In Vitro and In Vivo Biological Activities of Dipicolinate Oxovanadium(IV) Complexes

The work is focused on anticancer properties of dipicolinate (dipic)-based vanadium(IV) complexes [VO(dipic)(N∩N)] bearing different diimines (2-(1H-imidazol-2-yl)pyridine, 2-(2-pyridyl)benzimidazole, 1,10-phenanthroline-5,6-dione, 1,10-phenanthroline, and 2,2′-bipyridine), as well as differently 4,7-substituted 1,10-phenanthrolines. The antiproliferative effect of V(IV) systems was analyzed in different tumors (A2780, HCT116, and HCT116-DoxR) and normal (primary human dermal fibroblasts) cell lines, revealing a high cytotoxic effect of [VO(dipic)(N∩N)] with 4,7-dimethoxy-phen (5), 4,7-diphenyl-phen (6), and 1,10-phenanthroline (8) against HCT116-DoxR cells. The cytotoxicity differences between these complexes can be correlated with their different internalization by HCT116-DoxR cells. Worthy of note, these three complexes were found to (i) induce cell death through apoptosis and autophagy pathways, namely, through ROS production; (ii) not to be cytostatic; (iii) to interact with the BSA protein; (iv) do not promote tumor cell migration or a pro-angiogenic capability; (v) show a slight in vivo anti-angiogenic capability, and (vi) do not show in vivo toxicity in a chicken embryo.


Table of Contents Experimental
Experimental data for V(IV) complexes prepared according to literature procedures page S2 Experimental methods page S2 X-ray studies Crystal data and structure refinement for 1-7 Table S1 page S8 Selected bond lengths and angles for complexes 1-7 Table S2-S8 page S9 Short intra-and intermolecular hydrogen bonds in structures 1-7 Table S9 page S12 Short π•••π interactions in structures 1-7 Table S10 page S13 View of the intermolecular interactions and packing in structures 1-7 Figure S1-S7 page S13 IR spectroscopy IR spectra of the V(IV) complexes in comparison to dipicolinic acid and free ligands (a-k) Figure S8 page S17 EPR spectroscopy Magnetic moments of 1-7 Table S11 page S22 X-band EPR spectra of 1-9 at 77 K together with simulated spectra Figure S9 page S23 EPR frozen solution spectra (at 77 K) of 1-9 in DMSO solvent together with simulated spectra Figure S10 page S24

Absorption spectroscopy
The absorption maxima and molar extinction coefficients for 1-10 Table S12 page S25 Electronic absorption spectra of 1-10 in DMSO and MeOH Figure S11 page S26 Electronic absorption spectra of 1-10 in MeOH in comparison to the spectra of the dipicolinic acid, [VO(dipic)(H 2 O) 2 ] and corresponding ligand Figure S12 page S27 UV-Vis stability spectra in DMSO for compounds 1-10 Figure S13 page S29 Biological studies Cell viability of A2780, HCT116, HCT116-DoxR and fibroblasts cell lines after exposure to different concentrations of the vanadium complexes 1-4, 7, 9 and 10 Figure S14 page S31 Cell viability of HCT116, A2780 and fibroblasts cells after exposure to different concentrations of doxorubicin and cisplatin for 48 hours Figure S15 page S32 Cell viability of HCT116-DoxR cells after exposure to different concentrations of the vanadium complexes' ligands for 48 hours Figure S16 page S33 Cell viability of fibroblasts after exposure to different concentrations of the vanadium complexes' ligands for 48 hours Figure S17 page S34 Stability and solubility of complexes 5, 6 and 8 by UV-Vis spectroscopy in RPMI Figure S18 page S35 Data obtained with the ICP-AES experiment after 3 hours of exposure of HCT116-DoxR cells to 10x IC50 of complexes 5, 6 and 8 Table S13 page S36 Western Blot bands used for the quantification of proteins BAX, BCL-2 and cleaved PARP1 in HCT116-DoxR after their exposure to complexes 5, 6 or DMSO Figure S19 page S36

S2
Viability of HCT116-DoxR cells after exposure to TBHP for 48 hours Figure S20 page S37 UV-Vis spectra of complexes 5, 6 and 8 with its concentrations increasing and of DMSO, used as the control, measured and recorded after 24 h of exposure Figure S21 page S38 Fluorescence spectra of complexes 5, 6 and 8 with its concentrations increasing, measured and recorded after 24 h of exposure Figure

X-ray Crystal Structure Determination
The X-ray diffraction data for complexes 1-7 were collected using Oxford Diffraction four-circle diffractometer Gemini A Ultra with Atlas CCD detector using graphite monochromated MoKα radiation ( = 0.71073 Å) at room temperature. Diffraction data collection, cell refinement and data reduction were performed using the CrysAlis Pro software 1 . The structures were solved by the direct methods using SHELXS and refined by full-matrix least-squares on F 2 using SHELXL-2014 2 . All the non-hydrogen atoms were refined anisotropically, and hydrogen atoms were placed in calculated positions and refined with riding constrains: d(C-H) = 0.93 Å, U iso (H) = 1.2 U eq (C) (for aromatic) and d(C-H) = 0.96 Å, U iso (H) = 1.5 U eq (C) (for methyl). The methyl groups were allowed to rotate about their local threefold axis. For structures 1, 4 and 7 which show severe disorder of solvent molecules, OLEX2 solvent mask command was employed 3 . Details of the crystallographic data collection, structural determination, and refinement for 1-7 are given in Table S1, whereas selected bond lengths  and angles for these systems are listed in Tables S2-S8.

Physical Measurements
IR spectra were recorded on a Nicolet iS5 FT-IR spectrophotometer in the spectral range 4000-400 cm 1 with the samples in the form of KBr pellets ( Figure S8). Elemental analyses (C, H, N) were performed on a Perkin-Elemer CHN-2400 analyzer. The electronic spectra were obtained using Nicolet Evolution 220 in the range 240-1000 nm in DMSO and MeOH (Figures S11-S13 and Table  S12).

Magnetic measurement
The magnetic measurements were made on a Quantum Design MPMS SQUID magnetometer in the room temperature and magnetic field of 0.5 T. The susceptibilities of 1−7 have been corrected for the diamagnetic contribution by using the Pascal's constants.

EPR spectroscopy
The X-band electron paramagnetic resonance (EPR) spectra of the dipicolinate oxovanadium(IV) complexes were measured using a Bruker ELEXYS E 500 Spectrometer equipped with NMR teslametr and X-band frequency (9.7 GHz, microwave frequency 6.231 GHz, power of 10 mW and modulation amplitude of 8 G). The solid compound 1, 2 dissolved in water and a few drops of DMSO were added to the samples to ensure good glass formation at liquid nitrogen temperature. Anisotropic spectra were recorded on frozen solutions at 77 K using quartz Dewar and glass capillary tubes at room temperature. The experimental spectra were simulated with the DoubletExact (S=1/2) computer program 4 .

Lipophilicity
The n-octanol/PBS buffer partition coefficients (logP) were determined by shake-flash approach, and the transfer of V(IV) compounds from the aqueous environment to the organic phase was monitored by UV-Vis spectroscopy 5,6 . Solutions of 1-10 of concentrations in the range 10-100 M were prepared in PBS buffer saturated with n-octanol from the freshly-prepared stock solution in DMSO (5mM or 50 mM), and their UV-Vis spectra were recorded. The solution of 1-10 in aqueous buffer (3 ml) were mixed with n-octanol saturated with pH 7.4 aqueous buffer (3 ml), and the obtained mixtures were stirred in a mechanical shaker for 30 min. The mixtures were allowed to stand at room temperature for 24 h, and after this time, the aqueous phases were separated, and the concentrations of 1-10 were determined by measuring their UV-Vis spectra. For each complex, the highest energy absorption band was taken into consideration to determine the sample concentration.

Stability of vanadium complexes in biological medium
The stability of vanadium complexes in biological medium was evaluated by UV-Vis spectroscopy (between 220-700 nm). Complexes were initially solubilized in 100 % (v/v) DMSO and afterwards each complex was diluted with colourless (without phenol red) RPMI medium to a final concentration of 50 μM. A quartz cuvette with 1 cm path length was used and spectra were obtained after 0 h, 24 h and 48 h on the spectrophotometer Evolution 300 UV-Vis (Thermo Fischer Scientific, Waltham, MA, USA).

Cell viability assays
HCT116, HCT116-DoxR and A2780 cancer cell lines and normal human primary fibroblasts were used in cellular viability assays. Cells were seeded in a density of 0.75×10 5 cells/mL into 96 wellplates and incubated at 37ºC and 5% (v/v) CO 2 for 24 h. After the 24 h of incubation, complexes were weight and immediately solubilized in 100% (v/v) DMSO and then, diluted for the final concentration with cell culture medium. The medium in each 96 well-plates will be replaced by fresh medium containing each vanadium complex (concentrations ranging from 0.01 µM to 50 µM). Culture medium with 0.1% (v/v) DMSO and 0.4 µM Dox were used as negative and positive controls, respectively. Following a second incubation period of 48 h in the same conditions, cells' viability was assessed with the CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, USA) 8 . When cells are metabolically active, its mitochondrial dehydrogenases can reduce 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) to formazan, whose absorbance can be measured at 490 nm in a microplate reader, Tecan Infinite M200 (Tecan, Männedorf, Switzerland). Thus, formazan's absorbance is directly proportional to the number of viable cells 9,10 . By analysing the cell viability vs. concentration graphics, obtained using Prism 8 (GraphPad software), it is possible to determine the IC 50 (complex concentration which induces a 50% reduction in cell viability) of each complex in the respective cell line. The selective index (SI) for every complex was also determined, dividing the IC 50 of human fibroblasts by the IC 50 of HCT116, HCT116-DoxR or S4 A2780 cancer cell lines, respectively.

3D-spheroid formation and cell viability assays
HCT116-DoxR cells were seeded at 5000 cells/well in super low Attachment 96-well plates (Nunclon TM Sphera TM U-Shaped-Bottom Microplate, Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37°C, in a humidified atmosphere with 5% (v/v) CO 2 , to allow spheroids formation and growth. For MTS assay, HCT116-DoxR spheroids with 6 to 8 days were used. At the 6 th day of growth, culture media was replaced by medium with vanadium complexes at desired concentrations. Spheroids were then incubated for 48 h in a humidified atmosphere at 37ºC and 5% (v/v) CO 2 and, after the incubation time, medium was replaced by a mixture containing the MTS reagent and DMEM medium (20:100). Spheroids were incubated for another 4 h period and later transferred into a 96-well plate with flat bottom in order to be analysed in the microplate reader Tecan Infinite M200 (Tecan, Männedorf, Switzerland).

ICP-AES (Inductively coupled plasma -atomic emission spectrometry)
Internalization of vanadium complexes in the HCT116-DoxR cell line was assessed by ICP-AES. To do so, HCT116-DoxR cell line was seeded in 25 cm 2 T-flasks in a density of 5×10 5 cells/T-flask and incubated for 24 h in a humidified atmosphere at 37ºC and 5% (v/v) CO 2 . After the 24 h incubation, the culture media was replaced by fresh medium containing different vanadium complexes with 10 times its IC 50 concentrations or 0.1% (v/v) DMSO, and cells were later incubated for another 3 h period in the same conditions. Then, culture media was recovered, and cells were washed with PBS 1x, also recovered. The recovered culture media and PBS 1x were centrifuged at 800 xg (Sigma 3-16K Sartorius, Germany) for 5 min to separate the supernatant and some cells that might be present on them. Cells were detached from each T-flask with 2 mL of TrypLE Express and were centrifuged at 750 xg for 5 min. Fresh aqua regia (3:1 HCl/HNO 3 ) was added to supernatants and cellular pellets, and every sample were incubated at RT for 24 h in a hood fume. Samples were later delivered to Laboratório de Análises/LAQV and the levels of vanadium were evaluated by ICP-AES.

Evaluation of apoptosis induction in HCT116-DoxR cell line by flow cytometry
The induction of apoptosis in HCT116-DoxR cell line was evaluated by flow cytometry, using the Alexa Fluor ® 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen, Thermo Fisher Scientific, MA, USA). HCT116-DoxR cell line was seeded at a cell density of 1×10 5 cells/mL in 6-well plates and incubated for a 24 h period. Later, culture media was replaced by medium with different vanadium complexes at its IC 50 concentrations. Culture medium with 0.1% (v/v) DMSO, 6 μM Dox or 5 μM Cisplatin were used as controls. After 48 h of incubation, cells were washed with PBS 1x, detached from each well with TrypLE Express (Invitrogen) and washed again with PBS 1x. Cells were then resuspended in 5x annexin-binding buffer and incubated at RT (room temperature) for 15 min with Alexa ® Fluor 488 annexin V and 100 μg/mL PI. All samples were analysed by an Attune ® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA) and results were processed with Attune ® Cytometric software.

Western Blot for BAX, Bcl-2 and PARP-1 Quantification
HCT116-DoxR cell line was cultivated in 25 cm 2 T-flasks at a cell density of 2×10 6 cells/T-flask and incubated for 24 h in the conditions previously described. After 24 h, the culture media was replaced by fresh medium with different vanadium complexes at its IC 50 concentrations or 0.1% (v/v) DMSO. It was additionally prepared a 25 cm 2 T-flask containing untreated cells as a control. Cells were incubated for another 48 h period and were later washed and collected using cold PBS 1x and a cell scraper. The samples were centrifuged for 5 min at 700 xg (Sigma 3-16K Sartorius, Germany) and resuspended in fresh lysis buffer (150 mM NaCl; 5 mM ethylenediaminetetraacetic acid (EDTA); 50 mM Tris-HCl pH 8.0; 2% (v/v) NP-40 (Thermo Fisher Scientific, Waltham, MA, USA); 1x phosphatase inhibitors (PhosStop, Roche); 1x protease inhibitors (complete ULTRA tablets, Mini, EASYpack, Roche, Switzerland); 1 mM phenylmethylsulfonyl fuoride (PMSF, Sigma, St. Louis, USA) and 0.1% 1,4-dithiothreitol (v/v) (DTT, Amresco, USA). Cells were then submitted to 5 ultrasound cycles on ice (2 min on ultrasounds followed by a 30 s period on ice; Elma D-78224; Singen/Htw, Germany) and later centrifuged at 1000 xg for 5 min. Total protein extract (from supernatant) was quantified with Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA).
For the SDS-PAGE of BAX and Bcl-2, 20 μg of protein was loaded on 10% polyacrylamide gel and transferred to a 0.45 μm PVDF membrane (GE Healthcare Life Sciences, Germany). Each membrane was incubated 2 h with 5% (w/v) non-fat milk or Bovine Serum Albumin (BSA) solubilized in TBST 1x buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20) and later incubated for 1 h, at RT and constant agitation, with a solution containing the respective primary antibody in 5% non-fat milk in TBST 1x, specifically anti-BAX (1:5000; Abcam, United Kingdom) and anti Bcl-2 (1:1000; Sigma, St. Louis, USA). PARP 1 was first transferred into a 0.45 μm nitrocellulose membrane (GE Healthcare Life Sciences, Germany) and later, the membrane was incubated overnight at 4 ºC under constant agitation, with a solution containing the respective primary antibody in 5% BSA in TBST 1x (1:750; Thermo Fisher Scientific, Waltham, MA, USA). After that, membranes were washed three times with TBST 1x buffer for 5 min each, under constant agitation. This procedure was repeated in the incubation with the secondary antibody (1:3000, Antimouse IgG, horseradish peroxidase (HPR)-linked Antibody or 1:2000, Anti-rabbit IgG, HPR-linked Antibody; Cell Signalling Technology, USA). In order to identify the protein bands, membranes were treated with WesternBright ECL substrate (Advansta, USA) for 5 min and film was exposed to the membrane on a dark room. Membranes were later incubated for two times with Stripping buffer (0.1 M glycine, 20 mM magnesium acetate, 50 mM KCl, pH 2.0) during 10 and 20 min, respectively, under agitation and then, incubated with anti-β actin (1:5000; Sigma, St. Louis, USA) as a control to normalize the results. Proteins' quantification was made by densitometry with Image J software.

Evaluation of mitochondrial membrane potential ( m ) in HCT116-DoxR cell line by
∆ flow cytometry m was evaluated in HCT116-DoxR cell line by using the JC-1 Mitochondrial Membrane Potential ∆Ψ Assay Kit (Abnova Corporation, Walnut, CA, USA). Cells were seeded at a density of 1×10 5 cells/mL in 6-well plates and, after 24 h of incubation, culture media was replaced by medium with different vanadium complexes at its IC 50 concentrations. 0.1% (v/v) DMSO was used as a negative (vehicle) control while 6 μM Dox and 5 μM Cisplatin were used as positive controls. Cells were then incubated for 48 h and after that, cells were washed with PBS 1x, detached with TrypLE Express and washed again with DMEM medium. Cells were later resuspended in DMEM medium without phenol red + 5% (v/v) FBS and incubated with JC-1 in DMEM medium without phenol red + 5% (v/v) FBS for 20 min at 37ºC. The cells were then resuspended in DMEM medium without phenol red + 5% (v/v) FBS and the analysis of mitochondrial membrane potential was performed in the Attune ® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA).

Caspase-8 activity
The Caspase-8 activity in HCT116-DoxR cells exposed to vanadium complexes was evaluated using the Caspase-8 Assay kit (ab39700) (Abcam, Cambridge, UK). HCT116-DoxR cell line was cultivated in 25 cm 2 T-flasks at a cell density of 2×10 6 cells/T-flask and incubated for 24 h in a CO 2 incubator at 37ºC, 5% (v/v) CO 2 and 99% (v/v) relative humidity. After 24 h, medium was replaced with fresh medium supplemented with 0.1% (v/v) DMSO, 5 μM Cisplatin or the IC 50 concentration of the desired vanadium complexes. After 48 h of incubation, cells were detached with a cell scraper, collected into eppendorfs, and centrifuged at 4ºC and 500 xg for 5 min. Cells were then washed with 1 mL of cold PBS and centrifuged at 750 xg and 4ºC for 5 min. Right after, the pellet was resuspended in 50 μL of cold Lysis Buffer and the cells were incubated on ice for 20 min, being then centrifuged at 1000 xg and 4ºC for 10 min. Duplicate wells were prepared in a 96-well plate with 200 μg of protein in Lysis Buffer for a final volume of 50 μL of sample. It was added to each well 50 μL of the freshly prepared Reaction mix as well as 5 μL of IETD-pNA. The plate was then incubated under the same conditions as described above for 2 h and then absorbance was read at 400 nm on a Tecan Infinite M200 microplate reader (Männedorf, Switzerland).

Evaluation of autophagy induction in HCT116-DoxR cell line by flow cytometry
Autophagy induction in HCT116-DoxR cell line was evaluated using the Autophagy Assay Kit (ab139484) (Abcam, Cambridge, United Kingdom) according to the manufacturer's instructions. Briefly, HCT116-DoxR cell line was seeded in a density of 1×10 5 cells/mL in 6-well plates and, after an initial incubation time of 24 h, culture media was replaced by medium with different vanadium complexes at its IC 50 concentrations. DMSO 0.1% (v/v), 6 μM Dox and 5 μM Cisplatin were also used as controls. Cells were incubated for another 48 h, and 1,5 μM Rapamycin was added 18 h before finishing the 48 h period. After that, cells were firstly washed with PBS 1x and then detached with TrypLE Express, followed by another wash with DMEM medium without phenol red + 5% (v/v) FBS. Cells were then incubated with Green Stain solution in DMEM medium without phenol red + 5% (v/v) FBS for 30 min at RT. After the incubation period, cells were washed and later resuspended with Assay Buffer 1x. The evaluation of autophagy induction occurred due to the Attune ® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA) and the results were analysed via the Attune ® Cytometric software.

Evaluation of reactive oxygen species (ROS) production in HCT116-DoxR cell line by flow cytometry
HCT116-DoxR cell line was seeded in 6-well plates in a density of 1×10 5 cells/mL and incubated for 24 h. After that time, the culture media was replaced by medium with different vanadium complexes at its IC 50 concentrations and DMSO 0.1% (v/v), 6 μM Dox, 5 μM Cisplatin and 42 μM TBHP (Tert-Butyl hydroperoxide) were used as controls. After that, cells were incubated for another 48 h period. Later, cells were washed with PBS 1x, detached from each well with TrypLE Express, washed again with PBS 1x and later incubated with 10 μM of 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) (Thermo Fisher Scientific, Waltham, MA, USA) in PBS 1x, for 20 min. Cells were later analysed with the Attune ® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA) and the obtained data was processed by the respective software (Attune ® Cytometric software).

Cell cycle progression
HCT116-DoxR cells were seeded in 6-well plates in a density of 1×10 5 cells/mL, incubated for 8 h in a humidified atmosphere at 37ºC and 5% (v/v) CO 2 , and later synchronized in early S-phase with a 2 mM thymidine solution (Sigma, St. Louis, USA) for a 16 h period. Media with thymidine was replaced by fresh media and, after 8 h, another 2 mM thymidine solution was added to the wells (double thymidine block). Cells were incubated for 16 h and then culture media was replaced by medium with the vanadium complexes at its IC 50 concentrations. 0.1% (v/v) DMSO was used as the vehicle control while 5 μM cisplatin and 6 μM doxorubicin as the positive controls. To normalize the results, untreated cells were collected right after the double thymidine block (0 h). Treated cells were then incubated for 3 h, 9 h, 24 h or 32 h and, after each time point, cells were treated with TrypLE Express and centrifuged at 650 xg for 5 min, at 4ºC. The resultant pellet was resuspended in 1 mL of cold PBS 1x and centrifuged at 3000 xg for 5 min and 4ºC. After this, the pellet was resuspended in 100 μL of PBS 1x, and 1 mL of a solution of 80% (v/v) ethanol was gently added to the cells. Cells were stored at 4°C for at least 12 h and, after incubation, cells were centrifuged for 10 min at 7500 xg at 4°C, treated with 250 μL of 50 μg/mL RNase A and incubated for 30 min at 37°C. After incubation, 100 μL of PI (25 μg/mL) was added to the cells as well as 650 μL of PBS 1x. The DNA content was evaluated on Attune ® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA) and results were analysed by the respective software (Attune ® Cytometric software).

BSA binding assays
All solutions used in the BSA interaction studies were diluted in filtered buffer (10 mM phosphate and 150 mM NaCl) at pH 7.0, with BSA at a fixed final concentration of 20 μM and the concentrations of the complexes varying from 10 μM to 100 μM. Control solutions were also prepared, namely a solution containing only BSA, a solution consisting of BSA + DMSO and a solution consisting of DMSO only. Solutions were analysed after 24 hours of incubation at 37ºC. The UV-Vis spectra were obtained from 245 nm to 500 nm on the spectrophotometer Evolution 300 UV-Vis (Thermo Fischer Scientific, Waltham, MA, USA) while the fluorescence spectra were recorded through an emission range of 290 to 500 nm after an excitation wavelength of 278 nm (excitation and emission slit widths of 5 nm), on a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, CA, USA).

Cell Migration assay
Fibroblasts were seeded in 24-well plates in a density of 4×10 5 cells/mL and grown in the same conditions as described before until a confluent monolayer was obtained. Cells were later exposed to 0.1% (v/v) DMSO or the respective IC 50 concentrations of vanadium complexes and, with a sterile 200 μL micropipette tip, a scratch was made on the surface of each well. Fibroblasts were photographed right after exposure (0 h) and 24 h after incubation at 37ºC and 5% (v/v) CO 2 in a humidified atmosphere, using a Ti-U Eclipse inverted microscope (Nikon, Japan). The scratches were analysed S7 via ImageJ software.

Ex-ovo Chick Chorioallantoic Membrane (CAM) assay
The ex-ovo chorioallantoic membrane (CAM) assay was performed in order to evaluate the angiogenic potential of vanadium complexes, as already described in literature [11][12][13] . Firstly, chicken embryos were incubated for a 24 h period, to stabilize the embryos and later, a solution containing the IC 50 concentration of each complex dissolved in PBS 1x was placed in the centre of O-rings. Beside each complex, DMSO 0.1% (v/v) was used as a control and complex distribution in chicken embryos was always performed in a different order. The embryos were incubated for 48 h at 37ºC and images were captured after 0 h, 24 h and 48 h, using a digital USB Microscope Camera (Opti-Tekscope OT-V1). Newly formed blood vessels were later manually counted, via ImageJ software. The ex-ovo CAM assay fulfils the Directive 2010/63/ EU of the European Parliament for protection of animal models for scientific purposes.

Statistical analysis
All data are presented as Mean ± SEM from at least two independent experiments unless otherwise specified. One-way ANOVA or Student's t-test were used to determine statistical significance (p < 0.05) using the GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA).

HPLC
HPLC measurements were carried out on the apparatus Kanuer liquid chromatograph with UV-VIS detection and Agilent 1100 liquid chromatograph, as well as Waters 600 liquid chromatograph equipped with UV-Vis detector. In reference to limited reports on this subject [14][15][16][17] , the methanol-water, acetonitrile-water or methanol-acetonitrile-water phases were used. As demonstrated by selected chromatograms below, however, this method was found to be unsuitable for systems of our type, most probably due to their poor affinity for the methanol-water, acetonitrile-water or methanol-acetonitrile-water phases.    Table S9. Short intra-and intermolecular hydrogen bonds in structures 1-7      Figure S9. The X-band EPR spectra of dipicolinate oxovanadium(IV) complexes at 77 K together with the spectra calculated by computer simulation of the experimental spectra with average g parameters: 1.993 (1) Figure S10. EPR frozen solution spectra (at 77 K) of dipicolinate oxovanadium(IV) complexes in DMSO solvent together with the theoretical spectra calculated using the parameters: g x = g y =1.997, g z = 1.962, A x = A y = 65.4 G, Az = 165 G for 1, g x = g y =1.979, g z = 1.958, A x = A y = 75.3 G, A z = 186 G for 2, g x = g y =1.981, g z = 1.942, A x = A y = 65.4 G, A z = 180 G for 3, g x = g y =1.984, g z = 1.946, A x = A y = 68.4 G, A z = 169 G for 4, g x = g y =1.977, g z = 1.958, A x = A y = 62 G, A z = 1688.5 G for 5, g x = g y =1.984, g z = 1.956, A x = A y = 71.5 G, A z = 179 G for 6, g x = g y =1.979, g z = 1.950, A x = A y = 75, 2 G, A z = 165 G for 7, g x = g y =1.978, g z = 1.942, A x = A y = 65.5 G, A z = 180 G for 8 and g x = g y =1.977, g z = 1.958, A x = A y = 72 G, A z = 169 G for 9.