Novel platinum(II) compounds with O,S bidentate ligands: synthesis, characterization, antiproliferative properties and biomolecular interactions.

View Article Online View Journal

Dalton Transactions Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: W. Weigand, C. Mügge, H. Görls, R. Liu, C. Gabbiani, E. Michelucci, N. Rüdiger, J. H. Clement and L. Messori, Dalton Trans., 2013, DOI: 10.1039/C3DT52284A.

Volume 39 | Number 3 | 2010

This is an Accepted Manuscript, which has been through the RSC Publishing peer review process and has been accepted for publication.

Dalton Transactions An international journal of inorganic chemistry www.rsc.org/dalton

Volume 39 | Number 3 | 21 January 2010 | Pages 657–964

Dalton Transactions

Accepted Manuscripts are published online shortly after acceptance, which is prior to technical editing, formatting and proof reading. This free service from RSC Publishing allows authors to make their results available to the community, in citable form, before publication of the edited article. This Accepted Manuscript will be replaced by the edited and formatted Advance Article as soon as this is available. To cite this manuscript please use its permanent Digital Object Identifier (DOI®), which is identical for all formats of publication. More information about Accepted Manuscripts can be found in the Information for Authors.

Pages 657–964

ISSN 1477-9226

PAPER Manzano et al. Experimental and computational study of the interplay between C–H/p and anion–p interactions

COMMUNICATION Bu et al. Zinc(ii)-boron(iii)-imidazolate framework (ZBIF) with unusual pentagonal channels prepared from deep eutectic solvent

1477-9226(2010)39:1;1-K

Please note that technical editing may introduce minor changes to the text and/or graphics contained in the manuscript submitted by the author(s) which may alter content, and that the standard Terms & Conditions and the ethical guidelines that apply to the journal are still applicable. In no event shall the RSC be held responsible for any errors or omissions in these Accepted Manuscript manuscripts or any consequences arising from the use of any information contained in them.

www.rsc.org/dalton Registered Charity Number 207890

Page 1 of 15

Journal Name

Dalton Transactions

Dynamic Article Links ► View Article Online

DOI: 10.1039/C3DT52284A

Cite this: DOI: 10.1039/c0xx00000x

ARTICLE TYPE

www.rsc.org/xxxxxx

Carolin Mügge,a Ruiqi Liu,a Helmar Görls,a Chiara Gabbiani,b Elena Michelucci,c Nadine Rüdiger,d d e a,f 5 Joachim H. Clement, Luigi Messori,* Wolfgang Weigand* 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

10

15

20

25

Cisplatin and its analogues are first-line chemotherapeutic agents for the treatment of numerous human cancers. 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 O,S bidentate β-hydroxy 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 squareplanar 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 time-course UV-visible absorption spectra. Their anti-proliferative and pro-apoptotic activities were tested against the Cisplatin-resistant lung cancer cell line A549. Assays revealed significant effects of the sample drugs at low concentrations (in the µmolar range); initial structure-activity-relationships are proposed. The activity of the apoptosispromoting 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 the model proteins cytochrome c, lysozyme and albumin were studied by ESI MS and ICP-OES 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. 45

30

35

40

Introduction Since 1978, when it was first approved for clinical use, Cisplatin is one of the few metal-based anticancer agents exhibiting “true” curative potential: cure rates beyond 90 % have been reported for the treatment of testicular germ cell cancer1. Subsequent, tremendous efforts in research have led to a handful of platinum-based 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 doselimiting side effects often occur during chemotherapy which have not been overcome yet. Hence, the development of new anticancer metallo-chemotherapeutics 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 This journal is © The Royal Society of Chemistry [year]

50

55

60

structure-activity-relationships (SARs) defining the requirements that are considered essential for the activity of a “classical” platinum-based anticancer compound2–5. A number of platinum complexes have been synthesized based on these criteria; some of them did even enter clinical trials. Amongst them, all (worldwide 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. Satraplatin7,8 or Picoplatin9–11. One of the central aspects of these SARs is the kinetics of metal-ligand 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 seems to establish their high anticancer activity, since the [journal], [year], [vol], 00–00 | 1

Dalton Transactions Accepted Manuscript

Published on 11 October 2013. Downloaded by Laurentian University on 26/10/2013 05:56:48.

Novel Platinum(II) Compounds with O,S Bidentate Ligands: Synthesis, Characterization, Antiproliferative Properties and Biomolecular Interactions

Dalton Transactions

Page 2 of 15 View Article Online

5


10

15

20

25

30

35

40

45

50

55

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. Based on these arguments a new, more general set of structureactivity-relationships can be generated for the development of new platinum-based anticancer agents15. - Kinetically labile, i.e. fast hydrolyzing complexes can bind to biomolecules available in the blood (e.g. sulfur-containing 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, water-soluble 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 tumor-inhibiting 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 Pt-S 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. - Finally, the non-leaving 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. It 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) concept: 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 years, various research groups have addressed the question of protein binding and consequences for the activity of platinumbased drugs to a great extent (for recent reviews see e.g. refs19–21). Binding of Pt-based drugs to proteins is known to have a major impact on their activity. It is discussed that these reactions are not only the source of severe toxic side effects and of resistance to the drug. Moreover, some platinum-sulfur adducts could serve as reservoir for the platination of DNA or play a crucial role in detoxification processes17,22,23; indeed, even Phase-1 clinical trials have been conducted with Cisplatin-HSA adducts24. Overall, platinum-sulfur interactions are kinetically preferred, but the binding of platinum to guanine-N7 is believed to be thermodynamically favored17,25. This circumstance makes the anticancer activity of platinum compounds possible at all26–31. In spite of all the detailed insight that has been gained on 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

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

60

65

70

75

80

85

90

substances which might fulfill those requirements. In this work, a system based on O,S-chelating β-hydroxydithiocinnamic 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, especially with respect to their nucleoside binding capacities32–34 (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 biological sulfur donors and is active against MCF-7 breast cancer cells not via DNA attack, but through the so-called mitochondrial pathway, thus providing a new approach in cancer therapy36,37.

Fig. 1 Platinum(II) complexes bearing DMSO as ligand and interesting in vitro properties32,33,35.

The aim of this work is to derivatize the chelating system of β-hydroxydithiocinnamic esters in order to gain more insight into its potential as antitumor agent (Fig. 2). We expect the chelating subunit 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 milieu. 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 hydro-/lipophilicity of the compounds and, in the latter case, to allow for future modification of the core system. We attempt the inference of primary structure-activity relationships making further variations possible in a well-planned way. The reactivity towards established model proteins shall give a primary insight into their possible mode of action.

95

Fig. 2 Ligands 1-6 and synthesized complexes 7-12 as well as isolated side products 13,14.

100

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


DOI: 10.1039/C3DT52284A

Page 3 of 15

Dalton Transactions View Article Online

DOI: 10.1039/C3DT52284A


10

15

20

30

35

40

45

50

55

65

70

75

Results and Discussion Chemistry

25

60

Ligands: β-hydroxydithiocinnamic esters 1-6 For ligand synthesis, starting materials were either acetophenone or 4-bromo-acetophenone as enolate precursors. Methyl or ethyl iodide and hexyl bromide served as alkylation reagents. The deprotonation of the acetophenone derivative was achieved with two molar equivalents of potassium tert-buylate (KOtBu) in diethyl ether at -70 °C. Addition of carbon disulfide (CS2) gave the dithiolate anion, which was then reacted with one molar equivalent 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 1-6 with yields ranging from 10 % (for 3) to 72 % (for 2) (Fig. 2, left). All compounds were characterized by NMR spectroscopy, mass spectrometry, FTIR spectroscopy and elemental analyses. 1 H NMR spectroscopy confirmed the postulated cis-enolic 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 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 spectra. The resonance signals for the thiocarbonyl carbon atoms are located at fairly low field (217 ppm), signals for the β-oxo carbon atoms appear at 168 ppm and 169 ppm for the p-bromo substituted and the unsubstituted compounds, respectively. Carbon signals for the methine groups are found at 108 ppm (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 1-6. Further characteristic peaks are found at m/z = 243 and m/z = 163 belonging to ions after cleavagef of the S-Alk group in the p-bromo substituted and the unsubstituted compounds, respectively. In the FTIR spectra, characteristic bands are detected around This journal is © The Royal Society of Chemistry [year]

3400 resp. 3300 cm-1 for the hydroxyl group in the bromosubstituted and unsubstituted compounds, correspondingly. Both broad shape and position indicate the existence of hydrogen bonds. Very strong absorption bands around 1590, 1560, and 1490 cm-1 are assigned to stretching vibrations of C=C and C=O, whereas a very strong band at 1235 cm-1 belongs to the stretching vibration of the C=S group.

80

85

90

95

100

The O,S-chelating platinum(II) complexes 7-12 A number of methods for the preparation of platinum(II)-DMSO complexes is known47–49. Those compounds seem to play an activating role in the course of synthesizing platinum complexes with a variety of chelating ligands. Once coordinated to the Pt(II) center, DMSO as ligand can be kept in the complex50,32,51,52 or be substituted against a more stable ligand during the progression of the reaction53,54,35. A new synthesis towards target compounds 7-12 was developed (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 The platinum-DMSO complex K2-x[PtCl4-x(DMSO)x]. β-hydroxydithiocinnamic ester was deprotonated with sodium hydride 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 7-12 could be isolated in moderate yields (10- 33 %; Fig. 2, middle). All compounds were characterized by elemental analyses, multinuclear NMR and FTIR spectroscopy as well as mass spectrometry.

Scheme 1 Synthesis of O,S-chelating Pt(II)-DMSO complexes 7-12. Conditions: i, 2eq. DMSO, H2O, r.t.; ii, 1 eq. NaH, thf, r.t.; iii, 1.1:1, r.t., 1-7d.

As unavoidable side product, the analogous bischelate was formed for all complexations but could be separated from the product via column chromatography. Two representative compounds, 13 and 14, were isolated and characterized (Fig. 2, right and ESI† for syntheses and X-ray analyses). 1 H NMR spectra of compounds 7-12 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. The latter can be assigned to the methyl groups of the DMSO ligand, which constitutes a “marker” for the formation of Journal Name, [year], [vol], 00–00 | 3


5

The synthesis of β-hydroxydithiocinnamic ester derivatives has already been described by several researchers38–40, but a versatile “one-pot” synthesis was not described until 1998. Since then, the procedure has been refined and well-established41–45. 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 a 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. β-Hydroxydithiocinnamic acid esters serve as monoanionic ligands after deprotonation of the hydroxyl group. In this work, a series of Pt(II) complexes with one O,S-chelating 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.

Dalton Transactions


DOI: 10.1039/C3DT52284A


10

15

20

25

30

35

40

45

50

55


60

65

70

75

80

85

variable positions -R and -X might allow a distinct fine tuning of the properties of the complexes with regard to transport and solution properties without influencing the binding or release of this ligand.

Fig. 3 Depiction of solid-state molecular structures of 7 (top) and 11 (below). Thermal ellipsoids are given at the 50 % probability level and hydrogen atoms are omitted for clarity. Selected bond lengths [Å] and angles [°] for 7: Pt-O1 2.013(5), Pt-S1 2.253(2), Pt-S3 2.200(2), Pt-Cl 2.336(2), S3-O2 1.457(6), O1-Pt-S1 96.11(17), S1-Pt-S3 88.32(8), S3-Pt-Cl 91.33(9), O1-Pt-Cl 84.21(17), S1-Pt-Cl 177.94(9), O1-Pt-S3 175.49(17) Selected bond lengths [Å] and angles [°] for 11: Pt-O1 2.006(5), Pt-S1 2.249(2), Pt-S3 2.186(2), Pt-Cl 2.3597(19), S3-O2 1.472(6), O1-Pt-S1 96.02(16), S1-Pt-S3 88.50(7), S3-Pt-Cl 90.20(7), O1-Pt-Cl 85.28(16), S1-Pt-Cl 178.47(7), O1-Pt-S3 175.39(16).

Molecular structures It was possible to attain single-crystal X-ray structures from 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 square-planar geometry of Pt(II) complexes and 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 β-hydroxydithiocinnamic ester. As This journal is © The Royal Society of Chemistry [year]


5

monochelates in contrast to the respective bischelates with almost identical NMR peak positions. Both the occurrence of platinum satellites with 3JPt-H ≈ 23.6 Hz and a downfield shift of around 1 ppm compared to free DMSO55 give 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 previously described Pt-DMSO complexes51,56. 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 the coordinated metal ion due to good delocalization of electron density in the six-membered chelating ring. 13 C{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 6 ppm compared to free DMSO55. Platinum-carbon coupling can be observed for the methyl group of DMSO with 2JPt-C ≈ 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 β-oxo-carbon are slightly downfield shifted, by ≈ 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 β-hydroxydithiocinnamic esters towards the Pt(II) center. Oxygen atoms posses σ-donating properties towards the Pt(II) center. This results in a deshielding effect on the β-oxo-carbon atom. In contrast, the sulfur atom can act as an electron acceptor for π-back bonding from the Pt(II) center; the increased electron density shields the thiocarbonyl carbon. The overall delocalization of electron density in the six-membered 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 7-12 show characteristic absorption bands for the Pt-bound ligand. As expected, no O-H, C-O or C=S stretching vibrations are found. One characteristic broad band at ≈ 1146 cm-1 is 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 than 40 cm-1 58,59. 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 S-Pt bond has both σ-donating and π-donating character. Thus, most spectroscopic phenomena can be explained by a prevailing S→Pt σ bond49. By donating electron density to platinum, the sulfur-oxygen bond in DMSO is polarized. Consequently, the frequency of the S-O vibration is increased in the IR spectra. The distribution of electron density also affects the α-bound carbon atoms. Due to an 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}, 1 H NMR, IR) spectra of the different complexes. This means that up to the degree applied, variation of the O,S-chelating ligand has very little influence on the binding properties of DMSO towards the platinum center. As a consequence, derivatization at the

Page 5 of 15


5


10

15

20

25

30

35

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 order: Pt-Cl > Pt-S1 > Pt-S3 > Pt-O1. The angle in the coordination sphere comes close to 90° in the case of S1-Pt-S3 and S3-Pt-Cl, is smaller for O1-Pt-Cl and larger for O1-Pt-S1. The sulfur atom of the DMSO ligand is enclosed in a distorted tetrahedral geometry due to the tilting of the oxygen atom towards the methyl groups. When compared to free DMSO, the S3-O2 bond length is shortened whereas it is elongated for the S3-C 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. When compared to similar β-hydroxydithiocinnamic esters42,61, the C7-O1 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 square-planar coordination to the platinum center, the angles in the chelating unit are widened. The angle Pt-O1-C7 is the widest (130.9(6)° in 7 and 130.5(5)° in 11) and the angle Pt-S1-C9 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 six-membered chelating ring.

40

45

50

55

Fig. 4 Solubility of 11 (100 µM) in phosphate buffered solutions (pH 7.4) over 24 h, depicted by UV-Vis measurements. Top, 10 % DMSO, bottom, 50 % DMSO content. Spectra were recorded in 10 min intervals during the first 60 min and hourly afterwards.

60

Solution chemistry of the investigated compounds The obtained compounds were dissolved in DMSO and their solution behavior in buffered aqueous solutions was analyzed through UV-visible absorption spectroscopy. The UV-vis spectra

65


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 data for 11 are shown in Fig. 4, more information is given in the 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 dissolved as 100 µM solutions containing 50 % DMSO, and partially dissolved in solutions containing 10 % DMSO. Qualitative structure-solubility dependence could be assessed and revealed higher solubility for bromo-substituted compounds compared to their unsubstituted counterparts with equal alkyl substituent 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 c, HEWL, BSA, GMP, vide infra) investigated. For in vitro screenings of antiproliferative properties, the drug concentrations used are far lower and therefore do not require DMSO contents of the tested extent.

Fig. 5 UV-Visible 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).

Journal Name, [year], [vol], 00–00 | 5


DOI: 10.1039/C3DT52284A

Dalton Transactions


20

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 bidentate ligand, even over a period of days – sufficiently long for them to reach their biological target (Fig. 5, top). Some minor alterations were noticed in the long-wave band of the DMSO-PB 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 AgNO3 and gave a blue-shift of the long-wave band from 430 to 409 nm. This indicates that exchange of the monodentate ligands mainly leads to shifts in this band. These results point to DMSO or chloride being 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, e.g. varied pH or the presence of proteins or other potential ligands, the process might indeed be much faster.

25

Fig. 6 IC50 values of the novel Pt(II) compounds and CDDP as reference compound. At the given concentrations, the influence of DMSO from stock solutions was neglectably low.

5


10

15

50

55

60

65

70

Biology 75

30

35

40

45

Antiproliferative effects The antiproliferative effects of the compounds under investigation were evaluated against the Cisplatin-resistant lung cancer cell line A549, using the resazurin-resurofin-based PrestoBlue® assay. The cell-permeant blue dye resazurin is virtually nonfluorescent. When added to the reducing environment of viable cells, resazurin is reduced to red resurofin 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 IC50 values determined to be lower than those of Cisplatin (Fig. 6, Fig. 7 and 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” IC50 values determined for these complexes: IC50 values strongly depend on the experimental conditions used to determine cell viability and can therefore only be compared with results from equal assay conditions. For example, time6 | Journal Name, [year], [vol], 00–00

dependent cytotoxic assays on Pt(II) compounds by Muscella et al. ( Fig. 1) had shown that indeed, IC50 values strongly depend on drug incubation times (in this case 12-96 h), but the relative 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 presented Pt(II) compounds 7-12 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, control solutions with the respective DMSO content were also 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 applications63. Furthermore, excess DMSO in the liquid sample can prevent dissociation of the compounds so that application of the intact molecule can be expected, as shown by UV-visible absorption spectroscopy, vide supra.

80

85

90

Fig. 7 Dose-response curve for A549 cells incubated with different drug concentrations, determined with PrestoBlue assay. The dose-response 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.

A general structure-activity relationship can roughly be deduced from the tendency of the obtained IC50 values. 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 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 – 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 short-chained compounds have appreciable This journal is © The Royal Society of Chemistry [year]


DOI: 10.1039/C3DT52284A

Page 7 of 15


5


10

15

water solubility, the hexyl chains increase the compounds’ 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 that those compounds were excluded from further activity investigations (data not shown). Induction of Apoptosis Microscope-based visual inspection of the cells after incubation 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.

20

25

40

45

55

60

65

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 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.

70

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 caspases 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 chemotherapeutics65,66. Its activation can be measured with the substrate Z-DEVD-rhodamine 110, which by 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 obtained in viability assays, we believe the results are quite This journal is © The Royal Society of Chemistry [year]

Fig. 9 Activation of caspase 3/7 by Pt(II) compounds at different drug concentrations. For the sake of overview, standard deviations are not depicted but given in the ESI†.

Mechanistic studies 75

30

35

50

remarkable. Hexyl compounds 9 and 12, giving low IC50 values, 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 caspase 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 time span applied for the viability assays. This thesis is further corroborated by the fact that methyl substituted compounds 7 and 10 with higher IC50 values 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 the determined IC50 value. 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 incubated with DMSO or CDDP do not display concentrationdependent but rather intrinsic, natural protein activation, just as would be expected from results of the cell viability data.

80

85

90

Reactions with model proteins 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 ICP-OES in the latter case in order to gain more precise mechanistic information. These model proteins were chosen on the basis of several rationales: Lysozyme is a small protein well-suitable for ESI MS analytic invenstigation, since ionization occurs well due to the presence of several positively charged groups at its surface. Only 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 straight-forward interaction pattern is expected in ESI MS spectra of drug-incubated proteins. Similar factors count for cytochrome c, i.e. its small size and the outcome of well-resolved 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 Journal Name, [year], [vol], 00–00 | 7


DOI: 10.1039/C3DT52284A

Dalton Transactions


DOI: 10.1039/C3DT52284A


10

15

20

25

30

35

40

45

50

55

60

65

It is noteworthy to state that there seems to be a structuredependence of the amount and intensity of complex-protein interaction. Based on the relative peak intensities and the stoichiometries found in the deconvoluted ESI MS spectra, shortchained complexes, i.e. 7 and 10, give higher adduct abundancies and metal:protein ratios than long-chained 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, ICP-OES measurements were conducted to determine the relative Platinum content of the solutions 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. The strength of the formed Pt-protein adducts has been challenged against the very competitive sulfur-containing and ubiquituous molecule glutatione (GSH). As has been witnessed by ICP-OES, 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 might be considered as a hint that binding of this kind of platinum complexes is not reversible, other than has been discussed for Cisplatin and analogues 76,21,77. Table 1. Upper part: Results of ICP-OES measurements of incubating BSA with the Pt(II) compounds 7-12. Lower part: Results of protein-GSH 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. Compound 7 8 9 10 11 12 7/cyt c + GSH 7/BSA + GSH 8/BSA + GSH

70

75

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.


80

c (Pt) [ppb] UP DOWN 14285 167 30395 281 42191 92 56594 165 18100 104 34572 146 556 136 24165 487 20625 588

Ratio UP vs. total content [%] 98,84% 99,08% 99,78% 99,71% 99,43% 99,58% 80,37% 98,02% 97,23%

Conclusion After the successful synthesis of six β-hydroxydithiocinnamic ester derivatives 1-6, they were coordinated to a platinum(II) center in an O,S-chelating manner together with chloride and DMSO as monodentate ligands. Complexation yielded compounds 7-12 in moderate yields. Structure determination was possible for compound 7 and 11 as well as for the bischelates 13 and 14. Nearly square-planar 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 β-hydroxydithiocinnamic esters. In the structures of 13 and 14, cis coordination of the two ligands is observed. As it was introduced earlier, the properties of both leaving This journal is © The Royal Society of Chemistry [year]


5

8020,28. Cys 14 and Cys 17, which bind the heme unit, should not be available for platinum binding. The proteins’ role as electroncarrier with important function in the apoptotic process makes it even more of interest for the here-presented investigations. Both proteins have been well-investigated in view of their interaction with platinum-based drugs and some very interesting results have been reported68,69. Serum Proteins such as albumin are amongst the most abundant proteins a drug encounters when infused into the blood stream 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 well-resolved ESI MS-based evaluation of the whole protein. Hence, ICP-OES can give quantitative feedback on metal-protein interactions. 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 that the described reactions lead to formation of appreciable quantities of metallodrug- protein adducts. The position of the peaks allows us to determine the nature of the protein-bound metallic fragments. These correspond to the Pt(O,S) fragment (O,S is hereby the complete O,S-chelating ligand). These results imply that adduct formation takes place through chloride and DMSO aquation and subsequent coordination of the resulting 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 with cyt c seems to be more intense than is the case with HEWL.

Page 9 of 15


5


10

15

20

25

30

35

40

45

50

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 synthesized, indicating that the Pt-S(DMSO) bond is of constant strength. This bears the advantage that derivatization of the β-hydroxydithiocinnamic ester derivatives has no or little effect on the dissociation behavior of DMSO or chloride as leaving groups. Thus, the properties of the β-hydroxydithiocinnamic esters can be distinctly fine-tuned to define e.g. the solubility and activity 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. Solution and hydrolysis experiments showed that these substances 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 unfavourable side reactions (vide supra). Hydrolysis appears to be 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 extent. Structure-dependence was observed in the form that compounds with short alkylic substituents gave more intense interaction than their long-chained 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 platinum complexes introduced in this work, reduced protein interaction 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 IC50 values than the reference compound, Cisplatin. Hints were found suggesting that elongation of the alkylic chain lead to more 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 results 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 short-chained analogs. In a mammalian system, this might reduce unfavourable protein interaction, promote clearance from the blood stream and cell membrane permeation; on the contrary, administration of non-water-soluble drugs is in general not easy. Introduction of the electronwithdrawing bromo group does not significantly influence the cytotoxic activity, yet enhances solubility in polar solvents and might thus facilitate the compounds’ usage as anticancer active compound.

60

65

70

75

80

85

90

95

100

105

Experimental Section 110

Materials and methods 55

All Syntheses were carried out under argon atmosphere using conventional Schlenk technique. Solvents were dried according to


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 gel of the type VWR Kieselgel 60 was used. 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, 100.63 MHz resp. 150.94 MHz (13C). The deuterated solvent served as an internal standard for 1H 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 FT-IR-Spectrometer by PerkinElmer. Mass spectra (FAB, ESI, DEI) were recorded on an instrument SSQ 710 (Finnigen MAT) and MAT95XL. Ionization was performed at 70 eV. Elemental analyses (C, H, S) were carried out on a LECO CHNS-931 instrument. Halogen determination was performed by titration. Melting points were determined on a Stuart melting point SMP3 instrument and are 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 ESI†. The intensity data were collected on a Nonius KappaCCD diffractometer using graphite-monochromated Mo-Kα 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 full-matrix least squares techniques against Fo2 (SHELXL-9781). All hydrogen atoms were included at calculated positions with fixed thermal parameters. All non-disordered, non-hydrogen atoms were refined anisotropically81. 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 potassium-tertbutoxylate (KOtBu, 2 eq.) in diethyl ether (40 mL) at -78 °C. 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 sulfuric acid (50 mL 2 M solution in water) and the evolving twophased 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 chromatography on silica gel and dried under vacuum. Ligands 1-6 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). General Procedure 2: Platinum(II) complexes with O,Schelating β-hydroxydithiocinnamic esters, chlorido and DMSO ligands Sodium hydride (NaH, 17.5 mg 60 % sup. in mineral oil, 0.438 mmol, 1 eq.) was suspended in tetrahydrofuran (THF, Journal Name, [year], [vol], 00–00 | 9


DOI: 10.1039/C3DT52284A

Dalton Transactions


5


10

15

20

25

30

35

40

45

50

55

5 mL) and transferred into a solution of the desired β-hydroxydithiocinnamic 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. Potassium tetrachloroplatinate (K2PtCl4, 200 mg, 0.482 mmol, 1.1 eq.) was dissolved in degassed water (2 mL). After the addition of dimethylsulfoxide (DMSO, 62.2 µL, 0.876 mmol, 2 eq.), the reaction mixture was stirred at room temperature for 30 minutes or until the precipitation of a white solid indicated the formation of DMSO-platinum-species. 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 exclusion of light for 3 to 7 days, until TLC control showed no starting material. Workup included removal of the solvents in vacuo, uptake of the remainder in a proper solvent, filtration of the salts and column chromatography. Chloro-(1-(4-bromophenyl)-3-(methylthio)-3-thioxo-prop-1en-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 20 mL at room temperature, 1 x 100 mL at reflux for 2 hours) and filtered off each time. The remaining solid was identified as bischelate. The solution was reduced in volume and purified via column chromatography on silica gel (mobile phase: hexane/dichloromethane 3:2, Rf ≈ 0.1). The fraction which included the product was crystallized from dichloromethane and pentane. The yellow needles were then again purified via column chromatography (mobile phase: dichloromethane, Rf ≈ 0.5). The pure product (30.1 mg, 9 % as orange powder) was dried in vacuum. Single crystals were obtained by diffusion of pentane into a solution of the product in dichloromethane. M.p.: 193 °C (decomp.); 1H NMR (200 MHz, CDCl3): δ = 2.67 (s, 3H, CH3), 3.66 (s w/ Pt satellites, 3JPt-H = 23.4 Hz, 6H, CH3(DMSO)), 7.05 (s, 1H, CHCS2), 7.53 (d, 3JH2-H3 = 8.6 Hz, 2H, Ar-H2), 7.73 (d, 3JH2-H3 = 8.6 Hz, 2H, Ar-H3); 13 C{1H} NMR (50 MHz, CDCl3): δ = 17.7 (CH3), 46.9 (s w/ Pt satellites, 2JPt-C = 58.3 Hz, CH3(DMSO)), 111.1 (CHCS2), 126.9 (Ar-C4), 129.4 (Ar-C2), 132.0 (Ar-C3), 135.9 (Ar-C1), 172.9 (CO), 181.9 (CS2); FTIR (KBr): = 3011, 2914, 1583, 1504, 1482, 1463, 1318, 1292, 1263, 1146, 1071, 1025, 1008, 833, 793 cm-1, MS (DEI): m/z =598 [M+1], 596 [M-1]; MS (FAB in nba): m/z =597 [M]+; elemental analysis calcd for C12H14BrClOPtS3: C, 24.15; H, 2.36; S, 16.12 %; found: C, 24.34; H, 2.56; S, 16.09 %. Chloro-(1-(4-bromophenyl)-3-(ethylthio)-3-thioxo-prop-1-en1-olate-O,S)-(dimethylsulfoxide-S)-platinum(II) (8) Synthesis was performed from 2 according to general procedure 2. The solution was stirred for 7 days. All solvents were evaporated and the solid residue was dissolved in 10 mL acetone. The remaining precipitate was filtered off and dissolved in 10mL dichloromethane, the solid residue filtered off. All solutions were combined, dried over sodium sulfate and after filtration, the solvents were removed. The crude product was purified by column chromatography on silica gel with 10 | Journal Name, [year], [vol], 00–00

60

65

70

75

80

85

90

95

100

105

110

115

hexane/dichloromethane 2:3 as eluent (Rf ≈ 0.1). Removal of the solvents and subsequent column chromatography with dichloromethane/acetone 10:0.5 as mobile phase (Rf ≈ 0.6) afforded pure 8 (89 mg, 33 %) as orange powder. M.p.: 152 °C (decomp.); 1H NMR (200 MHz, CDCl3): δ = 1.42 (t, 3H, CH3), 3.24 (q, 2H, SCH2), 3.63 (s w/ Pt satellites, 3JPtH = 23.8 Hz, 6H, CH3(DMSO)), 7.00 (s, 1H, CHCS2), 7.50 (d, 3 JH2-H3 = 8.8 Hz, 2H, Ar-H2), 7.79 (d, 3JH2-H3 = 8.8 Hz, 2H, ArH3); 13C{1H} NMR (50 MHz, CDCl3): δ = 13.1 (CH3), 28.8 (8), 46.9 (s w/ Pt satellites, 2JPt-C = 59.6 Hz, CH3(DMSO)), 111.2 (CHCS2), 126.9 (Ar-C4), 129.4 (Ar-C2), 132.0 (Ar-C3), 136.0 (Ar-C1), 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 [M-1]+; elemental analysis calcd for C13H16BrClOPtS3: C, 25.56; H, 2.64; S, 15.75 %; found: C, 25.60; H, 2.53; S, 15.71 %.

Chloro-(1-(4-bromophenyl)-3-(hexylthio)-3-thioxo-prop-1-en1-olate-O,S)-(dimethylsulfoxide-S)-platinum(II) (9) According 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 sodium sulfate. The crude product was purified via column chromatography on silica gel (mobile phase: dichloromethane/hexane 3:2, Rf ≈ 0.2). The fraction containing the product was again purified via column chromatography on silica gel using dichloromethane as eluent (Rf ≈ 0.5). The product was crystallized from dichloromethane/pentane and dried under vacuum to yield 9 (34 mg, 12 %) as orange powder. Mp.: 150 °C (decomp.); 1H NMR (200 MHz, CDCl3): δ = 0.88 (m, 3H, CH3), 1.24-1.48 (m, 6H, 3x CH2), 1.77 (qui, 2H, SCH2CH2), 3.23 (t, 2H, SCH2), 3.64 (s w/ Pt satellites, 3JPtH = 23.4 Hz, 6H, CH3(DMSO)), 7.03 (s, 1H, CHCS2), 7.52 (d, 3 JH2-H3 = 8.8 Hz, 2H, Ar-H2), 7.81 (d, 3JH2-H3 = 8.6 Hz, 2H, ArH3); 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), 126.0 (Ar-C4), 129.4 (Ar-C2), 132.0 (Ar-C3), 136.0 (Ar-C1), 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 [M-Cl+H]+; elemental analysis calcd for C17H24BrClOPtS3: C,30.61, H, 3.63, S, 14.42 %, found: C, 30.77, H, 3.91, S, 13.94 %. Chloro-(1-phenyl-3-(methylthio)-3-thioxo-prop-1-en-1-olateO,S)-(dimethylsulfoxide-S)-platinum(II) (10) K2PtCl4 was conversed with 4 according to general procedure 2 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 eluent (Rf ≈ 0.1) and crystallized from dichloromethane/pentane. 10 (62 mg, 27 %)was yielded as orange needles. Mp.: 152 °C (decomp.); 1H NMR (200 MHz, CDCl3): δ = 2.67 (s, 3H, CH3), 3.65 (s w/ Pt satellites, 3JPt-H = 23.6 Hz, 6H, This journal is © The Royal Society of Chemistry [year]


DOI: 10.1039/C3DT52284A

Page 11 of 15


DOI: 10.1039/C3DT52284A

60

65


10

15

20

25

30

35

Chloro-(1-phenyl-3-(ethylthio)-3-thioxo-prop-1-en-1-olateO,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 bright 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 dichloromethane/hexane 3:2 as mobile pase (Rf ≈ 0.1). Evaporation of the solvents and drying in vacuum afforded pure 11 (29 mg, 12 %) as orange crystals. Single crystals suitable for X-ray analyses were obtained by slow evaporation of dichloromethane. Mp.: 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, 3 JPt-H = 23.6 Hz, 6H, CH3(DMSO)), 7.07 (s, 1H, CHCS2), 7.337.54 (m, 3H, Ar-H3,4), 7.89-7.96 (m, 2H, Ar-H2); 13C{1H} NMR (50 MHz, CDCl3): δ = 13.2 (CH3), 28.7 (SCH2), 46.8 (s w/ Pt satellites, 2JPt-C = 59.7 Hz, CH3(DMSO)), 111.6 (CHCS2), 128.0 (Ar-C2), 128.8 (Ar-C3), 132.0 (Ar-C4), 137.2 (Ar-C1), 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 [M-1]+; elemental analysis 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%.

70

75

80

85

90

95

40

45

50

55

Chloro-(1-phenyl-3-(hexylthio)-3-thioxo-prop-1-en-1-olateO,S)-(dimethylsulfoxide-S)-platinum(II) (12) Following general procedure 2, K2PtCl4 was conversed with 6 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 was reduced by evaporation and the crude product was purified via column chromatography on silica gel with dichloromethane/hexane 3:2 (Rf ≈ 0.1). After crystallization from dichloromethane/pentane, the pure product (49mg, 19 % of brownish powder) was dried in vacuum. Mp.: 150 °C (decomp.); 1H NMR (200 MHz, CDCl3): δ = 0.88 (m, 3H, CH3), 1.24-1.48 (m, 6H, 3x CH2), 1.77 (qui, 2H, SCH2CH2), 3.23 (t, 2H, SCH2), 3.64 (s w/ Pt satellites, 3JPtH = 23.6 Hz, 6H, CH3(DMSO)), 7.01 (s, 1H, CHCS2), 7.35-7.51 (m, 3H, Ar-H3,4), 7.81 (d, 3JH2-H3 = 7.2 Hz, 2H, Ar-H2); 13 C{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 (Ar-C2), 128.8 (Ar-C3), 132.0 (Ar-C4), 137.2 (Ar-C1), 174.6 (CO), 180.5 (CS2); FTIR (KBr): = 3005, 2955, 2926, This journal is © The Royal Society of Chemistry [year]

100

105

110

115

2856, 1505, 1488, 1468, 1294, 1265, 1223, 1144, 1023, 832, 772 cm-1; MS (FAB in nba): m/z =588 [M]+, 552 [M-Cl]+; elemental analysis calcd for C17H25ClOPtS3: C,34.72; H, 4.28; S, 16.36 %; found: C, 34.99; H, 3.73; S, 14.25 % 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 Scientific Inc.) under standard conditions (37°C, 5% CO2, H2Osaturation). Cells were cultivated in 75 cm2 cell culture flasks with Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with or without 10% (v/v) Fetal Calf Serum (FCS, Biochrom-Seromed). A549 cells were obtained from DSMZ GmbH (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 25th passage at most. The adherent cell cultures were divided every 2-3 days following standard procedures. Medium was removed and D-PBS (Dulbecco’s Phosphate-Buffered Saline) was used 3 x to wash off the medium. To detach the cells from the surface, Trypsin-EDTA (3 mL) was added and incubated for 3 min. The reaction was stopped with fresh medium containing FCS. Depending on the passaging ratio, the respective portions of cell suspension were transferred into new 75 cm2 cell culture flasks. For assays, cells were harvested at a density of 70-90 % confluency. Medium was removed and D-PBS was used as washing agent. Detachment with trypsin-EDTA (3 mL) was stopped with fresh medium containing FCS after 3 min. The desired number of cells (10,000 cells/well) was seeded into 96well plates. Wells were then supplied with medium (DMEM + 10 % FCS) to a final volume of 100 µL. Plates were preincubated for 1 day before adding drug samples. Drug Treatment Stock solutions were prepared in DMSO (Sigma Hybri-Max) at 4 mg/mL. Samples were prepared by diluting the DMSO stock solutions in D-PBS 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 96-well plate were supplemented with drug to reduce local growth effects. Preparing triplicates for each concentration, 10 µL sample or control solutions were added to the pre-incubated cells. For caspase 3/7 assays, 50 µL sample or control solutions were added to the wells after removal of 50 µL supernatant from the initial volume of 100 µL cell culture. In this case, duplicates of each concentration were prepared. 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 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


5

CH3(DMSO)), 7.11 (s, 1H, CHCS2), 7.35-7.55 (m, 3H, Ar-H3,4), 7.93-7.97 (m, 2H, Ar-H2); 13C{1H} NMR (50 MHz, CDCl3): δ = 17.6 (CH3), 46.9 (s w/ Pt satellites, 2JPt-C = 59.7 Hz, CH3(DMSO)), 111.4 (CHCS2), 127.9 (Ar-C2), 128.8 (Ar-C3), 132.0 (Ar-C4), 137.1 (Ar-C1), 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 %

Dalton Transactions


DOI: 10.1039/C3DT52284A


10

15

Apoptosis Assay For caspase 3/7 assay, the Apo-ONE Caspase-3/7 Buffer and Apo-ONE Caspase-3/7 Reagent (Promega Corporation) was used according to the supplier’s protocol. 100 µL caspase reagent mixture was added to each well and the liquids quickly mixed on a plate shaker. Cells were incubated for 2 h before reading the fluorescence at excitation wavelength of 499nm and emission wavelength of 521nm.

60

65

70

75

20

25

30

35

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 without 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. IC50 values were determined separately for each of three independent experiments, averaged and converted to molar concentration. For their calculation, single fluorescence intensities were used instead of their mean values. Regression was performed with the ReaderFit online tool (Hitachi Solutions America, Ltd.), applying four parameter logistics (4PL) model equations. The IC50 value obtained resembles the inflection point of the respective curve. For further information, cf. the ESI†.

80

45

50

55

UV-visible spectrophotometry UV-Vis absorption spectra were recorded on a Varian Cary 50 UV-Vis spectrophotometer in the range of 200-800 nm. Stock solutions (10 mM) of each compound were prepared by dissolving the complex under investigation in DMSO. For all experiments, the final complex concentration was 100 µM. For solubility studies, UV-vis measurements were carried out by diluting the compounds' stock solutions to 100 µM in phosphate buffer (PB, 25 mM, pH 7.4) with varying content of DMSO (final percentages 1 %, 10 %, 50 %). Spectra were 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. UV-Vis absorption spectra were periodically recorded by diluting aliquots to the final concentration of 100 µM in DMSO. Equally, samples were kept at 37 °C for 24h and the UV-Vis absorption spectra recorded before and after incubation. 12 | Journal Name, [year], [vol], 00–00

Protein interaction studies with cyt c and HEWL: ESI mass spectrometry Samples were prepared by dissolving horse heart cyt c (Sigma C7752) (100 µM) resp. chicken egg white lysozyme (Sigma L7651) (100 µM) in tetramethylammonium acetate buffer (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. After a 20-fold dilution with water, the ESI MS spectrum was recorded in an LTQ-Orbitrap high-resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with a conventional ESI source (direct introduction, flow rate 5 µL/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 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.

85

90

95

Mechanistic investigations 40

For hydrolysis studies, a stock solution of compound 11 was diluted to 1 mM in DMSO or DMSO/water 50%/50% and a 10fold excess of AgNO3, dissolved in DMSO, added. Spectra were recorded from 100µM solutions (diluted in the respective solvent composition) before and directly after addition of the silver salt.

100

105

110

Protein interaction studies with albumin: ICP-OES Bovine serum albumin (BSA, Fluka BioChemika 05470) (100 µM) was dissolved in TMAA (25 mM, pH 7.4) and the Pt compound was added from a DMSO stock solution (10mM) to give a final metal:protein ratio 1:1. Samples were then incubated at 37 °C for 96 h and then ultrafiltration was performed 3 times in Centricon YM-10 centrifugal filters (Amicon Bioseparations, Millipore Corporation) for 30 min at 3500 rpm in using water as diluent. All solutions were separately investigated by ICP-OES measurement on a Varian 720-OES instrument with 1.25 ppm of Ge as internal standard. Samples were diluted 16-fold and digested with HNO3 prior 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 UV-visible spectrophotometry according to the above-stated 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. 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 incubated 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 ICP-OES.

Acknowledgements 115

The Authors wish to thank Federica Scaletti and Lara Massai at University of Florence for help with UV-visible This journal is © The Royal Society of Chemistry [year]


5

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 i-control 1.6) at an excitation wavelength of 560nm and emission wavelength of 590nm.

Page 13 of 15


5

spectrophotometry and as well as Patricia Marques-Gallego at Massachusetts Institute of Technology for helpful discussions. COST Actions CM1105 and D39 (STSM 250310) as well as the DAAD Vigoni Program are gratefully acknowledged. C.M. thanks Carl-Zeiss-Stiftung as well as Studienstiftung des Deutschen Volkes for scholarships. C.G. gratefully acknowledges Beneficentia Stiftung for financial support. Heraeus Precious Metals is acknowledged for a generous gift of K2PtCl4.

70

75


Notes and references 10

15

20

25

30

35

40

45

50

55

60

65

a

Institute of Inorganic and Analytical Chemistry, Friedrich-SchillerUniversity Jena, Humboldtstraße 8, 07743 Jena, Germany, Fax: (+49) 3641 948102, E-mail: [email protected] b Department of Chemistry and Industrial Chemistry, University of Pisa, via Risorgimento 35, 56126 Pisa, Italy c Mass Spectrometry Center (CISM), University of Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy d Department of Internal Medicine II, Jena University Hospital, Erlanger Allee 101, 07740 Jena, Germany e Laboratory of Metals in Medicine, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy, Fax: (+39) 055 4573385, E-mail: [email protected] f Jena Center for Soft Matter (JCSM), Philosophenweg 7, 07743 Jena, Germany † Electronic Supplementary Information (ESI) available: Additional syntheses, Crystallographic data of 7, 11, 13 and 14, Behavior of 7-12 in solution, Biological assay data and Mass spectrometric data of 7-12. See DOI: 10.1039/b000000x/ Crystallographic data (excluding structure factors) has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication CCDC-915584 for 7, CCDC-915585 for 11, CCDC-915586 for 13, and CCDC-915587 for 14. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [E- mail: [email protected]]. ○ Deeper insight could in principle be gained by recording 1H NMR spectra of the complexes under the desired conditions. The solubility of the compounds in water is unfortunately too low for adequate data aquisition and when using DMSO as deuterated solvent, residual water overlays the peaks of coordinated DMSO. 1H NMR signals of coordinated or uncoordinated DMSO would however give the only reliable reference signal to prove ligand substitution of the monodentate ligands. The same solubility issues limit the applicability of 195Pt NMR spectroscopy.

1. 2.

M. Galanski, Anti-Cancer Agents Med. Chem., 2007, 7, 1–2. M. J. Cleare and J. D. Hoeschele, Platinum Met. Rev., 1973, 17, 2– 13. 3. M. J. Cleare and J. D. Hoeschele, Bioinorg. Chem., 1973, 2, 187– 210. 4. E. Wong and C. M. Giandomenico, Chem. Rev., 1999, 99, 2451– 2466. 5. M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger, and B. K. Keppler, Dalton Trans., 2008, 183–194. 6. A. M. Thayer, Chem. Eng. News, 2010, 88. 7. U.S. National Library of Medicine, http://clinicaltrials.gov/ct2/results?term=satraplatin&Search=Searc h, accessed: 2013-08-16. 8. A. O. Sartor, D. P. Petrylak, J. A. Witjes, W. R. Berry, G. S. Chatta, D. J. Vaughn, J. Ferrero, T. Demkow, J. C. Eymard, and C. N. Sternberg, J. Clin. Oncol., 2008, 26, 5003. 9. U.S. National Library of Medicine, http://clinicaltrials.gov/ct2/results?term=picoplatin&Search=Search , accessed 2013-08-16. 10. J. R. Eckardt, D. L. Bentsion, O. N. Lipatov, I. S. Polyakov, F. R. MacKintosh, D. A. Karlin, G. S. Baker, and H. B. Breitz, J. Clin. Oncol., 2009, 27, 2046–2051.


80

85

90

95

100

105

110

115

120

125

130

135

11. T. Ciuleanu, M. Samarzjia, Y. Demidchik, V. Beliakouski, M. Rancic, D. L. Bentsion, S. V. Orlov, B. A. Schaeffler, R. L. De Jager, and H. B. Breitz, J. Clin. Oncol., 2010, 28, 7002. 12. H. Taube, Chem. Rev., 1952, 50, 69–126. 13. J. Reedijk, Platinum Met. Rev., 2008, 52, 2–11. 14. J. Reedijk, Macromol. Symp., 2008, 270, 193–201. 15. M. Galanski and B. K. Keppler, Anti-Cancer Agents Med. Chem., 2007, 7, 55–73. 16. S. E. Sherman and S. J. Lippard, Chem. Rev., 1987, 87, 1153–1181. 17. J. Reedijk, Chem. Rev., 1999, 99, 2499–2510. 18. R. G. Pearson, J. Am. Chem. Soc., 1963, 85, 3533–3539. 19. A. Casini and J. Reedijk, Chem. Sci., 2012, 3, 3135–3144. 20. D. Esteban-Fernández, E. Moreno-Gordaliza, B. Canas, M. Antonia Palacios, and M. Milagros Gómez-Gómez, Metallomics, 2010, 2, 19– 38. 21. A. R. Timerbaev, C. G. Hartinger, S. S. Aleksenko, and B. K. Keppler, Chem. Rev., 2006, 106, 2224–2248. 22. X. Wang and Z. Guo, Anti-Cancer Agents Med. Chem., 2007, 7, 19– 34. 23. Y. Kasherman, S. Sturup, and D. Gibson, J. Med. Chem., 2009, 52, 4319–4328. 24. J. D. Holding, W. E. Lindup, C. van Laer, G. C. Vreeburg, V. Schilling, J. A. Wilson, and P. M. Stell, Br. J. Clin. Pharmacol., 1992, 33, 75–81. 25. J. Reedijk, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 3611. 26. J. Reedijk, Inorg. Chim. Acta, 1992, 198, 873–881. 27. C. Rothenburger, M. Galanski, V. B. Arion, H. Görls, W. Weigand, and B. K. Keppler, Eur. J. Inorg. Chem., 2006, 2006, 3746–3752. 28. A. Casini, C. Gabbiani, G. Mastrobuoni, L. Messori, G. Moneti, and G. Pieraccini, ChemMedChem, 2006, 1, 413–417. 29. A. Casini, G. Mastrobuoni, C. Temperini, C. Gabbiani, S. Francese, G. Moneti, C. T. Supuran, A. Scozzafava, and L. Messori, Chem. Commun., 2007, 156–158. 30. J. Kozelka, Inorg. Chim. Acta, 2009, 362, 651–668. 31. J. Kozelka, F. Legendre, F. Reeder, and J.-C. Chottard, Coord. Chem. Rev., 1999, 190-192, 61–82. 32. K.-L. Lu, J.-R. Hwu, S.-C. Tsay, S.-F. Yu, J.-T. Hung, and J.-J. Huang, US6458833 (B1), EP1394169 (A1), 2002. 33. M. D. Reily, K. Wilkowski, K. Shinozuka, and L. G. Marzilli, Inorg. Chem., 1985, 24, 37–43. 34. P.-C. Kong, D. Iyamuremye, and F. D. Rochon, Bioinorg. Chem., 1976, 6, 83–89. 35. A. Muscella, N. Calabriso, S. A. De Pascali, L. Urso, A. Ciccarese, F. P. Fanizzi, D. Migoni, and S. Marsigliante, Biochem. Pharmacol., 2007, 74, 28–40. 36. A. Muscella, N. Calabriso, F. P. Fanizzi, S. A. De Pascali, L. Urso, A. Ciccarese, D. Migoni, and S. Marsigliante, Br. J. Pharmacol., 2008, 153, 34–49. 37. S. A. De Pascali, F. Lugoli, A. De Donno, and F. P. Fanizzi, MetalBased Drugs, 2011, 2011, 1–10. 38. R. Gompper and H. Schaefer, Chem. Ber., 1967, 100, 591–604. 39. F. C. V. Larsson and S. O. Lawesson, Tetrahedron, 1972, 28, 5341– 5357. 40. G. Singh, S. S. Bhattacharjee, H. Ila, and H. Junjappa, Synthesis, 1982, 693–694. 41. R. Saumweber, C. Robl, and W. Weigand, Inorg. Chim. Acta, 1998, 269, 83–90. 42. K. Schubert, R. Saumweber, H. Görls, and W. Weigand, Z. Anorg. Allg. Chem., 2003, 629, 2091–2096. 43. K. Schubert, H. Goerls, and W. Weigand, Heteroat. Chem., 2005, 16, 369–378. 44. K. Schubert, T. Alpermann, T. Niksch, H. Görls, and W. Weigand, Z. Anorg. Allg. Chem., 2006, 632, 1033–1042. 45. K. Schubert, H. Görls, and W. Weigand, Z. Naturforsch., 2007, 62b, 475–482. 46. F. D. Rochon and C. Tessier, Inorg. Chim. Acta, 2008, 361, 2591– 2600. 47. Kukushkin, Y. N., Vyaz’menskii, Y. E., and Zorina, L. I., Russ. J. Inorg. Chem., 1968, 13, 1573–1576. 48. J. H. Price, A. N. Williamson, R. F. Schramm, and B. B. Wayland, Inorg. Chem., 1972, 11, 1280–1284.

Journal Name, [year], [vol], 00–00 | 13


DOI: 10.1039/C3DT52284A

Dalton Transactions


DOI: 10.1039/C3DT52284A


10

15

20

25

30

35

40

45

50

55

60

65



5

49. F. D. Rochon, C. Bensimon, and C. Tessier, Inorg. Chim. Acta, 2008, 361, 16–28. 50. D. J. Aitken, H.-P. Husson, D. Nguyen-Huy, S. Ongeri, F. Vergne, and B. Viossat, Inorg. Chem. Commun., 1998, 1, 314–316. 51. N. Farrell, WO 8909598, 1989. 52. N. Farrell, D. M. Kiley, W. Schmidt, and M. P. Hacker, Inorg. Chem., 1990, 29, 397–403. 53. H. Motschi, P. S. Pregosin, and H. Ruegger, J. Organomet. Chem., 1980, 193, 397–405. 54. S. F. Kaplan, V. Y. Kukushkin, and A. J. L. Pombeiro, J. Chem. Soc., Dalton Trans., 2001, 3279–3284. 55. G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb, A. Nudelman, B. M. Stoltz, J. E. Bercaw, and K. I. Goldberg, Organometallics, 2010, 29, 2176–2179. 56. S. A. De Pascali, P. Papadia, A. Ciccarese, C. Pacifico, and F. P. Fanizzi, Eur. J. Inorg. Chem., 2005, 2005, 788–796. 57. K. Schubert, PhD. Thesis, Friedrich-Schiller-University Jena, 2006. 58. W. D. Horrocks and F.A. Cotton, Spectrochim. Acta, 1961, 17, 134– 147. 59. G. Geiseler and G. Hanschmann, J. Mol. Struct., 1971, 8, 293–306. 60. S. E. McLain, A. K. Soper, and A. Luzar, J. Chem. Phys., 2006, 124, 074502. 61. I. García-Orozco, M. C. Ortega-Alfaro, J. G. López-Cortés, R. A. Toscano, and C. Alvarez-Toledano, Inorg. Chem., 2006, 45, 1766– 1773. 62. A. L. Niles, R. A. Moravec, and T. L. Riss, Curr Chem Genomics, 2009, 3, 33–41. 63. L. Di and E. H. Kerns, Drug Discovery Today, 2006, 11, 446–451. 64. B. Zhivotovsky, A. Samali, and S. Orrenius, in Current Protocols in Toxicology, John Wiley & Sons, Inc., 2001. 65. X. Yang, F. Zheng, H. Xing, Q. Gao, W. Wei, Y. Lu, S. Wang, J. Zhou, W. Hu, and D. Ma, J. Cancer Res. Clin. Oncol., 2004, 130, 423–428. 66. X.-H. Yang, T. L. Sladek, X. Liu, B. R. Butler, C. J. Froelich, and A. D. Thor, Cancer Res., 2001, 61, 348–354. 67. J. J. Cali, A. Niles, M. P. Valley, M. A. O’Brien, T. L. Riss, and J. Shultz, Expert Opin. Drug Metab. Toxicol., 2007, 4, 103–120. 68. C. Mügge, E. Micheucci, F. Boscaro, C. Gabbiani, L. Messori, and W. Weigand, Metallomics, 2011, 3, 987–990. 69. A. Casini, A. Guerri, C. Gabbiani, and L. Messori, J. Inorg. Biochem., 2008, 102, 995–1006. 70. J. Ghuman, P. A. Zunszain, I. Petitpas, A. A. Bhattacharya, M. Otagiri, and S. Curry, J. Mol. Biol., 2005, 353, 38–52. 71. F. Kratz, J. Controlled Release, 2008, 132, 171–183. 72. A. I. Ivanov, J. Christodoulou, J. A. Parkinson, K. J. Barnham, A. Tucker, J. Woodrow, and P. J. Sadler, J. Biol. Chem., 1998, 273, 14721–14730. 73. A. R. Timerbaev, S. S. Aleksenko, K. Polec-Pawlak, R. Ruzik, O. Semenova, C. G. Hartinger, S. Oszwaldowski, M. Galanski, M. Jarosz, and B. K. Keppler, Electrophoresis, 2004, 25, 1988–1995. 74. J. Will, D. A. Wolters, and W. S. Sheldrick, ChemMedChem, 2008, 3, 1696–1707. 75. C. Brauckmann, C. A. Wehe, M. Kieshauer, C. Lanvers-Kaminsky, M. Sperling, and U. Karst, Anal Bioanal Chem, 2013, 405, 1855– 1864. 76. C. Gabbiani, A. Casini, G. Mastrobuoni, N. Kirshenbaum, O. Moshel, G. Pieraccini, G. Moneti, L. Messori, and D. Gibson, J. Biol. Inorg. Chem., 2008, 13, 755–764. 77. T. Peleg-Shulman, Y. Najajreh, and D. Gibson, J. Inorg. Biochem., 2002, 91, 306–311. 78. Nonius, Collect Data collection software, Nonius BV, Delft, The Netherlands, 1998. 79. Z. Otwinowski and W. Minor, in Methods in Enzymology, ed. J. Charles W. Carter, Academic Press, 1997, vol. 276, pp. 307–326. 80. R. H. Blessing, J. Appl. Crystallogr., 1997, 30, 421–426. 81. G. M. Sheldrick, Acta Cryst.allogr., Sect. A: Found. Crystallogr., 2007, 64, 112–122.


Page 15 of 15


DOI: 10.1039/C3DT52284A

table of contents entry.

Carolin Mügge,a Ruiqi Liu,a Helmar Görls,a Chiara Gabbiani,b Elena Michelucci,c Nadine Rüdiger,d Joachim H. Clement,d Luigi Messori,*e Wolfgang Weigand*a,f

A new building block system of Pt(II) compounds with a O,S-binding moiety exhibits anticancer activity and promising structure-activity relationships.



Novel Platinum(II) Compounds with O,S Bidentate Ligands: Synthesis, Characterization, Antiproliferative Properties and Biomolecular Interactions

New ruthenium(II) coordination compounds possessing bidentate aminomethylphosphane ligands: synthesis, characterization and preliminary biological study in vitro.

Synthesis, characterization and biological evaluation of transition metal complexes derived from N, S bidentate ligands.

Novel antiproliferative chimeric compounds with marked histone deacetylase inhibitory activity.

Light-stable bis(norharmane)silver(I) compounds: synthesis, characterization and antiproliferative effects in cancer cells.

Synthesis and antiproliferative activity of novel polynuclear heterocyclic compounds derived from 2,3-diaminophenazine.

Synthesis and antiproliferative activity of novel methylselenocarbamates.

Crystal structures and spectral properties of two polyoxometalate-based inorganic-organic compounds from silver-azine building blocks with bis-bidentate and tridentate ligands.

Synthesis, antiproliferative activity and DNA binding properties of novel 5-aminobenzimidazo[1,2-a]quinoline-6-carbonitriles.

Platinacycloalkane complexes containing [P,N] bidentate ligands: synthesis and decomposition studies.

Synthesis, spectral and catalytic dehydrogenation studies of ruthenium complexes containing NO bidentate ligands.

Synthesis, characterization and photophysical properties of novel azole derivatives.

Determining the electron-donating properties of bidentate ligands by (13)C NMR spectroscopy.

Aluminum complexes with bidentate amido ligands: synthesis, structure and performance on ligand-initiated ring-opening polymerization of rac-lactide.

Synthesis and Antiproliferative and Metabolic Evaluations of Novel Securinine Derivatives.

Antiproliferative novel isoxazoles: modeling, virtual screening, synthesis, and bioactivity evaluation.

New heterocyclic compounds: Synthesis and antitrypanosomal properties.

MM Quadruply Bonded Complexes Supported by Vinylbenzoate Ligands: Synthesis, Characterization, Photophysical Properties and Application as Synthons.

Synthesis and Antiproliferative Activity of Novel Heterocyclic Indole-Trimethoxyphenyl Conjugates.

Two novel extraction chromatography resins containing multiple diglycolamide-functionalized ligands: preparation, characterization and actinide uptake properties.

Synthesis, characterization and biological evaluation of novel Ru(II)-arene complexes containing intercalating ligands.

Design and synthesis of novel antileishmanial compounds.

Modeling-Enabled Characterization of Novel NLRX1 Ligands.

Synthesis, characterization and luminescent properties of lanthanide complexes with a novel multipodal ligand.

Synthesis, characterization, and antioxidant properties of novel inulin derivatives with amino-pyridine group.