Photostimulated Anticancer Activity of Mitochondria Localized Rhenium(I) Tricarbonyl Complexes Bearing 1H-imidazo[4,5-f][1,10]phenanthroline Ligands Against MDA-MB-231 Cancer Cells.

{"full_text": " Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202401720\n\n www.chemeurj.org\n\n\n Photostimulated Anticancer Activity of Mitochondria\n Localized Rhenium(I) Tricarbonyl Complexes Bearing 1H-\n imidazo[4,5-f][1,10]phenanthroline Ligands Against MDA-\n MB-231 Cancer Cells\n Binoy Kar[a] and Priyankar Paira*[a]\n\n We have introduced Re(I) tricarbonyl complexes (ReL1 - ReL6) (IC50~6 \u03bcM, PI > 9) under yellow light irradiation compared to\n [Re(CO)3(N^N)Cl] where N^N = extensive \u03c0 conjugated imidazo- dark conditions. Importantly, extremely lipophilic complex ReL6\n [4,5-f][1,10]-phenanthroline derivatives that helps in strong DNA showed effective penetration through the cell membrane and\n intercalation, enhanced photophysical behavior, increase the 3\u03c0- localized primarily in mitochondria (Pearson\u2019s correlation coef-\n \u03c0* character of T1 state for PDT and high value of lipophilicity ficient, PCC = 0.918) of MDA-MB-231 cells. Complex ReL6\n for cell membrane penetration. These complexes exhibited exhibited more than 9 times higher photo-toxicity in normoxic\n prominent intraligand/ligand-centered (\u03c0-\u03c0*/1LC) absorption and hypoxic environment of tumor by inducing 1O2 generation\n bands at \u03bb 260\u2013350 nm and relatively weak metal-to-ligand (type II PDT), radical generation triggered by NADH oxidation\n charge-transfer (1MLCT) bands within the \u03bb 350\u2013550 nm range. (type I PDT). This complex is a promising candidate for TNBC\n Among the six synthesized complexes, [(CO)3ReICl(K2-N,N-2-(4- treatment in hypoxic tumors, with efficacy comparable to\n (1-benzyl-1H-tetrazol-5-yl)phenyl)-1H-imidazo[4,5- photofrin and have demonstrated CO release ability under UV\n f][1,10]phenanthroline] (ReL6) exhibited outstanding potency light irradiation.\n\n\n 1. Introduction natives to traditional agents like cisplatin and its congeners due\n to several potential applications and properties such as\n Since cancer is reckoned as the second main cause of death significant stokes shifts, prolonged emission states, tunable\n worldwide after cardiovascular disease (CVD), there is relentless emission through ligand modifications, high quantum yields,\n research being done to find novel anticancer medications. The ability to bind to proteins and DNA.[7\u201310] Initially, the diverse cell\n platinum-based complexes cisplatin, lobaplatin, oxaliplatin, and death mechanisms triggered by rhenium complexes, including\n carboplatin are among the frontline anticancer drugs. Although paraptosis, apoptosis, and necroptosis, offer a potential solution\n they exhibited significant results in cellular apoptosis, they have to certain constraints associated with current medications.[11,12]\n serious adverse effects and have limited effectiveness against Secondly, they find utility in photoactivatable therapy and\n tumors that are resistant to platinum drugs. All-encompassing photodynamic therapy (PDT) by employing CO-releasing mech-\n research has been conducted on organometallic and inorganic anisms that can be activated through appropriate irradiation in\n compounds as reasonable choices to pharmaceuticals derived tumor regions.[13,14] Additionally, Re tricarbonyl complexes play a\n from organic substances.[1,2] In this context, the promise of Ru(II/ role in bioimaging using fluorescence or vibration, aiding in\n III) and Ir(III) complexes emerges from their reduced side effects visualizing cellular distribution and understanding the mecha-\n and favorable characteristics. These include targeted actions on nism of action.[15]\n DNA or proteins, accumulation within various cellular organ- Historically, cancer treatment has predominantly relied on\n elles, amplified oxidative stress, photoactivation, improved conventional methods such as surgery, radiotherapy, and\n cytotoxicity specifically against cancer cells, and disruption of chemotherapy.[16] While contemporary research has introduced\n the cellular redox balance.[3-5] Notably, several ruthenium innovative therapeutic approaches like hormone-based therapy,\n complexes, such as NAMI-A, KP1019, KP1339, and TLD1433 stem cell therapy, and immunotherapy, photodynamic therapy\n have advanced into clinical trials.[6] In addition, in the realm of (PDT) has emerged as a notable and well-established comple-\n anticancer drug research, there has been a notable surge of mentary or alternative approach in cancer treatment.[17,18] The\n interest in organometallic tricarbonyl rhenium(I) complexes and development of noninvasive PDT aimed to mitigate the well-\n these compounds are being considered as promising alter- documented side effects associated with often non-selective\n chemotherapy. PDT involves the synergetic action of three\n components i. e. the utilization of light, a photosensitizer (PS),\n [a] B. Kar, P. Paira and cellular oxygen. The absorption of light by the photo-\n Department of Chemistry, School of Advanced Sciences, Vellore Institute of\n sensitizer (PS) generates a responsive excited state (PS*)\n Technology, Vellore, Tamil Nadu 632014, India\n E-mail: priyankar.paira@vit.ac.in capable of engaging in either electron transfer (Type I) or\n Supporting information for this article is available on the WWW under energy transfer (Type II) with the ground-state molecular oxy-\n https://doi.org/10.1002/chem.202401720 gen, generating reactive oxygen species (ROS) like singlet\n\n\n Chem. Eur. J. 2025, 31, e202401720 (1 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n oxygen (1O2), hydroxyl radical (HO ), superoxide radical (O2 ),\n * *\n chelating ligand results in strong DNA intercalation, enhances\n and hydrogen peroxide (H2O2).[19,20] The lifetime of 1O2 in a photophysical behavior, increases the 3\u03c0-\u03c0* character of T1 state\n biological environment is very short, the damage to unaffected and high value of lipophilicity.[32,33] The labile chlorine and CO\n cell proximity to the irradiation zone will be insignificant. A group enhance the covalent interaction with DNA and facilitate\n desirable photosensitizer (PS) possesses the capability to absorb interactions with other biomolecules such as human serum\n light within the therapeutic range (600-900 nm), an optimal albumin (HSA), and glutathione (GSH).[34,35] The strong lipophilic\n energy level for the triplet state, and a sufficiently prolonged rhenium component is anticipated to enhance both the\n triplet state lifetime, facilitating the generation of reactive efficiency of cellular uptake and the property of targeting\n oxygen species.[21,22] mitochondria. After well characterization and purification of all\n Nowadays, we observe a notable scarcity of approved PSs these complexes, we performed photophysical characterization\n for such PDT treatments. The majority of these PSs rely on to evaluate their emission quantum yield and phototoxic\n porphyrin and porphyrinoid structures, encompassing chlorins, screening against triple-negative human breast cell line MDA-\n bacteriochlorin\u2019s, many organic compounds containing C^N MB-231 which demonstrated complex ReL6 as the best\n bidentate, N^N bidentate, tridentate ligand, and similar potential compound. Subsequently, we conducted thorough\n configurations.[23\u201325] Challenges like short excited state lifetime, investigations with complex ReL6, such as binding studies with\n insufficient selectivity, low water solubility, potential toxicity to DNA, proteins, and biomolecules, as well as studies on singlet\n healthy tissues, and skin sensitivity associated with certain oxygen and ROS generation, along with colocalization assess-\n photosensitizers pose difficulties to the clinical adoption of ments and theoretical analysis. The CO release attributes under\n various photodynamic therapeutic strategies.[22,26] The ap- light which have the potential to trigger an additional cytotoxic\n proaches to solve these problems are (i) increasing the energy effect, were also investigated using electronic absorption and\n gap between the nonradioactive d-d state (MC) and 3MLCT; (ii) emission spectroscopy, and Fourier transform infrared (FT-IR)\n increasing the 3\u03c0-\u03c0* character to increase the T1 lifetime. The techniques.\n ligands with extended \u03c0-systems lower the energy of ligand\n centered (LC) 3\u03c0-\u03c0* state to below, or close to, that of the 3MLCT\n state, extraordinarily accumulating the 3\u03c0-\u03c0* character of T1 2. Results and Discussion\n state.\n In recent years, many research teams have endeavored to Synthesis and Characterization: The preparation of L5 and L6\n address these concerns and produce new classes of PSs. In this involves a multi-step procedure, whereas the syntheses of L1-\n category, recently, rhenium(I) tricarbonyl complexes have L4 follow a one-step pathway. The ivory white compound 5\n garnered attention as viable contenders for PDT because of was synthesized by dissolving a mixture of 4-bromobenzalde-\n their varied photophysical and biochemical characteristics, high hyde (1 equiv.), 4-biphenyl boronic acid (1 equiv.),\n photostability, and adaptability in terms of biocompatibility, tetrakis(triphenylphosphine)palladium(0), and K2CO3 (5 equiv.)\n which can be fine-tuned by altering the ligands.[27,28] Also, in toluene under reflux for 4 h. To prepare white-colored\n tricarbonylrhenium(I) complexes featuring N^N bidentate li- compound 6, in the first step (preparation of tetrazole 6\u2019), 4-\n gands also emerge as highly promising candidates for serving formylbenzonitrile and sodium azide (excess) were liquefied in\n as \u201cphoto-activated CO-releasing molecules\u201d (photoCORMs) dimethylformamide (DMF), and treated in the presence of\n with potential therapeutic applications. In addition to their role copper iodide (CuI) at 120 \u00b0C for 30 h whereas, in the second\n in CO release, these complexes can serve as luminescent step (formation of compound 6), we treated tetrazole 6\u2019\n indicators, aiding in the identification and evaluation of the (1 equiv.) with benzyl bromide (1.1 equiv.) in the presence of\n intracellular photochemical release of CO, both terms of K2CO3 in DMF at 0 \u00b0C until the conversion was finished. The 1H-\n location and magnitude.[29] In these complexes, carbon mon- imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol ligands, denoted\n oxide (CO) release is commonly initiated through ligand- as L1-L6, were synthesized by reacting an equimolar mixture of\n substitution reactions in aqueous solutions, an alternative 1,10-phenanthroline-5,6-dione and different derivatives of ben-\n approach explores the light-induced liberation of carbon zaldehyde (1-6) in the presence of ammonium acetate in glacial\n monoxide from initially dark stable carbonyl complexes. Specif- acetic acid, as illustrated in Scheme 1. The ligands (L1-L6)\n ically, tricarbonyl rhenium complexes have been demonstrated underwent complete characterization through 1H and 13C NMR,\n to release CO under UV radiation and display cytotoxic effects FT-IR, and ESI-HRMS analyses, while their purity was verified\n against cancer cells, remaining inactive even after extended using elemental analysis. Then, 1 equiv. of\n exposure in the absence of light.[13,29-31] pentacarbonylchlororhenium(I) and 1.1 equiv. of the previously\n Taking these factors into account, our endeavor in this prepared ligand (L1-L6) were dissolved in toluene and was then\n research was to formulate new PDT active compounds that refluxed over the course of 6 h to prepare tricarbonylrhenium(I)\n might possess the capacity to surmount common challenges. In complexes in high yield. The characterization of the metal\n this context, we have designed and synthesized six organo- complexes was done by 1H and 13C NMR spectroscopy, FT-IR\n metallics Re(I) complexes (ReL1-ReL6) [Re(CO)3(N^N)Cl] where spectroscopy, and ESI-HRMS. Elemental analysis was employed\n N^N = extensive \u03c0 conjugated imidazo-[4,5-f][1,10]-phenanthro- to confirm the purity of the complexes. In the 1H NMR spectra\n line derivatives. The introduction of (imidazo[4,5- of the metal complexes, the ligand proton peaks are observed\n f][1,10]phenanthrolin-2-yl)phenol as an extensively flat N^N to shift downfield in comparison to the free ligand, indicating\n\n\n Chem. Eur. J. 2025, 31, e202401720 (2 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n\n\n Scheme 1. Schematic representation for the formation of ligands (N^N) and [ReI(CO)3(N^N)Cl] complexes.\n\n\n\n\n coordination with the Re center. In ReL6 (considered arbitrarily ing the metal complex formation. Likewise, all other complexes\n for discussion), ligand proton peaks are observed at \u03b4 6.06 to are well characterized by NMR, Mass and IR spectroscopy.\n 9.38 ppm whereas at \u03b4 6.04 to 9.02 ppm in the free ligand L6. Theoretical Calculations: A computational study was also\n The peaks corresponding to the ligand are detected in the performed on the most potent complex ReL6 to explore the\n range of \u03b4 56.8 to 164.4 ppm, and peaks attributed to carbonyl photophysical characteristics. In this study, we employed\n carbon are identified at \u03b4 190.6 to 198.3 ppm in the 13C NMR Density Functional Theory (DFT) calculations to derive the\n spectrum. Further confirmation of the complex formation was energy-minimized three-dimensional structure of the analyzed\n supported by the FT-IR spectrum, where the characteristic peaks complex. Our approach involved utilizing computational meth-\n of CO stretching frequencies at 2021.57 cm 1 and 1899.01 cm 1 ods to elucidate the molecular geometry of the complex and\n were observed. In the ESI-HRMS analysis of complex ReL6, the optimize its energy state for further analysis of its properties.\n observed m/z value of 766.1305 for [M Cl + CH3CN] + precisely Theoretical calculations were conducted using the B3LYP\n corresponded to the calculated m/z value of 766.1325, confirm- exchange-correlation function. The energy-minimized opti-\n\n\n Chem. Eur. J. 2025, 31, e202401720 (3 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n mized structure of the complex unveiled specific bond lengths: transitions. Additionally, there were relatively weak bands\n 1.9243 \u00c5 for Re C, 2.5468 \u00c5 for Re Cl, and 2.1771 \u00c5 for Re N. within the \u03bb 350-550 nm range, associated with a metal-to-\n The structural geometry of complex ReL6 was identified as a ligand charge-transfer (1MLCT) process (Figure S2). We observed\n distorted octahedron with a C1 point group, as evidenced by the experimental results are moderately correlated with the\n angles such as N Re Cl (82.66\u00b0), N Re C (93.48\u00b0), Caxial Re Cl experimental results. The emission spectra of these complexes\n (175.50\u00b0), Cequatorial Re Cl (91.51\u00b0), and Caxial Re Cequatorial (91.75\u00b0). were observed in the range of \u03bbems 320-480 nm under \u03c0 - \u03c0*\n Furthermore, the HOMO-LUMO energy gap was computed to excitation, and under MLCT excitation, they occurred at \u03bbems\n be 2.7935 eV, providing valuable information about the elec- 500-750 nm (Table 1, Figure S2). The quantum yields (\u03a6f) of\n tronic characteristics of the complex. Examining the distribution these complexes were determined using the emission spectral\n of electron density revealed that the HOMO orbital predom- data, offering insights for applications in photocytotoxicity and\n inantly localized over the Re-center and CO molecules, whereas cellular imaging. It was apparent that all the compounds\n the electron density of the LUMO was concentrated on the displayed significant fluorescence, exhibiting a high \u03a6f value of\n bidentate ligand (Figure 1). TD-DFT calculations anticipated two approximately 0.22-0.51. The \u03a6f value of complex ReL6 reached\n bands in the UV-visible spectra, aligning with experimental the highest value at 0.508 among them (Equation (i), Table 1).\n findings. The UV band was assigned to the ILCT transition from Lipophilicity (log P), Cellular Uptake, Solubility, and\n HOMO-3 to LUMO + 1, while the visible band originated from Conductivity Study: The cellular uptake and anticancer mecha-\n the MLCT transition between HOMO-1 and LUMO (Figure S1). nisms of a compound are influenced by its lipophilicity (log P)\n Electronic Absorption (UV-visible) and Emission Study: A and hydrophilic nature. The partition coefficient (log P) is an\n majority of Re(I) tricarbonyl complexes exhibit prolonged indicator of how a compound, in its ionized state, distributes\n phosphorescence, significant Stokes shifts, and elevated quan- between organic and water phases, which serves as a suitable\n tum yields, providing advantages for intracellular sensing and estimation of physiological conditions.[38,39] Considering this, we\n bioimaging.[36] Various electronic states, encompassing config- assessed the lipophilicity of these complexes by determining\n urations like ligand-field (d-d*) or metal-centered (MC), intrali- the log P value in the n-Octanol-water mixture using a shake-\n gand (\u03c0-\u03c0*, IL), and charge-transfer (CT), are employed to flask method. The acquired experimental log Po/w values for\n elucidate the electronic UV-vis absorption spectra of metal these complexes ranged from 0.61 \ufffd 0.02 to 1.34 \ufffd 0.04, indicat-\n complexes.[37] To underscore the cellular imaging attributes of ing their hydrophobic characteristics (Table 2). Among them,\n the prepared complexes, investigations involving absorption complex ReL6 exhibited the highest log Po/w value of 1.34 \ufffd 0.04\n (UV-visible) and emission were carried out in a 10 % DMSO- because of the presence of hydrophobic \u03c0-extensive aromatic\n water (1 : 9, v/v). Rhenium complexes (ReL1-ReL6) exhibited group. The order of lipophilicity of the synthesized complexes\n prominent absorption peaks in the range of \u03bb 260-350 nm, were followed as ReL6 > ReL5 > ReL4 > ReL2 > ReL1 > ReL3. In\n which are ascribed to intraligand/ligand-centered (\u03c0-\u03c0*/ 1LC) general, the log Po/w values of the synthesized complexes line\n\n\n\n\n Figure 1. (a) Optimized structure of the complex ReL6, (b) HOMO electron density distribution (ISO = 0.02), (c) LUMO electron density distribution (ISO = 0.02).\n\n\n Chem. Eur. J. 2025, 31, e202401720 (4 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n Table 1. Photophysical profile of all the synthesized complexes (ReL1-ReL6).\n Samples \u03bba(nm)[a] \u03bbf (nm)[b] Stoke\u2019s shift O.D[c] \u025b[d] (\u03d5f)[e]\n \u03c0-\u03c0* MLCT \u03c0-\u03c0* MLCT \u03c0-\u03c0* MLCT\n\n ReL1 283 428 375 593 92 165 0.48 15900 0.217\n ReL2 288 427 360 589 72 162 0.47 15533 0.421\n ReL3 281 430 344 591 63 161 0.32 10633 0.334\n ReL4 276 420 352 518 76 98 0.31 10466 0.295\n ReL5 270 418 386 570 116 152 0.28 9300 0.315\n ReL6 297 438 391 591 94 153 0.38 12833 0.508\n Quinine Sulphate 350 452 102 0.26 8000 0.546\n 1 1\n [a] Absorption wavelength maxima. [b] Emission wavelength maxima. [c] Optical density. [d] Extinction coefficient(M cm ). [e] Quantum yield.\n\n\n\n Table 2. Table for lipophilicity, cellular uptake, and conductivity study of all the complexes (ReL1-ReL6).\n Samples ReL1 ReL2 ReL3 ReL4 ReL5 ReL6\n [a]\n logPo/w 0.78 \ufffd 0.03 0.82 \ufffd 0.04 0.61 \ufffd 0.02 0.93 \ufffd 0.05 1.18 \ufffd 0.03 1.34 \ufffd 0.04\n Cellular uptake (pmole/106 cells) MDA-MB 231 650 \ufffd 8.2 700 \ufffd 12.5 580 \ufffd 11.4 800 \ufffd 14.0 870 \ufffd 10.2 920 \ufffd 12.8\n MRC-5 119 \ufffd 5.6 120 \ufffd 7.2 116 \ufffd 8.5 140 \ufffd 6.5 180 \ufffd 7.5 220 \ufffd 6.4\n \u25a0M DMSO (\ufffd 0.5) 12.6 11.1 10.2 10.9 8.6 9.8\n (S.m2. mol 1)b\n 10 % DMSO (\ufffd 1) 35 28 31 33 25 29\n\n [a] n-Octanol/water partition coefficient. [b] Conductance in DMSO and 10 % aqueous DMSO.\n\n\n\n\n up favorably with those of other Re(I) tricarbonyl compounds Mass Spectrometry (ICP-MS). MDA-MB-231 Cells were preserved\n described in the literature.[40] As cellular uptake is one of the key with 4 \u03bcM complexes for 6 h and garnered, and total metal\n determinants of antitumor activity, the cellular accumulation of content was quantified by ICP-MS. As presented in Figure 2, the\n the synthesized Re(I) complexes were determined by measuring order of cancer cell accumulations (metal per 106 MDA-MB-231\n intracellular metal content using Inductively Coupled Plasma cells) were followed similarly with the log Po/w values.\n\n\n\n\n Figure 2. Cellular uptake of complexes (ReL1-ReL6) in MDA-MB-231 and MRC-5 cells. Data reflect the average \ufffd SD of two independent experiments each\n performed in triplicate.\n\n\n Chem. Eur. J. 2025, 31, e202401720 (5 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n Subsequently, we compared the cellular uptake of synthesized Figure S6, and Figure S7). This observation was further con-\n Re(I) complexes in non-cancerous MRC-5 cells to know the firmed by the ESI-HRMS study after a 24 h reaction of complex\n effect of cellular accumulation on cancer cell selectivity. ReL6 with NAC and GSH respectively (1 : 3) in methanol. The\n Eventually, all these complexes displayed similar uptake m/z values of 885.6244 and 1032.2177 are indicating the\n tendencies in normal cells, but uptake was much lower than formation of ReL6-NAC and ReL6-GSH adducts respectively\n cancer cells. The solubility of all these complexes was assessed (Figure 3, Figure S8, and Figure S9). This study also revealed\n through a diverse range of solvents. The complexes exhibited that the conversion rate of ReL6-NAC formation is slightly\n outstanding solubility in dimethyl sulfoxide, acetonitrile, and higher than the ReL6-GSH formation depending on the size of\n dimethylformamide while demonstrating fair solubility in water, biomolecules.\n ethanol, and methanol. The complexes displayed molar con- Oxidation and Reduction property of the complex by\n ductance values of approximately 8-15 S.m2 mol 1 in pure Cyclic Voltammetry: Given the pivotal role redox processes\n DMSO and in the range of 25-35 S.m2 mol 1 in 10 % aqueous often play in influencing the anticancer properties of metal-\n DMSO (Table 2). These results indicate their non-electrolytic based drug candidates, an examination of the electrochemical\n behavior in DMSO and an electrolytic nature with a 1 : 1 ratio in features of the compound ReL6 was carried out through cyclic\n an aqueous medium, attributed to the dissociation of the voltammetry (CV) in acetonitrile (CH3CN), utilizing a 0.1 M\n chloride ligand.[32,33,41] solution of tetrabutylammonium perchlorate (TBAP) as the\n Stability Study: Compounds capable of creating aqua supporting electrolyte to assess the redox traits of the metal in\n complexes within cellular environments are indicated to involve the Re(I) complex. Figure S10 displays the acquired CV features,\n in interactions with DNA base pairs through a covalent mode of providing insights into the redox behavior of the complex. The\n action. To ascertain the dissociation rate of the complex ReL6, quasi-reversible anodic peak identified at 1.54 V (I) in the case\n the stability was investigated through UV-visible spectroscopy. of complex ReL6 was associated with the metal-centric\n This analysis was carried out in a 5 % DMSO-PBS (1 : 19, v/v) oxidation process from Re1 + to Re2 +.[42] Additionally, the quasi-\n solution, with a pH of 7.4, as well as in a 5 % DMSO-DMEM reversible anodic peak at 2.46 V (II) was ascribed to the\n solution, a commonly utilized culture medium containing oxidation of chloride ions. The other cathodic peaks observed\n miscellaneous biological nucleophiles like amino acids, glucose, at 1.16 V (III), 1.15 V (IV), and 1.63 V (V) for complex ReL6\n and nucleotides, along with elevated concentrations of salts, were related to the ligand-centric reduction processes. More-\n both performed at room temperature. The absorption spectra over, in the case of complex ReL6, there is an additional\n of the complex ReL6 exhibited a negligible decrease of reduction wave at 1.63 V, confirming the presence of a ReI/Re0\n absorbance over 30 h duration in the absence of light, redox couple.[42,43] This behavior aligns with observations made\n suggesting excellent stability in these media (Figure S3). in previous studies involving tricarbonyl Re(I) complexes\n Furthermore, a study on the stability of the complex ReL6 was incorporating different diimine derivatives. Significantly, it is\n conducted in 2 : 3 (v/v) DMSO-d6 : 10 mM PBS solution (pD = 7.4) worth noting that all these reduction events occur at potentials\n (Figure S4) and 2 : 3 (v/v) DMSO-d6:D2O with 10 mM DMEM beyond the biologically relevant range.[44] Despite being outside\n solution, utilizing 1H NMR spectroscopy, recorded at various this range, the recorded reduction potentials provide valuable\n time intervals (0-30 h) at 37 \u00b0C (Figure S5). Nevertheless, no insights into the electronic characteristics of these complexes.\n significant changes in NMR spectra are the indications of As expected, these potentials align with the electron-donating\n excellent stability of the complex ReL6 reported from UV-visible properties of the diimine ligands.\n absorption spectroscopy previously. Molecular Docking and Molecular Dynamics Study: De-\n Effect of Kinetic Lability on Reactivity towards Biomole- tailed molecular docking experiments were conducted to\n cules (GSH and NAC): A primary objective behind maintaining investigate the binding modes and affinity of the studied\n the labile Cl with metal in the rhenium(I) tricarbonyl com- complex ReL6 with Human Serum Albumin (HSA) and DNA.\n plexes was to expedite the covalent interaction rate with Using the Autodock 4.2 computational program with the\n biomolecules containing nitrogen (N) and sulfur (S), such as Lamarckian genetic algorithm (LGA), valuable insights were\n guanine, adenine, ct-DNA, GSH, N-acetyl cysteine, and proteins. obtained through meticulous molecular docking experiments.\n The investigation into the interaction between complex ReL6, Notably, the crystal structures of DNA (PDB ID: 1BNA) and HSA\n recognized as the most potent among the compounds, and (PDB ID: 1AO6) sourced from the Protein Data Bank underwent\n pertinent biomolecules (GSH, NAC) was conducted through ESI- a comprehensive protein preparation process. This process\n HRMS and 1H NMR spectroscopy (Figure 3). This approach included the removal of water molecules, the addition of polar\n aimed to gain deeper insights into the underlying mechanisms hydrogens, and the computation of Gesteiger charges, ensuring\n contributing to the in vitro anticancer activity exhibited by the a robust foundation for subsequent analyses. Given the specific\n rhenium complexes. For the 1H NMR study, a mixture of focus on DNA as a pivotal target for anticancer drugs, molecular\n complex ReL6 and respective biomolecules (1 : 3) was prepared docking study of the complex ReL6 with DNA was conducted\n in DMSO-d6/D2O (2 : 1, v/v) at 37 \u00b0C, and the spectrum was to elucidate its binding affinity. The obtained outcomes,\n recorded at several time intervals over 24 h. The implementa- indicating a binding energy of 16.02 kcal/mol and an\n tion of time-dependent 1H NMR measurements led to the inhibition constant of 1.81 pM, underscored the remarkable\n observation of distinct changes in NMR signals, suggesting the binding capability of the metal complex with DNA (Figure 4,\n formation of a novel covalent complex adducts (Figure 3, Table S1). The molecular interactions that contribute to this\n\n\n Chem. Eur. J. 2025, 31, e202401720 (6 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n\n\n Figure 3. (a) Formation of adducts (ReL6-NAC and ReL6-GSH) with NAC and GSH through covalent interaction of complex ReL6. (b) Time-dependent 1H NMR\n spectra of a mixture of complex ReL6 and NAC (1 : 3) prepared in DMSO-d6/D2O (2/1, v/v) at 37 \u00b0C. (c) ESI-HRMS spectrum of a mixture of complex ReL6 and\n NAC (1 : 3) prepared in MeOH after 24 h stirring at r.t. (d) Time-dependent 1H NMR spectra of a mixture of complex ReL6 and GSH (1 : 3) prepared in DMSO-d6/\n D2O (2/1, v/v) at 37 \u00b0C. (e) ESI-HRMS spectrum of a mixture of complex ReL6 and GSH (1 : 3) prepared in MeOH after 24 h stirring at r.t. Full ESI-HRMS spectra\n are in supporting information.\n\n\n\n\n enhanced affinity were carefully outlined, highlighting the complex with HSA (Figure 5, Table S1). The detailed molecular\n creation of eight hydrogen bonds with T7, T20, G22, A6, T8, interactions contributing to this sharp binding affinity were\n T19, A18, and C9 residues of DNA, where the complex elucidated, encompassing five hydrogen bonds with LEU115,\n effectively acted as both a donor and an acceptor. Moreover, LYS190, ARG186, LYS519, and SER517 residues, where the\n the sophisticated binding profile was further enriched by complex functioned as both a donor and an acceptor. Addition-\n electrostatic interaction with T7 residues. ally, the complex binding profile was characterized by nine\n The molecular docking of complex ReL6 with HSA unveiled hydrophobic \u03c0-alkyl interactions with LYS137, ALA126, PRO118,\n a binding energy of 13.89 kcal/mol and an inhibition constant MET123, ARG117, and LEU115 residues, along with three\n of 66.27 pM, confirming the exceptional binding capacity of the\n\n\n Chem. Eur. J. 2025, 31, e202401720 (7 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n\n\n Figure 4. (a) The best dock pose of the complex ReL6 exhibiting all the interactions between complex and DNA residues. (b) The best dock pose for exhibiting\n hydrogen bonding interactions between complex and DNA residues. (c) The best dock pose for exhibiting hydrophobic interactions between complex and\n DNA residues. (d) Schematic 2D diagram of the DNA-complex interaction.\n\n\n\n electrostatic interactions involving ARG185 and ARG117 resi- the docking studies. A detailed analysis of the contact residues\n dues. unveiled a comprehensive network involving 18 specific\n To investigate the stability of ligand-protein interactions residues, thereby providing additional affirmation of the robust-\n derived from the docking outcomes, Molecular Dynamics (MD) ness and accuracy of the docking results (Figure 6).[45]\n simulations were performed for the optimal docking pose of DNA Binding Assay by UV-visible Study: UV-vis absorption\n the complex ReL6 with HSA. The simulation results were spectroscopy is widely used to assess the interaction between\n influenced by various intricate factors, such as the conformation molecules and ct-DNA through the formation of complexes.\n of a complex, the presence of water molecules, ions, cofactors, Typically, alterations in absorbance and/or shifts in peak\n complex protonation, conformational changes, and solvent positions are detected when small molecules interact with DNA\n entropy, all of which were carefully considered. The subsequent to create a complex. Metal complexes exhibit two distinct\n MD simulation revealed robust stereochemical geometry of binding modes with DNA: covalent and noncovalent interac-\n residues in the ultimate structure, and the RMSD plot indicated tions, encompassing intercalation, groove binding, and electro-\n initial fluctuations within the first 10 ns, succeeded by a trend static interactions. To validate the interaction of complex ReL6\n toward stabilization. The observed stabilization, as depicted in with DNA, the absorption spectrum of the compound was\n the RMSD plot, emphasized the sustained stability of the HSA examined in the presence of varying concentrations of ct-DNA\n complex, supporting the molecular interactions outlined during within the 280\u2013600 nm range. As the concentration of ct-DNA\n\n\n Chem. Eur. J. 2025, 31, e202401720 (8 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n\n\n Figure 5. (a) The best dock pose of the complex ReL6 exhibiting all the interactions between complex and HSA protein residues. (b) The best dock pose for\n exhibiting hydrogen bonding interactions between complex and HSA residues. (c) The best dock pose for exhibiting hydrophobic interactions between\n complex and HSA residues. (d) Schematic 2D diagram of the protein-complex interaction.\n\n\n\n\n increased, the absorbance spectrum of the complex ReL6 Ethidium Bromide (EtBr) Displacement Assay: To examine\n exhibited a hypochromicity of 39 % for the peak at 298 nm and the mechanism of interaction between the metal complex and\n an isosbestic point was observed at 284 nm, along with a 2 nm DNA, complex ReL6 was further subjected to an Ethidium\n hypsochromic shift in the peak at 298 nm. This spectral pattern Bromide (EtBr) displacement assay using fluorescence spectro-\n is commonly associated with the intercalation as well as an scopy. EtBr exhibits intense fluorescence when intercalated with\n electrostatic mode of the molecule with the DNA base pairs DNA base pairs, and this fluorescence may be diminished when\n (Figure S11). The intrinsic binding constant (kb) for the complex another scaffold replaces EtBr and subsequently binds to the\n ReL6 was determined as 4.4\u00d7105 M 1 (Equation (ii), Table 3, and same DNA site.[47] A solution containing EtBr bound to ct-DNA\n Figure S11) which is of a similar magnitude to that of EtBr-DNA displayed a strong fluorescence peak at 600 nm when excited\n (7\u00d7105 M 1).[4,32,33] The interaction of complexes with nucleo- at 485 nm. However, this fluorescence peak was diminished\n bases like guanine and adenine base was observed using UV- upon incubation with increasing concentrations of the complex\n visible spectroscopy. The spectral variations of these complexes, ReL6 (Figure S13). The calculated apparent binding constant\n as the concentration of nucleobase increased, provided evi- (Kapp) for complex ReL6 was determined to be 2.28\u00d7106 M 1\n dence that the complexes effectively form covalent bonds with using equation (iii). The Stern-Volmer quenching constant (KSV)\n DNA base pairs (Figure S12).[46,47] was computed from Equation (iv) as 0.349\u00d7106 M 1 for complex\n ReL6. The notable shift in spectral band position (hypochrom-\n\n\n\n Table 3. Binding parameters for the interaction of complex ReL6 with ct-DNA.\n [a] [b]\n Complex \u03bbmax [nm] Change in absorbance \u0394\u025b Kb(\u00d7106 M 1) [c]\n KSV(\u00d7106 M 1) [d]\n Kapp(\u00d7106 M 1)\n (%)\n\n ReL6 298 Hypochromism 39 0.44 \ufffd 0.03 0.349 \ufffd 0.06 2.28 \ufffd 0.25\n\n [a] \u0394\u025b, percentage of hypochromism. [b] kb, intrinsic DNA binding constant from UV-vis absorption titration. [c] KSV, Stern-Volmer quenching constant. [d]\n Kapp, apparent DNA binding constant from competitive displacement.\n\n\n\n Chem. Eur. J. 2025, 31, e202401720 (9 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n\n\n Figure 6. (a) RMSD plot of complex ReL6 bound HSA in MD simulation (0\u201350 ns). (b) The histogram of the contact residues in complex ReL6 bound HSA.\n\n\n\n\n ism, \u0394\u025b ~ 39 %), coupled with a substantial intrinsic binding or covalent binding results in a reduction in the effective DNA\n constant (Kb = 0.44\u00d7106 M 1) and elevated apparent binding length, contributing to a slight decrease in the viscosity of the\n constant (Kapp = 2.28\u00d7106 M 1) signifies the good intercalation DNA solution. The relative viscosity of DNA remains unaffected\n with ct-DNA.[32,48] when a drug interacts through groove binding or electrostatic\n Assessment of Viscosity: This approach offers compelling interaction, as the binding process does not result in any\n evidence for elucidating the DNA binding mechanism of small change in the length of the DNA molecule. Metal complexes\n molecules in solution. Intercalation induces a longitudinal form connections with the external surface of DNA molecules\n expansion of DNA, enhancing its stiffness, and thereby elevat- through either Van der Waals forces or hydrophobic interac-\n ing the viscosity in the solution. Conversely, partial intercalation tions in groove binding or electrostatic binding. The relative\n\n\n Chem. Eur. J. 2025, 31, e202401720 (10 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n viscosity of the DNA solution for complex ReL6 was determined The plotted graph depicts the relative changes in DPBF\n in comparison to Ethidium Bromide using an Ostwald viscom- absorbance at 417 nm (A/A0) against the exposure time to\n eter following a standardized protocol.[32,49] The inclusion of visible light in seconds, where A symbolizes the DPBF\n complex ReL6 in the ct-DNA solution leads to an increase in absorbance at a specific time, while A0 denotes the DPBF\n DNA viscosity, illustrating mainly the intercalative binding of absorbance at t = 0 s. This analysis was conducted for both Rose\n the compound to ct-DNA with some degree of other inter- Bengal and complex ReL6 (Figure 7). The calculated singlet\n actions at varying concentration levels (Figure S14). oxygen quantum yield for complex ReL6 (\u03d5\u0394ReL6) is 0.61, in\n HSA Binding Study: Serum albumin (SA) is the major comparison to the established singlet oxygen quantum yield of\n protein found in blood plasma, and interactions between drugs Rose Bengal (\u03d5\u0394RB = 0.76). Under similar experimental conditions,\n and proteins play a crucial role in drug transport, biodistribu- in the dark condition, there was no decline in DPBF absorbance\n tion, and potential toxicity. It is noteworthy that serum albumin at 417 nm (\u03bbmax) with complex ReL6, suggesting that singlet\n contains a free thiol group from cysteine at position 34, and Re oxygen generation did not occur under these circumstances.\n complexes demonstrate a remarkably high binding affinity for The high value of \u03d5\u0394 of complex ReL6 confirmed that these\n sulfur donors.[4,48] To assess the binding affinity of complex ReL6 types of complexes endured the PDT type II mechanism.\n with HSA, fluorescence spectra of HSA was measured both in To further verify the generation of singlet oxygen, electron\n the absence and presence of metal complex. The excitation paramagnetic resonance (EPR) measurements were performed\n wavelength used was 280 nm, with emission recorded at using 2,2,6,6-tetramethylpiperidine (TEMP) as spin trap\n 330 nm (Figure S15). The gradual decline in the emission agents.[51,52] As depicted in Figure S18a of the supporting\n intensity of HSA, correlating with an elevation in the concen- information, a distinct 1O2-induced triplet signal of TEMPO was\n tration of the complex ReL6, indicates a substantial interaction observed following visible light irradiation for 60 min, indicating\n between the metal complex and HSA. The Stern-Volmer the 1O2 formation, whereas no signal was detected in the\n quenching constant (KHSA), binding affinity (K), the number of absence of irradiation.\n binding sites (n), and bimolecular quenching constant (Kq) for Photo-oxidation of NADH and Detection of H2O2, O2 , and *\n\n\n\n\n the complex ReL6 was determined as 0.177\u00d7106 M 1, OH : The cytoplasm contains a substantial amount of the\n *\n\n\n\n\n 1.49\u00d7104 M 1, 1.63, and 1.77\u00d71013 M 1 s 1 respectively, using the reduced form of nicotinamide adenine dinucleotide (NADH), a\n Stern-Volmer equation (v), equation (vi) and corresponding coenzyme. This plays a pivotal role in regulating energy\n Stern-Volmer plots (Table 4). The determined Kq value for the production within mitochondria to a certain extent. An\n complex ReL6 exceeded the maximum conceivable value for imbalance in the ratio of NADH to NAD + can lead to the\n dynamic quenching (2.0\u00d71010 L.mol 1 s 1), indicating the in- suppression of the electron transport chain within the mito-\n volvement of static quenching, effective bimolecular quench- chondria, causing cellular dysfunction.[53,54] To assess the ability\n ing, and bimolecular binding. In the Stern-Volmer plots, a of the complex to initiate photocatalytic oxidation of the\n concave-like upward curvature toward the Y-axis was noted at coenzyme in oxygenated solutions, complex ReL6 at a concen-\n elevated quencher concentrations, signifying the establishment tration of 10 \u03bcM was placed in the presence of NADH (100 \u03bcM)\n of a ground state complex between the complex and within a solvent mixture comprising 20 % MeOH and 80 % H2O\n HSA.[32,33,49] (1 : 4, v/v). As shown in Figure S16, the UV-vis spectra of NADH\n (Photo) generation of Singlet Oxygen: The efficiency of a showed no alterations when exposed to the complex in the\n photosensitizer (PS) in inducing the generation of singlet absence of light. Nevertheless, under low concentrations, the\n oxygen (1O2) species upon light excitation is crucial for the absorbance of NADH gradually diminished after exposure to\n effectiveness of the photosensitizer in type II photodynamic visible light irradiation (400-700 nm, 10 J.cm 2) for the metal\n therapy (PDT). Molecular oxygen (3O2) becomes excited through complex. Through assessing alterations at \u03bb = 339 nm, the\n energy transfer from the excited T1 state of PS, resulting in the absorption peak of NADH, and turn over number (TON) were\n generation of 1O2. Singlet oxygen has an extremely short-term computed, revealing a remarkable value of 6.57 for 20 min light\n lifespan (< 0.04-3 \u03bcs) and exhibits exceptionally high duration which favors Type I PDT pathway. Consequently,\n reactivity.[22,37] The efficiency of complex ReL6 in generating during photo-oxidation of NADH, complex ReL6 procured\n singlet oxygen through photosensitization was determined electrons from NADH to form NADH +. This radical species\n *\n\n\n\n\n using a UV spectrophotometer with 1,3-diphenylisobenzofuran subsequently reacted with O2, and producing H2O2. The\n (DPBF) and Rose Bengal (RB) following standard protocol.[50] In presence of H2O2 was confirmed using peroxide test strips,\n this study, under visible light irradiation (400-700 nm, which indicated its production (Figure S16c).[55] Furthermore, we\n 10 J.cm 2), we observed a consistent decline in the absorbance assessed the ability of complex ReL6 to generate OH radical *\n\n\n\n\n of DPBF at 417 nm over time in the presence of complex ReL6. using Methylene Blue (MB) as a probe.[56] Similar to the\n\n\n Table 4. Binding parameters for the interaction of complex ReL6 with HSA.\n Complex KHSA [\u00d7106 M 1][a] kq[\u00d71013 M 1 s 1][b] K [\u00d7104 M 1][c] N[d]\n\n ReL6 0.177 \ufffd 0.01 1.77 \ufffd 0.20 1.49 \ufffd 0.17 1.63 \ufffd 0.08\n\n [a] KHSA, Stern-Volmer quenching constant. [b] Kq, quenching rate constant. [c] K, binding constant with HSA. [d] n, number of binding sites.\n\n\n\n Chem. Eur. J. 2025, 31, e202401720 (11 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n\n\n Figure 7. UV-visible spectroscopy investigations were conducted to examine the singlet oxygen generation in the presence of DPBF by Rose Bengal (RB)\n under dark conditions (a) and in the presence of light (b), as well as by complex ReL6 under dark conditions (c) and in the presence of light (d) at 298 K. A\n competitive plot depicting singlet oxygen generation between Rose Bengal and complex ReL6, expressed as (A0\u2013A)/A0 versus time, was also generated (e).\n Additionally, the relative change in absorbance by DPBF for Rose Bengal and complex ReL6 at 417 nm (A/A0) over time in seconds was analyzed (f).\n\n\n\n\n production of 1O2, complex ReL6 did not show significant OH *\n ReL6 under visible light exposure was further confirmed using\n generation in the absence of light. However, there was a EPR spectroscopy. To achieve this, 5,5-dimethyl-1-pyrroline N-\n noticeable decrease in absorbance of MB after exposure to oxide (DMPO) was utilized as a probe or radical trapping\n visible light, indicating the OH generation (Figure S17). Addi-\n *\n agent.[52] A prominent six-line electron spin resonance signal\n tionally, the formation of OH and O2 radicals by complex\n * *\n originating from the DMPO/O2 adduct was seen in the spectra\n *\n\n\n\n\n Chem. Eur. J. 2025, 31, e202401720 (12 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n under light irradiation (Figure S18b). Furthermore, in DMF-H2O under photo-irradiation indicating oxidative stress and subse-\n solution (2 : 98, v/v), a characteristic four-line resonance of the quent cell death. The formation of green fluorescent 2\u2019, 7\u2019-\n DMPO/OH adduct was identified in the spectra (Figure S18c).\n *\n dichlorofluorescein (DCF) by deacetylation and subsequent\n Hence, these results designated that complex ReL6 is extremely oxidation of nonfluorescent 2\u2019, 7\u2019-dichlorofluorescin diacetate\n effective photosensitizer for PDT against both normoxic and (DCFDA) by cellular esterases and ROS designated the degree of\n hypoxic tumors as it was accomplished for both Type I and ROS generation. The generation of ROS was less under dark\n Type II PDT pathways. condition which showed less intense cellular images at \u03bbems\n Intracellular ROS Generation by H2DCFDA: Reactive oxygen 530 nm. However, upon light irradiation, significantly increase\n species (ROS) are naturally generated as byproducts during in fluorescence intensity was observed as higher rate of DCF\n various physiological processes within cellular metabolism. formation, revealing the notable phototoxicity of complex ReL6\n Elevated levels of ROS can lead to oxidative stress or (Figure 8).\n inflammation, causing damage to cells and their components.[55] Co-localization Study: In exploring the subcellular local-\n In photoactivated cancer therapy, controlled ROS production is ization of complex ReL6, cells treated with complex ReL6 were\n utilized to selectively target and destroy cancer cells while subjected to dual staining with Hoechst and Mitotracker Red.\n preserving healthy ones. In photocatalytic cancer therapy, Subsequently, a thorough examination of their distribution\n photoresponsive metal complexes have been found to generate within the cellular milieu was carried out using fluorescence\n hydrogen peroxide (H2O2), hydroxyl radicals (OH ), and super-\n *\n microscopy and in which complex ReL6 (10 \u03bcM) exhibited\n oxide anions (O2 ) as byproducts of NADH oxidation, along\n *\n cytoplasmic localization. The subcellular positioning was ob-\n with singlet oxygen (1O2) produced through a type II energy served through the green fluorescence emission of the complex\n transfer pathway.[56] Here, in MDA-MB-231 breast cancer cells, with Hoechst and the red fluorescence emission with Mitotrack-\n the complex ReL6 produced reactive oxygen species (ROS) er Red, providing insights into their respective distributions\n\n\n\n\n Figure 8. DCFDA assay for assessing the intracellular ROS production in MDA-MB-231 cells: (a) Untreated control cells (b) complex ReL6 treatment of cells in\n dark condition (c) complex ReL6 treatment of cells under visible light irradiation (400\u2013700 nm, 10 J.cm 2) for 1 h (incubation time of 4 h and concentration of\n 10 \u03bcM) (d) bar graph representing the average green fluorescence intensity of DCF under dark and light condition after ReL6 treatment. Scale bar 75 \u03bcm.\n One-way ANOVA was utilised to get the p value between the groups. The observed p value was < 0.0001 (***). The error bar represents the \ufffd standard error\n of mean (SEM).\n\n\n Chem. Eur. J. 2025, 31, e202401720 (13 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n within the cellular context (Figure 9). The co-localization study intensity of the CO stretching band at 2021 cm 1 upon 30 min\n using Mitotracker Red revealed the intracellular localization of of UV illumination in the IR study indicated the liberation of\n complex ReL6 within the mitochondria of MDA-MB-231 cells, as only one CO molecule which is trans to a \u03c3-donating ligand Cl\n indicated by Pearson\u2019s coefficient of correlation (r = 0.918). (Figure S20a).[13,58] The complex ReL6 dissolved in acetonitrile\n Given the known impact of these complexes on cellular was subjected to periodic exposure of UV light (305 nm).\n energetics and the mitochondria being a primary target, our Subsequently, the photolyzed sample was dried, and IR spectra\n focus shifted to assessing mitochondrial damage correlated were obtained within KBr matrices.[13] The release of CO from\n with elevated reactive oxygen species (ROS) generation. complex ReL6 was further confirmed by myoglobin assay.[13,53]\n Photoinduced CO Release Property: Further exploration In this standard assay, a noticeable alteration was observed in\n has been undertaken about the use of carbon monoxide (CO) the soret band of reduced Mb, shifting from 432 nm to 421 nm\n as a cytotoxic agent for eliminating malignant cells. The (a blue shift of 11 nm) (Figure S20b). Additionally, new bands\n probable mechanism involves the high-affinity binding of CO to emerged at 540 and 577 nm. This spectral shift served as\n cytochrome c oxidase, leading to the inhibition of the confirmation of the formation of the Mb CO complex, affirming\n mitochondrial respiration pathway. By impeding mitochondrial the release of CO induced by UV light (305 nm).\n respiration through CO binding, the proliferation of cancer cells In vitro Phototoxicity of Re(I) Complexes: The cell viability\n is diminished, ultimately depleting cells metabolically.[30,56,57] In assays of the synthesized Re(I) complexes were investigated in\n this work, initially, we performed the photoinduced CO Release triple-negative breast cancer cells (MDA MB-231) besides\n study under visible light exposure (400-700 nm, 10 J.cm 2). normal MRC-5 cells under dark and light exposure in the\n Unfortunately, no significant spectral changes were observed in presence and absence of GSH in triplicates. The typical 3-(4,5-\n the electronic spectrum of the complex ReL6 under these dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as-\n conditions. Additionally, the myoglobin assay, conducted under say protocol was implemented for the cytotoxicity assay with\n reduced conditions, did not detect any photoinduced CO photofrin as a control PDT drug. The cells were incubated with\n release when exposed to visible light. However, we observed the complexes along with photofrin and irradiated by yellow\n notable changes when the complex ReL6 was exposed to UV light (0-2 J.cm 2) from a 400 W tungsten lamp fitted with a heat\n light (\u03bb = 305 nm, 5 mW cm ), which suggests that the complex isolation filter and 500 nm long pass filter at an intensity of\n may require higher energy light (shorter wavelengths) to trigger 4 mW/cm2 for 4 h. The normalized cell viability vs log [complex]\n CO release.[56] The photoinduced CO release of complex ReL6 was plotted, and a nonlinear regression analysis was performed\n was systematically performed by exposing the complex to using Origin 8.5 to determine the IC50 values (Figure S21,\n higher-energy UV light irradiation (\u03bb = 305 nm, 5 mW cm 2) over Table 5). The light and dark toxicity of all these Re(I) complexes\n a specified time duration. The ensuing alterations in the was reconnoitered in both normoxia and hypoxia environ-\n complex were meticulously examined through UV-Vis and IR ments. As shown in Table 5, all these complexes displayed\n spectroscopy in CH3CN solution. After light exposure at several insignificant dark toxicity against the TNBC cells (IC50~61-\n time intervals, there was a decrease in absorbance at 297 nm 79 \u03bcM). Interestingly, the dark toxicity was fond to be lower in\n indicating the photoinduced CO release from the complex. The hypoxic environment than normoxia might be due to strong\n CO release rate (KCO = 0.44 \ufffd 003 min 1, conc. 100 \u03bcM) was complex-GSH adduct overcome the GSH-GSSG redox. However,\n calculated from the changes in absorbance with time (Fig- a significant increase of toxicity (IC50~6-29 \u03bcM) against TNBC\n ure S19). The KCO value and a substantial decrease in the was observed in yellow light irradiation under both normoxia\n\n\n\n\n Figure 9. In the co-localization study, cytoplasmic localization of complex in MDA-MB-231 cells was observed. Co-staining with Mitotracker Red and Hoechst\n suggested potential mitochondrial localization. The calculated Pearson coefficient was 0.918 for complex ReL6. The scale bar in the image corresponds to\n 25 \u03bcm.\n\n\n Chem. Eur. J. 2025, 31, e202401720 (14 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n Table 5. Table for Phototoxicity of all the synthesized complexes (ReL1\u2013ReL6).\n Complex IC50 (\u03bcM)[a]\n MDA-MB-231[b] MRC-5[c]\n [d] [e]\n Normoxia Hypoxia Normoxia\n Dark In light[f] PI[g] Dark In light PI Dark In light SF[h]\n\n ReL1 62.55 \ufffd 3.3 29.35 \ufffd 4.1 2.13 79.91 \ufffd 2.3 32.46 \ufffd 3.6 2.46 99.02 \ufffd 1.2 97.02 \ufffd 1.1 3.30\n ReL2 67.76 \ufffd 5.3 25.27 \ufffd 1.4 2.68 75.18 \ufffd 0.5 28.81 \ufffd 2.4 2.60 98.06 \ufffd 0.7 96.81 \ufffd 1.6 3.88\n ReL3 69.54 \ufffd 3.3 31.73 \ufffd 4.3 2.19 72.19 \ufffd 1.3 31.15 \ufffd 1.3 2.31 > 100 > 100 > 3.15\n ReL4 64.36 \ufffd 2.6 23.08 \ufffd 0.5 2.78 73.90 \ufffd 3.1 21.15 \ufffd 1.5 3.49 > 100 > 100 > 4.33\n ReL5 65.61 \ufffd 1.4 17.90 \ufffd 0.5 3.66 75.08 \ufffd 1.2 19.38 \ufffd 1.7 3.87 96.06 \ufffd 0.6 95.06 \ufffd 1.6 5.31\n ReL6 61.76 \ufffd 1.2 6.24 \ufffd 1.6 9.89 61.54 \ufffd 2.1 6.40 \ufffd 1.5 9.61 97.06 \ufffd 1.3 94.06 \ufffd 0.9 15.07\n Photofrin 38.3 \ufffd 0.5 3.02 \ufffd 0.2 12.7 43.3 \ufffd 0.56 6.92 \ufffd 0.4 6.25 70.3 \ufffd 1.2 65.22 \ufffd 0.3 21.59\n\n [a] 50 % of cells experience cell death. [b] Triple negative human breast cancer cell line. [c] Normal Fibroblast. [d] 20\u201321 % oxygen. [e] 2\u20136 % oxygen. [f]\n Yellow light (0\u20132 J cm 2) from a 400 W tungsten lamp fitted with a heat isolation filter and 500 nm long pass filter at an intensity of 4 m W/cm2 for 4 h. [g]\n Phototoxicity Index. [h] Selectivity Factor. Based on the Student\u2019s t-test with Origin 8.5 software, statistical significance is denoted as P < 0.05. The error bar\n represents the \ufffd standard error of mean (SEM).\n\n\n\n\n and hypoxia environments. In sharp contrast, complex ReL6 plexes. The presence of substituted imidazo[4,5-\n displayed the highest cytotoxic behavior (IC50~6 \u03bcM, PI > 9) f][1,10]phenanthrolin-2-yl)phenol moiety imparted attractive\n under yellow light irradiation compared to dark conditions. The photo-physical properties and increase the 3\u03c0-\u03c0* character of T1\n order of light toxicity was observed as ReL6 > RrL5 > ReL4 > state for effective PDT. The dark and light cytotoxicity of all\n ReL2 > ReL1 > ReL3 which has been pointedly correlated with these complexes was explored in both normoxia and hypoxia\n the lipophilicity and cellular uptake of these complexes. The conditions. Notably, complex ReL6 exhibited outstanding\n drug transportation, cellular uptake, and DNA damaging ability potency and selectivity (IC50~6 \u03bcM, PI > 9) under yellow light\n of these complexes are the key factor in their antitumor irradiation compared to dark. The order of light toxicity was\n potency evaluation. The significant potency of complex ReL6 found to be ReL6 > RrL5 > ReL4 > ReL2 > ReL1 > ReL3 which\n under photo irradiation can be ascribed to (i) potential binding has been pointedly correlated with the lipophilicity and cellular\n with serum albumin for transportation, (ii) highest level of uptake (via ICP-MS) of these complexes. Interestingly, these\n lipophilicity for cellular entry, (iii) strong complex-cysteine complexes are 3-15 folds more selective in TNBC cells compared\n interaction and excessive production of ROS under normoxia to normal MRC-5 cells. The role of most potent complex ReL6 in\n and hypoxia environments (PDT type I and II) stop GSH biosyn- type I and type II PDT was investigated by singlet oxygen (1O2)\n thesis and triggered GSH to GSSG conversion respectively, (iv) generation study using a DBPF probe, photooxidation of NADH\n strong DNA intercalation, cellular DNA damage by singlet to NAD + measurement, and Intracellular ROS generation study\n oxygen (PDT type II, normoxia) and several radicals (PDT Type I, with H2DCFDA. Photoinduced CO release of the complex ReL6\n hypoxia), (v) significant mitochondrial accumulation and mt- was thoroughly performed by exposing the complex to higher-\n DNA damage by oxidative stress, (vi) Photoinduced CO release energy UV light irradiation (\u03bb = 305 nm, 5 mW.cm 2) over a\n encumber mitochondrial respiration through CO binding and specified time duration. The high value of fluorescence\n deplete cells metabolically. In contrast, photofrin exhibited quantum yield (\u03a6f = 0.50) and substantial emission in the range\n good photo-selectivity in TNBC under normoxia but a loss in of \u03bbems 500-750 nm (visible to NIR range) of complex ReL6\n activity was observed against hypoxic tumors (Table 5). There- certainly helps in bio-imaging application as well. The exten-\n fore, complex ReL6 is bringing forth a valuable drug in cancer sively flat N^N chelating ligand also enhanced the DNA\n therapy in the presence of light under hypoxic tumor ruling intercalation property of the complex. The labile chlorine and\n over the prevailing PDT drug, photofrin. The antitumor potency CO group improved the covalent interaction with DNA and\n of the molecules also depends on their selectivity towards facilitated interactions with other biomolecules such as human\n annihilating cancer cells over noncancerous cells. Herein, we serum albumin (HSA), and glutathione (GSH). The strong\n observed that all these complexes are 3\u201315 folds more selective lipophilic rhenium component and hydrophobic N^N chelating\n in TNBC cells compared to normal MRC-5 cells (Table 5). ligands increased the cellular uptake and localization in\n mitochondria which was measured by co-localization study\n using Mitotracker Red. The higher phototoxicity of complex\n 3. Conclusions ReL6 is due to its high absorbance within the visible region,\n high 1O2 quantum yield (type II PDT), and higher TON for NADH\n In summary, we have synthesized and characterized novel Re(I) photo-oxidation (type I PDT). Although, this complex formed\n tricarbonyl complexes (ReL1-ReL6) to achieve GSH stable, stable adduct with GSH under dark, it could not inhibit the light\n hypoxia efficient, highly cytoselective anti-TNBC metal com- toxicity of the complex because the excess GSH in tumor cells\n\n\n Chem. Eur. J. 2025, 31, e202401720 (15 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n was depleted by strong complex-cysteine interaction, excessive Synthesis\n ROS production under photo-irradiation. The transportation,\n cellular entry and mechanism of action of complex ReL6 in the General Synthetic Procedure of Ligands [L1\u2013L6]\n cell were triggered by the following steps: (i) strong complex-\n The ligands were synthesized based on our previously reported\n albumin adduct facilitates cellular transportation, (ii) cell procedure.[32,33] The 1H-imidazo[4,5-f][1,10]phenanthrolin-2-\n penetration by high lipophilic character and accumulation in yl)phenol ligands, denoted as L1-L6, were synthesized by reacting\n Mitochondria (PCC = 0.918) and expedites the Mitochondrial an equimolar combination of 1,10-phenanthroline-5,6-dione and\n pathway, (iii) nuclear and mitochondrial DNA intercalation and different derivatives of benzaldehyde (1-6) in the presence of\n ammonium acetate in glacial acetic acid, as illustrated in Scheme 1.\n damage by ROS under light irradiation, (iv) photo induced\n released CO bind with cytochrome c oxidase and inhibit the 2,4-dichloro-6-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol\n mitochondrial respiration. It\u2019s noteworthy to mention that Re(I) (L1): Yield: 92 %; Colour: Peach White; Mp: 230 \u00b0C; Rf (pure\n tricarbonyl complexes have not been well explored for photo- methanol): 0.65; IR (cm 1): \u03c5 Ar O H stretching (3504.74), N H\n stretching (3327.40), Ar C H stretching (3054.43), C=N stretching\n oxidation of NADH in aqua-rich conditions, and this study might\n (1679.81), Ar C=C stretching (1472.23), C N stretching (1348.01),\n open an opportunity to discover the efficacy of Re(I) tricarbonyl C Cl stretching (797.18), Ar C H bending (731.80); 1H NMR (DMSO-\n complexes to oxidize NADH more effectively for photocatalytic d6, 400 MHz): \u03b4 8.93 (s, 2H, H1, H10), 8.72 (d, 2H, H3, H8, J = 4.0 Hz),\n cancer therapy. Therefore, complex ReL6 emerges as a promis- 8.07 (s, 1H, H15), 7.72 (s, 2H, H2, H9), 7.41 (s, 1H, H17); 13C NMR (DMSO-\n ing option for cancer treatment using light therapy against d6, 100 MHz): \u03b4 152.89, 151.73, 148.35, 143.53, 136.61, 134.73,\n 131.14, 130.48, 130.19, 129.56, 126.78, 123.99, 122.03; ESI-MS\n hypoxic tumors, surpassing the current PDT drug, photofrin,\n (MeOH): observed m/z = 382.0147 [M + H] +, 403.9970 [M + Na] +,\n which loses effectiveness in such conditions. Overall, complex calcd m/z = 382.0202 [M + H] +, 403.0129 [M + Na] +.\n ReL6 represents a typical class of combined PDT and PACT\n agent that can generate 1O2 as well as oxidize NADH and could 2-(4-bromophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (L2):\n Yield: 96 %; Colour: Peach Pink; Mp: 210 \u00b0C; Rf (pure methanol): 0.60;\n also be developed as TNBC phototheranostic agent in near\n IR (cm 1): \u03c5 N H stretching (3391.96), Ar C H stretching (3083.85),\n future. C=N stretching (1685.53), Ar C=C stretching (1455.07), C N\n stretching (1371.71), Ar C H bending (737.52), C Br stretching\n (653.98); 1H NMR (DMSO-d6, 400 MHz): \u03b4 8.99 (s, 2H, H1, H10), 8.92 (d,\n Experimental Section 2H, H3, H8, J = 7.6 Hz), 8.24 (d, 2H, H15, H19, J = 6.4 Hz), 7.79 (t, 2H, H2,\n H9, J = 3.6 Hz), 7.57 (d, 2H, H16, H18, J = 6.0 Hz); 13C NMR (DMSO-d6,\n Materials and Method: We hired a variety of reagents and solvents, 100 MHz): \u03b4 149.58, 148.38, 143.68, 134.26, 132.58, 132.48, 131.65,\n all with the highest commercial grade and purity, in all our tests. 130.24, 128.93, 125.54, 123.85, 122.74, 121.77; ESI-MS (MeOH):\n Every chemical and organic solvent used in chemical synthesis and observed m/z = 375.0258 [M + H] +, 397.0068 [M + Na] +, calcd m/z =\n chromatography was purchased from Sigma Aldrich, E-Merck 375.0245 [M + H] +, 397.0065 [M + Na] +.\n (India), TCI Chemicals India, Sisco Research Laboratories Pvt. Ltd.\n (SRL) - India, and Spectrochem. A mixture of ethyl acetate and 2-bromo-6-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-4-nitro-\n methanol was used as the solvent system for thin-layer chromatog- phenol (L3): Yield: 90 %; Colour: Chrome Yellow; Mp: 240 \u00b0C, Rf\n raphy using pre-coated silica gel 60 F254 aluminum sheets from E. (pure methanol): 0.58; IR (cm 1): \u03c5 Ar O H stretching (3605.26), N H\n Merck in Germany. Sigma Aldrich Chemical Limited provided ct- stretching (3386.25), Ar C H stretching (3083.86), C=N stretching\n DNA, Human serum albumin (HSA), L-Glutathione reduced, N- (1590.74), Ar C=C stretching (1478.77), N=O stretching (1401.13),\n Acetyl-L-cysteine, Ethidium bromide, and myoglobin. We bought all C N stretching (1330.04), N O stretching (1282.63), C Br stretching\n the cell lines from the National Centre for Cell Science (NCCS), (802.90), Ar C H bending (737.52); 1H NMR (DMSO-d6, 400 MHz): \u03b4\n Pune, India. Tetramethylsilane (TMS) served as the internal standard 9.04 (s, 3H, H1, H10, H17), 8.98 (d, 2H, H3, H8, J = 6.4 Hz), 8.37 (s, 1H,\n as the 1H NMR and 13C NMR spectra were collected on a 400 MHz H15), 7.89 (t, 2H, H2, H9, J = 4.0 Hz); 13C NMR (DMSO-d6, 100 MHz): \u03b4\n Advanced Bruker DPX spectrometer. In ppm units, the chemical 159.44, 158.29, 149.09, 146.44, 143.67, 139.60, 135.57, 132.21,\n shifts (\u03b4) were displayed. For example, s stands for singlet; brs for 129.45, 129.24, 126.41, 121.65, 120.84, 119.79, 116.90, 114.01; ESI-\n broad singlet; d for doublet; dd for double doublet; t for triplet; and MS (MeOH): observed m/z = 355.2791 [M Br] +, 389.2638 [M NO2] +,\n m for multiplet. EPR spectra were recorded using an EMX Plus X- 433.2906 [M H] +, calcd m/z = 355.3130 [M Br] +, 389.2120 [M-\n Band spectrometer (BRUKER BIOSPIN, Germany). Using an open NO2] +, 433.9889 [M H] +.\n capillary tube and an Elchem Microprocessor-based DT equipment, 2-(2-chloro-8-methylquinolin-3-yl)-1H-imidazo[4,5-\n the melting points of the complexes were assessed. TDS conduc- f][1,10]phenanthroline (L4): Yield: 92 %; Colour: Raw Sienna; Mp:\n tometer-307 and Ostwald Viscometer were used to measure 220 \u00b0C; Rf (pure methanol): 0.69; IR (cm 1): \u03c5 N H stretching\n conductivity and viscosity respectively. A Shimadzu Affinity FT-IR (3351.64), Ar C H stretching (3167), sp3 CH stretching (3036.45),\n spectrometer recorded IR spectra in the 4000-400 cm 1 range. On a C=N stretching (1644.68), Ar C=C stretching (1437.09), C N\n Shimadzu ESI-MS-4000 mass spectroscopic apparatus with a 4000 stretching (1377.43), Ar C H bending (737.52), C Cl stretching\n triple quadrupole MS, the mass spectra of the synthesized (619.02); 1H NMR (DMSO-d6, 400 MHz): \u03b4 9.05 (d, 4H, H1, H3, H8, H10,\n compounds were computed. For elemental analysis, PerkinElmer J = 3.2 Hz), 9.03 (s, 1H, H1, H22), 7.84 (dd, 3H, H2, H9, H19, J = 8.0 Hz),\n equipment was used. Fluorescence spectra were taken using a 7.47 (d, 1H, H18, J = 7.2 Hz), 7.22 (t, 1H, H20, J = 7.6 Hz), 2.54 (s, 3H,\n Hitachi F7000 fluorescence spectrophotometer coupled with a Methyl protons); 13C NMR (DMSO-d6, 100 MHz): \u03b4 161.49, 156.30,\n xenon lamp, and UV-visible spectra were taken using a JASCO V- 150.96, 148.37, 147.75, 143.91, 139.65, 137.71, 133.63, 133.20,\n 730 spectrophotometer utilizing a 1 cm quartz cell. Utilizing an Elisa 127.61, 124.42, 123.75, 122.99, 120.46, 119.83, 17.85; ESI-MS\n reader and a 96-well plate, the cytotoxicity (MTT) assay was (MeOH): observed m/z = 396.1038 [M + H] +, calcd m/z = 396.1016\n conducted. [M + H] +.\n\n\n\n\n Chem. Eur. J. 2025, 31, e202401720 (16 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n 2-([1,1\u2019 : 4\u2019,1\u2019\u2019-terphenyl]-4-yl)-1H-imidazo[4,5- product, the organic layer was dried over Na2SO4, filtered, and then\n f][1,10]phenanthroline (L5): Yield: 92 %; Colour: Cream Orange; Mp: the solvent was removed under vacuum. The reaction product was\n 200 \u00b0C; Rf (pure methanol): 0.74; IR (cm 1): \u03c5 N H stretching then purified over a brief pad of SiO2. Yield: 80 %; Colour: Yellowish\n (3356.82), Ar C H stretching (3078.14), C=N stretching (1668.38), Ar white; Mp: 104 \u00b0C; Rf (1 : 4 Ethyl acetate: n-Hexane): 0.41; 1H NMR\n C=C stretching (1478.77), C N stretching (1395.41), Ar C H bending (CDCl3, 400 MHz): \u03b4 10.06 (s, 1H, CHO proton), 8.34 (d, 2H, H2, H6,\n (737.52); 1H NMR (DMSO-d6, 400 MHz): \u03b4 9.04 (d, 2H, H1, H10, J = J = 8.4 Hz), 8.07 (s, 1H, NH proton), 8.01 (d, 2H, H3, H5, J = 8.0 Hz);\n 13\n 2.8 Hz), 8.97 (d, 2H, H3, H8, J = 7.6 Hz), 8.42 (d, 2H, H15, H19, J = C NMR (CDCl3, 100 MHz): \u03b4 191.55, 162.85, 156.97, 137.57, 130.27,\n 8.0 Hz), 7.99 (d, 2H, H27, H31, J = 8.0 Hz), 7.91 (d, 2H, H2, H9, J = 127.80.\n 8.0 Hz), 7.86-7.80 (m, 4H, H21, H22, H24, H25), 7.75 (d, 2H, H16, H18, J =\n 7.6 Hz), 7.49 (t, 2H, H28, H30, J = 7.6 Hz), 7.39 (t, 1H, H29, J = 7.2 Hz);\n 13\n C NMR (DMSO-d6, 100 MHz): \u03b4 157.67, 153.94, 151.54, 148.41, Synthesis of 4-(1-benzyl-1H-tetrazol-5-yl) Benzaldehyde (6)\n 143.56, 140.26, 137.93, 134.55, 130.45, 130.26, 129.58, 126.78,\n Firstly, in a 50 mL pear-shaped round-bottom flask, 0.2 g tetrazole\n 124.01, 121.94; ESI-MS (MeOH): observed m/z = 449.1767 [M + H] +,\n 6\u2019 (1.15 mmol), benzyl bromide (1.15 mmol), and K2CO3 (2.87 mmol)\n 471.1588 [M + Na] +, calcd m/z = 449.1766 [M + H] +, 471.1586 [M +\n were combined in 10 mL of DMF and agitated at 0 \u00b0C until the\n Na] +.\n conversion was finished. The progression of the reaction was\n analyzed using TLC (1 : 4 ethyl acetate: n-hexane). After completion\n Synthesis of [1,1\u2019 : 4\u2019,1\u2019\u2019-terphenyl]-4-carbaldehyde (5) of the reaction, the mixture was added with 100 mL of ethyl acetate\n and stirred vigorously. Then the combined organic layer was\n The compound 5 was synthesized by modifying our previously washed several times with water and brine. To obtain the relevant\n reported procedure.[46] In a round-bottomed flask, A mixture of reaction product 6, the organic layer was dried over Na2SO4,\n 0.2 g of 4-bromobenzaldehyde (1.08 mmol), 4-biphenylboronic acid filtered, and then the solvent was dried under a vacuum. The crude\n (1.30 mmol), tetrakis(triphenylphosphine)palladium(0) (0.05 mmol), product was then purified over a brief pad of SiO2. Yield: 85 %;\n and K2CO3 (5.40 mmol) was dissolved in 15 mL of toluene and kept Colour: White; Mp: 110 \u00b0C; Rf (1 : 4 Ethyl acetate: n-Hexane): 0.75;\n 1\n for stirring at 110 \u00b0C for 4 h. After completion, the reaction mixture H NMR (CDCl3, 400 MHz): \u03b4 10.06 (s, 1H, CHO proton), 8.32 (d, 2H,\n was allowed to cool down to room temperature and added with H2, H6, J = 8.4 Hz), 7.99 (d, 2H, H3, H5, J = 8.4 Hz), 7.42 (t, 2H, H11, H13,\n 100 mL of ethyl acetate. The combined organic layer was washed J = 9.6 Hz), 7.39 (d, 3H, H10, H12, H14, J = 7.2 Hz), 5.83 (s, 2H, H8);\n 13\n with water and saturated NaHCO3 solution. Under reduced C NMR (DMSO-d6, 100 MHz): \u03b4 191.66, 164.38, 137.40, 133.06,\n pressure, the mixed organic layer was concentrated after being 132.79, 130.20, 129.14, 129.11, 128.50, 127.43, 57.07.\n dried with anhydrous Na2SO4 and purified by column chromatog-\n raphy on silica gel. Yield: 75 %; Colour: Ivory White; Mp: 120 \u00b0C; Rf\n (1 : 4 Ethyl acetate - Hexane): 0.64; 1H NMR (CDCl3, 400 MHz): \u03b4 10.07 General Procedure for the Synthesis of Rhenium (I) Metal\n (s, 1H, CHO proton), 7.99 (d, 2H, H2, H6, J = 8.4 Hz), 7.82 (d, 2H, H3, Complexes (ReL1-ReL6)\n H5, J = 8.0 Hz), 7.73 (s, 4H, H8, H9, H11, H12), 7.67 (d, 2H, H14, H18, J =\n The metal complexes were synthesized by modifying our previously\n 6.8 Hz), 7.48 (t, 2H, H15, H17, J = 7.6 Hz), 7.39 (t, 1H, H16, J = 7.2 Hz);\n 13 reported procedure.[46] Initially, 40 mg (0.110 mmole, 1 equiv.) of\n C NMR (CDCl3, 100 MHz): \u03b4 191.98, 146.71, 141.40, 140.33, 138.52,\n pentacarbonylchlororhenium(I) and 1.1 equiv. of the previously\n 135.24, 130.37, 128.93, 127.77, 127.75, 127.69, 127.55, 127.10.\n produced ligand (L1-L6) were added to the reaction vessel\n 2-(4-(1-benzyl-1H-tetrazol-5-yl)phenyl)-1H-imidazo[4,5- containing 15 ml of toluene. Afterward, the entire mixture was\n f][1,10]phenanthroline (L6): Yield: 90 %; Colour: Orange; Mp: subjected to reflux for 6 hours while being stirred constantly. The\n 260 \u00b0C; Rf (pure methanol): 0.65; IR (cm 1): \u03c5 N H stretching progress of the reaction was evaluated using TLC with 100 %\n (3394.77), Ar C H stretching (3020.83), sp3 CH stretching (2857.23), methanol as the solvent solution. Once the reaction was complete,\n C=N stretching (1560.45), Ar C=C stretching (1425.95), C N the reaction mixture was allowed to cool, and the resulting solid\n stretching (1332.46), Ar C H bending (721.17); 1H NMR (DMSO-d6, was filtered out and washed with hexane several times. After\n 400 MHz): \u03b4 9.02 (d, 2H, H1, H10, J = 2.8 Hz), 8.96 (d, 2H, H3, H8, J = purifying the product and recrystallizing it, the yellow-colored fine\n 8.0 Hz), 8.48 (d, 2H, H15, H19, J = 8.0 Hz), 8.24 (d, 2H, H16, H18, J = crystals were obtained with a yield of 85-95 %. The synthesized\n 8.4 Hz), 7.83 (dd, 2H, H2, H9, J = 8.0 Hz), 7.45 \u2013 7.39 (m, 5H, H23, H24, complexes (ReL1-ReL6) were characterized using various spectro-\n H25, H26, H27), 6.04 (s, 2H, H21); 13C NMR (DMSO-d6, 100 MHz): \u03b4 scopic techniques to confirm their structure.\n 164.52, 150.66, 148.20, 144.12, 134.58, 132.77, 130.19, 129.41,\n [(CO)3ReICl(K2-N,N-2,4-dichloro-6-(1H-imidazo[4,5-\n 129.12, 128.84, 127.72, 127.61, 127.38, 123.74, 122.25, 56.69; ESI-MS\n f][1,10]phenanthrolin-2-yl)phenol)] (ReL1): Yield: 88 %; Color:\n (MeOH): observed m/z = 455.1733 [M + H] +, 477.1542 [M + Na] +,\n Chrome Yellow; Mp: 218 \u00b0C; Rf (pure methanol): 0.72; IR (cm 1): \u03c5 Ar\n calcd m/z = 455.1733 [M + H] +, 477.1552 [M + Na] +.\n O H stretching (3611.44), N H stretching (3391.64), Ar C H\n stretching (3058.61), C=O stretching (2024.39), C=O stretching\n Synthesis of 4-(1H-tetrazol-5-yl) benzaldehyde (6\u2019) (1922.58, 1891.62), C=N stretching (1615.65), Ar C=C stretching\n (1410.19), C N stretching (1257.63), C Cl stretching (814.41), Ar\n The method used to create tetrazole 6\u2019 (as seen in Scheme 1) serves C H bending (725.89); 1H NMR (DMSO-d6, 400 MHz): \u03b4 9.39 (s, 2H,\n as an example. Initially, 0.3 g 4-formylbenzonitrile (2.29 mmol) was H1, H10), 9.12 (s, 2H, H3, H8), 8.12 (s, 2H, H2, H9), 7.99 (s, 1H, H15), 7.37\n taken in a 100 mL pear-shaped round-bottom flask. Then 0.6 g (s, 1H, H17); 13C NMR (DMSO-d6, 100 MHz): \u03b4 198.35, 195.19, 190.61,\n sodium azide (9.23 mmol), 100 mg copper iodide (CuI) as a catalyst, 151.57, 144.19, 141.76, 133.17, 132.85, 131.70, 129.18, 126.87,\n and 30 mL dimethylformamide (DMF) were added to it. Subse- 125.78, 124.61, 122.79; ESI-MS (MeCN): observed m/z = 685.9802\n quently, the reaction mixture was kept for stirring at 120 \u00b0C for [M H] +, 687.9813 [M + H] +, calcd m/z = 685.9325 [M H] +, 687.9110\n 30 h. The progression of the reaction was analyzed using TLC (1 : 4 [M + H] + ; Anal. Calcd for C22H10Cl3N4O4Re: C, 38.47; H, 1.47; N, 8.16.\n ethyl acetate: n-hexane). After completion of the reaction, the Found: C, 38.41; H, 1.46; N, 8.19.\n mixture was allowed to cool down to room temperature and was\n extracted with acidified water (acidified with 20 % aq. HCl) and [(CO)3ReICl(K2-N,N-2-(4-bromophenyl)-1H-imidazo[4,5-\n ethyl acetate. The combined organic layer was separated and f][1,10]phenanthroline] (ReL2): Yield: 86 %; Color: Yellow; Mp:\n washed with water and brine. To obtain the relevant reaction 220 \u00b0C; Rf (pure methanol): 0.68; IR (cm 1): \u03c5 N H stretching\n\n\n\n Chem. Eur. J. 2025, 31, e202401720 (17 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n (3439.12), Ar C H stretching (3130.27), C=O stretching (2023.53), C34H20ClN4O3Re: C, 54.15; H, 2.67; N, 7.43. Found: C, 54.08; H, 2.60; N,\n C=O stretching (1884.73), C=N stretching (1573.64), Ar C=C 7.40.\n stretching (1453.89), C N stretching (1366.09), Ar C H bending\n (692.71), C Br stretching (644.17); 1H NMR (DMSO-d6, 400 MHz): \u03b4 [(CO)3ReICl(K2-N,N-2-(4-(1-benzyl-1H-tetrazol-5-yl)phenyl)-1H-\n imidazo[4,5-f][1,10]phenanthroline] (ReL6): Yield: 92 %; Color:\n 9.38 (d, 2H, H1, H10, J = 3.6 Hz), 9.25 (d, 2H, H3, H8, J = 7.6 Hz), 8.25 (d,\n Lemon Yellow; Mp: 278 \u00b0C; Rf (pure methanol): 0.70; IR (cm 1): \u03c5\n 2H, H15, H19, J = 7.6 Hz), 8.17 (d, 2H, H2, H9, J = 4.0 Hz), 7.88 (d, 2H,\n H16, H18, J = 6.4 Hz); 13C NMR (DMSO-d6, 100 MHz): \u03b4 198.19, 195.76, N H stretching (3330.29), Ar C H stretching (3184.43), sp3 CH\n 190.63, 152.18, 151.58, 144.19, 137.64, 133.12, 132.95, 132.44, stretching (3026.47), C=O stretching (2021.57), C=O stretching\n 131.67, 129.15, 126.81, 125.70, 122.77; ESI-MS (MeCN): observed (1899.01), C=N stretching (1607.27), Ar C=C stretching (1437.23),\n m/z = 680.9338 [M + H] +, 685.9855 [(M Cl)(CH3CN)] +, 702.9177 [M + C N stretching (1367.12), Ar C H bending (724.81); 1H NMR (DMSO-\n Na] +, calcd m/z = 680.9160 [M + H] +, 685.9838 [(M Cl)(CH3CN)] +, d6, 400 MHz): \u03b4 14.47 (s, 1H, -NH proton), 9.38 (d, 2H, H1, H10, J =\n 702.9158 [M + Na] +; Anal. Calcd for C22H11BrClN4O3Re: C, 38.81; H, 4.8 Hz), 9.27 (d, 2H, H3, H8, J = 7.6 Hz), 8.48 (d, 2H, H15, H19, J =\n 1.63; N, 8.23. Found: C, 38.78; H, 1.61; N, 8.17. 8.4 Hz), 8.33 (d, 2H, H16, H18, J = 8.0 Hz), 8.15 (s, 2H, H2, H9), 7.46\u20137.39\n (m, 5H, H23, H24, H25, H26, H27), 6.06 (s, 2H, H21); 13C NMR (DMSO-d6,\n [(CO)3ReICl(K2-N,N-2-bromo-6-(1H-imidazo[4,5- 100 MHz): \u03b4 198.29, 195.62, 190.62, 164.37, 152.18, 151.94, 144.38,\n f][1,10]phenanthrolin-2-yl)-4-nitrophenol] (ReL3): Yield: 90 %; Col- 134.54, 133.36, 131.61, 129.43, 129.16, 128.88, 128.67, 128.57,\n or: Dark Yellow; Mp: 258 \u00b0C; Rf (pure methanol): 0.66; IR (cm 1): \u03c5 Ar 127.75, 127.63, 127.09, 125.78, 56.76; ESI-MS (MeCN): observed\n O H stretching (3579.11), N H stretching (3351.23), Ar C H m/z = 766.1305 [(M Cl)(CH3CN)] +, calcd m/z = 766.1325\n stretching (3150.03), C=O stretching (2017.24), C=O stretching [(M Cl)(CH3CN)] +; Anal. Calcd for C30H18ClN8O3Re: C, 49.72; H, 2.50;\n (1894.48), C=N stretching (1593.41), Ar C=C stretching (1430.79), N, 15.46. Found: C, 49.68; H, 2.59; N, 15.42.\n N=O stretching (1401.23), C N stretching (1363.14), N O stretching\n (1179.48), Ar C H bending (722.98), C Br stretching (691.87);\n 1\n H NMR (DMSO-d6, 400 MHz): \u03b4 9.39 (s, 1H, H17), 9.26 (s, 1H, -OH Biology\n proton), 9.09 (d, 2H, H1, H10, J = 21.2 Hz), 8.14 (s, 1H, -NH proton),\n 7.24 (d, 2H, H2, H9, J = 6.8 Hz), 7.18 (d, 3H, H3, H8, H15, J = 7.6 Hz); Cell Culture\n 13\n C NMR (DMSO-d6, 100 MHz): \u03b4 197.41, 194.66, 190.54, 157.81,\n 152.63, 148.04, 143.89, 136.85, 135.08, 131.12, 130.15, 126.52, The cells were cultured in DMEM media (Gibco) with 10 % fetal\n 123.69; 121.94, 119.40, 117.47; ESI-MS (MeCN): observed m/z = bovine serum (Himedia, India), 1 % penicillin and streptomycin, and\n 741.7419 [M + H] +, calcd m/z = 741.7139 [M + H] +; Anal. Calcd for 1 % Glutmax (Gibco, Thermo Scientific, USA) at 37 \u00b0C in 5 % CO2. The\n C22H10BrClN5O6Re: C, 35.62; H, 1.36; N, 9.44. Found: C, 35.58; H, 1.29; cells were trypsinized with 0.25 percent trypsin-EDTA when they\n N, 9.38. achieved 70 %\u201380 % confluency (Thermo Fisher Scientific, USA).\n\n [(CO)3ReICl(K2-N,N-2-(2-chloro-8-methylquinolin-3-yl)-1H-\n imidazo[4,5-f][1,10]phenanthroline] (ReL4): Yield: 85 %; Color: In Vitro Cytotoxic Study\n Yellow Ochre; Mp: 240 \u00b0C; Rf (pure methanol): 0.76; IR (cm 1): \u03c5 N H\n stretching (3306.92), Ar C H stretching (3178.78), sp3 CH stretch- The in vitro cytotoxicity of complexes (ReL1-ReL6) was investigated\n ing (3044.19), C=O stretching (2027.96), C=O stretching (1922.38, by standard MTT assay protocol. The complexes were first dissolved\n 1875.64), C=N stretching (1641.93), Ar C=C stretching (1443.68), in 0.1 % DMSO, and then diluted in DMEM medium. The triple\n C N stretching (1361.47), C Cl stretching (899.69), Ar C H bending negative breast cancer cell line, MDA-MB-231, as well as one normal\n (730.45); 1H NMR (DMSO-d6, 400 MHz): \u03b4 11.50 (s, 1H, -NH proton), fibroblast (MRC-5) were used for the investigation. In 96-well plates,\n 9.36 (d, 1H, H1, J = 4.4 Hz), 9.12 (d, 1H, H22, J = 4.4 Hz), 8.98 (d, 1H, approximately 1\u00d7104 cells per well were cultivated in 100 \u03bcL of\n H20, J = 12.0 Hz), 8.11 (d, 2H, H3, H8, J = 8.0 Hz), 7.73 (d, 1H, H18, J = growth media and then incubated at 37 \u00b0C in a 5 % CO2 environ-\n 7.2 Hz), 7.43 (d, 1H, H10, J = 7.2 Hz), 7.35 (d, 1H, H19, J = 7.2 Hz), 7.15 ment. The cells were subjected to a variety of concentrations,\n (t, 1H, H2, J = 7.6 Hz), 7.08 (t, 1H, H9, J = 7.6 Hz), 2.40 (s, 3H, Methyl commencing with an initial concentration of 200 \u03bcM, and sub-\n protons); 13C NMR (DMSO-d6, 100 MHz): \u03b4 197.75, 196.91, 190.75, sequently, a serial dilution was performed for all the complexes.\n 161.23, 150.82, 146.37, 140.47, 137.68, 133.54, 129.34, 127.70, This treatment was carried out in a 0.1 % DMSO-containing media\n 125.68, 124.24, 122.99, 119.52, 17.82; ESI-MS (MeCN): observed for duration of 4 h, all while being conducted in a light-restricted\n m/z = 706.0426 [(M Cl)(CH3CN)] +, 722.0082 [M + Na] +, 689.1051 environment. In one set of cells, was maintained in darkness with\n [(M CO)(OH)] +, calcd m/z = 706.0530 [(M Cl)(CH3CN)] +, 722.9851 the fresh DMEM medium for an additional 44 h; thus, cell cultures\n [M + Na] +, 689.0031 [(M CO)(OH)] +; Anal. Calcd for C26H14Cl2N5O3Re: were incubated for 5 h at 37 \u00b0C with 100 \u03bcL of MTT reagent (1 mg/\n C, 44.51; H, 2.01; N, 9.98. Found: C, 44.46; H, 1.96; N, 9.92. mL). The cells in the control wells also consumed the same volume\n of DMSO-DMEM. In another set of cells, following the exclusion of\n [(CO)3ReICl(K2-N,N-2-([1,1\u2019 : 4\u2019,1\u2019\u2019-terphenyl]-4-yl)-1H-imidazo[4,5- the compound-containing medium, 150 \u03bcL of DPBS was added to\n f][1,10]phenanthroline] (ReL5): Yield: 90 %; Color: Sandal Yellow; each well for light irradiation. Subsequently, the cells were exposed\n Mp: 224 \u00b0C; Rf (pure methanol): 0.78; IR (cm 1): \u03c5 N H stretching under yellow light (0\u20132 J.cm 2) from a 400 W tungsten lamp fitted\n (3367.47), Ar C H stretching (3152.51), C=O stretching (2019.55), with heat isolation filter and 500 nm long pass filter at an intensity\n C=O stretching (1885.09), C=N stretching (1591.30), Ar C=C of 4 m W/cm2 for 4 hours, and the cells were incubated in a CO2\n stretching (1436.65), C N stretching (1358.67), Ar C H bending incubator for 40 hours. After that, the suspension from both the\n (722.11); 1H NMR (DMSO-d6, 400 MHz): \u03b4 14.34 (s, 1H, -NH proton), sets was placed on a micro vibrator for 10 minutes, and the\n 9.38 (d, 2H, H1, H10, J = 4.0 Hz), 9.27 (d, 2H, H3, H8, J = 8.0 Hz), 8.41 (d, absorbance was measured in an ELISA plate reader at 570 nm.\n 2H, H15, H19, J = 8.4 Hz), 8.15 (t, 2H, H27, H31, J = 7.6 Hz), 8.05 (d, 2H, Similar protocol has been followed in hypoxic environment. The\n H2, H9, J = 8.4 Hz), 7.94 (d, 2H, H16, H18, J = 8.4 Hz), 7.84 (d, 2H, H21, experiment was likewise carried out three times. Photofrin was\n H25, J = 8.4 Hz), 7.76 (d, 2H, H22, H24, J = 7.6 Hz), 7.50 (t, 2H, H28, H30, used as a standard positive control in this experiment. The data\n J = 7.6 Hz), 7.39 (t, 1H, H29, J = 7.6 Hz); 13C NMR (DMSO-d6, 100 MHz): were reported as a percentage of growth inhibition, i. e. % growth\n \u03b4 198.26, 196.95, 190.54, 155.96, 152.97, 151.80, 144.27, 134.0, inhibition = 100 [(AD\u00d7100)/AB], where AD represents measured\n 133.42, 131.09, 130.77, 128.90, 128.78, 127.83, 127.11, 126.97, absorbance in sample wells and AB represents measured absorb-\n 126.41, 125.71, 123.17; ESI-MS (MeCN): observed m/z = 755.4911 [M ance in blank wells (cells with a medium and a vehicle).\n + H] +, calcd m/z = 755.4240 [M + H] +; Anal. Calcd for\n\n\n Chem. Eur. J. 2025, 31, e202401720 (18 of 20) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 6, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202401720 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.202401720\n\n\n Statistical Analysis [3] W. Ma, L. Guo, Z. Tian, S. Zhang, X. He, J. Li, Y. Yang, Z. Liu, Dalt. Trans.\n 2019, 48, 4788\u20134793.\n All experiments were conducted in triplicate, and appropriate [4] J. Li, L. Guo, Z. Tian, S. Zhang, Z. Xu, Y. Han, R. Li, Y. Li, Z. Liu, Inorg.\n statistical tests were used for comparisons. One-way ANOVA was Chem. 2018, 57, 13552\u201313563.\n utilised to compare the various groups, and multiple comparison [5] R. Ramos, J. F. 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