Design of a Tris-Heteroleptic Ru(II) Complex with Red-Light Excitation and Remarkably Improved Photobiological Activity.

pubs.acs.org/IC Article Design of a Tris-Heteroleptic Ru(II) Complex with Red-Light Excitation and Remarkably Improved Photobiological Activity Shuang Li, Jian Zhao,* Xinyi Wang, Gang Xu, Shaohua Gou,* and Qiang Zhao* Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Downloaded via FLORIDA INTL UNIV on July 24, 2020 at 02:31:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Ru(II)-polypyridyl complexes are of increasing interest in photodynamic therapy (PDT) due to their easily tunable photophysical and photochemical properties. However, short-wavelength absorption of Ru(II)-polypyridyl complexes has limited their penetration depth in PDT. Herein, the series of Ru(II)-polypyridyl complexes 1−4 was designed by replacing one bipyridine in [Ru(bpy)3]Cl2 with Schiﬀ bases (iminopyridine or iminoquinoline analogues) to achieve red-shifted absorption of Ru(II)polypyridyl photosensitizers. To further shift the absorption to longer wavelength and improve the photobiological activity of Ru(II)-polypyridyl complexes, the three tris-heteroleptic Ru(II) complexes 5−7 with benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (dppn) as a ligand were designed to achieve long-lived intraligand (3IL) excited states. Cytotoxicity data against A549 and HepG2 cells revealed that complex 7 showed extraordinarily high cytotoxicity under 650 nm irradiation, resulting in IC50 values of 56 and 63 nM with exceptionally large phototoxicity index (PI) values of 763 and 613, respectively. Thus, the resulting complex 7 with considerable red-light photocytotoxicity and high PI values shows a promising potential for therapeutic applications, which represents a new scaﬀold of Ru(II)-polypyridyl photosensitizers for PDT in the “therapeutic window”. This study delivers a rational strategy for the design of tris-heteroleptic Ru(II) complexes as promising photosensitizers for cancer therapy. ■ tissue treatment.27−31 However, the longest-wavelength absorption band (generally a metal to ligand charge transfer (MLCT) transition) of most Ru(II) complexes is located in the blue region (<500 nm), which has limited their phototherapeutic applications for deep-tissue diseases.32−34 Consequently, exploring novel Ru(II) complexes with onephoton red-light excitation is highly desired for PDT. Schiﬀ bases are useful for chelating ligands to metal ions, resulting in complexes with diﬀerent physical and chemical properties.35 Thus, metal complexes of Schiﬀ bases have been extensively designed and investigated for biomedical applications, such as antitumor, anti-inﬂammatory, antibacterial, and INTRODUCTION The successful application of platinum-based anticancer drugs in the clinic has stimulated increasing interest in discovering new metallodrugs, in which ruthenium complexes have been thought to be promising alternatives.1−6 To date, two ruthenium(III) complexes (NAMI-A, KP1019 and its sodium salt KP1339) have been evaluated in clinical trials for cancer chemotherapy.7 To further improve the therapeutic eﬃcacy, Ru(II)-polypyridyl complexes with unique photophysical and photochemical properties have been designed as photodynamic therapy (PDT) agents.8−20 A well-known example is TLD1433, which successfully completed phase Ib clinical trials in 2018.21 In PDT, the photosensitizer (PS) used is generally nontoxic in the dark but can be activated to produce cytotoxic reactive oxygen species (ROS) by light of a suitable wavelength.22−26 As red and near-infrared lights enable maximum tissue penetration with minimum damage, PS activated in the “therapeutic window” of 600−850 nm is suitable for deep© XXXX American Chemical Society Received: June 23, 2020 A https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC Article Figure 1. Ru(II)-polypyridyl complexes studied in this work. Figure 2. Frontier molecular orbital diagrams and energy proﬁles for the HOMOs and LUMOs of [Ru(bpy)3]2+ and complexes 1−7. B https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC Article Figure 3. UV−vis absorption spectra of complexes 1−7 and [Ru(bpy)3]Cl2 in methanol. The insets show expansions of the absorption above 460 nm. ■ antifungal activities.36 Recently, our group reported that dinuclear Ru(II)-arene complexes containing Schiﬀ base ligands (iminopyridine or iminoquinoline) exhibited unique biological characteristics.37 Moreover, replacing pyridine ligands with imine ligands in Ru(II) complexes can result in a red shift of the 1MLCT absorption band.38,39 Therefore, we intend to introduce iminopyridine and iminoquinoline ligands to Ru(II)-polypyridyl complexes with improved photophysical and biological properties for PDT. Herein we utilized [Ru(bpy)3]Cl2 as a model complex and replaced its one bipyridine with imine ligands to obtain complexes 1−4 (Figure 1). Prior to undertaking the experiments, we performed DFT (density functional theory) calculations to investigate the HOMO/LUMO energy levels of complexes 1−4. As shown in Figure 2, the HOMO−LUMO energy gaps of complexes 1−4 are smaller than that of [Ru(bpy)3]2+, which may lead to red shifts of the 1MLCT bands of complexes 1−4, providing a theoretical foundation for the following study.40 In addition, complexes 2 and 4 possess donor (dimethylamino)−acceptor (pyridine or quinoline) structures, which have relatively lower band gap energies in comparison with complexes 1 and 3, respectively. However, according to the energy gap law, redlight-absorbing metal complexes often exhibit a short excitedstate lifetime that may compromise the therapeutic eﬃciency of these complexes.41 Actually, the features of the triplet excited states of metal-based PSs can be tuned for diﬀerent application purposes such as phototherapy, catalysis, and electroluminescence. Subtle ligand modiﬁcations on metalbased PSs can lead to diﬀerent photophysical and photochemical properties and result in signiﬁcant changes in the anticancer eﬃcacy.42 McFarland has demonstrated that Ru(II) complexes with long-lived intraligand (3IL) excited states are extremely sensitive to O2 and can result in considerable photocytotoxicity.21,43−46 Moreover, the 3IL excited states of Ru(II)-polypyridyl complexes can be achieved by π-expansive ligands,21 such as benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (dppn),47−49 which can greatly improve the photocytotoxicity of the resulting Ru(II) complexes, as reported by Turro and co-workers.50 Thus, in order to improve the photobiological activity of Ru(II)-imine complexes, the three tris-heteroleptic Ru(II) complexes 5−7 were further designed to achieve longlived 3IL excited states with dppn as a ligand. It was anticipated that complex 7 with a remarkable red-shifted absorption could exhibit signiﬁcantly high photocytotoxicity in the PDT therapeutic window. RESULTS AND DISCUSSION Synthesis and Photophysical Properties. Complexes 1−7 were synthesized and characterized as described in the Figures S1−S21 and Scheme S1in the Supporting Information. The UV−vis absorption spectra of these complexes are presented in Figure 3. These complexes exhibit intense absorptions in the ultraviolet region, which are typical absorption bands of Ru(II)-polypyridyl complexes due to the 1 ππ* transitions of the ligands. Notably, the longest absorption maxima of complexes 1−7 are located above 470 nm, which are red-shifted in comparison to that of [Ru(bpy)3]Cl2 at 450 nm. Moreover, complexes 3, 4, and 7 with iminoquinoline ligands showed longer wavelength absorption in the visible region in comparison to the corresponding complexes 1, 2, and 6 with iminopyridine ligands due to the highly delocalized πsystem of the iminoquinoline ligand. In addition, a close examination of the UV−vis absorption spectra of complexes 4 and 7 revealed that the absorption tails of both complexes extend over 650 nm, indicating that they have the potential to be excited by red light. In particular, complex 7 displayed weak but clearly observable absorption bands above 700 nm (700 nm, ε = 200 M−1 cm−1). It was reported that Ru(II) complexes with long-lived 3IL excited states are highly sensitive to redlight (630 nm) excitation, which could be excited at wavelengths even where their molar extinction coeﬃcients are very low (ε < 100 M−1 cm−1).21,46 The luminescence spectra of complexes 1−7 and [Ru(bpy)3]Cl2 were studied and are presented in Figure S22. All of the complexes showed nearinfrared emission spectra between 700 and 900 nm in methanol, which are red-shifted in comparison to the emission of [Ru(bpy)3]Cl2, which might be attributed to CN bond distortion in the excited state.38 It is noted that the emission intensity of complexes 1−7 is so low that the quantum yields are below 1% in deaerated methanol solutions. In addition, the emission of complexes 1−7 can be quenched by O2, demonstrating that the luminescence can be attributed to phosphorescence from the triplet excited state. Theoretical Calculations. DFT calculations were undertaken to gain insight into the electronic transitions of these Ru(II) complexes, and the fully optimized geometries can be found in Table S1 in the Supporting Information. The selected vertical transition energies between the ground state (S0) and the singlet excited states (Sn) are presented in Table 1. For complexes 2, 4, 6, and 7, it is found that the molecular HOMOs are mostly localized on the donor group (dimethylamino), while the LUMOs (LUMO+1 for complexes 6 and 7) are more localized on the acceptor core (pyridine or C https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC Article Table 1. Selected Calculated Singlet Excited-State Transitions for Complexes 1−7 and [Ru(bpy)3]Cl2 quinoline). Therefore, the absorption bands (S0−Sn) of complexes 2, 4, 6, and 7 are composed of ILCT (donor to acceptor) or LLCT (Schiﬀ base ligands to bipyridine/dppn) along with MLCT transitions, while complexes 1, 3, and D https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC Article Figure 4. Absorption spectra of ABDA (50 μM) in the presence of 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), 6 (f), 7 (g), and [Ru(bpy)3]Cl2 (h) at concentrations of 10 μM upon 465 nm irradiation. Table 2. Photobiological Activity of Complexes 1−7 and [Ru(bpy)3]Cl2 toward A549 and HepG2 Cancer Cells A549 compound 1 2 3 4 5 6 7 [Ru(bpy)3] Cl2 IC50, μM (dark) IC50, μM (465 nm) 64.3 ± 2.9 51.6 ± 3.1 52.5 ± 2.6 49.6 ± 3.7 51.8 ± 3.3 48.7 ± 2.8 42.7 ± 3.5 54.4 ± 4.7 24.9 ± 3.4 29.5 ± 2.6 22.6 ± 1.3 16.2 ± 1.1 0.089 ± 0.008 0.073 ± 0.011 0.038 ± 0.003 8.5 ± 0.6 HepG2 PIa IC50, μM (650 nm) 2.6 1.7 2.3 3.1 582 667 1124 6.4 43.8 ± 3.1 35.5 ± 3.5 30.4 ± 1.7 18.8 ± 1.2 0.242 ± 0.006 0.149 ± 0.005 0.056 ± 0.004 53.1 ± 1.6 PIa IC50, μM (dark) IC50, μM (465 nm) PIa IC50, μM (650 nm) PIa 1.5 1.5 1.7 2.6 214 327 763 1.0 59.4 ± 1.9 48.5 ± 2.4 55.7 ± 4.6 45.9 ± 2.7 46.4 ± 3.0 40.4 ± 2.5 38.6 ± 2.1 53.2 ± 4.2 23.6 ± 1.7 25.8 ± 2.3 34.1 ± 3.1 11.5 ± 1.0 0.065 ± 0.008 0.045 ± 0.006 0.040 ± 0.003 7.5 ± 0.4 2.5 1.9 1.6 4.0 713 897 965 7.1 40.3 ± 3.1 37.1 ± 2.2 33.6 ± 2.5 17.3 ± 1.4 0.231 ± 0.012 0.132 ± 0.007 0.063 ± 0.004 49.1 ± 1.8 1.5 1.3 1.7 2.7 201 306 613 1.1 a PI = dark IC50 value/light IC50 value. [Ru(bpy)3]2+ are composed of MLCT transitions (Table 1 and Figures S23−S30). In addition, the calculated energy level of the S1 state of [Ru(bpy)3]2+ is 2.72 eV (456 nm), which is higher than those of complexes 1−7, in accordance with the experimental results that the absorption tails of complexes 1−7 terminate at longer wavelength in comparison with [Ru(bpy)3]2+. 1 O2 Generation. The singlet oxygen (1O2) generation ability was evaluated for complexes 1−7 together with [Ru(bpy)3]Cl2 as a control by using 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) as a probe, which can react with 1O2 and convert to a steady-state endoperoxide product, thereby leading to a decrease in absorption intensity at around 378 nm. As shown in Figure 4, the absorbance of ABDA decreased dramatically in the presence of complexes 5− 7 and [Ru(bpy)3]Cl2 upon 465 nm irradiation (1.26 J cm−2), whereas a slight decrease in ABDA absorbance was observed for complexes 1−4. By using [Ru(bpy)3]Cl2 as a reference (ΦΔ = 0.41 in water),51 the 1O2 quantum yields of complexes 1−7 were determined to be 0.025, 0.036, 0.061, 0.046, 0.27, 0.33, and 0.36, respectively, revealing that complexes 5−7 and [Ru(bpy)3]Cl2 are capable of producing 1O2 more eﬃciently than complexes 1−4 under 465 nm irradiation. Considering the red-light absorption ability of complexes 1− 7, we further investigated their 1O2 generation with irradiation at 650 nm (19.2 J cm−2) (Figure S31). As expected, complexes 4 and 7 can lead to an obvious decrease in ABDA absorbance, indicating that they are capable of producing 1O2 under 650 nm light irradiation. Strikingly, complexes 5 and 6 with low molar absorption coeﬃcients at 650 nm can cause severe reduction in ABDA, which is probably due to their highly photosensitizing 3IL excited states.45 In addition, little decrease in ABDA absorbance was observed for complexes 1−3 and [Ru(bpy)3]Cl2 due to their negligible molar absorption coeﬃcients in the red-light region. This study indicates that complexes 4−7 could be excited by red light, which provided the conditions for these complexes to realize PDT. In Vitro Photocytotoxicity. The in vitro photocytotoxicity of complexes 1−7 was evaluated against A549 and HepG2 cancer cells by using an MTT assay, together with [Ru(bpy)3]Cl2 for comparison (Table 2 and Figure S32). Upon blue-light irradiation (465 nm), the photocytotoxicity enhancement of complexes 1−4 and [Ru(bpy)3]Cl2 toward the tested cell lines is modest with PI (phototoxicity index) values ranging from 1.6 to 7.1 (Table 2). Unexpectedly, complexes 5−7 exhibited greatly enhanced cytotoxicity against A549 and HepG2 cancer cells with blue-light irradiation, resulting in IC50 values ranging from 38 and 89 nM with exceptionally large PI values from 582 to 1124, respectively, which are 2 orders of magnitude larger than those of complexes 1−4. This may be attributed to the highly photosensitizing 3IL excited state of complexes 5−7. Importantly, upon red-light irradiation (650 E https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC nm), considerable cytotoxicity was also observed for complexes 5−7 against A549 and HepG2 cells with PI values ranging from 201 to 763. In contrast, complexes 1−4 and [Ru(bpy)3]Cl2 produced no discernible photodynamic activities (PI = 1.0−2.7) upon irradiation with red light. When these results are taken together, complex 7 with the most considerable redlight photocytotoxicity as well as the highest PI value within these complexes shows a promising potential for therapeutic applications. To further conﬁrm the considerable photocytotoxicity of complex 7, a calcein AM and propidium iodide (PI) costaining assay was carried out to label the living and dead cells as indicators by staining the cytoplasm with the green ﬂuorescence generated by the enzymatic hydrolysis of calcein AM and the nucleus with the red ﬂuorescence of PI, respectively (Figure 5 and Figure S33). In addition, complex Article Figure 6. Confocal ﬂuorescence images of ROS generation in A549 cells incubated with complexes 4 and 7 and [Ru(bpy)3]Cl2 upon 465 and 650 nm irradiation. Cellular Accumulation. To investigate the possible mechanism of the considerable photocytotoxicity of complex 7 on A549 cancer cells, the intracellular content of Ru was detected using ICP-MS (inductively coupled plasma mass spectrometry) together with complex 4 and [Ru(bpy)3]Cl2 for comparison. As shown in Figure S34, there was no obvious diﬀerence of the intracellular Ru contents for complex 4 (91.7 ± 8.3 ng/105 cells) and 7 (98 ± 7.2 ng/105 cells). Thus, it is rational to conclude that the cellular accumulation is not the main reason for the markedly improved photocytotoxicity of complex 7. DNA Photocleavage. Ru(II)-polypyridyl complexes are known to induce DNA photocleavage, which may be responsible for the observed photocytotoxicity to some extent. Thus, the DNA photocleavage ability of complex 7 was investigated under blue (465 nm)- and red-light (650 nm) irradiation by agarose gel electrophoresis together with complex 4 and [Ru(bpy)3]Cl2 for comparison. As shown in Figure 7A, complex 4 did not show any observable DNA Figure 5. Confocal ﬂuorescence images of calcein AM (green, live cells)/propidium iodide (PI; red, dead cells) costained A549 cells after treatment with complexes 4, 7, and [Ru(bpy)3]Cl2 at concentrations of 1 μM: (a, left) without irradiation, (b, right) irradiation at 650 nm. 4 and [Ru(bpy)3]Cl2 were also studied for comparison. As shown in Figure 5a, no signiﬁcant cell death was observed without irradiation for A549 cells after treatment with complexes 4, 7 and [Ru(bpy)3]Cl2. Once irradiation was conducted under 465 or 650 nm, cells treated by complex 7 were eﬀectively killed as revealed by the intense red ﬂuorescence, whereas complex 4 or [Ru(bpy)3]Cl2 treated cells were negligibly damaged, demonstrating the robust PDT ability of complex 7 again. Intracellular ROS Production. The intracellular ROS generation in A549 cells after treated with complexes 4, 7, and [Ru(bpy)3]Cl2 was evaluated using a 2,7-dichlorodihydroﬂuorescein diacetate (DCFH-DA) staining method. DCFHDA is a nonﬂuorescent cell-permeable indicator for ROS, which can be converted to the highly ﬂuorescent DCF upon intracellular oxidation by ROS. As shown in Figure 6, no ROS production was observed in A549 cells without light irradiation. However, after they were exposed to 465 nm light irradiation, the compound-treated A549 cells showed obvious green ﬂuorescence, demonstrating the successful production of ROS. Notably, in the presence of 650 nm illumination, ROS production was detected after the cells were treated with complexes 4 and 7. In contrast, negligible ROS signals were observed in [Ru(bpy)3]Cl2-treated cells, matching well with the extracellular 1O2 generation results. Figure 7. (A) Gel electrophoretic mobility pattern of pBR322 plasmid DNA incubated with various concentrations of Ru(II) complexes under blue and/or red light irradiation. Lanes 1−9 (0, 1, 5, 20, 40, 80, 160, 320, and 640 μM) + DNA: (a) 4 + 465 nm; (b) 7 + 465 nm; (c) [Ru(bpy)3]Cl2 + 465 nm; (d) 4 + 650 nm; (e) 7 + 650 nm; (f) [Ru(bpy)3]Cl2 + 650 nm. (B) Stereoview of the molecular docking of complex 7 with DNA duplex (PDB ID: 4JD8). F https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC Article Figure 8. Flow cytometry analysis for apoptosis of A549 cancer cells in the presence of (a) blue light and (b) red light. ascribed to its strong DNA binding ability as well as high 1O2 generation eﬃciency. Cell Death Study. The potential of complexes 4 and 7 and [Ru(bpy)3]Cl2 to induce cell death was determined with blue (465 nm)- and red-light (650 nm) irradiation by using an Annexin V-FITC/propidium iodide (PI) assay. A549 cancer cells were treated with the Ru(II) complexes at concentrations of their IC50 values. As shown in Figure 8, all Ru(II) complexes can induce obvious incidences of early- to late-stage apoptosis in A549 cancer cells in comparison with untreated cells (control) under blue- and red-light irradiation with apoptotic rates of ∼50%. This result was in accordance with the results of the MTT assay. Overall, this study indicates that the two Ru(II) complexes produced cancer cell death through an apoptotic pathway under light exposure. cleavage at the tested concentrations under blue light irradiation. In contrast, complex 7 exhibited considerable DNA photocleavage activity, which can completely convert supercoiled DNA form (form I) to nicked circular form (form II) at an extremely low concentration of 5 μM (drug to nucleotide ratio: 0.032). Moreover, the plasmid DNA gradually disappeared with increasing concentrations of complex 7, indicating that it can inhibit the intercalation of EtBr in plasmid DNA at high concentrations. In addition, [Ru(bpy)3]Cl2 displayed much weaker DNA photocleavage activity in comparison to complex 7 under the experimental conditions. For the red-light irradiation, a concentration-dependent DNA cleavage pattern was also observed for complex 7, while complex 4 and [Ru(bpy)3]Cl2 did not show any obvious cleavage, which is indicative of the eﬃcient DNA photocleavage ability of complex 7 upon red-light irradiation. When these results are taken together, the considerable DNA photocleavage activity of complex 7 may be one of the causes for its high photocytotoxicity. The binding of the PS to DNA is a key step for DNA photocleavage.52 It has been reported that dppz-containing Ru(II) complexes (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) can intercalate between DNA base pairs and serve as DNA molecular light switches.53 The dppn ligand in complex 7 is a derivative of dppz, which may have the potential to intercalate into DNA due to the excellent planar conjugated structure of dppn.54,55 Thus, a molecular docking study was carried out on a DNA duplex structure (PDB ID: 4JD8) to elucidate the DNA binding mode of complex 7 using the AutoDock 4.2 package.56,57 Obviously, complex 7 showed an intercalation behavior similar to that of dppz-containing Ru(II) complexes (Figure 7B), and the binding energy was calculated to be −10.78 kcal/mol, indicating that complex 7 can eﬀectively bind to the DNA. When these results are taken together, the considerable DNA photocleavage activity of complex 7 may be ■ CONCLUSION In summary, complexes 1−7 were designed as analogues to [Ru(bpy)3]Cl2 with the aim to red-shift the 1MLCT absorption of Ru(II)-polypyridyl complexes into the PDT window (600−850 nm). DFT calculations indicated that the HOMO−LUMO energy gaps of complexes 1−7 are much smaller than that of [Ru(bpy)3]2+, which is responsible for the red shifting of the 1MLCT absorption band of these complexes. Cytotoxicity data against A549 and HepG2 cells revealed that complex 7 showed extraordinarily high cytotoxicity under 650 nm irradiation, resulting in IC50 values of 56 and 63 nM with exceptionally large phototoxicity index (PI) values of 763 and 613, respectively. Thus, the resulting complex 7 with considerable red-light photocytotoxicity and high PI values shows a promising potential for therapeutic applications, which represents a new scaﬀold of Ru(II)polypyridyl photosensitizers for PDT in the “therapeutic window”. This study delivers a rational strategy for the design of tris-heteroleptic Ru(II) complexes as promising photosensitizers for cancer therapy. G https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry ■ pubs.acs.org/IC Article Complex 4. Yield: 0.48 g (63.5%), reddish brown powder. Anal. Calcd for C38H33Cl2N7Ru: C, 60.08; H, 4.38; N, 12.91. Found: C, 60.19; H, 4.35; N, 12.94. ESI mass spectrum data: m/z 344.5973 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 2.81 (s, 6H), 6.30−6.32 (d, 2H, J = 8.9 Hz), 6.50−6.51 (d, 2H, J = 8.9 Hz), 7.15− 7.16 (d, 1H, J = 8.9 Hz), 7.30−7.32 (t, 1H, J = 6.6 Hz), 7.34−7.36 (m, 1H), 7.52−7.53 (d, 1H, J = 5.4 Hz), 7.58−7.60 (t, 1H, J = 5.4 Hz), 7.62−7.65 (q, 2H, J = 6.4 Hz), 7.67−7.70 (t, 1H, J = 7.5 Hz), 7.80−7.81 (t, 1H, J = 5.3 Hz), 7.95−7.97 (t, 1H, J = 7.6 Hz), 7.98− 7.99 (d, 1H, J = 5.4 Hz), 8.15−8.19 (m, 2H), 8.24−8.27 (t, 2H, J = 7.8 Hz), 8.54−8.55 (d, 1H, J = 5.4 Hz), 8.56−8.60 (d-d, 2H, J1 = 8.5 Hz, J2 = 12.4 Hz), 8.79−8.82 (m, 2H), 8.90−8.91 (d, 1H, J = 8.2 Hz), 8.99−9.01 (d, 1H, J = 8.2 Hz), 9.67 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 40.34, 111.68, 122.65, 123.96, 124.33, 124.66, 125.08, 125.41, 125.52, 127.88, 128.29, 128.62, 128.64, 129.60, 129.75, 130.38, 132.18, 138.39, 138.55, 138.89, 139.10, 139.23, 139.30, 149.73, 150.43, 151.39, 151.58, 152.42, 153.43, 156.77, 156.85, 157.05, 157.21, 159.49, 169.01. Complex 5. Yield: 0.28 g (33.2%), reddish brown powder. Anal. Calcd for C44H30Cl2N8Ru: C, 62.71; H, 3.59; N, 13.30. Found: C, 62.73; H, 3.53; N, 13.31. ESI mass spectrum data: m/z 386.0775 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 1H NMR (600 MHz, DMSO): δ 6.75 (d, J = 7.6 Hz, 2H), 7.11 (t, J = 7.8 Hz, 2H), 7.25−7.16 (m, 2H), 7.53−7.48 (m, 1H), 7.77−7.73 (m, 4H), 7.83 (d, J = 5.2 Hz, 1H), 7.89−7.85 (m, 1H), 7.91 (d, J = 5.6 Hz, 1H), 8.02 (dd, J = 8.1, 5.4 Hz, 1H), 8.25−8.15 (m, 3H), 8.29 (td, J = 8.1, 1.2 Hz, 1H), 8.50−8.41 (m, 3H), 8.62 (d, J = 7.8 Hz, 1H), 8.71 (d, J = 8.3 Hz, 1H), 9.19 (dd, J = 5.2, 0.9 Hz, 1H), 9.23 (d, J = 8.7 Hz, 2H), 9.49 (s, 1H), 9.59 (dd, J = 8.1, 1.0 Hz, 1H), 9.66 (dd, J = 8.1, 1.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δ 121.78, 123.98, 124.33, 127.60, 128.28, 128.45, 128.62, 128.74, 129.10, 129.39, 131.13, 131.22, 133.82, 133.95, 135.08, 138.06, 138.29, 138.37, 138.96, 141.61, 141.64, 148.99, 151.21, 151.32, 152.44, 152.51, 152.80, 154.00, 155.63, 156.55, 157.20, 169.93. Complex 6. Yield: 0.35 g (39.5%), reddish brown powder. Anal. Calcd for C46H35Cl2N9Ru: C, 63.41; H, 4.17; N, 13.91. Found: C, 63.38; H, 4.19; N, 13.94. ESI mass spectrum data: m/z 407.5984 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 2.35 (s, 6H), 6.01 (d, J = 9.1 Hz, 2H), 6.43 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 6.4 Hz, 1H), 7.67−7.62 (m, 1H), 7.72−7.67 (m, 2H), 7.80−7.76 (m, 1H), 7.87 (t, J = 6.2 Hz, 2H), 7.95 (d, J = 5.4 Hz, 1H), 8.14−8.13 (m, 1H), 8.16 (dd, J = 8.0, 5.3 Hz, 1H), 8.21−8.18 (m, 2H), 8.26−8.22 (m, 1H), 8.32 (dd, J = 11.7, 4.4 Hz, 1H), 8.38−8.35 (m, 2H), 8.59 (d, J = 7.8 Hz, 1H), 8.69 (d, J = 5.2 Hz, 1H), 9.04 (d, J = 11.1 Hz, 2H), 9.10−9.06 (m, 2H), 9.33−9.31 (m, 1H), 9.35 (s, 1H), 9.54 (dd, J = 8.0, 1.1 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δ 40.55, 111.59, 122.82, 128.29, 128.44, 129.06, 130.09, 130.15, 135.02, 138.09, 138.21, 138.73, 140.84, 150.08, 150.90, 151.11, 156.90, 157.09, 157.98, 166.10. Complex 7. Yield: 0.22 g (24.0%), reddish brown powder. Anal. Calcd for C50H37Cl2N9Ru: C, 64.17; H, 3.99; N, 13.47. Found: C, 64.22; H, 4.02; N, 13.49. ESI mass spectrum data: m/z 432.6187 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 2.33 (s, 6H), 5.93−5.94 (d, 2H, J = 8.9 Hz), 6.26−6.27 (d, 2H, J = 8.8 Hz), 7.28− 7.29 (d, 1H, J = 8.9 Hz), 7.40−7.43 (t, 1H, J = 7.8 Hz), 7.51−7.53 (t, 1H, J = 6.6 Hz), 7.68−7.70 (d, 1H, J = 6.5 Hz), 7.73−7.75 (t, 1H, J = 7.5 Hz), 7.78−7.81 (m, 3H), 7.98−7.99 (t, 1H, J = 5.0 Hz), 8.07− 8.10 (d-d, 1H, J = 5.4 Hz, J = 8.1 Hz), 8.19−8.27 (m, 4H), 8.35−8.36 (d, 1H, J = 5.0 Hz), 8.46−8.47 (m, 2H), 8.61−8.62 (d, 1H, J = 8.4 Hz), 8.65−8.66 (d, 1H, J = 5.4 Hz), 8.86−8.88 (d, 1H, J = 8.4 Hz), 8.94−8.95 (d, 1H, J = 8.2 Hz), 9.02−9.04 (d, 1H, J = 8.2 Hz), 9.21 (s, 1H), 9.24 (s, 1H), 9.36−9.37 (d, 1H, J = 8.0 Hz), 9.64−9.65 (d, 1H, J = 8.0 Hz), 9.70 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 63.28, 111.46, 122.37, 124.18, 125.10, 125.40, 125.58, 128.44, 128.53, 128.59, 128.74, 129.15, 129.89, 130.05, 130.44, 132.33, 133.73, 134.15, 135.15, 135.17, 138.24, 138.31, 138.33, 138.73, 139.18, 139.56, 140.83, 141.01, 149.87, 149.96, 151.21, 151.32, 152.55, 152.81, 154.11, 156.00, 156.81, 157.05, 159.68, 169.52. DFT Calculations. All calculations were performed using the Gaussian 09 suite of programs.58 Full geometry optimizations were EXPERIMENTAL SECTION Materials and Measurements. All analytical grade chemicals and solvents were used without further puriﬁcation. cis-[Ru(bpy)(dppn)Cl2] was prepared according to previous literature methods.49 1 H and 13C NMR spectra were recorded on a Bruker Avance III-HD 600 MHz spectrometer. Elemental analysis of C, H, and N used a Vario MICRO CHNOS elemental analyzer (Elementar). UV−vis absorption and luminescence spectra were measured on a Shimadzu UV2600 instrument and a FluoroMax-4 ﬂuorometer, respectively. Mass spectrometry was performed using an Agilent 6224 ESI/TOF MS instrument. Cell accumulation was conducted on a PerkinElmer NexION 1000G ICP mass spectrometer. Preparation of 1. Complex 1 was prepared according to previous literature.38 Yield: 0.57 g (71.3%), yellowish brown powder. Anal. Calcd for C32H26Cl2N6Ru: C, 57.66; H, 3.93; N, 12.61. Found: C, 57.71; H, 3.91; N, 12.64. ESI mass spectrum data: m/z 298.0647 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 6.63 (d, J = 7.6 Hz, 2H), 7.07 (t, J = 7.8 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 7.35 (t, J = 6.6 Hz, 1H), 7.53 (t, J = 6.2 Hz, 1H), 7.61−7.65 (m, 2H), 7.67−7.73 (m, 3H), 7.75 (d, J = 5.4 Hz, 1H), 7.85 (d, J = 5.5 Hz, 1H), 7.90 (m, 1H), 8.26−8.28 (m, 4H), 8.44 (d, J = 8.1 Hz, 1H), 8.58 (d, J = 7.7 Hz, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.69 (d, J = 5.3 Hz, 1H), 8.94− 9.00(m, 2H), 9.42 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 121.60, 124.06, 124.27, 125.15, 125.23, 127.79, 128.29, 128.55, 128.58, 128.70. 129.33, 129.55, 131.25, 138.02, 138.17, 138.60, 138.68, 138.77, 148.98, 151.64, 151.73, 152.05, 152.18, 153.43, 156.54, 156.76, 156.82, 156.93, 157.31, 169.88. General Procedure for the Synthesis of Complexes 2−7. A methanol solution (30 mL) of aniline (for the synthesis of complexes 3 and 5) or N,N-dimethyl-1,4-phenylenediamine (for the synthesis of complexes 2, 4, 6, and 7) (1.2 mmol) and the corresponding aldehydes (1.2 mmol) was heated at reﬂux for 12 h to obtain a yellowish brown solution. The solution was used without puriﬁcation. Then cis-[Ru(bpy)2Cl2] or cis-[Ru(bpy)(dppn)Cl2] (1.0 mmol) was added, and the resulting mixture was stirred under reﬂux for 12 h, during which time the mixture turned dark brown. The solvent was then removed by evaporation under reduced pressure. The crude product was puriﬁed using dichloromethane/methanol (20/1, v/v) through preparative column chromatography (basic Al2O3) to aﬀord the product. Complex 2. Yield: 0.58 g (68.11%), yellowish brown powder. Anal. Calcd for C34H31Cl2N7Ru: C, 57.55; H, 4.40; N, 13.82. Found: C, 57.58; H, 4.39; N, 13.86. ESI mass spectrum data: m/z 319.5857 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 2.81 (s, 6H), 6.31 (d, J = 9.0 Hz, 2H), 6.59 (d, J = 8.6 Hz, 2H), 7.41 (t, J = 6.6 Hz, 1H), 7.52−7.58 (m, 2H), 7.64 (d, J = 5.0 Hz, 1H), 7.73−7.65 (m, 3H), 7.73−7.77 (m, 2H), 7.96−8.02 (m, 1H), 8.24−8.15 (m, 3H), 8.25 (m, 1H), 8.52−8.63 (m, 3H), 8.74 (t, J = 9.3 Hz, 1H), 9.02 (m, 2H), 9.29−9.34 (m, 1H). 13C NMR (150 MHz, DMSO-d6): δ 48.99, 111.66, 122.95, 124.33, 128.52, 137.97, 138.53, 138.67, 150.56, 151.47, 151.70, 151.73, 151.81, 153.31, 156.75, 156.88, 157.02, 157.82, 165.42. Complex 3. Yield: 0.45 g (52.33%), reddish brown powder. Anal. Calcd for C36H28Cl2N6Ru: C, 60.34; H, 3.94; N, 11.73. Found: C, 60.40; H, 3.92; N, 11.75. ESI mass spectrum data: m/z 323.0726 ([M/2 − Cl]+). 1H NMR (600 MHz, DMSO-d6): δ 6.59 (d, J = 7.3 Hz, 2H), 7.08 (t, J = 7.2 Hz, 2H), 7.16 (t, J = 7.0 Hz, 1H), 7.24 (d, J = 8.9 Hz, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.49 (d, J = 4.8 Hz, 1H), 7.61− 7.67 (m, 3H), 7.73 (t, J = 7.2 Hz, 1H), 7.88 (d, J = 12.7 Hz, 2H), 8.00 (d, J = 4.8 Hz, 1H), 8.15 (t, J = 7.6 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.24−8.30 (m, 2H), 8.48 (d, J = 7.8 Hz, 1H), 8.65 (d, J = 7.5 Hz, 2H), 8.77 (d, J = 7.8 Hz, 1H), 8.84−8.93 (m, 2H), 8.99 (d, J = 7.9 Hz, 1H), 9.83 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 121.37, 124.15, 124.71, 125.05, 125.41, 125.05, 125.41, 125.97, 127.93, 128.34, 128.41, 128.71, 128.80, 129.26, 129.99, 130.04, 130.42, 132.39, 138.42, 138.66, 139.14, 139.21, 139.55, 149.21, 149.66, 151.29, 151.81, 152.33, 153.78, 156.68, 156.84, 157.08, 158.99, 172.53. H https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry pubs.acs.org/IC Article red light (650 ± 10 nm, 72.0 J cm−2). After 24 h of incubation, the mixtures (5 μL) with loading buﬀer (1 μL) were submitted to electrophoresis in agarose gel in TA buﬀer at 100 V for 90 min. Agarose gels were then dyed with ethidium bromide (0.5 mg/L) for 20 min. Bands were imaged by using a Molecular Imager (Bio-Rad, USA) under UV light. Molecular Docking. A molecular docking simulation was carried out using AutoDock 4.2.57 The crystal structure of the DNA duplex was obtained from the Protein Data Bank (PDB ID: 4JD8).58 The docking procedure was conducted using a Lamarckian genetic algorithm for 200 docking runs. Visualization results were performed by PyMOL. Apoptosis Analysis by Flow Cytometry. Complexes 4, 7, and [Ru(bpy)3]Cl2 with concentrations of IC50 values were added to the A549 cells. After incubation for 4 h, cells were irradiated with 465 ± 10 nm LED irradiation (25.2 J cm−2) or red light (650 ± 10 nm, 144.0 J cm−2). Then the cells were incubated in the dark for a further 24 h and collected by centrifugation (5 min, 25 °C, 2000 rpm). Afterward, the A549 cells were dyed by Annexin V-FITC/PI and analyzed with a ﬂow cytometer. carried out for complexes 1−7 and [Ru(bpy)3]Cl2 by using the B3LYP density functional with the LanL2DZ basis set and an eﬀective core functional used for the ruthenium atom, while the 6-31G(d,p) basis set was used for the other atoms.59,60 The time-dependent density functional theory (TD-DFT) calculations were performed at the same level to predict the singlet electronic transitions and the UV−visible spectra. 1 O2 Generation. The 1O2 generation of complexes 1−7 was evaluated through monitoring the absorption spectral change at 378 nm of ABDA, and [Ru(bpy)3]Cl2 (ΦΔ = 0.41 in water) was used as a standard in water. The experiment was conducted for complexes 1−7 (10 μM) in DMSO/water (1/99, v/v) containing ABDA (50 μM). The absorption spectra were recorded every 30 s under 465 ± 10 nm LED irradiation (1.26 J cm−2) or every 4 min with red-light irradiation (650 ± 10 nm, xenon lamp with a band-pass ﬁlter, 19.2 J cm−2). The ΦΔ values were calculated with the equation ΦΔ(PS) = ΦΔ(Std)SPS × FStd/(Sstd × FPS), where PS designates the complexes 1−7 and Std designates [Ru(bpy)3]Cl2; S is the decomposition rate of ABDA at 378 nm, and F is the correction factor of absorption, which is given by F = 1 − 10−OD (OD denotes the optical density of complexes 1−7 and [Ru(bpy)3]Cl2 at 465 nm). Cytotoxicity Assay of PDT. The photocytotoxicity of complexes 1−7 and [Ru(bpy)3]Cl2 against A549 and HepG2 cells was determined by means of an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. Cells (104 per well) were seeded in 96-well plates and allowed to adhere for 24 h. After that, complexes 1−7 and [Ru(bpy)3]Cl2 were dissolved with DMF and diluted with the medium to the required concentrations (the ﬁnal concentration of DMF was less than 0.4%). After being incubated in the dark for 4 h, cells were irradiated with 465 ± 10 nm nm LED irradiation (25.2 J cm−2) or red light (650 ± 10 nm, 144.0 J cm−2), and then the cells were incubated in the dark for a further 48 h. After that, the cells were stained with MTT (5 mg/mL) for another 5 h. The inhibition of cell growth was detected using an enzyme-labeling instrument. The IC50 values were calculated by SPSS software. Cellular Accumulation. A549 cells with good viability were transferred into 6-well plates and cultured overnight at 37 °C. Then, complexes 4, 7, and [Ru(bpy)3]Cl2 were added with a concentration of 20 μM and incubated with the cells for 12 h. Then the supernatants were removed, and the cells were washed three times with ice-cold PBS. The cells were then digested with HNO3 (65%), and the Ru contents were measured by ICP-MS. Calcein AM and Propidium Iodide (PI) Costaining. For the calcein AM and propidium iodide (PI) costaining assay, A549 cells (105 per well) were seeded and cultured in confocal dishes overnight at 37 °C. Then complexes 4, 7, and [Ru(bpy)3]Cl2 were added to the cells with a ﬁnal concentration of 1 μM. After 4 h of incubation, the cells were exposed to LED light (465 ± 10 nm, 25.2 J cm−2) or red light (650 ± 10 nm, 144.0 J cm−2). Thereafter, the cells were stained with Calcein AM/PI Double Stain Kit according to the instruction manual. Fluorescence images of the stained cells were then taken using a confocal microscope. Intracellular ROS Production. The ROS generation in A549 cells was measured by DCFH-DA staining. A549 cells were seeded in a 6-well plate at a density of 2 × 105 cells/well and cultured for 12 h at 37 °C. Then, the tested complexes were added with a ﬁnal concentration of 30 μM. After 4 h of incubation, DCFH-DA was added and the cells were incubated for another 30 min. Thereafter, the cells were washed with fresh medium three times followed by irradiation with 465 ± 10 nm LED light (25.2 J cm−2) or red light (650 ± 10 nm, 144.0 J cm−2). The photos were captured using a confocal microscope. Gel Electrophoresis Study. DNA photocleavage activities of complexes 4, 7, and [Ru(bpy)3]Cl2 were evaluated by agarose gel electrophoresis. The tested complexes were ﬁrst dissolved in DMF (10 mM) and then diluted to the desired concentrations with TrisH3PO4 (50 mM, pH 7.2) buﬀer. The tested complexes (5 μL; lanes 1−9 0, 1, 5, 20, 40, 80, 160, 320, and 640 μM) and the ﬁnal concentration of pBR322 plasmid DNA (50 ng/μL, 5 μL) were mixed together and irradiated with blue light (465 ± 10 nm, 12.5 J cm−2) or ■ ASSOCIATED CONTENT sı Supporting Information * The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01860. 1 ■ H and 13C NMR and ESI mass spectra of complexes 1− 7, frontier molecular orbitals of complexes 1−7 and [Ru(bpy)3]Cl2, production of 1O2 by complexes 1−7 and [Ru(bpy)3]Cl2 upon 650 nm irradiation and dosedependent cell viability curves and synthetic route of 1− 7, confocal ﬂuorescence images of calcein AM and propidium iodide (PI) costained A549 cells, cell accumulation of complexes 4, 7, and [Ru(bpy)3]Cl2 on A549 cells, and Cartesian coordinates of all optimized structures of complexes 1−7 and [Ru(bpy)3]2+ (PDF) AUTHOR INFORMATION Corresponding Authors Jian Zhao − Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China; Key Laboratory for Organic Electronics and Information Displays, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications (NUPT), Nanjing 210023, People’s Republic of China; orcid.org/ 0000-0002-9365-7727; Email: zhaojianzhaokuan@163.com Shaohua Gou − Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China; orcid.org/0000-0003-0284-5480; Email: sgou@seu.edu.cn Qiang Zhao − Key Laboratory for Organic Electronics and Information Displays, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications (NUPT), Nanjing 210023, People’s Republic of China; orcid.org/0000-0002-3788-4757; Email: iamqzhao@njupt.edu.cn Authors Shuang Li − Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Pharmaceutical Research Center, I https://dx.doi.org/10.1021/acs.inorgchem.0c01860 Inorg. 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School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China Xinyi Wang − Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China Gang Xu − Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.0c01860 Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS We are grateful to the National Natural Science Foundation of China (Grants 21601034 and 21571033) and Jiangsu Province Natural Science Foundation (Grant BK20160664) for ﬁnancial aid for this work. 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