Highly Effective and Hydrolytically Stable ... - ACS Publications

Dec 23, 2015 - Institute of Chemistry, the Hebrew University of Jerusalem, Jerusalem ... and related ligands is due to decomposition in cell culture m...
1 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/IC

Highly Effective and Hydrolytically Stable Vanadium(V) Amino Phenolato Antitumor Agents Lilia Reytman,† Ori Braitbard,‡ Jacob Hochman,‡ and Edit Y. Tshuva*,† †

Institute of Chemistry, the Hebrew University of Jerusalem, Jerusalem 9190404, Israel Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel

‡

S Supporting Information *

ABSTRACT: Vanadium(V) oxo complexes with no labile ligands, including six octahedral complexes with pentadentate diaminotris(phenolato) ligands and one pentacoordinate complex with a tetradentate aminotris(phenolato) ligand, were synthesized in high yields. All octahedral complexes demonstrated high hydrolytic stability with no signs of decomposition after days in the presence of water, whereas the pentacoordinate complex decomposed within minutes to release the free ligand, demonstrating the marked impact of coordination number and geometry on the complex electrophilicity. All complexes showed marked cytotoxicity toward human colon HT-29 and ovarian OVCAR-3 cells. In particular, the octahedral complexes exhibited especially high activity, higher than that of cisplatin by up to 200fold. Selected complexes demonstrated similarly high activity also toward the A2780 and the A2780cis cisplatin-resistant line. High cytotoxicity was also recorded after prolonged incubation in a DMSO solution at 4 and 37 °C temperatures and in biological medium. In vivo studies pointed to high efficacy in reducing tumor size, where no clinical signs of toxicity were detected in the treated mice. These results overall indicate high potential of the tested compounds as antitumor agents.

■

INTRODUCTION The utilization of the successful antitumor drug cisplatin and its derivatives is often hampered by severe side effects, narrow activity range, and resistance development,1 inspiring the search for other metal based antitumor compounds.2 Vanadium, which is naturally prevalent in the human body,3 has great potential for therapeutic purposes, as it introduces influence over several possible cellular pathways;4 although some toxic effects are occasionally encountered, they are often not major3,5 and can be modified to produce a reassuring toxicity profile, depending on the concentration of vanadium ions, route of administration, and the nature of the ligands; for instance, no obvious toxic effects were observed after vanadium supplementation in humans.3,6 Accordingly, vanadium compounds are examined for treatment of various diseases,7 among which are diabetes mellitus,8 leishmaniasis and trypanosomiasis,6a and cancer.4a,5,9 Recently, vanadium complexes as anticancer agents have received increasing attention, with reports of novel vanadium phenolato coordination compounds possessing cytotoxic activity.10 Additionally, a promising oxovanadium complex exerted a favorable pharmacokinetic profile in mice, by maintaining a highly cytotoxic therapeutic plasma concentration for at least 24 h after injection of a nontoxic dose.11 This characteristic is further supported by encouraging findings, including evidence of decrease in breast cancer risk in women with higher urinary vanadium concentrations,12 and increased © XXXX American Chemical Society

cytotoxic effects of vanadium toward cancer cells relative to normal cells,13 thus encouraging the search for additional, particularly effective, vanadium based anticancer compounds. The limitation of vanadium compounds in biological systems is their tendency to undergo rapid hydrolysis to produce a variety of vanadium-oxo species,14 thus impairing their shelf lives while making it difficult to determine the active species. The pursuit of vanadium complexes with increased hydrolytic stability and defined hydrolysis products has led us to develop oxovanadium(V) complexes coordinating a tetradentate diamino bis(phenolato) “salan” ligand and a labile isopropoxo ligand, as labile groups were thought to be essential for interaction with the cellular target.15 These vanadium−salan complexes rapidly released the labile ligand to produce welldefined stable products, and exhibited marked cytotoxic activity comparable to that of cisplatin. The hydrolysis product of a representative complex was characterized by X-ray crystallography as an oxo-bridged dimer containing two oxovanadium centers, each connected to a salan ligand. Unexpectedly, this dimeric product displayed cytotoxicity despite having lost the labile groups, evincing that labile ligands are not essential, thus prompting the current research on the development of oxovanadium complexes coordinating amino-phenolato ligands Received: August 1, 2015

A

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

was synthesized as previously described from hexamethylenetetramine, 2,4-dimethylphenol, and p-toluenesulfonic acid hydrate.19 The corresponding complex LtVO was also prepared through reaction of VO(OiPr)3 precursor with equimolar amounts of H3Lt ligand in the presence of triethylamine (Scheme 2).17 1H NMR of the complex included two broad peaks and singlet peaks, indicating high symmetry of the complex. Single crystals suitable for X-ray analysis of a representative complex Lp3VO were obtained from a 1:2 mixture of 1,2dichloroethane and heptane, after sitting at 4 °C for several days. Selected bond lengths and angles are given in Table 1.

with no labile ligands. This paper presents the cytotoxic properties and hydrolytic stability of oxovanadium(V) complexes varying in ligand denticity: namely, pentadentate diamino tris(phenolato) (LpVO) and tetradentate amino tris(phenolato) (LtVO) ligands, with varying aromatic substitutions, all bearing no labile groups. Extremely stable complexes were developed with activity higher than that of cisplatin by up to 200-fold, with indications of in vivo efficacy, all supporting the high potential of the tested compounds for medicinal applications.

■

RESULTS AND DISCUSSION Synthesis and Characterization. To accommodate V(V) oxo complexes lacking labile ligands, diamino tris(phenolato) ligand type (H3Lp) was selected, to form presumably stable16 hexacoordinate oxovanadium(V) complexes (Scheme 1).

Table 1. Selected Bond Lengths (Å) and Angles (deg) for Lp3VO Atoms

Scheme 1. Synthesis of LpVO Complexes

O(1)−V O(2)−V O(3)−V O(4)−V O(1)−V−O(2) O(1)−V−O(3) O(1)−V−O(4) O(2)−V−O(3) O(2)−V−O(4) O(3)−V−O(4) N(1)−V−N(2)

Additionally, a tetradentate amino tris(phenolato) ligand (H 3L t ) was employed for formation of an analogous pentacoordinate oxovanadium(V) complex (Scheme 2), as

Value

Atoms

Lengths 1.601(4) N(1)−V 1.884(4) N(2)−V 1.855(4) 1.887(4) Angles 97.8(2) O(1)−V−N(1) 104.5(2) O(2)−V−N(1) 93.4(2) O(3)−V−N(1) 89.8(2) O(4)−V−N(1) 163.8(2) O(1)−V−N(2) 98.8(2) O(2)−V−N(2) 77.7(2) O(3)−V−N(2) O(4)−V−N(2)

Value 2.131(5) 2.386(5)

96.1(2) 80.9(2) 158.4 (2) 86.32(2) 172.0(2) 86.3(2) 82.4(2) 81.3(2)

Scheme 2. Synthesis of LtVO Complex

was previously reported, to shed light on the role of ligand denticity.17 H3Lp ligands were synthesized according to a known synthetic procedure by condensation of the commercially available substituted benzaldehyde with ethylenediamine.18 LpVO complexes were prepared similarly to a previously published procedure by reacting the commercially available VO(OiPr)3 precursor with equimolar amounts of the corresponding H3Lp ligand (Scheme 1).15 1H NMR has verified that the desired compounds had been obtained. While 1H NMR spectra of the ligands suggested symmetrical and achiral structure by displaying two sets of aliphatic and aromatic hydrogens, with singlet signals attributed to the benzylic hydrogen atoms, the analogous spectra of the complexes suggested low symmetry, as six doublet signals characterized these benzylic hydrogen atoms. To evaluate the effect of structural features on the performance of these complexes, a series of LpVO complexes were prepared, differing in the electronic and steric properties of the substituent at the para position relative to the O-donor (Scheme 1). The H3Lt ligand

Figure 1. ORTEP drawing of Lp3VO in 50% probability ellipsoids; H atoms and disordered solvent were omitted for clarity.

The structure, as displayed in Figure 1, featured an octahedral geometry of C1 symmetry, in accordance with the 1H NMR features, in which the oxygen donors of the Lp3 ligand are in cis configuration to the single oxo ligand. Interestingly, the bond length between the vanadium atom and O(4) is very similar to the bond length to O(2), but slightly longer than the bond length to O(3), trans to the N donor. Additionally, the V−NH bond is markedly shorter than the analogous bond to the tertiary N donor, trans to the oxo group. Hydrolysis. The hydrolytic stability of the complexes was assessed by reacting the compound with 1000 equiv of water B

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry for 3 days and analyzing the products by 1H NMR. For all the examined LpVO complexes the 1H NMR spectrum of the obtained solution contained the LpVO complex as the main species with no detectable signals of a hydrolysis product, consequently suggesting that LpVO did not undergo hydrolysis (Figure 2). For a representative complex Lp1VO, the results

Figure 2. 1H NMR spectra of Lp4VO before (a) and after (b) the reaction with 1000 equiv of water for 3 days.

were verified using 1,4-dinitrobenzene as an internal standard, by comparing the integration ratio of the products with that of the reactants, which ruled out the formation of hydrolysis products not soluble in the NMR solvent. Therefore, it is apparent that LpVO complexes are indeed highly hydrolytically stable, and may be preserved for days in the presence of water. Additionally, the similar stability observed for all complexes studied indicate negligible influence of the para-substitution, despite the different electronic characteristics of the substituents employed. A similar analysis of the complex LtVO revealed rapid hydrolysis, where release of the free ligand H3Lt was detected by 1H NMR. Therefore, a kinetic experiment was performed in order to assess the stability of the complex by monitoring the integration of 1H NMR signals overtime following addition of D2O as previously described.15 The first 1H NMR measurement subsequent to the addition of D2O revealed only signals relating to the free ligand and none relating to the complex, thereupon indicating that LtVO decomposes completely to release the free ligand within several minutes, despite having no labile groups. It is thus obvious that coordination number and geometry strongly impact the hydrolytic stability; a single additional donor giving a pentadentate rather than tetradentate chelating ligand was sufficient to dramatically increase the hydrolytic stability for the LpVO type complexes, presumably by increasing the electron density on the electrophilic metal center. Cytotoxicity. In vitro cytotoxic activity measurements were performed on colon HT-29 and ovarian OVCAR-3 human cancer cell lines as previously described.20 The examined cells were treated with the tested complex dissolved in DMSO at 10 different concentrations and their viability was evaluated following 3 days of incubation by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Plots of cell viability dependence on the administered concentrations of LpVO and LtVO complexes are presented in Figure 3 and Figure 4 respectively. In order to reflect in a comparable manner the results when the cell growth inhibition is not 100%, relative IC50 values were extracted, i.e the concentration required to bring down the inhibition curve to a point half way between the top and bottom plateaus of the curve. The relative IC50 values, together with the bottom plateau of the curve presented as

Figure 3. Dependence of HT-29 (top) and OVCAR-3 (bottom) cell viability on administered concentration of Lp1−6VO following a three day incubation obtained by the MTT assay.

Figure 4. Dependence of HT-29 cell viability on administered concentration of LtVO, its ligand H3Lt, and the vanadium precursor VO(OiPr)3 following a three day incubation obtained by the MTT assay.

maximum cell growth inhibition values are summarized in Table 2. The cytotoxic activity of the oxovanadium complexes Lp1−6VO is exceptionally high toward both examined cell lines, with IC50 values up to 2 orders of magnitude lower than those of cisplatin. Complexes Lp1−6VO, differing in the aromatic para substituents exhibit similar IC50 values toward the examined cells, indicating a small influence of this position on the cytotoxic activity, similarly to oxovanadium(V)-salan complexes.15 A slight variation was observed for the chlorosubstituted complex Lp3VO that displayed particularly high activity toward OVCAR-3 cells with IC50 as low as 50 ± 20 nM. The higher activity of this complex may be attributed to its somewhat higher solubility, or relate to aspects particular to the cell type derived from specific interactions in the cellular intake process or cytotoxic pathway. The cytotoxic activity measured for LtVO, although still higher than that of cisplatin, was lower than those of the LpVO complexes (Table 2). This observation is consistent with its low hydrolytic stability, implying that some hydrolysis product/s is/ C

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry Table 2. Relative IC50 Values (μM) and Maximum Cell Growth Inhibition (MI, %)a Values for Lp1−6VO, LtVO, and Reference Compoundsb toward Colon HT-29 and Ovarian OVCAR-3 Cell Lines

Table 3. Relative IC50 values (μM), Maximum Cell Growth Inhibition (MI, %)a Values, and RFb Values for Lp1VO, Lp6VO, and Cisplatin toward A2780 and A2780cis (cisplatinresistant) Cell Lines

IC50 (μM) (MI (%)a) Complex Lp1VO Lp2VO Lp3VO Lp4VO Lp5VO Lp6VO LtVO H3Lt VO(OiPr)3 cisplatin

HT-29

OVCAR-3

1.4 ± 0.2 (96) 1.3 ± 0.2 (97) 1.0 ± 0.1 (95) 0.6 ± 0.1 (97) 1.70 ± 0.06 (95) 1.6 ± 0.4 (99) 4.6 ± 0.8 (96) 10.9 ± 1.8 (93) 25 ± 9 (87)15 12.2 ± 2.3 (90)15

0.7 ± 0.3 (86) 0.4 ± 0.2 (77) 0.05 ± 0.02 (80) 0.5 ± 0.1 (85) 0.9 ± 0.1 (87) 0.40 ± 0.04 (91) 2.8 ± 0.5 (87) 3.9 ± 1.2 (78) 25 ± 7 (85) 10.2 ± 2.9 (71)

IC50 (μM) (MI (%)a) Complex 1

Lp VO Lp6VO cisplatin

A2780

A2780cis

RFb

1.1 ± 0.2 (98) 0.6 ± 0.1 (97) 1.6 ± 0.3 (98)

1.3 ± 0.3 (96) 0.8 ± 0.1 (97) 26 ± 4 (95)

1.2 1.3 16

a

MI is the maximal percent of cell growth inhibition at the highest concentration applied. bResistance factor indicates the degree of resistance: the ratio between IC50 values of the resistant line and the sensitive line

two cell lines is similarly high, and the difference between the IC50 values falls within the margin of error (RF = 1.2−1.3, Table 3). This suggests a potentially broad spectrum of activity for these complexes, thus increasing their therapeutic potential. Additionally, this observation implies a different cytotoxic pathway invoked by LpVO complexes than that of cisplatin, as the different resistance mechanisms involved in A2780cis cells22 had no impact on the cytotoxicity of LpVO compounds. Durability in Organic and Aqueous Media. In light of the hydrolytic stability and high cytotoxic activity of LpVO type complexes, the durability of a representative complex Lp1VO was further evaluated; the complex was separately preincubated in DMSO at 37 °C and at 4 °C for increasing periods up to 6 weeks, prior to its administration to the cells. A summary of the IC50 and maximal cell growth inhibition values is presented in Table 4 (Figure S3).

a

MI is the maximal percent of cell growth inhibition at the highest concentration applied. bMeasurements for the reference compounds toward both cell lines were performed with a similar procedure.

are active inside the cell. In light of the indication of rapid hydrolysis of the complex to release the free ligand H3Lt in the presence of water, the cytotoxic activity of the free ligand was measured. Interestingly, the cytotoxic activity of LtVO is somewhat higher than those of the free ligand H3Lt, as well as the labile precursor VO(OiPr)3 (Table 2, Figures 4 and S1). This may either imply some different active vanadium oxo species operating in the cellular environment, or support the proclaimed assumption that in the presence of biomolecules vanadium complexes may form mixed ligand complexes with small bioligands, and thus their activity does not necessarily result from that of the hydrolysis products of the complex.21 To learn more about the potential activity range of the LpVO complexes, reactivity against cisplatin-resistant cells was also evaluated. Ovarian A2780, parent cisplatin-sensitive cells, and A2780cis cells maintaining acquired cisplatin-resistance, were treated with different concentrations of representative compexes Lp1,6VO for 3 days, after which their viability was recorded. Cell growth inhibition plots are depicted in Figures 5,

Table 4. Relative IC50 Values (μM) and Maximum Cell Growth Inhibition (MI, %)a Values for Lp1VO Following Preincubation in DMSO at 37 and 4 °C for Varying Periods Prior to Cell Addition IC50 (μM) (MI (%)a) 37 °C

Incubation time (weeks) 0 1 3 6

1.4 2.4 2.9 3.3

± ± ± ±

0.2 0.8 0.5 0.8

4 °C

(96) (96) (95) (97)

1.4 1.1 1.4 2.3

± ± ± ±

0.2 0.1 0.5 0.9

(96) (96) (95) (95)

a

MI is the maximal percent of cell growth inhibition at the highest concentration applied.

Preincubation of Lp1VO in DMSO at the biologically relevant temperature of 37 °C for several weeks only slightly diminished the cytotoxic activity of the compound; nevertheless, even after 6 weeks of suspension in the organic solvent, the cytotoxic activity remained extremely high, producing IC50 values significantly higher than that of cisplatin. Inspection of the 1 H NMR and 51V NMR spectra of the contents of the vial of Lp1VO following this incubation period confirmed that Lp1VO remained the main species in the vial (Figures S4 and S5). Preincubation of Lp1VO in DMSO under a storage-relevant temperature of 4 °C, at which the solution solidifies, did not impact the complex reactivity; the cytotoxic activity of Lp1VO was mostly preserved for up to 6 weeks of preincubation. These results imply high sustainability of LpVO compounds through prolonged periods of time, thus granting these complexes a substantial advantage, considering that the cytotoxic activity of even relatively stable complexes such as cisplatin is diminished

Figure 5. Dependence of A2780 and A2780cis cell viability on administered concentration of Lp1VO and cisplatin following a three day incubation obtained by the MTT assay.

S2, and the relative IC50 values are summarized in Table 3. Lp1VO and Lp6VO complexes exhibited excellent cytotoxic activity, similar and even slightly higher than that displayed by cisplatin even toward the cisplatin-sensitive A2780 cells. Moreover, unlike for cisplatin featuring a resistance factor (RF) of 16, the activity of the vanadium complexes against the D

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry after short preincubation periods in the administration solvent DMSO.23 Further investigation of the complex durability under biologically relevant conditions was carried out through preincubation of Lp1VO in culture medium for up to 3 days prior to its addition to HT-29 cells. The viability of the cells was then evaluated following additional 3 days of incubation (Figure 6). The IC50 and maximal cell growth inhibition values

Figure 6. Dependence of HT-29 cell viability on administered concentration of Lp1VO, following preincubation in culture medium at 37 °C for varying periods prior to cell addition and a three day incubation with cells, obtained by MTT assay.

Figure 7. In vivo results of Lp1VO: Immune deficient (nude) mice (N = 5) were inoculated S.C. with 5 × 106 HT-29 human colon adenocarcinoma cells and subsequently subjected to I.P. injections of Lp1VO (10 μg), every other day for 3 weeks. Control mice (N = 5) were injected with PBS/1% DMSO. (a) Tumor growth in Lp1VO treated mice relative to control. (b) Solid tumors at the termination of the experiment: upper pannel, nontreated mice; lower panel, treated mice (2/5 were devoid of tumors).

Table 5. Relative IC50 Values (μM) and Maximum Cell Growth Inhibition (MI, %)a Values for Lp1VO Following Preincubation in Medium at 37 °C for Varying Periods Prior to Cell Addition Incubation time (hrs) 0 24 48 72

■

CONCLUSIONS In this work we presented highly cytotoxic oxovanadium(V) complexes bearing no labile ligands, with high activity also toward cisplatin-resistant cells, implying high activity range and high potential in anticancer therapy. The exceptional cytotoxic activity manifested by LpVO complexes confirms the hypothesis that labile ligands are not necessary for the cytotoxic properties of vanadium(V) complexes. Furthermore, unlike the previously reported diaminobis(phenolato) isopropoxo vanadium oxo complexes with a labile lignad,15 the absence of labile ligands in LpVO affords complexes resistance toward hydrolysis for days; this promotes the longevity of the compounds, while avoiding unnecessary dissociation steps in the cellular environment and the accompanying undesired release of potentially toxic side products. Noteworthy, the absence of labile ligands is not the only perquisite for hydrolytic stability, as coordination number and geometry are apparently also significant factors for the stability of vanadium(V) complexes toward hydrolysis, where an octahedral structure is markedly favored over a pentacoordinate one. Nevertheless, structural factors involving substitutions at the para position of the phenolato rings did not produce any detectable effect on the hydrolytic stability, confirming that the hydrolytic stability gained by the electronrich chelating ligand is sufficient to render any electronic impact of the substituents negligible. The structure activity studies demonstrated generally similar cytotoxic activity for the studied LpVO complexes toward the colon cell line, with some slight variations toward the slightly more sensitive ovarian lines. Interestingly, the cytotoxic activity of para tert-butylated Lp5VO is appreciable, despite its large steric bulk, previously reported to induce negative effects on

IC50 (μM) (MI (%)a) 1.4 2.4 2.7 2.3

± ± ± ±

0.2 0.5 1.3 0.1

(96) (98) (97) (97)

a

MI is the maximal percent of cell growth inhibition at the highest concentration applied.

are summarized in Table 5. The results exhibit no significant decrease of the IC50 values following extended periods of preincubation in the biological medium, for up to 72 h, suggesting high stability and sustainability of Lp1VO in biological environment, unlike the less stable oxovanadium(V)-salan complexes.15 In-vivo Efficacy. To further probe into the efficacy of vanadium complexes in mice models, an in vivo analysis was performed. Nude mice were inoculated with human colon HT29 cells, and treated with L p1 VO. The treated mice demonstrated significant decrease in the rate of tumor growth relative to control untreated mice (Figure 7); the average weight of tumors in the control group was 0.57 ± 0.16 g whereas for the experimental group, the average weight of 3/5 of tumors was 0.04 ± 0.05 g, with the rest of the animals not developing any tumors. An additional independent experiment supported the observed results (Figure S6). These findings demonstrate a significant growth-retarding effect of Lp1VO. Notably, although no specific tests on toxicity were carried out, no clinical signs of toxicity were detected in the treated mice; the mice appeared healthy, with no weight or hair loss, no grooming or behavioral changes, no swellings or eye infections, and no mortality occurring during the period of the experiment. E

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry cytotoxicity;15 the slight decrease in this compound activity may be attributed to reduced solubility and impaired biological accessibility.24 Overall, activity as high as 200-fold higher than that of cisplatin was recorded for the most effective derivatives. High cytotoxic activity of representative LpVO complexes toward cisplatin-resistant A2780cis cells implied, along with the great medicinal potential, a different cellular pathway than that of cisplatin. A relevant mechanism may, for instance, take advantage of the redox chemistry of vanadium, through formation of reactive oxygen species,25 but this remains to be deduced. The extended durability of LpVO complexes supports the identification of these compounds as stable anticancer agents. On the more practical side, this quality should enable easier handling and storage of these compounds, in the open air and in solution, thus prolonging their shelf life. Furthermore, the high effectiveness of the complexes following 3 days of preincubation in biological medium suggests prolonged activity once inside the biological environment, which may potentially contribute to reduced frequency of treatments. These results overall disclose the high potential of the vanadium(V) complexes presented herein, especially considering the clear in vivo efficacy as established in nude mice. Additionally, the lack of observable signs of toxicity to the treated mice is particularly encouraging, especially as vanadium compounds are known as potentially cytotoxic to normal cells as well.26 Overall, the results presented herein should promote future development of anticancer vanadium(V) compounds, that should hopefully be safe and effective, either as single agents or in combination with other chemotherapeutic drugs. Accordingly, future perspective for LpVO complexes incorporates investigation of their cellular pathways along with development of additional desired qualities.

■

118.7, 118.5, 115.4, 115.2, 54.1, 51.7, 49.7, 45.0. Anal. Calcd for C23H26N2O3: C, 72.99; H, 6.92; N, 7.40. Found: C, 72.63; H, 6.97; N, 7.08%. 2-(((2-((2-Hydroxy-4-methylbenzyl)(2-hydroxy-5-methylbenzyl)amino)ethyl)amino) methyl)-4-methylphenol, H3Lp2. This ligand was prepared similarly from 5-methylsalicylaldehyde (1.02 g, 7.48 mmol), ethylene diamine (0.25 mL, 3.74 mmol) and sodium borohydride (0.29 g, 7.66 mmol), which formed the p-Me substituted salan (0.83 g, 74%). The salan (0.83 g, 2.76 mmol) then was reacted similarly with 5methylsalicylaldehyde (0.38 g, 2.76 mmol) and sodium borohydride (0.11 g, 2.91 mmol) to produce a colorless precipitant (0.98 g, 84%). 1 H NMR (500 MHz, DMSO-d6): δ = 6.92 (d, J = 1.9 Hz, 2H, Ar), 6.88 (dd, J = 8.1, 1.9 Hz, 2H, Ar), 6.84 (dd, J = 8.1, 1.9 Hz, 1H, Ar), 6.79 (d, J = 1.8 Hz, 1H, Ar), 6.63 (d, J = 8.1 Hz, 2H, Ar), 6.58 (d, J = 8.1 Hz, 1H, Ar), 3.64 (s, 2H, CH2), 3.58 (s, 4H, CH2), 2.70 (t, J = 6.5 Hz, 2H, CH2), 2.54 (t, J = 6.5 Hz, 2H, CH2), 2.17 (s, 6H, CH3), 2.15 (s, 3H, CH3). 13C NMR (500 MHz, DMSO-d6): δ = 154.6, 154.2, 130.6, 129.1, 128.7, 128.1, 127.0, 126.7, 123.9, 123.0, 115.2, 115.0, 54.3, 51.7, 49.8, 45.0, 20.1, 20.1. Anal. Calcd for C26H32N2O3: C, 74.26; H, 7.67; N, 6.66. Found: C, 73.86; H, 7.53; N, 6.64%. 4-Chloro-2-(((2-((4-chloro-2-hydroxybenzyl)(5-chloro-2-hydroxybenzyl)amino)ethyl)amino)methyl)phenol, H3Lp3. This ligand was prepared similarly from 5-chloroysalicylaldehyde (1.74 g, 11.11 mmol), ethylenediamine (0.37 mL, 5.56 mmol) and sodium borohydride (0.45 g, 11.95 mmol) to form the p-Cl substituted salan (1.47 g, 77%). The salan precipitant (0.94 g, 2.75 mmol) was refluxed with 5-chlorosalicylaldehyde (0.43 g, 2.75 mmol) in methanol for 1 h, after which the solution was cooled and sodium borohydride (0.11 g, 2.91 mmol) was added. After addition of water and extraction with ethyl acetate H3Lp3 was recrystallized from cold methanol (0.42 g, 32%). 1H NMR (500 MHz, DMSO-d6): δ = 7.21 (d, J = 2.7 Hz, 2H, Ar), 7.10 (dd+d, J = 8.5, 2.7 Hz, 3H, Ar), 7.07 (dd, J = 8.5, 2.7 Hz, 1H, Ar), 6.76 (d, J = 8.6 Hz, 2H, Ar), 6.71 (d, J = 8.5 Hz, 1H, Ar), 3.67 (s, 2H, CH2), 3.60 (s, 4H, CH2), 2.70 (t, J = 6.2 Hz, 2H, CH2), 2.56 (t, J = 6.2 Hz, 2H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 155.5, 155.3, 129.4, 128.1, 127.9, 127.3, 126.8, 125.8, 122.1, 122.0, 116.9, 116.7, 53.2, 52.1, 48.6, 45.1. Anal. Calcd for C23H23Cl3N2O3: C, 57.34; H, 4.81; N, 5.81. Found: C, 57.23; H, 4.64; N, 5.65%. 2-(((2-((2-Hydroxy-4-methoxybenzyl)(2-hydroxy-5-methoxybenzyl)amino)ethyl)amino)methyl)-4-methoxyphenol, H3Lp4. This ligand was prepared similarly from 5-methoxysalicylaldehyde (1.00 mL, 8.01 mmol), ethylenediamine (0.27 mL, 4.00 mmol) and sodium borohydride (0.66 g, 17.44 mmol) to form the p-OMe substituted salan (0.95 g, 71%). The salan precipitant (0.95 g, 2.86 mmol) was refluxed with 5-methoxysalicylaldehyde (0.36 mL, 2.86 mmol) in methanol for 1 h, after which the solution was cooled and sodium borohydride (0.11 g, 2.91 mmol) was added. After addition of water and extraction with ethyl acetate H3Lp4 was recrystallized from cold methanol (0.60 g, 45%).1H NMR (500 MHz, DMSO-d6): δ = 6.76 (s, 2H, Ar), 6.64−6.68 (m, 5H, Ar), 6.59−6.63 (m, 2H, Ar), 3.64 (s, 2H, CH2), 3.63 (s, 9H, CH3), 3.59 (s, 4H, CH2), 2.71 (t, J = 6.4 Hz, 2H, CH2), 2.56 (t, J = 6.4 Hz, 2H, CH2). 13C NMR (500 MHz, DMSOd6): δ = 151.9, 151.8, 150.4, 150.1, 125.2, 124.2, 115.8, 115.6, 115.3, 114.1, 113.4, 112.8, 55.2, 55.2, 54.0, 52.1, 49.6, 45.2. Anal. Calcd for C26H32N2O6: C, 66.65; H, 6.88; N, 5.98. Found: C, 66.65; H, 6.64; N, 5.93%. 4-(tert-Butyl)-2-(((2-((4-(tert-butyl)-2-hydroxybenzyl)(5-(tertbutyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)phenol, H3Lp5. This ligand was prepared similarly from 5-tert-butylsalicylaldehyde (0.60 mL, 3.50 mmol), ethylenediamine (0.12 mL, 1.75 mmol) and sodium borohydride (0.13 g, 3.44 mmol) to form the p-tBu substituted salan (0.45 g, 67%). The salan precipitant (0.45 g, 1.17 mmol) was refluxed with 5-tert-butylsalicylaldehyde (0.2 mL, 1.17 mmol) in methanol for 2 h, after which the solution was cooled and sodium borohydride (0.06 g, 1.59 mmol) was added. After addition of water and extraction with ethyl acetate H3Lp5 was recrystallized from cold methanol and water (0.11 g, 17%).1H NMR (500 MHz, DMSO-d6): δ = 7.15 (d, J = 2.4 Hz, 2H, Ar), 7.08 (dd, J = 8.4, 2.4 Hz, 2H, Ar), 7.04 (dd, J = 8.4, 2.4 Hz, 1H, Ar), 7.01 (d, J = 2.4 Hz, 1H, Ar), 6.66 (d, J = 8.4 Hz, 2H, Ar), 6.59 (d, J = 8.4 Hz, 1H, Ar), 3.66 (s, 2H, CH2), 3.62

EXPERIMENTAL SECTION

Synthesis of H3L1−6 was performed through a slight modification of a known procedure,18 from the “salan” ligands.27 Synthesis of H3Lt and was performed according to a known procedure,19 after which H3Lt was crystallized from cold methanol. LtVO was prepared via a modified published procedure.17 Materials were purchased from Fluka & Riedel-de Haën, Aldrich Chemical Co., Strem Chemicals Inc., and Acros Organics, and used without further purification. Solvents used in the complex synthesis were dried over alumina columns on M. Braun SPS-800 solvent purification system. All experiments requiring dry atmosphere were performed in a M. Braun glovebox under nitrogen atmosphere. NMR data was recorded using an AMX-500 MHz Bruker spectrometer. Xray diffraction data was obtained with Bruker Smart Apex diffractometer, running the SMART software package. Synthesis. 2,2′-(((2-((2-Hydroxybenzyl)amino)ethyl)azanediyl)bis(methylene))diphenol, H3Lp1. A solution of salicylaldehyde (1.56 mL, 14.96 mmol) and ethylenediamine (0.50 mL, 7.48 mmol) in methanol was stirred at room temperature for 30 min, after which sodium borohydride (0.59 g, 15.59 mmol) was added and a precipitant was formed. The precipitant was filtered, dried and identified as the symmetrical diamino bis(phenolato) “salan” molecule (1.54 g, 54%). The salan (0.18 g, 0.66 mmol) was refluxed with salicylaldehyde (0.07 mL, 0.66 mmol) in methanol for 2 h, after which the solution was cooled and sodium borohydride (0.02 g, 0.66 mmol) was added. A colorless precipitant formed (0.19 g, 76%) that was collected and dried under vacuum. 1H NMR (500 MHz, DMSO-d6): δ = 7.12 (d, J = 7.7 Hz, 2H, Ar), 7.09 (dt, J = 7.7, 1.5 Hz, 2H, Ar), 7.05 (dt, J = 7.7, 1.5 Hz, 1H, Ar), 6.99 (d, J = 7.6 Hz, 1H, Ar), 6.71−6.77 (m, 4H, Ar), 6.69 (d, J = 7.7 Hz, 2H, Ar), 3.68 (s, 2H, CH2), 3.63 (s, 4H, CH2), 2.72 (t, J = 6.5 Hz, 2H, CH2), 2.56 (t, J = 6.5 Hz, 2H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 156.9, 156.5, 130.1, 128.6, 128.4, 127.8, 124.2, 123.3, F

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry (s, 4H, CH2), 2.72 (t, J = 6.5 Hz, 2H, CH2), 2.56 (t, J = 6.5 Hz, 2H, CH2), 1.21 (s, 18H, tBu), 1.20 (s, 9H, tBu). 13C NMR (500 MHz, DMSO-d6): δ = 154.5, 154.1, 140.8, 140.5, 126.6, 125.4, 124.8, 124.3, 123.3, 122.5, 114.8, 114.6, 54.3, 51.9, 50.2, 45.3, 33.6, 31.4, 31.4. Anal. Calcd for C35H50N2O3: C, 76.88; H, 9.22; N, 5.12. Found: C, 76.74; H, 9.17; N, 5.15%. 4-Fluoro-2-(((2-((4-fluoro-2-hydroxybenzyl)(5-fluoro-2-hydroxybenzyl)amino)ethyl)amino)methyl)phenol, H3Lp6. This ligand was prepared similarly from 5-fluorosalicylaldehyde (1.73 g, 12.35 mmol), ethylenediamine (0.41 mL, 6.17 mmol) and sodium borohydride (0.47 g, 12.42 mmol), after which water was added to precipitate the p-F substituted salan (1.34 g, 70%). The salan precipitant (0.71 g, 2.30 mmol) was refluxed with 5-fluorosalicylaldehyde (0.32 g, 2.30 mmol) in methanol for 1 h, after which the solution was cooled and sodium borohydride (0.09 g, 2.38 mmol) was added. After addition of water and extraction with ethyl acetate H3Lp6 was recrystallized from cold methanol and water (0.68 g, 50%).1H NMR (500 MHz, DMSO-d6): δ = 7.03 (dd, J = 9.3, 3.2 Hz, 2H, Ar), 6.86−6.92 (m, 3H), 6.84 (dd, J = 8.6, 3.2 Hz, 1H, Ar), 6.72 (dd, J = 8.9, 4.8 Hz, 2H, Ar), 6.68 (dd, J = 8.8, 4.8 Hz, 1H, Ar), 3.66 (s, 2H, CH2), 3.60 (s, 4H, CH2), 2.69 (t, J = 6.5 Hz, 2H, CH2), 2.56 (t, J = 6.5 Hz, 2H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 156.1 (d, J = 12 Hz), 154.3 (d, J = 10 Hz), 152.7 (d, J = 7 Hz), 152.5 (d, J = 7 Hz), 126.4 (d, J = 27 Hz), 125.3 (d, J = 27 Hz), 116.1 (d, J = 89 Hz), 116.0 (d, J = 33 Hz), 115.7 (d, J = 31 Hz), 114.7 (d, J = 92 Hz), 114.3 (d, J = 90 Hz), 113.6 (d, J = 89 Hz), 53.3, 52.3, 48.9, 45.2. 19F NMR (500 MHz, DMSO-d6): δ = −125.8 (sextet, J = 5 Hz, 2F), −125.9 (sextet, J = 5 Hz, 1F). Anal. Calcd for C23H23F3N2O3: C, 63.88; H, 5.36; N, 6.48. Found: C, 63.59; H, 5.21; N, 6.51%. LpVO complex synthesis. Synthesis of Lp1−6VO was conducted similarly to previously published methods.15 A solution of H3L1−6 in dry THF (1 mL) was added to a stirred solution containing equimolar amounts of VO(OiPr)3 in dry THF (3 mL) under inert atmosphere. The reaction mixture was stirred at room temperature for 15 min, and the volatiles were removed under vacuum. All complexes were obtained as dark purple powder in quantitative yields (>95%). Lp1VO. This complex was prepared from H3L1 and VO(OiPr)3 in the manner described above. 1H NMR (500 MHz, DMSO-d6): δ = 7.16−7.28 (m, 3H, Ar), 7.10 (t, J = 8.4 Hz, 2H, Ar), 7.02 (t, J = 8.4 Hz, 1H, Ar), 6.89−6.95 (m, 2H, Ar), 6.83 (t, J = 7.2 Hz, 1H, Ar), 6.66− 6.74 (m, 2H, Ar), 6.27−6.32 (br, 1H, CH2),6.26 (d, J = 7.2 Hz, 1H, Ar), 4.81 (d, J = 15.5, 1H, CH2), 4.56 (d, J = 14.1, 1H, CH2), 4.04 (d, J = 15.5, 1H, CH2), 3.64 (d, J = 14.2, 1H, CH2), 3.20 (d, J = 14.1, 1H, CH2), 3.19 (d, J = 14.3, 1H, CH2), 2.51−2.64 (m, 2H, CH2), 2.18− 2.25 (m, 1H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 163.0, 161.3, 129.8, 129.5, 129.5, 128.8, 128.1, 127.6, 127.3, 126.9, 126.9, 121.2, 121.0, 120.4, 118.6, 118.0, 116.9, 115.9, 60.0, 57.0, 55.8, 52.3, 47.2. 51V NMR (500 MHz, DMSO-d6): δ = −493. Anal. Calcd for C23H23N2O4V: C, 62.45; H, 5.24; N, 6.33. Found: C, 62.56; H, 5.41; N, 5.95%. Lp2VO. This complex was prepared from H3L2 and VO(OiPr)3 in the manner described above. 1H NMR (500 MHz, DMSO-d6): δ = 7.03 (dd, J = 8.3, 2.0 Hz, 1H, Ar), 7.00 (dd, J = 8.2, 2.0 Hz, 1H, Ar), 6.98 (s, 1H, Ar), 6.89 (s, 2H, Ar), 6.81 (dd, J = 8.3, 2.0 Hz, 1H, Ar), 6.80 (d, J = 8.2, 1H, Ar), 6.56 (d, J = 8.2, 1H, Ar), 6.15 (d, J = 8.2, 1H, Ar), 6.05−6.11 (br, 1H, CH2), 4.75 (d, J = 15.5, 1H, CH2), 4.50 (d, J = 13.9, 1H, CH2), 3.95 (d, J = 15.5, 1H, CH2), 3.60 (d, J = 13.9, 1H, CH2), 3.10 (d, J = 14.2, 1H, CH2), 3.09 (d, J = 14.2, 1H, CH2), 2.53− 2.61 (m, 1H, CH2), 2.29 (s, 3H, CH3), 2.20−2.26 (m, 4H, CH3, CH2), 2.18 (s, 3H, CH3). 13C NMR (500 MHz, DMSO-d6): δ = 162.3, 161.1, 159.6, 130.1, 129.9, 129.9, 129.8, 129.0, 128.4, 128.0, 127.8, 126.8, 126.7, 126.6, 120.1, 117.7, 116.6, 115.6, 60.0, 57.0, 55.8, 52.3, 47.1, 20.4, 20.4, 20.1. 51V NMR (500 MHz, DMSO-d6): δ = −479. Anal. Calcd for C26H29N2O4V: C, 64.46; H, 6.03; N, 5.78. Found: C, 64.15; H, 6.09; N, 5.57%. Lp3VO. This complex was prepared from H3L3 and VO(OiPr)3 in the manner described above. 1H NMR (500 MHz, DMSO-d6): δ = 7.26−7.30 (m, 2H, Ar), 7.23 (dd, J = 8.6, 2.6 Hz, 1H, Ar), 7.21 (d, J = 2.5 Hz, 1H, Ar), 7.17 (d, J = 2.5 Hz, 1H, Ar), 7.04 (dd, J = 8.7, 2.6 Hz, 1H, Ar), 6.92 (d, J = 8.7, 1H, Ar), 6.71 (d, J = 8.6, 1H, Ar), 6.47−6.54

(br, 1H, CH2), 6.28 (d, J = 8.7, 1H, Ar), 4.78 (d, J = 15.5, 1H, CH2), 4.46 (d, J = 14.6, 1H, CH2), 4.01 (d, J = 15.5, 1H, CH2), 3.56 (d, J = 14.4, 1H, CH2), 3.27 (d, J = 14.5, 1H, CH2), 3.20 (d, J = 14.7, 1H, CH2), 2.55−2.63 (m, 2H, CH2), 2.20−2.26 (m, 1H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 162.6, 161.7, 160.1, 129.3, 128.9, 128.6, 128.5, 128.2, 127.7, 127.4, 127.0, 124.5, 124.4, 122.4, 121.9, 119.7, 118.8, 117.7, 59.2, 56.3, 55.3, 52.2, 47.2. 51V NMR (500 MHz, DMSOd6): δ = −448. Anal. Calcd for C23H20Cl3N2O4V: C, 50.62; H, 3.69; N, 5.13. Found: C, 50.52; H, 3.60; N, 4.81%. Crystal data for Lp3VO. Lp3VO was crystallized from a cold 1:2 1,2dichloroethane/heptane mixture, and crystallographic data and structure refinement details are shown in Table 6.

Table 6. Crystallographic Data and Structure Refinement Details for Lp3VO Empirical formula Formula weight Crystal system Unit cell dimensions

Volume Temperature Space group Z Absorption coefficient Reflections collected Independent reflections Final R indices [I > 2σ (I)]

C23H20Cl3N2O4V·(CH2Cl) 595.18 monoclinic a = 14.354(8) Å b = 10.238(5) Å c = 17.524(9) Å 2574(2) Å3 295(1) K P2(1)/n 4 0.835 mm−1 23417 4506 [R(int) = 0.0559] R1 = 0.0999, wR2 = 0.1986

Lp4VO. This complex was prepared from H3L4 and VO(OiPr)3 in the manner described above. 1H NMR (500 MHz, DMSO-d6): δ = 6.87 (d, J = 8.8 Hz, 1H, Ar), 6.75−6.83 (m, 3H, Ar), 6.70 (s, 1H, Ar), 6.69 (s, 1H, Ar), 6.59−6.63 (m, 2H, Ar), 6.20 (d, J = 8.9, 1H, Ar), 6.00−6.06 (br, 1H, CH2), 4.75 (d, J = 15.4, 1H, CH2), 4.52 (d, J = 14.0, 1H, CH2), 3.97 (d, J = 15.4, 1H, CH2), 3.75 (s, 3H, CH3), 3.69 (s, 3H, CH3), 3.65 (s, 3H, CH3), 3.60 (d, J = 14.0, 1H, CH2), 3.15 (d, J = 14.3, 1H, CH2), 3.10 (d, J = 14.3, 1H, CH2), 2.53−2.65 (m, 2H, CH2), 2.12−2.19 (m, 1H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 159.2, 157.5, 156.3, 153.4, 153.3, 151.6, 127.7, 127.7, 120.7, 118.7, 117.6, 116.4, 114.8, 114.2, 113.3, 113.1, 113.0, 112.8, 60.1, 57.1, 55.9, 55.4, 55.3, 55.3, 52.3, 47.0. 51V NMR (500 MHz, DMSO-d6): δ = −458. Anal. Calcd for C26H29N2O7V: C, 58.65; H, 5.49; N, 5.26. Found: C, 58.29; H, 5.49; N, 5.15%. Lp5VO. This complex was prepared from H3L5 and VO(OiPr)3 in the manner described above. 1H NMR (500 MHz, DMSO-d6): δ = 7.24 (dd, J = 8.7, 2.3 Hz, 1H, Ar), 7.23 (dd, J = 8.7, 2.3 Hz, 1H, Ar), 7.17 (d,, J = 2.3 Hz, 1H, Ar), 7.07−7.12 (m, 2H, Ar), 7.03 (dd, J = 8.6, 2.3 Hz, 1H, Ar), 6.84 (d, J = 8.5 Hz, 1H, Ar), 6.58 (d, J = 8.5 Hz, 1H, Ar), 6.24 (d, J = 8.5 Hz, 1H, Ar), 6.04−6.11 (br, 1H, CH2), 4.78 (d, J = 15.2, 1H, CH2), 4.59 (d, J = 13.9, 1H, CH2), 4.02 (d, J = 15.1, 1H, CH2), 3.59 (d, J = 14.0, 1H, CH2), 3.20 (d, J = 14.0, 1H, CH2), 3.14 (d, J = 14.0, 1H, CH2), 2.53−2.67 (m, 2H, CH2), 2.16−2.23 (m, 1H, CH2), 1.29 (s, 9H, tBu), 1.23 (s, 9H, tBu), 1.22 (s, 9H, tBu). 13C NMR (500 MHz, DMSO-d6): δ = 162.2, 161.1, 159.4, 143.3, 143.3, 140.5, 126.3, 126.2, 126.1, 125.9, 125.0, 124.6, 124.4, 124.0, 119.5, 117.4, 116.2, 115.4, 60.3, 57.4, 56.1, 52.4, 47.3, 34.0, 33.9, 33.6, 31.4, 31.4, 31.3. 51V NMR (500 MHz, DMSO-d6): δ = −477. Anal. Calcd for C35H47N2O4V: C, 68.83; H, 7.76; N, 4.59. Found: C, 68.43; H, 7.85; N, 4.46%. Lp6VO. This complex was prepared from H3L6 and VO(OiPr)3 in the manner described above. 1H NMR (500 MHz, DMSO-d6): δ = 6.95−7.11 (m, 5H, Ar), 6.93 (dd, J = 9.1, 5.2 Hz, 1H, Ar), 6.83 (dt, J = 8.6, 3.2 Hz, 1H, Ar), 6.70 (dd, J = 8.9, 5.2 Hz, 1H, Ar), 6.36−6.43 (br, 1H, CH2), 6.25 (dd, J = 9.0, 5.1 Hz, 1H, Ar), 4.78 (d, J = 15.8, 1H, CH2), 4.50 (d, J = 14.5, 1H, CH2), 4.07 (d, J = 15.6, 1H, CH2), 3.59 (d, J = 14.0, 1H, CH2), 3.25 (d, J = 14.5, 1H, CH2), 3.18 (d, J = 14.6, G

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry 1H, CH2), 2.55−2.66 (m, 2H, CH2), 2.20−2.26 (m, 1H, CH2). 13C NMR (500 MHz, DMSO-d6): δ = 160.4 (d, J = 5.1 Hz), 159.4 (d, J = 4.7 Hz), 157.8, 157.2 (d, J = 62 Hz), 156.0, 155.3 (d, J = 64 Hz), 154.1, 128.0 (d, J = 31 Hz), 121.5 (d, J = 31 Hz), 119.3 (d, J = 31 Hz), 118.3 (d, J = 33 Hz), 116.8 (d, J = 32 Hz), 116.0 (d, J = 93 Hz), 115.4 (d, J = 92 Hz), 114.6 (d, J = 93 Hz), 114.4 (d, J = 90 Hz), 114.1 (d, J = 91 Hz), 113.7 (d, J = 90 Hz), 59.5, 56.6, 55.5, 52.2, 47.2. 19F NMR (500 MHz, DMSO-d6): δ = −121.8 (m, 1F), −122.1 (m, 1F), −126.1 (m, 1F). 51V NMR (500 MHz, DMSO-d6): δ = −492. Anal. Calcd for C23H20F3N2O4V: C, 55.66; H, 4.06; N, 5.64. Found: C, 55.65; H, 4.13; N, 5.48%. LtVO. Was prepared similarly to a known procedure,17 by reacting H3Lt (0.04 g, 0.10 mmol) with VO(OiPr)3 (0.02 g, 0.10 mmol) and 3 drops of triethylamine, in dry THF (3 mL) for 1 h at room temperature. The volatiles were removed under vacuum to produce a dark purple powder. Anal. Calcd for C27H30NO4V: C, 67.07; H, 6.25; N, 2.90. Found: C, 67.22; H, 6.26; N, 2.90%. Hydrolysis. Hydrolysis studies for LpVO complexes were performed by reacting the reagent with 1000 equiv of water in THF for 3 days. Afterward the volatiles were removed under reduced pressure, and the reaction mixture was dissolved in DMSO-d6 and analyzed by 1H NMR spectroscopy. The results were verified by including 1,4-dinitrobenzene as an internal standard for a representative complex Lp1VO. For LtVO complex hydrolysis measurements were performed in a previously described manner,15,28 by reacting the compound dissolved in THF-d8 with over 1000 equiv of D2O and monitoring the integration of the peaks over time. Cytotoxicity. Cytotoxicity was measured using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay20,28 against HT-29 colon and OVCAR-3 ovarian cells obtained from ATCC Inc. and A2780 and A2780cis cells obtained from ECACC. Cells (0.6 × 106) in medium (contains: 1% penicillin/streptomycin antibiotics; 1% Lglutamine; 10% fetal bovine serum (FBS) and 88% medium RPMI1640 for HT-29, A2780 and A2780cis cells, and similar medium containing 20% FBS, 0.01 mg/mL human recombinant insulin and 78% medium RPMI-1640 for OVCAR-3 cells, all purchased from Biological Industries Inc.) were seeded into a 96-well plate and allowed to attach for a day. The cells were consequently treated with the reagent tested at 10 different concentrations. Solution of reagent was prepared by dissolving 4 mg of the reagent in 800 μL of DMSO and diluting 20 μL of this solution with 180 μL of medium. From the resulting solution, 10 μL was added to each well already containing 200 μL of the above solution of cells in the medium to give final concentration of up to 200 mg/L in 0.5% DMSO. Despite low aquatic solubility of the complexes, no precipitation or other solubility issues were observed under these conditions. After a standard of 3 days incubation at 37 °C in 5% CO2 atmosphere, MTT (0.1 mg in 20 μL) was added and the cells were incubated for additional 3 h. The MTT solution was then removed, and the cells were dissolved in 200 μL isopropanol. The absorbance at 550 nm was measured by a Bio-Tek EL-800 microplate reader spectrophotometer. Each experiment was repeated at least 3 × 3 times, namely, three repeats of the experiment per plate, all repeated at least 3 times on different days (9 repeats altogether). Relative IC50 values were determined by a nonlinear regression of a variable slope (four parameters) model, and are presented as mean ± SD values. During measurements of the durability in organic medium of Lp1VO, 4 mg of the complex were dissolved in 800 μL of sterile DMSO and preincubated in sterile conditions at 37 °C in 5% CO2 atmosphere or at 4 °C in a refrigerator. Subsequent cytotoxic activity measurements on HT-29 cells were conducted as described above. Durability measurements in biological medium were performed by preparing 10 concentrations of Lp1VO in sterile DMSO, diluting 20 μL of the DMSO solutions with 180 μL of medium and incubating the solutions at 37 °C in 5% CO2 atmosphere for up to 3 days. Afterward, 10 μL of each solution was added to HT-29 cells and cytotoxicity was measured following 3 days of incubation, as described above. In Vivo. Nude female Balb/C mice (6−8 weeks old) were obtained from Harlan (Israel), held in a SPF facility (AAALAC accreditation #1285) and treated in accordance with NIH guidelines and approval

by the institutional committee for ethics in animal experimentation. Mice were injected S.C. with 5 × 106 HT-29 Human colon adenocarcinoma cells. Starting 24 h after the challenge, mice were subjected to I.P. injections of a PBS solution containing 1% DMSO and 10 μg of Lp1VO (0.5 mg/1 kg mouse body weight each injection), every other day for 3 weeks. Mice were followed up to 28 days post inoculation, after which the experiment was terminated.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02519. An analogous cytotoxicity plot to that presented in Figure 4 but toward OVCAR-3 cells, an analogous cytotoxicity plot to that presented in Figure 5 but for Lp6VO, a cytotoxicity plot of HT-29 cell vialbility dependence on concentration of Lp1VO after incubation in DMSO at 37 and 4 °C, 1H and 51V NMR spectra of Lp1VO before and after incubation in DMSO at 37 °C, a repeat of in vivo results displayed in Figure 7, and demonstration of tumor size results of in vivo studies. (PDF) CIF data (CIF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Funding was received from the European Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement (No. 239603).

■

REFERENCES

(1) (a) Kelland, L. Nat. Rev. Cancer 2007, 7 (8), 573−584. (b) Jung, Y. W.; Lippard, S. J. Chem. Rev. 2007, 107 (5), 1387−1407. (c) Harper, B. W.; Krause-Heuer, A. M.; Grant, M. P.; Manohar, M.; GarbutcheonSingh, K. B.; Aldrich-Wright, J. R. Chem. - Eur. J. 2010, 16 (24), 7064− 7077. (d) Dasari, S.; Tchounwou, P. B. Eur. J. Pharmacol. 2014, 740, 364−378. (2) (a) Ott, I.; Gust, R. Arch. Pharm. (Weinheim, Ger.) 2007, 340 (3), 117−126. (b) Fricker, S. P. Dalton Trans. 2007, 43, 4903−4917. (c) Muhammad, N.; Guo, Z. Curr. Opin. Chem. Biol. 2014, 19, 144− 153. (d) Bruijnincx, P. C. A.; Sadler, P. J. Curr. Opin. Chem. Biol. 2008, 12 (2), 197−206. (e) Trudu, F.; Amato, F.; Vanhara, P.; Pivetta, T.; Pena-Mendez, E. M.; Havel, J. J. Appl. Biomed. 2015, 13 (2), 79−103. (f) Medici, S.; Peana, M.; Nurchi, V. M.; Lachowicz, J. I.; Crisponi, G.; Zoroddu, M. A. Coord. Chem. Rev. 2015, 284, 329−350. (3) Anke, M. Anal. Real Acad. Nac. F. 2004, 70 (4), 961−999. (4) (a) Evangelou, A. M. Cr. Rev. Oncol-Hem. 2002, 42 (3), 249−265. (b) Costa Pessoa, J.; Garribba, E.; Santos, M. F. A.; Santos-Silva, T. Coord. Chem. Rev. 2015, 301, 49−86. (5) Bishayee, A.; Waghray, A.; Patel, M. A.; Chatterjee, M. Cancer Lett. 2010, 294 (1), 1−12. (6) (a) Gambino, D. Coord. Chem. Rev. 2011, 255 (19−20), 2193− 2203. (b) Badmaev, V.; Prakash, S.; Majeed, M. J. Altern. Complement. Med. 1999, 5 (3), 273−291. (7) (a) Pessoa, J. C.; Etcheverry, S.; Gambino, D. Coord. Chem. Rev. 2015, 301, 24−48. (b) Costa Pessoa, J. J. Inorg. Biochem. 2015, 147, 4−24. (8) Thompson, K. H.; Lichter, J.; Lebel, C.; Scaife, M. C.; McNeill, J. H.; Orvig, C. J. Inorg. Biochem. 2009, 103 (4), 554−558. H

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry (9) (a) Korbecki, J.; Baranowska-Bosiacka, I.; Gutowska, I.; Chlubek, D. Acta Biochim. Polym. 2012, 59 (2), 195−200. (b) Kioseoglou, E.; Petanidis, S.; Gabriel, C.; Salifoglou, A. Coord. Chem. Rev. 2015, 301, 87−105. (10) (a) Gomathi Sankareswari, V.; Vinod, D.; Mahalakshmi, A.; Alamelu, M.; Kumaresan, G.; Ramaraj, R.; Rajagopal, S. Dalton Trans. 2014, 43 (8), 3260−3272. (b) Lu, J.; Guo, J.; Sang, W.; Guo, H. Transition Met. Chem. (Dordrecht, Neth.) 2013, 38 (5), 481−488. (11) D’Cruz, O. J.; Uckun, F. M. Expert Opin. Invest. Drugs 2002, 11 (12), 1829−1836. (12) Tang, L.-y.; Su, Y.; He, J.-r.; Chen, W.-q.; Su, F.-x.; Wu, B.-h.; Lin, Y.; Ren, Z.-f. Nutr. Cancer 2012, 64 (3), 368−376. (13) (a) Wozniak, K.; Blasiak, J. Arch. Toxicol. 2004, 78 (1), 7−15. (b) Wang, Q.; Liu, T.-T.; Fu, Y.; Wang, K.; Yang, X.-G. JBIC, J. Biol. Inorg. Chem. 2010, 15 (7), 1087−1097. (14) (a) Rehder, D. Bioinorganic Vanadium Chemistry; John Wiley & Sons, Ltd: 2008. (b) Kustin, K. J. Inorg. Biochem. 2015, 147, 32−38. (15) Reytman, L.; Braitbard, O.; Tshuva, E. Y. Dalton Trans. 2012, 41 (17), 5241−5247. (16) Colpas, G. J.; Hamstra, B. J.; Kampf, J. W.; Pecoraro, V. L. Inorg. Chem. 1994, 33 (21), 4669−4675. (17) Groysman, S.; Goldberg, I.; Goldschmidt, Z.; Kol, M. Inorg. Chem. 2005, 44 (14), 5073−5080. (18) Schmitt, H.; Lomoth, R.; Magnuson, A.; Park, J.; Fryxelius, J.; Kritikos, M.; Martensson, J.; Hammarstrom, L.; Sun, L. C.; Akermark, B. Chem. - Eur. J. 2002, 8 (16), 3757−3768. (19) Chandrasekaran, A.; Day, R. O.; Holmes, R. R. J. Am. Chem. Soc. 2000, 122 (6), 1066−1072. (20) Ganot, N.; Meker, S.; Reytman, L.; Tzubery, A.; Tshuva, E. Y. J. Visualized Exp. 2013, 81, e50767. (21) (a) Rehder, D.; Pessoa, J. C.; Geraldes, C.; Castro, M.; Kabanos, T.; Kiss, T.; Meier, B.; Micera, G.; Pettersson, L.; Rangel, M.; Salifoglou, A.; Turel, I.; Wang, D. R. JBIC, J. Biol. Inorg. Chem. 2002, 7 (4−5), 384−396. (b) Kiss, T.; Kiss, E.; Micera, G.; Sanna, D. Inorg. Chim. Acta 1998, 283 (1), 202−210. (c) Sanna, D.; Serra, M.; Micera, G.; Garribba, E. Inorg. Chem. 2014, 53 (3), 1449−1464. (22) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Morris, R. E.; Chen, H.; Sadler, P. J.; Jodrell, D. I. Br. J. Cancer 2002, 86 (10), 1652− 1657. (23) (a) Ganot, N.; Redko, B.; Gellerman, G.; Tshuva, E. Y. RSC Adv. 2015, 5 (11), 7874−7879. (b) Hall, M. D.; Telma, K. A.; Chang, K.-E.; Lee, T. D.; Madigan, J. P.; Lloyd, J. R.; Goldlust, I. S.; Hoeschele, J. D.; Gottesman, M. M. Cancer Res. 2014, 74 (14), 3913−3922. (24) (a) Meker, S.; Margulis-Goshen, K.; Weiss, E.; Braitbard, O.; Hochman, J.; Magdassi, S.; Tshuva, E. Y. ChemMedChem 2014, 9 (6), 1294−1298. (b) Glasner, H.; Meker, S.; Tshuva, E. Y. J. Organomet. Chem. 2015, 788, 33−35. (25) Jungwirth, U.; Kowol, C. R.; Keppler, B. K.; Hartinger, C. G.; Berger, W.; Heffeter, P. Antioxid. Redox Signaling 2011, 15 (4), 1085− 1127. (26) Sabbioni, E.; Pozzi, G.; Devos, S.; Pintar, A.; Casella, L.; Fischbach, M. Carcinogenesis 1993, 14 (12), 2565−2568. (27) Whiteoak, C. J.; Britovsek, G. J. P.; Gibson, V. C.; White, A. J. P. Dalton Trans. 2009, 13, 2337−2344. (28) Peri, D.; Meker, S.; Manna, C. M.; Tshuva, E. Y. Inorg. Chem. 2011, 50 (3), 1030−1038.

I

DOI: 10.1021/acs.inorgchem.5b02519 Inorg. Chem. XXXX, XXX, XXX−XXX