Effects of Decavanadate Salts with Organic and Inorganic Cations on

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Effects of Decavanadate Salts with Organic and Inorganic Cations on Escherichia coli, Giardia intestinalis, and Vero Cells Juliana M. Missina,† Bruno Gavinho,‡ Kahoana Postal,† Francielli S. Santana,† Glaucio Valdameri,§ Emanuel M. de Souza,‡ David L. Hughes,∥ Marcel I. Ramirez,⊥ Jaísa F. Soares,† and Giovana G. Nunes*,†

Inorg. Chem. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/10/18. For personal use only.

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Departamento de Química and ‡Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas, 81530-900 Curitiba, Paraná, Brazil § Departamento de Análises Clínicas, Universidade Federal do Paraná, Campus Jardim Botânico, Jardim Botânico, 80210-170 Curitiba, Paraná, Brazil ∥ School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom ⊥ Fundaçaõ Osvaldo Cruz, Av. Brazil, Manguinhos, 4365 Rio de Janeiro, Brazil S Supporting Information *

ABSTRACT: Decavanadate salts with nicotinamide (3pyridinecarboxamide, 3-pca) and isonicotinamide (4-pyridinecarboxamide, 4-pca) in both neutral and protonated forms, (3-Hpca)4[H2V10O28]·2H2O·2(3-pca) (complex I) and (4Hpca)4[H2V10O28]·2(4-pca) (complex II), have been synthesized and characterized by vibrational spectroscopy (infrared and Raman), thermogravimetric analysis (TGA), 51V NMR, and single-crystal X-ray diffraction analysis. The effects of sodium decavanadate (henceforth called NaV10) and compounds I and II on Escherichia coli, Giardia intestinalis, and Vero (African green monkey epithelial kidney) cells were evaluated. Enhanced growth inhibitory activity against E. coli cultures was observed upon treatment with products I and II when compared to that with NaV10 (GI50 values of 2.8, 4.0, and 11 mmol L−1, respectively), as well as lower cell viability as measured by the intake of propidium iodide (PI). Exposure of Giardia trophozoites to NaV10 and II revealed reduction in trophozoite viability (GI50 values of ca. 10 μmol L−1) and affected the parasite adherence to both polystyrene culture tubes and a monolayer of Vero cells, even at low concentrations. A lesser effect on Giardia was shown for I. Furthermore, all three compounds were significantly less toxic to Vero cells than the reference drug, albendazole, employed in the treatment of giardiasis. Toxicity reports of oxidovanadium compounds toward Giardia are unprecedented and open a path to the development of new therapeutic agents.

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INTRODUCTION Decavanadate, [HnV10O28](6−n)− (V10), has recently attracted attention as a potential precursor of therapeutic agents against a number of maladies, and much effort has been made to shed light on the pathways through which treatment with V10 affects lipidic structures, cell surface proteins and microbial targets.1−5 Toxicology studies with this polymetallic aggregate have also increased since 2005 and have recently been reviewed.6 Additionally, the anion has been evaluated for its applications in catalysis,7 protein crystallography,8 and material sciences.9 Many crystal structures of simple inorganic decavanadates and of V10 associated with organic cations have been reported.10 Though such data have provided valuable insights on possible interactions of V10 with biological targets, only recently the effect of pharmacologically relevant counterions on the biological activity of decavanadate has been addressed. As examples, V10 with zwitterionic betaines such as trimethylammonium acetate and trigonelline11 inhibited the © XXXX American Chemical Society

proliferation of MCF-7 breast cancer and A549 lung adenocarcinoma cells. Decavanadate with carnitine,12 in turn, was demonstrated to be 6−10 times more potent than orthovanadate and decavanadate with simpler organic counterions toward murine and human tumor cell lines. Lastly, studies with murine models of both type I and II diabetes have shown that the association of metforminium cations with decavanadate elicited higher glycemic and lipidemic responses,2 lower toxicity and increased insulin production. Whether these biological effects are caused by V10 or by lower-nuclearity vanadates generated from it in the course of the experiments is yet to be clarified;6 however, such enhanced responses in the presence of different cations prompts additional studies involving new salts of V10 with organic counterparts. Received: May 11, 2018

A

DOI: 10.1021/acs.inorgchem.8b01298 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Even though the potential antibacterial,13−16 antiparasitic,17 and antiviral18 effects of decavanadate have been frequently mentioned, these pharmacological activities remain scantly explored when compared to anticancer, antidiabetic, and interaction studies with potential targets such as mitochondria and proteins.6 Promising results have been obtained with chitosan-Ca3V10O28 membranes against Staphylococcus aureus and Escherichia coli.13 The increased toxicity was primarily assigned to a combination of the chitosan antimicrobial activity and the decavanadate capability to inhibit ion pumps, causing a severe disturbance in cell metabolism. As far as protozoa models are concerned, (NH4)6(V10O28)·5H2O demonstrated in vitro toxicity against promastigotes of Leishmania tarentolae.17 The reduction of cell viability by 50% was attributed to the increase in the intracellular level of the superoxide radical. E. coli is a flagellated, Gram-negative, facultative anaerobic bacterium that is a normal constituent of the mammalian gut microbiome.19 Despite this, its relationship with humans is not entirely benign; some pathogenic strains have evolved from the acquisition of virulence factors encoded on plasmids, transposons, and bacteriophages.20 In fact, E. coli is the cause of several diseases in humans such as urinary tract infections, bacteremia, colitis, and diarrhea. Infections due to E. coli have largely been related to poor hygienic conditions21 or healthcare associated phenomena, the antimicrobial therapy of which costs billions of dollars each year to treat around 2 million infected individuals throughout the globe.19 Giardia intestinalis (G. lamblia or G. duodenalis), in turn, is one of the most common protozoal causes of diarrheal disease in humans worldwide, infecting hundreds of millions of people every year.22 As part of its living cycle, after ingestion and contact with the acid environment of the stomach, Giardia cysts turn into trophozoites in the proximal portion of the small intestine.23 The adherence of these trophozoites to the intestinal epithelium is accompanied by excretion of parasite toxins; this damages the host cells, causes reduction of the enzymatic functions of the microvilli, and may lead to apoptosis. These changes affect the electrolytic balance and increase intestinal permeability, causing diarrhea.23 As no vaccine is available for the prevention of giardiasis, treatment remains based on chemotherapy. Among the few therapeutic agents, the most generally efficient 5-nitroheterocyclic drugs, albendazole and metronidazole, fail in up to 20% of cases.22 Resistance and high toxicity of commercial drugs have been reported, opening the way to the development of novel potent drugs that are less toxic to patients.22 Among these, auranofin, a gold-containing coordination compound originally used for the treatment of rheumatoid arthritis, has recently achieved phase I clinical trials.24 In addition, silverbased compounds that are well-known for their antibacterial and antiparasitic properties have also demonstrated promising results.25 Because giardiasis is not a chronic disease, the problems that are foreseen with using metal complexes for treating chronic conditions (diabetes, for example) may be circumvented in short-term treatments such as those employed for Giardia. The present work investigates the possible growth inhibitory and/or cytotoxic effects of the addition of V10 associated with organic counterions to E. coli and G. intestinalis cultures. These biological models have in common the capacity to induce pathological responses following colonization of the human gut, but their very distinct physiology and host−pathogen interactions, which in turn determine different clinical

outcomes, could lead to distinctive responses upon treatment with V10 salts. Decavanadate was synthesized in the presence of nicotinamide (3-pyridinecarboxamide), one of the forms of the hydrosoluble vitamin complex B3,26 and its isomer isonicotinamide (4-pyridinecarboxamide), producing compounds I and II, respectively. Even though isonicotinamide is not recognized as a substance of biological significance, it was chosen on the basis of being considered one of the most effective molecules in the cocrystallization of drugs.27 After characterization, the toxic effects of synthesized V10 salts I and II and of sodium decavanadate, [{Na6(H2O)20V10O28·4H2O}n] (NaV10),28 were evaluated on E. coli and G. intestinalis, the latter attached to polystyrene culture tubes or to a layer of mammalian Vero cells. This has been the first attempt to assess the effect of an oxidovanadium compound against Giardia trophozoites. Results with E. coli were more significant for I and II as compared with that for NaV10, while in G. intestinalis all compounds were effective in the detachment of trophozoites without being toxic to Vero cells. These findings suggest a potentially relevant role of oxidovanadium(V) compounds as new chemotherapeutic tools, particularly against Giardia infections.

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EXPERIMENTAL SECTION

General. The syntheses of I and II and the preparation of solutions were carried out in ultrapure water (Milli-Q, Millipore type 1, resistivity of 18.2 MΩ cm at 25 °C). Reactants vanadium(V) oxide (V2O5, ≥99.6%), 3-pyridinecarboxamide (nicotinamide, ≥99.5%), and 4-pyridinecarboxamide (isonicotinamide, 99%) were purchased from Sigma-Aldrich and used without further purification. Sodium decavanadate [{Na6(H2O)20V10O28·4H2O}n] (NaV10) was prepared according to the literature.28 Analytical Methods and Instruments. Vanadium(V) contents were determined by reduction titration with an aqueous solution of (NH4)2[Fe(SO4)2]·6H2O (0.01 mol L−1).29 Carbon, hydrogen, and nitrogen contents were determined by combustion analysis on a Thermal Scientific Flash EA 1112 Series Elemental Analyzer run by MEDAC Laboratories, Ltd. (Chobham, Surrey, UK). Absorption spectra in the medium infrared region (400 to 4000 cm−1) were obtained in KBr pellets from a BIORAD FTS 3500GX spectrophotometer, with resolution of 4 cm−1. Raman spectra were obtained on a Renishaw Raman Image spectrophotometer coupled to an optical microscope that focuses the incident radiation down to a 1 μm spot. A He−Ne laser (632.8 nm) with an incident potency of 0.2 mW was used over the 200−2000 cm−1 region. Electronic spectra were obtained from aqueous solutions on a PerkinElmer Lambda 1050 UV−vis−NIR spectrophotometer. 51V NMR (105.25 MHz) spectra were recorded at 295 K in D2O solutions with a Bruker AVANCE 400 spectrometer operating at 9.4 T and equipped with a direct detection multinuclear probe (5 mm). VOCl3 (pure, capillary) was used as external reference for 51V spectra, and its signal was fixed at 0.00 ppm. Spectra were acquired using calibrated 90° pulses and 1024−2048 scans with a recycling delay of 0.1 s and acquisition times of 0.157 s, on a spectral width of 990 ppm (+44 to −946 ppm). Signal intensities were normalized in each experiment by comparison with the reference signal. Thermogravimetric analyses (TGA) were run on a Netzsch STA449 F3 Jupiter analyzer instrument equipped with a silicon carbide furnace. Samples (ca. 4 mg) were heated in aluminum pans using a mixture of N2/O2 as carrier gas, from 25 to 800 °C at a heating rate of 10 °C min−1. Syntheses. Preparation of (3-Hpca)4[H2V10O28]·2H2O·2(3-pca) (I). To a suspension of V2O5 (0.46 g, 2.5 mmol) in 40 mL of water, a solution of nicotinamide (0.37 g, 3.0 mmol) in 10 mL of water was added. The resulting suspension was stirred and heated under reflux for 3 h. The content of the flask was then filtered under vacuum in order to remove the excess of V2O5. The filtrate was then carefully B

DOI: 10.1021/acs.inorgchem.8b01298 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry topped with a layer of isopropanol and was stored at 4 °C. Yellow crystals suitable for single-crystal X-ray diffraction analysis were formed after 3 days. The mother liquid was transferred to another flask, and the crystals were dried in air (0.33 g, 0.19 mmol). The product was soluble in hot water and dimethyl sulfoxide. Yield: 38% based on the formulation (3-Hpca)4[H2V10O28]·2H2O·2(3-pca). Elemental anal. calcd for C36H46N12O36V10 (1732.25 g mol−1): V, 29.4; C, 24.96; H, 2.68; N, 9.70%. Found: V, 30.3; C, 24.93; H, 2.69; N, 9.81%. FTIR (KBr, cm−1, s = strong, m = medium, w = weak, br = broad): 3541(br), 3379(m), 3215(m), 1676(s), 1628(m), 1421(w), 1198(w), 1140(w), 957(s), 829(m), 745(m), 602(m), 550(m). 51V NMR (D2O, ppm): −425(s), −507(s), −525(s). Preparation of (4-Hpca)4[H2V10O28]·2(4-pca) (II). A solution of 0.37 g (3.0 mmol) of isonicotinamide in 10 mL of water was added to a suspension of V2O5 (0.46 g, 2.5 mmol) in 40 mL of water. After stirring and heating under reflux for 3 h, the suspension was filtered under vacuum. Yellow crystals formed as soon as the solution cooled down to room temperature and were dried in air (0.17 g, 0.10 mmol). The product was soluble in hot water and dimethyl sulfoxide. Yield: 20% based on the formulation (4-Hpca)4[H2V10O28]·2(4-pca). Elemental anal. calcd for C36H42N12O34V10 (1696.21 g mol−1): V, 30.0; C, 25.49; H, 2.50; N, 9.91%. Found: V, 30.6; C, 25.55; H, 2.59; N, 9.87%. FTIR (KBr, cm−1, s = strong, m = medium, w = weak, br = broad): 3400(br), 3092(w), 1686(s), 1607(m), 1398(w), 1240(w), 1113(w), 962(s), 831(m), 741(m), 575(m). 51V NMR (D2O, ppm): −425(s), −507(s), −525(s). Single-Crystal X-ray Diffraction Analyses. Product I. Experimental conditions, X-ray diffraction data, and crystal structure diagrams are presented Tables S1−S3 and Figures S1 and S2). Product II. From a sample under oil, one yellow block, ca. 0.129 × 0.091 × 0.034 mm3, was mounted on a MiTeGen micromount/mesh and fixed in the cold nitrogen stream on a Bruker D8 Venture diffractometer equipped with a Photon 100 CMOS detector, Mo Kα radiation and graphite monochromator. Intensity data were measured by thin-slice ω- and φ-scans. Total number of reflections recorded, to θmax = 27.5°, was 57 076 of which 6081 were unique (Rint = 0.092); 4752 were observed with I > 2σ(I). Data were processed using the APEX3 program.30 The structure was determined by the direct methods routines in the SHELXS program31 and refined by fullmatrix least-squares methods, on F2, in SHELXL.31,32 The nonhydrogen atoms were refined with anisotropic thermal parameters. All hydrogen atoms were located in difference maps and were refined isotropically and freely; restraints were applied only to the H atoms bonded to O(4) and to N(2). At the conclusion of the refinement, Rw = 0.078 and R = 0.05631,32 for all 6081 reflections weighted w = [σ2(Fo2) + (0.0299P)2 + 1.9201P] −1 with P = (Fo2 + 2Fc2)/3; for the observed data only, R = 0.035. In the final difference map, the highest peak (ca. 0.48 eÅ−3) was on the C13−C14 bond. Scattering factors for neutral atoms were taken from the literature.33 Computer programs used in this analysis were noted above and run through WinGX.34 Biological Assays. General. All solutions, flasks and materials employed in the biological assays were autoclave-sterilized. The optical density (OD) of the bacterial cultures was measured at 600 nm (OD600) on a UV/vis Pharmacia LKB − Ultraspec III spectrophotometer. Cell viability assays with propidium iodide (PI) were performed for E. coli using a BD Accuri C5 Flow Cytometer (Becton Dickinson), with laser excitation at 488 nm and FL2 detector that captures fluorescence at 582/40 nm. Data were collected with the BC Accuri C6 software. Ampicillin was purchased from Invitrogen and propidium iodide (PI, C27H34I2N4) from Sigma-Aldrich. Albendazole (Sigma-Aldrich) was solubilized in dimethyl sulfoxide and used to keep final concentration within 0.1% range. WB isolates of G. intestinalis (ATCC 50803) were grown in TYI-S-33 medium prepared from Sigma-Aldrich substrates and supplemented with 10% adult bovine serum (Cripion), penicillin/streptomycin (10 000 UI) in 5% CO2 and 95% air. Giardia trophozoites were counted on a hemocytometer under a Bioval optical microscope. Vero cells were grown in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Cultilab) supplemented with 10% fetal bovine serum

(Thermo-Fisher Scientific), penicillin/streptomycin (10 000 UI, Thermo-Fisher Scientific). In the cytotoxicity assay, the plates were read on a Biorad 550 spectrophotometer. Studies on E. coli Cultures: Growth Inhibition Assay. Nalidixic acid resistant (NalR) E. coli DH5α colonies were transferred to 10 mL of Luria−Bertani (LB) broth, to which was added 10 μL of Nal (10 μg mL−1). The bacterial cells were grown at 37 °C in a rotary shaker (120 rpm) to OD600 close to 1.0. Cell pellets were obtained by centrifugation (4900 × g, 10 min) and resuspended, after supernatant removal, in 1.0 mL of 0.9% saline (negative control) or 1.0 mL of solutions of products I, II, and NaV10 (0.025, 0.1, 2.5, 5.0, and 10 mmol L−1). These solutions were prepared by dilution in 0.9% saline of an aqueous stock solution (10 mmol L−1) of each compound. The bacterial suspensions were incubated at 37 °C for 15 min and were then transferred to culture flasks containing 5.0 mL of fresh LB medium. The final concentrations of the compounds in LB were 0.0042, 0.017, 0.42, 0.83, and 1.7 mmol L−1, respectively. The bacterial cultures thus treated were incubated at 37 °C (120 rpm) for 2 h. Culture growth was evaluated by OD600 using LB medium as blank, and the growth of the treated cells was compared to that of the negative control, whose value was taken as 100%. The optical density measurements at the selected wavelength did not suffer interference from the decavanadate orange color because there are no absorptions in that region of the electronic spectrum (Figure S8). Ampicillin at concentrations ranging from 0.025 to 10 mmol L−1 was used as positive control. The bacterial assays were performed in triplicate using three independent batch cultures for each concentration of the products. GI50 values (50% growth inhibition) were calculated on the GraphPad Prism 7 software.35 Studies on E. coli Cultures: Cell Viability Assay by Flow Cytometry. Bacterial cultures were treated with 0.9% saline (negative control) and with products I, II, and NaV10 at 5.0 mmol L−1 according to the procedure described in the protocol above. After dilution, the final concentration of the vanadium compounds in LB was 0.83 mmol L−1. Pellets of the bacterial cells were obtained after centrifugation of 300 μL of each culture at 13 000 × g for 1 min. After supernatant removal, the pellets were resuspended in 300 μL of phosphate-buffered saline (PBS). Propidium iodide (50 μg mL−1, red fluorescent dye marker for membrane permeability in nonviable bacteria) was added and the sample incubated for 5 min at room temperature. Fluorescent cells were counted on the Accuri flow cytometer, using the FL2 detector, at the excitation (λex) and emission (λem) wavelengths of 488 and 585 nm, respectively. Each experiment sampled approximately 3000 cells. Studies on G. intestinalis Cultures: Time-Course Inhibition Assay of Both Adherence and Growth. A trophozoite suspension (100 μL at 1.0 × 105 trophozoites mL−1) in TYI-S-33 medium was added to polystyrene culture tubes containing aqueous solutions of I, II, and NaV10 (1.0 mmol L−1 in water) diluted with TYI-S-33. The final concentrations of the oxidovanadium compounds were 0.025, 0.50, 10, and 200 μmol L−1 in a total volume of 13 mL for each tube. The TYI-S-33 medium and serial dilutions of albendazole (0.025 to 200 μmol L−1) were used as negative and positive controls, respectively. Cultures were incubated for 4 days at 37 °C in 5% CO2 and 95% air, and cell growth was determined by optical microscopy on a hemocytometer. Every 24 h, a 10 μL aliquot of each culture was removed to determine the supernatant trophozoite population (nonadhered cells). After that, 10 μL of the medium was added to the remaining volume of each culture, which was then chilled in ice for 15 min to promote detachment. New aliquots of 10 μL were subsequently taken from the culture tubes to determine the total trophozoite populations, and the cultures were incubated again at 37 °C after receiving a new addition of 10 μL of the TYI-S-33 medium. The estimation of the adhered trophozoites was obtained as total population−supernatant. Experiments were performed as two independent duplicates. Growth inhibition by 50% (GI50) was determined with Graphpad Prism 7 software35 by comparing the sum of adhered and nonadhered trophozoites in the treated cultures with the negative control. C

DOI: 10.1021/acs.inorgchem.8b01298 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 1. ORTEP-338 representation of (4-Hpca)4[H2V10O28]·2(4-pca), complex II, indicating the atom numbering scheme. Hydrogen atoms were omitted for clarity, except those on the protonated decavanadate and isonicotinamidium ions. Thermal ellipsoids were drawn at the 50% probability level. The dark bonds in decavanadate refer to the longest V−O bonds found in the anion. sodium citrate solution (0.05 μmol L−1, 10 min), and absorbance was determined at 540 nm on a plate spectrophotometer.

Studies on G. intestinalis Cultures: The Effect of Polyoxidovanadates on Trophozoites Adhered to Polystyrene Tubes. G. intestinalis trophozoites were grown in polystyrene culture tubes in 13 mL of TYI-S-33 until confluence (1 × 106 cells mL−1). Products I, II, and NaV10 were added thereafter in concentrations ranging from 0.025 to 200 μmol L−1. TYI-S-33 and albendazole (0.025 to 200 μmol L−1) were used as controls. Incubation, sampling, and counting were performed every 24 h for 4 days as described in the protocol above. Studies on G. intestinalis Cultures: The Effect on Trophozoites Adhered to Vero Cells Monolayers. African green monkey kidney (Vero) cells (ATCC CCL-81) were seeded into 24-well plates and grown for 48 h at 37 °C in 5% CO2 and 95% air until 100% confluence in RPMI-1640 medium containing 10% fetal bovine serum and 1% of penicillin/streptomycin (10 000 UI). The supernatants were removed, the Vero cells monolayers were rinsed with RPMI, after which was added 100 μL of 3.0 × 105 trophozoites mL−1 in 1 mL of RPMI and the cells incubated at 37 °C until adherence of trophozoites. In each well, the supernatant was taken out, and NaV10, I, II, or albendazole was added to a final concentration of 10 μmol L−1 in 1 mL of RPMI. After incubation at 37 °C for 24, 48, or 72 h, nonadhered trophozoites were quantified from a 10 μL sample of the supernatant on a hemocytometer. Thereafter, the supernatant was removed, and the monolayer rinsed with RPMI. The remaining attached trophozoites were detached by adding 1 mL of cold medium (at 0 to 4 °C) and then counted after 15 min as previously described. The number of trophozoites that remained attached to Vero cells was calculated from the difference between adhered and nonadhered trophozoites. Study on Vero Cells: Cytotoxicity Assay of Decavanadates toward Vero Cells. Vero cells (1 × 104) were seeded into a 96-well plate and grown at 37 °C in 5% CO2 and 95% air until confluence in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (10 000 UI). Cells were treated with the vanadate derivatives (NaV10, I, II) and albendazole at 0.025, 10, and 200 μmol L−1, and the volume was adjusted to 100 μL of RPMI per well. After 96 h, the supernatant was removed, and cells were washed with 100 μL of PBS (50%). Cells were fixed with methanol (50 μL) for 10 min, after which 50 μL of crystal violet 0.2% in ethanol/water (2% V/V) were added to each tube. After 2 min, the wells were exhaustively washed with 200 μL of PBS. Elution was made with a

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RESULTS AND DISCUSSION

Syntheses of Decavanadate Associated with Organic Counterparts. The decavanadate complexes of nicotinamidium, (3-Hpca)4[H2V10O28]·2H2O.2(3-pca) (I, Figure S1), and isonicotinamidium, (4-Hpca)4[H2V10O28]·2(4-pca) (II, Figure 1), were prepared using V2O5 as starting material. Attempts to use NH4VO3 and NaVO3 resulted either in the crystallization of products contaminated with the inorganic cations or solely the respective inorganic decavanadates. Product II has been obtained for the first time in this work. I had a similar crystal structure previously reported without mention of purity or yield, and no further studies were published.36 In this work, I and II were both obtained pure and with moderate yield, by a methodology that employs mild reaction conditions37 without hydrogen peroxide; the latter is commonly applied when V2O5 is used as starting material. Single-Crystal X-ray Diffraction Analyses. The structures of I and II present four organic cations balancing the charge of the [H2V10O28]4− anion, together with two cocrystallized, neutral organic molecules. Two water molecules complete the crystal packing of I, whereas II was obtained as an anhydrous salt. Complete crystallographic data for II are presented in Tables 1, 2, and S4, while Tables S1−S3 contain the corresponding data for I in the Supporting Information. The polyoxidoanions in I and II consist of 10 edge-sharing, distorted VO6 octahedra, and exhibit angles and bond lengths similar to those of other decavanadates.10 Differences in V−O bond lengths in the fragments VO (1.60 Å) < V−μ2-O (1.85 Å) < V−μ2-OH (1.92 Å) < V−μ3-O (2.00 Å) < V−μ3-OH (2.05 Å) < V−μ6-O (2.23 Å) arise from the decreasing electronic density about the terminal and bridging oxygen atoms.39 V−O bond lengths in V(1)−O(9), V(2)−O(9)i, V(3)−O(9), and V(5)−O(9)i, in the range of 2.22−2.35 Å, D

DOI: 10.1021/acs.inorgchem.8b01298 Inorg. Chem. XXXX, XXX, XXX−XXX

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A crystal structure similar to that of product I was reported earlier by Rakovský and co-workers.36 The formerly published compound, (3-Hpca)6[V10O28]·2H2O, in spite of sharing the same unit cell parameters, differs from I in that it comprises a nicotinamidium salt of the fully deprotonated decavanadate anion, [V10O28]6−, without cocrystallized (neutral) organic molecules. In the present work, the amino and hydroxyl hydrogen atoms were positively identified from difference maps, whereas in the previously described structure the amino hydrogen atoms were included in idealized positions [Uiso(H) = 1.2Ueq(C,N)]. Support for the formulations proposed for I and II comes from the pKa difference between the acidic precursors of the chemical species in the crystal.41 In our case, the pKa of [H3V10O28]3− is equal to 1.21 (in an ionic strength of 0.6 mol L−1 of NaCl),42−44 while those for isonicotinamidium and nicotinamidium cations are 3.67 and 3.35, respectively.41 This indicates that the acidic [H3V10O28]3− anions can effectively protonate (iso)nicotinamide molecules to produce the 3-Hpca+ or 4-Hpca+ ions described in this work, together with [H 2 V 1 0 O 2 8 ] 4 − . Once formed, [H2V10O28]4− anions present a pKa of 3.61, close to those of the pyridinium cations; therefore, the pKa difference does not favor further deprotonation. In this context, remaining neutral nicotinamide or isonicotinamide molecules in the aqueous medium could then cocrystallize with the ionic product, as observed for I and II. However, [HV10O28]5− (pKa = 6.07 in the same ionic strength) and nicotinamidium have a large pKa difference (2.72) in favor of the more acidic pyridinium cation. This indicates the opposite direction in the protonation reaction, producing [H2V10O28]4− (and neutral nicotinamide) instead of the fully deprotonated [V10O28]6− anion. Therefore, pKa differences appear to favor the (3-Hpca)4[H2V10O28]·2(3pca) formulation proposed here for I over the previously reported (3-Hpca)6[V10O28]·2H2O. However, variations in pH in each reaction could perhaps justify the different degrees of protonation in the products. In recent years, synthetic strategies to combine distinct chemical entities aiming at specific structures or activities have been widely considered. Decavanadate has been employed to create such materials; however, the cocrystallization observed in the present work, of V10 with both the organic cations and the corresponding neutral organic molecules is less common, and most of the structures reported to date include other inorganic species.10 Examples similar to I and II are (C6N2H14)2·(C6N2H13)[V10O27(OH)]·(C6N2H12)·2H2O, obtained from 1,4-diazabicyclo-[2.2.2]octane in different protonation states45 and (H2en)3[H3V10O28]·(en)·7H2O, with en = ethylenediamine.46 In comparison, one decavanadate cocrystal has been reported that contains a biologically relevant molecule, cytosine, in both protonated and deprotonated forms, but in this case together with sodium cations.47 Vibrational Spectroscopy. The FTIR spectra registered for products I and II (Figure S4 and assignments in Table S5) show bands characteristic of oxidovanadium compounds from 970 to 550 cm−1 attributed to ν(V = O), νas(VOV), νs(VOV), and δ(VOV).14,15,48 Bands related to the organic cations can also be observed between 1670 and 1630 cm−1 for ν(CO) and δ(NH2), and three bands in the range of 1390 to 1240 cm−1 refer to δ(C−H) from the pyridine ring.49 In the Raman spectra (Figure S5 and Table S6), the decavanadate bands are found from 960 to 200 cm−1.48 Both Raman spectra also present bands coming from the organic molecules between 3500 and 1700 cm−1, assigned to the νas(NH), νs(NH), ν(C

Table 1. Crystallographic and Refinement Data for (4Hpca)4[H2V10O28]·2(4-pca) (II) elemental formula, molar mass (g mol−1), crystal system, space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) temperature (K) volume (Å3) Z density (g cm−3) F(000) absorption coefficient (mm−1) crystal size (mm) θ range (deg) reflections collected unique data observed data [I > 2σ(I)] number of parameters goodness of fit on F2 R [I > 2σ(I)], Rw [I > 2σ(I)]a R, Rw (all data)a largest diff. peak and hole (e Å−3)

H2O28V10, 4(C6H7N2O), 2(C6H6N2O), 1696.21, triclinic, P1̅ (no. 2) 9.3942(9) 10.2468(9) 14.0204(13) 82.984(4) 81.607(4) 88.817(4) 100(2) 1325.2(2) 1 2.126 844 1.795 0.129 × 0.091 × 0.034 3.6 to 27.5 57076 6081 [R(int) = 0.092] 4752 499 1.032 R = 0.035, Rw = 0.072 R = 0.056, Rw = 0.078 0.48 and −0.48

w = [σ2(Fo2) + (0.0299P)2 + 1.9201P]−1, where P = (Fo2 + 2Fc2)/3.

a

are longer than expected from the sum of van der Waals radii but are inside the range observed for several vanadium(V) oxides.40 The V10 aggregates in I and II are protonated on different sites (μ3-O in I and μ2-O in II) with an average O−H distance of 0.84(±0.01) Å [refined with constraints]. Reported quantum mechanical calculations by the Hartree−Fock method have shown that the triply bridging oxygens in the decavanadate anion are those of highest basic character, therefore being the natural candidate sites for protonation.39 However, it is well-known that the presence of different counterions generates distinct hydrogen bond networks10 that affect the preferred protonation sites on V10, as observed in I and II. In II, the three-dimensional hydrogen bonding network involves the polyoxidoanion, the cations, and the isonicotinamide molecules in the vicinity (Figure S3). The organic moieties interact with each other through their amide moieties and with decavanadate through hydrogen bonds involving the pyridine nitrogen. Different from anhydrous II, the crystal packing of product I includes two water molecules per complex anion, which promote a more extensive three-dimensional hydrogen-bonding network also involving the polyoxidoanion, the nicotinamidium cations, and the nicotinamide molecules (Figure S2). In I, the position of the amide group in nicotinamide prevents head-to-head interaction between the amide moieties, also impacting the supramolecular arrangement of the different components, charged, and neutral, in the crystals. In both solid-state structures, the organic counterparts are also stabilized by π−π stacking interactions, with centroidcentroid distances of ca. 3.5 Å between the essentially parallel aromatic rings. E

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Table 2. Selected Bond Lengths (Å) and Angles (deg) for (4-Hpca)4[H2V10O28]·2(4-pca) (Product II) with Estimated Standard Deviations in Parenthesesa V(1)−O(1) V(1)−O(3) V(1)−O(5) V(1)−O(6) V(1)−O(9) V(2)−O(2) V(2)−O(7) V(2)−O(4) V(2)−O(6) V(2)−O(9)#1 V(2)−O(14)#1 V(3)−O(10) V(3)−O(5) V(1)−O(3)−V(5)#1 V(1)−O(9)−V(3) V(1)−O(9)−V(5)#1 V(1)#1−O(14)−V(2)#1 V(2)−O(4)−V(3)#1 V(2)−O(4)−H(1O4) V(2)−O(6)−V(1) V(2)−O(7)−V(5) V(2)#1−O(9)−V(1) V(2)#1−O(9)−V(3) V(2)#1−O(9)−V(5)#1 V(3)#1−O(4)−H(1O4) V(3)−O(5)−V(1) V(3)−O(9)−V(5)#1

1.6065(18) 1.8118(18) 1.8272(17) 2.0080(18) 2.2506(18) 1.6010(19) 1.7571(18) 1.9019(18) 1.9388(17) 2.2268(18) 2.0824(18) 1.6034(18) 1.8262(18) 115.23(9) 84.36(6) 84.64(6) 98.71(7) 113.89(9) 122(4) 103.13(8) 114.10(9) 168.69(9) 90.24(6) 84.79(6) 124(4) 113.74(9) 82.35(6)

Bond Lengths (Å) V(3)−O(8) 1.8312(18) V(3)−O(9) 2.3057(17) V(3)−O(11) 2.0599(18) V(4)−O(12) 1.6811(18) V(4)−O(11) 1.6814(17) V(4)−O(6) 1.9303(18) V(4)−O(14) 1.9477(18) V(4)−O(9) 2.1153(18) V(5)−O(13) 1.5969(18) V(5)−O(7) 1.9174(19) V(5)−O(9)#1 2.3465(18) V(5)−O(12) 2.0568(18) O(3)−V(5)#1 1.8542(18) Angles (deg) V(4)−O(6)−V(1) 107.58(8) V(4)−O(6)−V(2) 106.04(8) V(4)−O(9)−V(1) 93.37(7) V(4)#1−O(9)−V(1) 93.06(7) V(4)−O(9)−V(2)#1 96.39(7) V(4)#1−O(9)−V(2)#1 90.56(7) V(4)−O(9)−V(3) 88.09(6) V(4)#1−O(9)−V(3) 170.01(9) V(4)−O(9)−V(4)#1 101.71(7) V(4)−O(9)−V(5)#1 170.38(9) V(4)#1−O(9)−V(5)#1 87.82(6) V(4)−O(11)−V(3) 110.23(9) V(4)−O(12)−V(5) 111.82(9) V(4)−O(14)−V(1)#1 107.36(8)

O(4)−V(3)#1 O(4)−H(1O4) O(8)−V(5)#1 O(9)−V(4)#1 O(14)−V(1)#1 C(1)−O(1A) C(1)−N(1) C(1)−C(2) C(4)−N(2) C(13)−N(5) C(13)−C(14) C(16)−N(6) N(2)−H(1N2)

1.9307(19) 0.83(2) 1.8123(18) 2.1216(17) 1.9918(17) 1.226(3) 1.316(4) 1.517(3) 1.341(3) 1.335(4) 1.520(4) 1.338(3) 0.856(18)

V(4)−O(14)−V(2)#1 V(5)#1−O(8)−V(3) O(7)−V(2)−O(4) O(8)−V(3)−O(11) O(9)−V(4)−O(9)# O(1A)−C(1)−N(1) O(1A)−C(1)−C(2) O(2A)−C(7)−N(3) O(2A)−C(7)−C(8) O(3A)−C(13)−N(5) O(3A)−C(13)−C(14) C(4)−N(2)−H(1N2) C(5)−N(2)−C(4) C(5)−N(2)−H(1N2) N(5)−C(13)−C(14)

106.85(8) 114.41(9) 95.06(8) 155.93(8) 78.29(7) 124.4(3) 118.8(2) 124.3(3) 118.6(2) 124.2(3) 119.9(2) 116(2) 121.7(2) 122(2) 115.9(3)

Symmetry transformation used to generate equivalent atoms: #1 − x + 1, −y + 1, −z + 1.

a

O), and δ(NH2) vibrations, and in the range of 1000−390 cm−1 relative to ν(C−C), ν(C−N), δ(C−H) and γ(C−C−C) of the pyridine ring.49 Thermogravimetric Analysis. The thermogram of product I evidences two decomposition steps (Figure S6). The first one, up to 261 °C, corresponds to the combined loss of lattice water and two neutral nicotinamide molecules that cocrystallize with (3-Hpca)4[H2V10O28]. The difference between the experimental and calculated mass losses (22.5 and 16.1%, respectively) can be accounted for by oxidation of the organic molecules during thermal analysis.50 The second mass loss finishes at a much higher temperature, 683 °C, and corresponds to the thermal loss of four nicotinamidium cations. This interpretation is supported by the good agreement between calculated and experimental values (28.2 and 28.5%, respectively). Product II presents a slightly different thermal decomposition profile (Figure S6) in which again two mass losses are evident. However, in this case the first step, from 190 to 290 °C, corresponds to the loss of the two isonicotinamide molecules and two isonicotinamidium cations that form selfcomplementary, strongly hydrogen-bonded R22(8) homomeric amide···amide dimers (Figure S7b, left). These dimers also interact electrostatically with V10 and do not occur in product I. The second mass loss for II, up to 390 °C, happens at a lower temperature than that observed for I and comes from the loss of the remaining isonicotinamidium cations. These cations

also form amide homodimers similar to those described above (Figure S7b, right) but do not participate in hydrogen bond interactions with the other organic species; hence, their loss up to 390 °C mainly depends on their interactions with V10. The calculated figures of 28.8 and 14.4% for II in each temperature range are in good agreement with the experimental values (30.8 and 13.0%, respectively). The distinct temperatures observed in I and II for the loss of the organic moieties seem to be determined not only by their noncovalent interactions with V10 but also by clear differences in the hydrogen bond patterns between amide moieties in the two products. In complex I, the 1,3-position of the substituents on the pyridine ring prevents the formation of homodimers, and this, added to the presence of the crystallized water molecules, determines the formation of multiple interactions involving all unit cell components, either neutral or ionic (Figure S7a). Therefore, as a consequence of both electrostatic forces and the extensive hydrogen bond network thus formed, the temperature in which the organic cations are lost is higher in I than in II. Solution Studies: EPR and 51V NMR Spectroscopy. As expected, products I and II were EPR-silent, confirming the absence of redox reactions between the organic molecules and the vanadium(V) centers in the anion. Both compounds were also analyzed by 51V NMR spectroscopy at the concentration of 1.0 mmol L−1 to evaluate their stability in aqueous solution (Figure 2). F

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Solutions of NaV10, in turn, showed 51V NMR signals at δ − 424, − 500, and −515 ppm, also related to the three different chemical environments of vanadium(V) in [V10O28]6−. Resonance peaks at lower frequencies are consistent with the presence of [H2VO4]− (V1), [HV2O7]3− (V2), and [V4O12]− (V4) at −560, − 573 and −578 ppm, respectively. Such speciation of V10 at pH = 7 is well described in a number of literature reviews.3,44 Effects of Decavanadate Salts on E. coli Cultures. Growth Inhibition Assay. Assays with NaV10, I, and II in E. coli DH5α cultures demonstrated concentration-dependent toxicity that varied with the nature of the V10 sample employed (Figure 3a). Treatment of the cultures with NaV10 only resulted in significant inhibition at the highest concentration tested (GI50 of 1.8 mmol L−1). Also, in accordance with early reports (for other antibiotics),13,14 NaV10 was less potent than ampicillin, which gave a GI50 value of 0.1 mmol L−1. Compounds I and II, in turn, showed higher toxicity toward E. coli (GI50 of 0.47 and 0.67 mmol L−1, respectively) when compared to NaV10. There are few literature studies on the toxicity of V10 salts with organic cations, or their breakdown products in culture media, toward Gram-positive and/or Gram-negative bacteria; some of them are qualitative only. Inhibitory activity was observed when solutions of decavanadate with 4-methoxybenzylammonium15 and tert-butylammonium16 were added respectively to Gram-positive Enterococcus feacium and Streptococcus pneumoniae. For S. pneumoniae, the response was dependent on the nature of the oxidovanadium compound,16 with MIC (minimum inhibitory concentrations) of 4−8 μg mL−1 for (tert-BuNH3)6[V10O28] and of 8−16 μg mL−1 for Na3VO4. Also, [4-pic]4[H2V10O28]·2H2O (4-Hpic = 4-picolinium) was moderately more toxic than sodium decavanadate to Gram-positive Bacillus cirroflagellosus and two species of fungi, Aspergillus niger and Penicillium notatum.14

Figure 2. (a) Representation of the pH-dependent speciation of the decavanadate anion into VV oligomers. For decavanadate, VA, VB, and VC notations are explained in the text. (b) 51V NMR spectra (105.25 MHz) recorded in D2O for NaV10 in comparison with I and II (1.0 mmol L−1 solutions). The observed signals are assigned as V1 = H2VO4−, V2 = H2V2O72−, and V4 = V4O124−,3,44 according to the speciation equilibrium. The measured pHs of the samples were equal to 5 for I and II and 7 for NaV10.

Products I and II generated similar spectra, showing signals of [H2V10O28]4− at −425, −507, and −525 ppm that correspond to the central vanadium (VA), the four outer vanadium atoms (VB) on the central plane of the structure, and to the four capping vanadium atoms (VC) on the anion, respectively (V10 representation in Figure 2a).3 As the pH measured in the analyzed samples of I and II was equal to 5, the absence of signals given by lower-nuclearity vanadate species (Figure 2a) is well-accounted for by the stability of decavanadate in acidic medium (pH 2−6).43,44

Figure 3. Toxicity of different polyoxidovanadates toward E. coli cells. (a) Bacterial growth inhibition caused by [{Na6(H2O)20V10O28·4H2O}n] (NaV10), (3-Hpca)4[H2V10O28]·2H2O·2(3-pca) (I), and (4-Hpca)4[H2V10O28]·2(4-pca) (II). OD600 values of 1.092 ± 0.050, 1.077 ± 0.030, and 1.107 ± 0.024 were taken as 100% for each treatment but have been omitted from the representation for clarity. (b) Percentages of E. coli cells permeable to propidium iodide (PI+ cells) after a 2 h incubation with polyoxidovanadates NaV10, I, and II at 0.83 mmol L−1. (c) Counts of nonviable E. coli cells measured by PI staining (PI+ cells) through flow cytometry. From left to right: cells treated with saline (negative control); NaV10, I, and II (all at 0.83 mmol L−1). G

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cell membrane models such as micelles, reverse micelles, and vesicles.55 NMR and infrared spectroscopy studies on the interaction of HMetV10 (HMet = metforminium cation) with sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles56 point to the rearrangement of water molecules around the negatively charged micelle surface in the presence of HMetV10, due to the formation of a new hydrogen bond network involving the cations, V10, and water molecules. This result was recently reinforced by a detailed review on the role of sodium cations in solid-state structures versus Na+-AOT reverse micellar water pools containing V10.57 In the present work, although the interactions in biological medium are far from understood, it does not seem that the position of substituents on the cation pyridine ring plays a crucial role. However, the higher lipophilicity of the organic cations as compared to Na+, and the formation of hydrogen bond networks involving amide groups, V10, and water molecules may help facilitate the interaction between the negatively charged bacterial membrane and the V10 anion. Evaluation of the Effects of the Decavanadate Salts on G. intestinalis Cultures. Time-Course Inhibition of Both Adherence and Growth. Studies on a more complex biological model, the protozoan G. intestinalis, were started by evaluating the effect of NaV10, I, and II on the overall growth and on adhesion of trophozoites to polystyrene culture tubes, using a medium that simulates the physicochemical environment of the small intestine58,59 (Figure 4). The pathogenesis of G. intestinalis is not completely understood, but it is well-known that attachment of the parasite to the intestinal epithelium, by means of the ventral disk, is of fundamental importance for the persistence of the population in the host.60 Factors that interfere with cell attachment, such as the presence of cytotoxic

These earlier studies did not evaluate the nature of the chemical species present in the solutions nor did they provide a systematic study on the effect of varying the cations. A 4-fold enhanced toxicity as compared to (NH4)6V10O28· 6H2O has previously been observed for chitosan-Ca3V10O28 composite membranes against E. coli and S. aureus,13 apparently due to a combination of the antibacterial effects of V10 and the chitosan biopolymer. In the present work, in turn, the cytotoxic activity appears to be potentialized by association of V10 with nontoxic organic components. In this context, treatment of E. coli DH5α with solutions of nicotinamide and isonicotinamide, in the same conditions and concentration range used for I and II (from 0.17 to 1.7 mmol L−1) did not result in any growth inhibition (Figure S9). In addition to I, II, and the chitosan system,13 only [4Hpic]4[H2V10O28]·2H2O (4-pic = 4-picoline) was tested against a Gram-negative species, Pseudomonas aeruginosa, although with negligible activity.14 In a related work, the exposure of E. coli DH5α cultures to polyoxidovanadates other than V10 also revealed an inhibitory growth effect when [H6V14O38(PO4)]5− (V14) was applied, while [V15O36(Cl)]6− (V15) was shown to be nontoxic.51 Spectroscopic analysis by 51V NMR revealed that V14 breaks and rearranges to produce decavanadate, whereas V15 speciates to a lesser extent to give H2VO4−, H2V2O72−, V4O124−, and V5O155− but not V10.52 According to those results and the present studies, the formation or presence of V10 in the culture media appears to be relevant to the observed toxicity, although the determination of the chemical species effectively responsible for the reported effects is a complex task. This is not only because of chemical speciation and the establishment of a complex equilibrium between oxidovanadates in biological medium but also because of the number of possible biological targets they could, in principle, interact with. Additionally, even though V10 has been detected in some acid cell organelles, a suitable mechanism to transport intact decavanadate into the cells is not known, and its existence has been the subject of discussion.3,4 Cell Viability Assay by Flow Cytometry. The results of the turbidimetric growth inhibition assays, as described above, provided an overall view of the bacterial culture response to NaV10, I, and II. In order to confirm these toxicity results and gain deeper insight into the effect of the different decavanadates upon cell viability in Gram-negative bacterial subpopulations, the propidium iodide (PI) incorporation assay by flow cytometry was performed (Figure 3b,c). In this assay, cells with damaged membranes are permeable to PI and are nonviable.53 Cells treated with NaV10 showed an overall 32% damage to cell membranes, while treatment with I and II rendered practically 100% of cells nonviable (totally permeable to PI) after 2 h. Earlier literature reports indeed suggested that the main targets of V10 in prokaryotic cells are membrane proteins,6,54 causing inhibition of the transport of small inorganic cations and other molecules, eventually leading to cell death.16 Our results are in full agreement with these findings; however, the open question remains of whether the observed effect in E. coli DH5α cultures was caused by the action on cell membranes of intact V10 itself or of breakdown products of V10 generated in physiological medium, or even both. In this context, much effort has already been made to shed light on the role of organic counterions of [HnV10O28](6−n)− in

Figure 4. (a) Adherence kinetics of G. intestinalis trophozoites to a polystyrene surface after treatment with NaV10, I, and II (0.025 to 200 μmol L−1) and albendazole (ABZ, 0.5 μmol L−1). Standard deviations were smaller than ±5 × 104, ± 4 × 104, and ±8 × 104 adhered trophozoites mL−1 for NaV10, I, and II, respectively; in several points, error bars are within the size of the symbols. (b) Percentage of growth inhibition of G. intestinalis trophozoites upon treatment with albendazole, NaV10, I, and II (0.025−200 μmol L−1) after 96 h. Total trophozoite count of 138.5 × 104 was considered as 100% growth (negative control). The total number of trophozoites was calculated as the sum of adhered and nonadhered parasites. H

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Figure 5. Detachment kinetics of G. intestinalis trophozoites from a polystyrene surface (after 100% confluence) with serial dilution treatments with NaV10, I, and II (0.025 to 200 μmol L−1) and albendazole (ABZ, 0.5 μmol L−1). Adhered trophozoites were counted on a hemocytometer every 24 h. Standard deviations were lower than ±5, ±3, and ±5% for NaV10, I, and II, respectively, and in several points, error bars are within the size of the symbols. Trophozoites grown in the presence of culture medium alone were considered the negative control.

substances or intestinal hypermobility,61,62 are known to help the elimination of trophozoites from the gut. An inhibitory effect on trophozoite adherence was observed for all three polyoxidovanadates after 48 h of treatment, achieving 40−50% of adherence inhibition at the lowest concentration (0.025 μmol L−1, Figure 4a). Interestingly, the kinetics of inhibition varied with the different compounds. Treatment with NaV10 solutions seemed to be the most effective, reaching 70−90% inhibition of adherence at 200 μmol L−1 from 48 h onward. The effectiveness of inhibition by NaV10 was maintained over time when 200 μmol L−1 was used, but lower concentrations seemed to level off at 50% inhibition for longer treatments. The addition of product II showed inhibition from early time points, but did not show concentration dependence up to 72 h. However, at 96 h adherence decreased markedly depending on the concentration of II, getting down to a minimum of ca. 20% at 200 μmol L−1. Treatment with complex I, in turn, produced a more clearly concentration-dependent inhibitory effect after 48 h, reaching ca. 30% adherence after 96 h at the highest concentration. This complex inhibition profile may be due to distinct interactions of the polyoxidovanadates and their respective counterions, and/or their speciation products, with G. intestinalis under physiological conditions. In spite of the differences observed during the time course of the experiments performed with NaV10, I, and II, similar effects were achieved at the concentration of 200 μmol L−1 after 96 h, with an average maximum adherence of ca. 20% compared to the control conditions. On the basis of this result, the total parasite density in the cultures was estimated after 96 h as the sum of adhered and nonadhered trophozoites. As shown in Figure 4b, culture growth was inhibited by incubation with NaV10 and II even at the lowest concentration evaluated, exhibiting GI50 values of 10 μmol L−1. However, the overall response caused by the addition of I was less pronounced, with growth inhibition of only 33% at 200 μmol L−1. The difference in the performance of I when adherence (Figure 4a) and growth inhibition (Figure 4b) are compared suggests that the treatment with I affects the adhesion mechanism of trophozoites to the polystyrene surface without lysing the cell, different from the possible effect of II and NaV10. Effect of Polyoxidovanadates on Trophozoites Adhered to Polystyrene Tubes. Assays that more closely resemble giardiasis conditions were performed to verify the capability of the polyoxidovanadates to detach a well-established adherent culture from polystyrene tubes and from Vero cells monolayers. Experiments with trophozoites adhered to the

polystyrene surface showed that the three oxidocompounds increase detachment as a function of time for all concentrations employed, achieving at least 80% of detachment after 96 h (Figure 5). A marked decrease in the adhered trophozoites was already observed from 48 to 72 h for cells treated with NaV10 and II, while the main response of I occurred only from 72 to 96 h. The metabolic pathways and virulence factors of G. intestinalis are less understood than those of bacterial cells and even other protozoa.63,64 With regard to the activity of vanadium-containing complexes, few oxidovanadium(IV) compounds have been studied in their potential effect against protozoa such as Trypanosoma spp., Leishmania spp., and Entamoeba histolytica.65 The action mechanisms of those compounds are far from being completely elucidated, but the existing studies assigned the activity to DNA intercalation and changes in mitochondria-related biological pathways. Since Giardia contains a mitochondria-like organelle called the mitosome,63 further studies on the action mechanism of V10 salts could access this potential target. Specifically concerning decavanadate, (NH4)6[V10O28]· 6H2O has been evaluated in vitro in its growth inhibitory effect on Leishmania tarentolae.17 In that case, although only a mild promastigote growth inhibitory activity was detected, the addition of V10 to the culture medium resulted in reduction in parasite mobility, decreasing its potential virulence. A more recent study has shown that both decavanadate and orthovanadate are excellent inhibitors of L. tarentolae secreted acid phosphatases (SAP),66 which are potential pharmaceutical targets for the treatment of leishmaniasis. According to that report, when both speciation and total vanadium concentration are taken into account, decavanadate is consistently a better inhibitor of SAP than orthovanadate. In the present work, both antiadherence and growth inhibitory activities on G. intestinalis were observed to some extent in all concentrations evaluated for the three decavanadate salts. The observed effect was moderate as compared to that of the reference drug albendazole up to 48 h, intensifying from 48 to 96 h for NaV10 and II and from 72 to 96 h for I, time when ca. 100% of trophozoites were detached when the highest concentration (200 μmol L−1) was used. In Vitro Studies: Cytotoxicity of Decavanadates toward Vero Cells and Their Effect on Trophozoites Adhered to Vero Cells Monolayers. In the in vitro assay, trophozoites adhered to a monolayer of Vero cells were treated with albendazole, NaV10, I, and II only at the GI50 concentration (10 μmol L−1), and the trophozoites that remained attached after rinsing the monolayer with RPMI at 24-h intervals were counted for up to I

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toward E. coli DH5α, with I and II being, respectively, 3.8 and 2.7 times more effective than NaV10. The relative positions of the carboxamide substituents on the pyridine ring in I and II apparently did not affect the microenvironment of V10 or its breakdown products in aqueous solution so as to clearly explain their different activities toward E. coli cells. It is possible that the counterions act mainly by facilitating the uptake of vanadium species by the bacterial cells or by disturbing the bacterial cell membrane, instead of determining the chemical speciation of the polymetallic aggregate under physiological conditions. In addition, all compounds had antigiardial activity, exhibiting cytotoxic effect against G. intestinalis trophozoites and inhibiting their adherence to both polystyrene surfaces and monolayers of Vero cells. This is a novel activity of oxidovanadium compounds. Moreover, the addition of V10 salts was much less toxic to Vero cells than the reference drug albendazole. Although the mechanism of the antimicrobial effect exhibited by vanadates continues to be elusive, further studies comparing polyoxidoanions with lower nuclearity oxidovanadates(V) and mixed-valence vanadium(IV)/(V) species may yield significant mechanistic insight. In any case, the activities shown in this work as well as the low toxicity in Vero cells suggest that decavanadates have pharmacological potential as new antimicrobial agents.

72 h (Figure 6a). The treatment with all compounds led to lower counts of trophozoites adhered to Vero cells when

Figure 6. (a) Effect of NaV10, I, and II on Giardia trophozoites attached to Vero cells monolayers, after incubation of the eukaryotic cells with 3 × 105 trophozoites and then with 10 μmol L−1 each oxidovanadium compound. At the indicated times, adhered trophozoites were quantified. For comparison, trophozoites were incubated with media (Ctrl) or 10 μmol L−1 albendazole (ABZ). (b) Cytotoxic effect of polyoxidovanadates on Vero cells. Cells were incubated for 96 h with albendazole, NaV10, I, and II at 200, 10, and 0.025 μmol L−1, or with culture medium. Cell viability was determined by the crystal violet method.

compared to the negative control, indicating their antigiardial effect. Interestingly, after 24 h the effect of all polyoxidovanadium complexes was similar to that of albendazole, but after 48 h, albendazole was significantly more effective, with NaV10 presenting a more pronounced effect than I and II. In contrast to the observations made with E. coli, the overall antigiardial effect seems less dependent on the chemical nature of the counterion, and perhaps more related to the interactions of V10 (or breakdown products) with the biological targets in the culture. As the number of oxidovanadates employed in protozoa studies is still very small, the identification of the chemical species generated from them in physiological media, as well as the effect of their combination with other organic cations on trophozoite adherence and viability are still to be evaluated before a broader view can be reached. The toxicity to Vero cells upon treatment with all three oxidovanadium(V) compounds was lower than that with albendazole. At 0.025 and 10 μmol L−1, the oxidovanadium compounds did not affect the viability of Vero cells, while the same concentrations of albendazole reduced viability to ca. 50 and 20%, respectively. Only at the highest concentration (200 μmol L−1) of the vanadium compounds reduced Vero cells viability to about 50%, while albendazole brought it down to less than 20% (Figure 6b). Similar results were reported for sodium decavanadate and chitosan-decavanadate, which exhibited low cytotoxic toward Vero cells while significantly affecting HeLa (cancerous cervical tumor) cells.67 In contrast, some vanadium compounds, including decavanadate, have been found to be cytotoxic to both cancer1 and normal cell lines such as hepatocytes L02.68 Although there is still much to be investigated, under our experimental conditions the use of the decavanadate salts NaV10, I, and II proved to be safe to Vero cells and effective against Giardia trophozoites, suggesting interesting perspectives for the design of new therapeutic candidates.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01298. Complementary crystallographic tables and additional ORTEP diagrams for products I and II, FTIR and Raman spectra and band assignment tables, TGA profiles, UV−vis absorption spectra and E. coli growth inhibition data for nicotinamide and isonicotinamide (PDF) Accession Codes

CCDC 1839623−1839624 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Giovana G. Nunes: 0000-0001-7052-2523 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Fundaçaõ Araucária (grants 20171010 and 283/2014, protocol 37509), Conselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, grant 308426/2016-9), Coordenaçaõ de Aperfeiçoá Superior (CAPES, PVE A099/ mento de Pessoal de Nivel 2013), the National Institute of Science and Technology−

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CONCLUSIONS In this work, the polyoxidovanadates NaV10, I, and II have shown a significant, dose-dependent growth inhibition effect J

DOI: 10.1021/acs.inorgchem.8b01298 Inorg. Chem. XXXX, XXX, XXX−XXX

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Biological Nitrogen Fixation (INCT−FBN) and Universidade Federal do Paraná (UFPR). Authors thank Mr. Angelo Roberto dos Santos Oliveira and Dr. André Luis Rüdiger (UFPR) for TGA and 51V NMR analyses, respectively. B.G., D.L.H., E.M.S., F.S.S., G.G.N., G.V., J.F.S., J.M.M., K.P., and M.I.R. thank CNPq, CAPES and Fundaçaõ Araucária for research grants and scholarships.

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