Building up PtII?Thiosemicarbazone?Lysine?sC18 Conjugates

Combining Chemistry and Biology

Accepted Article

Title: Building up Pt(II)-thiosemicarbazone-lysine-sC18 conjugates

Authors: Axel Klein, Alexander Haseloer, Tamara Lützenburg, Joss

Pepe Strache, Jörg Neudörfl, and Ines Neundorf

This manuscript has been accepted after peer review and appears as an

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of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemBioChem 10.1002/cbic.202000564

Link to VoR: https://doi.org/10.1002/cbic.202000564

01/2020

10.1002/cbic.202000564

ChemBioChem

Building up Pt(II)-thiosemicarbazone-lysine-sC18 conjugates

Alexander Haseloer,^[a]Tamara Lützenburg,^[b]Joss Pepe Strache,^[a]Jörg Neudörfl,^[c]Ines Neundorf,^[b],

and Axel Klein ^[a],

*

[a] Universität zu Köln, Department für Chemie, Institut für Anorganische Chemie, Greinstraße 6, D-50939

Köln, Germany.

[b] Universität zu Köln, Department für Chemie, Institut für Biochemie, Zülpicher Str. 47a, D-50674 Köln,

Germany.

[c] Universität zu Köln, Department für Chemie, Institut für Organische Chemie, Greinsstraße 4, D-50939

Köln, Germany.

*Correspondence: axel.klein@uni-koeln.de; Tel.: +49-221-470-4006;

ORCID: Alexander Haseloer 0000-0003-0480-7568, Tamara Lützenburg 0000-0003-0629-4022, Joss Pepe

Strache 0000-0001-7321-8375, Jörg Neudörfl 0000-0002-2918-0565, Ines Neundorf 0000-0001-6450-3991,

Axel Klein 0000-0003-0093-9619

Supplementary Information (SI) available: Figures showing crystal and molecular structures, cyclic

voltammograms, HPLC-ESI MS spectrometry and results of the antiproliferative activity, alongside

with tables containing structural data, NMR data, UV-vis absorption data, and electrochemical data.

See DOI: xxx

ChemBioChem accepted

Three

chiral

tridentate

N^N^S

coordinating

pyridine-carbaldehyde

(S)-N4-(α-

methylbenzyl)thiosemicarbazones (HTSCmB) were synthesised alongside with lysine-modified

derivatives. One of them was selected and covalently conjugated to the cell-penetrating peptide sC18

by solid phase peptide synthesis. The HTSCmB model ligands, the HTSCLp derivatives and the

peptide conjugate form rapidly and quantitatively, very stable Pt(II) chlorido complexes [Pt(TSC)Cl]

when reacted with K

2

PtCl in solution. From one TSCmB model complex and the peptide conjugate

4

complex the Pt(CN) derivatives were obtained through Cl^‒g CN^‒exchange. Ligands and complexes

were characterised using NMR, IR spectroscopy, HR-ESI-MS and single crystal XRD. Intriguingly, no

decrease in cell viability was observed when testing the biological activity of the lysine-tagged

HdpyTSCLp, its sC18 conjugate HdpyTSCL-sC18, or the PtCl and Pt(CN) conjugate complexes in

three different cell-lines. Thus, given the facile and effective preparation of such Pt-TSC-peptide

conjugates, these systems might pave the way for future use of late-stage labelling with Pt

radionuclides and application in nuclear medicine.

Keywords: Thiosemicarbazone; Platinum; cell penetrating peptide; conjugates; antiproliferative

Introduction

Thiosemicarbazones (TSC) have been studied in the last 70 years with regard to their use as versatile

ligands in coordination chemistry [1-17] with applications in transition metal catalysis,[1,4-6] and as

materials for biomedical imaging and treatment.[5-17] TSCs of heterocycles, especially pyridine, form

a vast group within the class of TSC. They can act as tridentate N^N^S chelate ligands forming stable

complexes with many metals [1-3] and many studies have shown their cytotoxic behaviour with and

without a coordinated metal ion.[14-20]

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Many Pt(II) complexes, the most prominent being cisplatin, exhibit high anti-proliferative

activity.[21-23] This holds also true for TSC and related Pt(II) complexes,[10-13,23-29] which affect

even cisplatin resistant cells. It is assumed that they act with high activity by a different mode of

action than cisplatin.[12,24,25]

However, although promising, metal complexes are generally only less developed as

pharmaceutics owing to their poor water solubility and limited cellular uptake.[30] One way to

overcome these issues is the combination of metal complexes with bioactive peptides with bioactive

peptides, which has gained considerable attention during the last decade.[31-41] In this context, so-

called cell penetrating peptides (CPPs) are promising tools to deliver metal complexes into various

types of cells.[41-49] For instance, the Pt(IV) analogue of oxaliplatin was successfully conjugated to the

CPP Tat(48-58) protein providing increased water solubility and bioavailability.[23,39,40]

Herein, we present the synthesis of three chiral (S)-N4-(α-methylbenzyl) thiosemicarbazone

(HTSCmB) protoligands (ligand precursors prior to deprotonation upon coordination) and their lysine

derivatives (HTSCL and HTSCLp). Moreover, the lysine-tagged di-2-pyridylketone TSC (HdpyTSCL)

was successfully coupled to the recently described cell-penetrating peptide sC18 (Gly-Leu-Arg-Lys-

Arg-Leu-Arg-Lys-Phe-Arg-Asn-Lys-Ile-Lys-Glu-Lys-NH ),[40-48] via solid phase peptide synthesis

2

(HTSCL-sC18; Scheme 1). We discuss the complex formation with Pt(II) and their detailed

characterisation using NMR, MS, UV-vis absorption spectroscopy, electrochemical methods and DFT

calculations. Finally, for selected compounds we present details about their stability in biological

conditions, and their anti-proliferative activity towards human cancer and non-cancerous cells.

Scheme 1. Compounds in this study and their nomenclature.

Results and Discussion

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Syntheses of the HTSCmB protoligands, lysine derivatives HTSCLp, and the conjugate HTSCL-

sC18

S-benzyl methylamine isothiocyanate was synthesised by reacting the amine with CS followed by a

2

tosylchloride mediated decomposition derived from a method reported by Wong et al.[50] (Scheme

2A, details in the Experimental Section in the Supporting Information, SI). Upon reaction with

hydrazine, the thiosemicarbazide was obtained in excellent yields (99%). The thiosemicarbazide was

then reacted with three different N-heterocyclic carbonyls (Scheme 2B) to yield the three HTSCmB

derivatives (analytical details and characterisation including single crystal XRD in the SI). The

HTSCmB molecules were soluble in solvents like THF, MeCN and MeOH but not in H O.

2

H

NH9 NH7

N

CH₃

R

Py3

Py6

HOAc

6

H₂N

H

im

1

5

N

CH₃

Py4

Py5

S

N

MeOH

2

u.s., 50 °C, 2 h

N

S

+

3

4

R

R = H

R = CH₃

R = Py

HfpyTSCmB

HapyTSCmB

HdpyTSCmB

O

N

(A)

(B)

Scheme 2. Multi-step synthesis of the (S)-N4-(α-methylbenzyl)thiosemicarbazone ligands (HTSCmB)

with NMR nomenclature.

L-Lysine was converted into t-butyl-(t-butoxycarbonyl)-L-lysine (Scheme 3) in four-steps in an overall

yield of 52% using using standard procedures (Scheme 3; details see SI). The amino function of the

boc-protected lysine was converted into the isothiocyanate which was then reacted with hydrazine

hydrate yielding the corresponding thiosemicarbazide. Condensation with the pyridine carbonyls

yielded the HTSCLp protoligands (Lp stands for protected lysine) in overall yields of 23, 36, and 37%,

respectively (Scheme 3).

Scheme 3. Synthesis of the protected lysine HTSCLp ligands (with NMR nomenclature).

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Then, the boc-protected 2-(2-pyridyl)thiosemicarbazone-lysine derivative HdpyTSCLp was prepared

for peptide coupling using a sequence of de-protection and re-protection steps (Scheme 4, details in

the SI). Meanwhile, sC18 was synthesised by solid-phase peptide synthesis (SPPS) as previously

described.[51] Subsequently, HdpyTSCL was coupled to the N-terminus of sC18 using a mixture of

HATU/DIPEA (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphate/N,N-

diisopropyl-ethyl-amine) as activating reagents. Finally, the resulting conjugate was cleaved from the

resin with concentrated trifluoroacetic acid and purified, yielding HdpyTSCL-sC18 in high purity of

>95% (Scheme 4). HdpyTSCL-sC18 was characterised using HPLC and ESI-MS(+) (Figure S1, SI).

Scheme 4. Synthesis of the HdpyTSCL-sC18 peptide conjugate from HdpyTSCLp (SPPS: solid phase

peptide synthesis).

Synthesis and characterisation of the Pt(II) complexes

In a next set of reactions, we let react all of the different protoligands HTSCmB and HTSCLp with

K2

PtCl using either organic solvents or aqueous solutions, respectively. Notably, the complexes

4

[Pt(TSC)Cl] in very short reaction times and excellent yields.

Syntheses and characterisation of the “model” complexes [Pt(fpyTSCmB)Cl], [Pt(apyTSCmB)Cl],

[Pt(dpyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)]. The three [Pt(TSCmB)Cl] complexes (Schemes 1 and 2)

were obtained from MeCN reaction mixtures in about 30 min with yields ranging from 93 to 97%. The

CN complex was synthesised in 81% yield after workup through mixing a MeCN solution of the Cl

derivative with an aqueous solution of KCN. NMR data and the crystal structure of the complexes

[Pt(apyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)] (two independent molecules in the unit cell) (Figure 1)

proved a square planar coordination of Pt(II) through the deprotonated TSC ligand (N^N^S^thiolate) and

a chlorido or cyanido coligand, respectively. The two experimental structures show C‒S bond lengths

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in the range of a single bond, whereas the C8‒N3 lengths lie in the typical range of a double bond

(Table 1) in line with already reported data and related structures.[10,24,25,27,29] A closer look at the

angles around Pt(II) reveal distorted planar coordination with almost perfect Cl/C21‒Pt‒N2 angles of

about 175 and 176°. However, the N1‒Pt‒N2 and N2‒Pt‒S bite angles of the five-membered chelates,

which are markedly smaller than 90°, resulted in S‒Pt‒N1 angles of 166° and 165°. Furthermore, trans

influence of the stronger CN^‒coligand compared with Cl^‒might be the reason for the elongated Pt‒N2

bond length of about 2.00 Å in [Pt(dpyTSCmB)(CN)] (Table S20) compared with 1.966(5) found for the

complex [Pt(apyTSCmB)Cl] (Table 1).

(A)

(B)

(C)

Figure 1. Molecular structures of [Pt(dpyTSCmB)(CN)] (A, one out of two independent molecules)

and [Pt(apyTSCmB)Cl] (B) from single crystal XRD; atoms shown with 50% probability and H atoms

omitted for clarity. DFT calculated molecular structure of [Pt(dpyTSCmB)Cl] (C) at B3LYP def2-TZVP

level (C,H,N,S,Cl) and B3LYP LANL2DZ level with ecp60 Hay & Wadt (Pt).[52,53]

The experimental and DFT calculated metrical parameters of [Pt(apyTSCmB)Cl] were essentially the

same (Table 1, and SI). Only the distances in the ligand backbone vary slightly between the

experimental and theoretical values. This might be the result of overestimation of the conjugation of

the TSC system. On the other hand the DFT optimised structure represents the geometry in the gas

phase, while the XRD data has been obtained from the crystalline solid in which bond lengths and

angles might be modified by crystal packing. From this we concluded that suitable basis-sets to model

the Pt complexes were found.

Table 1. Selected experimental and DFT-calculated bond lengths and angles of [Pt(apyTSCmB)Cl].^a

bond lengths / Å

Pt‒Cl

XRD ^a

DFT ^b

2.34

2.07

1.99

2.33

1.34

1.77

angles / °

N1‒Pt‒N2

N2‒Pt‒S

S‒Pt‒Cl

XRD ^a

DFT ^b

80.69

84.79

98.52

96.01

176.88

165.32

2.3173(2)

2.055(6)

1.966(5)

2.2623(2)

1.293(9)

1.391(8)

1.317(1)

1.775(8)

80.2(2)

Pt‒N1

85.75(2)

96.75(6)

97.35(2)

175.23(1)

166.05(1)

Pt‒N2

Pt‒S

Cl‒Pt‒N1

Cl‒Pt‒N2

N1‒Pt‒S

C10‒N2

N2‒N3

N3‒C8

C8‒S

Ʃ of angles Pt

360.05(3)

360.01

a

From single crystal XRD data measured at 100(2) K, solved in orthorhombic P 2

1

2

1

21; full

b

crystallographic and metric date in the SI.

DFT calculations on B3LYP def2-TZVP level (for

C,H,N,S,Cl) and B3LYP LANL2DZ level with ecp60 Hay & Wadt (for Pt).[51,52]

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Cyclic voltammograms (CV) of [Pt(dpyTSCmB)Cl] recorded in MeCN (Figure 2) showed two fully

reversible one-electron reduction waves at E1/2 = ‒1.6 V and ‒2.25 V, and a further irreversible wave at

E

pc = ‒2.9 V (not shown). On the anodic scan an irreversible oxidation is observed at Epa = 0.74 V. For

the apy and fpy derivatives very similar values were observed (Figure S16-S18, Table S24, SI). For the

[Pt(dpyTSCmB)(CN)] complex the oxidation is markedly anodically shifted (+0.23 V) compared with

the Cl derivative, while the reduction potential is only slightly shifted (+0.1 V) (Figure S18, Table S24,

SI). When taking a conversion factor of 0.62 V,[54] the two dpyTSCmB complexes are stable within a

range from 0.1 (0.33 for CN) to approx. ‒2.1 V vs. NHE. In conclusion, the complexes were stable in a

range of more than 2 V, giving hope for adequate redox stability in water and other biological

conditions.

2 µA

1

0

-1

E / V vs. ferrocene/ferrocenium

-2

Figure 2. Cyclic voltammetry of [Pt(dpyTSCmB)Cl] in 0.1 M n-Bu

4

NPF /MeCN solution at 100 mV/s

6

scan rate. Square wave voltammograms in black, cathodic CV waves in blue, anodic waves in red.

We have seen that for [Pt(dpyTSCmB)Cl] the DFT-calculated highest occupied molecular orbital

(HOMO) was located on the Pt(II) atom (dxy orbital) and on the thiolate S atom and also obtained a

minor contribution from the chlorido coligand (Figure 3). The electrochemical oxidation at 0.74 V can

thus be attributed to a Pt(II)/Pt(III) redox pair with contributions from a thiolate(S^‒)/thiyl-radical(S^•)

pair. This is completely consistent with the observed anodic shift when changing the weak Cl^‒

coligand for the strong CN^‒. The lowest unoccupied molecular orbital (LUMO) had essentially the

character of the lowest pyridine π* orbital extending into the TSC backbone (Figure 3, Figure S19, SI).

Thus, the first electrochemical reduction was directed into this orbital (ligand-centred).

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LUMO:

HOMO:

Figure 3. DFT-calculated composition of LUMO and HOMO for [Pt(dpyTSCmB)Cl] at B3LYP def2-

TZVP level (C,H,N,S,Cl) and B3LYP LANL2DZ level with ecp60 Hay & Wadt (Pt). Iso-surface level at

0.06.

The UV-vis absorption spectra of the four Pt(II) TSCmB complexes are all quite similar (Figure 4

and Figures S13 and S14, data in Table S23, SI). We assign the long-wavelength absorption to metal(d)-

to-ligand(π*) (MLCT) excited states based on our DFT calculations, while the UV bands were of π-π*

type and were also observed in the protoligands HTSCmB (Figures 4, S13 and S14, Table S23, SI).

25

[Pt(dpyTSCmB)Cl]

[Pt(dpyTSCmB)(CN)]

HdpyTSCmB

20

2

15

1

10

0

500

600

700

5

0

300

400

500

600

700

800

wavelength / nm

Figure 4. UV-vis absorption spectra of HdpyTSCmB (blue), [Pt(dpyTSCmB)Cl] (black), and

[Pt(dpyTSCmB)(CN)] (red) in MeCN.

When comparing complex [Pt(dpyTSCmB)(CN)] with the Cl derivative we recognised for the CN

complex a slight blue-shift for the long-wavelength MLCT band and the absorptions at around 400

and 320 nm received changes in intensity and resolution (Figure 4). We assigned these changes to the

stronger σ-donating and π-accepting CN ligand stabilising the metal-centred HOMO. Notably, this

data was consistent with the observed markedly higher oxidation and slightly higher reduction

potentials, as well as the DFT results.

Upon cathodic reduction of [Pt(dpyTSCmB)Cl] in 0.1 M n-Bu

4

NPF /MeCN solution, a narrow

6

unstructured EPR signal of approximate axial symmetry was observed in glassy frozen solution at 110

K (Figure 5 inset). The missing hyperfine splitting to the ¹⁹⁵Pt isotope (I = ½, 33.8 % nat. abundance)

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pointed to a negligible contribution of Pt orbitals to the singly occupied molecular orbital (SOMO).

The g and g values at 2.0066 and 2.0079 supported the ligand centred character.[54-58] At ambient T

‖

no signal was observed. Efforts to get EPR spectra of oxidised species were not successful.

Spectroelectrochemical UV-vis absorption measurements on cathodic scans showed the growth of

a strong and broad absorption band centred at 490 nm for the first reduction step obscuring the long-

wavelength MLCT band (Figure 5). Furthermore, the two π-π* absorption bands at 400 and 330 nm

moved to 380 and 260 nm, respectively. Importantly, this process is fully reversible; the spectrum of

the parent compound (green line in Figure 5) can be fully recovered upon anodic back-scan. The

spectroscopic changes are strongly indicative for a singly reduced pyridine moiety indicating a ligand-

centred reduction, and were thus, fully in line with our DFT calculations and related examples.[55-60]

field / G

3300

3350

3400

300

400

500 600

wavelength / nm

700

800

Figure 5. UV-vis absorption spectra recorded during the first reduction of [Pt(dpyTSCmB)Cl] in 0.1 M

n-Bu NPF /MeCN solution (0.05 V increments). Inset: X-band EPR spectrum recorded during the first

4

6

reduction of [Pt(dpyTSCmB)Cl] (cathodic reduction in 0.1 M n-Bu

4

NPF /MeCN solution at 298 K,

6

measured at 110 K).

The second reduction process shows the formation of new long-wavelength bands between 550 nm

and 800 nm as well as a hypochromic shift of the bands at 490 and 340 nm (Figure S20, SI) and turns

out to be not reversible on the timescale of this experiment (5-20 min).

Syntheses and characterisation of the complexes [Pt(fpyTSCLp)Cl], [Pt(apyTSCLp)Cl], and

[Pt(dpyTSCLp)Cl] containing protected lysin (Lp). The reaction of the boc-protected HTSCLp

protoligands and K

2

PtCl was carried out in a mixture of MeCN and water. After 2 h stirring at

4

ambient temperature and column chromatographic purification the three complexes were obtained in

75-87% yield. Good solubility of [Pt(apyTSCLp)Cl] in organic solvents allowed ¹⁹⁵Pt,¹H HMBC NMR

experiments showing several ^xJPt-H couplings between Pt and the TSC ligand backbone (Figure 6, Table

S22, SI). The complexes were not soluble in water.

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Figure 6. 300 MHz ¹⁹⁵Pt, ¹H HMBC NMR spectra of [Pt(apyTSCLp)Cl] in DMSO-d

.

6

Since the UV-vis absorption spectra (Figure S13, Table S23, SI) as well as the redox behaviour (Figure

S18, Table S24, SI) of the [Pt(TSCLp)Cl] complexes are very similar to those of the α-methyl benzyl

(TSCmB) derivatives, we supposed the same complex entity [Pt(N^N^S)Cl]. Moreover, we did not

observe any influence on the photophysical and electrochemical properties of the complexes when

changing the side residue from α-methyl benzyl to protected lysine.

Syntheses and characterisation of the Pt conjugate complexes [Pt(dpyTSCL-sC18)Cl] and

[Pt(dpyTSCL-sC18)(CN)]. The conjugate protoligand HdpyTSCL-sC18 was reacted with K

2

PtCl in

4

H2

O for 2 h at ambient T forming the complex [Pt(dpyTSCL-sC18)Cl]. The crude complex was

desalted using a C18ec cartridge (Chromafix C18ec, Macherey-Nagel), evaporated from solvent and

lyophilised. HPLC analysis and ESI-MS confirmed quantitative product formation, while unreacted

HdpyTSCL-sC18 was not observed (Figure 7).

Figure 7. HPLC analysis (left) and ESI-MS(+) analysis (right) of [Pt(dpyTSCL-sC18)Cl] after desalting.

Detected masses for [M+nH]ⁿ⁺are labelled in blue, the calculated mass for [Pt(dpyTSCL-sC18)Cl] is

2666.40 (found: 2666.26). Detected masses for [M‒Cl+nH]⁽ⁿ⁺¹⁾⁺are labelled in black representing the

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complex [Pt(dpyTSCL-sC18)]⁺without coordinated chloride. For [M‒Cl]⁺2631.43 were calculated,

2630.47 were found.

The ESI-MS analysis showed the target complex [Pt(dpyTSCL-sC18)Cl] alongside with the species

[Pt(dpyTSCL-sC18)]⁺having lost the chloride coligand. The UV-vis absorption spectrum of the

product proved the quantitative formation of a [Pt(N^N^S)Cl] complex as shown before for the other

TSC ligand derivatives (Figure S21, S13-S15, SI). Interestingly, changing from organic solvents to

water did not lead to alterations of the long-wavelength MLCT band energy. The observed energy

fitted well into the solvatochromic behaviour recorded for the [Pt(dpyTSCLp)Cl] complexes which is

in good correlation to the Reichardt E

Complex [Pt(dpyTSCL-sC18)(CN)] was synthesised in 2 h from KCN, HdpyTSCL-sC18 and

PtCl in quantitative yield. HPLC analysis and ESI-MS(+) of the purified complex showed

T

(30) solvent parameters[61] (Figure S22, SI).

K2

4

exclusively the product (Figure 8). In this case, the species [Pt(dpyTSCL-sC18)]⁺having lost the anionic

coligand was not observed. This observation underlined the stronger binding of the CN^‒coligand to

Pt compared to Cl^‒.

Figure 8

.

HPLC analysis and ESI-MS(+) analysis of [Pt(dpyTSCL-sC18)(CN)] conjugate after desalting.

O (incl. 0.1% trifluoroacetic

The sample was recorded using a linear gradient from 10-60% MeCN in H

2

acid) within 15 min. The identified molecular ions agree well with the calculated molecular weight

(calculated: 2657.44 g/mol; experimental: 2659.13 g/mol).

The stability of [Pt(dpyTSCL-sC18)Cl] and [Pt(dpyTSCL-sC18)(CN)] was tested for 72 h in fetal bovine

serum (FBS) containing medium using UV-vis absorption spectroscopy (Figure S23, SI). The long-

wavelength MLCT band of the complexes was used as an indicator for structural integrity. During the

first hours, a decay of about 2% of the [Pt(dpyTSCL-sC18)Cl] complex was observed, but no further

changes were detected (Figure S23, SI). Compared to this, a loss of 19% signal intensity was observed

for [Pt(dpyTSCL-sC18)(CN)] (Figure S23, SI).

Anti-proliferative activity

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Next, we investigated the anti-proliferative activity of several of our TSC ligands, as well as their Pt(II)

complexes (Figure 9). As control we included Dp44mT (di-2-pyridylketone-4,4,-dimethyl-3-

thiosemicarbazone, Figure S24, SI), which is structurally very similar to HdpyTSCmB,[19,20,62-65] the

We chose two different cancer cell lines, namely breast adenocarcinoma MCF-7 (Michigan Cancer

Foundation 7) and colorectal carcinoma HT-29 cells, since those have been already used in former

experiments, thus allowing for comparing the activity or our novel compounds with recent results

from our own studies and literature.[44-46,66-70] HEK-293 (embryonic kidney) cells were used as non-

cancerous control cell line (Figure 9).

HdpyTSCmB exhibited quite strong anti-proliferative activity against HT-29 cells, while MCF-7

cells were less harmed (Figure 9A, B). However, also HEK-293 cells were strongly affected (Figure 9C)

letting assume only less selectivity of this compound. Overall, the activity was in the range of Dp44mT

[72-75] and DpC (di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone).[18-20] Notably, the

Pt complexes [Pt(dpyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)] exhibited reduced activity compared with

the ligand alone. Only at high concentrations of 10-25 µM markedly reduced cell viability was

observed. Thereby it seemed that the CN complex was more active against HT-29 cells, while the Cl

derivative demonstrated higher activity against HEK cells. Interestingly, there was no difference

detectable when incubating the complexes with MCF-7 cells. Recently, it was hypothesised that TSC

molecules coordinate in the cell to redox-active metals such as Fe or Cu, and that these formed

complexes would lead to the production of reactive oxygen species, damaging the cell.[8,15,20,63-

65,71-75] In fact, this would explain also our observation of higher activity of the free ligands

compared with the Pt complexes. Obviously, Pt(II) is only a poor substitute to these redox-active

metals that are known to quickly react with H

2

O2 forming hydroxyl radicals in Fenton-type

reactions.[63-65,71-75]. Considering that we have measured high electrochemical stability for both Pt

model complexes [Pt(dpyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)], particularly against reduction, would

further substantiate our assumption.

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Figure 9. Anti-proliferative activity of HdpyTSCmB, [Pt(dpyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)]

against MCF-7 (A), HT-29 (B) and HEK-293 (C) cells after incubating them for 72 h with different

concentrations of the compounds. Anti-proliferative activity of sC18, HdpyTSCLp, HdpyTSCL-sC18,

[Pt(dpyTSCL-sC18)Cl], and [Pt(dpyTSCL-sC18)(CN)] against MCF-7 (D), HT-29 (E) and HEK-293 (F).

Cells treated with 70% EtOH served as a positive control. Data were normalised to untreated cells

(100% viability). Shown are mean ± SD values of three independent experiments, each performed in

triplicate.

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Interestingly, no or only minor decrease in cell viability was determined after incubating all other

compounds for 72 h with the three cell lines (Figure 9D, E, F). In fact, the non-complexed HdpyTSCL-

sC18 showed a significant anti-proliferative activity compared to the parent peptide sC18 when

applied at 25 µM to MCF-7 (p=0.0449) or HT-29 (p=0.0226) cells. Surprisingly, the ligand HdpyTSCLp

alone only exhibited significant toxicity towards HEK-293 cells (p=0.0058), also when at the highest

concentration studied. For the corresponding platinum complexes, exclusively [Pt(dpyTSCL-sC18)Cl]

was determined to significantly reduce the cell viability of HT-29 (p=0.0200) and HEK-293 (p=0.0409)

cells when added at 25 µM. That sC18 did not display cytotoxic effects agrees to already published

results and demonstrates again that sC18 is well-tolerated.[43,47,51]. For the other tested substances

including the HdpyTSCL-sC18 ligand and the complex [Pt(dpyTSCL-sC18)Cl], we found this more

surprising. However, since we have already demonstrated that sC18 is a highly efficient carrier for

various cargos, including also metal complexes,[42-48,51] we were very confident that the whole

conjugate entered the cells. The results let rather concluded that there might be a dramatic influence of

the linker structure that bridges the TSC with the carrier molecule (in this case sC18). This hypothesis

is probably proven by the observation that we did also not detect any activity of HdpyTSCLp being

structurally highly divers to recently reported toxic binuclear Pt(II)Cl complexes containing triazole-

bridged bisthiosemicarbazone ligands.[10] In addition, we have already seen similar effects when

coupling Rh(III) polypyridyl complexes to sC18.[44] Generally, the potency of such metal complexes is

often dependent on specific properties like DNA intercalation. For sC18 conjugates it is already

known that they are mainly taken up by endocytotic pathways, restricting efficient cytosolic release,

and thus, transfer of the cargo to its final target.[43,47] One way to overcome this limitation is to

include specific proteolytic cleavage sites within the ligand to peptide structure, e.g. for cathepsin B.

This protease is abundant in the lysosomes and may induce enhanced endosomal release.[42]

On the other side, the good tolerability of the novel Pt(II) conjugates paves the way for future

studies, in which we will use these and related Pt-TSC-peptide conjugates for late-stage radiolabelling

with Pt radionuclides , such as ¹⁸⁹Pt, ¹⁹¹Pt, ^193mPt, and ^195mPt that are very interesting candidates for

Auger-electron radionuclide therapy.[76-79] The advantage of this method is the emission of low

energy electrons which are able to ionise material in a very confined space, thus reducing radiation

damage to healthy cells.[80,81] Tagged by a ligand containing biological information, the radionuclide

can be transported to a desired location or cell, thus focussing the decay and cell damage in a certain

area (targeted radiotherapy). The facile, rapid and stable binding of Pt(II) from a simple source such as

K2

PtCl

4

together with the observed high stability and virtual non-toxicity make our systems very

suitable for late-stage labelling.

13

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Conclusions

L-lysine-based thiosemicarbazone (TSC) ligands were successfully conjugated to the cell-penetrating

peptide sC18, using standard solid phase peptide synthesis. The overall synthesis yield for the peptide

conjugate is 10% over 10 steps. The tridentate N^N^S binding site in of the pyridine-based TSC bound

rapidly and quantitatively to Pt(II) forming Pt chlorido complexes of the type [Pt(TSC)Cl]. This was

shown for dpyTSCL-sC18 as well as for model compounds with N4-(α-methylbenzyl) termination

(TSCmB) or protected L-lysinate (TSCLp). Increasing the terminating chain from dpyTSCmB to

dpyTSCL-sC18 drastically enhanced their solubility. The sC18 conjugated Pt complexes [Pt(dpyTSCL-

sC18)X] with X = Cl or CN were soluble in solvents ranging from rather non-polar CH

2

Cl to water.

2

The uncoordinated HdpyTSCmB showed antiproliferative activity towards the human cancer cell

lines MCF-7 and HT-29 as well as the non-cancer cell line HEK-293. For the two non-conjugated Pt

complexes containing this ligand [Pt(dpyTSCmB)X] (X = Cl or CN) the activity was markedly reduced

and only for the HEK cells IC50 values lower than 25 µM were found. Interestingly, none of the sC18-

conjugated compounds as well as HdpyTSCLp exhibited significant anti-proliferative activities.

Future studies will be directed to get unequivocal proof that the conjugates have entered the cells.

In summary, we highlighted these non-toxic and biologically very stable Pt(II) complexes as very

interesting candidates for a late stage labelling with the radioisotopes ¹⁸⁹Pt, ¹⁹¹Pt, ^193mPt, or ^195mPt.

Experimental Section

Materials and syntheses

The syntheses of the three HTSCmB protoligands and the boc-protected lysine derivatives HTSCLp

are outlined in detail in the Supporting Information (SI).

Synthesis of the Pt complexes [Pt(TSCmB)Cl] – general description

The protoligands HTSCmB and K

2

[PtCl ] were dissolved in MeCN and stirred for 2 h at ambient

4

temperature. The colour changes from yellow to dark red. Upon evaporation of the solvent a red solid

forms, which was isolated by filtration.

[Pt(fpyTSCmB)Cl]. Yield: 119.9 mg (0.23 mmol, 97%). C15

H

15ClN PtS (513.90) C: 35.06, H 2.94, N:

4

10.90, S: 6.24. Found C: 35.03, H: 3.02, N: 10.90, S 6.29%. ¹H NMR: (300 MHz, DMSO-d

6

) δ = 8.87 (d, 1H,

J = 8.0 Hz, NH), 8.70 (d, 1H, J = 5.6 Hz, ³JPtH = 27 Hz, HPy6), 8.35 (s, 1H, ³JPtH = 12 Hz Him), 8.13 (t, 1H, J =

7.7 Hz, HPy4), 7.70 (t, 1H, J = 7.9 Hz, HPy5), 7.33 (d, 5H, J = 5.9 Hz, H3,4,5), 7.25 (dd, 1H, J = 6.1, 2.1 Hz,

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ChemBioChem

HPy3), 5.06 (quint, 1H, J = 7.4 Hz, H1), 1.42 (d, 3H, J = 7.1 Hz, H6) ppm (nomenclature as in Scheme 2)

EI-MS(+) m/z = 514 [M]⁺, 478 [M-Cl]⁺.

[Pt(apyTSCmB)Cl]. Yield: 0.121 g (0.23 mmol, 96%). Anal. Calc. for C16

H

17ClN PtS (527.93) C: 36.40, H

4

3.25, N: 10.61, S: 6.07. Found C: 36.39, H: 3.24, N: 10.60, S 6.08%. ¹H NMR: (300 MHz, DMSO-d

6

) δ =

8.76 (m, 2H, NH7, ³JPtH = 16 Hz, HPy6), 8.16 (t, 1H, J = 7.9 Hz, HPy4), 7.74 (d, 1H, J = 8.1 Hz, HPy5), 7.68

(d, 1H, J = 6.8 Hz, HPy3), 7.41-7.29 (m, 4H, H3,4), 7.24 (m, 1H, H5), 5.13-4.93 (m, 1H, H1), 2.26 (d, 3H, J

= 6.9 Hz, CH3im), 1.43 (d, 3H, J = 7.0 Hz, H6) ppm. EI-MS(+) m/z = 528 [M]⁺, 492 [M-Cl]⁺.

[Pt(dpyTSCmB)Cl]. Yield: 0.131 g (0.22 mmol, 93%). Anal. Calc. for C²⁰

H

18ClN PtS (590.99) C: 40.65, H

5

3.07, N: 11.85, S: 5.42. Found C: 40.66, H: 3.08, N: 11.88, S 5.41 %. ¹H NMR: (300 MHz, DMSO-d

) δ =

6

3

9.02 (d, 1H, J = 7.2 Hz, NH7), 8.84 (d, 1H, J^HH= 5.0 Hz, JPtH = 27 Hz, HPy6), 8.72 (d, 1H, J = 4.9 Hz

HPy’6), 8.06 (d, 1H, J = 5.0 Hz, HPy4), 7.97 (t, 1H, J = 4.9 Hz, HPy’4), 7.70 (t, 1H, J = 7.4 Hz, HPy5), 7.60

(t, 1H, J = 7.5 Hz, HPy’5), 7.45 (d, 1H, J = 7.9 Hz, HPy3), 7.40-7.15 (m, 5H, H3, H4, H5), 7.36 (d, 1H, J =

7.9 Hz, HPy’3), 4.65 (quint, 1H, J = 6.9 Hz, H1), 1.32 (d, 3H, J = 7.0 Hz, 3H, H6) ppm. EI-MS(+) m/z =

591 [M]⁺, 555 [M-Cl]⁺.

Synthesis of [Pt(dpyTSCmB)(CN)]. 65 mg (0.11 mmol) of [Pt(dpyTSCmB)Cl] were dissolved in 5 mL

MeCN. 20 mg (0.3 mmol) KCN were dissolved in 1 mL H O of which 0.5 mL were added to the

2

complex solution. The mixture rapidly changed colour from a dark red to bright red. After 2 h at room

temperature, the solvent was removed and the crude mixture was purified by silica column

chromatography (100% EtOAc). A bright red/pinkish band was isolated containing the desired

complex. Yield: 46 mg (0.081 mmol, 81%). C21

H18

N

6

PtS (581.55). ¹H NMR (300 MHz, DMSO-d

) δ = 9.19

6

(d, 1H, J = 7.1 Hz, NH7), 8.85 (d, 1H, J = 5.4 Hz, ³JPtH = 35 Hz, HPy6), 8.77 (d, 1H, J = 4.9 Hz, HPy’6), 8.10

(t, 1H, J = 8.0 Hz, HPy4), 7.99 (t, 1H, J = 7.2 Hz, HPy’4), 7.66 (t, 1H, J = 7.7, 1.8 Hz, HPy5’), 7.59 (t, 1H,

J

HH = 7.8 Hz, HPy5), 7.49 (m, 1H, HPy’3), 7.44 (m, 1H, HPy3), 7.35-7.19 (m, 3H, H4,5), 7.04 (d, 2H, J =

8.1 Hz, H3), 4.77-4.62 (m, 1H, H1), 1.34 (d, 3H, J = 7.0 Hz, H6) ppm. EI-MS(+) m/z = 581 [M]⁺.

Synthesis of the Pt complexes [Pt(TSCLp)Cl] – general description

1 eq. of the ligand was dissolved in MeCN and added to a solution of 1 eq. K

2

[PtCl ]. The colour

4

changes from yellow to dark red. After 2 h the solvent was evaporated under reduced pressure and

the residue was purified by column chromatography.

[Pt(fpyTSCLp)Cl]. Yield: 127 mg (0.18 mmol, 75%). Anal. Calc. for C22

H

34ClN

5

O

4

PtS (695.13) C: 38.01,

(300 MHz, DMSO-d ) δ =

im), 8.30 (d, 1H, J = 4.5 Hz, HPy3),

1

.

6

H 4.93, N: 10.07, S: 4.61. Found C: 38.02, H: 4.95, N: 10.09, S 4.60 % H NMR:

3

8.72 (d, 1H,

HPy6), 8.40 (s, 1H

H

J = 5.3 Hz, JPtH = 24 Hz,

, JPtH = 12 Hz,

8.15 (t, 1H, J = 8.2 Hz, HPy4), 7.71 (t, 1H, J = 9.0 Hz, HPy5), 7.08 (d, 1H,

NH7), 5.07 (d, 1H, J = 8.3

J = 7.8 Hz,

Hz, NH), 3.75 (q, 2H, J = 6.7 Hz, H1), 3.65-3.55 (m, 1H, H5), 1.69-1.47 (m, 4H, H3,4), 1.39 (m, 20H,

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ChemBioChem

EI-MS(+) m/z = 695 [M]⁺, 659 [M-Cl]⁺, 465

H2,9,10,11,13,14,15) ppm (nomenclature as in Scheme 3).

[HfpyTSCLp]⁺.

[Pt(apyTSCLp)Cl]. Yield: 141 mg (0.2 mmol, 83%). Anal. Calc. for C23

H36ClN

5

O4

PtS (709.16) C: 38.95, H

1

(300 MHz, CDCl₃) δ =

5.12, N: 9.88, S: 4.52. Found C: 38.98, H: 5.12, N: 9.87, S 4.55 %. H NMR:

8.88 (d,

3

1H, J = 5.5 Hz, JPtH = 14 Hz, HPy6), 7.90 (t, 1H, J = 7.6 Hz, HPy4), 7.78 (d, 1H, , J = 9.0 Hz, HPy3), 7.69

(d, 1H , J = 6.7 Hz, HPy5), 7.07 (d, 1H, J = 8.3 Hz, NH7), 4.24-4.01 (m, 1H, H5), 3.47 (q, 2H, J = 6.7 Hz,

H1), 2.29 (s, 3H, CH

3

im), 1.86-1.52 (m, 6H, H2,3,4), 1.45 (s, 9H, H13,14,15), 1.43 (s, 9H, H9,10,11) ppm.

¹H/¹⁹⁵Pt-HMBC NMR: (300 MHz, CDCl

3

) δ = ‒3108 (³JPt-HPy6 = 14 Hz, JPt-HCH3 obscured, JPt-HPy4 = 21 Hz)

4

5

‒

1

ppm. HR-ESI-MS(+): m/z calculated [M+Na]⁺: 732.17167; found: 732.16964. FTIR-ATR: ν [cm ] 3853

(w), 3675 (m), 3424 (w), 2987 (s), 2901 (s), 2359 (w), 1715 (m), 1558 (w), 1507 (m), 1455 (m), 1406 (m),

1394 (m), 1380 (m), 1250 (m), 1230 (m), 1154 (m), 1075 (s), 1066 (s), 1056 (s), 1028 (m), 891 (w), 789 (w),

706 (w), 642 (w), 626 (w), 618 (m), 606 (m), 591 (w), 581 (w), 570 (w), 560 (w), 546 (w), 540 (w), 526 (w).

[Pt(dpyTSCLp)Cl]. Yield: 162 mg (0.21 mmol, 87%). Anal. Calc. for C²⁷

H37ClN

6

O4

PtS (772.22) C: 42.00,

1

(300 MHz, CDCl₃) δ =

H 4.83, N: 10.88, S: 4.15. Found C: 42.02, H: 4.82, N: 10.90, S 4.15 %. H NMR:

8.90 (d, 1H, J = 5.6 Hz, ³JPtH = 15 Hz, HPy6), 8.75 (d, 1H, J = 4.7 Hz, HPy'6), 8.08 (td, 1H, J = 7.9, 1.6 Hz,

HPy4), 7.98 (td, 1H, J = 7.8, 1.7 Hz, HPy'4), 7.85 (d, 1H, J = 7.9 Hz, HPy3), 7.73 (td, 1H, J = 7.9, 4.1 Hz,

HPy'5), 7.53 (m, 1H, HPy5), 7.43 (d, 1H, J = 8.1 Hz, HPy’3), 7.06 (d, 1H, J = 7.7 Hz, NH7), 4.03 (quint,

1H, J = 7.1 Hz, H5), 3.16 (d, 2H, J = 6.4 Hz, H1), 1.49 (m, 6H, J = 7.4 Hz, H2,3,4), 1.38 (d, 18H, J = 3.0 Hz,

H9,10,11,13,14,15) ppm.HR-ESI-MS(+): m/z calculated [M+Na]⁺: 795.18271; found: 795.18364.

Synthesis of HdpyTSCL-sC18. sC18 (Gly-Leu-Arg-Lys-Arg-Leu-Arg-Lys-Phe-Arg-Asn-Lys-Ile-Lys-

Glu-Lys-NH ) was synthesised as previously described.[51] HdpyTSCL (15 µmol, 2 eq) was coupled

2

manually to the resin using 2 eq HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate) and 2 eq DIPEA (N,N-diisopropylethylamine) in DMF for 2 h at r.t. The

resulting HdpyTSCL-sC18 conjugate was cleaved from the resin with trifluoroacetic

acid/triisopropylsilane/H

purified by preparative HPLC (column: Nucleodur C18ec; 100-5; Macherey-Nagel; solvent: 10-60%

MeCN in H O (incl. 0.1% TFA) over 45 min, 6.0 mL/min flow rate). The product was identified via

HPLC-ESI MS (column: Nucleodur C18ec; 100-5; Macherey-Nagel; gradient: 10-60% MeCN in H

2

O (95/2.5/2.5, v/v/v) and precipitated in ice cold diethyl ether. Then it was

2

O

(incl. 0.1 % formic acid) over 15 min; 0.6 mL/min flow rate). After purification a yield of 19.7 mg (8.08

µmol, 53.8%) was determined.

Synthesis of [Pt(dpyTSCL-sC18)Cl]. 500 mL 5 mM stock solution of HdpyTSCL-sC18 in H

2

O were

added to 250 µL of a freshly prepared 10 mM aqueous solution of K PtCl (41.5 mg in 10 mL) and

2

4

incubated for 2 h under shaking at r.t. The complex formation reaction was UV-vis monitored through

the absorption at 500 nm. The crude complex was desalted using a C18ec cartridge (Chromafix C18ec,

16

10.1002/cbic.202000564

ChemBioChem

Macherey-Nagel), solvent was evaporated and the red residue lyophilised. The product was analysed

by ESI-MS(+) (Figure 7 and Figure S2, SI) and UV-vis absorption spectroscopy (Figure S21, SI).

Synthesis of [Pt(dpyTSCL-sC18)(CN)]. 75 µL of a 10 mM aqueous KCN solution (6.52 mg in 10 mL)

were added to a 500 µL 1 mM stock solution of HdpyTSCL-sC18 and incubated under shaking for 1 h

at r.t. Then, 50 µL aqueous K

2

PtCl solution (4.15 mg in 1 mL) was added and the reaction mixture

4

incubated for 1 h at r.t. The complex formation was UV-vis monitored through the absorption at 500

nm. The crude complex was desalted using a C18ec cartridge (Chromafix C18ec, Macherey-Nagel),

solvent was evaporated and the red residue lyophilised. Figure 8 shows HPLC analysis and ESI-MS(+)

spectrum of [Pt(dpyTSCL-sC18)CN]. Final HPLC chromatogram was recorded using a linear gradient

from 10-60% MeCN in H O (incl. 0.1% trifluoroacetic acid) within 15 min (Nucleodur C18ec; 100-5

2

(Macherey-Nagel) column (1 mL/min flow rate)). UV-vis absorption spectroscopy in Figure S21, SI.

Instrumentation

NMR spectra were recorded on a Bruker Avance II 300 MHz spectrometer, using a triple resonance

n

1

¹H, BB inverse probe head. The unambiguous assignment of the H and ¹³C resonances was obtained

1

from 1H NOESY, H COSY, gradient selected H, ¹³C HSQC and H, ¹⁹⁵Pt HMBC experiments. All 2D

NMR experiments were performed using standard pulse sequences from the Bruker pulse program

library. Chemical shifts were relative to TMS (¹H, ¹³C) or H

2

PtCl (

¹⁹⁵Pt). UV-vis absorption spectra

6

were measured using a Varian 50 Scan UV-visible photometer. EPR spectra were recorded in the X-

band on a Bruker System ELEXSYS 500E equipped with a Bruker Variable Temperature Unit ER

4131VT (500 to 100 K); the g values were calibrated using a dpph sample. Electrochemical experiments

were carried out in 0.1 M n-Bu

4

NPF solutions using a three-electrode configuration (glassy carbon

6

working electrode, Pt counter electrode, Ag/AgCl pseudo reference) and an Autolab PGSTAT30

potentiostat and function generator. Experiments were run at a scan rate of 100 mV/s, at ambient

temperature and the ferrocene/ferrocenium couple served as internal reference. UV-vis

spectroelectrochemical measurements were performed with an optical transparent thin-layer

electrochemical (OTTLE) cell.[82,83] EI-MS(+) spectra were recorded on a Thermo Quest Finnigan

MAT 95 at 70 eV. HR-ESI-MS(+) spectra were measured at the Thermo Scientific LTQ Orbitrap XL

mass spectrometer via electron spray ionisation and a FTMS Analyzer. IR spectra were obtained on a

Si crystal Fourier-Transform spectrometer by Thermo Scientific (Nicolet 380 FT-IR).

Crystal structure determination

The measurements were performed at 293(2) K on IPDS II (STOE and Cie.) or at 100(2) K on a Bruker

Apex II, both using graphite-monochromatised Mo-Kα radiation (λ = 0.71073 Å). These structures

were solved by direct methods using SHELX-97 and WinGX (SHELXS-97)[84-86] and refined by full-

matrix least-squares techniques against F²(SHELXL-2017/1).[87,88] The numerical absorption

17

10.1002/cbic.202000564

ChemBioChem

corrections (X-RED V1.22; Stoe & Cie, 2001) were performed after optimising the crystal shapes using

X-SHAPE V1.06 (Stoe & Cie, 1999).[89,90] The non-hydrogen atoms were refined with anisotropic

displacement parameters. H atoms were included by using appropriate riding models. Further

measurements were performed on SC-XRD Bruker D8 Venture at 100(2) K in kappa geometry

equipped with a copper micro focus source and a Photon100 detector, or at the beamline P24 of the

synchrotron PETRA III at DESY in Hamburg, Germany. These structures were resolved using OLEX2

and refined by charge flipping.[91] CCDC: 1954934 (HfpyTSCmB), 1954950 (HapyTSCmB), 1954933

(HdpyTSCmB^.MeOH), 1954935 ([Pt(apyTSCmB)Cl]), and 2022743 ([Pt(dpyTSCmB)(CN)]), contain the

full

crystallographic

data.

These

data

can

be

obtained

free

of

charge

at

www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12

Union Road, Cambridge, CB2 1EZ UK. Fax: +44-1223-336-033; Email: deposit@ccdc.cam.ac.uk.

Quantumchemical DFT calculations

DFT-calculations on the geometries, were carried out on def-SV(P)[92]/B3LYP[93-95] level using the

TURBOMOLE[96] program package and TmoleX user interface.[97] HOMO and LUMO energies were

calculated on def2-TZVP[98]/B3LYP level for H, C, N, S, and Cl atoms and LANL2DZ[99]/B3LYP with

ecp60 Hay&Watt[52,53] for Pt.

Peptide conjugates

The cell-penetrating peptide was synthesised by automated solid phase peptide synthesis on a Syro I

peptide synthesiser (MultiSynTech). HPLC chromatograms were recorded on an Agilent 1260 HPLC

system (column: Nucleodur C18ec; 100-5; Macherey-Nagel; gradient: 10-60% MeCN in water (incl. 0.1

% formic acid) over 15 min; 0.6 mL/min flow rate). ESI-MS(+) spectra were recorded on a LTQ-XL

mass spectrometer via electron spray ionisation (Thermo Scientific). Data was evaluated with

XcaliburTM Software (Thermo Scientific).[100]

Antiproliferative activities

Antiproliferative activity was determined by photospectrometric measurements. The conversion of

resazurin (λ^ex= 550 nm) into the resorufin product (λex = 595 nm) was measured [101] on a Tecan

infinite M200 plate reader (Tecan Group AG). Data were normalised to untreated control cells were

(100% viability). Statistical analysis was performed via Mircosoft Excel T-test with 2 tails, type 2 (two-

sample, equal variances).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

18

10.1002/cbic.202000564

ChemBioChem

Prof. Dr. Mathias Wickleder and Dr. Daniel Werner, Institute for Inorganic Chemistry, Department of

Chemistry, University of Cologne are gratefully acknowledged for the beamline time at DESY. Prof.

Dr. Mathias Schäfer, Department of Chemistry, University of Cologne is thanked for HR-ESI-MS

measurements and Prof. Dr. Bernd Neumaier, Department of Chemistry, Institute for Nuclear

Chemistry, Cologne University Clinics, Institute of Radiochemistry, and Research Center Jülich for

financial support and consulting. We also like to thank the Regional Computing Center of the

University of Cologne (RRZK) for providing computing time on the DFG-funded High Performance

Computing (HPC) system CHEOPS as well as for the support. Tamara Lützenburg acknowledges

financial support by the Jürgen-Manchot Stiftung.

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