Aminoquinoline-based Re(I) tricarbonyl complexes: Insights into their antiproliferative activity and mechanisms of action.

{"full_text": " European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nAminoquinoline-based Re(I) tricarbonyl complexes: Insights into their\nantiproliferative activity and mechanisms of action\nPaige S. Zinman a, Athi Welsh a, Reinner O. Omondi a, Saif Khan b, Sharon Prince b,\nEbbe Nordlander c, Gregory S. Smith a, *\na\n Department of Chemistry, University of Cape Town, Rondebosch, 7701, South Africa\nb\n Department of Human Biology, University of Cape Town, Faculty of Health Science, Observatory, 7925, South Africa\nc\n Chemical Physics, Department of Chemistry, Lund University, Box 124, SE-221 00, Lund, Sweden\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: In an effort to develop new potent anticancer agents, two Schiff base rhenium(I) tricarbonyl complexes, con\u00ad\nRhenium(I) taining the ubiquitous aminoquinoline scaffold, were synthesized. Both aminoquinoline ligands and Re(I)\nAminoquinoline complexes showed adequate stability over a 48-h incubation period. Furthermore, the cytotoxic activity of the\nCytotoxicity\n precursor ligands and rhenium(I) complexes were evaluated against the hormone-dependent MCF-7 and\nDNA/BSA binding\nMolecular docking\n hormone-independent triple negative MDA-MB-231 breast cancer cell lines. Inclusion of the [Re(CO)3Cl]+ entity\n significantly enhanced the cytotoxicity of the aminoquinoline Schiff base ligands against the tested cancer cell\n lines. Remarkably, the incorporation of the Schiff-base iminoquinolyl entity notably enhanced the cytotoxic\n activity of the Re(I) complexes, in comparison with the iminopyridyl entity. Notably, the quinolyl-substituted\n complex showed up to three-fold higher activity than cisplatin against breast cancer cell lines, underpinning\n the significance of the quinoline pharmacophore in rational drug design. In addition, the most active Re(I)\n complex showed better selectivity towards the breast cancer cells over non-tumorigenic FG-0 cells. Western\n blotting revealed that the complexes increased levels of \u03b3H2AX, a key DNA damage response protein. Moreover,\n apoptosis was confirmed in both cell lines due to the detection of cleaved PARP. The complexes show favourable\n binding affinities towards both calf thymus DNA (CT-DNA), and bovine serum albumin (BSA), and the order of\n their interactions align with their cytotoxic effects. The in silico molecular simulations of the complexes were also\n performed with CT-DNA and BSA targets.\n\n\n\n\n1. Introduction tuning of their electronic and steric properties [8\u201310]. Schiff base mol\u00ad\n ecules have shown pertinence in a range of applications and industries,\n Despite numerous clinical successes and innovative research break\u00ad from analytical chemistry [11] to catalysis [9] and biomedical appli\u00ad\nthroughs, the incidence of cancer continues to increase worldwide. cations [11\u201314], with their notable biological activity attributed to the\nBreast cancer is the most frequently diagnosed form of cancer, globally, characteristic imine \u2013C\u2013 \u2013N functionality [15]. Numerous reports high\u00ad\naccounting for 1 in 8 cancer diagnoses across both sexes [1,2]. light the versatility of Schiff base metallodrugs, showcasing substantial\nFurthermore, it is the second leading cause of death due to cancer in anticancer properties [16\u201320], and demonstrating effectiveness as\nwomen, particularly burdening those living in developing countries antibacterial [21\u201323], antifungal [24\u201326], and antiparasitic agents [15,\n[1\u20133]. There is a dire need for new and improved cancer chemothera\u00ad 17,27]. These attributes have earned them recognition as \u2018privileged\npeutic agents, ones that possess unique mechanisms of action, have scaffolds\u2019 [10]. The following metal Schiff base complexes, exemplifying\nsuperior selectivity, are less toxic, and can withstand chemoresistance potent biological activity, highlight their dominance in medicinal\n[4]. chemistry research. Garza-Oritz et al. demonstrated Ru(II)-bis\n Schiff-base ligands [5\u20137] are renowned for being privileged scaffolds (arylimino)pyridine complexes\u2019 potent anticancer activity against\nin the drug design of robust drug candidates owing to their facile syn\u00ad seven cancer cell lines [28]. Sua\u0301rez-Oritz et al. reported selective cyto\u00ad\nthesis, favourable denticity for chelation, and the relatively simple toxicity of chiral fac-tricarbonyl(iminopyridine) rhenium(I) complexes\n\n\n * Corresponding author.\n E-mail address: gregory.smith@uct.ac.za (G.S. Smith).\n\nhttps://doi.org/10.1016/j.ejmech.2023.116094\nReceived 13 October 2023; Received in revised form 13 December 2023; Accepted 21 December 2023\nAvailable online 27 December 2023\n0223-5234/\u00a9 2023 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license\n(http://creativecommons.org/licenses/by-nc-nd/4.0/).\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\nagainst glioblastoma cells [20]. Bulatov et al. found an Isatin-Schiff agents due to their compact size compared to larger polypyridyl com\u00ad\nbase-copper(II) complex inhibiting p53-positive MCF-7 cell prolifera\u00ad plexes, kinetic stability, easy modular synthesis, and rich spectroscopic\ntion, activating p53, and inducing apoptosis [29]. A series of Ru properties [46,54]. In fact, various Re(I) tricarbonyl complexes, some of\n(II)-arene Schiff base complexes exhibited low micromolar anticancer which encompass a quinoline moiety (Fig. 1), have already emerged to\nactivity via a p53-independent pathway against colorectal and gastric display notable cytotoxic activity against a range of cancerous cell lines\ncancer cell lines [30]. Novicidin attached to a zinc-Schiff base carrier [24,46,54\u201361]. Despite the promising potency demonstrated by\neffectively penetrated prostate PC3 cancer cells, suggesting its potential rhenium(I) complexes, there has been limited research into elucidating\nas a novel anticancer drug [31]. their mechanism of action and further investigations are highly war\u00ad\n The aminoquinoline structure in itself is also a versatile and ubiq\u00ad ranted [46,62].\nuitously bioactive scaffold. This structural motif is present in a variety of The study reported herein, endeavoured to develop 4-aminoquino\u00ad\nbiologically active molecules and displays activity against a myriad of line Schiff base ligands and their corresponding N,N-chelated rhenium\nhuman diseases [32]. This is best exemplified by the clinical malaria (I) tricarbonyl complexes. The synthesized compounds were evaluated\ndrug, chloroquine [33]. Other than malaria, chloroquine has also been in vitro as potential anticancer drug leads against the hormone-\nutilized to treat several autoimmune diseases including systemic lupus, dependent MCF-7 and hormone-independent MDA-MB-231 breast can\u00ad\nerythematosus and rheumatoid arthritis [34,35]. More recently, cer cell lines. In addition, the selective cytotoxicity of the complexes was\nquinoline-based drugs, such as chloroquine, have been studied for can\u00ad assessed against the non-malignant dermal fibroblast cell line, FG-0. To\ncer treatment [36,37]. As a pivotal pharmacophoric scaffold, the gain further insight into the complexes\u2019 possible mechanism of action,\nincorporation of the quinoline structure constitutes a well-founded this study investigated their effect on the expression of key molecular\nrationale for the design and synthesis of more selective anticancer markers associated with DNA damage and apoptosis. Furthermore, the\nagents [32]. Interestingly, recent research reveals that this nitrogen interactions of the rhenium complexes with biomolecules such as calf\nheterocycle induces anticancer effects by intercalating between DNA thymus DNA (CT-DNA) and bovine serum albumin (BSA) was evaluated\nbase pairs or disrupting proteins that regulate DNA structure and cell through spectroscopic and in silico methods.\nproliferation [32]. For example, a recurrent scaffold in intercalating\nagents is amsacrine, a quinoline-containing molecule, and one of the 2. Results and discussion\nfirst DNA intercalators investigated and used to treat blood malignancy\n[32,38]. 2.1. Synthesis and characterization\n With the pivotal discovery of the platinum-based anticancer drug,\ncisplatin [39], the hybridization of bioactive organic moieties with The 4-aminoquinoline Schiff base ligands 2 and 3 were synthesized\nmetals is documented to display significant pharmacological advantages via two steps (Scheme 1), initially involving a nucleophilic aromatic\nand is a prevalent strategy in rational drug design of novel chemother\u00ad substitution (SNAr) reaction with 4,7-dichloroquinoline and 1,3-diami\u00ad\napeutics [40,41]. Transition metal complexes have been used to improve nopropane to synthesize N1-(7-chloroquinolin-4-yl)propane-1,3-\nmany traditional organic-based medical treatments by enhancing the diamine (1) [63].\nbioavailability of drugs through increased lipophilicity, effectively The second step, following an amended literature method, involved\ninhibiting enzymes, functioning as biocatalysts through reversible redox reacting compound 1 with an aromatic aldehyde via a Schiff base\nbehaviour and decreasing the required dosage for effective treatment condensation reaction [64]. Compound 2, encompassing the iminopyr\u00ad\n[41\u201345]. Even with the widespread success of platinum-based anti\u00ad idyl substituent, has been synthesized previously by Maurya et al. [64],\ncancer drugs, many serious clinical limitations have developed which while the quinolyl-substituted compound 3 has not been previously re\u00ad\nsignify a poor patient prognosis [46\u201349], leading to the ever-current ported, at the time of writing. The selected aromatic aldehydes, 2-pyri\u00ad\nexploration for alternative metal-based anticancer agents [50]. Ruthe\u00ad dinecarboxaldehyde and 2-quinolinecarboxaldehyde, were chosen to\nnium, titanium and gold anticancer agents have been at the forefront of investigate the effect of an extended \u03c0-system. Furthermore, the pres\u00ad\nresearch efforts, revealing extremely promising results with some com\u00ad ence of a second quinolyl entity was intended to ascertain the resulting\nplexes progressing to phase I and II clinical trials [42,51,52]. These effects on the overall biological activity of the compounds. Overall, the\nadvances propelled the investigations into metals further down the Pe\u00ad facile synthesis of the ligands afforded light brown (2) or light yellow (3)\nriodic Table such as the third-row transition metals of iridium, osmium powders in yields of 51% and 70%, respectively.\nand rhenium which have been found to also exhibit encouraging anti\u00ad The synthesis of the new neutral N,N-chelated rhenium(I) tri\u00ad\ncancer properties [43,46,53]. Relatively unexplored, rhenium-based carbonyl aminoquinoline Schiff base complexes 4 and 5 was achieved\ncomplexes with the stable rhenium(I) tricarbonyl motif exhibit prom\u00ad via a simple ligand substitution reaction (Scheme 1) [65]. The com\u00ad\nising features for designing theranostic (therapeutic and diagnostic) plexes, 4 and 5, were purified via recrystallisation and isolated as orange\n\n\n\n\n Fig. 1. Selected chemical structures of Re(I) tricarbonyl complexes with reported anticancer activity [24,55,56].\n\n 2\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nScheme 1. Synthetic procedure of the 4-aminoquinoline precursor 1, the Schiff base ligands (2\u20133) and the corresponding N,N-chelated Re(I) complexes 4 and 5.\nReagents and conditions: (i) 1,3-diaminopropane, 120\u2013130 \u25e6 C, 6\u20138 h; (ii) Aromatic aldehyde, MeOH, molecular sieves, 60 \u25e6 C, 48 h; (iii) [Re(CO)5Cl] (1.1 eq.), MeOH,\nreflux in the dark, 18 h.\n\n\npowders in yields of 68% and 59%, respectively. Both complexes are air- multiplet proton signals correlate to one carbon peak. The splitting of\nand moisture-stable as well as soluble in a range of polar and this aliphatic signal is due to the chirality induced by the introduction of\nalcohol-based solvents. Light exposure was minimized during the syn\u00ad a metal-based stereogenic centre, causing the hydrogen atoms to\nthetic procedure and the complexes were stored in the dark to prevent become diastereotopic, i.e., magnetically inequivalent.\npotential light sensitivity and the release of carbon monoxide molecules, The FT-IR spectra of ligands 2 and 3 depict further evidence for the\nas has been reported for analogous Re(I) tricarbonyl complexes [66\u201368]. formation of the desired Schiff base ligands owing to the characteristic\nThe rhenium(I) tricarbonyl complexes 4 and 5 were used for in vitro FT-IR absorption bands of the imine functionality v(C\u2013 \u2013N) observed at\nbiological evaluations as racemic mixtures. 1581 cm\u2212 1 (for 2) and at 1572 cm\u2212 1 (for 3) (Figs. S8\u2013S9). The observed\n The 4-aminoquinoline Schiff base ligands (2 and 3) and the corre\u00ad absorption bands and associated wavenumbers in the FT-IR spectra of\nsponding Re(I) complexes (4 and 5) were all characterized in solution ligands 2 and 3 agree well with those reported in the literature for\nusing 1H, 13C{1H}, COSY, HSQC and HMBC NMR spectroscopy, together similarly structured compounds [69\u201371]. In the FT-IR spectrum of\nwith FT-IR spectroscopy and high-resolution electrospray ionisation complex 4 (Fig. S8), the v(C\u2013 \u2013N)imine and v(C\u2013 \u2013N)pyridyl stretching vi\u00ad\nmass spectrometry (ESI-MS). The spectra obtained from these charac\u00ad brations are observed as overlapping absorption bands at 1573 cm\u2212 1,\nterization and analyses techniques are displayed in the Supplementary agreeing with that reported in literature for other Re(I) tricarbonyl\nInformation. 1H NMR spectroscopy (Figs. S1 and S2) revealed singlets at diimine complexes [72,73]. The v(C\u2013 \u2013N)imine stretching frequencies of\n8.42 ppm (2) and 8.59 ppm (3) corresponding to the imine hydrogen complex 4 is observed at a lower wavenumber than that of the unco\u00ad\n(Hn), which attests to the formation of the respective C\u2013 \u2013N bonds of ordinated ligand 2, being at 1581 cm\u2212 1, while the v(C\u2013 \u2013N)pyridyl ab\u00ad\nthese Schiff base compounds. Further, the 1H and 13C{1H} NMR analyses sorption band of complex 4 appears at a higher wavenumber than that of\nof the Re(I) complexes (4 and 5) confirmed successful bidentate coor\u00ad ligand 2 (1564 cm\u2212 1). This phenomenon is, in fact, commonly reported\ndination to the imine and aromatic nitrogen atoms of ligands 2 and 3 for diimine Re(I) complexes [72,73], and is indicative of successful\n(Figs. S1\u2013S6). Fig. S1 depicts the stacked 1H NMR spectra of the pyridyl- bidentate coordination of the imine and pyridyl nitrogen atoms to the\nsubstituted Schiff base ligand, 2, and the respective Re(I) complex, 4. In metal. Additional evidence of complexation is seen by the appearance of\nspectrum (b) of Re(I) complex 4, a downfield shift of most of the aro\u00ad three strong v(C\u2013 \u2013O) absorption bands in the range 2014\u20131862 cm\u2212 1,\nmatic and aliphatic signals is observed, with respect to that in spectrum which are absent in the spectrum of the corresponding ligand, 2. These\n(a) of the corresponding ligand, 2, indicative of metal complexation. The absorption frequencies, attributed to the three carbonyl ligands bonded\nmost significant downfield shifts are seen for the proton signal Hs from to rhenium, are consistent with literature reports for similarly structured\n8.65 ppm to 9.10 ppm and proton signal Hn from 8.42 ppm to 9.33 ppm. Re(I) complexes [24,72,74]. In addition, the appearance of the three\nThis observation substantiates a bidentate coordination to the imine and strong carbonyl absorption bands also suggests a facial arrangement of\nthe pyridyl nitrogen atoms of the ligand 2, as the hydrogens most these ligands around the Re(I) metal centre.\naffected after complexation (Hs and Hn) are, indeed, those closest to the High-resolution ESI-MS data was obtained for the ligands (2 and 3)\nexpected sites of coordination. Further evidence of complexation is the and the corresponding Re(I) complexes (4 and 5). A base peak corre\u00ad\nsplitting of the aliphatic proton signals corresponding to the propylene sponding to the protonated molecular ion [M+H]+ is observed in the\nhydrogen atom (Hl) of the propyl chain. The single multiplet, integrating spectrum for ligand 2 (Fig. S10). The positive-ion mode mass spectrum\nfor two hydrogen atoms, in the range 2.29\u20132.11 ppm in the spectrum (a) for ligand 3 shows a base peak indicative of the cationic fragment\nof ligand 2, appears as two multiplets (2.64\u20132.48 ppm) each integrating [M+Na]+ (Fig. S11). Lastly, the most abundant peak in the ESI mass\nfor one proton in the spectrum (b) of complex 4. This phenomenon has spectra of Re(I) complexes 4 and 5 correlate with the protonated mo\u00ad\nbeen confirmed by HSQC NMR spectroscopy (Fig. S7), i.e., the two lecular ion peak, i.e., [M+H]+ (Figs. S12\u2013S13).\n\n\n 3\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n The UV\u2013Vis absorption spectra of Re(I) complexes 4 and 5, in DMSO, This supports and agrees with the appearance of the three strong\nare shown in Fig. 2. The intense absorption bands in the UV region at ca. carbonyl absorption bands in the corresponding FT-IR spectrum\n250\u2013330 nm are attributed to the spin-allowed \u03c0L \u2192\u03c0\u2217L intraligand (Fig. S8). The adoption of the fac-isomeric structure is commonly\ntransitions of the diimine ligands. The broader lower energy peaks at ca. observed in other Re(I) tricarbonyl diimine complexes reported in\n330\u2013460 nm can most likely be assigned to the spin-allowed metal-to- literature [24,73,74,84]. The diagram also depicts coordination of a\nligand charge-transfer (1MLCT) absorptions, representative of the tran\u00ad chlorine atom and the two nitrogen atoms (pyridine-N and imine-N) of\nsitions from the d-orbitals of the Re(I) metal to the \u03c0* orbitals of the ligand 2 to the Re(I) metal ion. Moreover, noticeable disorder is\nrespective diimine Schiff base ligands [75\u201379]. However, further observed at Cl1, C21 and O3, with a site occupancy factor (S.O.F.) of\ndetailed spectrophotometric analysis and molecular modelling studies <0.1. The apparent disorder is owing to the co-crystallisation of more\nwould need to substantiate and confirm this postulation. The obtained than one isomer, resulting in a crystallographic structure that is an\nelectronic absorption spectra (Fig. 2) are comparable to those of simi\u00ad overlay and average of all the asymmetric units present. Table S2\nlarly structured diimine Re(I) tricarbonyl complexes reported in the summarises the crystal data and refinement parameters for both ligand 2\nliterature [80\u201382]. Interestingly, a slight red shift of approximately 35 and complex 4. Ligand 2 crystallizes in a P-1 space group with a triclinic\nnm is observed in the electronic absorbance spectrum of the system, while complex 4 crystallizes in a P21/c space group with a\nquinolyl-substituted complex 5 relative to the pyridyl-substituted com\u00ad monoclinic system. A total of two and four molecules per unit cell are\nplex 4, suggesting that the extended conjugated \u03c0-system of the quinolyl observed for ligand 2 and complex 4, respectively.\nversus the pyridyl entity influences the wavelength at which the 1MLCT Selected bond distances and angles for both compounds are listed in\nband appears. Table S3. Complex 4 adopts a distorted octahedral geometry around the\n The extended \u03c0-system of the quinolyl substituent 5 slightly lowers rhenium metal ion. The deviation of the rhenium coordination sphere\nthe antibonding \u03c0* orbital of the diimine ligand, which, in turn, de\u00ad from the ideal octahedron is due to the compression brought about by\ncreases the energy gap between the HOMO that is based on the metal d- the five-membered chelate ring (Re1-N1-C13-C14-N4), leading to a\norbital and the LUMO, which is a ligand \u03c0* orbital. As a result, the en\u00ad small bite angle of 74.88(8)\u25e6 . The observed bond angles and resulting\nergy required to effect electron promotion from the metal d-orbital to geometry around the Re(I) metal centre are comparable with other Re(I)\nthe ligand \u03c0* orbital is lowered, resulting in a bathochromic shift of the tricarbonyl diimine complexes reported in literature [50,72,84]. The\nabsorption band [83]. Re-Npyridyl bond length, Re1-N4, 2.170(2) A\u030a is almost equivalent to the\n Re-Nimine, Re1-N1, bond length of 2.175(2) A\u030a, which is corroborated by\n the FT-IR data collected (Fig. S8), where the v(C\u2013 \u2013N)pyridyl and v\n2.2. Molecular structure of compounds 2 and 4\n (C\u2013N)imine are seen to be overlapping and resonating at very similar\n \u2013\n The molecular structures of the pyridyl-substituted Schiff base ligand frequencies. In fact, these bonding parameters are consistent with that\n2 and its corresponding Re(I) tricarbonyl complex 4 were elucidated by reported in literature for structurally similar N,N-Re(I) tricarbonyl\nsingle crystal X-ray diffraction (Fig. 3). Single crystals of ligand 2 and complexes [50,72]. Additionally, the C=Nimine bond of complex 4,\ncomplex 4 were grown by layering diethyl ether over a concentrated C13\u2013N1, 1.274(3) A\u030a is slightly longer than that of the respective ligand\nsolution of either compound in dichloromethane. Complex 4 was kept in 2, C13\u2013N13, 1.264(2) A\u030a, which is expected due to the Re (d-\u03c0) \u2013 imine\nthe dark, to avoid light exposure. (\u03c0*) backdonation. This observation is also corroborated by the FT-IR\n From the ORTEP diagram of complex 4 (Fig. 3), it is evident that the\n spectrum (Fig. S8) where there is a small shift in the v(C\u2013 \u2013N)imine\ncarbonyl ligands adopt a facial arrangement around the metal centre.\n\n\n\n\nFig. 2. Stacked UV\u2013Vis absorption spectra of Re(I) complexes 4 (green) and 5 (orange) measured in DMSO at concentrations of ca. 1 \u00d7 10\u2212 4 M and ca. 0.5 \u00d7 10\u2212 4 M,\nrespectively, under ambient conditions.\n\n 4\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 3. ORTEP diagrams of (a) 4-aminoquinoline pyridyl-substituted Schiff base ligand 2 and (b) Re(I) tricarbonyl N,N-chelated complex 4, with hydrogen atoms\nomitted from both structures for clarity. Thermal ellipsoids are drawn at 30% probability level for both structures.\n\n\nabsorption band from a higher wavenumber, for ligand 2, to a lower affinity of the Pt(II) centre for the nucleophilic sulfur donor atom of\nwavenumber, for the complex 4. For complex 4, the new 5-membered DMSO [91,92]. For the biological studies to be relevant, the stability of\nring formed by chelation is essentially planar with a torsion angle of the aminoquinoline Schiff base ligands 2 and 3 and Re(I) complexes 4\n\u2212 1.1(4)\u25e6 , which has been commonly observed for closely related com\u00ad and 5 was monitored using UV\u2013Vis spectroscopy. Complexes 4 and 5\nplexes in literature [85]. In the case of the uncoordinated ligand, 2, the were dissolved in PBS supplemented with 1% DMSO and the absorption\npyridyl and imine functionalities are not co-planar, but rotated 172.69\u25e6 spectra were recorded over 48 h, while maintaining the physiological\nfrom each other, as has been noted for similarly structured Schiff base temperature of 37 \u25e6 C. The testing conditions mimicked the environment\ncompounds [86]. Furthermore, the Re-Cl, Re-Ccarbonyl and C-Ocarbonyl of the in vitro cytotoxicity screenings (vide infra).\nbond distances of complex 4 are within the expected range as reported in The obtained temporal UV\u2013Vis spectra of the complexes 4 and 5 are\nliterature for this class of Re(I) complex [72,73,84]. shown in Fig. 4. The complexes showed negligible changes in the elec\u00ad\n tronic bands, pointing to their stability. Additionally, no considerable\n2.3. Solution stability studies changes in the absorption spectra of ligands 2 and 3 were observed in\n DMSO over 48 h (Fig. S14) signifying their stability and structural\n Metal-based complexes have, in the past, been known to show integrity. This is pertinent, as the compounds are incubated with the\ninstability under physiological conditions, leading to undesirable side cancer cell lines for precisely 48 h during the in vitro biological assays.\neffects [87\u201389]. The stability of a compound is a crucial factor in the\nclassification and discernment of viable drug leads for biological ap\u00ad 2.4. In vitro cytotoxicity evaluation\nplications. Stability studies commonly involve the use of DMSO, which is\narguably the most frequently used organic solvent for in vitro biological The cytotoxic activity of the aminoquinoline Schiff base ligands 2\nscreenings of compounds [90]. There have been reports that cisplatin and 3 and the corresponding N,N-mononuclear complexes 4 and 5 were\nand related platinum-based complexes (carboplatin and oxaliplatin) assessed against two breast cancer cell lines (MCF-7 and MDA-MB-231)\nundergo ligand displacement when dissolved in DMSO, due to the and a non-tumorigenic cell line (FG-0). The compounds were pre-\n\n\n\n\n Fig. 4. The temporal UV\u2013Vis spectra of (a) iminopyridyl Re(I) complex 4 and (b) iminoquinolyl Re(I) complex 5 in PBS (with 1% DMSO) at 37 \u25e6 C for 48 h.\n\n 5\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\nscreened in vitro against the hormone-dependent MCF-7 and hormone- The MDA-MB-231 breast cancer cell line is a triple-negative cancer\nindependent MDA-MB-231 cell lines at 10 \u03bcM and 20 \u03bcM using the cell subtype, which lacks the oestrogen, progesterone, and human\nMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) epidermal growth factor 2 hormone receptors [94,95]. This cell line\nassay [93]. In all assays performed, DMSO was used as the biological represents a highly invasive form of breast cancer that is notoriously\nvehicle; therefore, it was included as a negative control when diluted in difficult to treat [95]. The data obtained from the cytotoxic evaluation of\ngrowth medium (0.1% DMSO in growth medium). Additionally, a clin\u00ad the ligands (2, 3) and corresponding N,N-complexes (4, 5) against the\nically used chemotherapeutic metallodrug, cisplatin, was included as the MDA-MB-231 cell line, at 10 \u03bcM and 20 \u03bcM, is shown in Fig. 6.\npositive control and for comparison purposes. The preliminary cytotoxicity data for the compounds, against the\n The percentage cell viability of MCF-7 cells following treatment with MDA-MB-231 cell line, somewhat resembles the trends observed for the\nthe aminoquinoline Schiff base ligands (2, 3) and the corresponding Re MCF-7 cell line (Fig. 5). The ligands 2 and 3 and Re(I) complexes 4 and\n(I) tricarbonyl complexes (4, 5) is herein represented in Fig. 5. Overall, 5, again, show activity in a dose-dependent manner and, in fact, inhibit\nthe ligands and complexes all show moderate to good activity against cell viability to a greater extent than cisplatin, 12% (10 \u03bcM) and 36%\nthis cell line, at both tested concentrations. At 10 \u03bcM, the compounds (20 \u03bcM). Comparatively, the Re(I) complexes, 4 and 5, exhibit superior\n2\u20135 reduce cell viability by between 38% and 80% while at 20 \u03bcM the activity relative to the uncoordinated ligands 2 and 3, with cell viability\ncell viability is further reduced by 42%\u201394%. Therefore, these com\u00ad inhibitions of 42% and 60%, at 10 \u03bcM, and 70% and 98%, at 20 \u03bcM,\npounds generally show activity in a dose-dependent manner, i.e. there is respectively. This phenomenon reiterates the beneficial role of transition\nan enhancement of cytotoxicity upon doubling the compound concen\u00ad metal complexes in giving rise to effective medical treatments for critical\ntration from 10 \u03bcM to 20 \u03bcM. Moreover, the tested ligands and com\u00ad human diseases. Furthermore, the results highlight the promising anti\u00ad\nplexes show superior cytotoxic activity in comparison with cisplatin at cancer properties of the rhenium(I) tricarbonyl core.\nboth tested concentrations in the MCF-7 breast cancer cell line. Owing to the promising activity in the pre-screen assays the N,N-\n Notably, the introduction of the rhenium(I) ion appears to enhance chelated Re(I) complexes (4,5) were selected for further in vitro multi\u00ad\nthe biological activity of the ligands, 2 and 3, as seen by the comparably dose screenings against the two breast cancer cell lines (MCF-7 and\nlower percentage cell viability of the respective Re(I) tricarbonyl com\u00ad MDA-MB-231) and a non-tumorigenic cell line (FG-0) to further ascer\u00ad\nplexes, 4 and 5. For example, complex 5 inhibits the cell viability by tain their potential as therapeutic agents. The concentrations of com\u00ad\n34% (at 10 \u03bcM) and 42% (at 20 \u03bcM), more than the corresponding ligand plexes 4 and 5 required to reduce cell viability to 50% of the control\n3. In fact, the quinolyl-substituted complex 5 is the most active com\u00ad (IC50) are summarized in Table 1. In these experiments, the relevant\npound boasting a 20% and 6% cell survival at 10 \u03bcM and 20 \u03bcM, cancer cells were treated with the test compounds (4, 5) over a range of\nrespectively. This remarkable cytotoxic activity is likely attributed to the concentrations from 0 \u03bcM to 35 \u03bcM for 48 h and the cell viability was\npresence of the additional quinolyl entity. Comparatively, its analogue, measured using the MTT assay. For comparison, cisplatin was included\nthe pyridyl-substituted complex 4, shows a lower potency, reducing cell as a positive control and a 0.1% DMSO in cell culture media solution was\nsurvival to 52% (at 10 \u03bcM) and 36% (at 20 \u03bcM). A similar observation is included as a negative control. Furthermore, the experiments were\nmade between the pyridyl-substituted ligand 2 and the quinolyl- conducted in the dark, with limited white light exposure, for the entire\nsubstituted ligand 3, where there is a clear improvement in cytotoxic duration of the assay.\nactivity upon incorporation of the quinolyl moiety ligand 3 over the As observed from the pre-screen data, the Re(I) complexes (4, 5)\npyridyl moiety ligand 2. At 10 \u03bcM, the quinolyl-substituted ligand 3 display a dose-dependent cytotoxic activity against the MCF-7 breast\nreduced cell survival by 46%, while the pyridyl-substituted ligand 2 cancer cell line (Fig. S15). Both complexes 4 and 5 display enhanced\nreduced cell survival by only 38%. This observation highlights the cytotoxicity relative to clinically used cisplatin in this cancer cell line.\nubiquitous pharmacological activity of the quinoline structure, sug\u00ad Impressively, complex 5 displays up to 3.6 times greater activity relative\ngesting that the presence of the iminoquinolyl motif, in addition to the 4- to cisplatin (IC50 = 24.92 \u03bcM). From the results, it is apparent that the\naminoquinoline core, positively influences the cytotoxic activity of complexes 4 and 5 show statistically comparable cytotoxicity with an\ntested compounds, 3 and 5. IC50 value of 6.82 \u00b1 1.03 \u03bcM for the quinolyl-substituted Re(I) complex,\n\n\n\n\nFig. 5. In vitro cytotoxic effect of aminoquinoline Schiff base ligands (2, 3) and corresponding N,N-bidentate Re(I) complexes (4, 5) towards the MCF-7 breast cancer\ncell line at 10 \u03bcM and 20 \u03bcM. Results displayed as the percentage cell survival as measured by MTT assays, cisplatin and the vehicle (0.1% DMSO) are included as\ncontrols. * = p \u2264 0.05, thus statistically significant and ** = p \u2264 0.01, thus very statistically significant.\n\n 6\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 6. In vitro cytotoxic effect of aminoquinoline Schiff base ligands (2, 3) and corresponding N,N-bidentate Re(I) complexes (4, 5) towards the MDA-MB-231 breast\ncancer cell line at 10 \u03bcM and 20 \u03bcM. Results displayed as the percentage cell survival as measured by MTT assays, cisplatin and the vehicle (0.1% DMSO) are included\nas controls. * = p \u2264 0.05, thus statistically significant and ** = p \u2264 0.01, thus very statistically significant.\n\n\n selectivity indices summarized in Table 1 show that complex 5 which is\nTable 1\n overall more active against the breast cell lines is also the most selective,\nIC50 values of the Re(I) complexes (4, 5) against two breast cancer cell lines\n with an S.I. that is greater than 1. This indicates that complex 5 shows\n(MCF-7 and MDA-MB-231) and a non-tumorigenic cell line (FG-0). a\n selective cytotoxicity towards the breast cancer cells over non-\n Compound IC50 (\u03bcM) S.I. tumorigenic cells.\n MCF-7 MDA-MB-231 FG-0\n\n 4 8.55 \u00b1 1.08 17.70 \u00b1 0.75 7.09 \u00b1 0.17 0.83\n 5 6.82 \u00b1 1.03 10.73 \u00b1 0.53 10.86 \u00b1 0.23 1.59 2.5. Western blot analysis\n Cisplatin 24.92 \u00b1 1.03 24.45 \u00b1 1.03 43.67 \u00b1 1.05* 1.75\n\nS.I.: Selectivity Index = IC50 FG-0/IC50 MCF-7.\n In a bid to gain insight on the potential mechanism of action of\n a\n The results are presented as mean values \u00b1 standard deviations (SD) out of complexes 4 and 5 in the breast cancer cell lines (MCF-7 and MDA-MB-\ntwo independent experiments and the cell viability was assessed after 48 h of 231), Western blotting was used to investigate the ability of the com\u00ad\nincubation. plexes to elicit DNA damage and apoptosis. This was achieved by\n *\n IC50 value extrapolated by Graphpad Prism V 8.0.2 from experimental data. assessing the protein levels of key markers of DNA damage and\n apoptosis, \u03b3H2AX and cleaved PARP, respectively. \u03b3H2AX is a central\n5, and 8.55 \u00b1 1.08 \u03bcM for the pyridyl-substituted analogue 4. biomarker of double-strand DNA breaks, while the cleavage of PARP\n A slightly different trend can be delineated for the cytotoxicity of the during apoptosis is a key hallmark of this type of cell death [96,97]. As\ncomplexes in the MDA-MB-231 cell line to that observed in the MCF-7 such, DNA damage and apoptosis are indicated by increased protein\ncell line (Fig. S16), with the iminoquinolyl complex 5 (IC50 = 10.73 \u03bcM) levels of \u03b3H2AX and cleaved PARP.\nbeing more cytotoxic than the iminopyridyl complex 4 (IC50 = 17.70 The data obtained from Western blotting experiments are summa\u00ad\n\u03bcM). However, the complexes (4, 5) prove to be less potent against the rized in Fig. 7, below. Generally, addition of complexes 4 or 5 does\naggressive triple-negative MDA-MB-231 cell line than against the MCF- indeed result in double-strand DNA breaks and significant DNA damage\n7 cell line, by a factor of approximately 2 for both complexes. Never\u00ad in both the MCF-7 and MDA-MB-231 breast cancer cell lines. This is\ntheless, this observation is noteworthy in that it shows that the com\u00ad evidenced by the notable increase in \u03b3H2AX protein levels upon treat\u00ad\npounds must have a well-defined mechanism of action and are not ment of the cells with complexes 4 and 5. Furthermore, the DNA damage\nsimply broad-spectrum toxins. Intriguingly, complex 4 still shows elicited by complexes 4 and 5 is noted to be concentration-dependent in\nslightly greater potency than the positive control, cisplatin (IC50 = both breast cancer cell lines, as the \u03b3H2AX protein levels of cells treated\n24.45 \u03bcM), while the iminoquinolyl complex 5 shows up to 2 times with the IC50 concentrations of 4 and 5 is generally higher than those\ngreater cytotoxicity than the clinical metallodrug. Once again, this treated with a lower concentration of the complexes (1/2 IC50). Inter\u00ad\nobservation emphasizes that the addition of the second quinolyl entity to estingly, complex 5 induced greater DNA damage in the MCF-7 cell line\nthe aminoquinoline Schiff base ligand in complex 5 evidently enhances relative to the clinically used, DNA-damaging metallodrug cisplatin.\nthe cytotoxic activity of the rhenium(I) complex against the breast The treatment of the cells with complexes 4 and 5 resulted in a sig\u00ad\ncancer cell line. Furthermore, this suggests that the additional quinoline nificant increase in cleaved PARP protein levels in both investigated\nsubstituent contributes to the observed cytotoxic activity and is, itself, breast cancer cell lines (MCF-7 and MDA-MB-231). This observation\neliciting a cytotoxic effect on the cancer cells. However, without a indicates that 4 and 5 do indeed induce apoptosis as a pathway of cell\ndetailed structure-activity based assessment, it is difficult to say whether death in these breast cancer cell lines. Furthermore, the densitometric\nthis is an additive or synergistic effect. readings for cleaved PARP in both cell lines show that complex 4 may be\n Both tested complexes 4 and 5 show concentration-dependent a more potent inducer of apoptosis relative to clinically used cisplatin, as\ncytotoxicity towards the non-tumorigenic FG-0 cell line (Fig. S17). the levels of cleaved PARP in cells treated with 4 are significantly higher\nPyridyl-substituted complex 4 shows enhanced cytotoxicity relative to than those treated with cisplatin (Fig. 7). Interestingly, 1/2 IC50 treat\u00ad\nquinolyl-substituted complex 5 and, overall, cisplatin is noted to show ment of complex 5 induced greater PARP cleavage compared to IC50\nmilder cytotoxicity relative to both tested Re(I) complexes. The treatment which may indicate that complex 5 induces additional forms\n of cell death in MCF-7 cells at this concentration. Taken altogether, these\n\n 7\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 7. Complexes 4 and 5 initiate the DNA damage and apoptosis pathways. Western blot analysis with antibodies to indicate protein levels of \u03b3H2AX, PARP and\ncleaved PARP in (a) MCF-7 and (b) MDA-MB-231 cells treated with \u00bd IC50 and IC50 of complex 4 and 5 for 48 h. \u03b2-actin was used as a loading control. Densitometry\nreadings were obtained from three independent experiments using ImageJ and protein expression levels are represented as a ratio of protein of interest/\u03b2-actin\nnormalized to the control sample (where possible).\n\n\nmechanistic studies indicate that complexes 4 and 5 induce apoptosis complex] curve. The resulting binding constants for the interaction be\u00ad\nthrough a process involving DNA damage in the investigated breast tween CT-DNA and the rhenium(I) complexes (4, 5) are given in Table 2.\ncancer cells. The high magnitude Kb values of complexes 4 and 5 (104-105), depict\n intercalation with the DNA.\n The order of the calculated binding constants (Kb) shows that com\u00ad\n2.6. DNA binding studies\n pound 5 exhibits greater binding affinity than compound 4, demon\u00ad\n strating that the additional quinolyl moiety in 5 contributes to stronger\n2.6.1. UV\u2013vis spectroscopy\n interactions with DNA in comparison to the pyridine moiety. This result\n Investigations into possible binding interactions between the Re(I)\n follows the trend from the in vitro cytotoxicity experiments where the\ncomplexes (4, 5) and CT-DNA, as a potential mechanistic route, were\n quinolyl-substituted complex 5 exhibited greater cytotoxic activity to\u00ad\nconducted by absorption titration experiments. The electronic absorp\u00ad\n wards the cancer cell lines than the pyridyl-substituted complex 4. This\ntion spectra of CT-DNA kept at a constant concentration of 50 \u03bcM in PBS\n suggests that the stronger DNA interactions attributed to the quinolyl\nbuffer solution (pH 7.2), with increasing amounts of metal complexes\n substituent may play an important role in the cytotoxic action of this\n(0\u2013350 \u03bcM) are given in Fig. 8. The hyperchromic effect is observed at\n rhenium complex. Notably, reports have described the structural and\n260 nm by consecutive addition of the metal complexes to CT-DNA. This\n electronic properties of quinoline, such as its planarity and highly con\u00ad\nobservation suggests that the complexes specifically bind to the purine\n jugated system, to contribute to its impressive ability to insert itself\nand pyridine bases of the DNA, leading to a subsequent moderate change\n between the DNA base pairs. This action disrupts the double helix of\nin the conformation of DNA [98\u2013101]. The binding constant Kb was\n DNA and causes strand breaks, ultimately leading to cytotoxicity [32,\nevaluated from the ratio of the slope in the plots of 1/A-A0 vs 1/[metal\n\n 8\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 8. Electronic absorption spectra of CT-DNA [50 \u03bcM] in the presence of increasing concentration of (a) complex 4 [0\u2013350 \u03bcM] and (b) complex 5 [0\u2013350 \u03bcM] in\nPBS buffer (pH = 7.2, 25 \u25e6 C). The arrows indicate absorbance changes with the increasing concentration of metal-complexes.\n\n\n respectively, and are due to short-range interactions between the\nTable 2\n rhenium complexes and the CT-DNA-EtBr adduct [118]. The dynamic\nThe binding and thermodynamic parameters for the interactions of complexes 4\n quenching process in various diffusion-controlled biomolecular re\u00ad\nand 5 with CT-DNA.\n actions has been reported to have a maximum value of 2 \u00d7 1010 M\u2212 1 s\u2212 1\n Compound Kb (105 L.mol\u2212 1) -\u0394G\u2215 \u2212 1\n 25\u25e6 C (kJ\u22c5mol )\n =\n [120]. Thus, considering that the kq values for complexes 4 and 5 exceed\n 4 0.88 \u00b1 0.33 28.20 this limit, it may be deduced that a static mechanism is in operation\n 5 2.67 \u00b1 0.81 30.95 [121,122]. Generally, complexes 4 and 5 exhibit KSV and kq values\n which are similar to related rhenium(I)-based complexes reported in the\n102]. The positive relationship between the strength of DNA binding literature, implying that complexes 4 and 5 experience a relatively\nand the degree of anticancer activity of tested compounds has also comparable intercalative interaction with CT-DNA [120,123,124]. In\nregularly been observed and reported in the literature [61,103]. The Kb addition, the KSV and kq results indicate that the\nvalues of 4 and 5 compare well with those of similar complexes reported iminoquinolyl-coordinated Re(I) complex 5 binds more favourably to\nin the literature [103\u2013111]. CT-DNA in comparison to the iminopyridyl-coordinated Re(I) complex\n Gibb\u2019s free energy of activation (\u0394G) values were derived from the 4.\nvan\u2019t Hoff Eq. (2) [112], as indicated in the experimental procedures The apparent association constant (Kapp), given in Table 3, was\nand the results are presented in Table 2. The negative values for 4 and 5 calculated for each complex (4, 5) using Eq. (4) [125]. For the\nsignify that the interactions between the complexes and CT-DNA occur iminopyridyl-coordinated rhenium(I) complex (4) Kapp is calculated to\nspontaneously [113]. be 5.51 \u00d7 106 M\u2212 1 whereas, for the iminoquinolyl-coordinated rhenium\n (I) complex (5), it is 14.06 \u00d7 106 M\u2212 1. The magnitude of the Kapp (106\n2.6.2. Ethidium bromide competitive studies M\u2212 1) are relatively lower compared to the binding constants of classical\n To further explore and validate the mode of interactions between the intercalators (107 M\u2212 1), indicating moderate intercalative interaction\nrhenium complexes (4, 5) and CT-DNA, a fluorescent-quenching assay abilities [104].\nbased on the ethidium bromide (EtBr) adduct was conducted [114\u2013117]. The Scatchard Eq. (5) was applied to determine the binding constant\nFigs. 9 and 10 depict the fluorescence spectra of the CT-DNA-EtBr (KF) and the number of binding sites (n) on CT-DNA for each Re(I)\ncomposite with varying concentrations (0\u2013150 \u03bcM) of complexes 4 complex [126]. The corresponding results are presented in Table 3.\nand 5, respectively. The emission intensities at 600 nm show significant Complexes 4 and 5 display moderate quenching efficiencies, as shown\nhypochromic shift. by the KF values (magnitude 102 M\u2212 1) which are in tandem with me\u00ad\n These results suggest that the rhenium complexes (4, 5) quench the dium interactions. The number of binding sites (n) for both complexes 4\nCT-DNA-EtBr fluorescence by intercalating with the DNA base pairs and 5 are approximately equal to one, indicating that there is a single\nwhile simultaneously displacing the EtBr molecule (due to the decrease binding site on the DNA molecule for the Re(I) complexes. Generally,\nof the binding sites available to EtBr). The Stern-Volmer quenching there is a strong correlation observed between the binding strength\nconstant (Ksv) and bimolecular quenching rate constant (kq) were values calculated from the UV\u2013Vis titration experiments and those from\ndetermined from the Stern-Volmer Eq (3) [118], and the data are pre\u00ad the fluorescence measurements.\nsented in Table 3.\n The KSV values for complexes 4 and 5 are 2.88 \u00d7 105 M\u2212 1 and 7.91 \u00d7\n 2.7. Protein interaction studies\n10 M\u2212 1, respectively, and the magnitude of 105 M\u2212 1 indicates that the\n 5\n\ncomplexes can displace the EtBr bound to CT-DNA via an intercalative\n Serum albumin is an essential plasma protein responsible for the\nmode of interaction. Notably, the KSV values show a striking 102 -fold\n transportation of small molecules, including vitamins, hormones and\nreduction compared to the classical intercalator EtBr (107 M\u2212 1), signi\u00ad\n drugs within the bloodstream. Moreover, it plays a crucial role in\nfying a relatively moderate intercalative interaction [119]. The kq values\n maintaining fluid balance within the circulatory system [127]. In this\nare 1.30 \u00d7 1013 M\u2212 1s\u2212 1 and 3.04 \u00d7 1013 M\u2212 1s\u2212 1 for complexes 4 and 5,\n study bovine serum albumin (BSA) was used as a relevant model as it is\n\n 9\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 9. (a) The effects of the addition of 4 on the emission intensity of EtBr bound to CT-DNA in the presence of varying amounts of 4 in PBS buffer (at pH = 7.2,\n25 \u25e6 C). [EtBr] = 20.0 \u03bcM, [CT-DNA] = 20.0 \u03bcM, and [4] = 0\u2013150 \u03bcM. The arrow indicates the changes upon the addition of the metal complex. (b) Stern-Volmer plot\nof Io/I vs. [Q]. (c) Scatchard plot of log[(Io\u2013I)/I] vs. log[Q].\n\n\nstructurally homologous to human serum albumin. BSA possesses an and 5 are similar to those reported for a series of Re(I) complexes [110,\nintrinsic fluorescence due to unique properties of the tyrosine, trypto\u00ad 133,134].\nphan and phenylalanine residues contained within the protein [128]. The KF values of complexes 4 and 5, which are of the order of 102\nFluorescence quenching has been widely used to evaluate the interac\u00ad M , are lower than the value of the association constant (KF = 1015\n \u2212 1\n\ntion of complexes with various proteins. Reduction in the fluorescence M\u2212 1) of protein-ligand adducts with the highest binding capability\nintensity of the fluorophore, specifically the tryptophan residue of BSA, [135]. In addition, the KF magnitude strongly suggests that the in\u00ad\nis brought about by strong interactions between the complexes of in\u00ad teractions of 4 and 5 with BSA amino acids are predominantly due to\nterest and the protein [129,130]. The fluorescence spectra of BSA, hydrophobic interactions, specifically within subdomain IIA of the\nmaintained at a constant concentration of 1.5 \u03bcM in the presence of protein. Consequently, complexes 4 and 5 can easily be released from\nincreasing amounts (0\u201330 \u03bcM) of the rhenium(I) complexes (4, 5) are the protein upon reaching the target cells. Hence both complexes prove\ndepicted in Figs. 11 and 12. The spectra show a significant decrease in to be well-suited for facilitating drug-cell interactions. Furthermore,\nthe fluorescence intensity of BSA as the concentration of the Re(I) both 4 and 5 display n values that are close to unity, indicating the\ncomplex increases, with no changes in the position of the emission presence of a single binding on BSA.\nwavelengths and shape of the peaks. As a result, complexes 4 and 5\ndemonstrate a remarkable ability to bind to BSA, inducing considerable\n 2.8. Molecular docking\nconformational changes in its secondary structure while effectively\nquenching its intrinsic fluorescence [131]. This observation reveals a\n We employed molecular docking to gain an insight into the drug-\ndefinite interaction occurring between both of the rhenium(I) complexes\n receptor interactions. Complexes 4 and 5 were simulated in the DNA\nand BSA.\n dodecamer with a sequence of d(CGCGAATTCGCG)2 (PDB ID: 1BNA)\n The changes in fluorescence intensities fit well with the Stern-Volmer\n binding pockets as shown in Fig. 13. The intermolecular interactions and\n(Ksv and kq) and Scatchard (KF and n) equations, and their corresponding\n binding affinities of the complexes within specific distances are given in\nvalues are shown in Table 4. The values of KSV of magnitude 105 M\u2212 1\n Table 5. The docked poses of 4 and 5 depict intercalative modes of\n(Table 4), signify that the process of interaction between the complexes\n binding, consistent with the experimental results. Moreover, the binding\nand the BSA protein is not fully controlled by diffusion, resulting in\n scores of \u2212 6.6 kcal/mol (4) and \u2212 6.9 kcal (5) show a positive correla\u00ad\nlarger kq values. The magnitude of the kq values of 4 and 5 (1013 M\u2212 1s\u2212 1)\n tion with the experimentally determined binding constant values in\nexceed the maximum scattering collision rate observed in most known\n Tables 2 and 3. Complex 4 forms carbon hydrogen bond and conven\u00ad\nquenchers (2.0 \u00d7 1010 M\u2212 1s\u2212 1), suggesting the existence of a static\n tional hydrogen bond interactions with nucleotides DC23 (3.39 \u00c5) and\nquenching mechanism [132]. The values of KSV and kq for complexes 4\n DG22 (2.33 \u00c5), respectively. As for complex 5, it interacts with DC3\n\n 10\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 10. (a) The effects of the addition of 5 on the emission intensity of EtBr bound to CT-DNA in the presence of varying amounts of 5 in PBS buffer (at pH = 7.2,\n25 \u25e6 C). [EtBr] = 20.0 \u03bcM, [CT-DNA] = 20.0 \u03bcM, and [5] = 0\u2013150 \u03bcM. The arrow indicates the changes upon the addition of the metal complex. (b) Stern-Volmer plot\nof Io/I vs. [Q]. (c) Scatchard plot of log[(Io\u2013I)/I] vs. log[Q].\n\n\n pi-pi-T-shaped interactions (with HIS145, 4.86 \u00c5). The docked energies\nTable 3 of both complexes are relatively the same (\u2212 8.0 \u00b1 0.1 kcal/mol), with\nFluorescence quenching parameters for the interactions of complexes 4 and 5\n highly favourable best-docked conformations.\nwith CT-DNA.\n Compound KSV kq Kapp KF n 3. Conclusions\n (105 M\u2212 1) (1013 M\u2212 1 (106 M\u2212 1) (102 M\u2212 1)\n s\u2212 1 )\n A pair of 4-aminoquinoline Schiff base ligands (2, 3) containing an\n 4 2.88 \u00b1 1.30 \u00b1 0.25 5.51 \u00b1 0.42 1.19 \u00b1 0.96\n iminoquinolyl or iminopyridyl entity were successfully synthesized and\n 0.38 0.10\n 5 7.91 \u00b1 3.04 \u00b1 0.31 14.06 \u00b1 2.12 \u00b1 1.01 coordinated to the Re(I) tricarbonyl core via N,N-chelation, producing\n 0.67 0.90 0.14 the corresponding metal complexes (4, 5) in good yields. The synthe\u00ad\n sized ligands (2, 3) and complexes (4, 5) were fully characterized using\n various spectroscopic and analytical techniques. Single crystals of the\n(2.62 \u00c5) and DG4 (2.03 \u00c5) via conventional hydrogen bond interactions. pyridyl-substituted ligand (2) and the corresponding Re(I) complex (4)\nIn addition, the stability of 5 is considerably contributed by an attractive were analysed by X-ray diffraction, confirming numerous structural\ncharge interaction with DG24 (5.38 \u00c5). characteristics that were corroborated by spectroscopic measurements.\n The interactions of 4 and 5 with the BSA protein are illustrated in Results from the cytotoxicity assays suggest that metal complexation,\nFig. S18. Binding energies and a list of intermolecular interactions with with the [Re(CO)3Cl]+ core (4, 5), significantly enhanced the activity of\nthe active amino acids are provided in Table S4. Notably, hydrophobic the corresponding, uncoordinated ligands (2, 3). Both Re(I) complexes\ninteractions were observed between complexes 4 and 5 and amino acids (4, 5) showed superior cytotoxic activity compared to cisplatin against\nresidues. The most stable pose of 4 depicts interactions with: ALA349, both the hormone-dependent (MCF-7) and hormone-independent\n4.92 \u00c5 and VAL215, 4.53 \u00c5 (alkyl interactions); LEU330, 4.89 \u00c5 and (MDA-MB-231) breast cancer cell lines, with the iminoquinolyl-\nARG208 5.49 \u00c5 (pi-alkyl interactions); GLU353, 2.91 \u00c5 and VAL481, coordinated complex 5 being up to 3.6 times more potent than the\n2.19 \u00c5 (conventional hydrogen bond interactions); PHE205, 5.44 \u00c5 (pi- clinical drug. The Re(I) complexes (4, 5) displayed moderate activity\npi T-shaped interactions) and LYS211, 4.81 \u00c5 (amide-pi stacked in\u00ad against the highly aggressive and invasive MDA-MB-231 cell line;\nteractions). Similarly, complex 5 establishes pi-alkyl interactions (with however, they were far more potent against the hormone-dependent\nLYS114, 5.14 \u00c5; ARG144, 4.35 \u00c5; LEU112, 5.09 \u00c5; ARG196,3.71 \u00c5; MCF-7 cell line. Overall, the iminoquinolyl-coordinated complex 5\nALA193, 4.46 \u00c5; LEU189, 4.10 \u00c5), conventional hydrogen bond in\u00ad was more potent, relative to the iminopyridyl-coordinated analogue 4,\nteractions (with ASP108, 2.70 \u00c5; ARG458, 2.96 \u00c5; ASP111, 2.94 \u00c5) and towards the tested breast cancer cell lines. Of significance, the more\n\n\n 11\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 11. (a) Fluorescence emission spectra of BSA protein in the absence and presence of complex 4. BSA [1.5 \u03bcM] in the presence of consecutive quantities of 4\n[0\u201330 \u03bcM]. The arrow shows the intensity changes upon increasing the concentration of 4. (b) Stern\u2013Volmer plot of Io/I vs. [Q]. (c) Scatchard plot of log[(Io\u2013I)/I] vs.\nlog[Q].\n\n\ncytotoxic complex 5 was also the more selective complex with an S.I. 4. Experimental\nvalue over 1, showing selectivity towards the MCF-7 breast cancer cell\nline over the FG-0 non-tumorigenic cell line. Due to rhenium-based 4.1. General methods and chemicals\ncomplexes being relatively underexplored in terms of their promising\nanticancer activity, further investigations are warranted to understand All reagents and solvents were commercially sourced (Sigma-\ntheir mechanism of actions. In that regard, western blot experiments Aldrich, Merck and Kimix) and used without further purification.\nrevealed that complexes 4 and 5 triggered the upregulation of principal Compounds 1 and 2 were synthesized following literature methods [63,\nmolecular markers for DNA damage and apoptosis. Absorption spectral 64]. All solvents used were reagent grade and dried over molecular\nstudies established that the interactions between the complexes and sieves, where required. All aqueous solutions (NaOH, NaCl, NaHCO3)\nDNA to be of an intercalative nature; these observations were further were prepared using deionized water. Synthetic procedures were carried\nsupported by competitive binding studies with ethidium bromide. In out under nitrogen atmosphere using standard Schlenk line techniques\naddition, protein interaction studies monitored by fluorescence emission unless otherwise stated. Reactions were monitored by thin-layer chro\u00ad\nspectroscopy showed favourable interactions between BSA and both Re matography (TLC) using aluminium-backed precoated silica gel 60 F254\n(I) complexes. The DNA and protein binding results were confirmed or neutral alumina oxide 60 F254 plates and viewed under ultraviolet\nthrough in silico molecular docking simulations where the complexes (UV) light at 254 nm.\nexhibited strong intermolecular interactions with double-stranded DNA\nand similarly with the amino acid residues of BSA. It is noteworthy that\nthe quinolyl-substituted complex 5 exhibits greater binding affinity to 4.2. Spectroscopic and analytical techniques\nDNA and the highest cytotoxic activity against the tested breast cancer\ncell lines. These observations suggest that a superior interaction with Nuclear magnetic resonance (NMR) spectra were obtained using a\nDNA may be associated with an enhanced activity against the cancer cell Bruker XR600 MHz spectrometer (1H at 599.95 MHz and 13C{1H} at\nlines. Further, the additional quinoline scaffold appears to contribute 151.0 MHz), a Bruker XR400 spectrometer (1H at 399.95 MHz and 13C\nsignificantly to the cytotoxic activity and biomolecule interactions dis\u00ad {1H} at 100.58 MHz) or a Varian Mercury 300 (1H at 300.08 MHz)\nplayed by the complex, thus emphasizing the invaluable pharmaco- spectrometer. Chemical shifts are recorded in ppm (\u03b4) and J-coupling\nproperties of the quinoline structure. values reported in Hz with tetramethylsilane (TMS) used as the internal\n standard. The signals were assigned as follows: s, singlet; d, doublet; dd,\n doublet of doublets; dt, doublet of triplets; t, triplet; m, multiplet; br.s,\n broad singlet. Fourier-transform infrared (FT-IR) spectroscopy was\n performed on a Perkin-Elmer Spectrum 100 FT-IR spectrometer using a\n\n 12\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n\n\nFig. 12. (a) Fluorescence emission spectra of BSA protein in the absence and presence of complex 5. BSA [1.5 \u03bcM] in the presence of consecutive quantities of 5\n[0\u201330 \u03bcM]. The arrow shows the intensity changes upon increasing the concentration of 5. (b) Stern\u2013Volmer plot of Io/I vs. [Q]. (c) Scatchard plot of log[(Io\u2013I)/I] vs.\nlog[Q].\n\n\nTable 4 Table 5\nFluorescence quenching parameters for the interaction between complexes 4 Interaction properties of complexes 4 and 5 analysed by Discovery Studio\nand 5 and BSA protein. Visualizer.\n Compound KSV kq KF n Compound Docking score Carbon H- Conventional H- Attractive\n (105 M\u2212 1) (1013 M\u2212 1 s\u2212 1) (102 M\u2212 1) (kcal/mol) bond (\u00c5) bond (\u00c5) charge\n (\u00c5)\n 4 3.37 \u00b1 0.19 1.47 \u00b1 0.25 1.93 \u00b1 0.12 0.99\n 5 9.79 \u00b1 0.44 4.19 \u00b1 0.32 2.58 \u00b1 0.11 1.13 4 \u2212 6.6 DC23 (3.39 DG22 (2.33) \u2013\n \u00c5)\n 5 \u2212 6.9 \u2013 DC3 (2.62) DG24 (5.38)\n DG4 (2.03)\n\n The more negative the free binding energy, the stronger the binding affinity.\n Numbers indicated within brackets represent the distance of interacting atoms in\n Angstrom (\u00c5).\n\n\n Electronic absorption (UV\u2013Vis) spectra were recorded using an\n Agilent Cary 8454 UV\u2013Vis spectrometer, recording in the range of 250\n nm and 800 nm. Fluorescence emission spectra were recorded using a\n Perkin Elmer LS 45 Fluorescence Spectrometer using 1 cm path length\n cuvettes at room temperature. Melting points were measured using a\n B\u00fcchi Melting Point Apparatus B-540 and are uncorrected. High reso\u00ad\n lution (HR) electrospray ionisation mass spectrometry (ESI-MS) was\n performed, using the positive ion-mode, on a Waters Synapt G2 mass\nFig. 13. The best docked conformer of (a) complex 4 and (b) complex 5 with spectrometer equipped with an ESI probe. The Agilent HPLC 1260\nDNA duplex depicting intercalative binding. equipped with an Agilent DAD 1260 UV\u2013Vis detector and an Agilent\n Pursuit 5C18 column (5 \u03bcM pore size, 150 mm \u00d7 4.6 mm) was utilized to\nKBr pellet and fitted with an Attenuated Total Reflectance (ATR) unit. evaluate the purity of the complexes prepared and used in this study.\nThe measurements of bond vibrations were recorded in reciprocal cen\u00ad The compounds were eluted using a mixture of solvent A (H2O) and\ntimetres (cm\u2212 1). solvent B (MeOH) at a flow rate of 1.0 mL min\u2212 1, with the detection\n\n\n 13\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\nwavelength set at 254 nm. The gradient elution conditions were as fol\u00ad methanol (20.0 mL), after which [Re(CO)5Cl] (0.101 g, 0.280 mmol)\nlows: 30% solvent A between 0 and 1 min, 30\u201390% solvent from 1 to 10 was added. The reaction was refluxed at 65 \u25e6 C overnight. The reaction\nmin, 90 to 10% solvent A from 10 to 20 min. flask was covered in foil for the duration of the reaction. After\n completion of the reaction, the mixture was cooled to room temperature\n and the solvent was reduced to dryness by rotary evaporation. The crude\n4.3. Synthesis\n compound was re-dissolved in minimal CH2Cl2 and then precipitated\n with diethyl ether to yield an orange solid (5). Yield: 0.111 g (59%). 1H\n4.3.1. 7-Chloro-N-(3-((quinolin-2-ylmethylene)amino)propyl)quinolin-4-\n NMR (600 MHz, CD2Cl2): \u03b4 (ppm) = 8.92 (1H, s, Hn), 8.84 (1H, d, J =\namine (3)\n 8.9 Hz, Hv), 8.46 (1H, d, J = 5.5 Hz, Hb), 8.36 (1H, d, J = 8.2 Hz, Hq),\n Precursor compound 1 (0.291 g, 1.23 mmol) and 2-quinoline car\u00ad\n 8.09\u20138.04 (1H, m, Hu), 8.01 (1H, d, J = 8.2 Hz, Hs), 7.86 (1H, m, Ht),\nboxaldehyde (0.221 g, 1.41 mmol) were dissolved in anhydrous meth\u00ad\n 7.83 (1H, d, J = 2.0 Hz, Hd), 7.71 (1H, d, J = 8.9 Hz, Hg), 7.44 (1H, d, J\nanol (20.0 mL). Dry 3 A\u030a molecular sieves were then added to the reaction\n = 8.2 Hz, Hp),6.70 (1H, dd, J = 8.9, 2.1 Hz, Hf), 6.46 (1H, d, J = 5.5 Hz,\nflask. This was allowed to stir at 30 \u25e6 C for 48 h. The reaction mixture was\n Ha), 6.08 (1H, s, Hj), 4.58 (1H, m, Hm), 4.39 (1H, m, Hm), 3.62 (2H, m,\nthen cooled to room temperature and the molecular sieves were\n Hk), 2.91\u20132.81 (1H, m, Hl), 2.52\u20132.42 (1H, m, Hl). 13C{1H} NMR (151\nremoved via filtration and washed with excess methanol. The solvent\n MHz, CD2Cl2): \u03b4 (ppm) = 197.93, 196.34, 186.99, 170.30, 156.36,\nwas removed under reduced pressure, the resulting residue was re-\n 151.56, 150.53, 148.16, 141.11, 135.18, 133.88, 130.56, 130.35,\ndissolved in dichloromethane (50.0 mL) and subsequently washed\n 130.06, 129.41, 128.01, 125.24, 123.28, 123.04, 117.46, 99.23, 65.91,\nwith water (3 \u00d7 20.0 mL). The organic layers were collected and dried\n 42.72, 27.85. IR (ATR): (\u03bdmax/cm\u2212 1) 3224 (sec. NH), 2011 (C\u2013 \u2013O), 1910\nover anhydrous MgSO4, filtered and the solvent was then removed in\n (C\u2013\u2013O), 1863 (C\u2013 \u2013O), 1574 (C\u2013 \u2013N)imine, quinolyl, 1524 (C\u2013\u2013N)quinoline.\nvacuo. Purification was performed via recrystallisation by dissolving the\n UV\u2013Vis (DMSO): \u03bbmax nm (10\u2212 4 \u03b5, mol\u2212 1 L cm\u2212 1) 333 (1.39), 420\ncrude in minimal CH2Cl2 followed by inducing precipitation by layering\n (0.330). Melting point: >170.2 \u25e6 C dec. without melt. MS (HR-ESI, m/z):\nthe solution with diethyl ether. Compound 3 was isolated as a light-\n 679.0280 (100%, [M \u2212 H]-), calculated 679.0392; 681.0483 (80%, [M +\nyellow solid. Yield: 0.323 g (70%). 1H NMR (300 MHz, CD2Cl2) \u03b4\n H]+), calculated 681.0552. HPLC purity: 99% (tr\u2019 = 2.05 min).\n(ppm) = 8.59 (1H, s, Hn), 8.48 (1H, d, J = 5.3 Hz, Hb), 8.28 (1H, d, J =\n8.6 Hz, Hg), 8.17 (1H, d, J = 8.5 Hz, Hp), 8.10 (1H, d, J = 8.4 Hz, Hq),\n 4.4. Single crystal X-ray crystallography\n7.97\u20137.85 (2H, m, Hu,v) 7.77 (2H, m, Hd,s), 7.62 (1H, t, J = 7.5 Hz, Ht)\n7.23 (1H, d, J = 8.9 Hz, Hf), 6.45 (1H, d, J = 5.3 Hz, Ha), 6.12 (1H, s, Hj),\n Suitable single crystals of ligand 2 and complex 4 were grown from\n3.95 (2H, t, J = 5.9 Hz, Hm), 3.53 (2H, q, J = 5.9 Hz, Hk), 2.29\u20132.16 (2H,\n the layering of diethyl ether into a concentrated dichloromethane so\u00ad\nm, Hl), 1.98 (1H, s, N-Hj\u22c5\u22c5\u22c5N\u2013 \u2013C). 13C{1H} NMR (101 MHz, CD2Cl2): \u03b4 lution, followed by slow evaporation of solvent at room temperature. A\n(ppm) = 163.91, 154.90, 152,50, 150,29, 149.65, 148.32, 136.93, Bruker D8 Venture diffractometer with graphite-monochromated Cu-K\u03b1\n134.77, 130.31, 130.02, 129.17, 129.00, 128.19, 128.00, 125.09, radiation (\u03bb = 1.54178 \u00c5) was utilized to obtain single-crystal X-ray\n122.05, 118.54, 117.75, 99.30, 60.62, 43.24, 29.93. IR (ATR): (\u03bdmax/ diffraction data of the prepared crystals. The data was collected at a\ncm\u2212 1) 3238 (sec. NH), 1576 (C\u2013 \u2013N)imine, quinolyl, 1558 (C\u2013\u2013N)quinoline. temperature of 100(2) K and controlled by an Oxford Cryostream\nUV\u2013Vis (DMSO): \u03bbmax nm (10\u2212 4 \u03b5, mol\u2212 1 L cm\u2212 1) 334 (0.601). Melting cooling system (Oxford Cryostat). The program SAINT was used to\npoint: 168.5\u2013169.4 \u25e6 C. MS (HR-ESI, m/z): 397.0992 (70%, [M + Na]+), conduct cell refinement and data reduction [136]. The data were scaled,\ncalculated 397.1196. and absorption correction carried out using SADABS [137]. The struc\u00ad\n tures were solved using SHELXS-97 [137] by means of direct methods,\n4.3.2. Re(I) aminoquinoline-pyridyl complex (4) refined via full-matrix least-squares methods based on F2 by operating\n A solution of compound 2 (0.0688 g, 0.212 mmol) and [Re(CO)5Cl] both SHELXL-2014 [137] and the graphics interface program, X-Seed\n(0.0813 g, 0.225 mmol) was prepared in anhydrous methanol (20.0 mL), [138,139]. The X-Seed and POV-Ray [138,140] programs were utilized\nand refluxed at 65 \u25e6 C for 18 h. The reaction flask was covered in foil for to produce graphical images of the molecular structures. All\nthe duration of the reaction. After completion of the reaction, the non-hydrogen atoms contained within the structures were refined\nmixture was cooled to room temperature and the solvent was reduced to anisotropically. Additionally, all hydrogen atoms, except the amino\ndryness by rotary evaporation. The crude compound was re-dissolved in hydrogen H2, were arranged in idealised positions and refined in riding\na minimal volume of CH2Cl2 and then precipitated with diethyl ether. models where, Uiso was assigned 1.2 or 1.5 times Ueq of the parent\nThe orange precipitate was isolated by filtering under vacuum followed hydrogen atoms. Further, the C\u2013H bond distances were constrained to\nby washing it with diethyl ether. Recrystallisation of the isolated orange the range between 0.95 \u00c5 to 0.99 \u00c5. The hydroxyl hydrogens and the\npowder in minimal CH2Cl2 followed by slow diffusion of diethyl ether hydrogens on N2, of ligand 2, were located in the difference density\nproduced the pure compound (4) as orange needles. Yield: 0.0903 g maps and refined independently. The structure of ligand 2 was refined to\n(68%). 1H NMR (600 MHz, (CD3)2(CO)): \u03b4 (ppm) = 9.33 (1H, s, Hn), 9.10 R factor of 0.0433. In addition, the hydrogen H2 on N2, of complex 4,\n(1H, d, J = 5.3 Hz, Hs) 8.45 (1H, d, J = 5.5 Hz, Hb), 8.29 (2H, td, J = 7.8, was found in the difference density maps and refined independently. The\n1.4 Hz, Hq), 8.24 (1H, d, J = 9.0 Hz, Hg), 8.17 (1H, d, J = 7.7 Hz, Hp), structure of complex 4 was refined to R factor of 0.0215.\n7.87 (1H, d, J = 2.1 Hz, Hd), 7.83 (1H, m, Hr), 7.33 (1H, dd, J = 9.0, 2.2\nHz, Hf), 7.16 (1H, s, Hj), 6.6 (1H, d, J = 5.6 Hz, Ha), 4.42 (2H, t, J = 6.5 4.5. Solution stability studies\nHz, Hm), 3.72\u20133.56 (2H, m, Hk), 2.64 (1H, m, Hl), 2.48 (1H, m, Hl). 13C\n{1H} NMR (151 MHz, (CD3)2(CO)): \u03b4 (ppm) = 198.77, 198.47, 188.43, The stability of complexes 4 and 5 was carefully observed in a so\u00ad\n170.64, 156.36, 154.01, 151.82, 141.08, 135.47, 130.01, 129.82, lution of PBS supplemented with 1% DMSO over a 48-hour period at\n128.05, 125.58, 124.65, 118.55, 100.08, 66.23, 64.09, 41.51, 28.86, 37\u25e6 C, employing UV-Vis spectroscopy.\n15.68. IR (ATR): (\u03bdmax/cm\u2212 1) 3214 (sec. NH), 2014 (C\u2013 \u2013O), 1862\n(C\u2013\n \u2013O), 1573 (C\u2013 \u2013N)imine, pyridyl, 1549 (C\u2013\u2013N)quinoline. UV\u2013Vis (DMSO): 4.6. Biological studies\n\u03bbmax nm (10\u2212 4 \u03b5, mol\u2212 1 L cm\u2212 1) 336 (0.890), 385 sh (0.378). Melting\npoint: 213.8\u2013214.9 \u25e6 C. MS (HR-ESI, m/z): 631.0297 (100%, [M + H]+), 4.6.1. Cell culture\ncalculated 631.0235. HPLC purity: 98% (tr\u2019 = 2.36 min). Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-\n Aldrich, USA) was used to maintain the human breast adenocarcinoma\n4.3.3. Re(I) aminoquinoline-quinolyl complex (5) cell line, MCF-7, which is oestrogen-receptor positive (ER+). While the\n Ligand 3 (0.104 g, 0.276 mmol) was dissolved in anhydrous human breast adenocarcinoma cell line, MDA-MB-231, a triple negative\n\n 14\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\nbreast cancer (TNBC), and the non-malignant dermal fibroblast cells, of 250\u2013400 nm. The intrinsic binding constant, Kb, for each complex is\nFG-0, were maintained in Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) estimated using Eq. (1) 101.\n(Sigma-Aldrich, USA). The culture media were supplemented with 10%\n 1/A - A0 = 1/Kb. \u0394\u03b5. 1/[complex] + 1/A0 (1)\nheat-inactivated foetal bovine serum (FBS), 100 U/mL of penicillin and\n100 \u03bcg/mL streptomycin. To maintain physiological pH and tempera\u00ad where A is the recorded absorption at different metal concentrations;\nture, all cells were nurtured in a 95% air and 5% CO2 humidified A0 is the initial absorption of CT-DNA at 260 nm; \u0394\u03b5 is the molar ab\u00ad\nincubator and maintained at 37 \u25e6 C. Additionally, the culture media were sorptivity change upon complex formation; [complex] is the concen\u00ad\nreplenished with fresh media every 48\u201372 h. tration of the metal complex.\n The standard Gibb\u2019s free energy (\u0394G) for each experiment was\n4.6.2. Cytotoxicity assays calculated following the van\u2019t Hoff Eq. (2) [112].\n The MCF-7, MDA-MB-231 and FG-0 cells were seeded on a 96-well\nplate at densities of 4500 cells/well, 3000 cells/well and 1500 cells/ \u0394G = \u2212 RT ln Kb. (2)\nwell, respectively. Under physiological conditions, the MCF-7 and FG-0\ncells were incubated for 48 h and the MDA-MB-231 cells for 24 h, to\nallow for adhesion. Once adhered the MCF-7 and MDA-MB-231 cells\n 4.7.2. Ethidium bromide (EtBr) displacement studies\nwere treated with either the vehicle (0.1% DMSO in growth media) or\n The CT-DNA-EtBr (calf thymus DNA \u2013 ethidium bromide) composite\nthe test compounds at 10 \u03bcM or 20 \u03bcM and incubated for 48 h. The 3-\n was prepared at a molar ratio of 1:1 EtBr:CT-DNA (10 \u03bcM each) in PBS\n(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)\n buffer solution (pH = 7.2, 25 \u25e6 C) and kept in the dark for the experiment.\nassay was used to determine the effect of the test compounds on the\n The concentration of the CT-DNA-EtBr composite was kept constant\ncancer cell viability, as described in literature [93]. The MTT colori\u00ad\n while the quencher (the Re(I) complex) was added in increments of\nmetric assay is based on the NAD(P)H-dependent oxidoreductase en\u00ad\n 0\u2013150 \u03bcM, at ambient temperature. The fluorescence emission was\nzymes produced by actively growing cells. These cells catalyse the\n excited at 525 nm, and the emission recorded, at ambient temperature,\nreduction of MTT to purple formazan crystals [141]. The absorbance of\n at a range of 530 nm\u2013700 nm, following a 10 min incubation period. The\neach well was measured at 600 nm using a spectrophotometer (Glo\u00ad\n Stern-Volmer binding constant (KSV), and the bimolecular quenching\nMax\u00ae Explorer Multimode Microplate Reader GM3500, Promega) and\n rate constant (kq) were computed from the classical Stern-Volmer Eq. (3)\nnormalized to the RPMI medium (for the MCF-7 cell line) or the DMEM\n [118].\ngrowth media (for the MDA-MB-231 and FG-0 cell lines) [142]. The\nMCF-7, MDA-MB-231 and FG-0 cell lines were treated with concentra\u00ad Io/I = 1 + Ksv[Q] = 1 + kq\u03c40[Q]w (3)\ntions ranging from 0 to 35 \u03bcM of the tested complexes to determine their\nIC50 (concentration required for 50% viability). All experiments were here Io and I are the emission intensities in the absence and presence of\nperformed twice in triplicate, and the mean cell viability determined the Re(I) complex (quencher), respectively; [Q] is the concentration of\nusing the GraphPad Prism V.5.01 software. the Re(I) complex, and \u03c40 is the average lifetime of the fluorophore\n (10\u2212 8 s) without the Re(I) complex.\n4.6.3. Western blotting The apparent association constant, Kapp, was obtained using Eq. (4)\n MCF-7 and MDA-MB-231 cells were seeded in 6 cm dishes and [125].\ntreated for 48 h with complexes 4 and 5 at the \u00bd IC50 and IC50 con\u00ad KEtBr[EtBr] = Kapp[Q] (4)\ncentrations. The relevant proteins were extracted and prepared from the\ntreated cells, thereafter, subjected to SDS-PAGE and immunoblotting where [Q] is the concentration of the Re(I) complex that causes a 50%\nfollowing the procedure formerly described by Bleloch et al. [143] The reduction in the intensity of the CT-DNA-EtBr fluorescence, and KEtBr =\nprimary antibodies utilized in this study, rabbit polyclonal antibodies to 107 M\u2212 1. The binding constant, KF, and the number of binding sites (n)\nPhospho-Histone \u03b3H2A.X (Ser139) (#2577), PARP (#9542) and mouse were estimated from the Scatchard Eq. (5) [126].\nmonoclonal anti-\u03b2-Actin (sc-47778), were supplied by Cell Signaling\n log(Io \u2212 I)/I = logKF + n log[Q] (5)\nTechnology (USA). The secondary antibodies were used at a dilution of\n1:5000 and comprised of horseradish peroxidase\u2013conjugated goat anti\u00ad\nrabbit (Bio-Rad) and goat anti-mouse (Bio-Rad). Densitometry was\nexecuted with the image analysis software Fiji (Version 4.8. Protein interaction studies\n2.0.0-rc-68/1.52e). The levels of protein expression were represented as\na ratio of the protein of interest/\u03b2-actin loading control and normalized The studies were conducted in a similar manner as outlined above,\nto the control sample, where appropriate. All blots are indicative of no for the CT-DNA-EtBr emissions. The concentration of PBS buffer was\nless than three independent repeats. spectrophotometrically determined at a molar extinction coefficient of\n \u03b5280 = 44,300 mol\u2212 1 cm\u2212 1. The concentration of BSA protein was\n4.7. DNA interaction studies maintained at 1.5 \u03bcM, while the quencher, i.e. Re(I) complex, was added\n in increments from 0 \u03bcM to 30 \u03bcM, at ambient temperature. Following a\n4.7.1. Absorption spectroscopic studies 10 min period of incubation, at ambient temperature, the samples were\n All experiments regarding DNA interactions with complexes 4 and 5 excited at a wavelength of 280 nm and the emission spectra were\nwere carried out in PBS buffer with pH = 7.2, at ambient temperature. recorded from 300 nm to 450 nm.\nThe concentration of CT-DNA solution in PBS buffer was determined Following literature procedures [144], data corrections of the spec\u00ad\nspectrophotometrically with a molar extinction coefficient \u03b5260 = 6600 trophotometric titrations were employed, to account for the existing\nmol\u2212 1 cm\u2212 1. The purity of the CT-DNA solution in PBS buffer was primary and/or secondary inner filter effects, by applying Eq. (6).\nconfirmed from the ratio of electronic absorption at 260 nm. Standard\nstock solutions of the rhenium complexes (4\u20135) were prepared with 5% Fcorr = Fobs10(Aex + Aem)/2w (6)\nDMSO and 95% ultrapure water. here Fcorr and Fobs are the corrected and observed fluorescence in\u00ad\n Absorption spectra of CT-DNA maintained at constant concentra\u00ad tensities, respectively; whilst Aex and Aem are the absorbance values at\ntions of 50 \u00b5M, were recorded with increasing amounts of metal- the excitation and emission wavelengths, respectively.\ncomplexes (0\u2013350 \u00b5M). The solutions were incubated for 8 min at\nambient temperature prior to recording the absorption spectra at a range\n\n 15\n\fP.S. Zinman et al. European Journal of Medicinal Chemistry 266 (2024) 116094\n\n\n4.9. Molecular docking [3] A.N. Giaquinto, H. Sung, K.D. Miller, J.L. Kramer, L.A. Newman, A. Minihan,\n A. Jemal, R.L. Siegel, Breast cancer statistics, 2022, CA, Cancer J. Clin. 72 (2022)\n 524.\n The crystal structures of DNA (PDB ID: 1Z3F) and BSA (PDB ID:4F5S) [4] M. Vanneman, G. Dranoff, Combining immunotherapy and targeted therapies in\nwere obtained from the RCSB protein data bank (http://www.rcsb.org/) cancer treatment, Nat. Rev. Cancer 12 (2012) 237.\nat a solution of 1.60 and 2.47 \u00c5, respectively. The structures were [5] H. Schiff, Ann. Chem. Suppl. 3 (1864) 343.\n [6] H. Schiff, Schiff base reaction, Justus Liebigs Ann. Chem. 131 (1864) 118.\nrefined by removing any co-crystallized hetero ligands, waters, and/or [7] P. Sykes, A Guidebook to Mechanism in Organic Chemistry, sixth ed., Pearson\ncofactors. Thereafter, polar hydrogen atoms and Kollman charges were Education Limited, Essex, England, 1986.\nadded to the structures. Gasteiger charges were calculated and assigned [8] S. Parveen, Recent advances in anticancer ruthenium Schiff base complexes,\n Appl. Organomet. Chem. 34 (2020) e5687.\nto each atom accordingly, and non-polar hydrogen atoms were merged [9] P.G. Cozzi, Metal\u2013Salen Schiff base complexes in catalysis: practical aspects,\ninto carbon atoms. The coordination spheres of the metal complexes Chem. Soc. Rev. 33 (2004) 410.\nwere generated from DFT calculations, and the GaussView 5.0 software [10] T.P. Yoon, E.N. Jacobsen, Privileged chiral catalysts, Science 299 (2003) 1691.\n [11] K. Brodowska, E. Lodyga-Chruscinska, Schiff bases - interesting range of\nwas used to convert the complexes\u2019 optimized geometry and lowest applications in various fields of science, Chemik 68 (2014) 129.\nenergy conformations to the appropriate PDB format. The complexes [12] G. Moustafa, E. Sabry, E.M. Zayed, G.G. Mohamed, Structural characterization,\nand receptors (i.e. DNA and BSA) were prepared using AutoDock Tools. spectroscopic studies, and molecular docking studies on metal complexes of new\n hexadentate cyclic peptide ligand, Appl. Organomet. Chem. 36 (2022) e6515.\nThe molecular docking investigations were performed using the PyRx [13] A. Singh, P. Barman, Recent advances in Schiff base ruthenium metal complexes:\nprogram [145]. During the docking analyses, the binding sites were synthesis and applications, Top. Curr. Chem. 379 (2021) 29.\nconstrained to the entire receptor, where a grid spacing of 0.375 \u00c5 was [14] D.N. Dhar, C. Taploo, Schiff-bases and their applications, J. Sci. Ind. Res. 41\n (1982) 501.\napplied. The BIOVIA Discovery Studio Visualizer 2022 package was used\n [15] S. Shekhar, A.M. Khan, S. Sharma, B. Sharma, A. Sarkar, Schiff base metallodrugs\nto visualize the predicted interactions and prepare the structural in antimicrobial and anticancer chemotherapy applications: a comprehensive\ngraphics, molecular animations, and atomic interaction measurements. review, Emergent Mater 5 (2022) 279.\nThe predicted binding affinities were obtained in terms of binding en\u00ad [16] K. Sztanke, A. Maziarka, A. Osinka, M. Sztanke, An insight into synthetic Schiff\n bases revealing antiproliferative activities in vitro, Bioorg. Med. Chem. 21 (2013)\nergies in kcal/mol and assigned as their docking scores. 3648.\n [17] M.A. Malik, O.A. Dar, P. Gull, M.Y. Wani, A.A. Hashmi, Heterocyclic Schiff base\n transition metal complexes in antimicrobial and anticancer chemotherapy,\nCRediT authorship contribution statement\n MedChemComm 9 (2018) 409.\n [18] G. Puthilibai, S. Vasudhevan, Synthesis, DNA binding, anticancer and cytotoxic\n Paige S. Zinman: Writing \u2013 original draft, Validation, Investigation, evalutation of novel ruthenium(II) isatin based Schiff base complex, Rasayan J.\n Chem. 12 (2019) 855.\nFormal analysis, Data curation. Athi Welsh: Investigation. Reinner O.\n [19] A. Garza-Ortiz, P. Uma Maheswari, M. Siegler, A.L. Spek, J. Reedijk, A new family\nOmondi: Writing \u2013 review & editing, Investigation. Saif Khan: Inves\u00ad of Ru(ii) complexes with a tridentate pyridine Schiff-base ligand and bidentate\ntigation. Sharon Prince: Supervision, Resources, Investigation. Ebbe co-ligands: synthesis, characterization, structure and in vitro cytotoxicity studies,\nNordlander: Writing \u2013 review & editing. Gregory S. Smith: Writing \u2013 New J. Chem. 37 (2013) 3450.\n [20] G.A. Sua\u0301rez-Ortiz, R. Herna\u0301ndez-Correa, M.D. Morales-Moreno, R.A. Toscano, M.\nreview & editing, Supervision, Resources, Project administration, T. Ramirez-Apan, A. Hernandez-Garcia, M. Ame\u0301zquita-Valencia, D. Araiza-\nFunding acquisition, Conceptualization. Olivera, Diastereomeric separation of chiral fac-Tricarbonyl(iminopyridine)\n rhenium(I) complexes and their cytotoxicity studies: approach toward an action\n mechanism against glioblastoma, J. Med. Chem. 65 (2022) 9281.\nDeclaration of competing interest [21] A. Lapasam, V. Banothu, U. Addepally, M.R. Kollipara, Synthesis, structural and\n antimicrobial studies of half-sandwich ruthenium, rhodium and iridium\n complexes containing nitrogen donor Schiff-base ligands, J. Mol. Struct. 1191\n The authors declare that they have no known competing financial (2019) 314.\ninterests or personal relationships that could have appeared to influence [22] J. Shi, H. Ge, F. Song, S. Guo, Synthesis, characterization, crystal structure and\n antibacterial activity studies of cobalt(II) and zinc(II) complexes containing\nthe work reported in this paper. halogen quinoline Schiff base ligand, J. Mol. Struct. 1253 (2022) 132263.\n [23] E. Yousif, A. Majeed, K. Al-Sammarrae, N. Salih, J. Salimon, B. Abdullah, Metal\nData availability complexes of Schiff base: preparation, characterization and antibacterial activity,\n Arab. J. Chem. 10 (2017) S1639.\n [24] R.K. Mohapatra, A.K. Sarangi, M. Azam, M.M. El-ajaily, M. Kudrat-E-Zahan, S.\n Data will be made available on request. B. Patjoshi, D.C. 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