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Synthesis, cytotoxic and hydrolytic studies of titanium complexes anchored by a tripodal diamine bis( phenolate) ligand† Sónia Barroso,*a Ana M. Coelho,a Santiago Gómez-Ruiz,*b Maria José Calhorda,c Željko Žižak,d Goran N. Kaluđeroviće and Ana M. Martinsa The reactivity, cytotoxic studies and hydrolytic behaviour of diamine bis(phenolate) titanium complexes are reported. The reactions of [Ti(tBu2O2NN’)Cl]2(µ-O) (1) with LiOiPr or HOiPr in the presence of NEt3, aiming at the synthesis of the alkoxido derivative of 1 led to no reaction or to the synthesis of the monomeric complex [Ti(tBu2O2NN’)(OiPr)2] (3), respectively. A small amount of the alkoxidotitanium dimer [Ti(tBu2O2NN’)(OiPr)]2(μ-O) (2) crystallized out of a solution of 3 and DFT calculations showed that the transformation of 1 into 3 is a thermodynamically favorable process in the presence of a base (NEt3) (ΔG = −14.7 kcal mol−1). 2 was quantitatively obtained through the direct reaction of the ligand precursor H2(tBu2O2NN’) with titanium tetra(isopropoxido). Further reaction of 2 with an excess of TMSCl was revealed to be the most suitable method for the preparation of [Ti(tBu2O2NN’)Cl2] (4). 1 and 3 disclosed cytotoxic activity towards HeLa, Fem-x, MDA-MB-361 and K562 cells and 1 exhibited moderate binding affinity to

Received 2nd April 2014, Accepted 19th September 2014

FS-DNA. 1H NMR hydrolysis studies attested the fast decomposition of 4 in the presence of D2O. The hydrolysis of 3 is slower and proceeds through the formation of [Ti(tBu2O2NN’)(OH)]2(µ-O) (5) that was

DOI: 10.1039/c4dt00975d

crystallographically characterized. Upon D2O addition 1 immediately forms complex new species, stable

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in solution for long periods (weeks).

1.

Introduction

Cisplatin was one of first inorganic complexes exhibiting cytotoxic activity and is considered one of the most efficient drugs for the treatment of certain types of cancer.1 However, due to its toxicity and resistance, there has been growing interest in the development of alternative non-toxic anti-cancer drugs. In this respect, titanium complexes have the advantage of being

a Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. E-mail: [email protected], [email protected] b Departamento de Química Inorgánica y Analítica, Escuela Superior de Ciencias Experimentales y Tecnología, Universidad Rey Juan Carlos, Calle Tulipán s/n, 28933 Móstoles, Madrid, Spain c Departamento de Química e Bioquímica, CQB, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Ed. C8, 1749-016 Lisboa, Portugal d Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia e Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany † Electronic supplementary information (ESI) available: Detailed ESI-MS data for compounds 3, 4 and 5, tables with atomic coordinates of the optimized molecules 1*, 3*, 5*, HOiPr, Net3, NEt3·HCl and H2O and detailed X-ray data for compound 4. Data for structures 2, 4 and 5 were deposited at the CCDC. CCDC 994885–994887. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt00975d

17422 | Dalton Trans., 2014, 43, 17422–17433

relatively biologically compatible and this circumstance led to the investigation of their biological properties.2 Titanocene dichloride (Scheme 1a), budotitane (Scheme 1b) and their derivatives2,3 showed promising activity towards cisplatinsensitive and resistant tumor cells but their hydrolytic instability limited their applicability and further investigation about the nature of the active species.4 Tshuva and co-workers have reported cytotoxic studies on alkoxidotitanium(IV) compounds supported by tripodal

Scheme 1

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Scheme 2

ESI-MS spectra were acquired on a Bruker HCT mass spectrometer operated in the positive and negative ion modes. The optimized operating parameters were: solution flow rate, 2.5 μL min−1; capillary voltage, ±4 kV; skimmer voltage, ±40 V; capillary exit voltage, ±129 V; nebulizer gas pressure, 8 psi; dry gas flow rate, 4 L min−1; dry gas temperature, 250 °C. The spectra were recorded in the range 100–1500 Da. Spectra typically correspond to the average of 20–35 scans. 2.2.

(Scheme 1d) and salan-type (Scheme 1c) diamine bis( phenolate) ligands.5 The two families of complexes were found to have cytotoxic activity against colon HT-29 and ovarian OVCAR-1 cancer cell lines. However, in both cases, complexes having bulky tert-butyl substituents in the phenolate groups presented negligible activity or were inactive against the cancer cell lines studied. The study of transition metal complexes supported by a tripodal diamine bis( phenolate) ligand bearing tert-butyl substituents in the phenolate group6 led some of us to explore radical reactions of titanium(III) diamine bis( phenolate) complexes of the general formula [Ti(tBu2O2NN′)Cl(S)] (S = THF, py; tBu2 O2NN′ = Me2N(CH2)2N(CH2-2-O-3,5-tBu2C6H2)2). Complex [Ti(tBu2O2NN′)Cl]2(μ-O), obtained from the reaction of [Ti(tBu2O2NN′)Cl(S)] (S = THF, py) with O2, is one of the few examples of diamine bis( phenolate) μ-oxo-complexes (Scheme 2).6f Having in mind the high stability of this compound in the presence of air and moisture, we became interested in its cytotoxic properties and carried out a study that extends the cytotoxic properties of titanium complexes to tripodal tBu2O2NN′ derivatives.

2. Experimental procedures 2.1.

General

All preparations and subsequent manipulations of air/moisture sensitive compounds were performed under a nitrogen atmosphere using standard Schlenk line and glovebox techniques. CH2Cl2 and Et2O were dried by standard methods (sodium/benzophenone for Et2O and calcium hydride for CH2Cl2) and distilled prior to use. C6D6 was dried over Na and distilled under reduced pressure. THF-d8 was dried with 4 Å molecular sieves and degassed by the freeze–pump–thaw method. Unless stated otherwise, all reagents were purchased from commercial suppliers (e.g. Aldrich, Acrös, Fluka) and used as received. H2(tBu2O2NN′)7 was prepared according to literature procedures and [Ti(tBu2O2NN′)Cl]2(µ-O) (1) was prepared as previously described by us.6f NMR spectra were recorded on a Bruker Advance II+ 300 MHz (UltraShield Magnet) instrument at ambient temperature. 1H and 13C chemical shifts (δ) are expressed in ppm relative to Me4Si. Carbon, hydrogen and nitrogen analyses were performed in-house using an EA110 CE Instruments automatic analyser. The results presented are, in general, the average of two independent measurements.

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Synthetic procedures

2.2.1. [Ti(tBu2O2NN′)(OiPr)]2(μ-O), 2. Compound 2 is an intermediate product of the alcoholysis of 1 that gives 3 as the main product. A small amount of yellow crystals of 2 was obtained from the slow evaporation of a toluene solution of 3 (see below) in air. Although the characterization of 2 is limited to X-ray diffraction, the data are included because they confirm the role of the compound in the overall reaction. 2.2.2. Synthesis of complex [Ti(tBu2O2NN′)(OiPr)2], tBu2 3. Method 1 – A solution of H2( O2NN′) (1.57 g, 3 mmol) in Et2O was slowly added to a 1 M solution of Ti(OiPr)4 (3 mL, 3 mmol) in the same solvent. The solution was stirred for 2 h at room temperature. Evaporation of all the volatiles under reduced pressure led to a yellow solid. Yield: 1.86 g, 96%. Method 2 – Triethylamine (0.22 mL, 1.600 mmol) was added to a solution of [Ti(tBu2O2NN′)Cl]2(µ-O) (1.00 g, 0.814 mmol) in isopropanol and the mixture was stirred for 48 h. The yellow solution obtained was evaporated to dryness, and the residue was extracted in Et2O and filtered. Evaporation of the Et2O solution under vacuum led to a yellow powder. Yield: 0.85 g, 75%. 1H NMR (300 MHz, C6D6, ppm): δ 7.58 (d, 2H, 4JHH = 2.3 Hz, p-CH-Ar), 7.06 (d, 2H, 4JHH = 2.1 Hz, o-CH-Ar), 5.23 (sept, 3 JHH = 5.9 Hz, 1H, CH(CH3)2), 4.81 (sept, 3JHH = 5.9 Hz, 1H, CH(CH3)2), 4.24 (d, 2H, 2JHH = 13.1 Hz, NCH2Ar), 3.12 (d, 2H, 2 JHH = 13.1 Hz, NCH2Ar), 2.28 (m, 2H, NCH2CH2NMe2), 2.09 (s, 6H, N(CH3)2), 1.81 (m, 2H, NCH2CH2NMe2), 1.76 (s, 18H, C(CH3)3), 1.50 (d, 3JHH = 6.0 Hz, 6H, CH(CH3)2), 1.43 (s, 18H, C(CH3)3), 0.98 (d, 3JHH = 6.0 Hz, 6H, CH(CH3)2). 13C-{1H} NMR (75 MHz, C6D6, ppm): δ 161.2, 138.9, 135.2 and 124.6 (Cipso-Ar), 124.1 and 124.0 (CH-Ar), 77.5 and 77.4 (CH(CH3)2), 65.2 (NCH2Ar), 58.4 (NCH2CH2NMe2), 52.4 (NCH2CH2NMe2), 49.8 (N(CH3)2), 35.6 and 34.3 (C(CH3)3), 32.1 and 31.0 (C(CH3)3), 27.2 and 26.3 (CH(CH3)2). EA calculated for C40H68N2O4Ti·1.5(C3H8O): C, 68.61; H, 10.35; N, 3.60. Found: C, 68.27; H, 9.86; N, 3.90. ESI-MS (CH3CN): m/z = 629.5 ([Ti(tBu2O2NN′)(OiPr)]+). 2.2.3. Synthesis of complex [Ti(tBu2O2NN′)Cl2], 4. TMSCl (0.84 mL, 6.6 mmol) was added to a solution of 3 (1.86 g, 2.9 mmol) in CH2Cl2 and the mixture was stirred for 2 h at room temperature. All the volatiles were removed under reduced pressure. The dark orange residue was extracted with toluene and this solution was evaporated to dryness under vacuum to give a reddish-orange powder. Yield: 1.60 g, 89%. Dark orange crystals suitable for X-ray diffraction were obtained from toluene at −4 °C. 1H NMR (300 MHz, C6D6, ppm): δ 7.59 (d, 2H, 4JHH = 2.3 Hz, p-CH-Ar), 6.97 (d, 2H, 4JHH = 2.3 Hz, o-CH-Ar), 4.35 (d, 2JHH = 13.8 Hz, 2H, NCH2Ar), 2.79 (d, 2 JHH = 13.3 Hz, 2H, NCH2Ar), 2.14 (m, 2H, CH2), 1.94 (s, 6H,

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Table 1

Selected crystallographic experimental data and structure refinement parameters for 2, 4 and 5

Empirical formula Formula weight Temperature (K) Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Z, ρcalc. (g cm−3) μ (mm−1) Crystal size Crystal colour Crystal shape Refl. collected Unique refl. [R(int)] R1 [I > 2σ(I)] wR2 [I > 2σ(I)] GooF

2

4

5

C74H122N4O7Ti2 1275.56 150(2) Monoclinic C2/c 21.183(3) 10.885(1) 32.283(4) 90 102.157(8) 90 7276.8(17) 4, 1.164 0.272 0.40 × 0.40 × 0.20 Yellow Prism 40 097 6454 [0.0522] 0.064 0.1572 1.049

C34H54Cl2N2O2Ti 641.59 150(2) Monoclinic P21/c 16.7000(11) 15.6390(10) 15.1740(10) 90 115.821(2) 90 3567.3(4) 4, 1.195 0.420 0.18 × 0.18 × 0.04 Orange Plate 15 392 6294 [0.0811] 0.0590 0.1093 0.906

C68H110N4O7Ti2·4(C4H8O) 1479.82 150(2) Monoclinic P21/c 13.080(2) 13.894(3) 23.193(4) 90 95.620(9) 90 4194.7(12) 2, 1.172 0.248 0.30 × 0.20 × 0.04 Yellow Plate 31 375 7447 [0.1430] 0.0693 0.1282 0.926

N(CH3)2), 1.80 (s, 18H, C(CH3)3), 1.61 (m, 2H, CH2), 1.39 (s, 18H, C(CH3)3). 13C-{1H} NMR (75 MHz, C6D6, ppm): δ 160.2, 143.6, 136.7 and 127.6 (Cipso-Ar), 125.0 ( p-CH-Ar), 124.4 (o-CH-Ar), 67.2 (NCH2Ar), 60.6 (CH2), 54.3 (CH2), 51.3 (N(CH3)2), 36.1 and 35.0 (C(CH3)3), 32.2 and 31.4 (C(CH3)3). EA calculated for C34H54Cl2N2O2Ti·0.5(C7H8): C, 65.51; H, 8.50; N, 4.07. Found: C, 65.47; H, 8.67; N, 3.83. ESI-MS (THF): m/z = 675.8 ([Ti(tBu2O2NN′)Cl3]−). 2.2.4. [Ti(tBu2O2NN′)(OH)]2(μ-O), 5. A small amount of yellow crystals of 5 was obtained from the slow evaporation of a THF-d8/D2O solution of 3 (THF-d8 : D2O ≈ 9 : 1) in air. 5 is an intermediate product of the hydrolysis of 3. 1H NMR (300 MHz, THF-d8, ppm): δ 7.11 (s, 4H, p-CH-Ar), 7.84 (s, 4H, o-CH-Ar), 5.12 (d, 2JHH = 13.3 Hz, 4H, NCH2Ar), 3.18 (d, 2JHH = 13.8 Hz, 4H, NCH2Ar), 2.60 (m, 4H, CH2), 2.25 (s, 12H, N(CH3)2), 1.44 (m, 4H, CH2), 1.22 (s, 36H, C(CH3)3), 1.10 (s, 36H, C(CH3)3). 13C-{1H} NMR (75 MHz, THF-d8, ppm): δ 154.1, 140.9, 136.3 and 123.2 (Cipso-Ar), 125.7 ( p-CH-Ar), 123.6 (o-CH-Ar), 66.4 (NCH2Ar), 57.1 (CH2), 49.8 (CH2), 45.1 (N(CH3)2), 35.6 and 34.7 (C(CH3)3), 32.1 and 30.1 (C(CH3)3). EA calculated for C68H110N4O7Ti2·4.5(HOiPr): C, 66.96; H, 10.07; N, 3.83. Found: C, 66.77; H, 9.74; N, 4.01. ESI-MS (CH2Cl2/CH3CN): m/z = 1174.3 ([(Ti(tBu2O2NN′)(OH))-µ-O-(Ti(tBu2O2NN′))]+). 2.3.

General procedures for X-ray crystallography

Crystals suitable for single-crystal X-ray analysis were obtained for 2, 4 and 5 as described in the synthetic procedures. The data were collected using graphite monochromated Mo-Kα radiation (α = 0.71073 Å) on a Bruker AXS-KAPPA APEX II diffractometer equipped with an Oxford Cryosystem open-flow nitrogen cryostat. Cell parameters were retrieved using Bruker SMART software and refined using Bruker SAINT on all observed reflections. Absorption corrections were applied using SADABS.8 The structures were solved and refined using

17424 | Dalton Trans., 2014, 43, 17422–17433

direct methods with the program SIR2004 9 using WINGXVersion 1.80.01 10 SHELXL11 system of programs. All nonhydrogen atoms were refined anisotropically and the hydrogen atoms were inserted in idealized positions and allowed to refine riding on the parent carbon atom. The molecular diagrams were drawn with ORTEP-3 for Windows12 included in the software package. For crystallographic experimental data and structure refinement parameters see Table 1. Data for structures 2, 4 and 5 are deposited at the CCDC under the deposit numbers CCDC 994885–994887.

2.4.

Computational details 13

DFT calculations for 1*, 3*, 5*, HOiPr, Net3, NEt3·HCl and H2O were performed using the GAUSSIAN 03 software package14 and the PBE1PBE functional, without symmetry constraints. That functional uses a hybrid generalized gradient approximation (GGA), including a 25% mixture of Hartree– Fock15 exchange with DFT13 exchange correlation, given by the Perdew, Burke, and Ernzerhof functional (PBE).16 The optimized geometries were obtained with a standard 6-31G(d,p)17 basis set for all elements (basis b1). The electronic energies obtained were converted to standard free Gibbs energies at 298.15 K using zero point energy and thermal energy corrections based on structural and vibrational frequency data calculated at the PBE1PBE/b1 level of theory. Single point energy calculations were performed using an improved basis set (basis b2) and the geometries were optimized at the PBE1BPE/b1 level. Basis b2 consisted of a standard 6-311++G(d,p).18 Solvent effects (HOiPr) were considered in specified PBE1BPE/b2// PBE1BPE/b1 energy calculations, using the polarizable continuum model (PCM) initially devised by Tomasi and coworkers19 as implemented in Gaussian 03.20 The molecular cavity was based on the united atom topological model

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applied on UAHF radii, optimized for the HF/6-31G(d) level. Three-dimensional structures were obtained with Chemcraft.21

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2.5.

Cytotoxicity and DNA-binding studies

2.5.1. Preparation of drug solutions. Stock solutions of the studied titanium complexes were made in dimethyl sulfoxide (DMSO) at a concentration of 20 mM, filtered through a Millipore filter (0.22 μm) before use, and diluted with nutrient medium to various working concentrations. DMSO was used due to solubility problems. RPMI 1640 nutrient medium was used, without phenol red, supplemented with L-glutamine (3 mM), streptomycin (100 µg mL−1), and penicillin (100 IU mL−1), 10% fetal bovine serum (FBS) and 25 mM Hepes, and was adjusted to pH = 7.2 with bicarbonate solution. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was dissolved (5 mg mL−1) in phosphate buffered saline pH = 7.2, and filtered through a Millipore filter (0.22 μm) before use. 2.5.2. Cell culture. Human cervix adenocarcinoma HeLa, malignant melanoma Fem-x and human breast carcinoma MDA-MB-361 cells were cultured as monolayers in the nutrient medium, while human myelogenous leukemia K562 cells were maintained as suspension culture. The cells were grown at 37 °C under a 5% CO2 and humidified air atmosphere. For the growth of MDA-MB-361 cells and all subsequent experiments, the complete medium was enriched with 1.11 g L−1 glucose. 2.5.3. Cell sensitivity analysis. HeLa, Fem-x (2000 cells per well) and MDA-MB-361 cells (10 000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after the cell adherence, five different concentrations of the studied compounds were added to the wells. The final concentrations were in the range of 12.5 to 200 μM. The studied compounds were added to a suspension of leukemia K562 cells (3000 cells per well) 2 h after cell seeding, in the same final concentrations applied to HeLa and Fem-x cells. All experiments were carried out in triple triplicate. Only nutrient medium was added to the cells in the control wells. Nutrient medium with the corresponding concentrations of compounds, but void of cells, was used as the blank. All survival points were done in triplicate and experiments were conducted three times. Dose–response curves were plotted (values expressed as the percentage of control optical density, see details in the ESI†) and IC50 values (50% inhibitory concentration) were estimated by regression analysis with the Microsoft EXCEL (2003) software. 2.5.4. Determination of target cell survival. Cell survival was determined by the MTT test according to the method of Mosmann22 and modified by Ohno and Abe,23 72 hours after drug addition. 20 µL of MTT solution (5 mg mL−1 in phosphate buffered saline) was added to each well. Samples were incubated for a further four hours at 37 °C under a humidified atmosphere with 5% CO2. Then, 100 µL of 10% SDS was added to the wells. The absorbance was measured at 570 nm the next day. To obtain cell survival percentages, the absorbance at 570 nm of a sample with cells grown in the presence of various concentrations of the agent was divided by the absorbance of the control sample (the absorbance of cells grown only in

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nutrient medium), having subtracted the absorbance of the blank from the absorbance of the corresponding sample with target cells. 2.5.5. DNA-binding experiments monitored by UV-Vis spectroscopy. Fish sperm DNA (FS-DNA) was kindly provided by Departamento de Ciencias de la Salud from Universidad Rey Juan Carlos (Spain). The spectroscopic titration of FS-DNA was carried out in the buffer (50 mM NaCl – 5 mM Tris-HCl, pH = 7.1) at room temperature. A solution of FS-DNA in the buffer gave a ratio of UV absorbance 1.8–1.9 : 1 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein.24 MilliQ water was used to prepare the solutions. The DNA concentration per nucleotide was determined by absorption spectroscopy using the known molar extinction coefficient value of 6600 M−1 cm−1 at 260 nm.25 Absorption titrations were performed at 260 nm using a fixed concentration of complex 3 (in DMSO) to which increments of the DNA stock solution were added. Complex-DNA adduct solutions were incubated at 37 °C for 30 minutes before the absorption spectra were recorded. UV-Vis measurements were performed at room temperature with an Analytik Jena Specord 200 spectrophotometer between 190 and 900 nm.

3. Results and discussion 3.1.

Synthesis and characterization of the complexes

As previously reported,6f [Ti(tBu2O2NN′)Cl]2(µ-O) (1) is prepared in high yield by reacting titanium(III) complexes [Ti(tBu2O2NN′)Cl(S)] (S = THF, py) with oxygen in THF solutions. 1 is a very stable complex, not susceptible to hydrolysis in air although it reacts with water when it is added to THF solutions, as described below. Aiming at the preparation of an alkoxido derivative of [Ti(tBu2O2NN′)Cl]2(µ-O), the reaction of 1 with 2 equiv. of LiOiPr in toluene was attempted. However, no reaction occurred, even when the mixture was heated to 60 °C. In a second attempt, 1 was dissolved in isopropanol in the presence of triethylamine at room temperature. Surprisingly, the product obtained was the monomer [Ti(tBu2O2NN′)(OiPr)2] (3) that formed as a bright yellow solid in 75% yield (Scheme 3). The formation of 3 probably resulted from the reaction of [Ti(tBu2O2NN′)(OiPr)]2(μ-O) (2) with an excess of isopropanol and triethylamine, which are essential for the outcome of the reaction. Indeed, a small amount of yellow crystals of [Ti(tBu2O2NN′)(OiPr)]2(μ-O) (2) grew from a toluene solution of 3, indicating that some amount of 2, which is likely to have a higher crystallinity than 3, was still present in solution (see Scheme 1). Alternatively, compound 3 was quantitatively obtained through the direct reaction of the ligand precursor H2(tBu2O2NN′) with titanium tetra(isopropoxido) as previously reported by Kol and co-workers.26 The NMR data of 3 are consistent with a formulation of two isopropoxido groups and one tBu2 O2NN′ ligand per titanium centre and are in agreement with those reported by Kol and co-workers. As mentioned above, crystals of [Ti(tBu2O2NN′)(OiPr)]2(μ-O) (2) grew from a toluene solution of 3. An ORTEP view of 2 is

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Scheme 3

Table 2

Selected structural parameters for 2

Distances (Å) Ti(1)–O(1) Ti(1)–O(2) Ti(1)–O(3) Ti(1)-eq. planea Angles (°) O(1)–Ti(1)–O(2) O(1)–Ti(1)–O(4) O(1)–Ti(1)–N(1) O(1)–Ti(1)–N(2) O(3)–Ti(1)–O(4) O(3)–Ti(1)–O(1) O(3)–Ti(1)–O(2) O(4)–Ti(1)–O(2) θb

1.910(2) 1.915(2) 1.827(1) 0.236(1)

Ti(1)–O(4) Ti(1)–N(1) Ti(1)–N(2)

1.833(2) 2.279(3) 2.451(3)

164.0(1) 94.1(1) 84.1(1) 84.2(1) 104.9(1) 94.9(1) 95.9(1) 94.4(1) 154.5(1)

O(3)–Ti(1)–N(1) O(4)–Ti(1)–N(1) O(2)–Ti(1)–N(1) O(3)–Ti(1)–N(2) O(4)–Ti(1)–N(2) O(2)–Ti(1)–N(2) N(1)–Ti(1)–N(2) Ti(1)–O(3)–Ti(1)#

88.8(1) 166.4(1) 84.5(1) 162.8(1) 92.3(1) 82.0(1) 74.1(1) 180.0(0)

The equatorial plane is defined by atoms O1, O2, O3 and N2. b θ is the dihedral angle between the planes containing the phenolate rings.

a

Fig. 1 ORTEP-3 diagram of [Ti(tBu2O2NN’)(OiPr)]2(μ-O) (2), using 30% probability level ellipsoids. The equivalent atoms labelled with # are generated using the symmetry transformations −x + 1, −y + 1, −z. Hydrogen and disordered carbon atoms are omitted for clarity.

depicted in Fig. 1 and selected bond distances and angles are listed in Table 2. The compound crystallizes in the monoclinic system, space group C2/c, with a half molecule in the asymmetric unit. The structure features an oxygen-bridged dinuc-

17426 | Dalton Trans., 2014, 43, 17422–17433

lear titanium(IV) complex with slightly distorted octahedral geometry around each metal centre. The equatorial planes are defined by O1, O2 and N2 of the diamine bis( phenolate) ligand and the bridging oxygen O3, while the axial positions are occupied by the tripodal nitrogen N1 and atom O4 of the isopropoxido ligand. The two sides of the molecule are structurally related across a linear oxido bridge by a rotation of 180°. The overall bond distances and angles compare well to those

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found for 1 6f and for other titanium(IV) diamine bis( phenolate) complexes.27–29 The linearity of the Ti–O–Ti fragment is in agreement with a sp oxygen atom and a Ti–O multiple bond with a significant π contribution from the oxygen. The Ti–(μ-O) distance and Ti–(μ-O)–Ti angle compare well with the values found for 1 6f and with those reported for the analogous μ-oxido titanium(IV) complex [Ti(tBu2O2NN′)(OEt)]2(μ-O) (1.81 Å and 177°, respectively).27 The phenolate groups are in the trans configuration defining a dihedral angle of 154.5(1)° between the planes containing the phenolate rings. The Ti1–O4 bond length of the alkoxido ligand [1.833(2) Å] is significantly shorter than the Ti–O bond lengths for the phenoxido ligands [1.910(2) and 1.915(2)°] probably due to more pronounced oxygen-to-metal π-donation of the isopropoxido ligand. All attempts to obtain suitable crystals of 3 for X-ray diffraction failed. Thus, a geometry optimization at the PBE1PBE/ 6-31G** level was carried out in order to complement the experimental results. A model of 3 (3*), with tBu groups replaced by Me, is depicted in Fig. 2. The geometry optimization was performed only for the trans-phenolate isomers in accordance with the NMR data of 3, which reveals a Cs-symmetric species. Among the diamine-bis(phenolate) family of compounds these isomers are always more stable than the cis-phenolate analogues often leading to the formation of one single isomer.6b,f The structural calculated parameters are in good agreement with those found in 2 and reported for analogous bis(isopropoxido) titanium(IV) diamine bis(phenolate) complexes.5c,7,30 The reaction of 3 with an excess of TMSCl led to formation of [Ti(tBu2O2NN′)Cl2] (4) as a dark orange powder in 89% yield. Compound 4 had been previously reported by us as one of the products of the reaction of the titanium(III) complex [Ti(tBu2O2NN′)Cl(THF)] with [Cp2Fe][BPh4].6f Furthermore, the reaction of TiCl4(THF)2 with a related amine bis( phenolate) having a sidearm THF donor also gave a mixture of two products that were not fully characterized.7 The preparation of 4 directly from the reaction of [TiCl2(acac)2] with H2(tBu2O2NN′)

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Scheme 4

in THF was also attempted but a mixture of products was obtained. The latter reactions do not constitute efficient methods for the preparation of 4 since they do not provide a pure compound and proceed with low and irreproducible yields. The reaction of TMSCl with 3 thus revealed the most suitable method for the preparation of [Ti(tBu2O2NN′)Cl2] (4). Orange crystals of 4 suitable for X-ray diffraction were grown from a toluene–hexane double layer solution. The compound obtained under these conditions crystallizes in the monoclinic system, space group P21/a, in contrast to the tetragonal system, space group P4/nnc, found when crystals of 4 are grown from a C6D6 solution.6f The X-ray data for the monoclinic system crystallized 4 can be found in the ESI.† The conversion of 1 in 3 through the cleavage of the oxygen bridge is unexpected since Ti–(µ-oxido)–Ti bonds are very stable and frequently the result of the hydrolysis of titanium complexes. Thus, DFT calculations were performed in order to help understand this transformation. The overall reaction A shown in Scheme 4 (values in kcal mol−1) is thermodynamically unfavourable (ΔG = 29.7 kcal mol−1), which is in agreement with the experimental observation that the reaction does not occur in the absence of a base. If triethylamine is considered in the overall reaction (B in Scheme 4), the Gibbs free energy value slightly decreases (ΔG = 21.8 kcal mol−1) but the reaction is still unfavourable. When solvent effects (HOiPr) are considered, the overall reaction B becomes favourable, with a free Gibbs energy of −14.7 kcal mol−1. The results point out that the driving force for the reaction is the presence of triethylamine to neutralise the HCl formed and the large excess of HOiPr. 3.2.

Fig. 2 DFT optimized structure of 3*. Selected bond lengths (Å) and angles (°): Ti1–O1 1.89, Ti1–O2 1.89, Ti1–O3 1.81, Ti1–O4 1.82, Ti1–N1 2.37, Ti1–N2 2.44, O1–Ti1–O2 163.3, N1–Ti1–N2 74.3, O3–Ti1–O4 106.7.

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Cytotoxicity studies

The cytotoxic effects of complexes [Ti(tBu2O2NN′)Cl]2(µ-O) (1) and [Ti(tBu2O2NN′)(OiPr)2] (3) were measured on human cervical carcinoma HeLa cells, human melanoma Fem-x cells, human breast cancer MDA-MB-361 cells and human chronic myelogenous leukemia K562 cells. Cisplatin effects on the same cell lines were measured for comparison. The results are presented in Table 3. Complexes 1 and 3 exhibited moderate cytotoxic effects against all tested human cancer cells. Estimations based on the IC50 values show similar cytotoxic activity for both complexes. Complex 1 seems to be slightly more active against HeLa and MDA-MB-361 cells while complex 3 presents more activity against Fem-x cells, however the IC50 values are in the

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Table 3 Concentrations of complexes 1 and 3 that induced 50% decrease (IC50) in malignant cell survival

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IC50 ± SDa (μM) Cell line

Cisplatin

1

3

HeLa Fem-x MDA-MB-361 K562

4.4 ± 0.3 4.7 ± 0.3 13.0 ± 1.7 5.7 ± 0.3

22.4 ± 1.2 36.4 ± 2.8 37.7 ± 1.5 33.2 ± 1.5

32.9 ± 0.4 27.1 ± 3.4 48.9 ± 0.8 32.8 ± 11.5

a

From three independent experiments.

same order of magnitude being difficult to highlight one of the compounds to the other in each cancer cell line. Although the IC50 values measured for complexes 1 and 3 are higher than the values measured for cisplatin, they are in the range found by Tshuva and co-workers for analogous compounds in other cell lines (HT-29 and OVCAR-1).5c In addition, compound 3, which is active against all tested cell lines, was found to be inactive against HT-29 and OVCAR-1 cells used by Tshuva.5c Additional studies such as DNA-laddering or apoptosis tests need to be carried out, in order to gain more insight into the anticancer mechanisms of action of the studied complexes on the cancer cells investigated in this work. This mechanism is unknown in detail up to now, although it seems that these complexes may follow similar pathways to those described for the anticancer mechanism of action of titanocene(IV) complexes,3c which include the proposal that titanium may reach the cells assisted by the major iron transport protein “transferrin”,31 binding to DNA and leading to cell death.32 Absorption spectroscopy is one of the most useful techniques to study the binding of any drug to DNA.33 Thus the binding of 1 to the DNA helix was followed by absorption spectral titrations, recording the absorption spectra of the complex in the absence and in the presence of FS-DNA (fish sperm DNA). The increasing concentration of FS-DNA resulted in hyperchromism and a slight blue shift of the LMCT (ligand-tometal charge-transfer) absorption band of complex 1 at ca. 260 nm. Fig. 3 shows the absorption spectra of complex 1 in the presence of increasing amounts of FS-DNA. Complex 1, or most likely its hydrolysis product, may bind to DNA in different modes on the basis of its structure, charge in the medium and type of ligand. DNA has several hydrogen bonding sites in the minor and major grooves and complex 1 contains N-donor atoms that may facilitate hydrogen bonding.34 In addition, complex 1 contains small phenolate rings, which can induce classical intercalations. The hyperchromism effect shown in the present study suggests that hydrophobic associations of aromatic rings with the hydrophobic interior of DNA may also be possible, as well as classical electrostatic interactions. The binding strength of the complex was evaluated by the calculation of the intrinsic binding constant, Kb, which was determined using eqn (1).35 ½DNA ½DNA 1 ¼ þ εa  εf ε0  εf K b ðε0  εf Þ

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ð1Þ

Fig. 3 Absorption spectra of 1 in the presence of increasing amounts of DNA, [complex] = 2.5 µM, [DNA] = 0.75–3 µM. The arrow indicates that the absorbance changes upon increasing DNA concentrations. Inset: plot of eqn (1), experimental data points; solid line, linear fitting of the data.

[DNA] is the concentration of DNA in base pairs, εa, εf and ε0 correspond to Aobs/[complex], the extinction coefficient of the free titanium complex and the extinction coefficient of the complex in the fully bound form, respectively, and Kb is the intrinsic binding constant. The ratio of slope to intercept in the plot of [DNA]/(εa − εf ) versus [DNA] gives the value of Kb (inset Fig. 3). Thus, the intrinsic binding constant of 3.09 × 105 M−1 for 1 was successfully calculated and indicates moderate affinity to DNA. 3.3.

Hydrolysis studies

Since the DNA-binding study involves the addition of DNA aqueous solutions to DMSO aqueous solutions of titanium complex 1, and the cytotoxic activity is determined in aqueous medium over a long period, it is sensible to assume that the results obtained are related to the hydrolysis product(s) of complex 1. Thus, UV/Vis and NMR measurements upon water addition were monitored over time for several hours to investigate the hydrolytic behaviour of complex 1 in order to assess its stability under biologically relevant conditions. UV-Vis absorption spectra of a solution of complex 1 in DMSO, to which 10% water was added, taken during 7 days, are presented in Fig. 4. A slight shift of the LMCT band of the Ti–OAr ligand (from ca. 400 nm to 350 nm) is observed after the addition of water to the solution (no decay to zero is observed) indicating the formation of a new species with a bound bis( phenolate) chelating ligand.5 The regular decay in the absorbance up to 7 days after addition of water indicates that the decomposition of the new species formed is very slow. To gain more information on species that may form in the presence of water, the hydrolytic behaviour of complexes 1, 3 and 4 was followed by 1H NMR upon addition of D2O to a d8THF solution of the complex (THF-d8 : D2O ≈ 9 : 1). 1H NMR measurements were performed every hour for several hours. The results of the hydrolysis of complex 1 (Fig. 5) show that new species form immediately after the addition of D2O to 1. The multiple signals of the new species formed are very broad

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Fig. 4 UV-Vis absorption over time for 1 (in DMSO) upon addition of water.

and small indicating that no defined species is formed at that point. On the other hand, no immediate hydrolysis with ligand release occurs. Only 1 h after the addition of D2O did small signals of the free ligand start to appear. The shift to low field observed for some of the ligand signals, namely the signals due to ArCH2N, NCH2CH2N and NMe2 protons, is most probably due to the protonation of the NMe2 group. Actually, the fast hydrolysis of the chloride ligands leads to the release of HCl to the medium and concomitant protonation of the amine(s). After 10 h a considerable amount of free ligand is present in solution and a white precipitate, possibly TiO2, was visible in the NMR tube. However, even 72 h after the addition of D2O only small peaks characteristic of the free ligand are visible, accompanied by several new resonances that reveal the presence of new species. The complicated spectrum of the

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latter may correspond to polymetallic structures with bridging oxygens of the type [Tin(tBu2O2NN′)n(µ2-O)n] related to the [Ti3(L)3(µ2-O)3] cluster obtained by Tshuva and co-workers when studying the hydrolytic behaviour of [Ti(L)(OiPr)2] complexes having diamine bis( phenolate) ligands of the SALAN type.5b A 1H NMR measurement performed 3 weeks later revealed the same composition. These results are consistent with those obtained by UV-Vis spectroscopy as they indicate the formation of new species immediately after the addition of D2O to 1 that are then stable in solution for long periods (weeks). In the case of complex 4 (Fig. 6), immediate hydrolysis and free ligand release is observed. Similar to what happens in the case of complex 1, there is also a significant shift to low field of the signals of the ArCH2N, NCH2CH2N and NMe2 protons. In this case the shift of those signals is even larger than observed for complex 1 probably due to release of two equiv. of HCl per titanium centre. The hydrolysis of complex 3 (Fig. 7) follows a different path. A new species forms immediately after the addition of D2O to 3. As expected, the isopropoxido ligands are the first to hydrolyse and immediate formation of isopropanol is observed. The new species formed presents an AB system for the methylenic ArCH2N protons at similar chemical shifts to the ones observed for the dimeric complex 1 suggesting a similar O-bridged species. 1 h after the addition of D2O to 3 there is a 50 : 50 mixture of that new species and free ligand and after 10 h all the ligands were released. Despite the relatively fast decomposition of 3 a small amount of yellow crystals that formed in the NMR tube walls could be isolated and analysed by X-ray diffraction. The molecular structure obtained,

Fig. 5 1H NMR spectra of (a) 1; (b) 1 + D2O, immediately after D2O addition; (c) 1 + D2O, 10 h after D2O addition; (d) 1 + D2O, 3 weeks after D2O addition; and (e) ligand precursor + D2O, in THF-d8. * and Δ denote the residual THF and H2O, respectively.

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Fig. 6 1H NMR spectra of (a) 4; (b) 4 + D2O, immediately after D2O addition; (c) 4 + D2O, 10 h after D2O addition; and (d) ligand precursor + D2O in THF-d8. *, Δ and ● denote the residual THF, H2O and toluene, respectively.

Fig. 7 1H NMR spectra of (a) 3; (b) 3 + D2O, immediately after D2O addition; (c) 3 + D2O, 10 h after D2O addition; and (d) ligand precursor + D2O in THF-d8. * and Δ denote the residual THF and H2O, respectively.

depicted in Fig. 8, is the oxo-bridged dimer [Ti(tBu2O2NN′)(OH)]2(µ-O) (5) that corresponds, most probably, to the intermediate species formed immediately following the addition of D2O to 3 (Scheme 5). DFT calculations have shown that the formation of the oxygen bridged dimer 5 and four molecules of HOiPr from the reaction of two molecules of 3 with three molecules of H2O is a favourable process from the thermodynamic point of view, corresponding to a global ΔG = −7.0 kcal mol−1. Compound 5 crystallises in the monoclinic system, space group P21/c. Selected bond distances and angles are listed in

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Table 4. The structure is very similar to that described above for 2 featuring an oxygen-bridged dinuclear titanium(IV) complex with slightly distorted octahedral geometry around each metal centre. The equatorial planes are defined by O1, O2 and N2 of the diamine bis( phenolate) ligand and the bridging oxygen O3, while the axial positions are occupied by the tripodal nitrogen N1 and atom O4 of the hydroxido ligand. The overall bond distances and angles compare well to those found for 1 6f and 2 and for other titanium(IV) diamine bis( phenolate) complexes such as [Ti(Cl2O2NN′)(CH2Ph)2].6c,f,7,27–29 The Ti–

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Fig. 8 Left: ORTEP-3 diagram of [Ti(tBu2O2NN’)(OH)]2(μ-O) (5), using 30% probability level ellipsoids. The equivalent atoms labelled with # are generated using the symmetry transformations −x, −y + 1, −z + 1. Hydrogen atoms and disordered carbon atoms are omitted for clarity. Right: wireframe diagram showing 5 and hydrogen-bonded THF.

characterized titanium complexes with terminal hydroxido groups restricted mostly to those supported by cyclopentadienyl ligands,36 an example with a chelating bis( phosphine oxide) ligand37 and an example with a tripodal amine tris ( phenolate) ligand.38 The Ti1–O4 bond length of the hydroxido ligand [1.819(3) Å] compares well with the value reported for the tripodal amine tris( phenolate) complex.38 The crystal structure shows that the complex contains two terminal hydroxido groups that form hydrogen bonds with co-crystallized THF (d(O4⋯OTHF) = 2.752(5) Å). This is the first crystallographically characterized Ti–(µ-O)–Ti complex with terminal hydroxido ligands.

Scheme 5

Table 4 Selected structural parameters for 5

Distances (Å) Ti(1)–O(1) Ti(1)–O(2) Ti(1)–O(3) Ti(1)-eq. planea

1.904(2) 1.890(2) 1.811(1) 0.221(1)

Ti(1)–O(4) Ti(1)–N(1) Ti(1)–N(2)

1.819(3) 2.288(3) 2.429(3)

Angles (°) O(1)–Ti(1)–O(2) O(1)–Ti(1)–O(4) O(1)–Ti(1)–N(1) O(1)–Ti(1)–N(2) O(3)–Ti(1)–O(4) O(3)–Ti(1)–O(1) O(3)–Ti(1)–O(2) O(4)–Ti(1)–O(2) θb

164.4(1) 94.7(1) 84.1(1) 86.0(1) 104.9(1) 96.4(1) 93.7(1) 94.2(1) 154.3(2)

O(3)–Ti(1)–N(1) O(4)–Ti(1)–N(1) O(2)–Ti(1)–N(1) O(3)–Ti(1)–N(2) O(4)–Ti(1)–N(2) O(2)–Ti(1)–N(2) N(1)–Ti(1)–N(2) Ti(1)–O(3)–Ti(1)#

90.4(1) 164.7(1) 84.0(1) 164.4(1) 90.3(1) 81.2(1) 74.5(1) 180.0(0)

The equatorial plane is defined by atoms O1, O2, O3 and N2. b θ is the dihedral angle between the planes containing the phenolate rings.

a

(μ-O) distance and the Ti–(μ-O)–Ti angle are analogous to the values found for 1 6f and 2 and for the analogous μ-oxido titanium(IV) complex [Ti(tBu2O2NN′)(OEt)]2(μ-O).27 The phenolate groups are in the trans configuration as in 2 defining a dihedral angle of 154.3(2)° between the planes containing the phenolate rings. There are few examples of crystallographically

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4.

Conclusions

Both [Ti(tBu2O2NN′)Cl]2(µ-O) and [Ti(tBu2O2NN′)(OiPr)2] revealed cytotoxic activity towards HeLa, Fem-x, MDA-MB-361 and K562 cells, with IC50 ranging between ∼22 and 49 μM and [Ti(tBu2O2NN′)Cl]2(µ-O) showed moderate binding affinity to FS-DNA (k = 3.09 × 105 M−1). The hydrolysis of [Ti(tBu2O2NN′)(OiPr)2] originated [Ti(tBu2O2NN′)(OH)]2(µ-O). Although a few crystals of the latter compound were obtained and its molecular structure determined by X-ray diffraction, [Ti(tBu2O2NN′)(OH)]2(µ-O) is not stable and suffers from further hydrolysis that is accompanied by ligand release. A different result was obtained from the reaction of [Ti(tBu2O2NN′)Cl]2(µ-O) with water. In this case, immediate conversion to complex new species, which are stable in solution for long periods (weeks), was observed. Those may correspond to poly-metallic structures with bridging oxygens of the type [Tin(tBu2O2NN′)n(µ2-O)n] that are likely related to the cytotoxic species. Despite the structural analogy between [Ti(tBu2O2NN′)Cl]2(µ-O) and [Ti(tBu2O2NN′)(OH)]2(µ-O) they follow different hydrolysis paths

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which may be attributed to the formation of HCl in the first case. The synthesis of [Ti(tBu2O2NN′)(OiPr)]2(μ-O) by reaction of [Ti(tBu2O2NN′)Cl]2(µ-O) with LiOiPr was unsuccessful while the dissolution of [Ti(tBu2O2NN′)Cl]2(µ-O) in HOiPr led to the cleavage of the oxygen bridge with formation of the monomeric complex [Ti(tBu2O2NN′)(OiPr)2]. DFT calculations showed that this process is thermodynamically favorable only in the presence of a base and a large excess of HOiPr (ΔG = −14.7 kcal mol−1). However, a few crystals of [Ti(tBu2O2NN′)(OiPr)]2(μ-O) were obtained from the previous reaction revealing that this compound is an intermediate in the synthesis of [Ti(tBu2O2NN′)(OiPr)2].

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7 8

9

Acknowledgements The authors thank the Fundação para a Ciência e a Tecnologia, Lisbon, Portugal, for funding (SFRH/BPD/7394/2010, PEst-OE/ QUI/UI0100/2013 and PEst-OE/QUI/UI0612/2013 and RECI/ QEQ-QIN70189/2012) and IST-UL Centers of the Portuguese NMR and Mass Spectrometry Networks (REM2013, RNNMR).

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