EPR and photophysical characterization of six bioactive oxidovanadium(IV) complexes in the conditions of in vitro cell tests

EPR AND PHOTOPHYSICAL CHARACTERIZATION OF SIX BIOACTIVE OXIDOVANADIUM(IV) COMPLEXES IN THE CONDITIONS OF IN VITRO CELL TESTS

Marta Lovisari1_{, Giorgio Volpi}1_{, Domenica Marabello}1_{, Silvano Cadamuro}1_{, Annamaria}

Deagostino1_{, Eliano Diana}1_{, Alessandro Barge}2_{, Margherita Gallicchio}2_{, Valentina Boscaro}2_,

Elena Ghibaudi1*

1- Dip.to di Chimica, University of Torino - Via Giuria 7, I-10125 Torino (Italy)

2- Dip.to di Scienza e Tecnologia del farmaco, University of Torino - Via Giuria 9, I-10125 Torino (Italy)

* _{Elena Ghibaudi}

Dip.to di Chimica, University of Torino - Via Giuria 7, I-10125 Torino (Italy)

E-mail: elena.ghibaudi@unito.it; Tel. ++39-(0)11-6707951; Fax. ++39-(0)11-6707855

Abstract

A number of oxidovanadium(IV) complexes have been reported to display anticancer activity. A theranostic approach, based on the simultaneous observation of both the effect of oxidovanadium(IV) complexes on cell viability and the disclosure of their intracellular fate, is possible by using oxidovanadium(IV) complexes functionalized with fluorescent ligands. In the present study we accomplished the characterisation of six oxidovanadium(IV) complexes in conditions close to those employed for in vitro administration. In particular, we investigated the light harvesting properties of such complexes in the presence of a dimethylsulphoxide/aqueous buffer mixture, and we found that one complex exhibits a quantum yield suitable for confocal microscopy investigations. EPR investigations in the same conditions provide information about the presence of ligands’ substitution processes. Finally, the electrochemical properties of all 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

complexes were determined by cyclic voltammetry. The overall results show that these complexes exhibit an average stability in solution; EPR data confirm that DMSO enter the first coordination sphere of oxidovanadium(IV) and suggest the occurrence of partial ligand substitution in the dimethylsulphoxide/aqueous buffer mixture.

Keywords

Oxidovanadium(IV) compounds; antitumoral metal complexes; fluorescence; cyclic voltammetry; EPR spectroscopy; theranostic.

List of abbreviations

EPR Electron Paramagnetic Resonance DMEM Dulbecco's Modified Eagle's medium

DMF Dimethylformamide

DMSO Dimethylsulphoxide

SCE Saturated Calomel Electrode

TBAPF6 Tetrabutylammonium hexafluorophosphate

TLC Thin Layer Chromatography 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Introduction

Vanadium compounds constitute an important family of pharmacologically-active compounds displaying a range of therapeutic effects (e.g. insulin-mimetic, cardiovascular, etc.) [1-3], that are characterised by relatively low toxicity. Some vanadium complexes have been found to exhibit antitumoral, antiproliferative and pro-apoptotic properties [2, 4-10]. One of the oldest vanadium therapeutic agent ever synthesized is oxidovanadium(IV) bis-acetylacetonate VO(acac)2 that

-apart from its insulin-mimetic activity [7,11,12] - was found effective against human hepatoma cell lines [7,8]. VO(acac)2 is an efficient DNA cleaving agent at submicromolar concentration

[12] and it acts as a stimulator of the activity of a cytosolic protein kinase [13, 14], blocking cell cycle progression at G1 phase [8] and inducing mitochondrial toxicity through oxidative stress mechanisms [15].

Despite the interest raised by vanadium compounds as therapeutic agents, little is known about their intracellular fate and distribution; in addition, vanadium complexes are known to undergo complex speciation equilibria in aqueous solution, a phenomenon that may generate a range of chemical species with distinct pharmacological effects [16-19]. A theranostic approach, based on the simultaneous observation of both the effect of oxidovanadium(IV) complexes on cell viability and the disclosure of their intracellular fate, is possible by using oxidovanadium(IV) complexes functionalized with fluorescent ligands. In a previous study [20] we reported the synthesis and characterisation of six new oxidovanadium(IV) complexes with asymmetric derivatives of the acetylacetonate ligand. The structures of complexes A-F are shown in Figure 1. Through the application of several complementary techniques (X-ray crystallography, electronic absorption, vibrational and EPR spectroscopies), we were able to show that these compounds adopt a distorted square pyramidal geometry and, in the presence of strongly coordinating solvents, the cis-planar isomer is formed preferentially, whereas weakly coordinating solvents favour the trans-planar isomer. Further evidence of this behaviour is reported in the present work. In addition, we argued about the presence of mono- and bis-44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

chelated forms of complexes D and E. We also investigated the effect of complexes A-F on cell viability, showing that the responsiveness of tumour cells is related to the ligands’ properties rather than the oxidovanadium(IV) moiety. In the present study we accomplish the characterisation of complexes A-F in conditions close to those employed for in vitro cell tests. The light harvesting properties of complexes A-F were investigated by electronic absorption and emission spectroscopy in the presence of a DMSO/aqueous buffer mixture; we found that complex D exhibits a quantum yield suitable for confocal microscopy investigations. EPR investigations in similar conditions provided further information about the presence of ligands’ substitution processes. Finally, the redox behaviour of complexes A-F was investigated by cyclic voltammetry.

2. Experimental

All solvents and raw materials were used as received from commercial suppliers (Sigma-Aldrich and Alfa Aesar) without further purification. TLC was performed on Fluka silica gel TLC-PET foils GF 254, particle size 25 nm, medium pore diameter 60 Å. 1_{H and} 13_{C NMR spectra of the}

ligands were recorded on a Bruker Avance 200 spectrometer at 200 MHz and 50 MHz, respectively, in CDCl3.

The synthesis of the ligands and complexes was performed according to the protocol described by Sgarbossa and coworkers [20].

2.1 Spectroscopic characterization

UV-Vis spectra of the complexes were recorded on a UNICAM UV 300 (Thermo Spectronic) spectrophotometer in the presence of acetone and DMSO/aqueous buffer (NaHCO3 3,7 g/l; NaCl

6,4 g/l; NaH2PO4 0,109 g/l – pH 7.0), in order to check the stability of complexes A-F in

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

solution. The buffer composition was aimed at mimicking the DMEM buffer employed for in

vitro cell tests [20] that was not suitable for absorption measurements.

Absorption and emission spectra of each oxidovanadium(IV) complex in DMSO/aqueous buffer were recorded on diluted solutions (30 M), freshly prepared from a stock solution of each complex (18 mM) in DMSO.

Fluorescence measurements were carried out on a Cary Eclipse Varian V (Varian Instruments) spectrophotometer. Fluorescence emission was recorded in the 360-750 nm wavelength range. Fluorescence quantum yields were determined with the same instrument through a comparative method, using quinin sulphate as standards [21-23].

2.2 Electrochemical characterization

Electrochemistry was performed on a PC-controlled AMEL 430 electrochemical analyzer, using a standard three-electrode cell configuration (glassy carbon working electrode, Pt counter electrode, aqueous 3 M KCl Calomel reference electrode). All measurements were carried out under N2 atmosphere, in acetonitrile solution containing 0.1 M TBAPF6 as the supporting

electrolyte. Scan rate = 200 mV s-1 _{within the -2V to +2V potential range. Positive feedback iR}

compensation was applied routinely and ferrocene (Fc) was used as an internal standard (half-wave potentials are reported against the Fc(0/+1) redox couple).

2.3 EPR characterization

77 K EPR spectra of solutions of each complex were recorded on a CW-EPR spectrometer ESP300E (Bruker) equipped with a cylindrical cavity.

EPR spectra of complexes A-F in DMSO were recorded on ~18 mM solutions at 77K. EPR spectra of each oxidovanadium(IV) complex in DMSO/aqueous buffer were recorded on diluted solutions (~1.0 mM), freshly prepared from a 18 mM stock solution in DMSO. The buffer was degassed by fluxing argon for 15 min. Experimental settings were as follows: microwave 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

frequency ~9.5 GHz; modulation frequency 100 KHz; modulation amplitude 4 G; microwave power 5 mW; time constant 163 ms. All spectra were simulated with the EPRSim32.03 software [24] whose spin Hamiltonian takes into account second order effects typical of oxidovanadium(IV) systems.

2.4 X-ray diffraction

Crystals suitable for X-ray analysis were obtained at room temperature, by very slow evaporation of the solvent acetone at room temperature. The intensity data were collected at 153 K on an Oxford Diffraction Gemini R-Ultra diffractometer equipped with nitrogen low temperature device and Enhanced Ultra Cu X-ray Source. The intensities were corrected for absorption with the numerical correction based on gaussian integration over a multifaceted crystal model. Software used: CrysAlisPro (Agilent Technologies, Version 1.171.37.35) for data collection, data reduction and absorption correction; SHELXT [25] for structure solution using Direct Methods and ShelXL [26] for refinement through least squares minimization; Olex2 [27] for graphics. All non-hydrogen atoms were anisotropically refined, except for the C(2) and the CF3 group, that is disordered over two positions. Hydrogen atoms were calculated and refined

riding with Uiso=1.2 or 1.5 Ueq of the connected carbon atom. The data/parameter ratio is low

(6.6), being the crystal very little (0.14 x 0.04 x 0.02) and low diffracting, but data were sufficient for a satisfying resolution and refinement.

Details of crystal data, data collection and refinement parameters are given in Table S1. Crystallographic data for the structure reported in this paper were deposited with the Cambridge Crystallographic Data Centre (CCDC 1446833). Copies of the data can be obtained free of

charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: +44-1223-336408; Fax: +44-1223-336003; e-mail: deposit@ccdc.cam.ac.uk; Web site: http://www.ccdc.cam.ac.uk).

3. Results and discussion

Oxidovanadium(IV) compounds are known to undergo complex kinetic equilibria when brought into solution, depending on the nature of the ligands and the solvent employed [16, 17, 28]. This aspect is especially important whenever one seeks for oxidovanadium(IV) complexes with potential pharmacological applications, as speciation equilibria may determine their pharmacological effectiveness or their failure [18]. Therefore, a detailed knowledge of the complexes’ behavior in the conditions of their administration during in vitro cell tests is needed. The present study aims at accomplishing the characterization of six new oxidovanadium(IV) complexes, designed by our research group, through the investigation of their behavior in solution, in the presence of pure DMSO or DMSO/aqueous buffer mixture (whose chemical composition mimics the medium employed in cell tests). Electronic absorption and EPR spectroscopies allowed investigating the integrity of complexes A-F in the above-described conditions, checking the presence of ligands’ exchange phenomena and/or disruption of the complexes. The fluorescence properties of the complexed ligands were also explored in view of the possibility of tracking their intracellular fate by confocal microscopy. In addition, we characterized complexes A-F from the electrochemical viewpoint. Finally, on completion of previously published structural data, the crystallographic structure of a complex D in the presence of acetone was solved.

3.1 X-ray crystal structure analysis

The determination of crystallographic structures of oxidovanadium(IV) complexes with asymmetric ligands is crucial for finding out whether trans-planar or cis-planar isomers are formed; it can also provide information about the influence of the solvent on the stabilization of specific isomers. A number of X-ray structures of complexes A-F have already been reported; namely complex B in DMF, complex F in DMSO and complex E in DMSO and in acetone [20]. We found out that strongly coordinating solvents are able to stabilize the cis-planar isomer, whereas weakly coordinating solvents seem to favor the trans-planar isomer. This finding is 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172

further confirmed by the X-ray structure of complex D crystallized from acetone that is reported in Figure 2.

The asymmetric unit of complex D contains one-half molecule, lying the molecule in a crystallographic 2-fold axis, and the CF3 group is disordered over two very close positions. As

expected, the complex shows a square pyramidal geometry with the oxidovanadium(IV) moiety in the apical position and the two β-dicarbonyl moieties lying in the equatorial plane. The other axial coordination site is not occupied by the solvent, as expected in the case of weakly coordinating solvents [20]. Nevertheless, a strong intermolecular contact with a partial dative character is established between the oxygen atom of a neighbor oxidovanadium(IV) moiety and the vanadium center. In fact a very short V∙∙∙O distance (2.32(1) Å) is found between two proximal oxidovanadium(IV) centers, to the point that the oxygen atom of a close oxidovanadium(IV) center seems to act as a donor atom that saturates the sixth coordination position of the D complex (Figure S1). Noteworthy is also the prominent bending of the β-dicarbonyl ligands toward the sixth coordination position (the angle between the V1-O1 bond and the V1-O2-C2-C3-C4-O3 ring is 110°). This finding - in line with our previous structural studies of complexes A-F [20] - is likely due to the repulsion exerted by the oxidovanadium(IV)-oxygen electron couplets on the four equatorial carbonyl oxidovanadium(IV)-oxygens as well as to a more favorable crystal packing of the strictly connected molecules along the [001] direction (Figure 2). The methoxynaphtile moiety is completely planar (mean deviation from plane 0.018 Å) and is rotated by only 6° with respect to the -dicarbonyl fragment, so that a wide delocalization of charge density through the whole ligand is not prevented, as witnessed by the lower C(4)-C(5) distance with respect to a typical localized single bond (Tables S2 and S3).

3.2 Optical absorption spectra and complex stability

The photophysical features of oxidovanadium(IV) complexes are well characterized [29,30]. The absorption maxima of complex A-F in acetone and DMSO/buffer solution in the UV range are 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198

compared in Table 1. Complexes A-F dissolved in acetone usually exhibit two bands: a main one in the 340-360 nm range and a shoulder in the 360-380 nm range. This spectral region exhibits overlapping contributions from both the ligands and the oxidovanadium(IV) band III, associated with the dxy→dz2 transition. The comparison between compounds A and B (or C and

D), whose ligands differ by the presence of a methoxyl substituent on the aromatic ring, highlights a considerable red-shift likely due to the presence of the strong electron-donor group. The change to DMSO/aqueous buffer brings about a blue-shift of the absorption bands that, in addition, are better resolved as compared to those in acetone. These findings agree with the expectations and are consistent with the coordination of a solvent molecule to the metal centre and the consequent bending of the two ligands towards DMSO [20, 31]. The UV absorption pattern of complexes A-F either in acetone or DMSO/aqueous buffer does not change dramatically with respect to the free ligands, apart from a general increase of the intensity of absorption bands upon complexation (data not shown). This confirms that the contribution to this spectral region is mainly due to the organic ligands or to metal-ligand CT bands.

In order to check the stability of complexes A-F in DMSO/aqueous buffer, their solutions were monitored spectrophotometrically vs. time at RT: the results are summarised in the last column of Table 1.

Only complexes D and E exhibited kinetic instability, witnessed by significant changes of the spectral pattern. In both cases, an isosbestic point was detected (at 360 nm for complex D; at 361 nm for complex E). Figure 3 reports the time-dependent spectra of complex E dissolved in DMSO/aqueous buffer: the absorption at =358 nm increases progressively, whereas the band centered at =390 nm decreases gradually and finally disappears (after ~60 minutes). The single isosbestic point suggests the occurrence of equilibrium between two distinct species, with a progressive conversion of one species into the other. This behavior is consistent with a process of ligand substitution or rearrangement, in line with previously reported data on these oxidovanadium(IV) complexes in solution [17,20]. In fact, previously published EPR evidence 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224

showed that complexes D and E dissolved in acetone are likely to form mono- and bis-chelated species. In this case, the simultaneous presence of DMSO and water has a stronger destabilizing effect as compared to acetone; it favors ligands’ replacement processes with relatively rapid kinetics. Conversely, all other compounds do not exhibit any evident spectral changes upon dissolution in DMSO/water; this is consistent with either complete kinetic stability or rapid kinetic exchange leading to new stable species within a few seconds from dissolution.

3.3 Optical emission spectra and quantum yield

The emission spectra of compounds A-F dissolved in acetone are shown in Figure 4A whereas Table 2 reports the emission wavelengths upon excitation at ~380 nm (with the only exception of complex E). Upon dissolution in acetone, all products exhibit a structured emission that falls approximately at 410 nm with their corresponding vibronic bands at ~430 nm and ~460 nm. The methoxyl substitution in compound D results in important changes of the vibronic profile distribution as compared to its homologous complex C. Such difference was not detected with the other two homologous compounds A and B.

In line with the expectations, a comparison between the spectra of complexes and organic ligands (data not shown) showed that fluorescence is essentially due to the organic moiety; moreover, in all cases the complexation process lowers the yield of fluorescence emission. In the unique case of complex D, a concentration-dependence quenching effect was highlighted: this is likely due to self-absorption phenomena determined by the partial overlap between absorption and emission spectra reported in Figure 4B.

Emission spectra were also recorded in the presence of DMSO/aqueous buffer (Figure 4B and Table 2), by excitation at ~ 330 nm. The switch towards a more polar environment resulted in much weaker emissions; in fact only complexes C and D showed detectable emission peaks at 435 nm and 453 nm, respectively. Further, in both cases the emission profile was no longer structured as the vibronic profile was not observable.

225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250

As compound D dissolved in DMSO/aqueous buffer exhibited good emission intensity upon excitation at 380 nm, we decided to determine its quantum yield. A comparative method in DMSO/aqueous buffer was applied [23]. Compound D turned out to have = 5.34%, a result comparable to other metal complexes [32,33]; this value is quite significant, considering the polar environment, and will allow confocal microscopy investigations. In fact, the photophysical properties of compound D (abs: 337 nm; em: 450 nm) are similar to those of commercial

fluorophores widely used in confocal microscopy, e.g. AlexaFluor (abs: 350 nm em: 442

nm), DeadBlue (abs: 350 nm em: 450 nm), AMCA coumarin (abs: 350 nm em: 442 nm).

Conversely, its homologous complex C exhibited a quantum yield lower than 0.1 %, a difference that is likely related with the distinct electronic structure of the two chromophores.

3.4 EPR spectroscopy

The protocol of cell test performed on complexes A-F implies their dissolution in a small volume of DMSO, followed by dilution with an aqueous medium. As solvent changes have been reported to affect the structure of VO(acac)2 complexes [17,31,34], it is relevant to find out

whether the structure of complexes A-F is affected by such protocol, and how. More in details, it is crucial to establish whether the oxidovanadium(IV) ion is finally set free in solution or the complexes keep their structural integrity. In order to answer these questions, the 9 GHz EPR spectra of complexes A-F dissolved either in DMSO (Figure 5A) or in DMSO/aqueous buffer (Figure 5B) were recorded at 77K. All spectra underwent simulation and the EPR parameters are reported in Table 3.

Dissolution of complexes A-F in DMSO invariably resulted in a complex 77K EPR spectral patterns of complexes A-F, with a pronounced baseline distorsion. This feature was absent upon dissolution of complexes A-F in acetone or methanol [20] and appears to be related with the strongly coordinating character of DMSO. The best simulation of the EPR pattern was obtained by positing the presence of a single species with rhombic symmetry: this choice allowed getting 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276

a fairly accurate reproduction of the hyperfine pattern of the experimental spectrum (Figure 5A). In all cases, both gx and gy values fall around ~1.98 whereas gz is ~1.94, consistent with the

presence of distorted octahedral geometry [1,35]. Rhombicity (expressed as |gx -gy|) varies

between 0.001 and 0.003. The values of the hyperfine splitting constant (with Ax and Ay ~65∙10-4

cm-1 _{and A}

z ~173 ∙10-4 cm-1) confirm the slight octahedral distorsion and are consistent with

previously published data, collected on complexes A-F dissolved in acetone [20]. The substantial homogeneity of this set of EPR data throughout the complexes suggests a similar structure for all of them. As the X-ray structure of complex E crystallized in DMSO shows very clearly the coordination of one DMSO molecule to the metal centre [20], we conclude that complexes A-F dissolved in DMSO incorporate one DMSO molecule into the first coordination sphere, without affecting the other ligands. Based on the EPR values, we exclude the formation of a cis isomer: DMSO binds at the axial position, trans to the oxidovanadium(IV) moiety, in agreement with the expectations [16,17]. In addition, evidence from electron absorption and X-rays support the assignment of a cis-planar arrangement of the acac-derived ligands.

Dilution of the DMSO adducts of complexes A-F with water resulted in meaningful spectral changes. The quality of the experimental data was lower as compared to the DMSO series, due to the dilution with an aqueous medium. Hence spectral simulations turned out to be more problematic and less accurate with respect to the previous set of data. Contrary to the previous case, the present spectral set highlights the heterogeneous behavior of complexes A-F in the presence of water. In all cases but complex F baseline distortion was very pronounced. As such distorsion was found only in the presence of metal-bound DMSO, we took it this as an evidence of the persistence of a bound DMSO molecule in complexes A-E, whereas complex F seems to undergo important structural changes.

Despite all our attempts to simulate the EPR spectra as the sum of 2 or more species, we managed to get the simulation process to convergence only by positing the presence of a single rhombic species with higher rhombicity (0.002-0.01) as compared to the previous set of data. In 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302

no case we found evidence of the presence of [VO(H2O)5]2+, that would imply complete

disruption of the complexes. None of the EPR data fully matches the expected values for mono-and bis-chelated species, thus preventing a clear identification of the species involved: data provided by simulation are likely to represent an average of the EPR parameters of distinct species. In facts, the overall evidence deriving from electronic absorption and EPR suggest - at least for complex D and E - the presence of speciation equilibria that might derive from the replacement/rearrangement of ligands brought about by water [17,28,36]. The distinct behavior of complex F in cell tests (where it turned out to be almost ineffective towards tumor cells) [20] is mirrored by a peculiar behavior in DMSO/aqueous buffer.

3.5 Electrochemistry

The electrochemical properties of complexes A-F were investigated by cyclic voltammetry (CV) in acetonitrile solution. The results of CV measurements, performed at 0.2 V/s, are summarized in Table 4. Potentials were reported vs. the ferrocene/ferrocenium redox couple used as an internal standard. Oxidovanadium(IV) acetylacetonate complexes are known to undergo typical one-electron metal-ligand-based reduction, one-electron metal-based reduction (VIV_O2+ _{+ e}− _⇌

VIII_O+ _{) and one-electron metal-based oxidation V}IV_O2+ _{⇌ V}V_O3+ _{+ e}− _{at E° = +0.81 V for}

VIV_O(acac)

2 [35,37-40].

Quasi-reversible metal-centered first oxidation was observed for all complexes A-F. A second oxidation step was found as reversible peak for complex D and as irreversible peak for complex E.

In the present work, the reduction of oxidovanadium(IV) acetylacetonate complexes was investigated and results similar to those reported by Nawi and coworkers on acetylacetonate complexes were obtained: VIV_O(acac)

2 + e-  [VIIIO(acac)2]- at Epc = -1.90 V vs. SCE [37,38,41].

Almost all complexes showed two reductions at ~ E = -1.4 V and E = -1.8 V. The first reversible step, at rather positive potential was not found with complex E. The further irreversible 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328

reduction sequence (reversible for complex B) was found with all complexes. A comparison between homologous complexes C and D does not highlight significant differences; conversely, differences were detected between complexes A and B, as a shift of the reduction peaks towards more positive values (about 0.25 V for each peak) between A and B was found.

4. Conclusions

The overall data show that complex A-F dissolved in DMSO coordinate a solvent molecule in the axial position and undergo a symmetry distorsion that is responsible for changes of both electronic absorption and EPR spectral patterns. Crystallographic evidence shows that the distortion is due to bending of the two acac-derivative ligands towards the bound DMSO moiety. All DMSO adducts exhibit a fair stability when dissolved in DMSO. Upon water dilution, the DMSO adducts of complexes A-F are partially destabilized and ligand replacement/rearrangement processes are likely to occur, although the oxidovanadium(IV) moiety is never set free in solution. Electronic absorption shows that the kinetic of ligand replacement is variable and it is strongly dependent on the type of ligand. The overall data are consistent with previously published results on the cytotoxic effect of complexes A-F and support the conclusion that the biological activity of this family of complexes is modulated by the ligands and cannot be uniquely ascribed to the oxidovanadium(IV) ion. The electrochemical behaviour of complexes A-F was assessed by cyclic voltammetry and it is in line with previously reported data on similar complexes. Finally, the emission behaviour of complex D in aqueous medium makes it a good probe for confocal microscopy studies aimed at establishing its intracellular fate.

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380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405

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406 407 408 409 410 411

TABLES, FIGURES, LEGENDS

Table 1 - Absorption maxima of 30M solutions of complexes A-F dissolved in acetone or DMSO/aqueous buffer, in the UV region. The last column shows the behavior of the complexes vs. time. Complex Acetone max (nm) DMSO/ buffer max (nm)

Stability over time in DMSO/buffer (4 min cycles) A 358 (sh) 322 Unchanged B 346 362 (sh) 326 Unchanged C 343 367 (sh) 329 Unchanged D 345 (sh) 370 337 401 (sh) Slight increase Slight decrease Isosbestic at 360 nm Stabilized after 30 min

E 368

382 (sh)

358 390

Increase and blue shift Decrease and red shift Isosbestic at 361 nm Stabilized after 60 min

F 350 370 (sh) 339 Unchanged 412 413 414 415 416 417

Table 2 - Fluorescence emission data relative to 30 μM solutions of complexes A-F in acetone or DMSO/Buffer.

Acetone DMSO/buffer

Complex λexc (nm) Emission (nm) λexc(nm) Emission (nm) A 380 409 432 458 - -B 380 409 432 459 - -C 382 409 433 460 329 435 D 380 412 435 461 337 453 E 405 413 434 461 - -F 380 410 433 459 - -418 419 420 421

Table 3 - EPR parameters of complexes A-F in DMSO or DMS0/aqueous buffer at 77 K Complex Solvent gx gy gz Ax (cm-1_.104₎ Ay (cm-1_.104₎ Az (cm-1_.104₎ A DMSO 1.9814 1.9824 1.9425 65.60 65.60 173.50 DMSO/Buffer 1.9839 1.9934 1.9606 64.85 66.03 183.36 B DMSO 1.9842 1.9813 1.9423 62.80 65.70 172.90 DMSO/Buffer 1.9772 1.9609 1.9465 55.38 74.99 168.04 C DMSO 1.9809 1.9825 1.9418 65.40 65.00 172.90 DMSO/Buffer 1.9818 1.9799 1.9495 59.89 59.59 168.95 D DMSO 1.9814 1.9831 1.9423 65.50 65.30 172.70 DMSO/Buffer 1.9754 1.9862 1.9418 54.46 72.73 171.39 E _DMSO/BufferDMSO 1.9833_2.0776 1.9816_1.9693 1.9426_{1.9344 114.07}65.30 65.50_56.15 172.50_165.80 F DMSO 1.9841 1.9820 1.9446 65.17 65.58 171.44 DMSO/Buffer 1.9767 1.9799 1.9466 67.46 57.06 167.15 [VO(H2O)5]2+ _{[42] 1.978 1.978 1.933 70.7 70.7 182.6} 422

Table 4. Cyclic voltammetry data relative to complexes A-F. E1/2 of reversible process, Ep of irreversible process.

Complex Reduction potentials

(Volts) Oxidation potentials (Volts ) A Ep = -1.523 Ep = - 1.975 Ep = - 2.177 Ep = 1.125 B E1/2 = - 1.289 E1/2 = - 1.751 E1/2 = -1.957 E1/2 = 0.790 C E1/2 = -1.387 Ep = - 1.744 Ep = 1.032 D Ep = - 1.398 Ep = - 1.728 Ep = 0.923 E1/2 = 1.268 E Ep = - 1.760 Ep = 0.735 Ep = 1.095 F Ep = - 1.405 Ep = - 1.831 Ep = - 2.011 Ep = 1.009 423 424 425 426

Figure 1 - Structural formulas of complexes A-F1

1 – The ligands are, respectively: A) 1-phenyl-4,4,4-trifluorobutane-1,3-dione; B) 1-(4-methoxyphenyl)-4,4,4-trifluorobutane-1,3-dione; C) 1-(2-naphtyl)-4,4,4-trifluorobutane-1,3-dione; D) 1-(6-methoxy-2-naphtyl)-4,4,4-trifluorobutane-1,3-1-(2-naphtyl)-4,4,4-trifluorobutane-1,3-dione; E) 1-(N-methyl-3-indolyl)-4,4,4-trifluorobutane-1,3-dione; F) 1-(3-thienyl)-4,4,4-trifluorobutane -1,3-dione

A B E C D F 427 428 429 430 431 432 433 434

Figure 2 – X-ray structure of complex D crystallized from acetone, with atom labeling 435

436

437 438

Figure 3 - Absorption spectra of a 30M solution of complex E in DMSO/aqueous buffer vs. time (spectra recorded each 4 minutes)

439 440 441

442 443

Figure 4 – Panel A) Emission spectra of 30M solutions of complexes A-F in acetone; Panel B) Absorption ( ____ _{) and normalized emission (} _ _ _ _{) spectra of complex D in} DMSO/aqueous buffer. A B 444 445 446 447

Figure 5 – Experimental (_____{) and simulated (}….._{) EPR spectra of complexes A-F dissolved in: panel A) DMSO; panel B) DMSO/aqueous} buffer A _B 448 449 450