DNA binding and antitumor activities of platinum(IV) and zinc(II) complexes with some S-alkyl derivatives of thiosalicylic acid

Article abstract of DOI:10.1007/s11243-018-0260-2

A series of complexes of platinum(IV) (C1–C5) and zinc(II) (C6–C10) with S-alkyl derivatives of thiosalicylic acid were prepared and characterized. The interactions of the complexes with calf thymus DNA were analyzed by absorption (UV–Vis) and emission spectral studies (ethidium bromide displacement studies). The cytotoxic activities of complexes C1–C10 were determined against mouse B cell lymphocytic leukemia cells (BCL1), human B-prolymphocytic leukemia (JVM-13), mouse mammary carcinoma cells (4T1), and human mammary carcinoma cells (MDA-MB-468) and compared to the activities of the free ligand precursors and cisplatin. The cytotoxicities of the platinum(IV) and zinc(II) complexes toward mouse tumor cell lines were higher compared with their effects on human tumor cell lines. The zinc(II) complex C9 showed the highest antitumor activity toward the tested human cell lines, while the platinum(IV) complex C4 exhibited the highest antitumor activity toward mouse BCL1 and 4T1 cells. Both C4 and C9 have ligands derived from S-propyl thiosalicylic acid.

Full text of DOI:10.1007/s11243-018-0260-2

Transition Metal Chemistry
https://doi.org/10.1007/s11243-018-0260-2
DNA binding and antitumor activities of platinum(IV) and zinc(II)
complexes with some S‑alkyl derivatives of thiosalicylic acid
Zana Besser Silconi1 · Sasa Benazic2 · Jelena Milovanovic3,6 · Milena Jurisevic4,6 · Dragana Djordjevic4,6
·
Milos Nikolic4 · Marina Mijajlovic4 · Zoran Ratkovic5 · Gordana Radić4 · Snezana Radisavljevic5 · Biljana Petrovic5 ·
Gordana Radosavljevic6 · Marija Milovanovic6 · Nebojsa Arsenijevic6
Received: 21 April 2018 / Accepted: 13 July 2018
© Springer Nature Switzerland AG 2018
Abstract
A series of complexes of platinum(IV) (C1–C5) and zinc(II) (C6–C10) with S-alkyl derivatives of thiosalicylic acid were
prepared and characterized. The interactions of the complexes with calf thymus DNA were analyzed by absorption (UV–Vis)
and emission spectral studies (ethidium bromide displacement studies). The cytotoxic activities of complexes C1–C10 were
determined against mouse B cell lymphocytic leukemia cells (BCL1), human B-prolymphocytic leukemia (JVM-13), mouse
mammary carcinoma cells (4T1), and human mammary carcinoma cells (MDA-MB-468) and compared to the activities of
the free ligand precursors and cisplatin. The cytotoxicities of the platinum(IV) and zinc(II) complexes toward mouse tumor
cell lines were higher compared with their efects on human tumor cell lines. The zinc(II) complex C9 showed the highest
antitumor activity toward the tested human cell lines, while the platinum(IV) complex C4 exhibited the highest antitumor
activity toward mouse BCL1 and 4T1 cells. Both C4 and C9 have ligands derived from S-propyl thiosalicylic acid.
Introduction
Platinum(II) complexes present a valuable starting point
Zana Besser Silconi and Sasa Benazic contributed equally to this
in the development of metal complex-based anticancer
study.
therapy. The original such platinum(II) complex, cisplatin,
exhibits limited activity against several tumors, including
colon and breast cancer, and is frequently accompanied
with the development of resistance [1]. Furthermore, cis-
platin therapy is accompanied by severe side efects such
as potentially fatal renal failure, bone marrow suppression,
severe nausea, and immunodefciency [2]. Problems asso-
ciated with cisplatin application, including high reactivity
and thus poor biological stability [3, 4], have resulted in
the synthesis of new transition metal complexes with bio-
active ligands [5–8]. Platinum(IV) complexes have greater
inertness compared with corresponding platinum(II) com-
plexes, and undesirable side reactions with proteins in vivo
can be avoided using platinum(IV) complexes [9]. The
inertness of platinum(IV) gives rise to several advan-
tages such as oral administration, reduced toxicity, and a
decrease in the amount of the complex being lost or deac-
tivated in the path to the target cell [10–12]. The reactivity
of platinum(IV) complexes toward DNA is enhanced in
the presence of a reducing agent such as ascorbic acid,
glutathione, and proteins with sulfhydryl groups [13]. This
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11243-018-0260-2) contains
supplementary material, which is available to authorized users.
* Marija Milovanovic
marijaposta@gmail.com
* Nebojsa Arsenijevic
arne@medf.kg.ac.rs
1
Department of Cytology, Pula General Hospital, Pula,
Croatia
2
Department of Transfusiology, Pula General Hospital, Pula,
Croatia
3
Department for Histology, Faculty of Medical Sciences,
University of Kragujevac, Kragujevac, Serbia
4
Department of Pharmacy, Faculty of Medical Sciences,
University of Kragujevac, Kragujevac, Serbia
5
Department of Chemistry, Faculty of Science, University
of Kragujevac, Kragujevac, Serbia
6
Center for Molecular Medicine and Stem Cell Research,
Faculty of Medical Sciences, University of Kragujevac,
Kragujevac, Serbia
Vol.:(0123456789)
1 3

Transition Metal Chemistry
suggests that platinum(IV) complexes are reduced by cel-
lular components to form the platinum(II) analogs that
bind to DNA [14–16].
Synthesis of the S‑alkyl thiosalicylic acids
The thioacid ligands benzyl-(L1), methyl-(L2), ethyl-(L3),
propyl-(L4), and butyl-(L5) were prepared by alkylation
of thiosalicylic acid using the corresponding alkyl halo-
genides in alkaline water–ethanol solution. Thiosalicylic
acid (1 mmol) was added to a 30% solution of ethanol in
water (50 mL) and stirred. A solution of NaOH (2 mmol)
in water (5 mL) was added to the acid suspension, where-
upon the solution became clear. The corresponding alkyl
halogenide (2 mmol) was dissolved in ethanol (5 mL) and
transferred to the stirred solution. The resulting mixture
was kept overnight at 60 °C. Ethanol was then evaporated
of by gentle heating. Dilute hydrochloric acid (2 mol/dm3)
was added to the resulting aqueous solution, whereupon
the S-alkyl thiosalicylic acid was precipitated as a white
powder. The product was fltered of, washed with plenty
of distilled water, and dried under vacuum overnight.
Yield was 85–95%.
The eforts of several research groups, which were ini-
tially focused on the evaluation of platinum-based anticancer
drugs, have now shifted to non-platinum metal-based agents
in order to minimize the side efects [17–19]. Based on the
assumption that endogenous metals may be less toxic for
normal cells, the investigation of zinc-based complexes has
started only recently. The link between zinc defciency and
cancer has been established in human, animal, and cell cul-
ture studies [20, 21]. Zinc defciency causes oxidative DNA
damage [22], and chromosome breaks have been reported
in zinc-defcient diet-fed animals [23]. During the past two
decades, a number of zinc(II) complexes with anticancer
activity have been reported, such as zinc(II) phthalocyanine
tumor photosensitizers [24], and semicarbazone and thio-
semicarbazone [25, 26] zinc(II) complexes.
Previously, copper(II) complexes with S-alkyl derivatives
of thiosalicylic acid have been shown to exert potential anti-
tumor activity and to interact with DNA [27–29]. There-
fore, in order to investigate the possibility of even greater
cytotoxicities, the central metal was changed and analogous
platinum(IV) and zinc(II) complexes were synthetized [30,
31].
S‑benzyl‑thiosalicylic acid (L1) M.p. 179–180 °C,
white powder. Anal. Calc. for S-bz-thiosal = C14H12O2S
(Mr=244.24): C, 68.84; H, 4.95; S, 13.10. Found: C, 68.50;
H, 4.82; S, 13.07%. IR (KBr, cm−1): 3414w, 3061m, 2920m,
2648m, 2559m, 1674s, 1584w, 1562m, 1463m, 1412m,
1317m, 1272s, 1255s 1154w, 1062m, 1046m, 897m, 743m,
Herein, the interactions with DNA and cytotoxic activ-
ity against murine and human leukemia and breast carci-
noma cells of fve S-alkyl derivatives of thiosalicylic acid,
together with fve platinum(IV) and fve zinc(II) complexes,
are described.
1
711w, 652w, 551w. H NMR (200mHz, CDCl3, δ ppm):
4.17 (s, 2H, CH2), 7.21–8.14 (m, 9H, Ar and bz). 13C NMR
(50 MHz, DMSO-d6, δ ppm): 35.9 (CH2), 124.1, 125.9,
126.7, 127.3, 127.9, 128.3, 128.6, 129.3, 131.0, 132.4,
136.8, 141.3 (Ar and bz), 167.5 (COOH).
S‑methyl‑thiosalicylic acid (L2) M.p. 165–166 °C,
white powder. Anal. Calc. for S-met-thiosal = C8H8O2S
(Mr=168.144): C, 57.14; H, 4.80; S, 19.03. Found: C, 57.72;
H, 4.76; S, 19.50%. IR (KBr, cm−1): 3446w, 3068m, 2916m,
2652m, 2560m, 1674s, 1586w, 1561m, 1466m, 1412m,
1308m, 1291s, 1270s, 1255s, 1151w, 1062m, 1048m, 892m,
Experimental
Materials and measurements
All chemicals were obtained commercially and used with-
out further purification. Elemental microanalyses were
obtained with a Vario III CHNOS Elemental Analyzer,
Elemental Analysensysteme GmbH. For infrared spectra,
a Perkin-Elmer FTIR 31725-X spectrophotometer and KBr
pellet technique were employed. 1H and 13C NMR spectra
were recorded on a Varian Gemini-200 NMR spectrometer
using TMS in DMSO-d6 as an internal reference, at 22 °C.
Ethidium bromide (EB) and calf thymus DNA (CT-DNA)
were obtained from Sigma-Aldrich. The stock solution of
CT-DNA was prepared in 0.01 M phosphate-bufered saline
(PBS) (Sigma-Aldrich) at pH=7.4, which gave a ratio of UV
absorbances at 260 and 280 nm (A260/A280) of ca. 1.8–1.9,
indicating that the DNA was sufciently free of proteins.
The concentration of DNA solution was determined by UV
absorbance at 260 nm (ε=6600 M−1 cm−1) [32–34].
1
743m, 699w, 652w, 556w. H NMR (200 MHz, CDCl3, δ
ppm): 2.48 (s, 3H, CH3), 7.16–8.18 (m, 4H, Ar). 13C NMR
(50 MHz, CDCl3, δ ppm): 15.6 (CH3), 123.5, 124.4, 125.4,
132.5, 133.6, 144.4 (Ar), 171.6 (COOH).
S‑ethyl‑thiosalicylic acid (L3) M.p. 133–134 C, white pow-
der. Anal. Calc. S-et-thiosal = C9H10O2S (Mr = 182.17):
C, 59.33; H, 5.53; S, 17.57. Found: C, 59.21; H, 5.49; S,
17.68%. IR (KBr, cm−1): 3435w, 3066m, 2972m, 2652m,
2562m, 1682s, 1588w, 1563m, 1466m, 1414m, 1315m,
1275s, 1252s, 1152w, 1063m, 1049m, 884m, 740m, 704w,
1
690w, 651w, 550w. H NMR (200 MHz, CDCl3, δ ppm):
1.42 (t, 3H, CH3), 2.97 (q, 2H, CH2), 7.16–8.17 (m, 4H,
Ar). 13C NMR (50 MHz, CDCl3, δ ppm): 13.1 (CH3), 26.2
1 3

Transition Metal Chemistry
(CH2), 124.0, 125.9, 126.4, 132.6, 133.2, 142.9 (Ar), 171.4
(COOH).
(Mr = 600.40): C, 32.00; H, 2.35; S, 10.68. Found: C,
31.54; H, 2.59; S, 10.22%. IR (KBr, cm−1): 3436m, 2923w,
2794w, 2439w, 1693s, 1581s, 1552s, 1469w, 1423m, 1361s,
1290s, 1274s, 1149w, 1116w, 1056w, 970m, 858w, 798w,
754s, 697w, 653w, 568w. 1H NMR (200 MHz, DMSO-d6, δ
ppm): 2.47 (s, 6H, CH3), 7.41–8.30 (m, 8H, Ar). 13C NMR
(50 MHz, DMSO-d6, δ ppm): 15.6 (CH3), 125, 125.5, 126.4,
129.9, 134.1, 137.2 (Ar), 169.3 (COO−).
S‑propyl‑thiosalicylic acid (L4) M.p. 104 °C, white powder.
Anal. Calc. for S-pr-thiosal = C10H12O2S (Mr = 196.196):
C, 61.21; H, 6.16; S, 16.31. Found: C, 60.18; H, 6.50; S,
16.18%. IR (KBr, cm−1): 3414w, 3056m, 2979m, 2641m,
2555m, 1678s, 1588w, 1562m, 1462m, 1405m, 1310m,
1271s, 1257s, 1150w, 1062m, 1053m, 811m, 740m, 704w,
691w, 653w, 554w. 1H NMR (200 MHz, CDCl3, δ ppm): 1.1
(t, 3H, CH3), 1.74 (m, 2H, CH2), 2.92 (t, 2H, CH2), 7.15–
8.15 (m, 4H, Ar). 13C NMR (50 MHz, CDCl3, δ ppm): 13.8
(CH3), 21.6 (CH2), 34.1 (CH2), 123.8, 125.6, 126.2, 132.5,
133.1, 143.1 (Ar), 171.6 (COOH).
Platinum(IV) complex of S‑ethyl thiosalicylic acid
[PtCl2(S‑et‑thiosal)2] (C3) Yield: 0.13 g (58%). Anal. Calc.
for [PtCl2(S-et-thiosal)2]=PtC18H18O4S2Cl2 (Mr=828.46):
C, 34.40; H, 2.89; S, 10.20. Found: C, 34.14; H, 2.71; S,
10.11%. IR (KBr, cm−1): 3436s, 2521w, 1692m, 1634s,
1563w, 1437w, 1404w, 1274w, 1143w, 1122w, 1050w,
1
S‑butyl‑thiosalicylic acid (L5) M.p. 82–83 °C, white pow-
der. Anal. Calc. for S-bz-thiosal C11H14O2S (Mr=210.222):
C, 62.84; H, 6.71; S, 15.22. Found: C, 62.66; H, 6.81; S,
15.30%. IR (KBr, cm−1): 3420w, 2955m, 2869m, 2641m,
2556m, 1674s, 1586w, 1560m, 1462m, 1408m, 1320m,
1270s, 1250s, 1153w, 1060m, 1048m, 924m, 810m,
997w, 872w, 794w, 749w, 693w, 643w, 568w. H NMR
(200 MHz, DMSO-d6, δ ppm): 1.27 (t, 6H, CH3), 2.81 (q,
4H, CH2), 7.42–8.28 (m, 8H, Ar). 13C NMR (50 MHz,
DMSO-d6, δ ppm): 12.9 (CH3), 13.8 (CH2), 125.1, 126.4,
126.6, 133.3, 133.8, 137.1 (Ar), 169.2 (COO−).
1
738m, 704w, 651w, 553w. H NMR (200 MHz, CDCl3, δ
Platinum(IV) complex of S‑propyl thiosalicylic acid
[PtCl2(S‑pr‑thiosal)2] (C4) Yield: 0.13 g (56%). Anal. Calc.
for [PtCl2(S-pr-thiosal)2]=PtC20H22O4S2Cl2 (Mr=656.51):
C, 36.59; H, 3.38; S, 9.77. Found: C, 36.17; H, 3.30; S,
9.61%. IR (KBr, cm−1): 3444m, 3061w, 2963w, 2930w,
2873w, 2600w, 1706m, 1639s, 1586m, 1562m, 1461w,
1436w, 1416w, 1293s, 1253m, 1138w, 1091w, 1052w, 863w,
798w, 753m, 691w, 652w. 1H NMR (200 MHz, DMSO-d6,
δ ppm): 0.90 (t, 6H, CH3), 1.34 (m, 4H, CH2), 2.75 (t, 4H,
CH2), 7.40–8.31 (m, 8H, Ar). 13C NMR (50 MHz, DMSO-
d6, δ ppm): 13.0 (CH3), 23,2 (CH2), 12 (CH2), 125.3, 126.1,
126.4, 133.2, 133.9, 136.9 (Ar), 169.1 (COO−).
ppm): 0.96 (t, 3H, CH3), 1.46 (m, 2H, CH2), 1.78 (m, 2H,
CH2), 2.94 (t, 2H, CH2), 7.15-8.16 (m, 4H, Ar). 13C NMR
(50 MHz, CDCl3, δ ppm): 13.7 (CH3), 22.3 (CH2), 30.2
(CH2), 31.9 (CH2), 123.8, 125.7, 126.3, 132.5, 133.1, 143.1
(Ar), 171.4 (COOH).
Synthesis of the platinum(IV) complexes
Potassium hexachloroplatinate(IV) (0.1000 g, 0.2056 mmol)
was dissolved in hot water (10 mL), and the appropriate
thiosalicylic acid L1–L5 was added. The resulting mixture
was stirred for 2 h, and during this time, an aqueous solu-
tion of LiOH (0.0099 g, 0.4112 mmol) in water (10 mL) was
introduced. The resulting yellow precipitate was fltered of,
washed with water, and air-dried [30]
Platinum(IV) complex of S‑butyl thiosalicylic acid
[PtCl2(S‑bu‑thiosal)2] (C5) Yield: 0.14 g (59%). Anal. Calc.
for [PtCl2(S-bu-thiosal)2]=PtC22H26O4S2Cl2 (Mr=684.56):
C, 38.60; H, 3.83; S, 9.37. Found: C, 38.35; H, 3.71; S,
9.28%. IR (KBr, cm−1): 3437w, 3054w, 2956m, 2931m,
2869m, 2629w, 1673m, 1644s, 1635s, 1583m, 1561m,
1462m, 1433w, 1410w, 1318w, 1286m, 1250m, 1137w,
1100w, 1060w, 1049w, 916w, 863w, 754m, 738m, 698w,
652w, 551w. 1H NMR (200 MHz, DMSO-d6, δ ppm): 0.89
(t, 6H, CH3), 1.43 (m, 4H, CH2), 1.60 (m, 4H, CH2), 2.73
(t, 4H, CH2), 7.41–8.28 (m, 8H, Ar). 13C NMR (50 MHz,
DMSO-d6, δ ppm): 13.4 (CH3), 21.5 (CH2), 31(CH2), 10
(CH2), 124.9, 125.9, 126.5, 133.1, 134.2, 136.9 (Ar), 168.9
(COO−).
Platinum(IV) complex of S‑benzyl derivative of thiosali‑
cylic acid [PtCl2(S‑bz‑thiosal)2] (C1) Yield: 0.15 g (58%).
Anal. Calc. for [PtCl2(S-bz-thiosal)2] = PtC28H22O4S2Cl2
(Mr==752.59): C, 44.68; H, 2.95; S, 8.52. Found: C, 44.26;
H, 2.88; S, 8.60%. IR (KBr, cm−1): 3437m, 3062w, 3028w,
2924w, 1629s, 1561s, 1493w, 1463w, 1412w, 1318s, 1254m,
1142w, 1046w, 868w, 799w, 750m, 697m, 667w, 652w,
552w. 1H NMR (200 MHz, DMSO-d6, δ ppm): 4.01 (s, 4H,
CH2), 7.23–8.24 (m, 18H, Ar и bz). 13C NMR (50 MHz,
DMSO-d6, δ ppm): 17 (CH2), 124, 125.5, 127.1, 127.4,
127.7, 133.1, 133.9, 136.2 (Ar и bz), 169.1 (COO−).
Synthesis of the zinc(II)complexes
Platinum(IV) complex of S‑methyl thiosalicylic acid
[PtCl2(S‑met‑thiosal)2] (C2) Yield: 0.12 g (56%). Anal.
Calc. for [PtCl2(S-met-thiosal)2] = PtC16H14O4S2Cl2
Zinc(II) chloride (0.1000 g, 0.7337 mmol) was dissolved
in hot water (10 mL), and the required S-alkyl thiosalicylic
1 3

Transition Metal Chemistry
acid L1–L5 was added. The reaction mixture was heated at
50 °C for 2 h, and during this period, a solution of LiOH
(0.0352 g, 1.4674 mmol) in water (10 mL) was added in
small portions. The solution was then fltered and evapo-
rated to a small volume. The white precipitate of zinc(II)
complex was separated by fltration, washed with water,
and air-dried [31].
22 (CH2), 125.3, 126.6, 126.5, 133.4, 134.2, 138.7 (Ar),
169.1 (COO−).
Zinc(II) complex of S‑butyl thiosalicylic acid [Zn(S‑bu‑thi‑
osal)2] (C10) Yield: 0.21 g (60%) Anal. Calc. for [Zn(S-
bu-thiosal)2]=ZnC22H26O4S2 (Mr=483.950): C, 54.60; H,
5.42; S, 13.25. Found: C, 54.33; H, 5.19; S, 13.17%. IR
(KBr, cm−1): 3436m, 3053w, 2952w, 2934w, 2867w, 1614s,
1594s, 1582s, 1565m, 1467m, 1430m, 1408s, 1312s, 1291w,
1252w, 1139w, 1098w, 1063w, 1051w, 918w, 853w, 754m,
733m, 697w, 652w, 551w. 1H NMR (200 MHz, DMSO-d6,
δ ppm): 0.88 (t, 6H, CH3), 1.44 (m, 4H, CH2), 1.60 (m, 4H,
CH2), 2.77 (t, 4H, CH2), 7.42–8.28 (m, 8H, Ar). 13C NMR
(50 MHz, DMSO-d6, δ ppm): 13.4 (CH3), 21.6 (CH2), 32
(CH2), 20 (CH2), 124.9, 126.6, 126.8, 133.3, 134.2, 138.5
(Ar), 168.8 (COO−).
Zinc(II) complex of S‑benzyl thiosalicylic acid [Zn(S‑bz‑thi‑
osal)2] (C6) Yield: 0.25 g (61%). Anal. Calc. for [Zn(S-bz-
thiosal)2] = ZnC28H22O4S2 (Mr = 551.978): C, 60.92; H,
4.02; S, 11.62. Found: C, 60.53; H, 3.94; S, 11.71%. IR
(KBr, cm−1): 3428m, 3059w, 2922w, 1598s, 1580s, 1437m,
1403s, 1282w, 1259w, 1063w, 1044w, 846w, 744m, 697w.
1H NMR (200 MHz, DMSO-d6, δ ppm): 4.03 (s, 4H, CH2),
7.26–8.25 (m, 18H, Ar и bz). 13C NMR (50 MHz, DMSO-
d6, δ ppm): 26 (CH2), 125, 126.4, 126.8, 127.2, 127.3, 128.9,
133.5, 134.3, 137.7, 138.6 (Ar и bz), 169.2 (COO−).
Absorption spectroscopic studies
Zinc(II) complex of S‑methyl thiosalicylic acid [Zn(S‑met‑thi‑
osal)2] (C7) Yield: 0.19 g (64%) Anal. Calc. for [Zn(S-met-
thiosal)2] = ZnC16H14O4S2 (Mr = 399.794): C, 48.06; H,
3.53; S, 16.04. Found: C, 47.69; H, 3.78; S, 15.71%. IR
(KBr, cm−1): 3436m, 2917w, 2859w, 1593s, 1576s, 1435m,
1399s, 1280w, 1256w, 1156w, 1065w, 953w, 847w, 744m,
654w. 1H NMR (200 MHz, DMSO-d6, δ ppm): 2.47 (s, 6H,
CH3), 7.42-8.30 (m, 8H, Ar). 13C NMR (50 MHz, DMSO-d6,
δ ppm): 15.6 (CH3), 126, 126.3, 126.8, 133.4, 134.3, 137.2
(Ar), 169.4 (COO−).
The interactions of the complexes with CT-DNA were
studied using UV–Vis spectroscopy at 310 K. Absorp-
tion titrations in bufer (0.01 M PBS, pH 7.4) were per-
formed using a fxed complex concentration (8 µM) to
which increments of the DNA stock solution were added
(2.2 mM).
Fluorescence quenching measurements
Zinc(II) complex of S‑ethyl thiosalicylic acid [Zn(S‑et‑thi‑
osal)2] (C8) Yield: 0.18 g (58%) Anal. Calc. for [Zn(S-et-
thiosal)2] = ZnC18H18O4S2 (Mr = 427.846): C, 50.53; H,
4.24; S, 14.99. Found: C, 50.17; H, 4.07; S, 14.88%. IR
(KBr, cm−1): 3432m, 2954w, 2765w, 1595s, 1563s, 1436m,
1404s, 1273w, 1149w, 1122w, 1054w, 995w, 871w, 784w,
739m, 693w, 655w. 1H NMR (200 MHz, DMSO-d6, δ ppm):
1.27 (t, 6H, CH3), 2.81 (q, 4H, CH2), 7.43–8.27 (m, 8H,
Ar). 13C NMR (50 MHz, DMSO-d6, δ ppm): 13.9 (CH3), 13
(CH2), 125.1, 126.6, 126.5, 133.3, 134.2, 137.1 (Ar), 169.3
(COO−).
EB-competitive studies for the complexes were carried out
by fuorescence emission spectroscopy. DNA–EB was ini-
tially prepared by mixing 21 μM EB and 21 μM CT-DNA in
0.01 M PBS bufer solution (pH 7.4). The binding efect of
the complexes was investigated step by step after the addi-
tion of a certain amount of complex solution into the solu-
tion of DNA–EB. The fuorescence intensities were meas-
ured with an excitation wavelength of 527 nm and detection
of emission at 612 nm. Before measurements, each system
was shaken and incubated at room temperature for 5 min.
Zinc(II) complex of S‑propyl thiosalicylic acid [Zn(S‑pr‑thi‑
osal)2] (C9) Yield: 0.20 g (62%) Anal. Calc. for [Zn(S-pr-
thiosal)2] = ZnC20H22O4S2 (Mr = 455.898): C, 52.69; H,
4.86; S, 14.07. Found: C, 52.27; H, 4.78; S, 13.91%. IR
(KBr, cm−1): 3443m, 3061w, 2965w, 2931w, 2863w, 1591s,
1562s, 1462m, 1435m, 1312w, 1294w, 1252w, 1137w,
1092w, 1055w, 867w, 794w, 755m, 692w, 652w. 1H NMR
(200 MHz, DMSO-d6, δ ppm): 0.90 (t, 6H, CH3), 1.35 (m,
4H, CH2), 2.77 (t, 4H, CH2), 7.41–8.32 (m, 8H, Ar). 13C
NMR (50 MHz, DMSO-d6, δ ppm): 13.0 (CH3), 24.2 (CH2),
Stock solutions for cytotoxicity studies
The complexes were dissolved in 10% DMSO in distilled
water at a concentration of 10 mM and fltered through
a 0.22-mm Millipore filter. These stock solutions were
diluted in culture medium immediately before use. MTT,
3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide, was dissolved (5 mg/mL) in a phosphate-bufered
saline having a pH of 7.2 and fltered through a 0.22-mm
Millipore flter before use. All reagents were purchased from
Sigma.
1 3

Transition Metal Chemistry
A high control, leading to 100% cytotoxicity by lysing the
cells completely, was included in the assay. Cells exposed
to medium only were used as a low control. After 24 h of
treatment, the supernatant (50 µL) was transferred to a new
multiplate and incubated with previously prepared substrate
solution (100 µL). The plates were protected from light and
incubated at room temperature for 30 min. The reaction
was stopped by addition of stop solution, and the data were
acquired by spectrophotometry at 490 nm. Experiments were
repeated three times. The percentage of dead cells was cal-
culated using the formula:
Cell culture
BCL1 (mouse B cell leukemia), 4T1 (mouse mammary
carcinoma), JVM-13 (human B-prolymphocytic leukemia),
and MDA-MB-468 (human mammary gland adenocarci-
noma, metastatic) cells were purchased from the American
Type Culture Collection (ATCC, Manassas, USA). The
cells were maintained in DMEM (Sigma-Aldrich) or RPMI
1640 (Sigma-Aldrich) supplemented with 10% fetal bovine
serum (FBS, Sigma-Aldrich), penicillin (100 IU/mL), and
streptomycin (100 μg/mL) in a humidifed atmosphere of
95% air/5% CO2 at 37 °C. Subconfuent monolayers, in a
log growth phase, were harvested by a brief treatment with
0.25% trypsin and 0.02% EDTA in phosphate-bufered saline
(PBS, Sigma-Aldrich) and washed three times in serum-free
PBS. The number of viable cells was determined by trypan
blue exclusion assay.
% dead cells = (exp . value − low control)∕
(high control − low control) × 100.
Results and discussion
Synthesis and characterization
Cell viability assay
Thiosalicylate ligands (L1–L5) were synthesized by the
reaction of thiosalicylic acid and the corresponding alkyl
halides in alkaline water–ethanol solution [36]. The corre-
sponding platinum(IV) complexes (C1–C5) were obtained
by the direct reaction of K2PtCl6 with thiosalicylic acids
L1–L5 in water (Scheme 1) [30], while the zinc(II) com-
plexes (C6–C10) were obtained by the direct reaction of
ZnCl2 with the thiosalicylic acids in water (Scheme 1) [31].
On the basis of microanalysis results, IR and NMR spec-
tra of the proligands and the corresponding Pt(IV) and Zn(II)
complexes, we conclude that the ligands are bidentately
coordinated to the metal centers. However, we could not
conclude anything about the exact geometries of the com-
plexes. We were unable to obtain crystals of the complexes
suitable for X-ray difraction.
The efects of the test compounds on cell viability were
determined by MTT colorimetric technique [35]. The cells
were diluted in growth medium to (5×104 cells/mL), and
aliquots (5 × 103 cells/100 mL) were placed in individual
wells in 96-well multiplates. The test compounds were seri-
ally diluted twofold in the medium to give concentrations
raging from 1000 to 7.8 μM in growth medium and then
added to wells with JVM-13 cells; the medium in the wells
with adherent cells was exchanged the next day with 100 μL
of fresh compound. Each compound was tested in triplicate.
The cells were incubated at 37 °C in 5% CO2 for 72 h. After
incubation, the supernatant was removed and 15% MTT
solution (5 mg/mL in PBS, 10 μL) in DMEM medium with-
out FBS was added to each well. After an additional 4 h of
incubation at 37 °C in 5% CO2, the medium with MTT was
removed and DMSO (150 μL) with glycine bufer (20 μL)
was added to dissolve the crystals. The plates were shaken
for 10 min. The optical density of each well was determined
at 595 nm using a microplate Zenyth 3100 Multimode detec-
tor. The cytotoxicity was calculated using the formula:
DNA binding studies
Absorption spectroscopy is one of the most universally
employed methods for the determination of both the mode
and extent of binding metal complexes with DNA. In gen-
eral, transition metal complexes can bind to DNA via cova-
lent (replacement of a labile ligand of the complex by a
nitrogen base of DNA, e.g., guanine N7) and/or non-covalent
(intercalation, electrostatic or groove binding) interactions
[37]. The absorption intensity of a complex in the presence
of DNA may either decrease (hypochromism) or increase
(hyperchromism), with slight increase in the absorption
wavelength (bathochromism). The absorption spectra of
complex C9 in the absence and in the presence of CT-DNA
are shown in Fig. 1, and the spectra for the other complexes
are presented in Fig. S1 of ESI.
% cytotoxicity = 100 − ((E − B)∕(S − B) ∗ 100),
where B is the background of medium alone, S is the total
viability/spontaneous death of untreated target cells, and E
is the value for the experimental well.
LDH (lactic dehydrogenase) assay
Cytotoxicities of the test compounds were assayed using an
In Vitro Toxicology Assay Kit (lactic dehydrogenase based)
(Sigma-Aldrich). 4T1 cells were prepared and treated with
the test compound in the same way as for the MTT assays.
The addition of CT-DNA to a solution of complex
C9 resulted in a significant hyperchromic effect with
1 3

Transition Metal Chemistry
Scheme 1 Preparation of the
S-alkyl derivatives of thio-
salicylic acid. R=benzyl (L1),
methyl (L2), ethyl (L3), propyl
(L4), butyl (L5), preparation
of the platinum(IV) complexes
C1–C5 and the preparation of
the zinc(II) complexes C6–C10
Table 1 The DNA binding constants (Kb) and calculated Stern–Vol-
mer constants (Ksv) for complexes C1–C9
0.9
0.6
0.3
0.0
Complex
Kb (M−1
)
KSV (M−1
)
C1
C2
C3
C4
C5
C6
C7
C8
C9
(1.13 0.01)×104
(9.86 0.01)×103
(2.50 0.02)×104
(5.51 0.01)×103
(2.22 0.02)×104
(1.43 0.01)×104
(1.18 0.02)×104
(5.01 0.05)×103
(3.33 0.02)×104
(1.9 0.1)×104
(7.4 0.1)×103
(1.04 0.08)×104
(6.12 0.09)×103
(7.83 0.04)×103
(2.50 0.05)×103
(5.21 0.03)×103
(1.13 0.02)×103
(2.53 0.02)×103
240
260
280
λ nm
300
320
Fig. 1 Absorption spectra of complex C9 in 0.01 M PBS (pH
7.4) upon addition of CT-DNA. [Zn complex]=8×10−6 M,
[DNA]=(0.8–3.2)×10−5 M. The arrow shows the change in absorb-
ance upon increasing the DNA concentrations. Inset: Plot of [DNA]/
(εA−εf) versus [DNA]
hyperchromic efect with the appearance of a new signal in
the Uv–Vis spectra indicates an interaction between these
complexes and CT-DNA [38].
The intrinsic binding constant of the complexes with CT-
DNA (Kb) can be used to estimate the magnitude of binding
strength. The values of Kb (M−1) for all of the complexes
calculated by equation (S1) are given in Table 1.
the appearance of a new band at 262 nm, as shown in
Fig. 1, and the spectrum of C9 is shown in ESI, Fig. S4,
λmax = 260 nm. Considering that DNA also shows an
absorption at 260 nm, this result confrms the presence
of interactions as explained before. The behavior of the
other complexes was quite similar upon addition of increas-
ing amounts of CT-DNA (Fig. S1, ESI). A signifcant
The high values for Kb confrm the binding of all of these
complexes to CT-DNA. Complex C9 exhibits the highest
Kb value compared with the other Zn(II) complexes, while
C3 is the strongest binder among the Pt(II) complexes. In
1 3

Transition Metal Chemistry
comparison with the classical intercalator ethidium bromide
(EB), for which Kb = (1.23 0.07) × 105 M−1 [39], it can
be seen that the present complexes exhibit lower binding
constants.
between the complexes and EB for DNA binding (Fig. 2
and Fig. S2).
The Stern–Volmer plots for EB–DNA displacement
(Fig. 2 and Fig. S2) confirm the fluorescence emission
quenching. The Stern–Volmer quenching constants (Ksv)
were calculated from the slopes of the plots of I0/I versus
[Q] ([Q] is the total concentration of quencher, Eq. (S2)
ESI), inset Fig. 2 and Fig. S2) and are reported in Table 1.
All of these complexes showed high quenching constants,
indicating that they efciently displace EB and bind to DNA.
Complex C7 exhibits the highest KSV value in comparison
with the other zinc(II) complexes, while C1 exhibits the
highest KSV value in comparison with the other platinum(IV)
complexes.
The binding of the complexes to DNA was also investi-
gated by fuorescence quenching. EB is a classical intercala-
tor that gives intense fuorescence emission when it interca-
lates into the base pairs of DNA. Specifcally, the maximum
fuorescence emission is at 612 nm, when excited at 527 nm,
due to the formation of an EB–DNA complex. If a test com-
pound, which is also capable of DNA intercalation, is added
to the EB–DNA complex, the fuorescence should decrease
due to competitive intercalation.
The fluorescence quenching curves of EB bound to
DNA in the absence and in the presence of the complexes
are shown in Fig. 2 and Fig. S2, ESI. Increasing complex
concentrations resulted in signifcant decreases in intensity
of the emission band at 612 nm, indicating competition
Antitumor activity
In order to investigate in vitro cytotoxic potential of the
platinum(IV) and zinc(II) complexes, various concentrations
of the complexes were applied to JVM-13 and pre-seeded
BCL1, 4T1 and MDA-MB-468 cells, and the cell survival
was determined after 24-h exposure by MTT assay. The
results of these experiments, including cisplatin as a control
substance, are presented in Table 2. All complexes showed
a dose-dependent cytotoxic efect against all tested tumor
cell lines (Fig. 3). Also these complexes, including cisplatin,
had higher cytotoxic activities toward murine tumor cell
lines (4T1 and BCL1) in comparison with human cell lines
(MDA-MB-468 and JVM-13). Previously it was shown that
copper(II) complexes of thiosalicylic acids had signifcantly
lower cytotoxic activity toward murine and human colon
carcinoma cell lines than cisplatin [28]. The free thiosali-
cylic acids had mild cytotoxicity only in the highest tested
concentrations (500 and 1000 µM) (Fig. S3). It seems that
coordination to a metal center greatly changes the antitumor
activity of S-alkyl derivatives of thiosalicylic acid.
100
80
60
40
20
0
560
600
640
680
720
λ / nm
Fig. 2 Emission spectra of EB bound to DNA in the presence of
complex C5. [EB]=21 μM, [DNA]=21 μM; [C5]=0–73.5 μM;
λex =527 nm. Arrows show the change in the intensity upon increas-
ing the concentration of the complex. Inset: Stern–Volmer quenching
plot of DNA–EB for complex C5
Table 2 IC50 values (µM) for
Compound
BCL1
79.56 12.11
4T1
38.81 7.96
133.62 19.62
56.13 8.77
2.9 1.01
42.88 6.89
59.27 10.21
85.31 12.54
78.78 10.12
49.41 8.13
7883.58 251.14
3.44 1.24
JVM-13
MDA-468
24 h of action of platinum(IV)
and zinc(II) complexes and
cisplatin on BCL1, 4T1, JVM-
13, and MDA-468 cells, as
determined by MTT assay
C1
513.31 87.74
433.29 65.44
255.45 18.78
88.78 12.03
632.86 87.96
115.06 16.57
99.08 14.74
224.40 23.54
67.16 10.02
416.35 74.11
94.26 9.89
305.66 41.25
211.37 31.41
187.53 28.78
354.96 52.17
252.91 35.47
120.94 15.71
125.86 14.78
271.19 24.96
72.73 9.56
C2
65.83 9.87
60.79 8.11
2.47 0.84
70.59 11.24
109.08 10.23
183.83 24.55
187.53 18.78
104.14 14.11
2383.19 121.26
2.04 0.79
C3
C4
C5
C6
C7
C8
C9
C10
Cisplatin
191.31 21.11
27.38 5.32
The data are presented as mean values SD (standard deviation) from three experiments
1 3

Transition Metal Chemistry
C6
4T1
C1
4T1
C7
120
100
80
60
40
20
0
120
100
80
60
40
20
0
C2
C8
C3
C9
C4
C10
cisplatin
C5
cisplatin
7,8
31,25
125
500
7,8 15,625 31,25 62,5 125 250 500 1000
concentration µM
concentration µM
C6
C1
BCL1
BCL1
C7
C2
120
100
80
60
40
20
0
120
100
80
60
40
20
0
C8
C9
C10
cisplatin
C3
C4
C5
cisplatin
7,8 15,625 31,25 62,5 125 250 500 1000
7,8
31,25
125
500
concentration µM
concentration µM
C1
C6
C7
C8
C9
C10
cisplatin
JVM-13
JVM-13
C2
120
120
100
80
60
40
20
0
C3
C4
100
80
60
40
20
0
C5
cisplatin
7,8 15,625 31,25 62,5 125 250 500 1000
7,8 15,625 31,25 62,5 125 250 500 1000
concentration µM
concentration µM
C6
C1
MDA-MB-468
MDA-MB-468
C7
C2
120
100
80
60
40
20
0
120
100
80
60
40
20
0
C8
C3
C9
C4
C10
C5
cisplatin
cisplatin
7,8 15,625 31,25 62,5 125 250 500 1000
7,8 15,625 31,25 62,5 125 250 500 1000
concentration µM
concentration µM
Fig. 3 Graphs of 4T1, BCL, MDA-MB-468, and JVM-13 cell survival after 24-h cell growth in the presence of the platinum(IV) and zinc(II)
complexes. Mean values from three experiments are shown
Based on the obtained IC50 values, platinum(IV) com-
plex C4 had the highest cytotoxic potential toward murine
cell lines. The cytotoxic potential of C4 toward murine B
cell leukemia cell line BCL-1 was similar to that of cispl-
atin (2.47 0.84 versus 2.04 0.79) and even higher toward
murine mammary carcinoma cell line 4T1 (2.90 1.01
versus 3.44 1.24). These results are in accordance with pre-
vious fndings, indicating high cytotoxicities of platinum(IV)
complexes toward leukemia [40, 41] and breast cancer cell
lines [42–44].
Among the zinc(II) complexes, propyl-S derivative C9
had the highest activity toward murine cell lines 4T1 and
1 3

Transition Metal Chemistry
35
50
45
40
35
30
25
20
15
10
5
C1
C6
C2
30
25
20
15
10
5
C7
C3
C8
C9
C10
cisplatin
C4
C5
cisplatin
0
0
7,8 15,625 31,25 62,5 125 250 500 1000
7,8 15,625 31,25 62,5 125 250 500 1000
concentration µM
concentration µM
Fig. 4 LDH assay of 4T1 cells treated with platinum(IV) and zinc(II) complexes for 24 h. Mean values from three experiments are shown
BCL1. However, the activity of C9 toward 4T1 and BCL1
was several times lower than that of cisplatin. The high cyto-
toxicity of platinum(IV) complex C4 toward murine chronic
lymphocytic leukemia cells is in agreement with previous
studies [6, 40, 41]. The highest activity toward human tumor
cell lines JVM-13 and MDA-MB-468 was shown by C9.
Furthermore, the IC50 value for C9 toward JVM-13 was
lower than IC50 value for cisplatin (67.16 10.02 versus
94.26 9.89). High cytotoxicities of diferent zinc(II) com-
plexes against diferent leukemia cells have been observed
previously [45–47]. The S-propyl platinum(IV) complex C4
also had higher cytotoxic activity toward JVM-13 than cis-
platin. Importantly, the free propyl-S thiosalicylic acid had
no cytotoxic potential toward tumor cell lines (Fig. S3), with
very mild cytotoxicity only at a concentration of 1000 µM
(cell viability approximately 80%).
can efectively displace EB from DNA. Among the zinc(II)
complexes, C7 showed the highest Ksv values, while among
the platinum(IV) complexes, C1 exhibited the highest Ksv
values.
Both the platinum(IV) and zinc(II) complexes of these
S-alkyl derivatives of thiosalicylic acid strongly interact with
DNA. The platinum(IV) complexes exhibited the strong-
est antitumor activities toward murine cell lines, while the
zinc(II) complexes showed the strongest cytotoxic activities
toward human cell lines. Based on these results, the tumori-
cidal potential of S-propyl derivatives of thiosalicylic acid
complexed with platinum(IV) and zinc(II) merits further
investigation.
Acknowledgements This work was funded by grants from the Ser-
bian Ministry of Science and Technological Development (Grants Nos.
ON175069, ON172016, and ON172011), Serbia, and the Faculty of
Medical Sciences, University of Kragujevac (MP 01/14 and MP 02/14).
The potential of these platinum(IV) and zinc(II) com-
plexes to induce cell death was further investigated on 4T1
cells by LDH assay and found to follow a dose-dependent
response (Fig. 4). Additionally, the zinc(II) complexes
induced higher release of LDH from 4T1 cells in comparison
with the platinum(IV) complexes. Platinum(IV) complexes
C1 and C3 induced lower LDH release from 4T1 cells than
cisplatin. On the other hand, all of the zinc(II) complexes
in concentrations from 15.625 to 125 µM and C6–C9 in
all tested concentrations induced higher release of LDH in
comparison with cisplatin (Fig. 4), indicating that these zinc
complexes can induce necrotic cell death. Among the tested
zinc(II) complexes, C9 induced the highest release of LDH
from 4T1 cells (Fig. 4).
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