. The enzymatic

activity was calculated as the maximum absorbance

observed in 4 min in each well. The experiments were

performed in triplicate.

Results and discussion

Characterization of gold(III) and platinum(II,IV)

complexes

For the gold(III) complexes microanalyses and molar

conductivity data are compatible with the formation of

[Au(2Ac4oT)Cl][AuCl2] (1), [Au(Hpy2Ac4mT)Cl2]

Cl�H2O (2) and [Au(Hpy2Ac4pT)Cl2]Cl (3).

In 1 an anionic thiosemicarbazone and a chloride

ion are attached to the gold(III) center and [AuCl2]-

acts as counter ion. The presence of [Au(2Ac4oT)Cl]?

and [AuCl2]- was observed in the mass spectra of 1 in

the positive (ESI(?)-MS) m/z 515.0357 (calcd. m/z

515.0321) and negative (ESI(-)-MS) m/z 266.9003

(calcd. m/z 266.9043) modes, respectively. The per-

centage of gold obtained by atomic absorption anal-

ysis, 52.34 % (calcd. 50.27 %), confirms the proposed

formulation. In addition, the literature shows other

gold complexes with thiosemicarbazones containing

[AuCl2]- as counter ion (Sreekanth et al. 2004).

In 2 and 3 a neutral thiosemicarbazone is attached to

the metal center together with two chlorides. The

crystallization water molecule in 2 was confirmed by its

thermogravimetric curve (TG) that showed a weight

loss of 2.94 % (calcd. 3.15 %). The infrared spectrum

682 Biometals (2013) 26:677–691

123

of 2 also supported the presence of a hydration water

molecule (see ‘‘Synthesis of gold(III) complexes with

H2Ac4oT, H2Ac4mT and H2Ac4pT’’ section).

Microanalyses of the platinum(II,IV) complexes

indicate the formation of [Pt(H2Ac4oT)Cl]Cl (4),

[Pt(2Ac4mT)Cl]�H2O (5), [Pt(2Ac4pT)Cl] (6) and

[Pt(L)Cl2OH], L = 2Ac4mT (7), 2Ac4oT (8), 2Ac4pT

(9). Molar conductivity data suggested that the com-

pounds are non-electrolytes or weak electrolytes

[complex (4)]. Complex [Pt(H2Ac4oT)Cl]Cl (4) is

formed by a neutral thiosemicarbazone and one

chloride ligand coordinated to the metal center. A

second chloride ion probably acts as counter ion as

shown by NMR data (‘‘Synthesis of platinum(II)

complexes with H2Ac4oT, H2Ac4mT and H2Ac4pT’’

section), which revealed that the thiosemicarbazone is

neutral and behaves as a tridentate chelating agent.

Other groups attributed molar conductivities lower

than the expected in metal complexes with thiosemi-

carbazones to hydrogen bond association between the

chloride ions and some hydrogen donor group from the

ligand (Casas et al. 2004).

In complex [Pt(2Ac4mT)Cl]�H2O (5) an anionic

thiosemicarbazone and a chloride ion are coordinated

to the metal center. The presence of the hydration water

molecule was suggested by CHN analyses as well as by

the TG curve of 5 which showed a weight loss of 3.30 %

(calcd. 3.38 %) close to 180 �C. For complex

[Pt(2Ac4pT)Cl] (6) an anionic thiosemicarbazone is

coordinated to the metal center together with a chloride

ligand.

In complexes [Pt(L)Cl2OH] (7–9) one anionic

thiosemicarbazone is coordinated to Pt(IV) along with

two chloride ions and a hydroxyl group. The use of

water as solvent in the syntheses could have favored

coordination of the hydroxyl ion.

Spectroscopic characterization

In the infrared spectra of [Au(Hpy2Ac4mT)Cl2]

Cl�H2O (2) and [Au(Hpy2Ac4pT)Cl2]Cl (3) a strong

and broad band at 2808 and 2841 cm-1, respectively,

was assigned to the m(Npy–H) vibration, indicating the

formation of a pyridinium ion (Lessa et al. 2011;

Chenon and Sandorfy 1958). This absorption is absent

in the spectrum of 1.

The m(C=S) stretching at 870–815 cm-1 in the

spectra of free thiosemicarbazones shifts to

859–826 cm-1 in the spectra of 2 and 3, in accordance

with the presence of a thione sulfur (Lessa et al. 2011;

Lobana et al. 2008; Khanye et al. 2010). In the

spectrum of 1 the shift of 49 cm-1 is compatible with

complexation of thiolate sulfur (Da Silva et al. 2009).

Shifts in the band assigned to the m(C=N) vibration,

from 1590–1581 cm-1 in the spectra of the free

thiosemicarbazones to 1599–1537 cm-1 in the spectra

of 1–3 indicate coordination of the imine nitrogen

(Ferraz et al. 2011; Lessa et al. 2011).

Upon coordination the in-plane deformation mode

of the pyridine ring shifts from 655 cm-1 in the free

base to 617 cm-1 in 1, according to coordination

through the pyridine nitrogen (Ferraz et al. 2011). No

significant shift was observed in this band for 2–3,

suggesting that the heteroaromatic nitrogen is not

attached to the metal center (Lessa et al. 2011).

New bands in the spectra of 1–3 at 398–374, 351 and

328–315 cm-1 were assigned to the m(Au–N) (Lessa

et al. 2012), m(Au–S) (Lessa et al. 2011) and m(Au–Cl)

(Lobana et al. 2008) vibrations, respectively.

Hence the infrared data indicate that coordination

of the thiosemicarbazone occurs through the Npy–N–S

chelating system in 1 and through the N–S bidentate

system in 2 and 3.

Significant displacements were observed in the

m(C=N) absorption, from 1590–1581 cm-1 in the spectra

of the free thiosemicarbazones to 1592–1557 cm-1 in the

spectra of all platinum(II,IV) complexes, indicating

coordination through the imine nitrogen (Ferraz et al.

2011). The m(C=S) vibration at 870–815 cm-1 in the

spectra of the thiosemicarbazones shifts to 774–760

cm-1 in 4–9 in accordance with coordination of a thiolate

sulfur (Mishra et al. 2007).

The in-plane deformation mode of the pyridine ring

at 655–621 cm-1 in the spectra of the thiosemicarba-

zones shifts to 637–618 cm-1 in the spectra of all

platinum complexes (4–9) (Kovala-Demertzi et al.

2009), indicating coordination through the heteroaro-

matic nitrogen.

All together the results suggest that the thiosemi-

carbazone is coordinated through the Npy–N–S system

in 4–9.

Absorptions at 431–419, 390–340, 343–313 and

307–290 cm-1 in the spectra of 4–9 were assigned to

the m(Pt–N), m(Pt–S), m(Pt–Cl) and m(Pt–Npy) vibra-

tions, respectively (Perez-Rebolledo et al. 2005;

Kovala-Demertzi et al. 2009; Amani et al. 2009).

The NMR spectra of all thiosemicarbazones and

gold(III) complexes were recorded in methanol-d4 due

Biometals (2013) 26:677–691 683

123

to the instability of the complexes in DMSO-d6.

However, the spectra of 2 and 3 were obtained as well

in DMSO-d6 immediately after dissolution. The 1H

resonances were assigned on the basis of chemical

shifts and multiplicities. We were unable to obtain 13C

NMR spectra of 1–3 due to their low solubility in

methanol-d4. The spectra of the platinum(II,IV) com-

plexes were recorded in DMSO-d6. The 1H resonances

were assigned on the basis of chemical shifts and

multiplicities. The carbon type (C, CH) was deter-

mined by using distortionless enhancement by polar-

ization transfer (DEPT135) experiments. The

assignments of the protonated carbons were made by

2D hetero-nuclear multiple quantum coherence exper-

iments (HMQC).

Duplicated signals were observed for each hydro-

gen in the 1H NMR spectrum of all free thiosemicar-

bazones, indicating the presence of E (80 %) and

Z (20 %) configurational isomers in solution. In the

first N3–H is hydrogen-bonded to the pyridine nitro-

gen, while in the latter N3–H is hydrogen-bonded to

the solvent (Mendes et al. 2006, 2007; Da Silva et al.

2009; Perez-Rebolledo et al. 2005; Ferraz et al. 2009).

In the spectra of all gold(III) and platinum(II,IV)

complexes only one signal was observed for each

hydrogen, suggesting the presence of only one isomer.

In the 1H NMR spectra of 1–9 the signals of all

hydrogens undergo significant shifts in relation to their

positions in the free bases. In the spectra of 2 and 3

recorded immediately after dissolution in DMSO-d6 a

signal at d 16.33 for 2 and d 16.29 for 3 was attrib-

uted to protonation at the pyridine nitrogen (Lessa

et al. 2011). This signal is absent in the spectrum of

1 and 4–9.

The signal of N3–H is absent in the spectra of 1–3.

This signal appears in the spectrum of 4 but not in

those of 5–9. Hence the ligand is protonated at N3–H

in 4 and deprotonated in all other complexes.

The signals of all hydrogens in the pyridine ring

undergo significant shifts on coordination in all

complexes (1–9). Protonation at the pyridine nitrogen

is probably responsible for the shifts in 2 and 3. In 1

and 4–9 the shifts are in accordance with complexation

through the pyridine nitrogen. In addition the signals

of all carbons in the heteroaromatic ring undergo

significant shifts in 4–9, also indicating coordination

through the pyridine nitrogen. Significant shifts occur

as well in the signals of C=N and C=S. Hence the

NMR data are compatible with complexation through

the Npy–N–S chelating system in 1 and 4–9 (Lessa

et al. 2012).

The signal of O–H was not observed separately in

the spectra of 7–9 but appeared as a broad weak peak

involving the hydrogen of O–H together with the

hydrogens of water from DMSO-d6. Upon addition of

D2O this peak undergoes a 0.5 ppm shift and appears

as an intense signal, in accordance with the presence of

coordinated hydroxyl ion.

The presence of hydration water in 5 was confirmed

by the appearance of a signal at 5.46 ppm (integrating

for two hydrogens) which shifts to 3.3 ppm on

addition of D2O.

X-ray crystallography

The crystal structures of [Pt(2Ac4mT)Cl] (5a),

[Pt(2Ac4oT)Cl] (7a) and [Pt(2Ac4pT)Cl] (9a) were

determined. 5a was obtained from re-crystallization of

[Pt(2Ac4mT)Cl]�H2O (5) in 1:9 DMSO:acetone. 7a

and 9a were obtained from re-crystallization of

[Pt(2Ac4oT)Cl2OH] (7) and [Pt(2Ac4pT)Cl2OH] (9),

respectively, in the same mixture of solvents. During

the crystallization process reduction from plati-

num(IV) to a platinum(II) occurred.

ORTEP drawing of these compounds are shown in

Fig. 2. Selected bond lengths and angles around the

metal are given in Table 2. The crystal structures of

H2Ac4oT and H2Ac4mT have been previously deter-

mined by some of us (Mendes et al. 2007). Selected

bond distances and angles in both structures are also

reported in Table 2 for the sake of comparison.

In all complexes the thiosemicarbazone is attached

to the metal center through the Npy–N–S coordination

system and a chloride ion occupies the fourth coor-

dination position (Fig. 2). This coordination mode is

similar to the observed in platinum(II) complexes

containing anionic N(4)-o-tolyl-2-benzoylpyridine

thiosemicarbazone (Ferraz et al. 2011).

The thiosemicarbazone conformation in relation to

the C7–N2 and C8–N3 bonds changes from EE in the

free thiosemicarbazones (H2Ac4oT and H2Ac4mT) to

EZ in 7a and 9a. Hence, the N2–N3–C8 angle

decreases from 118.25(16)� in H2Ac4oT to

117.4(5)� in 7a, and from 118.5(2)� in H2Ac4mT to

112.4(2)� in 5a; the N3–C8–S angle varies from

120.83(14)� in H2Ac4oT to 125.8(4)� in 7a, and from

120.3(2)� in H2Ac4mT to 125.1(2)� in 5a.

684 Biometals (2013) 26:677–691

123

C8–S, which is a predominantly double bond in the

free thiosemicarbazones, increases from 1.6740(19) A

(in H2Ac4oT) and 1.671(3) A (in H2Ac4mT) to

1.767(3) A (in 5a) and 1.757(6) A (in 7a), as a

consequence of deprotonation at N3 with formation of

a new predominantly single bond. The N3–C8 bond

goes from 1.359(2) A (in H2Ac4oT) and 1.359(3) A

(in H2Ac4mT) to 1.305(4) A in 5a and 1.310(6) A in

7a A due to this same effect (Perez-Rebolledo et al.

2005; Ferraz et al. 2009). Significant modifications

were observed as well in the bond angles involving the

sulfur atom in the free thiosemicarbazone and its

complex.

It is interesting to notice that 5a is almost planar,

with an angle between the N(4)-tolyl-ring and the

thiosemicarbazone chain (C7–N2–N3–C8–S–N4) of

Fig. 2 Molecular plot of [Pt(2Ac4mT)Cl] (5a), [Pt(2Ac4oT)Cl] (7a) and [Pt(2Ac4pT)Cl] (9a) showing the labeling scheme of the non-

H atoms and their displacement ellipsoids at the 50 % probability level

Table 2 Bond lengths (A) and angles (�) for H2Ac4mT, [Pt(2Ac4mT)Cl] (5a), H2Ac4oT, [Pt(2Ac4oT)Cl] (7a) and [Pt(2Ac4pT)Cl]

(9a)

Atoms H2Ac4mT 5a H2Ac4oT 7a 9a

Bond lengths (A)

C2–C7 1.488(4) 1.467(4) 1.493(3) 1.474(7) 1.470(1)

N2–C7 1.289(3) 1.308(3) 1.287(2) 1.312(6) 1.307(13)

N2–N3 1.381(3) 1.382(3) 1.380(2) 1.386(6) 1.374(11)

N3–C8 1.359(3) 1.305(4) 1.359(2) 1.310(6) 1.308(12)

N4–C8 1.339(3) 1.356(4) 1.344(2) 1.362(7) 1.355(12)

N4–C9 1.429(3) 1.415(3) 1.433(2) 1.416(7) 1.421(12)

S–C8 1.671(3) 1.767(3) 1.6740(19) 1.757(6) 1.768(9)

Pt–N2 – 1.956(2) – 1.941(4) 1.948(8)

Pt–N1 – 2.041(2) – 2.041(4) 2.045(8)

Pt–S – 2.2601(7) – 2.2504(14) 2.251(2)

Pt–Cl – 2.3110(7) – 2.3197(14) 2.307(2)

Angles (�)

C7–N2–N3 119.0(2) 119.0(2) 118.99(16) 117.4(5) 118.6(8)

N2–N3–C8 118.5(2) 112.4(2) 118.25(16) 117.4(5) 112.3(8)

N4–C8–N3 114.8(2) 120.4(3) 114.46(17) 119.8(5) 121.1(8)

N4–C8–S 124.9(2) 114.5(2) 124.71(15) 114.4(4) 114.0(7)

N4–C8–C9 125.7(2) 130.6(2) 127.09(17) 127.9(5) 129.8(8)

N3–C8–S 120.3(2) 125.1(2) 120.83(14) 125.8(4) 124.9(7)

N2–Pt–N1 – 80.61(9) – 80.88(19) 80.6(3)

N1–Pt–S – 165.35(7) – 165.88(14) 165.6(2)

N2–Pt–S – 84.82(7) – 84.99(13) 85.0(2)

N1–Pt–Cl – 96.77(7) – 96.55(14) 97.2(2)

N2–Pt–Cl – 177.38(7) – 177.01(14) 177.2(2)

Biometals (2013) 26:677–691 685

123

4.99(11)�. 7a and 9a, on the other hand, are clearly

non-planar molecules, with angles of 30.13(9)� and

23.05(22)�, respectively. In the molecular packing of

all three complexes dimers are generated by pairs of

N4–H4���Cl hydrogen bonds in 7a, and N4–H4���Shydrogen bonds in 5a and 9a (see Table 3; Fig. 3).

Cytotoxic activity against malignant U-87

and T-98 glioma cells and against MRC5 cells

All thiosemicarbazones were cytotoxic against glioma

cells in a dose-dependent manner. The concentrations

of the studied compounds that inhibit 50 % of cell

survival (IC50) are listed in Table 4. All thiosemicar-

bazones were highly cytotoxic against malignant

glioma with IC50 values in the 0.042–2.73 and

0.003–0.12 lM ranges against U-87 and T-98 cells,

respectively. The thiosemicarbazones were more

active than cisplatin, an antineosplastic drug currently

used in clinics and than auranofin, a gold-based drug

clinically used against rheumatoid arthritis, which has

demonstrated high cytotoxic activity (Tiekink 2008).

Among the gold(III) complexes [Au(2Ac4oT)-

Cl][AuCl2] (1) was the most active against both cell

lines (IC50 = 0.032 and 0.076 lM) and was also more

active than HAuCl4, cisplatin and auranofin. 1 was

slightly more active than the free thiosemicarbazone

against U-87 cells.

Upon coordination, the cytotoxic activity increased

about tenfold in [Au(Hpy2Ac4mT)Cl2]Cl�H2O (2)

against U-87 cells, suggesting that complexation was

a good strategy to improved the cytotoxic activity in

this case. All gold(III) complexes were more active

than HAuCl4 and cisplatin against U-87 cells. 3

exhibited cytotoxic activity similar to that of auran-

ofin, while 1 was approximately four times more

active than auranofin against U-87 cells. All gold(III)

complexes were more active than HAuCl4, auranofin

and cisplatin against T-98 cells.

Low therapeutic indexes [TI = IC50(MRC5)/

IC50(tumor cell)] were found for all studied compounds

in both cell lines, including cisplatin and auranofin.

The best TI values were determined for cisplatin

(TI = 2.8 in U-87 cells) and for complex [Au(H-

py2Ac4pT)Cl2]Cl (3) (TI = 2.7 in T-98 cells). Cyto-

toxicity of 3 was similar to that of the free

thiosemicarbazone against T-98 cells. However 3

showed a TI value about 27 times greater than that of

the ligand, suggesting that, in this case, coordination

seems to be an efficient strategy to decrease toxicity.

Platinum(II,IV) complexes were less active than

their gold(III) analogues against both U-87 and T-98

cell lines. Except for 8, in general the platinum(IV)

complexes were more active than their platinum(II)

analogues.

Upon coordination the cytotoxic activity against

U-87 cells increased in complexes [Pt(2Ac4mT)Cl]�H2O (5) and [Pt(2Ac4mT)Cl2OH] (8). 5, 7 and 8 proved

to be more cytotoxic than cisplatin against T-98 cells. 5

and 7 revealed to be more cytotoxic than cisplatin and 8

proved to be as cytotoxic as cisplatin against U-87 cells.

The calculated values of TI revealed that all plati-

num(II,IV) complexes were very toxic. However, it is

worth mentioning that although cisplatin presented

TI = 2.8 against U-87 cells, it presented TI = 1 against

T-98 cells.

Changes such as cell shrinkage, round shapes and

blebs formation are associated with apoptotic effects.

These features were observed under contrast phase

microscopy for H2Ac4mT and 2, 5 and 8 (see Fig. 4)

suggesting induction of programmed cell death in all

cases.

U-87 cells express functional p53 protein while

T-98 cells express mutant p53. It has been shown that

cells with mutant or absent p53 are less sensitive than

cells with wild-type p53 to the majority of clinically

used anticancer agents, including DNA alkylating

agents (O’Connor et al. 1997).

The thiosemicarbazones presented distinct cyto-

toxic activities against U-87 and T-98 cells, indicating

that the p53 status influenced the sensitivity of the

glioma cells to the studied compounds. However,

Table 3 Hydrogen bond distances (A) and angles (�) for [Pt(2Ac4mT)Cl] (5a), [Pt(2Ac4oT)Cl] (7a) and [Pt(2Ac4pT)Cl] (9a)

Compound D–H���A d(D���A) d(H���A) d(D–H) \(D���H���A)

5a N4–H4A���S11 3.512(2) 2.66 0.86 171.0

7a N4–H4���Cl12 3.792(5) 2.94 0.86 171.0

9a N4–H4N���S3 3.497(8) 2.83 0.86 135.5

Symmetry operations: (1) 1 - x, 1 - y, (2) -x, 1 - y, -z; (3) 2 - x, 1 – y, 1 - z

686 Biometals (2013) 26:677–691

123

similar cytotoxicities were observed for the gold(III)

and platinum(II,IV) complexes against both glioma

cell lineages, indicating that these complexes can

activate apoptotic cell death pathways by mechanisms

that are both dependent and independent of p53, and

can probably recruit more than one pathway to trigger

cell death (O’Connor et al. 1997).

Inhibition of TrxR activity

Inhibition of TrxR activity could be a mode of action

of the studied complexes. Since 2, 5 and 8 were in

general more cytotoxic than H2Ac4mT, their effects

on the activity of TrxR were evaluated. Hence,

H2Ac4mT 2, 5, 8, HAuCl4, K2PtCl4, K2PtCl6, cis-

platin and auranofin were evaluated at 10 lM using

isolated rat liver TrxR, by means of the DTNB

reduction assay (see Fig. 5). This assay makes use of

the fact that TrxR reduces the disulfide bonds of

DTNB with formation of TNB, which can be detected

photometrically.

H2Ac4mT, the platinum complexes (5) and (8)

and the platinum precursor salts inhibited about 10 %

of TrxR activity, whereas the gold(III) complex

Fig. 3 Intermolecular

interactions via hydrogen

bond (indicated by dashes

lines) of [Pt(2Ac4mT)Cl]

(5a) and [Pt(2Ac4oT)Cl]

(7a)

Table 4 Cytotoxic effect of 2-acetylpiridine-derived thiosemicarbazones and their gold(III) and platinum(II,IV) complexes against

malignant glioma (U-87 and T-98) and fibroblast (MRC-5) cells

Compound IC50 ± 95 % confidence interval in parenthesis (lM)

U-87 T-98 MRC-5

H2Ac4oT 0.042 ± 0.002 0.003 ± 0.001 0.00898 ± 0.00001

[Au(2Ac4oT)Cl2][AuCl2] (1) 0.032 ± 0.002 0.076 ± 0.001 0.01 ± 0.01

[Pt(H2Ac4oT)Cl]Cl (4) 4.7 ± 1.4 7 ± 2 7.7 ± 1.7

[Pt(2Ac4oT)Cl2OH] (7) 1.1 ± 0.2 1.5 ± 0.3 0.73 ± 0.07

H2Ac4mT 2.73 ± 0.6 0.057 ± 0.006 0.0662 ± 0.0007

[Au(Hpy2Ac4mT)Cl2]Cl�H2O (2) 0.32 ± 0.05 0.33 ± 0.02 0.062 ± 0.011

[Pt(2Ac4mT)Cl]�H2O (5) 0.7 ± 0.2 0.8 ± 0.1 0.74 ± 0.02

[Pt(2Ac4mT)Cl2OH] (8) 1.7 ± 0.4 1.7 ± 0.3 1.21 ± 0.07

H2Ac4pT 0.051 ± 0.006 0.12 ± 0.01 0.012 ± 0.002

[Au(Hpy2Ac4pT)Cl2]Cl (3) 0.25 ± 0.03 0.11 ± 0.02 0.3 ± 0.1

[Pt(2Ac4pT)Cl] (6) 29 ± 11 38 ± 13 16.5 ± 0.7

[Pt(2Ac4pT)Cl2OH] (9) 3.1 ± 0.8 4.0 ± 1.2 2.6 ± 0.2

HAuCl4 [100 [100 4.3 ± 0.8

K2PtCl4 122 ± 40 129 ± 58 69 ± 5

K2PtCl6 32 ± 12 34 ± 21 74 ± 9

Cisplatin 1.8 ± 0.2 5 ± 2 5.1 ± 0.7

Auranofin 0.188 ± 0.009 5.6 ± 0.6 0.30 ± 0.02

Biometals (2013) 26:677–691 687

123

[Au(Hpy2Ac4mT)Cl2]Cl�H2O (2) inhibited 75 % of

TrxR activity. HAuCl4 and auranofin inhibited 90 %

of the enzyme’s activity.

These results indicate that the cytotoxic activities of

the free thiosemicarbazones, their gold(III) complexes

and the platinum(II,IV) analogues involve different

modes of action. The studied thiosemicarbazones and

their platinum(II,IV) complexes acted as weak inhib-

itors of TrxR while the gold(III) complexes strongly

inhibited the enzyme’s activity.

Studies of interactions with supercoiled plasmid

DNA

The effects of H2Ac4mT, 2, 5, 8, HAuCl4, K2PtCl4,

and K2PtCl6 on DNA conformation was evaluated by

the electrophoretic mobility of the plasmid DNA-

pUC19 after interaction with the compounds (see

Fig. 6). Cisplatin and auranofin were used as controls.

At the employed concentration (200 lM), the free

thiosemicarbazone, its gold(III) complex (2), HAuCl4and auranofin did not interact with DNA, indicating

that direct binding to DNA is not the mechanism of

cytotoxic action of these compounds.

At the same concentration platinum complexes (5)

and (8) strongly interacted with DNA hindering its

electrophoretic mobility. Cisplatin also significantly

interacted with DNA. The precursor K2PtCl4, and

K2PtCl6 salts interacted weakly with DNA.

These results corroborate those obtained in the

study of inhibition of TrxR. The platinum compounds

may target primarily DNA, while the gold compounds

mainly act by inhibiting TrxR activity. The mecha-

nism of action of the free thiosemicarbazones likely

occurs through inhibition of the enzyme ribonucleo-

side diphosphate reductase (RDR), as already

Fig. 4 Morphological changes induced by treatment with

H2Ac4mT, 2, 5 and 8 (concentration 1 lM): Photomicrographs

of T-98 (expressing p53 mutant) and U-87 (p53 wild type)

glioma cell lines. Changes such as cell shrinkage, round shapes

and blebs formation suggest the induction of programmed cell

death. Amplification 9400

Fig. 5 Inhibitory effect of H2Ac4mT, [Au(Hpy2Ac4mT)Cl2]Cl�H2O (2), [Pt(2Ac4mT)Cl]�H2O (5), [Pt(2Ac4mT)Cl2OH] (8),

HAuCl4, K2PtCl4, K2PtCl6, cisplatin (Cisp) and auranofin (Aur)

on TrxR’s activity. Enzyme (0.10 unit) was treated without

(control) and with compounds (10 lM) for 1 h and TrxR

activity was evaluated by the DTNB assay as described in

methods. Representative data of experiments performed in

triplicate

688 Biometals (2013) 26:677–691

123

suggested in the literature for a(N)-heterocyclic thio-

semicarbazones (Finch et al. 2000).

Conclusions

The studied thiosemicarbazones proved to be highly

cytotoxic to human glioma cells. However the com-

pounds were also very toxic. Upon coordination to

gold(III) and platinum(II,IV) the cytotoxic effect

improved in some cases. In general platinum(IV)

complexes showed higher cytotoxic activities than the

platinum(II) analogues and the gold(III) complexes

were better cytotoxic agents than their platinum(II,IV)

counterparts. 3 presented therapeutic index TI = 2.7

against T-98 cells while for cisplatin TI = 1.

Preliminary investigation on the mode of action of

the compounds revealed that the platinum(II,IV)

complexes bind to DNA and are poor inhibitors of

TrxR activity while the gold(III) complexes strongly

inhibited the enzyme’s activity and did not interact

with DNA. 2, as well as HAuCl4 and auranofin were

able to inhibit TrxR activity, whereas H2Ac4mT acted

as a poor inhibitor of the enzyme. Hence inhibition of

TrxR is due to the presence of gold. However, as

previously observed by some of us (Lessa et al. 2011,

2012), in spite of the fact that HAuCl4 strongly

inhibited the enzyme’s activity, it does not show any

cytotoxic effect, indicating that the thiosemicarba-

zone, besides its own cytotoxic properties, probably

acted as a carrier of the metal into the cell.

Supplementary material

CCDC reference number 912353, 912355 and 912354

for 5a, 7a and 9a, respectively, contain the supple-

mentary crystallographic data. These data can be

obtained free of charge from the CCDC via

www.ccdc.cam.ac.uk/data_request/cif.

Acknowledgments The authors are grateful to Capes, CNPq

and INCT-INOFAR/CNPq (Proc. CNPq 573.364/2008-6) for

financial support.

References

Amani V, Safari N, Khavasi HR, Akkurt M (2009) Synthesis,

characterization and crystal structure determination of

platinum(IV) complexes containing, bipyridine derivatives

and chloride, [Pt(5,50-dmbipy)Cl4] and [Pt(6-mbipy)Cl4].

Polyhedron 28:3026–3030

Arsenijevic M, Milovanovic M, Volarevic V, Djekovic A,

Kanjevac T, Arsenijevic N, Dukic S, Bugarcic ZD (2012)

Cytotoxicity of gold(III) complexes on A549 human lung

carcinoma epithelial cell line. Med Chem 7:2–8

Beraldo H, Gambino D (2004) The wide pharmacological ver-

satility of thiosemicarbazones, semicarbazones and their

metal complexes. Mini Rev Med Chem 4:31–39

Fig. 6 Agarose gel electrophoresis of plasmid DNA pUC-19

treated without (control) and with compounds H2Ac4mT,

[Pt(2Ac4mT)Cl]�H2O (5), [Pt(2Ac4mT)Cl2OH] (8), K2PtCl4,

K2PtCl6, cisplatin (Cisp), [Au(Hpy2Ac4mT)Cl2]Cl�H2O (2),

HAuCl4 and auranofin (Aur) at 200 lM in TRIS–HCl buffer

(NaCl 50 mM, Tris–HCl 5 mM, pH 7.2) incubated at 37 �C for

24 h

Biometals (2013) 26:677–691 689

123

Bindoli A, Rigobello MP, Scutari G, Gabbiani C, Casini A,

Messori L (2009) Thioredoxin reductase: a target for gold

compounds acting as potential anticancer drugs. Coord

Chem Rev 253:1692–1707

Casas JS, Castano MV, Cifuentes MC, Garcıa-Monteagudo JC,

Sanchez A, Sordo J, Abram U (2004) Complexes of

dichloro[2-(dimethylaminomethyl)phenyl-C1, N]gold(III),

[Au(damp-C1, N)Cl2], with formylferrocene thiosemicar-

bazones: synthesis, structure and cytotoxicity. J Inorg

Biochem 98:1009–1016

Casini A, Hartinger C, Gabbiani C, Mini E, Dyson PJ, Keppler

BK, Messori L (2008) Gold(III) compounds as anticancer

agents: relevance of gold–protein interactions for their

mechanism of action. J Inorg Biochem 102:564–575

Chenon B, Sandorfy C (1958) Hydrogen bonding in the amine

hydrohalides: I. General aspects. Can J Chem 36:

1181–1206

Cox AG, Brown KK, Arner ES, Hampton MB (2008) The thi-

oredoxin reductase inhibitor auranofin triggers apoptosis

through a Bax/Bak-dependent process that involves

peroxiredoxin 3 oxidation. Biochem Pharmacol 76:

1097–1109

Da Silva JG, Azzolini LS, Wardell SMV, Wardell JL, Beraldo H

(2009) Increasing the antibacterial activity of gallium(III)

against Pseudomonas aeruginosa upon coordination

to pyridine-derived thiosemicarbazones. Polyhedron 28:

2301–2305

Dhar S, Lippard SJ (2009) Mitaplatin, a potent fusion of cis-

platin and the orphan drug dichloroacetate. Proc Natl Acad

Sci 106:22199–22204

Dilovic I, Rubcic M, Vrdoljak V, Pavelic SK, Kralj M, Pian-

tanida I, Cindric M (2008) Novel thiosemicarbazone

derivatives as potential antitumor agents: synthesis, phys-

icochemical and structural properties, DNA interactions

and antiproliferative activity. Bioorg Med Chem 16:

5189–5198

Farrugia LJ (1997) ORTEP-3 for Windows—a version of OR-

TEP-III with a graphical user interface (GUI). J Appl Cryst

30:565–566

Ferraz KSO, Ferandes L, Carrilho D, Pinto MCX, Leite MF,

Souza-Fagundes EM, Speziali NL, Mendes IC, Beraldo H

(2009) 2-Benzoylpyridine-N(4)-tolyl thiosemicarbazones

and their palladium(II) complexes: cytotoxicity against

leukemia cells. Bioorg Med Chem 17:7138–7144

Ferraz KOS, Cardoso GMM, Bertollo CM, Souza-Fagundes

EM, Speziali N, Zani CL, Mendes IC, Gomes MA, Beraldo

H (2011) N(4)-Tolyl-2-benzoylpyridine-derived thiosemi-

carbazones and their palladium(II) and platinum(II) com-

plexes: cytotoxicity against human solid tumor cells.

Polyhedron 30:315–321

Finch RA, Liu M, Grill SP, Rose WC, Loomis R, Vasquez KM,

Cheng Y, Sartorelli AC (2000) Triapine (3-aminopyridine-

2-carboxaldehyde-thiosemicarbazone): a potent inhibitor

of ribonucleotide reductase activity with broad spectrum

antitumor activity. Biochem Pharmacol 59:983–991

Freshney RI (2000) Culture of animal cells: a manual of basic

technique. Wiley-Liss, New York

Giannopoulou E, Dimitropoulos K, Argyriou AA, Koutras AK,

Dimitrakopoulos F, Kalofonos HP (2010) An in vitro

study, evaluating the effect of sunitinib and/or lapatinib on

two glioma cell lines. Invest New Drugs 28:554–560

Hall MD, Mellor HR, Callaghan R, Hambley TW (2007) Basis

for design and development of platinum(IV) anticancer

complexes. J Med Chem 50:3403–3411

Henderson W, Nicholson BK, Faville SJ, Fan D, Ranford JD

(2001) Gold(III) thiosalicylate complexes containing cy-

cloaurated 2-arylpyridine, 2-anilinopyridine and 2-ben-

zylpyridine ligands. J Organomet Chem 631:41–46

Jung Y, Lippard S (2007) Direct cellular responses to platinum-

induced DNA damage. Chem Rev 107:1387–1407

Kaps L, Biersack B, Muller-Bunz H, Mahal K, Munzner J,

Tacke M, Mueller T, Schobert R (2012) Gold(I)–NHC

complexes of antitumoral diarylimidazoles: structures,

cellular uptake routes and anticancer activities. J Inorg

Biochem 106:52–58

Khanye SD, Bathori NB, Smith GS, Chibale K (2010)

Gold(I) derived thiosemicarbazone complexes with rare

halogen–halogen interaction–reduction of [Au(damp-C1,

N)Cl2]. Dalton Trans 39:2697–2700

Kiaris H, Schally AV, Sun B, Armatis P, Groot K (1999) Inhi-

bition of growth of human malignant glioblastoma in nude

mice by antagonists of bombesin/gastrin-releasing peptide.

Oncogene 18:7168–7173

Kouodom MN, Ronconi L, Celegato M, Nardon C, Marchio L,

Dou QP, Aldinucci D, Formaggio F, Fregona D (2012)

Toward the selective delivery of chemotherapeutics into

tumor cells by targeting peptide transporters: tailored gold-

based anticancer peptidomimetics. J Med Chem 55:

2212–2226

Kovala-Demertzi D, Papageorgiou A, Papathanasis L, Alexan-

dratos A, Dalezis P, Miller JR, Demertzis MA (2009)

In vitro and in vivo antitumor activity of platinum(II)

complexes with thiosemicarbazones derived from 2-formyl

and 2-acetyl pyridine and containing ring incorporated at

N(4)-position: synthesis, spectroscopic study and crystal

structure of platinum(II) complexes with thiosemicarba-

zones, potential anticancer agents. Eur J Med Chem

44:1296–1302

Langner EHG, Swarts JC, Tuchscherer A, Lang H, Joone GK,

Van Rensburg CEJ (2012) Superior cytotoxicity of hyd-

rophylic gold carboxylato complexes overhydrophylic

silver carboxylates. Anticancer Res 32:2697–2701

Lessa JA, Mendes IC, da Silva RO, Soares MA, dos Santos RG,Speziali NL, Romeiro NC, Barreiro EJ, Beraldo H (2010)

2-Acetylpyridine thiosemicarbazones: cytotoxic activity in

nanomolar doses against malignant gliomas. Eur J Med

Chem 45:5671–5677

Lessa JA, Guerra JC, Miranda LF, Romeiro CFD, Da Silva JG,

Mendes IC, Speziali NL, Souza-Fagundes EM, Beraldo H

(2011) Gold(I) complexes with thiosemicarbazones: cyto-

toxicity against human tumor cell lines and inhibition of

thioredoxin reductase activity. J Inorg Biochem 105:

1729–1739

Lessa JA, Ferraz KSO, Guerra JC, Miranda LF, Romeiro CFD,

Souza-Fagundes EM, Barbeira PJS, Beraldo H (2012)

Spectroscopic and electrochemical characterization of

gold(I) and gold(III) complexes with glyoxaldehyde

bis(thiosemicarbazones): cytotoxicity against human

tumor cell lines and inhibition of thioredoxin reductase

activity. Biometals 25:587–598

Lobana TS, Khanna S, Butcher RJ (2008) Synthesis of a fluo-

rescent gold(I) complex with a thiosemicarbazone, [Au2(3-

690 Biometals (2013) 26:677–691

123

NO2-Hbtsc)4]Cl2�2CH3CN. Inorg Chem Commun

11:1433–1435

Maia PIS, Nguyen HH, Ponader D, Hagenbach A, Bergemann S,

Gust R, Deflon VM, Abram U (2012) Neutral gold com-

plexes with tridentate SNS thiosemicarbazide ligands.

Inorg Chem 51:1604–1613

Marzano C, Gandin V, Folda A, Scutari G, Bindoli A, Rigobello

MP (2007) Inhibition of thioredoxin reductase by auranofin

induces apoptosis in cisplatin-resistant human ovarian

cancer cells. Free Radic Biol Med 42:872–881

Mendes IC, Moreira JP, Speziali NL, Mangrich AS, Takahashi

JA, Beraldo H (2006) N(4)-Tolyl-2-benzoylpyridine thio-

semicarbazones and their copper(II) complexes with sig-

nificant antifungal activity. Crystal structure of N(4)-para-

tolyl-2-benzoylpyridine thiosemicarbazone. J Braz Chem

Soc 17:1571–1577

Mendes IC, Moreira JP, Mangrich AS, Balena SP, Rodrigues

BL, Beraldo H (2007) Coordination to copper(II) strongly

enhances the in vitro antimicrobial activity of pyridine-

derived N(4)-tolyl thiosemicarbazones. Polyhedron

26:3263–3270

Mishra AK, Mishra SB, Manav N, Kumar R, Sharad, Chandra R,

Saluja D, Kaushik NK (2007) Platinum (IV) thiohydrazide,

thiodiamine and thiohydrazone complexes: a spectral,

antibacterial and cytotoxic study. Spectrochim Acta A

66:1042–1047

O’Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M,

Scudiero DA, Monks A, Sausville EA, Weinstein JN,

Friend S, Fornace AJJ, Kohn KW (1997) Characterization

of the p53 tumor suppressor pathway in cell lines of the

National Cancer Institute anticancer drug screen and cor-

relations with the growth-inhibitory potency of 123 anti-

cancer agents. Cancer Res 57:4285–4300

Ott I, Qian X, Xu Y, Vlecken DHW, Marques IJ, Kubutat D,

Will J, Sheldrick WS, Jesse P, Prokop A, Bagowski CPJ

(2009) A gold(I) phosphine complex containing a naph-

thalimide ligand functions as a TrxR inhibiting antiprolif-

erative agent and angiogenesis inhibitor. J Med Chem

52:763–770

Perez-Rebolledo A, Vieites M, Gambino D, Piro OE, Castellano

EE, Zani CL, Souza-Fagundes EM, Teixeira LR, Batista

AA, Beraldo H (2005) Palladium(II) complexes of 2-ben-

zoylpyridine-derived thiosemicarbazones: spectral char-

acterization, structural studies and cytotoxic activity.

J Inorg Biochem 99:698–706

Prast-Nielsen S, Cebula M, Pader I, Arner ESJ (2010) Noble

metal targeting of thioredoxin reductase—covalent com-

plexes with thioredoxin and thioredoxin-related protein of

14 kDa triggered by cisplatin. Free Radic Biol Med

49:1765–1778

Quiroga AG, Perez JM, Montero EI, Masaguer JR, Alonso C,

Navarro-Ranninger C (1998) Palladated and platinated

complexes derived from phenylacetaldehyde thiosemicar-

bazone with cytotoxic activity in cis-DDP resistant tumor

cells. Formation of DNA interstrand cross-links by these

complexes. J Inorg Biochem 70:117–123

Roberts JJ, Friedlos F (1981) Quantitative aspects of the for-

mation and loss of DNA interstrand crosslinks in Chinese

hamster cells following treatment with cis-diamminedi-

chloroplatinum(II) (Cisplatin). Biochim Biophys Acta

655:146–151

Rubbiani R, Can S, Kitanovic I, Alborzinia H, Stefanopoulou M,

Kokoschka M, Moenchgesang S, Sheldrick WS, Wolfl S,

Ott I (2011) Comparative in vitro evaluation of N-hetero-

cyclic carbene gold(I) complexes of the benzimidazoly-

lidene type. J Med Chem 54:8646–8657

Soares MA, Lessa JA, Mendes IC, Da Silva JG, Santos RG,

Salum LB, Daghestani H, Andricopulo AD, Day BW, Vogt

A, Pesquero JL, Rocha WR, Beraldo H (2012) N4-Phenyl-

substituted 2-acetylpyridine thiosemicarbazones: cytotox-

icity against human tumor cells, structure–activity rela-

tionship studies and investigation on the mechanism of

action. Bioorg Med Chem 20:3396–3409

Sreekanth A, Fun HK, Kurup MRP (2004) Formation of first

gold(III) complex of an N(4)-disubstituted thiosemicarba-

zone derived from 2-benzoylpyridine: structural and

spectral studies. Inorg Chem Commun 7:1250–1253

Tiekink ERT (2008) Anti-cancer potential of gold complexes.

Inflammopharmacol 16:138–142

Biometals (2013) 26:677–691 691

123