Increasing Anticancer Activity with Phosphine Ligation in Zwitterionic Half-Sandwich Iridium(III), Rhodium(III), and Ruthenium(II) Complexes.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Increasing Anticancer Activity with Phosphine Ligation in\n Zwitterionic Half-Sandwich Iridium(III), Rhodium(III), and\n Ruthenium(II) Complexes\n Xueyan Hu, Lihua Guo,* Mengqi Liu, Qiuya Zhang, Yuwen Gong, Mengru Sun, Shenghan Feng,\n Youzhi Xu, Yiming Liu, and Zhe Liu*\n Cite This: Inorg. Chem. 2022, 61, 20008\u221220025 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:19:57 (UTC).\n\n\n\n\n ABSTRACT: The synthesis and biological assessment of neutral or cationic platinum group metal-based anticancer complexes have\n been extremely studied, whereas there are few reports on the corresponding zwitterionic complexes. Herein, the synthesis,\n characterization, and bioactivity of zwitterionic half-sandwich phosphine\u2212imine iridium(III), rhodium(III), and ruthenium(II)\n complexes were presented. The sulfonated phosphine\u2212imine ligand and a group of zwitterionic half-sandwich P,N-chelating\n organometallic complexes were fully characterized by nuclear magnetic resonance (NMR), mass spectrum (electrospray ionization,\n ESI), elemental analysis, and X-ray crystallography. The solution stability of these complexes and their spectral properties were also\n determined. Notably, almost all of these complexes showed enhanced anticancer activity against model HeLa and A549 cancer cells\n than the corresponding zwitterionic pyridyl\u2212imine N,N-chelating iridium(III) and ruthenium(II) complexes, which have exhibited\n inactive or low active in our previous work. The increase in the lipophilic property and intracellular uptake levels of these zwitterionic\n P,N-chelating complexes appeared to be associated with their superior cytotoxicity. In addition, these complexes showed\n biomolecular interactions with bovine serum albumin (BSA). The flow cytometry studies indicated that the representative complex\n Ir1 could induce early-stage apoptosis in A549 cells. Further, confocal microscopy imaging analysis displayed that Ir1 entered A549\n cells through the energy-dependent pathway, targeted lysosome, and could cause lysosomal damage. In particular, these complexes\n could impede cell migration in A549 cells.\n\n\n 1. INTRODUCTION plexes (Scheme 1). Over the past few years, cyclometalated\n Cancer has been a common disease with high incidence, which complexes have been gaining popular attention due to their\n is widely perceived as one of the leading reasons of death.1,2 excellent luminescence nature and pretty good cytotoxic\n Although a series of platinum antitumor drugs such as cisplatin efficacy under light, which afforded quite a wide range of\n and its derivatives have been applied in clinical practice, the applications for photodynamic therapy (PDT), molecular\n serious side effects have made scientists commit themselves to imaging, and bioprobes.23,24 Recently, half-sandwich iridium-\n searching for more efficient and less toxic antitumor drugs.3\u22128 (III), rhodium(III), ruthenium(II), and osmium(II) organo-\n The tumor-killing agents of a number of platinum family metallic anticancer complexes with the structure type [(\u03b76-\n metals with reduced side effects and excellent anticancer arene)/(\u03b75-Cp*)M(XY)Cl]0/+ (Cp*: C5(CH3)5; M: Ir, Rh,\n activity have been widely concerned.9\u221220 The ruthenium Ru, Os; XY: bidentate chelating ligands) have attracted\n complexes NAMI-A and KP1019 have shown the most\n promising results in preclinical and clinical trials.21,22 Recently, Received: September 15, 2022\n the organometallic platinum group metal compounds offer rich Published: November 25, 2022\n versatility for the rational design of anticancer complexes.\n According to the structure type, these platinum group metal-\n based anticancer complexes can be divided into two main\n groups: cyclometalated complexes and half-sandwich com-\n\n \u00a9 2022 American Chemical Society https://doi.org/10.1021/acs.inorgchem.2c03279\n 20008 Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Cyclometalated Complexes and Half-Sandwich In the last few years, organometallic anticancer complexes\nComplexes bearing phosphine-containing chelating ligand have become a\n new approach to adjust the chemical and biological proper-\n ties.42\u221245 A typical example is RAPTA family (e.g., RAPTA-C;\n Scheme 2, V) containing 1,3,5-triaza-7-\n phosphatricyclo[3.3.1.1.]decane ligand and Ru\u2212arene com-\n plexes,46,47 which has been at an advanced preclinical trial.48\n Previous studies have showed that the presence of phosphine\n ligands could increase membrane permeability and the\n lipophilicity of the anticancer complexes to gain cytotox-\n icity.49\u221251 Consequently, we have become interested in\n whether the introduction of phosphine into zwitterionic\nwidespread attention due to their amenable coordination complexes could increase cytotoxicity. Additionally, the\nstructure and different mechanisms of action (MoAs) with introduction of lipophilic phenyl rings in phosphine ligands\nplatinum drugs.25\u221228 Most of these studies concentrated on the may also give rise to the enhanced anticancer activity of the\npreparation and application of neutral and cationic anticancer corresponding complexes. In particular, the zwitterionic\ncomplexes with various bidentate XY ligands.29\u221234 For coordination mode with lipophilic phosphine ligation may\nexample, the Sadler group has shown that switching on the increase the cellular accumulation and even the possibility to\ncytotoxic potency of the corresponding iridium(III) complexes target organelles. Based on these considerations, we herein\ncould be realized by replacing neutral N,N-chelating ligand describe the preparation, characterization, and biological\n(2,2\u2032-bipyridine) with the negatively charged anionic C,N- assessment of a group of zwitterionic phosphine\u2212imine half-\nchelating ligand (2-phenylpyridine) (Scheme 2, I and sandwich iridium(III), rhodium(III), and ruthenium(II)\nII).33,35\u221238 Our group have also systematically investigated organometallic complexes (Scheme 2). Furthermore, the\nthe counteranion effect on the cytotoxicity and biological phosphine-enhanced cytotoxicity of zwitterionic complexes,\nactivity of the cationic half-sandwich iridium(III) complexes mechanisms of action, and molecular imaging have also been\n(Scheme 2, III).39 The above-mentioned studies indicated that discussed.\nthe charge of the metal center, the substitution model of the\nchelate ligands, and the counteranion have a great influence on 2. RESULTS AND DISCUSSION\nchemical reactivity and anticancer activity of the complexes.\nHence, our group subsequently have prepared a group of rarely 2.1. Syntheses and Characterizations. Sulfonated\nreported zwitterionic iridium(III) and ruthenium(II) com- phosphine\u2212imine ligand L was synthesized in a 58% isolated\nplexes and investigated their biological activity (Scheme 2, yield by a Schiff base reaction of sodium 2,6-diisopropylaniline\nIV).40 Unfortunately, these zwitterionic half-sandwich com- sulfonate with 2-(diphenylphosphinyl) benzaldehyde (Scheme\nplexes were inactive, which may be mainly due to their low 3). This ligand can be readily purified by recrystallization from\nhydrophobicity.40 The introduction of fluorinated substituents methanol and subsequent diethyl ether washing. In the 1H\ninto zwitterionic complexes seemed to be a feasible strategy to NMR spectra, the characteristic peak of L was at 8.72 ppm\nimprove the hydrophobicity, thus leading to increased (Figure S3), which was assigned to the hydrogen of double\nanticancer activity.41 However, these pyridyl\u2212imine zwitter- bonds of the CH\ufffdN group. The structure of L was also\nionic complexes still showed no or low anticancer activity confirmed by 31P{1H} NMR (Figure S4), 13C{1H} NMR\ncompared to the commercial cisplatin. Therefore, this (Figure S5), and mass spectrometry (Figure S36).\nprompted us to increase their anticancer activity through The bimetallic iridium(III), rhodium(III), and ruthenium-\nfurther structural modification. (II) precursors [(\u03b75-C5Me5)MCl2]2 (M = Ir (D1); Rh (D5)),\n\nScheme 2. Reported Organometallic Half-Sandwich Complexes and Our Current Work\n\n\n\n\n 20009 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 3. Syntheses of Ligand L and Complexes Ir1\u2212Ir4, Rh1\u2212Rh3, and Ru1\u2212Ru3\n\n\n\n\nFigure 1. X-ray crystal structures of complexes (a) Ir1, (b) Ir2, and (c) Rh2 with the thermal ellipsoids drawn at the 50% probability level. The\nhydrogen atoms have been omitted for clarity. (a) Bond angles around Ir(III) ion (deg): N1\u2212Ir1\u2212P1 = 88.2(5), P1\u2212Ir1\u2212Cl1 = 90.2(2), N1\u2212Ir1\u2212\nCl1 = 88.0(5). Bond lengths (\u00c5): Ir1\u2212C(centroid) = 2.0144, Ir1\u2212P1 = 2.260(6), Ir1\u2212N1 = 2.06(2), Ir1\u2212Cl1 = 2.360(6). (b) Bond angles around\nIr(III) ion (deg): N1\u2212Ir1\u2212P1 = 85.2(2), P1\u2212Ir1\u2212Cl1 = 88.17(9), N1\u2212Ir1\u2212Cl1 = 85.3(2). Bond lengths (\u00c5): Ir1\u2212C(centroid) = 1.8448, Ir1\u2212P1\n= 2.280(3), Ir1\u2212N1 = 2.132(8), Ir1\u2212Cl1 = 2.409(2). (c) Bond angles around Rh(III) ion (deg): N1\u2212Rh1\u2212P1 = 84.56(6), P1\u2212Rh1\u2212Cl1 =\n88.64(3), N1\u2212Rh1\u2212Cl1 = 87.30(6). Bond lengths (\u00c5): Rh1\u2212C(centroid) = 1.8587, Rh1\u2212P1 = 2.3128(7), Rh1\u2212N1 = 2.181(2), Rh1\u2212Cl1 =\n2.4292(7).\n\n[(\u03b75-CpR)MCl2]2 (CpR = C5(CH3)4R, R = Cy (M = Ir (D2); methods40,41,52\u221258 or using the modified procedure (D6; see\nM = Rh (D6)), 2-methylbenzene (M = Ir (D4)), 3,5- the Supporting Information). It should be noted that the arene\nbis(trifluoromethyl)benzene) (M = Ir (D3); Rh (D7)) and ligand of ruthenium complexes is different from that of iridium\n[(\u03b76-arene)RuCl2]2 (D8\u2212D10) were prepared by the reported and rhodium complexes since the presence of \u03b76-arene\n 20010 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 2. UV\u2212vis spectra for complexes Ir1, Ir2, Rh1, and Ru1 recorded over a period of 24 h at 37 \u00b0C, (a) solution in 25% DMSO/75% PBS (v/\nv) of Ir1; (b) solution in 20% DMSO/80% PBS (v/v) of Ir2; (c) solution in 25% DMSO/75% PBS (v/v) of Rh1; and (d) solution in 20%\nDMSO/80% PBS (v/v) of Ru1.\n\nsubstituent was identified to stabilize ruthenium complexes in also confirmed by the X-ray crystallography (Figure 1 and\nthe +2 oxidation state by being relatively inert to displace- Tables S1\u2212S3).\nment.59\u221262 Moreover, another motivation for the development The single-crystal samples of Ir1, Ir2, and Rh2 were\nof air-stable \u03b76-arene ruthenium(II) complexes was the obtained by the slow diffusion of n-hexane (or a mixture of\nactivation by reduction hypothesis, which suggested that active diethyl ether and n-hexane) into its solution in dichloro-\nruthenium(II) species may be formed in vivo from ruthenium- methane (or acetonitrile) at room temperature. The selected\n(III) complexes.63\u221265 Scheme 3 shows the synthetic procedure bond lengths, bond angles, and the crystalline structure of\nof zwitterionic phosphine\u2212imine half-sandwich iridium(III), complexes Ir1, Ir2, and Rh2 are given in Figure 1. Ir1, Ir2, and\nrhodium(III), and ruthenium(II) complexes containing Rh2 adopted a pseudo-octahedral \u201cthree-legged piano-stool\u201d\nsulfonate moiety. Treatment of precursors D1\u2212D10 with the geometry configuration. The \u201cseat\u201d was composed of the CpR\nligand L in methanol for 72 h afforded the complexes Ir1\u2212Ir4, ring. Moreover, sulfonated phosphine\u2212imine ligand and\nRh1\u2212Rh3, and Ru1\u2212Ru3 in 58\u221269% isolated yields. These chloride occupied the three legs in a \u201cpiano-stool\u201d config-\ncomplexes were fairly stable in air and soluble in methanol, uration. Furthermore, the central metallic iridium(III) or\ndichloromethane, and DMSO. All of the desired compounds rhodium(III) ion is a portion of a six-membered metallacycle\nwere first reported and determined by 1H, 13C{1H}, and system. The Ir1\u2212Cl1 or Rh1\u2212Cl1 distances of Ir1, Ir2, and\n31 1\n P{ H} NMR (Figures S6\u2212S35), mass spectrum (ESI, Figures Rh2 are 2.360(6), 2.409(2), and 2.4292(7) \u00c5, respectively.\nS37\u2212S46), and elementary analyses (C, H, N). The 1H NMR The Ir1\u2212Cp*(centroid) distances of Ir2 (1.8448 \u00c5) are\nspectra of these complexes showed the characteristic peaks of slightly shorter than those of Ir1 (2.0144 \u00c5). The Ir1\u2212P1 or\nthe CpR/arene ring and the P,N-chelating ligands. Compared Rh1\u2212P1 bond distances (Ir1: 2.260(6) \u00c5; Ir2: 2.280(3) \u00c5;\nto the spectra of the free ligand L, the chemical shift of CH\ufffd Rh2: 2.3128(7) \u00c5) of Ir1, Ir2, and Rh2 are longer than Ir1\u2212\nN peaks for these complexes changed and appeared at ca. \u03b4 N1 or Rh1\u2212N1 bond distances (Ir1: 2.06(2) \u00c5; Ir2: 2.132(8)\n8.58\u22128.80 ppm, which supported the ligand coordination to \u00c5; Rh2: 2.181(2) \u00c5). Clearly, the cationic centers Ir(III) and\nthe metal center. The splitting of the 31P signal was observed in Rh(III) are bounded covalently through the phosphine\u2212imine\nthe 13P{1H} spectrum of the complexes Rh1, Rh2, and Rh3, ligand to the terminational negative sulfonate moiety. Hence,\nwhich was attributed to rhodium\u2212phosphine coupling. The the zwitterionic structure of these complexes was successfully\n13\n C{1H} NMR spectra for these complexes showed the signals identified.\nof CH\ufffdN in the range of \u03b4 175\u2212176 ppm, which appeared 2.2. Stability in Solution. It is meaningful to assess the\nmore downfield than that (161 ppm) in the free ligand L. stability of the metal complexes in aqueous media or\nFurther, the molecular structure of some typical complexes was physiological conditions for drug development. The hydrolysis\n 20011 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 3. (a) UV/vis spectra of complexes Ir1\u2212Ir4, Rh1\u2212Rh3, and Ru1\u2212Ru3 (20 \u03bcM) in methanol solutions at 37 \u00b0C. The inset represents the\noffset spectra for clarity. (b) Normalized emission spectra of complexes Ir1\u2212Ir4, Rh1\u2212Rh3, and Ru1\u2212Ru3 (20 \u03bcM) in methanol at 37 \u00b0C (\u03bbex =\n404\u2212407 nm; \u03bbem = 460\u2212466 nm). The inset represents the locally enlarged spectra for clarity. Inset: wavelength from 450 to 490 nm.\n\nof M\u2212Cl bonds, i.e., the exchange of Cl/H2O, generally at ca. 260 and 350 nm were observed, respectively. The\nrepresented an important activation step for some transition- absorption bands below 350 nm were attributed to spin-\nmetal-based anticancer complexes since M-OH2 aqua com- allowed \u03c0\u2212\u03c0* ligand-centered transitions. The wide and low-\nplexes were often more active than the corresponding chloride intensity bands at ca. 410 nm were detected in these\ncomplexes.33,66 The stability of these complexes had been complexes, which was considered to be related to spin-allowed\nassessed in a 20% DMSO/80% PBS (pH \u2248 7.2, prepared from charge transfer from metal to ligand (MLCT) since the peak\nH2O) (v/v) or 25% DMSO/75% PBS (v/v) using UV\u2212vis above 350 nm could not be found in the absorption spectra of\nspectroscopy at 37 \u00b0C at different time intervals (Figures 2 and the free ligand.\nS47). Minor changes of absorption intensities were detected in Upon excitation at 404\u2212407 nm, Ir1\u2212Ir4, Rh1\u2212Rh3, and\nthe spectrum of complex Ir1 for a period of 8 h (Figure 2a). Ru1\u2212Ru3 exhibited blue emissions with maxima at 460\u2212466\nHowever, only negligible changes were found in the absorption nm (Ir1: 463 nm, Ir2: 465 nm, Ir3: 460 nm, Ir4: 464 nm,\nspectra of complexes Ir2\u2212Ir4, Rh1\u2212Rh3, and Ru1\u2212Ru3, Rh1: 465 nm, Rh2: 461 nm, Rh3: 461 nm, Ru1: 464 nm, Ru2:\nwhich suggested their sufficient stability in aqueous solutions 466 nm, Ru3: 466 nm) at 37 \u00b0C in methanol (Figure 3b).\n(e.g., Ir2 in Figure 2b). It seemed that the change in the metal They showed similar emission spectra, indicating that the\ncenter from Ir(III) to Rh(III) increased their stability (Ir1 vs variation of the CpR/arene ring and the metal center has a\nRh1). Further, the stability of several typical zwitterionic minor influence on the spectral emission bands. The emission\nphosphine\u2212imine complexes (Ir1, Rh1, and Ru1) in 90% quantum yields (\u03a6) of the representative complexes Ir1, Rh1,\nDMSO-d6/10% PBS (pH \u2248 7.2, prepared from D2O) (v/v) or and Ru1 were determined, respectively, with a calibrated\n85% DMSO-d6/15% PBS (v/v) was also monitored through integrating sphere system. The absolute quantum yields of\n1\n H NMR spectroscopy (Figures S48\u2212S50). The spectra data these complexes are in the order Rh1 (0.26) > Ru1 (0.18) >\nof the above-mentioned complexes under the test conditions Ir1 (0.14). Further, the average lifetimes of Ir1, Rh1, and Ru1\npresented no changes over time at 37 \u00b0C, evidencing that no are 4.61, 4.27, and 4.50 \u03bcs, respectively (Figure S52),\nhydrolysis occurred in the concentrated solutions. Additionally, indicating that these complexes are fluorescent. The photo-\nthe NMR analysis of these complexes was in agreement with luminescent characteristic could make it possible to investigate\ntheir corresponding molecular structure over 24 h, indicating the mechanism of actions of the complexes by bioimaging.\nthat no ligand dissociation and decomposition occurred under 2.4. Cytotoxicity. With cisplatin and half-sandwich\nthe experimental conditions. The stability of these complexes complex RAPTA-C as control, the cytotoxicity of Ir1\u2212Ir4,\nin pure DMSO was also evaluated. The 1H NMR spectra of Ir1 Rh1\u2212Rh3, and Ru1\u2212Ru3 toward lung cancer A549 cells and\nexhibited no obvious change over 24 h, and the assignment of cervical carcinoma HeLa cells was determined using MTT\nprotons was completely in agreement with its molecular assay (Table 1). Notably, no cytotoxicity (IC50 > 100 \u03bcM,\nstructure (Figure S51), suggesting that this type of zwitterionic IC50: half-maximal inhibitory concentration) was observed by\ncomplex showed sufficient stability in DMSO. Overall, the the free ligand (L) and dimer precursors (D1\u2212D10) (Table\nexperiments demonstrated that these zwitterionic complexes S4), suggesting that the cytotoxicity of the complexes in this\nwere fairly stable and could be subjected to perform further system was ascribed to the chelation. Notably, these\nbiological studies under physiological conditions. Notably, our zwitterionic phosphine\u2212imine iridium(III) complexes Ir1\u2212\npreviously reported zwitterionic pyridyl\u2212imine N,N-chelating Ir4 exhibited a significant increase in cytotoxic behaviors than\niridium(III) or ruthenium(II) complexes also demonstrated our previously reported zwitterionic pyridyl\u2212imine iridium-\ngood aqueous stability.35 (III) complexes against all of the tested cancer cell lines, which\n 2.3. Spectroscopic Studies. The UV\u2212visible absorbance have been shown to be inactive (e.g., Figure 4, Ir1 vs A).40,41\nspectra of complexes Ir1\u2212Ir4, Rh1\u2212Rh3, and Ru1\u2212Ru3 in Moreover, the zwitterionic P,N-chelating iridium(III) com-\nmethanol solutions at 37 \u00b0C are shown in Figure 3a. The plexes also showed higher cytotoxicity than the fluorinated\nmaximum of a sharp band appeared at ca. 210 nm for all of the zwitterionic N,N-chelating iridium(III) complexes (Figure 4,\ncomplexes. In addition, two weak and broad bands maximum Ir1 vs B).41 Similarly, the title ruthenium(II) complexes\n 20012 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. IC50 Values of Complexes Ir1\u2212Ir4, Rh1\u2212Rh3, and coefficients between octanol and water) for the above-\nRu1\u2212Ru3 Tested toward Cancer and Normal Cell Lines and mentioned complexes were measured through a general\nComparison with Cisplatin and RAPTA-C \u201cshake flask\u201d method. The trend in lipophilicity has been\n revealed using the log P values as follows: Ir1 (2.19) > Rh1\n IC50 (\u03bcM)\n (1.44) > Ru1 (1.28) > Ru2 (1.02) > Ru3 (0.78). The log P\n complex A549 HeLa BEAS-2B values of the zwitterionic phosphine\u2212imine iridium(III) and\n Ir1 14.7 \u00b1 0.4 9.1 \u00b1 0.2 25.3 \u00b1 0.6 ruthenium(II) complexes were significantly higher than those\n Ir2 15.6 \u00b1 0.1 8.0 \u00b1 0.1 27.6 \u00b1 0.1 of the corresponding zwitterionic pyridyl\u2212imine iridium(III)\n Ir3 15.2 \u00b1 0.1 8.5 \u00b1 0.4 25.6 \u00b1 0.2 and ruthenium(II) complexes (log P values: Figure 4, Ir1\n Ir4 16.1 \u00b1 0.2 7.2 \u00b1 0.7 29.3 \u00b1 0.3 (2.19) vs A (0.26) and B (1.43); Ru1 (1.28), Ru2 (1.02) and\n Rh1 21.1 \u00b1 0.6 10.3 \u00b1 1.1 35.3 \u00b1 0.1 Ru3 (0.78) vs C (\u22120.71)).40,41,67 Clearly, compared with\n Rh2 23.1 \u00b1 0.2 9.5 \u00b1 0.8 34.7 \u00b1 1.2 zwitterionic pyridyl\u2212imine iridium(III) and ruthenium(II)\n Rh3 22.6 \u00b1 0.5 12.2 \u00b1 0.3 35.7 \u00b1 0.9 complexes, the lipophilicity of zwitterionic phosphine\u2212imine\n Ru1 88.2 \u00b1 0.3 81.5 \u00b1 0.1 93.3 \u00b1 0.3 iridium(III) and ruthenium(II) complexes greatly increased.\n Ru2 90.9 \u00b1 0.1 79.6 \u00b1 0.2 98.3 \u00b1 0.3 Thus, the strategic incorporation of phosphine into zwitter-\n Ru3 121.3 \u00b1 0.4 98.6 \u00b1 0.5 115.5 \u00b1 0.4 ionic complexes could increase their lipophilicity. Meanwhile,\n Cisplatin 21.3 \u00b1 1.7 7.5 \u00b1 0.2 42.0 \u00b1 2.3 the introduction of lipophilic phenyl rings adjacent to\n RAPTA-C >150 87.1 \u00b1 0.3 >150 phosphine (PPh2) may also contribute to the enhanced\n lipophilicity of these zwitterionic complexes. In general, cell\nshowed the improvement in the cytotoxicity compared with uptake and cytotoxicity were correlated with lipophilicity.\nthe inactive zwitterionic N,N-chelating ruthenium(II) com- Therefore, the total cell uptake accumulation of some\nplexes (Figure 4, Ru2 vs C).67 Moreover, the cytotoxicity of zwitterionic complexes was tested using inductively coupled\nthese ruthenium(II) complexes toward HeLa and A549 cells plasma-mass spectrometry (ICP-MS) by exposure to these\nwas comparable to or even higher than the reference half- complexes (5 \u03bcM) for 48 h. The intracellular contents (ng/\u03bcg\nsandwich phosphine ligation complex RAPTA-C. The protein) are as follows: Ir1 (0.653) > Rh1 (0.622) > Ru2\nzwitterionic phosphine\u2212imine rhodium(III) complex also (0.325) > Ru1 (0.311) > Ru3 (0.267). The order of level of\ndisplayed the cytotoxicity comparable to the cisplatin (Figure cell uptake was basically consistent with the above-mentioned\n4, Rh1). Obviously, the introduction of phosphine into lipophilicity and their cytotoxic activity. As a result, it seemed\nzwitterionic complexes could significantly increase the that the increased lipophilic property and cellular uptake of\ncytotoxic potency. Previous studies have shown that the lack these zwitterionic phosphine\u2212imine half-sandwich complexes\nof cytotoxicity of zwitterionic pyridyl\u2212imine iridium(III) and were likely to be one of the key factors of their enhanced\nruthenium(II) complexes appeared to arise from their low cytotoxicity. The modifications of substituents on the \u03b75-CpR\nlipophilic property and the lipophilic fluorinated substituents ring have little influence on the cytotoxic activity (Ir1 vs Ir2 vs\ncould increase their anticancer activity.40,41 This prompted us Ir3 vs Ir4). In particular, the presence of lipophilic fluorinated\nto investigate the lipophilicity of these novel zwitterionic substituents in the \u03b75-CpR did not result in a significant\nphosphine\u2212imine complexes. The log P values (partition increase in cytotoxicity (Ir3 vs Ir1, Ir2 and Ir4; Rh3 vs Rh1\n\n\n\n\nFigure 4. IC50 values of previously reported complexes and the title complexes in this work toward A549 cells.\n\n 20013 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nand Rh2), which is different from the tendency of our mentioned complexes ranged from 9.06 \u00d7 102 to 3.02 \u00d7 103\npreviously reported zwitterionic pyridyl\u2212imine system.41 The M\u22121 (Ru1: Kb = 3.020 \u00d7 103 M\u22121, Ir1: Kb = 1.427 \u00d7 103 M\u22121,\nmajor role of the phosphine ligation may have offset the Rh1: Kb = 9.062 \u00d7 102 M\u22121), showing that the binding efficacy\nadvantages of having the fluorinated phenyl rings in the to CT-DNA was not significant for these complexes. These\nzwitterionic phosphine\u2212imine system. The iridium(III) and results suggested that these complexes displayed a weak\nrhodium(III) complexes exhibited higher cytotoxicity than the interaction with CT-DNA and it seemed not to be the major\ncorresponding ruthenium(II) complexes, suggesting that the MoAs for these zwitterionic phosphine\u2212imine complexes.\nmetal center has a significant effect on the cytotoxicity of these 2.6. Protein Binding Studies. The serum albumin usually\ncomplexes. As mentioned above, ruthenium(II) complexes plays a major role in transportation and metabolism of drugs in\nwith \u03b76-arene ligands in this system gave rise to a decreased cancer cells.72 BSA (bovine serum albumin) serving as a target\nhydrophobicity compared to \u03b75-CpR iridium(III) and rhodium- protein is commonly used to study the binding of drugs with\n(III) complexes, thus leading to a decreased cell uptake and plasma proteins on account of the structural similarity to HSA\ncytotoxicity. The cytotoxic action of Ir1\u2212Ir4, Rh1\u2212Rh3, and (human serum albumin). Herein, the binding potency of Ir1,\nRu1\u2212Ru3 against the noncancerous BEAS-2B has also been Rh1, and Ru1 with BSA could be assessed using UV\u2212vis\ninvestigated. Unfortunately, there was no obvious selectivity absorption spectrum and fluorescence spectrum (Figure 6).\nfor normal and cancer cells. Ir1 showed the highest UV\u2212vis absorption spectra of BSA were observed before and\ncytotoxicity against A549 cells, and thus it was selected for after the addition of complexes (Figure 6a\u2212c). The absorbance\nfurther investigations of MoAs. effect of complexes could be eliminated by adding drug\n 2.5. DNA Binding Results. Binding to DNA was relevant solutions in the same concentration to the reference cells. The\nto the cytotoxic activity of antitumor agent since DNA was intensities of the absorption peak at 229 nm regularly\nconsidered the most potential target site for metal-based decreased and red-shifted (229\u2212233 nm) with the increased\nantitumor complexes.68 The time-dependent 1H NMR spectra concentration of the drugs, which can be attributed to induce\nof Ir1, Rh1, or Ru1 mixed with the model nucleobase 9- \u03b1-helix perturbation and the role of ambient polarity.73\u221276 In\nmethyladenine (9-MeA) in 90% DMSO-d6/10% D2O or 85% addition, at the weak absorption peak at ca. 276 nm, a subtle\nDMSO-d6/15% D2O solutions at 37 \u00b0C were utilized to detect increase in the intensity and hardly any shift for the absorbance\nthe coordination of 9-MeA with Ir1, Rh1, or Ru1 (Figures peak position were observed, suggesting a kind of tiny variation\nS53\u2212S55). There was no coordination reaction occurring with of microenvironment of aromatic amino acid residues (Phe,\n9-MeA at various time intervals within 24 h. Moreover, the Tyr, and Trp) in BSA.75\u221279\nresult of mass spectrum also showed that no nucleobase adduct Moreover, the interaction between the sample and BSA\nof these complexes was generated. could be further investigated by studying the fluorescence\n Further, the interaction between complexes Ir1, Rh1, and quenching of BSA as the concentration of the drugs increased\nRu1 and CT-DNA was monitored via UV\u2212vis absorption at room temperature. The correction for the inner filter effect\nspectroscopy (Figures 5 and S1). With the fixed concentration was applied to calibrate the measured fluorescence.80 In\n general, the fluorescent characteristic of BSA is mainly due to\n two residues of the protein named tryptophan and tyrosine.\n The release of fluorescence arising from aromatic amino acid\n residues was much sensitive to microenvironmental variety.\n Therefore, the fluorescence emission could be attenuated by\n binding small-molecule complexes near the residues.75 The\n increasing concentration of the complexes led to a regular\n decline in the fluorescence intensity of the BSA at ca. 350 nm,\n which could be attributed to the static quenching mechanism\n after the complexes bound to the protein. The values of Ksv, Kq,\n Kb, and n could be obtained using the conventional Stern\u2212\n Volmer equation and Scatchard equation in a static quenching\n process (Table 2).81,82 The calculated value of Kq for Ir1 was\n 5.39 \u00d7 1012 M\u22121 s\u22121, which was hundreds of times greater than\n that of the dynamic type of quenching (2.0 \u00d7 1010 M\u22121 s\u22121),\n further indicating that these complexes could bind to BSA\n mainly through static quenching process. The calculated value\n of Kb of Ir1 and Rh1 reached the order of 104 M\u22121, suggesting\nFigure 5. UV\u2212vis titration spectra of Rh1 complexes (60 \u03bcM) in 5\nmM tris\u2212HCl/10 mM NaCl buffer solution (pH = 7.2) with intense binding to BSA. Notably, the calculated value of Kb of\nincreasing concentration of CT-DNA (0\u2212163.4 \u03bcM). (Inset) Plots of Ru1 was 1 order of magnitude smaller than that of Ir1 and\nA0/(A \u2212 A0) vs 1/[DNA]. Rh1, which might be associated with the low anticancer\n activity of ruthenium complexes. In addition, the number of\n binding sites (n) in BSA approximates 1.0 for Ir1, Rh1, and\nof these complexes, CT-DNA solution was added regularly. As Ru1, showing that only one site in BSA reacted with the\nshown in Figure 5, the hyperchromism and red shift (ca. 1.5 corresponding complexes.\nnm) were observed for Rh1 in a slow increase of CT-DNA Synchronous fluorescence spectroscopy was also monitored\nconcentration, suggesting noncovalent binding modes of to further clarify the conformational changes that occurred to\nelectrostatic binding.69\u221271 Also, the Benesi\u2212Hildebrand BSA after adding complexes (Figures S56 and S57). The\nequation is solved numerically to calculate the binding spectra of tryptophan residues (Trp) and tyrosine residues\nconstants (Kb). The calculated Kb values for the above- (Tyr) were given. With an incremental addition of typical\n 20014 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. UV\u2212vis spectrum of BSA (10 \u03bcM) in 5 mM tris\u2212HCl/10 mM NaCl buffer solution (pH = 7.2) upon addition of the complex (a) Ir1,\n(b) Rh1, and (c) Ru1 (0\u221210 \u03bcM). Inset: wavelength from 255 to 290 nm. Fluorescence spectra of BSA (10 \u03bcM; \u03bbex = 288 nm; \u03bbem = 350 nm) in\nthe absence and presence of the complexes (d) Ir1, (e) Rh1, and (f) Ru1 (0\u221210 \u03bcM). The arrow shows the intensity changes when the\nconcentration of the iridium(III), rhodium(III), or ruthenium(II) complex increases.\n\nTable 2. Quenching Parameters and Binding Parameters for\nthe Interaction of Complexes Ir1, Rh1, and Ru1 with BSA\ncomplex ksv (104 M\u22121) Kq (1012 M\u22121 s\u22121) Kb (104 M\u22121) n\n Ir1 5.39 \u00b1 0.06 5.39 1.43 0.874\n Rh1 5.82 \u00b1 0.29 5.82 1.38 0.868\n Ru1 4.96 \u00b1 0.12 4.96 0.21 0.707\n\n\n\ncomplexes, the fluorescence intensities corresponding to both\nTrp (\u0394\u03bb = 60, 286 nm) and Tyr (\u0394\u03bb = 15, 291 nm) decreased\nand a 1\u22122 nm red shift occurred to tryptophan at the\nmaximum emission wavelength. Overall, these results have\nestablished that the introduction of the title complexes altered\nthe conformation of BSA, decreased the microenvironmental\nhydrophobicity of the tryptophan residues, and enhanced the\nextension degree of the BSA macromolecule peptide chain.\n 2.7. ROS Determination. Highly oxidizing drugs could\nproduce reactive oxygen species (ROS) to induce cell oxidative Figure 7. Analysis of ROS levels by fluorescence microscope after\ndeath. The ROS assay kit was used to determine the generation A549 cells were treated with Ir1 for 24 h at 37 \u00b0C and stained with\nof ROS in A549 cells induced by Ir1 (Figures 7 and S58). DCFH-DA. P values were calculated after a t test against the negative\nCompared to control cells, the obvious concentration-depend- control data, *p < 0.05, **p < 0.01.\nent increase in ROS levels was observed in the treated A549\ncells, indicating that complex Ir1 could result in the cells for up to 48 h, and then the treated cells were examined\naccumulation of intracellular ROS. It has been reported that using flow cytometry. The proportion of early apoptosis\nthe potency of anticancer agents to induce apoptosis was increased after treatment with Ir1 (Figure 8 and Table S5).\nassociated with the potency of generating ROS in cancer cell Hence, the exposure to these zwitterionic phosphine\u2212imine\nlines. Thus, the induction of cell apoptosis was also studied in complexes could induce apoptosis and accordingly lead to\nthe next experiment. cancer cell death.\n 2.8. Apoptosis and Cell Cycle Studies. One of the To demonstrate whether the induction of apoptosis was the\npotential goals of treating anticancer agents is targeting primary pathway for the cancer cell death, the cytotoxicity of\ncancerous cells and inducing cell death by apoptotic pathways. Ir1 was determined in the presence of various inhibitors\nThe annexin V/PI technique was used to reveal apoptotic cell including autophagy inhibitor 3-methyladenine (3-MA),\ndeath. Ir1 was continuously incubated with lung cancer A549 necroptosis inhibitor necrostatin-1 (Nec-1), the protease\n 20015 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 8. (a) Apoptosis analysis of A549 cells after 48 h of exposure to complex Ir1 at 37 \u00b0C determined by flow cytometry using annexin V-FITC\nvs PI staining. (b) Histograms of early apoptosis analysis for A549 cells after treated with complex Ir1 (0.25 \u00d7 IC50, 0.5 \u00d7 IC50 and 1 \u00d7 IC50) for 48\nh. Data are quoted as mean \u00b1 SD of three replicates.\n\ninhibitor leupeptin (LPT), and the protein synthesis inhibitor\ncycloheximide (CHX) (Table S6). The negligible changes\nwere observed in the cytotoxic efficacy, indicating that these\ninhibitors were inoperative and apoptosis was the primary\npathway for cancer cell death.\n To investigate the role of zwitterionic phosphine\u2212imine\ncomplexes in cell cycle arrest, the flow cytometry was also used\nto analyze the blocking of cell cycle progression by Ir1 (Figure\nS59). The results showed that G0/G1, S, and G2/M phases\nhad a trivial change in A549 cells at the concentrations of 0.25,\n0.5, and 1 \u00d7 IC50. Therefore, these complexes seemed not to\neffectively disrupt the cell cycle progression.\n 2.9. Cellular Uptake Mechanisms. Based on the\nconsideration of the fluorescence properties of the above-\nmentioned products, the intracellular uptake principle of the\nmost active Ir1 could be detected by laser confocal\nmicroscopy. Energy-dependent and energy-independent path-\nways are two kinds of ways for small-molecule complexes to\nenter cells.83 As Figure 9 illustrates, the appearance of punctate\ngreen fluorescence indicated that Ir1 entered into A549 cells\nafter a 1 h incubation. A marked decline of fluorescence\nstrength was observed at the time when A549 cells were\n Figure 9. Effects of temperatures (37 or 4 \u00b0C), chloroquine (50 \u03bcM),\nincubated with Ir1 under 4 \u00b0C or CCCP conditions (CCCP:\n and CCCP (50 \u03bcM) on the cellular uptake of Ir1 (2 \u03bcM). Scale bar:\ncarbonyl cyanide 3-chloro-phenylhydrazone, a kind of 20 \u03bcm, \u03bbex = 405 nm, \u03bbem = 430\u2212490 nm.\nmetabolic inhibitor), in comparison with the control group\nof 37 \u00b0C (Figure 9). These results suggested that the cellular\nuptake of complex Ir1 is energy-dependent. In addition, there accumulation. A549 cells and complex Ir1 were cotreated with\nwas a negligible change in intracellular fluorescence intensity variable concentrations of amphotericin B. The level of cell\nafter treatment with chloroquine (a kind of endocytosis accumulation of iridium exhibited no significant variation\ninhibitor) compared to that of the untreated group (Figure 9), (Figure 10b and Table S8), suggesting that facilitated diffusion\nindicating that endocytosis was not involved in the cellular (an energy-independent pathway) was not responsible for the\nuptake. uptake pathway of complex Ir1.\n Antimycin A1, which played a role in lowering ATP levels, 2.10. Cellular Localization. Whether these complexes\nwas used to coincubate with complex Ir1 to further validate the were able to target organelles was further assessed by the\nenergy-dependent cellular uptake mechanisms (Figure 10a). cellular localization analysis via confocal microscopy (Figure\nThe experimental results showed that the accumulation of 11). The A549 cells were dual-stained with Ir1 and nucleus\niridium in A549 cells was affected after the changes of ATP fluorescent probe (4,6-diamino-2-phenyl indole, DAPI),\nlevels, which was consistent with the observation of significant mitochondrial fluorescent probe (Mito Tracker Red\ncellular uptake at relatively high temperatures (Figure 10a and CMH2XRos, MTDR), and lysosome fluorescent probe\nTable S7; 37 \u00b0C vs 4 \u00b0C). (LysoTracker Red DND-99, LTDR). In the cytoplasm, the\n Moreover, the means of protein-mediated uptake were also punctate and intense green fluorescence showed that Ir1 could\ninvestigated. Amphotericin B could disrupt cell membrane effectively penetrate A549 cells after a 1 h incubation. The\nintegrity to form pores, which may lead to increased cell degree of mergence between Ir1 and DAPI or MTDR was\n 20016 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 10. Total accumulation of Ir in A549 cells when coincubated with complex Ir1 (5 \u03bcM) and (a) antimycin A1 (0 and 5 \u03bcM) or (b)\namphotericin B (0, 1, 5 and 10 \u03bcM) after exposure to Ir1 for 48 h at 37 \u00b0C with no recovery time.\n\n\n\n\nFigure 11. Determination of the intercellular localization of Ir1 by\nconfocal microscopy. A549 cells were incubated with Ir1 (2 \u03bcM) for 1\nh at 37 \u00b0C and then coincubated with DAPI (1 \u03bcg/mL), MTDR (500\nnM), or LTDR (75 nM) for 1 h (Ir1, \u03bbex = 405 nm, \u03bbem = 460\u2212520\nnm; DAPI, \u03bbex = 345 nm, \u03bbem = 410\u2212455 nm; MTDR, \u03bbex = 644 nm, Figure 12. Iridium content of the cytoplasm, nucleus, and\n\u03bbem = 660\u2212720 nm; LTDR, \u03bbex = 594 nm, \u03bbem = 600\u2212660 nm). Scale cytoskeleton fractions (Ir ng per \u03bcg protein) of A549 cells after 48\nbar: 20 \u03bcm. The green, red, and blue fluorescence represent Ir1, h of exposure to 5 \u03bcM Ir1.\nmitochondria or lysosome, and nucleus, respectively.\n nucleus. These results are consistent with the above-mentioned\nnegligible with a low Pearson correlation coefficient (PCC) observations of cellular localization experiments.\nvalue (DAPI: PCC = 0.11; MTDR: PCC = \u22120.04), indicating 2.11. Lysosomal Damage. The functional state of\nthat Ir1 cannot effectively localize in the nucleus and lysosomes could be monitored by acridine orange (AO),\nmitochondria. The weak binding efficacy of these complexes which was a kind of probe of lysosome dysfunction with red or\nto CT-DNA (see above; the low Kb value in Section 2.5) may green photoluminescence in lysosomes or in the cytosol and\nbe associated with the low accumulation in the nucleus. nuclei.91 Hence, the lysosome integrity could be further\nHowever, the concordance with a PCC value of 0.78 was found investigated by AO to study cell death pathway mediated by\nbetween the merged image of Ir1 and LTDR, suggesting that lysosomes. As shown in Figure 13, A549 cells stained with AO\nIr1 primarily targeted lysosome. Thus, lysosome-mediated cell have strong red photoluminescence in lysosomes. However,\ndeath could be responsible for the cytotoxic potency of these the intensity of red photoluminescence showed a significant\nzwitterionic phosphine\u2212imine complexes. Possibly, the high decrease with the increased concentration of Ir1, indicating\nlipophilicity of these zwitterionic P,N-chelating complexes may that lysosomal integrity was damaged after the treatment of\nbe related to the lysosome targeting. However, the previous Ir1. Since the disruption of lysosomal integrity could result in\nstudy has shown that lysosome accumulation and targeting are the release of cathepsin B from the lysosomes into the cytosol,\nassociated with various factors including lipophilicity, type and lysosomal damage was further determined by the fluorogenic\nnumber of charges of complexes, aromatic area, and charge/ magic red MR-(RR)2 to monitor the intracellular activity of\nmass ratio.84\u221290 cathepsin B (Figure S60). The control cells exhibited red\n The biodistribution in different subcellular compartments of fluorescence that was predominantly localized in the\nthe A549 cells was also quantitatively studied by ICP-MS. After lysosomes. The red fluorescence gradually spread with the\n48 h of exposure to Ir1, the iridium content of the cytoplasm, increased concentration of Ir1, suggesting that cathepsin B\nnucleus, and cytoskeleton fractions isolated from A549 cells gradually entered into the cytosol from lysosomes. The above-\nwas determined (Figure 12 and Table S9). As shown in Figure mentioned results further suggested that these zwitterionic\n12, the highest concentration of iridium was in the cytoplasm phosphine\u2212imine complexes might target lysosomes and thus\nsection and more iridium entered the cytoplasm than the cause lysosomal damage.\n 20017 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n reaction with 9-MeA, and the weak affinity between these\n zwitterionic phosphine\u2212imine complexes and CT-DNA was\n observed. In addition, the spectroscopic analysis supported the\n binding of the typical complexes to BSA. The mechanistic\n study showed that the most reactive complex Ir1 could\n increase the ROS levels, induce apoptosis, target, and damage\n lysosomes. Meanwhile, this type of zwitterionic phosphine\u2212\n imine complex could also suppress cancer cell migration.\n\n 4. EXPERIMENTAL SECTION\n 4.1. General Information. All solvents and reagents were\n commercially sourced. Unless otherwise stated, they were used\n without further purification. The sodium 2,6-diisopropylaniline\n sulfonate and RAPTA-C were prepared using the literature\nFigure 13. Lysosomal disruption of A549 cells verified with AO assay. procedure.92,93 The 1H, 13C{1H}, and 31P{1H} NMR spectroscopy\n(a) Cells only treated with AO, as a control; (b,c) cells treated with were determined via Bruker DPX 500 spectrometers (Figures S2\u2212\nAO and Ir1 (1 and 3 equipotent concentrations of IC50), AO green S35). And absorption spectroscopy was performed by a TU-1901\nfluorescence, \u03bbex = 488 nm and \u03bbem = 510 \u00b1 20 nm; AO red UV\u2212visible recording spectrophotometer. MS of these new products\nfluorescence, \u03bbex = 488 nm and \u03bbem = 625 \u00b1 20 nm. Scale bar: 20 \u03bcm. was recorded on a Thermo LTQ Orbitrap XL (ESI+; Figures S36\u2212\n S46). XRD analysis results were collected by a Bruker Apex SMART\n CCD area detector (Tables S1\u2212S3) with graphite-monochromated\n 2.12. Inhibition of Cell Migration. Malignant cancer cells Mo K\u03b1 radiation. Elemental analysis (C, H, N) was determined by\ntended to metastasize to adjacent or distant tissues due to the vario El cube.\nreduced superficial adhesion. Herein, the wound-healing assay 4.2. Synthesis of L (Sulfonated Phosphine\u2212Imine Ligand).\nwas conducted to observe the inhibition action of complex Ir1 The sodium 2,6-diisopropylaniline sulfonate (1.22 g), HCOOH (ca.\non the migration of A549 cells. Compared to 45.3% of the 0.30 g of an 88% aqueous solution), and Na2SO4 (1.10 g) were added\ncontrol group, the wound closure rate (WCR) of A549 cells to a solution of 2-(diphenylphosphinyl)benzaldehyde (1.17 g) in\ntreated with Ir1 not only significantly decreased to 28.7% at CH2Cl2 and MeOH (1:1, v/v) mixed solvents (60 mL). The\n suspension was vigorously stirred at room temperature for 24 h. The\n0.25 \u00d7 IC50 but also presented a concentration-dependent mixture was filtered. The filtrate was collected and concentrated under\ncharacteristic (Figure 14). This result implied that the complex reduced pressure. Recrystallization from MeOH afforded yellow\nIr1 could inhibit the migration of A549 cells. Consequently, crystals. The yellow crystal was washed using Et2O under ultrasound,\nIr1 has both pretty good anticancer ability and antimetastatic filtered, and dried to obtain L as a light-yellow powder.\npotential, which might contribute to addressing the issue of\nadvanced malignancy metastasis.\n\n3. CONCLUSIONS\nIn summary, with an easy access to the sulfonated phosphine\u2212\nimine ligand, the synthesis and characterization of a family of\nzwitterionic phosphine\u2212imine half-sandwich iridium(III), Light-yellow powder, yield: 1.30 g (58.3%). 1H NMR (500 MHz,\nrhodium(III), and ruthenium(II) complexes have been DMSO-d6) (ppm): \u03b4 8.72 (d, J = 5.1 Hz, 1H, CH\ufffdN), 8.21\u22128.19\npresented. These new complexes performed basically stable (m, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.52 (t, J = 7.4 Hz, 1H), 7.41 (s,\n 6H), 7.31 (s, 2H), 7.24\u22127.14 (m, 4H), 6.88\u22126.86 (m, 1H), 2.62\u2212\nin aqueous solution and have a detectable photoluminescence. 2.54 (m, 2H, CH(CH3)2), 0.92 (d, J = 6.8 Hz, 12H, CH(CH3)2).\nNotably, the presence of phosphine ligation was beneficial for 13\n C{1H} NMR (126 MHz, DMSO-d6) (ppm): \u03b4 161.05 (CH\ufffdN),\nimproving the lipophilic properties and cellular uptake effects 160.88, 149.28, 144.16, 138.73, 138.59, 138.42, 136.55, 136.47,\nof these zwitterionic complexes, further affording an enhanced 136.39, 134.08, 133.92, 133.64, 131.99 129.86, 129.64, 129.39,\ncytotoxic efficacy against A549 and HeLa cell lines. Moreover, 129.34, 128.95, 128.92, 120.64, 27.77 (CH(CH3)2), 23.61 (CH-\nthe representative complexes exhibited no coordination (CH3)2). 31P{1H} NMR (202 MHz, DMSO-d6) (ppm): \u03b4 14.84. ESI-\n\n\n\n\nFigure 14. Wound-healing assay of A549 cells treated with Ir1 for 24 h. (a) Typical images were taken at 0 and 24 h. The widths of the wounds are\nindicated with the lines (\u03bcm). Scale bar: 100 \u03bcm. (b) Histograms of wound-healing assay after 24 h. Data are quoted as the mean \u00b1 SD of three\nreplicates. Wound closure rate = (R0 \u2212 R1)/R0 \u00d7 100%.\n\n 20018 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nMS (m/z): calcd. for C31H33NO3PS: 530.1924, found: 530.0800, [M 0.96 (m, 1H, Cy-CH), 0.81 (d, J = 2.6 Hz, 3H, CH(CH3)2),\n\u2212 Na + 2H]+; ESI-MS (m/z): calcd. for C31H31NNa2O3PS 574.1558, 0.18 (d, J = 6.5 Hz, 3H, CH(CH3)2). 13C{1H} NMR (151\nfound: 573.9900 [M + Na]+. MHz, DMSO-d6) (ppm): \u03b4 175.94 (CH\ufffdN), 150.72, 148.68,\n 4.3. Synthesis of Ir1\u2212Ir4, Rh1\u2212Rh3, and Ru1\u2212Ru3 (Sulfo- 140.57, 139.41, 137.53, 135.44, 134.79, 133.94, 133.84, 133.77,\nnated Phosphine\u2212Imine Complexes). 4.3.1. Common Method. 133.22, 132.83, 130.28, 130.21, 129.66, 129.59, 128.23, 127.85,\nPrecursors (D1\u2212D10) and ligand L (2 equiv) were dissolved in\n 127.36, 127.03, 126.99, 126.63, 121.93, 121.25, 107.26, 104.65,\nMeOH and vigorously stirred for 72 h and monitored by thin-layer\n 94.39, 94.33, 90.07, 36.79 (Cy-CH), 30.26 (Cy-CH2), 29.32\nchromatography (TLC). Then, the solvent was removed under\nreduced pressure. The residue was dissolved in CH2Cl2 and filtered to\n (Cy-CH2), 27.55 (CH(CH3)2), 26.97 (CH(CH3)2), 26.88\nremove NaCl. The filtrate was concentrated, and an excess of Et2O (Cy-CH2), 26.66 (Cy-CH2), 25.80 (Cy-CH2), 25.77 (CH-\nwas added. The precipitate was washed using Et2O, filtered, and dried (CH3)2), 25.54 (CH(CH3)2), 23.26 (CH(CH3)2), 22.40\nto afford an orange-yellow/orange-red/orange-brown/brown powder. (CH(CH3)2), 10.07 (CpR-CH3), 10.04 (CpR-CH3), 9.92\n 4.3.2. Ir1. (CpR-CH3), 8.44 (CpR-CH3). 31P{1H} NMR (202 MHz,\n DMSO-d6) (ppm): \u03b4 7.09. ESI-MS (m/z): calcd. for\n C46H55ClIrNO3PS 960.2958, found: 960.3144 [M + H]+;\n ESI-MS (m/z): calcd. for C46H54ClIrNNaO3PS 982.2777,\n found: 982.2943 [M + Na]+. Anal. calcd for\n C46H54ClIrNO3PS: C, 57.57; H, 5.67; N, 1.46. Found: C,\n 57.23; H, 5.59; N, 1.52.\n\n\n\nOrange-yellow powder, yield: 69.0 mg (60.9%). 1H NMR (500\nMHz, DMSO-d6) (ppm): \u03b4 8.71 (s, 1H, CH\ufffdN), 8.00 (s,\n1H), 7.82\u22127.69 (m, 6H), 7.64 (s, 2H), 7.59 (s, 1H), 7.41 (s,\n3H), 7.20 (s, 2H), 6.99\u22126.95 (m, 1H), 3.39\u22123.35 (m, 1H,\nCH(CH3)2), 2.08\u22121.97 (m, 1H, CH(CH3)2), 1.33 (d, J = 6.4\nHz, 3H, CH(CH3)2), 1.15 (s, 15H, CpR-CH3), 1.05 (d, J = 6.3\n 4.3.4. Ir3.\nHz, 3H, CH(CH3)2), 1.00 (d, J = 6.3 Hz, 3H, CH(CH3)2),\n0.16 (d, J = 6.3 Hz, 3H, CH(CH3)2). 13C{1H} NMR (151\nMHz, DMSO-d6) (ppm): \u03b4 175.25 (CH\ufffdN), 150.31, 147.99,\n143.14, 140.04, 139.15, 137.01, 135.22, 135.15, 134.89, 133.74,\n133.07, 132.56, 129.98, 129.91, 129.16, 127.57, 127.19, 127.08,\n126.72, 126.11, 125.71, 121.54, 120.69, 119.67, 96.84, 96.83,\n92.77, 92.76, 92.17, 27.03 (CH(CH3)2), 26.31 (CH(CH3)2),\n25.35 (CH(CH3)2), 25.20 (CH(CH3)2), 22.85 (CH(CH3)2),\n22.01 (CH(CH3)2), 8.41 (CpR-CH3), 8.31 (CpR-CH3), 8.01\n(CpR-CH3). 31P{1H} NMR (202 MHz, DMSO-d6) (ppm): \u03b4\n6.58. ESI-MS (m/z): calcd. for C41H47ClIrNO3PS 892.2332,\nfound: 892.2515 [M + H]+; ESI-MS (m/z): calcd. for Orange-yellow powder, yield: 174.2 mg (63.7%). 1H NMR\nC41H46ClIrNNaO3PS 914.2151, found: 914.2290 [M + Na]+. (400 MHz, DMSO-d6) (ppm): \u03b4 8.77 (d, J = 2.3 Hz, 1H,\nAnal. calcd for C41H46ClIrNO3PS: C, 55.24; H, 5.20; N, 1.57. CH\ufffdN), 8.56 (s, 2H), 8.29 (s, 1H), 8.05 (d, J = 2.1 Hz, 1H),\nFound: C, 54.95; H, 5.24; N, 1.65. 7.85\u22127.79 (m, 5H), 7.61 (m, 1H), 7.50\u22127.40 (m, 6H), 7.29\u2212\n 4.3.3. Ir2. 7.17 (m, 2H), 7.03\u22126.90 (m, 1H), 3.25\u22123.17 (m, 1H,\n CH(CH3)2), 1.99\u22121.84 (m, 1H, CH(CH3)2), 1.44 (d, J = 1.7\n Hz, 3H, CpR-CH3), 1.35 (s, 3H, CpR-CH3), 1.27 (s, 3H, CpR-\n CH3), 0.96 (d, J = 5.9 Hz, 6H, CH(CH3)2), 0.88 (d, J = 2.3\n Hz, 3H, CpR-CH3), 0.54 (d, J = 6.5 Hz, 3H, CH(CH3)2), 0.26\n (d, J = 6.5 Hz, 3H, CH(CH3)2). 13C{1H} NMR (101 MHz,\n DMSO-d6) (ppm): \u03b4 176.53 (CH\ufffdN), 150.21, 149.00,\n 140.29, 140.02, 137.66, 137.57, 135.56, 135.45, 135.32,\n 135.25, 134.15, 134.04, 133.70, 133.63, 133.52, 133.35,\n 133.25, 131.29, 130.96, 130.62, 130.51, 129.59, 129.48,\nOrange-yellow powder, yield: 140.3 mg (58.3%). 1H NMR 128.13, 127.24, 127.03, 126.81, 126.65, 126.47, 125.80,\n(500 MHz, DMSO-d6) (ppm): \u03b4 8.66 (d, J = 2.6 Hz, 1H, 125.65, 125.18, 124.98, 122.31, 122.26, 121.83, 121.31, 27.51\nCH\ufffdN), 8.00\u22127.98 (m, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.75\u2212 (CH(CH3)2), 27.12 (CH(CH3)2), 26.05 (CH(CH3)2), 25.55\n7.72 (m, 5H), 7.65\u22127.63 (m, 2H), 7.60 (d, J = 1.5 Hz, 1H), (CH(CH3)2), 22.63 (CH(CH3)2), 21.45 (CH(CH3)2), 10.12\n7.58\u22127.44 (m, 2H), 7.43 (d, J = 1.5 Hz, 1H), 7.29\u22127.23 (m, (CpR-CH3), 9.98 (CpR-CH3), 9.35 (CpR-CH3), 8.84 (CpR-\n2H), 7.06\u22127.03 (m, 1H), 3.49\u22123.41 (m, 2H, CH(CH3)2), CH3). 31P{1H} NMR (162 MHz, DMSO-d6) (ppm): \u03b4 7.11.\n2.10\u22122.05 (m, 2H, Cy-CH2), 1.71\u22121.68 (m, 2H, Cy-CH2), ESI-MS (m/z): calcd. for C48H47ClF6IrNO3PS 1090.2236,\n1.64\u22121.56 (m, 2H, Cy-CH2), 1.46 (s, 3H, CpR-CH3), 1.41\u2212 found: 1090.2216 [M + H]+; ESI-MS (m/z): calcd. for\n1.39 (m, 1H, Cy-CH2), 1.37\u22121.34 (m, 6H, CpR-CH3), 1.30 (d, C48H46ClF6IrNNaO3PS 1112.2056, found: 1112.2030 [M +\nJ = 3.7 Hz, 3H, CpR-CH3), 1.25\u22121.23 (d, 1H, Cy-CH2), 1.09 Na]+. Anal. calcd for C48H46ClF6IrNO3PS: C, 52.91; H, 4.26;\n(m, 2H, Cy-CH2), 1.04 (d, J = 6.5 Hz, 6H, CH(CH3)2), 0.99\u2212 N, 1.29. Found: C, 52.68; H, 4.19; N, 1.36.\n 20019 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n 4.3.5. Ir4. 802.1865 [M + H] + ; ESI-MS (m/z): calcd. for\n C41H46ClNNaO3PRhS 824.1577, found: 824.1677 [M +\n Na]+. Anal. calcd for C41H46ClNO3PRhS: C, 61.39; H, 5.78;\n N, 1.75. Found: C, 61.68; H, 5.69; N, 1.67.\n 4.3.7. Rh2.\n\n\n\n\nOrange-yellow powder, yield: 149.8 mg (61.7%). 1H NMR\n(500 MHz, DMSO-d6) (ppm): \u03b4 8.80 (d, J = 2.5 Hz, 1H,\nCH\ufffdN), 8.09\u22128.07 (m, 1H), 7.85\u22127.74 (m, 6H), 7.65\u22127.63\n(m, 1H), 7.57 (t, J = 5.7 Hz, 4H), 7.49 (d, J = 1.4 Hz, 1H),\n7.41 (d, J = 1.3 Hz, 1H), 7.31\u22127.21 (m, 4H), 7.19\u22127.16 (t, Orange-red powder, yield: 137.8 mg (63.1%). 1H NMR (500\n1H), 7.03 (m, 1H), 3.52 (m, 1H, CH(CH3)2), 2.08\u22122.00 (m, MHz, DMSO-d6) (ppm): \u03b4 8.58 (s, 1H, CH\ufffdN), 7.96\u22127.94\n1H, CH(CH3)2), 1.90 (s, 3H, aryl-CH3), 1.54 (d, 3H, CpR- (m, 1H), 7.77 (s, 1H), 7.76\u22127.73 (m, 5H), 7.72\u22127.69 (m,\nCH3), 1.22 (s, 3H, CpR-CH3), 1.10 (s, 1H, CpR-CH3), 1.09\u2212 2H), 7.66 (t, J = 6.1 Hz, 2H), 7.60 (s, 1H), 7.42 (s, 1H), 7.28\u2212\n1.07 (m, 4H, CpR-CH3), 1.02 (d, 3H, CH(CH3)2), 0.97 (d, 7.24 (m, 2H), 7.04\u22127.00 (m, 1H), 3.64\u22123.55 (m, 1H,\n3H, CH(CH3)2), 0.95 (s, 1H, CpR-CH3), 0.65 (d, 3H, CH(CH3)2), 2.07 (s, 6H, Cy-CH2), 1.96\u22121.88 (m, 1H,\nCH(CH3)2), 0.29 (d, 3H, CH(CH3)2). 13C{1H} NMR (126 CH(CH3)2), 1.83\u22121.81 (m, 1H, Cy-CH2), 1.69\u22121.66 (m, 1H,\nMHz, DMSO-d6) (ppm): \u03b4 176.52 (CH\ufffdN), 150.40, 148.66, Cy-CH2), 1.64\u22121.58 (m, 2H, Cy-CH2), 1.39\u22121.37 (m, 6H,\n140.70, 139.96, 137.58, 135.56, 135.49, 135.15, 134.51, 134.43, CpR-CH3), 1.27 (d, J = 3.4 Hz, 3H, CpR-CH3), 1.24 (d, J = 5.2\n133.58, 133.49, 133.11, 133.07, 130.37, 129.64, 129.62, 129.59, Hz, 3H, CpR-CH3), 1.01 (t, J = 6.8 Hz, 7H, (CH(CH3)2, 6H)+\n129.54, 129.36, 129.33, 128.88, 128.82, 128.80, 127.30, 127.28, (Cy-CH, 1H)), 0.91 (d, J = 3.1 Hz, 3H, CH(CH3)2), 0.17 (d, J\n126.84, 126.83, 126.57, 126.33, 126.30, 126.08, 126.06, 125.60, = 6.5 Hz, 3H, CH(CH3)2). 13C{1H} NMR (126 MHz,\n121.90, 121.17, 27.55 (CH(CH3)2), 27.06 (CH(CH3)2), 26.03 DMSO-d6) (ppm): \u03b4 176.44 (CH\ufffdN), 150.10, 149.21,\n(CH(CH3)2), 25.77 (CH(CH3)2), 23.57 (CH(CH3)2), 22.67 148.23, 144.23, 140.86, 139.14, 136.51, 135.21, 134.08,\n(CH(CH3)2), 19.61 (aryl-CH3), 9.86 (CpR-CH3), 9.60 (CpR- 133.92, 130.47, 129.69, 129.40, 129.35, 127.89, 127.47,\nCH3), 9.21 (CpR-CH3), 8.78 (CpR-CH3). 31P{1H} NMR (202 126.94, 126.55, 121.97, 121.22, 120.62, 120.07, 111.02,\nMHz, DMSO-d6) (ppm): \u03b4 6.21. ESI-MS (m/z): calcd. for 106.93, 104.93, 103.66, 99.35, 98.61, 97.98, 35.63 (Cy-CH),\nC47H51ClIrNO3PS 968.2645, found: 968.2819 [M + H]+; ESI- 30.33 (Cy-CH2), 29.99 (Cy-CH2), 27.75 (CH(CH3)2), 27.36\nMS (m/z): calcd. for C47H50ClIrNNaO3PS 990.2464, found: (CH(CH3)2), 26.85 (Cy-CH2), 25.99 (Cy-CH2), 25.80 (Cy-\n990.2629 [M + Na]+. Anal. calcd for C47H50ClIrNO3PS: C, CH2), 25.66 (CH(CH3)2), 23.60 (CH(CH3)2), 23.37 (CH-\n58.34; H, 5.21; N, 1.45. Found: C, 58.13; H, 5.29; N, 1.31. (CH3)2), 22.25 (CH(CH3)2), 10.60 (CpR-CH3), 10.40 (CpR-\n CH3), 10.15 (CpR-CH3), 9.07 (CpR-CH3). 31P{1H} NMR (202\n 4.3.6. Rh1.\n MHz, DMSO-d6) (ppm): \u03b4 37.63, 36.94. ESI-MS (m/z):\n calcd. for C46H55ClNO3PRhS 870.2384, found: 870.2367 [M +\n H] + ; ESI-MS (m/z): calcd. for C 46 H54 ClNNaO 3PRhS\n 892.2203, found: 892.2175 [M + Na]+. Anal. calcd for\n C46H54ClNO3PRhS: C, 63.48; H, 6.25; N, 1.61. Found: C,\n 63.75; H, 6.17; N, 1.58.\n 4.3.8. Rh3.\n\n\nOrange-red powder, yield: 70.4 mg (69.3%). 1H NMR (500\nMHz, DMSO-d6) (ppm): \u03b4 8.61 (s, 1H, CH\ufffdN), 7.97\u22127.95\n(m, 1H), 7.82\u22127.71 (m, 7H), 7.71\u22127.63 (m, 3H), 7.59 (d, J =\n1.7 Hz, 1H), 7.40 (d, J = 1.7 Hz, 1H), 7.23\u22127.19 (m, 2H),\n6.99\u22126.95 (m, 1H), 3.54\u22123.47 (m, 1H, CH(CH3)2), 1.91\u2212\n1.82 (m, 1H, CH(CH3)2), 1.36 (d, J = 6.7 Hz, 3H,\nCH(CH3)2), 1.15 (d, J = 3.8 Hz, 15H, CpR-CH3), 1.03 (d, J\n= 6.6 Hz, 3H, CH(CH3)2), 0.97 (d, J = 6.6 Hz, 3H,\nCH(CH3)2), 0.16 (d, J = 6.6 Hz, 3H, CH(CH3)2). 13C{1H}\nNMR (151 MHz, DMSO-d6) (ppm): \u03b4 176.08 (CH\ufffdN), Orange-red powder, yield: 166.7 mg (66.4%). 1H NMR (400\n149.93, 148.44, 140.66, 139.07, 137.76, 135.66, 134.39, 134.32, MHz, DMSO-d6) (ppm): \u03b4 8.69 (s, 1H, CH\ufffdN), 8.31 (s,\n133.59, 133.52, 133.17, 132.92, 130.50, 130.43, 129.75, 129.68, 1H), 8.05\u22127.99 (m, 1H), 7.85\u22127.68 (m, 7H), 7.61\u22127.59 (m,\n127.95, 127.65, 127.50, 127.16, 126.46, 126.13, 122.02, 121.08, 3H), 7.39 (d, J = 2.8 Hz, 3H), 7.26\u22127.17 (m, 3H), 6.96\u22126.91\n103.24, 27.73 (CH(CH3)2), 27.02 (CH(CH3)2), 25.76 (CH- (m, 1H), 3.32\u22123.26 (m, 1H, CH(CH3)2), 2.60\u22122.55 (m, 1H,\n(CH3)2), 25.68 (CH(CH3)2), 23.26 (CH(CH3)2), 22.26 CH(CH3)2), 1.36 (s, 3H, CpR-CH3), 1.34 (d, J = 5.7 Hz, 3H,\n(CH(CH3)2), 9.25 (CpR-CH3), 9.03 (CpR-CH3). 31P{1H} CpR-CH3), 1.25 (s, 3H, CpR-CH3), 0.90 (d, J = 6.4 Hz, 6H,\nNMR (202 MHz, DMSO-d6) (ppm): \u03b4 37.61, 36.91. ESI-MS CH(CH3)2), 0.75 (d, J = 3.3 Hz, 3H, CpR-CH3), 0.40 (d, J =\n(m/z): calcd. for C41H47ClNO3PRhS 802.1758, found: 6.5 Hz, 3H, CH(CH3)2), 0.29 (d, J = 6.4 Hz, 3H, CH(CH3)2).\n 20020 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n13\n C{1H} NMR (101 MHz, DMSO-d6) (ppm): \u03b4 176.81 Brown powder, yield: 122.6 mg (65.7%). 1H NMR (500 MHz,\n(CH\ufffdN), 149.56, 148.66, 140.47, 139.79, 135.75, 135.63, DMSO-d6) (ppm): \u03b4 8.61 (d, J = 2.1 Hz, 1H, CH\ufffdN), 7.94\u2212\n135.02, 134.60, 134.50, 133.73, 133.66, 133.55, 133.33, 133.16, 7.92 (m, 1H), 7.81\u22127.62 (m, 11H), 7.42\u22127.38 (t, 3H), 6.85\u2212\n132.00, 131.34, 131.01, 130.81, 130.70, 129.98, 129.88, 129.69, 6.81 (m, 1H), 5.62 (s, 6H, arene-H), 3.93\u22123.86 (m, 1H,\n129.45, 129.39, 127.68, 127.47, 127.01, 126.68, 126.14, 125.63, CH(CH3)2), 1.76\u22121.68 (m, 1H, CH(CH3)2), 1.46 (d, J = 6.7\n125.12, 124.97, 123.46, 122.25, 121.86, 121.37, 121.26, 27.70 Hz, 3H, CH(CH3)2), 1.11 (d, J = 6.7 Hz, 3H, CH(CH3)2),\n(CH(CH3)2), 27.49 (CH(CH3)2), 26.32 (CH(CH3)2), 25.50 0.71 (d, J = 6.7 Hz, 3H, CH(CH3)2), 0.60 (d, J = 6.6 Hz, 3H,\n(CH(CH3)2), 22.55 (CH(CH3)2), 21.18 (CH(CH3)2), 10.47 CH(CH3)2). 13C{1H} NMR (126 MHz, DMSO-d6) (ppm): \u03b4\n(CpR-CH3), 10.16 (CpR-CH3), 9.76 (CpR-CH3), 9.21 (CpR- 175.29 (CH\ufffdN), 155.35, 148.62, 139.39, 138.80, 137.39,\nCH3). 31P{1H} NMR (162 MHz, DMSO-d6) (ppm): \u03b4 40.16, 136.29, 135.40, 135.03, 133.40, 133.16, 132.82, 132.75, 132.64,\n39.29. ESI-MS (m/z): calcd. for C48H47ClF6NO3PRhS 132.23, 130.26, 130.18, 130.04, 129.96, 129.39, 128.96, 124.79,\n1000.1662, found: 1000.1649 [M + H]+; ESI-MS (m/z): 124.40, 122.29, 120.95, 93.62 (arene-C), 27.75 (CH(CH3)2),\ncalcd. for C 48 H 46 ClF 6 NNaO 3 PRhS 1022.1481, found: 27.54 (CH(CH3)2), 26.53 (CH(CH3)2), 26.17 (CH(CH3)2),\n1022.1463 [M + Na]+. Anal. calcd for C48H46ClF6NO3PRhS: 22.64 (CH(CH3)2), 21.56 (CH(CH3)2). 31P{1H} NMR (202\nC, 57.64; H, 4.64; N, 1.40. Found: C, 57.48; H, 4.69; N, 1.33. MHz, DMSO-d6) (ppm): \u03b4 39.40. ESI-MS (m/z): calcd. for\n C37H38ClNO3PRuS 744.1042, found: 744.1140 [M + H]+;\n 4.3.9. Ru1. ESI-MS (m/z): calcd. for C37H37ClNNaO3PRuS 766.0861,\n found: 766.0952 [M + Na]+. Anal. calcd for\n C37H37ClNO3PRuS: C, 59.79; H, 5.02; N, 1.88. Found: C,\n 59.98; H, 5.08; N, 1.79.\n\n\n\n 4.3.11. Ru3.\n\nOrange-brown powder, yield: 65.8 mg (65.5%). 1H NMR (500\nMHz, DMSO-d6) (ppm): \u03b4 8.61 (d, J = 2.4 Hz, 1H, CH\ufffdN),\n8.00\u22127.97 (m, 1H), 7.94\u22127.86 (m, 2H), 7.78\u22127.74 (m, 1H),\n7.74\u22127.63 (m, 7H), 7.61 (t, J = 7.6 Hz, 1H), 7.48\u22127.38 (m,\n3H), 7.02\u22126.98 (m, 1H), 6.36\u22126.29 (m, 1H, arene-H), 5.71\n(d, J = 6.2 Hz, 1H, arene-H), 5.11 (d, J = 6.0 Hz, 1H, arene-\nH), 4.65 (d, J = 6.4 Hz, 1H, arene-H), 4.04\u22123.97 (m, 1H,\nCH(CH3)2), 2.57\u22122.52 (m, 1H, CH(CH3)2), 1.91\u22121.83 (m,\n1H, CH(CH3)2), 1.49 (d, J = 6.7 Hz, 3H, CH(CH3)2), 1.39 (s,\n3H, arene-CH3), 1.22 (d, J = 7.0 Hz, 3H, CH(CH3)2), 1.15 (d, Orange-brown powder, yield: 68.0 mg (66.4%). 1H NMR (500\nJ = 6.9 Hz, 3H, CH(CH3)2), 1.05 (d, J = 6.6 Hz, 3H, MHz, DMSO-d6) (ppm): \u03b4 8.59 (d, J = 2.3 Hz, 1H, CH\ufffdN),\nCH(CH3)2), 0.87 (d, J = 6.7 Hz, 3H, CH(CH3)2), 0.78 (d, J = 7.94\u22127.91 (m, 1H), 7.82\u22127.56 (m, 11H), 7.45\u22127.31 (m, 3H),\n6.5 Hz, 3H, CH(CH3)2). 13C{1H} NMR (151 MHz, DMSO- 6.83\u22126.79 (m, 1H), 6.22\u22126.19 (m, 1H, arene-H), 5.43 (d, J =\nd6) (ppm): \u03b4 176.88 (CH\ufffdN), 154.99, 148.54, 139.75, 6.0 Hz, 1H, arene-H), 5.32 (d, J = 6.1 Hz, 1H, arene-H), 5.11\n139.25, 137.76, 136.41, 135.63, 135.56, 135.04, 133.41, 133.01, (t, J = 5.5 Hz, 1H, arene-H), 4.49 (t, J = 6.0 Hz, 1H, arene-H),\n132.69, 132.58, 132.11, 131.13, 130.81, 129.71, 128.96, 128.59, 4.43 (t, J = 5.2 Hz, 1H, (CH2)4OH), 3.89\u22123.77 (m, 1H,\n125.62, 125.30, 124.94, 122.39, 121.38, 100.41 (arene-C), CH(CH3)2), 3.41\u22123.38 (m, 2H, (CH2)4OH), 2.55\u22122.53 (m,\n100.30 (arene-C), 91.58 (arene-C), 91.52 (arene-C), 90.39 1H, CH(CH3)2), 2.39\u22122.31 (m, 1H, (CH2)4OH), 1.74\u2212\n(arene-C), 78.33 (arene-C), 31.31 (CH(CH3)2), 27.58 (CH- 1.69(m, 1H, (CH2)4OH), 1.67\u22121.57 (m, 2H, (CH2)4OH),\n(CH3)2), 27.40 (CH(CH3)2), 26.80 (CH(CH3)2), 26.36 1.46 (d, J = 6.7 Hz, 3H, CH(CH3)2), 1.45\u22121.36 (m, 2H,\n(CH(CH3)2), 22.89 (CH(CH3)2), 22.57 (CH(CH3)2), 21.98 (CH2)4OH), 1.08 (d, J = 6.6 Hz, 3H, CH(CH3)2), 0.67 (d, J =\n(CH(CH 3 ) 2 ), 21.92 (CH(CH 3 ) 2 ), 16.48 (arene-CH 3 ). 6.8 Hz, 3H, CH(CH3)2), 0.59 (d, J = 6.6 Hz, 3H, CH(CH3)2).\n 13\n31 1\n P{ H} NMR (202 MHz, DMSO-d6) (ppm): \u03b4 39.03. ESI- C{1H} NMR (101 MHz, DMSO-d6) (ppm): \u03b4 175.42\nMS (m/z): calcd. for C41H46ClNO3PRuS 800.1668, found: (CH\ufffdN), 154.85, 148.83, 139.16, 139.01, 137.27, 136.49,\n800.1791 [M + H] + ; ESI-MS (m/z): calcd. for 135.33, 135.23, 133.71, 133.11, 132.80, 132.56, 132.20, 130.31,\nC41H45ClNNaO3PRuS 822.1487, found: 822.1604 [M + 130.21, 130.08, 129.97, 129.66, 129.12, 125.20, 124.71, 122.23,\nNa]+. Anal. calcd for C41H45ClNO3PRuS: C, 61.60; H, 5.67; 122.12, 121.08, 99.86 (arene-C), 95.99 (arene-C), 95.89\nN, 1.75. Found: C, 61.72; H, 5.63; N, 1.70. (arene-C), 91.23 (arene-C), 83.34 (arene-C), 82.14 (arene-\n C), 60.74 ((CH 2 ) 4 OH), 33.39 ((CH 2 ) 4 OH), 32.53\n 4.3.10. Ru2. ((CH2)4OH), 27.68 ((CH2)4OH), 27.59 (CH(CH3)2), 26.46\n (CH(CH3)2), 26.25 (CH(CH3)2), 25.96 (CH(CH3)2), 22.80\n (CH(CH3)2), 21.76 (CH(CH3)2). 31P{1H} NMR (202 MHz,\n DMSO-d6) (ppm): \u03b4 39.21. ESI-MS (m/z): calcd. for\n C41H46ClNO4PRuS 816.1617, found: 816.1742 [M + H]+;\n ESI-MS (m/z): calcd. for C41H45ClNNaO4PRuS 838.1437,\n found: 838.1549 [M + Na]+. Anal. calcd for\n C41H45ClNO4PRuS: C, 60.40; H, 5.56; N, 1.72. Found: C,\n 60.11; H, 5.63; N, 1.82.\n 20021 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. Chem. 2022, 61, 20008\u221220025\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\u25a0 ASSOCIATED CONTENT\n* Supporting Information\ns\u0131\n \u25a0 REFERENCES\n (1) Cao, Y.; Giovannucci, E. L. Alcohol as a Risk Factor for Cancer.\nThe Supporting Information is available free of charge at Semin. Oncol. Nurs. 2016, 32, 325\u2212331.\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.2c03279. (2) Tsai, H.-J.; Chang, J. S. Environmental Risk Factors of Pancreatic\n Cancer. J. Clin. Med. 2019, 8, 1427.\n Additional experimental details and methods, 1H, 13C- (3) Dyson, P. J.; Sava, G. Metal-based antitumour drugs in the post\n {1H}, and 31P{1H} NMR spectra, and ESI-MS spectra genomic era. Dalton Trans. 2006, 1929\u22121933.\n for all compounds (Figures S1\u2212S60 and Tables S1\u2212S9) (4) Torigoe, T.; Izumi, H.; Ishiguchi, H.; Yoshida, Y.; Tanabe, M.;\n (PDF) Yoshida, T.; Igarashi, T.; Niina, I.; Wakasugi, T.; Imaizumi, T.;\nAccession Codes Momii, Y.; Kuwano, M.; Kohno, K. Cisplatin Resistance and\n Transcription Factors. Curr. Med. Chem.: Anti-Cancer Agents 2005,\nCCDC 2205155, 2205157, and 2216236 contain the 5, 15\u221227.\nsupplementary crystallographic data for this paper. These (5) Matei, D.; Fang, F.; Shen, C.; Schilder, J.; Arnold, A.; Zeng, Y.;\ndata can be obtained free of charge via www.ccdc.cam.ac.uk/ Berry, W. A.; Huang, T.; Nephew, K. P. Epigenetic Resensitization to\ndata_request/cif, or by emailing data_request@ccdc.cam.ac. Platinum in Ovarian Cancer. Cancer Res. 2012, 72, 2197\u22122205.\nuk, or by contacting The Cambridge Crystallographic Data (6) Florea, A.-M.; Bu\u0308sselberg, D. Cisplatin as an Anti-Tumor Drug:\nCentre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 Cellular Mechanisms of Activity, Drug Resistance and Induced Side\n1223 336033. Effects. Cancers 2011, 3, 1351\u22121371.\n (7) Allardyce, C. S.; Dyson, P. J. Metal-based drugs that break the\n\u25a0 AUTHOR INFORMATION\nCorresponding Authors\n rules. Dalton Trans. 2016, 45, 3201\u22123209.\n (8) Oun, R.; Moussa, Y. E.; Wheate, N. J. The side effects of\n platinum-based chemotherapy drugs: a review for chemists. Dalton\n Lihua Guo \u2212 School of Chemistry and Chemical Engineering, Trans. 2018, 47, 6645\u22126653.\n Qufu Normal University, Qufu 273165, P. R. China; (9) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,\n orcid.org/0000-0002-0842-9958; Email: guolihua@ Z.-S. The development of anticancer ruthenium(II) complexes: from\n qfnu.edu.cn single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,\n Zhe Liu \u2212 School of Chemistry and Chemical Engineering, 46, 5771\u22125804.\n Qufu Normal University, Qufu 273165, P. R. China; (10) Zhang, P.; Sadler, P. J. Redox-Active Metal Complexes for\n orcid.org/0000-0001-5796-4335; Email: liuzheqd@ Anticancer Therapy. Eur. J. Inorg. Chem. 2017, 2017, 1541\u22121548.\n 163.com (11) Truong, D.; Sullivan, M. P.; Tong, K. K. H.; Steel, T. R.;\n Prause, A.; Lovett, J. H.; Andersen, J. W.; Jamieson, S. M. F.; Harris,\nAuthors H. H.; Ott, I.; Weekley, C. M.; Hummitzsch, K.; S\u00f6hnel, T.; Hanif,\n Xueyan Hu \u2212 School of Chemistry and Chemical Engineering, M.; Metzler-Nolte, N.; Goldstone, D. C.; Hartinger, C. G. Potent\n Qufu Normal University, Qufu 273165, P. R. China Inhibition of Thioredoxin Reductase by the Rh Derivatives of\n Anticancer M(arene/Cp*)(NHC)Cl2 Complexes. Inorg. Chem. 2020,\n Mengqi Liu \u2212 School of Chemistry and Chemical Engineering,\n 59, 3281\u22123289.\n Qufu Normal University, Qufu 273165, P. R. China (12) P\u00e9rez-Arnaiz, C.; Acun\u0303a, M. I.; Busto, N.; Echevarr\u00eda, I.;\n Qiuya Zhang \u2212 School of Chemistry and Chemical Mart\u00ednez-Alonso, M.; Espino, G.; Garc\u00eda, B.; Dom\u00ednguez, F.\n Engineering, Qufu Normal University, Qufu 273165, P. R. Thiabendazole-based Rh(III) and Ir(III) biscyclometallated com-\n China plexes with mitochondria-targeted anticancer activity and metal-\n Yuwen Gong \u2212 School of Chemistry and Chemical sensitive photodynamic activity. Eur. J. Med. Chem. 2018, 157, 279\u2212\n Engineering, Qufu Normal University, Qufu 273165, P. R. 293.\n China (13) Rubio, A. R.; Gonz\u00e1lez, R.; Busto, N.; Vaquero, M.; Iglesias, A.\n Mengru Sun \u2212 School of Chemistry and Chemical Engineering, L.; Jal\u00f3n, F. A.; Espino, G.; Rodr\u00edguez, A. M.; Garc\u00eda, B.; Manzano, B.\n Qufu Normal University, Qufu 273165, P. R. China R. Anticancer Activity of Half-Sandwich Ru, Rh and Ir Complexes\n with Chrysin Derived Ligands: Strong Effect of the Side Chain in the\n Shenghan Feng \u2212 School of Chemistry and Chemical\n Ligand and Influence of the Metal. Pharmaceutics 2021, 13, 1540.\n Engineering, Qufu Normal University, Qufu 273165, P. R. (14) Konkankit, C. C.; Marker, S. C.; Knopf, K. M.; Wilson, J. J.\n China Anticancer activity of complexes of the third row transition metals,\n Youzhi Xu \u2212 School of Chemistry and Chemical Engineering, rhenium, osmium, and iridium. Dalton Trans. 2018, 47, 9934\u22129974.\n Qufu Normal University, Qufu 273165, P. R. China (15) Lord, R. M.; Zegke, M.; Basri, A. M.; Pask, C. M.; McGowan, P.\n Yiming Liu \u2212 School of Chemistry and Chemical Engineering, C. Rhodium(III) Dihalido Complexes: The Effect of Ligand\n Qufu Normal University, Qufu 273165, P. R. China Substitution and Halido Coordination on Increasing Cancer Cell\n Potency. Inorg. Chem. 2021, 60, 2076\u22122086.\nComplete contact information is available at: (16) Biancalana, L.; Kostrhunova, H.; Batchelor, L. K.; Hadiji, M.;\nhttps://pubs.acs.org/10.1021/acs.inorgchem.2c03279 Degano, I.; Pampaloni, G.; Zacchini, S.; Dyson, P. J.; Brabec, V.;\n Marchetti, F. Hetero-Bis-Conjugation of Bioactive Molecules to Half-\nNotes Sandwich Ruthenium(II) and Iridium(III) Complexes Provides\nThe authors declare no competing financial interest. Synergic Effects in Cancer Cell Cytotoxicity. Inorg. Chem. 2021, 60,\n\n\u25a0 ACKNOWLEDGMENTS\nThe authors thank the Natural Science Foundation of\n 9529\u22129541.\n (17) \u0141omzik, M.; Hanif, M.; Budniok, A.; B\u0142auz\u0307, A.; Makal, A.;\n Tchon\u0301, D. M.; Les\u0301niewska, B.; Tong, K. K. H.; Movassaghi, S.;\n S\u00f6hnel, T.; Jamieson, S. M. F.; Zafar, A.; Reynisson, J.; Rychlik, B.;\nShandong Province (ZR2022MB038), the Young Talents Hartinger, C. G.; Plaz\u0307uk, D. Metal-Dependent Cytotoxic and Kinesin\nInvitation Program of Shandong Provincial Colleges and Spindle Protein Inhibitory Activity of Ru, Os, Rh, and Ir Half-\nUniversities, the Taishan Scholars Program, the Youth Tutor Sandwich Complexes of Ispinesib-Derived Ligands. Inorg. Chem.\nVisiting Program of Shandong Province, and the Student\u2019s 2020, 59, 14879\u221214890.\nPlatform for Innovation and Entrepreneurship Training (18) Navale, G.; Singh, S.; Agrawal, S.; Ghosh, C.; Roy Choudhury,\nProgram (S202110446047 and XJ20210056) for support. A.; Roy, P.; Sarkar, D.; Ghosh, K. DNA binding, antitubercular,\n\n 20022 https://doi.org/10.1021/acs.inorgchem.2c03279\n Inorg. 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Chem. 2022, 61, 20008\u221220025\n\f", "pages_extracted": 18, "text_length": 118331}