Novel Unsymmetrical Ru(III) and Mixed-valence Ru(III)/Ru(II) Dinuclear Compounds Related to the Antimetastatic Ru(III) Drug NAMI-A

In this paper we report the stepwise preparation and the characterization of new unsymmetrical monoanionic Ru(III) dinuclear compounds, [NH4][{trans-RuCl4(Me2SO-S)}(μ-L){mer-RuCl3(Me2SO-S)(Me2SO-O)}] (L = pyz (1), pym (2)). By a similar synthetic approach we also prepared new mixed-valence Ru(III)/Ru(II) dinuclear compounds of formula [NH4][{trans-RuCl4(Me2SO-S)}(μ-pyz){cis,cis,cis-RuCl2(Me2SO-S)2(CO)}] (L = pyrazine (pyz, 3), pyrimidine (pym, 4)). Moreover, we describe the chemical behavior of compounds 1-4 in physiological solution, also after complete reduction (with ascorbic acid) to the corresponding Ru(II)/Ru(II) species. Overall, the chemical behavior of 1 and 2 after reduction resembles that of the corresponding dianionic and neutral dinuclear species, [{trans-RuCl3(Me2SO-S)}2(μ-L)]2−and [{mer-RuCl3(Me2SO-S)(Me2SO-O)}2 (μ-L)]. On the other hand, the mixed-valence dinuclear compounds 3 and 4, owing to the great inertness of the cis,cis,cis-RuCl2(Me2SO-S)2(CO)(1/2μ-L) fragment, behave substantially like the mononuclear species [trans-RuCl4(Me2SO-S)(L)]− in which the terminally bonded L ligand can be considered as bearing a bulky substituent on the other N atom.


INTRODUCTION
The introduction of the Ru(IlI)-dimethylsulfoxide complex ImH[trans-RuCl4(Me2SO-S)Im] (NAMI-A, Im imidazole, [1]) into phase I clinical trials as antimetastatic drug (October 1999) prompted us to investigate also other closely related ruthenium compounds. Within this frame, stimulated by the excellent results obtained by the platinum antieaneer dimers and trimers developed by Farrell  The coordination environment of each ruthenium(III) center in the dianionic species a is essentially the same as in the mononuclear compound NAMI-A (four trans chlorides, one S-bonded dimethylsulfoxide and one heterocyclic N-ligand). Preliminary results from in vivo tests on the murine MCa carcinoma model showed that some of the dianionic species were as effective as NAMI-A in reducing the spontaneous metastases (to about 5% with respect to controls) at a dosage 3.5 times lower in terms of ruthenium compared to that normally used for NAMI-A [4]. The neutral dinuclear compounds b, based on the known chemical behavior of the corresponding mononuclear species mer-RuCI3(Me2SO-S)(MezSO-O)(L) [5], are expected to Dinuclear Compounds Related of the Antimetastatic Ru(III) Drug Nami-A. behave differently compared to the dianionic analogs a and might therefore give different biological responses as well; however, they could not be tested owing to very low solubility in aqueous solution . We thought thus of combining the chemical environments of both neutral and dianionic species and prepared a new series of unsymmetrical dinuclear compounds, [NI][{trans-RuCh(Me2SO-S)}Ot-L){mer-RuCI3(MezSO-S)(Me2SO-O)}] (L pyz (1), pym (2), Figure 2), whose negative charge is expected to provide appreciable solubility in aqueous solution. By a similar synthetic approach we also prepared new mixed-valence Ru(III)/Ru(II) dinuclear  Beside the preparation and characterization of the novel unsymmetrical dinuclear compounds 1-4, in this paper we also describe their chemical behavior in physiological solution and after complete reduction (with ascorbic acid) to the corresponding Ru(II)/Ru(II) species.

MATERIALS AND METHODS
The bridging ligands were purchased from Aldrich and used as received. Electronic absorption spectra were recorded in quartz cells with a Jasco UV/vis V500 spectrophotometer equipped with a Peltier thermostatic unit. Infrared spectra were obtained on a Perkin-Elmer 983 G spectrometer. H-NMR spectra (D20, Aldrich) were collected at 400 MHz on a Jeol EX 400 FT spectrometer; spectra were recorded at room temperature with 2,2-dimethyl-2,2-silapentane-5-sulfonate (DSS) as an internal standard. An inversion recovery pulse sequence (n r-n/2) was applied for recording the spectra of the paramagnetic compounds (spectral window 30.000 Hz, pulse delay 0.5 s); with : 100 ms the resonances of molecules not coordinated to Ru(III) were negative, while those of the coordinated ligands were positive. A 0.1 mol dm 3 pH* 7.4 phosphate-D20 buffer (pH meter reading from D20 solutions) containing 0.9% NaCI was used for NMR experiments aimed to monitor the chemical behavior of the dimers in physiological solution. [7], cis, cis, cis-RuCl2(MezSO-S)2(CO)(pyz) [7], and cis, cis, cis-RuClz(MezSO-S)z(CO)(pym) [7] were prepared according to the reported procedures. precipitate formed within two days at ambient temperature, and was then collected by filtration, washed with cold methanol and diethyl ether, and vacuum dried at ambient T (0.044 g, 47%). According to elemental analysis and NMR spectroscopy the product contains one CH3OH molecule of crystallization; found C, 16

Structure determination
The dataset was collected at room temperature on an Enraf-Nonius CAD4 diffractometer, equipped with graphite monochromator and Mo-Kct radiation (k 0.71073 A). Intensities data were corrected for Lorentzpolarization effects and absorption through an empirical ap-scan method. The structure was solved by conventional Patterson and Fourier technique [8] and refined by full-matrix anisotropic least-squares method [9]. A molecule of methanol was located on the AF map with the oxygen atom disordered over two positions refined to occupancies of 0.76 and 0.24. All the calculations were performed using the WinGX System, Vet 1.63 [lO]. Regardlesss of the pathway chosen, in the crucial step an anionic Ru(III) species has to react with a neutral species and the choice of the most appropriate solvent is of paramount importance. We focused on pyz and pym ligands and found that their coordination to the neutral precursor mer-RuCl3(MezSO)3, followed by treatment with the anionic fragment [trans-RuCl4(Me2SO-S)2], gives best results. Firstly we found that pyrazine protons are broadened beyond detection by the two close paramagnetic Ru(III) centers. The NMR spectrum of the corresponding dinuclear species with pyrimidine, 2, shows also a rather broad and weak resonance at 6 -9 attributed to H5, i.e. the proton of the bridging pym ligand which is further removed from the Ru(III) nuclei. The X-ray structure of I was also determined. 2.291(2) A) [11]. In fact, in one case the conformation is that usually found in Ru-(Me2SO-S) complexes  1(4) , respectively. The S-coordinated ligands, like the free sulfoxides, are able to give rise to H-bonding with protic solvents [13]. In the present case, the sulfoxide oxygen O(1) interacts electrostatically with two ammonium ions at 2.93 and 2.96 A, leading to a lengthening of the S(I)-O(1) bond distance (1.489(6) A), with respect to the value of 1.451(7) A observed for S(2)-O(2). These interactions might be also responsible for the elongation detected in the Ru-S(I) bond length. As for the conformation of the Me2SO-O, the torsion angles Ru-O-S-C of about +128 indicate a trans-trans orientation of the methyl groups, which correponds to a minimum in the strain energy profile [12]. The structure of the anion results slightly bowed along the Ru-pyrazine-Ru vector, as evidenced by the dihedral angles made by the best fit planes through the N base (r.m.s. 0.013) and the Ru (1) and Ru(2) equatorial mean planes of 81.4(2) and 87.8(2) , respectively, the former being considerably apart from the ideal value of 90

RESULTS AND DISCUSSION
Overall, the structural parameters of compound 1 are comparable to those previously reported by us for the symmetrical dinuclear Ru complexes of type a and b [3].
Chemical behavior in physiological solution.
Aqueous solutions of the dimeric species 1-4 are characterized by a rather strong absorption band 5000 / 9000 dm mol "1 cm1) in the visible region of the electronic spectrum (380+400 nm), attributed to a charge-transfer from the chloride ligands to Ru(III). The time profiles of the visible spectra were used to investigate the chemical behavior of 1-4 at physiological pH.
The spectral changes of the symmetrical dinuclear species a at pH 7.4 were relatively simple and attributed to stepwise chloride hydrolysis from both Ru(III) centers [3]; conversely, for 1 and 2 a general decrease of the main absorption band, not accompanied by the formation of any new clear spectral feature, was observed. When the chemical behavior of I was monitored by H NMR spectroscopy, the resonance of free dimethylsulfoxide at 6 2.72 increased with time; dissociation of the O-bonded ligand prevailed, as its resonance decreased faster than that of Me2SO-S. Moreover, even though no relevant fragmentation of the dinuclear structure was observed, relatively sharp resonances increased with time at b 2.55 and 1.90, which might be attributed to Me2SO coordinated through O and through S, respectively, to a Ru(II) center in a mixed-charge dinuclear species; thus, partial self-reduction of the RuCI3(MezSO)2 half of the dimeric compound is likely to occur. A similar self-reduction process has been already hypothesized for explaining the chemical behavior of the mononuclear species RuCI3(MezSO)3 in aqueous solution [6]. Overall, the spectral behaviors of 1 and 2 were attributed to multiple and contemporaneous hydrolytic processes concerning chloride and dimethylsulfoxide ligands, and occurring with relatively slow rates (hours), accompanied by partial self-reduction ofthe neutral RuCI3(Me2SO)2 fragment.
The spectral changes observed for 3 and 4 were more straightforward and qualitatively very similar to those observed for NAMI-A under the same conditions and were attributed to stepwise chloride hydrolysis from the Ru(III) fragment only. Figure 5 reports the spectral variations for 4; in the first step, which is complete in ca. 90 min at 25.0 C, the main absorption band at 400 nm is gradually replaced by a new band at 350 rim, typical of a mer-RuC13 unit, which was thus attributed to the neutral [{mer-RuCl3(HzO)(Me2SO-S)}(t-L){cis, cis, cis-RuCl2(Me2SO-S)2(CO)}] species. The clean isosbestic point at 375 nm in the visible spectra, supported by time-driven H NMR spectra under the same conditions, indicates that no relevant fragmentation of the dinuclear structure occurs during this step. The first hydrolysis is followed by a slower step that involves a decrease of the 350 nm band and was attributed to the hydrolysis of a second chloride from the Ru(III) fragment. The behavior of the pyrimidine compound 2 after reduction is qualitatively very similar to that of 1, except that the chloride hydrolytic processes are ca. 20% faster and also the formation of monomeric species is more pronounced.
In both cases, the reduction of the dimeric compounds is also accompanied by a remarkable increase in solubility due to the increased charge and by a dramatic change in color, from orange to dark red (the main absorption band at 380 nm is replaced by an intense and broad band at 447 nm, attributable to a t2g --* g* charge-transfer absorption as in the case of the symmetrical dimeric species [3]).
Treatment. of physiological solutions of the mixed-valence dinuclear species [{trans-RuCl4(MeSO-S)}(t-L){cis, cis, cis-RuCb(Me2SO-S)2(CO)}] (L pyz (3) and pym (4)) with one equivalent of ascorbic acid led similarly to the complete and rapid reduction of the Ru(III) fragment. Also in this case the chemical behavior of the reduced species was investigated mainly by IH NMR spectroscopy. The behavior of 3 after reduction is summarized in Scheme 4 and is substantially similar to that of the corresponding monomeric species [trans-RuCl4(MeSO-S)(pyz)]" [3] because the cis, cis,cis-RuCl2(MeSO-S)(CO) fragment is inert and all hydrolytic processes occur on the other half of the dimer. Also fracture of the Ru(II)-pyz bond occurs exclusively at the site of the reduced Ru atom, generating the mononuclear species cis, cis, cis-RuC12(MeSO-S)2(CO)(pyz) (identified by comparison with the resonances of a pure sample). In more details, the dianionic species 3A disappears within 20 min after reduction, generating the monoanion [{mer-RuCl3(MezSO-S)(HzO)}(t-pyz){cis,cis,cis-RuCl2(MezSO-S)z(CO)}]" (3B), which is the prevailing species in the first hour after reduction. 3B either hydrolyzes a further chloride to give the trans and cis geometric isomers 3C and 3C' in a ca. 1:2 ratio (in this case the pyz resonances of the two isomers are distinguishable, but assignment to each isomer was not possible) or splits into mononuclear species which represent 20% of the total after 2.5 h. The pyrimidine dinuclear species 4 behaves similarly to 3 upon reduction, except that the hydrolytic processes are more rapid and the splitting of the Ru-N bond, which yields selectively cis, cis, cis-RuClz(MeSO-S)z(CO)(pym), occurs to a larger extent (50% of mononuclear species 3h after reduction). The reduction of 3 and 4 involves an increase in solubility and a change of color similar to that observed upon reduction of the Ru(III)/Ru(III) dimeric species 1 and 2.
In , respectively. As reported in Scheme 3 (and not considering fragmentation) reduced 1 and 2, after chloride and/or sulfoxide hydrolysis, are bi-functional binders at one site and mono-functional binders at the other (coordinated water is a labile ligand); thus, their adducts with biological targets are likely to be different from those obtained upon coordination of the corresponding mononuclear species. On the other hand, the mixedvalence dinuclear compounds [{trans-RuCh(MezSO-S)}(t-L){cis, cis,cis-RuClz(Me2SO-S)2(CO)}] (L pyz (3) and pyre (4)), owing to the great inertness of the cis, cis, cis-RuCl2(Me2SO-S)2(CO)(1/2tt-L) fragment, both before and after reduction behave substantially like the corresponding mononuclear species [trans-RuCI4(Me2SO-S)(L)]" in which the terminally bonded L ligand can be considered as bearing a bulky substituent on the other N atom.