Structural and solution equilibrium studies on half-sandwich organorhodium complexes of (N,N) donor bidentate ligands

Complex formation equilibrium processes of [Rh(  5 -C 5 Me 5 )(H 2 O) 3 ] 2+ with N,N’-dimethylethylenediamine (dmen), N,N,N′,N′-tetramethylethylenediamine (tmeda), 2-picolylamine (pin) and 1,10-phenanthroline (phen) were studied in aqueous solution by 1 H NMR spectroscopy, UV-vis spectrophotometry and pH-potentiometry. Formation and deprotonation of [Rh(  5 -C 5 Me 5 )(L)(H 2 O)] 2+ complexes and exchange process of the aqua to chlorido ligand were characterized in addition to single-crystal X-ray diffraction analysis of [Rh(  5 -C 5 Me 5 )(L)(Cl)] + complexes (L = dmen, tmeda and pin). Formation of complexes with significantly high stability was found except tmeda due to the sterical hindrance between the methyl groups of the chelating ligand and the arenyl ring resulting in an increased methyl group‒ring plane torsion angle. [Rh(  5 C 5 Me 5 )(L)(H 2 O)] 2+ complexes of dmen, pin, phen predominate at pH 7.4 without decomposition even in the micromolar concentration range. The complexes were characterized by relatively high chloride affinity and a strong correlation was obtained between the logK’ (H 2 O/Cl ‒ ) and pK a of [Rh(  5 -C 5 Me 5 )(L)(H 2 O)] 2+ constants for a series of (O,O), (O,N) and (N,N)-chelated complexes. For this set of 12 complexes a relationship between logK’ (H 2 O/Cl ‒ ) values and certain crystallographic parameters was found using multiple linear regression approach. DNA binding of these complexes was also monitored and compared by ultrafiltration and fluorimetry.


Introduction
The tremendous success of Pt(II) anticancer drugs, which currently belong to the best sold and most widely used antitumor compounds, has stimulated the exploration of other effective metal-based compounds. In this context Ru-based antineoplastic metal complexes with low side effects have been developed, e.g. trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339/IT-139), which is currently under development against numerous human tumour types. 1 complexes are considered as prodrugs that are activated by reduction that provides the impetus for the development of various Ru(II) anticancer compounds. Ru is often stabilized in the +2 oxidation state by the coordination of  6 -arene type ligands. 4 Besides the numerous half-sandwich Ru(II) organometallics of the type [Ru( 6 -arene)(X,Y)(Z)], in which (X,Y) is a chelating ligand and Z is leaving co-ligand, analogous complexes of the heavier congener Os(II) are also extensively being investigated. 5,6 In addition a large number of the isoelectronic Rh(III) and Ir(III)  5 -bound arenyl complexes were also developed showing promising in vitro anticancer activity. 7 Notably, the half-sandwich organometallic compounds have attracted increasing attention not just as potential therapeutic agents, but this type of compounds offers a broad scope for the design of water-soluble catalysts for transfer hydrogenation reactions as well. In general, the type of the metal ion, the arene ring, the chelating bidentate ligand and the leaving group have a strong impact on the biological or the catalytic activity. Some structure-activity relationships have already been established [8][9][10][11] considering for instance the anticancer potency of Ru( 6 -arene) compounds bearing ligands providing (N,N), (N,O) and (O,O) donor sets, 8 or catalytic activity of Rh, Ir and Ru complexes containing 1,10-phenanthroline (phen) or its derivatives for the regeneration of NADH in the chemoenzymatic reduction of ketones. 9 However, the knowledge on the aqueous solution (N,N) 20 donor containing Rh( 5 -pentamethylcyclopentadienyl) (Rh( 5 -C 5 Me 5 )) coordination compounds were reported in our previous works. These results revealed that the chloride affinity of the [Rh( 5 -C 5 Me 5 )(L)H 2 O)] 2+/+ complexes seems to be a crucial factor, just like in case of analogous Ir( 5 -C 5 Me 5 ) and some Ru( 6 arene) compounds. 6,21 While the Rh( 5 -C 5 Me 5 ) complexes of the simplest bidentate (N,N) donor ethylenediamine and the aromatic diimine bpy exhibited only poor anticancer activity, 7 the analogous complexes of phen, 7 polypyridyl ligands 7 and their various derivatives 22 with more extended aromatic systems are reported to show remarkable cytotoxic properties in various human cancer cell lines. Due to the lack of solution equilibrium data on the latter complexes herein we investigate Rh( 5 -C 5 Me 5 ) complex of phen in addition to methylated derivatives of ethylenediamine. 2-picolylamine was also involved as a representative of a mixed (N,N) donor ligand containing an aliphatic amine and an aromatic imine (Chart 1). The main aim of our study is to reveal correlations between complex architectures and thermodynamic data regarding their solution behavior.

Synthesis and X-ray structures of the organometallic rhodium(III) complexes
The rhodium(III) precursor [Rh( 5 -C 5 Me 5 )(-Cl)Cl] 2 used for the complex preparation was synthesized according to literature. 23 The     (17) in our former work (Table 1). 20 Regarding the Rh-to-ring centroid distances in [Rh( 5 -C 5 Me 5 )(en)Cl]ClO 4 (1.763 Å), 1•CF 3 SO 3 (1.778 Å) and 2•CF 3 SO 3 (1.812 Å) we can conclude that it is increasing with the higher number of the methyl substituents. The bond lengths between Rh and the nitrogen donor atoms show a similar trend. However, not only these bond lengths represent considerable differences, as the methyl group-ring plane torsion angles become higher and higher in the order of the complexes of en, dmen and tmeda as well (Table 1) It is worth mentioning that a significant difference is also observed between the N1-C-C-N2 torsion angles in the case of the various (N,N) donor ligands. Compounds bearing only aliphatic amines (en, dmen, tmeda) have torsion angle falling in the range of 53.82-56.62⁰, while for the rigid bpy and phen fairly low torsion angles (0.00⁰, 0.24⁰ respectively) were observed. This torsion angle for the complex of 2-picolylamine (3•Cl) falls between these extremities (25.63⁰).

Proton dissociation processes of the ligands and hydrolysis of the organometallic cation
Proton dissociation constants (pK a ) of dmen, tmeda, pin and phen ( Table 2) were determined herein by pH-potentiometry in a chloride-free medium and values are in good agreement with those reported in the literature [29][30][31] when account is taken of the different ionic strengths. Notably, the tertiary diamine (tmeda) has significantly lower pK a values compared to the secondary (dmen) and primary diamine ethylenediamine. The pK (H 2 L 2+ ) and pK (HL + ) of 2-picolylamine are attributed to the deprotonation of the pyridinium and the primary amine nitrogens, respectively. In the case of phen only pK a of HL + species could be determined in the studied pH range with adequate accuracy. H NMR spectra recorded for the dmen complex reveal slow ligand-exchange processes on the NMR time scale (t 1/2(obs) ~ 1 ms) and as a consequence the peaks belonging to the free or bound metal fragment (and ligand) could be detected separately (Fig. 3). Based on the integrated peak areas of the C 5 Me 5 protons in the unbound and bound fractions a logK [ML] 2+ constant could be also calculated from data collected at pH < 7.5 ( (2.80 ppm) protons and they turn to be doublet of triplets and doublet, respectively in the metal-bound forms.  These secondary amine nitrogen atoms have three different substituents and when coordinating to Rh they become chirality centers, thus formation of four different isomers is possible. This phenomenon was also observed in the case of [Pt(dmen)Cl 2 ] complexes and the (S,S') and (R,R') isomers crystallized from aqueous solution. 32 Based on the 1 H NMR spectra two isomers are formed and their ratio is ca. 1:1. The ratio of the doublets represents the ratio of the nitrogens in the different chemical environment and configuration. On the other hand the ratio of the methyl protons of the C 5 Me 5 fragment of the two complexes is also ca. 1:1. One of the isomers is most probably the (R,S) complex that was crystallized from the solution (vide supra), while the other is assumed to be the (S,R) isomer. (Otherwise the ratio cannot be 1:1.) The peaks of the CH 3 protons of the coordinated ligand and the C 5 Me 5 moiety are found at higher and at lower chemical shift (δ) values, respectively in the (R,S) isomer as compared to the other isomer, as a results of the stronger steric hindrance between the Me groups in the (R,S) isomer. An upfield shift of all peaks belonging to both [ML] 2+ isomers is observed in the basic pH range due to the fast exchange process between the aquated and the mixed hydroxido [ML(OH)] + species. Therefore, pK a of the aqua isomers as microscopic constants could be determined on the basis of the pH-dependent  values ( Table 2). The spectra recorded undoubtedly reveal that neither the free organometallic ion nor the free ligand is present at pH > 5.3, which means that the dmen complexes do not suffer from decomposition at pH 7.4. The decomposition is negligible even at  M concentration at this pH on the basis of the stability constants determined.
On the contrary unbound ligand and organometallic fragment are detected by 1 H NMR spectroscopy in the whole pH range studied (2-11.5) 2+ -tmeda (1:1) system even at 1 mM concentration (Fig. 4). Notably, only one kind of [ML] 2+ complex is formed in the pH range from 4 to 10 reaching the maximum fraction (85%) at pH 7.0 ( Fig. 4 (Table 2). These data undoubtedly indicate the formation of complexes with much lower stability in the case of tmeda as compared to dmen (or en) as it was expected on the basis of the findings of the X-ray structure analysis (vide supra).
The complex formation with the aromatic nitrogen containing ligands (pin, phen) was found to be fast, although only bound fractions of the ligands and the metal ion could be detected by 1 H NMR titrations in the pH range 2-11.5 ( Fig. S3 for pin complex). This is the consequence of the formation of complexes with outstandingly high solution stability. Based on the spectral changes only pK a [ML] 2+ constants were computed (

Chloride ion affinity and correlations between equilibrium constants and crystallographic data
The Rh( arene)(X,Y)Cl] (M= Ru(II), Os(II)). 6 In order to characterize the chloride ion affinity of these organorhodium complexes the following equilibrium process was monitored spectrophotometrically:   Table S2) clearly reveals the strong correlation between these values as shown in Fig. 6. The coordinated ligands in the complexes are: deferiprone 16 as (O,O) donor, 2-picolinic acid, 16 6methylpicolinic acid, 17 quinoline-2-carboxylic acid, 17 3isoquinolinecarboxylic acid, 17 8-hydroxyquinoline, 18 8hydroxyquinoline-5-sulfonate   The hindrance of the EB binding might be a consequence of a structural distortion of the DNA due to the covalent (coordinative) binding of the studied Rh( 5 -C 5 Me 5 ) complexes to the donor atoms of the macromolecule. Therefore their binding to adenosine and guanosine was also compared using 1 H NMR spectroscopy at 1:1 Rh: nucleoside ratio at pH 7.4 (Fig. 9).
We have found that only [Rh( The structures of dmen, tmeda and pin complexes were determined by single-crystal X-ray diffraction showing a pseudo-octahedral 'piano-stool' geometry. Solution equilibrium processes were studied via a combined approach using 1 H NMR spectroscopy, UVvis spectrophotometry and pH-potentiometry and were compared to literature data of ethylenediamine and 2,2′-bipyridine. Complex formation with ligands possessing aliphatic nitrogens (dmen, tmeda) was found to be much slower compared to 2-picolylamine and phen.

Experimental Chemicals
All solvents were of analytical grade and used without further purification. Dmen, en, phen, pin, tmeda, [Rh( 5 -C 5 Me 5 (-Cl)Cl] 2 , adenosine, guanosine, EB, DNA from calf thymus, KCl, KNO 3 , AgNO 3 , HCl, HNO 3 , KOH, KH-phthalate, 4,4-dimethyl-4-silapentane-1sulfonic acid (DSS), KH 2 PO 4 , NaH 2 PO 4 and Na 2 HPO 4 were purchased from Sigma-Aldrich in puriss quality. Milli-Q water was used for sample preparation. The exact concentration of the ligand stock solutions together with the proton dissociation constants were determined by pH-potentiometric titrations with the use of the computer program Hyperquad2013. 36 The aqueous [Rh( represents the overall goodness-of-fit derived from the sum of squared residuals (calculated-experimental titration data). The model was accepted when  was close to one (< 1.5). The standard deviation of the log values of species included into the model was always lower than 0.1. Samples were degassed by bubbling purified argon through them for about 10 min prior to the measurements and the inert gas was also passed over the solutions during the titrations.
Log  values for the various hydroxido complexes [(Rh( 5 -C 5 Me 5 )) 2 (-OH) i ] (4-i)+ (i = 2 or i = 3) were calculated based on the pHpotentiometric titration data in the absence of chloride ions and were found to be in good agreement with our previously published data. 15 Stability constants for M p L q H r complexes cannot be determined by pH-potentiometry because of several problems. In the case of dmen, complex formation was too slow to use pH-potentiometry. Also the dissociation of the tmeda complex was slow. H NMR spectra were recorded with the WATERGATE water suppression pulse scheme using DSS internal standard. 1 H NMR spectra were recorded after 24 h waiting time. Stability constants for the complexes were calculated by the computer program PSEQUAD. 33 Fluorescence spectra were recorded on a Hitachi-F4500 fluorimeter in 1 cm quartz cell at 25.0 ± 0.1 °C. All DNA-containing solutions were prepared in 20 mM phosphate buffer with 4 mM KCl, which mimics the chloride concentration of the nucleus. The concentration of DNA from calf thymus (as nucleobases) was 20 μM, 5 μM for ethidium bromide and the EB-to-metal ion/or metal complex ratio was varied between 1:10 and 1:50. The excitation wavelength was 510 nm and the emission was read in the range of 530-680 nm, where the absorption of the metal ion and the metal complex is negligible. All samples were incubated for 24 h. This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins
In the first series the DNA from calf thymus and metal complex concentration was 100-100 μM. Eppendorf Minispin Plus centrifuge and 10 kDa membrane filters (Millipore Amicon Ultra-0.5 centrifugal filter unit) were used. Samples were centrifuged for 10 min with 10000 rpm. UV-vis spectra of LMM fraction were recorded by a Hewlett Packard 8452A diode array spectrophotometer.
In the case of 2-picolylamine there was no need for chloride ion abstraction. Two equivalents of pin (31 L) was added to suspension of [Rh( 5 -C 5 Me 5 )(-Cl)Cl] 2 (92.71 mg, 0.15 mmol) in dichloromethane (30 mL). The mixture was stirred for 3 h at room temperature. Subsequent solvent removal under vacuum afforded 3•Cl as orange powder. The complexes were characterized by 1 H NMR spectroscopy and elemental analysis in addition to X-ray crystallography. Elemental analysis of all compounds was performed with a Perkin-Elmer 2400 CHN Elemental Analyser (Perkin-Elmer, Waltham, MA) at the Microanalytical Laboratory of the University of Vienna. ESI-MS measurements were performed using a Micromass Q-TOF Premier (Waters MS Technologies) mass spectrometer equipped with electrospray ion source (Fig. S7). Single crystals suitable for X-ray diffraction experiment of compound 1•CF 3 SO 3 , 2•CF 3 SO 3 and 3•Cl were grown from water/methanol solution mixture (1:1, 2.0 mL). Please do not adjust margins and 1590518. Crystal data and structure refinement details for complexes 1-3 are given in Table S1.