Novel platinum(ii) compounds with O,S bidentate ligands: Synthesis, characterization, antiproliferative properties and biomolecular interactions

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ARTICLE TYPE

Novel Platinum(II) Compounds with O,S Bidentate Ligands:

Synthesis, Characterization, Antiproliferative Properties and

Biomolecular Interactions

Carolin Mügge,^aRuiqi Liu,^aHelmar Görls,^aChiara Gabbiani,^bElena Michelucci,^cNadine Rüdiger,^d

5

Joachim H. Clement,^dLuigi Messori,*^eWolfgang Weigand*^a,f

Dedicated to Prof. Werner Uhl on the occasion of his 60th birthday.

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

DOI: 10.1039/b000000x

Cisplatin and its analogues are firstꢀline chemotherapeutic agents for the treatment of numerous human

¹⁰cancers. A major inconvenience in their clinical use is their strong tendency to link to sulfur compounds,

especially in kidneys, ultimately leading to severe nephrotoxicity. To overcome this drawback we

prepared a variety of platinum complexes with sulfur ligands and analyzed their biological profiles. Here,

a series of six platinum(II) compounds bearing a conserved O,S binding moiety have been synthesised

and characterized as experimental anticancer agents. The six compounds differ in the nature of the

15 O,S bidentate βꢀhydroxy dithiocinnamic acid ester ligand where both the substituents on the aromatic ring

and the length of the alkyl chain may be varied. The two remaining coordination positions at the squareꢀ

planar platinum(II) center are occupied by a chloride ion and a DMSO molecule. These novel platinum

compounds showed an acceptable solubility profile in mixed DMSO/buffer solutions and an appreciable

stability at physiological pH as judged from analysis of their timeꢀcourse UVꢀvisible absorption spectra.

20 Their antiꢀproliferative and proꢀapoptotic activities were tested against the Cisplatinꢀresistant lung cancer

cell line A549. Assays revealed significant effects of the sample drugs at low concentrations (in the

ꢀmolar range); initial structureꢀactivityꢀrelationships are proposed. The activity of the apoptosisꢀ

promoting protein caspase 3/7 was determined; results proved that these novel platinum compounds,

under the chosen experimental conditions, preferentially induce apoptosis over necrosis. Reactions with

25 the model proteins cytochrome c, lysozyme and albumin were studied by ESI MS and ICPꢀOES to gain

preliminary mechanistic information. The tested compounds turned out to metalate the mentioned

proteins to a large extent. In view of the obtained results these novel platinum complexes qualify

themselves as promising cytotoxic agents and merit, in our opinion, a deeper pharmacological evaluation

as prospective anticancer agents.

⁴⁵structureꢀactivityꢀrelationships (SARs) defining the requirements

that are considered essential for the activity of a “classical”

platinumꢀbased anticancer compound^2–5. A number of platinum

complexes have been synthesized based on these criteria; some of

them did even enter clinical trials. Amongst them, all (worldwide

50 or regionally) approved compounds, i.e. Cisplatin, Carboplatin

and Oxaliplatin, as well as Nedaplatin, Lobaplatin and

Heptaplatin, comply with these requirements. Others have

entered clinical trials⁶, e.g. Satraplatin^7,8or Picoplatin^9–11. One of

the central aspects of these SARs is the kinetics of metalꢀligand

55 exchange in the complexes. It has been discussed decades ago

that the kinetic inertness or lability of a complex is markedly

determined by the nature of the central ion (as well as by the

ligand)¹². In Pt(II) complexes, ligand exchange processes are

slow, i.e. generally occur within several hours¹³. This property

60 seems to establish their high anticancer activity, since the

30 Introduction

Since 1978, when it was first approved for clinical use,

Cisplatin is one of the few metalꢀbased anticancer agents

exhibiting “true” curative potential: cure rates beyond 90 % have

been reported for the treatment of testicular germ cell cancer¹.

35 Subsequent, tremendous efforts in research have led to a handful

of platinumꢀbased drugs that are in widespread clinical use today.

Nevertheless, only a quite limited number of solid tumors can

successfully be treated by platinum based drugs and severe doseꢀ

limiting side effects often occur during chemotherapy which have

40 not been overcome yet. Hence, the development of new

anticancer metalloꢀchemotherapeutics is still an important issue

of today’s research.

Efforts to understand the main aspects in the therapeutic

efficacy of Cisplatin have led to the formulation of some precise

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substitution rate is comparable to that of many cellular division

processes. Hence, an effective amount of the drug is able to reach

the biological target before it is defragmented¹⁴.

substances which might fulfill those requirements.

In this work, system based on O,Sꢀchelating

βꢀhydroxydithiocinnamic esters coordinated to Pt(II), also

including dimethyl sulfoxide (DMSO) as monodentate ligand, is

exploited. Investigations on related compounds have revealed

interesting results for Pt(II) complexes with a DMSO moiety,

60

a

Based on these arguments a new, more general set of structureꢀ

activityꢀrelationships can be generated for the development of

new platinumꢀbased anticancer agents¹⁵.

5

10

15

20

25

ꢀ

Kinetically labile, i.e. fast hydrolyzing complexes can bind to ⁶⁵especially with respect to their nucleoside binding capacities^32–34

biomolecules available in the blood (e.g. sulfurꢀcontaining

amino acids, peptides, proteins). This fast metabolization is

believed to foremost cause severe systemic toxicity and

hinders the substances from significantly participating in the

anticancer process.

Kinetically inert, waterꢀsoluble platinum complexes

hydrolyze slower. Due to their longer persistence in the blood

and good excretion, they are expected to have less side effects

but also lower tumorꢀinhibiting properties. Interaction via the

kidneys might take place to some extent.

Kinetically inert, lipophilic platinum complexes are

distributed into the tissue rapidly and cleared from the blood

quite fast. As a consequence, fewer PtꢀS interactions can

occur. Renal excretion would be reduced and some typical

side effects might be absent.

Consequently, the leaving ligand in platinum complexes is

the one that essentially influences the biodistribution as well

as toxic side effects.

(Fig. 1). The cytotoxicity of a platinum(II) complex with an

O,O’ꢀchelated acetylacetonate and DMSO was recently

demonstrated to be higher than Cisplatin³⁵. There is evidence

indicating that this type of compounds has great affinity towards

⁷⁰biological sulfur donors and is active against MCFꢀ7 breast

cancer cells not via DNA attack, but through the soꢀcalled

mitochondrial pathway, thus providing a new approach in cancer

ꢀ

therapy^36,37

.

75 Fig. 1 Platinum(II) complexes bearing DMSO as ligand and interesting in

vitro properties^32,33,35

.

ꢀ

The aim of this work is to derivatize the chelating system of

βꢀhydroxydithiocinnamic esters in order to gain more insight into

its potential as antitumor agent (Fig. 2). We expect the chelating

⁸⁰subunit to coordinate tightly towards the platinum(II) center

whilst keeping the DMSO and chlorido ligands labile as much as

necessary for substitution against desired biological molecules,

yet retaining them with the Pt(II) center enough to prevent

unforeseeable reactions with all available ligands in the biological

⁸⁵milieu. The nature of the O,S chelating unit can be varied in wide

ranges. Here we present the first examples of such derivatization

by varying the length of the alkylic chain and by introducing a

bromo substituent to the aromatic ring. Both bear the rationale of

tuning hydroꢀ/lipophilicity of the compounds and, in the latter

⁹⁰case, to allow for future modification of the core system. We

attempt the inference of primary structureꢀactivity relationships

making further variations possible in a wellꢀplanned way. The

reactivity towards established model proteins shall give a primary

insight into their possible mode of action.

Finally, the nonꢀleaving ligand, might it be an amine or not,

controls the anticancer properties by forming structurally

different adducts with the proposed final target, i.e. DNA¹⁶.

³⁰It is nowadays established that Cisplatin, once infused into the

bloodstream, rapidly diffuses into tissues. A large amount of the

drug reacts with plasma proteins, especially with those containing

sulfur binding sites¹⁷. Platinum affinity to sulfur is high; when

taking into account the HSAB (hard and soft acids and bases)

³⁵concept: both platinum(II) and sulfur are concerned to be “soft”

and are therefore expected to form stable bonds¹⁸. A variety of

sulfur containing biomolecules is available in the blood and

cytoplasm, e.g. methionine, cysteine, gluthathione (GSH),

metallothionein (MT), albumin (HSA) and many more. In recent

⁴⁰years, various research groups have addressed the question of

protein binding and consequences for the activity of platinumꢀ

based drugs to a great extent (for recent reviews see e.g. refs^19–21).

Binding of Ptꢀbased drugs to proteins is known to have a major

impact on their activity. It is discussed that these reactions are not

45 only the source of severe toxic side effects and of resistance to

the drug. Moreover, some platinumꢀsulfur adducts could serve as

reservoir for the platination of DNA or play a crucial role in

detoxification processes^17,22,23; indeed, even Phaseꢀ1 clinical trials

have been conducted with CisplatinꢀHSA adducts²⁴. Overall,

50 platinumꢀsulfur interactions are kinetically preferred, but the

binding of platinum to guanineꢀN7 is believed to be

thermodynamically favored^17,25. This circumstance makes the

95

anticancer activity of platinum compounds possible at all^26–31

.

In spite of all the detailed insight that has been gained on

55 metallodrug activity and the consequent synthesis of new drug

candidates, it has not been possible to bring selective drugs with a

reconcilable set of side effects into clinical application. It is

therefore one of the major aims in cancer research to create

Fig. 2 Ligands 1ꢀ6 and synthesized complexes 7ꢀ12 as well as isolated

side products 13,14.

100

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The synthesis of βꢀhydroxydithiocinnamic ester derivatives has

already been described by several researchers^38–40, but a versatile

3400 resp. 3300 cm^ꢀ1for the hydroxyl group in the bromoꢀ

substituted and unsubstituted compounds, correspondingly. Both

“oneꢀpot” synthesis was not described until 1998. Since then, the ⁶⁰broad shape and position indicate the existence of hydrogen

procedure has been refined and wellꢀestablished^41–45. Applying

this method, the restriction to methyl esters is overcome: The

dithiolate anion can be reacted in situ with alkyl halides to form

the respective dithiocinnamic acid ester. Apart from that,

isolation of the intermediately formed dithiocinnamic acids is

possible and provides us with a “building block” system allowing

¹⁰a multitude of possible variations by subsequent conversion with

alkyl halides of great structural diversity. The nature of the arylic

substituent is also variable and can bear diverse substituents.

βꢀHydroxydithiocinnamic acid esters serve as monoanionic

ligands after deprotonation of the hydroxyl group. In this work, a

¹⁵series of Pt(II) complexes with one O,Sꢀchelating ligand and one

DMSO molecule as monodentate ligand was synthesized. DMSO

is known to coordinate towards Pt(II) using its sulfur atom as

neutral electron donor⁴⁶. The coordination sphere and charge are

saturated through one chlorido ligand.

bonds. Very strong absorption bands around 1590, 1560, and

1490 cm^ꢀ1are assigned to stretching vibrations of C=C and C=O,

whereas a very strong band at 1235 cm^ꢀ1belongs to the stretching

vibration of the C=S group.

5

65

The O,S-chelating platinum(II) complexes 7-12

A number of methods for the preparation of platinum(II)ꢀDMSO

complexes is known^47–49. Those compounds seem to play an

activating role in the course of synthesizing platinum complexes

⁷⁰with a variety of chelating ligands. Once coordinated to the Pt(II)

center, DMSO as ligand can be kept in the complex^50,32,51,52or be

substituted against a more stable ligand during the progression of

the reaction^53,54,35

.

A

new synthesis towards target compounds 7ꢀ12 was

⁷⁵developed (Scheme 1). Potassium tetrachloroplatinate (K₂PtCl₄)

was reacted with excess DMSO in a minimal amount of water.

The formation of a white precipitate indicated the formation of a

platinumꢀDMSO

complex

K_2ꢀx[PtCl_4ꢀx(DMSO)_x].

The

20 Results and Discussion

βꢀhydroxydithiocinnamic ester was deprotonated with sodium

⁸⁰hydride and the obtained sodium salt slowly added to the

platinum compound in slight deficit and stirred until TLC showed

no starting material. After workup through column

chromatography, the desired complexes 7ꢀ12 could be isolated in

moderate yields (10ꢀ 33 %; Fig. 2, middle). All compounds were

⁸⁵characterized by elemental analyses, multinuclear NMR and

FTIR spectroscopy as well as mass spectrometry.

Chemistry

Ligands: β-hydroxydithiocinnamic esters 1-6

For ligand synthesis, starting materials were either acetophenone

or 4ꢀbromoꢀacetophenone as enolate precursors. Methyl or ethyl

²⁵iodide and hexyl bromide served as alkylation reagents. The

deprotonation of the acetophenone derivative was achieved with

two molar equivalents of potassium tertꢀbuylate (KO^tBu) in

diethyl ether at ꢀ70 °C. Addition of carbon disulfide (CS₂) gave

the dithiolate anion, which was then reacted with one molar

³⁰equivalent of the desired alkyl halide. To ensure tautomerization

towards the enolic form, the reaction mixture was worked up with

an excess of diluted sulfuric acid. Column chromatography

afforded the pure products 1ꢀ6 with yields ranging from 10 % (for

3) to 72 % (for 2) (Fig. 2, left). All compounds were

³⁵characterized by NMR spectroscopy, mass spectrometry, FTIR

spectroscopy and elemental analyses.

¹H NMR spectroscopy confirmed the postulated cisꢀenolic

form of the compounds. Characteristic signals can be found at

6.9 ppm for the methine proton and at 15.1 ppm for the proton

40 belonging to the hydroxyl group (average values given). Since the

resonance for the hydroxyl group shows a strong downfield shift,

the formation of an intramolecular hydrogen bond can be

postulated and is in accordance with the literature⁴².

Further distinctive signals can be found in the ¹³C{¹H} NMR

45 spectra. The resonance signals for the thiocarbonyl carbon atoms

are located at fairly low field (217 ppm), signals for the βꢀoxo

carbon atoms appear at 168 ppm and 169 ppm for the pꢀbromo

substituted and the unsubstituted compounds, respectively.

Carbon signals for the methine groups are found at 108 ppm

50 (average values given).

DEI Mass spectrometric analyses allow the assignment of

characteristic fragmentation peaks. Either the [M]⁺or the [M+H]⁺

peak can be found for all compounds 1ꢀ6. Further characteristic

peaks are found at m/z = 243 and m/z = 163 belonging to ions

55 after cleavagef of the SꢀAlk group in the pꢀbromo substituted and

the unsubstituted compounds, respectively.

Scheme 1 Synthesis of O,Sꢀchelating Pt(II)ꢀDMSO complexes 7-12.

90 Conditions: i, 2eq. DMSO, H₂O, r.t.; ii, 1 eq. NaH, thf, r.t.; iii, 1.1:1, r.t.,

1ꢀ7d.

As unavoidable side product, the analogous bischelate was

formed for all complexations but could be separated from the

product via column chromatography. Two representative

95 compounds, 13 and 14, were isolated and characterized (Fig. 2,

right and ESI^†for syntheses and Xꢀray analyses).

¹H NMR spectra of compounds 7ꢀ12 are similar to the spectra

of their corresponding ligands. Major differences are loss of the

OH signal and the occurrence of an additional singlet at 3.6 ppm.

100 The latter can be assigned to the methyl groups of the DMSO

ligand, which constitutes a “marker” for the formation of

In the FTIR spectra, characteristic bands are detected around

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monochelates in contrast to the respective bischelates with almost

variable positions ꢀR and ꢀX might allow a distinct fine tuning of

identical NMR peak positions. Both the occurrence of platinum 60 the properties of the complexes with regard to transport and

3

satellites with J_PtꢀH≈ 23.6 Hz and a downfield shift of around

1 ppm compared to free DMSO⁵⁵give proof of the coordination

of DMSO to platinum. The values of chemical shift and coupling

constant for the DMSO protons are in good accordance with

solution properties without influencing the binding or release of

this ligand.

5

previously described PtꢀDMSO complexes^51,56

.

A

slight

downfield shift (≈ 0.18 ppm) can also be observed for the signal

of the methine proton. This is assigned to a deshielding effect of

¹⁰the coordinated metal ion due to good delocalization of electron

density in the sixꢀmembered chelating ring.

¹³C{¹H} NMR spectra of the complexes also show similarity

to the ligand spectra. Coordinated DMSO can be detected as one

signal at 46.9 ppm for all complexes. This is downfield shifted by

¹⁵6 ppm compared to free DMSO⁵⁵. Platinumꢀcarbon coupling can

2

be observed for the methyl group of DMSO with J_PtꢀC≈ 59 Hz.

These findings give further proof for the coordination of DMSO

towards the Pt(II) center. The characteristic signals of the methine

carbon and the βꢀoxoꢀcarbon are slightly downfield shifted, by

²⁰≈ 3.5 and ≈ 5 ppm, respectively. In contrast, the quarternary

thiocarbonyl carbon experiences a considerable upfield shift by

≈ 36 ppm to 181 ppm. The shift of those signals can be explained

through the coordination of the βꢀhydroxydithiocinnamic esters

towards the Pt(II) center. Oxygen atoms posses σꢀdonating

²⁵properties towards the Pt(II) center. This results in a deshielding

effect on the βꢀoxoꢀcarbon atom. In contrast, the sulfur atom can

act as an electron acceptor for πꢀback bonding from the

Pt(II) center; the increased electron density shields the

thiocarbonyl carbon. The overall delocalization of electron

³⁰density in the sixꢀmembered chelating ring causes the slight

deshielding effect on the methine carbon⁵⁷.

Mass spectrometric analyses gave the pseudo molecular peak

with matching isotope pattern for all complexes.

The FTIR spectra of compounds 7ꢀ12 show characteristic

³⁵absorption bands for the Ptꢀbound ligand. As expected, no OꢀH,

CꢀO or C=S stretching vibrations are found. One characteristic

broad band at ≈ 1146 cm^ꢀ1is assigned to the S=O stretching

vibration in coordinated DMSO. Compared to free DMSO, the

band shifted to the hypsochromic region of the spectrum by more

65

70

75

Fig. 3 Depiction of solidꢀstate molecular structures of 7 (top) and 11

(below). Thermal ellipsoids are given at the 50 % probability level and

hydrogen atoms are omitted for clarity.

⁴⁰than 40 cm^{ꢀ1 58,59}

.

Selected bond lengths [Å] and angles [°] for 7: PtꢀO1 2.013(5),

PtꢀS1 2.253(2), PtꢀS3 2.200(2), PtꢀCl 2.336(2), S3ꢀO2 1.457(6),

O1ꢀPtꢀS1 96.11(17), S1ꢀPtꢀS3 88.32(8), S3ꢀPtꢀCl 91.33(9),

O1ꢀPtꢀCl 84.21(17), S1ꢀPtꢀCl 177.94(9), O1ꢀPtꢀS3 175.49(17)

Selected bond lengths [Å] and angles [°] for 11: PtꢀO1 2.006(5),

PtꢀS1 2.249(2), PtꢀS3 2.186(2), PtꢀCl 2.3597(19), S3ꢀO2 1.472(6),

O1ꢀPtꢀS1 96.02(16), S1ꢀPtꢀS3 88.50(7), S3ꢀPtꢀCl 90.20(7),

O1ꢀPtꢀCl 85.28(16), S1ꢀPtꢀCl 178.47(7), O1ꢀPtꢀS3 175.39(16).

Through the spectroscopic analyses, it could be shown that the

DMSO ligand bears some peculiar properties when bound to the

platinum center. It is discussed that the SꢀPt bond has both

σꢀdonating and πꢀdonating character. Thus, most spectroscopic

45 phenomena can be explained by a prevailing S→Pt σ bond⁴⁹. By

donating electron density to platinum, the sulfurꢀoxygen bond in

DMSO is polarized. Consequently, the frequency of the SꢀO

vibration is increased in the IR spectra. The distribution of

electron density also affects the αꢀbound carbon atoms. Due to an

50 overall polarization effect and the donation of electrons towards

platinum, electron density is reduced on those carbon atoms and a

shift to lower fields can be detected for the respective carbon and

hydrogen signals. It is further perceptible that signals assigned to

the DMSO ligand have a constant position in all (¹³C{¹H},

55 ¹H NMR, IR) spectra of the different complexes. This means that

up to the degree applied, variation of the O,Sꢀchelating ligand has

very little influence on the binding properties of DMSO towards

the platinum center. As a consequence, derivatization at the

Molecular structures

It was possible to attain singleꢀcrystal Xꢀray structures from

80 complexes 7 and 11, as depicted in Fig. 3. In both cases, two

independent molecules were found in the structure. For

simplicity, only one molecule is represented. Selected bond

lengths and angles are given in Fig. 3 and ESI^†. The compounds

show the typical squareꢀplanar geometry of Pt(II) complexes and

85 are planar with the exception of the methyl groups of DMSO.

In compounds 7 and 11, the DMSO ligand is coordinated cis to

the sulfur atom S1 of the βꢀhydroxydithiocinnamic ester. As

4

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discussed previously, the donor atom is sulfur. The chloride anion

is coordinated trans to the sulfur atom S1 of the chelating ligand.

Bond lengths of the donor atoms to platinum decrease in the

of these compounds are characterized by three main bands

centered at 290, 360 and 420 nm, respectively, and do not show

great differences amongst one another. Representative spectral

order: PtꢀCl > PtꢀS1 > PtꢀS3 > PtꢀO1. The angle in the ⁴⁰data for 11 are shown in Fig. 4, more information is given in the

5

coordination sphere comes close to 90° in the case of S1ꢀPtꢀS3

and S3ꢀPtꢀCl, is smaller for O1ꢀPtꢀCl and larger for O1ꢀPtꢀS1.

The sulfur atom of the DMSO ligand is enclosed in a distorted

tetrahedral geometry due to the tilting of the oxygen atom

ESI^†.

To elucidate the best experimental conditions for further work

and to assess compounds’ stability in solution, spectra were

recorded over time spans of 24 h. Compounds proved to stay

towards the methyl groups. When compared to free DMSO, the ⁴⁵dissolved as 100 ꢁM solutions containing 50 % DMSO, and

¹⁰S3ꢀO2 bond length is shortened whereas it is elongated for the

S3ꢀC bonds⁶⁰. This is in good accordance with the spectroscopic

observations of these compounds. In the chelating ligand,

structural changes can be observed upon coordination as well.

partially dissolved in solutions containing 10 % DMSO.

Qualitative structureꢀsolubility dependence could be assessed and

revealed higher solubility for bromoꢀsubstituted compounds

compared to their unsubstituted counterparts with equal alkyl

⁵⁰substituent and increasing lipophilicity with elongation of the

chain.

At the given concentration, which is sufficient for activity

investigations, a DMSO content of 10 % is fully acceptable and

should not interfere with any activity of the target molecules (cyt

When compared to similar βꢀhydroxydithiocinnamic esters^42,61

,

15 the C7ꢀO1 bond is significantly shortened whereas all other

bonds belonging to the chelating unit are equal within the range

of standard deviation. The character of all four bonds of the

chelating unit lies between single and double bond.

To achieve squareꢀplanar coordination to the platinum center, ⁵⁵c, HEWL, BSA, GMP, vide infra) investigated. For in vitro

²⁰the angles in the chelating unit are widened. The angle PtꢀO1ꢀC7

is the widest (130.9(6)° in 7 and 130.5(5)° in 11) and the angle

PtꢀS1ꢀC9 the smallest (108.6(3)° in both structures). The angles

of the carbon skeleton are almost equal and therefore once again

affirm the delocalization of electrons in the sixꢀmembered

²⁵chelating ring.

screenings of antiproliferative properties, the drug concentrations

used are far lower and therefore do not require DMSO contents of

the tested extent.

Fig. 4 Solubility of 11 (100 ꢁM) in phosphate buffered solutions (pH 7.4)

30 over 24 h, depicted by UVꢀVis measurements. Top, 10 % DMSO, bottom,

50 % DMSO content. Spectra were recorded in 10 min intervals during

the first 60 min and hourly afterwards.

60

65

Fig. 5 UVꢀVisible absorption spectra of 11, recorded in DMSO. Top,

spectra of 11 after being kept in DMSO / PB 50% / 50% at r.t. after 0 h

(black), 24, 48 h(blue), 72 h (pink) and 96 h (green line). Bottom, spectra

of 11 recorded before (black) and after addition of 10 eq. AgNO₃(red

line).

Solution chemistry of the investigated compounds

The obtained compounds were dissolved in DMSO and their

35 solution behavior in buffered aqueous solutions was analyzed

through UVꢀvisible absorption spectroscopy. The UVꢀvis spectra

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From spectra inspection it emerges that the various compounds

manifest a substantial stability of their platinum(II) chromophores

with no evidence of major changes in the coordination of the

dependent cytotoxic assays on Pt(II) compounds by Muscella et

al. (

Fig. 1) had shown that indeed, IC₅₀values strongly depend on

bidentate ligand, even over a period of days – sufficiently long ⁵⁰drug incubation times (in this case 12ꢀ96 h), but the relative

5

for them to reach their biological target (Fig. 5, top). Some minor

alterations were noticed in the longꢀwave band of the DMSOꢀPB

solutions that may be ascribed to partial detachment of the weak

chlorido and DMSO ligands in the platinum(II) coordination

sphere.

To confirm this hypothesis, complete detachment of the

chlorido ligand was achieved by addition of excess AgNO₃and

gave a blueꢀshift of the longꢀwave band from 430 to 409 nm. This

indicates that exchange of the monodentate ligands mainly leads

behaviour of HeLa cells towards sample compounds and

Cisplatin was comparable over time, indicating that also short

exposure of drugs to cells can yield reliable information on the

drugs’ activity³⁵. Overall, the antiproliferative activity of the here

55 presented Pt(II) compounds 7ꢀ12 is evident and can be

corroborated by further investigations (vide infra).

Stock solutions were prepared in DMSO and diluted with PBS

to give sample solutions with the desired concentrations. To

establish the effect of remaining DMSO on the cells’ viability,

10

to shifts in this band. These results point to DMSO or chloride ⁶⁰control solutions with the respective DMSO content were also

¹⁵being released from the complex in aqueous solution and as part

of their biological mode of action°. Nevertheless, under the given

conditions the hydrolysis process seems to be slow and occurs

only to a small extent. Under different experimental conditions,

tested. It could be shown that at the given concentrations, the

cytotoxic effect of DMSO is very low and can thus be neglected

(Fig. 7). The use of DMSO as solvent for stock solutions is a

common practice in many medicinal and biological

e.g. varied pH or the presence of proteins or other potential ⁶⁵applications⁶³. Furthermore, excess DMSO in the liquid sample

²⁰ligands, the process might indeed be much faster.

can prevent dissociation of the compounds so that application of

the intact molecule can be expected, as shown by UVꢀvisible

absorption spectroscopy, vide supra.

Fig. 6 IC₅₀values of the novel Pt(II) compounds and CDDP as reference

compound. At the given concentrations, the influence of DMSO from

70

75

25

stock solutions was neglectably low.

Fig. 7 Doseꢀresponse curve for A549 cells incubated with different drug

concentrations, determined with PrestoBlue assay. The doseꢀresponse

curve for DMSO is plotted to correspond to the amount of DMSO used to

prepare the respective drug sample; contents ranging from 2.5 % to

0.00025 % (v/v). Graphs represent the results of one out of three

independent experiments. Further results are given in the supporting

information.

Biology

Antiproliferative effects

The antiproliferative effects of the compounds under

30 investigation were evaluated against the Cisplatinꢀresistant lung

cancer cell line A549, using the resazurinꢀresurofinꢀbased

PrestoBlue® assay. The cellꢀpermeant blue dye resazurin is

virtually nonfluorescent. When added to the reducing

environment of viable cells, resazurin is reduced to red resurofin

35 and becomes highly fluorescent. The conversion is proportional

to the number of metabolically active cells and can therefore be

detected through fluorescence measurements⁶².

All drugs show a cytotoxicity profile with IC₅₀values

determined to be lower than those of Cisplatin (Fig. 6, Fig. 7 and

40 ESI^†). It is instructive to state that cells were incubated with drug

samples for 24 h to give us a first insight into the acute toxicity

and potential of the drug candidates. This fact can explain the

relatively “high” IC₅₀values determined for these complexes:

IC₅₀values strongly depend on the experimental conditions used

45 to determine cell viability and can therefore only be compared

with results from equal assay conditions. For example, timeꢀ

A general structure-activity relationship can roughly be

80 deduced from the tendency of the obtained IC₅₀values.

Elongation of the alkylic chain from methyl to hexyl seems to

increase the cytotoxic effect of the drug. To further investigate

this tendency, compounds with intermediate chain length are

subject of ongoing investigations. Whether the existence of

85 bromine at the aromatic ring has an effect on the drugs’ activity is

not directly deducible from the results presented here. These

findings are not surprising when considering the effects the

substituents have towards the physical properties of the molecule.

Substitution at the aromatic site – to the extent performed here –

90 does not alter the solution behavior or binding of the ligands

towards the Pt(II) center. In contrast, the length of the apolar

alkyl chain does influence the solution behavior in aqueous

solutions. Whilst the shortꢀchained compounds have appreciable

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water solubility, the hexyl chains increase the compounds’

remarkable. Hexyl compounds 9 and 12, giving low IC₅₀values,

show decreasing caspase activity, reaching almost zero from

2.5 mg/mL on. Their concentration dependence seems to be

reversed as compared to the expected results, i.e. increasing

⁵⁰caspase activation with higher drug concentration. We believe

that the drugs’ cytotoxicity in this range is so high that all cells

have undergone apoptosis at the time the assay was performed,

thus leading to actually decreasing caspase activity with rising

concentration. The incubation time is in fact 24 hours; the same

lipophilicity and they may therefore have an advantage in cell

proliferation. This might in last consequence improve transport to

possible targets, i.e. DNA or proteins inside the cell, but also

hamper administration via aqueous systems.

To assess whether the bischelates that were obtained as side

products would give potent drug candidates, one sample, i.e. 14,

was incubated under the same conditions. No significant

antiproliferative activity was noticed in this first experiment so

5

¹⁰that those compounds were excluded from further activity ⁵⁵time span applied for the viability assays. This thesis is further

investigations (data not shown).

corroborated by the fact that methyl substituted compounds 7 and

10 with higher IC₅₀values give rising apoptotic activity in the

low concentration range followed by an abrupt loss of caspase

activation at higher drug concentrations; roughly in the range of

⁶⁰the determined IC₅₀value.

Induction of Apoptosis

Microscopeꢀbased visual inspection of the cells after incubation

15 with drugs revealed a small and round appearance characteristic

for apoptotic cells instead of their natural, spindle shaped

adherent form (Fig. 8). To clearly establish whether the drugs’

toxicity leads to apoptosis, we measured caspase 3/7 activity as

landmark for the apoptotic pathway.

When considering caspase activity induced by CDDP and

DMSO, it is evident that they show a similar and roughly

constant level of caspase

3 activation, giving an overall

intermediate fluorescence intensity. This shows us that cells

⁶⁵incubated with DMSO or CDDP do not display concentrationꢀ

dependent but rather intrinsic, natural protein activation, just as

would be expected from results of the cell viability data.

20

Fig. 8 Microscopic evaluation of antiproliferative activity of Cisplatin

(left) and compound 9 (middle) at different final drug concentrations.

DMSO (right) was used as control and applied in the concentrations

Fig. 9 Activation of caspase 3/7 by Pt(II) compounds at different drug

70

concentrations. For the sake of overview, standard deviations are not

depicted but given in the ESI^†.

25

corresponding to the amounts used for respective Pt(II) samples. Only

compound 9 shows clear antiproliferative properties and a considerable

amount of cells have the typical morphology of apoptotic bodies. Pictures

were taken after 24 h incubation at 37 °C, percentages given resemble

estimated proportions of living cells upon visual evaluation.

Mechanistic studies

Reactions with model proteins

75 The investigational platinum compounds were reacted with

model proteins, hen egg white lysozyme (HEWL), horse heart

cytochrome c (cyt c), and bovine serum albumin (BSA). The

resulting samples were analyzed by ESI mass spectrometry in the

first two cases and by ICPꢀOES in the latter case in order to gain

80 more precise mechanistic information.

These model proteins were chosen on the basis of several

rationales: Lysozyme is a small protein wellꢀsuitable for ESI MS

analytic invenstigation, since ionization occurs well due to the

presence of several positively charged groups at its surface. Only

85 one possible binding site, His 15, is located at the surface of the

protein (two Met sites, Met 12 and Met 105, are difficult to

access)²⁹. Thus, a straightꢀforward interaction pattern is expected

in ESI MS spectra of drugꢀincubated proteins. Similar factors

count for cytochrome c, i.e. its small size and the outcome of

90 wellꢀresolved ESI MS spectra as well as few “free” binding sites

at the protein surface, i.e. His 18, His 26, His 33, Met 65 or Met

30

Caspases are a family of proteases that play a key role in

apoptosis. They are abundant in cells as precursor forms and are

activated through apoptotic and death signal cascades to form the

mature enzyme. Up to now, there has been no evidence of

³⁵caspases taking part in necrosis, so that measuring the activation

of caspases can be considered a strong indicator for apoptotic

behavior of cells⁶⁴. Caspase 3 is a central enzyme of the apoptotic

pathway induced by chemotherapeutics^65,66. Its activation can be

measured with the substrate ZꢀDEVDꢀrhodamine 110, which by

40 activated caspase 3 (or 7) is cleaved at the DEVD site and

converted to the green fluorescent rhodamine 110⁶⁷.

Results of the assay, conducted exemplarily with methyl and

hexyl compounds, are shown in Fig. 9 and in the ESI^†. At first

glance, results might seem inconclusive, but in light of the results

45 obtained in viability assays, we believe the results are quite

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80^20,28. Cys 14 and Cys 17, which bind the heme unit, should not

35

be available for platinum binding. The proteins’ role as electronꢀ

carrier with important function in the apoptotic process makes it

even more of interest for the hereꢀpresented investigations. Both

proteins have been wellꢀinvestigated in view of their interaction

It is noteworthy to state that there seems to be a structureꢀ

dependence of the amount and intensity of complexꢀprotein

interaction. Based on the relative peak intensities and the

stoichiometries found in the deconvoluted ESI MS spectra, shortꢀ

5

with platinumꢀbased drugs and some very interesting results have ⁴⁰chained complexes, i.e. 7 and 10, give higher adduct abundancies

been reported^68,69

Serum Proteins such as albumin are amongst the most

abundant proteins a drug encounters when infused into the blood

.

and metal:protein ratios than longꢀchained compounds (9 and 12)

do (for comparative graphs cf. the ESI^†). Mass spectra are indeed

not eligible for quantitative evaluation, but the peak intensity can

be considered as an indicator.

To study the interaction of the platinum compounds with BSA,

the model protein was incubated in a 1:1 ratio with the complexes

under investigation for 96 h at 37 °C. After ultracentrifugation to

remove unbound platinum complex, ICPꢀOES measurements

were conducted to determine the relative Platinum content of the

⁵⁰solutions containing BSA and the washing solutions. Results are

reported in Fig. 10. It is evident that practically complete

conversion has taken place for all compounds, since less than 1 %

of platinum is found in the washing solutions.

¹⁰stream an can bind a number of drugs¹⁹. It can either reduce the

concentration of free drug in the molecule or serve as

“chaperone”^70,71; various binding sites of CDDP with HSA have

been reported ^72–75. It is a rather large protein and therefore

unfavorable for wellꢀresolved ESI MSꢀbased evaluation of the

15 whole protein. Hence, ICPꢀOES can give quantitative feedback

on metalꢀprotein interactions.

45

For cyt c and HEWL interaction, samples were incubated with

the protein in a 3:1 metal:protein ratio for 72 h. Representative

ESI MS spectra are reported in Fig. 10 and the ESI^†. It is evident

20 that the described reactions lead to formation of appreciable

The strength of the formed Ptꢀprotein adducts has been

quantities of metallodrugꢀ protein adducts. The position of the ⁵⁵challenged against the very competitive sulfurꢀcontaining and

peaks allows us to determine the nature of the proteinꢀbound

metallic fragments. These correspond to the Pt(O,S) fragment

(O,S is hereby the complete O,Sꢀchelating ligand). These results

²⁵imply that adduct formation takes place through chloride and

ubiquituous molecule glutatione (GSH). As has been witnessed

by ICPꢀOES, almost no detachment of compounds 7 or 8 through

GSH occurs in the case of BSA adducts and only marginal

release of 7 is witnessed from cyt c, cf. Table 1. This finding

DMSO aquation and subsequent coordination of the resulting ⁶⁰might be considered as a hint that binding of this kind of platinum

aqua species to protein side chains. Typically Pt(O,S)/cyt c

stoichiometries of 1:1 and 2:1 are found, whilst predominantly

1:1 Pt(O,S)/HEWL adducts are witnessed. Overall, interaction

³⁰with cyt c seems to be more intense than is the case with HEWL.

complexes is not reversible, other than has been discussed for

Cisplatin and analogues ^76,21,77

.

Table 1. Upper part: Results of ICPꢀOES measurements of incubating

65 BSA with the Pt(II) compounds 7ꢀ12. Lower part: Results of proteinꢀGSH

detachment experiments of 7 and 8 bound to BSA resp. cyt c. UP

represents the samlple solution containing the protein with bound Pt(II)

complex. DOWN represents all washing solutions.

c (Pt) [ppb]

Compound

Ratio UP vs. total content [%]

UP

DOWN

7

8

9

10

11

12

14285

30395

42191

56594

18100

34572

556

167

281

92

165

104

146

136

487

588

98,84%

99,08%

99,78%

99,71%

99,43%

99,58%

7/cyt c + GSH

7/BSA + GSH

8/BSA + GSH

80,37%

98,02%

97,23%

24165

20625

70 Conclusion

After the successful synthesis of six βꢀhydroxydithiocinnamic

ester derivatives 1ꢀ6, they were coordinated to a platinum(II)

center in an O,Sꢀchelating manner together with chloride and

DMSO as monodentate ligands. Complexation yielded

75 compounds 7ꢀ12 in moderate yields. Structure determination was

possible for compound 7 and 11 as well as for the bischelates 13

and 14. Nearly squareꢀplanar coordination was found in all

compounds. In 7 and 11, the DMSO ligand is bound via the sulfur

atom, cis to the sulfur donor site at the βꢀhydroxydithiocinnamic

⁸⁰esters. In the structures of 13 and 14, cis coordination of the two

ligands is observed.

Fig. 10 Deconvoluted ESI MS spectra of cyt c (top) and HEWL (bottom)

incubated with complex 11 in a 3:1 Pt:protein ratio for 72 h.

As it was introduced earlier, the properties of both leaving

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and non-leaving groups in platinum compounds define the mode

of action toward biological targets. The DMSO ligand shows

nearly identical spectroscopic properties in all complexes

known procedures and distilled prior to use. Most starting

materials were purchased from common suppliers (ABCR, Acros,

Aldrich, Fluka and Merck). For column chromatography, Silica

synthesized, indicating that the PtꢀS(DMSO) bond is of constant ⁶⁰gel of the type VWR Kieselgel 60 was used.

5

strength. This bears the advantage that derivatization of the

βꢀhydroxydithiocinnamic ester derivatives has no or little effect

on the dissociation behavior of DMSO or chloride as leaving

groups. Thus, the properties of the βꢀhydroxydithiocinnamic

NMR-Spectra were recorded on a 200 MHz, 400 MHz or

600 MHz instrument Bruker AVANCE 200, AVANCE 400 or

Ultrashield+ AVANCE 600. Measurements took place at

200.13 MHz, 400.13 MHz resp 600.13 MHz (¹H), or 50.33 MHz,

esters can be distinctly fineꢀtuned to define e.g. the solubility and ⁶⁵100.63 MHz resp. 150.94 MHz (¹³C). The deuterated solvent

1

¹⁰activity of the target compounds. Together with the fact that

structurally modified compounds are easily accessible through

the “building block” system of synthons, the here presented novel

compounds bear potential for further developments.

served as an internal standard for H and ¹³C{¹H} NMR spectra.

Signal assignment was confirmed by H,H COSY, HMBC and

HSQC experiments. FT-IR spectra were measured in form of

pressed discs (KBr) on a 2000 FTꢂIRꢂSpectrometer by Perkinꢂ

Solution and hydrolysis experiments showed that these ⁷⁰Elmer. Mass spectra (FAB, ESI, DEI) were recorded on an

¹⁵substances are sufficiently soluble in aqueous buffered solutions.

Partial lipophilicity might account for their high cytotoxic profile,

since this is claimed to be one of the properties kinetically inert

compounds should have to show decreased activity towards

instrument SSQ 710 (Finnigen MAT) and MAT95XL. Ionization

was performed at 70 eV. Elemental analyses (C, H, S) were

carried out on

a LECO CHNSꢂ931 instrument. Halogen

determination was performed by titration. Melting points were

unfavourable side reactions (vide supra). Hydrolysis appears to ⁷⁵determined on a Stuart melting point SMP3 instrument and are

²⁰be slow under the conditions applied. Still, substantial

interaction with proteins was witnessed that can be considered a

landmark for a possible mode of action. Model proteins cyt c,

HEWL and BSA interacted with the novel compounds to a great

uncorrected.

Crystal structure determination. Single crystals were

obtained from CH₂Cl₂/pentane. Crystallographic data as well as

structure solution and refinement details are summarized in the

extent. Structureꢀdependence was observed in the form that ⁸⁰ESI^†. The intensity data were collected on a Nonius KappaCCD

²⁵compounds with short alkylic substituents gave more intense

interaction than their longꢀchained homologues. As has been

stated earlier, the role of the model proteins in the overall

anticancer process has not yet been fully elucidated. For the

diffractometer using graphiteꢀmonochromated MoꢀKα radiation.

Data were corrected for Lorentz effects, polarization effects and

for absorption effects ^78–80. The structures were solved by direct

methods (SHELXS⁸¹) and refined by fullꢀmatrix least squares

platinum complexes introduced in this work, reduced protein ⁸⁵techniques against Fo²(SHELXLꢀ97⁸¹). All hydrogen atoms were

³⁰interaction might be a favourable trait. Overall, the compounds

revealed substantial in vitro activity towards the A549 lung

cancer cell line. The novel compounds showed significantly

lower IC₅₀values than the reference compound, Cisplatin. Hints

were found suggesting that elongation of the alkylic chain lead to

³⁵more cytotoxic agents. It was further assessed that the drug

candidates induce apoptosis in favour of necrosis, as determined

by caspase 3/7 activation assay and morphological inspection of

incubated cells.

Nevertheles, evaluation of these first structural and biological

⁴⁰results allows for the formulation of rough structure-activity

relationships of these compounds. Longer alkylic chains lead to

more hydrophobic complexes and give higher antiproliferative

activity compared to their shortꢀchained analogs. In a mammalian

system, this might reduce unfavourable protein interaction,

45 promote clearance from the blood stream and cell membrane

permeation; on the contrary, administration of nonꢀwaterꢀsoluble

drugs is in general not easy. Introduction of the electronꢀ

withdrawing bromo group does not significantly influence the

cytotoxic activity, yet enhances solubility in polar solvents and

50 might thus facilitate the compounds’ usage as anticancer active

compound.

included at calculated positions with fixed thermal parameters.

All nonꢀdisordered, nonꢀhydrogen atoms were refined

anisotropically⁸¹.

90 Syntheses

General Procedure 1: β-hydroxy dithiocinnamic alkyl esters

The acetophenone derivative (2 g, 1 eq.) was dissolved in diethyl

ether (40 mL) and transferred into a suspension of potassiumꢀtertꢀ

butoxylate (KO^tBu, 2 eq.) in diethyl ether (40 mL) at ꢀ78 °C.

95 Carbon disulfide (CS₂, 1.4 eq.) was slowly dropped into the

solution and the mixture stirred at ꢀ78 °C for 3 hours and then

warmed up to room temperature. The alkyl halide (1 eq.) was

added and the mixture stirred in darkness at room temperature for

further 15 hours. For workup, the suspension was acidified with

100 sulfuric acid (50 mL 2 M solution in water) and the evolving twoꢀ

phased system separated. The aquatic phase was extracted with

dichloromethane (3 x 5 mL) and the combined organic solutions

washed with water (3 x 5 mL), followed by drying with sodium

sulfate. After filtration, the crude product was purified by column

105 chromatography on silica gel and dried under vacuum.

Ligands 1ꢀ6 were prepared according to general procedure 1

and are described in detail in the ESI^†(2,5, and 6) or have been

reported earlier (1³⁸, 3⁴⁵, and 4³⁹).

Experimental Section

110 General Procedure 2: Platinum(II) complexes with O,S-

chelating β-hydroxydithiocinnamic esters, chlorido and

DMSO ligands

Materials and methods

55 All Syntheses were carried out under argon atmosphere using

conventional Schlenk technique. Solvents were dried according to

Sodium hydride (NaH, 17.5 mg 60 % sup. in mineral oil,

0.438 mmol, 1 eq.) was suspended in tetrahydrofuran (THF,

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5 mL) and transferred into

a

solution of the desired 60 hexane/dichloromethane 2:3 as eluent (R_f≈ 0.1). Removal of the

βꢀhydroxydithiocinnamic ester (0.438 mmol, 1 eq. in 15mL

THF). The formation of gas bubbles and intensification of the

ligand color indicated the deprotonation of the ligand. The

solution was stirred at room temperature for 30 minutes.

solvents and subsequent column chromatography with

dichloromethane/acetone 10:0.5 as mobile phase (R_f≈ 0.6)

afforded pure 8 (89 mg, 33 %) as orange powder.

1

5

M.p.: 152 °C (decomp.); H NMR (200 MHz, CDCl₃): δ = 1.42

3

Potassium tetrachloroplatinate (K₂PtCl₄, 200 mg, 0.482 mmol, ⁶⁵(t, 3H, CH₃), 3.24 (q, 2H, SCH₂), 3.63 (s w/ Pt satellites, J_Ptꢂ

1.1 eq.) was dissolved in degassed water (2 mL). After the

addition of dimethylsulfoxide (DMSO, 62.2 ꢀL, 0.876 mmol,

2 eq.), the reaction mixture was stirred at room temperature for

¹⁰30 minutes or until the precipitation of a white solid indicated the

formation of DMSOꢀplatinumꢀspecies.

The solution of the ligand sodium salt was then dropped into the

suspension of the platinum complex over the period of 1 hour.

The reaction mixture was stirred at room temperature under the

¹⁵exclusion of light for 3 to 7 days, until TLC control showed no

_H= 23.8 Hz, 6H, CH₃(DMSO)), 7.00 (s, 1H, CHCS₂), 7.50 (d,

3

³J_H2ꢂH3= 8.8 Hz, 2H, ArꢀH2), 7.79 (d, J_H2ꢂH3= 8.8 Hz, 2H, Arꢀ

H3); ¹³C{¹H} NMR (50 MHz, CDCl₃): δ = 13.1 (CH₃), 28.8 (8),

2

46.9 (s w/ Pt satellites, J_PtꢂC= 59.6 Hz, CH₃(DMSO)), 111.2

70 (CHCS₂), 126.9 (ArꢀC4), 129.4 (ArꢀC2), 132.0 (ArꢀC3), 136.0

(ArꢀC1), 173.1 (CO), 181.2 (CS₂); FTIR (KBr): ꢀꢁ = 3003, 2924,

1584, 1505, 1483, 1457, 1311, 1291, 1263, 1148, 1071, 1022,

1008, 832, 794 cm^ꢀ1; MS (DEI): m/z =612 [M+1], 610 [Mꢀ1]⁺;

elemental analysis calcd for C₁₃H₁₆BrClOPtS₃: C, 25.56; H,

starting material. Workup included removal of the solvents in 75 2.64; S, 15.75 %; found: C, 25.60; H, 2.53; S, 15.71 %.

vacuo, uptake of the remainder in a proper solvent, filtration of

the salts and column chromatography.

Chloro-(1-(4-bromophenyl)-3-(hexylthio)-3-thioxo-prop-1-en-

1-olate-O,S)-(dimethylsulfoxide-S)-platinum(II) (9)

²⁰Chloro-(1-(4-bromophenyl)-3-(methylthio)-3-thioxo-prop-1-

en-1-olate-O,S)-(dimethylsulfoxide-S)-platinum(II) (7)

Synthesis was performed from 1 according to general procedure

2, stirring was for 3 days. All solvents were evaporated to

dryness. The solid was extracted with acetone three times (2 x

²⁵20 mL at room temperature, 1 x 100 mL at reflux for 2 hours) and

⁸⁰According to general procedure 2, the synthesis was performed

from 3 by stirring for 3 days. THF and water were removed in

vacuo, the precipitate was dissolved in dichloromethane and

water. The aqueous phase was extracted with dichloromethane

(3 x 3 mL) and the combined organic solutions were dried with

filtered off each time. The remaining solid was identified as ⁸⁵sodium sulfate. The crude product was purified via column

bischelate. The solution was reduced in volume and purified via

column chromatography on silica gel (mobile phase:

hexane/dichloromethane 3:2, R_f≈ 0.1). The fraction which

30 included the product was crystallized from dichloromethane and

chromatography

on

silica

gel

(mobile

phase:

dichloromethane/hexane 3:2, R_f≈ 0.2). The fraction containing

the product was again purified via column chromatography on

silica gel using dichloromethane as eluent (R_f≈ 0.5). The product

pentane. The yellow needles were then again purified via column 90 was crystallized from dichloromethane/pentane and dried under

chromatography (mobile phase: dichloromethane, R_f≈ 0.5). The

pure product (30.1 mg, 9 % as orange powder) was dried in

vacuum. Single crystals were obtained by diffusion of pentane

vacuum to yield 9 (34 mg, 12 %) as orange powder.

Mp.: 150 °C (decomp.); H NMR (200 MHz, CDCl₃): δ = 0.88

(m, 3H, CH₃), 1.24ꢀ1.48 (m, 6H, 3x CH₂), 1.77 (qui, 2H,

SCH₂CH₂), 3.23 (t, 2H, SCH₂), 3.64 (s w/ Pt satellites, J_Ptꢂ

1

3

³⁵into a solution of the product in dichloromethane.

1

M.p.: 193 °C (decomp.); H NMR (200 MHz, CDCl₃): δ = 2.67 95 _H= 23.4 Hz, 6H, CH₃(DMSO)), 7.03 (s, 1H, CHCS₂), 7.52 (d,

3

(s, 3H, CH₃), 3.66 (s w/ Pt satellites, J_PtꢂH= 23.4 Hz, 6H,

³J_H2ꢂH3= 8.8 Hz, 2H, ArꢀH2), 7.81 (d, J_H2ꢂH3= 8.6 Hz, 2H, Arꢀ

3

CH₃(DMSO)), 7.05 (s, 1H, CHCS₂), 7.53 (d, J_H2ꢂH3= 8.6 Hz,

H3); ¹³C{¹H} NMR (50 MHz, CDCl₃): δ = 13.9 (CH₃), 22.4

(CH₂CH₃), 27.9 (CH₂CH₂CH₃), 28.5 (SCH₂CH₂CH₂), 31.2

(SCH₂CH₂), 34.5 (SCH₂), 46.9 (CH₃(DMSO)), 111.2 (CHCS₂),

3

2H, ArꢀH2), 7.73 (d, J_H2ꢂH3

=

8.6 Hz, 2H, ArꢀH3);

40 ¹³C{¹H} NMR (50 MHz, CDCl₃): δ = 17.7 (CH₃), 46.9 (s w/ Pt

satellites, J_PtꢂC= 58.3 Hz, CH₃(DMSO)), 111.1 (CHCS₂), 126.9 ¹⁰⁰126.0 (ArꢀC4), 129.4 (ArꢀC2), 132.0 (ArꢀC3), 136.0 (ArꢀC1),

2

(ArꢀC4), 129.4 (ArꢀC2), 132.0 (ArꢀC3), 135.9 (ArꢀC1), 172.9

(CO), 181.9 (CS₂); FTIR (KBr): ꢀꢁ = 3011, 2914, 1583, 1504,

1482, 1463, 1318, 1292, 1263, 1146, 1071, 1025, 1008, 833,

45 793 cm^ꢀ1, MS (DEI): m/z =598 [M+1], 596 [Mꢀ1]; MS (FAB in

172.9 (CO), 181.6 (CS₂); FTIR (KBr): ꢀꢁ = 3000, 2955, 2927,

2856, 1585, 1505, 1482, 1463, 1310, 1290, 1262, 1144, 1071,

1023, 1008, 833, 792 cm^ꢀ1; MS (FAB in nba): m/z =667 [M]⁺,

631

[MꢀCl+H]⁺;

elemental

analysis

calcd

for

nba): m/z =597 [M]⁺; elemental analysis calcd for 105 C₁₇H₂₄BrClOPtS₃: C,30.61, H, 3.63, S, 14.42 %, found: C,

C₁₂H₁₄BrClOPtS₃: C, 24.15; H, 2.36; S, 16.12 %; found: C,

30.77, H, 3.91, S, 13.94 %.

24.34; H, 2.56; S, 16.09 %.

Chloro-(1-phenyl-3-(methylthio)-3-thioxo-prop-1-en-1-olate-

O,S)-(dimethylsulfoxide-S)-platinum(II) (10)

¹¹⁰K₂PtCl₄was conversed with 4 according to general procedure 2

50 Chloro-(1-(4-bromophenyl)-3-(ethylthio)-3-thioxo-prop-1-en-

1-olate-O,S)-(dimethylsulfoxide-S)-platinum(II) (8)

Synthesis was performed from 2 according to general procedure

and stirred for 7 days. All solvents were evaporated, the solid

residue was dissolved in THF and the inorganic precipitates were

filtered off. The crude product was purified by column

chromatography on silica gel and hexane/dichloromethane 2:3 as

2. The solution was stirred for 7 days. All solvents were

evaporated and the solid residue was dissolved in 10 mL acetone.

55 The remaining precipitate was filtered off and dissolved in 10mL

dichloromethane, the solid residue filtered off. All solutions were 115 eluent (R_f≈ 0.1) and crystallized from dichloromethane/pentane.

combined, dried over sodium sulfate and after filtration, the

solvents were removed. The crude product was purified by

10 (62 mg, 27 %)was yielded as orange needles.

1

Mp.: 152 °C (decomp.); H NMR (200 MHz, CDCl₃): δ = 2.67

3

column

chromatography

on

silica

gel

with

(s, 3H, CH₃), 3.65 (s w/ Pt satellites, J_PtꢂH= 23.6 Hz, 6H,

10 | Journal Name, [year], [vol], 00–00

This journal is © The Royal Society of Chemistry [year]

Page 11 of 15

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DOI: 10.1039/C3DT52284A

CH₃(DMSO)), 7.11 (s, 1H, CHCS₂), 7.35ꢀ7.55 (m, 3H, ArꢀH3,4), 60 2856, 1505, 1488, 1468, 1294, 1265, 1223, 1144, 1023, 832,

7.93ꢀ7.97 (m, 2H, ArꢀH2); ¹³C{¹H} NMR (50 MHz, CDCl₃): δ =

17.6 (CH₃), 46.9 (s w/ Pt satellites, J_PtꢂC= 59.7 Hz,

772 cm^ꢀ1; MS (FAB in nba): m/z =588 [M]⁺, 552 [MꢀCl]⁺;

elemental analysis calcd for C₁₇H₂₅ClOPtS₃: C,34.72; H, 4.28;

S, 16.36 %; found: C, 34.99; H, 3.73; S, 14.25 %

2

CH₃(DMSO)), 111.4 (CHCS₂), 127.9 (ArꢀC2), 128.8 (ArꢀC3),

132.0 (ArꢀC4), 137.1 (ArꢀC1), 174.5 (CO), 180.8 (CS₂);

FTIR (KBr): ꢀꢁ = 2999, 2911, 1510, 1469, 1429, 1315, 1295,

1267, 1146, 1023, 833, 770 cm^ꢀ1; MS (DEI): m/z =518 [M]⁺;

elemental analysis calcd for C₁₂H₁₅ClOPtS₃· 0.5 CH₂Cl₂: C,

26.79; H, 2.88; S, 17.16 %; found: C, 26.84; H, 2.94; S, 17.17 %

5

65 Biological experiments

Cell Culture

Handling of cultivated cells generally occurred at sterile

conditions under a laminar airflow cabinet. Cell cultures were

maintained in microbiological incubators (Thermo Fisher

70 Scientific Inc.) under standard conditions (37°C, 5% CO₂, H₂Oꢀ

saturation). Cells were cultivated in 75 cm²cell culture flasks

with Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen)

supplemented with or without 10% (v/v) Fetal Calf Serum (FCS,

BiochromꢀSeromed). A549 cells were obtained from DSMZ

⁷⁵GmbH (Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH, German Collection of Microorganisms and

Cell Cultures DSMZ No. ACC 107). Cells were counted with a

cell counter (Z2 Coulter Particle Count and Size Analyzer).

Cultivation was performed until the 25^thpassage at most. The

80 adherent cell cultures were divided every 2ꢀ3 days following

standard procedures. Medium was removed and DꢀPBS

(Dulbecco’s PhosphateꢀBuffered Saline) was used 3 x to wash off

the medium. To detach the cells from the surface, TrypsinꢀEDTA

(3 mL) was added and incubated for 3 min. The reaction was

⁸⁵stopped with fresh medium containing FCS. Depending on the

passaging ratio, the respective portions of cell suspension were

transferred into new 75 cm²cell culture flasks.

10

Chloro-(1-phenyl-3-(ethylthio)-3-thioxo-prop-1-en-1-olate-

O,S)-(dimethylsulfoxide-S)-platinum(II) (11)

According to general procedure 2, preparation was performed

from 5 and stirring took 3 days. THF was evaporated from the

¹⁵bright red solution and the remainder was dissolved in

dichloromethane (3 mL) and water (3 mL). The aqueous phase

was washed with dichloromethane (3 x3 mL), the combined

organic solutions with water (3 x 3 mL). Purification was

accomplished

via

column

chromatography

applying

²⁰dichloromethane/hexane 3:2 as mobile pase (R_f≈ 0.1).

Evaporation of the solvents and drying in vacuum afforded pure

11 (29 mg, 12 %) as orange crystals. Single crystals suitable for

Xꢀray analyses were obtained by slow evaporation of

dichloromethane.

²⁵Mp.: 150°C (decomp.); ¹H NMR (200 MHz, CDCl₃): δ = 1.41 (t,

3H, CH₃), 3.24 (q, 2H, SCH₂), 3.62 (s w/ Pt satellites,

³J_PtꢂH= 23.6 Hz, 6H, CH₃(DMSO)), 7.07 (s, 1H, CHCS₂), 7.33ꢀ

7.54 (m, 3H, ArꢀH3,4), 7.89ꢀ7.96 (m, 2H, ArꢀH2); ¹³C{¹H} NMR

(50 MHz, CDCl₃): δ = 13.2 (CH₃), 28.7 (SCH₂), 46.8 (s w/ Pt

2

30 satellites, J_PtꢂC= 59.7 Hz, CH₃(DMSO)), 111.6 (CHCS₂), 128.0

For assays, cells were harvested at a density of 70ꢀ90 %

confluency. Medium was removed and DꢀPBS was used as

⁹⁰washing agent. Detachment with trypsinꢀEDTA (3 mL) was

stopped with fresh medium containing FCS after 3 min. The

desired number of cells (10,000 cells/well) was seeded into 96ꢀ

well plates. Wells were then supplied with medium (DMEM +

10 % FCS) to a final volume of 100 ꢁL. Plates were preꢀ

⁹⁵incubated for 1 day before adding drug samples.

(ArꢀC2), 128.8 (ArꢀC3), 132.0 (ArꢀC4), 137.2 (ArꢀC1), 174.7

(CO), 180.1 (CS₂); FTIR (KBr): ꢀꢁ = 3011, 2965, 2924, 2869,

1507, 1488, 1470, 1430, 1312, 1297, 1266, 1223, 1142, 1127,

1023, 832, 772 cm^ꢀ1; MS (DEI): m/z =531 [Mꢀ1]⁺; elemental

³⁵analysis calcd for C₁₃H₁₇ClOPtS₃· 0.1 C₆H₁₄: C, 31.21; H,

3.43; S, 17.79 %; found: C, 31.25; H, 3.27; S, 17.21%.

Chloro-(1-phenyl-3-(hexylthio)-3-thioxo-prop-1-en-1-olate-

O,S)-(dimethylsulfoxide-S)-platinum(II) (12)

40 Following general procedure 2, K₂PtCl₄was conversed with 6

Drug Treatment

Stock solutions were prepared in DMSO (Sigma HybriꢀMax) at

4 mg/mL. Samples were prepared by diluting the DMSO stock

¹⁰⁰solutions in DꢀPBS to the desired test concentrations. To

eludicate the effect of DMSO on the cells’ viability, respective

controls were prepared to give corresponding DMSO contents

(v/v). All samples were prepared freshly before each assay.

For viability assays, only the interior wells of each 96ꢀwell

105 plate were supplemented with drug to reduce local growth effects.

Preparing triplicates for each concentration, 10 ꢁL sample or

control solutions were added to the preꢀincubated cells. For

caspase 3/7 assays, 50 ꢂL sample or control solutions were added

to the wells after removal of 50 ꢂL supernatant from the initial

110 volume of 100 ꢂL cell culture. In this case, duplicates of each

concentration were prepared.

and stirred for 24 hours. THF was removed from the red reaction

mixture in vacuo, the residue was dissolved in dichloromethane

and water, whereupon the aqueous phase was extracted with

dichloromethane (3 x 3 mL). The volume of the organic solution

45 was reduced by evaporation and the crude product was purified

via

column

chromatography

on

silica

gel

with

dichloromethane/hexane 3:2 (R_f≈ 0.1). After crystallization from

dichloromethane/pentane, the pure product (49mg, 19 % of

brownish powder) was dried in vacuum.

1

50 Mp.: 150 °C (decomp.); H NMR (200 MHz, CDCl₃): δ = 0.88

(m, 3H, CH₃), 1.24ꢀ1.48 (m, 6H, 3x CH₂), 1.77 (qui, 2H,

3

SCH₂CH₂), 3.23 (t, 2H, SCH₂), 3.64 (s w/ Pt satellites, J_Ptꢂ

_H= 23.6 Hz, 6H, CH₃(DMSO)), 7.01 (s, 1H, CHCS₂), 7.35ꢀ7.51

3

(m, 3H, ArꢀH3,4), 7.81 (d, J_H2ꢂH3= 7.2 Hz, 2H, ArꢀH2);

Plates were then incubated under standard conditions for 24 h.

Before adding the respective assay reagent, the plates were

microscopically inspected (Zeiss Axiovert 25 Inverted

55 ¹³C{¹H} NMR (100 MHz, CDCl₃): δ = 13.9 (CH₃), 22.4

(CH₂CH₃), 27.9 (CH₂CH₂CH₃), 28.5 (SCH₂CH₂CH₂), 31.2

(SCH₂CH₂), 34.4 (SCH₂), 46.9 (CH₃(DMSO)), 111.6 (CHCS₂),

128.0 (ArꢀC2), 128.8 (ArꢀC3), 132.0 (ArꢀC4), 137.2 (ArꢀC1),

174.6 (CO), 180.5 (CS₂); FTIR (KBr): ꢀꢁ = 3005, 2955, 2926,

115 Microscope;

camera:

AxioCam

HRc,

software:

AxiovisionRel.4.6), cell morphology recorded and the number of

living cells estimated. No evidence of necrotic cells was found.

Journal Name, [year], [vol], 00–00 | 11

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Page 12 of 15

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DOI: 10.1039/C3DT52284A

For hydrolysis studies, a stock solution of compound 11 was

60 diluted to 1 mM in DMSO or DMSO/water 50%/50% and a 10ꢀ

fold excess of AgNO₃, dissolved in DMSO, added. Spectra were

recorded from 100ꢁM solutions (diluted in the respective solvent

composition) before and directly after addition of the silver salt.

Cell Viability Assays

For viability assays, PrestoBlue Cell Viability Reagent

(Invitrogen Corporation) was used according to the supplier’s

protocol. After adding the reagent solutions (11 % relative

volume), the liquid phase was quickly mixed on a plate shaker

and subsequently incubated under standard conditions for 2 h.

Fluorescence was read on a TECAN infinite M200 plate reader,

(Tecan Group Ltd., software iꢀcontrol 1.6) at an excitation

5

⁶⁵Protein interaction studies with cyt c and HEWL: ESI mass

spectrometry

Samples were prepared by dissolving horse heart cyt c (Sigma

C7752) (100 ꢁM) resp. chicken egg white lysozyme (Sigma

L7651) (100 ꢁM) in tetramethylammonium acetate buffer

⁷⁰(TMAA, 25 mM, pH 7.4). The metal complex was added from a

DMSO stock solution (10 mM, final metal:protein ratio 3:1) and

incubated at 37 °C for 72 h.

¹⁰wavelength of 560nm and emission wavelength of 590nm.

Apoptosis Assay

For caspase 3/7 assay, the ApoꢀONE Caspaseꢀ3/7 Buffer and

ApoꢀONE Caspaseꢀ3/7 Reagent (Promega Corporation) was used

¹⁵according to the supplier’s protocol. 100 ꢁL caspase reagent

mixture was added to each well and the liquids quickly mixed on

After a 20ꢀfold dilution with water, the ESI MS spectrum was

recorded in an LTQꢀOrbitrap highꢀresolution mass spectrometer

a plate shaker. Cells were incubated for 2 h before reading the ⁷⁵(Thermo, San Jose, CA, USA), equipped with a conventional ESI

fluorescence at excitation wavelength of 499nm and emission

wavelength of 521nm.

source (direct introduction, flow rate

5

ꢂL/min). The

spectrometer’s working conditions were the following: spray

voltage 3.1 kV, tube lens voltage 230V, capillary voltage 45 V

and capillary temperature 220 °C. The sheath and the auxiliary

80 gases were set at 17 and 1 (arbitrary units), respectively. For

acquisition, Xcalibur 2.0. software (Thermo) was used.

Monoisotopic and average deconvoluted masses were obtained

by using the integrated Xtract tool. For spectrum acquisition a

nominal resolution of 100,000 (at m/z 400) was used.

20

Data handling

Data obtained from the in vitro assays were evaluated with

Microsoft Office Excel 2007. Fluorescence intensity was

determined in arbitrary units (a.u.) and corrected by blanks

²⁵without cells as determined separately for each plate. Triplicates

for each drug concentration were averaged, standard deviations

determined and possible outliers elucidated by Grubbs’ test at a

significance level of 0.05.

85

Protein interaction studies with albumin: ICP-OES

IC₅₀values were determined separately for each of three

30 independent experiments, averaged and converted to molar

concentration. For their calculation, single fluorescence

Bovine serum albumin (BSA, Fluka BioChemika 05470)

(100 ꢁM) was dissolved in TMAA (25 mM, pH 7.4) and the Pt

compound was added from a DMSO stock solution (10mM) to

intensities were used instead of their mean values. Regression ⁹⁰give a final metal:protein ratio 1:1. Samples were then incubated

was performed with the ReaderFit online tool (Hitachi Solutions

America, Ltd.), applying four parameter logistics (4PL) model

³⁵equations. The IC₅₀value obtained resembles the inflection point

of the respective curve.

at 37 °C for 96 h and then ultrafiltration was performed 3 times in

Centricon YMꢀ10 centrifugal filters (Amicon Bioseparations,

Millipore Corporation) for 30 min at 3500 rpm in using water as

diluent. All solutions were separately investigated by ICPꢀOES

⁹⁵measurement on a Varian 720ꢀOES instrument with 1.25 ppm of

Ge as internal standard. Samples were diluted 16ꢀfold and

digested with HNO₃prior to injection. Detection wavelengths

were λ = 238, 214 and 209 nm for Fe, Pt and Ge, respectively.

Parallel, equally prepared sample solutions were studied with

100 UVꢀvisible spectrophotometry according to the aboveꢀstated

procedure (24 h data collection in 1 h intervals at r.t.) to eludicate

the solvolytic status of the sample. It was shown that all samples

stayed dissolved over time.

For further information, cf. the ESI^†.

Mechanistic investigations

⁴⁰UV-visible spectrophotometry

UVꢀVis absorption spectra were recorded on a Varian Cary 50

UVꢀVis spectrophotometer in the range of 200ꢀ800 nm. Stock

solutions (10 mM) of each compound were prepared by

dissolving the complex under investigation in DMSO. For all

45 experiments, the final complex concentration was 100 ꢁM.

For solubility studies, UVꢀvis measurements were carried out

by diluting the compounds' stock solutions to 100 ꢁM in

phosphate buffer (PB, 25 mM, pH 7.4) with varying content of

DMSO (final percentages 1 %, 10 %, 50 %). Spectra were

50 collected for 24 hours at r.t., operating in 1 min intervals up to

10 min, 10 min intervals during the first hour and in 1 h intervals

afterwards.

For stability studies, compound 11 was kept in 1 mM DMSO

or PB/DMSO solutions and kept at room temperature over 5 d.

55 UVꢀVis absorption spectra were periodically recorded by diluting

aliquots to the final concentration of 100 ꢁM in DMSO. Equally,

samples were kept at 37 °C for 24h and the UVꢀVis absorption

spectra recorded before and after incubation.

105 Competitive interaction with cytochrome c and GSH

Samples of compounds 7 and 8 were preincubated for 24 h with

cyt c resp. BSA as described above. Then, GSH was added to the

sample solutions to give a final ratio GSH:protein:Pt of 6:1:3

resp. 2:1:3 for cytochrome c and BSA. The solutions were again

¹¹⁰incubated at 37 °C for 24 h and then ultrafiltrated (3 times,

30 min at 3500 rpm, using water as diluent). Sample and washing

solutions were then investigated with ICPꢀOES.

Acknowledgements

The Authors wish to thank Federica Scaletti and Lara Massai at

115 University of Florence for help with UVꢀvisible

12 | Journal Name, [year], [vol], 00–00

Page 13 of 15

Dalton Transactions

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DOI: 10.1039/C3DT52284A

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thanks CarlꢀZeissꢀStiftung as well as Studienstiftung des

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10 ^aInstitute of Inorganic and Analytical Chemistry, FriedrichꢂSchillerꢂ

University Jena, Humboldtstraße 8, 07743 Jena, Germany, Fax: (+49)

3641 948102, Eꢂmail: wolfgang.weigand@uniꢂjena.de

^bDepartment of Chemistry and Industrial Chemistry, University of Pisa,

via Risorgimento 35, 56126 Pisa, Italy

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15 ^cMass Spectrometry Center (CISM), University of Florence, Via U. Schiff

6, 50019 Sesto Fiorentino, Firenze, Italy

^dDepartment of Internal Medicine II, Jena University Hospital, Erlanger

Allee 101, 07740 Jena, Germany

^eLaboratory of Metals in Medicine, Department of Chemistry, University

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table of contents entry

.

Novel Platinum(II) Compounds with O,S Bidentate Ligands:

Synthesis, Characterization, Antiproliferative Properties and

Biomolecular Interactions

Carolin Mügge,^aRuiqi Liu,^aHelmar Görls,^aChiara Gabbiani,^bElena Michelucci,^cNadine

Rüdiger,^dJoachim H. Clement,^dLuigi Messori,*^eWolfgang Weigand*^a,f

A new building block system of Pt(II) compounds with a O,S-binding moiety exhibits anti-

cancer activity and promising structure-activity relationships.

Article Doi

Novel platinum(ii) compounds with O,S bidentate ligands: Synthesis, characterization, antiproliferative properties and biomolecular interactions

DOI: 10.1039/c3dt52284a

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c3dt52284a

Full text of DOI:10.1039/c3dt52284a

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