Trans–cis–cis-[RuCl2(DMSO)2(2-amino-5-methyl-thiazole)2], (PMRu52), a novel ruthenium(II) compound acting as a strong inhibitor of cathepsin B

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Abstract

A novel ruthenium(II) compound, trans–cis–cis–[Ru(II)Cl2(DMSO)2(2-amino-5-methyl-thiazole)2], (I), PMRu52 hereafter, that may be obtained from the previously described (cis and trans)-[Ru(II)Cl2(DMSO)4] complexes, was designed, synthesized and characterised. The single crystal X-ray structure shows a roughly regular octahedral environment for the ruthenium(II) center with the two chloride ligands in trans and the other two pairs of identical ligands in cis. The behaviour of PMRu52 in phosphate buffer, at pH = 7.4, was characterised spectroscopically as well as its interactions with a few representative biomolecules. Tight ruthenium binding to serum albumin was established by joint use of spectroscopic and separation methods. Afterward, the reactions of PMRu52 with the model proteins ubiquitin and cytochrome c were monitored through electrospray ionisation mass spectrometry (ESI-MS) methods: the formation of metallodrug–protein adducts was documented in detail and the fragmentation patterns of PMRu52 were defined. Finally, the ability of PMRu52 to affect the activity of cathepsin B, a well known cysteine protease, was evaluated in vitro and a pronounced enzyme inhibition highlighted, with an IC50 value of 5.5 μM. This latter finding is of particular interest as cathepsin B constitutes an attractive “druggable” target for cancer, rheumatoid arthritis and other important diseases.

Introduction

Ruthenium compounds form a class of metallopharmaceuticals, of potential wide application in various disease areas [1]. An extensive overview of the possible medical uses of ruthenium compounds was provided, a few years ago, by Michael Clarke [2]. A number of studies have exploited the use of ruthenium compounds as experimental anticancer agents [3]. For instance, Keppler and coworkers developed a large family of anionic ruthenium(III) compounds, of general formula [Ru(III)Cl4L2] (where L is an heterocycle), most of which showing outstanding activity against colorectal cancer models [4], [5]. In turn, Alessio and Mestroni prepared and characterised NAMI A (imidazolium trans-[tetrachloro(DMSO)(imidazole)ruthenate(III)]), a mixed-ligand ruthenium(III)–DMSO complex that manifested outstanding antimetastatic properties [6]. More recently, a conspicuous number of ruthenium(II) arene and ruthenium(II) arene–PTA compounds, endowed with encouraging anticancer properties, were prepared and tested by Peter Sadler [7], [8] and Paul Dyson [9], respectively. As a result of the numerous research activities concerning their potential anticancer properties, two ruthenium compounds – i.e. NAMI A and KP1019 – have already entered clinical trials while a few others, in particular the ruthenium arenes, are currently undergoing advanced preclinical testing.

A mononuclear mixed-ligand ruthenium(II) complex, trans-[RuCl2(DMSO)4], (II), was the subject of much attention starting from the late 1980s. Mestroni and Alessio determined its crystal structure, analysed its solution behaviour and described some of its biological properties [10]. Afterward, extensive pharmacological studies were carried out by Sava et al., working both on the cis and trans isomers, and rather encouraging anticancer properties were disclosed [11], [12]. However, in subsequent years, owing to the success of NAMI A, the interest of those researchers rapidly shifted toward ruthenium(III) compounds [13], so that the studies on the antitumor properties of ruthenium(II) complexes were nearly completely abandoned.

Upon consideration of those early promising results and of the usually favourable pharmacological profile of ruthenium drugs, we thought that additional ruthenium(II) compounds might be designed and prepared starting from cis-[Ru(II)Cl2(DMSO)4], III [10], [14] and trans-[Ru(II)Cl2(DMSO)4], II [15] through (partial) substitution of the DMSO ligands. Specifically, we hypothesized that progressive replacement of DMSO ligands with aromatic heterocycles might favourably affect the chemical and biological properties of the resulting species and modify its interactions with biomolecular targets. Indeed, at least in principle, it may be assumed that heterocycles are somewhat stronger and less labile ruthenium ligands than DMSO leading to a higher stability of the resulting complexes. These aspects were considered in depth in some previous literature [16], [17], [18] and in a few recent computational studies [19], [20].

With this in mind, we designed a novel compound, trans–cis–cis-[Ru(II) Cl2(DMSO)2(2-amino-5-methyl-thiazole)2] (I), PMRu52 hereafter, formally derived from trans-[Ru(II)Cl2(DMSO)4], in which two adjacent DMSO ligands are replaced by two 2-amino-5-methyl–thiazole ligands (Chart 1). PMRu52 can be obtained starting from either II or III, under the same reaction conditions, with nearly equal yields; this possibility was already stated by other authors 15working with different ligands. Notably, it was previously reported that cis-[RuCl2(DMSO)4] and trans-[RuCl2(DMSO)4] typically react with excess azole heterocycles to produce six-coordinate complexes of the general formula RuCl2(DMSO)2(azole)2, where azole = imidazole, 4-nitroimidazole, dimethylimidazole, benzimidazole, 1,5,6-trimethylbenzimidazole, pyrazole and indazole (often as individual isomers) [21], [22], [23], [24], [25], [26].

We decided to use the cis isomer (III) as starting material only for the ease of its preparation; in this case, as already suggested, substitution of two DMSO ligands is accompanied by a rearrangement of the RuCl2 fragment from cis to trans configuration [16]. Remarkably, compound I was the only isomer – out of the five theoretically possible – resulting from the various synthetic procedures we carried out with no appreciable amounts of secondary products.

The choice of 2-amino-5-methyl-thiazole was suggested to us by previous use of this ligand in the synthesis of ruthenium complexes and by the attractive properties of one of its compounds. Indeed, the “Keppler-type” ruthenium(III) complex bearing two 2-amino-5-methyl-thiazole molecules as axial ligands was previously characterised in our laboratory and found to act as a strong inhibitor of thioredoxin reductase [27].

However, PMRu52 bears significant differences with respect to that previous compound owing to the presence of ruthenium in the oxidation state +2, to the cis type configuration of the two heterocyclic ligands and to the presence of two DMSO moieties as additional ligands.

Section snippets

Materials

Solvents and reagents were used as received without any further purification.

Physical measurements

Solid state infrared spectra (CsI pellets) were recorded on a Perkin–Elmer 16PC FT IR spectrophotometer. Electronic absorption spectra were carried out with a Lambda 20 Bio Perkin–Elmer instrument.

Synthesis of the starting materials

Cis-[Ru(II)Cl2(DMSO)4] [10], [14] and trans-[Ru(II)Cl2(DMSO)4] [15] were synthesized as reported in the literature.

Synthesis of trans,cis,cis,-dichlorobis (dimethyl sulfoxide) bis(2-amino-5-methyl-thiazole) ruthenium(II) ½ C2H5OH, trans–cis–cis-[Ru(II)Cl2(DMSO)2(2-amino-5-methyl-thiazole)2]·½ C2H5OH, (I, PMRu52)

This new complex can be synthesized either starting from cis- or trans-Ru(II)Cl2(DMSO)4, obtaining the same

Structural analysis

The crystal structure of PMRu52 (I), (Fig. 1), was solved [29] by examining the nice crystals directly obtained in the course of the synthesis; relevant crystal data for I are given in Table 1 (full crystallographic details are provided in the supplementary material, CIF file). Upon inspection of the structure it is evident that the two chloride ligands are positioned in trans whereas the two dimethylsulfoxide and the two 2-amino-5-methyl-thiazole groups are in cis each other. The overall

Concluding remarks

In conclusion, with the present study, we have designed, prepared and characterised a novel ruthenium(II) compound, PMRu52, exhibiting quite promising biological properties. Notably, PMRu52 has been found to manifest a high propensity to react with proteins as it emerges from studies on serum albumin and on the model proteins cyt c and ubq. Protein binding of PMRu52 takes place after ruthenium(II) activation through release of some of its original ligands. Even more remarkably, PMRu52,

Acknowledgements

Thanks are due to Prof. Marcello Colapietro, Department of Chemistry, Rome1 University “La Sapienza” – Rome – Italy, for the X-ray data collection of (I, PMRu52). Thanks are also due to the referees for theirs very useful suggestions. We thank Dr. Elena Michelucci from CISM (University of Florence) for recording the ESI-MS spectra. A.C. thanks SNSF for funding (Ambizione project n°PZ00P2-121933).

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