Substitution Kinetics, DNA/BSA Interactions, Cytotoxicity Evaluation and Computational Analysis of [bis‐(azaaryl)amine)Pt(II)/Pd(II)Cl] Complexes, Azaaryl = Quinoline or Phenanthridine

Abstract

The search for metal‐based anticancer agents with improved efficacy and reduced side effects is ongoing. The activities of these anticancer drugs depend on their aqueous stability, substitutional reactivity at target sites (cytotoxicity) and nontarget sites (toxicity), as well as their transportation and cell bioavailability. In this study, six square‐planar Pt(II) and Pd(II) complexes (Pt/PdL1Cl‐3), all bearing the bis(azaaryl)amine (azaaryl = quinoline or phenanthridine) chelating ligands, were synthesised and characterised by various spectroscopic methods. Their biochemical interactions with bovine serum albumin (BSA)/deoxyribonucleic acid (DNA) and rates of ligand exchange with biological nucleophiles (guanine and thiourea) were probed spectrophotometrically. DFT‐optimised molecular structures in Gaussian 9 were computed. Molecular docking simulations of the optimised structures at the receptors of CT‐DNA, BSA and relevant enzymes that upregulate cancer progression were conducted. The metal complexes showed moderate to strong interactions (K _b ca.10⁴) with calf thymus DNA (CT‐DNA) and BSA. On BSA, the metal complexes were predominantly bound in Subdomain IIIA. Ethidium bromide’s (EtBr) competitive binding titrations and docking simulations suggested that these complexes are bimodal DNA binders, functioning both as groove binders and partial intercalators. The rates of chloride substitutions decreased in the order: PdL1Cl > PdL2Cl > PdL3Cl and PtL1Cl > PtL2Cl > PtL3Cl. Molecular docking of PtL1Cl predicted stronger binding affinity towards proteins associated with the inhibition of proteases for cervical (PDB: 5VBN), breast (4DRH) and prostate cancers (PDB: XPO1). The in vitro cytotoxic effects of uncoordinated ligands (L1-L3) and their respective Pt/PdL1Cl-3 metal complexes were tested at a single dose of 10 μM in the human breast (MCF‐7, T47D and MDA‐MB‐231), cervical (HeLa and CaSki) and pancreatic (PANC‐1 and CFPAC‐1) cancer cell lines, as well in a noncancerous human dermal fibroblasts (FG‐0) cell line. The Pt/PdL1Cl complexes showed promise as lead inhibitory compounds against breast (T47D, MDA‐MB‐231) and pancreatic (PANC‐1) cancer cells. The efficacy of PtL1Cl against the T47D cell line was superior to that of the anticancer drug cisplatin.

1. Introduction

Platinum‐based complexes have been the mainstay of metallodrugs for chemotherapy. The clinical success of cisplatin, particularly its pronounced efficacy against a wide range of malignancies, has inspired the development of numerous analogues aimed at enhancing its selectivity and improving its therapeutic index [1, 2]. However, despite extensive clinical application and decades of research, platinum drugs continue to suffer from poor tumour selectivity, dose‐limiting side effects, and acquired resistance, which constrains their broader clinical utility [3]. Owing to the similar square‐planar coordination geometry of the Pd(II) and Pt(II) complexes, the former metal complexes have recently been explored as promising anticancer alternatives to the latter. However, Pd(II) complexes are 10³−10⁵ times more labile than their Pt(II) counterparts [4]. Consequently, they undergo rapid and indiscriminate ligand substitutions [5]. If the rapid and uncontrolled ligand exchange reactions were to occur in vivo, they could cause premature deactivation of the metal complexes by nontarget biological nucleophiles and heightened systemic toxicity than Platinum II analogues [6, 7]. The design of high efficacy Pd(II)‐based chemotherapeutics should aim at ensuring an optimal reactivity balance between the biological targets for anticancer proliferation, such as DNA, and nonsystemic toxicity through minimising nondiscriminate and high reactivity with other nucleophiles. A solution to attaining this balance is to design Pd(II) complexes with chelating ligands that confer thermodynamic stability and, at the same time, ensure predictably lower substitutional reactivity [8–11]. Pd(II) complexes of tridentate N^∧N^∧N‐donor ligands, typically constructed from pyridine, pyrimidine or other nitrogen‐containing heterocycles linked via amine or imine bridges, have appeared and have been reviewed extensively in the literature [12]. Tridentate N^∧N^∧N‐donor ligands such as bis‐(8‐quinolinyl)amine and bis‐(phenanthridine)amine ligands [13–18] or the relatively more flexible analogues with methylene arms are based on the bis(azaaryl)amine chelate framework and possess attractive coordination attributes that significantly influence the structural, electronic and reactive properties of their metal complexes. Structurally, the former type of ligands features planar and extended π‐conjugated azaarenes as lateral N‐donor rings, with nitrogen donor atoms situated within rigid planar systems. Quinoline comprises fused benzene and pyridine rings, while phenanthridine, an azaarene acridine and derivative of phenanthrene, offers an additional phenyl ring which further extends delocalisation of π‐electron density, thereby enhancing chemical stability and π‐acceptor capacity. The azaarenes promote planar chelation at d ⁸‐metal centres. Both quinoline and phenanthridine act as strong σ/π‐donors as well as strong π‐acceptor ligands [13]. Their chelate coordination in square‐planar complexes ensures efficient donation of electron density to the metal centres as back‐donation from filled metal d‐orbitals into their delocalised π∗ molecular orbitals. This dual donor/acceptor character enables strong Pt/Pd─N bonds and, at the same time, relieves electron density at the metal centres into the N^∧N^∧N‐donor ligands, thereby making the complexes more electrophilic. Furthermore, the incorporation of electron‐donating/‐withdrawing substituents on the N^∧N^∧N‐donor framework scales the reactivity of the d ⁸‐metal complexes. Consequently, these electronic interactions affect the rate of ligand substitution of the metal complexes in a way that reduces reactivity towards nontarget nucleophiles, which causes toxicity while promoting anticancer activity at the DNA targets [19–21]. Onunga et al. demonstrated that extension of the π‐conjugated framework of Pd(II) complexes coordinated by 1,3‐bis‐(2‐arylimino)isoindolate as the N^∧N^∧N‐core donor chelate lowers the metal complexes’ reactivity towards nucleophiles [11]. Moreover, the extended conjugation and planarity of these ligands may enhance stacking interactions in biological systems, which is especially relevant for anticancer complexes with DNA‐targeting properties. In essence, these N^∧N^∧N‐donor ligands form flexible six‐membered chelates at the d ⁸ metal centre, which not only confer stability but also cause a significant decrease in the lability of the chloride ligand in the fourth position of the plane [11].

Structural modifications to the N^∧N^∧N chelators allow precise modulation of steric profile and lipophilicity properties that are critical for optimising the pharmacokinetics, cellular uptake and target engagement [11]. Beyond imparting basic stability, some N^∧N^∧N ligands offer secondary functionality such as pH‐responsive protonation, redox‐switchable coordination or incorporation of targeting moieties that further refine drug delivery and activation [22]. Collectively, these ligand features underscore the promise of N^∧N^∧N‐donor ligands in overcoming the limitations associated with both Pt/Pd(II)‐based chemotherapeutics. By fine‐tuning stability and reactivity of the metal complexes, such ligands pave the way for the development of next‐generation metallodrugs with improved efficacy, reduced systemic toxicity and enhanced tumour selectivity [23]. This work aimed at evaluating and correlating the substitutional reactivity and associated anticancer biological interactions of Pt/Pd(II) analogues endowed with N^∧N^∧N bis‐(azaaryl)amine chelates, see Figure 1. The anticipated outcome was to reconcile the often‐opposing demands of kinetic control and biological reactivity, offering a viable pathway towards more effective and safer metal‐based anticancer agents.

FIGURE 1.

Structures of Pt/Pd(L1-L3)Cl complexes.

2. Experimental

2.1. Materials and Methods

The following reactants and reagents such as N‐iodosuccinimide, 8‐bromoquinoline, 2‐bromo‐2‐methylaniline, trifluoracetic acid, 8‐bromoquinoline, 2‐formyl phenylboronic acid, 1,2‐dimethoxyethane, Pd₂(dba)₃, sodium tert‐butoxide, ammonium hexafluorophosphate, phosphate‐buffered saline (PBS) tablets, calf thymus (CT)‐DNA, bovine serum albumin (BSA), guanine (Guan), d₆‐dimethyl sulfoxide (DMSO), CDCl₃, thiourea (Tu), lithium chloride and the metal precursors (Pt(COD)Cl₂ and Pd(COD)Cl₂) were all procured from Sigma‐Aldrich. Organic solvents were also purchased from Sigma‐Aldrich and used without further purification. All cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, United States of America). These included human breast MCF‐7 (HTB‐22), T‐47D (HTB‐133), MDA‐MB‐231 (CRM‐HTB‐26), pancreatic PANC‐1 (CRL‐1469), CFPAC‐1 (CRL‐1918), cervical CaSki (CRL‐1550) and HeLa (CRM‐CCL‐2) cancer cells. The key aryl halide/amine precursors for the subsequent C‐N cross‐coupling reactions to form the NNN ligands, viz., 4‐amino‐2‐methylphenanthridine and 4‐bromo‐2‐methylphenanthridine, were synthesised according to the previously reported procedures [14].

2.2. Instrumentation

NMR spectra of ligands and complexes (in CDCl₃) were collected on a Bruker 400 MHz spectrometer. FTIR measurements were recorded (in the range 4000–400 cm⁻¹) with a Bruker Alpha FTIR equipped with an ATR Platinum Diamond accessory. UV‐visible absorption data were obtained on an Agilent Cary 60 spectrophotometer. High‐resolution mass spectra were acquired on a Waters Micromass LCT Premier instrument using electrospray ionisation and a TOF mass analyser. Fluorescence emission and excitation spectra were measured in a 1‐cm quartz cell on a Perkin Elmer LS‐45 spectrometer with a xenon lamp. Elemental (CHNS) analyses were performed on a Thermo Scientific Flash 2000 analyser. A Glomax® microplate spectrophotometer was used to read absorbance for the cell viability assay at 600 nm.

2.3. Synthesis of N^∧N^∧N Chelate Ligands

The precursors for the synthesis of the bis‐(aryl)amine chelate ligands (L2-L3) were all synthesised following the procedures described in the literature [14]. Ligands N‐(quinolin‐8‐ylmethyl)quinolin‐8‐amine, L1 [17], N‐(quinolin‐8‐yl)‐2‐methylphenanthridin‐4‐amine (L2) [13, 14] and bis‐(2‐methylphenanthridin‐4‐yl)amine (L3) [13, 14] were synthesised following the literature procedures with minor modifications. Detailed procedures are given in the SI document.

2.3.1. N‐(Quinolin‐8‐Ylmethyl)Quinolin‐8‐Amine (L1)

¹H NMR (CDCl₃, 500 MHz): [δ ppm]: 8.76 (q, 2H, Ha), 8.19 (q, H, Hb), 8.14 (m, 2H, Hc), 5.77 (s, H, Hd), 5.03 (s, 2H, He). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 148.94, 146.87, 146.58, 136.33, 136.01, 128.73, 128.40, 128.19, 127.83, 127.42, 126.86, 126.61, 126.44, 121.33, 121.06, 113.86, 105.35, 43.81. IR (cm⁻¹): 3369 ν(NH), 1569 ν(C=C), 1369 ν(C‐CH₃ stretch), 1498 ν(C=N), 1113 ν(C‐N), 2921 ν(C‐N‐C), 755 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for C₁₉H₁₅N₃ (285.8462), found 286.1431 [M+H]⁺. EA calculated for C₁₉H₁₅N_3: C 79.98; N 14.73; H 5.30, found C 80.43; N 15.10; H 4.90.

2.3.2. N‐(Quinolin‐8‐Yl)‐2‐Methylphenanthridin‐4‐Amine (L2)

¹H NMR (CDCl₃, 500 MHz): 9.36 (s, 2H, Ha), 8.78 (d, H, Hb), 8.32 (s, 2H, Hc), 8.14 (t, H, Hd), 7.85 (q, 2H, He); 7.76 (t, H, Hf); 7.46 (m, H, Hg); 7.38 (d, H, Hh); 5.46 (d, H, Hi); 2.63 (s, H, Hj). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 148.94, 146.87, 146.58, 136.33, 136.01, 128.73, 128.40, 128.19, 127.83, 127.42, 126.86, 126.61, 126.44, 121.33, 121.06, 113.86, 105.35, 43.81. IR (cm⁻¹): 3623 ν(NH), 1540 ν(C=C), 1371 ν(C‐CH₃ stretch), 1457 ν(C=N), 1105 ν(C‐N), 2921 ν(C‐N‐C), 749 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for C₂₃H₁₇N₃ (335.40), found m/z 335.8571 [M]⁺. EA calculated for C₂₃H₁₇N_3: C 82.36; N 12.53; H 5.11, found C 81.91; N 12.05; H 4.51.

2.3.3. bis‐(2‐Methylphenanthridin‐4‐Yl)Amine (L3)

¹H NMR (CDCl₃, 500 MHz): 9.72 (s, H, Ha); 9.36 (s, 2H, Hb), 9.18 (s, H, Hc), 8.52 (t, 2H, Hd), 8.34 (s, H, He), 5.25 (m, 2H, Hf); 2.60 (s, H, Hg); 2.53 (s, H, Hh). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 153.35, 139.78, 137.72, 135.18, 133.95, 131.96, 131.26, 128.94, 128.82, 128.37, 128.00, 126.53, 126.39, 125.51, 125.19, 125.04, 122.00, 121.66, 100.30, 32.20, 30.82, 29.68, 26.40, 23.42, 22.67, 21.56, 18.42, 14.11. IR (cm⁻¹): 3355 ν(NH), 1608 ν(C=C), 1345 ν(C‐CH₃ stretch)1444 ν(C=N), 1173 ν(C‐N), 2918 ν(C‐N‐C), 750 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for C₂₈H₂₁N₃ (399.17), found 478.1318 (M + DMSO + H), found 477.62 [(M + DMSO)]⁺, 478.1318 [(M + DMSO + H)]⁺. EA calculated for C₂₈H₂₁N_3: C 84.18; N 10.82; H 5.30, found C 84.37; N 10.46; H 5.08.

2.4. Synthesis of Pt/Pd(II) Metal Complexes [14]

2.4.1. [(Chlorido) (N‐(Quinolin‐8‐Ylmethyl)Quinolin‐8‐Amine)Pt(II)] Hexafluorophosphate (PtL1Cl)

A solution of (0.0292 g, 7.8 mmol) Pt(COD)Cl₂, COD = cyclooctadiene, in chloroform was placed in a round‐bottom flask, followed by the addition of NaOtBu (0.00750 g, 7.8 mmol) in drops. With the solution under moderate stirring, 25 mL of L1 (0.0222 g, 7.8 mmol) in chloroform was added dropwise. The mixture was stirred at a maximum rate for 72 h at 40°C under an inert nitrogen atmosphere. To induce lyophilisation, NH₄PF₆ (0.00150 g, 1.8 mmol) was added towards the end of the reaction, and the stirring continued for a further 2 h. Thereafter, the solvent was removed by microfiltration (0.45 μm nitrocellulose) under partial vacuum. The resulting precipitate was washed with small amounts of cold ethanol, water and diethyl ether. Yield: 79%.

¹H NMR (CDCl₃, 500 MHz); δ (ppm): 9.36 (s, 2H, Ha), 8.78 (d, H, Hb), 8.32 (s, 2H, Hc), 8.14 (t, H, Hd), 7.85 (q, 2H, He); 7.76 (t, H, Hf); 7.46 (m, H, Hg); 7.38 (d, H, Hh); 6.82 (d, H, Hi); 2.63 (s, H, Hj). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 148.94, 146.87, 146.58, 136.33, 136.01, 128.73, 128.40, 128.19, 127.83, 127.42, 126.86, 126.61, 126.44, 121.33, 121.06, 113.86, 105.35, 43.81. IR (cm⁻¹): 3355 ν(NH), 1608 ν(C=C), 1345 ν (C‐CH3 stretch), 1444 ν(C=N), 1173 ν(C‐N), 2918 ν(C‐N‐C), 750 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for [C₁₉H₁₄N₃ClPt]⁺ (514.05). Found 516.91 [(M+2H)]⁺. EA calculated for [C₁₉H₁₄N₃ClPt](PF₆): C 44.32; N 8.16; H 2.74. Found C 43.84; N 7.83; H 2.99.

PtL2Cl, PtL3Cl and Pd(L1-L3)Cl were synthesised following a similar procedure described for PtL1Cl by using Pt(COD)Cl₂ and Pd(COD)Cl₂, respectively [13, 14]. Notably, these complexes precipitated from the solution as neutral complexes.

2.4.2. [(Chlorido)N‐(Quinolin‐8‐Yl)‐2‐Methylphenanthridin‐4‐Amine)Pt(II)] (PtL2Cl)

¹H NMR (CDCl₃, 500 MHz); δ (ppm): 9.32 (s, H, Ha); 8.58 (d, H, Hb), 8.33 (s, H, Hc), 8.08 (t, 2H, Hd), 7.90 (t, H, He), 7.74 (t, H, Hf). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 158.20, 157.97, 144.47, 142.61, 138.73, 138.68, 136.68, 136.20, 133.61, 132.93, 131.14, 130.30, 129.85, 126.85, 126.56, 105.24, 82.61, 82.29, 81.97, 35.60, 34.32, 26.30. IR (cm⁻¹): 1722 ν(C=C), 1505 ν(C=N), 1266 ν(C‐N), 2922 ν(C‐N‐C), 838 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for [C₂₃H₁₆N₃ClPt] (628.10), found 627.38 [(M‐H)]. EA calculated for [C₂₃H₁₆N₃ClPt]: C 48.90; N 7.44; H 2.85. Found C 48.35; N 7.84; H 3.20.

2.4.3. [(Chlorido)‐bis‐(2‐Methylphenanthridin‐4‐yl)Amine)Pt(II)] (PtL3Cl)

¹H NMR (CDCl₃, 500 MHz; δ (ppm): 9.35 (s, H, Ha); 8.62 (d, H, Hb), 8.36 (s, H, Hc), 8.10 (d, 2H, Hd), 7.92 (m, H, He), 6.11 (t, H, Hf); 2.54 (s, H, Hg). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 148.94, 146.87, 146.58, 136.33, 136.01, 128.73, 128.40, 128.19, 127.83, 127.42, 126.86, 126.61, 126.44, 121.33, 121.06, 113.86, 105.35, 43.81. IR (cm⁻¹): 1100 ν(C‐N), 1261 ν (C‐CH₃ stretch), 1454 ν(C=N), 1723 ν(C=C), 2919 ν(C‐N‐C), 752 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for [C₂₈H₂₀N₃ClPt] (628.10). Found 629.08 [(M + H)]⁺. EA calculated [C₂₈H₂₀N₃ClPt]: C 53.46; N 6.68; H 3.20. Found C 52.98; N 7.03; H 3.57.

2.4.4. [(Chlorido) (N‐(Quinolin‐8‐Ylmethyl)Quinolin‐8‐Amine)Pd(II)] Hexafluorophosphate (PdL1Cl)

¹H NMR (CDCl₃, 500 MHz); δ (ppm): 9.32 (s, H, Ha); 8.58 (d, H, Hb), 8.33 (s, H, Hc), 8.08 (t, 2H, Hd), 7.90 (t, H, He), 7.74 (t, H, Hf). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 158.20, 157.97, 144.47, 142.61, 138.73, 138.68, 136.68, 136.20, 133.61, 132.93, 131.14, 130.30, 129.85, 126.85, 126.56, 105.24, 82.61, 82.29, 81.97, 35.60, 34.32, 26.30. IR (cm⁻¹): 1507 ν(C=C), 1089 ν (C‐N stretch), 2920 ν(C‐N‐C), 1507 ν(C=C), 838 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for [C₁₉H₁₄N₃ClPd]⁺ (426.99). Found 425.13 ([M‐H]⁺), EA calculated for [C₁₉H₁₄N₃ClPd](PF₆): C 53.54; N 9.86; H 3.31. Found C 53.86; N 9.37; H 3.43.

2.4.5. [(Chlorido)N‐(Quinolin‐8‐Yl)‐2‐Methylphenanthridin‐4‐Amine)Pd(II)] Hexafluorophosphate (PdL2Cl)

¹H NMR (CDCl₃, 500 MHz); δ (ppm): 9.30 (s, H, Ha); 8.54 (d, H, Hb), 8.29 (s, H, Hc), 8.10 (d, 2H, Hd), 7.87 (m, H, He), 7.71 (t, H, Hf); 7.12 (s, H, Hg); 2.59 (s, H, Hh). ¹³C NMR (500 MHz, CDCl₃, 303 K); δ (ppm): [δ ppm]: 153.41, 139.84, 137.74, 134.02, 132.03, 131.27, 128.88, 128.00, 126.45, 125.55, 125.24, 122.02, 121.66, 29.69, 28.03, 21.58. IR (cm⁻¹): 1610 ν(C=C), 1256 ν (C‐CH3 stretch), 1447 ν(C=N), 1104 ν(C‐N), 2918 ν(C‐N‐C), 751 ν(aromatic C‐H vibrations). ESI⁺‐TOF‐MS calculated for [C₂₃H₁₆N₃ClPd] (475.01). Found 478.38 [(M+3H)]⁺, EA calculated for [C₂₃H₁₆N₃ClPd]: C 58.00; N 8.82; H 3.39. Found C 58.31; N 8.79; H 3.64.

2.4.6. [(Chlorido)‐bis‐(2‐Methylphenanthridin‐4‐yl)Amine)Pd(II)] Hexafluorophosphate (PdL3Cl)

¹H NMR (CDCl₃, 500 MHz)δ (ppm): 9.35 (s, H, Ha); 8.61 (d, H, Hb), 8.36 (s, H, Hc), 8.09 (d, 2H, Hd), 7.90 (m, H, He), 7.75 (t, H, Hf); 7.45 (s, H, Hg); 3.74 (s, H, Hh); 2.64 (s, H, Hi). ¹³C NMR (500 MHz, CDCl₃, 303 K), [δ ppm]: 153.35, 139.78, 137.72, 135.18, 133.95, 131.96, 131.26, 128.94, 128.82, 128.37, 128.00, 126.53, 126.39, 125.51, 125.19, 125.04, 122.00, 121.66, 100.30, 32.20, 30.82, 29.68, 26.40, 23.42, 22.67, 21.56, 18.42, 14.11. IR (cm⁻¹): 1100 ν(C‐N), 1261 ν (C‐CH₃ stretch), 1454 ν(C=N), 1723 ν(C=C), 2919 ν(C‐N‐C), 752 ν(aromatic C‐H vibrations). ESI⁺‐MS calculated for [C₂₈H₂₀N₃ClPd] (541.04), found 542.41 [(M + H)]⁺. EA calculated for [C₂₈H₂₀N₃ClPd]: C 62.14; N 7.78; H 3.73, found C 62.40; N 7.57; H 3.78.1.2.

2.5. Interactions and Reactivity of the Pt/Pd(II) Metal Complexes (Pt/Pd(L1‐L3)Cl)

2.5.1. Stability of Complexes in DMSO and Buffer Solution

The aqueous stability of each metal complex was assessed under simulated physiological conditions (37°C). Solutions (0.1 mM) of each metal complex were prepared in PBS containing 2% DMSO (v/v), and their UV‐visible spectra were recorded hourly for 24 h. To evaluate resistance to DMSO solvolysis, the UV‐visible absorption spectra of the metal complexes dissolved in anhydrous DMSO were recorded at 1 h intervals for 24 h.

2.5.2. Computational Studies

Geometry‐optimised structures of Pt/Pd(II) complexes were computed at the density functional theory (DFT) level using the B3LYP/LanL2DZ method in the Gaussian 09 suite programs [24]. The 6‐311 core‐valence basis set was applied to all nonmetal atoms, while the LANL2DZ basis set, which uses effective core potentials, was applied to the metal centres [25]. In addition, time‐dependent DFT (TD‐DFT) was performed in DMSO, for which its solvent effects on the geometry of the complexes are taken into account according to a conductor polarisable continuum model (C‐PCM) [26]. The calculations were carried out to obtain geometry‐optimised molecular structures, trends in the energetics of the frontier molecular orbitals, and structural metrics such as bond angles and lengths, atomic charges, and predictive electronic reactivity descriptors. The optimised structures of the Pt/Pd(II) complexes were also used as input structures in simulated docking experiments to gauge the strength of their noncovalent interactions and affinities for receptors of selected macromolecules such as CT‐DNA and BSA, which are relevant for the treatment of cancers by molecular and metallodrugs. Molecular docking simulations are particularly valuable in the screening, predictive design and development of therapeutic drugs. The aqueous stability, substitutional reactivity of the metal complexes and molecular interactions during transportation and with relevant receptor sites correlate with their delivery efficiency, pharmacokinetic profiles and hence their therapeutic effect in diseased tissues [27]. Molecular docking simulations were carried out using Autodock tools 1.5.7 [28]. The Autodock parameter execution files were modified by adding parameters of Pt/Pd(II) ions since the default files do not have them. The metal complexes were docked on the receptor biomolecules relevant to the treatment of cancers, viz., B‐DNA (PDB:1bna), BSA (PDB:4f5s), RIP1‐kinase (PDB:6RLN), human aromatase cytochrome P450 (PDB:3EQM), human DNA polymerase epsilon (PDB: 5VBN), Exportin (PDB: XPO1) and the SARS‐CoV‐2 main protease (Mpro:8YA5), a crucial enzyme for the retroviral of COVID‐19 virus and related respiratory morbidities. The crystal structures of DNA and the proteins were retrieved from the Protein Data Bank (The Rutgers Artificial Intelligence and Data Science (RAD) Collaboratory). Before docking, all the receptor molecules were modified by removing water molecules. In the case of BSA, the second cocrystallised strand was deleted. These receptor molecules were further altered by adding nonpolar hydrogens, Gasteiger and Kollman charges. A generic algorithm was used as a search parameter, while the output files were expressed in a Lamarckian GA. Docked conformers of the Pt/Pd(II) complex‐receptor molecule were analysed and visualised using Bovia Discovery Studio 2024.

2.5.3. Rates and Mechanism of Chloride Substitution From the Pt/Pd(II) Complexes

Stock solutions of Pt/Pd(II) complexes and the nucleophiles Tu and Guan were prepared by dissolving accurately weighed amounts of each in methanol containing 0.10 M LiCl. The added LiCl suppresses the spontaneous solvolysis of the chlorometal complexes [29]. Solutions for kinetic analysis of the substitution reactions were prepared by diluting the stock solutions of the metal complexes to 0.1 mM, and the concentration of nucleophiles was prepared at 10–50‐fold excess to ensure pseudo‐first‐order kinetic conditions. Equal volumes of metal complexes and nucleophiles (in duplicates) were thermally equilibrated (298–313 K) in tandem cuvettes in the sample holder of a UV‐visible absorption spectrophotometer. The reaction was initiated by quickly mixing the reactants and recording the absorbance of the solution as a function of time at a set wavelength. Concentration‐dependent observed rate constants at 298 K were used to determine the bimolecular rate constant, k ₂, for the chloride substitution from each complex. Temperature‐dependent rate constants of the 30‐fold nucleophile solution were measured in the temperature range of 298–313 K, at 5 K increment intervals. Data of k ₂ were plotted to obtain Eyring plots from which the activation parameters (ΔH ^≠ and ΔS ^≠) were estimated.

2.5.4. DNA/BSA Spectroscopic Titrations With the Metal Complexes

Detailed DNA/BSA spectroscopic (absorption/emission) titrations to probe the binding affinities of the metal complexes are provided in the Supporting Information section (available here).

2.5.5. Cell Culture Growth, Maintenance and Treatments

The MDA‐MB‐231 triple negative breast cancer, PANC‐1 pancreatic cancer, HeLa cervical cancer and nonmalignant FG0 fibroblasts cell lines were cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM), the CFPAC‐1 pancreatic cancer cell line in Iscove’s Modified Dulbecco’s Medium (IMDM) and the oestrogen receptor–positive MCF‐7 and T47D breast cancer cell lines and CaSki cervical cancer cell line in Roswell Park Memorial Institute 1640 medium (RPMI‐1640). The cell culture media were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Each growth medium was supplemented with 10% heat‐inactivated foetal bovine serum (FBS) and 1% penicillin–streptomycin (Pen/Strep) (Gibco, MD, USA) to support optimal cell growth and prevent microbial contamination. Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. Culture media were replenished routinely, and cells were passaged before they reached 100% confluency. All cell lines were routinely screened for mycoplasma contamination, and only mycoplasma‐free cells were used in all experiments.

2.5.6. Drug Treatments

The uncoordinated ligands (L1-L3) and their respective Pt/PdL1Cl-3 metal complexes were dissolved in DMSO to achieve a stock concentration of 10 mM and subsequently stored at −20°C before use. For cell culture treatments, the 10‐mM stock solutions were diluted to final concentrations of 10 μM using the respective supplemented cell culture media. The percentage of DMSO in the 10‐μM solution (0.1%) was used as a vehicle control, and cisplatin (CDDP) was included as a reference‐positive control drug.

2.5.7. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltrazolium Bromide (MTT) Cell Viability Assay

To evaluate the cytotoxicity of the Pt/Pd(II) complexes, selected cell lines were treated at 10 μM (0.1% DMSO) of each of the Pt/Pd(II) complexes or the free ligands; LI-L3 and the viability of the cultured cells were measured using the MTT assay. Briefly, cells were plated in a 96‐well plate at densities of 3 × 10³–1 × 10⁴ cells per well and allowed to adhere for 24–48 h. After incubation, cells were treated for 48 h with 10 μM of each ligand (L1-3) or Pt/Pd(II) complexes, along with 10 μM CDDP and 0.1% DMSO. Thereafter, 10 μL of 5 mg/mL MTT solution was added to each well, and the plates were incubated at 37°C for 4 h to allow the formation of the purple formazan crystals. After incubation, 100 μL of the solubilising reagent was added to each well, followed by overnight incubation to dissolve the crystals. Absorbance was measured at 600 nm using a Glomax® microplate spectrophotometer, and mean cell viability was calculated and expressed as a percentage of the vehicle control.

3. Results and Discussion

3.1. Synthesis and Characterisation

L1 was prepared through a base‐catalysed nucleophilic substitution reaction of 8‐aminoquinoline with 8‐(bromomethyl)quinoline [17]. The reactants were stirred in a K₂CO₃ solution for 72 h at 90°C, resulting in the SN₂ nucleophilic substitution of the bromo on the methylene carbon bonded to the bridging amino‐N donor of 8‐quinoline to form the N^∧N^∧N ligand. Resultantly, a flexible methylene spacer in one arm of the bis‐(azaaryl)methylamine ligand was formed. The precursors needed for the C‐N cross‐coupling of the aryl halides and amino arenes to form L2-L3 were all synthesised via a multistep procedure [14]. The reaction of N‐iodosuccinimide with either 2‐bromo‐p‐toluidine or 2‐nitro‐p‐toluidine in TFA (0°C) afforded 2‐bromo‐6‐iodo‐p‐toluidine, [(2‐bromo‐6‐iodo‐4‐methyl)aniline] or 2‐iodo‐4‐nitro‐p‐toluidine [(2‐iodo‐4‐nitro‐4‐methyl)aniline] quantitatively. In the presence of Pd₂(dba)₃, the iodo‐p‐toluidine intermediates (in basic DME) coupled with 2‐formylphenyl boronic acid with concomitant in situ cyclic condensation to form 4‐bromo‐2‐methyl‐phenanthridine and 4‐nitro‐2‐methyl‐phenanthridine, respectively, in moderate yields of 65%–74%. The nitro derivative was further reduced with Zn dust in the presence of hydrazine formate and catalytic amounts of formic acid to form 4‐amino‐2‐methyl‐phenanthridine, one of the key intermediates to access L2-L3, in a good yield of 81%. The characterisation data of these intermediates are presented and listed in the SI section (Figures S1–S4). The bis‐(azaaryl)amine ligands, L2 (2‐methyl‐N‐(quinolin‐8‐yl)phenanthridin‐4‐amine) and L3 (bis‐(2‐methylphenanthridin‐4‐yl)amine) were formed from the Pd‐catalysed cross‐coupling reactions [14, 17] of either 4‐amino‐2‐methyl‐phenanthridine or 8‐aminoquinoline with 4‐bromo‐2‐methyl‐phenanthridine at 90°C and in a basic solution of K₂CO₃ and catalytic amounts of Pd(OAc)₂ over 72 h of reaction. The reactions afforded brown oils in yields of 76%–84%. The ¹H NMR spectra of uncoordinated ligands L1-L3 all display the expected diagnostic singlet amide (N‐H) resonances at 5.03 (L1), 5.47 (L2) and 5.45 ppm (L3), respectively, confirming the formation of the bis‐(azaaryl)mine ((R‐N)₂NH) ligands. Also, spectra of L2-L3 featured the characteristic singlet at 2.62 and 2.58 ppm for the 2‐methyl substituent of the phenanthridine arm(s) of the two N^∧N^∧N tridentates (Figures S5–S10). In addition, the total number of carbon resonances in the ¹³C NMR spectra of (L1 and L3) correlates with the theoretical carbon count corresponding to the molecular formula of each ligand. As expected, the distinctive pseudo‐imine (C=N) carbons of the middle ring on the phenanthridine arms resonate at 138.7 ppm (L2) and 136.2 ppm (L3) in the respective ¹³C NMR spectra of L2 and L3. This further confirms the formation of the bis‐(azaaryl)NH ligands bearing the 2‐methyl‐substituted bis‐(azaaryl)amine donor arm(s) (Figures S11–S16).

The Pt/Pd(II) complexes were prepared in yields of 68%–79% from equimolar reactions of L1–L3 with the respective metal precursors [Pt(COD)Cl₂ or Pd(COD)Cl₂] in (NaOtBu)/CH₂Cl₂ solution, as reported in the literature [14]. The metal complexes are soluble in chlorinated and high‐boiling aprotic solvents such as CHCl₃, DCM, DMSO and DMF, while they are poorly soluble in organic solvents. The metal complexes were characterised by NMR, FTIR, ESI⁺‐TOF‐mass spectrometry and elemental analysis. LI (quinolin‐8‐ylmethyl)quinolin‐8‐amine)) coordinates the metal centres as a neutral N^∧N^∧N tridentate chelate, forming monocationic Pt/Pd(II) analogues, as demonstrated in its single‐crystal structures of dinuclear Cd(II) [17]. Contrastingly, L2 or L3 coordinates to the Pt/Pd(II) metal centres as anionic tridentate, and their ¹H NMR spectra are characterised by the absence of the singlet peak of the bridging amino (N–H) protons. Their analogous Pt/Pd(II) complexes are therefore neutral. The rigid bis‐(azaaryl)amine ligand has to first undergo solvent‐assisted deprotonation at its donor amine nitrogen before its coordination to the metal centre takes place. Also, the chemical shifts of some of the ¹H signals of the free N^∧N^∧N ligands (L2-L3) are shifted upfield upon chelating the metal centres. For example, some ¹H signals, especially for protons close to the N^∧N^∧N donor atoms for L1–L3, are shifted upfield by 0.1–0.3 ppm. The aromatic protons of the quinoline/phenanthridine moieties in the free tridentate ligands appear in the range 7.2–8.5 ppm but appear as coalesced broad multiplexes between 6.9 and 8.2 ppm in the spectra of the respective complexes (Figures S5–S10). This upfield shift is consistent with a slight increase in electron density within the ligand π‐system following metal chelation. Corroboratively, the ¹⁹⁵Pt NMR spectra of Pt(L1-L3)Cl complexes exhibit a single broad resonance at −3700, −3350 and −3050 ppm, respectively (Figures S17–S19). These chemical shifts are typical of ¹⁹⁵Pt resonances for the [Pt(II) (N^∧N^∧N)Cl] square‐planar coordination environment, thus further validating successful ligand chelation of the Pt(II) ion, forming square planar pincer complexes as reported in the literature for similarly structured tridentate chelate ligands [15, 30, 31].

The FTIR spectra of the free ligands (L1–L3), all exhibit a broad vibronic band between 3335 and 3623 cm⁻¹, attributable to the stretching vibrations of the primary amine (N‐H) moiety, along with a sharp, intense C=N imine stretch at 1352 cm⁻¹ (L1), 1563 cm⁻¹ (L2) and 1724 cm⁻¹ (L3), confirming successful ligand formation. The strong peaks at 783 (L1), 764 (L2) and 749 cm⁻¹ (L3) correlate with the C‐C/N in‐plane vibration within the conjugated aromatic skeleton [32]. The methyl C–CH₃ bending vibrations appear at 1128 cm⁻¹ (L1) and shift slightly upwards to 1132 cm⁻¹ (L2) and 1136 cm⁻¹ (L3), which is consistent with the difference in the aryl ring to which it is attached. Upon ligand chelation of the Pt/Pd(II) ion, the N‐H stretching of L2-L3 disappears, indicating deprotonation of the amino group and metal coordination of the headgroup N as an amido group. At the same time, the imine C=N stretch shifts to lower wavenumbers 1567 cm⁻¹ (PtL1Cl), 1571 cm⁻¹ (PtL2Cl) and 1716 cm⁻¹ in PtL3Cl, reflecting a reduction in the C=N bond order, as electron density is drawn towards the metal centre. This is further corroborated by DFT‐simulated spectra, which predict C=N stretches at 1605 cm⁻¹ (PtL1Cl), 1611 cm⁻¹ (PtL2Cl) and 1686 cm⁻¹ (PtL3Cl) (Figures S20–S25). The C‐CH₃ bending modes also migrate slightly to higher frequencies 1131, 1133 and 1138 cm⁻¹ for Pt(L1-L3)Cl, respectively, consistent with altered ligand vibrational coupling upon coordination. Analogous spectral shifts are observed for the Pd(II) congeners, collectively validating successful chelation and the attendant electronic perturbation of the ligand framework.

The mass‐to‐charge (m/z) ratios and elemental compositions of the synthesised chelating ligands and their corresponding metal complexes were verified using electrospray ionisation–time of flight–mass spectrometry (ESI⁺‐TOF‐MS) and elemental analysis (CHNS). The m/z values obtained for all the ligands and Pt/Pd(II) complexes compare well (within experimental error) with the theoretical nominal values of the pseudomolecular ion, confirming the synthesis of the compounds (Figures S26–S31). Additionally, the experimental values of the elemental compositions of the ligands and metal complexes were within ±0.5% of the calculated values, further validating their purity (Figures S32–S37). Detailed mass spectrometry and elemental analysis data are presented in Table S1.

The electronic absorption spectra of the chelating ligands L1–L3 and their corresponding Pt/Pd(II) complexes (Pt/Pd(L1-L3)Cl) were recorded in DMSO. The UV‐visible absorption spectra of the free ligands are dominated by π-π∗ transitions below 350 nm, with absorption maxima that exhibit a systematic hypsochromic shift from 362 nm (for L1) and 346 nm (for L2) to 334 nm (for L3). This trend is attributed to the progressive extension of the π‐conjugated system along the ligand backbone of L1 to L3. In addition to the primary absorption bands, L2 and L3 exhibit shoulder peaks at approximately 335 and 350 nm, which are assigned to n-π∗ transitions localised on the phenanthridinyl termini. These bands are shifted to longer wavelengths (with bathochromic shifts of up to 150 nm) in the metal complexes’ spectra, a reflection that Pt/Pd(II) chelation is accompanied by π-π∗ stacking of the square planar cations. Besides these bands, the spectra of Pt/Pd(L1-L3)Cl complexes display less intense and broad charge transfer bands with maxima between 500 and 700 nm (Figure 2). These bands are tentatively assigned to weak to moderate metal‐to‐ligand charge transfer (MLCT) for these electronic transitions [33].

FIGURE 2.

Overlay UV‐visible absorption spectra of (a) PtL1Cl and the free ligand L1, (b) PtL2Cl and L2, (c) PtL3Cl and L3, (d) PdL1Cl and L1, (e) PdL2Cl and L2 and (f) PdL3Cl and L3.

(a)

(b)

(c)

(d)

(e)

(f)

3.2. Stability of Pt/Pd(L1‐L3)Cl in DMSO and Aqueous Solution

The aqueous stabilities of Pt/Pd(L1-L3)Cl in a 98:2% (PBS: DMSO) solvent mixture and in pure DMSO were monitored by recording time‐dependent spectra of the metal complexes using UV‐visible absorption spectrophotometry. These two test media represent the solvent system conditions that were used in the biomolecular evaluations and in vitro cytotoxicity assays of the metal complexes, respectively. Electronic absorption spectra were recorded at physiological temperature (37°C) over 24 h, with measurements taken at 1 h intervals. In DMSO, no meaningful changes were observed in the UV‐visible spectra of all complexes over the specified time, indicating that the metal complexes remain stable and maintain their structural integrity under these conditions (Figures S38–S43). In contrast, for an aqueous PBS: DMSO (98:2) solvent mixture, the time‐dependent spectra of most metal complexes changed significantly over time. As shown in Figures S38–S43, for the stacked spectra of PdL3Cl in PBS: DMSO (98:2) solution, there is a gradual decrease in absorption intensity over 24 h, consistent with hydrolytic displacement of the coordinated chloro‐ligand [34]. This is an affirmation that the metal complexes undergo slow and partial hydrolysis in aqueous media, which can also activate them towards their ultimate covalent binding at the target site of action, such as DNA.

3.3. DFT‐Optimised Structures and Data of the Pt/Pd(L1‐L3)Cl Complexes

DFT calculations were carried out to obtain optimised input‐molecular structures, trends in the energetics of the frontier molecular orbitals, structural metrics such as bond angles and lengths, atomic charges and predictive electronic reactivity descriptors. The DFT‐optimised structures and calculated HOMO‐LUMO frontier orbitals of the Pt/Pt(II) complexes are presented in Table 1. In all cases, the optimised geometry is a distorted square planar, in which L1 coordinates as a neutral N^∧N^∧N tridentate ligand, forming unsymmetrical 6‐ and 5‐membered chelates while L2 or L3 coordinates as a monoanionic N^∧N^∧N tridentate ligand, forming twin 5‐membered rigid chelates at the Pt/Pd(II) ions. As a result, in the structures of the Pt/PdL1Cl complexes, the six‐membered chelates are more flexible and slightly tipped off the square plane defined by the Pt/Pd‐centres and the other quinoline ring. However, L2 or L3 lies entirely on the square plane. The simulated structures of the (Pt/Pd(L1-L3)Cl) complexes are comparable to those reported for respective single‐crystal X‐ray structures [14, 15]. A look at the respective highest‐occupied molecular orbitals (HOMOs) of all the metal complexes shows that they lie predominantly on the d‐orbitals of the Pt/Pd(II) centres, with a significant admixture on the p‐orbitals of chloride and with the π‐group molecular orbitals of the phenanthridine chelating ligand arms. The HOMO mapping is also mirrored in the molecular electrostatic potential surfaces (MEPS) of the metal complexes (Figure 3). In particular, analysis of the HOMO‐electron density gradient MEPS reveals that electron‐rich regions (red/yellow regions) are also predominantly localised along the Pt/Pd‐Cl bond and over the phenanthridine chelated arms. In contrast, the lowest‐unoccupied molecular orbitals (LUMOs) are concentrated on the tridentate N^∧N^∧N ligands. These are group π∗ molecular orbitals of quinoline/phenanthridine moieties. The electron density mapping of these frontier MOs indicates that these metal complexes are stabilised by MLCT processes that can relieve excess electron density from the metal‐centred d‐orbitals into the ligands’ π∗‐acceptors group MOs. If the electron density mapping is considered according to the MEPs, the rate of MLCT would be predicted to increase in the order Pt/PdL1 < Pt/PdL2 < Pt/PdL3, which follows the order of increase in benzannulation, π‐conjugation and delocalisation of electron density within the N^∧N^∧N chelates of the (bis‐aryl)amine ligands.

TABLE 1.

Geometry‐optimised structures and mappings of the HOMO and LUMO surfaces for Pt/Pd(L1-L3)Cl.

Complex	Optimised structure	HOMO map	LUMO map
PtL1Cl
PdL1Cl
PtL2Cl
PdL2Cl
PtL3Cl
PdL3Cl

FIGURE 3.

Colour‐coded molecular electrostatic potential surfaces (MEPS), overlaid onto the molecular structures of (Pt/Pd(L1-L3)Cl).

The influence of the stereoelectronic properties of the N‐donor chelators is shown in the metric data in Table 2. For example, metal complexes of L1, which bear a flexible 6‐membered chelate in one arm of the quinoline ligand, exhibit longer Pt/Pd─Cl bond lengths and wider bite angles of PtL1Cl (2.489 Å, 174.26°) and PdL1Cl (2.467 Å, 173.48°). In comparison, the metal complexes incorporating bis‐(azaaryl)amido ligands L2 or L3 (in which asymmetric or symmetric 5‐membered rigid chelates are formed by the amido and methyl‐substituted phenanthridine/8‐quinolyl ligands) feature relatively shorter Pt/Pd─Cl bond lengths and narrow bite angles as exemplified in the structural metrics of PtL3Cl (2.457 Å, 165.27°) and PdL3Cl (2.428 Å, 164.81°). The longer Pt/Pd─Cl bond lengths make these metal complexes the most reactive towards chloride substitution in comparison to their respective congeners. Furthermore, the phenanthridine metal complexes have higher positive natural population analysis (NPA) charges at the metal centres, indicating enhanced electron donation from both the ligand backbone and the methyl substituents relative to the quinoline analogues. The LUMO‐HOMO (band) gap energy decreases in the order Pt/PdL1Cl > Pt/PdL2Cl > Pt/PdL3Cl and correlates inversely with the increase in the extension of π‐conjugation. For the Pt(II) series, the order is 2.152 eV. (for PtL1Cl) < 2.752 eV (for PtL2Cl) < 2.799 eV (for PtL3Cl). These trends predict that Pt/PdL1Cl complexes would be more reactive towards ligand substitution with incoming nucleophiles compared to their Pt/Pd(L2-L3)Cl congeners due to their lower associated activation energies. The trends of computed electrophilicity indices (ω) of the two sets of complexes decrease in the order of PtL1Cl > PtL2Cl > PtL3Cl and PdL1Cl > PdL2Cl > PdL3Cl. These trends correlate well with the increasing order in the band gap, predicting higher chemical reactivity of the dicationic Pt/PdL1Cl analogues in comparison to their monocationic analogues featuring the more rigid bis‐(aryl)amine nonleaving ligands, L2 or L3.

TABLE 2.

Summary of DFT‐calculated data for the optimised structures of Pt/Pd(L1-L3)Cl.

Property	Complex
	PtL1Cl	PdL1Cl	PtL2Cl	PdL2Cl	PtL3Cl	PdL3Cl
Bond lengths (Å)
M(Pt/Pd)─Cl	2.489	2.467	2.460	2.425	2.457	2.428
M─N1	2.004	2.006	2.004	2.007	2.001	2.003
M─N2	2.040	2.110	2.027	2.039	2.026	2.037
M─N3	2.082	2.061	2.026	2.037	2.025	2.037
Bond angles (°)
N1─Pt─N2	91.02	90.95	88.27	88.34	87.15	87.32
N2─Pt─N3	174.26	173.48	165.36	164.85	165.27	164.81
N1─Pt─N3	89.24	89.50	88.38	88.42	87.24	87.36
Natural charges
M	0.746	0.554	0.809	0.596	0.812	0.597
Cl	−0.418	−0.505	−0.399	−0.481	−0.392	−0.488
N1	−0.886	−0.765	−1.093	−0.984	−1.102	−0.993
N2	−0.821	−0.683	−0.836	−0.701	−0.847	−0.709
N3	−0.809	−0.690	−0.847	−0.709	−0.847	−0.710
HOMO (eV)	−4.710	−4.826	−5.034	−4.832	−4.954	−5.031
LUMO (eV)	−2.558	−2.529	−2.235	−2.176	−2.202	−2.139
ΔE (eV)	2.152	2.297	2.752	2.939	2.799	2.892
μ	−3.634	−3.677	−3.635	5.115	−3.578	5.031
η	1.081	1.156	1.399	1.469	1.376	1.446
ω	6.111	5.848	4.721	4.523	4.652	4.372
Dipole moment (Debye)	5.917	5.922	5.609	5.569	5.456	5.405

3.4. Chloride Substitution From Pt/Pd(L1‐L3)Cl by the Model Bionucleophiles, Guan and Tu

The substitution rate of the coordinated chloride from Pt/Pd(L1-L3)Cl by Guan and Tu nucleophiles was measured under pseudo-first‐order kinetics. To probe structure‐reactivity relationships of the bis‐(aryl)amine nonleaving ligands, the neutral N^∧N^∧N donor ligand L1 and more rigid N^∧N^∧N ligands, L2 and L3, were used as chelating ligands. By removing the flexible (spacer) methylene group in one of the chelate arm L1, and replacing one of the 8‐quinolinyl rings with a phenanthridine donor moiety, the rigid chelate analogue, L2, coordinates the Pt/Pd(II) ion as a monoanionic ligand. The phenanthridine donor arm is structurally similar to the 8‐quinolinyl ring but with an additional side‐fused phenyl ring, which expands its π‐surface via benzannulation. This extension is doubled in the bis‐(phenanthridine)amine ligand, L3. These modifications were used as a design strategy to assess how electronic and steric changes in the ancillary ligands affect the Pt/Pd(II)’s electrophilicity, substitution reactivity, and ultimately gauge the metal complexes’ susceptibility towards unintended deactivation by biological nucleophiles. Kinetic measurements were performed by monitoring UV‐visible absorbance changes at convenient wavelengths (where the largest changes were observed) as a function of time. The chosen wavelengths are presented in Table S2. Kinetic traces fit to first‐order exponential decay functions, yielding the observed pseudo‐first‐order rate constants (k _obs) (Tables S3–S14), for each nucleophile concentration and temperature, as exemplified by a fit of the experimental data to appropriate standard exponential growth polynomials in Figure 4, and S44–S45.

FIGURE 4.

(a) A kinetic trace at 275 nm for the substitution of chloride from PdL1Cl (0.3 mM) by guanine (guan, 10‐fold excess), (b) a kinetic trace at 280 nm for the substitution of chloride from PdL1Cl (0.5 mM) by thiourea (Tu, at 10‐fold excess) at 298 K.

(a)

(b)

Plots of k _obs versus (nucleophile)yielded linear correlations passing through the origin, indicating negligible contribution from the solvolytic or reverse (dissociative) pathways [35], see Figure 5. Second‐order rate constants (k ₂) were determined from the slopes of these plots and are compiled in Table 3.

FIGURE 5.

Concentration‐dependent plots of (k _obs, s⁻¹) for the chloride substitution from (a) Pt(L1-L3)Cl by guan, (b) Pd(L1-L3)Cl by guan, (c) Pt(L1-L3)Cl by Tu, and (d) Pd(L1-L3)Cl by Tu, I = 0.1 M (LiCl) in methanol, T = 298 K.

(a)

(b)

(c)

(d)

TABLE 3.

Second‐order rate constants, k ₂ (M⁻¹ s⁻¹), for the chloride substitution from Pt/Pd(L1-L3)Cl by guan and Tu at 298 K.

Complex	Tu	Guan
	k 2 (M−1 s−1)	k 2 (M−1 s−1)
PtL1Cl	23.6 ± 0.2	2.82 ± 0.08
PtL2Cl	19.5 ± 0.3	2.41 ± 0.06
PtL3Cl	16.4 ± 0.3	1.97 ± 0.04
PdL1Cl	1179 ± 20	485 ± 10
PdL2Cl	968 ± 10	392 ± 4
PdL3Cl	875 ± 6	302 ± 4

The chloride substitution rates for the two sets of metal complexes by both nucleophiles decrease in the order, PdL1Cl > PdL2Cl > PdL3Cl, and PtL1Cl > PtL2Cl > PtL3Cl. For both sets of metal complexes, analogues of L1 are more reactive than those of L2 or L3. Contrary to literature trends, the extension of π‐conjugation in L3 metal complexes led to a reduction in substitutional reactivity. The increase in the number of fused aromatic rings and chelate rigidity was anticipated to promote efficient delocalisation of metal d‐electron density into the ligand’s extended π‐system, thereby increasing metal electrophilicity and accelerating chloride displacement as reported in some previous studies [36–38]. This anomalous trend is similarly reported for the rate of Cl substitution from Pt(II) complexes, wherein the terpyridine (N^∧N^∧N) core ligand was expanded to incorporate 1,10‐phenanthrolinyl or isoquinolinyl rings, with the latter showing lower rates of substitution despite the overall increase in π‐conjugation and π‐back bonding of the ligands of the latter metal complexes [27]. However, for the panel studied herein, the higher reactivity of L1 metal complexes is rooted in the larger formal charge, to some extent, the relative flexibility of one of its chelates, which is 6‐membered. L1 binds the metal centres as a neutral N^∧N^∧N tridentate, forming mono‐cationic Pt/Pd(L1)Cl complexes. In the cases of its rigid bis‐(azaaryl)amine counterparts, L2 and L3, they deprotonate first, binding as monoanionic N^∧N^∧N tridentates and forming neutral Pt/Pd(L2-L3)Cl complexes. Thus, the higher formal charge on the metal ions of Pt/Pd(L1)Cl complexes makes these metal complexes more reactive towards nucleophiles since the substitution process is accompanied by a higher ion‐dipole moment between the lone pairs on the S of Tu or N of Guan and the metal centre of the monocationic Pt/Pd(II) complexes. The metal complexes, Pt/Pd(L1)Cl, also exhibit longer Pt/Pd─Cl bond lengths (see Table 2). The reason for the high trans‐influence of this bond relative to those of Pt/Pd(L2-L3)Cl is unclear. One would have expected a stronger trans-effect and influence on the Pt/Pd─Cl of the amido N of L2 and L3 compared to the amino of L1. This may also suggest a weaker Pt/Pd─N_amido (that would lead to a longer trans‐Pt/Pd─Cl bond) due to the more acute bite angles of L2 or L3. Despite an increase in π‐conjugation and π‐back bonding capacity of L3 relative to L2, Pt/Pd(L2)Cl complexes are more reactive than Pt/Pd(L3)Cl, suggesting that methylation of their phenanthridine at C4 counteract the acceptor capacity by direct σ‐donation of electron density into the π∗ molecular orbitals of the phenanthridine ring system [19]. Without the methyl substituents, Pt/Pd(L3)Cl complexes would relieve their metal‐centres of electron density more efficiently by π‐back‐bonding into the π∗‐molecular orbitals of the ligands, making the metal centres more electrophilic and hence more reactive towards incoming nucleophiles. For L2, only one arm is methylated and has its ring π‐acceptor system disrupted, hence the slightly better reactivity of its complexes. Additionally, the shorter Pt/Pd(II)–Cl bond lengths and narrow N─M─N bite angles of PtL3Cl and PdL3Cl induce stronger metal‐ligand orbital overlaps, increasing the energy barrier for chloride dissociation and consequently slowing the substitution reactions. These trends are also correlated to lower DFT‐calculated electrophilicity indices for the L3 complexes relative to L1 analogues (Table 2). In all these metal complexes, the steric demand of the N^∧N^∧N chelates within the plane and along the Pt/Pd─Cl plays little to no effect since the N donor rings of both 8‐quinolinyl and 2‐methyl phenanthridine are positioned conveniently (inward) for bonding to the Pd/Pt(II) with little hindrance to the approach of the incoming nucleophile. Comparing rate constants for Pt/Pd(L1-L3)Cl reveals that the rate of chloride substitutions from the Pd(II) complexes is more than 170‐fold higher than their Pt(II) analogues. This trend has been reported in the literature [4, 12, 39]. While both are isoelectronic d ⁸ ions, Pt(II)’s 5d orbitals are larger and more readily polarisable compared to Pd(II)’s compact or less diffuse 4d orbitals. Consequently, in the associative substitution process at a square‐planar coordination sphere, the larger and easily polarisable orbitals stabilise the transition state, [Nu…Pt─Cl] better, leading to relatively lower rates of substitution compared to the Pd(II) analogues [12]. In all substitutions, the S‐donor nucleophile, Tu substitutes the chloride labile ligand much faster (with reactivity factors of > 50) than the N‐donor nucleophile, Guan. The intrinsic nucleophilicity constant of the S‐donor atom of Tu (S _N  ≈ 7.2) is larger than that of N (Guan, S _N  ≈ 3.5) due to its larger atomic size of the former and hence more polarisable (“softness”) of its donor valence orbitals. Thus, the lone pair of electrons in the more diffuse orbitals is easily polarised and thus readily donated. The diffuse orbitals are compatible for bonding with the ‘softer’ and larger orbitals of the Pt(II) ions (more polarisable electrophile), leading to faster rates of Cl substitution of the labile ligand. Thus, Tu proceeds more swiftly towards a product‐tipped SN₂ transition state. The observed trend is per the principle of the Hard‐Soft Acid Base Theory, for which ‘soft Lewis bases’ rapidly react with ‘soft Lewis acids’ [40, 41].

The temperature dependence of k ₂ was evaluated by measuring the rate of chloride substitution over a temperature range of 298–313 K at a constant nucleophile to Pt/Pd(II) complex concentration ratio of 30‐fold. Linearising the correlation between the rate constants and reaction temperature using the Eyring equation afforded activation enthalpies (ΔH ^≠) and entropies (ΔS ^≠) from the slopes and intercepts of the plots, respectively. Their values are summarised in Table 4, and the Eyring plots are shown in Figure 6.

TABLE 4.

Activation parameters for chloride substitution from Pt/Pd(L1-L3)Cl by nucleophiles (Tu and guan).

Complex	Nucleophile
	Tu (S‐donor)			Guan (N donor)
	ΔH ≠ kJ mol−1	ΔS ≠ J K−1 mol−1	ΔG ≠ kJ mol−1	ΔH ≠ kJ mol−1	ΔS ≠ J K−1 mol−1	ΔG ≠ kJ mol−1
PtL1Cl	20 ± 1	−195 ± 6	−78	33 ± 1	−98 ± 6	−62
PtL2Cl	25 ± 1	−147 ± 3	−69	35 ± 1	−152 ± 3	−80
PtL3Cl	37 ± 2	−172 ± 3	−88	39 ± 2	−81 ± 3	−63
PdL1Cl	16 ± 1	−97 ± 3	−44	20 ± 1	−94 ± 3	−48
PdL2Cl	18 ± 2	−159 ± 6	−65	24 ± 2	−157 ± 6	−70
PdL3Cl	21 ± 2	−181 ± 5	−75	29 ± 2	−172 ± 5	−80

FIGURE 6.

Eyring plots for the chloride substitution from (a) Pt(L1-L3)Cl by guan, (b) Pd(L1-L3)Cl by guan, (c) Pt(L1-L3)Cl by Tu, and (d) Pd(L1-L3)Cl by Tu, temperature range of 298–313 K, in methanol.

(a)

(b)

(c)

(d)

The reactions are characterised by markedly negative activation entropies (ΔS ^≠ < 0, in the range −81 to −195 J K⁻¹ mol⁻¹) and relatively low activation enthalpies (ΔH ^≠), and very negative free Gibbs’ energy of activation ranging from −44 to −80 kJ mol⁻¹. Thus, their transition states for the chloride substitutions are more compact than the overall microstates of the reactants. Consequently, the transition complex is rapidly attained and occurs spontaneously. These kinetic attributes are the hallmarks of an associative mechanism substitution in square‐planar Pd/Pt(II) complexes in which the reactions proceed via a trigonal bipyramidal transition state.

3.5. Molecular Docking of (Pt/Pd(L1‐L3)Cl) on Selected Receptive Biomolecules

Molecular docking is an invaluable tool in the drug discovery process. It provides essential insights into the structural interactions between potential drugs and their biological target molecules, guiding the design and optimisation of new therapeutic agents. Through these in silico simulations, key noncovalent interactions can be characterised and quantified, such as hydrogen bonding, hydrophobic interactions, van der Waals forces, and electrostatic forces, all of which contribute to the intrinsic binding affinities of the drugs for the receptors in the biological molecules [42]. In addition, computational screening enables rapid vetting of library compounds before resource‐intensive experimental assays are conducted on the lead and promising compounds. The docking of Pt/Pd(L1/L2/L3)Cl to preferred receptors and domains of BSA afforded stable simulated ligand‐complex adducts, which are shown in Figure 7 and S46–S49. The calculated stability energy scores for the complexes are summarised in Table 5. Binding scores range from −6.97 to −8.05 eV and follow the increasing order of stability: PdL2Cl < PdL1Cl < PdL3Cl and PtL2Cl < PtL1Cl < PtL3Cl. The noncovalent interactions are stabilised by multiple hydrogen bonds and π-π stacking interactions involving key amino acid residues of BSA, including glutamic acid (Glu), leucine (Leu), phenylalanine (Phe), arginine (Arg), histidine (His), alanine (Ala) and proline (Pro) interacting with chloride atoms, nitrogen’s, hydrogens and metal centres of complexes. These short‐contact bonds are considered moderate to strong contributors to the overall stability of the poses. For all cases, the metal complexes predominantly bind at Site II (Subdomain IIIA) of the protein. Subdomain IIIA is located near the N‐terminal region of BSA. It facilitates rapid initial uptake and distribution of the metal complexes through allosteric modulation of the protein’s conformation, which in turn stabilises Subdomain IIIA. It also forms strong and stable interactions with hydrophobic and planar moieties of the metal complexes, thereby elongating their pharmacokinetic half‐lives, sustaining their circulation in the bloodstream, as well as enhancing their delivery and bioavailability at the site of action. The interactions of BSA with small complex ions/compounds model the reversible uptake, transportation and release of many metal‐based drugs throughout the bloodstream. It is the most abundant small‐molecule peddling protein in blood plasma. Thus, the predicted medium to strong affinity of the Pt/Pd(L1-L3)Cl complexes for certain receptors and domains of BSA will have a positive impact on their efficient transportation, tissue distribution and pharmacokinetic profiles [43].

FIGURE 7.

(a) 3D simulated images of PtL1Cl docked into BSA Subdomain IIIA, (b) PtL1Cl localised noncovalent bonding to BSA’s amino acid residues, (c) 3D simulated images of PtL3Cl docked into BSA Subdomain IIIA and (d) PtL3Cl localised noncovalent bonding to BSA’s amino acid residues.

(a)

(b)

(c)

(d)

TABLE 5.

Calculated binding affinities of Pt/Pd(L1-L3)Cl on receptors of BSA.

Complexes	Binding scores (eV)	RMSD (Å) of the pose	BSA binding site domain
PtL1Cl	−7.60	±0.66	IIIA
PtL2Cl	−7.07	±1.39	IIIA
PtL3Cl	−7.97	±1.62	IIIA
PdL1Cl	−7.09	±0.52	IIIA
PdL2Cl	−6.97	±0.57	IIIA
PdL3Cl	−8.05	±0.94	IIIA

Pt/Pd(L1-L3)Cl were also docked onto DNA to have insights into their noncovalent interactions with the biopolymer. Typical simulated ligand‐complex adducts are illustrated in Figure 8 and S51–S54. All adducts show stability energy scores in the range of −9.25 to −10.52 eV. The calculated binding affinity scores are summarised in Table 6. The simulated docked poses of the adducts showed preferred noncovalent interactions of the Pt/Pd(L1-L3)Cl complexes with DNA groups in the minor grooves of DNA, as previously reported for similarly structured Pt/Pd(II) complexes [44, 45]. In the predicted binding models, the chloride atoms, Pt/Pd(II) centres and atoms of the aromatic moieties of the metal complexes engage in electrostatic interactions, van der Waals forces, and hydrogen bonding interactions with DNA’s phosphates and nucleobases, forming stable structures. The computed binding affinity scores and the stability of the formed DNA–metal complex adducts progressively increase in the order: PdL2Cl < PdL1Cl < PdL3Cl and PtL2Cl < PtL1Cl < PtL3Cl.

FIGURE 8.

(a) 3D simulated images of PtL1Cl inserted in the groove of DNA, (b) key intermolecular short contact bonds in the noncovalent interactions of PtL1Cl with the N‐bases of DNA, (c) 3D simulated images of PtL3Cl inserted in the groove of DNA and (d) key intermolecular short contact bonds in the noncovalent interactions of PtL3Cl with the N‐bases of DNA.

(a)

(b)

(c)

(d)

TABLE 6.

Calculated stability score (eV.) for noncovalent binding of Pt/Pd(L1-L3)Cl complexes at the preferred sites of DNA receptors.

Complexes	Binding scores (eV.)	RMSD (Å) of the pose
PtL1	−10.18	±0.41
PtL2	−9.39	±0.52
PtL3	−10.52	±0.97
PdL1	−9.66	±0.58
PdL2	−9.25	±0.61
PdL3	−10.02	±1.67

The cytotoxic lead metal complex, PtL1Cl (vide infra), was further docked against a panel of selected proteins/enzymes known to mediate the progression or antiproliferation of various cancer types (PDB ID), including prostate cancer (XPO1), cervical cancer (5VBN), breast cancer (4DRH) and 3EQM) and pancreatic cancer (6RLN). The simulation was extended to the SARS‐Coronavirus‐2 (CoV‐2) main protease (Mpro: 8YA5), a protein that upregulates the replication of the virus, to evaluate the potential of PtL1Cl as a dual‐function therapeutic agent. The poses and interaction diagrams with these proteins are presented in Figures S56–S59. Figure 9 shows, as an example, the simulated pose of the adduct of PtL1Cl and the enzyme, RIP1 kinase, an enzyme that is overexpressed in pancreatic cancer cells. The corresponding binding energy scores for each protein target are summarised in Table 7. PtL1Cl exhibits the highest binding affinity towards enzymes/proteins associated with the inhibition of prostate (XPO1) and cervical cancers (5VBN), and breast (4DRH). The binding affinity score for SARS‐CoV‐2’s Mpro: 8YA5 is, however, moderate to strong. By inhibiting the overexpressed proteins/enzymes, Mpro disrupts key survival mechanisms unique to cancer cells through several interconnected mechanisms. The selective toxicity, coupled with the induction of apoptosis and inhibition of metastasis, is the key to reducing off‐target toxicity, side effects, and drug resistance of cancer cells. PtL1Cl has the potential to serve as a dual‐purpose drug, that is, to inhibit cancer as well as serve as a COVID‐19 antiretroviral/inhibitor in patients with comorbidities. The calculated RMSD values for all docking poses were within the acceptable range of < ±2.0 Å, relative to the geometry‐optimised reference structures of the complexes, indicating good precision and accuracy of the used docking method. The superimposed poses of the docked and DFT‐optimised structures of the complexes are depicted in Figures S50, S55 and S60.

FIGURE 9.

(a) 3D‐simulated image of PtL1Cl docked into a preferred receptor of RIP1 kinase (PDB: 6RLN), an enzyme that is overexpressed in pancreatic cancer cells, (b) PtL1Cl bonded to the protein amino acid residues. (c) 3D image of PtL1Cl docked into a preferred receptor of peptidyl‐prolyl isomerase (PDB: 4DRH), an enzyme that is overexpressed in breast cancer cells and (d) PtL1Cl bonded to the protein amino acid residues.

(a)

(b)

(c)

(d)

TABLE 7.

Calculated stability score (eV.) for noncovalent binding of PtL1Cl at the preferred receptor site of selected cancer‐mediating proteins.

Complex	Cancer and SARS‐CoV‐2‐mediating proteins
	XPO1 (prostate)	5VBN (cervical)	4DRH (breast)	3EQM (breast)	6RLN (pancreatic)	8YA5 (SARS‐CoV‐2)
PtL1Cl	−9.56	−8.76	−8.16	−7.35	−7.49	−7.23
RMSD (Å) of the pose	±0.49	±1.32	±0.61	±1.59	±0.52	±0.66

3.6. Spectroscopic Titrations of Pt/Pd(L1‐L3)Cl With BSA

In this study, we investigated the reversible binding interactions between BSA and Pt/Pd(L1-L3)Cl complexes, employing UV‐visible absorption spectroscopy. Changes observed in the absorption spectra are an indication of some conformational alterations in the structure of BSA induced by the binding of a small molecule (metal complex), whereas wavelength shifts reveal the changes in the energetics of the bound adduct ([Pt/Pd(L1-L3)Cl…BSA]) and its effect on the absorbing chromophores in hydrophobic or hydrophilic environments. A gradual increase (hyperchromism in the range 47%–54%, Table 8) accompanied by blue shifts was observed in the absorption spectra of all the metal complexes upon the addition of increasing concentrations of the aforementioned complexes to a fixed concentration of BSA, see Figure 10 and S61–S62. The observed hyperchromism in the band of BSA upon its interaction with Pt/Pd(L1-L3)Cl suggests minor unfolding and unwinding of the protein and more exposure of its tryptophan residues for noncovalent interactions with the Pt/Pd(II) complexes, forming adducts with stronger molar absorptivities than BSA [46]. The values of the apparent association binding constants (K _app) are within the range 10⁴–10⁶ M⁻¹ (see Table 8), which is generally regarded as moderate and ideal for the reversible binding and releasing of small drug molecules by the transporter protein [47]. The binding constants decrease in the following order: PdL1Cl > PdL2Cl > PdL3Cl and PtL1Cl > PtL2Cl > PtL3Cl. Complexes such as Pt/PdL1Cl, which are monocationic and bear relatively small and compact 8‐quinoline moieties, exhibited the highest K _app values, and they formed more stable and stronger absorbing adducts than those bearing the bulkier phenanthridine ligand. A combination of strong electrostatic (higher formal charge) and noncovalent interactions contributed to this stability. The smaller size of 8‐quinoline and the flexibility of the structure of L1 enable better dynamic penetration of the Pt/PdL1Cl complexes into BSA. This enabled the metal complexes to snugly fit into the preferred receptor domains of the protein, thereby forming stronger noncovalent bonds. Thus, the observed trends in the values of K _app are influenced both by electrostatic charges as well as by the steric effects of the bis‐(azaaryl)amine ligands. Competitive displacement of the BSA site markers, ibuprofen (Site II) and warfarin (Site I) by the lead cytotoxic complexes Pt/PdL1Cl were conducted (Figures S70–S74). A comparison of the binding constants (Tables S16–S17) with and without site markers reveals competitive dislodging of the site markers from their preferred receptor sites on BSA. The Scatchard plots of the data gave the number of docking sites per ligand, n = 1. These results suggested that the lead complexes, as well as their other congeners, preferentially bind to Site II (Subdomain IIIA) of BSA in a 1:1 ratio.

TABLE 8.

Calculated binding association constants (K _app) of Pt/Pd(L1-L3)Cl for the preferred BSA receptor and the accompanying % hyperchromism observed in the UV‐visible absorption titrations.

Complex	K app, 104 M−1	Hypercromism (%)
PtL1Cl	9.58	54
PtL2Cl	5.98	50
PtL3Cl	4.45	47
PdL1Cl	8.40	53
PdL2Cl	7.13	51
PdL3Cl	5.99	49

FIGURE 10.

UV‐visible absorption spectra for the titration of (a) PtL1Cl, (b) PtL3Cl, (c) PdL1Cl and (d) PdL3Cl with BSA in PBS buffer at pH = 7.2. The inset is the double reciprocal plot of 1/(A ₀–A) vs. 1/[Complex].

(a)

(b)

(c)

(d)

3.7. Spectroscopic Titrations of Pt/Pd(L1‐L3)Cl With DNA and Pd/PtL1Cl With Hoechst‐DNA

DNA serves as the primary repository of genetic information; it is responsible for the development and function of all cellular systems [48]. Therefore, DNA is the primary target in the development of anticancer therapeutics. Planar and aromatic metal complexes can interact with DNA via different binding modes, such as minor and major groove binding, and also through intercalation with DNA base pairs [49]. The interactions of complexes with DNA induce deformities in DNA structures, leading to antiproliferation signalling and eventual death of the targeted malignant cells. In this study, the binding of Pt/Pd(L1-L3)Cl to CT‐DNA was evaluated by electronic absorption spectroscopy. Incremental additions of CT‐DNA to a fixed concentration of each metal complex produced spectral changes exemplified in Figure 11 and S63–S64. Specifically, hyperchromic effects (26%–40%, Table 9) are observed in the absorption band (maximum wavelength (λ _max = 268 nm) due to the π-π∗ transitions and indicate partial unwinding and elongation of the helix upon complex binding. This is consistent with more intimate interactions between the guest metal complexes and the more exposed nucleobases of DNA. Concurrent bathochromic shifts of 2‐3 nm suggest perturbations in the electronic environment of the DNA bases, attributable to localised electrostatic attractions, partial π-π‐stacking and hydrogen‐bond interactions between the DNA’s functional groups and the metal complexes. In addition, the appearance of well‐defined isosbestic points (e.g., at 312 and 358 nm for PtL1Cl) indicates a systematic (noncovalent) binding equilibrium between the metal complex and DNA. However, the electronic spectral profile of other metal complexes had no clear isosbestic points, which is indicative of nonspecific binding with DNA. The intrinsic binding constants of complexes lie in the range of 2.93–8.40 × 10⁴ M⁻¹ (see Table 9), and the trends in their magnitudes correlate with the binding scores (relative stability) predicted by molecular docking. These values are significantly lower than those of aromatic planar DNA intercalators (K _b  = 10⁶ M⁻¹) [50]. The mode of binding was confirmed by titrating DNA‐Hoechst solution with the most cytotoxic promising complexes, Pd/PtL1Cl, as illustrated in the competitive fluorescence titrations in Figure S68. These two complexes, as well as their analogues, can competitively displace (K _B  ≈ 10³) Hoechst, a known DNA groove binder, in a 1:1 ratio (Table S15 and Figure S69). Thus, they interact with DNA mainly via groove binding, as already discussed in the molecular docked structures and the binding strength of complexes towards CT‐DNA decreases in the order: PtL3Cl < PtL2Cl < PtL1Cl and PdL3Cl < PdL2Cl < PdL1Cl. The monocationic complexes (Pt/PdL1Cl) show the highest affinity for the negatively charged phosphate backbone of DNA, while Pt/PdL3Cl had the smallest DNA binding constants. This trend is contrary to expectation if the complexes were interacting only by insertion between the nucleobases, for which the planar rigid structure of phenanthridine moieties in L3 would result in a larger insertion and, hence, stronger binding. However, in the minor groove binding mode, the bulk of L3 impedes and limits accessibility of its complexes to the groups within the groove. Moreover, Pt(II) analogues consistently display higher binding constants than their Pd(II) analogues, underscoring the significantly higher polarisability and diffusivity of the molecular orbitals of Pt and how they, in turn, influence the noncovalent interactions of the metal ion in interactions with biomolecules such as DNA.

FIGURE 11.

UV‐visible absorption spectra of (a) PdL1Cl, (b) PdL3Cl, (c) PtL1Cl and (d) PtL3Cl in PBS buffer at pH = 7.2 upon addition of increasing CT‐DNA concentration (0–18 μM). Inset is the linear plot of [CT‐DNA] versus [CT‐DNA]/(ε _b  − ε _f ).

(a)

(b)

(c)

(d)

TABLE 9.

Binding affinities of the Pt/Pd(L1-L3)Cl for DNA and the accompanying % hyperchromism observed in the UV‐visible absorption titrations.

Complex	Binding constant (K b ), 104 M−1	Hyperchromism (%)
PtL1Cl	8.40	36
PtL2Cl	5.73	33
PtL3Cl	3.15	26
PdL1Cl	6.51	40
PdL2Cl	4.24	35
PdL3Cl	2.93	34

To evaluate the binding modes of the metal complexes with DNA, competitive binding titrations were performed using ethidium bromide (EtBr) as a fluorescence probe. EtBr, a cationic dye based on the phenanthridine scaffold, has been widely employed as a standard/reference intercalator for evaluating DNA‐binding affinities and modes of interaction [51]. Its utility arises from pronounced fluorescence enhancement upon intercalation into DNA. In an aqueous solution, EtBr exhibits weak fluorescence; however, upon intercalation between DNA base pairs, its fluorescence intensity increases significantly. The addition of a competing DNA intercalating complex results in a quenched fluorescence intensity that is proportional to the extent of displacement of the bound EtBr, thus providing a convenient spectroscopic assay for monitoring complex–DNA interactions. In this study, we investigated the binding mode of Pt/Pd(L1-L3)Cl complexes by a competitive‐displacement assay of EtBr. A fixed concentration of [EtBr‐CT‐DNA] adduct was titrated with increasing concentrations of each metal complex. In all cases, progressive fluorescence quenching (25%–36%, Table 10) was observed, which is consistent with displacement of the bound EtBr (Figure 12 and S65–S66). Stern–Volmer plot analysis yielded quenching constants (K _SV) in the range (5.54–7.98) × 10³ M⁻¹, apparent bimolecular quenching rate constants (k _q ) of (1.11–1.59) × 10¹² M⁻¹ s⁻¹ and a Scatchard DNA receptor‐to‐complex ratio, n, close to 1 (Table 10 and Figure S67). This is indicative of a predominantly static quenching mechanism involving a competitive intercalative displacement of EtBr [52]. The assay confirms that, as expected from their almost square planar geometry in which the tridentate ligands are also planar, these Pt/Pd(II) complexes are moderate DNA‐intercalators. However, results from absorption titrations and competitive displacement titrations of Hoechst‐DNA by Pd/PtL1Cl (Table S15) indicate that the complexes are moderate to strong 1:1 DNA groove binders, forming ground‐state DNA‐complex adducts. Moreover, stable DNA‐complex groove binding poses were predicted from molecular docking. Taken together, the results suggest that these metal complexes are bimodal DNA binders, functioning both as groove binders and partial intercalators. The binding strength (K _SV) and quenching rate constants (k _q ) follow the trend: PtL3Cl > PtL2Cl > PtL1Cl and PdL3Cl > PdL2Cl > PdL1Cl. Notably, the phenanthridine‐based metal complexes (PtL3Cl and PdL3Cl) exhibited higher binding affinities and quenching efficiencies compared to their quinoline analogues (PtL1Cl and PdL1Cl). Their enhanced binding may be attributed to the increased π-surface of the phenanthridine moiety, which allows stronger noncovalent interactions with the DNA bases, thereby facilitating more efficient and rapid EtBr displacement.

TABLE 10.

Binding and quenching rate constants for EtBr displacement by Pt/Pd(L1-L3)Cl from DNA and the observed % quenching of emission during the fluorescence titrations.

Complexes	K sv, 103 M−1	K q , 1012 M−1 s−1	n	Emission quenching (%)
PtL1Cl	6.34	1.27	0.93	25
PtL2Cl	7.82	1.56	0.96	28
PtL3Cl	7.98	1.59	0.67	31
PdL1Cl	5.54	1.11	0.67	30
PdL2Cl	5.74	1.15	1.21	32
PdL3Cl	6.07	1.21	1.03	36

FIGURE 12.

Fluorescence emission spectra depicting the quenching upon addition of (a) PdL1Cl, (b) PdL3Cl, (c) PtL1Cl and (d) PtL3Cl to CT‐DNA‐EB. The inset is the Stern–Volmer plot of (I _o /I) versus [Q], Q = Pt/Pd(II) complex.

(a)

(b)

(c)

(d)

3.8. In Vitro Cytotoxic Effects of Ligands L1–L3 and Their Respective Complexes Pt/Pd(L1–L3)Cl in Human Cancer Cell Lines

In vitro evaluation of the effect of candidate drugs on the viability of cancer cells is generally the first cytotoxicity test that is conducted to identify lead compounds that can be taken for further evaluation in vitro and subsequently in an animal model. The short‐term toxic effects of the ligands (L1–L3) and their respective Pt/Pd(L1–L3)Cl complexes at 10 μM were therefore evaluated against a panel of seven human cancer cells and a nontumorigenic human dermal fibroblast (FG‐0) cell line using the in vitro MTT assay. The tested cancer cell lines included the human breast cancer cell lines (MCF‐7, T47D and MDA‐MB‐231), the pancreatic cancer cell lines (PANC‐1 and CFPAC‐1) and the cervical cancer cell lines (CaSki and HeLa). The % cell viability (inhibition‐to‐growth) data after 48 h of incubation with the cancer and the benign (FG‐0) cell lines at 10 μM of the metal complexes are presented as bar graphs in Figures 13(a), 13(b) (for cancer cells) and 13(c) (for the FG‐0 cell line). The cell viability (%CV) and the accompanying statistical data are summarised in Tables S18‐S19. Their cell inhibition is also ranked in an attribute grid in Table 11, where x represents (< 50%), while √ represents (> 50%) cell inhibition. The majority of the test compounds could not inhibit the viability of most of the selected cancer cells by more than 20% after 48 h of treatment. This was also true for the nonmalignant human dermal fibroblast (FG‐0) cells, where neither the complexes nor their ligands could inhibit their growth by more than 20%. Surprisingly, Pd/PtL3Cl, analogues bearing phenanthridine moieties (which were meant to offer more extended surface moieties in the bis‐(azaary)amine N^∧N^∧N nonleaving ligand, L3) are all inactive against the tested cell lines, unlike the high activity against breast cancer reported for dinuclear palladacycles bearing the C, N amphiphilic chelates [53]. Their inactivity correlates with their relatively small rate constants for their ligand exchange reactions when compared to the analogous complexes of L1. The poor cytotoxicity of the phenanthridine‐bearing complexes contrasts with the wide range of anticancer activity of transplatin, which bears a phenanthridine as one of the amine ligands of cisplatin configuration [54, 55]. Encouragingly, at the micromolar level, metal complexes, Pt/PdL1Cl could reduce cell viability by more than 50% in the T47D breast cancer cell line and were better than the positive control drug, CDDP, which had an inhibitory effect of 14%. However, Pt/PdL1Cl were inactive against MCF‐7 or MDA‐MB‐231. The latter is a triple‐negative breast cancer cell line, known to be resistant to CDDP therapy [56]. PtL1Cl reduced the T47D cell viability to approximately 52%, while PdL1Cl produced a slightly smaller effect, to about 48%. The free ligand, L1, reduced T47D cell viability by less than 25%, showing that chelation to Pd/Pt(II) is a required condition for cytotoxicity against this cell line by these complexes. Curiously, L1 inhibited the growth of MDA‐MB‐231 by approximately 50%, and this performance was only comparable to that of CDDP. However, its metal complexes are almost inactive against this cell line. Of the three compounds of L2, only PtL2Cl reduced T47D cell viability by about 32%. Of the two pancreatic cell lines, only PANC‐1 cells showed marked susceptibility to PtL1Cl. PtL1Cl significantly reduced PANC‐1 cell viability by as much as 56%, while the viability of CFPAC‐1 cells was reduced by less than 20%. None of the tested compounds significantly reduced the viability of the two cervical cancer cell lines by more than 15% when compared to the vehicle control (Figure S74). Across most cell lines, the Pt/Pd(L1-L3)Cl metal complexes reduce cell viability slightly better than the free ligands. This suggests chelation and formation of metal complexes as a requisite condition for better activity, especially in susceptible cell lines such as PANC‐1 and T47D in the case of PtL1Cl. The observed cytotoxicity profiles of complexes in some cases correlate with their DNA/BSA binding affinities and their substitutional reactivity with biologically relevant molecules. While Pt/Pd(L1)Cl showed best cytotoxicity against PANC‐1 and T47D, they were inactive against the benign FG‐0 cell line, portraying good selectivity indices.

TABLE 11.

Test compounds (Pt/Pd(L1–L3)Cl) exhibiting more than 50% antiproliferation of seven selected cancer cell lines and a nonmalignant fibroblast (FGO) (c) cell line by the MTT assay.

Compound	Breast			Pancreatic		Cervical		Benign cells
	MCF‐7	T‐47D	MDA‐MB‐231	PANC‐1	CFPAC‐1	CaSki	HeLa	FG‐0
L1	x∗	x	√ ∗	x	x	x	x	x
L2	x	x	x	x	x	x	x	x
L3	x	x	x	x	x	x	x	x
PtL1Cl	x	√	x	√	x	x	x	x
PtL2Cl	x	√	x	x	x	x	x	x
PtL3Cl	x	x	x	x	x	x	x	x
PdL1Cl	x	√	x	x	x	x	x	x
PdL2Cl	x	x	x	x	x	x	x	x
PdL3Cl	x	x	x	x	x	x	x	x
CDDP (+ve control)	x	x	√	√	x	√	x	x

^∗Attribute legend: more than 50% (√) or less than 50% (x) inhibition (antiproliferation) ranking of compounds in cancer and benign FG‐0 cells.

FIGURE 13.

Single‐dose treatments of the breast (a), pancreatic (b) cancer cells and a nonmalignant fibroblast (FGO) (c) cell line, with the ligands (L1–L3) and Pt/Pd(L1–L3)Cl complexes. Cells were treated with vehicle (0.1% DMSO) or 10 μM of the test compounds for 48 h, followed by an MTT cell viability assay. Graphs represent the mean cell viability (±SD) of three independent experiments performed in quadruplicate. Data were analysed using the parametric unpaired t‐test by GraphPad Prism 8.0 software, where ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗0.001 and ^∗∗∗∗0.0001.

(a)

(b)

(C)

4. Conclusions

In this study, Pt/Pd(II) complexes bearing a relatively flexible (L1) and more rigid (L2-L3) bis‐(azaaryl)amine ligands were synthesised and characterised, and their rates of chloride substitution by biological nucleophiles (Tu and Guan) were measured. Pt/PdL1Cl displayed the highest rate of chloride substitution. Metal complexes of L3, despite the increase in π‐conjugation, have the lowest ligand exchange rates. The complexes’ binding affinities towards BSA and DNA decreased in the same order as the rates of ligand exchange in the two sets of complexes, PtL1Cl > PtL2Cl > PtL3Cl and PdL1Cl > PdL2Cl > PdL3Cl. Competitive binding studies supported by molecular docking simulations indicated that the complexes preferably bind DNA both as groove binders and partial intercalators, with the former mode seemingly prominent. Most of the metal complexes and their ligands showed poor in vitro cytotoxicity against a noncancerous human dermal fibroblast (FG‐0) cell line, as well as across a panel of three breast, two pancreatic, and two cervical cancer cell lines. However, PtL1Cl complex exerted significant antiproliferative activity, particularly against the breast cancer cell line, T‐47D and pancreatic cell line, PANC‐1, with potency surpassing that of the clinical standard cisplatin. PdL1Cl showed a similar activity, albeit only against T‐47D. Furthermore, PtL1Cl exhibits the highest binding affinity towards enzymes/proteins associated with the inhibition of prostate (XPO1), cervical (5VBN) and breast cancer (4DRH). Its binding affinity score for SARS‐CoV‐2’s Mpro: 8YA5 is, however, moderate to strong. The selective targeting and inhibition of these proteins by PtL1Cl may enhance its anticancer selectivity and activity, thus potentially circumventing cisplatin‐intrinsic resistance in T‐47D breast cancer, where the clinical drug showed poor activity.

Author Contributions

Phakamani C. Dlamini: laboratory and computational investigations, data analysis and visualisation, data curation, drafting and review. Thato T. Medupe: conduction of in vitro cytotoxic experiments, data analysis, visualisation and procedure writing. Karabo Serala: conduction of in vitro cytotoxic experiments, data analysis, visualisation and procedure writing. Lucy W. Macharia: conduction of in vitro cytotoxic experiments, data analysis, visualisation and procedure writing. Sharon Prince: partial funding (in vitro‐cytotoxic studies), data formal analysis and manuscript review. Gregory S. Smith: partial funding (in vitro‐cytotoxic studies), data formal analysis and manuscript review. Irvin N. Booysen: research supervision, resourcing, data validation, writing and review. Allen Mambanda: conceptualisation, funding acquisition, resourcing and administration, research supervision, data validation, writing and review.

Funding

This study was supported by the South African Medical Research Council (SAMRC).

Disclosure

The views expressed are those of the authors and should not be attributed to the SAMRC or the affiliated universities.

Conflicts of Interest

The author(s) declare no conflicts of interest regarding the publication of this article.

Supporting Information

Supporting Information (SI) available: Synthesis of precursors and tridentate NNN ligands, DNA/BSA‐metal complex titration procedures, 1H and 13C NMR spectra of precursors of ligands and the Pt/Pd(II) complexes, mass and IR spectra of ligands and Pt/Pd(II) complexes, elemental composition, spectral and tabled data on the DFT‐docked metal‐protein structures, BSA/DNA‐EtBr‐metal and DNA‐Hoechst‐metal absorption/emission titrations, substitution kinetics, cytotoxicity and metal stability in aqueous solution.

Supporting information

Supporting Information Additional supporting information can be found online in the Supporting Information section.

Dlamini, Phakamani C. , Medupe, Thato T. , Macharia, Lucy W. , Serala, Karabo , Prince, Sharon , Smith, Gregory S. , Booysen, Irvin N. , Mambanda, Allen , Substitution Kinetics, DNA/BSA Interactions, Cytotoxicity Evaluation and Computational Analysis of [bis‐(azaaryl)amine)Pt(II)/Pd(II)Cl] Complexes, Azaaryl = Quinoline or Phenanthridine, Bioinorganic Chemistry and Applications, 2026, 6206843, 25 pages, 2026. 10.1155/bca/6206843

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding authors. The data are not publicly available due to privacy or ethical restrictions.