Photoactivatable Ruthenium Complexes Containing Minimal Straining Benzothiazolyl-1,2,3-triazole Chelators for Cancer Treatment.

Ruthenium(II) complexes containing diimine ligands have contributed to the development of agents for photoactivated chemotherapy. Several approaches have been used to obtain photolabile Ru(II) complexes. The two most explored have been the use of monodentate ligands and the incorporation of steric effects between the bidentate ligands and the Ru(II). However, the introduction of electronic effects in the ligands has been less explored. Herein, we report a systematic experimental, theoretical, and photocytotoxicity study of a novel series of Ru(II) complexes Ru1 – Ru5 of general formula [Ru(phen) 2 (N ∧ N′)] 2+ , where N ∧ N′ are different minimal strained ligands based on the 1-aryl-4-benzothiazolyl-1,2,3-triazole (BTAT) scaffold, being CH 3 ( Ru1 ), F ( Ru2 ), CF 3 ( Ru3 ), NO 2 ( Ru4 ), and N(CH 3 ) 2 ( Ru5 ) substituents in the R4 of the phenyl ring. The complexes are stable in solution in the dark, but upon irradiation in water with blue light (λ ex = 465 nm, 4 mW/cm 2 ) photoejection of the ligand BTAT was observed by HPLC-MS spectrometry and UV–vis spectroscopy, with t 1/2 ranging from 4.5 to 14.15 min depending of the electronic properties of the corresponding BTAT, being Ru4 the less photolabile (the one containing the more electron withdrawing substituent, NO 2 ). The properties of the ground state singlet and excited state triplet of Ru1–Ru5 have been explored using density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations. A mechanism for the photoejection of the BTAT ligand from the Ru complexes, in H 2 O, is proposed. Phototoxicity studies in A375 and HeLa human cancer cell lines showed that the new Ru BTAT complexes were strongly phototoxic. An enhancement of the emission intensity of HeLa cells treated with Ru5 was observed in response to increasing doses of light due to the photoejection of the BTAT ligand. These studies suggest that BTAT could serve as a photocleavable protecting group for the cytotoxic bis-aqua ruthenium warhead [Ru(phen) 2 (OH 2 ) 2 ] 2+ . ## Introduction Introduction Cancer continues to be one of the main causes of death worldwide, only behind cardiovascular diseases. 1 Different therapeutic approaches can be selected depending on their aggressiveness, stage, and accessibility, but in general terms, the standard strategy for malignant tumors usually involves resection of the tumor tissue, followed by localized radiotherapy and immunotherapy or chemotherapy. Systemic chemotherapy exhibits considerable limitations related to its typically low selectivity, which leads to numerous undesirable side effects, and drug resistances, relapses and metastatic tumors. 2 , 3 Metal-based therapeutics, with their diverse coordination structures, and high tunability, possess unique properties, when compared to organic compounds. 3 − 10 The use of the stimulus-responsive “prodrug approach” is very appealing to reduce the systemic toxicity, 11 , 12 and in recent years, the photochemical and photophysical properties of precious metal complexes, such as strong spin–orbit coupling (SOC) effects, and tunable excited-state electronic configurations, have been exploited to make light-activated drugs for use as photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) agents that allow to minimize effects on normal tissue through the use of light directed to the tumor, achieving a high temporal and spatial control. 13 − 23 PDT requires the combination of three fundamental components, namely, a nontoxic photosensitizer (PS), ground state molecular oxygen ( 3 O 2 ), and light, to generate highly toxic singlet oxygen ( 1 O 2 ) in a photocatalytic manner and subsequently to induce cancer eradication with low systemic toxicity. 24 Thus, it is important to highlight that the polypyridyl Ru(II) complex TLD-1433, reported by McFarland and co-workers, 25 is being currently studied as a photosensitizer for PDT in Phase II clinical trials for the treatment of nonmuscle invasive bladder cancer (NMIBC). However, despite recent promising research advances, 18 , 26 − 30 due to the hypoxic nature of many native tumors, PDT is frequently limited in its therapeutic effect. 31 − 33 Additionally, oxygen consumption during PDT may exacerbate the tumor’s hypoxic condition, which stimulates tumor proliferation, metastasis, and invasion, resulting in poor treatment outcomes. 34 Compared with traditional PDT, PACT offers an oxygen-independent mechanism, requiring only a photosensitive complex and light to operate, being therefore more suitable for hypoxic tumors. 35 − 37 In addition, PACT can be a useful technique to deliver already known chemotherapeutic or bioactive agents, notably in the case of enzyme inhibitors, 38 where the effect of the desired molecule is directed only to its target after light irradiation. 39 , 40 Octahedral bis- and tris-heteroleptic ruthenium(II) scaffolds represent a highly promising family of uncaging molecules via a photosubstitution reaction, due in part to their ease of modification and tunability as well as their biological and photophysical profiles. 29 , 39 , 41 , 42 For most ruthenium-based PACT agents, once the complex is excited to its triplet metal-to-ligand charge transfer ( 3 MLCT state), it rapidly populates the metal-centered triplet ( 3 MC) state, which removes the ruthenium photocage. The octahedral Ru(II) systems proposed for PACT are often based on the incorporation of distortion into the structure of the coordination complex through the use of, for example, hindering polypyridyl ligands, lowering the energy of dissociative excited states, and increasing the yield of the photosubstitution reaction. 37 , 42 − 44 Alternatively, the photorelease of monodentate ligands can be easier, as they are not subject to the chelate effect. 45 − 47 On the other hand, benzothiazoles represent privileged scaffolds in medicinal chemistry with many applications as anticancer agents. 48 Thus, the 5-fluorobenzothiazole prodrug (Phortress) is a suitable candidate for Phase I clinical trials. 49 We have recently reported a series of benzothiazolyl-1,2,3-triazole molecules ( L1–L5 , Figure 1 A), possessing a push–pull architecture, and exhibiting moderate to high selective antiproliferative activity in A2780 and HeLa cancer cells, together with interesting optical properties based on charge-transfer emission depending on the substituent in the 1,2,3-triazole moiety. 50 Based on this background, herein, we developed a series of novel photoactive Ru(II) octahedral complexes containing benzothiazolyl-1,2,3-triazole (BTAT) chelators lacking steric hindrance ( Ru1–Ru5 , Figure 1 B) in order to explore their potential action as phototherapeutic anticancer agents, together with their photochemistry and theoretical calculations. Figure 1 BTAT ligands (A). New synthesized ruthenium complexes and possible photosubstitution reaction when complex is irradiated with visible light in water (B). ## Results and Discussion Results and Discussion Synthesis and Characterization of Ru(II) Complexes ( Ru1–Ru5 ) The benzothiazolyl-1,2,3-triazole ligands L1–L5 ( Figure 1 A) were prepared by condensation reactions between the respective 1,2,3-triazole-4-carbaldehydes and ortho -aminothiophenol, as recently reported by us ( Scheme S1 ). 50 Important to note, these BTAT derivatives allowed intramolecular charge transfer tuning, apart from exhibiting solvatofluorochromism and selective antiproliferative properties, whereas their coordination chemistry is still unexplored. The synthesis of the new orange air- and moisture-stable complexes Ru1–Ru5 was carried out ( Scheme 1 ) by the reaction of Ru(phen) 2 Cl 2 with the corresponding BTAT ligand in an ethanol–water mixture (1:1) and potassium triflate under microwave in 2 min. Ru1–Ru5 complexes were characterized using multinuclear 1 H and 13 C{ 1 H} NMR spectroscopy ( Figures S1–S32 in the Supporting Information). Final evidence of the correct formation of the compounds has been obtained from the high-resolution ESI + mass spectra ( Figures S33–S37 ). Scheme 1 Synthesis of Complexes Ru1–Ru5 Hydrogen and carbon labels used in 1 H NMR assignments of complexes Ru1–Ru5 are shown in Chart 1 . In the 1 H NMR spectra, the signals of the aromatic protons appear between 5.5 and 11 ppm, being the proton H9 of the 1,2,3-triazole ring ( Chart 1 ) which appears at more downfield as a singlet resonance. In the aliphatic region of the 1 H NMR spectra, only signals from complexes 2 and 5 are observed, due to the CH 3 and N(CH 3 ) 2 substituents on the phenyl ring of the 1-(aryl)-4-(benzothiazolyl)-1,2,3-triazole, respectively. The stacked 1 H NMR spectra of all complexes for comparison is shown in Figure 2 . Chart 1 Hydrogen and Carbon Labels Used for BTAT Ligands in 1 H NMR Assignments of Complexes Ru1–Ru5 Figure 2 1 H NMR spectra of complexes Ru1–Ru5 in DMSO- d 6 at 293 K, aromatic region. 1 H NMR signals of the benzothiazole fragment (H2–H5) appear as two doublets (H2 and H5) and two pseudotriplets (H3 and H4). The resonances of the protons of the phenyl ring H11, H12, H14, and H15 appear as doublets at different chemical shift values since they are influenced, like the H9 proton, by the electron-donating or electron-withdrawing capacity of the substituent on the ligand 1-aryl-4-benzothiazolyl-1,2,3-triazole. With these characteristic signals as starting point, the resonances of the 1 H and 13 C NMR were assigned via the observed 1 H– 1 H COSY, 1 H– 1 H NOESY, 13 C– 1 H HSQC, and 13 C– 1 H HMBC correlations ( Figures S1–S32 ). The resonances of the benzothiazole fragment of the H2 and H5 protons, the most shielded and the most unshielded, respectively have been assigned from the 13 C– 1 H HMBC spectra. In these spectra, the correlations of C1 with H2, C6 with H5, and C8 with H9 were observed. From these assignments, the resonances of all the protons and carbons of the BTAT ligands can be assigned and are listed in Table S9 . These assignments are also supported by the 1 H– 1 H NOESY spectra in which correlations between the H5 proton and phen rings protons are observed. X-ray Crystallography The coordination geometries of cations in complexes Ru1 , Ru2 , and Ru5 were confirmed by single-crystal X-ray crystallography ( Figure 3 and Table S1 ). The complexes crystallize in the triclinic space group P 1̅. The ORTEP plots of the structures of the cations are shown in Figure 3 and Table 1 contains selected bond lengths and angles. There are two interstitial ethanol molecules in the asymmetric unit of Ru2 and one acetonitrile molecule in the asymmetric unit of Ru5 . The cations exhibit distorted octahedral geometries with ruthenium–nitrogen (phen) bond lengths similar to the values reported for a ruthenium(II) tris–diimine complex with values between 2.044 and 2.067 Å for the two phen ligands. 51a The bond lengths Ru–N1 (benzothiazole moiety) are longer (approximately 2.11 Å), while the Ru–N2 (triazole moiety) are the shortest (approximately 2.03 Å), 51b maybe due to the fact that the smaller ring size for a triazole donor and the absence of a C–H proton adjacent to the coordinating N atom, which is present for other N-heterocyclic ligands, and making the triazole donor less sterically demanding. The N–M–N bite angles range from 77.4° for BTAT to 80.2° for phen, being the values found for the BTAT ligands similar in the three complexes. The dihedral angles N1–C7–C8–N2 are −1.47, −0.81, and −0.22° for complexes Ru1 , Ru2 , and Ru5 , respectively, showing the quasi-planar coordination of the BTAT ligands. Apart from the cation–anion triflate Coulomb interactions, the packing in the structures of Ru1 , Ru2 , and Ru5 are organized by C–H···N, C–H···O, and O–H···O intra- and intermolecular interactions ( Tables S2 and S3 and Figures S38–S39 ). Intermolecular π–π interactions involving the phen rings are also observed ( Figures S40–S42 ). The usual π-interaction is an offset or slipped stacking and the ring normal and the vector between the ring centroids form an angle of about 20° up to centroid–centroid distances of 3.8 Å. 52 As all the π–π interactions in our compounds have shorter centroid distances (3.5300(12) to 3.7770(17) Å; Table S4 ) and the angle between the ring normal and the vector Cg–Cg is in the range of 19.2 to 28.9° ( Table S4 ), the π–π interactions in these compounds belong to strong π–π interactions. In these complexes, the π–π interactions form the chains along the c axis. Figure 3 ORTEP plots of the cations of complexes Ru1 (left), Ru2 (middle), and Ru5 (right). For clarity, counterions and hydrogen atoms have been omitted. Ellipsoids have been represented at 50% probability. CCDC reference numbers are 2284059 for Ru1 , 2284060 for Ru5 , and 2284061 for Ru2 . Table 1 Selected Bond Lengths (Å) and Angles (deg) for Complexes Ru1 , Ru2 , and Ru5   Ru1 Ru2 Ru5 Ru(01)–N(1) 2.117(2) 2.1167(18) 2.1117(14) Ru(01)–N(2) 2.027(2) 2.0359(18) 2.0374(14) Ru(01)–N(5) a 2.063(2) 2.0678(19) 2.0622(14) Ru(01)–N(6) b 2.044(2) 2.0501(18) 2.0677(14) Ru(01)–N(7) c 2.059(2) 2.0651(18) 2.0451(13) Ru(01)–N(8) d 2.065(2) 2.0607(19) 2.0666(13) N(1)–Ru(01)–N(2) 77.53(9) 77.91(7) 77.36(5) N(5) a –Ru(01)–N(6) b 79.68(9) 79.95(7) 79.90(5) N(7) c –Ru(01)–N(8) d 80.14(9) 80.17(7) 79.88(5) a In complex Ru5 : N(6). b In complex Ru5 : N(7). c In complex Ru5 : N(8). d In complex Ru5 : N(9). Photophysical Properties The UV–visible absorption spectra of complexes Ru1 – Ru5 have been recorded in ACN and water solutions 10 –5 M ( Table 2 and Figures 4 and S43 and S54 ). All complexes display sharp and intense bands in the region below 350 nm corresponding to singlet intraligand 1 ππ* transitions that are allocated to the polypyridyl and BTAT ligands. On the other hand, the broad bands of lower intensity between 400 and 500 nm could be assigned to singlet metal-to-ligand charge transfer transitions ( 1 MLCT), from dπ orbitals of Ru to the π* orbitals of the ligand(s). Our complexes contain two phen ligands and the BTAT ligand contains a delocalized π system that in some cases could have an intraligand charge transfer (ILCT) character due to the more polarizing groups. Therefore, as expected, the absorption spectra showed characteristics of the BTAT ligands. The calculated UV–vis spectra ( Figure 4 bottom, in water, and Figure S54 , in ACN) were found to be in accordance with the experimental data ( Figure 4 top). Observing the absorption spectra along the series of complexes Ru1–Ru5 allows us to conclude that the contributions of the BTAT ligands to the LUMO in complexes Ru1–Ru3 is very small and therefore these orbitals are mainly delocalized on the other N ∧ N ligands (phen). The differences are greater in the spectra of complexes Ru4 and Ru5 that are blue-shifted, which could indicate a greater contribution of the BTAT ligands in the LUMO or in HOMO, respectively (see below, Figure 5 ). Table 2 UV–Visible Absorption Data for Complexes Ru1–Ru5 complex   λ abs /nm (ε/dm 3  mol – 1  cm – 1 ) Ru1 ACN 224 (65,150), 263 (76,690), 293 sh (29,850), 313 sh (25,640), 327 sh (18,700), 416 (14,200)   H 2 O 221 (67,110), 262 (77,840), 294 sh (29,950), 312 sh (25,710), 322 sh (20,200), 417 (14,280) Ru2 ACN 224 (71,110), 263 (80,960), 293 sh (30,070), 312 sh (25,030), 327 sh (16,880), 415 (14,910)   H 2 O 221 (64,380), 262 (72,390), 290 sh (28,400), 310 sh (22,660), 324 sh (16,240), 423 (13,380) Ru3 ACN 224 (74,470), 263 (81,440), 293 sh (29,230), 312 sh (24,580), 326 sh (17,780), 410 (15,100)   H 2 O 221 (73,820), 262 (80,270), 290 sh (30,330), 314 sh (23,340), 325 sh (17,380), 417 (14,900) Ru4 ACN 222 (70,220), 263 (83,260), 290 sh (30,920), 314 sh (22,530), 329 sh (18,700), 406 (16,870)   H 2 O 219 (82,830) sh, 262 (92,260), 288 sh (37,510), 313 sh (26,480), 326 sh (22,210), 407 (19,420) Ru5 ACN 221 (7500), 264 (80,630), 316 sh (27,370), 325 sh (27,000), 345 sh (21,660), 433 (15,360)   H 2 O 221 (81,490), 262 (88,120), 291 sh (34,630), 314 sh (31,160), 406 (17,390) Figure 4 Experimental (top) and calculated (bottom) UV–vis absorption spectra of complexes Ru1–Ru5 in H 2 O. Figure 5 HOMO–LUMO orbitals of the Ru complexes Ru1 – Ru5 in H 2 O, singlet ground state, S 0 , obtained by DFT calculations (top). Energies (in au) and energy gaps (in kJ/mol) of the HOMO and LUMO of compounds Ru1–Ru5 , obtained by DFT calculations in H 2 O solution (bottom). The complexes Ru1–Ru3 present dual emission at room temperature when the excitation was made by light with the appropriate wavelength see Table 3 and Figure S44 . Fluorescence in the ultraviolet region corresponding to 1 IL states and weak yellow phosphorescence from 3 MLCT states. This behavior has been previously observed in Ru(II) complexes. 53 − 57 Table 3 Luminescence Data, 1 O 2 Generation Quantum Yields (Φ Δ ), and Half-Life ( t 1/2 ) for Photoejection f for Complexes Ru1–Ru5 complex λ em , nm (λ exc ) a τ em , ns (%) b Φ P (λ em /nm) b , c Φ Δ (air) d t 1/2  (min) e Ru1 362 (305) 8.4 (56); 275 (44) <0.01 (532) 0.02 4.85   532 (390)         Ru2 362 (305) 1.8 (3); 419 (97) <0.01 (579) 0.02 4.55   579 (400)         Ru3 370 (320) 7.8 (83); 202 (17) 0.01 (514) 0.03 5.54   514 (320)         Ru4 517 (320) 7.9 (88); 171 (12) <0.01 (517) 0.04 14.15 Ru5 516 (320) 7.1 (80); 95 (20) 0.09 (516) 0.01 6.78 a In ACN solutions, 294 K. b In ACN solutions, 294 K, Ar. c Absolute emission quantum yield. d Reference [Ru(bpy) 3 ] 2+ : ACN Φ Δ = 0.56. 58 e In water. f Measured using a 3 mW cm –2 465 nm LED. The 1 IL fluorescent emission observed at 362 nm in ACN for complexes Ru1 and Ru2 was more intense than that observed for complex Ru3 . All the complexes present weak yellow phosphorescence from 3 MLCT states at room temperature in ACN solutions, see Table 3 . The emission from 3 MLCT varies little from one compound to another, which could be indicate of the π* acceptor orbital of 3 MLCT is similar in each complex. Although the emission intensity around 500 nm is not very intense, the decay profile of the lifetime of the excited state was found to be biexponential in nature. The biexponential decay shows a short (2–8 ns) and a long (95–420 ns) component. This biexponential decay indicates that multiple triplet excited states are involved in the emission profile. DFT Calculations The properties of the ground state singlet and excited state triplet of complexes Ru1–Ru5 have been explored using DFT and TD-DFT calculations. Optimized geometries were obtained in ACN and water. The structures of the singlet ground and triplet excited states, in water, are shown in Figures S52–S53 . The structures obtained in ACN are essentially the same as those obtained in water. Interestingly, for all Ru1–Ru5 complexes, the N1–Ru distance, between the Ru atom and the BTAT nitrogen, is consistently larger in the T 1 state, about 0.49 Å, compared to that in the S 0 state ( Table S5 ). This indicates that the excitation to the triplet spin state induces a weakening of such a bond. The electronic structures of the ground state of the complexes were characterized according to their frontier molecular orbitals HOMO and LUMO, Figure 5 top. The nature of the HOMO changes with the substituents of the BTAT ligand, as can be seen in Figure 5 . Thus, the HOMO of complexes Ru1–Ru4 was predominantly ruthenium d-orbital character but with some additional BTAT π-contribution and in complex Ru5 was instead located on the triazole rings and its phenyl–N(CH 3 ) 2 . The LUMO was dominated by π* contribution from the phen ligands in complexes Ru1–Ru3 . However, in complex Ru4 it comprises the triazole ring and its 4-NO 2 -phenyl substituent. The LUMO was progressively stabilized from Ru5 , Ru1 , Ru2 , Ru3 , to Ru4 as the electron-withdrawing character of the substituent of BTAT ligands increases ( Figure 5 bottom). The HOMO was also stabilized in similar extent leading the shorter HOMO–LUMO gap for Ru4 (164.5 kJ/mol) following by Ru5 (173.6 kJ/mol) and compared to Ru1–Ru3 (202–205 kJ/mol) ( Figure 5 bottom). Very similar orbital shapes and energy values were obtained also in ACN. In all complexes, the T 1 state mainly originates from HOMO → LUMO (70%) transition ( Table S6 ) and characterized as 3 MLCT. The other high energy emission band are derived from triplet states which are predominantly ligand centered 3 LLCT/ 3 ILCT. Finally, it is interesting to note that the S 0 –T 1 transition is accompanied by a change in the dipole moment ( Table S8 ), which is greater for Ru5 and Ru1 , in particular in water and Ru4 exhibited the lowest change in the dipolar moment in the S 0 –T 1 transition. Photochemistry Studies The capacity of complexes Ru1–Ru5 to undergo photosubstitution reactions was explored by UV–vis. In the dark, UV–vis measurements revealed a remarkable stability in water at 310 K of Ru1–Ru5 over a period of 120 h, as shown in Figure S45 no changes in their UV–vis spectra were observed. Conversely, light irradiation (λ = 465 nm, 4 mW/cm 2 ) of water solutions of the new complexes provokes with the time noticeable changes in their absorption spectra, as can be seen in Figure 6 for Ru1 and in Figure S46 for complexes Ru2–Ru5 . Upon light irradiation, the metal to ligand charge transfer (MLCT) band centered between 410 and 425 nm was bathochromically shifted to a broad MLCT band centered at 478 nm, with time a clear isosbestic point around 450 nm was observed along with decreasing absorption intensity within 250–350 nm. Figure 6 Changes in absorption spectra of Ru1 in H 2 O (10 –5 M) as observed upon irradiation with blue light (λ ex = 465 nm, 4 mW/cm 2 ). Parallel HPLC experiments for the Ru complexes (vide infra) evidenced the existence of a photoejection process of the corresponding BTAT ligand ( Figures S47–S51 for Ru1–Ru5 ). The evaluation of these changes in the absorption spectra demonstrated variation in t 1/2 values through all complexes ( Figure 6 and Table 3 ). Under these conditions complexes Ru1–Ru3 became more labile, t 1/2 between 4.5 and 5.5 min, instead complex Ru4 containing the BTAT ligand with the more electron-withdrawing substituent (−NO 2 ) was the less active, t 1/2 = 14.15 min. Complex Ru5 with BTAT ligand with the substituent more electron donating [−N(CH 3 ) 2 ] shows t 1/2 around 6.8 min. Except for complex Ru4 , these complexes exhibit similar efficient photosubstitution than other complexes with bidentate ligands and steric hindrance which have t 1/2 < 5 min. In these photolabile compounds the 3 MLCT excited states generated photochemically are quenched by low lying metal-centered ( 3 MC) triplet excited states that lead to nonradiative decay and photosubstitution. 59 , 60 As stated above, the release of the BTAT ligand from the corresponding prodrug Ru1–Ru5 could be confirmed by HPLC using ACN solutions of the complexes at time zero and after 1 h of irradiation with blue light (λ ex = 465 nm, 4 mW/cm 2 ) ( Figures S47–S51 ). As observed in Figure 7 A, the chromatograms show the disappearance of complex Ru1 peak ( t R = 9.9 min), while a new signal appears at a higher retention time ( t R = 15.6 min) corresponding to the BTAT ligand, together with the peak at a lower retention time ( t R = 6.7 min), assigned to the Ru photoproduct [Ru(phen) 2 (ACN) 2 ] 2+ (calcd m / z = 272.05, Figure 7 B), a labile complex that could potentially bind to DNA or other biomolecules within the cell. 61 Figure 7 Determination of photoejection products of Ru1 by HPLC-MS. (A) HPLC chromatogram (DAD = 280 nm) of Ru1 at t = 0 (top) and after being irradiated for 1 h with blue light, λ ex = 465 nm, 4 mW/cm 2 . (B) Mass spectra of the peaks of interest extracted from the chromatograms. The remarkable N1–Ru bond weakening in the triplet state ( Table S5 ) suggests a possible mechanism involved in the photoejection of the BTAT ligand. An example of such a mechanism, considering compound Ru5 , is shown in Figure 8 . The proposed mechanism of ligand photoejection for complex Ru5 , in H 2 O, was obtained by DFT calculations. The ground state reactant, S 0 , in the presence of two water molecules, is excited into the triplet state T 1 ; in the excited state, the two water molecules substitute the chelating ligand and finally the substitution product decays from T 1 into the ground state S 0 . Analogous result is obtained for compound Ru4 ( Figure S55 ), showing that the slower reactivity observed of compound Ru4 is attributable to the larger t 1/2 photoejection half time ( Table 3 ). Figure 8 Proposed mechanism of ligand photoejection for complex Ru5 , in H 2 O, obtained by DFT calculations. For ruthenium polypyridyl compounds photosubstitution reactions compete with phosphorescence and 1 O 2 generation. 62 The capacity of Ru1–Ru5 to generate 1 O 2 upon irradiation at 465 nm (4 mW/cm 2 ) in aerated CH 3 CN was examined by measuring the absorption of 1,3-diphenylisobenzofuran (DPBF) at 413 nm in acetonitrile solution. The complexes showed comparatively low but nonzero Φ Δ values ( Table 3 ), suggesting that a PDT type II mechanism can hardly explain the phototoxicity observed in normoxic conditions. 63 Important to note, the related bis-aqua ruthenium complex [Ru(Ph 2 phen) 2 (H 2 O) 2 ] 2+ has been probed able to bind to proteins or nucleic acids and subsequently generate ROS. 63 Photobiological Studies Cell Uptake First, the cellular uptake of two representative Ru(II) complexes were investigated in HeLa cells (15 μM, 2 h incubation) by using confocal microscopy. Excitation was carried out with a blue light laser (λ exc = 450 nm). As shown in Figure 9 , a strong fluorescence signal was clearly observed inside cancer cells, thereby confirming an excellent and rapid cellular uptake. Worthy of note, no cell toxicity was appreciated during the experiments. The pattern of staining of Ru4 and Ru5 was similar and excluded nuclei as their preferential target. Indeed, the distribution of the red fluorescence emission of both compounds was found across the cytoplasm in punctate deposits, suggesting selective accumulation within intracellular vesicles. Important to note, it has been previously shown that the free ligand L5 was observed (λ exc = 405 nm) evenly distributed around cell cytoplasm but not within nuclei of Hela cells, although only a partial correlation with MitoTracker Green (MTG) was observed. 50 Figure 9 Confocal microscopy images of HeLa cells incubated with Ru(II) complexes Ru4 and Ru5 [15 μM] for 2 h. Scale bar 30 μm. Dark Cytotoxicity Ideally, the nonirradiated form of a given PACT compound should display low toxicity, while the irradiated form should exert biological activity. 64 To assess the photoactivity of the present Ru(II) PACT compounds under biologically relevant conditions, we performed a screening in a panel of cell lines including both cancerous and noncancerous cells under dark conditions. For this screening, ovarian cancer (A2780), cervical cancer (HeLa), and melanoma (A375) cells along with nontumorigenic ovarian (CHO) cells were used; the clinical drug cisplatin was included for comparative purposes. As shown in Table 4 , none of the complexes displayed antiproliferative activity after 48 h incubation, with negligible toxicity up to 100 μM, in contrast to cisplatin, which exerted cytotoxic activity against both normal and cancer cells in the low micromolar range. Importantly, no cell toxicity was found in normal CHO cells, which is an important requirement of anticancer drug development. Overall, these results validated our compounds as potential PACT agents since minimal dark toxicity is highly desirable in anticancer phototherapy. Table 4 IC 50 Values (μM) of Complexes Ru1–Ru5 and CDDP at 48 h in the Dark compound A2780 HeLa A375 CHO Ru1 >100 >100 >100 >100 Ru2 >100 >100 >100 >100 Ru3 >100 >100 >100 >100 Ru4 >100 >100 >100 >100 Ru5 >100 >100 >100 >100 CDDP 1.9 ± 0.1 21 ± 2   7.8 ± 0.4 Photoactivation Studies Once demonstrated that the compounds were not active under dark conditions, their ability to become toxic to cancer cells was evaluated using low doses of a visible light trigger (LED source at 465 nm, 4 mW/cm 2 , 1 h). Two cancer cell lines, namely, HeLa and A375 cells, were employed to illustrate this effect, and the phototoxic index (PI), defined as the ratio of dark to light IC 50 values, served as a measure of the photoactivity. Since the complexes were deemed as inactive up to 100 μM, we included higher concentrations both in the presence or absence of light trigger to better characterize their photoactivation. To our delight, no cell inhibition was found with any complex, which corroborates their suitability for PACT ( Table 4 ). In the presence of light irradiation, all the complexes showed antiproliferative activity in normoxic conditions (21% O 2 ) against cancer cells, with IC 50 values within the same micromolar range ( Table 5 ). In general, higher photoactivation was found in A375 cells than in HeLa cells (PI values ranging from >3.6 to >29.1 compared to >6.8 to >14.8, respectively). Compound Ru4 , bearing the nitro moiety, exhibited the higher phototoxic action in both cancer cell lines (PI values of >26.6 in A375 and >14.8 in HeLa), followed by compound Ru1 . Interestingly, complex Ru3 was barely photoactivated upon visible light irradiation against these cancer cell lines (PI values of >6.8 in both cancer cell lines). Overall, these results indicate that photoactivation of Ru(II) complexes bearing benzothiazolyl-1,2,3-triazole BTAT chelators can be achieved inside cancer cells. In contrast, light treatment had very little effect on the viability of cells treated with BTAT ligands, which exhibited very low cytotoxicity in each of the cell lines tested after 1 h treatment followed by 1 h irradiation under blue light (IC 50 > 100 μM, Table S8 ). While our study demonstrates the noncytotoxic nature of the ligands after a 2 h treatment, in a previous study 50 we reported cytotoxic effects after a 48 h exposure in the absence of light. This difference could be due to the prolonged accumulation of the ligands within HeLa cells over 48 h compared to the 2 h duration in our study. In our current research, we have demonstrated that the ruthenium complexes facilitate the entry of the ligands into the cells, which subsequently become cytotoxic upon irradiation and breakdown under blue light irradiation. Table 5 IC 50 Values (μM) and Phototoxicity Index (P.I.) under Normoxia of Ru1–Ru5 a   HeLa A375 compound dark blue light P.I HeLa dark blue light P.I A375 Ru1 >500 48.0 ± 3.4 >10.4 >500 20.55 ± 1.5 >24.4 Ru2 >500 58.5 ± 4.6 >8.6 >500 17.2 ± 3.0 >29.1 Ru3 >500 73.5 ± 4.2 >6.8 >500 73.5 ± 4.3 >6.8 Ru4 >500 33.8 ± 5.5 >14.8 >500 18.8 ± 3.7 >26.6 Ru5 >500 41.8 ± 3.9 >12.0 >500 27.1 ± 6.2 >18.5 a Compounds were incubated with the cells 1 h and then irradiated with blue light (λ = 465 nm, 4 mW/cm 2 ) for 1 h. The phototoxicity index (P.I.) was calculated as IC 50 dark /IC 50 λ=465nm . We have also performed the phototoxicity test in both A375 and HeLa cell lines under hypoxic conditions. The results reveal a contrasting pattern, where the compounds exhibit partially greater toxicity under normoxia conditions compared to hypoxia ( Table 6 ). Table 6 IC 50 Values (μM) and Phototoxicity Index (P.I.) under Hypoxia (2% O 2 ) of the Compounds Ru1 – Ru5 a   HeLa A375 compound dark blue light P.I HeLa dark blue light P.I A375 Ru1 >500 70.2 ± 5.0 >7.1 >500 66.9 ± 6.1 >7.5 Ru2 >500 46.0 ± 3.9 >10.9 >500 >100   Ru3 >500 85.2 ± 4.4 >5.9 >500 >100   Ru4 >500 41.7 ± 2.7 >12.0 >500 51.9 ± 2.4 >9.6 Ru5 >500 57.2 ± 6.4 >8.7 >500 >100   a Compounds were incubated with the cells 1 h and then irradiated with blue light (λ = 465 nm, 4 mW/cm 2 ) for 1 h. The phototoxicity index (P.I.) was calculated as IC 50 dark /IC 50 λ=465nm . Lower phototoxicity of light-activated drugs in oxygen-deprived environments can be a sign either that the phototoxicity under normoxia involves some form of a photodynamic effect, or that the hypoxic cells are more difficult to kill than normoxic cells, as hypoxia triggers a range of resistance effects. 64 , 65 Confocal microscopy imaging of HeLa cells treated with Ru5 under two different irradiation timings reveals distinct cellular responses. After 1 min of irradiation under blue light, no cell cytotoxicity was observed ( Figure 10 , first row, bright field image). In contrast, following 1 h irradiation, discernible cell damage is evident, characterized by the presence of cytoplasmic blebbing and notable morphology changes ( Figure 10 , orange triangles). These imaging data provide robust support for the photocytotoxicity results previously described, demonstrating the heightened cytotoxic effects of Ru BTAT compounds after blue light irradiation, as opposed to their relative dark condition or cells treated with the ligands alone. Figure 10 Confocal microscopy images of HeLa cells treated with Ru5 at 15 μM for 1 h followed by blue light irradiation for 1 min and 1 h. λ ex : 405 nm, λ em : 516 ± 30 nm. The control group was maintained under untreated conditions. Scale bar: 10 μm. The orange triangles indicate the cellular membrane irregularities or blebs associated with apoptosis. The higher fluorescence signal observed in cells treated for 1 h under blue light, in comparison to 1 min of irradiation, is attributed mainly to the photoejection of the ligand after irradiation. 50 This comprehensive examination through confocal microscopy provides valuable insights into the photoresponsive behavior of Ru5 and its impact on cellular structures, further reinforcing its potential as an effective photoactive agent in phototherapy applications. ## Synthesis and Characterization of Ru(II) Complexes ( Synthesis and Characterization of Ru(II) Complexes ( Ru1–Ru5 ) The benzothiazolyl-1,2,3-triazole ligands L1–L5 ( Figure 1 A) were prepared by condensation reactions between the respective 1,2,3-triazole-4-carbaldehydes and ortho -aminothiophenol, as recently reported by us ( Scheme S1 ). 50 Important to note, these BTAT derivatives allowed intramolecular charge transfer tuning, apart from exhibiting solvatofluorochromism and selective antiproliferative properties, whereas their coordination chemistry is still unexplored. The synthesis of the new orange air- and moisture-stable complexes Ru1–Ru5 was carried out ( Scheme 1 ) by the reaction of Ru(phen) 2 Cl 2 with the corresponding BTAT ligand in an ethanol–water mixture (1:1) and potassium triflate under microwave in 2 min. Ru1–Ru5 complexes were characterized using multinuclear 1 H and 13 C{ 1 H} NMR spectroscopy ( Figures S1–S32 in the Supporting Information). Final evidence of the correct formation of the compounds has been obtained from the high-resolution ESI + mass spectra ( Figures S33–S37 ). Scheme 1 Synthesis of Complexes Ru1–Ru5 Hydrogen and carbon labels used in 1 H NMR assignments of complexes Ru1–Ru5 are shown in Chart 1 . In the 1 H NMR spectra, the signals of the aromatic protons appear between 5.5 and 11 ppm, being the proton H9 of the 1,2,3-triazole ring ( Chart 1 ) which appears at more downfield as a singlet resonance. In the aliphatic region of the 1 H NMR spectra, only signals from complexes 2 and 5 are observed, due to the CH 3 and N(CH 3 ) 2 substituents on the phenyl ring of the 1-(aryl)-4-(benzothiazolyl)-1,2,3-triazole, respectively. The stacked 1 H NMR spectra of all complexes for comparison is shown in Figure 2 . Chart 1 Hydrogen and Carbon Labels Used for BTAT Ligands in 1 H NMR Assignments of Complexes Ru1–Ru5 Figure 2 1 H NMR spectra of complexes Ru1–Ru5 in DMSO- d 6 at 293 K, aromatic region. 1 H NMR signals of the benzothiazole fragment (H2–H5) appear as two doublets (H2 and H5) and two pseudotriplets (H3 and H4). The resonances of the protons of the phenyl ring H11, H12, H14, and H15 appear as doublets at different chemical shift values since they are influenced, like the H9 proton, by the electron-donating or electron-withdrawing capacity of the substituent on the ligand 1-aryl-4-benzothiazolyl-1,2,3-triazole. With these characteristic signals as starting point, the resonances of the 1 H and 13 C NMR were assigned via the observed 1 H– 1 H COSY, 1 H– 1 H NOESY, 13 C– 1 H HSQC, and 13 C– 1 H HMBC correlations ( Figures S1–S32 ). The resonances of the benzothiazole fragment of the H2 and H5 protons, the most shielded and the most unshielded, respectively have been assigned from the 13 C– 1 H HMBC spectra. In these spectra, the correlations of C1 with H2, C6 with H5, and C8 with H9 were observed. From these assignments, the resonances of all the protons and carbons of the BTAT ligands can be assigned and are listed in Table S9 . These assignments are also supported by the 1 H– 1 H NOESY spectra in which correlations between the H5 proton and phen rings protons are observed. ## X-ray Crystallography X-ray Crystallography The coordination geometries of cations in complexes Ru1 , Ru2 , and Ru5 were confirmed by single-crystal X-ray crystallography ( Figure 3 and Table S1 ). The complexes crystallize in the triclinic space group P 1̅. The ORTEP plots of the structures of the cations are shown in Figure 3 and Table 1 contains selected bond lengths and angles. There are two interstitial ethanol molecules in the asymmetric unit of Ru2 and one acetonitrile molecule in the asymmetric unit of Ru5 . The cations exhibit distorted octahedral geometries with ruthenium–nitrogen (phen) bond lengths similar to the values reported for a ruthenium(II) tris–diimine complex with values between 2.044 and 2.067 Å for the two phen ligands. 51a The bond lengths Ru–N1 (benzothiazole moiety) are longer (approximately 2.11 Å), while the Ru–N2 (triazole moiety) are the shortest (approximately 2.03 Å), 51b maybe due to the fact that the smaller ring size for a triazole donor and the absence of a C–H proton adjacent to the coordinating N atom, which is present for other N-heterocyclic ligands, and making the triazole donor less sterically demanding. The N–M–N bite angles range from 77.4° for BTAT to 80.2° for phen, being the values found for the BTAT ligands similar in the three complexes. The dihedral angles N1–C7–C8–N2 are −1.47, −0.81, and −0.22° for complexes Ru1 , Ru2 , and Ru5 , respectively, showing the quasi-planar coordination of the BTAT ligands. Apart from the cation–anion triflate Coulomb interactions, the packing in the structures of Ru1 , Ru2 , and Ru5 are organized by C–H···N, C–H···O, and O–H···O intra- and intermolecular interactions ( Tables S2 and S3 and Figures S38–S39 ). Intermolecular π–π interactions involving the phen rings are also observed ( Figures S40–S42 ). The usual π-interaction is an offset or slipped stacking and the ring normal and the vector between the ring centroids form an angle of about 20° up to centroid–centroid distances of 3.8 Å. 52 As all the π–π interactions in our compounds have shorter centroid distances (3.5300(12) to 3.7770(17) Å; Table S4 ) and the angle between the ring normal and the vector Cg–Cg is in the range of 19.2 to 28.9° ( Table S4 ), the π–π interactions in these compounds belong to strong π–π interactions. In these complexes, the π–π interactions form the chains along the c axis. Figure 3 ORTEP plots of the cations of complexes Ru1 (left), Ru2 (middle), and Ru5 (right). For clarity, counterions and hydrogen atoms have been omitted. Ellipsoids have been represented at 50% probability. CCDC reference numbers are 2284059 for Ru1 , 2284060 for Ru5 , and 2284061 for Ru2 . Table 1 Selected Bond Lengths (Å) and Angles (deg) for Complexes Ru1 , Ru2 , and Ru5   Ru1 Ru2 Ru5 Ru(01)–N(1) 2.117(2) 2.1167(18) 2.1117(14) Ru(01)–N(2) 2.027(2) 2.0359(18) 2.0374(14) Ru(01)–N(5) a 2.063(2) 2.0678(19) 2.0622(14) Ru(01)–N(6) b 2.044(2) 2.0501(18) 2.0677(14) Ru(01)–N(7) c 2.059(2) 2.0651(18) 2.0451(13) Ru(01)–N(8) d 2.065(2) 2.0607(19) 2.0666(13) N(1)–Ru(01)–N(2) 77.53(9) 77.91(7) 77.36(5) N(5) a –Ru(01)–N(6) b 79.68(9) 79.95(7) 79.90(5) N(7) c –Ru(01)–N(8) d 80.14(9) 80.17(7) 79.88(5) a In complex Ru5 : N(6). b In complex Ru5 : N(7). c In complex Ru5 : N(8). d In complex Ru5 : N(9). ## Photophysical Properties Photophysical Properties The UV–visible absorption spectra of complexes Ru1 – Ru5 have been recorded in ACN and water solutions 10 –5 M ( Table 2 and Figures 4 and S43 and S54 ). All complexes display sharp and intense bands in the region below 350 nm corresponding to singlet intraligand 1 ππ* transitions that are allocated to the polypyridyl and BTAT ligands. On the other hand, the broad bands of lower intensity between 400 and 500 nm could be assigned to singlet metal-to-ligand charge transfer transitions ( 1 MLCT), from dπ orbitals of Ru to the π* orbitals of the ligand(s). Our complexes contain two phen ligands and the BTAT ligand contains a delocalized π system that in some cases could have an intraligand charge transfer (ILCT) character due to the more polarizing groups. Therefore, as expected, the absorption spectra showed characteristics of the BTAT ligands. The calculated UV–vis spectra ( Figure 4 bottom, in water, and Figure S54 , in ACN) were found to be in accordance with the experimental data ( Figure 4 top). Observing the absorption spectra along the series of complexes Ru1–Ru5 allows us to conclude that the contributions of the BTAT ligands to the LUMO in complexes Ru1–Ru3 is very small and therefore these orbitals are mainly delocalized on the other N ∧ N ligands (phen). The differences are greater in the spectra of complexes Ru4 and Ru5 that are blue-shifted, which could indicate a greater contribution of the BTAT ligands in the LUMO or in HOMO, respectively (see below, Figure 5 ). Table 2 UV–Visible Absorption Data for Complexes Ru1–Ru5 complex   λ abs /nm (ε/dm 3  mol – 1  cm – 1 ) Ru1 ACN 224 (65,150), 263 (76,690), 293 sh (29,850), 313 sh (25,640), 327 sh (18,700), 416 (14,200)   H 2 O 221 (67,110), 262 (77,840), 294 sh (29,950), 312 sh (25,710), 322 sh (20,200), 417 (14,280) Ru2 ACN 224 (71,110), 263 (80,960), 293 sh (30,070), 312 sh (25,030), 327 sh (16,880), 415 (14,910)   H 2 O 221 (64,380), 262 (72,390), 290 sh (28,400), 310 sh (22,660), 324 sh (16,240), 423 (13,380) Ru3 ACN 224 (74,470), 263 (81,440), 293 sh (29,230), 312 sh (24,580), 326 sh (17,780), 410 (15,100)   H 2 O 221 (73,820), 262 (80,270), 290 sh (30,330), 314 sh (23,340), 325 sh (17,380), 417 (14,900) Ru4 ACN 222 (70,220), 263 (83,260), 290 sh (30,920), 314 sh (22,530), 329 sh (18,700), 406 (16,870)   H 2 O 219 (82,830) sh, 262 (92,260), 288 sh (37,510), 313 sh (26,480), 326 sh (22,210), 407 (19,420) Ru5 ACN 221 (7500), 264 (80,630), 316 sh (27,370), 325 sh (27,000), 345 sh (21,660), 433 (15,360)   H 2 O 221 (81,490), 262 (88,120), 291 sh (34,630), 314 sh (31,160), 406 (17,390) Figure 4 Experimental (top) and calculated (bottom) UV–vis absorption spectra of complexes Ru1–Ru5 in H 2 O. Figure 5 HOMO–LUMO orbitals of the Ru complexes Ru1 – Ru5 in H 2 O, singlet ground state, S 0 , obtained by DFT calculations (top). Energies (in au) and energy gaps (in kJ/mol) of the HOMO and LUMO of compounds Ru1–Ru5 , obtained by DFT calculations in H 2 O solution (bottom). The complexes Ru1–Ru3 present dual emission at room temperature when the excitation was made by light with the appropriate wavelength see Table 3 and Figure S44 . Fluorescence in the ultraviolet region corresponding to 1 IL states and weak yellow phosphorescence from 3 MLCT states. This behavior has been previously observed in Ru(II) complexes. 53 − 57 Table 3 Luminescence Data, 1 O 2 Generation Quantum Yields (Φ Δ ), and Half-Life ( t 1/2 ) for Photoejection f for Complexes Ru1–Ru5 complex λ em , nm (λ exc ) a τ em , ns (%) b Φ P (λ em /nm) b , c Φ Δ (air) d t 1/2  (min) e Ru1 362 (305) 8.4 (56); 275 (44) <0.01 (532) 0.02 4.85   532 (390)         Ru2 362 (305) 1.8 (3); 419 (97) <0.01 (579) 0.02 4.55   579 (400)         Ru3 370 (320) 7.8 (83); 202 (17) 0.01 (514) 0.03 5.54   514 (320)         Ru4 517 (320) 7.9 (88); 171 (12) <0.01 (517) 0.04 14.15 Ru5 516 (320) 7.1 (80); 95 (20) 0.09 (516) 0.01 6.78 a In ACN solutions, 294 K. b In ACN solutions, 294 K, Ar. c Absolute emission quantum yield. d Reference [Ru(bpy) 3 ] 2+ : ACN Φ Δ = 0.56. 58 e In water. f Measured using a 3 mW cm –2 465 nm LED. The 1 IL fluorescent emission observed at 362 nm in ACN for complexes Ru1 and Ru2 was more intense than that observed for complex Ru3 . All the complexes present weak yellow phosphorescence from 3 MLCT states at room temperature in ACN solutions, see Table 3 . The emission from 3 MLCT varies little from one compound to another, which could be indicate of the π* acceptor orbital of 3 MLCT is similar in each complex. Although the emission intensity around 500 nm is not very intense, the decay profile of the lifetime of the excited state was found to be biexponential in nature. The biexponential decay shows a short (2–8 ns) and a long (95–420 ns) component. This biexponential decay indicates that multiple triplet excited states are involved in the emission profile. ## DFT Calculations DFT Calculations The properties of the ground state singlet and excited state triplet of complexes Ru1–Ru5 have been explored using DFT and TD-DFT calculations. Optimized geometries were obtained in ACN and water. The structures of the singlet ground and triplet excited states, in water, are shown in Figures S52–S53 . The structures obtained in ACN are essentially the same as those obtained in water. Interestingly, for all Ru1–Ru5 complexes, the N1–Ru distance, between the Ru atom and the BTAT nitrogen, is consistently larger in the T 1 state, about 0.49 Å, compared to that in the S 0 state ( Table S5 ). This indicates that the excitation to the triplet spin state induces a weakening of such a bond. The electronic structures of the ground state of the complexes were characterized according to their frontier molecular orbitals HOMO and LUMO, Figure 5 top. The nature of the HOMO changes with the substituents of the BTAT ligand, as can be seen in Figure 5 . Thus, the HOMO of complexes Ru1–Ru4 was predominantly ruthenium d-orbital character but with some additional BTAT π-contribution and in complex Ru5 was instead located on the triazole rings and its phenyl–N(CH 3 ) 2 . The LUMO was dominated by π* contribution from the phen ligands in complexes Ru1–Ru3 . However, in complex Ru4 it comprises the triazole ring and its 4-NO 2 -phenyl substituent. The LUMO was progressively stabilized from Ru5 , Ru1 , Ru2 , Ru3 , to Ru4 as the electron-withdrawing character of the substituent of BTAT ligands increases ( Figure 5 bottom). The HOMO was also stabilized in similar extent leading the shorter HOMO–LUMO gap for Ru4 (164.5 kJ/mol) following by Ru5 (173.6 kJ/mol) and compared to Ru1–Ru3 (202–205 kJ/mol) ( Figure 5 bottom). Very similar orbital shapes and energy values were obtained also in ACN. In all complexes, the T 1 state mainly originates from HOMO → LUMO (70%) transition ( Table S6 ) and characterized as 3 MLCT. The other high energy emission band are derived from triplet states which are predominantly ligand centered 3 LLCT/ 3 ILCT. Finally, it is interesting to note that the S 0 –T 1 transition is accompanied by a change in the dipole moment ( Table S8 ), which is greater for Ru5 and Ru1 , in particular in water and Ru4 exhibited the lowest change in the dipolar moment in the S 0 –T 1 transition. ## Photochemistry Studies Photochemistry Studies The capacity of complexes Ru1–Ru5 to undergo photosubstitution reactions was explored by UV–vis. In the dark, UV–vis measurements revealed a remarkable stability in water at 310 K of Ru1–Ru5 over a period of 120 h, as shown in Figure S45 no changes in their UV–vis spectra were observed. Conversely, light irradiation (λ = 465 nm, 4 mW/cm 2 ) of water solutions of the new complexes provokes with the time noticeable changes in their absorption spectra, as can be seen in Figure 6 for Ru1 and in Figure S46 for complexes Ru2–Ru5 . Upon light irradiation, the metal to ligand charge transfer (MLCT) band centered between 410 and 425 nm was bathochromically shifted to a broad MLCT band centered at 478 nm, with time a clear isosbestic point around 450 nm was observed along with decreasing absorption intensity within 250–350 nm. Figure 6 Changes in absorption spectra of Ru1 in H 2 O (10 –5 M) as observed upon irradiation with blue light (λ ex = 465 nm, 4 mW/cm 2 ). Parallel HPLC experiments for the Ru complexes (vide infra) evidenced the existence of a photoejection process of the corresponding BTAT ligand ( Figures S47–S51 for Ru1–Ru5 ). The evaluation of these changes in the absorption spectra demonstrated variation in t 1/2 values through all complexes ( Figure 6 and Table 3 ). Under these conditions complexes Ru1–Ru3 became more labile, t 1/2 between 4.5 and 5.5 min, instead complex Ru4 containing the BTAT ligand with the more electron-withdrawing substituent (−NO 2 ) was the less active, t 1/2 = 14.15 min. Complex Ru5 with BTAT ligand with the substituent more electron donating [−N(CH 3 ) 2 ] shows t 1/2 around 6.8 min. Except for complex Ru4 , these complexes exhibit similar efficient photosubstitution than other complexes with bidentate ligands and steric hindrance which have t 1/2 < 5 min. In these photolabile compounds the 3 MLCT excited states generated photochemically are quenched by low lying metal-centered ( 3 MC) triplet excited states that lead to nonradiative decay and photosubstitution. 59 , 60 As stated above, the release of the BTAT ligand from the corresponding prodrug Ru1–Ru5 could be confirmed by HPLC using ACN solutions of the complexes at time zero and after 1 h of irradiation with blue light (λ ex = 465 nm, 4 mW/cm 2 ) ( Figures S47–S51 ). As observed in Figure 7 A, the chromatograms show the disappearance of complex Ru1 peak ( t R = 9.9 min), while a new signal appears at a higher retention time ( t R = 15.6 min) corresponding to the BTAT ligand, together with the peak at a lower retention time ( t R = 6.7 min), assigned to the Ru photoproduct [Ru(phen) 2 (ACN) 2 ] 2+ (calcd m / z = 272.05, Figure 7 B), a labile complex that could potentially bind to DNA or other biomolecules within the cell. 61 Figure 7 Determination of photoejection products of Ru1 by HPLC-MS. (A) HPLC chromatogram (DAD = 280 nm) of Ru1 at t = 0 (top) and after being irradiated for 1 h with blue light, λ ex = 465 nm, 4 mW/cm 2 . (B) Mass spectra of the peaks of interest extracted from the chromatograms. The remarkable N1–Ru bond weakening in the triplet state ( Table S5 ) suggests a possible mechanism involved in the photoejection of the BTAT ligand. An example of such a mechanism, considering compound Ru5 , is shown in Figure 8 . The proposed mechanism of ligand photoejection for complex Ru5 , in H 2 O, was obtained by DFT calculations. The ground state reactant, S 0 , in the presence of two water molecules, is excited into the triplet state T 1 ; in the excited state, the two water molecules substitute the chelating ligand and finally the substitution product decays from T 1 into the ground state S 0 . Analogous result is obtained for compound Ru4 ( Figure S55 ), showing that the slower reactivity observed of compound Ru4 is attributable to the larger t 1/2 photoejection half time ( Table 3 ). Figure 8 Proposed mechanism of ligand photoejection for complex Ru5 , in H 2 O, obtained by DFT calculations. For ruthenium polypyridyl compounds photosubstitution reactions compete with phosphorescence and 1 O 2 generation. 62 The capacity of Ru1–Ru5 to generate 1 O 2 upon irradiation at 465 nm (4 mW/cm 2 ) in aerated CH 3 CN was examined by measuring the absorption of 1,3-diphenylisobenzofuran (DPBF) at 413 nm in acetonitrile solution. The complexes showed comparatively low but nonzero Φ Δ values ( Table 3 ), suggesting that a PDT type II mechanism can hardly explain the phototoxicity observed in normoxic conditions. 63 Important to note, the related bis-aqua ruthenium complex [Ru(Ph 2 phen) 2 (H 2 O) 2 ] 2+ has been probed able to bind to proteins or nucleic acids and subsequently generate ROS. 63 ## Photobiological Studies Photobiological Studies Cell Uptake First, the cellular uptake of two representative Ru(II) complexes were investigated in HeLa cells (15 μM, 2 h incubation) by using confocal microscopy. Excitation was carried out with a blue light laser (λ exc = 450 nm). As shown in Figure 9 , a strong fluorescence signal was clearly observed inside cancer cells, thereby confirming an excellent and rapid cellular uptake. Worthy of note, no cell toxicity was appreciated during the experiments. The pattern of staining of Ru4 and Ru5 was similar and excluded nuclei as their preferential target. Indeed, the distribution of the red fluorescence emission of both compounds was found across the cytoplasm in punctate deposits, suggesting selective accumulation within intracellular vesicles. Important to note, it has been previously shown that the free ligand L5 was observed (λ exc = 405 nm) evenly distributed around cell cytoplasm but not within nuclei of Hela cells, although only a partial correlation with MitoTracker Green (MTG) was observed. 50 Figure 9 Confocal microscopy images of HeLa cells incubated with Ru(II) complexes Ru4 and Ru5 [15 μM] for 2 h. Scale bar 30 μm. Dark Cytotoxicity Ideally, the nonirradiated form of a given PACT compound should display low toxicity, while the irradiated form should exert biological activity. 64 To assess the photoactivity of the present Ru(II) PACT compounds under biologically relevant conditions, we performed a screening in a panel of cell lines including both cancerous and noncancerous cells under dark conditions. For this screening, ovarian cancer (A2780), cervical cancer (HeLa), and melanoma (A375) cells along with nontumorigenic ovarian (CHO) cells were used; the clinical drug cisplatin was included for comparative purposes. As shown in Table 4 , none of the complexes displayed antiproliferative activity after 48 h incubation, with negligible toxicity up to 100 μM, in contrast to cisplatin, which exerted cytotoxic activity against both normal and cancer cells in the low micromolar range. Importantly, no cell toxicity was found in normal CHO cells, which is an important requirement of anticancer drug development. Overall, these results validated our compounds as potential PACT agents since minimal dark toxicity is highly desirable in anticancer phototherapy. Table 4 IC 50 Values (μM) of Complexes Ru1–Ru5 and CDDP at 48 h in the Dark compound A2780 HeLa A375 CHO Ru1 >100 >100 >100 >100 Ru2 >100 >100 >100 >100 Ru3 >100 >100 >100 >100 Ru4 >100 >100 >100 >100 Ru5 >100 >100 >100 >100 CDDP 1.9 ± 0.1 21 ± 2   7.8 ± 0.4 Photoactivation Studies Once demonstrated that the compounds were not active under dark conditions, their ability to become toxic to cancer cells was evaluated using low doses of a visible light trigger (LED source at 465 nm, 4 mW/cm 2 , 1 h). Two cancer cell lines, namely, HeLa and A375 cells, were employed to illustrate this effect, and the phototoxic index (PI), defined as the ratio of dark to light IC 50 values, served as a measure of the photoactivity. Since the complexes were deemed as inactive up to 100 μM, we included higher concentrations both in the presence or absence of light trigger to better characterize their photoactivation. To our delight, no cell inhibition was found with any complex, which corroborates their suitability for PACT ( Table 4 ). In the presence of light irradiation, all the complexes showed antiproliferative activity in normoxic conditions (21% O 2 ) against cancer cells, with IC 50 values within the same micromolar range ( Table 5 ). In general, higher photoactivation was found in A375 cells than in HeLa cells (PI values ranging from >3.6 to >29.1 compared to >6.8 to >14.8, respectively). Compound Ru4 , bearing the nitro moiety, exhibited the higher phototoxic action in both cancer cell lines (PI values of >26.6 in A375 and >14.8 in HeLa), followed by compound Ru1 . Interestingly, complex Ru3 was barely photoactivated upon visible light irradiation against these cancer cell lines (PI values of >6.8 in both cancer cell lines). Overall, these results indicate that photoactivation of Ru(II) complexes bearing benzothiazolyl-1,2,3-triazole BTAT chelators can be achieved inside cancer cells. In contrast, light treatment had very little effect on the viability of cells treated with BTAT ligands, which exhibited very low cytotoxicity in each of the cell lines tested after 1 h treatment followed by 1 h irradiation under blue light (IC 50 > 100 μM, Table S8 ). While our study demonstrates the noncytotoxic nature of the ligands after a 2 h treatment, in a previous study 50 we reported cytotoxic effects after a 48 h exposure in the absence of light. This difference could be due to the prolonged accumulation of the ligands within HeLa cells over 48 h compared to the 2 h duration in our study. In our current research, we have demonstrated that the ruthenium complexes facilitate the entry of the ligands into the cells, which subsequently become cytotoxic upon irradiation and breakdown under blue light irradiation. Table 5 IC 50 Values (μM) and Phototoxicity Index (P.I.) under Normoxia of Ru1–Ru5 a   HeLa A375 compound dark blue light P.I HeLa dark blue light P.I A375 Ru1 >500 48.0 ± 3.4 >10.4 >500 20.55 ± 1.5 >24.4 Ru2 >500 58.5 ± 4.6 >8.6 >500 17.2 ± 3.0 >29.1 Ru3 >500 73.5 ± 4.2 >6.8 >500 73.5 ± 4.3 >6.8 Ru4 >500 33.8 ± 5.5 >14.8 >500 18.8 ± 3.7 >26.6 Ru5 >500 41.8 ± 3.9 >12.0 >500 27.1 ± 6.2 >18.5 a Compounds were incubated with the cells 1 h and then irradiated with blue light (λ = 465 nm, 4 mW/cm 2 ) for 1 h. The phototoxicity index (P.I.) was calculated as IC 50 dark /IC 50 λ=465nm . We have also performed the phototoxicity test in both A375 and HeLa cell lines under hypoxic conditions. The results reveal a contrasting pattern, where the compounds exhibit partially greater toxicity under normoxia conditions compared to hypoxia ( Table 6 ). Table 6 IC 50 Values (μM) and Phototoxicity Index (P.I.) under Hypoxia (2% O 2 ) of the Compounds Ru1 – Ru5 a   HeLa A375 compound dark blue light P.I HeLa dark blue light P.I A375 Ru1 >500 70.2 ± 5.0 >7.1 >500 66.9 ± 6.1 >7.5 Ru2 >500 46.0 ± 3.9 >10.9 >500 >100   Ru3 >500 85.2 ± 4.4 >5.9 >500 >100   Ru4 >500 41.7 ± 2.7 >12.0 >500 51.9 ± 2.4 >9.6 Ru5 >500 57.2 ± 6.4 >8.7 >500 >100   a Compounds were incubated with the cells 1 h and then irradiated with blue light (λ = 465 nm, 4 mW/cm 2 ) for 1 h. The phototoxicity index (P.I.) was calculated as IC 50 dark /IC 50 λ=465nm . Lower phototoxicity of light-activated drugs in oxygen-deprived environments can be a sign either that the phototoxicity under normoxia involves some form of a photodynamic effect, or that the hypoxic cells are more difficult to kill than normoxic cells, as hypoxia triggers a range of resistance effects. 64 , 65 Confocal microscopy imaging of HeLa cells treated with Ru5 under two different irradiation timings reveals distinct cellular responses. After 1 min of irradiation under blue light, no cell cytotoxicity was observed ( Figure 10 , first row, bright field image). In contrast, following 1 h irradiation, discernible cell damage is evident, characterized by the presence of cytoplasmic blebbing and notable morphology changes ( Figure 10 , orange triangles). These imaging data provide robust support for the photocytotoxicity results previously described, demonstrating the heightened cytotoxic effects of Ru BTAT compounds after blue light irradiation, as opposed to their relative dark condition or cells treated with the ligands alone. Figure 10 Confocal microscopy images of HeLa cells treated with Ru5 at 15 μM for 1 h followed by blue light irradiation for 1 min and 1 h. λ ex : 405 nm, λ em : 516 ± 30 nm. The control group was maintained under untreated conditions. Scale bar: 10 μm. The orange triangles indicate the cellular membrane irregularities or blebs associated with apoptosis. The higher fluorescence signal observed in cells treated for 1 h under blue light, in comparison to 1 min of irradiation, is attributed mainly to the photoejection of the ligand after irradiation. 50 This comprehensive examination through confocal microscopy provides valuable insights into the photoresponsive behavior of Ru5 and its impact on cellular structures, further reinforcing its potential as an effective photoactive agent in phototherapy applications. ## Cell Uptake Cell Uptake First, the cellular uptake of two representative Ru(II) complexes were investigated in HeLa cells (15 μM, 2 h incubation) by using confocal microscopy. Excitation was carried out with a blue light laser (λ exc = 450 nm). As shown in Figure 9 , a strong fluorescence signal was clearly observed inside cancer cells, thereby confirming an excellent and rapid cellular uptake. Worthy of note, no cell toxicity was appreciated during the experiments. The pattern of staining of Ru4 and Ru5 was similar and excluded nuclei as their preferential target. Indeed, the distribution of the red fluorescence emission of both compounds was found across the cytoplasm in punctate deposits, suggesting selective accumulation within intracellular vesicles. Important to note, it has been previously shown that the free ligand L5 was observed (λ exc = 405 nm) evenly distributed around cell cytoplasm but not within nuclei of Hela cells, although only a partial correlation with MitoTracker Green (MTG) was observed. 50 Figure 9 Confocal microscopy images of HeLa cells incubated with Ru(II) complexes Ru4 and Ru5 [15 μM] for 2 h. Scale bar 30 μm. ## Dark Cytotoxicity Dark Cytotoxicity Ideally, the nonirradiated form of a given PACT compound should display low toxicity, while the irradiated form should exert biological activity. 64 To assess the photoactivity of the present Ru(II) PACT compounds under biologically relevant conditions, we performed a screening in a panel of cell lines including both cancerous and noncancerous cells under dark conditions. For this screening, ovarian cancer (A2780), cervical cancer (HeLa), and melanoma (A375) cells along with nontumorigenic ovarian (CHO) cells were used; the clinical drug cisplatin was included for comparative purposes. As shown in Table 4 , none of the complexes displayed antiproliferative activity after 48 h incubation, with negligible toxicity up to 100 μM, in contrast to cisplatin, which exerted cytotoxic activity against both normal and cancer cells in the low micromolar range. Importantly, no cell toxicity was found in normal CHO cells, which is an important requirement of anticancer drug development. Overall, these results validated our compounds as potential PACT agents since minimal dark toxicity is highly desirable in anticancer phototherapy. Table 4 IC 50 Values (μM) of Complexes Ru1–Ru5 and CDDP at 48 h in the Dark compound A2780 HeLa A375 CHO Ru1 >100 >100 >100 >100 Ru2 >100 >100 >100 >100 Ru3 >100 >100 >100 >100 Ru4 >100 >100 >100 >100 Ru5 >100 >100 >100 >100 CDDP 1.9 ± 0.1 21 ± 2   7.8 ± 0.4 ## Photoactivation Studies Photoactivation Studies Once demonstrated that the compounds were not active under dark conditions, their ability to become toxic to cancer cells was evaluated using low doses of a visible light trigger (LED source at 465 nm, 4 mW/cm 2 , 1 h). Two cancer cell lines, namely, HeLa and A375 cells, were employed to illustrate this effect, and the phototoxic index (PI), defined as the ratio of dark to light IC 50 values, served as a measure of the photoactivity. Since the complexes were deemed as inactive up to 100 μM, we included higher concentrations both in the presence or absence of light trigger to better characterize their photoactivation. To our delight, no cell inhibition was found with any complex, which corroborates their suitability for PACT ( Table 4 ). In the presence of light irradiation, all the complexes showed antiproliferative activity in normoxic conditions (21% O 2 ) against cancer cells, with IC 50 values within the same micromolar range ( Table 5 ). In general, higher photoactivation was found in A375 cells than in HeLa cells (PI values ranging from >3.6 to >29.1 compared to >6.8 to >14.8, respectively). Compound Ru4 , bearing the nitro moiety, exhibited the higher phototoxic action in both cancer cell lines (PI values of >26.6 in A375 and >14.8 in HeLa), followed by compound Ru1 . Interestingly, complex Ru3 was barely photoactivated upon visible light irradiation against these cancer cell lines (PI values of >6.8 in both cancer cell lines). Overall, these results indicate that photoactivation of Ru(II) complexes bearing benzothiazolyl-1,2,3-triazole BTAT chelators can be achieved inside cancer cells. In contrast, light treatment had very little effect on the viability of cells treated with BTAT ligands, which exhibited very low cytotoxicity in each of the cell lines tested after 1 h treatment followed by 1 h irradiation under blue light (IC 50 > 100 μM, Table S8 ). While our study demonstrates the noncytotoxic nature of the ligands after a 2 h treatment, in a previous study 50 we reported cytotoxic effects after a 48 h exposure in the absence of light. This difference could be due to the prolonged accumulation of the ligands within HeLa cells over 48 h compared to the 2 h duration in our study. In our current research, we have demonstrated that the ruthenium complexes facilitate the entry of the ligands into the cells, which subsequently become cytotoxic upon irradiation and breakdown under blue light irradiation. Table 5 IC 50 Values (μM) and Phototoxicity Index (P.I.) under Normoxia of Ru1–Ru5 a   HeLa A375 compound dark blue light P.I HeLa dark blue light P.I A375 Ru1 >500 48.0 ± 3.4 >10.4 >500 20.55 ± 1.5 >24.4 Ru2 >500 58.5 ± 4.6 >8.6 >500 17.2 ± 3.0 >29.1 Ru3 >500 73.5 ± 4.2 >6.8 >500 73.5 ± 4.3 >6.8 Ru4 >500 33.8 ± 5.5 >14.8 >500 18.8 ± 3.7 >26.6 Ru5 >500 41.8 ± 3.9 >12.0 >500 27.1 ± 6.2 >18.5 a Compounds were incubated with the cells 1 h and then irradiated with blue light (λ = 465 nm, 4 mW/cm 2 ) for 1 h. The phototoxicity index (P.I.) was calculated as IC 50 dark /IC 50 λ=465nm . We have also performed the phototoxicity test in both A375 and HeLa cell lines under hypoxic conditions. The results reveal a contrasting pattern, where the compounds exhibit partially greater toxicity under normoxia conditions compared to hypoxia ( Table 6 ). Table 6 IC 50 Values (μM) and Phototoxicity Index (P.I.) under Hypoxia (2% O 2 ) of the Compounds Ru1 – Ru5 a   HeLa A375 compound dark blue light P.I HeLa dark blue light P.I A375 Ru1 >500 70.2 ± 5.0 >7.1 >500 66.9 ± 6.1 >7.5 Ru2 >500 46.0 ± 3.9 >10.9 >500 >100   Ru3 >500 85.2 ± 4.4 >5.9 >500 >100   Ru4 >500 41.7 ± 2.7 >12.0 >500 51.9 ± 2.4 >9.6 Ru5 >500 57.2 ± 6.4 >8.7 >500 >100   a Compounds were incubated with the cells 1 h and then irradiated with blue light (λ = 465 nm, 4 mW/cm 2 ) for 1 h. The phototoxicity index (P.I.) was calculated as IC 50 dark /IC 50 λ=465nm . Lower phototoxicity of light-activated drugs in oxygen-deprived environments can be a sign either that the phototoxicity under normoxia involves some form of a photodynamic effect, or that the hypoxic cells are more difficult to kill than normoxic cells, as hypoxia triggers a range of resistance effects. 64 , 65 Confocal microscopy imaging of HeLa cells treated with Ru5 under two different irradiation timings reveals distinct cellular responses. After 1 min of irradiation under blue light, no cell cytotoxicity was observed ( Figure 10 , first row, bright field image). In contrast, following 1 h irradiation, discernible cell damage is evident, characterized by the presence of cytoplasmic blebbing and notable morphology changes ( Figure 10 , orange triangles). These imaging data provide robust support for the photocytotoxicity results previously described, demonstrating the heightened cytotoxic effects of Ru BTAT compounds after blue light irradiation, as opposed to their relative dark condition or cells treated with the ligands alone. Figure 10 Confocal microscopy images of HeLa cells treated with Ru5 at 15 μM for 1 h followed by blue light irradiation for 1 min and 1 h. λ ex : 405 nm, λ em : 516 ± 30 nm. The control group was maintained under untreated conditions. Scale bar: 10 μm. The orange triangles indicate the cellular membrane irregularities or blebs associated with apoptosis. The higher fluorescence signal observed in cells treated for 1 h under blue light, in comparison to 1 min of irradiation, is attributed mainly to the photoejection of the ligand after irradiation. 50 This comprehensive examination through confocal microscopy provides valuable insights into the photoresponsive behavior of Ru5 and its impact on cellular structures, further reinforcing its potential as an effective photoactive agent in phototherapy applications. ## Conclusions Conclusions This work shows the electronic, physicochemical, and biological properties of a new series of Ru(II) heteroleptic photocages [Ru(phen) 2 (BTAT)] 2+ containing a novel type of minimal strained N,N-chelator, based on substituted 2-(1-(aryl)-1,2,3-triazol-4-yl)benzothiazoles possessing a push–pull architecture. The crystal structures of Ru1 , Ru2 , and Ru5 showed the quasi-planar coordination of the BTAT ligands. Upon irradiation in water with blue light (λ ex = 465 nm, 4 mW/cm 2 ) photoejection of the ligand BTAT was observed by HPLC-MS spectrometry and UV–vis spectroscopy, with t 1/2 ranging from 4.5 to 14.15 min depending of the electronic properties of the corresponding BTAT, being the one containing the more electron withdrawing substituent, Ru4 , the less photolabile. A DFT mechanism for the photoejection of the BTAT ligand from Ru the complexes has been proposed. The new complexes showed very low toxicity in the dark even at high concentrations against the human cancer cells A375, HeLa, and A2780, and nontumorigenic ovarian CHO cells. They showed high phototoxicity toward cancer cells by blue light irradiation with high phototoxicity indexes in both normoxic and hypoxic conditions. An enhancement of the emission intensity of HeLa cells treated with Ru5 was observed in response to increasing doses of light confirming the photoejection of the BTAT ligand. These studies suggest that BTAT could serve as a photocleavable protecting group for the cytotoxic bis-aqua ruthenium warhead [Ru(phen) 2 (OH 2 ) 2 ] 2+ . ## Experimental Section Experimental Section Materials Potassium trifluoromethanesulfonate and 1,10-phenanthroline monohydrate were acquired from Sigma-Aldrich (Merk, Spain) and ruthenium trichloride trihydrate (RuCl 3 ·3H 2 O) from Johnson Matthey. Deuterated solvents were obtained from Eurisotop, except for deuterated trifluoroacetic acid, which was obtained from Sigma-Aldrich. All chemicals were used as received without further purification. Characterization Techniques 1 H NMR and 13 C NMR experiments have been recorded on Bruker AV 400 or Buker AV 600 spectrometers, and 19 F NMR experiments were carried out on a Bruker AV200 spectrometer. The 1 H NMR and 13 C NMR chemical shifts have been referenced to tetramethylsilane and were determined by referencing the residual 1 H and 13 C signal of the deuterated solvent. The 19 F NMR chemical shifts have been referenced to the deuterated trifluoroacetic acid signal. UV–visible absorption spectra were recorded on a PerkinElmer Lambda 750 S spectrometer. Emission spectra were recorded on a Jobin Yvon Fluorolog 3–22 spectrofluorometer with a 450 W xenon lamp, two double-slit monochromators and a TBX-04 photomultiplier. Measurements were made in a fluorescence quartz cuvette with a 10 mm optical path. The lifetimes of the excited state were determined using an IBH FuoroHub TCSPC controller and a NanoLED pulse diode as the driving source, with an estimated measurement error of ±10% or better. Emission quantum yields (Φ) were measured using a Hamamatsu C11347 Absolute PL Quantum Yield spectrometer; the estimated uncertainty is ±5% or better. For measurements in deaerated conditions, solutions of the samples were previously degassed by bubbling argon for 30 min. ESI-MS spectra (positive mode) were recorded on an Agilent 6220 HPLC-MS TOF or an Agilent 1290 Series II HPLC coupled to an Agilent 6550 i-Funnel Q-TOF MS. HPLC experiments were carried out in a VWR-Hitachi, Elite LaChrom model, with a DAD detector, using a Teknokroma C18 chromatography column (25 × 0.46 cm, 5 μm). FT-IR spectra were recorded on a Jasco FT-IR-4600 spectrometer with a bounce-only ATR-PRO ONE system with a monolithic diamond prism. Elemental analysis experiments for carbon, hydrogen, nitrogen, and sulfur were performed on a LECO CNHS-932 microanalyzer. Reactions were carried out in 10 mL glass reaction vials in an Anton Paar Monowave 50 microwave (315 W). General Procedure of Synthesis of Ru(II) Complexes In a glass reaction vial, 6 mL of 1:1 (v/v) EtOH/H 2 O mixture was poured and Ru(phen) 2 Cl 2 ·2H 2 O (0.2 mmol), 66 the respective BTAT ligand ( L1–L5 ) (1 equiv, 0.2 mmol) and potassium triflate (3 equiv, 0.6 mmol) were added. The suspension is microwaved and heated at 120 °C for 2 min. The residue is taken to dryness, after that it was dissolved in dichloromethane and filtered to eliminate the salts. The solvent was then removed and the residue was purified by column chromatography on alumina using an ACN/DCM/MeOH 7:2:1 (v/v/v) mixture as eluent. The solution was taken to dryness and the residue was dissolved in dichloromethane and precipitated with hexane. The solid was filtered, washed with ethyl ether (2 × 5 mL), and dried in vacuo. [Ru(phen) 2 (L1)][OTf] 2 ( Ru1 ) The compound was isolated as an orange solid. Yield 79%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.61 (s, 1H), 8.88 (dd, J = 8.4, 3.2, 1.2 Hz, 2H), 8.8 (dd, J = 8.3 Hz, 1H), 8.75 (d, J = 8.3 Hz, 1H), 8.62 (d, J = 5.3 Hz, 1H), 8.50 (dd, J = 5.3, 1.3 Hz, 1H), 8.45–8.34 (m, 5H), 8.32 (d, J = 5.4 Hz, 1H), 7.98 (d, J = 5.3 Hz, 1H), 7.97–7.91 (m, 2H), 7.85 (dd, J = 8.3, 5.3 Hz, 1H), 7.69 (dd, J = 8.3, 5.3 Hz, 1H), 7.61–7.53 (d, J = 8.2 Hz, 2H), 7.45 (dd, J = 7.6, 1.3 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.10 (dd, J = 7.6, 1.3 Hz, 1H), 5.90 (d, J = 8.5 Hz, 1H), 2.33 (s, 3H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.4(q), 154.3, 153.9, 153.4, 153.3, 150.7(q), 148.1(q), 147.8(q), 147.4(q), 147.3(q), 144.4(q), 140.1(q), 137.3, 137.2, 137.0, 134.2(q), 133.4(q), 130.5(q), 130.4, 130.0(q), 128.3, 128.2, 128.0, 127.9, 127.0, 126.8, 126.7, 126.6, 126.4, 125.8, 125.1, 120.3, 118.2, 20.7. TOF-HRMS (ESI+) m / z : [M] 2+ calcd for C 40 H 28 N 8 RuS, 377.0595; found, 377.0596. Anal. Calcd for C 42 H 28 F 6 N 8 O 6 RuS 3 : C, 47.95; H, 2.68; N, 10.65; S, 9.14. Found: C, 47.84; H, 2.73; N, 10.65; S, 9.07. [Ru(phen) 2 (L2)][OTf] 2 ( Ru2 ) The compound was isolated as an orange solid. Yield 70%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.68 (s, 1H), 8.89 (ddd, J = 8.3, 2.8, 1.3 Hz, 2H), 8.84 (dd, J = 8.3, 1.3 Hz, 1H), 8.75 (dd, J = 8.4, 1.3 Hz, 1H), 8.64 (dd, J = 5.2, 1.2 Hz, 1H), 8.50 (dd, J = 5.2, 1.3 Hz, 1H), 8.45–8.34 (m, 5H), 8.32 (dd, J = 5.3, 1.3 Hz, 1H), 8.00–7.92 (m, 3H), 7.86 (dd, J = 8.1, 5.3 Hz, 1H), 7.80–7.72 (m, 2H), 7.69 (dd, J = 8.1, 5.3 Hz, 1H) 7.49–7.41 (m, 3H), 7.10 (dd, 8.2, 1.3 Hz, 1H), 5.91 (d, J = 8.4 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 162.3 (d, J = 247.7 Hz), 159.3(q), 154.2, 153.8, 153.3, 153.3, 150.7(q), 148.1(q), 147.7(q), 147.3(q), 147.3(q), 144.4(q), 137.3, 137.1, 137.0, 134.2(q), 132.2(q), 130.4(q), 130.4(q), 130.3(q), 130.0(q), 128.3, 128.2, 128.0, 127.9, 127.0, 126.7, 126.6, 126.4, 125.8, 125.0, 123.1 (d, J = 9.3 Hz), 118.2, 116.9 (d, J = 23.6 Hz). 19 F RMN (188 MHz, DMSO- d 6 , δ): −76.40 (s), −109.21 (tt, J = 8.5, 4.1 Hz). TOF-HRMS (ESI+) m / z : [M] 2+ calcd for C 39 H 25 FN 8 RuS, 379.0470; found, 379.0453 ( m / z ). Anal. Calcd for C 41 H 25 F 7 N 8 O 6 RuS 3 : C, 46.64; H, 2.39; N, 10.61; S, 9.11. Found: C 46.41; H, 2.44; N, 10.62; S, 9.06. [Ru(phen) 2 (L3)][OTf] 2 ( Ru3 ) The compound was isolated as an orange solid. Yield 71%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.89 (s, 1H), 8.89 (ddd, J = 8.4, 3.5, 1.3 Hz, 2H), 8.85 (dd, J = 8.3, 1.3 Hz, 1H), 8.77 (dd, J = 8.3, 1.3 Hz, 1H), 8.67 (dd, J = 5.3, 1.3 Hz, 1H), 8.51 (dd, J = 5.2, 1.3 Hz, 1H), 8.46–8.34 (m, 5H), 8.32 (dd, J = 5.4, 1.3 Hz, 1H), 8.04–7.97 (m, 3H), 7.97–7.91 (m, 4H), 7.86 (dd, J = 8.2, 5.3 Hz, 1H), 7.70 (dd, J = 8.3, 5.3 Hz, 1H), 7.47 (dd, J = 8.4, 7.3, 1.1 Hz, 1H), 7.11 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 5.91 (d, J = 8.5 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.2(q), 154.3, 153.9, 153.3, 150.7(q), 148.1(q), 147.7(q), 147.3(q), 144.7(q), 138.4(q), 137.4, 137.2, 137.1, 134.3(q), 130.5(q), 130.4(q), 130.4(q), 130.0(q), 129.9 (q, J = 33.3 Hz), 128.3, 128.2, 128.0, 127.9, 127.4, 127.4, 127.2, 127.1, 126.8, 126.7, 126.4, 125.9, 125.1, 123.6 (q, J = 272.9 Hz), 121.1, 118.3. 19 F RMN (188 MHz, DMSO- d 6 , δ): −59.86 (s), −76.40 (s). TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 40 H 25 F 3 N 8 RuS, 404.0453; found, 404.0434 ( m / z ). Anal. Calcd for C 42 H 25 F 9 N 8 O 6 RuS 3 : C, 45.61; H, 2.28; N, 10.13; S, 8.70. Found: C, 45.60; H, 2.29; N, 9.95; S, 8.72. [Ru(phen) 2 (L4)][OTf] 2 ( Ru4 ) The compound was isolated as an orange solid. Yield 68%. 1 H RMN (600 MHz, DMSO- d 6 , δ): 10.59 (s, 1H), 8.86 (ddd, J = 8.3, 3.0, 1.2 Hz, 2H), 8.83 (dd, J = 8.3, 1.3 Hz, 1H), 8.75 (dd, J = 8.3, 1.2 Hz, 1H), 8.65 (dd, J = 5.2, 1.3 Hz, 1H), 8.51 (dd, J = 5.3, 1.3 Hz, 1H), 8.48–8.43 (m, 2H), 8.42–8.33 (m, 5H), 8.30 (dd, J = 5.3, 1.3 Hz, 1H), 7.98 (dd, J = 5.3, 1.3 Hz, 1H), 7.97–7.91 (m, 4H), 7.85 (dd, J = 8.3, 5.3 Hz, 1H), 7.69 (dd, J = 8.3, 5.3 Hz, 1H), 7.47 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.11 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 5.91 (d, J = 8.4 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.1(q), 154.4, 154.0, 153.4, 150.7(q), 148.1(q), 147.7(q), 147.5(q), 147.4(q), 145.0(q), 139.7(q), 137.5, 137.4, 137.2, 134.4(q), 130.6(q), 130.5(q), 130.4(q), 130.4(q), 130.1(q), 128.4, 128.1, 128.0, 127.3, 127.3, 126.9, 126.7, 126.5, 126.0, 125.7, 125.1, 121.4, 118.3. TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 39 H 25 N 9 O 2 RuS, 392.5442; found, 392.5452. Anal. Calcd for C 41 H 25 F 6 N 9 O 8 RuS 3 : C, 45.47; H, 2.33; N, 11.64; S, 8.88. Found: C, 45.30; H, 2.40; N, 11.38; S, 8.66. [Ru(phen) 2 (L5)][OTf] 2 ( Ru5 ) The compound was isolated as an orange solid. Yield 62%. 1 H-RMN (600 MHz, DMSO- d 6 , δ): 10.18 (s, 1H), 8.85 (ddd, J = 8.3, 2.6, 1.2 Hz, 2H), 8.81 (dd, J = 8.3, 1.3 Hz, 1H), 8.72 (dd, J = 8.3, 1.2 Hz, 1H), 8.57 (dd, J = 5.3, 1.3 Hz, 1H), 8.49 (dd, J = 5.3, 1.3 Hz, 1H), 8.42–8.37 (m, 3H), 8.37–8.32 (m, 3H), 7.97 (dd, J = 5.3, 1.3 Hz, 1H), 7.94 (ddd, J = 8.3, 5.3, 1.9 Hz, 2H), 7.84 (dd, J = 8.3, 5.2 Hz, 1H), 7.67 (dd, J = 8.3, 5.3 Hz, 1H), 7.45 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.43–7.39 (m, 2H), 7.09 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 6.81–6.75 (m, 2H), 5.91 (d, J = 8.5 Hz, 1H), 2.93 (s, 6H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.4(q), 154.2, 153.8, 153.4, 153.3, 151.0(q), 150.7(q), 148.1(q), 147.8(q), 147.4(q), 147.3(q), 144.0(q), 137.2, 137.0, 136.8, 134.1(q), 130.4(q), 130.4(q), 130.3(q), 129.9(q), 128.2, 128.1, 127.9, 127.9, 126.9, 126.7, 126.3, 125.8, 125.6, 125.4, 124.9, 124.7(q), 121.4(q), 118.1, 111.9, 39.8. TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 41 H 31 N 9 RuS, 391.5728; found, 391.5738. Anal. Calcd for C 43 H 31 F 6 N 9 O 6 RuS 3 : C, 47.78; H, 2.89; N, 11.66; S, 8.90. Found: C, 46.43; H, 2.96; N, 11.10; S, 8.78. X-ray Structure Determinations Crystals suitable for X-ray diffraction of Ru1 , Ru2 , and Ru5 were obtained by diffusion of Et 2 O into a diluted acetonitrile solution of complex Ru1 and Ru5 and in diluted EtOH solution of complex Ru2 , respectively. Intensities were registered at low temperature on a Bruker D8QUEST diffractometer using monochromated Mo Kα radiation (λ = 0.71073 Å). Absorption corrections were based on multiscans (program SADABS). 67 Structures were refined anisotropically using SHELXL-2018. 68 Hydrogen atoms were included using rigid methyl groups or a riding model. A summary of crystal data collection and refinement parameters are given in Tables S1 . CCDC reference numbers are 2284059 for Ru1 , 2284060 for Ru5 , and 2284061 for Ru2 . Special Features Ru1 : The structure contains poorly resolved regions of residual electron density; this could not be adequately modeled and so was “removed” using the program SQUEEZE, 69 which is part of the PLATON system. The void volume per cell was 445 Å 3 , with a void electron count per cell of 105 in one void per unit cell. This could be consistent with the presence of 1 acetonitrile per unit cell which accounts for 88 electrons per unit cell because of this electron difference the additional solvent was not taken account of when calculating derived parameters such as the formula weight because the nature of the solvent was not certain. In this structure, one triflate anion is disordered over two positions, ca. 52:48%. Ru2 : the OH hydrogen atom on the two ethanol molecules were found from a difference map and were refined with SADI restrain. Ru5 : the structure contains poorly resolved regions of residual electron density; this could not be adequately modeled and so was “removed” using the program SQUEEZE, which is part of the PLATON system. The void volume per cell was 382 Å 3 , with a void electron count per cell of 96. This additional solvent was not taken account of when calculating derived parameters such as the formula weight, because the nature of the solvent was uncertain. The solvent could be consistent with the presence of one Et 2 O per unit cell which just accounts for 84 electrons per unit cell. One triflate anion is disordered over two positions, ca. 66:34%. Solution Stability The stability of complexes was evaluated by UV–vis spectra after 24 and 120 h at 37 °C. Complexes were dissolved in ACN or water at concentration 10 μM. Photoejection by UV–Vis Photoejection experiments were carried out in duplicate in 10 mm quartz cuvettes on 3 mL solutions of 10 μM in H 2 O-mQ, and blue light irradiation λ = 465 nm, 4 mW/cm 2 . Irradiation intervals were as short as 60 s at early times and more than 150 s at later ones; experiments were considered complete when 150 s irradiation intervals produced no further discernible changes in the absorption spectrum. The normalized change in absorbance was plotted to determine the half-life of ejection using Graph Pad Prism 5.0 software as the published method by Glazer and co-workers. 60 , 70 , 71 Half-life in this context refers to the time it takes to reach 1/2 of the maximum change in the signal used to monitor the process. Ligand Photoejection Experiments by HPLC-MS-TOF Solutions 10 –4 M in ACN of complexes Ru1–Ru5 were prepared and divided into two aliquots: one aliquot was irradiated in a UV–vis spectroscopy cuvette with blue light (λ = 465 nm, 4 mW/cm 2 ) for 60 min and the second aliquot was protected from light. The samples were analyzed in an Agilent 1290 series II HPLC equipment, with a DAD detector, coupled to an Agilent 6550 i-Funnel Q-TOF MS mass spectrometer. The column used is a C18 Zorbax Eclipse Plus column (10 × 2.1 cm, 1.8 μm). The method used uses as mobile phase: Milli-Q water (0.1% HCOOH) as phase A and ACN (0.1% HCOOH) as phase B. The flow is 0.4 mL/min in a gradient of: 0 min (99% A), 2 min (99% A), 22 min (100% B), 24 min (100% B), 25 min (99% A), 30 min (99% A). Singlet Oxygen Quantum Yield (Φ Δ ) Procurement was adapted from literature. 72 , 73 Samples were prepared in an air-saturated acetonitrile solution 5 × 10 –6 M. Absorbance of 1,3-diphenylisobenzofuran (DPBF) at 411 nm (5 × 10 –5 M) was plotted against irradiation times (465 nm, 4 mW/cm 2 ). Slope and linear regression were calculated. Singlet oxygen quantum yield where determined using the equation: , where Φ Δr is the reference singlet oxygen quantum yield [Ru(bpy) 3 ](PF 6 ) 2 , Φ Δ = 0.57 in aerated acetonitrile, 74 m are the slopes of samples and reference, and A λ are the absorbance of compounds and reference at irradiation wavelength. Phototoxicity Testing For dark cytotoxic screening, A2780, CHO, HeLa, and A375 cells were cultured in 96-well plates at a density of 5 × 10 3 cells/well in complete medium and incubated for 24 h at 310 K and 5% CO 2 in a humidified incubator. Serial dilutions of tested compounds in cell culture media were then added at final concentrations in the range of 0 to 100 μM in a final volume of 100 μL/well (%v/v DMSO below 0.4%) for 48 h prior to MTT test. For photoactivation studies, HeLa and A375 cells were used. Treatments were added at final concentrations in the range of 0 to 500 μM. After 1 h incubation with the compounds, light irradiation treatments were applied using a LED photoreactor (Luzchem; Canada) fitted with LED lamps centered at 465 nm (final intensity 4 mW/cm 2 ) for 1 h. Dark control analogues were directly kept in the dark for 2 h. After incubation periods, cells were washed with saline PBS buffer and loaded with 50 μL of MTT solution (1 mg/mL) for additional 4 h, then removed and 50 μL DMSO was added to solubilize the purple formazan crystals formed in active cells. The absorbance was measured at 570 nm using a microplate reader (FLUOstar Omega) and the IC 50 values were calculated based on the inhibitory rate curves using the next the equation where I represents the percentage inhibition of viability observed, I max is the maximal inhibitory effect, IC 50 is the concentration that inhibits 50% of maximal growth, C is the concentration of the treatment, and n is the slope of the semilogarithmic dose–response sigmoidal curves. The nonlinear fitting was performed using SigmaPlot 14.0 software. Two independent experiments were performed with triplicate points per concentration level ( n = 3). Confocal Microscopy HeLa cells were seeded onto Ibidi μ-slides at 10 4 cells/cm 2 in a complete medium and incubated for 24 h at 310 K and 5% CO 2 in a humidified incubator. Compounds were added at indicated concentrations for 2 h. Cells were then washed with PBS twice and imaged under confocal microscopy (STELLARIS Leica Microsystems) using 405 nm excitation laser. Computational Details DFT calculations have been carried out on compounds Ru1–Ru5 with full geometry optimization, by using the M06-L functional, 75 the Lanl2tz(f) basis set 76 , 77 for Ru and the 6-311G(d,p) basis set 78 , 79 for the lighter atoms. The structure of the first triplet excited states, T 1 , was obtained by imposing an open shell triplet spin multiplicity. The solvents ACN and water were implicitly considered by using the PCM method. 80 Frequency calculations, in the normal oscillator approximation, were carried out to check that the optimized geometries corresponded to energy minima on the potential energy surface. TD-DFT calculations have been performed using the same methods and models described above to calculate the electronic absorption spectra of the considered ruthenium compounds. All calculations have been performed by the Gaussian16 program package. 81 ## Materials Materials Potassium trifluoromethanesulfonate and 1,10-phenanthroline monohydrate were acquired from Sigma-Aldrich (Merk, Spain) and ruthenium trichloride trihydrate (RuCl 3 ·3H 2 O) from Johnson Matthey. Deuterated solvents were obtained from Eurisotop, except for deuterated trifluoroacetic acid, which was obtained from Sigma-Aldrich. All chemicals were used as received without further purification. ## Characterization Techniques Characterization Techniques 1 H NMR and 13 C NMR experiments have been recorded on Bruker AV 400 or Buker AV 600 spectrometers, and 19 F NMR experiments were carried out on a Bruker AV200 spectrometer. The 1 H NMR and 13 C NMR chemical shifts have been referenced to tetramethylsilane and were determined by referencing the residual 1 H and 13 C signal of the deuterated solvent. The 19 F NMR chemical shifts have been referenced to the deuterated trifluoroacetic acid signal. UV–visible absorption spectra were recorded on a PerkinElmer Lambda 750 S spectrometer. Emission spectra were recorded on a Jobin Yvon Fluorolog 3–22 spectrofluorometer with a 450 W xenon lamp, two double-slit monochromators and a TBX-04 photomultiplier. Measurements were made in a fluorescence quartz cuvette with a 10 mm optical path. The lifetimes of the excited state were determined using an IBH FuoroHub TCSPC controller and a NanoLED pulse diode as the driving source, with an estimated measurement error of ±10% or better. Emission quantum yields (Φ) were measured using a Hamamatsu C11347 Absolute PL Quantum Yield spectrometer; the estimated uncertainty is ±5% or better. For measurements in deaerated conditions, solutions of the samples were previously degassed by bubbling argon for 30 min. ESI-MS spectra (positive mode) were recorded on an Agilent 6220 HPLC-MS TOF or an Agilent 1290 Series II HPLC coupled to an Agilent 6550 i-Funnel Q-TOF MS. HPLC experiments were carried out in a VWR-Hitachi, Elite LaChrom model, with a DAD detector, using a Teknokroma C18 chromatography column (25 × 0.46 cm, 5 μm). FT-IR spectra were recorded on a Jasco FT-IR-4600 spectrometer with a bounce-only ATR-PRO ONE system with a monolithic diamond prism. Elemental analysis experiments for carbon, hydrogen, nitrogen, and sulfur were performed on a LECO CNHS-932 microanalyzer. Reactions were carried out in 10 mL glass reaction vials in an Anton Paar Monowave 50 microwave (315 W). ## General Procedure of Synthesis of Ru(II) Complexes General Procedure of Synthesis of Ru(II) Complexes In a glass reaction vial, 6 mL of 1:1 (v/v) EtOH/H 2 O mixture was poured and Ru(phen) 2 Cl 2 ·2H 2 O (0.2 mmol), 66 the respective BTAT ligand ( L1–L5 ) (1 equiv, 0.2 mmol) and potassium triflate (3 equiv, 0.6 mmol) were added. The suspension is microwaved and heated at 120 °C for 2 min. The residue is taken to dryness, after that it was dissolved in dichloromethane and filtered to eliminate the salts. The solvent was then removed and the residue was purified by column chromatography on alumina using an ACN/DCM/MeOH 7:2:1 (v/v/v) mixture as eluent. The solution was taken to dryness and the residue was dissolved in dichloromethane and precipitated with hexane. The solid was filtered, washed with ethyl ether (2 × 5 mL), and dried in vacuo. [Ru(phen) 2 (L1)][OTf] 2 ( Ru1 ) The compound was isolated as an orange solid. Yield 79%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.61 (s, 1H), 8.88 (dd, J = 8.4, 3.2, 1.2 Hz, 2H), 8.8 (dd, J = 8.3 Hz, 1H), 8.75 (d, J = 8.3 Hz, 1H), 8.62 (d, J = 5.3 Hz, 1H), 8.50 (dd, J = 5.3, 1.3 Hz, 1H), 8.45–8.34 (m, 5H), 8.32 (d, J = 5.4 Hz, 1H), 7.98 (d, J = 5.3 Hz, 1H), 7.97–7.91 (m, 2H), 7.85 (dd, J = 8.3, 5.3 Hz, 1H), 7.69 (dd, J = 8.3, 5.3 Hz, 1H), 7.61–7.53 (d, J = 8.2 Hz, 2H), 7.45 (dd, J = 7.6, 1.3 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.10 (dd, J = 7.6, 1.3 Hz, 1H), 5.90 (d, J = 8.5 Hz, 1H), 2.33 (s, 3H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.4(q), 154.3, 153.9, 153.4, 153.3, 150.7(q), 148.1(q), 147.8(q), 147.4(q), 147.3(q), 144.4(q), 140.1(q), 137.3, 137.2, 137.0, 134.2(q), 133.4(q), 130.5(q), 130.4, 130.0(q), 128.3, 128.2, 128.0, 127.9, 127.0, 126.8, 126.7, 126.6, 126.4, 125.8, 125.1, 120.3, 118.2, 20.7. TOF-HRMS (ESI+) m / z : [M] 2+ calcd for C 40 H 28 N 8 RuS, 377.0595; found, 377.0596. Anal. Calcd for C 42 H 28 F 6 N 8 O 6 RuS 3 : C, 47.95; H, 2.68; N, 10.65; S, 9.14. Found: C, 47.84; H, 2.73; N, 10.65; S, 9.07. [Ru(phen) 2 (L2)][OTf] 2 ( Ru2 ) The compound was isolated as an orange solid. Yield 70%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.68 (s, 1H), 8.89 (ddd, J = 8.3, 2.8, 1.3 Hz, 2H), 8.84 (dd, J = 8.3, 1.3 Hz, 1H), 8.75 (dd, J = 8.4, 1.3 Hz, 1H), 8.64 (dd, J = 5.2, 1.2 Hz, 1H), 8.50 (dd, J = 5.2, 1.3 Hz, 1H), 8.45–8.34 (m, 5H), 8.32 (dd, J = 5.3, 1.3 Hz, 1H), 8.00–7.92 (m, 3H), 7.86 (dd, J = 8.1, 5.3 Hz, 1H), 7.80–7.72 (m, 2H), 7.69 (dd, J = 8.1, 5.3 Hz, 1H) 7.49–7.41 (m, 3H), 7.10 (dd, 8.2, 1.3 Hz, 1H), 5.91 (d, J = 8.4 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 162.3 (d, J = 247.7 Hz), 159.3(q), 154.2, 153.8, 153.3, 153.3, 150.7(q), 148.1(q), 147.7(q), 147.3(q), 147.3(q), 144.4(q), 137.3, 137.1, 137.0, 134.2(q), 132.2(q), 130.4(q), 130.4(q), 130.3(q), 130.0(q), 128.3, 128.2, 128.0, 127.9, 127.0, 126.7, 126.6, 126.4, 125.8, 125.0, 123.1 (d, J = 9.3 Hz), 118.2, 116.9 (d, J = 23.6 Hz). 19 F RMN (188 MHz, DMSO- d 6 , δ): −76.40 (s), −109.21 (tt, J = 8.5, 4.1 Hz). TOF-HRMS (ESI+) m / z : [M] 2+ calcd for C 39 H 25 FN 8 RuS, 379.0470; found, 379.0453 ( m / z ). Anal. Calcd for C 41 H 25 F 7 N 8 O 6 RuS 3 : C, 46.64; H, 2.39; N, 10.61; S, 9.11. Found: C 46.41; H, 2.44; N, 10.62; S, 9.06. [Ru(phen) 2 (L3)][OTf] 2 ( Ru3 ) The compound was isolated as an orange solid. Yield 71%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.89 (s, 1H), 8.89 (ddd, J = 8.4, 3.5, 1.3 Hz, 2H), 8.85 (dd, J = 8.3, 1.3 Hz, 1H), 8.77 (dd, J = 8.3, 1.3 Hz, 1H), 8.67 (dd, J = 5.3, 1.3 Hz, 1H), 8.51 (dd, J = 5.2, 1.3 Hz, 1H), 8.46–8.34 (m, 5H), 8.32 (dd, J = 5.4, 1.3 Hz, 1H), 8.04–7.97 (m, 3H), 7.97–7.91 (m, 4H), 7.86 (dd, J = 8.2, 5.3 Hz, 1H), 7.70 (dd, J = 8.3, 5.3 Hz, 1H), 7.47 (dd, J = 8.4, 7.3, 1.1 Hz, 1H), 7.11 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 5.91 (d, J = 8.5 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.2(q), 154.3, 153.9, 153.3, 150.7(q), 148.1(q), 147.7(q), 147.3(q), 144.7(q), 138.4(q), 137.4, 137.2, 137.1, 134.3(q), 130.5(q), 130.4(q), 130.4(q), 130.0(q), 129.9 (q, J = 33.3 Hz), 128.3, 128.2, 128.0, 127.9, 127.4, 127.4, 127.2, 127.1, 126.8, 126.7, 126.4, 125.9, 125.1, 123.6 (q, J = 272.9 Hz), 121.1, 118.3. 19 F RMN (188 MHz, DMSO- d 6 , δ): −59.86 (s), −76.40 (s). TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 40 H 25 F 3 N 8 RuS, 404.0453; found, 404.0434 ( m / z ). Anal. Calcd for C 42 H 25 F 9 N 8 O 6 RuS 3 : C, 45.61; H, 2.28; N, 10.13; S, 8.70. Found: C, 45.60; H, 2.29; N, 9.95; S, 8.72. [Ru(phen) 2 (L4)][OTf] 2 ( Ru4 ) The compound was isolated as an orange solid. Yield 68%. 1 H RMN (600 MHz, DMSO- d 6 , δ): 10.59 (s, 1H), 8.86 (ddd, J = 8.3, 3.0, 1.2 Hz, 2H), 8.83 (dd, J = 8.3, 1.3 Hz, 1H), 8.75 (dd, J = 8.3, 1.2 Hz, 1H), 8.65 (dd, J = 5.2, 1.3 Hz, 1H), 8.51 (dd, J = 5.3, 1.3 Hz, 1H), 8.48–8.43 (m, 2H), 8.42–8.33 (m, 5H), 8.30 (dd, J = 5.3, 1.3 Hz, 1H), 7.98 (dd, J = 5.3, 1.3 Hz, 1H), 7.97–7.91 (m, 4H), 7.85 (dd, J = 8.3, 5.3 Hz, 1H), 7.69 (dd, J = 8.3, 5.3 Hz, 1H), 7.47 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.11 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 5.91 (d, J = 8.4 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.1(q), 154.4, 154.0, 153.4, 150.7(q), 148.1(q), 147.7(q), 147.5(q), 147.4(q), 145.0(q), 139.7(q), 137.5, 137.4, 137.2, 134.4(q), 130.6(q), 130.5(q), 130.4(q), 130.4(q), 130.1(q), 128.4, 128.1, 128.0, 127.3, 127.3, 126.9, 126.7, 126.5, 126.0, 125.7, 125.1, 121.4, 118.3. TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 39 H 25 N 9 O 2 RuS, 392.5442; found, 392.5452. Anal. Calcd for C 41 H 25 F 6 N 9 O 8 RuS 3 : C, 45.47; H, 2.33; N, 11.64; S, 8.88. Found: C, 45.30; H, 2.40; N, 11.38; S, 8.66. [Ru(phen) 2 (L5)][OTf] 2 ( Ru5 ) The compound was isolated as an orange solid. Yield 62%. 1 H-RMN (600 MHz, DMSO- d 6 , δ): 10.18 (s, 1H), 8.85 (ddd, J = 8.3, 2.6, 1.2 Hz, 2H), 8.81 (dd, J = 8.3, 1.3 Hz, 1H), 8.72 (dd, J = 8.3, 1.2 Hz, 1H), 8.57 (dd, J = 5.3, 1.3 Hz, 1H), 8.49 (dd, J = 5.3, 1.3 Hz, 1H), 8.42–8.37 (m, 3H), 8.37–8.32 (m, 3H), 7.97 (dd, J = 5.3, 1.3 Hz, 1H), 7.94 (ddd, J = 8.3, 5.3, 1.9 Hz, 2H), 7.84 (dd, J = 8.3, 5.2 Hz, 1H), 7.67 (dd, J = 8.3, 5.3 Hz, 1H), 7.45 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.43–7.39 (m, 2H), 7.09 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 6.81–6.75 (m, 2H), 5.91 (d, J = 8.5 Hz, 1H), 2.93 (s, 6H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.4(q), 154.2, 153.8, 153.4, 153.3, 151.0(q), 150.7(q), 148.1(q), 147.8(q), 147.4(q), 147.3(q), 144.0(q), 137.2, 137.0, 136.8, 134.1(q), 130.4(q), 130.4(q), 130.3(q), 129.9(q), 128.2, 128.1, 127.9, 127.9, 126.9, 126.7, 126.3, 125.8, 125.6, 125.4, 124.9, 124.7(q), 121.4(q), 118.1, 111.9, 39.8. TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 41 H 31 N 9 RuS, 391.5728; found, 391.5738. Anal. Calcd for C 43 H 31 F 6 N 9 O 6 RuS 3 : C, 47.78; H, 2.89; N, 11.66; S, 8.90. Found: C, 46.43; H, 2.96; N, 11.10; S, 8.78. ## [Ru(phen) [Ru(phen) 2 (L1)][OTf] 2 ( Ru1 ) The compound was isolated as an orange solid. Yield 79%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.61 (s, 1H), 8.88 (dd, J = 8.4, 3.2, 1.2 Hz, 2H), 8.8 (dd, J = 8.3 Hz, 1H), 8.75 (d, J = 8.3 Hz, 1H), 8.62 (d, J = 5.3 Hz, 1H), 8.50 (dd, J = 5.3, 1.3 Hz, 1H), 8.45–8.34 (m, 5H), 8.32 (d, J = 5.4 Hz, 1H), 7.98 (d, J = 5.3 Hz, 1H), 7.97–7.91 (m, 2H), 7.85 (dd, J = 8.3, 5.3 Hz, 1H), 7.69 (dd, J = 8.3, 5.3 Hz, 1H), 7.61–7.53 (d, J = 8.2 Hz, 2H), 7.45 (dd, J = 7.6, 1.3 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.10 (dd, J = 7.6, 1.3 Hz, 1H), 5.90 (d, J = 8.5 Hz, 1H), 2.33 (s, 3H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.4(q), 154.3, 153.9, 153.4, 153.3, 150.7(q), 148.1(q), 147.8(q), 147.4(q), 147.3(q), 144.4(q), 140.1(q), 137.3, 137.2, 137.0, 134.2(q), 133.4(q), 130.5(q), 130.4, 130.0(q), 128.3, 128.2, 128.0, 127.9, 127.0, 126.8, 126.7, 126.6, 126.4, 125.8, 125.1, 120.3, 118.2, 20.7. TOF-HRMS (ESI+) m / z : [M] 2+ calcd for C 40 H 28 N 8 RuS, 377.0595; found, 377.0596. Anal. Calcd for C 42 H 28 F 6 N 8 O 6 RuS 3 : C, 47.95; H, 2.68; N, 10.65; S, 9.14. Found: C, 47.84; H, 2.73; N, 10.65; S, 9.07. ## [Ru(phen) [Ru(phen) 2 (L2)][OTf] 2 ( Ru2 ) The compound was isolated as an orange solid. Yield 70%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.68 (s, 1H), 8.89 (ddd, J = 8.3, 2.8, 1.3 Hz, 2H), 8.84 (dd, J = 8.3, 1.3 Hz, 1H), 8.75 (dd, J = 8.4, 1.3 Hz, 1H), 8.64 (dd, J = 5.2, 1.2 Hz, 1H), 8.50 (dd, J = 5.2, 1.3 Hz, 1H), 8.45–8.34 (m, 5H), 8.32 (dd, J = 5.3, 1.3 Hz, 1H), 8.00–7.92 (m, 3H), 7.86 (dd, J = 8.1, 5.3 Hz, 1H), 7.80–7.72 (m, 2H), 7.69 (dd, J = 8.1, 5.3 Hz, 1H) 7.49–7.41 (m, 3H), 7.10 (dd, 8.2, 1.3 Hz, 1H), 5.91 (d, J = 8.4 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 162.3 (d, J = 247.7 Hz), 159.3(q), 154.2, 153.8, 153.3, 153.3, 150.7(q), 148.1(q), 147.7(q), 147.3(q), 147.3(q), 144.4(q), 137.3, 137.1, 137.0, 134.2(q), 132.2(q), 130.4(q), 130.4(q), 130.3(q), 130.0(q), 128.3, 128.2, 128.0, 127.9, 127.0, 126.7, 126.6, 126.4, 125.8, 125.0, 123.1 (d, J = 9.3 Hz), 118.2, 116.9 (d, J = 23.6 Hz). 19 F RMN (188 MHz, DMSO- d 6 , δ): −76.40 (s), −109.21 (tt, J = 8.5, 4.1 Hz). TOF-HRMS (ESI+) m / z : [M] 2+ calcd for C 39 H 25 FN 8 RuS, 379.0470; found, 379.0453 ( m / z ). Anal. Calcd for C 41 H 25 F 7 N 8 O 6 RuS 3 : C, 46.64; H, 2.39; N, 10.61; S, 9.11. Found: C 46.41; H, 2.44; N, 10.62; S, 9.06. ## [Ru(phen) [Ru(phen) 2 (L3)][OTf] 2 ( Ru3 ) The compound was isolated as an orange solid. Yield 71%. 1 H-RMN (400 MHz, DMSO- d 6 , δ): 10.89 (s, 1H), 8.89 (ddd, J = 8.4, 3.5, 1.3 Hz, 2H), 8.85 (dd, J = 8.3, 1.3 Hz, 1H), 8.77 (dd, J = 8.3, 1.3 Hz, 1H), 8.67 (dd, J = 5.3, 1.3 Hz, 1H), 8.51 (dd, J = 5.2, 1.3 Hz, 1H), 8.46–8.34 (m, 5H), 8.32 (dd, J = 5.4, 1.3 Hz, 1H), 8.04–7.97 (m, 3H), 7.97–7.91 (m, 4H), 7.86 (dd, J = 8.2, 5.3 Hz, 1H), 7.70 (dd, J = 8.3, 5.3 Hz, 1H), 7.47 (dd, J = 8.4, 7.3, 1.1 Hz, 1H), 7.11 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 5.91 (d, J = 8.5 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.2(q), 154.3, 153.9, 153.3, 150.7(q), 148.1(q), 147.7(q), 147.3(q), 144.7(q), 138.4(q), 137.4, 137.2, 137.1, 134.3(q), 130.5(q), 130.4(q), 130.4(q), 130.0(q), 129.9 (q, J = 33.3 Hz), 128.3, 128.2, 128.0, 127.9, 127.4, 127.4, 127.2, 127.1, 126.8, 126.7, 126.4, 125.9, 125.1, 123.6 (q, J = 272.9 Hz), 121.1, 118.3. 19 F RMN (188 MHz, DMSO- d 6 , δ): −59.86 (s), −76.40 (s). TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 40 H 25 F 3 N 8 RuS, 404.0453; found, 404.0434 ( m / z ). Anal. Calcd for C 42 H 25 F 9 N 8 O 6 RuS 3 : C, 45.61; H, 2.28; N, 10.13; S, 8.70. Found: C, 45.60; H, 2.29; N, 9.95; S, 8.72. ## [Ru(phen) [Ru(phen) 2 (L4)][OTf] 2 ( Ru4 ) The compound was isolated as an orange solid. Yield 68%. 1 H RMN (600 MHz, DMSO- d 6 , δ): 10.59 (s, 1H), 8.86 (ddd, J = 8.3, 3.0, 1.2 Hz, 2H), 8.83 (dd, J = 8.3, 1.3 Hz, 1H), 8.75 (dd, J = 8.3, 1.2 Hz, 1H), 8.65 (dd, J = 5.2, 1.3 Hz, 1H), 8.51 (dd, J = 5.3, 1.3 Hz, 1H), 8.48–8.43 (m, 2H), 8.42–8.33 (m, 5H), 8.30 (dd, J = 5.3, 1.3 Hz, 1H), 7.98 (dd, J = 5.3, 1.3 Hz, 1H), 7.97–7.91 (m, 4H), 7.85 (dd, J = 8.3, 5.3 Hz, 1H), 7.69 (dd, J = 8.3, 5.3 Hz, 1H), 7.47 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.11 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 5.91 (d, J = 8.4 Hz, 1H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.1(q), 154.4, 154.0, 153.4, 150.7(q), 148.1(q), 147.7(q), 147.5(q), 147.4(q), 145.0(q), 139.7(q), 137.5, 137.4, 137.2, 134.4(q), 130.6(q), 130.5(q), 130.4(q), 130.4(q), 130.1(q), 128.4, 128.1, 128.0, 127.3, 127.3, 126.9, 126.7, 126.5, 126.0, 125.7, 125.1, 121.4, 118.3. TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 39 H 25 N 9 O 2 RuS, 392.5442; found, 392.5452. Anal. Calcd for C 41 H 25 F 6 N 9 O 8 RuS 3 : C, 45.47; H, 2.33; N, 11.64; S, 8.88. Found: C, 45.30; H, 2.40; N, 11.38; S, 8.66. ## [Ru(phen) [Ru(phen) 2 (L5)][OTf] 2 ( Ru5 ) The compound was isolated as an orange solid. Yield 62%. 1 H-RMN (600 MHz, DMSO- d 6 , δ): 10.18 (s, 1H), 8.85 (ddd, J = 8.3, 2.6, 1.2 Hz, 2H), 8.81 (dd, J = 8.3, 1.3 Hz, 1H), 8.72 (dd, J = 8.3, 1.2 Hz, 1H), 8.57 (dd, J = 5.3, 1.3 Hz, 1H), 8.49 (dd, J = 5.3, 1.3 Hz, 1H), 8.42–8.37 (m, 3H), 8.37–8.32 (m, 3H), 7.97 (dd, J = 5.3, 1.3 Hz, 1H), 7.94 (ddd, J = 8.3, 5.3, 1.9 Hz, 2H), 7.84 (dd, J = 8.3, 5.2 Hz, 1H), 7.67 (dd, J = 8.3, 5.3 Hz, 1H), 7.45 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.43–7.39 (m, 2H), 7.09 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 6.81–6.75 (m, 2H), 5.91 (d, J = 8.5 Hz, 1H), 2.93 (s, 6H). 13 C RMN (101 MHz, DMSO- d 6 , δ): 159.4(q), 154.2, 153.8, 153.4, 153.3, 151.0(q), 150.7(q), 148.1(q), 147.8(q), 147.4(q), 147.3(q), 144.0(q), 137.2, 137.0, 136.8, 134.1(q), 130.4(q), 130.4(q), 130.3(q), 129.9(q), 128.2, 128.1, 127.9, 127.9, 126.9, 126.7, 126.3, 125.8, 125.6, 125.4, 124.9, 124.7(q), 121.4(q), 118.1, 111.9, 39.8. TOF-HRMS (ESI + ) m / z : [M] 2+ calcd for C 41 H 31 N 9 RuS, 391.5728; found, 391.5738. Anal. Calcd for C 43 H 31 F 6 N 9 O 6 RuS 3 : C, 47.78; H, 2.89; N, 11.66; S, 8.90. Found: C, 46.43; H, 2.96; N, 11.10; S, 8.78. ## X-ray Structure Determinations X-ray Structure Determinations Crystals suitable for X-ray diffraction of Ru1 , Ru2 , and Ru5 were obtained by diffusion of Et 2 O into a diluted acetonitrile solution of complex Ru1 and Ru5 and in diluted EtOH solution of complex Ru2 , respectively. Intensities were registered at low temperature on a Bruker D8QUEST diffractometer using monochromated Mo Kα radiation (λ = 0.71073 Å). Absorption corrections were based on multiscans (program SADABS). 67 Structures were refined anisotropically using SHELXL-2018. 68 Hydrogen atoms were included using rigid methyl groups or a riding model. A summary of crystal data collection and refinement parameters are given in Tables S1 . CCDC reference numbers are 2284059 for Ru1 , 2284060 for Ru5 , and 2284061 for Ru2 . ## Special Features Special Features Ru1 : The structure contains poorly resolved regions of residual electron density; this could not be adequately modeled and so was “removed” using the program SQUEEZE, 69 which is part of the PLATON system. The void volume per cell was 445 Å 3 , with a void electron count per cell of 105 in one void per unit cell. This could be consistent with the presence of 1 acetonitrile per unit cell which accounts for 88 electrons per unit cell because of this electron difference the additional solvent was not taken account of when calculating derived parameters such as the formula weight because the nature of the solvent was not certain. In this structure, one triflate anion is disordered over two positions, ca. 52:48%. Ru2 : the OH hydrogen atom on the two ethanol molecules were found from a difference map and were refined with SADI restrain. Ru5 : the structure contains poorly resolved regions of residual electron density; this could not be adequately modeled and so was “removed” using the program SQUEEZE, which is part of the PLATON system. The void volume per cell was 382 Å 3 , with a void electron count per cell of 96. This additional solvent was not taken account of when calculating derived parameters such as the formula weight, because the nature of the solvent was uncertain. The solvent could be consistent with the presence of one Et 2 O per unit cell which just accounts for 84 electrons per unit cell. One triflate anion is disordered over two positions, ca. 66:34%. ## Solution Stability Solution Stability The stability of complexes was evaluated by UV–vis spectra after 24 and 120 h at 37 °C. Complexes were dissolved in ACN or water at concentration 10 μM. ## Photoejection by UV–Vis Photoejection by UV–Vis Photoejection experiments were carried out in duplicate in 10 mm quartz cuvettes on 3 mL solutions of 10 μM in H 2 O-mQ, and blue light irradiation λ = 465 nm, 4 mW/cm 2 . Irradiation intervals were as short as 60 s at early times and more than 150 s at later ones; experiments were considered complete when 150 s irradiation intervals produced no further discernible changes in the absorption spectrum. The normalized change in absorbance was plotted to determine the half-life of ejection using Graph Pad Prism 5.0 software as the published method by Glazer and co-workers. 60 , 70 , 71 Half-life in this context refers to the time it takes to reach 1/2 of the maximum change in the signal used to monitor the process. ## Ligand Photoejection Experiments by HPLC-MS-TOF Ligand Photoejection Experiments by HPLC-MS-TOF Solutions 10 –4 M in ACN of complexes Ru1–Ru5 were prepared and divided into two aliquots: one aliquot was irradiated in a UV–vis spectroscopy cuvette with blue light (λ = 465 nm, 4 mW/cm 2 ) for 60 min and the second aliquot was protected from light. The samples were analyzed in an Agilent 1290 series II HPLC equipment, with a DAD detector, coupled to an Agilent 6550 i-Funnel Q-TOF MS mass spectrometer. The column used is a C18 Zorbax Eclipse Plus column (10 × 2.1 cm, 1.8 μm). The method used uses as mobile phase: Milli-Q water (0.1% HCOOH) as phase A and ACN (0.1% HCOOH) as phase B. The flow is 0.4 mL/min in a gradient of: 0 min (99% A), 2 min (99% A), 22 min (100% B), 24 min (100% B), 25 min (99% A), 30 min (99% A). ## Singlet Oxygen Quantum Yield (Φ Singlet Oxygen Quantum Yield (Φ Δ ) Procurement was adapted from literature. 72 , 73 Samples were prepared in an air-saturated acetonitrile solution 5 × 10 –6 M. Absorbance of 1,3-diphenylisobenzofuran (DPBF) at 411 nm (5 × 10 –5 M) was plotted against irradiation times (465 nm, 4 mW/cm 2 ). Slope and linear regression were calculated. Singlet oxygen quantum yield where determined using the equation: , where Φ Δr is the reference singlet oxygen quantum yield [Ru(bpy) 3 ](PF 6 ) 2 , Φ Δ = 0.57 in aerated acetonitrile, 74 m are the slopes of samples and reference, and A λ are the absorbance of compounds and reference at irradiation wavelength. ## Phototoxicity Testing Phototoxicity Testing For dark cytotoxic screening, A2780, CHO, HeLa, and A375 cells were cultured in 96-well plates at a density of 5 × 10 3 cells/well in complete medium and incubated for 24 h at 310 K and 5% CO 2 in a humidified incubator. Serial dilutions of tested compounds in cell culture media were then added at final concentrations in the range of 0 to 100 μM in a final volume of 100 μL/well (%v/v DMSO below 0.4%) for 48 h prior to MTT test. For photoactivation studies, HeLa and A375 cells were used. Treatments were added at final concentrations in the range of 0 to 500 μM. After 1 h incubation with the compounds, light irradiation treatments were applied using a LED photoreactor (Luzchem; Canada) fitted with LED lamps centered at 465 nm (final intensity 4 mW/cm 2 ) for 1 h. Dark control analogues were directly kept in the dark for 2 h. After incubation periods, cells were washed with saline PBS buffer and loaded with 50 μL of MTT solution (1 mg/mL) for additional 4 h, then removed and 50 μL DMSO was added to solubilize the purple formazan crystals formed in active cells. The absorbance was measured at 570 nm using a microplate reader (FLUOstar Omega) and the IC 50 values were calculated based on the inhibitory rate curves using the next the equation where I represents the percentage inhibition of viability observed, I max is the maximal inhibitory effect, IC 50 is the concentration that inhibits 50% of maximal growth, C is the concentration of the treatment, and n is the slope of the semilogarithmic dose–response sigmoidal curves. The nonlinear fitting was performed using SigmaPlot 14.0 software. Two independent experiments were performed with triplicate points per concentration level ( n = 3). ## Confocal Microscopy Confocal Microscopy HeLa cells were seeded onto Ibidi μ-slides at 10 4 cells/cm 2 in a complete medium and incubated for 24 h at 310 K and 5% CO 2 in a humidified incubator. Compounds were added at indicated concentrations for 2 h. Cells were then washed with PBS twice and imaged under confocal microscopy (STELLARIS Leica Microsystems) using 405 nm excitation laser. ## Computational Details Computational Details DFT calculations have been carried out on compounds Ru1–Ru5 with full geometry optimization, by using the M06-L functional, 75 the Lanl2tz(f) basis set 76 , 77 for Ru and the 6-311G(d,p) basis set 78 , 79 for the lighter atoms. The structure of the first triplet excited states, T 1 , was obtained by imposing an open shell triplet spin multiplicity. The solvents ACN and water were implicitly considered by using the PCM method. 80 Frequency calculations, in the normal oscillator approximation, were carried out to check that the optimized geometries corresponded to energy minima on the potential energy surface. TD-DFT calculations have been performed using the same methods and models described above to calculate the electronic absorption spectra of the considered ruthenium compounds. All calculations have been performed by the Gaussian16 program package. 81