Traceable platinum(II) complexes with alkylene diamine-derived ligands: synthesis, characterization and in vitro studies

Article abstract of DOI:10.1080/00958972.2018.1431392

Diiodido- (6a/6b) and dichloridoplatinum(II) complexes (7a/7b) with fluorescent ligands 2-[(2-aminoethyl)amino]ethyl-2-(methylamino)benzoate (5a) and 2-amino-1-(aminoethyl)ethyl-2-(methylamino)benzoate (5b) were prepared and characterized by elemental ana

Full text of DOI:10.1080/00958972.2018.1431392

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Traceable platinum(II) complexes with
alkylene diamine derived ligands: Synthesis,
characterization and in vitro studies
Till Walther, Renate Herzog, Milena R. Kaluđerović, Christoph Wagner, Harry
Schmidt & Goran N. Kaluđerović
To cite this article: Till Walther, Renate Herzog, Milena R. Kaluđerović, Christoph Wagner, Harry
Schmidt & Goran N. Kaluđerović (2018): Traceable platinum(II) complexes with alkylene diamine
derived ligands: Synthesis, characterization and in vitro studies, Journal of Coordination Chemistry,
DOI: 10.1080/00958972.2018.1431392
To link to this article: https://doi.org/10.1080/00958972.2018.1431392
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Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://doi.org/10.1080/00958972.2018.1431392
Traceable platinum(II) complexes with alkylene diamine derived ligands:
Synthesis, characterization and in vitro studies
TILL WALTHER†, RENATE HERZOG†, MILENA R. KALUĐEROVIƇ, CHRISTOPH WAGNER†,
HARRY SCHMIDT† and GORAN N. KALUĐEROVIĆ*§
†
Institute of Chemistry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle (Saale),
Germany
Department of Oral, Maxillary, Facial and Reconstructive Plastic Surgery, University Hospital of Leipzig,
Liebigstr. 12, 04103 Leipzig, Germany
Department of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale),
‡
§
Germany
Diiodido- (6a/6b) and dichloridoplatinum(II) complexes (7a/7b) with fluorescent ligands 2-[(2-
aminoethyl)amino]ethyl-2-(methylamino)benzoate (5a) and 2-amino-1-(aminoethyl)ethyl-2-
(methylamino)benzoate (5b) were prepared and characterized by elemental analysis, ESI-MS
analysis, fluorescence spectrometry, as well as H, C and 195Pt NMR spectroscopy. All
compounds have been tested against A2780 ovarian cancer, A549 lung carcinoma and HT-29
colon cancer cell lines using sulforhodamine-B assay. The activity increased from ligand
precursors, diiodido- to dichloridoplatinum(II) complexes, except against HT-29 cell line where
diiodido and dichlorido expressed similar activity. These compounds enter the tumor cells and
emit a bright fluorescence at ca. 470 nm, mainly targeting nuclei.
1
13
Keywords: Cisplatin; Platinum(II) complexes; Cytotoxicity; Fluorescence
1
. Introduction
Cis-[diamminedichloridoplatium(II)], cisplatin, is one of the leading anticancer drugs since
approval granted by the US Food and Drug Administration in 1978 [1-3]. However, cisplatin as a
chemotherapeutic agent has several disadvantages. The side-effects and the occurring resistance
of some tumors against cisplatin are big challenges medicinal chemistry has to face [4-6]. Taking
this into consideration, it is necessary to search for new chemotherapeutic agents without these
*
Corresponding author. Email: goran.kaluderovic@ipb-halle.de

disadvantages and more effectivity on tumors. During the past few decades, many platinum
complexes have been synthesized which were hoped to minimize mentioned problems [7]. Some
recent platinum anticancer drugs are already in use such as nedaplatin (only in Japan), oxaliplatin
and carboplatin (figure 1(a)). Some other compounds are still undergoing clinical investigations,
for example satraplatin and picoplatin [8-10].
In order to increase the effectiveness of anticancer drugs, it is necessary to find out the
fate of the compounds in cells [11-13]. For this reason, different approaches have been
developed. One approach is to synthesize metal complexes with fluorescent ligands [14-16].
Cells exposed to such compounds can be investigated with fluorescence microscopy and it can
be determined directly where the fluorescent compound is allocated in the living cell. There are
many potential ligands which can be used as fluorescent markers in complexes. Some examples
are shown in figure 1(b).
Several fluorescent platinum(II) and platinum(IV) complexes are already described in the
literature [17-25]. A cisplatin derivative in which one ammine ligand is substituted by coumarin
1
20 and its corresponding platinum(IV) compound having two hydroxido ligands in mutual trans
positions are described [26]. The complexes have been investigated on A2780 human ovarian
cancer cell line via fluorescence microscopy. Both complexes enter the cells (located in
lysosomes) but do not enter the nuclei. Furthermore, the complexes are at least 10 times less
cytotoxic than cisplatin. Kalayda et al. described binuclear platinum(II) complex with
carboxyfluorescein derivative [27]. In this case, the fluorescence microscopic investigations of
the treated U2-OS and U2-OS/Pt tumor cells with synthesized platinum(II) complex showed that
the compound could be found in the nuclei (after 1 h treatment) and in the Golgi apparatuses
(
24 h after treatment) of the cells. Also here, the IC value of the platinum(II) complex is quite
50
low (> 50 µM) against the investigated cell lines.
Beside platinum compounds, other metal complexes containing fluorescent ligands, for
example copper(II), zinc(II), palladium(II), and ruthenium(II), may also be of interest [28-35].
Zobi et al. described the synthesis of a ruthenium(II) complex with an isatoic acid derivative as
fluorescent ligand, whereas, although the fluorescence properties of this compound seem to be
satisfying, no results about cytotoxicity have been reported [36]. On the other hand, the work of
Navas et al. (and references therein) reported that changing of chlorido with iodido ligands in
platinum(II) complexes influences the biological activity [37].

The objective of this report is to synthesize two ligand precursors, 5a already known
from the literature [36] and new ligand precursor 5b, both containing a fluorescent moiety. These
ligand precursors are designed as fluorescence markers for the synthesis of cisplatin analogues.
Furthermore, the ligand precursors as well as corresponding platinum(II) complexes should be
investigated on human cancer cell lines and monitored by fluorescence microscopy and
cytotoxicity assay.
2
2
. Experimental
.1. General manipulations
NMR spectra were recorded on Varian Gemini 200, Gemini 400 and Unitiy 500. The residual
proton signals from deuterated solvent and the solvent signals were used as internal reference for
1
13
H NMR and C NMR spectroscopy, respectively (CDCl : δ 7.24, δ 77.0 ppm, DMSO-d ,
3
H
C
6
δ 2.50, δ 39.5 ppm). Na [PtCl ] was used as external standard for the 195Pt NMR spectroscopy
H
C
2
6
(
δPt 0.0 ppm). ESI mass spectra (positive ion polarity mode) were obtained using a Finnigan
LCQ spectrometer (Thermo Electron) under the following conditions: MeOH, solvent flow:
μL/min; ESI spray voltage: 4.1 kV; capillary temperature: 150 °C; capillary voltage: +4 V;
tube lens offset: +3 V; sheath gas N ; damping gas He.
8
2
2
2
.2. Synthesis of the ligand precursors, 5a and 5b
.2.1. Synthesis of Boc-protected amino alcohols, 2a [33] and 2b. The corresponding amino
alcohol was dissolved in a suspension of NaHCO in THF/H O (1:1). Tert-butoxycarbonyl
3
2
anhydride was added slowly at 0 °C. The mixture was stirred overnight at room temperature. By
slow addition of AcOH at 0 °C the mixture was then acidified to pH = 4 and extracted with
CH Cl (1×30 mL, 2×25 mL). The combined organic phases were dried over Na SO , filtered
2
2
2
4
and the solvent was removed by evaporation. The crude product still contained acetic acid. The
amount of residual acetic acid was determined by integrals arising from hydrogen atoms
1
(
CH COOH) in the H NMR spectra. The compounds were used for further reactions without
3
additional purification. Residual acetic acid: 18–33.0%.
Boc-protected N-(2-hydroxyethyl)ethylenediamine (2a): For the synthesis of 2a, the
following was used: 1a (1.00 g, 9.6 mmol), NaHCO (2.42 g, 28.8 mmol), THF/H O (60 mL),
3
2
Boc O (5 g, 23.0 mmol). Residual AcOH: 17.8%. Yield: 2.94 g (95%). Colorless oily liquid.
2

1
H NMR (400 MHz, CDCl ), δ (ppm): 1.40 (s, 9H, 3CH ), 1.43 (s, 9H, 3CH ), 3.27 (br, 4H,
3
3
3
1
3
2
CH ), 3.33 (br, 2H, CH ), 3.70 (br, 2H, CH ). C NMR (400 MHz, CDCl ), δ (ppm): 28.39 (s,
2 2 2 3
CH ), 28.40 (s, CH ), 39.8 (s, CH ), 48.5 (s, CH ), 51.3 (s, CH ), 62.9 (s, CH ), 79.5, 80.4 (s,
3
3
2
2
2
2
OC(CH ) ), 156.4 (br, N(CO)O).
3
3
Boc-protected 2-hydroxy-1,3-propanediamine (2b): For the synthesis of 2b, the following
was used: 1b (0.90 g, 10.0 mmol), NaHCO (2.52 g, 30.0 mmol), THF/H O (60 mL), Boc O
3
2
2
1
(
5.24 g, 24.0 mmol). Residual AcOH: 33.0%. Yield: 2.60 g (67%). Colorless oily liquid. H
NMR (500 MHz, CDCl ), δ (ppm): 1.43 (s, 18H, 6CH ), 3.20 (m, 4H, 2CH ), 3.73 (quin,
3
3
2
3
13
J(H/H) = 5.18 Hz, 1H, CH). C NMR (500 MHz, CDCl ), δ (ppm): 28.3 (s, CH ), 43.5 (s,
3
3
CH ), 71.0 (s, CH), 79.8 (s, OC(CH ) ), 157.2 (s, N(CO)O).
2
3 3
2
.2.2. Synthesis of Boc-protected N-methylisatoic esters, 3a and 3b. The corresponding Boc-
protected aminoalcohol was dissolved in dry acetonitrile. N-methylisatoic anhydride and K CO
2
3
were added to the solution. The mixture was stirred for 48 h at room temperature. The solvent
then was removed by evaporation and the crude product was purified by centrifugally
accelerated, radial, thin-layer chromatography. The eluent was changed from n-hexane to
n-hexane/acetone to acetone. The fractions which contained the product (TLC) were combined
and the solvent was removed by evaporation.
3
a: For the synthesis of 3a, the following was used: 2a (2.94 g, 9.7 mmol), CH CN
3
(
100 mL), N-methylisatoic anhydride (2.60 g, 14.7 mmol), K CO (6.9 g, 50 mmol). Yield:
2 3
1
1
.26 g (31%). White powder. H NMR (200 MHz, CDCl ), δ (ppm): 1.40 (s, 18H, 6CH ), 2.88
3 3
3
(
s, 3H, CH ), 3.27 (br, 2H, CH ), 3.38 (br, 2H, CH ), 3.55 (br, 2H, CH ), 4.32 (t, J(H/H) = 5.43
3 2 2 2
Ar
3
Hz, 2H, CH ), 6.54 (ddd, J(H/H) = 8.1, 7.2, 1.0 Hz, 1H, C H), 6.63 (d, J(H/H) = 8.5 Hz, 1H,
2
Ar
Ar
C H), 7.35 (ddd, J(H/H) = 8.6, 7.1, 1.7 Hz, 1H, C H), 7.85 (dd, J(H/H) = 8.0, 1.6 Hz, 1H,
Ar
13
C H). C NMR (200 MHz, CDCl ), δ (ppm): 28.51 (s, CH ), 28.57 (s, CH ), 29.7 (s, CH ), 39.7
3
3
3
3
Ar
(
s, CH ), 46.7 (s, CH ), 62.4 (s, CH ), 80.5 (s, OC(CH ) ), 109.8 (s, C COO), 111.0 (s, C ),
2 2 2 3 3 ipso
Ar
Ar
Ar
1
14.6 (s, C ), 131.8 (s, C ), 135.0 (s, C ), 152.3 (s, C N), 156.1 (s, N(CO)O), 168.5 (s,
ipso
(CO)O).
3
b: For the synthesis of 3b, the following was used: 2b (2.60 g, 9.0 mmol), CH CN
3
(
100 mL), N-methylisatoic anhydride (2.70 g, 15.2 mmol), K CO (6.9 g, 50 mmol). Yield:
2 3
1
2
.40 g (63%). White viscid powder. H NMR (500 MHz, CDCl ), δ (ppm): 1.41 (s, 18H, 6CH ),
3 3

3
3
2
5
7
.88 (d, J(H/H) = 5.0 Hz, 3H, CH ), 3.33 (br, 2H, CH ), 3.47 (br, 4H, 2CH ), 5.00 (q, J(H/H) =
3 2 2
3
Ar
3
Ar
.1 Hz, 1H, CH), 6.54 (t, J(H/H) = 7.5 Hz, 1H, C H), 6.63 (d, J(H/H) = 8.5 Hz, 1H, C H),
3
Ar
3
Ar
13
.35 (t, J(H/H) = 7.7 Hz, 1H, C H), 7.86 (d, J(H/H) = 8.0 Hz, 1H, C H). C NMR
(
500 MHz, CDCl ), δ (ppm): δ 28.3 (s, CH ), 29.5 (s, CH ), 40.5 (s, CH ), 71.7 (s, CH), 79.6 (s,
3 3 3 2
Ar
Ar
Ar
Ar
OC(CH ) ), 109.3 (s, C COO), 110.7 (s, C ), 114.2 (s, C ), 131.6 (s, C ), 134.9 (s, C ),
3
3
ipso
1
52.2 (s, C N), 156.2 (s, N(CO)O), 167.7 (s, (CO)O).
ipso
2
.2.3. Deprotection of N-methylisatoic esters, 4a and 4b. The corresponding N-methylisatoic
ester, 3a or 3b, was dissolved in dry CH Cl . Trifluoroacetic acid (TFA) was added slowly to the
2
2
solution. The mixture was stirred for 48 h. Within this time, a second phase established at the
bottom of the flask. This new phase was collected and the residual solvent was removed by
evaporation.
4
a: The following was used: 3a (1.70 g, 3.9 mmol), CH Cl (150 mL), TFA (4.6 mL,
2 2
6
0.0 mmol). Amount of TFA residue was not determined. Yield: 2.85 g. Colorless viscid oil.
1
H NMR (400 MHz, DMSO-d ), δ (ppm): 2.84 (s, 3H, CH ), 3.18 (br, 2H, CH ), 3.29 (m, 2H,
6
3
2
Ar
CH ), 3.44 (m, 2H, CH ), 4.44 (m, 2H, CH ), 6.57 (ddd, J(H/H) = 8.1, 7.1, 1.0 Hz, 1H, C H),
2
2
2
3
Ar
Ar
6
.71 (d, J(H/H) = 8.0 Hz, 1H, C H), 7.42 (ddd, J(H/H) = 8.6, 7.1, 1.7 Hz, 1H, C H), 7.97 (dd,
Ar
13
J(H/H) = 8.0, 1.6 Hz, 1H, C H). C NMR (400 MHz, DMSO-d ), δ (ppm): 29.7 (s, CH ), 35.6
6
3
Ar
(
s, CH ), 44.8 (s, CH ), 46.4 (s, CH ), 60.2 (s, CH ), 109.0 (s, C COO), 111.4 (s, C ), 114.5 (s,
2 2 2 2 ipso
Ar
1
Ar
Ar
C ), 116.2 (q, J(F/C) = 291.9 Hz, CF COOH), 132.3 (s, C ), 135.6 (s, C ), 152.1 (C N),
3
ipso
3
1
59.2 (q, J(F/C) = 35.9 Hz, CF COOH), 167.7 (s, (CO)O).
3
4
b: The following was used: 3b (1.00 g, 2.4 mmol), CH Cl (100 mL), TFA (2.7 mL,
2 2
3
5.4 mmol). Amount of TFA residue was not determined. Yield: 1.30 g. Light brown viscid oil.
1
3
H NMR (400 MHz, D O), δ (ppm): 3.00 (s, 3H, CH ), 3.37 (d, J(H/H) = 2.1 Hz, 2H, CHH),
2
3
Ar
3
.38 (s, 2H, CHH), 5.60 (m, 1H, CH), 7.48 (dd, J(H/H) = 8.2, 0.8 Hz, 1H, C H), 7.51 (dt,
Ar
Ar
J(H/H) = 7.8, 1.1 Hz, 1H, C H), 7.73 (dt, J(H/H) = 7.9, 1.5 Hz, 1H, C H), 8.16 (dd, J(H/H) =
Ar
13
8
.0, 1.5 Hz, 1H, C H). C NMR (400 MHz, D O), δ (ppm): 36.9 (s, CH ), 40.2 (s, CH ), 69.3
2 3 2
1
Ar
(
s, CH), 116.0 (q, J(F/C) = 291.1 Hz, CF COOH), 119.9 (s, C COO), 123.5 (s, C ), 129.8 (s,
3 ipso
Ar
Ar
Ar
3
C ), 132.4 (s, C ), 136.8 (s, C ), 152.7 (s, C N), 162.5 (q, J(F/C) = 36.1 Hz, CF COOH),
ipso
3
1
66.0 (s, (CO)O).

2
.2.4. Synthesis of hydrochlorides of the N-methylisatoic esters, 5a and 5b. Hydrogen
chloride, evolved from the reaction of H SO (20 mL) with NaCl (35.0 g), dried with
2
4
concentrated H SO , was bubbled through a solution of the corresponding N-methylisatoic ester
2
4
(
4a: 2.51 g; 4b: 1.62 g) in 100 mL of dry diethylether (4b) or THF (4a). After a short period of
time, the product starts to precipitate. The product was filtered off and washed with diethylether
5a) or THF (5b).
a: Yield: 1.18 g. White viscid solid. ESI-MS(+): 238.4 [M+H] . H NMR (500 MHz,
DMSO-d ), δ (ppm): 2.86 (s, 3H, CH ), 3.27 (br, 2H, CH ), 3.33 (br, 2H, CH ), 3.41 (br, 2H,
(
+
1
5
6
3
2
2
Ar
3
Ar
CH ), 4.50 (m, 2H, CH ), 6.65 (m, 1H, C H), 6.78 (d, J(H/H) = 8.2 Hz, 1H, C H), 7.45 (ddd,
2
2
Ar
Ar
13
J(H/H) = 8.6, 7.1, 1.7 Hz, 1H, C H), 8.12 (dd, J(H/H) = 8.0, 1.6 Hz, 1H, C H). C NMR
(
(
(
500 MHz, DMSO-d ), δ (ppm): 30.2 (s, CH ), 35.6 (s, CH ), 44.9 (s, CH ), 46.1 (s, CH ), 60.3
6 3 2 2 2
Ar
Ar
Ar
Ar
s, CH ), 101.0 (s, C COO), 112.2 (s, C ), 115.6 (s, C ), 132.7 (s, C ), 135.5 (s, C ), 151.0
2
ipso
s, C N), 167.6 (s, (CO)O).
ipso
+
1
5
b: Yield: 0.94 g. White viscid solid. ESI-MS(+) m/z: 224.2 [M+H] . H NMR
3
(
400 MHz, DMSO-d ), δ (ppm): 2.85 (s, 3H, CH ), 3.27 (br, 4H, 2CH ), 5.41 (q, J(H/H) =
6
3
2
Ar
3
Ar
5
8
.4 Hz, 1H, CH), 6.63 (m, 1H, C H), 6.77 (d, J(H/H) = 8.2 Hz, 1H, C H), 7.43 (ddd, J(H/H) =
Ar
Ar
13
.6, 7.1, 1.7 Hz, 1H, C H), 8.07 (dd, J(H/H) = 8.0, 1.6 Hz, 1H, C H). C NMR (400 MHz,
DMSO-d ), δ (ppm): δ 30.2 (s, CH ), 39.8 (s, 2CH ), 68.1 (s, CH), 110.3 (s, C COO), 112.0 (s,
6
3
2
ipso
Ar
Ar
Ar
Ar
C ), 115.3 (s, C ), 133.1 (s, C ), 135.6 (s, C ), 151.1 (s, C N), 167.1 (s, (CO)O).
ipso
2
2
0
.3. Synthesis of the platinum(II) complexes, 6a-7b
.3.1. Synthesis of diiodidoplatinum(II) complexes, 6a and 6b. K [PtCl ] (100 mg,
2
4
.24 mmol) was dissolved in a small amount of water (2 mL) and a six-fold excess of potassium
iodide was added (240 mg, 1.45 mmol). The solution was then stirred for 40 min in the dark. The
corresponding ligand (5a: 75 mg, 0.24 mmol; 5b: 71 mg, 0.24 mmol) was dissolved in water
(1 mL) and triethylamine was added (67 µL, 0.48 mmol). This solution was added dropwise to
the K [PtCl ] solution and the mixture was stirred overnight. The precipitate was filtered off,
2
4
washed with water and dried in vacuum.
a·2H O: Yield: 138 mg (83%). Anal. Calc. (%) for C H I N O Pt (722.22): C, 19.96;
6
2
12 23 2
3
4
+
H, 3.21; N, 5.82. Found (%): C, 19.93; H, 3.26; N, 5.69. ESI-MS(+) m/z: 686.9 [M-2H O+H] .
2
1
H NMR (400 MHz, DMSO-d ), δ (ppm): 2.84 (s, 3H, CH ), 3.48 (br, 2H, CH ), 3.64 (br, 2H,
6
3
2

Ar
3
CH ), 4.35 (br, 2H, CH ), 4.47 (br, 2H, CH ), 6.57 (m, 1H, C H), 6.71 (d, J(H/H) = 8.6 Hz, 1H,
2
2
2
Ar
Ar
C H), 7.42 (ddd, J(H/H) = 8.5, 5.1, 1.5 Hz, 1H, C H), 7.98 (dd, J(H/H) = 8.0, 1.6 Hz, 1H,
Ar
13
C H). C NMR (400 MHz, DMSO-d ), δ (ppm): 29.3 (s, CH ), 43.8 (s, CH ), 45.7 (s, CH ),
6
3
2
2
Ar
Ar
Ar
5
5.2 (s, CH ), 60.8 (s, CH ), 108.8 (s, C N), 110.8 (s, C ), 114.0 (s, C ), 131.7 (s, C ), 135.0
2 2 ipso
Ar
195
(
s, C ), 151.5 (s, C N), 167.2 (s, (CO)O). Pt NMR (400 MHz, DMSO-d ), δ (ppm): -3326
ipso 6
(s, Pt).
6
b: Yield: 118.4 mg (70%). Anal. Calc. (%) for C H I N O Pt (672.16): C, 19.66; H,
11 17 2 3 2
+
1
2
.55; N, 6.25. Found (%): C, 19.87; H, 2.81; N, 5.81. ESI-MS(+) m/z: 695.0 [M+Na] . H NMR
(
400 MHz, DMSO-d ), δ (ppm): 2.75 (s, 3H, CH ), 3.19 (m, 4H, 2CH ), 5.30 (m, 1H, CH), 6.56
6
3
2
Ar
3
Ar
Ar
(
m, 1H, C H), 6.61 (d, J(H/H) = 8.4 Hz, 1H, C H), 7.28 (m 1H, C H), 7.52 (dd, J(H/H) = 7.8,
Ar
13
1
.5 Hz, 1H, C H). C NMR (400 MHz, DMSO-d ), δ (ppm): 29.3 (s, CH ), 42.5 (s, CH ), 50.4
6 3 2
Ar
Ar
Ar
(
s, CH ), 68.2 (s, CH), 109.5 (s, C COO), 110.4 (s, C ), 113.8 (s, C ), 128.3 (s, C ), 132.7 (s,
2 ipso
Ar
195
C ), 149.9 (s, C N), 169.4 (s, (CO)O). Pt NMR (400 MHz, DMSO-d ), δ (ppm): -3188 (s,
ipso
6
Pt).
2
.3.2. Synthesis of dichloridoplatinum(II) complexes, 7a and 7b. The corresponding
diiodidoplatinum(II) complex was suspended in water and silver nitrate was added to the
mixture. After stirring for 8 h the precipitated silver iodide was filtered off and potassium
chloride was added to the filtrate. The solution was stirred overnight, filtered, evaporated to one
third of original volume and left over several days at 4 °C. Obtained precipitate was filtered off,
washed with cold water and dried in vacuo.
7
a: Following was used: 6a (255 mg, 0.37 mmol), H O (6 mL), AgNO (126 mg,
2 3
0
.74 mmol), KCl (111 mg, 1.49 mmol). Yield: 80 mg (43%). Anal. Calc. (%) for
C H Cl N O Pt (503.28): C, 28.64; H, 3.81; N, 8.35. Found (%): C, 28.51; H, 3.97; N, 8.12.
1
2
19
2
3
2
+
1
ESI-MS(+) m/z: 526.3 [M+Na] . H NMR (400 MHz, DMSO-d ), δ (ppm): 2.83 (s, 3H, CH ),
6
3
3
.47 (br, 2H, CH ), 3.63 (br, 2H, CH ), 4.35 (br, 2H, CH ), 4.47 (br, 2H, CH ), 6.56 (m, 1H,
2 2 2 2
Ar
3
Ar
Ar
C H), 6.70 (d, J(H/H) = 8.6 Hz, 1H, C H), 7.41 (ddd, J(H/H) = 8.5, 5.1, 1.5 Hz, 1H, C H),
Ar
13
7
.99 (dd, J(H/H) = 8.0, 1.6 Hz, 1H, C H). C NMR (500 MHz, DMSO-d ), δ (ppm): 29.3 (s,
6
CH ), 45.3 (s, CH ), 49.7 (s, CH ), 54.4 (s, CH ), 60.8 (s, CH ), 108.8 (s, C COO), 110.9 (s,
3
2
2
2
2
ipso
Ar
Ar
Ar
Ar
C ), 114.1 (s, C ), 131.6 (s, C ), 135.0 (s, C ), 151.6 (s, C COO), 167.3 (s, (CO)O).
ipso
1
95
Pt NMR (400 MHz, DMSO-d ), δ (ppm): -2117 (s, Pt).
6

7
b: Following was used: 6b (150 mg, 0.22 mmol), H O (5 mL), AgNO (76 mg,
2 3
0
.45 mmol), KCl (66 mg, 0.89 mmol). Yield: 38 mg (35%). Anal. Calc. (%) for
C H Cl N O Pt (489.26): C, 27.00; H, 3.50; N, 8.59. Found (%): C, 26.89; H, 3.74; N, 8.43.
1
1
17
2
3
2
+
1
ESI-MS(+) m/z: 490.0 [M+H] . H NMR (400 MHz, DMSO-d ), δ (ppm): 2.76 (s, 3H, CH ),
6
3
Ar
3
3
.18 (m, 4H, 2CH ), 5.31 (m, 1H, CH), 6.55 (m, 1H, C H), 6.61 (d, J(H/H) = 8.4 Hz, 1H,
2
Ar
Ar
Ar
13
C H), 7.27 (m, 1H, C H), 7.51 (dd, J(H/H) = 7.8, 1.5 Hz, 1H, C H). C NMR (400 MHz,
DMSO-d ): δ 29.8 (s, CH ), 42.5 (s, CH ), 44.0 (s, CH ), 69.5 (s, CH), 109.4 (s, C COO),
6
3
2
2
ipso
Ar
Ar
Ar
Ar
1
11.1 (s, C ), 114.4 (s, C ), 133.3 (s, C ), 135.6 (s, C ), 152.2 (s, C N), 167.0 (s, (CO)O).
ipso
1
95
Pt NMR (400 MHz, DMSO-d ), δ (ppm): -2042 (s, Pt).
6
2
.4. X-ray structure determination
Crystals suitable for X-ray analysis were obtained from the mixture of 5a (7.5 mg, 0.025 mmol)
and Na [PtCl ] (10.0 mg, 0.024 mmol) in H O (10 mL) at room temperature over several weeks.
2
4
2
Intensity data were collected on a STOE IPDS 2 diffractometer at 200(2) K using graphite
monochromated Mo Kα radiation (λ = 0.71073 Å). A summary of the crystallographic data, the
data collection parameters, and the refinement parameters is given in table S1. Integrational
absorption correction was applied (T /Tmax: 0.06/0.17). The structures were solved by direct
min
2
methods with SHELXS-96 and refined using full-matrix least-squares routines against F with
SHELXL 2014/7 [38]. Non-hydrogen atoms were refined with anisotropic displacement
parameters. Hydrogens were placed in calculated positions with fixed displacement parameters
(riding model) and all of them were refined isotropically. CCDC 1502151 contains the
supplementary crystallographic data for this paper. These data can be obtained free of charge
from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
2
2
.5. In vitro studies
.5.1. Cell lines, culture conditions and preparation of drug solutions. Cell lines A2780,
A549 and HT-29 were included in these experiments trial. The cultures were maintained as
monolayer in RPMI 1640 (PAA Laboratories, Pasching, Germany) supplemented with 10% heat
inactivated fetal bovine serum (Biochrom AG, Berlin, Germany) and penicillin/streptomycin
(
PAA Laboratories) at 37 °C in a humidified atmosphere of 5% (v/v) CO . The stock solutions of
2
the investigated compounds were made in dimethylsulfoxide (DMSO, Sigma Aldrich)

immediately before use at a concentration of 10 mM, diluted by nutrient medium to various
working concentrations. The nutrient medium was RPMI-1640 (PAA Laboratories)
supplemented with 10% fetal bovine serum (Biochrom AG) and penicillin/streptomycin (PAA
Laboratories).
2
.5.2. Cytotoxicity assay. The cytotoxic activities of the platinum compounds were evaluated
using the sulforhodamine-B (SRB, Sigma Aldrich) micro-culture colorimetric assay [39]. In
short, exponentially growing cells were seeded into 96-well plates on day 0 at the appropriate
cell densities to prevent confluence of the cells during the period of experiment. After 24 h, the
cells were treated with several dilutions of the studied compounds for 96 h. The final
concentrations achieved in the treated wells were 0.1, 1.0, 10, 25, 50, 75, 100, 125 and 150 μM.
Each concentration was tested in three triplicates on each cell line. The final concentration of
DMSO solvent never exceeded 0.5%, which is non-toxic to the cells. The percentages of the
surviving cells relative to the untreated controls were determined 96 h after the beginning of the
drug exposure. After 96 h treatment, the supernatant medium from the 96-well plates was thrown
away and the cells were fixed with 10% TCA. For a thorough fixation, plates are now allowed to
stand at 4 °C for at least 2 h. After the fixation the cells were washed in a strip washer. The
washing was carried out four times with water using alternate dispensing and aspiration
procedures. The plates were then dyed with 100 µL of 0.4% SRB for 45 min. After dyeing, the
plates were washed to remove the excess of dye with 1% acetic acid and allowed to air dry
overnight. 100 µL of 10 mM Tris base solutions were added to each well of the plate and
absorbance was measured at 570 nm using a 96-well plate reader (Tecan Spectra, Crailsheim,
Germany). The IC value, defined as the concentration of the compound at which 50% cell
5
0
inhibition was observed, was estimated from the dose-response curves.
4
2
.5.3. Fluorescence microscopy. 1×10 cells of A2780 were seeded on eight chamber slides.
Following 24 h incubation, the medium was removed and the 10 µM and 100 µM concentrated
medium solutions of the studied compounds (5a, 5b, 6a, 6b, 7a and 7b) were added to the cells.
After 24 h, the supernatant was removed and the cells were washed with PBS, covered with a
cover slip and immediately examined as wet mounts with an Axioskop from Zeiss and
photographed with an AxioCam MRC camera.

3
3
. Results and discussion
.1. Synthesis and characterization of the ligands
The synthesis of the ligand precursors 5a is based on the route by Zobi et al. [36] But so far the
ligand was not used in the synthesis of platinum(II) complexes. The ethylenediamine and
propylenediamine derivatives that are used as starting materials contain free amino groups that
have to be protected (1a/1b → 2a/2b, scheme 1) to prevent their reaction with N-methylisatoic
anhydride in further reaction steps. Therefore, the Boc-protection group has been chosen due to
its easy implementation and cleavage. For the implementation of the Boc-group, the
corresponding amino alcohol is dissolved and treated with an excess of tert-butoxycarbonyl
anhydride for 24 h under stirring. To inactivate residual tert-butoxycarbonyl anhydride, the
mixture is acidified with acetic acid. The final product after extraction and evaporation of the
solvent still contains acetic acid but is used without further purification because the acetic acid is
neutralized in the following reaction step. The amount of residual acetic acid varies from 17.8
(2a) to 33.0% (2b). The reaction results in yields of 67 (2b) and 95% (2a). To implement the
fluorescent moiety N-methylisatoic anhydride was esterified with the hydroxyl groups of the
Boc-protected diamines (2a/2b → 3a/3b, scheme 1).
To obtain the esterified product, a solution of the corresponding Boc-protected amino
alcohol is treated with an excess of N-methylisatoic anhydride for 24 h under stirring. The crude
product, after evaporation of the solvent, was purified by centrifugally accelerated, radial, thin-
1
layer chromatography resulting in yields of 31 (3a) and 63% (3b). In H NMR spectra of 3a and
3
b methyl hydrogen atoms from isatoic moiety are found at 2.88 ppm, while corresponding
carbon atoms resonate at around 29.6 ppm. The integrated intensities of proton chemical shifts in
1
the H NMR spectra support structural assignments.
In order to enable the nitrogen atoms for coordination, the amino groups have to be
deprotected (3a/3b → 4a/4b, scheme 1). The cleavage of the Boc-group takes place under acidic
conditions but has to be free of water to prevent hydrolysis of the ester function. Therefore, the
corresponding Boc-protected amines are dissolved in dry methylene chloride and treated with an
excess of TFA for 48 h under stirring. Within this time, two phases separated. The phase with
1
3
higher density is collected and residual solvent is removed by evaporation. In the C NMR

spectra, chemical shifts of the carbon atoms of the residual TFA are observed meaning that all
products still contained TFA. Therefore, the yields were not determined.
It is essential to perform in vitro studies with the synthesized ligand precursors for
comparison with the complexes but TFA is toxic and, for that reason, ligand precursors
containing TFA should not be used in cell tests. To remove the residual TFA, the corresponding
compounds were dissolved in THF and precipitated as hydrochlorides by bubbling hydrogen
chloride through the solution (4a/4b → 5a/5b, scheme 1).
1
For all synthesized compounds (25), expected chemical shifts were found in H and
1
3
13
C NMR spectra. The resonances arising from the TFA could not be found in the C NMR
spectra for compounds 5a and 5b, confirming that the removal of TFA was successful. The
characteristic chemical shifts in the spectra of 5a and 5b for the protons of the methyl group of
1
the N-methylisatoic ester moiety in H NMR spectra can be found as singlets at 2.86 ppm (5a)
1
3
and 2.85 ppm (5b) [36]. Furthermore, in C NMR spectra, the resonances of the carbon atom of
the N-methyl group are located at 30.2 ppm for both 5a and 5b. The mass spectrometry analysis
+
(
ESI-MS) confirmed molecular ions [M+H] at 238.4 for 5a and 224.2 for 5b.
3
.2. Synthesis and characterization of the platinum(II) complexes
The dichloridoplatinum(II) complexes were synthesized analogously to the route for cisplatin,
described by Dhara (scheme 2) [40, 41]. Potassium tetrachloridoplatinate(II) is dissolved in
water and treated in the dark with potassium iodide to form the tetraiodidoplatinate(II). To form
complexes 6a and 6b, a triethylamine neutralized solution of the ligand in water was added
dropwise to the solution containing tetraiodidoplatinate(II). To prevent the formation of side-
products, the addition of ligand has to be done very slowly. The product precipitates readily as a
yellow to brown solid and can be filtered off. All steps are done in the dark to prevent possible
side reactions. The yields of the reaction vary between 70 (6b) and 83% (6a).
To obtain corresponding dichloridoplatinum(II) complexes, the diiodido complexes are
suspended in water and treated with silver nitrate forming precipitate of silver iodide and the
soluble diaqua platinum(II) complexes. Silver iodide was filtered off and by adding an excess of
sodium chloride, the dichlorido complexes were obtained after purification, as confirmed with
elemental analysis, in the yields of 35 (7b) and 43% (7a).

The formation of the diiodidoplatinum(II) complexes was confirmed by ESI-MS
+
+
measurements giving the [M+H] ion at 686.9 for 6a and the [M+Na] ion at 695.0 for 6b. In the
+
+
ESI-MS spectra of 7a and 7b, [M+Na] (526.3 m/z) and [M+H] ions (490.0 m/z), respectively,
could be detected. H, C and 195Pt NMR spectra confirmed the formation of 6a/6b and 7a/7b
1
13
1
with the expected chemical shifts. In H NMR spectra, the N-Me moiety of the platinum(II)
complexes was found around 2.85 ppm, similar to ligand precursors (5a and 5b). As expected by
κ2-N,N’-ethylenediamine/propylenediamine platinum(II) complexes [42, 43], coordination
induced shifts of hydrogen atoms from the CH groups (up to 0.31 ppm) were observed for
2
1
3
6
a-7b. Carbonyl carbon atoms in C NMR spectra resonate around 168 ppm, as for the ligand
precursors. In the 195Pt NMR spectra, only one chemical shift was observed at expected
resonances due to [Cl ,N ] (7a/7b) and [I ,N ] (6a/6b) donor sets [44]. Replacement of iodido
2
2
2
2
with chlorido ligands in the platinum(II) complexes (6a/6b7a/7b) induces changes on the
195Pt NMR shift which follows the predictable tendency moving to the lower field.
The fluorescence spectra of the ligand precursors (5a and 5b) exhibit intense fluorescence
with excitation and emission maxima at ca. 360 and 470 nm, respectively (figure 2). The
coordination of ligands to the platinum(II) center (6a-7b) does not induce any distinct shift in
excitation and emission. Moreover, platinum(II) complexes exhibit considerable and stable
fluorescence which will be used herein for uptake study by fluorescence microscopy.
Various crystallization approaches were performed in order to crystallize obtained
platinum(II) complexes. Only direct reaction between K [PtCl ] and 5a after several weeks
2
4
yielded a compound [5a–2Cl+H]2[PtCl ] (6H O) (figure 3) [45].
4 3
2
3
.3. In vitro cytotoxicity
The cytotoxicity of the six compounds 5a-7a and 5b-7b has been determined against three
different cell lines: A2780 (ovarian cancer), A549 (lung carcinoma) and HT-29 (colon cancer)
using sulforhodamine-B (SRB) micro-culture colorimetric assay. The resulting IC values
5
0
(concentration which inhibit 50% of cell growth inhibition) are shown in table 1. The
representative graphs of the survival of the cells in the presence of increasing concentrations of
5
a-7a and 5b-7b are shown in figure 4.
The ligand precursors 5a and 5b show the lowest activity (IC > 100 µM) against the
5
0
tested cell lines. The diiodidoplatinum(II) complexes 6a and 6b show higher activity than these

ligand precursors. As expected, the highest antitumor activity was observed when the cells were
treated with the dichloridoplatinum(II) complexes 7a and 7b. On all three tested cell lines, the
dichloridoplatinum(II) complexes 7a and 7b are the most active. The IC values were found
5
0
between 2.8 (7a against A2780) and 24.1 µM (7a against HT-29), except for 7b on the cell line
HT-29 where its activity is comparable to the activity of 6a and 6b against same cell line (IC ≈
5
0
1
00 µM). However, all the tested compounds expressed lower activity than cisplatin.
3
.4. In vitro fluorescence evaluation
The uptake of the ligand precursors and platinum(II) compounds, 5a-7a and 5b-7b, in the tumor
cells have been investigated by fluorescence microscopy. Therefore, A2780 tumor cells were
treated with 10 and 100 µM solution of each compound (figure 5). After 24 h of incubation, the
supernatant was removed and the cells were washed with PBS. The washing step guarantees that
the cells are free from compounds which were not taken from the cells and therewith avoiding an
interference with the fluorescence of the compounds inside the cells. Fluorescence microscopy
experiments revealed that 5a-7a and 5b-7b enter into the cells to a significant extent. Moreover,
it seems that the platinum(II) compounds as well as ligand precursors are mainly targeting
nuclei. Furthermore, a clear sign of apoptosis, fragmented rounded cells, upon treatment of
A2780 culture (figures 5 and S1) could be identified especially with platinum(II) complexes.
Consistent with cytotoxicity results, even with the higher concentration (100 µM), ligand
precursors 5a and 5b had a lower impact on cell death than appropriate platinum(II) complexes
in lower applied concentrations (10 µM).
4
. Conclusion
In this paper a novel fluorescent ligand precursor 5b as well as ethylenediamine
diiodidoplatinum(II) complex (6a), a fluorescent propylenediamine diiodidoplatinum(II)
complex (6b) and their corresponding dichlorido complexes (7a/7b) have been synthesized over
several reactions steps. The structures of the obtained compounds have been characterized by
1
13
elemental analysis, mass spectrometry, fluorescence spectrometry as well as H, C and
195Pt NMR spectroscopy. The cytotoxic activity of the ligand precursors (5a and 5b) and
platinum(II) complexes (6a-7b) has been investigated against A549, A2780 and HT-29 cancer
cell lines. It was shown that the ligand precursors (5a and 5b) are not as active as the

diiodidoplatinum(II) complexes (6a/6b) and that the dichloridoplatinum(II) complexes (7a and
b) are compounds with the highest activity. It is proven that the ligand precursors (5a and 5b)
7
and platinum(II) complexes (6a-7b) enter the tumor cells.
Supplementary material
Supplemental data for this article can be accessed on-line.
Acknowledgements
We thank Prof. Dr. S. Mijatović and Prof. Dr. D. Maksimović-Ivanić for helpful discussion.
Disclosure statement
No potential conflict of interest was reported by the authors.
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Figure captions
Figure 1. (a) Structures of some platinum(II) complexes clinically used. (b) Examples of some
fluorescence ligands.
Figure 2. Absorption (dotted lines) and emission spectra of compounds 5a-7b.
Figure 3. ORTEP presentation of cation and anion in [5a–2Cl+H]2[PtCl ] (6H O).
4 3
2
Figure 4. Dose-dependent cell response of the investigated cell lines treated with compounds
a-7b.
5
Figure 5. Fluorescence microphotographs of A2780 cells treated (24 h) with 10 or 100 µM of
compounds 5a-7b.
Table 1. IC values [µM] for tested ligand precursors 5a/5b and platinum(II)
5
0
complexes 6a–7b.
Compound
A2780
A549
HT-29
5
5
6
6
7
7
a
b
a
b
a
b
101.4 ± 1.3
121.8 ± 1.8
18.1 ± 1.0
35.6 ± 0.8
2.8 ± 0.3
>150
135.9 ± 3.7
144.4 ± 2.8
99.5 ± 1.6
105.6 ± 2.0
24.1 ± 4.0
102.1 ± 2.4
0.6 ± 0.1
>150
28.9 ± 3.5
51.6 ± 3.8
12.5 ± 1.1
12.1 ± 0.6
1.5 ± 0.1
3.7 ± 0.4
Cisplatin
0.6 ± 0.1