Ruthenium(II)-Arene Complexes: Biomolecular Binding, Thermodynamic Insights, and Selective Cytotoxicity

Abstract

Two novel dimeric ruthenium­(II)-arene complexes, PNRP and PNRB, incorporating a planar π-conjugated PNN ligand and different arene moieties (p-cymene and benzene, respectively), were synthesized and characterized. These complexes were designed to explore the role of arene identity in modulating biomolecular interactions and redox-mediated cytotoxicity. Comprehensive physicochemical and spectroscopic studies confirmed the structural integrity and solution stability of both complexes. Binding interactions with calf thymus DNA and human serum albumin were investigated using absorption and fluorescence spectroscopy, with thermodynamic parameters (ΔG°, ΔH°, ΔS°) determined from temperature-dependent binding studies. The results indicated spontaneous and moderately strong binding driven primarily by hydrophobic and electrostatic interactions. Viscosity analysis and guanine binding further supported partial intercalation. Cellular assays in A549 lung carcinoma and HEK293 normal kidney cells demonstrated that both complexes exhibit notable cytotoxic activity, with PNRB showing enhanced selectivity (IC₅₀ = 31.37 μM; SI = 7.58). Reactive oxygen species (ROS) generation was confirmed via fluorescence microscopy, suggesting oxidative stress as a contributing mechanism. Overall, the study highlights arene-dependent modulation of biological response in Ru­(II) complexes and supports the development of redox-active, π-conjugated metal-based agents as selective anticancer therapeutics.

1. Introduction

Nonsmall cell lung carcinoma (NSCLC) remains a major global health burden, with 2.2 million cases and 1.8 million deaths reported in 2020. In India, NCRP projects over 1.46 million new cancer cases in 2022, with lung cancer being one of the leading types. These trends highlight the urgent need for more effective and less toxic treatment options. ,

These concerning trends highlight the immediate requirement for better treatments, especially for lung cancers. Even with progress in treatment options, existing strategies frequently lead to minimal survival gains and are linked to considerable toxicity and the emergence of resistance. Conventional platinum-based drugs, including cisplatin and carboplatin, work by creating DNA cross-links that initiate apoptosis. Nonetheless, their unspecific nature results in significant side effects, such as nephrotoxicity, neurotoxicity, and hematological issues. These restrictions have led to an increasing interest in metal-containing anticancer agents that provide better selectivity, advantageous pharmacokinetics, and different modes of action.

Ruthenium­(II)-arene complexes are increasingly considered viable alternatives to platinum-based chemotherapeutic agents, owing to their unique redox activity, flexible coordination environments, and potential for selective tumor targeting. Complexes such as [Ru­(η⁶-p-cymene)­(phen)­Cl]⁺ and [Ru­(η⁶-benzene)­(bpy)­Cl]⁺ have demonstrated notable anticancer potential through DNA interaction and redox-mediated mechanisms in both in vitro and in vivo studies. Furthermore, preclinical compounds such as NKP-1339 have advanced to early phase clinical trials, highlighting the therapeutic potential of Ru-based drugs. In the present study, we synthesized two novel dimeric Ru­(II) complexes incorporating a planar, π-conjugated PNN ligand. The PNN ligand was selected for its extended π-conjugation (across 12 atoms), which facilitates enhanced DNA intercalation and redox modulation, potentially boosting cytotoxic effects. These were specifically designed to enhance biomolecular interactions and redox-driven cytotoxic effects. The two complexes, PNRP and PNRB, feature distinct arene ligands (p-cymene and benzene), enabling the exploration of arene-dependent structure–activity relationships.

However, most reported Ru­(II)-arene complexes utilize simple polypyridyl ligands, and the use of π-extended PNN scaffolds in dimeric architectures remains largely unexplored. Additionally, the influence of different arene rings on redox activity, DNA/HSA binding, and cytotoxicity in dimeric Ru­(II) systems has not been systematically investigated. This gap forms the core motivation of the present study.

Ruthenium’s toggle between Ru­(II) and Ru­(III) oxidation states, along with its iron-mimicking properties in biological systems, facilitates selective tumor targeting with reduced systemic toxicity compared to platinum-based agents. Additionally, ruthenium complexes exhibit tunable ligand exchange kinetics, allowing their stability and reactivity to be tailored within biological environments.

These limitations have prompted a growing interest in metal-based anticancer agents that offer improved selectivity, favorable pharmacokinetics, and alternative mechanisms of action. −

Ruthenium, with its ability to switch between multiple oxidation states and mimic iron-binding behavior, is well-suited for biomedical applications. , Herein, we present two novel dimeric Ru­(II)-arene complexes (PNRP and PNRB) incorporating a π-conjugated PNN scaffold designed to enhance redox activity, biomolecular interactions, and anticancer efficacy.

The dimeric Ru­(II) design was chosen based on the hypothesis that two metal centers, combined with a π-extended PNN scaffold, may enable stronger biomolecular interactions and enhanced redox behavior compared to monometallic analogues. This design approach was expected to enhance cytotoxicity via synergistic interactions, concurrently facilitating the methodical assessment of arene-related structure–activity relationships. Although higher molecular mass can limit oral absorption, this concern can be overcome by intravenous administration, which ensures direct delivery into systemic circulation, rapid bioavailability, and targeted cellular uptake, thereby maximizing therapeutic efficiency.

2. Experimental Section

2.1. Materials and Methods

All reagents and solvents were of analytical grade and used without further purification unless otherwise specified. Crucial chemicals like 1,10-phenanthroline-5,6-dione, N′-bis­(4-formylphenyl)-N,N′-diphenylbenzidine, ruthenium­(II)-(dichloro)-p-cymene dimer, ruthenium­(II) benzene dimer, and ammonium acetate were procured from BLD Pharma (India). Aluminum sheets precoated with silica gel for thin-layer chromatography were supplied by E. Merck, Germany. Human serum albumin was obtained from HIMEDIA (India), n-Octanol is from SD Fine-Chem (India).

The ¹H and ¹³C NMR spectra were obtained with a 400 MHz Advanced Bruker DPX spectrometer, employing tetramethyl silane (TMS) as the reference standard. Melting points were assessed using an open capillary tube and a DT device controlled by an Elchem Microprocessor. Viscosity was measured using an Ostwald viscometer. Conductivity was determined using a TDS Conductometer 307. Infrared (IR) spectra (4000–400 cm-1) were obtained utilizing a Shimadzu Affinity FT-IR spectrometer. Mass spectra were acquired with a Shimadzu ESI-MS-4000 mass spectrometer, utilizing methanol as the solvent. UV–visible spectra were recorded using a JASCO V-670 spectrophotometer equipped with a 1 cm quartz cuvette.

DPPH obtained from SRL Chem in methanol and ascorbic acid sourced from SD Fine Chemicals were employed. The human lung cancer cell line (A 549) was sourced from the National Centre for Cell Science (NCCS), Pune, and cultivated in A549 and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum and antibiotics. The cells were kept at 37 °C, 5% CO₂, 95% air, and 100% relative humidity. DMEM medium, Fetal Bovine Serum (FBS), and antibiotic solution were obtained from Gibco (USA), the Total reactive oxygen species (ROS) kit was sourced from Invitrogen (USA), and 1X PBS was acquired from Himedia (India). The 6-well tissue culture plate and wash beaker were sourced from Tarson (India).

2.2. Synthetic Procedure

2.2.1. Synthesis of Ligand

PNN ligand synthesis was carried out by using 1,10-Phenanthroline-5,6-dione and N,N′-Bis­(4-formylphenyl)-N,N′-diphenylbenzidine were mixed with ammonium acetate and acetic acid as the solvent for 30 h. The advancement of the reaction was observed using TLC TLC in Hexane: ethyl acetate (1:1) as mobile phase. TLC analysis of the synthesized PNN ligand showed a single spot with an Rf value of ∼0.38, indicating its purity. Upon completion, the reaction mixture was transferred into ice-cold water and neutralized with ammonium acetate. Upon neutralization, the product precipitated out of the reaction mixture. The precipitate was filtered off, washed several times with a hexane and ethyl acetate mixture to remove residual impurities, and then dried under reduced pressure to yield the pure ligand (Scheme ). Colar Pink powder. Yield 74%, M.P. 160 ° and Molecular Formula C₆₂H₄₀N₁₀. Elemental analysis PNN (C 80.73%, H 4.57%, N 15.03%). ¹H NMR (400 MHz, DMSO): δ 13.66 (s, 2H), 9.01 (dd, J = 4.4, 1.8 Hz, 4H), 8.90–8.88 (m, 4H), 8.19–8.88 (d, J = 8.3 Hz, 3H), 7.81 (s, 5H), 7.67–7.65 (dd, J = 11.6, 8.3 Hz, 4H), 7.43–7.39 (m, 4H), 7.20–7.15 (dq, J = 9.8, 5.4 Hz, 14H). ¹³C NMR (101 MHz, DMSO): δ 172.16, 151.14, 148.08, 146.96, 146.23, 143.58, 130.33, 130.23, 128.04, 127.93, 125.54, 125.07, 124.01, 123.80, 122.78, 40.52. Ir (cm^–1): 3320 (N–H stretching), 3060 (C–H Ar stretching), 1660 (CC stretching). HRMS (ESI) m/z: [M]⁺ calcd is M = 925.05; found, 925.40.

1. Synthesis of Ligand PNN.

2.2.2. Synthesis of Complex PNRP

The PNRP complex was synthesized by reacting the PNN ligand with ruthenium­(II) p-cymene dimer in methanol as the medium. The reaction was conducted at room temperature for 4 h, with progress observed through TLC in DCM: Methanol (9:1) as mobile phase. TLC analysis of PNRP displayed a single spot with an Rf value of ∼0.63, confirming successful complex formation. Upon completion, the precipitate was filtered and rinsed with hexane, resulting in a dark pink product. (Figure ). Upon completion, the precipitate was filtered and rinsed with hexane, resulting in a dark pink product. Yield 77%. M. P. > 300 °C and Molecular Formula C₈₂H₆₈C_l2N₁₀Ru₂. Elemental analysis PNRP (C 67.41%, H 4.91%, N 9.72%). ¹³C NMR (101 MHz, DMSO): δ 146.70, 132.83, 130.54, 126.90,122.12, 104.37, 86.66, 84.40, 30.88, 22.03, 18.71.¹H NMR (400 MHz, DMSO): δ 14.65 (s, 2H), 9.83 (s, 4H), 9.45 (s, 2H), 9.13 (s, 2H), 8.28 (d, J = 8.6 Hz, 4H), 8.26 (d, J = 18.9 Hz, 4H), 7.69 (d, J = 8.0 Hz, 4H), 7.43 (t, J = 7.9 Hz, 4H), 7.20 (d, J = 8.1 Hz, 14H), 6.34 (d, J = 6.1 Hz, 4H), 6.11 (d, J = 6.1 Hz, 4H), 2.23 (s, 2H), 2.19 (s, 6H), 0.91 (d, J = 6.9 Hz, 12H). Ir (cm^–1): 3364 (N–H stretching), 3067 (C–H Ar stretching), 1595 (CC stretching), 1195 (C–N stretching), 506 (N–Ru Stretching). HRMS (ESI⁺) m/z: [M + H]⁺ calcd 1466.22; found, 1466.43. A major peak at m/z = 734.19 corresponds to [M/2 + H]⁺ calcd M/2 = 733.11, [M/2 + H]⁺ = 734.11.

1.

Structures of complexes PNRP (p-cymene) and PNRB (benzene) with π-conjugated PNN ligand.

2.2.3. Synthesis of Complex PNRB

The PNRB complex was created by reacting the PNN ligand with ruthenium­(II) benzene dimer using methanol as the solvent. The reaction was conducted at room temperature for 4 h, and its advancement was tracked using TLC in DCM/Methanol (9:1) as mobile phase. TLC analysis of PNRB exhibited a single spot with an Rf value of ∼0.58, consistent with the expected polarity of the complex. Once the reaction was completed, the precipitate formed was gathered, thoroughly rinsed with hexane, yielding a rosewood-hued product (Figure ). Yield 82%. M. P. 210 °C and Molecular Formula C₇₄H₅₂C_l2N₁₀Ru₂. Elemental analysis PNRB (C 65.59%, H 4.23%, N 10.49%). ¹H NMR (400 MHz, DMSO): δ 14.55 (s, 2H), 9.92 (s, 4H), 9.39 (s, 2H), 9.10 (s, 2H), 8.27 (d, J = 8.3 Hz, 4H), 8.25 (d, J = 31.2 Hz, 4H), 7.70 (t, J = 10.4 Hz, 4H), 7.44 (t, J = 7.8 Hz, 4H), 7.21 (q, J = 10.2 Hz, 14H), 6.32 (s, 12H). ¹³C NMR (101 MHz, DMSO): δ 172.30, 153.21, 146.77, 143.51, 130.40, 128.78, 128.45, 128.05, 125.79, 122.24, 88.85, 88.11, 87.22, 83.11, 22.92. FT - Ir (cm^–1): 3320 (N–H stretching), 3060 (C–H Ar stretching), 1660 (CC stretching), 1195 (C–N stretching), 554 (C–F Stretching). HRMS (ESI⁺) m/z: [M + H]⁺ calcd 1354.22; found, 1354.32. A major peak at m/z = 677.11 corresponds to [M/2]⁺ calcd M/2 = 677.16.

3. Result and Discussion

3.1. Synthesis and Characterizations

The PNN ligand was synthesized by using 1,10-phenanthroline-5,6-dione and N,N′-Bis­(4-formylphenyl)-N,N′-diphenylbenzidine as the starting materials. The reaction took place in acetic acid as the solvent, with an excess of ammonium acetate added to facilitate the condensation, and the mixture was refluxed for a duration of 30 h. The structural validation of the ligand was performed utilizing techniques including ¹H NMR, ¹³C NMR, FT-IR, and HRMS. In the ¹H NMR spectrum, aromatic protons appeared in the range of δ 7.159–9.018 ppm, while NH protons displayed a distinct signal at δ 13.663 ppm. The ¹³C NMR spectrum displayed chemical shifts for the carbon atoms between δ 122.78 and δ 172.16 ppm. FT-IR analysis verified the presence of functional groups characteristic of the ligand, whereas HRMS confirmed its molecular weight, providing strong evidence of successful synthesis and structural integrity (Figures S1–S14).

The PNN ligand was additionally used to synthesize PNRP and PNRB complexes by reacting it with ruthenium­(II)-η⁶-p-cymene and ruthenium­(II)-η⁶-benzene in methanol at ambient temperature for 4 h. The progress of the reaction was tracked via thin-layer chromatography (TLC), and the resulting complexes were carefully characterized. In the PNRP complex, ¹H NMR spectra displayed signals for aliphatic protons from δ 0.897–2.238 ppm and aromatic protons in the range of δ 6.098–9.838 ppm. The ¹³C NMR signals were observed in the ranges of δ 18.71–30.88 ppm for aliphatic carbons and δ 84.40–146.70 ppm for aromatic carbons. For the PNRB complex, ¹H NMR signals for aromatic protons were detected between 6.324–9.926 ppm, while ¹³C NMR signals for aromatic carbons were found in the range of 83.10–172.30 ppm. Structural validation was additionally reinforced by HRMS for assessing molecular weight and FT-IR spectroscopy for analyzing functional groups.

3.2. Photophysical Properties

Absorption and emission studies were systematically carried out in two different solvent systems: pure DMSO (100%) and a diluted solution (1% DMSO in a suitable solvent), to examine how solvent composition affects the electronic characteristics of the complexes. In both scenarios, π → π* transitions were detected in the 250–297 nm range, ascribed to intra ligand electronic excitations stemming from the ligands’ conjugated π system. Additionally, metal-to-ligand charge transfer (MLCT) transitions were observed between 300 and 450 nm (Figures S15 and S16), emphasizing effective electronic interaction between the metal core and the ligands (Tables and ). ,

1. 1% and 100% DMSO Absorption and Emission Parameters in UV–vis.

sample	1% DMSO		100% DMSO
	π–π*	MLCT	π–π*	MLCT
PNRP	277 nm	380 nm	283 nm	379 nm
PNRB	268 nm	380 nm	281 nm	380 nm

^aAbsorption maxima in the π–π* resign in UV.

^bAbsorption maxima in MLCT resign in UV.

2. Photophysical Parameters and Partition Coefficient.

sample	λ (nm)		λ (nm)	stock shift	O.D.	Φ	Log P o/w
	π–π*	MLCT
PNRP	277	380	434	54	0.95	0.011	0.6030
PNRB	268	380	436	56	1.02	0.03	1.133
quinoline sulfate	350		442	92	0.46	0.31

^aAbsorption maxima.

^bEmission maxima.

^cOptical density.

^dQuantum yield.

^e n-octanol/water partition coefficient.

In fluorescence investigations, PNRP in 1% DMSO exhibited excitation at 380 nm, whereas in 100% DMSO, excitation took place at 379 nm, demonstrating a slight solvent-dependent variation. For the intricate PNRB, excitation was reliably detected at 380 nm in both solvents, indicating its stability and independence from the solvent’s nature. ,

The quantum yield (φ) of these complexes in 1% DMSO was assessed using quinoline sulfate as a standard reference (Table ). These transitions exhibited a clear sensitivity to the solvent environment, highlighting the influence of polarity and solvation effects in altering the photophysical behavior. ,

3.3. Study of Solubility and Lipophilicity

The solubility of the complexes PNRP and PNRB was evaluated in various organic solvents, such as acetonitrile, acetone, methanol, tetrahydrofuran (THF), toluene, ethyl acetate, dimethyl sulfoxide (DMSO), and ethanol. Both complexes showed impressive solubility in every solvent tested, highlighting their outstanding versatility and potential for use in different chemical and biological scenarios.

The shake-flask method, a well-known methodology for assessing compound distribution between aqueous and organic phases, was used to calculate the partition coefficient (log P) of the complexes PNRP and PNRB. In this study, n-octanol was used as the organic phase and deionized water as the aqueous phase.

PNRP and PNRB had computed log P values of 0.6030 and 1.133, respectively.(Table ) Its comparatively hydrophilic nature is indicated by its PNRP. This implies that PNRP is more suited for applications where water solubility is crucial because it has a greater affinity for watery conditions. PNRB, which reflects its increased lipophilicity. This characteristic suggests a stronger preference for surroundings based on lipids.

3.4. Stability Study

The stability of both complexes was tested in three distinct media phosphate-buffered saline (PBS, pH 7.4), glutathione (GSH), and aqueous medium at various time points (0, 1, 24, and 48 h). Understanding the interactions between metal complexes in biological systems depends on these stability investigations. PBS ensures that the complexes stay stable in the body by simulating physiological conditions. While the aqueous medium evaluates the complexes’ general stability in water-based systems, GSH, which represents a reducing intracellular environment, tests the complexes’ resilience in cellular circumstances. The findings demonstrated that neither complex changed significantly over the course of 48 h, demonstrating their strong stability under all conditions (Figures S17–S19). This shows that these complexes maintain their structural integrity and chemical composition even under settings similar to physiological and intracellular environments.

3.5. DNA Binding Experiments

3.5.1. Absorption Titration with DNA

DNA binding investigations were conducted to understand the interaction between metal complexes and DNA, which is essential for evaluating their therapeutic potential. In these experiments, DNA concentrations between 1 and 100 μM were incrementally introduced to solutions that maintained a constant concentration of each complex. With the rise in DNA concentration, the absorbance heightened as well, indicating a robust interaction between the complexes and DNA. This implies that the complexes probably interact with the DNA via groove-binding, a precise and stable connection where they adhere to the minor or major grooves of the DNA helix without altering its structure. The binding constants for the complexes 3.98 × 10⁵ M^–1 for PNRP and 3.19 × 10⁵ M^–1 for PNRB provide additional confirmation of this strong binding affinity (Figures and S20). These findings underscore the complexes’ ability to affect DNA functions, rendering them promising options for therapeutic use, particularly in cancer therapy, where DNA interaction can interfere with essential cellular activities such as transcription and replication. (Table ) ,

2.

DNA binding Plot of in Uv–vis A. PNRP B. PNRB.

4. DNA Binding Constants of Complexes Obtained from UV–Vis Absorption (K _b) and Fluorescence Quenching (K _SV, K _app) Studies.

sample	λ (nm)	change absorbance intensity	K b × (105 M–1)	K SV × (106 M–1)	K app × (106 M–1)
PNRP	603	hyperchromic	3.98	9.24	5.71
PNRB	603	hyperchromic	3.19	7.3	1.17

^aK_b, DNA binding constant derived from UV–visible absorption spectra.

^bK_SV, Stern–Volmer quenching constant.

^cK_app, apparent DNA binding constant, determined through competitive displacement in fluorescence spectroscopy.

3.5.2. Absorption Titration with Guanine

After establishing that the complexes PNRP and PNRB interact with DNA primarily through groove binding, we extended our investigation to understand their specific interaction with guanine using UV–visible spectroscopy. By gradually increasing guanine concentrations from 0 μM to 60 μM, we observed a consistent decrease in absorption (hypochromic), pointing to a strong and specific interaction between guanine and the complexes. The binding constants were calculated as 0.085 × 10⁵ M^–1 for PNRP and 1.045 × 10⁵ M^–1 for PNRB, clearly showing that PNRP has a stronger affinity for guanine (Figures and S21). These findings highlight guanine as a critical binding site for these complexes and emphasize their potential for selectively targeting nucleobases. The strong and specific binding observed here paves the way for deeper exploration into the biological applications of PNRP and PNRB (Table and ). ,

3.

Guanine binding plot of UV–vis spectroscopy for A. PNRP B. PNRB.

3. Binding Constants and Absorbance Changes of Complexes with Guanine.

sample	change absorbance intensity	K b × (105 M–1)
PNRP	hypochromic	0.085
PNRB	hypochromic	1.045

^aK_b, DNA binding constant derived from UV–visible absorption spectra.

3.5.3. Fluoresce Titration with DNA - EtBr

To gain more insight into the DNA binding characteristics of the complexes, a study on ethidium bromide (EtBr) displacement was conducted utilizing fluorescence spectroscopy. In this experiment, the concentration of the DNA-EtBr solution remained constant as the concentration of the metal complex was progressively raised. The system was excited at 485 nm, and the emission was recorded between 530 and 700 nm, with the peak emission detected at 603 nm (Figures and S22) Upon the introduction of the complexes, a significant reduction in fluorescence intensity was observed, indicating that the complexes effectively displaced EtBr from the DNA. This indicates a mode of intercalative binding, wherein the complexes position themselves between the DNA base pairs, vying with EtBr for binding locations. This form of interaction is extensively researched for its capacity to stabilize within the DNA framework and possibly affect essential DNA functions. The Stern–Volmer constants for PNRP and PNRB were determined to be 9.24 × 10⁴ M^–1 and 7.37 × 10⁴ M^–1, respectively, demonstrating notable promising candidate binding to DNA, PNRP demonstrates stronger DNA binding compared to PNRB, as indicated by its higher Stern–Volmer constant and apparent binding constant. Furthermore, the observed binding constants (K_app) were 5.71 M^–1 for PNRP and 1.17 M^–1 for PNRB, indicating the comparative binding strengths of each complex. These results offer important understanding of how the complexes engage with DNA, emphasizing their promise as agents that target DNA. This holds particular importance for therapeutic uses, such as in cancer therapies, where intercalative binding can interfere with crucial DNA functions like replication and transcription, thereby affecting cellular activity in a specific way. (Table ) ,

4.

DNA EtBr binding plot of fluorescence quenching study for complex A. PNRP B. PNRB.

3.5.4. Viscosity Measurement

The viscosity investigation examined the relationship between calf thymus DNA and two metal complexes, PNRP and PNRB, through hydrodynamic techniques. An Ostwald viscometer was utilized to assess variations in relative viscosity (η/η₀) as the molar ratio of the complexes to DNA was systematically changed. The data were examined by graphing η/η₀ ^1/3 as a function of the molar ratio, revealing information about the complexes’ binding behavior. Both PNRP and PNRB showed almost the same viscosity patterns, maintaining steady η/η₀ values throughout the range of complex-to-DNA ratios examined. ,

The steady viscosity findings suggest that PNRP and PNRB attach to DNA via processes like minor groove binding or electrostatic interactions, which maintain the structural integrity of the DNA helix (Figure ). This consistent behavior indicates that the complexes engage with DNA without inducing any notable structural alterations or distortions. These results emphasize how PNRP and PNRB can preserve the natural structure of DNA, positioning them as ideal choices for uses where DNA stability is critical. ,

5.

Viscosity measurements of ct-DNA in the presence of increasing amounts of complexes.

3.6. Protein Interaction Studies with Human Seram Albumin

3.6.1. Fluorescence Titration with HSA

The study on protein binding investigated the interaction of PNRP and PNRB complexes with human serum albumin, an essential protein that plays a key role in drug transport and distribution within biological systems. HSA was chosen as a model protein due to its capacity to replicate physiological conditions, offering insights into the biological behavior of these complexes. Fluorescence quenching studies were performed by titrating HSA solutions with higher concentrations of the complexes (0–20 μM) (Figure ) to ascertain their binding affinity, binding ratio, and the number of binding sites. The fluorescence data was analyzed using the Stern–Volmer equation, and its double logarithmic version was employed to determine the number of binding sites (n) on HSA.

6.

Fluorescence emission spectra of HSA upon titration with increasing concentrations of complexes (A) PNRP (B) PNRB.

The findings showed notable variations in how the complexes interacted with HSA. PNRP demonstrated a binding constant of 1.08 × 10⁶ M^–1, reflecting a robust binding affinity, whereas PNRB presented a reduced binding constant of 0.07 × 10⁶ M^–1. The Scatchard plot evaluation revealed that the quantity of binding sites was 1.076 for PNRP and 0.92 for PNRB, indicating that PNRP associates with HSA in an almost 1:1 ratio. The binding constants determined using HSA were 0.064 × 10⁶ M^–1 for PNRP and 0.0845 × 10⁶ M^–1 for PNRB. (Table , Figures S23 and S24) ,

5. Binding Parameters Complexes with HSA Determined from Fluorescence Quenching Analysis .

sample	KHSA × 106 M–1	K q × 1014 M–1	k × 106 M–1	n
PNRP	1.08	1.08	0.0642	1.07
PNRB	0.07	0.07	0.0845	0.92

^a K _HSA, Stern–Volmer quenching constant.

^b K _q, quenching rate constant (HSA).

^c K, binding constant with HSA.

^d n number of binding sites.

These results emphasize PNRP’s stronger binding affinity and consistent interaction with HSA in comparison to PNRB. This indicates that PNRP is a viable option for drug delivery and therapeutic uses, due to its capacity to create stable protein–ligand complexes in physiological environments. (Table )

3.6.2. Synchronous Spectroscopy of HSA

Synchronous fluorescence spectroscopy was utilized in this research to investigate the microenvironmental changes surrounding the key amino acid residues, tryptophan and tyrosine, in human serum albumin (HSA) when interacting with the complexes PNRP and PNRB. This sensitive technique was used to identify the binding locations of the complexes on HSA by examining fluorescence quenching close to the tryptophan and tyrosine residues, offering insights into their interaction mechanisms. ,

Synchronous mode fluorescence emission measurements were conducted with excitation and emission wavelengths differing by constant Δλ values of 15 and 60 nm, which are characteristic for tyrosine and tryptophan residues, respectively. A constant concentration of HSA was titrated with escalating concentrations of PNRP and PNRB complexes (0–100 μM), and the resulting changes in fluorescence intensity were measured. ,

The findings demonstrated notable fluorescence quenching at Δλ = 60 nm in contrast to Δλ = 15 nm, suggesting that both PNRP and PNRB mainly engage with tryptophan residues in HSA. The increased quenching at Δλ = 60 nm indicates tryptophan’s increased sensitivity to variations in its environment, implying that the complexes affect the microenvironment surrounding tryptophan. This backs the conclusion that PNRP and PNRB preferentially attach to tryptophan-rich areas of HSA, without considerably impacting tyrosine residues (Figures S25 and S26). These results offer important perspectives on the precise binding mechanism of the complexes, essential for comprehending their potential in drug transport and delivery systems. ,

3.7. Determination of Thermodynamic Parameters

3.7.1. Study of Binding Affinity (log K), Binding Capacity (n) and Free Energy (ΔG◦)

To determine the protein-binding properties of the synthesized metal complexes, PNRP and PNRB, we performed fluorescence quenching analysis utilizing a static quenching mechanism. From this analysis, we were able to determine the binding constants (log K), binding stoichiometry (n), and Gibbs free energy changes (ΔG°), at three biologically relevant temperatures; 298, 308, and 318 K. The PNRP complex showed a strong and stable binding affinity in all tests with log K values from 6.49 to 6.03 across all biological levels. The binding stoichiometry was nearly unity (n ≈ 1.2–1.3), which would indicate a highly specific binding. The ΔG° values were all negative (−3.706 to −3.672 × 10⁴ J mol^–1), indicating that the interaction is thermodynamically favorable, and spontaneous across all levels. This is further represented by the high R ² values, suggesting that the experimental data was very clean, and the binding model was consistent with high correlation.

The PNRB complex also exhibited higher protein affinity, evidenced by a consistent increase in log K from 4.48 at 298 K to 5.95 at 318 K, indicating increased protein recognition at physiological and elevated temperatures. In addition to stronger protein recognition, a subsequent increase in negative ΔG° values (−2.56 to −3.63 × 10⁴ J mol^–1) and larger binding stoichiometry, were observed at higher temperatures (Table ). At higher temperatures, the binding stoichiometry (n) increased to 1.2, indicating a more favorable protein interaction with HSA as well as more favorable and cooperative protein interaction (Figure ). Overall, both complexes exhibited high-affinity, site-specific, and spontaneous binding to HSA, supporting an ability to remain stable in vivo and effectively distribute via protein-mediated delivery. ,

6. Temperature-dependent Binding Constants (log K, n) and Thermodynamic Parameters (ΔG°, ΔH°, ΔS°) for Complexes HSA Interaction with Corresponding Linear Regression Coefficients.

sample	temp.	Log K	n	ΔG J mol–1 104	R 2	ΔH J mol–1 104	ΔS J mol–1 102	R 2
PNRP	298 K	6.49	1.3	–3.706	0.994	–1.17	–0.04	0.9949
	308 K	6.22	1.2	–3.672	0.994
	318 K	6.03	1.2	–3.672	0.998
PNRB	298 K	4.48	0.9	–2.557	0.988	3.73	1.49	0.9017
	308 K	5.66	1.2	–3.343	0.995
	318 K	5.95	1.2	–3.627	0.9878

7.

Log [(F ₀ – F)/F] vs log [Q] plots for complexes A. PNRP B. PNRB binding to HSA at different temp. (298, 308, and 318 K) for the determination of thermodynamic parameters.

3.7.2. Determination of Enthalpy (ΔH°) and Entropy (ΔS°) of Binding

In order to better understand these interactions at a mechanistic level, we derived thermodynamic parameters ΔH° and ΔS° from van’t Hoff plots based on temperature-dependent binding data. , The PNRP complex demonstrated a favorable thermodynamic signature, ΔH° = −1.17 × 10⁴ Jmol^–1 and ΔS° = −0.04 × 10² J mol^–1 K^–1, which is reflective of an enthalpy-dominated, highly structured interaction. These values suggest complex formation via stable associations with HSA at specific sites mediated by hydrogen bonding, van der Waals forces, adjacent metal–ligand coordination involving donor groups on the protein, and the subsequent release of loosely associated water. The exothermic nature of this process indicates that the PNRP complex has high affinity and is specifically fitting into binding pockets of HSA, which supports long-term serum stability. The force of hydrophobic associations that characterize PNRB also gave excellent thermodynamic parameters, ΔH° = +3.73 × 10⁴ Jmol^–1 and ΔS° = +1.49 × 10² J mol^–1 K^–1, indicating that hydrophobic associations drive PNRB binding (Table , Figures and ). The large positive entropy change indicates that there are not only many hydrophobic interactions, but there were considerable favorable conformational dynamics associated with displacement of structured water molecules, which characterized a highly favorable hydrophobic environment. Hydrophobic interactions grant PNRB the properties of strong protein binding when physiological stress is applied, indicating its adaptability and robustness in biological systems.

8.

Van’t Hoff plot (ln K vs 1/T) for the interaction of complexes A. PNRP B. PNRB with HSA used to determine thermodynamic parameters ΔH° and ΔS°.

9.

Visualization of intracellular ROS generation in A549 cells after treatment with PNRP and PNRB A. Control cells treated with 0.1% DMSO B. Cells treated with PNRP C. PNRB-treated cells.

These thermodynamic results show that there are two different but still very high-value types of protein interactions based on thermodynamic principles. PNRP achieves great structural recognition using specific binding unique to the protein and PNRB captures the protein in a dynamic and flexible manner using hydrophobic and entropic forces. Both of these pathways provided great protein-binding properties, emphasizing the wide-ranging therapeutic potential of these types of complexes in relevant biological environments. −

3.8. Antioxidant Activity

The antioxidant properties of the synthesized compounds PNRP and PNRB were assessed through the DPPH radical scavenging assay, resulting in IC₅₀ values of 30.2 μM and 39.2 μM, respectively, in contrast to 88.73 μM for ascorbic acid (Figures S27–S29). These results demonstrate that both substances show significant free radical scavenging ability, with PNRP showing marginally better performance. Although the assay relies on a chemical interaction, it accurately represents the redox properties of molecules and their capacity to stabilize or neutralize reactive species. This antioxidant ability is frequently linked to the existence of electron-donating groups, conjugated structures, or heteroatoms that can delocalize charge factors that may contribute to the observed activity of PNRP and PNRB. In general, the findings indicate that these substances have inherent radical-scavenging properties, enhancing their chemical stability and functional significance in oxidative conditions. (Table )

7. Antioxidant Value of Sample PNRP and PNRB.

S. No.	sample	IC50 ± SD (μM)
1	ascorbic acid	88.73 ± 0.058
2	PNRP	30.2 ± 8.12
3	PNRB	39.2 ± 10.37

3.9. In Vitro Cytotoxicity Study

The cytotoxicity of the synthesized ruthenium complexes PNRP and PNRB was evaluated against A549 lung cancer cells, with HEK293 normal cells used to assess selectivity. , Both complexes exhibited notable anticancer activity and a clear preference for targeting malignant cells over normal cells. PNRP reduced A549 cell viability with comparatively limited effects on HEK293 cells, reflecting a favorable therapeutic window. −

Among the two complexes, PNRB demonstrated superior potency, showing a lower IC₅₀ value for A549 cells while maintaining high IC₅₀ values for HEK293 cells. This enhanced selectivity underscores PNRB as a promising lead candidate for further biological investigation. Overall, the data indicate that both complexes particularly PNRB possess meaningful anticancer potential with reduced off-target toxicity (Figures S30–S35). IC₅₀ values were derived from nonlinear regression of dose–response curves, and the reported standard deviations represent triplicate experimental measurements. (Table ) ,−

8. Cytotoxicity of the Complexes and Cisplatin.

sample code	IC50 value (μM) of A549 ± SD	IC50 value (μM) of HEK293 ± SD	SIA549
PNRP	35.58 ± 0.4	222.72 ± 0.17	6.26
PNRB	31.37 ± 0.42	237.85 ± 0.13	7.58
cisplatin	16.7 ± 0.06	36.6 ± 0.02	2.2

^aIC 50% of cells undergo cell death cell line cancer.

^bSelectivity index of cells.

3.10. Assessment of ROS Production in A549 Cells

The synthesized complexes PNRP and PNRB notably increased the levels of intracellular reactive oxygen species in A549 lung cancer cells, evidenced by heightened fluorescence intensity under a 520 nm filter and semiquantified using fluorescence microscopy. ,

The initiation of oxidative stress is acknowledged as an important anticancer mechanism, particularly in cancer cells that are more vulnerable to ROS-induced harm because of their heightened baseline oxidative state. PNRB generated a significantly increased ROS signal in comparison to PNRP, reflecting its enhanced cytotoxicity. This relationship indicates that PNRB’s enhanced potency might stem from its greater capacity to disturb redox balance, causing oxidative harm to essential cellular elements like DNA and mitochondria. ,

The function of ROS in the action mechanism was additionally confirmed by pretreatment with reduced glutathione (GSH, 5 mM), a recognized intracellular antioxidant. GSH notably reduced fluorescence signals in both treatment groups, verifying that the ROS detected was intracellular and resulted from the treatment. Given that cancer cells depend greatly on antioxidant mechanisms such as the Nrf2/HO-1 pathway to endure oxidative stress, PNRB’s capacity to surpass these defenses emphasizes its significance in therapy. The increased ROS levels probably trigger mitochondrial-mediated apoptosis by disrupting membrane potential, activating pro-apoptotic proteins like Bax, and reducing antiapoptotic factors such as Bcl-2. While these downstream events were not directly assessed in this study, the detected redox disturbance strongly indicates the participation of oxidative apoptotic signaling. , The greater selectivity index (SI = 7.58) of PNRB in contrast to PNRP (SI = 6.26) might result from different ROS management in cancerous versus normal cells. These results emphasize PNRB as a selective redox-active option for lung cancer treatment. Additional research is being conducted to verify its role in the apoptotic pathway at the molecular level. −

4. Conclusions

This study reports two novel dimeric Ru­(II)-arene complexes, PNRP and PNRB, incorporating a redox-active, π-conjugated PNN scaffold designed to enhance DNA/protein interactions and promote oxidative stress in cancer cells. Spectroscopic and thermodynamic investigations confirmed spontaneous binding to both calf thymus DNA and HSA, with favorable ΔG° values (−27.9 to −30.2 kJ mol^–1) and binding constants exceeding 10⁵ M^–1, indicative of moderately strong noncovalent interactions predominantly governed by hydrophobic forces.

The complexes exhibited partial intercalative DNA binding, as evidenced by viscosity enhancement and guanine interaction studies, and quenched HSA fluorescence via a static quenching mechanism. ROS fluorescence imaging further confirmed redox-mediated oxidative stress as a major contributor to cytotoxicity.

Notably, both complexes demonstrated selective antiproliferative activity toward A549 lung carcinoma cells over normal HEK293 cells. PNRB showed the most potent activity with an IC₅₀ of 31.37 μM and a selectivity index of 7.58, while PNRP showed an IC₅₀ of 35.58 μM and SI of 6.26. These findings underscore the potential of π-conjugated, arene-tuned Ru­(II) scaffolds as redox-active chemotherapeutic agents capable of selective biomolecular engagement. This work lays a mechanistic foundation for the future development of rationally designed ruthenium complexes for targeted cancer therapy.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07975.

• Supporting Information is available and includes additional spectral data, supplementary figures, and other materials that support the findings presented in this manuscript (PDF)

The authors declare no competing financial interest.