Necrosis-Inducing High-Valent Oxo-Rhenium(V) Complexes with Potent Antitumor Activity: Synthesis, Aquation Chemistry, Cisplatin Cross-Resistance Profile, and Mechanism of Action.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Necrosis-Inducing High-Valent Oxo\u2212Rhenium(V) Complexes with\n Potent Antitumor Activity: Synthesis, Aquation Chemistry, Cisplatin\n Cross-Resistance Profile, and Mechanism of Action\n Shubhangi Das, Pulkit Joshi, and Malay Patra*\n Cite This: Inorg. Chem. 2023, 62, 19720\u221219733 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Chemotherapy with the cytotoxic platinum (Pt)\n drugs cisplatin, carboplatin, and oxaliplatin is the mainstay of\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 13:37:36 (UTC).\n\n\n\n\n anticancer therapy in the clinic. The antitumor activity of Pt drugs\n originates from their ability to induce apoptosis via covalent adduct\n formation with nuclear DNA. While the phenomenal clinical\n success is highly encouraging, resistance and adverse toxic side\n effects limit the wider applicability of Pt drugs. To circumvent these\n limitations, we embarked on an effort to explore the antitumor\n potential of a new class of oxo\u2212rhenium(V) complexes of the type\n [(N\u2227N)(EG)Re(O)Cl] (where EG = ethylene glycolate and N\u2227N =\n bipyridine, Bpy (1); phenanthroline, Phen (2); 3,4,7,8-tetramethyl-\n phenanthroline, Me4Phen (3)). Investigation of speciation chem-\n istry in aqueous media revealed the formation of [(N\u2227N)Re(O)-\n (OH)3] as the biologically active species. Complex 3 was found to be the most potent among the three, with IC50 values ranging\n from 0.1 to 0.4 \u03bcM against a panel of cancer cells, which is 5\u221270-fold lower when compared with cisplatin. The higher potency of 3\n is attributed to its higher lipophilicity, which enhanced cellular uptake. Importantly, complex 3 efficiently overcomes cisplatin\n resistance in ovarian, lung, and prostate cancer cells. In addition to reporting the aquation chemistry and identifying the active\n species in aqueous media, we performed in-depth in vitro mechanistic studies, which revealed that complex 3 preferentially\n accumulates in mitochondria, depletes mitochondrial membrane potential, and upregulates intracellular reactive oxygen species\n (ROS), leading to ER stress-mediated necrosis-mediated cancer cell death.\n\n\n \u25a0 INTRODUCTION\n Cancer is the second leading cause of death after\n obtained using ruthenium (Ru), gold (Au), iridium (Ir), and\n rhenium (Re) complexes.8\u221212\n cardiovascular diseases, accounting for 10 million deaths and In the past decade, Re(I) complexes of the type [(N\u2227N)-\n 20 million new cases in 2020.1 Even in the modern era of Re(CO)3X] (N\u2227N = polypyridyl ligands and X = Cl, H2O,\n personalized therapy and immunotherapy, chemotherapy with isonitriles, pyridyl, phosphines, etc.) have emerged as a\n platinum (Pt) drugs\ufffdcisplatin, carboplatin, and oxaliplatin promising class of antitumor agents.13,14 Wilson et al.\n remains the mainstay of cancer management in the clinic.2\u22124 In developed several highly in vitro as well as in vivo potent\n fact, approximately half of all patients receiving chemotherapy Re(I) complexes.15\u221219 Notably, this class of complexes are\n are being treated with Pt drugs either as a single agent or in endowed with the extraordinary ability of overcoming cisplatin\n combination with other drugs or treatment modalities.3,5 resistance in cervical, ovarian, and lung cancer cells, owing to\n However, in spite of this remarkable clinical success, Pt drugs their novel mechanism of action.15 Zobi and co-workers\n suffer from two critical limitations.4 Innate and acquired recently demonstrated potent antiangiogenic and antimeta-\n resistance against Pt drugs increasingly reduce their efficacy, static properties of the Re(CO)3 systems.20,21 Massi and\n leading to treatment failure.6 Further, severe side effects, such Falasca et al. introduced Re(CO)3 complexes bearing N-\n as nephrotoxicity, neurotoxicity, myelosuppression, etc., arising\n from the indiscriminate killing of cancer and normal cells, limit Received: September 6, 2023\n their applicability as well as dose escalation during the Revised: November 2, 2023\n treatment cycle.4,7 To overcome these limitations, tremendous Accepted: November 2, 2023\n efforts have been devoted toward the development of non-Pt Published: November 16, 2023\n metal-based drugs with a mechanism of action different from\n the DNA-targeted Pt drugs, and promising results were\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c03110\n 19720 Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nheterocyclic carbene ligands, which exerted antitumor activity et al., in vitro antitumor activity of phosphine-containing\nvia inhibiting important cell signaling processes.22,23 Re(V)\ufffdO complexes, such as Re-3 (Figure 1 for the\n Additionally, the rich spectroscopic properties of Re(I) structure), in combination with verapamil hydrochloride and\n 32\nsystems offer numerous unique advantages over the classical Pt L-buthionine-sulfoximine was investigated. However, this\ndrugs. For instance, the luminescent properties and distinct class of complexes presented poor in vitro potency with IC50 >\nRe\u2212C\ufffdO stretching enable real-time intracellular tracking of 50 \u03bcM. Besides the antitumor research, the thiophilic nature of\nRe(I) complexes via fluorescence and vibrational microscopies, Re(V)\ufffdO complexes was exploited for the development of\nwhich facilitates mechanistic investigation.24,25 Further, the covalent inhibitors of thiol-containing enzymes, such as cystine\npossibility of synthesizing the \u201chot\u201d 99mTc analogue provides proteases and cathepsins.33\u221236\nthe opportunity for pharmacokinetic assessment through in These aforementioned fascinating properties of Re(V)\ufffdO\nvivo imaging in the very early developmental stage of Re(I) complexes intrigued us to investigate the antitumor properties\nantitumor agents.15 of a new class of such complexes consisting of N\u2227N and O\u2227O\n By contrast to the Re(I) system, the antitumor potential of bidentate ligands (Figure 1). Herein, we report synthesis,\nhigh-valent Re complexes was poorly explored. Investigations characterization, aquation chemistry, and biological evaluation\non the antitumor properties of Re(III) clusters by Schtemenko of Re(V)\ufffdO complexes with general formula [(N\u2227N)(EG)-\net al. led to the identification of paddle wheel dirrhenate(III) Re(O)Cl] (where EG = ethylene glycolate and N\u2227N =\ncomplexes with remarkable in vivo anticancer properties and bipyridine, Bpy (1); phenanthroline, Phen (2); 3,4,7,8-\nlow neuro-, nephro- and hepato-toxicities.26\u221228 An octahedral tetramethyl-phenanthroline, Me4Phen (3)). While the O\u2227O\nRe(IV) complex was also shown to display potent in vitro and Cl ligands were kept unchanged in all complexes, the N\u2227N\nantitumor activity against breast, ovarian, and prostate cancer ligand was varied to tune the lipophilicity of this class of\ncells.29 The antitumor activity of Re(V)\ufffdO systems was first Re(V)\ufffdO complexes. It is worth noting that the EG ligand\ndemonstrated in 2013 by Abram et al. using an in vitro was chosen due to its proven ability to strongly coordinate with\nscreening of a few Re(V)\ufffdO complexes containing S,N,S- the Re(V)\ufffdO and Te(V)\ufffdO cores and form stable\ntridentate thiosemicarbazone/thiosemicarbazide ligands.30 complexes for medicinal and radiopharmaceutical applica-\nTheir best candidate Re-1 (Figure 1 for the structure) exerted tions.37,38 We showed that the most potent candidate complex\n 3 possesses outstanding antitumor activity over a panel of\n cancer cells and has the ability to overcome cisplatin resistance\n in ovarian, lung, and prostate cancer cells. The potency of 3\n was found to be comparable to or slightly higher than that of\n the previously reported most potent compound Re-2. In\n addition to reporting the aquation chemistry of these Re(V)\ufffd\n O complexes and identifying the active species in biological\n media for the first time, we performed in-depth mechanistic\n studies, which revealed that complex 3 preferentially\n accumulates in mitochondria, depletes mitochondrial mem-\n brane potential, and upregulates intracellular ROS, leading to\n ER stress-mediated necrosis-mediated cancer cell death.\n\n \u25a0 RESULTS AND DISCUSSION\n Synthesis, Characterization, and Theoretical Studies.\n The synthesis of Re(V)\ufffdO complexes 1\u22123 is presented in\n Scheme 1a, and details are provided in the Experimental\n Section.38 Briefly, a solution of [(Bu)4N][ReOCl4] in\n methanol was treated with ethylene glycol (H2EG). To this,\n addition of N\u2227N bidentate ligands Bpy, Phen, and Me4Phen\n afforded 1, 2, and 3, respectively, as a reddish brown solid. All\n complexes were fully characterized using 1H and 13C NMR\n spectroscopies and matrix-assisted laser desorption/ionization-\n time-of-flight (MALDI-TOF) mass spectrometry (Figures S1\u2212\n S9). The 1H and 13C NMR spectra of the complexes indicated\nFigure 1. Structures of Re(V)\ufffdO complexes investigated previously that both polypyridyl N\u2227N and EG ligands are coordinated to\nand in this work. the Re(V)\ufffdO center. For instance, while the 1H NMR\n spectrum of free Me4Phen showed two sets of signals for four\nan IC50 value (half inhibitory concentration) of 0.41 \u03bcM aromatic C\u2212H protons owing to the presence of symmetry, the\n 1\nagainst MCF-7 breast cancer cells. Soon after that, remarkably H NMR spectrum of complex 3 showed four peaks due to the\nin vitro potent Re(V)\ufffdO complexes of phenanthroline-based loss of symmetry upon coordination. Additionally, the EG\nligands, such as Re-2 (Figure 1 for the structure), were ligand protons in complex 3 appeared as four significantly\nreported by Lippard and co-workers.31 Importantly, in addition downfield-shifted signals as compared to the single peak\nto potent activity against cisplatin-sensitive and -resistant cells observed for the free H2EG. Purities of 1\u22123 were confirmed to\nwith IC50 vales 0.04\u22128.6 \u03bcM, Re-2 was shown to promote be >95% by elemental microanalysis.\nnecroptosis-mediated cancer cell death via oxidative stress In principle, the ligands in a complex of the [(N\u2227N)(O\u2227O)-\ninduction. Further, Re-2 possessed remarkable in vivo Re(O)Cl] type may adopt three different orientations,\ntolerability in C57BL/6 mice. In a recent paper by Poljarevic\u0301 resulting in two unsymmetric isomers A and B (which may\n 19721 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. (a) Synthesis of [(N\u2227N)(EG)Re(O)Cl] single crystals of these complexes for the determination of their\nComplexes 1\u22123. (b) Possible Ligand Orientations in molecular structures, which prevented us from determining\n[(N\u2227N)(O\u2227O)Re(O)Cl] Type Complexes unambiguous ligand orientation around the Re(V) center.\n In pursuit of this objective, we performed density functional\n theory (DFT) calculations using 3 as a representative of this\n class. Initially, we optimized the geometries in the gas phase,\n revealing that the 3B isomer is energetically more stable than\n the 3A isomer by approximately 16 kcal/mol (Figure 2a).\n Furthermore, we simulated the gas-phase 1H NMR spectra of\n 3A and 3B and observed an overall better agreement for the\n 3B isomer with the experimental spectrum than for 3A (Figure\n 2b and Table S1). Particularly, the two of the glycolate protons\n are upfield shifted in the case of 3A possibly due to the trans\n effect of the \u201c\ufffdO\u201d group, leading to only two peaks within the\n range of 4\u22126 ppm. Conversely, in the case of 3B, all four\n glycolate protons are in good agreement with the experimental\n spectrum (Figure 2b). Taken together, the outcomes derived\n from the DFT calculations collectively lead us to formulate the\n hypothesis that the compound synthesized according to\n Scheme 1 likely assumes the isomeric configuration of 3B.\n All of the computational details are provided later in the\n Experimental Section.\n Aquation Chemistry and Stability in the Cell Culture\n Medium. With the complexes in hand, we first explored their\n aquation chemistry using 1H NMR spectroscopy. Formation of\n an aqua complex (M\u2212OH2) through the exchange of the \u201cCl\u201d\n ligand with water is considered an important activation step for\n anticancer metal complexes containing M\u2212Cl bond(s).4,11,12 A\n few previous reports also showed the hydrolysis of the Re\u2212Cl\n bond in [(N\u2227N)(CO)3ReICl] type anticancer complexes in\n aqueous media.15,39 However, aquation chemistries of Re-\nexist as a mixture of enantiomers) and one symmetric isomer C (V)\ufffdO anticancer complexes including Re-1, Re-2, and Re-3\n(Scheme 1b). 1H and 13C NMR spectra of 1\u22123 suggested the (Figure 1) remain unexplored. To initiate the study, complex 3\npresence of a single unsymmetric species, either A or B, and was dissolved in a mixture of DMSO-d6 and D2O (2:1, v/v),\nthe absence of symmetric isomer C (Figures S1, S2, S4, S5, S7, and the 1H NMR spectrum was recorded at different time\nand S8). However, 1H NMR cannot distinguish between two points. Results are presented in Figure 3a. DMSO, a routinely\nunsymmetrical isomers A and B. Unfortunately, despite used solvent for preparation of a stock solution of drug\nrepeated efforts, we were unsuccessful in growing good-quality candidates with poor water solubility, was used to ensure the\n\n\n\n\nFigure 2. (a) DFT-optimized structures of 3A and 3B isomers. Color codes: cyan, Re; violet, N; red, O; green, Cl; gray, C. Hydrogen atoms are\nomitted for clarity. (b) Comparison of experimental 1H NMR spectra of 3 (4\u22126 ppm, diastertopic CH2 protons from the EG ligand) with\nsimulated 1H NMR spectra of isomers 3A and 3B. Full 1H NMR spectra are presented in Figure S10.\n\n 19722 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n 1\n H NMR spectra of 3 and the free Me4Phen ligand in aqueous\n media (Figure 3a). Although the 1H NMR data confirmed the\n release of the EG ligand from complex 3, it could not be\n ascertained from 1H NMR spectroscopy whether the \u201cCl\u201d\n ligand remains bound to the Re(V)\ufffdO core or replaced by\n water in the hydrolyzed species. To investigate this, we\n hypothesized that complex 3 may undergo hydrolysis via either\n the P1 or P2 pathway (Figure 3b). While the P1 pathway will\n lead to the formation of [(Me4Phen)(OH)2Re(O)Cl] through\n replacement of only the EG ligand by water, the P2 pathway\n will lead to the formation of [(Me4Phen)(OH)3Re(O)]\n through replacement of both EG and Cl ligands by water. As\n shown in Figure 3b, [(Me4Phen)(OH)2Re(O)Cl] can exist as\n four possible isomers in solution: T1, T2, T3, and T4.\n Similarly, [(Me4Phen)(OH)3Re(O)] can exist as two possible\n isomers, T5 and T6 (Figure 3b). Based on 1H NMR data, the\n possibility of the existence of T2, T3, T4, and T6 isomers,\n where the two pyridine rings of the Me4Phen N\u2227N ligand have\n different chemical environments, can be ruled out. Then, we\n analyzed the key structural difference between the remaining\n two isomers, T1 and T5, where both pyridine rings of the\n Me4Phen ligand have similar chemical environments (Figure\n 3b). While the labile \u201cCl\u201d ligand is oriented trans to the \u201c\ufffdO\u201d\n ligand in T1, T5 does not contain any labile \u201cCl\u201d ligand\n (Figure 3b). The high trans effect of the \u201c\ufffdO\u201d group is known\n to dramatically increase the lability of the trans \u201cCl\u201d ligand. In\n fact, the rapid hydrolysis of the [O\ufffdRe/Tc(V)\u2212Cl] system to\n [O\ufffdRe/Tc(V)\u2212OH] was observed even in the presence of\n moisture.40 Therefore, the existence of the T1 isomer of\n [(Me4Phen)(OH)2Re(O)Cl] in aqueous media is highly\nFigure 3. (a) Stacked 1H NMR plot of complex 3 in the nonaqueous\nCDCl3 medium and complex 3, Me4Phen, and ethylene glycol in unlikely. To this end, it is reasonable to speculate that\naqueous media. (b) Two plausible and distinct hydrolysis pathways, hydrolysis of 3 in aqueous media led to the formation of the\nP1 and P2, of [(N\u2227N)(EG)Re(O)Cl] type complexes 1\u22123. T5 isomer of [(Me4Phen)(OH)3Re(O)].\nHydrolysis via path P1 will result in the formation of [(N\u2227N)- Then, to further confirm the molecular composition of the\n(OH)2Re(O)Cl] that may exist as the T1, T2, T3, or T4 isomer. hydrolyzed species in aqueous media, we isolated the\nHydrolysis via path P2 will result in the formation of [(N\u2227N)- hydrolyzed species by lyophilizing a solution of 3 in a\n(OH)3Re(O)] that may exist as T5 or T6 isomers. Note: Only T1 DMSO/water mixture and characterized using 1H and 13C\nand T5 isomers, not T2, T3, T4, and T6, have identical chemical (or NMR spectroscopy, mass spectrometry, and elemental analysis.\nmagnetic) environments for both the pyridine rings of the N\u2227N As expected, 1H and 13C NMR spectroscopy confirmed the\nligands.\n absence of the EG ligand (removed during lyophilization) but\n the presence of the Me4Phen ligand (Figures S11 and S12).\nsolubility of complex 3 in mM concentrations. Although we Importantly, as noted in Table S2, elemental analysis\nanticipated the hydrolysis of the Re(V)\u2212Cl bond and confirmed the molecular composition of the isolated solid to\nformation of the corresponding hydrolyzed species, we were be [(Me4Phen)(OH)3Re(O)]\u00b7(DMSO)0.75. The presence of\nsurprised to observe that unsymmetric complex 3 transformed 0.75 equiv of residual DMSO was also evident in the 1H and\n 13\nto a single new symmetric species immediately after dissolving C NMR spectra (Figures S11 and S12). Further, ESI-mass\nin aqueous media, as evidenced by the two pyridine rings of spectroscopy of a solution of the solid in MeOH showed a\nthe N\u2227N ligand showing identical chemical environments. The peak at m/z = 519.1 with the expected isotopic pattern,\nnewly formed species remained unchanged over 72 h. While corresponding to the ([(Me4Phen)(OH)3Re(O)] + 2MeOH-\ncomplex 3 presented four aromatic CH (8.0\u22129.2 ppm) and 2H2O + H)+ adduct (Figure S13). Furthermore, we compared\nfour aliphatic CH3 (2.4\u22123.5 ppm) proton signals in non- the 1H NMR spectrum of compound 3 and the isolated\naqueous solvents such as CDCl3, the same complex showed hydrolyzed solid of complex 3 (Figure S14). It was evident that\ntwo aromatic CH (8.3 and 8.9 ppm) and two aliphatic CH3 the 1H NMR signals of complex 3 in DMSO-d6/D2O were\n(2.5 and 2.7 ppm) proton signals in the presence of D2O identical to those of the lyophilized solid of the hydrolyzed\n(Figure 3a). Further, the four diasterotopic proton signals (4.2, species, except for the signal from free H2EG for complex 3,\n4.6, 5.1, and 5.6 ppm) from the EG ligand in 3 disappeared in which was removed during lyophilization of the hydrolyzed\naqueous media with the appearance of a new upfield shifted species and was absent for the isolated hydrolyzed solid. Taken\nsinglet at 3.4 ppm (Figure 3a). This new singlet was assigned together, these data firmly confirmed that complex 3\nto be the uncoordinated H2EG by comparing it with the 1H underwent hydrolysis in aqueous media through the release\nNMR spectrum of an authentic sample of H2EG, indicating the of both EG and Cl ligands, resulting in formation of the\nrelease of the EG ligand from complex 3 in the presence of symmetric T5 isomer of [(Me4Phen)(OH)3Re(O)]. A similar\nwater. However, the Me4Phen ligand remained attached to the aquation chemistry was observed for complexes 1 and 2 in\nRe(V)\ufffdO core in aqueous media, as evident from different aqueous media (Figures S15 and S16). Notably, the hydro-\n 19723 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 4. Stability of hydrolyzed species [(Me4Phen)(OH)3Re(O)] from complex 3 in DMEM cell culture medium. (a) Full 1H NMR spectra at t\n= 0, 12, and 72 h and (b) zoomed aliphatic region of the same. # denotes complex 3, * denotes the hydrolyzed species from 3,\n[(Me4Phen)(OH)3Re(O)], and $ denotes free H2EG released upon the hydrolysis of 3.\n\nlyzed species for 1\u22123 [(N\u2227N)(OH)3Re(O)] presented robust\nstability over a period of 72 h (Figures 3a, S15 and S16).\n Next, we evaluated the stability of 1\u22123 in Dulbecco\u2019s\nmodified Eagle medium (DMEM), a routinely used culture\nmedium that contains various biological nucleophiles such as\nglucose, amino acids, and nucleotides, in addition to high\nconcentrations of salts. Similar to aqueous media, complexes\n1\u22123 underwent hydrolysis via releasing the EG ligand (Figures\n4, S17 and S18). Interestingly, in sharp contrast to the\nimmediate release of the EG ligand in aqueous media,\ncomplexes 1\u22123 released the EG ligand at a slower rate in\nDMEM (\u223c12 h for completion). Importantly, the hydrolyzed\nspecies [(N\u2227N)(OH)3Re(O)] remained stable for more than Figure 5. Log P values and cellular uptake in HeLa cells of 1\u22123 and\n72 h (Figures 4, S17 and S18). With this hydrolysis behavior in cisplatin (10 \u03bcM, 6 h incubation). Data presented as mean \u00b1 SD of\nmind, we prepared a stock solution of complexes 1\u22123 for two independent experiments each performed as triplicates. The log P\nbiological study in 2:1 DMSO/H2O. value of cisplatin was taken from a previously reported article.41\n Lipophilicity and Cellular Uptake. Lipophilicity is an\nimportant physicochemical property that determines the extent (HeLa). Free N\u2227N ligands and cisplatin were also included for\nof passive diffusion-mediated cellular uptake as well as the comparison purposes. Cells were treated with increasing doses\npotency of anticancer agents. To tune the lipophilicity of our of the test compounds and incubated for either 24 or 72 h, and\nRe(V)\ufffdO complex, the N\u2227N ligands were varied from Bpy to viability was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-\nPhen and Me4Phen in complexes 1\u22123. To investigate the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Repre-\ninfluence of N\u2227N ligands on lipophilicity, the octanol\u2212water sentative dose\u2212response plots are presented in Figures S19\npartition coefficient (P) of 1\u22123 was measured using a shake- and S20, and half inhibitory concentrations (IC50) are listed in\nflask method in combination with inductively coupled plasma Table 1. Complex 3 was identified to be the most potent in the\nmass spectrometry (ICP-MS). The log P values of 1\u22123 are series, with an IC50/72h value of 0.4 \u03bcM in the 72 h assay.\nsignificantly higher (0.3\u22121.6) than cisplatin (\u22122.2) and follow Notably, the potency of 3 is 9-fold higher than that of cisplatin\nthe expected order 1 < 2 \u226a 3 (Figure 5). Then, we measured (IC50/72h = 3.6 \u03bcM). While complex 2 presented an IC50/72h\nthe cellular uptake of compounds 1\u22123 and cisplatin (as a value of 2.7 \u03bcM, complex 1 turned out to be inactive up to the\ncontrol). Human cervical HeLa cancer cells were treated with maximum conc. tested (25 \u03bcM). A similar trend in the potency\n10 \u03bcM test compounds for 6 h, and the amount of the of 1\u22123 and cisplatin was observed in the 24 h incubation\nintracellular metal was quantified using ICP-MS. As shown in assays (IC50/24h values, Table 1). The better activity of 3 than\nFigure 5, the uptake of 3 (1329 pmole Re/mg protein) was 3\u2212 those of 1, 2, and cisplatin can be attributed to its significantly\n4-fold higher than those of 1 (333 pmole Re/mg protein), 2 higher cellular uptake (Figure 5). Interestingly, a comparison\n(413 pmole Re/mg protein), and cisplatin (386 pmole Pt/mg of IC50 values of 2 and 3 with their respective N\u2227N ligands\nprotein). Notably, the cellular uptake trend of Re(V)\ufffdO revealed that 2 and 3 possess dramatically higher potency than\ncomplexes, 1 < 2 \u226a 3, mirrors the order of their log P values. their free ligands Phen and Me4Phen, respectively. These data\n In Vitro Antitumor Activity and Cisplatin Cross- not only have implications in establishing the importance of\nResistance Profile. In vitro antitumor activity of complexes the Re(V)\ufffdO core in the potency of this class of compounds\n1\u22123 was first screened against human cervical cancer cell lines but also confirm that the N\u2227N ligands remain attached to the\n 19724 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. IC50 Values (\u03bcM) of 1\u22123, Bpy, Phen, Me4Phen, Table 2. IC50/72h Values (\u03bcM) of 3, Cisplatin, and Re-2 in\nCisplatin, and Re-2 in HeLa Cellsa Cisplatin-Sensitive (A549, A2780, and DU145) and their\n Matched Cisplatin-Resistant (A549 Cis, A2780 Cis, and\n IC50/24h (\u03bcM); 24 h IC50/72h (\u03bcM); 72 h\ncomplexes incubation incubation DU145 Cis) Cellsa\n 1 \u226525 \u226525 cell lines complex 3 cisplatin Re-2\n 2 10.5 \u00b1 0.4 2.7 \u00b1 0.6 A549 0.1 \u00b1 0.08 1.5 \u00b1 0.1 0.2 \u00b1 0.01\n 3 3.4 \u00b1 0.1 0.40 \u00b1 0.03 A549 Cis 0.4 \u00b1 0.03 27.8 \u00b1 0.5\n Bpy \u226525 \u226525 RF (A549) 4 18\n Phen \u226525 13.4 \u00b1 0.8 A2780 0.3 \u00b1 0.03 1.6 \u00b1 0.6 0.2 \u00b1 0.01\n Me4Phen \u226525 3.5 \u00b1 0.1 A2780 Cis 0.2 \u00b1 0.01 12 \u00b1 2.5\n H2EG \u226525 \u226525 RF (A2780) 0.7 7\n cisplatin 8.2 \u00b1 0.5 3.6 \u00b1 0.2 DU145 0.1 \u00b1 0.01 1.7 \u00b1 0.9 1.4 \u00b1 0.08\n b\n Re-2 0.69 \u00b1 0.02 DU145 Cis 0.1 \u00b1 0.01 3.1 \u00b1 1.3\na\n Data presented as mean \u00b1 SD from three independent experiments, RF (DU145) 1 2\neach performed in triplicates. bIC50 values of Re-2 were taken for a\n Data presented as mean \u00b1 SD from three independent experiments,\ncomparison purposes from ref 31. each performed in triplicates. Resistance factor (RF) = IC50/72h value\n in cisplatin-resistant cells/IC50/72h value in matched cisplatin-resistant\n cells. IC50/72h Re-2 was taken from for comparison purposes from ref\nRe(V)\ufffdO core in the active species that enter the cells. Given 31.\nthat the EG ligand is released from the complexes in aqueous\nmedia, we determined the cytotoxicity of free H2EG in HeLa\ncells. H2EG did not affect the cell viability up to the highest to their matched cisplatin-sensitive cells. For better comparison\nconc. tested (25 \u03bcM), suggesting a negligible contribution of purposes, we calculated the resistance factor (RF) as the ratio\nthe released EG ligand to the overall cytotoxicity of the of IC50/72h values in cisplatin-resistant cells and matched\nRe(V)\ufffdO complexes (Table 1). Further, a comparison of the cisplatin-sensitive cells. While the RF values determined for\nIC50/72h of our best complex 3 with the most potent Re(V)\ufffdO complex 3 were 4 in A549, 0.7 in A2780, and 1 for DU145, the\ncomplex Re-2 (Figure 1) reported to date suggested that RF determined for cisplatin were 18 in A549, 7 in A2780, and\ncomplex 3 has comparable or slightly better potency than Re-2 2 for DU145 cells (Table 2). The significantly lower RF values\nin HeLa cells.31 presented by complex 3 as compared to cisplatin in any given\n As mitochondrial damage and ROS induction contribute to matched pair of cell lines clearly demonstrated the ability of\nthe antitumor activity of complex 3 (discussed later), we complex 3 to overcome cisplatin resistance in cancer cells.\nredetermined the IC50 values 3 in HeLa cells using a To assess the cancer cell selectivity, we performed\nnonenzymatic crystal violet assay. It is worth noting that the cytotoxicity evaluation of the best candidate 3 and cisplatin\nMTT assay measures the mitochondrial dehydrogenase activity as control using the MTT assay on CCD18Co noncancerous\nand may provide unreliable IC50 values for mitochondria- colon epithelial cells and HT29 colon cancer cells.\ntargeted antitumor agents.42 A crystal violet assay, on the other Unfortunately, similar to cisplatin (IC50/72h, HT29 = 3.01\nhand, does not rely on any enzymatic activity and rather \u03bcM; IC50/72h, CCD18Co = 5.09 \u03bcM), complex 3 (IC50/72h,\nreports the IC50 value based on the biomass of the adherent HT29 = 0.4 \u03bcM; IC50/72h, CCD18Co = 0.3 \u03bcM) also presented\ncells.42 As presented in Table S3, we found that the IC50 values comparable IC50/72h values in cancerous and noncancerous\nof 3 obtained from the crystal violet assay were comparable to cells, suggestive of poor cancer cell selectivity (Figure S23).\nthose from the MTT assay for both 24 and 72 h (Figure S21 Therefore, further efforts are certainly required to improve the\nfor dose\u2212response plots). cancer cell selectivity of this class of complexes.\n Cisplatin and carboplatin are considered to be the Intracellular Organelle Distribution of Complex 3.\nworkhorses for the management of different types of cancers Next, we investigated the mechanism of the antitumor activity\nin the clinic. However, inherent and acquired resistance of our most potent complex 3. First, to obtain preliminary\nincreasingly reduces the effectiveness of these chemother- insights into the possible intracellular target, HeLa cells were\napeutics, leading to treatment failure.9,43,44 Intrigued by the treated with 10 \u03bcM 3 or cisplatin (for comparison purposes)\nexcellent potency of 3 against HeLa cells, we further evaluated for 24 h. Important organelles such as the nucleus, cytosol,\nits ability to circumvent cisplatin resistance. For this purpose, mitochondria, and insoluble membrane were isolated (detailed\nthe IC50/72h values of 3 and cisplatin were determined in the in the Experimental Section), and metal content (Re for\nwild-type cisplatin-sensitive and their matched cisplatin- complex 3 and Pt for cisplatin) in each organelle was measured\nresistant lung (A549 and A549 Cis), ovarian (A2780 and using ICP-MS. The organelle distribution profiles for 3 and\nA2780 Cis) and prostate (DU145 and DU145 Cis) cancer cisplatin are presented in Figure 6. For cisplatin-treated cells,\ncells. Dose\u2212response plots are presented in Figure S22, and nearly equal distribution of Pt among mitochondria (388\nIC50/72h values are listed in Table 2. As anticipated, complex 3 pmole Pt/\u03bcg protein), nucleus (274 pmole Pt/\u03bcg protein),\nexerted remarkably higher potency (IC50/72 = 0.1\u22120.4 \u03bcM) and membrane (325 pmole Pt/\u03bcg protein) was observed. In\nthan cisplatin (IC50/72h = 1.5\u221227.8 \u03bcM) against all six cancer sharp contrast, complex 3 is predominantly localized in\ncell lines. Further, potencies of 3 are comparable to those of mitochondria (1610 pmol Re/\u03bcg protein) and membrane\nRe-2 (Figure 1) in A549 and A2780 cells but are \u223c14-fold (2104 pmol Re/\u03bcg protein), and only a trace amount was\nhigher in DU145 cells.31 Importantly, complex 3 was nearly found in the nucleus (153 pmol Re/\u03bcg protein) and cytosol\nequipotent in all three cisplatin-sensitive and matched (116 pmol Re/\u03bcg protein). Importantly, the mitochondrial\ncisplatin-resistant cells. In sharp contrast, cisplatin\u2019s effective- accumulation of 3 was found to be 4-fold higher as compared\nness reduced drastically in cisplatin-resistant cells as compared to that of cisplatin, suggestive of mitochondria as one of the\n 19725 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n Mitochondrial Dysfunction and ROS Production.\n Owing to its high propensity to accumulate in mitochondria,\n we investigated the effect of complex 3 on mitochondria.\n Mitochondria are the main powerhouse and important\n signaling organelles for cells, which play a key role in cell\n survival as well as programmed cell death (PCD).45 As altered\n mitochondrial dynamics and/or functioning are closely linked\n to the initiation, progression, and metastasis of cancer,46,47\n mitochondria are an attractive target for the development of\n the next generation of antitumor agents.48 The potential of 3\n to cause mitochondrial damage was assessed by measuring\n mitochondrial membrane potential (\u0394\u03a8m) in HeLa cells\n treated with complex 3 using the JC-1 (5,5,6,6\u2032-tetrachloro-\n 1,1\u2032,3,3\u2032-tetraethyl-imidacarbocyanine iodide) probe in combi-\nFigure 6. Intracellular organelle distribution of complex 3 and\ncisplatin in HeLa cells (10 \u03bcM, 24 h). Data presented as mean \u00b1 SD nation with flow cytometry.49,50 In healthy and functional\nfor two independent experiments each performed in triplicates. mitochondria with intact \u0394\u03a8m, JC-1 forms an aggregate that\n emits in red. But in damaged and dysfunctional mitochondria\n with dissipated \u0394\u03a8m, JC-1 remains as a monomer that emits in\nprime targets for complex 3. Further, membrane accumulation green.51 As shown in Figure 7a,7b, complex 3 treatment\nof 3 is 6.5-fold as compared to cisplatin, which can be resulted in dramatic dose-dependent depletion of \u0394\u03a8m, similar\nattributed to the 3.8 log P unit higher lipophilicity of 3 as to the positive control carbonyl cyanide m-chlorophenyl\ncompared to cisplatin. Nevertheless, these results confirmed hydrazone (CCCP, Figure S24 for the dot plot).\nthat complex 3 had a different intracellular distribution profile Numerous earlier studies have demonstrated that mitochon-\nas compared to cisplatin, in addition to pointing to drial damage and/or dysfunction is known to enhance\nmitochondria and membrane as potential targets for complex intracellular oxidative stress through overproduction of reactive\n3. oxygen species (ROS).10,52 Elevated levels of ROS cause\n\n\n\n\nFigure 7. (a) Flow cytometry measurement of \u0394\u03a8m using the JC-1 dye in HeLa cells treated with increasing concentrations of complex 3 (0\u22126\n\u03bcM, 24 h) or CCCP (2 \u03bcM, 1 h). The dot plot for CCCP-treated cells is presented in Figure S24. (b) Quantification of relative \u0394\u03a8m as the ratio of\nJC-1 red/JC-1 green in untreated treated cells. (c) Fluorescence microscopy images of HeLa cells stained with the DCFH-DA ROS probe after\ntreatment with increasing concentrations of complex 3 (0\u22126 \u03bcM, 4 h) or H2O2 (3 mM, 1 h). The fluorescence microscopy image of H2O2-treated\ncells is presented in Figure S25. (d) Quantification of the relative ROS level in untreated and compound-treated cells. Note: concentrations of\ncomplex 3 were chosen to be 1.5 \u03bcM (0.5 \u00d7 IC50/24h), 3 \u03bcM (IC50/24h), and 6 \u03bcM (2 \u00d7 IC50/24h) based on IC50/24h = 3.4 \u03bcM obtained from a 24 h\nincubation assay. Data presented as mean \u00b1 SD from two independent experiments each performed in duplicates (***p < 0.001).\n\n 19726 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 8. (a) Western blot analysis of hallmark proteins of ER stress in Hela cells treated with complex 3 (24 h exposure). The uncropped full\nimages of all blots are presented in Figure S28. (b) Effect of cycloheximide on the viability of HeLa cells treated with complex 3.\n\n\n\n\nFigure 9. (a) Flow cytometry dot plots of apoptotic and necrotic population of HeLa cells left untreated or treated with 0.5 \u00d7 IC50/24h, IC50/24h, and\n2 \u00d7 IC50/24h concentrations of complex 3 for 24 h. (b) Percent of cell population determined from dot plots. Cisplatin (a known apoptosis inducer)\nand H2O2 (a known necrosis inducer) were used for gating purposes, and corresponding dot plots are presented in Figure S27. (c) Percent viability\nof HeLa cells treated with 6 \u03bcM complex 3 in the absence or presence of various cell death inhibitors. Z-Vad-FMK, pan-caspase inhibitor; 3-\nmethyladeniine, autophagy inhibitor; necrostatin-1, necroptosis inhibitor; IM-54, necrosis inhibitor. (d) Western blot analysis of necroptosis\nmarker proteins RIP-1 and RIP-3 in HeLa cells treated with complex 3 or the known necroptosis inducer shikonin for 24 h. Images of uncropped\nfull blots are presented in Figure S28.\n\noxidation of essential biomolecules and cellular reductants, activity through mitochondrial damage-mediated induction of\nleading to further enhancement of the ROS level in a vicious oxidative stress, which is consistent with the mechanism of\ncyclic pathway, which impairs essential cellular processes and action of Re(V)\ufffdO antitumor complex Re-2 reported\neventually triggers cell death. We employed a widely used ROS previously.31\nindicator DCFH-DA (2\u2032,7\u2032-dichlorodihydrofluorescein diace- ER Stress Induction. Since complex 3 caused mitochon-\ntate) to measure the level of ROS in cells treated with complex drial damage and elevated intracellular ROS, we investigated\n3.41 As presented in Figure 7c,7d, HeLa cells treated with the effect of complex 3 on the endoplasmic reticulum (ER).\ncomplex 3 for 4 h presented dose-dependent upregulation of ER is an important organelle responsible for various essential\nintracellular ROS, similar to that of positive control H2O2 cellular functions such as protein synthesis, folding, and\n(Figure S25). As anticipated, the level of intracellular ROS is transport, in addition to playing a key role in various signaling\nreduced significantly in the presence of N-acetyl cysteine processes.54 ER and mitochondria are closely spaced organelles\n(NAC), a known quencher of ROS (Figure S26).53 Taken that form multiple contact points through cholesterol-rich\ntogether, these data suggested that complex 3 exerts antitumor microdomains termed mitochondria-associated ER membranes\n 19727 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n(MAMs).55 MAMs-mediated cross-talk between ER and confirmed necrosis to be the major cell death mechanism\nmitochondria is crucial for the proper functioning of both evoked by complex 3.\norganelles. Therefore, mitochondrial damage, \u0394\u03a8m depletion,\nand ROS overproduction are expected to interfere with ER\nfunctioning and escalate ER stress.17 ER stress-related signaling\n \u25a0 CONCLUSIONS\n This work reports the synthesis, characterization, aquation\nis transduced through three main ER transmembrane chemistry, in vitro antitumor activity, and mechanism of action\nproteins\ufffdIRE1\u03b1, ATF6, and PERK. Our western blot analysis of a structurally new class of Re(V)\u2212oxo complexes 1\u22123 of the\nconfirmed that treatment with complex 3 led to significant type [(N\u2227N)(EG)Re(O)Cl]. In aqueous media, the complexes\nupregulation of ATF6 and p-eIF2\u03b1 (a downstream protein of underwent rapid hydrolysis through replacement of the EG\nPERK wing) but not IRE1\u03b1 (Figure 8a). Further, to investigate O\u2227O and Cl ligands with water. The resulting hydrolyzed\nwhether ER stress contributes to the antitumor activity of species [(N\u2227N)(OH)3Re(O)] presented robust stability in cell\ncomplex 3, we studied the effect of the ER stress inhibitor culture media DMEM over 72 h and was the active species\ncycloheximide on the potency of complex 3. In fact, we involved in executing the cancer cell death. Owing to its\nobserved an \u223c22% increase in cell viability in the presence of highest lipophilicity, the cellular uptake efficiency of 3 is the\ncycloheximide as compared to cells treated with complex 3 highest among all. Concomitantly, complex 3 was identified to\nalone (Figure 8b). Cumulatively, our mechanistic data be the most potent in the series with IC50/72h values ranging\nsuggested that antitumor activity of 3 involves mitochondrial from 0.1 to 0.4 \u03bcM against a panel of cisplatin-sensitive as well\ndamage, ROS production, and ER stress. as cisplatin-resistant cancer cells. Notably, complex 3 presented\n Characterization of the Cell Death Mechanism. 5\u221217-fold higher potency in cisplatin-sensitive HeLa, A549,\nThereafter, we turned our attention to obtaining preliminary A2780, and DU145 cells and 31\u221270-fold higher potency in\ninsights into the mechanism of cell death triggered by complex cisplatin-resistant A549 Cis, A2780 Cis, and DU145 Cis cells\n3. In general, oxidative stress, mitochondrial dysfunction, and as compared to the gold standard clinical drug cisplatin.\nER stress induce apoptosis, necrosis (or necroptosis), and Importantly, complex 3 was found to overcome cisplatin\nautophagy-mediated cell death.17,56 Except necrosis, all other resistance, which is considered to be one of the major issues in\ntypes of above-mentioned cell death are programmed cell managing cancers with Pt drugs in the clinic. An intracellular\ndeath. First, we performed flow cytometry analysis of dual organelle localization study revealed that unlike cisplatin that\nAnnexin V\u2212FITC (AV) and propidium iodide (PI) stained was equally distributed among the nucleus, mitochondria, and\nHeLa cells either left untreated or treated with increasing membrane, complex 3 mainly accumulated in mitochondria\nconcentrations of complex 3. Cisplatin and H2O2 were and membrane. Furthermore, we demonstrated that complex 3\nincluded as controls for gating purposes. While PI stains the caused mitochondrial damage through depletion of \u0394\u03a8m and\nnucleus of necrotic cells with compromised plasma mem- upregulation of oxidative stress via production of ROS, in\nbranes, AV binds to apoptotic cells with phosphatidyl serine addition to triggering ER stress. This multipronged mechanism\nflipped outside of the plasma membrane.57 As shown in of action induced necrosis-mediated cell death.\nFigures 9a,b and S27, similar to the positive control H2O2,\ntreatment of 3 caused a dose-dependent increase in AV\u2212/PI+\nnecrotic population (1.4% in untreated vs 4 and 20% in 3 and\n \u25a0 EXPERIMENTAL SECTION\n Materials and Methods. All chemicals and solvents used were\n reagent grade or better quality and were purchased from commercial\n6 \u03bcM 3-treated, respectively). No significant increase in the suppliers. Solvents were used as received or dried with molecular\npopulation of AV+/PI\u2212 early apoptotic and AV+/PI+ late sieves. All synthetic procedures were performed using standard\napoptosis populations was observed. These data indicated that Schlenk techniques.\nnot apoptosis but necrosis was one of the main mechanisms of Purities of newly synthesized complexes 1\u22123 used for the\ncell death induced by complex 3. In line with our expectation, biological study were \u226595%, confirmed using elemental microanalysis.\nthe pan-caspase inhibitor Z-Vad-FMK failed to inhibit the NMR spectroscopic measurements were done using Bruker 800\nviability reduction caused by complex 3 (Figure 9c), MHz and Varian 600 MHz spectrometers at 25 \u00b0C in the NMR\n facility, TIFR Mumbai. All of the measurements were carried out\nconfirming the absence of apoptosis. Necrosis has long been\n using deuterated solvents; chemical shifts \u03b4 were reported in ppm\nviewed as an accidental and unprogrammed cell death without (parts per million). The residual solvent peaks were used as an\nthe involvement of any molecular regulators. However, internal reference for 1H and 13C NMR spectra, and chemical shifts\ngrowing evidence in the recent past firmly established that were expressed relative to tetramethylsilane (SiMe4, \u03b4 = 0 ppm).\nnecrosis may also happen in a regulated fashion mediated Abbreviations for the peak multiplicities were as follows: s (singlet), d\nthrough RIP-1 and/or RIP-3 kinases.31,56 The regulated form (doublet), dd (doublet of doublets), t (triplet), q (quartet), m\nof necrosis is termed necroptosis. In order to investigate (multiplet), and br (broad). Mass spectra were recorded on a Bruker\nwhether complex 3 elicited necrosis or necroptosis, we ultrafleXtreme MALDI-TOF mass spectrometer or Thermo Q\n Exactive orbitrap mass spectrometer equipped with an electrospray\nperformed western blot analysis to check the level of RIP-1 ionization source. Simulated mass spectra were obtained from SISweb\nand RIP-3 proteins in HeLa cells treated with complex 3. As (https://www.sisweb.com/mstools.htm). Elemental microanalyses\nshown in Figure 9d, complex 3 treatment did not result in were performed on a Thermo Fisher FLASH2000 CHNS/O analyzer.\nsignificant upregulation of either RIP-1 or RIP-3, while the Inductively coupled mass spectrometry (ICP-MS) measurements\nknown necroptosis inducer shikonin significantly upregulated were carried out using an Agilent 7900 ICP-MS. All of the\nboth marker proteins. Further, the RIP-1 inhibitor necrost- colorimetric and imaging assays were performed using a Biotec\nstain-1 had a negligible effect on the potency of complex 3, but Cytation 5 Imager. Flow cytometry measurements were done using a\n BD FACS Aria Fusion instrument. We analyzed the immunoblots\nnecrosis inhibitor IM-54 significantly reduced the potency of using an Amersham Imager 6000.\ncomplex 3 by \u223c2.7 fold (Figure 9c). Further, 3-methyl adenine Synthesis and Characterization Data of Re(V)\ufffdO Com-\nhas no effect on the potency of complex 3, indicating the plexes. [(Bipy)(EG)Re(O)Cl] (1).38 [(Bu)4N][ReOCl4] (62 mg, 0.1\nabsence of autophagic cell death. Cumulatively, these data mmol) was dissolved in 1.5 mL of MeOH, followed by addition of 0.5\n\n 19728 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nmL of ethylene glycol, and stirred at 750 rpm for 5 min at room ppm (s, 2H), 2.98 ppm (s, 6H), 2.75 ppm (s, 6H). 13C {1H} NMR\ntemperature. The color of the solution changed from green to reddish (acetone-d6, 200 MHz): \u03b4 (ppm) = 150.2, 147.6, 136.5, 134.7, 128.0,\nbrown. Bipyridine (33 mg, 0.4 mmol) was then added to the solution 123.7, 16.8, 14.7. ESI-mass (in MeOH): 519.1 [M + 2MeOH\u2212H2O\u2212\nand stirred at 750 rpm for 30 min. The mixture was centrifuged to OH]\u00b7+ Elemental analysis: Anal. calcd for [(Me4Phen)(OH)3Re(O)]\u00b7\nisolate the solid precipitate. It was then dissolved in 2 mL of CHCl3 (DMSO)0.75, C17.5H23.5N2O4.75ReS0.75: C, 38.35; H, 4.32; N, 5.11;\nand reprecipitated using diethyl ether. The precipitate was isolated by found: C, 38.12; H, 4.20; N, 5.15.\nusing centrifugation and dried in high vacuum to afford complex 1 as Stability of Complexes in DMEM Cell Culture Medium.\na red-brown solid. Yield = 76% (34 mg). 1H NMR (CDCl3, 600 Phenol-free DMEM (1 mL) was lyophilized in order to obtain\nMHz): \u03b4 (ppm) = 9.32 (d, J = 5.8 Hz, 1H), 8.58 (d, J = 5.5 Hz, 1H), powdered DMEM. The powder was dissolved in 1 mL of D2O.\n8.33 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.90 (t, J = 7.8 Hz, Complexes 1\u22123 (\u223c1\u22122 mg) were dissolved in a mixture of 400 \u03bcL of\n1H), 7.60 (t, J = 6.7 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 6.7 DMSO-d6 and 200 \u03bcL of DMEM in D2O. Then, 1H NMR spectra\nHz, 1H), 5.57 (dd, J = 10.7, 5.6 Hz, 1H), 4.98 (td, J = 10.4, 5.2 Hz, were recorded at different time points until 72 h.\n1H), 4.75\u22124.69 (m, 1H), 4.25 (dt, J = 15.4, 7.8 Hz, 1H). 13C {1H} Log P Measurement. A freshly prepared stock solution of\nNMR (CDCl3, 200 MHz): \u03b4 (ppm) = 154.2, 151.9, 148.3, 142.2, complexes 1\u22123 in DMSO/H2O (2:1) was added to 600 \u03bcL of\n139.1, 125.7, 124.4, 123.20, 122.2, 120.1, 88.1, 87.6. MALDI-TOF: water. A 100 uL of this solution was taken out and diluted to 5 mL\n419.1 [1 \u2212 Cl]+, 391.1 [1 \u2212 Cl \u2212 C2H2]+. Anal. calcd For 1, using double deionized water, and the amount of Re was quantified\nC12H12ClN2O3Re: C, 31.75; H, 2.66; N, 6.17; found: C, 31.97; H, using ICP-MS. To the 500 \u03bcL of the remaining aqueous solution, 500\n3.07; N, 6.18. \u03bcL of octanol was added. The biphasic mixture was shaken for 2 h\n [(Phen)(EG)Re(O)Cl] (2). Complex 2 was synthesized following a using an orbital shaker. The mixture was then allowed to settle down\nsimilar procedure as described for complex 1, except that Phen was for effective phase separation, and the aqueous and organic layers\nused, instead of Bpy, as the N\u2227N ligand. The reaction was performed were carefully separated. 100 \u03bcL of the aqueous layer was carefully\non the same scale. Complex 2 was isolated as a brown solid. Yield = taken out and diluted to 5 mL using double deionized water for\n68% (32 mg). 1H NMR (CDCl3, 600 MHz): \u03b4 (ppm) = 9.41 (d, J = quantification of Re using ICP-MS. Log P was then calculated by the\n5.4 Hz, 1H), 8.89 (d, J = 5.2 Hz, 1H), 8.38 (d, J = 8.1 Hz, 1H), 8.04 following formula\n(d, J = 8.9 Hz, 1H), 8.00\u22127.95 (m, 1H), 7.88 (d, J = 8.9 Hz, 1H),\n log P = log((C0 C 2)/C 2)\n7.71 (d, J = 8.0 Hz, 1H), 7.66\u22127.59 (m, 1H), 5.72\u22125.61 (m, 1H),\n5.12 (td, J = 10.3, 5.4 Hz, 1H), 4.74\u22124.64 (m, 1H), 4.29 (dd, J = 15.5, where C0 = initial concentration of Re in ppb (preshaking) and C2 =\n9.9 Hz, 1H). 13C{1H} NMR (CDCl3, 200 MHz): \u03b4 (ppm) = 141.2, concentration of the metal in ppb after 2 h (postshaking).\n137.2, 130.4, 128.1, 128.1, 126.0, 124.7, 121.8, 88.5, MALDI-TOF: Whole Cell Uptake. HeLa cells (2 \u00d7 106) were seeded in 60 mm\n478.3 [2 + H]+, 443.5 [2 \u2212 Cl]+. Anal. calcd for 2\u00b7(diethyl ether)0.2, Petri dishes (four Petri dishes for each complex). After 16 h, the cells\nC14H12ClN2O3Re\u00b7(C4H10O)0.2: C, 36.08; H, 2.86; N, 5.69; found: C, were incubated with 3 mL of fresh media containing 10 \u03bcM of test\n35.94; H, 2.60; N, 5.97. complexes. All stock solutions were freshly prepared (cisplatin in PBS\n [(Me4Phen)(EG)Re(O)Cl] (3). Complex 3 was synthesized following and complexes 1\u22123 in 2:1 DMSO/H2O). After 6 h incubation at 37\na similar procedure as described for complex 1, except that 1 equiv of \u00b0C, the media was aspirated, and the cells were washed with PBS (3 \u00d7\nMe4Phen was used, instead of 4 equiv of Bpy, as the N\u2227N ligand. The 3 mL). For each complex, three Petri dishes were digested by\nreaction was performed on the same scale. Complex 3 was isolated as treatment with ICP-MS grade HNO3 (400 \u03bcL per Petri dish) for 2\na brown solid. Yield = 56% (30 mg). 1H NMR (CDCl3, 600 MHz): \u03b4 days at room temperature, followed by digestion with ICP-MS grade\n(ppm) 9.22 (s, 1H), 8.60 (s, 1H), 8.13 (d, J = 9.4 Hz, 1H), 8.00 (d, J H2O2 (400 \u03bcL per Petri dish) for 1 day at room temperature. The\n= 9.5 Hz, 1H), 5.62 (dd, J = 10.9, 5.5 Hz, 1H), 5.07 (td, J = 10.5, 5.3 volume of each sample was made to 10 mL by adding the required\nHz, 1H), 4.65 (dd, J = 9.6, 5.2 Hz, 1H), 4.29\u22124.22 (m, 1H), 3.51 (s, amount of double deionized water. The metal content in the digested\n3H), 2.73 (s, 3H), 2.56 (s, 3H), 2.41 (s, 3H). 13C {1H} NMR samples was measured using ICP-MS. The remaining one Petri dish\n(CDCl3, 200 MHz): \u03b4 (ppm) = 151.6, 149.9, 148.2, 141.1, 137.4, was treated with RIPA buffer (200 \u03bcL for 15 min incubation at 0 \u00b0C),\n132.9, 130.8, 128.8, 126.5, 124.0, 122.2. MALDI-TOF: 499.1 [3 \u2212 and then the Petri dish was scratched using a cell scraper, and the\nCl]+, 472.1 [3 \u2212 C2H2 \u2212 Cl]+. Anal. calcd for 3, C18H20ClN2O3Re: C, lysate was collected. The lysates were ultrasonicated in order to\n40.48; H, 3.78; N, 5.25; found: C, 40.26; H, 3.80; N, 5.03. dissolve the membrane proteins and then ultracentrifuged at 12,000\n DFT Calculations. All of the computations were performed using rpm, 4 \u00b0C for 15 min, and the supernatant was collected. It was then\nORCA 5.0.0.58 Geometry optimization for both Re(V) isomers, 3A used for measuring the total protein content using a BCA assay kit\nand 3B, was carried out using the DFT B3LYP exchange\u2212correlation (Thermo Fisher) following the supplier protocol. The metal content\nfunctional along with dispersion correction D3 (with Becke\u2212Johnson in each sample was then normalized to the total protein content of the\n(BJ) damping scheme).59\u221264 The basis set def2-TZVP was used for all respective sample.\natoms.65 60 core electrons of Re were replaced by def2-ECP.66 Energy Cytotoxicity Assay. The cytotoxicity of compounds was\nand gradient convergence criteria were set to their default values as evaluated using colorimetric MTT and nonenzymatic crystal violet\nprovided in the software. For geometry optimization, tolerance values assays. Cells (3 \u00d7 103/well for 72 h assay and 8 \u00d7 103/well for 24 h\nfor the convergence of the energy and the root-mean-square gradient assay, 100 \u03bcL of media) were seeded in a 96-well plate and allowed to\nwere 5 \u00d7 10\u22126 au and 1 \u00d7 10\u22124 au, respectively. For the 1H NMR attach overnight. The following day, freshly prepared stock solutions\nspectra, shielding constants for both isomers were computed using the of test complexes (cisplatin in PBS and complexes 1\u22123 in 2:1\nsame level of theory and basis sets. To obtain the NMR chemical DMSO/H2O) were serially diluted using complete media, 100 \u03bcL was\nshifts, we subtract the absolute shielding constant of protons in the added to each well, and the cells were incubated for the desired period\nRe(V) isomers from that of reference tetramethylsilane (TMS) (24 or 72 h). The final concentration of DMSO in each well was\nprotons (also computed using the same level of theory). \u22640.5%, which did not significantly affect the viability of the cell lines\n Aquation Chemistry. Approximately 1 mg of complexes 1\u22123 used. Then, the media were aspirated from all of the wells, fresh media\nwere dissolved in a mixture of 400 \u03bcL of DMSO-d6 and 200 \u03bcL of containing (0.5 mg/mL) MTT were added, and the cells were\nD2O, and 1H NMR spectra were recorded at different time points up incubated for 3 h. The media containing MTT were removed, 150 \u03bcL\nto 72 h at room temperature. The 1H NMR spectra of N\u2227N ligands of DMSO was added to lyse the cells and dissolve the purple\nand EG were also measured using identical experimental conditions. formazan crystals, and absorbance was measured at 570 nm. For the\n Isolation and Characterization of the Hydrolyzed Species of crystal violet assay, the cells were fixed with 4% PFA and treated with\nComplex 3. Complex 3 (10 mg, 0.02 mmol) was dissolved in 2 mL 0.1% Triton X in order to perforate the cells, followed by washing\nof DMSO, and 10 mL of water was added. The mixture was with PBS. Then, the cells were treated with a 0.04% crystal violet\nlyophilized in order to obtain a pink solid. Yield = 92% (8.8 mg). 1H solution in PBS. The crystal violet solution was aspirated from the\nNMR (acetone-d6, 600 MHz): \u03b4 (ppm) = 9.18 ppm (s, 2H), 8.56 wells, 200 \u03bcL of ethanol was added to lyse the cells and dissolve the\n\n 19729 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\ncrystal violet precipitate, and absorbance was measured at 570 nm. 5 min, 4 \u00b0C) and washed with PBS (2 \u00d7 100 \u03bcL). The cell pellets\nThe absorbance values were normalized to the untreated control wells were then resuspended in 100 \u03bcL of PBS and analyzed immediately\nand plotted against the concentration. The IC50 values were obtained using flow cytometry.\nfrom the resulting dose\u2212response curves. The reported IC50 values Immunoblotting Analysis. HeLa cells (3 \u00d7 106 cells/well) were\nwere the average of two or more independent experiments, each of seeded in a 60 mm Petri dish. The following day, the cells were\nwhich had three or six replicates. incubated with fresh media without or with 3 (1.5, 3, 6 \u03bcM) or with\n Intracellular Distribution. HeLa cells (2 \u00d7 107) were seeded in shikonin (10 \u03bcM) for 24 h at 37 \u00b0C. Then, the media were aspirated,\n100 mm Petri dishes (three Petri dishes per compound). The the cells were washed with PBS, and RIPA buffer was added (200 \u03bcL/\nfollowing day, the cells were incubated with 10 \u03bcM 3 or cisplatin. All Petri dish, 15 min incubation at 0 \u00b0C). Then, the Petri dishes were\nstock solutions were freshly prepared (cisplatin in PBS and complexes scratched using a cell scraper to facilitate the lysis, and the protein\n1\u22123 in 2:1 DMSO/H2O). After 24 h, the media containing content of the lysates was measured using a BCA assay. The lysates\ncompounds was aspirated and washed with PBS. The cells were were then mixed with SDS-PAGE loading buffer (64 mM Tris\u2212HCl\ntrypsinized and counted. The nucleus, cytoplasm, and insoluble (pH 6.8), 9.6% glycerol, 2% SDS, 5% \u03b2-mercaptoethanol, 0.01%\nmembrane fractions were separated using the NEPER kit following bromophenol blue). The mixed lysates were incubated at 95 \u00b0C for 10\nthe manufacturer\u2019s protocol (Thermo Fisher Scientific, #78835). The min and then cooled to room temperature. The lysates were loaded\nmitochondria, cytosol, and insoluble protein fractions were separated on 12% sodium dodecyl sulfate polyacrylamide gel such that each well\nusing a Mito kit following the manufacturer\u2019s protocol (Thermo contained 30 \u03bcg of the protein and resolved by electrophoresis (SDS-\nFisher Scientific, #89874). After the separation, 100 \u03bcL of RIPA was PAGE; 130 V for 100 min), followed by electro-transfer to the\nadded to the insoluble fraction. The lysates were ultrasonicated in poly(vinylidene difluoride) membrane (300 mA for 1 h). Membranes\norder to dissolve the insoluble proteins, if any, and then ultra- were blocked in 2% (w/v) nonfat milk in PBST (PBS, 0.1% Tween\ncentrifuged at 12,000 rpm and 4 \u00b0C for 15 min, and the supernatant 20) and incubated with the suitable primary antibodies in 1% (w/v)\nwas collected. Then, the protein content of each fraction was\n nonfat milk in PBST overnight at 4 \u00b0C. Then, the blots were washed\nmeasured using the BCA assay. Then, the remaining lyases were\n with PBST (2 \u00d7 3 mL). The product numbers of the primary\ndigested using HNO3 and H2O2 as described above, and the volume\n antibodies from Cell Signaling Technologies were D9G8 (p-eIF2\u03b1),\nof each sample was adjusted to 10 mL by adding the required double\n 14C10 (IRE1\u03b1), D4Z8 V (ATF6), D97C12 (RIP-1), E717F (RIP-3),\ndeionized water. The metal content in the digested samples was\nmeasured using ICP-MS. The metal content in different samples was and D16H11 (GAPDH).\nthen normalized to the total protein content of the samples. After incubation with rabbit secondary antibodies (Cell Signaling\n Measurement of Mitochondrial Membrane Potential Using Technology, product number 7074) for 1 h at room temperature, the\nthe JC-1 Assay. HeLa cells (0.75 \u00d7 106) were seeded in 35 mm Petri blots were again washed with PBST (3 \u00d7 3 mL). The immune\ndishes. The following day, media aspirated, and fresh medium without complexes were then detected with an ECL detection reagent\nor with 3 (1.5, 3, or 6 \u03bcM) were added and incubated for 24 h. CCCP (Thermo Fisher) and analyzed using an Amersham Imager 6000\n(2 mM, 1 h exposure) was used as a positive control. All stock instrument fitted with a chemiluminescence filter.\nsolutions were freshly prepared (CCCP in DMSO and complexes 1\u2212 Viability Assay in the Presence of Cell Death Inhibitors. The\n3 in 2:1 DMSO/H2O). The cells were harvested by trypsinization, viability of HeLa cells with complex 3, in both the presence and\npelleted using an ultracentrifuge (1100 rpm, 5 min, 4 C), and washed absence of various cell death inhibitors, was assessed using the MTT\nwith PBS (2 \u00d7 100 \u03bcL). Then, the cells were resuspended in 50 \u03bcL of assay. A total of 8 \u00d7 103 cells per well (100 \u03bcL of media) were seeded\nFBS-free media containing 10 \u03bcM of the JC-1 dye and incubated for in a 96-well plate and allowed to attach overnight. On the subsequent\n30 min at 37 C, protected from light. The cells were again pelleted by day, a 24 \u03bcM solution of complex 3 in cell culture media was prepared\nultracentrifugation (1100 rpm, 5 min, 4 C) and washed with PBS (2 \u00d7 from its freshly prepared stock solution in DMSO. Solutions of\n100 \u03bcL). Then, the cell pellet was resuspended in 100 \u03bcL of PBS, and different cell death inhibitors, namely, Z-Vad-FMK (apoptosis\nthe fluorescence was measured using flow cytometry immediately. inhibitor, 160 \u03bcM), necrostatin (necroptosis inhibitor, 160 \u03bcM), 3-\n Measurement of Intracellular ROS. HeLa cells (0.3 \u00d7 106) were methyl-adenine (autophagy inhibitor, 160 \u03bcM), and IM-54 (oxidative\nseeded in 35 mm Petri dishes. The following day, media was replaced stress-induced necrosis inhibitor, 20 \u03bcM), were prepared in complete\nwith fresh media without or with freshly prepared stock solution (in media (1.5 mL each). To 750 \u03bcL of the inhibitor solution was added\n2:1 DMSO/H2O) of test compound 3 (1.5, 3 or 6 \u03bcM) or with a 750 \u03bcL of fresh media, and to the remaining 750 \u03bcL was added a\nmixture of 3 (3 \u03bcM) and N-acetyl cysteine (NAC, 500 \u03bcM) and solution of complex 3. For the control group, 750 \u03bcL of complex 3\nincubated for 4 h. H2O2 (3 mM) was used as the positive control and solution was added to 750 \u03bcL of fresh media. Subsequently, 100 \u03bcL of\nincubated for 1 h. The media was then removed, and the cells were each final solution was added to individual wells, and the cells were\nincubated with FBS-free media containing DCFH-DA (10 \u03bcM) for 20 incubated for a desired period (24 h). The final concentration of\nmin. The media was then aspirated, the cells were washed with PBS, DMSO in each well was maintained at or below 0.5%, which did not\nphenol red-free complete DMEM was added, and imaging was significantly impact the viability of the utilized cell lines. Following\nperformed immediately. The intracellular DCF fluorescence was this, the media were aspirated from all of the wells, and fresh media\nimaged in the GFP channel using a Cytation 5 imager using identical containing 0.5 mg/mL MTT were added. The cells were then\nparameters for all Petri dishes. The experiments were performed in incubated for 3 h, after which the MTT-containing media were\nduplicates. We then quantified the normalized ROS by normalizing removed. To lyse cells and dissolve the purple formazan crystals, 150\nthe mean GFP fluorescence with a number of cells and plotted it as a \u03bcL of DMSO was added, and absorbance was measured at 570 nm.\nbar plot. The absorbance values for samples containing complex 3, with or\n Annexin V/Propidium Iodide Assay. HeLa cells (0.75 \u00d7 106) without inhibitors, were normalized to the respective control wells\nwere seeded in 35 mm Petri dishes. The following day, media was containing media with or without inhibitors in order to obtain the\nreplaced with fresh media without or with freshly prepared stock percentage viability. The reported percentage viability represents the\nsolution (in PBS for cisplatin and in 2:1 DMSO/H2O for 3) of test average of two independent experiments, each of which consisted of\ncompound 3 (1.5, 3, or 6 \u03bcM) and cisplatin (10 \u03bcM) incubated for 24\n three replicates.\nh, and H2O2 (3 mM) was incubated for 4 h. The cells were harvested\nby trypsinization and pelleted by centrifugation (1100 rpm, 5 min, 4\n\u00b0C). The cell pellet was resuspended and washed with PBS (2 \u00d7 100\n\u03bcL). Then, the cells were suspended in FBS-free media containing 10\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\n\u03bcM Annexin V\u2212FITC conjugate (Thermo Fisher) and 10 \u03bcM\npropidium iodide dye and incubated for 30 min at 37 \u00b0C, protected The Supporting Information is available free of charge at\nfrom light. The cells were again pelleted by centrifugation (1100 rpm, https://pubs.acs.org/doi/10.1021/acs.inorgchem.3c03110.\n 19730 https://doi.org/10.1021/acs.inorgchem.3c03110\n Inorg. Chem. 2023, 62, 19720\u221219733\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n 1\n H and 13C NMR spectra; mass spectra; aquation (8) Ott, I.; Gust, R. Non platinum metal complexes as anti-cancer\n chemistry and stability studies; dose\u2212response plots; drugs. Arch Pharm. 2007, 340, 117\u2212126.\n and uncropped western blot images (PDF) (9) Konkankit, C. C.; Marker, S. C.; Knopf, K. M.; Wilson, J. J.\n Anticancer activity of complexes of the third row transition metals,\n\n\u25a0 AUTHOR INFORMATION\nCorresponding Author\n rhenium, osmium, and iridium. Dalton Trans. 2018, 47, 9934\u22129974.\n (10) Cao, J.-J.; Zheng, Y.; Wu, X.-W.; Tan, C.-P.; Chen, M.-H.; Wu,\n N.; Ji, L.-N.; Mao, Z.-W. Anticancer Cyclometalated Iridium(III)\n Malay Patra \u2212 Laboratory of Medicinal Chemistry and Cell Complexes with Planar Ligands: Mitochondrial DNA Damage and\n Biology, Department of Chemical Sciences, Tata Institute of Metabolism Disturbance. J. Med. Chem. 2019, 62, 3311\u22123322.\n Fundamental Research, 400005 Mumbai, India; (11) De Palo, A.; Draca, D.; Murrali, M. G.; Zacchini, S.; Pampaloni,\n G.; Mijatovic, S.; Maksimovic-Ivanic, D.; Marchetti, F. A Comparative\n orcid.org/0000-0003-3373-6762; Email: malay.patra@\n Analysis of the In Vitro Anticancer Activity of Iridium(III) {\u03b7(5)-\n tifr.res.in C(5)Me(4)R} Complexes with Variable R Groups. Int. J. Mol. Sci.\nAuthors 2021, 22, No. 7422, DOI: 10.3390/ijms22147422.\n (12) Alessio, E.; Messori, L. NAMI-A and KP1019/1339, Two\n Shubhangi Das \u2212 Laboratory of Medicinal Chemistry and Cell Iconic Ruthenium Anticancer Drug Candidates Face-to-Face: A Case\n Biology, Department of Chemical Sciences, Tata Institute of Story in Medicinal Inorganic Chemistry. Molecules 2019, 24,\n Fundamental Research, 400005 Mumbai, India No. 1995, DOI: 10.3390/molecules24101995.\n Pulkit Joshi \u2212 Department of Chemical Sciences, Tata Institute (13) Leonidova, A.; Gasser, G. Underestimated Potential of\n of Fundamental Research, 400005 Mumbai, India; Organometallic Rhenium Complexes as Anticancer Agents. ACS\n orcid.org/0000-0003-2138-6662 Chem. Biol. 2014, 9, 2180\u22122193.\n (14) Huang, Z.; Wilson, J. J. Therapeutic and Diagnostic\nComplete contact information is available at:\n Applications of Multimetallic Rhenium(I) Tricarbonyl Complexes.\nhttps://pubs.acs.org/10.1021/acs.inorgchem.3c03110 Eur. J. Inorg. Chem. 2021, 2021, 1312\u22121324.\n (15) Knopf, K. M.; Murphy, B. L.; MacMillan, S. N.; Baskin, J. M.;\nAuthor Contributions Barr, M. P.; Boros, E.; Wilson, J. J. In Vitro Anticancer Activity and in\nM.P. conceptualized and supervised the project. S.D. Vivo Biodistribution of Rhenium(I) Tricarbonyl Aqua Complexes. J.\nsynthesized the complexes, performed all experiments, and Am. Chem. Soc. 2017, 139, 14302\u221214314.\ninterpreted the data. P.J. performed theoretical studies and (16) Konkankit, C. C.; Vaughn, B. A.; MacMillan, S. N.; Boros, E.;\ninterpreted the results. All authors contributed to the Wilson, J. J. Combinatorial Synthesis to Identify a Potent, Necrosis-\npreparation and editing of the manuscript and approved the Inducing Rhenium Anticancer Agent. Inorg. Chem. 2019, 58, 3895\u2212\nsubmission. 3909.\n (17) King, A. P.; Wilson, J. J. Endoplasmic reticulum stress: an\nNotes arising target for metal-based anticancer agents. Chem. Soc. Rev. 2020,\nThe authors declare no competing financial interest. 49, 8113\u22128136.\n\n\u25a0 ACKNOWLEDGMENTS\nThe authors gratefully acknowledge the financial support under\n (18) Konkankit, C. C.; King, A. P.; Knopf, K. M.; Southard, T. L.;\n Wilson, J. J. In Vivo Anticancer Activity of a Rhenium(I) Tricarbonyl\n Complex. ACS Med. Chem. Lett. 2019, 10, 822\u2212827.\nproject no. RTI4003 from the Department of Atomic Energy (19) Marker, S. C.; King, A. P.; Granja, S.; Vaughn, B.; Woods, J. J.;\n(DAE) and Tata Institute of Fundamental Research (TIFR), Boros, E.; Wilson, J. J. Exploring the In Vivo and In Vitro Anticancer\nIndia. M.P. thanks the Science and Engineering Research Activity of Rhenium Isonitrile Complexes. Inorg. Chem. 2020, 59,\n 10285\u221210303.\nBoard (SERB), India, for financial support under the project (20) Delasoie, J.; Pavic, A.; Voutier, N.; Vojnovic, S.; Crochet, A.;\nSRG/2019/002041. The authors thank Prof. Ankona Datta Nikodinovic-Runic, J.; Zobi, F. 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