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Preparation, structural characterization and cytotoxicity of hydrolytically stable Ti(IV) citrate complexes
Inorganica Chimica Acta 503 (2020) 119429
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Research paper
Preparation, structural characterization and cytotoxicity of hydrolytically stable Ti(IV) citrate complexes ⁎,1
Nathan Engelberg, Avi Bino
, Edit Y. Tshuva
T
⁎,1
Institute of Chemistry, The Hebrew University of Jerusalem, 9190401 Jerusalem, Israel
ABSTRACT
Hydrolytically stable and water soluble Ti(IV) complexes can be used for various applications, including industrial, medicinal and more. Herein, a novel tetra-nuclear Ti(IV) complex with citrate ligands was obtained by reacting Ti(IV) isopropoxide and citric acid in an aqueous solution. The three dimensional structure of the complex [Ti4O6(Hcit)3(cit)]9− 1, as it appears in the compound [Co(NH3)6]3[Ti4O6(Hcit)3(cit)]⋅20H2O was determined by single-crystal X-ray crystallography. Complex 1 possesses an adamantane-like {Ti4O6} core where each metal atom is coordinated to a tri-dentate citrate ligand with one dangling CH2COOH group. An additional already known Ti(IV) complex [Ti(Hcit3−)3]5− 2 was synthesized for comparison. The high hydrolytic stability of complexes 1 and 2 has been substantiated based on NMR techniques. The cytotoxicity of the complexes was tested on three cancer cell lines: human ovarian A2780 and drug-resistant variants A2780cis and A2780ADR. Complex 1 showed a slight cytotoxicity towards the ovarian cells, while complex 2 was inactive.
1. Introduction Titanium is commonly used, either as a metal or in its complexed form, in different fields such as material science and medicine, including for surgical equipment and prosthetics and for development of anti-cancer agents [1]. The low intrinsic toxicity of titanium makes it an attractive metal for use in biological systems and several titanium compounds were found to be bioactive [2]. However, due to its highly oxophilic nature, Ti(IV) is prone to fast hydrolytic processes when exposed to aqueous environments [3–5]. Hydrolysis of Ti(IV) produces numerous polynuclear Ti(IV) oxide species, which have been thoroughly studied for their potential as functional materials [6]. Preparation is usually performed under inert water-free conditions, with a few successes in aqueous environments to produce titanium oxides of large nuclearity (TixOy, x > 7) [7–11]. Titanium oxides with a nuclearity of x = 4 mostly contain either a {Ti4O4} core [11], or an adamantanelike {Ti4O6} [12,13] core. Similar {M4O6} cores have been previously observed with other metal-ions as well (M = Mn(IV), Ru(IV)) [14,15]. In order to enhance the hydrolytic stability of these complexes and enabling activity in aqueous solutions, citrate ligands are used to bind Ti(IV) and form relatively stable water soluble species. In the Pechini method to produce a high quality barium titanate ceramic, a Ti(IV) alkoxide precursor is added to an aqueous solution of citric acid, followed by the addition of barium salts [16]. Due to the abundance of titanium from prosthetics and food additives and citrate ions in the human body, the interactions between them have been of great interest. Citrate has been found to leach solid titanium and solubilize it as ions
⁎ 1
and assisting in regulating Ti(IV) concentration in the blood stream [17]. It has also been shown that a siderophore protein transferrin binds Ti(IV) using citrate as a co-anion [18-20]. Research of the bioactivity of Ti(IV) citrate showed modulation of the structure of human erythrocyte membrane [21]. The speciation of Ti(IV) citrate complexes has been the subject of numerous studies and several species were structurally characterized. Among them a series of mononuclear complexes with the general formula [Ti(Hxcit)3]n− x = 0–2 and n = 2–8 depending on citrate protonation [22–25], and an octanuclear complex [Ti8O10(cit)4]0 [26]. Several Ti(IV) complexes were found to be bioactive, with marked anti-cancer activity against a variety of cancer cell lines. Initial studies on budotitane ((bzac)2Ti(OEt)2) and titanocene dichloride (Cp2TiCl2) showed low effectivity due to their rapid hydrolysis upon exposure to aqueous solutions [27–31]. Later developments of Ti(IV) complexes utilizing aminephenlato-type ligands with different denticities [32] increased the hydrolytic stability, and thereafter showed significantly higher anti-cancer activity both in-vitro and in-vivo [33,34]. However, the introduction of relatively hydrophobic ligands into the complexes significantly decreased their water solubility. Herein we aimed at obtaining new hydrolytically stable and water soluble Ti(IV) complexes for anticancer medicinal applications. We thus report the preparation and structure of a novel tetra-Ti(IV) citrate complex, namely, [Ti4O6(Hcit)3(cit)]9− 1, featuring a tetranuclear adamantane-like core {Ti4O6}. We also report the structure of the sodium salt of the already known [Ti(Hcit)3]5− 2 complex. The hydrolytic stability of these complexes was examined using 1H NMR spectra of
Corresponding authors. Both authors contributed equally to this work.
https://doi.org/10.1016/j.ica.2020.119429 Received 6 October 2019; Received in revised form 8 January 2020; Accepted 8 January 2020 Available online 09 January 2020 0020-1693/ © 2020 Elsevier B.V. All rights reserved.
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N. Engelberg, et al.
D2O solutions, and the anti-cancer activity of the two complexes was tested in-vitro towards the human ovarian cancer cell line A2780 and drug-resistant variants A2780cis and A2780ADR.
Table 1 Crystal data and structure refinement parameters for [Co(NH3)6]3⋅1⋅20H2O and Na5⋅2⋅19.5H2O.
2. Experimental
Empirical formula Formula weight Temperature K Wavelength Å Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Z ρcalc (g cm−3) µ (mm−1) Crystal size (mm3) Range of θ (°) Total reflections Independent reflection (Rint) Parameters R1 [I > 2sigma(I)] wR2 (I)
2.1. Reagents and measurements All chemicals and solvents were purchased from commercial sources and were used without further purification. Ti(IV) tetraisopropoxide (98%) was purchased from Acros Organics, citric acid anhydrous (99.5%) was purchased from Sigma-Aldritch, tetrahydrofuran (99.8%) was purchased from Bio-Lab, sodium p-toluenesulfonate was purchased from Sigma-Aldritch, hexaamminecobalt(III) chloride was purchased from Alfa Aesar, sodium chloride was purchased from Frutarom. All reactions were carried out at room temperature and ambient conditions unless stated otherwise. Quantitative elemental analysis of C, H, N atom weight content was performed by the HUJI lab for microanalysis using a Thermo Flash 2000 CHN-O Elemental Analyzer. 1H NMR and 13C NMR spectra were obtained using a Bruker Avance Neo-500 spectrometer. Mass spectrometry was performed by the HUJI lab for microanalysis using Agilent 6520 QTOF analyzer. Data were collected on a Bruker SMART APEX CCD X-ray diffractometer system controlled by a pentium-based PC running the SMART software package [35]. Cytotoxicity measurements were performed on the A2780 cancer cell line obtained from ATCC and the A2780cis and A2780ADR cell lines obtained from ECACC. Cell viability was assessed using a Tecan Spark 10 M microplate reader spectrophotometer.
[Co(NH3)6]3⋅1⋅20H2O
Na5⋅2⋅19.5H2O
C24H70Co3N18O54Ti4 1843.37 293(2) 0.71073 Monoclinic C2/c
C18H51Na5O39.50Ti 1062.44 294(1) 0.71073 Cubic Pa-3
23.799(4) 18.965(3) 18.185(3) 90 106.348(2) 90 7876(2) 4 1.555 1.112 0.46 × 0.38 × 0.19 2.15–28.09 43,966 9436 (0.0565) 493 0.0512 0.1576
20.476(1) 20.476(1) 20.476(1) 90 90 90 8581(1) 8 1.644 0.369 0.25 × 0.18 × 0.14 2.22–24.98 75,445 2519 (0.0704) 205 0.0733 0.1542
2.2.3. Preparation of Na5[Ti(Hcit)3]⋅19.5H2O (Na5⋅2⋅19·.5H2O) Ti(OiPr)4 (0.074 mL, 0.25 mmol) was reacted with citric acid C6H8O7 (0.960 g, 5 mmol) dissolved in 2 mL THF, resulting in a cloudy solution. Water (8 mL) was added and the mixture was stirred for 1 h at 90 °C. After cooling to room temperature, the pH was adjusted to 4.75 by adding 4 M NaOH solution, followed by the addition of sodium ptoluensulfonate (CH3C6H4SO3Na) (0.291 g, 1.5 mmol). The solvent was evaporated slowly to yield colorless crystals after 7 days. The crystals were filtered, washed with cold water and air-dried. Yield 0.264 g (98%). Anal. Found: C, 20.27; H, 4.97%. Calc. for C18H54Na5O39.5Ti: C, 20.29; H, 5.11%. 1H-NMR (500 MHz, D2O): δ = 2.752 (dd; CH2), δ = 2.926 (d, CH2).
2.2. Preparation of Ti(IV) citrate complexes. 2.2.1. Preparation of [Co(NH3)6]3[Ti4O6(Hcit)3(cit)]⋅20H2O ([Co (NH3)6]3⋅1⋅20H2O) Ti(OiPr)4 (0.740 mL, 2.5 mmol) was reacted with citric acid C6H8O7 (2.401 g, 12.5 mmol) dissolved in 20 mL THF. Water (80 mL) was added and the solution was stirred for 1 h at 90 °C. After cooling to room temperature, the pH was adjusted to 6.40 by the addition of a 4 M NaOH solution, followed by the addition of [Co(NH3)6]Cl3 (0.668 g, 2.5 mmol). The solvent was evaporated slowly to yield orange crystals after three weeks. The crystals were filtered, washed with cold water and air-dried. The calculated values suggest the presence of some adsorbed water molecules that could not be removed by air-drying. Organic solvents or high vacuum lead to the loss of lattice water molecules and crystal disintegration. Yield 0.943 g (82%). Anal. Found: C, 15.44; H, 5.82; N, 12.05%. Calc. for [Co(NH3)6]3⋅1⋅23H2O C24H119Co3N18O57Ti4: C, 14.86; H, 6.18; N, 12.99%. HRMS: m/z calc. for [Ti4O6(Hcit)3(cit)]: [M]+ 1042.76866, [M + 2Na]+2 544.37382, [M + 3H]+3 348.59701; found: [M]+ 1042.76102, [M + 2Na]+2 544.37522, [M + 3H]+3 348.60101.
2.3. Single crystal X-ray crystallography A single crystal was coated with NVH oil, attached to a 400/50 MicroMeshes™ crystal mount (MiTeGen LLC), mounted on the threecircle goniometer with χ fixed at 54.76°. Measurement was performed at room temperature. MoKα radiation (λ = 0.71073 Å) with a graphite monochromator in the incident beam was used. Crystallographic data and other pertinent information are given in Table 1. Intensity data were corrected for absorption using the face-indexed absorption correction program incorporated into Bruker-XPREP. The structures were solved and refined by the SHELXTL software package [36]. The structures were refined to convergence using anisotropic thermal parameters for all non-hydrogen atoms. In [Co(NH3)6]3⋅1⋅20H2O, position of all hydrogen atoms of the complex were introduced in calculated positions using the riding model.
2.2.2. Synthesis of Na9[Ti4O6(Hcit)3(cit)] (Na9⋅ 1) Since crystals of [Co(NH3)6]3⋅1⋅20H2O are sparingly soluble in water, an ion-exchange chromatography was used in order to exchange the counter-cation of 1 from [Co(NH3)6]3+ to Na+. A powdered sample of [Co(NH3)6]3⋅1⋅20H2O was loaded on top of a DOWEX 50 W-X8 resin (Na+ form) column and left overnight until fully adsorbed as a bright orange band. The column was washed with water and the eluent was collected and lyophilized to yield a white powder. Elemental analysis confirms a full exchange of the nitrogen containing counter-cations by Na+ cations. Complex 1 can be recovered quantitatively from solution upon addition of [Co(NH3)6]Cl3 indicating that the tetra-nuclear species is kinetically stable for an extended period of time (a few days). Anal Found: C, 23.13; H, 2.16. Calcd. For Na9⋅1⋅1.5H2O C24H22O35.5Ti4Na9: C, 22.59; H, 1.74%. 1H NMR (500 MHz, D2O): δ = 2.622 (dd; CH2). 13C NMR (125 MHz, D2O): δ = 186.9, 180.3, 178.7, 177.3, 85.5, 74.5, 45.4.
2.4. Hydrolysis Hydrolysis studies were performed using NMR to monitor the formation of hydrolysis products over a period of 7 days in a D2O solution. NMR data were recorded using Bruker Avance Neo-500 spectrometer. 2.5. Cytotoxicity Cytotoxicity was measured against the ovarian cancer cell line A2780 and drug-resistant cell lines A2780cis (cisplatin-resistant) and 2
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A2780ADR (adriamycin-resistant). Cell viability was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay [37]. Cells were cultured in an RPMI-1640 biological medium according to known procedures (supplied either by ATCC or ECACC). Drug-resistant cell lines were cultured once a week in medium containing their respective drug to preserve resistance. Approximately 0.6x106 cells were seeded in a 96-well plate and allowed to attach overnight. The cells were consequently treated with the compound tested at 10 different concentrations, in a 2-fold dilution. Solution of 1 was prepared by dissolving 56 mg of the sodium salt in 80 μL of medium 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 700 mg/L. A solution of complex 2 was prepared with concentrations of up to 300 mg/L. After 3 days of 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 on a microplate reader spectrophotometer. Each experiment was repeated at least three times on different days, with three replicates in each repetition (nine 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.
Table 2 Significant bond distances (Å) and angles (°) in complex 1. Ti(1)–O(1) Ti(1)–O(3) Ti(1)–O(4) Ti(2)–O(1) Ti(2)–O(2) Ti(2)–O(3) C(15)–O(14) C(15)–O(15) C(25)–O(24) C(25)–O(25) Ti(1)–O(1)–Ti(2) Ti(2)–O(2)–Ti(2) Ti(1)–O(3)–Ti(2) Ti(1)–O(4)–Ti(1)
1.816(2) 1.871(2) 2.004(1) 1.871(2) 1.855(1) 1.970(2) 1.177(6) 1.280(7) 1.226(5) 1.260(5) 137.3(1) 133.4(1) 131.9(1) 128.0(1)
and three oxygen atoms, belonging to one citrate ligand, in an approximate octahedral geometry. The central {Ti4O6} unit, shown in Fig. 1, possesses an adamantane-like core. Two examples of complexes featuring a {Ti4O6} core exist in the literature, an organometallic complex [Ti4O6(η5-C5Me5)] [12] and a coordination complex [Ti4O6(C6H15N3)4] [13]. Their average Ti-O bond lengths are 1.837 Å and 1.834 Å, and the average Ti-O-Ti angles are 109.5° and 125.6° respectively. In 1, the average Ti-O length is 1.898 Å and the average Ti-O-Ti angle is 132.6°. These values are more similar to those in the coordination complex [Ti4O6(C6H15N3)4] than to those in the second complex, in both bond lengths and angles. This difference most likely stems from the similar distortion of the {Ti4O6} core structure by the citrate ligands, alike the classic coordination bonding of the TACN (C6H15N3) ligand in contrast to the η5-side-on bonding by the cyclopentadienyl ligands. Each Ti(IV) atom in 1 is coordinated to a citrate ligand through one alkoxido oxygen, one COO− and one CH2COO− group. The overall charge of complex 1 is 9-, as deduced from the presence of three [Co (NH3)6]3+ cations per each anionic complex. This charge implies a complete de-protonation of one citrate ligand and a partial de-protonation of three citrates. In other words, the complex can be formulated as [(Ti4O6)4+(Hcit3−)3(cit4−)]9−. As seen in Table 2, the CeO distances in the two crystallographically independent dangling CH2COO− groups of the citrate ligands are slightly different. In one group, the CeO distances C(15)–O(14) and C(15)–O(15) are 1.177(6) and 1.280(7)Å whereas in the second group the distances of C(25)–O(24) and C(25)–O(25) are 1.226(5) and 1.260(5)Å respectively . The smaller difference in the CeO distances in the latter indicates deprotonation of the CH2COO− group, and a crystallographic disorder between COOH and COO−. An extensive network of hydrogen bonding exists in the lattice of [Co(NH3)6]3⋅1⋅20H2O involving all water molecules, ammonia ligands and the various oxygen atoms of the citrate ligands. CCDC − 1953691 contains the crystallographic data for this material. The compound Na5⋅2⋅19.5H2O contains the already known [Ti (Hcit3−)3]5− complex [23] that has been previously crystallized as the potassium salt in a triclinic system and hence, without any imposed crystallographic symmetry. The sodium salt Na5⋅2⋅19.5H2O crystallizes in the cubic space group Pa-3 and the titanium atom of 2 resides on a crystallographic three-fold axis with only one unique bi-dentate (Hcit3−) ligand. The dimensions of complex 2 were found to be practically identical with literature structure and some crystallographic data for Na5⋅2⋅19.5H2O are presented in Table 1. CCDC − 1953901 contains the crystallographic data for this material.
3. Results and discussion 3.1. Synthesis Ti(IV) cations are highly oxophilic and therefore synthesis of Ti(IV) compounds is usually hampered by hydrolysis, which leads to the formation of polynuclear products. In order to prevent hydrolysis, syntheses are usually performed in an inert atmosphere such as a Schlenk line or glovebox, where water presence is negligible. In this work however, both Ti(IV) complexes were prepared in the open in THF, in which citric acid was dissolved followed by the addition of the Ti(IV) precursor Ti (O-iPr)4 with stirring. Water was then added and the solutions become cloudy, eventually yielding a clear solution. The process was accelerated upon heating the aqueous solution to 90 °C. It was found that solutions with citric acid:Ti(IV) molar ratios of 3:1 and above may produce either complex 1 or complex 2 by adjusting the pH and the various salts were obtained by using the appropriate counter-ion. The compound [Co(NH3)6]3⋅1⋅20H2O was crystallized from a solution at pH = 6.4 upon the addition of [Co(NH3)6]3+ whereas Na5⋅2⋅19.5H2O crystallized at pH = 4.75 when sodium ions were introduced into the solution. The procedures described in the experimental section produced the highest yields and purity. Complex 1 with bridging oxido ligands is obviously a hydrolysis product, whereas in complex 2 the Ti (IV) ion is surrounded by the three bi-dentate (Hcit)3− ligands that prevent hydrolysis and formation of poly-nuclear species. 1H NMR analysis of 1 and 2 (Figs. S1 and S2) showed a double doublet of methylene groups in the citrate ligand, with different chemical shifts due to coordination to the different Ti(IV) cores. 13C NMR of 1 (Fig. S3) shows peaks attributed to bound citrate ligands, and also peaks belonging to free citrate that were not detected in 1H NMR due to low concentrations. Both compounds were therefore free of notable contaminants or side products. 3.2. Structures The structures and the numbering schemes of 1 and 2, as found in the crystals [Co(NH3)6]3⋅1⋅20H2O and Na5⋅2⋅19.5H2O respectively, are presented in Table 1. Table 2 shows selected bond lengths and angles of 1. With Z = 4 in the unit cell of space group C2/c, complex 1 is bisected by a crystallographic two-fold axis passing through two oxygen atoms, O(2) and O(4). Each Ti(IV) atom is surrounded by three oxido bridges
3.3. Cell viability studies First, to confirm that there is no change in the complex structure in water within the time frame relevant to in-vitro cytotoxicity studies 3
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Fig. 1. Left, structure and numbering scheme of 1. Right, structure and numbering scheme of the {Ti4O6} core of 1.
(72 h), the sodium salt of complex 1 (Fig. S4) was solubilized in D2O. No new peaks appeared in the 1H NMR spectra recorded, supporting high hydrolytic stability. This can arise from the already partially-hydrolyzed {Ti4O6} core, and it is therefore reasonable to assume that complex 1 does not undergo additional hydrolysis at physiological pH. Complex 2 (Fig. S5) also showed high hydrolytic stability, with no new peaks appearing during the first 72 h. A new peak (2.23 ppm) appeared in the complex spectrum after 7 days, yet peaks belonging to the bound citrate (2.67–2.93 ppm) in the complex remained unchanged. Studies on the speciation of Ti(IV) citrate compounds [22,24] show that Ti(cit)3 species crystallize at near-physiological pH from water, implying some hydrolytic stability as well. The cytotoxicity of the complexes was measured based on the MTT assay on three human ovarian cancer cell lines: sensitive A2780, cisplatin-resistant A2780cis and multi-drug-resistant A2780ADR. Relative IC50 and growth inhibition values for compounds Na91 and Na52 and known complexes (bzac)2Ti(O-iPr)2 and Cp2TiCl2 are summarized in Table 3. Cytotoxicity plots are given in Fig. S6. The sodium salt of 1 showed low cytotoxicity compared with other Ti(IV) compounds such as budotitane or Ti(IV) amino phenolato complexes [3,34], as indicative by the high IC50 of 420 μM toward the parent line; yet 1 still inhibited cell viability to 18% or lower in A2780 cell lines. Complex 2 did not show significant activity. Drug-resistant cell lines were used to attempt to discern mechanistic similarity to either cisplatin or adriamycin. Complex 1 showed similar IC50 values towards A2780 and the adriamycin-resistant cell line A2780ADR, yet
the cisplatin-resistant cell line A2780cis was significantly less affected showing a higher IC50. This is a first observation of a cytotoxic Ti(IV) complex affected by cisplatin-resistance mechanisms [3,39,40], the reason for which is yet unclear [15,23,24]. The relatively low cytotoxic activity of complexes 1 and 2 can be attributed to their high negative charge that, along with their large overall size, decreases their ability to penetrate through membranes, or may relate to binding of the Ti(IV) cations to human serum transferrin [18]. In order to test the dependence of cytotoxicity on charge, the known neutral Ti(IV) citrate complex [Ti8O10(cit)4]0 [26] was prepared, yet it was practically insoluble in water and therefore could not be examined for potential cytotoxic activity. 4. Conclusions In this study we present the preparation and structure of a novel tetranuclear Ti(IV) citrate complex [Ti4O6(Hcit)3(cit)]9− 1, featuring an adamantane-like {Ti4O6} core, and the crystal structure of the sodium salt of the mono-nuclear titanium(IV) citrate [Ti(Hcit)3]5− 2. Since 1 and 2 are both hydrolytically stable and water-soluble, we examined their potential cytotoxicity and compared them with other known active reagents. Complex 1 showed slight cytotoxicity towards ovarian cancer cells, although not sufficient for potential applicability. Complex 2 did not exhibit any cytotoxicity. Lack of cytotoxic activity can be attributed to possible binding to transferrin or inability to pass the cell membrane due to high negative charge or large size. Future studies with other physiological ligands may be valuable for the development of stable, soluble, and potentially also cytotoxic – Ti(IV) compounds.
Table 3 Relative IC50 (μM) and Maximal Cell Growth Inhibition (%) Valuesa for A2780 cells.b. Compound
A2780
A2780cis
A2780ADR
Na9[Ti4O6(Hcit)3(cit)] (Na91) Na5[Ti(Hcit)3] (Na52) (bzac)2Ti(O-iPr)2 [38]
420 ± 60 (7%) Negligiblec 10.3 ± 0.2 (26%) 977 ± 1 (17%)
1300 ± 200 (18%) –d 7.5 ± 0.2 (18%) 811 ± 1 (3%)
420 ± 90 (16%) –d –
Cp2TiCl2 [38]
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
–
Acknowledgments
a Maximal cell growth inhibition refers to the % inhibition recorded at the highest compound concentration tested, relative to untreated control. b Error values are based on standard deviations. c Activity does not reach 50% inhibition. d Was not tested on resistant cell lies due to lack of activity on the parent line.
We are grateful to Dr. Shmuel Cohen and Dr. Benny Bogoslavsky for the crystallographic measurements. Funding was received from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681243). 4
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Appendix A. Supplementary data [22]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ica.2020.119429. References
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5
Structural effects of titanium citrate on the human erythrocyte membrane, J. Inorg. Biochem. 99 (2005) 764–770. P. Panagiotidis, E.T. Kefalas, C.P. Raptopoulou, A. Terzis, T. Mavromoustakos, A. Salifoglou, Delving into the complex picture of Ti(IV)–citrate speciation in aqueous media: synthetic, structural, and electrochemical considerations in mononuclear Ti(IV) complexes containing variably deprotonated citrate ligands, Inorganica Chim. Acta. 361 (2008) 2210–2224. Y.-F. Deng, Y.-Q. Jiang, Q.-M. Hong, Z.-H. Zhou, Speciation of water-soluble titanium citrate: synthesis, structural, spectroscopic properties and biological relevance, Polyhedron 26 (2006) 1561–1569. E.T. Kefalas, P. Panagiotidis, C.P. Raptopoulou, A. Terzis, T. Mavromoustakos, A. Salifoglou, Mononuclear titanium(IV)−citrate complexes from aqueous solutions: pH-specific synthesis and structural and spectroscopic studies in relevance to aqueous titanium(IV)−citrate speciation, Inorg. Chem. 44 (2005) 2596–2605. J.M. Collins, R. Uppal, C.D. Incarvito, A.M. Valentine, Titanium(IV) citrate speciation and structure under environmentally and biologically relevant conditions, Inorg. Chem. 44 (2005) 3431–3440. T. Kemmitt, N.I. Al-Salim, G.J. Gainsford, A. Bubendorfer, M. Waterland, Unprecedented oxo-titanium citrate complex precipitated from aqueous citrate solutions, exhibiting a novel bilayered Ti8O10 structural core, Inorg. Chem. 43 (2004) 6300–6306. S. Frühauf, W.J. Zeller, New platinum, titanium, and ruthenium complexes with different patterns of DNA damage in rat ovarian tumor cells, Cancer Res. 51 (1991) 2943–2948. M.J. Clarke, F. Zhu, D.R. Frasca, Non-platinum chemotherapeutic metallopharmaceuticals, Chem. Rev. 99 (1999) 2511–2534. F. Caruso, L. Massa, A. Gindulyte, C. Pettinari, F. Marchetti, R. Pettinari, M. Ricciutelli, J. Costamagna, J.C. Canales, J. Tanski, M. Rossi, (4-Acyl-5-pyrazolonato)titanium derivatives: oligomerization, hydrolysis, voltammetry, and DFT study, Eur. J. Inorg. Chem. 2003 (2003) 3221–3232. F. Caruso, M. Rossi, J. Tanski, R. Sartori, R. Sariego, S. Moya, S. Diez, E. Navarrete, A. Cingolani, F. Marchetti, C. Pettinari, Synthesis, structure, and antitumor activity of a novel tetranuclear titanium complex, J. Med. Chem. 43 (2000) 3665–3670. F. Caruso, M. Rossi, Antitumor titanium compounds, Mini-Rev. Med. Chem. 4 (2004) 49–60. M. Shavit, D. Peri, C.M. Manna, J.S. Alexander, E.Y. Tshuva, Active cytotoxic reagents based on non-metallocene non-diketonato well-defined C2-symmetrical titanium complexes of tetradentate bis(phenolato) ligands, J. Am. Chem. Soc. 129 (2007) 12098–12099. N. Ganot, O. Briaitbard, A. Gammal, J. Tam, J. Hochman, E.Y. Tshuva, In vivo anticancer activity of a nontoxic inert phenolato titanium complex: high efficacy on solid tumors alone and combined with platinum drugs, ChemMedChem 13 (2018) 2290–2296. S. Meker, O. Braitbard, M.D. Hall, J. Hochman, E.Y. Tshuva, Specific design of titanium(IV) phenolato chelates yields stable and accessible, effective and selective anticancer agents, Chem. - A Eur. J. 22 (2016) 9849. SMART-NT V5.6, BRUKER AXS GMBH, D-76181 Karlsruhe, Germany, 2002. SHELXTL-NT V6.1, BRUKER AXS GMBH, D-76181 Karlsruhe, Germany, 2002. N. Ganot, S. Meker, L. Reytman, A. Tzubery, E.Y. Tshuva, Anticancer metal complexes: synthesis and cytotoxicity evaluation by the MTT assay, J. Vis. Exp. (2013) e50767. C.M. Manna, O. Braitbard, E. Weiss, J. Hochman, E.Y. Tshuva, Cytotoxic salantitanium(IV) complexes: high activity toward a range of sensitive and drug-resistant cell lines, and mechanistic insights, ChemMedChem 7 (2012) 703–708. M. Miller, E.Y. Tshuva, Racemic vs. enantiopure inert Ti(IV) complex of a single diaminotetrakis(phenolato) ligand in anticancer activity toward human drug-sensitive and -resistant cancer cell lines, RSC Adv. 8 (2018) 39731–39734. M. Miller, E.Y. Tshuva, Synthesis of pure enantiomers of titanium(IV) complexes with chiral diaminobis(phenolato) ligands and their biological reactivity, Sci. Rep. 8 (2018) 9705.
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Research paper
Preparation, structural characterization and cytotoxicity of hydrolytically stable Ti(IV) citrate complexes ⁎,1
Nathan Engelberg, Avi Bino
, Edit Y. Tshuva
T
⁎,1
Institute of Chemistry, The Hebrew University of Jerusalem, 9190401 Jerusalem, Israel
ABSTRACT
Hydrolytically stable and water soluble Ti(IV) complexes can be used for various applications, including industrial, medicinal and more. Herein, a novel tetra-nuclear Ti(IV) complex with citrate ligands was obtained by reacting Ti(IV) isopropoxide and citric acid in an aqueous solution. The three dimensional structure of the complex [Ti4O6(Hcit)3(cit)]9− 1, as it appears in the compound [Co(NH3)6]3[Ti4O6(Hcit)3(cit)]⋅20H2O was determined by single-crystal X-ray crystallography. Complex 1 possesses an adamantane-like {Ti4O6} core where each metal atom is coordinated to a tri-dentate citrate ligand with one dangling CH2COOH group. An additional already known Ti(IV) complex [Ti(Hcit3−)3]5− 2 was synthesized for comparison. The high hydrolytic stability of complexes 1 and 2 has been substantiated based on NMR techniques. The cytotoxicity of the complexes was tested on three cancer cell lines: human ovarian A2780 and drug-resistant variants A2780cis and A2780ADR. Complex 1 showed a slight cytotoxicity towards the ovarian cells, while complex 2 was inactive.
1. Introduction Titanium is commonly used, either as a metal or in its complexed form, in different fields such as material science and medicine, including for surgical equipment and prosthetics and for development of anti-cancer agents [1]. The low intrinsic toxicity of titanium makes it an attractive metal for use in biological systems and several titanium compounds were found to be bioactive [2]. However, due to its highly oxophilic nature, Ti(IV) is prone to fast hydrolytic processes when exposed to aqueous environments [3–5]. Hydrolysis of Ti(IV) produces numerous polynuclear Ti(IV) oxide species, which have been thoroughly studied for their potential as functional materials [6]. Preparation is usually performed under inert water-free conditions, with a few successes in aqueous environments to produce titanium oxides of large nuclearity (TixOy, x > 7) [7–11]. Titanium oxides with a nuclearity of x = 4 mostly contain either a {Ti4O4} core [11], or an adamantanelike {Ti4O6} [12,13] core. Similar {M4O6} cores have been previously observed with other metal-ions as well (M = Mn(IV), Ru(IV)) [14,15]. In order to enhance the hydrolytic stability of these complexes and enabling activity in aqueous solutions, citrate ligands are used to bind Ti(IV) and form relatively stable water soluble species. In the Pechini method to produce a high quality barium titanate ceramic, a Ti(IV) alkoxide precursor is added to an aqueous solution of citric acid, followed by the addition of barium salts [16]. Due to the abundance of titanium from prosthetics and food additives and citrate ions in the human body, the interactions between them have been of great interest. Citrate has been found to leach solid titanium and solubilize it as ions
⁎ 1
and assisting in regulating Ti(IV) concentration in the blood stream [17]. It has also been shown that a siderophore protein transferrin binds Ti(IV) using citrate as a co-anion [18-20]. Research of the bioactivity of Ti(IV) citrate showed modulation of the structure of human erythrocyte membrane [21]. The speciation of Ti(IV) citrate complexes has been the subject of numerous studies and several species were structurally characterized. Among them a series of mononuclear complexes with the general formula [Ti(Hxcit)3]n− x = 0–2 and n = 2–8 depending on citrate protonation [22–25], and an octanuclear complex [Ti8O10(cit)4]0 [26]. Several Ti(IV) complexes were found to be bioactive, with marked anti-cancer activity against a variety of cancer cell lines. Initial studies on budotitane ((bzac)2Ti(OEt)2) and titanocene dichloride (Cp2TiCl2) showed low effectivity due to their rapid hydrolysis upon exposure to aqueous solutions [27–31]. Later developments of Ti(IV) complexes utilizing aminephenlato-type ligands with different denticities [32] increased the hydrolytic stability, and thereafter showed significantly higher anti-cancer activity both in-vitro and in-vivo [33,34]. However, the introduction of relatively hydrophobic ligands into the complexes significantly decreased their water solubility. Herein we aimed at obtaining new hydrolytically stable and water soluble Ti(IV) complexes for anticancer medicinal applications. We thus report the preparation and structure of a novel tetra-Ti(IV) citrate complex, namely, [Ti4O6(Hcit)3(cit)]9− 1, featuring a tetranuclear adamantane-like core {Ti4O6}. We also report the structure of the sodium salt of the already known [Ti(Hcit)3]5− 2 complex. The hydrolytic stability of these complexes was examined using 1H NMR spectra of
Corresponding authors. Both authors contributed equally to this work.
https://doi.org/10.1016/j.ica.2020.119429 Received 6 October 2019; Received in revised form 8 January 2020; Accepted 8 January 2020 Available online 09 January 2020 0020-1693/ © 2020 Elsevier B.V. All rights reserved.
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D2O solutions, and the anti-cancer activity of the two complexes was tested in-vitro towards the human ovarian cancer cell line A2780 and drug-resistant variants A2780cis and A2780ADR.
Table 1 Crystal data and structure refinement parameters for [Co(NH3)6]3⋅1⋅20H2O and Na5⋅2⋅19.5H2O.
2. Experimental
Empirical formula Formula weight Temperature K Wavelength Å Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Z ρcalc (g cm−3) µ (mm−1) Crystal size (mm3) Range of θ (°) Total reflections Independent reflection (Rint) Parameters R1 [I > 2sigma(I)] wR2 (I)
2.1. Reagents and measurements All chemicals and solvents were purchased from commercial sources and were used without further purification. Ti(IV) tetraisopropoxide (98%) was purchased from Acros Organics, citric acid anhydrous (99.5%) was purchased from Sigma-Aldritch, tetrahydrofuran (99.8%) was purchased from Bio-Lab, sodium p-toluenesulfonate was purchased from Sigma-Aldritch, hexaamminecobalt(III) chloride was purchased from Alfa Aesar, sodium chloride was purchased from Frutarom. All reactions were carried out at room temperature and ambient conditions unless stated otherwise. Quantitative elemental analysis of C, H, N atom weight content was performed by the HUJI lab for microanalysis using a Thermo Flash 2000 CHN-O Elemental Analyzer. 1H NMR and 13C NMR spectra were obtained using a Bruker Avance Neo-500 spectrometer. Mass spectrometry was performed by the HUJI lab for microanalysis using Agilent 6520 QTOF analyzer. Data were collected on a Bruker SMART APEX CCD X-ray diffractometer system controlled by a pentium-based PC running the SMART software package [35]. Cytotoxicity measurements were performed on the A2780 cancer cell line obtained from ATCC and the A2780cis and A2780ADR cell lines obtained from ECACC. Cell viability was assessed using a Tecan Spark 10 M microplate reader spectrophotometer.
[Co(NH3)6]3⋅1⋅20H2O
Na5⋅2⋅19.5H2O
C24H70Co3N18O54Ti4 1843.37 293(2) 0.71073 Monoclinic C2/c
C18H51Na5O39.50Ti 1062.44 294(1) 0.71073 Cubic Pa-3
23.799(4) 18.965(3) 18.185(3) 90 106.348(2) 90 7876(2) 4 1.555 1.112 0.46 × 0.38 × 0.19 2.15–28.09 43,966 9436 (0.0565) 493 0.0512 0.1576
20.476(1) 20.476(1) 20.476(1) 90 90 90 8581(1) 8 1.644 0.369 0.25 × 0.18 × 0.14 2.22–24.98 75,445 2519 (0.0704) 205 0.0733 0.1542
2.2.3. Preparation of Na5[Ti(Hcit)3]⋅19.5H2O (Na5⋅2⋅19·.5H2O) Ti(OiPr)4 (0.074 mL, 0.25 mmol) was reacted with citric acid C6H8O7 (0.960 g, 5 mmol) dissolved in 2 mL THF, resulting in a cloudy solution. Water (8 mL) was added and the mixture was stirred for 1 h at 90 °C. After cooling to room temperature, the pH was adjusted to 4.75 by adding 4 M NaOH solution, followed by the addition of sodium ptoluensulfonate (CH3C6H4SO3Na) (0.291 g, 1.5 mmol). The solvent was evaporated slowly to yield colorless crystals after 7 days. The crystals were filtered, washed with cold water and air-dried. Yield 0.264 g (98%). Anal. Found: C, 20.27; H, 4.97%. Calc. for C18H54Na5O39.5Ti: C, 20.29; H, 5.11%. 1H-NMR (500 MHz, D2O): δ = 2.752 (dd; CH2), δ = 2.926 (d, CH2).
2.2. Preparation of Ti(IV) citrate complexes. 2.2.1. Preparation of [Co(NH3)6]3[Ti4O6(Hcit)3(cit)]⋅20H2O ([Co (NH3)6]3⋅1⋅20H2O) Ti(OiPr)4 (0.740 mL, 2.5 mmol) was reacted with citric acid C6H8O7 (2.401 g, 12.5 mmol) dissolved in 20 mL THF. Water (80 mL) was added and the solution was stirred for 1 h at 90 °C. After cooling to room temperature, the pH was adjusted to 6.40 by the addition of a 4 M NaOH solution, followed by the addition of [Co(NH3)6]Cl3 (0.668 g, 2.5 mmol). The solvent was evaporated slowly to yield orange crystals after three weeks. The crystals were filtered, washed with cold water and air-dried. The calculated values suggest the presence of some adsorbed water molecules that could not be removed by air-drying. Organic solvents or high vacuum lead to the loss of lattice water molecules and crystal disintegration. Yield 0.943 g (82%). Anal. Found: C, 15.44; H, 5.82; N, 12.05%. Calc. for [Co(NH3)6]3⋅1⋅23H2O C24H119Co3N18O57Ti4: C, 14.86; H, 6.18; N, 12.99%. HRMS: m/z calc. for [Ti4O6(Hcit)3(cit)]: [M]+ 1042.76866, [M + 2Na]+2 544.37382, [M + 3H]+3 348.59701; found: [M]+ 1042.76102, [M + 2Na]+2 544.37522, [M + 3H]+3 348.60101.
2.3. Single crystal X-ray crystallography A single crystal was coated with NVH oil, attached to a 400/50 MicroMeshes™ crystal mount (MiTeGen LLC), mounted on the threecircle goniometer with χ fixed at 54.76°. Measurement was performed at room temperature. MoKα radiation (λ = 0.71073 Å) with a graphite monochromator in the incident beam was used. Crystallographic data and other pertinent information are given in Table 1. Intensity data were corrected for absorption using the face-indexed absorption correction program incorporated into Bruker-XPREP. The structures were solved and refined by the SHELXTL software package [36]. The structures were refined to convergence using anisotropic thermal parameters for all non-hydrogen atoms. In [Co(NH3)6]3⋅1⋅20H2O, position of all hydrogen atoms of the complex were introduced in calculated positions using the riding model.
2.2.2. Synthesis of Na9[Ti4O6(Hcit)3(cit)] (Na9⋅ 1) Since crystals of [Co(NH3)6]3⋅1⋅20H2O are sparingly soluble in water, an ion-exchange chromatography was used in order to exchange the counter-cation of 1 from [Co(NH3)6]3+ to Na+. A powdered sample of [Co(NH3)6]3⋅1⋅20H2O was loaded on top of a DOWEX 50 W-X8 resin (Na+ form) column and left overnight until fully adsorbed as a bright orange band. The column was washed with water and the eluent was collected and lyophilized to yield a white powder. Elemental analysis confirms a full exchange of the nitrogen containing counter-cations by Na+ cations. Complex 1 can be recovered quantitatively from solution upon addition of [Co(NH3)6]Cl3 indicating that the tetra-nuclear species is kinetically stable for an extended period of time (a few days). Anal Found: C, 23.13; H, 2.16. Calcd. For Na9⋅1⋅1.5H2O C24H22O35.5Ti4Na9: C, 22.59; H, 1.74%. 1H NMR (500 MHz, D2O): δ = 2.622 (dd; CH2). 13C NMR (125 MHz, D2O): δ = 186.9, 180.3, 178.7, 177.3, 85.5, 74.5, 45.4.
2.4. Hydrolysis Hydrolysis studies were performed using NMR to monitor the formation of hydrolysis products over a period of 7 days in a D2O solution. NMR data were recorded using Bruker Avance Neo-500 spectrometer. 2.5. Cytotoxicity Cytotoxicity was measured against the ovarian cancer cell line A2780 and drug-resistant cell lines A2780cis (cisplatin-resistant) and 2
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A2780ADR (adriamycin-resistant). Cell viability was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay [37]. Cells were cultured in an RPMI-1640 biological medium according to known procedures (supplied either by ATCC or ECACC). Drug-resistant cell lines were cultured once a week in medium containing their respective drug to preserve resistance. Approximately 0.6x106 cells were seeded in a 96-well plate and allowed to attach overnight. The cells were consequently treated with the compound tested at 10 different concentrations, in a 2-fold dilution. Solution of 1 was prepared by dissolving 56 mg of the sodium salt in 80 μL of medium 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 700 mg/L. A solution of complex 2 was prepared with concentrations of up to 300 mg/L. After 3 days of 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 on a microplate reader spectrophotometer. Each experiment was repeated at least three times on different days, with three replicates in each repetition (nine 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.
Table 2 Significant bond distances (Å) and angles (°) in complex 1. Ti(1)–O(1) Ti(1)–O(3) Ti(1)–O(4) Ti(2)–O(1) Ti(2)–O(2) Ti(2)–O(3) C(15)–O(14) C(15)–O(15) C(25)–O(24) C(25)–O(25) Ti(1)–O(1)–Ti(2) Ti(2)–O(2)–Ti(2) Ti(1)–O(3)–Ti(2) Ti(1)–O(4)–Ti(1)
1.816(2) 1.871(2) 2.004(1) 1.871(2) 1.855(1) 1.970(2) 1.177(6) 1.280(7) 1.226(5) 1.260(5) 137.3(1) 133.4(1) 131.9(1) 128.0(1)
and three oxygen atoms, belonging to one citrate ligand, in an approximate octahedral geometry. The central {Ti4O6} unit, shown in Fig. 1, possesses an adamantane-like core. Two examples of complexes featuring a {Ti4O6} core exist in the literature, an organometallic complex [Ti4O6(η5-C5Me5)] [12] and a coordination complex [Ti4O6(C6H15N3)4] [13]. Their average Ti-O bond lengths are 1.837 Å and 1.834 Å, and the average Ti-O-Ti angles are 109.5° and 125.6° respectively. In 1, the average Ti-O length is 1.898 Å and the average Ti-O-Ti angle is 132.6°. These values are more similar to those in the coordination complex [Ti4O6(C6H15N3)4] than to those in the second complex, in both bond lengths and angles. This difference most likely stems from the similar distortion of the {Ti4O6} core structure by the citrate ligands, alike the classic coordination bonding of the TACN (C6H15N3) ligand in contrast to the η5-side-on bonding by the cyclopentadienyl ligands. Each Ti(IV) atom in 1 is coordinated to a citrate ligand through one alkoxido oxygen, one COO− and one CH2COO− group. The overall charge of complex 1 is 9-, as deduced from the presence of three [Co (NH3)6]3+ cations per each anionic complex. This charge implies a complete de-protonation of one citrate ligand and a partial de-protonation of three citrates. In other words, the complex can be formulated as [(Ti4O6)4+(Hcit3−)3(cit4−)]9−. As seen in Table 2, the CeO distances in the two crystallographically independent dangling CH2COO− groups of the citrate ligands are slightly different. In one group, the CeO distances C(15)–O(14) and C(15)–O(15) are 1.177(6) and 1.280(7)Å whereas in the second group the distances of C(25)–O(24) and C(25)–O(25) are 1.226(5) and 1.260(5)Å respectively . The smaller difference in the CeO distances in the latter indicates deprotonation of the CH2COO− group, and a crystallographic disorder between COOH and COO−. An extensive network of hydrogen bonding exists in the lattice of [Co(NH3)6]3⋅1⋅20H2O involving all water molecules, ammonia ligands and the various oxygen atoms of the citrate ligands. CCDC − 1953691 contains the crystallographic data for this material. The compound Na5⋅2⋅19.5H2O contains the already known [Ti (Hcit3−)3]5− complex [23] that has been previously crystallized as the potassium salt in a triclinic system and hence, without any imposed crystallographic symmetry. The sodium salt Na5⋅2⋅19.5H2O crystallizes in the cubic space group Pa-3 and the titanium atom of 2 resides on a crystallographic three-fold axis with only one unique bi-dentate (Hcit3−) ligand. The dimensions of complex 2 were found to be practically identical with literature structure and some crystallographic data for Na5⋅2⋅19.5H2O are presented in Table 1. CCDC − 1953901 contains the crystallographic data for this material.
3. Results and discussion 3.1. Synthesis Ti(IV) cations are highly oxophilic and therefore synthesis of Ti(IV) compounds is usually hampered by hydrolysis, which leads to the formation of polynuclear products. In order to prevent hydrolysis, syntheses are usually performed in an inert atmosphere such as a Schlenk line or glovebox, where water presence is negligible. In this work however, both Ti(IV) complexes were prepared in the open in THF, in which citric acid was dissolved followed by the addition of the Ti(IV) precursor Ti (O-iPr)4 with stirring. Water was then added and the solutions become cloudy, eventually yielding a clear solution. The process was accelerated upon heating the aqueous solution to 90 °C. It was found that solutions with citric acid:Ti(IV) molar ratios of 3:1 and above may produce either complex 1 or complex 2 by adjusting the pH and the various salts were obtained by using the appropriate counter-ion. The compound [Co(NH3)6]3⋅1⋅20H2O was crystallized from a solution at pH = 6.4 upon the addition of [Co(NH3)6]3+ whereas Na5⋅2⋅19.5H2O crystallized at pH = 4.75 when sodium ions were introduced into the solution. The procedures described in the experimental section produced the highest yields and purity. Complex 1 with bridging oxido ligands is obviously a hydrolysis product, whereas in complex 2 the Ti (IV) ion is surrounded by the three bi-dentate (Hcit)3− ligands that prevent hydrolysis and formation of poly-nuclear species. 1H NMR analysis of 1 and 2 (Figs. S1 and S2) showed a double doublet of methylene groups in the citrate ligand, with different chemical shifts due to coordination to the different Ti(IV) cores. 13C NMR of 1 (Fig. S3) shows peaks attributed to bound citrate ligands, and also peaks belonging to free citrate that were not detected in 1H NMR due to low concentrations. Both compounds were therefore free of notable contaminants or side products. 3.2. Structures The structures and the numbering schemes of 1 and 2, as found in the crystals [Co(NH3)6]3⋅1⋅20H2O and Na5⋅2⋅19.5H2O respectively, are presented in Table 1. Table 2 shows selected bond lengths and angles of 1. With Z = 4 in the unit cell of space group C2/c, complex 1 is bisected by a crystallographic two-fold axis passing through two oxygen atoms, O(2) and O(4). Each Ti(IV) atom is surrounded by three oxido bridges
3.3. Cell viability studies First, to confirm that there is no change in the complex structure in water within the time frame relevant to in-vitro cytotoxicity studies 3
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Fig. 1. Left, structure and numbering scheme of 1. Right, structure and numbering scheme of the {Ti4O6} core of 1.
(72 h), the sodium salt of complex 1 (Fig. S4) was solubilized in D2O. No new peaks appeared in the 1H NMR spectra recorded, supporting high hydrolytic stability. This can arise from the already partially-hydrolyzed {Ti4O6} core, and it is therefore reasonable to assume that complex 1 does not undergo additional hydrolysis at physiological pH. Complex 2 (Fig. S5) also showed high hydrolytic stability, with no new peaks appearing during the first 72 h. A new peak (2.23 ppm) appeared in the complex spectrum after 7 days, yet peaks belonging to the bound citrate (2.67–2.93 ppm) in the complex remained unchanged. Studies on the speciation of Ti(IV) citrate compounds [22,24] show that Ti(cit)3 species crystallize at near-physiological pH from water, implying some hydrolytic stability as well. The cytotoxicity of the complexes was measured based on the MTT assay on three human ovarian cancer cell lines: sensitive A2780, cisplatin-resistant A2780cis and multi-drug-resistant A2780ADR. Relative IC50 and growth inhibition values for compounds Na91 and Na52 and known complexes (bzac)2Ti(O-iPr)2 and Cp2TiCl2 are summarized in Table 3. Cytotoxicity plots are given in Fig. S6. The sodium salt of 1 showed low cytotoxicity compared with other Ti(IV) compounds such as budotitane or Ti(IV) amino phenolato complexes [3,34], as indicative by the high IC50 of 420 μM toward the parent line; yet 1 still inhibited cell viability to 18% or lower in A2780 cell lines. Complex 2 did not show significant activity. Drug-resistant cell lines were used to attempt to discern mechanistic similarity to either cisplatin or adriamycin. Complex 1 showed similar IC50 values towards A2780 and the adriamycin-resistant cell line A2780ADR, yet
the cisplatin-resistant cell line A2780cis was significantly less affected showing a higher IC50. This is a first observation of a cytotoxic Ti(IV) complex affected by cisplatin-resistance mechanisms [3,39,40], the reason for which is yet unclear [15,23,24]. The relatively low cytotoxic activity of complexes 1 and 2 can be attributed to their high negative charge that, along with their large overall size, decreases their ability to penetrate through membranes, or may relate to binding of the Ti(IV) cations to human serum transferrin [18]. In order to test the dependence of cytotoxicity on charge, the known neutral Ti(IV) citrate complex [Ti8O10(cit)4]0 [26] was prepared, yet it was practically insoluble in water and therefore could not be examined for potential cytotoxic activity. 4. Conclusions In this study we present the preparation and structure of a novel tetranuclear Ti(IV) citrate complex [Ti4O6(Hcit)3(cit)]9− 1, featuring an adamantane-like {Ti4O6} core, and the crystal structure of the sodium salt of the mono-nuclear titanium(IV) citrate [Ti(Hcit)3]5− 2. Since 1 and 2 are both hydrolytically stable and water-soluble, we examined their potential cytotoxicity and compared them with other known active reagents. Complex 1 showed slight cytotoxicity towards ovarian cancer cells, although not sufficient for potential applicability. Complex 2 did not exhibit any cytotoxicity. Lack of cytotoxic activity can be attributed to possible binding to transferrin or inability to pass the cell membrane due to high negative charge or large size. Future studies with other physiological ligands may be valuable for the development of stable, soluble, and potentially also cytotoxic – Ti(IV) compounds.
Table 3 Relative IC50 (μM) and Maximal Cell Growth Inhibition (%) Valuesa for A2780 cells.b. Compound
A2780
A2780cis
A2780ADR
Na9[Ti4O6(Hcit)3(cit)] (Na91) Na5[Ti(Hcit)3] (Na52) (bzac)2Ti(O-iPr)2 [38]
420 ± 60 (7%) Negligiblec 10.3 ± 0.2 (26%) 977 ± 1 (17%)
1300 ± 200 (18%) –d 7.5 ± 0.2 (18%) 811 ± 1 (3%)
420 ± 90 (16%) –d –
Cp2TiCl2 [38]
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
–
Acknowledgments
a Maximal cell growth inhibition refers to the % inhibition recorded at the highest compound concentration tested, relative to untreated control. b Error values are based on standard deviations. c Activity does not reach 50% inhibition. d Was not tested on resistant cell lies due to lack of activity on the parent line.
We are grateful to Dr. Shmuel Cohen and Dr. Benny Bogoslavsky for the crystallographic measurements. Funding was received from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681243). 4
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Appendix A. Supplementary data [22]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ica.2020.119429. References
[23]
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