Variation of Arene Moiety in Ruthenium (II) Arene Halido Complexes of Quinoline and Benzazole‐Derived Ligands: Development and Antiprostate Cancer Potential

ABSTRACT

Castration‐resistant prostate cancer (CRPC) is an advanced, treatment‐refractory disease with high mortality and limited therapeutic options, necessitating novel metallodrugs with enhanced potency and reduced toxicity. Herein, we report the design and syntheses of two quinoline–benzazole hybrid bidentate ligands—2‐(1H‐benzo[d]imidazol‐2‐yl)quinoline (L1) and 2‐(quinolin‐2‐yl)benzo[d]thiazol (L2)—along with four new half‐sandwich ruthenium(II) arene chlorido complexes of the general formula [(η⁶‐arene)(L)RuCl]PF₆, where arene = benzene (Bz) or hexamethylbenzene (Hmb), yielding complexes 1–4. The ligands and complexes were fully characterized by spectroscopic (¹H/^{1 3}C NMR, IR, UV–vis) and analytical methods (elemental analysis, ESI‐MS), with aqueous stability profiles confirming their suitability for biological evaluation. Cytotoxicity assays against the PC‐3 CRPC cell line identified potent antiproliferative activity in selected complexes. Mechanistic studies revealed predominant G2/M phase cell cycle arrest, induction of apoptosis, and moderate‐to‐strong DNA binding affinities suggestive of intercalative/groove‐binding modes. These results highlight the potential of quinoline–benzazole Ru(II) arene scaffolds as promising candidates for CRPC therapy, meriting further optimization.

Novel quinoline–benzazole hybrid Ru(II) arene complexes were synthesized and characterized. They exhibit potent cytotoxicity against castration‐resistant prostate cancer (PC‐3) cells, inducing G2/M arrest, apoptosis, and DNA intercalation/groove binding. These scaffolds show promise as new therapeutic candidates for treatment‐refractory CRPC.

1. Introduction

Prostate cancer is the second most common cancer and the fifth leading cause of cancer‐related death among men worldwide [1]. In 10%–20% of patients, the disease progresses to castration‐resistant prostate cancer (CRPC), which continues to proliferate despite suppression of serum testosterone to castrate levels [1, 2, 3]. The majority of CRPC cases are metastatic (mCRPC) at diagnosis or rapidly become so, resulting in a highly treatment‐refractory state with limited therapeutic options [3, 4, 5]. Currently, taxane‐based chemotherapy with docetaxel (first‐line) or cabazitaxel (post‐docetaxel) remains a cornerstone for mCRPC, while platinum agents such as carboplatin show modest activity—primarily in combination regimens for aggressive variants—and are associated with increased toxicity and limited overall benefit in unselected patients [6, 7, 8]. In an effort to overcome these limitations, considerable attention has recently been directed toward the development of bimetallic complexes, particularly platinum‐based systems incorporating either a second platinum center or alternative metal ions, with the goal of enhancing anticancer efficacy and circumventing resistance mechanisms [9, 10, 11]. In this context, ruthenium(II) half‐sandwich complexes have also emerged as highly promising anticancer candidates due to their remarkable antineoplastic and antimetastatic properties, often superior to platinum drugs in resistant models [12, 13, 14, 15, 16, 17, 18, 19]. Although several ruthenium‐based compounds (e.g., NAMI‐A, KP1019/NKP‐1339, and TLD1433) have advanced to clinical trials for various malignancies, their evaluation in CRPC remains limited, underscoring the potential of new Ru(II)‐arene scaffolds to address this unmet clinical need [20]. Advanced prostate cancer, particularly CRPC, continues to pose a significant therapeutic challenge. While platinum‐based drugs have been extensively studied in this setting [21], relatively few metal complexes beyond well‐established Pt agents have been evaluated [22]. Moreover, only a limited number of non‐platinum complexes have demonstrated meaningful activity against prostate cancer models [23, 24].

Our group has previously reported a series of half‐sandwich Ru(II) arene complexes exhibiting potent antiproliferative activity against CRPC models, with one lead compound demonstrating approximately 45‐fold greater potency (IC₅₀ = 0.706 µM) than cisplatin (Chart 1A) [25, 26]. The anticancer efficacy of such half‐sandwich scaffolds can be finely tuned by modulating the bidentate chelating ligand and the η⁶‐arene moiety. Quinoline derivatives are renowned for their diverse pharmacological properties [12, 27, 28, 29, 30]. This promising activity prompted further structural optimization. Benzazoles represent privileged heterocyclic motifs in medicinal chemistry, owing to their versatile bioactivities and widespread utility in biological applications [31, 32, 33, 34, 35].

CHART 1.

Chemical structures of previously reported anticancer agents from our group (A and B) and complexes shown in this work (C).

Accordingly, we designed and developed quinoline–benzazole based ligands, 2‐(1H‐benzo[d]imidazol‐2‐yl)quinoline (L1), 2‐(quinolin‐2‐yl)benzo[d]thiazole (L2). Further, a series of four new half‐sandwich Ru(II) arene chlorido complexes based upon these ligands were developed. The Ru precursors [((η⁶‐benzene)Ru(μ‐Cl)Cl)₂] (P1) and [((η⁶‐hexamethylbenzene)Ru(μ‐Cl)Cl)₂] (P2) were utilized to synthesize organometallic complexes [(η⁶‐benzene)((L1))RuCl]PF₆ (1) [(η⁶‐benzene)((L2))RuCl]PF₆ (2), [(η⁶‐hexamethylbenzene)((L1))RuCl]PF₆ (3) and [(η⁶‐hexamethylbenzene)((L2))RuCl]PF₆ (4) along with ligand (L1) and (L2). These complexes were fully characterized using a range of spectroscopic and analytical techniques. Moreover, single‐crystal x‐ray diffraction analyses were used to confirm selected structures. Aqueous stability studies and structural analyses further elucidated their solution behavior and key coordination features. The results of these comprehensive investigations, along with structure–activity relationships and rationales for the observed biological outcomes, are discussed in detail herein.

2. Results and Discussion

2.1. Physico–Chemical Characterization

The syntheses of mononuclear arene‐Ru(II) complexes [(η⁶‐benzene)(L1)/(L2)RuCl]PF₆ (1 and 2) and [(η⁶‐hexamethylbenzene)(L1)/(L2)RuCl]PF₆ (3 and 4) were carried out by the single‐step reactions of the dinuclear Ru(arene) precursors (P1‐P2) and ligands (L1‐L2) with excellent yields (Scheme 1). The structural determination of 2 and 4 were done by single crystal x‐ray diffraction analyses. The physical state of the complexes were crystalline and nonhygroscopic showing the color ranged from yellowish orange to deep brown. All the complexes were partially soluble in water, but easily soluble in a 4% DMSO‐water mixture and other polar organic solvents. The conductivity values were measured in acetonitrile at 298 K, which were comparable to 1:1 electrolyte and indicating the monocationic nature (147 S cm² mole⁻¹ for 1, 153 S cm² mole⁻¹ for 2, 148 S cm² mole⁻¹ for 3, and 181 S cm² mole⁻¹ for 4) [36]. The elemental analysis data for these complexes were collected using powdered samples. Complexes 1 and 2 were purified using column chromatography, while complexes 3 and 4 were recrystallized. All complexes were vacuum‐dried before their elemental analysis was conducted. The results indicated the purity of the complexes, along with the presence of a few solvent molecules (Figures S1–S4).

SCHEME 1.

Scheme for syntheses of the complexes.

In FT‐IR studies, the ṽ(N–H) stretching vibrations of complex 1 and 3 were shifted as compared to L1 (3485.6 cm⁻¹) and observed at 3439.9 and 3423.0 cm⁻¹, respectively. The C–H, aliphatic and aromatic vibrations were observed in the range 3075–2800 cm⁻¹ for all the complexes. The vibrations corresponding to C═N and C═C aromatic stretching bands were observed in the range of 1650–1400 cm⁻¹. The vibrations related to ṽ(PF₆) were observed around 840 cm⁻¹ for all the complexes. The second typical band for PF₆ bending vibration was observed around 554–558 cm⁻¹ for all the complexes. It indicates the presence of PF₆ anion outside the coordination sphere balancing the cation counterpart. Moreover, the vibrations in the range 500–600 cm⁻¹ corresponded to the Ru–N bands, which are indication of existence of coordination between Ru and ligands [37]. The vibrations related to ṽ(Ru–Cl) were observed in the range 400–500 cm⁻¹ for all the complexes, showing the presence of Ru bound chloride inside the coordination sphere [37]. The peaks observed for these complexes, had shifted compared to that of the both ligands, as well as Ru(arene) precursors. The most relevant IR frequencies are given in the experimental section (Figures S5–S8).

The NMR spectra (¹H, ¹³C, and ¹H–¹H COSY) of these complexes were recorded in DMSO‐d₆. For complex 1 and 2 in ¹H‐NMR, δ 6.305 and δ 6.328 ppm signals corresponded to six protons of the benzene moiety coordinated to ruthenium, respectively (Figures S9 and S10). Similarly, complexes 3 and 4 showed the signals related to 18 protons of six methyl groups of the hexamethylbenzene moiety were observed at δ 1.871 and δ 1.834 ppm, respectively (Figures S11 and S12). The ¹H‐NMR spectra of 1 and 2 showed downfield shifts in the signals compared to that of P1. While the ¹H‐NMR spectra of 3 and 4 demonstrated up‐field shifts in the signals compared to that of P2. The signals found in the range of δ 7.542–8.991 ppm were related to the quinoline–benzazole‐based ligand part. The ¹H‐NMR spectra of the complexes showed downfield shifts with respect to those of ligands L1‐L2. The ¹H‐NMR spectral peaks were found to be in good agreement with the chemical structures of the complexes. ¹H─¹H COSY NMR spectroscopy of all the complexes shows the interactions of neighboring protons with each other, which further supports the elucidated structures (Figures 1 and S13–S15). The ¹³C‐NMR spectra of 1 and 2 showed signals at δ 85.54 and δ 87.37 ppm which corresponded to six carbons of the benzene moiety coordinated to ruthenium, respectively (Figures S16 and S17). Similarly, the ¹³C‐NMR spectra of 3 and 4 (Figures S18 and S19) showed the signals related to six methyl groups of the hexa‐methylbenzene moiety at δ 16.26 and δ 15.55 ppm, respectively. For complexes 3 and 4, the carbon peak related to benzene ring of hexamethylbenzene moieties was observed at δ 94.74 and δ 95.20 ppm, respectively. The other carbons related to the ligand parts of the complexes were observed in the range δ 114.17–166.87 ppm.

FIGURE 1.

¹H‐¹H‐COSY NMR spectrum of 4 recorded in DMSO‐d₆.

The high‐resolution ESI mass spectra of the complexes exhibited an intense peak molecular ion peak corresponding to the [(η⁶‐arene)(L)RuCl]⁺ fragment (Figures S20–S23). For [1‐PF₆]⁺, the observed m/z value is 424.0395 while for [3‐PF₆]⁺, the observed m/z value is 508.1331. In the cases of complexes 2 and 4, the molecular ion peak was the base peak in the high‐resolution ESI mass spectra. For complex [2‐PF₆]⁺, the observed m/z value was at 476.9767, while for [4‐PF₆]⁺, the observed m/z value is at 561.0705. All the spectroscopic techniques detailed above indicate the mono‐cationic form of all complexes in the solution phase.

2.2. Electronic Spectral Studies

In the absorption spectrum of the complexes recorded in DMSO, two and three absorption bands were observed for complexes 1 and 3, while for complexes 2 and 4, four absorption bands were observed, respectively. The high energy (HE) absorption band observed at ∼310 nm (303 nm for 1; 307 nm for 2; 312 nm for 3, and 336 nm for 4) corresponds to the intra‐ligand charge transfer (ILCT) transitions. The low energy (LE) metal to ligand charge transfer (MLCT) bands were observed in the range 360–450 nm (369 nm for 1; 362 nm, 379 nm, 425 nm for 2; 372 nm, 430 nm for 3; and 336 nm, 365 nm, 383 nm, 452 nm for 4; Figure 2). Similarly, for complexes 2 and 4, five absorption bands were shown in their respective electronic spectra in 4% DMSO: PBS (pH = 7.4). However, the complexes 1 and 3 exhibited to have three absorption bands in their electronic spectrum in 4% DMSO: PBS (pH = 7.4). In this medium, two strong HE band were observed at ∼260 nm (251 nm for 1; 260 nm for 2; 250 nm for 3, and 264 nm for 4) and ∼310 nm (302 nm for 1; 304 nm for 2; 306 nm for 3, and 311 nm for 4), which could be assigned to the ILCT transitions. Moreover, the LE strong absorption bands were demonstrated in the range 360–450 nm (373 nm for 1; 361 nm, 375 nm, 415 nm for 2; 375 nm for 3; and 363 nm, 379 nm, 434 nm for 4), would be for the MLCT.

FIGURE 2.

UV–vis absorption spectra of the complexes [30 µM in DMSO] at 298 K.

2.3. X‐Ray Crystallographic Studies

The structures of complexes 2 and 4 comprise [(η⁶‐bz)(L2)RuCl]PF₆ and [(η⁶‐Hmb)(L2)RuCl]PF₆, respectively, were determined using single crystal x‐ray diffraction (Figure 3). Both complexes crystallized in a monoclinic system with the P2₁ space group for complex 2 and P2₁/c space group for complex 4. In both complexes, the cationic units comprise the “piano‐stool” geometry of the Ru(II) arene complexes, in which η⁶‐π‐bonded arene rings form the seat and three other donor atoms (two nitrogen atoms, one of quinoline and the other of benzimidazole units and one chloride ion) form the three legs of the stool. For complex 2, the observed Ru─N bond lengths are 2.088(8) for N1 and 2.127(6) Å for N2 and the Ru─Cl bond length is 2.396(2) Å. The Ru─C(Bz) bond lengths are in the range of 2.147(10)–2.211(9) Å. For complex 4, the Ru─N bond lengths are 2.106(5) and 2.109(5) Å for N1 and N2, respectively and the Ru─Cl bond length is 2.3894(16) Å. The Ru─C(Hmb) bond lengths are in the range of 2.177(6)–2.263(7) Å. The bond parameters are comparable with those of similar systems reported earlier [37, 38, 39, 40, 41, 42]. Data collection and refinement details are given in the experimental section. The crystallographic data of both complexes are listed in Table S1, and selected bond lengths and angles are given in Table S2.

FIGURE 3.

ORTEP diagram of 2 (left) and 4 (right) with thermal ellipsoid probability 30% for all non‐hydrogen atoms. Counter anion and hydrogen atoms were omitted for clarity.

2.4. Electrochemical Properties

The electrochemical behavior of all the complexes was assessed employing cyclic voltammetry to get some insights into the stability of the Ru(II) state in the complexes. The representative cyclic voltammogram of complex 4 is shown in Figure 4. For complex 4, the E _1/2 value of the redox peak observed at 1.45 V (I) (ΔE _pI = 0.11 V) was related to the reversible redox process of Ru²⁺↔Ru³⁺. The anodic peaks at 2.06 V (II_a) and 2.52 V (III_a) were irreversible and corresponded chloride oxidation and Ru³⁺ to Ru⁴⁺ oxidation, respectively. The irreversible cathodic peak at −1.21 V (IV_c) as well as the E _1/2 values of the redox peaks at −0.78 V (V) (ΔE _pV = 0.08 V) and −1.90 V (VI) (ΔE _pVI = 0.12 V) correspond to the ligand centric redox processes. This may be due to the redox processes of heteroatom (S) and ─C═N─ groups present in the complex or could be for Ru²⁺ to Ru¹⁺ reduction process as suggested by some literature reports [43].

FIGURE 4.

Cyclic voltammograms of 4 (1 mM solution in CH₃CN with 0.1 M [nBu₄N][ClO₄]) at scan rates of 100 mV s⁻¹ at room temperature.

The complexes 1, 2, and 3 also showed different oxidation and reduction peaks shown in Figures S24–S26. The complexes 1 and 2 showed irreversible oxidation peaks (E _pa) for Ru²⁺ to Ru³⁺ at 1.76 V (I_a) and 1.86 V (I_a), respectively. While the complex 3 showed the E _1/2 value of the redox peak observed at 1.35 V (I) (ΔE _pI = 0.11 V) related to the Ru²⁺ ↔ Ru³⁺ redox process. The other irreversible anodic peak at 2.13 V (II_a) corresponded to the oxidation of chloride for the complex 1. Similarly, the different anodic peaks in the range 2.13–2.61 V (III_a) corresponded to the oxidation of Ru³⁺ to Ru⁴⁺ process, for the complexes 1, 2, and 3. The cathodic peaks observed in the range −0.39 to −1.75 V (ΔE _pV = 0.10–0.27 V and ΔE _pVI = 0.07–0.10 V) for complexes 1, 2, and 3 were related to the ligand centric reduction processes. All this data has been listed in Table S3 of supporting information. This data has been compared to the reported literature and found to be similar [43, 44, 45].

2.5. Stability in Aqueous and DMSO Media

The stability studies of the complexes were investigated using the UV–vis spectroscopy in 4% DMSO‐PBS (pH ∼ 7.4) and DMSO, as well as ¹H‐NMR spectroscopy in DMSO‐d₆. UV–vis spectra in 4% DMSO‐PBS (pH ∼ 7.4) at 0, 12, 24, and 48 h (Figures S27–S30) revealed characteristic absorption bands in the range 250–440 nm. All complexes exhibited good aqueous stability, with only minor spectral changes attributable to hydrogen bonding or solvation effects in the buffer. Notably, the hexamethylbenzene derivatives 3 and 4 displayed slightly lower stability compared to their benzene counterparts 1 and 2. In pure DMSO (Figures S31–S34), the complexes absorbed in the 300–370 nm range. 1, 2, and 4 remained highly stable, whereas 3 showed minor shifts in intensity and λ _max, likely due to coordination interactions with DMSO [46]. ¹H NMR monitoring in DMSO‐d₆ over 48 h (0, 2, 6, 12, 24, and 48 h; Figures S35–S38) confirmed excellent stability for all four complexes, including 3, with no signs of degradation. The free ligands L1 and L2 were similarly stable in DMSO, consistent with prior reports [15]. The dinuclear [Ru(η⁶‐arene)(μ‐Cl)Cl]₂ precursors (P1‐P2) also proved stable in both media, though minor changes in DMSO absorption bands were noted [46].

2.6. Distribution Coefficient

The distribution coefficient (Log D) is a key physicochemical parameter used to evaluate the lipophilicity of drug candidates and is commonly determined using the octanol/water shake‐flask method [47]. The Log D values of compounds 1 and 3 were found to be −0.43 and −0.194, respectively. These negative values indicate a greater preference for the aqueous phase over the organic phase, reflecting the predominantly hydrophilic nature of these compounds. In contrast, compounds 2 and 4 exhibited Log D values of 0.216 and 0.102, respectively, indicating a higher affinity for the organic phase and, therefore, relatively greater lipophilicity. The observed differences in lipophilicity can be attributed to the heterocyclic moieties present in the compounds. Compounds 1 and 3 contain a benzimidazole ring, whose nitrogen atoms can participate in hydrogen bonding with water molecules, thereby enhancing aqueous solubility. In contrast, compounds 2 and 4 contain a benzothiazole ring, in which the sulfur atom is less polar and a weaker hydrogen‐bonding participant than nitrogen, resulting in comparatively higher lipophilicity.

2.7. Anticancer Activity

Only a few Ru–arene complexes have been found to exhibit antiproliferative activity against prostate cancer cell lines and these studies are with DU‐145 [48, 49] and LNCaP cells [50], only a limited number have been studied in more aggressive PC‐3 cells [51]. Therefore, the antiproliferative activity of the Ru complexes along with ligands and Ru(arene) precursors, were evaluated against highly proliferative PC‐3 human prostatic adenocarcinoma cell line, HepG2 hepatocellular carcinoma cell line and HEK‐293 normal human epithelial cell line. A range of concentrations was screened in a concentration‐dependent study using MTT assay. The stock solution of the 1–4, L1‐L2 and precursors were prepared in DMSO maintaining a final DMSO concentration below 0.5%. Cells were seeded at a density of 10,000 cells per well in 96‐well plates and treated with the compound after 24 h of incubation. Untreated control cells were maintained under identical conditions for comparison of growth inhibition.

The results illustrated that all four complexes significantly inhibited the proliferation of PC‐3 cells at 72 h post‐treatment. Among the four complexes 1–4, complex 4 displayed highest cytotoxicity against PC‐3 cells with IC₅₀ value of 1.68 µM. Other complexes 1, 3, and 2 also exhibited significant anti‐proliferative activity with IC₅₀ values of 2.06, 3.25, and 6.42 µM, respectively (Figure 5). Additionally, all four complexes exhibited moderate to good anti‐proliferative activity against HepG2 cell lines. Complex 4 displaying maximum cytotoxicity with IC₅₀ value of 9.83 µM and 1, 3, and 2 displaying IC₅₀ values of 11.08, 16.60, and 31.18 µM, respectively, at 72 h of the treatment (Figure S39A). The order for potency against HepG2 cell line is similar to PC‐3 cell line, that is, 4 > 1 > 3 > 2. The cytotoxicity studies with normal HEK‐293 cells suggest that the compounds 1 and 4 exhibited lower cytotoxicity on these cells with IC₅₀ 24.25 and 30.08 µM, respectively, suggesting higher toxicity and selectivity toward cancer cells. However, compounds 2 and 3 have slightly lower IC₅₀ values on HEK‐293 cells 8.04 and 12.84 µM, respectively indicating moderate efficacy. Additionally, the selectivity index of compound 4 in PC‐3 cell lines suggests its good selectivity for targeting cancer cells (Table 1 and Figure S39B). Overall, MTT results indicated the prominent cytotoxicity of all four complexes against both PC‐3, HepG2, and HEK‐293 cell lines, (Table 1). It was interesting to note that ligand L2 studied as control exhibit good activity (Table S4), higher than complex 2 especially in HepG2 cell likely due to string DNA interactions. The cytotoxic behavior of Ru(arene) precursors P1 and P2 as control has been reported in our previous publication [25]. Additionally, the cytotoxicity of standard reference cisplatin (cis‐diamminedichloroplatinum(II), CDDP) was checked against both cell lines and found to be 31.42 µM for PC‐3 cells and 15.90 µM for HepG2 cells at 72 h post‐treatment. Overall, 4, 1, and 3 demonstrated significant anti‐proliferative activity and 2 exhibited moderate activity.

FIGURE 5.

Dose response curves of MTT cell viability assay of complexes 4 and 1, cisplatin against PC‐3 cell after 72 h of treatment (error bars represent standard deviation and statistical analysis was performed using one‐way ANOVA test in GraphPad Prism 9).

TABLE 1.

Cytotoxicity data for treatment of complexes (1–4) against PC‐3, HepG2, and HEK‐293 cells, along with the selectivity index values.

				Selectivity index (SI = IC50HEK cell line/IC50cancer cell line)
Compounds	IC50 value (µM) (PC‐3 cells)	IC50 value (µM) (HepG2 cells)	IC50 value (µM) (HEK‐293)	PC‐3 cells	HepG2 cells
1	2.06	11.08	24.25 µM	11.77	2.18
2	6.42	31.18	8.040 µM	1.25	3.87
3	3.25	16.60	12.84 µM	3.95	1.29
4	1.68	9.83	30.08 µM	17.90	3.06

2.8. Mechanistic Investigation

2.8.1. Evaluation of Cell Cycle Arrest in PC‐3 Cell Line

Results of the MTT assay clearly indicate the prominent anti‐proliferative activity of 4 and 1 against PC‐3 cells. Anticancer drugs have been shown to reduce cell viability either through cell cycle arrest or apoptosis [26]. Therefore, propidium iodide (PI)‐flow cytometry analysis was performed to determine cell progression and modes of action of 4 and 1. PC3 cells were treated with different concentrations of 4 and 1 for 24 h, stained with PI following the cell cycle analysis by flow cytometry. The flow cytometry results revealed a marked increase in sub‐G1 cell population accompanied by an increase in G0/G1 population after the treatment with 4, indicating that the compound suppresses the cell cycle progression at the G1 checkpoint and subsequently induces DNA fragmentation and drives arrest of cell cycle progression. Compared with control sample which showed minimal sub‐G1 content (1.5%) and G0/G1 (67.9%), while treated cells exhibited a dose‐dependent accumulation in sub‐G1 (2% at 10 µM, 5.4% at 20 µM and 5.8% at 40 µM) and G0/G1phase (72.1% at 10 µM, 72.3% at 20 µM, 72.5% at 40 µM) (Figure 6, Panel 1). For 1, the major inhibition can be seen at the sub‐G1 phase as the concentration increases (Figure 6, Panel 2). However, there is also an increase in G2/M cell population at high concentration 20 µM (34.3%) and 40 µM (31.2%) as compared to the control sample (17.9%). The cell cycle arrest at G2/M phase and sub‐G1 phase indicates the link between cell death through DNA interaction.

FIGURE 6.

Panel 1 represents the cell cycle arrest of 4 (A, B, C, D), while panel 2 represents the cell cycle arrest of 1 (E, F, G, H).

2.8.2. Apoptosis Assay

To detect and quantify programmed cell death, annexin V apoptosis assay was performed. Annexin V combined with propidium iodide (PI) can efficiently differentiate early and late apoptosis or necrosis. PC‐3 cells were treated individually with 4 and 1, followed by sequential staining with annexin V and PI. The results revealed a significant increase in both early and late apoptotic cell populations for both compounds compared to untreated controls. However, 1 exhibited markedly stronger cytotoxic effects, induced 22.4% early apoptosis and 12.3% late apoptosis, whereas 4 resulted in 12.3% early apoptosis and 9.1% late apoptosis. These findings highlight the enhanced apoptotic activity of 1 against PC‐3 cells (Figure 7).

FIGURE 7.

(A) and (B) represent the annexin V assay of 4, while (C) and (D) represent the annexin V assay of 1.

2.9. Interaction With DNA

2.9.1. Emission Spectral Studies for DNA Interaction

The interaction of complexes with DNA was assed using DNA‐EtBr assay. The emission intensity of the DNA‐EtBr complex was expressively quenched in our studies as complex concentrations elevated in 4% DMSO‐PBS (pH ∼ 7.4).

EtBr's migration from the EtBr‐DNA complex was the cause of this. At 480 nm, the EtBr‐DNA combination was stimulated, and at 607 nm, the fluorescence intensity was measured. The spectra (Figures S40–S43) obtained from the above titrations exhibited a noticeable redshift (13 nm for 1; 10 nm for 2; 9 nm for 3; and 5 nm for 4) along with a substantial decline in the emission value for complexes. In addition to this, significant quenching of the fluorescence intensity was observed for all the complexes (56.29%, 1; 41.85%, 2; 43.77%, 3; and 42.82%, 4). The data proposed that all the complexes were capable to attach with the DNA binding spots robustly and displace EtBr from the DNA‐EtBr complex [52, 53].

The values of K _sv, K _a, and n were derived by means of the Stern–Volmer equation and modified Stern–Volmer or Scatchard equation as shown in Table S4 (Figures 8 and 9 and S44–S51). The values of K _sv and K _a advised the affinity of the complexes with DNA in the following order: 4 > 3 > 2 > 1.

FIGURE 8.

Stern–Volmer plot to determine the quenching constant (K _SV) related to EtBr‐DNA and complexes (1‐4) titration at 298 K.

FIGURE 9.

Scatchard plot to determine the association constant (K _a) related to EtBr‐DNA and complexes (1‐4) titration at 298 K.

The data acquired from the above investigations revealed that all the complexes were adept of removing EtBr from DNA‐EtBr complex, and all compounds showed the pattern: 4 > 3 > 2 > 1. The data of the computed factors further endorse that all four complexes tightly bind with DNA, and further spectroscopic observations are in line with those of the EtBr displacement experiments.

The experiments were similarly done for Ru(arene) precursors P1 and P2 to check the binding affinity with DNA [25]. The studies related to the binding affinities of ligands L1 and L2 with DNA were described in our previous work [15]. The Quenching coefficients (K _sv) and association coefficients (K _a) of Ru(arene) precursors were lower compared to the respective complexes. This showed that Ru(arene) precursors had lesser capability to remove EtBr from DNA‐EtBr complex then their relevant complexes.

Specifically, it is clear from EtBr displacement experiments that all four compounds may bind with DNA sturdily. The affinity coefficients derived from the experimental data indicate that compounds attach to DNA via non‐covalent linkages. According to the DNA binding experiments, the compounds substitute EtBr in the EtBr‐DNA complex by intercalating and causing straight EtBr removal. However, compounds that cause conformational modifications to fit in the surface or groove of DNA may cause indirect displacement, which could also help to breakdown the EtBr‐DNA complex. The Ru(arene) precursors as well as the ligands showed weaker affinity with DNA to that of Ru complexes, perhaps via electrostatic and hydrogen bonding affinities.

3. Summary and Conclusions

In presented study, four new half sandwich Ru(II) arene chlorido compounds incorporating quinoline–benzazole hybrid ligands have been synthesized and comprehensively characterized. Spectroscopic, analytical, stability, lipophilicity, and electrochemical studies confirmed their structural integrity, good aqueous stability, predominantly hydrophilic character, and high stability of the Ru(II) oxidation state, with access to Ru(III) requiring very high potentials. Cytotoxicity evaluation against the castration‐resistant PC‐3 prostate cancer cell line revealed promising antiproliferative activity, with complex 4 exhibiting the highest potency (IC₅₀ = 1.68 µM). Mechanistic investigations demonstrated predominant G2/M phase cell cycle arrest, and strong interactions with CT‐DNA. DNA‐binding studies indicated moderate‐to‐strong affinities, with 4 displaying the highest binding constant, consistent with its superior cytotoxic profile. Overall, these Ru(II) arene compounds demonstrate substantial potential as antiproliferative agents against CRPC. In particular, the lead compounds 4 and 1 warrant further evaluation in animal models to assess their in vivo efficacy and therapeutic promise.

4. Experimental Section

4.1. Materials Used

The chemicals and solvents were acquired from Sigma Aldrich, Thermo–Fisher Scientific, Molychem, and CDH utilized without additional purification. Solvents used for spectroscopic investigations were refined and dried by typical techniques [54, 55]. Metal chloride (RuCl₃.3H₂O), cyclohexa‐1,3‐diene, hexamethylbenzene (Hmb), quinaldine, 2‐amino thiophenol, o‐phenylene diamine, ethidium bromide (EtBr), phosphate buffer saline (PBS), calf thymus‐deoxyribonucleic acid (CT‐DNA), and bovine serum albumin (BSA) were procured by Sigma Aldrich and TCI, India. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) was acquired from Sigma–Aldrich, MO, USA.

4.2. Physical Measurements

The melting point was obtained by the Büchi melting point B‐540 machine. A Perkin Elmer FTIR spectrometer operating in the 4000–400 cm⁻¹ window was used to record infrared spectra at ambient temperature. The Systronics Conductivity Meter 304 was utilized to obtain conductivity. A Thermo Scientific Flash 2000 elemental analyzer was used for the elemental study. A Bruker Avance III‐500 MHz spectrometer operating at 298 K was used to record the ¹H, ¹³C, and COSY NMR spectra in DMSO‐d₆ and CDCl₃ solvents.

A Bruker Ultrashield 500 Plus‐500 MHz spectrometer and a Jeol 400 MHz spectrometer operating at 298 K were used for certain ¹³C NMR investigations. An Agilent Technologies Cary 60 UV–vis spectrophotometer was used to conduct UV–vis spectrophotometric investigations. The Thermo Exactive Plus mass spectrometer was used to acquire the ESI‐mass spectra. Fluorescence experiments were done utilizing an Agilent Cary Eclipse fluorescence spectrophotometer. An AUTOLAB PGSTAT 302N (Metrohm Autolab B.V., Netherlands) electrochemical analyzer was used to conduct cyclic voltammetry studies.

4.3. Syntheses and Characterization

The ligands 2‐(1H‐benzo[d]imidazol‐2‐yl)quinoline (L1), and 2‐(quinolin‐2‐yl)benzo[d]thiazole (L2) were synthesized using literature methods [56]. The Ru(benzene) precursor (P1) and Ru(hexamethylbenzene) precursor (P2) were synthesized and purified using methods described in the literature [57]. All four Ru(arene) complexes were synthesized and purified by the following synthetic procedures.

4.3.1. Synthesis and Characterization of [(η⁶‐benzene)(L1)RuCl]PF₆ (1)

A round bottom flask was charged with P1 [((η⁶‐Bz)Ru(μ‐Cl)Cl)₂] (0.250 g, 0.5 mmol). It was dissolved in dry dichloromethane (25 mL), followed by the addition of 2‐(1H‐benzo[d]imidazol‐2‐yl)quinoline (L1) (0.245 g, 1.0 mmol). The resulting mixture was refluxed under nitrogen for 12 h. The reaction mixture was then allowed to attain room temperature (RT). Further, potassium hexafluorophosphate (KPF₆) (0.276 g, 1.5 mmol) dissolved in dry methanol was added and stirred at RT for 4–5 h. The yellowish orange solid product was obtained as a precipitate. The product was filtered to isolate. After methanol and diethyl ether wash it was dried using high vacuum. Yield = 95%. m.p. = 198–203°C decomp. ¹H NMR (500 MHz, DMSO‐d₆, δ ppm): 6.305 (6H, s, ─H_k ); 7.62–7.68 (2H, m, ─H_13,14 ); 7.92 (1H, d, J = 7 Hz, ─H₁₅ ); 7.97 (1H, t, J = 7.5 Hz, ─H₄ ); 8.18 (1H, t, J = 7.5 Hz, ─H₅ ); 8.24 (1H, d, J = 7.5 Hz, ─H₁₂ ); 8.29 (1H, d, J = 8.5 Hz, ─H₃ ); 8.59 (1H, d, J = 6.5 Hz, ─H₁₀ ); 8.95 (1H, d, J = 8.5 Hz, ─H₆ ); 8.98 (1H, d, J = 8.5 Hz, ─H₉ ). ¹³C NMR (101 MHz, DMSO‐d₆, δ ppm): 150.3, 148.4, 148.0, 141.7, 141.6, 134.2, 133.2, 129.4, 129.3, 128.9, 126.5, 125.3, 118.8, 118.1, 114.2, 85.5. Anal. Calcd. (%) for C₂₂H₁₇ClF₆N₃PRu ^. MeOH: C, 43.37; H, 3.32; N, 6.60. Found (%): C, 42.97; H, 3.00; N, 7.06. ESI‐MS(+) in CH₃OH (Calcd, found, m/z) 460.0154, 460.0162, [M]⁺ = [1‐PF₆]⁺. FTIR (KBr, cm⁻¹) ṽ(N‐H) 3439.89, ṽ(C–H aromatic + aliphatic) 3062.82, 2927.93, 2859.89, ṽ(C═N) 1569.53, 1505.24, ṽ(C═C) 1433.99, 1361.96, ṽ(PF6) 836.05, 557.22, ṽ(Ru–N) 512.30, ṽ(Ru–Cl) 476.67. UV–vis: (in DMSO), λ _max (nm) (ϵ, dm³mol⁻¹cm⁻¹): 303 (1.31 × 10⁴), 369 (1.76 × 10⁴); (in 4 % DMSO:PBS (pH = 7.4)) λ _max (nm): 251, 302, 373.

4.3.2. Synthesis and Characterization of [(η⁶‐benzene)(L2)RuCl]PF₆ (2)

A round bottom flask was charged with P1 [((η⁶‐Bz)Ru(μ‐Cl)Cl)₂] (0.250 g, 0.5 mmol). It was dissolved in dry dichloromethane (25 mL), followed by the addition of 2‐(quinolin‐2‐yl)benzo[d]thiazole (L2) (0.262 g, 1.0 mmol). The resulting mixture was refluxed under nitrogen for 12 h. The reaction mixture was then allowed to attain RT. Further, potassium hexafluorophosphate (KPF₆) (0.276 g, 1.5 mmol) dissolved in dry methanol was added and stirred at RT for 4–5 h. The reddish orange solid product was obtained as precipitate. The product was filtered to isolate. After methanol and diethyl ether wash it was dried using high vacuum. Yield = 83%. m.p. = 221°C–227°C decomp. ¹H NMR (500 MHz, DMSO‐d₆, δ ppm): 6.33 (6H, s, ─H_k ); 7.87 (1H, t, J = 7 Hz, ─H₁₃ ); 7.94 (1H, t, J = 7.5 Hz, ─H₁₄ ); 8.01 (1H, t, J = 7 Hz, ─H₄ ); 8.19 (1H, t, J = 7.5 Hz, ─H₅ ); 8.35 (1H, d, J = 7.5 Hz, ─H₃ ); 8.56 (1H, d, J = 8 Hz, ─H₁₂ ); 8.61 (1H, d, J = 8 Hz, ─H₁₅ ); 8.69 (1H, d, J = 8 Hz, ─H₁₀ ); 8.95 (1H, d, J = 8.5 Hz, ─H₆ ) 8.99 (1H, d, J = 8 Hz, ─H₉ ).¹³C NMR (101 MHz, DMSO‐d₆, δ ppm): 166.9, 152.3, 151.0, 149.2, 142.6, 134.2, 133.8, 130.6, 130.1, 130.1, 129.9, 129.8, 129.2, 125.5, 123.7, 121.7, 87.4. Anal. Calcd. (%) for C₂₂H₁₆ClF₆N₂PRuS ^. 1.5CH₂Cl₂: C, 37.67; H, 2.56; N, 3.74; S, 4.28. Found (%): C, 37.87; H, 2.24; N, 3.76; S, 4.66. ESI‐MS(+) in CH₃OH (Calcd, found, m/z) 476.9766, 476.9767, [M]⁺ = [2‐PF₆]⁺. FTIR (KBr, cm⁻¹) ṽ(C–H aromatic + aliphatic) 3051.97, 2920.31, 2856.02, ṽ(C═N) 1595.87, 1516.86, ṽ(C═C) 1437.86, 1349.56, ṽ(PF6) 837.60, 558.00. UV–vis: (in DMSO), λ _max (nm) (ϵ, dm³mol⁻¹cm⁻¹): 307 (1.47 × 10⁴), 362 (1.96 × 10⁴), 379 (2.28 × 10⁴), 425 (3.87 × 10³); (in 4 % DMSO:PBS (pH = 7.4)) λ _max (nm): 260, 304, 361, 375, 415.

4.3.3. Synthesis and Characterization of [(η⁶‐hexamethylbenzene)(L1)RuCl]PF₆ (3)

A round bottom flask was charged with P2 [((η⁶‐Hmb)Ru(μ‐Cl)Cl)₂] (0.250 g, 0.374 mmol). It was dissolved in dry dichloromethane (25 mL), followed by the addition of 2‐(1H‐benzo[d]imidazol‐2‐yl)quinoline (L1) (0.183 g, 0.748 mmol). The resulting mixture was refluxed under nitrogen for 12 h. The reaction mixture was then allowed to attain RT. Further, potassium hexafluorophosphate (KPF₆) (0.206 g, 1.122 mmol) dissolved in dry methanol was added and stirred at RT for 4–5 h. After removing the solvent under reduced pressure, it was subjected to silica gel (60–120 mesh) column chromatography (MeOH‐DCM eluent system) to obtain orange red solid. Yield = 77%. m.p. = 283°C–290°C decomp. ¹H NMR (500 MHz, DMSO‐d₆, δ ppm): 1.871 (18H, s, ─H_k ); 7.54–7.60 (2H, m, ─H_13,14 ); 7.77 (1H, d, J = 7.5 Hz, ─H₁₂ ); 7.88 (1H, d, J = 7 Hz, ─H₁₅ ); 7.91 (1H, t, J = 7.5 Hz, ─H₄ ); 8.13 (1H, t, J = 7 Hz, ─H₅ ); 8.25 (1H, d, J = 7.5 Hz, ─H₃ ); 8.48‐8.51 (2H, m, ─H_6,10 ); 8.91 (1H, d, J = 8 Hz, ─H₉ ). ¹³C NMR (101 MHz, DMSO‐d₆, δ ppm): 150.2, 148.9, 148.6, 141.3, 141.0, 135.5, 132.8, 130.1, 129.58, 129.3, 129.2, 126.6, 125.2, 119.4, 118.4, 115.2, 94.7, 16.3. Anal. Calcd. (%) for C₂₈H₂₉ClF₆N₃PRu ^. 0.33CH₃OH: C, 48.63; H, 4.37; N, 6.01. Found (%): C, 48.67; H, 4.32; N, 5.92. ESI‐MS(+) in CH₃OH (Calcd, found, m/z) 544.1093, 544.1097, [M]⁺ = [3‐PF₆]⁺. FTIR (KBr, cm⁻¹) ṽ(N–H) 3422.97, ṽ(C–H aromatic + aliphatic) 3075.21, 2924.14, 2859.89, ṽ(C═N) 1592.66, 1506.01, ṽ(C═C) 1437.86, 1383.72, ṽ(PF6) 843.52, 554.39, ṽ(Ru–N) 594.41, ṽ(Ru–Cl) 443.67. UV–vis: (in DMSO), λ _max (nm) (ϵ, dm³mol⁻¹cm⁻¹): 312 (1.66 × 10⁴), 372 (2.22 × 10⁴), 430 (3.74 × 10³); (in 4 % DMSO:PBS (pH = 7.4)) λ _max (nm): 250, 306, 375.

4.3.4. Synthesis and Characterization of [(η⁶‐hexamethylbenzene)(L2)RuCl]PF₆ (4)

A round bottom flask was charged with P2 [((η⁶‐Hmb)Ru(μ‐Cl)Cl)₂] (0.250 g, 0.374 mmol). It was dissolved in dry dichloromethane (25 mL), followed by the addition of 2‐(quinolin‐2‐yl)benzo[d]thiazole (L2) (0.196 g, 0.748 mmol). The resulting mixture was refluxed under nitrogen for 12 h. The reaction mixture was then allowed to attain RT. Further, potassium hexafluorophosphate (KPF₆) (0.206 g, 1.122 mmol) dissolved in dry methanol was added and stirred at RT for 4–5 h. After removing the solvent under reduced pressure, it was subjected to silica gel (60–120 mesh) column chromatography (MeOH‐DCM eluent system) to obtain deep brown solid. Yield = 74%. m.p. = 270–276°C decomp. ¹H NMR (500 MHz, DMSO‐d₆, δ ppm): 1.83 (18H, s, ─H_k ); 7.83 (1H, t, J = 7 Hz, ─H₁₃ ); 7.91 (1H, t, J = 7 Hz, ─H₁₄ ); 7.96 (1H, t, J = 7 Hz, ─H₄ ); 8.08 (1H, d, J = 8.5 Hz, ─H₁₅ ); 8.16 (1H, t, J = 7 Hz, ─H₅ ); 8.31 (1H, d, J = 7.5 Hz, ─H₃ ); 8.47 (1H, d, J = 8.5 Hz, ─H₆ ); 8.53 (1H, d, J = 8 Hz, ─H₁₂ ); 8.67 (1H, d, J = 8.5 Hz, ─H₁₀ ); 8.93 (1H, d, J = 8.5 Hz, ─H₉ ). ¹³C NMR (126 MHz, DMSO‐d₆, δ ppm): 164.80, 151.6, 148.7, 147.9, 141.0, 133.6, 132.9, 130.2, 129.2, 128.8, 128.7, 128.6, 128.5, 125.1, 122.8, 121.4, 95.2, 15.5. Anal. Calcd. (%) for C₂₈H₂₈ClF₆N₂PRuS: C, 47.63; H, 4.00; N, 3.97; S, 4.54. Found (%): C, 47.52; H, 4.01; N, 3.81; S, 4.67. ESI‐MS(+) in CH₃OH (Calcd, found, m/z) 561.0705, 561.0713, [M]⁺ = [4‐PF₆]⁺. FTIR (KBr, cm⁻¹) ṽ(C–H aromatic + aliphatic) 3065.14, 2923.93, 2854.57, ṽ(C═N) 1588.83, 1513.90, ṽ(C═C) 1451.98, 1385.99, ṽ(PF6) 841.51, 556.89, ṽ(Ru–N) 510.75, ṽ(Ru–Cl) 437.17. UV–vis: (in DMSO), λ _max (nm) (ϵ, dm³mol⁻¹cm⁻¹): 336 (1.67 × 10⁴), 365 (2.13 × 10⁴), 383 (2.43 × 10⁴), 452 (3.23 × 10³); (in 4% DMSO:PBS (pH = 7.4) λ _max (nm): 264, 311, 363, 379, 434.

4.4. UV–vis Spectral Studies

An Agilent Technologies Cary 60 UV–vis spectrophotometer operating at 298 K was used for all UV–vis spectrum analyses. The stock solutions of compounds with exact molarity (30 µM) were made in a 4% DMSO‐phosphate buffered saline (PBS, pH ∼ 7.4) system for aqueous stability investigations. Likewise, the equal molar solution (30 µM) was used to examine the compounds’ stability in DMSO. After 12, 24, and 48 h, same experiment were carried out.

4.5. Distribution Coefficient

The distribution coefficient (log D) of complexes 1–4 was assessed at pH 7.4 using the shake‐flask method employing fluorescence spectroscopy. Equal volumes of PBS and n‐octanol were stirred for 24 h to achieve saturation and then allowed to equilibrate and separate for an additional 24 h. Subsequently, 10 µL of a DMSO stock solution (5 mM) of complexes was added to 4 mL of a pre‐saturated 1:1 (v/v) PBS:n‐octanol mixture. Following continuous stirring at 1500 rpm for 6 h, the phases were separated and the fluorescence spectrum of the octanol layer was recorded (λ _ex = 294 nm). The residual PBS layer was back‐extracted with 2 mL of fresh pre‐saturated octanol and stirred for another 6 h at 1500 rpm. The second octanol layer was collected, and its fluorescence spectrum was recorded. The log D values were calculated from the ratio of fluorescence intensities of the first and second octanol extracts measured at the characteristic emission wavelength of the complexes.

4.6. X‐Ray Crystallography

Using graphite monochromated Mo_Kα radiation (λ = 0.7017 Å) and a CCD detector, the single crysal XRD study of complexes 2 and 4 were done on a Bruker APEX‐II. The structure was enhanced using a full‐matrix least‐squares method based on F2 with SHELXL2018 [58] after being unraveled using direct methods implemented in SHELXT 58. Every non‐hydrogen atom underwent anisotropic refinement. Hydrogen atoms were represented as riding atoms in geometrically idealized positions after being easily identified in various Fourier maps. Platon software was used to verify the space group.

4.7. Cyclic Voltammetry

An AUTOLAB PGSTAT 302N (Metrohm Autolab B.V., Netherlands) electrochemical analyzer was used to conduct cyclic voltammetry studies. For cyclic voltammetric studies, a three‐electrode setup comprising glassy carbon being working electrode (GCE), platinum wire being auxiliary electrode, and silver wire being pseudo‐reference electrode was employed. The cyclic voltammetry studies of compounds were investigated in acetonitrile having tetrabutylammoniumperchlorate (TBAP; 0.1 M), being supporting electrolyte. The GCE (3 mm diameter), was sophisticated by hand with 0.3 µm Al₂O₃ slurry using a polishing cloth and cleaned with double distilled water and acetonitrile. Argon was used to dry it. Before being measured, oxygen was eliminated from all solutions using pure argon. Every electrochemical test was performed with an argon blanket covering the solutions at ambient temperature.

4.8. Cell Viability Assay

PC‐3, HepG2, and HEK‐293 cells were acquired from the National Centre for Cell Sciences (NCCS), Pune. PC‐3 and HEK‐293 cell lines were cultured in Roswell Park Memorial Institute (RPMI) medium (Sigma–Aldrich, USA) and HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco). The cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO₂ under standard culture conditions. A stock solution of the complexes, ligands and precursors was prepared in DMSO and working concentrations were freshly diluted in the respective culture media, maintaining a final DMSO concentration below 0.5%. Cells were seeded at a density of 10,000 cells per well in 96‐well plates and treated with a two‐fold serial dilution of the compound after 24 h of incubation. Untreated control cells were maintained under identical conditions for comparison of growth inhibition. After 72 h of treatment, the media from all wells (test and control) were removed, and 10 µL of MTT reagent (0.5 mg/mL) was added to each well. Plates were incubated in the dark for 2 h at 37°C in a 5% CO₂ humidified incubator. The supernatant was then discarded, and 100 µL of DMSO was added to dissolve the formazan crystals by gentle shaking at 37°C. Absorbance was measured at 570 nm using a microplate reader. The IC₅₀ values were determined using GraphPad Prism 10. All experiments were performed in biological triplicates.

4.9. Cell Cycle Arrest Studies

PC‐3 cells (1 × 10⁶ cells/well) were seeded in 6‐well plates and incubated for 24 h, followed by treatment with complexes 4 and 1 individually at the specified concentrations. After 24 h of treatment, the cells were harvested and washed with 1× PBS (Gibco). Fixation was performed by the gradual, dropwise addition of ethanol to achieve a final concentration of 70%, followed by incubation for 2 h. The fixed cells were then centrifuged at 600 g for 5 min, washed with 1× PBS, and subsequently stained with a labelling solution containing RNase (100 µg/mL) and propidium iodide (50 µg/mL). After incubation in the dark for 15 min, the samples were analyzed by flow cytometry. All experiments were conducted in biological triplicates.

4.10. Apoptosis Assay

The apoptosis study was carried out With the eBioscience Annexin V‐FITC Apoptosis Detection Kit (Invitrogen) and analyzed by flow cytometry (BD Accuri). PC‐3 cells (1 × 10⁶ cells/well) were seeded in 6‐well plates and incubated for 24 h, followed by addition of complexes 4 and 1 individually at the desired concentrations. After 24 h of treatment, the cells were harvested, washed once With 1× PBS, and then washed again With 1× binding buffer. The cell pellet was resuspended in 200 µL of 1× binding buffer, and 5 µL of annexin V‐FITC was included. The suspension was incubated for 15 min at RT in the dark. Afterward, the cells were washed, resuspended in 200 µL of 1× binding buffer, and stained With 5 µL of propidium iodide (20 µg/mL). Samples were immediately analyzed by flow cytometry. All experiments were performed in biological triplicates.

4.11. Emission Spectral Studies for DNA Interaction

An Agilent Cary Eclipse fluorescence spectrophotometer was used to conduct the DNA interaction using EtBr. CT‐DNA (35 µM) and EtBr (35 µM) solutions having exact molarity were made in PBS buffer (pH ∼ 7.4). After intercalation of EtBr with DNA, the EtBr‐DNA combination revealed strong emission peaks. The gradual increase in amounts of compounds (0–694 µM for 1, 0–423 µM for 2, 0–391 µM for 3, and 0–251 µM for 4; 4% DSMO‐PBS buffer, pH ∼ 7.4) displayed substantial decrease in emission profile at 298 K. At 480 nm for the excitation wavelength (λ _ex.) and 607 nm for the emission wavelength (λ _em), the emission spectrum variations were spotted.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting Information

Single crystal XRD data has been submitted to Cambridge Crystallographic Data Centre with CCDC No. 2501641 (2) and 2501636 (4). Copy of this information can be obtained free of charge from, “The Director, CCDC, 12 Union Road,” Cambridge, CB2 1EZ, UK (fax: +44‐1223‐336033); email: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). Additional data and graphs of elemental analysis, IR spectra, NMR spectra, HRMS, UV–vis spectra, crystallographic figures and parameters, cyclic voltammetry graphs and tables, data and graphs for DNA binding studies, and MTT assay.

Supporting information

Supporting File 1: asia70946‐sup‐0001‐SuppMat.docx

Supporting File 2: asia70946‐sup‐0002‐SuppMat.cif

Supporting File 3: asia70946‐sup‐0003‐SuppMat.cif

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.