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Ruthenium(II)-Dithiocarbazates as Anticancer Agents: Synthesis, Solution Behavior, and Mitochondria-Targeted Apoptotic Cell Death.
{"full_text": " Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n www.chemeurj.org\n\n\n Ruthenium(II)-Dithiocarbazates as Anticancer Agents:\n Synthesis, Solution Behavior, and Mitochondria-Targeted\n Apoptotic Cell Death\n Gurunath Sahu,[a] Sushree Aradhana Patra,[a] Sudhir Lima,[a, b] Sanchita Das,[a] Helmar G\u00f6rls,[b]\n Winfried Plass,*[b] and Rupam Dinda*[a]\n\n\n Abstract: The reaction of the Ru(PPh3)3Cl2 with HL1 3 OH crystallography. The solution/aqueous stability, hydrophobic-\n ( OH stands for the oxime hydroxyl group; HL1 OH = ity, DNA interactions, and cell viability studies of 1\u20133 against\n diacetylmonoxime-S-benzyldithiocarbazonate; HL2 OH = diac- HeLa, HT-29, and NIH-3T3 cell lines were performed. Cell\n etylmonoxime-S-(4-methyl)benzyldithiocarbazonate; and viability results suggested 3 being the most cytotoxic of the\n HL3 OH = diacetylmonoxime-S-(4-chloro)benzyl-dithiocarbaz- series with IC50 6.9 \ufffd 0.2 \u03bcM against HeLa cells. Further, an\n onate) gives three new ruthenium complexes apoptotic mechanism of cell death was confirmed by cell\n [RuII(L1 3 H)(PPh3)2Cl] (1\u20133) ( H stands for imine hydrogen) cycle analysis and Annexin V-FITC/PI double staining techni-\n coordinated with dithiocarbazate imine as the final products. ques. In this regard, the live cell confocal microscopy results\n All ruthenium(II) complexes (1\u20133) have been characterized by revealed that compounds primarily target the mitochondria\n elemental (CHNS) analyses, IR, UV-vis, NMR (1H, 13C, and 31P) against HeLa, and HT-29 cell lines. Moreover, these ruthenium\n spectroscopy, HR-ESI-MS spectrometry and also, the structure complexes elevate the ROS level by inducing mitochondria\n of 1\u20132 was further confirmed by single crystal X-ray targeting apoptotic cell death.\n\n\n\n Introduction another Ru(II) complex that induces detachment from the\n primary tumor cell mass, migration and invasion, and activates\n Ruthenium-based therapeutics of the platinum-group metals mitochondrial apoptosis.[8] Both RM175 and ONCO4417 demon-\n have been the focus of significant interest because of their strate apoptosis by causing cell death by G2/M phase arrest.\n acceptable biological and rich anticancer properties.[1] Platinum- ONCO4417 caused DNA damage at similar levels to cisplatin.\n based anticancer drugs such as cisplatin, oxaliplatin, and (Figure 1).[9]\n carboplatin are potent against a variety of cancerous cells, but Besides the above ruthenium drugs, ruthenium-\n the lack of selectivity, solubility, and other side effects have polypyridyl[1i,10] and ruthenium-arene[11] complexes have also\n prompted researchers to develop anticancer agents that differ been reported extensively as potential chemotherapeutic\n from the stereotypical ones.[2] Thus, there are multiple reports agents. In spite of these ruthenium complexes, there may still\n on ruthenium complexes that have been explored for anti- be new opportunities for developing anticancer agents based\n cancer studies within the frame of a possible\n \u201cruthenotherapy\u201d.[3] NAMI-A,[4] KP1019,[5] and its sodium salt\n analogue (N)KP-1339,[6] are the ruthenium complexes that have\n progressed into the human and clinical testing.[7] RAPTA is\n\n\n [a] G. Sahu, S. A. Patra, S. Lima, S. Das, Prof. Dr. R. Dinda\n Department of Chemistry\n National Institute of Technology\n Rourkela 769008, Odisha (India)\n E-mail: rupamdinda@nitrkl.ac.in\n [b] S. Lima, Dr. H. G\u00f6rls, Prof. Dr. W. Plass\n Institut f\u00fcr Anorganische und Analytische Chemie\n Friedrich-Schiller-Universit\u00e4t Jena\n Humboldtstr. 8, 07743 Jena (Germany)\n E-mail: sekr.plass@uni-jena.de\n Supporting information for this article is available on the WWW under\n https://doi.org/10.1002/chem.202202694\n \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH\n GmbH. This is an open access article under the terms of the Creative\n Commons Attribution License, which permits use, distribution and re- Figure 1. Structures of ruthenium compounds in clinical, and preclinical\n production in any medium, provided the original work is properly cited. trials.\n\n\n Chem. Eur. J. 2023, 29, e202202694 (1 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n on ruthenium systems incorporated with some other bioactive potential in cells.[20] Thus, it is an important aspect to investigate\n ligands. the hydrophobicity, DNA interaction, and also intracellular\n In the development of anticancer agents, ligand design is target of the complexes in the motif of cytotoxicity studies.\n extremely important. In medicinal chemistry, dithiocarbazate Based on these factors and our past experiences with\n Schiff-base derivatives of S-alkyl and S-aryl groups are emerging transition metals in anticancer studies,[16,17,21] three new com-\n ligands with various pharmaceutical and biological properties, pounds of ruthenium(II)-4-R-aroyldithiocarbazoneimine have\n such as anticancer, antiamoebic, and antibacterial properties.[12] been synthesized and characterized by various physicochemical\n Like thiosemicarbazones, they possess NS-donors, enclosed in a techniques including the structure of 1, and 2 solved by single\n thioamide moiety (thione-thiol). In recent times, in vitro tests crystal X-ray analysis. Here, three 4-R-aroyldithiocarbazoneox-\n have shown some promising anticancer activity of non-Pt- imes (Scheme 1) were used as ligands, each with a different\n metallic complexes with dithiocarbamate Schiff-base ligands.[13] inductive effect (R=H, Me, and Cl), in order to examine their\n These ligands have a vital role in cytotoxicity against the human effects, if any, on the biological activity of the complexes.[22] The\n pancreatic cell lines namely, PANC-1, ASPC-1, and BxPc-3 as hydrophobicity of 1\u20133 was tested by partition coefficient\n reported by Gou et al.[14] during the investigation of a series of measurements (log Pow). The interaction of the complexes with\n fluorescent dithiocarbazate based Fe(II), Co(II), Ni(II), Cu(II), and calf thymus (CT) DNA was studied by UV-vis absorption titration\n Zn(II) complexes. Their complexes also displayed anticancer and fluorescence quenching experiments. Finally, the in vitro\n activity against a pancreatic cancer xenograft in mice with low cytotoxicity of 1\u20133 was tested against cancerous HeLa, and HT-\n toxicity. Thus, dithiocarbazate Schiff-base ligands containing 29, and the noncancerous NIH-3T3 cell lines. The apoptotic\n non-Pt-metal complexes offer great potential as anticancer pathway was tested by cell cycle arrest and Annexin V-FITC/\n agents. Propidium Iodide (PI) assays. Compounds being highly cyto-\n Furthermore, metal complexes coordinated with triphenyl- toxic, the intracellular target and apoptotic mode of complexes\n phosphine (PPh3) also have pharmacophoric interest as they were investigated through confocal microscopy and ROS\n exhibit fluorescent properties that provide valuable information (reactive oxygen species) analysis. It is the first detailed study,\n about the distribution, absorption, and uptake of anticancer to the best of our knowledge, to evaluate the effects of Ru(II)-\n drugs in living cells and are also excellent for chemotherapy dithiocarbazate complexes on cancer cells, where all complexes\n since they influence mitochondrial activity.[15] As a result of their cause apoptosis via mitochondrial dysfunction. Overall, this\n increased membrane crossing property,[15b,16] hydrophobic PPh3 study shows the success of mitochondria-targeted ruthenium-\n ligated systems have good cytotoxicity. Dithiocarbazate deriva- aroyldithiocarbazoneimine complexes as successful luminescent\n tives and PPh3 derivative complexes are therefore cytotoxic and anticancer agents.\n have drawn our attention to investigate new mixed dithiocarba-\n zate based Schiff base and PPh3 coordinated complexes. In\n recent times, two azo and PPh3 derived mixed ligand Results and Discussion\n ruthenium(II) complexes have been reported by us exhibiting\n impressive cytotoxicity against both HeLa and HT-29 cancer cell Synthesis and Characterization\n lines (IC50 values between 3.84 and 4.48 \u03bcM).[16] Significant in\n vitro cytotoxic results of dithiocarbazate Schiff-base-vanadium Refluxing an equimolar ratio of HL1 3 OH, with [Ru(PPh3)3Cl2] in\n complexes have also been reported in our very recent works,[17] ethanol under normal atmospheric conditions, afforded [RuII-\n which overall further stimulates us to design and investigate (L1 3 H)(PPh3)2Cl] (1\u20133), respectively (Scheme 1). Initial charac-\n some fluorescent active PPh3-coordinated Ru(II)-dithiocarbazate terizing tools such as elemental (C, H, and N) analyses, IR, NMR,\n complexes (being non-polypyridyl-ruthenium and arene-ruthe- UV-vis, and HR-ESI-MS identified the formation of the com-\n nium complex) to study their anticancer activity.\n DNA is an important target for the transition metal-based\n anticancer drugs.[7,10c,11c,18] Metallodrugs usually damage the\n DNA or disrupt the DNA repair process in cancer cells by\n preventing cell division and triggering cancer cell\n apoptosis.[7,10e,11l] Literature suggests that ruthenium complexes\n with higher DNA binding affinity seem to enhance the\n anticancer activity; some polypyridyl or arene-based Ru(II)\n complexes were reported to exhibit the anticancer activity in\n parallel with their ability to interact with DNA.[7,10c,e,g,11c] How-\n ever, in addition to DNA interaction, the cytotoxicity of Ru(II)\n compounds may attribute to other factors such as mitochon-\n dria-targeted apoptotic cell death via generation of reactive\n oxygen species (ROS) in tumor cells by targeting different\n mitochondrial enzymes.[10c,19] Few recent studies suggested that\n lipophilic Ru(II) complexes target the mitochondria and pro-\n mote apoptosis by disturbing the mitochondrial membrane Scheme 1. Preparation routes of [RuII(L1 3 H)(PPh3)2Cl] (1\u20133).\n\n\n Chem. Eur. J. 2023, 29, e202202694 (2 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n plexes, and the occurrence was further confirmed by X-ray X-ray Crystallography of 1\u20132\n crystallography.\n All complexes undergo an in situ ruthenium-assisted organic Two structures, [RuII(L1 H)(PPh3)2Cl] (1), [RuII(L2 H)(PPh3)2Cl] (2),\n transformation of the oxime to imine during metalation.[23] The were studied with the help of crystallographic technique. The\n spontaneous oxygen atom transfer processes involved in ORTEP diagrams are depicted in Figure 3, whereas relevant\n dithiocarbazate oxime ligands to their corresponding metal crystallographic parameters are given in Table S1 and selected\n coordinated dithiocarbazate imine species were investigated bond lengths and angles with estimated standard deviations\n with a proposed mechanism (Eq. (1), Scheme 2). During the are presented in Table 1. The X-ray structures can be best\n course of the reaction of Ru(PPh3)3Cl2 and HL1 3 OH, an oxygen described as distorted octahedral with RuIIN2P2SCl geometry for\n atom from the oxime ligand has been transferred to a PPh3, metal centers 1\u20132 (See Figure 3). The ruthenium (II) species are\n dissociated from [Ru(PPh3)3Cl2], and forms an imine ligand coordinated in an N, N, and S tri-dentate mode by the uni-\n coordinated ruthenium(II) complexes [RuII(L1 3 H)(PPh3)2Cl] (1\u2013 negative fashion. Out of the three remaining positions, the -Cl\n 3) along with OPPh3 elimination.[23a,24] Also, to establish this as a atom is present within the molecular plane whereas the other\n metal(Ru)-assisted transformation we carried out similar reac- two are occupied by the PPh3 groups present in axial positions\n tions using free PPh3 and also other metal precursor like to the molecular plane and are mutually trans to each other.\n [Ir(PPh3)3Cl]. However, no evidence of oxime to imine trans- The Ru P bond lengths are lengthened (nearly 2.3621 \u00c5) in the\n formations was observed[23a] (Figure S1).\n\n (1)\n\n\n\n\n Scheme 2. Proposed mechanisms for the formation of [Ru(PPh3)2(L1 3 H)Cl]\n (1\u20133) [R = benzyl for 1; 4-methylbenzyl for 2; and 4-chlorobenzyl for 3].\n\n\n\n\n IR Spectroscopy\n\n The infrared spectra of HL1 3 OH and 1\u20133 are performed and\n the results are added in the experimental section. 1\u20133 exhibit\n some bands at ~ 515, 695, and 745 cm 1 for \u03bd(P C) bonds of\n triphenylphosphine molecule and at ~ 330 cm 1 due to the\n \u03bd(Ru Cl) fragment coordinated to the metal centre.[16,23a]\n Further, a stretching band near ~ 3450 cm 1 may be assigned to\n the \u03bd(N H)imine in all three complexes.[23a,24] By using solution\n FTIR (Figure 2), OPPh3, produced during the synthesis of the\n complexes, was identified (\u03bdP=O observed at 1192 cm 1)[24] in\n the filtrate residue of 1 taking as representative.\n\n\n\n\n Figure 3. Molecular structures and selected atom numbering schemes of 1\n (top) and 2 (bottom). The ellipsoids represent a probability of 30 %, and H\n atoms bonded to N1 are drawn with arbitrary radii. All other Hydrogen\n Figure 2. IR spectra of A) Pure crystals; B) Filtrate residue of 1. atoms are omitted for clarity.\n\n\n Chem. Eur. J. 2023, 29, e202202694 (3 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n Table 1. Selected bond distances and angles for the ruthenium coordina- (MLCT).[16,20] Representative UV-vis spectra of 1\u20133 are shown in\n tion of 1\u20132. Figure S2.\n Bond lengths [\u00c5] The stabilities of 1\u20133 were examined through two inde-\n 1 2 pendent experiments under physiological conditions (H2O, and\n Ru1 N2 l.969(3) 1.9722(19) DMEM solutions) at 298 K with the help of UV-vis spectroscopy.\n Ru1 N1 2.079(3) 2.074(2) As shown in Figure S3, the absorbance of complexes was tested\n Ru1 P1 2.3638(8) 2.3651(6) with an increase in the percentage of H2O (up to 99 % H2O/1 %\n Ru1 P2 2.3730(8) 2.3754(6)\n Ru1 S1 2.3953(8) 2.3904(6) DMSO v/v) by keeping the concentration of 1\u20133 at 1 \u00d7 10 4 M.\n Ru1 Cl3 2.4773(8) 2.4743(6) From the results, complexes were found stable, and also no\n Bond angles (\u00b0) additional transitions were found in the absorbance spectra\n even in presence of excess H2O. The time-dependent absorb-\n N2 Rul Nl 77.01(11) 76.91(8)\n N2 Rul Pl 92.34(8) 91.80(6)\n ance spectra (Figure S4) of each complex were also performed\n Nl Rul Pl 89.86(8) 90.99(6) in 2 : 1 DMEM : DMSO (v/v) at different time intervals (0, 12, 24,\n N2 Rul P2 93.21(8) 92.70(6) and 48 h). The overall results indicated that there are no\n Nl Rul P2 90.59(8) 90.78(6)\n Pl Rul P2 174.39(3) 175.43(2)\n noticeable changes in the absorption spectra of 1\u20133 in presence\n N2 Rul Sl 81.99(8) 82.19(6) of aqueous/biological media in the mentioned time period.\n Nl Rul Sl 158.99(8) 159.09(6)\n Pl Rul Sl 90.48(3) 89.79(2)\n P2 Rul Sl 91.10(3) 90.05(2)\n N2 Rul Cl1 176.54(8) 175.77(6) NMR Spectroscopy\n Nl Rul Cl1 99.53(8) 98.89(6)\n Pl Rul Cl1 87.65(3) 87.76(2) 1\n P2 Rul Cl1 86.77(3) 87.80(2)\n H NMR spectra of HL1 3 OH have been recorded in DMSO-d6\n Sl Rul Cl1 101.47(3) 102.02(2) whereas 1\u20133 in CDCl3 and are depicted in Figures S5\u2013S10 in\n Supporting Information. The 1H and 13C{H} NMR spectral data\n (added in the experimental section) of all the compounds are in\n accordance with their corresponding compositions. The imine\n axial positions due to the bulkier triphenylphosphine group N H proton is observed at 8.41 ppm as a singlet peak\n whereas Ru Cl is found to be ~ 2.47 \u00c5. The observed Ru1 S1, suggesting the imine form of the ruthenium complexes.[24] Also,\n Ru1 N1, Ru1 N2, Ru1 Cl1, Ru1 P1, and Ru1 P1 bond lengths the 31P NMR data of 1\u20133 exhibits a single peak at ~ 29 ppm can\n are comparable with complexes reported in the literature.[23b] be assigned to the presence of the PPh3 group in the\n The ligand molecule with the metal center possesses two five- complex.[16] The detection of OPPh3 isolated from the filtrate\n membered rings i. e. with RuN2C2 ring through an angle residue (1) was identified by solution 31P NMR (\u03b4 29.4 ppm)\n \u2220N2 Ru1 N1 [77.01(11)\u00b0, 1; and 76.91(8)\u00b0, 2] and RuSN2C rings (Figure 4a).[24]\n through an angle \u2220N2-Ru1-S1 [81.99(8)\u00b0, 1; and 82.19(6)\u00b0, 2].\n Considering the conversion of oxime to imine groups, it can be\n confirmed by bond parameters obtained by X-ray analysis for HR-ESI-MS\n N1 H1 bond distances 0.85 \u00c5 for 1, and 0.87 \u00c5 for 2 comparable\n to normal N H bond distances reported earlier.[23\u201325] It is Masses of 1\u20133 have been performed in aqueous mixture\n noteworthy that the Ru1 N2 bonds (1.969 \u00c5, 1; 1.972 \u00c5, 2) are solvents (H2O, DMSO, and CH3CN) in positive ion mode. The HR-\n shorter than the Ru1 N1 bonds (2.079 \u00c5, 1; 2.074 \u00c5, 2), ESI-MS shows the molecular ion peaks [M] + at m/z = 925.1205\n indicating weak donor ability of the imine nitrogen group.[23b,25] for 1, m/z = 939.0727 for 2, and m/z = 959.0744 for 3. In addition\n As result, C2 N2 [1.321(4) \u00c5, 1; and 1.316(3) \u00c5, 2] were observed to the molecular ion peaks, some common solvent coordinated\n to be longer than that of the C1 N1 [1.302(4) \u00c5, 1; and masses have also been observed for all three complexes.\n 1.298(3) \u00c5, 2] bonds. Overall, the terminal M L bonds [Ru1 N1, Overall, ruthenium metal remained intact with the ligands even\n and Ru1 S1] were found to be longer than that of the central in the presence of water which indicates that 1\u20133 is stable in\n donor bond [Ru1 N2] of the tridentate ligand which is in the aqueous medium. A representative mass spectrum of 1 is\n agreement with earlier reports.[26] shown in Figure 5, along with the detailed analysis of 1\u20133 has\n been added in Supporting Information (Figure S11\u2013S13, Ta-\n ble S2\u2013S4). The detection of OPPh3 isolated from the filtrate\n Stability Studies in Solution/Aqueous Media residue (1) was also identified by HR-ESI-MS (m/z 279.1210;\n OPPh3 + H +) (Figure 4b).[24]\n UV-vis Spectroscopy\n\n The electronic spectra of 1\u20133 were recorded in the DMSO Partition Coefficients Measurements (log Pow)\n solution. The strong transitions in the range 350\u2013360 nm of 1\u20133\n are assigned to intra-ligand charge transfer transitions while the Partition coefficients, log Pow, were measured to establish a\n weaker transitions in the range 460\u2013480 nm of the spectra are possible relationship between the hydrophobicity/lipophilicity\n probably due to metal to ligand charge transfer transition and biological activity of the compounds.[21d,27] It has been\n\n Chem. Eur. J. 2023, 29, e202202694 (4 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n hydrophobic compound of the series. The positive log Pow\n values of 1\u20133 suggest that all complexes are hydrophobic,\n perhaps because of triphenylphosphine groups present in the\n complex environment.[3a] Further, the high hydrophobicity of\n complex 3 (log Pow = 1.63) may be due to the additional\n electron-withdrawing group present at the para position of the\n ligand moiety.[29]\n\n\n\n DNA-Binding Assays\n\n UV-vis spectroscopy titration is a frequently used technique for\n investigating the probable binding modes of complexes to CT-\n DNA and determining their binding constants (Kb).[30] So, here\n the binding of 1\u20133 with CT-DNA was performed through UV-vis\n spectroscopy. The extent of hypochromism/hyperchromism\n depends upon the binding affinity and mode of the complexes\n toward DNA. The DNA sample was sequentially added in\n aliquots (5 \u03bcM), and the absorbance were measured after each\n addition. The variations in the absorbance of 1\u20133 are presented\n in Figures 6 and S14. On gradual addition of CT-DNA,\n substantial decreases in the absorbance (hypochromic shift) at\n 370\u2013380 nm are observed.\n The calculated Kb ranged from 4.43 \u00d7 104 to 1.16 \u00d7 105 M 1,\n increased order as follows 1 < 2 < 3, suggesting that 3 has\n maximum interaction towards CT-DNA. Also, the binding\n Figure 4. a) Detection of OPPh3 in the filtrate of 1 by 31P NMR; b) Detection activity of HL1 3 OH was assayed and among the three ligands,\n of OPPh3 in the filtrate of 1 by HR-ESI-MS. HL3 OH showed the maximum binding constant at 2.33 \u00d7\n 104 M 1. However, as discussed above the actual ligands have\n been transformed into their corresponding imine species during\n the metalation reaction. Therefore, the results suggest that the\n\n\n Table 2. Partition coefficients (log Pow) of 1\u20133.\n Complex Partition coefficient (log Pow)\n\n 1 1.45\n 2 1.47\n 3 1.63\n\n\n\n\n Figure 5. HR-ESI-MS spectrum of [Ru(L1 H)(PPh3)2Cl] (1) recorded in\n H2O : CH3CN (10 : 90 v/v) in positive ion mode with (a) simulated and (b)\n observed isotopic distributions.\n\n\n\n\n reported that hydrophobic cations have a higher affinity to\n accumulate in mitochondria due to the negative potential\n difference across the mitochondrial membrane.[20,28] Generally,\n the values of log Pow range between 3 (very hydrophilic) to Figure 6. Absorption spectra of 1 in 50 mM Tris HCl buffer (pH = 7.4), 298 K\n in the presence of increasing amounts of CT-DNA. [Ru] = 10 \u03bcM, [CT-\n + 10 (extremely hydrophobic).[21d] In this work, the observed log DNA] = 0-50 \u03bcM from top to bottom. Arrows indicate the change in\n Pow values (Table 2) are found 1.45 for 1; 1.47 for 2; and 1.63 for absorbance upon increasing the DNA concentration. Inset shows a linear fit\n 3 by following the order 1 < 2 < 3; with 3 being the most plot of [DNA]/(\u025ba\u2013\u025bf) vs. [DNA].\n\n\n Chem. Eur. J. 2023, 29, e202202694 (5 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n better binding of 3 may be due to the presence of a chlorine Circular Dichroism (CD) Analysis with CT-DNA\n atom at the para position which may slightly shift the dipole of\n the molecule at the binding site, for enhanced interaction with CD is a powerful tool used to diagnose the conformational\n CT-DNA.[22b,31] Another probable reason is that the presence of changes in DNA helix in order to elucidate the groove and\n chlorine substituents in complexes/ligands may change their intercalation binding of the complexes.[21k,32] During groove or\n ability to strongly bind to CT-DNA by increasing their hydro- electrostatic interaction with CT-DNA, the intrinsic CD spectra\n phobic properties.[22b,29,31] In agreement with the preliminary are less or not perturbed, whereas during intercalation there is\n studies, the extent of intercalation was further investigated an increase in the intensity of both negative and positive\n using EB displacement studies. bands.[21k,32] It was observed that (Figure 8) both negative\n (helicity) and positive (ellipticity) bands increased significantly\n after complexes 1\u20133 were treated with CT-DNA, further\n Competitive DNA Binding Assays suggesting intercalation binding.\n\n Ethidium bromide (EB) is an effective fluorescent tool that binds\n to DNA through intercalation mode. EB (black spectral line in Cytotoxicity\n Figure 7a) upon binding with CT-DNA gets fluorescent active\n (can be seen as a red spectral line in Figure 7a). As shown in The in vitro cytotoxicity of HL1 3 OH, and [RuII(L1 3 H)(PPh3)2Cl]\n Figures 7 and S15, with the gradual addition of complexes to (1\u20133) was evaluated using standard MTT assays against two\n luminescent EB-DNA adduct the emission intensity was cancer cell line, i. e., human cervical cancer (HeLa), and human\n quenched significantly leading to the displacement of bound colon cancer (HT-29). The IC50 values of 1\u20133 are found in the\n EB from the adduct. As a result, the Ksv values of 1\u20133 for EB- ranges of 6.9\u201312.1 and 13.9\u201332.8 \u03bcM against HeLa and HT-\n bound CT-DNA complexes are found in the range 1.49 \u00d7 104 to 29 cells, respectively (Table 3, Figure 9, and Figure S16\u2013S17).\n 1.79 \u00d7 104 M 1 whereas the binding constant (Kb) in the range The cytotoxicity increased with compound order 1 < 2 < 3 and\n 8.29 \u00d7 104 to 1.31 \u00d7 105 M 1, with order 1 < 2 < 3. A higher Kb compound 3 was the most toxic with IC50 values of 6.9 \ufffd 0.2 \u03bcM\n value of 1.31 \u00d7 105 M 1 for complex 3 indicates better intercala- against HeLa, and 13.9 \ufffd 0.4 \u03bcM against HT-29 cell lines. An\n tion with CT-DNA. analogous trend is observed in the cell-specific selectivity of 1\u2013\n 3 against both cell lines. The results revealed that 1\u20133 exerts\n\n\n\n\n Figure 8. CD spectra of CT-DNA (150 \u03bcM) in the presence and absence of 1\u2013\n 3 in 50 mM Tris HCl buffer (pH 7.4). The path length of the cuvette was\n 5 mm.\n\n\n\n\n Table 3. Effect of complexes (1-3) against HeLa, HT-29, and NIH-3T3 cells.\n IC50 values were perceived by the MTT assay, post 48 h incubation.\n Complex IC50 [\u03bcM]\n HeLa HT-29 NIH-3T3\n\n 1 12.1 \ufffd 0.1 32.8 \ufffd 0.4 46.1 \ufffd 1.4\n 2 7.8 \ufffd 0.1 24.5 \ufffd 0.1 23.4 \ufffd 0.5\n 3 6.9 \ufffd 0.2 13.9 \ufffd 0.4 36.7 \ufffd 0.1\n Figure 7. (a) Fluorescence titration of the EB bound CT-DNA by increasing HL1 OH > 50 31.4 \ufffd 1.6 > 50\n concentrations of 1 (5\u201350 \u03bcM) (\u03bbex 520 nm; \u03bbem 530\u2013800 nm) with insets HL2 OH 31.8 \ufffd 2.7 28.5 \ufffd 0.1 35.8 \ufffd 0.9\n showing the Stern\u2013Volmer plot for the quenching of fluorescence intensity HL3 OH 25.7 \ufffd 4.6 11.8 \ufffd 0.3 > 50\n on the addition of the ruthenium complex. (b) Scatchard plot of complex 1. Cisplatin 25.5 \ufffd 0.8 32.7 \ufffd 0.6 \u2013\n\n\n Chem. Eur. J. 2023, 29, e202202694 (6 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n\n\n Figure 9. In vitro cytotoxicity profile of the tested complexes (1-3) against\n HeLa cell line after 48 h incubation. Data are reported as the mean \ufffd SD for Figure 10. Correlation between cytotoxicity (HeLa and HT-29) and partition\n n = 4 and ***p < 0.0001. coefficient (log Pow).\n\n\n\n effective toxicity in inhibiting the growth of cancer cells and with those of other ruthenium-dithiocarbazate species [Ru(H-\n were highly toxic even at lower concentrations. At their highest Nap-sbdtc)(PPh3)2(CO)Cl] and [Ru(H-Nap-sbdtc)(AsPh3)2(CO) Cl]\n dose (50 \u03bcM), almost 80 % cell inhibition was seen (Figure 9 and (IC50 between 22.9 to 26.4 \u03bcM against HeLa cells);[12i] ruthenium-\n S16\u2013S17) after 48 h of exposure. arene species [(\u03b76-p-cym)Ru(NN)Cl]PF6 (IC50 > 100 \u03bcM against\n Again, to determine the degree of selectivity on cancerous HeLa cells);[35] and Keppler-type ruthenium-trifluoromethyl spe-\n cells, the cytotoxicity of investigated molecules was also cies Na[RuCl4(CF3Hin)2] and Na[RuCl4(CF3Him)2] (IC50 > 24-100 \u03bcM\n evaluated against noncancerous cells NIH-3T3. According to the against HT-29 cells).[36]\n results (Table 3), complexes were less toxic to normal cells, NIH- According to the results of all the investigated compounds,\n 3T3 with IC50 values in the range of 36.7 to 46.1 \u03bcM. In the complex 3 exhibited the maximum anticancer activity against\n present study, 3 with a selectivity index (SI) of 5.3 folds was HeLa cells. Cancer cell proliferation may be inhibited by 3\n chosen as the most promising anticancer compound for HeLa through induction of either apoptosis or cell cycle arrest, or by\n cells. a combination of both. In order to examine the mode of cell\n As mentioned earlier, dithiocarbazate-based ligands also death induced by these complexes, flow cytometric analysis\n exhibited significant cytotoxicity.[12g\u2013h,14] In fact, HL3 OH with with propidium iodide (PI) and Annexin V-FITC/PI was\n IC50 11.8 \ufffd 0.3 \u03bcM showed better toxicity (Table 3) than metal performed taking 3 as representatives.\n complexes (1\u20133) against HT-29 cells; to be precise, the better\n cytotoxicity of HL3 OH and 3 may be due to the effect of the\n chlorine group present at the para position of ligand Cell Cycle Analysis\n molecule.[22b\u2013d] Further, from ESI-MS results some solvent\n coordinated ruthenium species are also observed (see HR-ESI-MS Cell cycle arrest determines the effectiveness of an anticancer\n section), so the overall cytotoxicity perceived from individual drug in stopping cancerous cell division.[35,37] The percentage of\n compounds may be originated from all the possible mixture accumulation of HeLa cells after treatment at different concen-\n species generated in the incubation media.[17a,21a\u2013b] trations (0.5 \u00d7 IC50, 1.0 \u00d7 IC50, 2.0 \u00d7 IC50) of 3 (the concentrations\n In addition, lipophilicity is a vital property of compounds were taken after performing the MTT assay against HeLa cells\n that is directly related to anticancer activity as it allows a for 24 h (Figure S18)) for 24 h in Sub-G0/G1, G0/G1, S, and G2/M\n compound to diffuse through the biological lipid layer leading has been shown in Figure 11. From the results, the number of\n to better availability of compounds in its target, and also useful cells in the Sub G0/G1 phase is increased with an increase in\n to the maximum cellular uptake of complexes leading to better doses of 3 from 4.7 % (untreated cells) to 9.0, 10.6, and 15.1 %,\n cytotoxicity activity.[21d,27,29,33] Based on the results of log Pow respectively; along with in the S phase there is a straight\n (Table 2), and IC50 values (Table 3), complexes with high hydro- increase from 14.3 % (untreated cells) to 19.1, 19.4, and 25.2 %,\n phobic behavior induced maximum effect on anticancer respectively. However, the G0/G1 phase is decreased from\n activity. As presented in Figure 10, with the increase in the 57.9 % (untreated cells) to 40.1 % (at the highest concentration\n hydrophobicity of 1\u20133, an increase in cytotoxicity activities (IC50) i. e. 2 \u00d7 IC50), whereas in G2/M no significant change is observed.\n was observed. Overall, it can be concluded that, the compound These results indicated that complex 3 mainly arrested the S\n (1-3) with higher hydrophobicity and stronger DNA interaction phase in a concentration dependent manner. Thus, the overall\n (as discussed in the DNA binding section above) has shown increase of the DNA content in Sub G0/G1 (sub-diploid) and S\n better anticancer activity.[18,20,21d,34] phase cells might result in apoptosis by disrupting the cell\n Nonetheless, under similar conditions, 1\u20133 show compara- cycle.[35,38] Overall, the treatment of HeLa cells with 3 for 24 h\n ble anticancer activity with respect to the existing chemo- resulted in a noticeable increase in the proportion of apoptotic\n therapeutic drug cisplatin (25.5 \ufffd 0.8 and 32.7 \ufffd 0.6 \u03bcM against cells in dose-dependent manner, as shown in the subdiploid\n HeLa and HT-29, respectively See Table 3). Moreover, the above region. Induction of apoptosis was further confirmed by\n in vitro cytotoxicity results of 1\u20133 are better than or comparable Annexin V-FITC/PI apoptosis assay.\n\n Chem. Eur. J. 2023, 29, e202202694 (7 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n\n\n Figure 11. (a) Cell cycle analysis of HeLa cancer cells post treatment of 3 for\n 24 h. Cell staining for flow cytometry was performed using PI/RNase. (b)\n Histogram of the cell cycle distribution.\n\n\n\n\n Double-Staining Apoptosis Assay\n\n An Annexin V-FITC/propidium iodide (PI) measurement is\n required to determine whether the complex induces cell death\n through apoptosis or necrosis. So, the HeLa cells were stained Figure 12. a) Induced apoptosis in HeLa cells upon treatment of three\n with Annexin V-FITC/PI reagents, and apoptosis was analyzed different concentrations of 3 for 24 h measured by flow cytometer using\n Annexin V-FITC/propidium iodide (PI) staining method. Lower left, viable\n using a flow cytometer. The different stages of cells, i. e., live cells; lower right, early apoptotic cells; upper right, late apoptotic cells; upper\n cells, early apoptotic cells, late apoptotic cells, and necrotic left, necrotic cells. (b) Histogram showing populations for HeLa cells in three\n were calculated using the fluorescence-activated cell sorting stages treated by complex 3. (c) Effect of NAC (5 mM) on induced apoptosis\n by complex 3.\n (FACS) methodology. So, at three different concentrations (0.5 \u00d7\n IC50, 1.0 \u00d7 IC50, and 2.0 \u00d7 IC50,) of complex 3, the cell population\n in the lower right quadrant represents cells undergoing early\n apoptosis, which increased from 4.5 % (Control) to 27.8, 36.1 Cellular Localization Assay\n and 41.3 % with an increase in concentrations of 3, respectively\n (Figure 12). However, no significant cell population was ob- In the process of apoptosis, cancer cells get stimuli from\n served in the late apoptotic and necrotic regions. Altogether, different intracellular organelles. Thus, apoptosis induced by\n the flow cytometry result concluded that the cell death induced Ru(II) complexes (1-3) requires a deeper understanding of their\n by 3 is mainly caused by apoptosis. (Figure 12) Further, the cellular targets and mechanisms.[18,35,40] Being fluorescence\n effect of ROS on apoptosis induced by 3, was investigated in active (Figure S19), the subcellular localization of 1\u20133 was\n presence of ROS scavenger N-acetylcysteine (NAC) before being explored along with the commercially available staining probes\n treated with the complex.[39] As shown in Figure 12c, about a such as Hoechst 33342 for the nucleus and MitoTracker Deep\n 26 % decrease in the apoptotic cells indicates that ROS plays a Red (MTDR) for mitochondria using live cell confocal micro-\n crucial role in cell apoptosis. scopy colocalization studies. Further, the colocalization effect of\n complexes was quantified using Pearson\u2019s correlation coeffi-\n cient (PCC) for both nucleus and mitochondria staining probes.\n So, initially, the confocal images of the compounds along with\n Hoechst 33342 were taken against HeLa and HT-29 cells. The\n\n Chem. Eur. J. 2023, 29, e202202694 (8 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n obtained PCC values of the green fluorescent signals of 1\u20133\n with blue fluorescent signals of Hoechst 33342 are very poor;\n that is 0.10, 1; 0.25, 2; and 0.18, 3 against Hela cells whereas\n 0.26, 1; 0.23, 2; and 0.28, 3 against HT-29 cells. This data,\n therefore, specifies that the nucleus is not a preferential target\n of the biological action of 1\u20133 tested in this present work. The\n representative confocal images of 3 along with Hoechst 33342\n against HeLa and HT-29 are given in Figure 13 whereas for 1,\n and 2 in Figure S20 of Supporting Information.\n In order to assess the actual target of 1\u20133, another\n colocalization experiment was performed with standard com-\n Figure 13. Confocal microscopy images of live HeLa and HT-29 cells treated\n mercially available MTDR dye. From the results, it is observed\n with 3 (10 \u03bcM for 1 h incubation) followed by counter staining with Hoechst\n that the green fluorescence of 1\u20133 overlapped with the red (0.5 \u03bcM for 5 min, stain nucleus). Hoechst, \u03bbex = 405 nm, \u03bbem = 420\u2013470 nm;\n fluorescence signals of MTDR, yielding the Pearson\u2019s correlation complex, \u03bbex = 488 nm, \u03bbem = 500\u2013550 nm. Inset scale bars: 20 \u03bcm.\n coefficients of 0.71, 0.73, and 0.75, respectively, on live HeLa\n cells. All the complexes exhibit characteristic colocalization\n (more than 70 %) with MTDR allowing complexes to specifically\n target mitochondria. Also, the colocalization results were\n acquired for HT-29 cells, yielding Pearson\u2019s correlation coeffi-\n cients of 0.78, 0.80, and 0.89 for 1, 2, and 3 respectively. From\n the colocalization results in HeLa and HT-29 cells, it can be\n stated that 1\u20133 are predominantly localized in mitochondria\n irrespective of the change of cancer cells. Among the series, 3\n exhibited the maximum colocalization coefficient which also\n exhibited higher hydrophobicity and fluorescence activity as\n discussed earlier. The representative confocal images of com-\n plex 3 along with MTDR against HeLa and HT-29 are given in\n Figure 14 whereas for 1, and 2 in Figure S21 of Supporting\n Information. However, it is well known in the literature that,\n Figure 14. Confocal microscopy images of live HeLa and HT-29 cells treated\n ruthenium(II) complexes preferentially target mitochondria, and with 3 (10 \u03bcM for 1 h incubation) followed by counter staining with MTDR\n also showed the potential to influence mitochondrial (0.5 \u03bcM for 15 min, stain mitochondria). MTDR, \u03bbex = 635 nm, \u03bbem = 650\u2013\n metabolism.[10f,15b,35] This will make it fascinating to study the 740 nm; complex, \u03bbex = 488 nm, \u03bbem = 500\u2013550 nm. Inset scale bars: 20 \u03bcm.\n mechanism of mitochondrial function when these complexes\n are present.\n\n\n\n\n Figure 15. (a) Time dependent fluorescence kinetic measurement of ROS (O2 level) generation upon treatment with 3 and DHE against HeLa cells for 24 h.\n *\n\n\n\n\n (b) The histogram shows the level of ROS upon treatment with 3 and DHE against HeLa cells measured by flow cytometer. (c) Live cell confocal images of\n HeLa cells upon treatment with 3 and DHE; Blue channel, \u03bbex = 405 nm, \u03bbem = 420\u2013470 nm; red channel, \u03bbex = 560 nm, \u03bbem = 570\u2013640 nm.\n\n\n Chem. Eur. J. 2023, 29, e202202694 (9 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n Reactive Oxygen Species Generation Studies Experimental Section\n Benzyl chloride, 4-methylbenzyl chloride, 4-chlorobenzyl chloride,\n From colocalization studies, 1\u20133 are found to localize at and 2,3-butanedionemonoxime were procured from Sigma Aldrich\n mitochondria, so the next logical step was to assess mitochon- and used as received. Ruthenium trichloride was obtained from\n drial function or respiratory competence in the presence of Arora Matthey. [Ru(PPh3)3Cl2] was synthesized from RuCl3 and\n these complexes. The accumulation of ROS (O2 level; as *\n triphenylphosphine.[43] The solvents were dried and distilled before\n being used for the reaction. Reagents for biological tests were\n mitochondria is the major contributor)[41] was investigated by\n acquired mostly from Sigma-Aldrich (USA) and HiMedia laborato-\n staining the HeLa cells with different concentrations (0.5 \u00d7 IC50, ries.\n 1.0 \u00d7 IC50, 2.0 \u00d7 IC50) of 3 and dihydroethidium (DHE, 5 \u03bcM) as a\n fluorescent probe. The results of the kinetic fluorescence IR spectra were recorded on a Perkin\u2013Elmer Spectrum RXI\n spectrophotometer. UV-visible absorption spectra were measured\n (Figure 15a) and flow cytometer (Figure 15b) study showed a\n on a Shimadzu spectrophotometer (UV-2450). 1H and 13C{1H}, and\n significant increase in ROS generation upon treatment of 3 31\n P NMR spectra were analyzed on a Bruker Ultrashield spectrom-\n against HeLa cells for 24 h. Confocal images (Figure 15c) eter (400 MHz) in presence of SiMe4 as the internal standard. HR-\n suggest the increase in the red emission upon the increase in ESI-MS data were collected on a Waters XEVO G2-XS QTOF MS\n the concentrations of 3 with respect to control (blue emission). instrument. Fluorescence emission spectra were recorded on a\n Perkin Elmer LS 55 spectrofluorometer.\n Further, the effect of NAC on ROS production was investigated\n by flow cytometer and confocal microscopy assays.[39] As shown Synthesis of Ligands (HL1 3 OH) and Complexes [RuII-\n in Figure S22, in presence of NAC (5 mM) the ROS generation (L1 3 H)(PPh3)2Cl] (1\u20133): The synthesis and spectroscopic details of\n was greatly reduced, suggesting that the increased cell the ligands (HL1 3 OH) are discussed in the Supporting Informa-\n tion.\n fluorescence (in absence of NAC) is certainly due to the\n production of ROS by the 3. Overall, the increase in ROS level [RuII(L1 H)(PPh3)2Cl] (1): An equimolar ratio of [Ru(PPh3)3Cl2]\n may be due to the direct action of the complexes on the (0.1 mmol) was refluxed with HL1 OH (0.1 mmol) in ethanol (30 mL)\n for 4 h. A dark green crystalline precipitate of [RuII(L1 H)(PPh3)2Cl]\n mitochondria, triggering mitochondrial dysfunction to produce\n (1) was separated out from the reaction mixture. At room temper-\n excess ROS, leading to the induction of apoptosis.[7,15b,35,42] ature, the crystals, suitable for X-ray analysis, were collected by\n filtration, washed with ethanol and dried in air. Yield: 0.068 g (73 %).\n Anal. calcd. for C48H44ClN3P2RuS2 (925.48): C, 62.29; H, 4.79; N, 4.54;\n Conclusion found C, 62.16; H, 4.86; N, 4.51. Main IR peaks (KBr, cm 1): 3453\n \u03bd(N H)imine, 742, 696, 515 \u03bd(3P Ph). UV-Vis (DMSO): \u03bbmax, nm (\u025b,\n M 1 cm 1): 478 (1448), 361 (12067). 1H NMR (400 MHz, CDCl3): \u03b4\n In this study, we demonstrate the synthesis and characterization\n (ppm) = 8.40 (s, 1H, N H), 7.74\u20137.21 (m, 35H, aromatic), 3.80 (s, 2H,\n of new Ru(II) dithiocarbazate complexes with one of the most SCH2), 1.87\u20131.29 (s, 6H, 2CH3). 13C{1H} NMR (100 MHz, CDCl3): \u03b4\n detailed investigations on biological activity highlighting their 190.74, 173.51, 152.34, 137.82\u2013126.86 (42 C, aromatic), 39.03, 23.47,\n effective anticancer potential and mechanism of cell death. 14.72. 31P NMR (120 MHz, CDCl3): \u03b4 27.32 (s, PPh3).\n In the present study, a notable instance of metal-mediated [RuII(L2 H)(PPh3)2Cl] (2): This complex was also prepared by\n oxime-imine transformation has been reported. following the same procedure as above. Yield: 0.059 g (63 %). Anal.\n The solution stability, hydrophobicity, DNA interaction, and calcd. for C49H46ClN3P2RuS2 (939.51): C, 62.64; H, 4.94; N, 4.47; Found:\n in vitro cytotoxicity against human cancer cells HeLa, and HT-29 C, 62.55; H, 5.00; N, 4.39. Main IR Peaks (KBr pellet, cm 1): 3437\n were performed. All complexes showed impressive cytotoxicity \u03bd(N H)imine, 750, 697, 516 \u03bd(3P Ph). UV-Vis (DMSO): \u03bbmax, nm (\u025b,\n M 1 cm 1): 471 (3650), 355 (22491). 1H NMR (400 MHz, CDCl3): \u03b4\n against both the cancer cell lines, while 3 was found to have\n (ppm) = 8.40 (s, 1H, N H), 7.75\u20137.04 (m, 34H, aromatic), 3.76 (s, 2H,\n maximum IC50 values of 6.9 \ufffd 0.2 \u03bcM against HeLa cells, SCH2), 2.33 (s, 3H, Ar CH3), 1.87\u20131.30 (s, 6H, 2CH3). 13C{1H} NMR\n relatively better than the clinically used anticancer drugs (100 MHz, CDCl3): \u03b4 190.86, 173.46, 152.26, 136.48\u2013127.58 (42 C,\n cisplatin, and also some selectivity for cancer cells over healthy aromatic), 38.85, 23.47, 21.13, 14.71. 31P NMR (120 MHz, CDCl3): \u03b4\n cells (NIH-3T3). Here, the different R-substituents present at the 27.58 (s, PPh3).\n para-position of the aromatic ring in ligand moiety play an [RuII(L3 H)(PPh3)2Cl] (3): This complex was also prepared by\n important role in cell cytotoxicity. following the same procedure as above. Yield: 0.056 g (59 %). Anal.\n Complex 3 with high IC50 values was chosen for detailed calcd. for C48H43Cl2N3P2RuS2 (959.93): C, 60.06; H, 4.52; N, 4.38;\n analysis of cell death mechanism (apoptosis) and intracellular Found: C, 60.13; H, 4.44; N, 4.39. Main IR Peaks (KBr pellet, cm 1):\n 3432 \u03bd(N H)imine, 746, 699, 511 \u03bd(3P Ph). UV-Vis (DMSO): \u03bbmax, nm\n targets by flow cytometry and live cell imaging confocal\n (\u025b, M 1 cm 1): 469 (3455), 357 (22910). 1H NMR (400 MHz, CDCl3): \u03b4\n microscopy. (ppm) = 8.41 (s, 1H, N H), 8.12\u20137.05 (m, 34H, aromatic), 3.79 (s, 2H,\n Overproduction of ROS (O2 level) was observed with\n *\n SCH2), 1.88\u20131.24 (s, 6H, 2CH3). 13C{1H} NMR (100 MHz, CDCl3): \u03b4\n exposure of 3 which resulting the induction of apoptosis 190.57, 173.59, 165.46, 136.74\u2013127.59 (42 C, aromatic), 37.96, 29.71,\n against HeLa cells. Altogether, results suggest that 1\u20133 are 14.69. 31P NMR (120 MHz, CDCl3): \u03b4 27.50 (s, PPh3).\n potentially active to induce a mitochondrial-targeted apoptotic Single crystal X-ray Structure Determination and Biological\n mode of cell death in human cancer cells. The positive Assays: The details of all the experimental procedures were added\n outcomes from this study provide great encouragement to in the Supporting Information. Deposition Number(s) 2192720 (for\n pursue further studies toward utilizing these ruthenium- 1), 2192721 (for 2) contain the supplementary crystallographic data\n for this paper. These data are provided free of charge by the joint\n aroyldithiocarbazoneimine complexes as anticancer agents in\n the near future.\n\n Chem. Eur. J. 2023, 29, e202202694 (10 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f 15213765, 2023, 18, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202202694 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202202694\n\n\n Cambridge Crystallographic Data Centre and Fachinformationszen- [10] a) A. Notaro, M. Jakubaszek, S. Koch, R. Rubbiani, O. D\u00f6m\u00f6t\u00f6r, \u00c9. A.\n trum Karlsruhe Access Structures service. Enyedy, M. Dotou, F. Bedioui, M. Tharaud, B. Goud, S. Ferrari, E. Alessio,\n G. Gasser, Chem. 2020, 26, 4997\u20135009; b) A.-C. Munteanu, A. Notaro, M.\n Jakubaszek, J. Cowell, M. Tharaud, B. Goud, V. Uivarosi, G. Gasser, Inorg.\n Chem. 2020, 59, 4424\u20134434; c) A. Notaro, A. Frei, R. Rubbiani, M.\n Acknowledgements Jakubaszek, U. Basu, S. Koch, C. Mari, M. Dotou, O. Blacque, J. Gouyon, F.\n Bedioui, N. Rotthowe, R. F. Winter, B. Goud, S. Ferrari, M. Tharaud, M.\n \u0158ez\u00e1\u010dov\u00e1, J. Humajov\u00e1, P. Tom\u0161\u00edk, G. Gasser, J. Med. Chem. 2020, 63,\n Rupam Dinda thanks CSIR, Govt. of India [Grant No. 01(3073)/ 5568\u20135584; d) N. Soliman, G. Gasser, C. M. Thomas, Adv. Mater. 2020, 32,\n 21/EMR-II] for funding and also the fellowship for SD. Open e2003294; e) H.-L. Huang, Z.-Z. Li, Z.-H. Liang, J.-H. Yao, Y.-J. Liu, Eur. J.\n Med. Chem. 2011, 46, 3282\u20133290; f) C.-C. Zeng, S.-H. Lai, J.-H. Yao, C.\n Access funding enabled and organized by Projekt DEAL. Zhang, H. Yin, W. Li, B.-J. Han, Y.-J. Liu, Eur. J. Med. Chem. 2016, 122,\n 118\u2013126; g) H. Huang, P. Zhang, B. Yu, Y. Chen, J. Wang, L. Ji, H. Chao, J.\n Med. Chem. 2014, 57, 8971\u20138983; h) N. Alatrash, E. S. 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Hallman, T. A. Stephenson, G. Wilkinson, Inorg. Synth. 1970, 12,\n Saswati, M. Mohanty, A. Banerjee, S. Biswal, A. Horn, G. Schenk, K. 237\u2013240.\n Brzezinski, E. Sinn, H. Reuter, R. Dinda, J. Inorg. Biochem. 2020, 203,\n 110908, 10.1016/j.jinorgbio.2019.110908; n) S. A. Patra, M. Mohanty, A.\n Banerjee, S. Kesarwani, F. Henkel, H. Reuter, R. Dinda, J. Inorg. Biochem.\n 2021, 224, 111582; o) S. Roy, M. B\u00f6hme, S. Lima, M. Mohanty, A.\n Banerjee, A. Buchholz, W. Plass, S. Rathnam, I. Banerjee, W. Kaminsky, R. Manuscript received: August 29, 2022\n Dinda, Eur. J. Inorg. Chem. 2022, 2022; p) S. Roy, M. Mohanty, S. Pasayat, Accepted manuscript online: January 4, 2023\n S. Majumder, K. Senthilguru, I. Banerjee, M. Reichelt, H. Reuter, E. Sinn, Version of record online: February 24, 2023\n\n\n\n\n Chem. Eur. J. 2023, 29, e202202694 (12 of 12) \u00a9 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH\n\f", "pages_extracted": 12, "text_length": 87865}