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. 2022 Oct 23;61(44):17653–17661. doi: 10.1021/acs.inorgchem.2c02737

Hydrolytically Stable and Cytotoxic [ONON]2Ti(IV)-Type Octahedral Complexes

Anastasia Pedko †, Eden Rubanovich ‡, Edit Y Tshuva †,*, Avital Shurki ‡,*
PMCID: PMC9644366  PMID: 36273341

Abstract

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A new family of titanium(IV) complexes based on [ONON] diaminobis(phenolato) ligands with Me, Br, Cl, and F ortho substitutions was synthesized and characterized. X-ray structures of three derivatives revealed homoleptic L2Ti-type compounds that exhibit an octahedral geometry without binding of the dangling amine unit. DFT calculations demonstrated that the preference of an L2Ti complex is not driven by solvent or ligand substitutions but rather by entropic effects. Except for the fluorinated derivative that was hydrolyzed immediately following water addition at room temperature and had the lowest biological activity of the series tested, all other complexes showed cytotoxic activity comparable to or higher than (up to 10-fold) that of cisplatin toward human ovarian A2780 and colon HT-29 cancer cell lines (IC50 values: 0.6–13 μM after incubation for 72 h). Activity was generally higher (up to 10-fold) toward the more sensitive ovarian line and similar for all active complexes, whereas differences were recorded toward the colon line that are attributed to bioavailability variations among the complexes analyzed. Particularly high hydrolytic stability was recorded for the brominated derivative with a t1/2 of 17 ± 1 days for ligand hydrolysis in 10% D2O at room temperature, relative to t1/2 of 56 ± 5 and 22 ± 6 h measured for the chlorinated and methylated derivatives, respectively. Altogether this series of compounds represent a promising family of anticancer agents, with the chlorinated derivative showing the best combination of stability, cytotoxicity, and bioavailability.

Short abstract

A new series of homoleptic [ONON]2Ti-type complexes with potential anticancer activity was synthesized, with the chlorinated derivative showing the best combination of stability, cytotoxicity, and bioavailability.

Introduction

The disadvantages of cisplatin related to toxicity and resistance development prompt the quest toward alternative drugs based on other transition metals,1−7 among which is titanium(IV).8−16 Titanium(IV) coordination complexes are biocompatible; their final hydrolysis product, titanium dioxide, is known as a safe material, often present in food and cosmetic products, presenting an advantage for medicinal use.17−19 Titanocene dichloride and budotitane were the first Ti(IV) complexes analyzed for antitumor applications and reached clinical trials due to several attractive features: (a) they are effective in vivo;11,13 (b) they show reduced toxic effects in animals;11,12 and (c) they are active toward cells resistant to cisplatin.11,12,20−22 Nevertheless, the hydrolytic instability of these complexes eventually led to their failure in clinical trials as rapid formation of inactive aggregates was clinically ineffective.14,23

As a part of the quest toward advanced more stable and cytotoxic Ti(IV) complexes, our group has introduced amine phenolato complexes for antitumor applications.24 “Salan” complexes of [ONNO]-type diaminobis(phenolato) ligands gave highly cytotoxic complexes, with enhanced stability relative to classical titanocene or diketonato complexes. In these complexes, a single tetradentate ligand wraps around the metal to leave room for two additional labile ligands (Scheme 1, top).25−31 The t1/2 values for labile ligand hydrolysis were between a few hours for complexes with NMe donors and with alkylated aromatic rings to a few days for ortho-halogenated compounds, that is, ortho-brominated or -chlorinated derivatives.27 Interestingly, a complex with ethylated amine groups was highly unstable and consequently inactive.26 Another related family of aminobis(phenolato) complexes includes the [ONON]-type ligands with a dangling amine donor (Scheme 1, middle).32−35 These ligands also generally bind Ti(IV) through all four atoms, including the dangling amine,33,34 but weaker Ti–N bonds for the side arm lead to less stable and somewhat less active complexes. A ligand with ortho- and para-dimethylated aromatic rings and a diethylated side amine donor arm (Scheme 1, middle: R = Me, R′ = Et) is bound similarly to give an LTiX2-type complex, despite the relatively long Ti–N bond (2.46 Å) leading to negligible stability and activity.32 Homoleptic L2Ti complexes of bis(phenolato) ligands were observed only for tridentate [ONO]-type ligands (Scheme 1, bottom)25,33,34 and were mostly unstable and inactive, although some related complexes of mono-phenolato ligands exhibit stability and cytotoxicity.25,36,37

Scheme 1. Aminephenolato Ligands of [ONNO] (Top), [ONON] (Middle), and [ONO] (Bottom) Type Give LTiX2-, LTiX2-, and L2Ti-Type Complexes upon Reaction with Ti(OiPr)4, Respectively; Hydrolytic Stability of Complexes: High t1/2 for Labile Ligand Hydrolysis in 10% D2O at Room Temperature > 5 h; Low t1/2 for All Ligand Hydrolysis at the Aforementioned Conditions < 10 min; Cytotoxicity of Complexes: High IC50 Values < 5 μM and Maximal Inhibition > 75%; Mediocre IC50 Values in the Range 5–50 μM and/or Maximal Inhibition < 75%; No IC50 Values > 50 μM and/or Maximal Inhibition < 20%.

Scheme 1

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Herein, we report on octahedral [ONON]2Ti complexes, obtained from [ONON]-type ligands without binding of the side amine donor (Scheme 2). In particular, heating provided an entropic drive for second ligand binding without coordination of the dangling N donor. Additionally, ortho-methylation, -bromination, or -chlorination provide complexes of this family that are highly stable and highly cytotoxic toward human cancer cells.

Scheme 2. Synthesis of Diaminebis(phenolato) Ligands and Their Complexes.

Scheme 2

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Experimental

Materials and Physical Measurements

Starting materials 2-chloro-4-methylphenol, 2-fluoro-4-methylphenol (Chem Scene), 2-bromo-4-methylphenol, titanium(IV) isopropoxide (Aldrich), 2,4-dimethylphenol (Fluka), N,N-diethylethylenediamine (Alfa Aesar), formaldehyde 37% (J.T. Baker), and methanol (Bio-lab) were used as received. Tetrahydrofuran (Bio-lab) was dried by solvent purifiers MB-SPS. Elemental analysis of C, H, and N was performed on a Thermo Flash 2000 CHN-O elemental analyzer; Elemental analysis of Cl, Br, and F was performed by Anton Paar Microwave Induced Oxygen Combustion (MIC) for the decomposition of organic samples by Dionex LC20 ion chromatography. High-resolution mass spectrometry was conducted using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled with a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific). Solutions for 1H and 13C NMR spectra were prepared in DMSO-d6 or THF-d8 (Cambridge Isotope Laboratories, INC), measured at 400 and 125 MHz, respectively, and analyzed with TopSpin 4.0.6, Bruker Corporation. The chemical shifts were referenced to Me4Si (δ 0.00 ppm) and DMSO-d6 (δ 2.5 ppm) or THF d8 (δ 3.58 and 1.72 ppm). Hydrolysis studies were performed by 1H NMR spectroscopy at 298 K with 32 scans using 1,4-dinitrobenzene (Sigma Aldrich) as the internal standard, recording the spectra at 500 MHz.

Ligand Synthesis

Ligands were synthesized by mixing different ortho-substituted meta-methylphenols, N,N-diethylethylenediamine, and formaldehyde in a molar ratio of 2:1:2 in methanol under reflux for 24 h, as described previously.34,38 For H2L4, the reagents were mixed for a week under similar conditions, then the solvent was evaporated, and the product was recrystallized from methanol. The ligands were characterized by elemental analysis, 1H NMR and 13C NMR spectroscopy, and HRMS.

H2L1: Obtained as white powder in 45.76% (2.2 gr) yield. Anal. Found: C, 74.68; H, 9.49; N, 7.23. Calcd for C24H36N2O2: C, 74.96; H, 9.44; N, 7.28. 1H NMR (C4D8O 400 MHz) δH: 8.99 (S, 2H, OH), 6.66 (d, J = 2 Hz, 2H, Ar–H), 6.55 (d, J = 2 Hz, 2H, Ar–H), 3.41 (S, 4H, CH2), 2.53 (t, J = 6 Hz, 2H, CH2), 2.44–2.34 (m, 6H, CH2), 2.04 (S, 6H, CH3), 2.00 (S, 6H, CH3), and 098 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR (C4D8O 125 MHz): 151.6, 132.4, 129.9, 128.7, 124.1, 110.3, 66.9, 54.9, 49.6, 49.1, 45.6, 19.1, and 9.6 ppm. HRMS: (C24H36N2O2 + H+) m/z Calcd: 385.28495. Found: 385.28497.

H2L2: Obtained as white powder in 65.75% (2.87 gr) yield. Anal. Found: C, 52.01; H, 5.97; N, 5.49; Br, 29.51. Calcd for C22H30Br2N2O2: C, 52.19; H, 5.97; N, 5.29; Br, 30.19. 1H NMR ((CD3)2SO 400 MHz) δH: 10.5 (S, 2H, OH), 7.21 (d, J = 2 Hz, 2H, Ar–H), 6.89 (d, J = 2 Hz, 2H, Ar–H), 3.59 (S, 4H, CH2), 2.61 (t, J = 5 Hz, 2H, CH2), 2.44 (q, J = 8 Hz 4H, CH2), 2.14 (S, 6H, CH3), and 0.96 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR ((CD3)2SO 125 MHz): 151.5, 132.4, 130.7, 125.3, 110.5, 54.6, 49.2, 45.6, 40.6, 20.1, and 10.5 ppm. HRMS: (C22H30Br2N2O2+H+) m/z Calcd: 515.07263. Found: 515.07227.

H2L3: Obtained as white powder in 30.07% (1.08 gr) yield. Anal. Found: C, 62.42; H, 6.94; Cl, 17.18; N, 6.59. Calcd forC22H30Cl2N2O2: C, 62.12; H, 7.11; Cl, 16.67; and N, 6.59. 1H NMR ((CD3)2SO 400 MHz) δH: 7.07 (d, J = 2 Hz, 2H, Ar–H), 6.8 (d, J = 2 Hz, 2H, Ar–H), 3.6 (S, 4H, CH2), 2.61 (t, J = 6 Hz, 2H, CH2), 2.52–2.51 (m, 2H, CH2), 2.43 (q, J = 7 Hz, 4H, CH2), 2.16 (S, 6H, CH3), and 096 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR ((CD3)2SO 125 MHz): 150.4, 129.9, 129.4, 128.7, 125.5, 120.3, 54.39, 49.43, 49.18, 45.7, 20.2, and 10.6 ppm. HRMS: (C22H30Cl2N2O2 + H+) m/z Calcd: 425.17571. Found: 425.17578.

H2L4: Obtained as white powder in 66.3% (1.2 gr) yield. Anal. Found: C, 67.10; H, 7.61; F, 9.13; N, 7.06. Calc. for C22H30F2N2O2: C, 67.32; H, 7.70; F, 9.68; N, 7.14. 1H NMR ((CD3)2SO 400 MHz) δH: 10.12 (S, 1H, OH), 6.87 (d, J = 2 Hz, 1H, Ar–H), 6.84 (d, J = 2 Hz, J = 2, 1H, Ar–H), 6.71 (S, 2H, Ar–H), 3.57 (S, 4H, CH2), 2.56 (t, J = 7 Hz, 2H, CH2), 2.49–2.45 (m, 2H, CH2), 2.37 (q, J = 7 Hz, 4H, CH2), 2.16 (S, 6H, CH3), and 091 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR (C4D8O 125 MHz): 152.33, 150.42, 142.62–142.52 (d, J = 12), 127.49–127.44 (d, J = 7), 125.81–125.55 (dd, J = 3, J = 30), 115.27–115.13 (d, J = 18), 53.96–53.94(d, J = 2), 49.66–49.48 (d, J = 22), 45.79, 19.48–19.47 (d, J = 2), and 10.05 ppm. HRMS: (C22H30F2N2O2:+H+) m/z Calcd: 393.23481. Found: 393.23483.

Complex Synthesis

Titanium(IV) complexes were synthesized by mixing titanium(IV) isopropoxide and the ligand precursor in a molar ratio of 1:2 in dry THF at 61 °C for 24 h in a Universal oven UF 30 under an inert environment. The solvent was evaporated, and a red powder was obtained. The complexes were characterized by elemental analysis, 1H NMR and 13C NMR spectroscopy, and HRMS.

Synthesis of L12Ti

The compound was obtained as red powder in quantitative yield. Anal. Found: C, 70.41; H, 8.46; N, 6.78. Calcd for C48H68N4O4Ti: C, 70.92; H, 8.43; N, 6.89. 1H NMR (C4D8O 400 MHz) δH: 6.85 (d, J = 2 Hz, 1H, Ar–H), 6.77 (d, J = 1 Hz, 1H, Ar–H), 6.68 (t, J = 3 Hz, 2H, Ar–H), 4.721 (d, J = 13 Hz, 1H, CH2), 4.51 (d, J = 12 Hz, 1H, CH2), 3.68 (q, J = 9 Hz, 2H, CH2), 2.86–2.78 (m, 1H, CH2), 2.73–2.65 (m, 1H, CH2), 2.62–2.60 (m, 2H, CH2), 2.42–2.34 (m, 2H, CH2), 2.10 (S, 3H, Ar-CH3), 2.07 (S, 3H, Ar-CH3), 2.06–2.04 (m, 2H, CH2), 1.98 (S, 3H, Ar-CH3), 1.4 (S, 3H, Ar-CH3), and 0.64 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR (C4D8O125 MHz): 160.6, 159.7, 130.9, 130.6, 127.4 (d, J = 4), 126.3, 126.1, 122.7, 122.26 (d, J = 2), 122.23, 59.1, 58.3, 55.4, 47.5, 46.4, 42.8, 19.7, 19.6, 16.3, 16.05, 11.8, and 0.38 ppm. HRMS: (C48H68N4O4Ti + H+) m/z Calcd: 813.47928. Found: 813.47931.

Crystal data for C48H68N4O4Ti (M =812.96 g/mol): triclinic, space group P1̅ (no. 2), a = 11.1327(2) Å, b = 12.8122(2) Å, c = 17.5751(3) Å, α = 72.901(2)°, β = 83.726(2)°, γ = 66.857(2)°, V = 2203.14(8) Å3, Z = 2, T = 149.99(10) K, μ(Mo Kα) = 0.242 mm–1, Dcalc = 1.225 g/cm3, 37,018 reflections measured (4.23° ≤ 2θ ≤ 64.854°), 12960 unique (Rint = 0.0346, Rsigma = 0.0365) which were used in all calculations. The final R1 was 0.0426 (I > 2σ(I)) and wR2 was 0.1144 (all data).

Synthesis of L22Ti

The compound was obtained as red powder in quantitative yield. Anal. Found: C, 49.58; H, 5.48; N, 5.06; Br, 29.69. Calc. for C44H56Br4N4O4Ti: C, 49.28; H, 5.26; N, 5.22; Br, 29.80. 1H NMR ((CD3)2SO 400 MHz) δH: 7.31 (d, J = 2 Hz, 2H, Ar–H), 7.27 (S, 4H, Ar–H), 7.23 (d, J = 2 Hz, 2H, Ar–H), 4.71 (d, J = 13 Hz, 4H, CH2), 4.06 (d, J = 13 Hz, 2H, CH2), 3.99 (d, J = 13 Hz, 2H, CH2), 3.14–3.05 (m, 2H, CH2), 2.70–2.63 (m, 4H, CH2), 2.22 (d, J = 7 Hz, 12H, Ar-CH3), 2.18–2.12 (m, 10H, CH2), and 0.71 (t, J = 7 Hz, 12H, CH3) ppm. 13C NMR (C4D8O 125 MHz): 158.3, 156.9, 132.9, 132.2, 129.4, 129.1, 128.7, 128.4, 124.47, 124.44, 109.3, 108.6, 67.2, 58.7, 58.1, 47.2, 46.5, 42.8, 25.39, 19.3, 19.2, and 11.7 ppm. HRMS: (C44H56Br4N4O4Ti + H+ (m/z Calcd: 1073.05464. Found: 1073.05627.

Crystal data for C53H80.5Br4N4O7.25Ti (M = 1257.25 g/mol): monoclinic, space group P21/n (no. 14), a = 13.4994(6) Å, b = 22.0291(7) Å, c = 19.9404(8) Å, β = 104.712(4)°, V = 5735.5(4) Å3, Z = 4, T = 149.99(10) K, μ(Mo Kα) = 2.987 mm–1, Dcalc = 1.456 g/cm3, 40031 reflections measured (3.626° ≤ 2θ ≤ 52°), 11,148 unique (Rint = 0.0681, Rsigma = 0.0675), which were used in all calculations. The final R1 was 0.1001 (I > 2σ(I)) and wR2 was 0.2040 (all data).

Synthesis of L32Ti

The compound was obtained as red powder in quantitative yield. Anal. Found: C, 58.72; H, 6.49; Cl, 15.09; N, 5.92. Calcd for C44H56N4Cl4O4Ti: C, 59.07; H, 6.31; Cl, 15.85; N, 6.26.1H NMR ((CD3)2SO 400 MHz) δH: 7.20 (d, J = 2 Hz, 1H, Ar–H), 7.17 (d, J = 2 Hz, 1H, Ar–H), 7.15 (d, J = 2 Hz, 1H, Ar–H), 7.09 (d, J = 2 Hz, 1H, Ar–H), 4.57 (dd, J = 13, J = 7 Hz, 2H, CH2), 4.06 (d, J = 14 Hz, 1H, CH2), 3.98 (d, J = 14 Hz, 1H, CH2), 3.02–2.94 (m, 1H, CH2), 2.64 (t, J = 8 Hz, 2H, CH2), 2.57–2.52 (m, 1H, CH2), 2.22–2.20 (d, J = 6 Hz, 6H, Ar-CH3), 2.18–2.15 (m, 1H, CH2), 2.14–2.13 (q, J = 4 Hz, 2H, CH2), 2.11–2.08 (m, 1H, CH2), and 0.7 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR (C4D8O 125 MHz): 157.2, 156.0, 129.88, 129.37, 128.61, 128.31, 128.19, 127.97, 124.64, 124.56, 119.32, 118.73, 67.23, 58.57, 57.89, 47.17, 46.47, 42.83, 25.40, 19.44, 19.36, and 11.74 ppm. HRMS: (C44H56N4Cl4O4Ti + H+) m/z Calcd: 895.25784. Found: 895.25867.

Crystal data for C44.8H54.9Cl4N4O4.5S0.4Ti (M =923.95 g/mol): triclinic, space group P1̅ (no. 2), a = 14.9277(2) Å, b = 17.5396(2) Å, c = 19.8460(3) Å, α = 98.5360(10)°, β = 111.6610(10)°, γ = 91.5980(10)°, V = 4756.40(12) Å3, Z = 4, T = 199.9(4) K, μ(Mo Kα) = 0.467 mm–1, Dcalc = 1.290 g/cm3, 78,070 reflections measured (4.324° ≤ 2θ ≤ 58°), 25,126 unique (Rint = 0.0397, Rsigma = 0.0504) which were used in all calculations. The final R1 was 0.0637 (I > 2σ(I)) and wR2 was 0.1749 (all data).

Synthesis of L42Ti

The compound was obtained as red powder in quantitative yield. Anal. Found: C, 63.19; H, 6.91; F, 8.70; N, 6.56. Calcd for C44H56N4F4O4Ti: C, 63.76; H, 6.81; F, 9.17; N, 6.76 (measurement affected by hydrolytic instability). 1H NMR ((CD3)2SO 400 MHz) δH: 6.99 (d, J = 9 Hz, 2H, Ar–H), 6.87 (d, J = 11 Hz, 2H, Ar–H), 4.44 (d, J = 13 Hz, 1H, CH2), 4.34 (d, J = 14 Hz, 1H, CH2), 4.01 (d, J = 14 Hz, 2H, CH2), 2.81–2.60 (m, 4H, CH2), 2.22 (S, 3H, Ar-CH3), 2.19 (S, 3H, Ar-CH3), 2.16 (q, J = 7 Hz, 4H, CH2), and 0.71 (t, J = 7 Hz, 6H, CH3) ppm. 13C NMR (C4D8O 125 MHz): 148.8, 148.3, 125.64, 124.5, 124.3, 115.91, 66.1, 58.06, 47.17, 47.06, 43.25, 19.59–19.62 (d), 19.33, and 11.59 ppm. HRMS: (C44H56N4F4O4Ti + H+) m/z Calcd: 829.37899. Found: 829.37817.

X-ray Crystallography

Single crystals of L12Ti for X-ray crystallography were obtained by slow evaporation of acetonitrile at 254 K; single crystals of L22Ti were obtained by slow evaporation of isopropanol at 254 K; and single crystals of L32Ti were obtained by slow evaporation of DMSO solution at 298 K. The crystals were analyzed using an XtaLAB Synergy-S, single source at offset/far, HyPix diffractometer. The crystal was kept at 150 K during data collection. The structure was solved with the Olex2 and SHELXT structure solution program using intrinsic phasing and refined with the SHELXL refinement package using least squares minimization.39−41

Computational Details

Molecular geometries of the Ti bound meta- and para-methylated N-methylated33 (referred to here as structure L5Ti(OiPr)2 and the corresponding L52Ti) and ortho- and para-methylated N-ethylated (structure L1Ti(OiPr)2 and the corresponding L12Ti) complexes as well as that of the free ligands H2L1,5 (Scheme 3), and isopropanol ligands were optimized at the PBE0-D3 level of theory42,43 in n-pentane (ε = 1.8) and in acetonitrile (ε = 35.7) using the PCM solvation model.44 All calculations were performed using the Gaussian 16 software package45 employing Pulay’s m6-31G* basis set for the titanium atom46 and the 6-31G* basis set for the rest of the atoms.47−49 The PBE0 functional with a combination of basis sets was shown to provide a good compromise between the accuracy and computational cost for non-anionic transition-metal complexes.50 Optimizations were followed by frequency calculations to verify that the structures obtained are indeed minima along the potential energy surface and to determine thermochemical data (at 298.15 K and 1.0 atm). Optimized geometries of different calculated compounds as well as their absolute energies are available in the Supporting Information.

Scheme 3. Ligands Employed for Analysis of Their Complexes in the Ligand-Exchange Reaction Presented in eq 1.

Scheme 3

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Stability

Kinetic hydrolytic stability was examined by 1H NMR spectroscopy at 298 K with 32 scans. Ti(IV) complexes and 1,4-dinitrobenzene (Sigma Aldrich) as the internal standard were dissolved in DMSO-d6 or THF-d8 and 10% D2O was added as reported previously.51 The chemical shifts were calibrated to DMSO-d6 or THF-d8 (δ—2.5, δ—3.58 and 1.72 ppm, respectively). The integration of the peaks was analyzed with TopSpin 4.0.6, Bruker Corporation. To determine the half-life time, first the decay constant(k) was calculated from the slope of plotting the ln of the peak integral versus time (Figure S1) and then t1/2 was calculated as t1/2 = ln(2)/k (given as an average and STD of two to three repeats). All complexes are stable on the shelf in air as powder for at least 6 months.

In Vitro Cytotoxicity

The cytotoxicity of Ti(IV) complexes was tested on A2780 human ovarian carcinoma cell line (European Collection of Authenticated Cell Cultures) and HT-29 human colorectal adenocarcinoma cell line (American Type Culture Collection). Cells were seeded in 96-well plates at 10,000 cells per well concentration, in complete RPMI 1640 medium, which contains 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin (Biological Industries, Beit Haemek, Israel), and allowed to attach overnight. On the next day, a series of 10 different concentrations of the examined substances were prepared in either DMSO or THF: L2,42Ti was diluted in DMSO and L1,32Ti in THF. They were further diluted in growth medium and finally added to the cells, so that the final DMSO/THF concentration was 0.5%. Following a 3 day incubation at 37 °C in a 5% CO2 atmosphere, the cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.52 In a standard experiment, 20 μL of solution containing 0.1 mg of MTT (Sigma-Aldrich) was added to the cells and incubated for 3 h. The medium was replaced with 200 μL of isopropyl alcohol (Daejung), and the absorbance was measured at 550 nm using a Spark 10 M multimode microplate reader. Relative IC50 values were calculated by nonlinear regression of a variable slope model using GraphPad Prism 5.04 software ([top + bottom plateaus]/2). Error values were determined based on standard deviation. All measurements were repeated at least 3 × 3 times: 3 repeats per plate, all repeated three times on different days (nine repeats in total). Statistical validity (p < 0.1) was obtained for L1,22Ti and L1,42Ti for HT-29 and for L22Ti for the two lines.

Results and Discussion

Structure

Ligands were synthesized by a Mannich condensation of substituted phenol, formaldehyde, and the diamine following procedures reported previously.34,38 The 1H NMR spectrum featured two aromatic peaks and new peaks at 3.6–3.41 ppm of four methylene protons. Elemental analysis, 13C NMR, and HRMS characterizations confirmed that the desired compounds had been obtained, with the expected symmetry manifested by the NMR results. The complexes were then synthesized by mixing the ligand and Ti(OiPr)4 in a molar ratio of 2:1 in dry THF at 61 °C for 24 h under an inert environment. The 1H NMR spectra confirmed the formation of L2Ti-type complexes (L1–42Ti, Scheme 2), with shifts in the ligand peaks relative to the free ligand, and the lack of signals corresponding to bound isopropoxo groups. Such complexes have been previously obtained from related [ONO]-type tridentate ligands,33,34 whereas tetradentate [ONON] ones with an extra dangling amine donor have given the LTi(OiPr)2-type complexes with binding of both N atoms.32−35 Interestingly, applying a 1:1 ligand to metal ratio at room temperature did not lead to the formation of LTi(OiPr)2-type complexes as observed previously with similar salan ligands. The spectra each featured two sets of aromatic and aliphatic ligand signals, unlike the single set previously obtained for the salan LTi(OiPr)2-type ligands,26,27 except for the dangling arm presented by a single set of signals. These results imply that the two bound ligands are symmetrically related, whereas the ligand symmetry was abolished by different chemical environments for the two ligand sides. Two AB signal systems at ∼4.0–5.0 ppm for the methylene benzylic units reflect the constrained geometry of the bound complex.33,3413C NMR exhibited similar features, and HRMS and elemental analyses supported the L2Ti-type complex formation. Nevertheless, it could not be unequivocally determined based on the NMR alone whether the dangling amine group was bound to the metal center, although no constrained geometry was observed for the related signals.

Single crystals suitable for X-ray crystallographic analyses were obtained from slow evaporation of a cold acetonitrile solution of L12Ti, a cold isopropanol solution of L22Ti, and a DMSO solution of L32Ti. Figure 1 depicts the ORTEP structure at 50% probability ellipsoids, and Table 1 summarizes selected bond lengths and angles.

Figure 1.

Figure 1

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ORTEP drawing of L12Ti (left), L22Ti (middle), and L32Ti (right) a 50% probability ellipsoids. Disorder in diethylamino groups was omitted for clarity.

Table 1. Selected Bond Lengths (Å) and Angles (°) for L22Ti.

  angle (deg)
   bond distances (Å)
atom L12Ti L22Ti L32Ti atom L12Ti L22Ti L32Ti
O1–Ti1–O2 166.27(4) 169.2(3) 168.38(7) Ti–O1 1.8795(9) 1.885(6) 1.895(2)
O1–Ti1–O4 89.97(4) 91.4(3) 92.41(8) Ti–O2 1.8970(9) 1.885(6) 1.889(2)
O1–Ti1–N1 81.75(4) 84.1(3) 83.99(8) Ti–O3 1.8764(9) 1.882(6) 1.879(2)
O1–Ti1–N3 95.47(4) 92.0(3) 91.73(7) Ti–O4 1.8990(9) 1.873(6) 1.870(2)
O2–Ti1–O4 89.63(4) 89.6(3) 89.39(8) Ti–N1 2.264(1) 2.254(8) 2.267(2)
O2–Ti1–N1 84.72(4) 85.1(3) 84.41(8) Ti–N3 2.262(1) 2.245(8) 2.248(2)
O2–Ti1–N3 98.16(4) 98.8(3) 99.87(7) Ti···N2 5.55 5.64 5.52
O1–Ti1–O3 94.05(4) 92.2(3) 91.75(8) Ti···N4a 5.56 5.51 5.43
O2–Ti1–O3 89.56(4) 88.9(3) 89.03(9)        
O3–Ti1–O4 166.19(4) 168.3(3) 166.94(7)        
O3–Ti1–N1 95.83(4) 94.2(3) 96.25(7)        
O3–Ti1–N3 81.72(4) 84.7(3) 83.09(7)        
O4–Ti1–N1 97.82(4) 97.3(3) 96.49(7)        
O4–Ti1–N3 84.75(4) 84.0(3) 84.42(7)        
N1–Ti1–N3 176.18(4) 175.9(3) 175.65(8)       Pl
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The structures of all three complexes are generally similar, featuring an octahedral metal center bound to two tridentate [ONO] ligands, supporting the NMR results with a solution symmetry of C2.33 The dangling amine groups did not bind the metal center, with >5 Å Ti–N distance and some disorder in the diethylamino groups. The structures evince that the two halves of each ligand are in different chemical environments, whereas both bound ligands are symmetrically related. The bond lengths and angles (Table 1) are like those found in related structures, indicative of strong covalent Ti–O and coordinative Ti–N bonds. As expected, the Ti–N bonds are similar to those previously reported for binding of the central amine and not to those found for the dangling amine in LTi(OiPr)2-type complexes, which are typically longer and indicative of weaker binding.33,36

Comparing the binding motif to that observed previously for [ONON]-type diaminobis(phenolato) ligands, it is interesting to note that homoleptic L2Ti complexes were obtained herein and not LTi(OiPr)2-type complexes as obtained previously.33−35 In particular, the exact same ligand with identical substitutions has previously produced an LTi(OiPr)2-type complex as analyzed crystallographically.32 One main difference in the reaction conditions stands out; the higher temperature applied herein, 61 °C versus room temperature, favoring the formation of the thermodynamic product. An additional difference involves the crystallization solvent: acetonitrile in the current study versus n-pentane in previous work.32,33 To determine whether the temperature is indeed the dominant factor in dictating the binding scheme, the change in enthalpy (ΔH) and the change in free energy (ΔG) were calculated at 298 K for the following ligand-exchange reaction in different solvents

graphic file with name ic2c02737_m001.jpg 1

Here, H2L is the free ligand. As various other similar salan complexes analyzed previously with different aromatic rings and N donor substituents gave similar LTi(OiPr)2-type complexes, similar calculations were conducted for a representative derivative bearing meta, para-, and N-methylation L5 (Scheme 3). The results of these calculations for both ligands are summarized in Table 2.

Table 2. Calculated Enthalpy and Free Energy (in kcal/mol) for Complexes in the Ligand-Exchange Reaction (eq 1) at 298.15 K and 1.00 atm in Different Solvents.

  acetonitrile
 n-pentane
ligand ΔH ΔG ΔH ΔG
L1 11.4 –1.7 12.6 –0.5
L5 11.1 –2.3 12.5 –0.7
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The differences between the calculated results for the two ligands as well as for the two solvents are very small, suggesting that the different structural aspects of the ligand as well as the different solvents did not dominate the resulting complex. The table also shows that enthalpy alone clearly favors the LTi(OiPr)2 over the L2Ti complexes for both ligands and in both solvents. Nevertheless, this clear preference disappears when free energy is considered, and the differences become virtually zero (with the L2Ti slightly favored in both cases). The large differences between the ΔH and the ΔG values suggest that the considered ligand-exchange reaction is entropy-driven, which indicates that heating is the main reason for obtaining the homoleptic L2Ti complexes in the current study.

Hydrolysis

Hydrolysis studies were performed by adding 10% D2O (>100 equiv) to the complexes at room temperature to afford a pseudo-first-order reaction and monitoring the signal change in the 1H NMR spectra (Table 3). These conditions do not presume to reflect the biological environment but rather serve as a comparable tool to assess the stability relative to that of compounds analyzed under similar conditions as previously reported.24−27,30−32 For L1–32Ti, decay in the integration of the complex signals was followed, ultimately indicating free ligand release. The methylated 2Ti complex exhibited a high stability with t1/2 of ca. 1 day, which is markedly higher than that reported for [ONNO]TiX2-type alkylated complexes analyzed under similar conditions (t1/2 of several hours).26,27 The two halogenated complexes, L2,32Ti, demonstrated an even higher stability with t1/2 values of several days, with the highest t1/2 recorded for the brominated derivative, L22Ti, of more than 2 weeks. These results are consistent with previous observations that ortho-bromination or -chlorination enhances the hydrolytic stability of phenolato-Ti(IV) complexes, which may be attributed to the combination of steric and electronic influences.27,32 Steric hindrance may pose a kinetic barrier to the approach of water molecules, whereby additional electronic influences are provided by halogenation. Interestingly, the fluorinated derivative L42Ti demonstrated low stability with instantaneous decomposition upon water addition (Figure S2), further emphasizing the importance of the steric parameter, with the potential contribution of H-bonding with water molecules in further reducing hydrolytic stability.

Table 3. T1/2 Values for Ligand Hydrolysis Measured for L1–42Ti at Room Temperature following the Addition of >100 Water Equivalents.

complex ortho substitution t1/2
L12Ti Me 22 ± 6 h
L22Ti Br 17 ± 1 days
L32Ti Cl 56 ± 5 h
L42Ti F —a
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a

Decomposes spontaneously.

Cytotoxicity

The cytotoxic activities of the complexes were tested on two types of cancer cell lines, A2780, human ovarian carcinoma cells and HT-29, human colorectal adenocarcinoma cells, and analyzed by the MTT assay.52Figure 2 depicts the dose–response curves of L1–42Ti. The IC50 values are summarized in Figure 2. The complexes were highly active against both cell lines with activity that is comparable to, or greater than, that of cisplatin. Activity was also recorded for free ligands (Figure S3); nevertheless, previous studies indicated that any activity of ligands is unrelated to that of the complexes, as even complexes that were unstable and rapidly released the free ligands were inactive despite the activity of the free ligand itself, supporting the notion that the activity does not result from dissociated ligands.26,27 Ligand release is in any case of reduced relevance for the stable derivatives, L1–32Ti, as similarly stable complexes have also been previously detected in their intact form in the cellular environment.53

Figure 2.

Figure 2

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Dependence of human ovarian A2780 (top) and colon HT-29 (bottom) cancer cell viability on different concentrations (shown on a logarithmic scale) of L1–42Ti, following a 3 day incubation period as analyzed by the MTT assay. Cisplatin IC50 = 1.6 ± 0.4 μM for A2780 and IC50 = 25 ± 4 μM for HT-29. H2L1–4 = 1.9 ± 0.7, 1.5 ± 0.1, 1.4 ± 0.6, 7.4 ± 1.5 μM for A2780 and IC50 = 1.3 ± 0.4, 10 ± 3, 10 ± 3, 26.9 ± 6.2 μM for HT-29 (see maximal inhibition values in Table S1). Relative IC50 values were calculated by nonlinear regression of a variable slope model by GraphPad Prism 5.04 software ([top + bottom plateaus]/2).

Comparing the activity among the complexes reveals that all but the fluorinated derivative show similarly high activity toward the more sensitive ovarian line; nevertheless, differences were recorded on the more resistant colon line. Inspecting the series L1–32Ti, activity is in opposite correlation to stability: the brominated complex is the least active despite its highest stability, which may be attributed to its large steric bulk, possibly affecting the bioavailability.26,30,31,54 Accordingly, the more lipophilic methylated derivative shows the highest activity of this series. The differences between the two lines may reflect different membrane permeability, affecting the insertion of bulkier, less bioavailable derivatives. As expected, L42Ti, decomposing rapidly in water, showed markedly reduced cytotoxicity, presumably due to the instantaneous formation of less active or less bioavailable hydrolysis products.26,27,30

Conclusions

A new series of homoleptic [ONON]2Ti-type complexes is presented, which did not include the extra donor binding and yet showed high anticancer activity. Interestingly, for most derivatives, the high activity is combined with exceptional hydrolytic stability when compared with analogous derivatives.24−27,32 In previously reported work, the homoleptic [ONO]2Ti complex with no ortho substitutions was unstable and inactive,25 as well as most of the [ONON]TiX2 derivatives that included binding of the dangling amine;32 particularly, the derivative with ortho-methylated aromatic rings and ethylated dangling amine gave at room temperature an [ONON]TiX2-type complex that was unstable and inactive. In contrast, in the work presented here for ortho-substituted derivatives, upon heating, the dangling donor did not bind the metal and homoleptic [ONO]2Ti complex driven by entropic effects were obtained as supported by the calculations and still mostly provided high stability; in fact, by tuning complexation conditions, the exact same ligand with ortho-methylated aromatic rings and ethylated dangling amine gave an [ONO]2Ti-type complex of high stability and high activity. Other derivatives with ortho-halogenations produced compounds that are even more stable and markedly cytotoxic, mostly more than the clinically employed cisplatin.

The hydrolytic stability of the complexes is obviously valuable, as often unstable complexes decompose rapidly into inactive or non-bioavailable species and are therefore inactive.26,30,32 Nevertheless, the fair cytotoxicity of the fluorinated unstable complex should be attributed to its hydrolysis products (free ligand or clusters as observed in previous cases for relatively small unstable derivatives).30,54,55 Published work suggests that hydrolyzed free ligands of related compounds, if active, are not the source of activity even of relatively unstable complexes, probably due to the formation of ligand-bound inactive clusters.26,27 Nevertheless, the activity of H2L4 implies that its contribution to the activity of L42Ti cannon be ruled out. For the stable derivatives L1–32Ti, ligand hydrolysis is of less relevance as previous work has detected similarly stable derivatives in their intact form in the cell.53 For these derivatives, the cellular penetration seems to be a parameter of influence, especially as active transportation of some Ti(IV) cytotoxic compounds has been suggested:55 The cellular penetration may be affected by large steric bulk and general hydrophobicity/lipophilicity of the compounds and thus, along with solubility issues, may explain the activity pattern observed. As similarly hydrolytically stable derivatives have been observed in their intact form for days in the cellular environment,55 stable active complexes are good candidates to be employed for mechanistic investigations and eventually serve as potential drugs.

Altogether, considering all aspects, the chlorinated complex is identified as the derivative featuring the best combination of cytotoxicity, hydrolytic stability, and bioavailability of the series presented herein. We are currently looking into mechanistic aspects of these compounds and whether they may be correlated to other promising Ti(IV)-based anticancer drugs.

Acknowledgments

Funding was received from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681243) (EYT) and the Israel Science Foundation (grant no. 1691/17) (AS).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c02737.

  • X-ray structures of L12Ti (CCDC 2123949), L22Ti(CCDC 2162882), and L32Ti (CCDC 2168724); calculated geometries and absolute energies of different compounds; hydrolysis measurements; and cytotoxicity of free ligands (PDF)

The authors declare no competing financial interest.

Supplementary Material

ic2c02737_si_001.pdf (1.9MB, pdf)

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