Synthesis and Structure of Arene Ru(II) N^∧O-Chelating Complexes: In Vitro Cytotoxicity and Cancer Cell Death Mechanism

Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2023 Supporting Information for: Triazenide-supported [Cu4S] structural mimics of CuZ that mediate N2O disproportionation rather than reduction Neal P. Mankad npm@uic.edu Table of Contents: GENERAL CONSIDERATIONS ............................................................................................................................... S2 SYNTHESIS & CHARACTERIZATION OF 1N ............................................................................................................ S3 SYNTHESIS & CHARACTERIZATION OF [1N][K(KRYPT222] ....................................................................................... S7 N2O REACTIVITY................................................................................................................................................ S10 REFERENCES ..................................................................................................................................................... S12 S1 GENERAL CONSIDERATIONS Synthesis. Synthetic procedures were carried out under N2 atmosphere inside a MBraun LabMaster glovebox or using standard Schlenk line techniques.1 Reaction solvents were purified of air and moisture using a Glass Contours solvent purification system2 built by Pure Process Technology, LLC, and stored over 3-Å molecular sieves in the glovebox. Deuterated solvents were degassed and then dried over 3-Å molecular sieves. The precursor compound, Cu2(NNN)2, was prepared according to literature procedures.3 All other reagents were purchased from commercial vendors and used without further purification unless otherwise stated. Instrumentation. 1H NMR spectra were recorded using a Bruker Avance DPX-400-MHz spectrometer, with chemical shifts referenced using residual solvent peaks, and plotted in MestReNova. IR spectra were recorded on solid samples using a Bruker ALPHA spectrometer with a diamond-ATR detection unit. UVVis-NIR data were obtained using an Ocean Optics HDX-XR spectrometer fitted with a transmission dip probe and plotted in Origin after applying Lowess smoothing; peaks positions were determined by deconvolution as implemented in Origin. Cyclic voltammetry data was collected using a WaveNow USB Potentiostat from Pine Research Instrumentation using a classic three-electrode system4 (glassy carbon working, Pt counter, Ag/AgNO3 reference) and referenced to external FeCp2. High-resolution EI mass spectra were recorded at the Mass Spectrometry Laboratory at the University of Illinois UrbanaChampaign using a Q-TOF MS instrument. X-band EPR spectra were recorded at 9.463473 GHz using a Bruker EMX EPR spectrometer and simulated using EasySpin.5 Elemental analysis data were obtained by Atlantic Microlab, Inc. X-ray diffraction data for 1N was collected at the X-ray diffraction facility at Cornell University using a Rigaku XtaLAB Synergy diffractometer, and for [1N][K(Krypt222)] at UIC using a Bruker D8 QUEST ECO diffractometer. Solution and refinement were done using the SHELX and OLEX2 software suites by standard methods.6,7 Crystallographic data is available in CIF format by download from the CCDC (deposition numbers 2299540-2299541). Computations. All DFT and TD-DFT calculations were performed using Gaussian16 (Revision B.01).8 Geometries were optimized with no symmetry constraints using the B3LYP functional with ultrafine integration grid.9,10 All atoms were treated using the def2TZVPP basis set.11 Implicit solvation effects were included using the CPCM solvation model with default dichloromethane parameters.12 Vibrational frequency analysis confirmed that all stationary points were correctly identified as either energy minima (zero imaginary frequencies) or saddle points (1 imaginary frequency). Transition states were further analyzed using implicit reaction coordinate scans along the imaginary frequency vector to verify that they were situated between reactant and product states on the potential energy surface. Mulliken population analysis was used to determine spin densities and molecular orbital coefficients. Reaction thermochemistry was calculated by including zero-point and thermal corrections to enthalpies and Gibbs free energies as implemented using default settings for vibrational frequency analysis in Gaussian16. Calculated UV-Vis-NIR spectra were generated in Gaussview using excited states calculated by TD-DFT after applying line broadening to qualitatively match experimental data. Coordinates for all relevant energy minima and saddle points are provided in XYZ format as Supporting Information attached to this manuscript. For TS2, unfortunately, the geometry was not optimized at the level of theory indicated above after repeated attempts. Instead, the geometry could only be optimized successfully to the point of having exactly 1 imaginary frequency only in the gas phase (i.e, no CPCM solvation correction). Thus, although bond metrics for TS2 are presented in the paper, reaction thermochemistry (i.e., activation energy) is omitted for this particular transition state. S2 SYNTHESIS & CHARACTERIZATION OF 1N Synthesis of Cu4(µ4-S)(NNN)4 (1N). A solution of S8 (10.7 mg, 0.0417 mmol) in toluene (2 mL) was added dropwise to a stirring solution of Cu2(NNN)2 (437 mg, 0.636 mmol) in THF (4 mL), causing an immediate color change from yellow to inky blue. After stirring the reaction overnight, volatiles were removed in vacuo. CH3CN (3 mL) was added to the residue, and the resulting blue solid was collected by filtration and washed with additional CH3CN (2 x 3 mL) followed by Et2O (2 x 3 mL). Yield: 389 mg, 0.276 mmol, 87%. X-ray quality crystals spontaneously precipitated during removal of the THF/toluene reaction solvent. In both solution and solid state, the compound was found to be air stable indefinitely. 1 H NMR (CDCl3, 𝛿): 6.81 (1H), 6.74 (1H), 6.68 (1H), 6.34 (1H), 6.32 (1H), 2.59 (3H), 2.49 (3H), 2.36 (3H), 2.28 (3H), 2.22 (6H, overlapping singlets), 1.47 (3H), 1.32 (3H), 1.25 (3H). IR (solid, cm-1): 2977, 2910, 2853, 1476, 1322, 1196, 883, 850. UV-Vis-NIR (CH2Cl2, 𝜆max): 602 (12000 M-1cm-1). Anal. calcd. for C72H88Cu4N12S: C, 61.43; H, 6.30; N, 11.94. Found: C, 61.70; H, 6.27; N, 11.81. 7.5 7.0 6.5 1 5.5 5.0 4.5 4.0 f1 (ppm) 3.5 3.0 2.0 1.32 1.25 2.97 1.5 1.99 1.47 3.02 2.36 2.28 2.22 2.5 7.67 2.49 2.63 2.59 6.0 3.06 3.00 1.89 0.92 0.97 0.97 6.34 6.32 6.81 6.74 6.68 7.26 CDCl3 NNN-15_Cu4SNNN4 Proton standard parameters, BBO probe 1.0 N Figure S1. H NMR spectrum of 1 in CDCl3. S3 Figure S2. IR spectrum of 1N in the solid state. Figure S3. X-ray crystallography data for 1N: asymmetric unit (left) and molecular structure including all disordered components (right). S4 Excited State 9: Singlet-A 2.3453 eV 528.66 nm f=0.0661 <S**2>=0.000 131 -> 145 -0.13427 134 -> 145 -0.19483 138 -> 145 0.58535 142 -> 145 0.28809 Figure S4. Calculated (TD-DFT) UV-Vis-NIR spectrum for 1N output by Gaussview, along with excitations comprising the main charge transfer state. Figure S5. MO145 for 1N, which is the acceptor orbital for all transitions admixed in excited state 9. Key orbital coefficients are given in the main text, Table 1. S5 2.00E-06 1.00E-06 Current (A) 0.00E+00 -1.00E-06 -2.00E-06 -3.00E-06 -4.00E-06 -3 -2.5 -2 -1.5 Potential (V vs. FeCp2) -1 -0.5 0 Figure S6. Cyclic voltammogram of 1N (2 mM) in THF with [nBu4N][PF6] supporting electrolyte (0.3 M). Initial potential was -0.4 V and scanning started in negative potential direction. S6 SYNTHESIS & CHARACTERIZATION OF [1N][K(Krypt222] Synthesis of [Cu4(µ4-S)(NNN)4][K(Krypt222)] ([1N][K(Krypt222])). An orange solution of K[FeCp(CO)2] (15.9 mg, 0.0736 mmol) in THF (2 mL) was added dropwise to a stirring blue suspension of 1N (99.5 mg, 0.0707 mmol) in THF (1 mL) at room temperature. Upon addition, the initial color was blue-green. A solution of Kryptofix-222 (28.1 mg, 0.0746 mmol) in THF (1 mL) was then added. The resulting solution was stirred for 1 h, over which time the color intensified to black. Volatiles were removed in vacuo. Toluene (3 mL) was added, and the resulting suspension was stirred for 5 min. Resulting solids were collected by filtration and washed with additional toluene (3 mL) and Et2O (2 x 3 mL). The filtrate started red and became colorless during these washes, indicating complete removal of [FeCp(CO)2]2. The desired product was isolated as a blue powder. Yield: 102 mg, 0.0559 mmol, 79%. X-ray quality crystals were grown by slow diffusion of hexane vapors into a concentrated THF solution. H NMR (acetone-d6, 𝛿): 3.62-3.58 (2H, overlapping singlets), 2.58 (1H). IR (solid, cm-1): 2906, 1475, 1320, 1204, 1103, 945, 846, 850. UV-Vis-NIR (CH2Cl2, 𝜆max): 620 (4100 M-1cm-1), 934 (3300 M-1cm-1). EPR (X-band, 1:1 CH3CN:CH2Cl2): giso = 2.071. Anal. calcd. for C90H124Cu4KN14O6S: C, 59.28; H, 6.85; N, 10.75. Found: C, 58.21; H, 6.73; N, 10.35. 1.00 0.46 2.58 3.62 3.58 NNN-23_Cu4SNNN4anion Proton standard parameters, BBO probe 11.0 10.5 10.0 1 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 2.05 (CD3)2CO 1 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 N Figure S7. H NMR spectrum of [1 ][K(Krypt222)] in acetone-d6. S7 Figure S8. IR spectrum of [1N][K(Krypt222)] in the solid state. Figure S9. X-ray crystallography data: asymmetric unit for [1N][K(Krypt222)]. Figure S10. Optimized geometry for the DFT model of [1N]- S8 Excited State 2: 2.012-A 1.2896 eV 961.42 nm f=0.0356 <S**2>=0.762 144B -> 145B 0.98273 Excited State 5: 2.009-A 1.9410 eV 638.77 nm f=0.1496 <S**2>=0.759 141B -> 145B 0.97963 Figure S11. Calculated (TD-DFT) UV-Vis-NIR spectrum for [1N]- output by Gaussview, along with excitations comprising the two main charge transfer states. S9 N2O REACTIVITY Reaction with N2O gas. A representative procedure is given here from among the various conditions that were examined. CH2Cl2 (6 mL) was added to [1N][K(Krypt222)] (96.5 mg, 0.0529 mmol) and CoCp2 (12.8 mg, 0.0677 mmol) in a Schlenk tube with a magnetic stir bar. The solution was degassed by three freezepump-thaw cycles, the flask was backfilled with N2O (1 atm), and then the reaction was stirred vigorously overnight. Over this time, the solution gradually darkened its blue color. The reaction mixture was filtered through a Celite pad to remove some dark-colored precipitate. After washing the Celite pad with additional CH2Cl2 (2 x 3 mL), the filtrate was concentrated in vacuo. The residue was washed with pentane (3 x 3 mL), removing a light orange supernatant each time. A 5:1 mixture of Et2O/toluene (10 mL) was added to the solid residue. After trituration, the solution was filtered into a tared vial. Volatiles were removed in vacuo to yield a brown solid. Yield: 9.5 mg, 0.0067 mmol, 13%. 1H NMR analysis of this solid revealed 2 with some small impurities including Krypt222 and trace 1N. Analysis of the solid by UV-Vis-NIR and HRMS provided the data shown in the main manuscript (Figure 3a). The remaining, insoluble materials from the combined previous filtrations were diluted with THF and analyzed by UVVis-NIR, showing that the major species is unreacted [1N][K(Krypt222)]. 1 H NMR for 2 (CDCl3, 𝛿): 6.81 (2H), 2.28 (6H), 2.21 (3H). Reaction with Me3NO. A representative procedure is given here from among the various conditions that were examined. A solution of CoCp2 (32.5 mg, 0.172 mmol) in CH2Cl2 (2 mL) was added to a suspension of 1N (109.6 mg, 0.0779 mmol) in CH2Cl2 (3 mL). Immediately, the reaction color changed from inky blue to dull blue with formation of a small amount of precipitate. After 5 min, a solution of Me3NO (8.2 mg, 0.11 mmol) in CH2Cl2 (2 mL) was added. The reaction was allowed to stand overnight, after which the reaction mixture had become cloudy and brown in color. An aliquot was dried in vacuo, reconstituted in CDCl3, and analyzed by 1H NMR, revealing predominantly a mixture of 2, [CoCp2]+ (presumably paired with [1N]-), and Me3NO (see figure below). For the remainder of the reaction mixture, volatiles were removed in vacuo, and toluene (5 mL) was added. After filtration, a yellow-brown filtrate and blue solid were obtained. Analysis of the solid by 1H NMR and UV-Vis-NIR indicated a mixture of 1N and [1N][CoCp2]. The filtrate was concentrated in vacuo. The residue was washed with pentane (3 x 3 mL) and dried in vacuo. The remaining trace solid (est. < 5 mg) was dissolved in CDCl3 and analyzed by 1H NMR, revealing a relatively pure sample of 2 (albeit with trace impurities, see figure below). S10 Me3NO NNN-35 Proton standard parameters, BBO probe 2 [CoCp2]+ crude 1 extracted into toluene washed with pentane 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 f1 (ppm) 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 Figure S12. Representative NMR data from O-atom transfer experiments: crude mixture from Me3NO experiment (top) and toluene-soluble fraction after washing with pentane (bottom). Figure S13. An alternative O2 binding mode calculated to be 13.6 kcal/mol higher in energy than the structure of 2 presented in the main manuscript. S11 REFERENCES (1) Borys, A. M. An Illustrated Guide to Schlenk Line Techniques. Organometallics 2023, 42 (3), 182– 196. https://doi.org/10.1021/acs.organomet.2c00535. (2) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Safe and Convenient Procedure for Solvent Purification. Organometallics 1996, 15 (5), 1518–1520. https://doi.org/10.1021/om9503712. (3) Alayoglu, P.; Chang, T.; Lorenzo Ocampo, M. V.; Murray, L. J.; Chen, Y.-S.; Mankad, N. P. Metal Site-Specific Electrostatic Field Effects on a Tricopper(I) Cluster Probed by Resonant Diffraction Anomalous Fine Structure (DAFS). Inorg. 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