Enhancing Bioactivity of <i>N,N,N</i>-Chelating Rhodium(III) Complexes with Ionic Liquids: Toward Targeted Cancer Therapy.

{"full_text": " pubs.acs.org/jmc Article\n\n\n\n Enhancing Bioactivity of N,N,N-Chelating Rhodium(III) Complexes\n with Ionic Liquids: Toward Targeted Cancer Therapy\n Angelina Cakovic,\u0301 Dus\u030can C\u0301 oci\u0301 c,\u0301 Marko Z\u030c ivanovic,\u0301 Nenad Jankovic,\u0301 Nevena Milivojevic,\u0301 Marija Delibas\u030cic,\u0301\n Marina Kostic,\u0301 Ivana Radojevic,\u0301 Mirjana Grujovic,\u0301 Katarina G. Markovic,\u0301 Olivera R. Klisuric,\u0301\n Milan Vranes\u030c, and Jovana Bogojeski*\n Cite This: J. Med. Chem. 2024, 67, 13349\u221213362 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n\n\n ABSTRACT: This study investigates the potential of using ionic\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 13:48:28 (UTC).\n\n\n\n\n liquids as cosolvents to enhance the solubility and activity of poorly\n soluble rhodium(III) complexes, particularly those with diene, pyridine\n derivatives, and camphor-derived bis-pyrazolylpyridine ligands, in\n relation to 5\u2032-GMP, CT-DNA, and HSA as well as their biological\n activity. Findings indicate that ionic liquids significantly increase the\n substitution activity of these complexes toward 5\u2032-GMP while only\n marginally affecting DNA/HSA binding affinities with molecular\n docking, further confirming the experimental results. Lipophilicity\n assessments indicated good lipophilicity. Notably, cytotoxicity studies show that Rh2 is selectively effective against HeLa cancer\n cells, with IL1 and IL10 modulating the cytotoxic effects. Redox evaluations indicate that rhodium complexes induce oxidative stress\n in cancerous cells while maintaining redox balance in noncancerous cells. By elucidating the role of ionic liquids in modulating these\n effects, the study proposes a promising avenue for augmenting the efficacy and selectivity of cancer treatments, thus opening new\n horizons in cancer therapeutics.\n\n\n \u25a0 INTRODUCTION\n Transitional metal complexes have played a crucial role in the\n including cancer treatment, is undeniable.20,21 The DNA\n molecule is one of the primary intracellular targets for the\n treatment and diagnosis of cancer, owing to the success of activity of anticancer transition metal complexes, with the\n possibility of interacting by intercalation, groove binding, or\n cisplatin in cancer therapy worldwide.1\u22123 However, not many\n electrostatic interactions.17,22\u221225 The potential use of transition\n transition complexes besides cisplatin are used in cancer\n metal complexes in therapy is significantly limited by a key\n therapy because of their toxic effects, poor solubility, and the\n drawback: the tendency of these metals to form sparingly\n development of resistance.4 The toxic effects of cisplatin and\n soluble complexes. This drawback severely restricts their\n the resistance to its application are the main reasons for the\n efficacy and is a major challenge that must be overcome to\n worldwide investigation of different metal complexes as\n realize their full potential in medical treatment. One way to\n anticancer agents.4 Metals like rhodium, ruthenium, osmium,\n enhance complexes\u2019 solubility is through using cosolvents.\n and iridium are especially interesting in this area of\n Among these, ionic liquids (ILs) have emerged as a particularly\n research.5\u221210 At the early stages of anticancer research,\n viable option, earning them the nickname \u201cgreen solvents of\n rhodium was not considered interesting to the public due to\n the future\u201d.26,27 Composed entirely of ions and possessing low\n its inert nature. However, since the 70s, there has been a\n melting points, ILs are less harmful than conventional organic\n growing interest in the potential of rhodium complexes as\n solvents and are considered ecologically sound. Thanks to their\n anticancer agents. This interest has evolved from binuclear\n ion-based composition, these solvents can be modified to be\n rhodium(II) complexes to mononuclear species that contain\n nontoxic and biocompatible with the human body. The most\n Rh(I) and finally Rh(III) ions.11\u221217 Rhodium complexes are famous ILs consist of cations like choline and different\n unequivocally one of the most promising potential anticancer biocompatible anions.27\u221229 Given their unique properties,\n agents available today.17\u221219 Their inertness provides the\n opportunity for a complex design that can specifically target\n molecules, such as DNA, proteins, or enzymes, making them Received: May 28, 2024\n highly versatile and effective in fighting cancer. Revised: July 12, 2024\n The interaction between transition metal complexes and Accepted: July 19, 2024\n DNA molecules is an essential and exciting research area in Published: July 26, 2024\n bioinorganic chemistry, medicine, and pharmacy. Its potential\n to unlock new treatment methods for various diseases,\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.jmedchem.4c01220\n 13349 J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nionic liquids have the potential to be highly effective cosolvents Table 1. Structures and Names of the Investigated Ionic\nfor sparingly soluble complexes that exhibit anticancer activity. Liquids\nThe fact that ILs are ecofriendly and less harmful than\nconventional organic solvents and that they can be modified to\nbe nontoxic and biocompatible with the human body makes\nthem a promising option for enhancing the solubility of such\ncomplexes.\n In this research, we explore the influence of nontoxic\ncosolvents (ILs) on the structure\u2212activity relationship of\nrhodium(III) complexes containing N,N,N-ligands toward\nDNA and HSA molecules, as well as 5\u2032-GMP molecules, and\nassess their impact on biological activity.\n\n\u25a0 RESULTS AND DISCUSSION\n Synthesis and Characterization. The structures of the\nexamined Rh(III) complexes are illustrated in Figure 1. The\n\n\n\n\nFigure 1. Structural formulas of the investigated Rh1\u22124 complexes.\n\ncomplex Rh2 was synthesized and characterized in this work,\nwhereas the complexes Rh1, Rh3, and Rh4 were synthesized\naccording to previously published procedures.14,30,31 The Rh2\ncomplex was synthesized by stirring equimolar amounts of\nRhCl3\u00b7xH2O and bpma (bis(pyridin-2-ylmethyl)amine) ligand\nin ethanol, followed by refluxing overnight. The synthesized\ncomplex was characterized by NMR spectroscopy, elemental\nanalysis, and X-ray diffraction. The elemental analysis results\nclosely matched the proposed composition of the complex.\nThe NMR spectra of the complex indicated the formation of a\nsingle, distinct species. Additionally, single crystals of the Rh2\ncomplex, suitable for X-ray diffraction analysis, were obtained.\nFurther details are provided in the Experimental Section.\n It is worth noting that the selection process began with the\ndiene type, followed by complexes that feature pyridine and\nthen camphor.14 Pyridine is known for its \u03c0-back-donation,32,33\nwhich heightens the electrophilicity of the metal center and\ncan improve its binding affinity. The fourth complex that is family of ionic liquids, whereas IL8\u221211 are classified as\nexamined contains camphor-derived bis-pyrazolylpyridine. agmatine-based ionic liquids.36\nCamphor, known for its historical use in certain cultures for Crystal Structure Analysis. The crystallographic structure\nperceived medicinal benefits, has found its way into topical of complex [Rh(bpma)Cl3] (Rh2) with the adopted atom-\nproducts due to its cooling sensation and is occasionally numbering scheme is shown in Figure 2. Complex Rh2\nutilized for its decongestant properties. crystallizes in the triclinic crystal system and P1\u0305 space group, in\n Table 1 contains information on the structures and names of which the asymmetric part of the unit cell contains one neutral\nthe examined ionic liquids (ILs). It is important to mention molecule. Selected bond lengths, bond angles, and dihedral\nthat IL1 was obtained commercially, and IL2\u221211 are nontoxic angles of Rh2 are listed in Table S1.\nionic liquids that were synthesized according to published The geometrical structure of complex Rh2 is best defined by\nprocedures.34,35 Notably, IL2\u22127 belong to the choline-based the coordination of Rh2 and the angle between the mean\n 13350 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n Scheme 1. Schematic Representation of the Substitution\n Reactions for the Complexes Rh1\u22124 with 5\u2032-GMP as a\n Nucleophile\n\n\n\n\n Table 2. Rate Constants of the Substitution Reactions of the\n Complexes Rh1\u22124 with 5\u2032-GMP in the Presence of Ionic\n Liquids IL1\u221211 and DMF as Cosolvents (Complex\u2212Ionic\n Liquid Ratio Was 1:1, 1 \u00d7 10\u22124 M) at pH = 7.2 (25 mM\nFigure 2. MERCURY37 drawing of the molecular structure of HEPES Buffer) in the Presence of 50 mM NaCl\ncomplex Rh2 with labeled non-H atoms. Displacement ellipsoids are\n Rh1 Rh2 Rh3 Rh4\nshown at 30% probability.\n 105k2 105k2 105k2 105k2\n \u22121 \u22121 \u22121 \u22121 \u22121 \u22121\n (M s ) (M s ) (M s ) (M\u22121 s\u22121)\nplanes of the phenyl rings (Table S1). The rhodium(III) DMF 0.32 \u00b1 0.01 0.19 \u00b1 0.02 0.40 \u00b1 0.01 0.78 \u00b1 0.01\ncenter in the [Rh(bpma)Cl3] complex is coordinated in a IL1 3.95 \u00b1 0.02 2.90 \u00b1 0.02 3.10 \u00b1 0.03 5.24 \u00b1 0.01\nslightly distorted octahedral geometry. The tridentate N,N,N- IL2 3.93 \u00b1 0.01 4.46 \u00b1 0.01 4.93 \u00b1 0.01 6.93 \u00b1 0.04\ndonor from the bpma (bis(pyridin-2-ylmethyl)amine) ligand IL3 4.11 \u00b1 0.03 3.29 \u00b1 0.01 3.60 \u00b1 0.01 4.84 \u00b1 0.03\ncoordinates facially to the rhodium(III) core, and the other IL4 4.13 \u00b1 0.01 1.52 \u00b1 0.01 1.70 \u00b1 0.01 3.16 \u00b1 0.03\nthree positions are occupied by chlorine atoms. The N,N,N- IL5 3.33 \u00b1 0.03 1.66 \u00b1 0.02 2.10 \u00b1 0.01 3.45 \u00b1 0.01\ndonor from the bpma ligand is involved in chelation to form IL6 4.06 \u00b1 0.01 3.44 \u00b1 0.04 3.80 \u00b1 0.01 5.34 \u00b1 0.02\ntwo five-membered rings. IL7 2.91 \u00b1 0.01 1.84 \u00b1 0.02 2.40 \u00b1 0.01 3.42 \u00b1 0.02\n Comparing the bond lengths (Table S1) of the coordinated IL8 0.71 \u00b1 0.02 0.72 \u00b1 0.04 1.30 \u00b1 0.01 2.35 \u00b1 0.01\nnitrogen atoms, one can notice slight variations in the lengths IL9 1.31 \u00b1 0.01 1.03 \u00b1 0.03 1.50 \u00b1 0.03 2.85 \u00b1 0.01\nof Rh\u2212N bonds [from 2.023 (3) to 2.053 (3) \u00c5] where the IL10 0.80 \u00b1 0.02 0.47 \u00b1 0.01 0.70 \u00b1 0.02 1.53 \u00b1 0.02\naxial nitrogen is placed at the longest distance from the metal IL11 0.93 \u00b1 0.03 0.63 \u00b1 0.01 0.90 \u00b1 0.02 2.17 \u00b1 0.01\ncenter. The same can be concluded when it comes to chlorine\natoms, where the Rh\u2212Cl bond lengths vary from 2.3519 (10) were obtained by plotting the linear relationship between kobsd\nto 2.3544 (11) \u00c5 (Table S1). and the total nucleophile concentration (eq S1). By\n The two phenyl rings of the bpma ligand are nearly examination of the slope of the kobsd vs nucleophile\nperpendicular, as confirmed by the value 83.37 (2)\u00b0 of the concentration graph, the second-order rate constant (k2) can\nangle between their mean planes. Other conformation be calculated, providing insight into product formation.\nparameters are given in Tables S1 and S2. Cosolvents play a crucial role in the solubility and stability of\n Kinetic Studies. The reactivity of complexes Rh1\u22124 sparingly soluble complexes. In this study, ionic liquids were\n(Figure 1) was measured by performing substitution reactions found to be effective cosolvents for Rh1\u22124 complexes. The\nwith 5\u2032-GMP under physiological conditions with different ILs substitution rate constant values for all studied complexes\nas cosolvents. By examining the substitution with 5\u2032-GMP, it is increased significantly when ionic liquids were used, surpassing\npossible to assess the potential for coordination with DNA even the standard organic solvent DMF. Notably, the addition\nmolecules. Consequently, DNA emerges as a prospective target of only a small amount of ionic liquids to the solution was\nfor the activity of the investigated complexes.14,23,35 enough to enhance the solubility of the tested complexes, and\n The substitution reaction of the labile chloride ligand was higher concentrations can be achieved if needed, owing to the\nfollowed by using UV\u2212vis spectroscopy, where the changes in nontoxic properties of the ionic liquids.\nabsorbance were measured as a function of time at pH = 7.2 Among the ionic liquids tested, IL2\u22127 containing choline\n(25 mM HEPES buffer/50 mM NaCl) and T = 310 K. The ions were found to be the most effective in increasing the\nproposed reaction pathways for all observed substitution constant values, specifically IL4 and IL3 for Rh1 and IL2 and\nprocesses of the examined complexes Rh1\u22124 are presented IL6 for Rh2\u22124. The electronic and steric impacts of the ionic\nin Scheme 1. The nucleophile concentrations were kept at least liquids were found to play a crucial role in substitution,\n10-fold in excess to ensure accurate results, allowing the facilitating the departure of chloride ions, stabilizing the\nreactions to be studied under pseudo-first-order conditions. complex, and improving coordination with the 5\u2032-GMP\n The experiments aimed to evaluate the effect of ionic liquids, molecule. Interestingly, the tested ILs did not stop but instead\nspecifically IL1\u221211, on the constant values of k2 of the enhanced the coordination of the 5\u2032-GMP molecule to Rh1\u22124\nsubstitution reactions. To achieve this, the reactions were complexes, making them ideal cosolvents for sparingly soluble\nconducted with ionic liquids as cosolvents, added in the same complexes. Furthermore, the tested ionic liquids are nontoxic,\namount as the studied complexes to maintain the same making them ideal for biological applications. IL2\u221211 contain\nconcentrations (ratio 1:1, conc. 1 \u00d7 10\u22124 M). Displayed in biologically friendly cations (choline and agmatine) and anions\nTable 2 are the substitution rate constants, which were (nicotinate, ascorbate, biotinate, etc.), which are nontoxic to\ndetermined using pseudo-first-order conditions. These values the human body, while IL1 was commercially obtained for\n 13351 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\ncomparison. The results show that the constant values k2 of observed from previously published results.13,14 The highest\ncomplexes Rh1\u22124 for IL2\u22127 were higher than those for IL1, value of the binding constant for complex Rh1 was observed in\nwhile those for IL8\u221211 were lower. In conclusion, the tested the presence of IL3 and IL4, while for complexes Rh2\u22124, the\nionic liquids are effective cosolvents for sparingly soluble highest value was observed in the presence of IL2 and IL6,\ncomplexes and can be used to enhance their solubility and which is consistent with the kinetic results. These findings\neven their biological activity. The biocompatible and nontoxic suggest that ionic liquids had a limited effect on the DNA\nproperties of these ionic liquids make them ideal for biological interaction of complexes Rh1\u22124, with Kb values remaining in\napplications, and their effectiveness in increasing constant the same order of reactivity of 104.\nvalues makes them a promising alternative to conventional The bulky structure of the DNA molecule is likely the\norganic solvents. primary factor that has limited the increase in the constant\n DNA Binding Studies. Electronic Absorption Method. value. However, increasing the concentration of ionic liquids\nElectronic absorption spectroscopy is frequently employed can potentially overcome this limitation and lead to a\nwhen investigating the bonding between complexes and DNA. substantial increase in the value. Although the DNA structure\nThe nature of the interaction between transition metal and the small concentration of the applied ILs present some\ncomplexes and DNA can be covalent or noncovalent.38,39 By challenges, they are still highly promising candidates for the\nexamining the changes in the absorption spectra of the cosolvents of the examined complexes.\ncomplex in response to increasing DNA concentration, it is Fluorescence Spectroscopic Methods. It is worth noting\npossible to determine the type of interaction occurring. These that while absorption spectroscopic measurements can confirm\nchanges may include a hypochromic shift, a hyperchromic shift the interaction between a complex and a DNA molecule,42\nin absorption intensity, and a slight shift in the absorption fluorescence measurements are a far more reliable method for\nwavelength maximum.20,40 To further explore the effect of determining the binding mode. Intercalation, a type of DNA\nusing ionic liquids as cosolvents in the interaction between interaction, can be determined using fluorescence quenching\ncomplexes Rh1\u22124 and DNA, absorption titration studies were titrations with ethidium bromide (EB), a classical intercalator.\nconducted. The measurements at room temperature were\n EB exhibits low fluorescence intensity in solution, but upon\ntaken with a fixed complex/ionic liquid concentration (ratio of\n intercalation between the DNA chains, it displays a significant\n1:2, 8 \u03bcM) and varying amounts of DNA up to a ratio of 5.35,41\n intensity of fluorescence emission at around 612 nm.40,43\nIt is important to note that ionic liquids do not interact with\n Confirming the interaction between the tested complex and\nDNA independently, which was considered to ensure the\nreliability of the results. DNA through intercalative mode relies heavily on the\n The complexes Rh1\u22124 were investigated and found to have complex\u2019s ability to intercalate more effectively than EB. By\na constant order of 104, indicating moderate interaction with displacing the EB molecule in a DNA\u2212EB cluster, the complex\nDNA. Previous research provided the Kb values for complexes causes a decrease in the fluorescence emission of the solution,\nRh3 and Rh4 in the presence of DMF as a cosolvent. The which serves as a trustworthy indicator of intercalation.42,44\u221246\nresults presented in Table 3 show that ionic liquids had a Hence, a reduction in fluorescence intensity is a reliable\n indicator of whether the examined complex interacts with\nTable 3. DNA Binding Constants, Kb, for the Examined DNA via an intercalative mode.\nComplexes Rh1\u22124 in the Presence of DMF and Ionic The investigation aimed to determine the impact of using\nLiquids IL1\u221211 as Cosolvents (Complex\u2212Ionic Liquid ionic liquids as cosolvents on the ability of complexes Rh1\u22124\nRatio Was 1:2) in PBS to interact with DNA. To achieve this objective, fluorescence\n quenching titrations with EB were conducted in the presence\n Rh1 Rh2 Rh3 Rh4 of ionic liquids IL1\u221211, with a constant concentration of\n 4\n 10 Kb (M ) \u22121 4\n 10 Kb (M ) \u22121 4\n 10 Kb (M ) \u22121\n 10 Kb (M\u22121)\n 4 DNA\u2212EB cluster solution (ratio of DNA to EB is 1:1, 5 \u03bcM)\n DMF 2.6 \u00b1 0.1 4.1 \u00b1 0.1 7.0 \u00b1 0.1 8.3 \u00b1 0.1 while increasing the concentration of Rh1\u22124 complexes (up to\n IL1 2.9 \u00b1 0.1 4.7 \u00b1 0.1 4.3 \u00b1 0.1 5.2 \u00b1 0.1 a ratio of 5). Throughout the process, the emission intensity of\n IL2 2.8 \u00b1 0.1 4.9 \u00b1 0.1 4.6 \u00b1 0.1 5.6 \u00b1 0.1 the DNA\u2212EB cluster solution was monitored, and the Stern\u2212\n IL3 3.1 \u00b1 0.1 4.6 \u00b1 0.1 4.5 \u00b1 0.1 5.4 \u00b1 0.1 Volmer constants for complexes Rh1\u22124 in the presence of\n IL4 3.4 \u00b1 0.1 4.3 \u00b1 0.1 4.1 \u00b1 0.1 5.2 \u00b1 0.1 cosolvents are listed in Table 4.\n IL5 2.8 \u00b1 0.1 4.5 \u00b1 0.1 4.2 \u00b1 0.1 5.3 \u00b1 0.1 The results in Table 4 demonstrate that all applied ILs\n IL6 2.6 \u00b1 0.1 4.7 \u00b1 0.1 4.5 \u00b1 0.1 5.5 \u00b1 0.1 exhibit a minor impact on the Ksv constant. The constants\n IL7 2.7 \u00b1 0.1 4.4 \u00b1 0.1 4.4 \u00b1 0.1 5.1 \u00b1 0.1 remained in the same order of reactivity, which was 104,\n IL8 2.2 \u00b1 0.1 4.3 \u00b1 0.1 3.8 \u00b1 0.1 4.7 \u00b1 0.1 indicating that the presence of ILs barely affected the\n IL9 2.5 \u00b1 0.1 4.1 \u00b1 0.1 3.9 \u00b1 0.1 4.9 \u00b1 0.1 interactions via the intercalative mode. The UV\u2212vis and\n IL10 2.4 \u00b1 0.1 4.2 \u00b1 0.1 3.7 \u00b1 0.1 4.6 \u00b1 0.1 fluorescence spectroscopic measurements showed a good\n IL11 2.5 \u00b1 0.1 4.2 \u00b1 0.1 4.1 \u00b1 0.1 4.5 \u00b1 0.1 stacking of acquired values, thereby confirming the reliability\n of the data. For the complexes Rh3 and Rh4, good value\nminor effect on the Kb values, with the constant order stacking was observed in line with already published results,\nremaining unchanged from when DMF was used. Additionally, further validating the findings. A slight increase in binding\nthe values in the presence of ionic liquids containing agmatine constant values when using IL1\u22127 and a slight decrease in the\nion, IL8\u221211, were significantly lower than those observed in value when using IL8\u221211 can be observed for all examined\nthe presence of ionic liquids containing choline ion, IL2\u22127. complexes. The highest constant value for IL4 (Rh1) and IL2\nFor complexes Rh1 and Rh2, all ILs resulted in a slight (Rh2\u22124) is in accordance with the kinetic and UV\u2212vis\nincrease in the binding constant values. However, for spectroscopic measurements, providing a more robust under-\ncomplexes Rh3 and Rh4, a slight decrease in value was standing of the study\u2019s findings.\n 13352 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nTable 4. Stern\u2212Volmer Constants, Ksv, for the Examined compared to Rh2\u22124, which have multiple. It is well-known\nComplexes Rh1\u22124 in the Presence of DMF and Ionic that, for a compound to intercalate between DNA strands, it\nLiquids IL1\u221211 as Cosolvents (Complex\u2212Ionic Liquid must have a voluminous and rigid structure. Usually,\nRatio Was 1:2, 16 \u03bcM) in PBS complexes intercalate between DNA strands with their organic\n Rh1 Rh2 Rh3 Rh4\n ligand part, so the results obtained were expected due to the\n structure of Rh1. As the number of rings in the tested\n 104Ksv (M\u22121) 104Ksv (M\u22121) 104Ksv (M\u22121) 104Ksv (M\u22121)\n complex\u2019s structure increased, the viscosity increased in the\n DMF 2.2 \u00b1 0.1 3.4 \u00b1 0.1 3.8 \u00b1 0.1 5.5 \u00b1 0.1 order of Rh2 < Rh3 < Rh4, and the difference between Rh2\n IL1 2.6 \u00b1 0.1 3.7 \u00b1 0.1 4.2 \u00b1 0.1 5.6 \u00b1 0.1 and Rh3 was only slight. Although the presence of ILs slightly\n IL2 2.5 \u00b1 0.1 3.8 \u00b1 0.1 4.4 \u00b1 0.1 5.9 \u00b1 0.1 increased the solution\u2019s viscosity, the data presented in Figure\n IL3 2.6 \u00b1 0.1 3.6 \u00b1 0.1 4.3 \u00b1 0.1 5.8 \u00b1 0.1\n 3 demonstrated that the ILs\u2019 presence did not significantly\n IL4 2.7 \u00b1 0.1 3.4 \u00b1 0.1 3.9 \u00b1 0.1 5.6 \u00b1 0.1\n increase the possibility of intercalation of the tested complexes.\n IL5 2.4 \u00b1 0.1 3.5 \u00b1 0.1 4.1 \u00b1 0.1 5.7 \u00b1 0.1\n In summary, the results obtained through UV\u2212vis,\n IL6 2.3 \u00b1 0.1 3.6 \u00b1 0.1 4.3 \u00b1 0.1 5.8 \u00b1 0.1\n fluorescence spectroscopy, and viscosity measurements con-\n IL7 2.3 \u00b1 0.1 3.5 \u00b1 0.1 4.2 \u00b1 0.1 5.5 \u00b1 0.1\n IL8 2.0 \u00b1 0.1 3.4 \u00b1 0.1 3.6 \u00b1 0.1 5.2 \u00b1 0.1\n firm that the tested ILs are appropriate cosolvents for the\n IL9 2.2 \u00b1 0.1 3.2 \u00b1 0.1 3.7 \u00b1 0.1 5.3 \u00b1 0.1\n examined complexes. The constant values of all applied\n IL10 2.1 \u00b1 0.1 3.3 \u00b1 0.1 3.5 \u00b1 0.1 5.2 \u00b1 0.1 biocompatible ion liquids were similar to those obtained in\n IL11 2.3 \u00b1 0.1 3.3 \u00b1 0.1 3.8 \u00b1 0.1 5.1 \u00b1 0.1 the presence of organic solvents. The presence of the selected\n ILs did not affect the way of binding of complexes to DNA.\n Viscosity Measurements. Viscosity measurements are The best results for the complex Rh1 were obtained in the\ncrucial in identifying DNA binding patterns. In classical presence of IL4, while the best results for the complexes Rh2\u2212\nintercalation, the insertion of a compound between DNA bases 4 were obtained in the presence of IL2. Although it should be\nunequivocally results in an increase in the length of the DNA mentioned that all ILs showed similar values, the mentioned\nchain, thereby leading to a corresponding rise in the solution\u2019s ones showed a slightly higher value.\nviscosity.23,47,48 The degree of intercalation is directly propor- Rhodium complexes were analyzed in two distinct DNA\ntional to the increase in DNA viscosity. Experiments were structures: canonical B-DNA (PDB ID: 1BNA) and DNA with\nconducted to confirm that the tested complexes bind via an intercalation gap (PDB ID: 1Z3F). This examination\nintercalation as their DNA interaction method and to test the allowed for the evaluation of two types of interactions: minor\neffect of using ILs on DNA interactions. To measure the groove and intercalation. The crystal structure of a synthetic\nviscosity of DNA solutions, complexes Rh1\u22124 were added in DNA dodecamer was represented by fragment 1BNA, while\nincreasing concentrations in the presence of IL4 as a cosolvent 1Z3F showcased the crystal structure of six base pairs of DNA\nfor Rh1 and IL2 for Rh2\u22124 (Figure 3). It was determined that fragments in a complex with an intercalating anticancer drug\n ellipticine (which was removed before docking). The highest-\n ranking poses are presented in Table S3, while the best-docked\n poses of complexes with DNA are depicted in Figures 4 and\n S1\u2212S3, based on the scoring functions employed.\n\n\n\n\nFigure 3. Relative viscosity of the DNA solution in 10 mM PBS in the\npresence of the complexes Rh1\u22124 in the absence and presence of IL\nof different concentrations (r).\n\nthese particular ILs (IL2 and IL4) were the most optimal\nchoice for the study based on the fact that they consistently\nproduced the highest constant values in fluorescence experi-\nments.\n The results showed that the relative viscosity of DNA\nmoderately increased with the addition of complex concen-\ntrations up to r = 1.0 in a DNA solution (0.01 mM) for Rh2\u22124\nand slightly increased for Rh1. These findings were consistent Figure 4. Computational docking model illustrating interactions\nwith the data obtained through UV\u2212vis and fluorescence between complex Rh2 and DNA (A) with the canonical gap (1BNA)\nmeasurements and the structures of the tested complexes. and (B) with the intercalation gap (1Z3F) (dotted lines display a\nComplex Rh1 does not have any rings in its structure possibility of forming hydrogen bonds).\n\n 13353 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n According to the scoring functions based on Molegro Virtual interactions of complexes Rh1\u22124, as they yielded the highest\nDocker (such as MolDock values) outlined in Table S3, the constants during the examination of DNA interactions in their\nexamined complexes appear to have a high degree of structural presence.\ncomplementarity with the DNA molecule. Across all The Ksv values in Table 5 indicate that the complexes\ncomplexes analyzed, minor groove binding was found to be analyzed interact favorably with HSA. The order of affinity\ngenerally more effective than intercalation. The scores toward HSA, i.e., Rh4 > Rh3 \u2265 Rh2 > Rh1, is like that of the\nobtained from Table S3 for intercalation and minor groove DNA interactions. Notably, rhodium complexes with larger\nbinding were ranked as follows: Rh4 > Rh3 > Rh2 > Rh1. ligands display higher binding constants for HSA interactions.\nComplexes containing planar ligands, such as Rh3 and Rh4, Additionally, the presence of ILs as cosolvents led to a slight\nhave a stronger affinity for DNA structures. Even the presence increase in constant values with the most significant enhance-\nof a relatively bulky camphor moiety in complex Rh4 ment observed in complex Rh2. Complexes Rh2 and Rh3\nhighlights the interactions. Complex Rh1, which has the exhibited nearly identical constant values in DMF as a\nlowest affinity for the DNA helix according to the MolDock cosolvent, but complex Rh2 had a higher constant value in\nscoring function, is best suited for the formation of potential the presence of IL2 than complex Rh3.\nhydrogen bonds that can further stabilize the complex\u2212DNA Competitive Experiments with HSA and Site Markers.\nsystem. Human serum albumin (HSA) possesses a distinctive heart-\n Fluorescence Spectroscopy of HSA. As the principal shaped structure comprising three domains (I\u2212III), each of\nprotein in the bloodstream, albumin is vital in facilitating the which encompasses two subdomains (A and B). 57,58\ntransportation of a wide range of substances throughout the Specifically, the subdomains IIA and IIIA of HSA are\nhuman body. These substances include hormones, fatty acids, recognized to harbor binding sites for metallo complexes.59\nions, drugs, and other essential components, which albumin Competitive experiments using site markers have been\ncarries through the blood plasma to various organs and conducted to identify the binding site of the analyzed\ntissues.49,50 Understanding the interactions between transi- complexes and determine whether they bind to sites I and/\ntional metal complexes and human serum albumin (HSA) or II of the HSA molecules using fluorescence titration. Eosin\nproteins is therefore crucial in studying potential drugs, given Y was utilized as a marker for site I of the subdomain IIA, while\nthe critical role albumin plays in this process.45,51\u221254 ibuprofen was a marker for site II of the subdomain IIIA.59,60\n In this research, the interaction between complexes Rh1\u22124 The fluorescence titration was carried out with an excitation\nand HSA was explored using fluorescence spectroscopy. To do wavelength of 295 nm and an emission range of 300\u2212500 nm.\nso, the HSA solution was excited at 295 nm, the wavelength at HSA and the markers were added in equimolar concentrations\nwhich fluorescence is solely caused by tryptophan. The (2 \u00d7 10\u22126 M), and the complexes were gradually added in\nexamined complexes were then added to the HSA solution increasing concentration. The reduction in the fluorescence\nin increasing concentrations, resulting in a reduction in intensity of the HSA solution following the introduction of the\nfluorescence, indicating an interaction. The reduction in site marker suggests that the marker molecule has successfully\nfluorescence observed is likely the result of alterations in the attached to the HSA. If the complex and corresponding marker\ntertiary structure of the protein, induced by modifications in bind to the same site in the site marker\u2212HSA system, the\nthe environment surrounding tryptophan within serum complex must compete with the marker for binding to the\nalbumin upon binding of the complex.55 The Stern\u2212Volmer HSA. This competition would result in a significant change in\nconstant (Ksv) values were calculated using the Stern\u2212Volmer the constant value. According to the data analysis in Table 5\nequation derived from the linear relationship between I0/I and using the Scatchard equation, there was a change in the\n[Q].56 constant value for both site markers. The site marker eosin Y\n For further reference, Table 5 presents the Ksv values for the showed a slightly higher effect, indicating that both the\ncomplexes\u2019 interaction with serum albumin, both independ- complex and corresponding marker bind to the same site in the\nently and in the presence of ionic liquids (ILs). Notably, it is site marker\u2212HSA system and compete for binding to the HSA.\nobserved that all ILs exerted a comparable influence on the This resulted in a significant change in the constant value.\nconstant values throughout the DNA interactions. Conse- However, the change in the constant value in the presence of\nquently, IL2 and IL4 were selected to investigate the HSA ibuprofen could not be excluded; therefore, docking measure-\n ments were performed. The measurements confirmed that the\nTable 5. Interaction Constants for the Examined Complexes complex has binding preferences for both binding sites of the\nRh1\u22124 with HSA and Site Markers in the Presence of DMF HSA molecule with a slight preference for site I (site IIA).\nand Ionic Liquids IL2 and IL4 as Cosolvents (Complex\u2212 Based on this information, it is believed that the examined\nIonic Liquid Ratio Was 1:2, 16 \u03bcM) in PBS complexes primarily bind to site I (site IIA) of the HSA\n molecule due to hydrophobic interaction, although the role of\n HSA HSA\u2212eosin Y HSA\u2212ibuprofen\n other interactions, such as hydrogen bonds, electrostatic\n interactions, van der Waals interactions, and steric contacts,\n 104Ksv (M\u22121) 104Ksv (M\u22121) 104Ksv (M\u22121)\n cannot be entirely ruled out.\n Rh1 DMF 1.2 \u00b1 0.1 0.5 \u00b1 0.1 0.8 \u00b1 0.1 A molecular docking simulation was also conducted to\n IL4 1.5 \u00b1 0.1 / / investigate the interaction between rhodium complexes and\n Rh2 DMF 2.3 \u00b1 0.1 1.1 \u00b1 0.1 1.7 \u00b1 0.1 HSA. The docking was carried out in subdomains IIA and IIIA\n IL2 3.5 \u00b1 0.1 / / of the HSA molecule. Subdomain IIA was found to have a large\n Rh3 DMF 2.4 \u00b1 0.1 1.6 \u00b1 0.1 1.9 \u00b1 0.1 hydrophobic cavity that can accommodate the drug molecule,\n IL2 2.6 \u00b1 0.1 / / which plays a significant role in biomolecule metabolism and\n Rh4 DMF 3.9 \u00b1 0.1 1.7 \u00b1 0.1 2.1 \u00b1 0.1 transportation. The top-scored values for the examined\n IL2 4.2 \u00b1 0.1 / / complexes with HSA protein docked into binding sites IIA\n 13354 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nand IIIA are presented in Table S4, based on the scoring for clinically used therapeutic agents, including metal\nfunctions used. Figures 5 and S4\u2212S6 illustrate the best poses complexes, generally fall within the range from \u22120.4 to 5.6.\naccording to the H bond scoring function with HSA docked Notably, all Rh1\u22124 complexes demonstrate log P values that\ninto subdomains IIA and IIIA for the investigated complexes. fall within the optimal lipophilicity range of 0 \u2264 log P \u2264 0.90.\n Of particular interest is that Rh2 and Rh4 complexes exhibit\n significantly higher log P values (0.64 and 0.87) than the other\n two, indicating their preference for the octanol phase. This\n distinctive characteristic of Rh2 and Rh4 complexes also\n implies their potential as highly efficient therapeutic agents\n given their superior cellular uptake efficiency.\n In Vitro Cytotoxicity Study. In our comprehensive study\n assessing the cytotoxic potential of novel rhodium complexes,\n we meticulously quantified their impact on the cell viability in\n HeLa and MRC-5 cell lines. The half-maximal inhibitory\nFigure 5. Best pose with HSA docked into a binding pocket for concentration (IC50) serves as a benchmark for cytotoxicity,\ncomplex Rh2 according to the H bond values: (A) complexes into the\n indicating the potency of a substance in inhibiting a biological\nsubdomain IIA of HSA protein and (B) complexes into the\nsubdomain IIIA of HSA protein. Selected amino acid residues process by 50%. Across the four rhodium complexes tested\n(selected by applying an energy threshold of 0.625) are represented (Rh1, Rh2, Rh3, and Rh4), we observed a range of IC50\nby stick models (hydrogen bonds are shown as blue dotted lines). values, revealing a diverse cytotoxic profile (Figure 6).\n Rhodium complex Rh2 demonstrated a particularly potent\n effect on HeLa cells with an IC50 of 85.13 \u03bcM after 72 h from\n Through investigation, it has been found that rhodium treatment, while its influence on MRC-5 was notably less\ncomplexes can be accommodated within subdomains IIA and pronounced. This disparity underscores the complex\u2019s ability\nIIIA of an HSA molecule. According to the gain simulation to selectively target cancerous cells while sparing normal,\nresults, subdomain IIA is the more favorable location for healthy cells. Such a differential could be indicative of the\nhosting the examined complex. This is due, in part, to the complex\u2019s mechanism of action, which may disrupt specific\nlarger cavity of subdomain IIA compared to that of the IIIA\n pathways that are more active in cancer cells, hinting at a\nsubdomain, allowing for more voluminous octahedra com-\n possibly safer therapeutic window. Complexes Rh1 and Rh3\nplexes to be accommodated more effectively. Molecular\ndocking simulations have shown that the affinity order of the were found to be nontoxic, as indicated by their IC50 values,\ncomplexes toward HSA is Rh4 > Rh3 \u2265 Rh2 > Rh1 for both which were greater than 500 \u03bcM for both HeLa and MRC-5\nsubdomains. While there is a minor possibility of a hydrogen cell lines. This suggests that these compounds do not exhibit\nbond forming between the complexes and amino acid residues significant cytotoxicity under the tested conditions. On the\nof the HSA protein, it was observed to be infrequent. other hand, Rh4 displayed a different profile; despite having\n Lipophilicity Assay. Extensive research has demonstrated very low IC50 values for both HeLa and MRC-5 cells, it\nthat the lipophilicity of metal complexes is a crucial factor in demonstrated a strong cytotoxic effect. However, it lacked\ndetermining their potential biological activity. It is well- selectivity between the two cell types, indicating that although\nestablished that compounds with a higher lipophilicity tend to Rh4 is toxic, it does not differentiate between cancerous and\nhave greater cellular uptake, making them more effective as normal cell lines. To further illustrate the cytotoxic effects, we\ntherapeutic agents. Furthermore, the partition coefficient (log conducted a time-course study, which revealed that the action\nP) between the hydrophobic octanol and hydrophilic water of Rh2 was both time-dependent and dose-dependent, with\nphases is a crucial indicator of a compound\u2019s lipophilicity and higher concentrations leading to a more rapid decline in cell\nability to traverse the cell membrane.61 Notably, log P values viability (Figure S7).\n\n\n\n\nFigure 6. Comparative IC50 values of rhodium complexes Rh1\u22124 on HeLa and MRC-5 cell lines at 24 and 72 h post-treatment.\n\n 13355 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n This granular analysis of the cytotoxic effects of rhodium rhodium complexes differed across the specific complex and\ncomplexes provides a clear indication of their potential as cell line, with some combinations suggesting the potential for\nselective anticancer agents. The numerical distinctions between selective targeting of cancer cells. This modulation by IL1 and\ntheir IC50 values offer insight into their therapeutic potential IL10 suggests that the choice of ionic liquid in cotreatment\nand lay the groundwork for further investigations into their use strategies could be critical in optimizing the therapeutic\nas targeted treatments for cancer. The specificity of Rh2 in efficacy and selectivity of rhodium-based cancer treatments.\nparticular points toward a promising avenue for the develop- In Vitro Redox Status Modulation. The modulation of\nment of drugs that could deliver maximum therapeutic effects the redox status by rhodium complexes provides pivotal\nwith minimal impact on healthy tissues. More details on the insights into their mechanistic underpinnings as potential\nrhodium complexes\u2019 viability influence on HeLa and MRC-5 anticancer agents. In our study, three key redox parameters,\ncells are presented in Figures S7 and S8. namely superoxide anion radicals, nitrite levels, and glutathione\n The assessment of ionic liquids (ILs) in our study was (GSH) content, were meticulously measured to ascertain the\npredicated on identifying any intrinsic cytotoxic properties that oxidative impact of these complexes on HeLa and MRC-5 cell\nthey might exhibit when used independently from rhodium lines.\ncomplexes. Employing a fixed concentration of 100 \u03bcM for Upon treatment with rhodium complexes, HeLa cells\neach ionic liquid, we aimed to discern any immediate cytotoxic exhibited a marked increase in superoxide anion radicals, a\neffects that could be critical for the selection process in further primary indicator of oxidative stress (Figure S11). These\ncotreatment assays (Figure S9). Upon analysis, it was observed radicals, often precursors to more reactive and damaging\nthat IL1 and IL10, out of the series tested, stood out\ufffdnot for species, suggest the initiation of an oxidative cascade that may\ntheir cytotoxicity but rather for their relatively lower impact on lead to cellular damage and apoptosis. Similarly, nitrite levels,\ncell viability, marking them as compounds of interest for which can serve as a surrogate marker for the presence of nitric\nsubsequent experiments. This benchmark of 100 \u03bcM was oxide, were elevated, as shown in Figure S12. This increase in\nchosen as a hallmark for potential toxicity, and the minimal nitrites further implicates the rhodium complexes in the\neffects observed suggest that these ILs are suitable candidates promotion of a nitrosative stress environment, which can lead\nfor further study in combination treatments without the to various forms of nitrosative damage within cellular\nconfounding factor of high inherent cytotoxicity. components. In stark contrast, GSH levels in HeLa cells\n The selection of IL1 and IL10 for cotreatment was not solely showed a significant decrease post-treatment, as shown in\nbased on their cytotoxic profile; their chemical significance was Figure S13. GSH is a critical antioxidant molecule that\nalso considered. According to the chemical properties and\n quenches free radicals and detoxifies reactive oxygen species\nresults that are presented in the current data set, it is implied\n (ROS). The depletion of GSH indicates a compromised\nthat these ILs possess chemical features that could potentially\n cellular defense mechanism against the onslaught of oxidative\ninteract synergistically with the rhodium complexes, altering\n stress, leaving the cells more susceptible to damage and\ntheir bioavailability, stability, or cellular uptake, which in turn\n dysfunction.\ncould influence the overall therapeutic efficacy of the\n The changes in redox parameters in MRC-5 cells, however,\ncotreatment.\n The rationale behind the selection process was two-fold: to were less pronounced, pointing toward a differential sensitivity\nensure that the ionic liquids themselves do not exert significant to the rhodium complexes. While there was a slight increase in\ntoxicity at the chosen concentration, which could confound the superoxide anion radicals and nitrite levels (Figures S14 and\nresults of cotreatment studies, and to identify ILs that might S15), suggesting the initiation of an oxidative response, the\npossess chemical properties conducive to enhancing the GSH levels did not decrease as substantially as in HeLa cells\nanticancer potential of rhodium complexes. The benign (Figure S16). This nuanced response underscores the selective\ncytotoxic profiles of IL1 and IL10 at 100 \u03bcM make them toxicity of rhodium complexes, capable of inducing significant\nideal candidates for these cotreatment assays, and their oxidative stress in cancerous cells while sparing noncancerous\nchemical significance paves the way for a more nuanced cells, thus maintaining a better redox homeostasis.\nunderstanding of how ILs can be used to modulate the activity The correlation between increased ROS production and cell\nof metal-based therapeutics in cancer treatment. death in HeLa cells supports the hypothesis that the\n The comparative cytotoxicity study of rhodium complexes cytotoxicity of rhodium complexes is mediated through an\nRh1\u22124 with and without the cotreatment of ionic liquids IL1 oxidative stress mechanism. The differential increase in\nand IL10 reveals a nuanced modulation of their effects on cell superoxide anion radicals and nitrites, coupled with the\nviability (Figure S10). The rhodium complexes alone showed a depletion of GSH in HeLa cells, suggests that the cancer\nbaseline cytotoxic effect on HeLa cells that was altered upon cells are less equipped to counteract the increased oxidative\nthe addition of ionic liquids. On HeLa cells, IL1 generally burden, leading to cell death. In contrast, the MRC-5 cells\u2019\nshowed a tendency to decrease cell viability compared to Rh1 ability to maintain higher levels of GSH in the face of rhodium\nalone, especially after 72 h. Cotreatment with Rh2 showed a complex-induced oxidative stress may explain their relative\ntendency to maintain the cell viability, while cotreatment with resistance to the cytotoxic effects.\nRh3 and Rh4 lead to an increase in cell viability. IL10 often This dichotomy in redox status between the cancerous and\nresulted in a maintained cell viability except for cotreatment noncancerous cells is critical for understanding the therapeutic\nwith Rh2, where decreased cell viability was observed, potential of rhodium complexes. It highlights the possibility of\nindicating an enhancement of the cytotoxic effects. In exploiting the heightened vulnerability of cancer cells to\nnoncancerous MRC-5 cells, the presence of IL1 and IL10 oxidative damage while minimizing the impact on normal cells.\nsometimes led to higher cell viability percentages compared to Such selectivity is desirable in anticancer therapy, aiming to\nthe treatment with rhodium complexes alone, hinting at a maximize the cytotoxic effect on malignant cells while reducing\nprotective effect. The interaction between the ionic liquids and the collateral damage to healthy tissue.\n 13356 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n Further investigations into the precise mechanisms by which and IL10 sometimes enhancing the cytotoxic effect of the\nrhodium complexes disrupt the redox balance and induce cell rhodium complexes. This ability to modulate cytotoxicity\ndeath could facilitate the design of targeted cancer treatments. through cotreatment opens new avenues for optimizing cancer\nUnderstanding the interplay among ROS generation, anti- therapies that are as effective as they are discerning.\noxidant depletion, and cellular defenses will be key to The investigation into the redox status of treated cells\noptimizing the use of rhodium complexes in clinical settings. provided additional insights, with rhodium complexes inducing\nThis redox-based approach to cancer therapy could form the oxidative stress in cancerous cells, as evidenced by increased\ncornerstone of new, more effective treatments that leverage the superoxide anion radicals and nitrite levels coupled with\ninherent biochemical disparities between cancerous and decreased GSH levels. In contrast, the noncancerous cells\nnoncancerous cells. maintained a better redox balance, supporting the idea of\n Microbiology. In addition to in vitro testing, the selective cytotoxicity. The distinct redox responses of cancer-\nantimicrobial activity of Rh1\u22124 complexes was investigated. ous vs noncancerous cells to rhodium complexes highlight the\nThe studied complexes exhibited moderate to negligible potential for exploiting the unique vulnerabilities of cancer\nantimicrobial activity, with further details provided in the cells. Such targeted therapies could revolutionize cancer\nSupporting Information (Tables S5 and S6 and Figures S17\u2212 treatment, minimizing harm to healthy cells while effectively\nS20). The antimicrobial activity of the Rh1\u22124 complexes was combating cancerous growths. Our findings lay the ground-\nnot tested in the presence of ionic liquids. These complexes work for future studies aimed at unlocking the full therapeutic\ndemonstrated greater antitumor potential compared to their potential of metal-based complexes in oncology.\nantimicrobial activity, suggesting that future research could\nfocus on their application in cancer treatment. \u25a0 EXPERIMENTAL SECTION\n\n\u25a0 CONCLUSION\nIn conclusion, research within this paper has delved into the\n Chemicals and Solutions. Phosphate buffer (PBS) (0.01 M,\n CNaCl = 0.137 M, CKCl = 0.0027 M, pH = 7.4), RhCl3\u00b7xH2O,\n bis(pyridin-2-ylmethyl)amine (bpma), DMF, 1-ethyl-3-methylimida-\n zolium ethyl sulfate (IL1), calf thymus DNA (CT-DNA), eosin Y,\nuntapped potential of biocompatible ionic liquids as ethidium bromide (EB), ibuprofen, and human serum albumin\ncosolvents, effectively amplifying the solubility of sparingly (HSA) were purchased from Sigma-Aldrich and were prepared by\nsoluble rhodium(III) complexes. Our primary aim was to dissolving the measured amount of the substance without prior\nscrutinize the interactions between octahedral rhodium(III) purification. The DNA solution was prepared in 0.01 M phosphate\ncomplexes\ufffdcomprising diene and pyridine derivative ligands, buffer at pH = 7.4, with the ratio of UV absorbance at 260 and 280\nas well as the camphor-derived bis-pyrazolylpyridine ligand\ufffd nm (A260/A280) between 1.8 and 1.9, indicating that the DNA was\nand essential biomolecules such as 5\u2032-GMP, DNA, and HSA in liberated from the protein. The concentration of the DNA solution\nthe presence of diverse ionic liquids (IL1\u221211) and DMF as was determined by UV absorption at 260 nm (\u03b5 = 6600 M\u22121 cm\u22121).\n All solutions were stored at 277 K and used within 5 days. Bidestilated\ncosolvents. The substitution reactions involving the examined\n water was used for preparing the solutions. Complexes Rh1, Rh3, and\ncomplexes Rh1\u22124 with 5\u2032-GMP revealed that ionic liquids Rh4 were synthesized according to the published procedures.14,30,31\nsubstantially increased the solubility of the complexes, thereby IL2\u221211 were synthesized according to the published procedures.34\u221236\nresulting in a notable augmentation of the constant Instrumental Methods. NMR spectra were recorded on a 200\nsubstitution values. Complexes Rh1\u22124 showed moderate MHz Varian Gemini-2000 instrument. NMR signals were referenced\ninteraction ability toward CT-DNA and HSA in the presence to residual proton or carbon signals of the deuterated solvent (1H\nof IL1\u221211 and DMF, indicating a minimal influence of the NMR) and are reported in parts per million relative to TMS.\nionic liquids on the complexes\u2212DNA or complexes\u2212HSA Elemental analyses (C, H, and N) were recorded on an Elementar\nbinding. Furthermore, assessments of the lipophilicity of the Vario MICRO elemental analyzer. UV\u2212vis and kinetic measurements\ncomplexes showed good lipophilicity. Docking studies revealed were conducted on a PerkinElmer Lamda 25 double-beam\n spectrophotometer with thermostated 1.00 cm quartz Suprasil cells,\nstrong structural complementarity between the complexes and with the temperature controlled to \u00b10.1 \u00b0C. Fluorescence was\nDNA, favoring minor groove binding. Also, the complexes measured on an RF-1501 PC spectrofluorometer (Shimadzu, Japan),\nexhibited notable affinity for HSA, localized mainly within with the excitation and emission bandwidths both 10 nm. HPLC\nHSA\u2019s subdomain IIA. A docking examination of DNA Gyr traces were done on am HPLC-LC20AT instrument, Shimadzu.\nand TyrRS interactions highlighted steric effects\u2019 significance, Synthesis and Characterization of Complex [Rh(bpma)Cl3],\nwith complex Rh4 showing higher affinity for both DNA Gyr Rh2. Complex [Rh(bpma)Cl3] (Rh2) (Figure 1) was synthesized by\nand TyrRS. The intensity of the antimicrobial effect of Rh1\u22124 stirring one equivalent of RhCl3\u00b7xH2O, with one equivalent of bpma\ncomplexes varied depending on the type of microorganisms (bis(pyridin-2-ylmethyl)amine) in ethanol and refluxing for four\n hours. Obtained yellow orange participants were filtered and dried\nand the type and concentration of complexes. In general, the under a vacuum. The newly synthesized Rh2 complex was\nrhodium(III) complexes showed highly selective and moderate characterized, and a single crystal of a DMSO adduct suitable for\nactivity. the X-ray analysis was also obtained.\n Research also elucidated the complex interplay between 1\n H NMR (500 MHz, DMSO-d6): \u03b4 = 4.34 (s; 4H; CH2), 7.47 (m;\nrhodium complexes and ionic liquids, shedding light on their 2H; py), 7.65 (m; 2H; py), 7.89 (m; 2H; py), 9.22 (d; 2H; py).\npotential to selectively target cancer cells. The cytotoxicity Anal. Calcd for C12H13Cl3N3Rh: C, 35.28; H, 3.21; N: 10.29.\nstudies revealed that among the rhodium complexes, Rh2 Found: C, 34.72; H, 3.06; N, 10.15.\nstands out with its unique ability to selectively diminish the The purity of the used complexes is greater than 95% (>95%), as\nviability of cancerous HeLa cells while exerting minimal effects confirmed by elemental microanalysis and HPLC (high-performance\n liquid chromatography). The HPLC trace chromatograms and\non noncancerous MRC-5 cells. This selectivity is a crucial elemental microanalysis data are presented in the Supporting\nfinding, as it suggests the possibility of targeted cancer Information (Figures S21\u2212S24).\ntreatment with reduced side effects on healthy tissue. The Crystal Structure Determination. X-ray diffraction data for\naddition of ionic liquids IL1 and IL10 further nuances this complex [Rh(bpma)Cl3] (Rh2) were collected at room temperature\neffect, with IL1 typically decreasing the viability of HeLa cells using graphite-monochromated Mo K\u03b1 radiation (\u03bb = 0.71073 \u00c5) on\n\n 13357 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nan Oxford Diffraction Gemini S diffractometer. The CrysAlisPro and left undisturbed for 24 h to enable phase separation. The\nCrysAlis RED software packages were employed for data collection concentrations of the complexes in both phases were determined by\nand data integration.62 Final cell parameters were determined by a spectrophotometric analysis using previously established calibration\nglobal refinement of 2215 reflections (3.2\u00b0 < q < 28.6\u00b0). Space group curves. Logarithmic partition coefficients (log P) were computed\ndetermination was based on analysis of the Laue class and employing the following formula:\nsystematically absent reflections. Collected data for Rh2 were\ncorrected for absorption effects using the Multiscan method, applying log P = log(C 0/C w )\nan empirical absorption correction using spherical harmonics63 as\nimplemented in the SCALE3 ABSPACK62 scaling algorithm. where C0 represents the concentration of the complex in n-octanol\n Structure solution and refinement for Rh2 were carried out with and Cw denotes the concentration in the aqueous phase.\nthe programs SHELXT64 and SHELXL-2018/3,65 respectively. Protein Binding Studies. The examination of interactions with\nMolecular geometry calculations were performed by PLATON.66 human serum albumin (HSA) was conducted through the application\nMERCURY37 was employed for molecular graphics, and the of fluorescence spectroscopy. Spectra were recorded within the\nOLEX267 software was used to prepare material for publication. wavelength range 300\u2212500 nm with the excitation wavelength set at\nNon-hydrogen atoms were refined freely with anisotropic displace- 295 nm. The binding affinity of the investigated complex to the\nment parameters. Hydrogen atoms attached to carbon atoms in protein was discerned by monitoring alterations in the emission\nphenyl rings and methylene groups were placed in geometrically intensity of albumin (2 \u03bcM) subsequent to the incremental addition\nidealized positions and refined as riding on their parent atoms, with of the complex (0\u221210 \u03bcM). This method enabled the qualitative\nC\u2212H = 0.93 \u00c5 and Uiso(H) = 1.2Ueq(C) and with C\u2212H = 0.97 \u00c5 and assessment of the binding effect with a particular focus on the\nUiso(H) = 1.5 Ueq(C), respectively. Crystal data and experimental diminishing emission intensity as a consequential outcome of the\ndetails of the structure determination are given in Tables S1 and S2. complex\u2212protein interaction.\n Kinetic Studies. The kinetics of the coordinated chloride Molecular Docking Simulations. Molecular docking simulations\nsubstitution were measured spectrophotometrically by following the have proven to be highly beneficial in predicting the atomic-level\nchange in absorbance at suitable wavelengths as a function of time. compatibility of small molecules and macromolecules. Scoring\nWorking wavelengths were determined by recording the spectra of the functions such as MolDock and Hbond, derived from Molegro\nreaction mixture from 220 to 450 nm. Kinetic measurements were Virtual Docker, can accurately measure the affinity between parent\nperformed under pseudo-first-order conditions. Reactions were scaffolds and their integration complexes with target macromolecules.\ninitiated by mixing 0.50 mL of a nucleophile\u2019s complex solution Moreover, the simulations\u2019 top-ranked poses provide valuable insights\nwith 2.50 mL of the thermally equilibrated complex solution in a into the optimal positioning of the investigated complexes.\nUV\u2212vis cuvette. Reactions were followed for at least 8 half-lives. The Structures of the investigated rhodium complexes were optimized\nobserved pseudo-first-order rate constants, kobsd, represent an average and characterized as a local minimum (calculating vibration\nvalue of three to four independent kinetic runs for each experimental frequencies), using a B3LYP functional69 in combination with the\ncondition. Values for constants and other thermodynamic parameters def2-SVP basis set70,71 implemented in the GAUSSIAN suite\nwere determined by using Microsoft Excel 2010 and OriginPro 8. program,72 which was used just for optimizing the starting structures\n Absorption Spectroscopic Studies. UV\u2212vis spectrophotomet- of the investigated rhodium complexes. These structures were docked\nric spectroscopy was used to determine the binding constant (Kb). into a rigid three-dimensional (3D) structural representation of\nThe binding of complexes to DNA was performed at 37 \u00b0C. canonical B-DNA (PDB ID: 1BNA), DNA with an intercalation gap\nPhosphate buffer (0.01 M, pH = 7.4) was used for absorption (PDB ID: 1Z3F), HSA protein (PDB ID: 1AO6), tyrosyl-tRNA\nmeasurements. synthetase (PDB: 1JIJ), topoisomerase II DNA gyrase (PDB: 2XCT),\n Ethidium bromide (EB) Displacement Studies. The fluo- and Bcl-2 protein (PDB ID: 2W3L). Structural coordinates of these\nrescence intensity was measured with the wavelength of excitation at macromolecules were obtained from the Protein Data Bank (PDB)\n527 nm and the wavelength of emission at 612 nm. The width of the (http://www.rcsb.org). If present, then all heteromolecules, ligands,\nexcitation and emission slits (10 nm) and scan rate were maintained and water molecules were removed. The docking was performed in a\nconstant throughout all experiments. The emission of the solution was rigid structure of target macromolecules with flexible complex\nrecorded in the range of 550\u2212750 nm. Interactions of the complex structures using Molegro Virtual Docker (MVD, version\nwith DNA were studied in the presence of EB to determine whether 2013.6.0.1).73 Docking parameters were as follows: grid resolution\nthe complex could replace EB from the DNA\u2212EB complex. The of the binding side, 0.3 \u00c5; maximum number of interactions, 1500;\nDNA\u2212EB complex was prepared by mixing 10 \u03bcM EB and 10 \u03bcM population size, 50; energy threshold, 100.00; and maximum number\nDNA (pH = 7.4). A series of solutions were prepared by mixing EB\u2212 of steps, 300. A maximum population of 100 and a maximum number\nDNA solutions with an increasing amount of complex. Possible effects of interactions of 10 000 were used for each run. The search algorithm\nof the complex binding to the DNA were studied by recording a was MolDock SE, with the number of runs set to 100, and the number\nchange in the fluorescence emission spectrum after the addition of the of generated poses at 10. The MVD-related scoring functions\ncomplex solution. described the estimation of the investigated complexes\u2019 affinity to\n Viscosity Measurements. DNA viscosity changes were assessed target macromolecules: MolDock, Docking, Rerank, and Hbond.\nin the presence of rising concentrations of the investigated complexes, Molegro scores were evaluated relatively (omitted from units, where\ndenoted as Rh1\u22124, both with and without selected ionic liquids (ILs) the more negative values suggest greater binding prosperity of a\nserving as cosolvents. Flow time was measured using a digital complex to a macromolecule).73 It is important to note that this\nstopwatch three times for each sample to determine the average flow approach of molecular docking simulation foremost presents the\ntime. Data are presented as (\u03b7/\u03b70)1/3 plotted against r, where \u03b7 and \u03b70 structural compatibility, emphasizing possible weak interactions,\nrepresent the DNA viscosities observed in the presence of the between selected macromolecules and investigated complexes and\ncomplex and under buffer conditions, respectively. Viscosity should be understood and discussed in that matter. It does not take\nquantifications were derived from the observed flow time of DNA- into account the presence and therefore influence of the solvent and\ncontaining solutions (t) normalized against the flow time of buffer possible bond breaking and/or bond formatting which may occur\nsolely (t0), expressed as \u03b7 = (t \u2212 t0)/t0. during the experimental investigation of these interactions. Docked\n Lipophilicity Assay. The lipophilicity of Rh1\u22124 complexes was poses were visualized using the CHIMERA (http://www.cgl.ucsf.\ndetermined utilizing the flask-shaking method.68 The examined edu/chimera/) molecular graphics program.\ncomplexes were dissolved in dimethyl sulfoxide (DMSO) and In Vitro Cytotoxicity Study and Redox Status Modulation.\nsubsequently introduced into the water/n-octanol system. Following This in vitro exploration focused on examining the cytotoxicity and\nthis, the mixture underwent vortexing for a duration of 1 h at room principal redox status markers in human cervical cancer cells (HeLa)\ntemperature to facilitate partitioning. Subsequently, the solutions were and normal lung-derived fibroblasts (MRC-5), which were sourced\n\n 13358 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nfrom the European Collection of Authenticated Cell Cultures. These 10 \u03bcL of samples from wells, where no indicator color change was\ncells were propagated in culture flasks, achieving approximately 80% recorded, on nutrient agar medium. At the end of the incubation\nconfluence, under rigorous laboratory conditions compliant with period, the lowest concentration with no growth (no colony) was\nGood Laboratory Practice (GLP) and a cell-centric approach to defined as the minimum microbicidal concentration.\nhandling.\n Cell viability was ascertained using the MTT assay, which relies on\nspectrophotometric measurement of the conversion of MTT to\nformazan, reflecting cellular metabolic activity. The cells, seeded in\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\n\nmicroplates, underwent a stabilization phase before treatment and\n The Supporting Information is available free of charge at\nwere cultured in high-glucose DMEM with essential supplements. https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01220.\nViability percentages were calculated by comparing the absorbance of Additional details of the experiments involving com-\ntreated cells to that of the control group across a broad range of plexes Rh1\u22124 including X-ray analyses; equations for\nsubstance concentrations. kinetic studies and DNA binding studies using electronic\n We quantified redox parameters, including superoxide anion absorption and fluorescence spectroscopic methods;\nradicals, nitrites, and glutathione (GSH), to evaluate the oxidative\nstates induced by the substances. These parameters were determined\n docking studies and computational docking models of\nspectrophotometrically at specific wavelengths for each compound complexes Rh1, Rh3, and Rh4 interacting with DNA\nwith assays conducted at varying substance concentrations. and HSA; cytotoxicity and redox potential studies as well\n Statistical analyses were carried out with six replicates per dose, as assessments of ionic liquids and cotreatment effects;\npresenting the data as an average of two distinct experiments with the superoxide anion radical, nitrite, and glutathione\nstandard error. The relationship between variables was analyzed using production data; antimicrobial investigations of Rh1\u22124\nthe SPSS software, and the dose\u2212response relationship was plotted to complexes; antimicrobial docking results with DNA Gyr\ndeduce the IC50 values, calculated via the CalcuSyn application. More and TyrRS enzymes; and HPLC chromatograms for\ndetails are available in our previously published studies.23,35 Rh1\u22124 complexes (PDF)\n In Vitro Antimicrobial Assay. Test substances, microorganisms,\nand suspension preparation: the tested compounds were dissolved in\n Complex\u2019sRh2 structure (CIF)\nDMSO and then diluted into a nutrient liquid medium to achieve a Molecular formula strings for Rh1\u22124 and IL1\u221211\nconcentration of 10%. The antibiotics doxycycline (Galenika A.D., (CSV)\nBelgrade) and fluconazole (Pfizer Inc., USA) were dissolved in Rh1\u22121AO6(IIA) system (PDB)\nTrypton Soy Broth (Torlak, Belgrade). The antimicrobial activity of Rh1\u22121AO6(IIIA) system (PDB)\nfour ligands and corresponding rhodium(III) complexes was tested Rh1\u22121BNA system (PDB)\nfor nine microorganisms including seven standard and clinical strains Rh1\u22121JIJ system (PDB)\nof pathogenic bacteria, one species of probiotic, and one clinical\nisolate yeast (Table S6). All clinical isolates were a generous gift from Rh1\u22121Z3F system (PDB)\nthe Institute of Public Health, Kragujevac. The other microorganisms Rh1\u22122XCT system (PDB)\nwere provided from the collection held by the Microbiology Rh2\u22121AO6(IIA) system (PDB)\nLaboratory Faculty of Science, University of Kragujevac. Rh2\u22121AO6(IIIA) system (PDB)\n Bacterial and yeast suspensions were prepared by the direct colony Rh2\u22121BNA system (PDB)\nmethod. The colonies were taken directly from the plate and were Rh2\u22121JIJ system (PDB)\nsuspended in 5 mL of sterile 0.85% saline. The turbidity of the initial\n Rh2\u22121Z3F system (PDB)\nsuspension was adjusted using a densitometer (DEN-1, BioSan,\nLatvia). When adjusted to the turbidity of a 0.5 McFarland standard, Rh2\u22122XCT system (PDB)\nthe bacteria suspension contained about 108 colony forming units Rh3\u22121AO6(IIA) system (PDB)\n(CFU)/mL and the suspension of yeast contained 106 CFU/mL. Rh3\u22121AO6(IIIA) system (PDB)\nTen-hold dilutions of the initial suspension were additionally prepared Rh3\u22121BNA system (PDB)\ninto sterile 0.85% saline. Rh3\u22121JIJ system (PDB)\n Microdilution Method. Antimicrobial activity was tested by Rh3\u22121Z3F system (PDB)\ndetermining the minimum inhibitory concentration (MIC) and\nminimum microbicidal concentration (MMC) by using the micro- Rh3\u22122XCT system (PDB)\ndilution plate method with resazurin.74 The 96-well plates were Rh4\u22121AO6(IIA) system (PDB)\nprepared by dispensing 100 \u03bcL of Trypton Soy Broth into each well. Rh4\u22121AO6(IIIA) system (PDB)\nA 100 \u03bcL portion from the stock solution of tested compound Rh4\u22121BNA system (PDB)\n(concentration of 2000 \u03bcg/mL) was added into the first row of the Rh4\u22121JIJ system (PDB)\nplate. Then, 2-fold serial dilutions were performed by using a Rh4\u22121Z3F system (PDB)\nmultichannel pipet. The obtained concentration range was from 1000\nto 7.8 \u03bcg/mL. A 10 \u03bcL of diluted bacterial and yeast suspension was\n Rh4\u22122XCT system (PDB)\nadded to each well to give a final concentration of 5 \u00d7 105 CFU/mL\nfor bacteria and 5 \u00d7 103 CFU/mL for yeast. Finally, 10 \u03bcL of\nresazurin solution was added to each well inoculated with bacteria and\n \u25a0 AUTHOR INFORMATION\n Corresponding Author\nyeast. The inoculated plates were incubated at 37 \u00b0C for 24 h for Jovana Bogojeski \u2212 University of Kragujevac, Faculty of\nbacteria and 28 \u00b0C for 48 h for the yeast. MIC was defined as the\nlowest concentration of the tested substance that prevented resazurin\n Science, 34000 Kragujevac, Serbia; orcid.org/0000-0002-\ncolor change from blue to pink. 3433-7774; Phone: +381 (0)34 336223;\n A solvent control test was performed to study the effect of 10% Email: jovana.bogojeski@pmf.kg.ac.rs; Fax: +381 (0)34\nDMSO on the growth of microorganisms. It was observed that 10% 335040\nDMSO did not inhibit the growth of the microorganisms.\nDoxycycline and fluconazole were used as positive control. Each Authors\ntest included a growth control and sterility control. All tests were Angelina Cakovic\u0301 \u2212 University of Kragujevac, Faculty of\nperformed in duplicate, and MICs were constant. The minimum Science, 34000 Kragujevac, Serbia; orcid.org/0000-0003-\nbactericidal and fungicidal concentrations were determined by plating 3909-4211\n 13359 https://doi.org/10.1021/acs.jmedchem.4c01220\n J. Med. Chem. 2024, 67, 13349\u221213362\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n Dus\u030can C\u0301 oc\u0301ic\u0301 \u2212 University of Kragujevac, Faculty of Science,\n 34000 Kragujevac, Serbia\n \u25a0 REFERENCES\n (1) Phillips, A. M. F.; Pombeiro, A. J. L. Tran-sition Metal-Based\n Marko Z\u030c ivanovic\u0301 \u2212 University of Kragujevac, Institute for Prodrugs for Anti-cancer Drug Delivery. Curr. Med. 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Acta\nThe manuscript was written through contributions of all 2012, 393, 84\u2212102.\nauthors. (13) Milutinovic\u0301, M. M.; Bogojeski, J. V.; Klisuric\u0301, O.; Scheurer, A.;\nNotes Elmroth, S. K. C.; Bugarc\u030cic\u0301, Z\u030c . D. Synthesis and Structures of a\nThe authors declare no competing financial interest. Pincer-Type Rhodium(III) Complex: Reactivity toward Biomolecules.\n Dalton Trans 2016, 45 (39), 15481\u221215491.\n\n\u25a0 ACKNOWLEDGMENTS\nThe authors gratefully acknowledge the financial support of the\n (14) Petrovic\u0301, A.; Milutinovic\u0301, M. M.; Petri, E. T.; Z\u030c ivanovic\u0301, M.;\n Milivojevic\u0301, N.; Puchta, R.; Scheurer, A.; Korzekwa, J.; Klisuric\u0301, O. R.;\n Bogojeski, J. Synthesis of Camphor-Derived Bis(Pyrazolylpyridine)\nMinistry of Science, Technological Development and In- Rhodium(III) Complexes: Structure-Reactivity Relationships and\nnovation of the Republic of Serbia, Grants 451-03-66/2024- Biological Activity. Inorg. 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