Trinuclear ruthenium(II) polypyridyl complexes: Evaluation as photosensitizers for enhanced cervical cancer treatment.

{"full_text": " Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nTrinuclear ruthenium(II) polypyridyl complexes: Evaluation as\nphotosensitizers for enhanced cervical cancer treatment\nAthi Welsh a, Refilwe Matshitse b, Saif F. Khan c, Tebello Nyokong b, Sharon Prince c, Gregory\nS. Smith a, *\na\n Department of Chemistry, University of Cape Town, Rondebosch 7700, ,South Africa\nb\n Institute for Nanotechnology Innovation, Rhodes University, Makhanda 6140, South Africa\nc\n Division of Cell Biology, Department of Human Biology, University of Cape Town, Faculty of Health Science, Observatory, 7925, South Africa\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: Trinuclear ruthenium(II) polypyridyl complexes anchored to benzimidazole-triazine / trisamine scaffolds were\nPolynuclear investigated as photosensitizers for photodynamic therapy. The trinuclear complexes were noted to produce a\nBenzimidazole significant amount of singlet oxygen in both DMF and aqueous media, are photostable and show appreciable\nRuthenium(II)\n emission quantum yields (\u0278em). In our experimental setting, despite the moderate phototoxic activity in the HeLa\nAnticancer\n cervical cancer cell line, the phototoxic indices (PI) of the trinuclear complexes are superior relative to the PIs of\nPhotodynamic therapy\n a clinically approved photosensitizer, Photofrin\u00ae, and the pro-drug 5-aminolevulinic acid (PI: >7 relative to PI:\n >1 and PI: 4.4 for 5-aminolevulinic acid and Photofrin\u00ae, respectively). Furthermore, the ruthenium complexes\n were noted to show appreciable long-term cytotoxicity upon light irradiation in HeLa cells in a concentration-\n dependent manner. Consequently, this long-term activity of the ruthenium(II) polypyridyl complexes em\u00ad\n bodies their ability to reduce the probability of the recurrence of cervical cancer. Taken together, this presents a\n strong motivation for the development of polymetallic complexes as anticancer agents.\n\n\n\n\n1. Introduction to the non-invasive characteristics of PDT agents which provide spatial\n and temporal control over cell death [8\u201312]. PDT is a form of local light-\n The development of new chemotherapeutic treatments to circum\u00ad based therapy in which Reactive Oxygen Species (ROS), generated\nvent drug resistance for long-term use, has become one of the important photochemically by nontoxic photosensitizers (PS), induce oxidative\nresearch thrusts in the battle against cancer. Since the discovery of damage to the essential cell and tissue components, bringing about cell\ncisplatin, several platinum-based metallodrugs have been developed, and tissue death [13\u201315]. PDT is a two-step procedure, consisting of the\nincluding the clinically used carboplatin and oxaliplatin [1,2]. However, administration of the PS, followed by exposure to light. This two-step\nthe use of these metallodrugs is hindered by the same hurdles that limit procedure significantly reduces side effects, as the nontoxic PS is\nthe application of cisplatin, including off-target effects, the development solely activated by light [16].\nof tumour drug resistance and a limited range of activity [3,4]. These Ruthenium(II) polypyridyl complexes have remained amongst the\nlimitations have resulted in a redirection in metallodrug discovery to most studied transition metal complexes in light-driven applications,\nother platinum-group metals, with ruthenium-based complexes repre\u00ad including PDT, photocatalysis and luminescent sensing [7,17\u201320]. The\nsenting the vanguard of research into the development of metallodrugs interest in ruthenium(II) complexes stems from their favourable and\nbased on alternative platinum-group metals. This is exemplified by conferrable characteristics, which can be fine-tuned to yield excited\nseveral ruthenium-based complexes including NAMI-A, NKP-1339 and states that are accessible to visible light [21,22]. Indeed, ruthenium(II)\nTLD1433 having been clinically investigated for the treatment of various polypyridyl complexes have garnered a considerable amount of interest\ncancers [5\u20137]. in their development as photoactive agents, in the treatment of cancer,\n In recent years, photodynamic therapy (PDT) has evolved as a suc\u00ad and continue to yield prolific results [21,23\u201327].\ncessful alternative or adjuvant treatment modality for cancer. This is due Combining photosensitizers with suitable pharmacophoric ligands is\n\n\n\n * Corresponding author.\n E-mail address: gregory.smith@uct.ac.za (G.S. Smith).\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112545\nReceived 6 December 2023; Received in revised form 23 March 2024; Accepted 24 March 2024\nAvailable online 26 March 2024\n0162-0134/\u00a9 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-\nnc-nd/4.0/).\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\nof paramount importance to metallodrug design in an effort to improve (II) in ethanol at 78 \u25e6 C overnight; ii) secondly, a salt metathesis reaction\nthe photophysical properties of photosensitizers. The broad biological using ammonium hexafluorophosphate to yield the desired complexes as\nproperties conferred by the benzimidazole scaffold and the benefit of hexafluorophosphate salts (Fig. 2). The ruthenium(II) complexes were\nincorporating this scaffold onto organometallic complexes is now well characterized by nuclear magnetic resonance (NMR) spectroscopy\ndocumented in the literature [28\u201331]. However, reports on the use of (Fig. S5-S7 in the ESI), high-resolution electrospray ionization mass\nthe benzimidazole scaffold as an ancillary ligand in the design of PSs for spectrometry (HR-ESI) and purity confirmed by liquid chromatography.\nPDT are sparse [32\u201334].\n The advantages of the incorporation of more than one metal center as\npart of a potential photosensitizer are well documented in the literature, 2.2. Photophysicochemical properties\nas ruthenium(II)-based homo- and hetero-polynuclear complexes\ngenerally show enhanced phototoxicity relative to their mononuclear The emission and singlet oxygen quantum yields (\u0278em and \u0278\u0394\ncounterparts [35\u201340]. Furthermore, a similar trend can be observed respectively) of the ruthenium(II) compounds were determined using\nwhen comparing the antiproliferative activity of cisplatin to that of its reported comparative methods [44,45]. Tris(bipyridine)ruthenium(II)\ntrinuclear conjugate, BBR3464, where the activity of trinuclear complex chloride ([Ru(bpy)3Cl2]) served as a benchmark with \u0278\u0394 and \u0278em values\nis superior relative to the analogous mononuclear counterpart [41,42]. of 0.57 and 0.018 obtained in aerated DMF and acetonitrile, respectively\nDespite this, the development of polymetallic metallodrugs and metal- [46\u201348].\norganic PSs for PDT remains in its infancy and relatively unexplored.\n Herein, we report the synthesis of a series of trinuclear 2-pyridyl- and 2.2.1. Emission spectra and Emission Quantum Yield (\u0278em)\n2-quinolylbenzimidazole-based ruthenium(II) complexes and discuss All the experiments were conducted in acetonitrile and the absor\u00ad\ntheir phototoxic activity as PDT agents in the HeLa cervical cancer cell bance of the complexes at the excitation wavelength was maintained at\nline. 0.05 a.u. The 2-pyridylbenzimidazole-based complexes (Ru-1, Ru-2 and\n Mono) were noted to show emission maxima in the range 650\u2013665 nm,\n2. Results and discussion however, the emission maximum of the 2-quinolylbenzimidazole-based\n complex (Ru-3) was noted to be significantly red-shifted (759 nm). This\n2.1. Synthesis and characterization of the ruthenium complexes is attributed to the extended aromatic ring inherent in complex Ru-3,\n which results in an emission maximum at a longer wavelength. The\n The synthesis of the key s-triazine-anchored 2-pyridylbenzimidazole calculated emission quantum yields for the complexes are as follows:\nligand (6) was carried out via a six-step procedure (Scheme 1) involving 8.6%, 2.6%, 5.1% and 0.95% for Mono, Ru-1, Ru-2 and Ru-3, respec\u00ad\nthe i) tert-butyloxycarbonyl (Boc) protection of 1,3-diaminopropane, tively (Table 1). The significantly lower emission quantum yields for the\nyielding 1; ii) nucleophilic aromatic substitution reaction of 1 with 1-flu\u00ad trinuclear complexes (Ru-1, Ru-2, Ru-3) may be ascribed to non-\noro-2-nitrobenzene, to yield the aryl nitro precursor (2); iii) reduction of radiative dissipation of the excited state of these trinuclear complexes,\nthe aryl nitro precursor (2), with zinc in the presence of ammonium and transient electron transfer between the excited ruthenium trisbi\u00ad\nchloride, to produce the aryl 1,2-diamine (3); iv) the cyclocondensation pyridyl metal centers and either the triazine or trisalkylamine cores of\nof 3 with 2-pyridinecarboxaldehyde in the presence of a catalytic the respective trinuclear complexes [49\u201353].\namount of trifluoracetic acid (TFA), yielding the 2-pyridylbenzimida\u00ad\nzole (4); v) Boc-deprotection of 4 using an excess of TFA to yield 5; 2.2.2. Singlet oxygen quantum yield (\u0278\u0394)\nvi) reaction of 5 with cyanuric chloride in N,N-dimethylformamide to Singlet oxygen is formed via an energy transfer process between the\nyield the s-triazine anchored 2-pyridylbenzimidazole ligand (6). triplet excited state and molecular oxygen, widely referred to as the\n In support of this study, the analogous trimeric and monomeric 2- Type II reaction [54]. The \u0278\u0394 values were determined using an indirect\npyridylbenzimidazole (9 and 13) and 2-quinolylbenzimidazole (10) li\u00ad method, in which the photobleaching of 9,10-dimethylanthracene\ngands (based on a trisamine core) were synthesized in parallel, as (DMA) was monitored in DMF after irradiation with blue light at 455\ndescribed in the literature (Fig. 1) [28,41,43]. nm (330 mW.cm\u2212 2). Solutions of the ruthenium(II) complexes, the [Ru\n The synthesis of the polymetallic ruthenium(II) polypyridyl com\u00ad (bpy)3Cl2] standard (at 15 \u03bcM, respectively) and DMA (at 25 \u03bcM) were\nplexes (Ru-1, Ru-2, Ru-3) and mononuclear complex (Mono) was prepared in DMF. The photodegradation of DMA was evidenced by the\nachieved in two steps: i) firstly, by the reaction of the appropriate decrease in absorbance at 402 nm, as illustrated in Fig. 3, in which DMA\nbenzimidazole-based ligand with dichloridobis(bipyridine)ruthenium is degraded in the presence of the mononuclear complex (Mono)\n (Fig. 3a) and the trinuclear complex (Ru-1) (Fig. 3b).\n\n\n\n\nScheme 1. Reagents and conditions: i) Diaminopropane/ Boc2(O)/ CHCl3/ RT/ 3 h; ii) 1-Fluoro-2-nitrobenzene/ DMF/ 100 \u25e6 C/ 24 h; iii) Zn/ NH4Cl/ MeOH/ RT/ 1\nh; iv) 2-pyridinecarboxaldehyde/ TFA/ EtOH/ MgSO4/ RT/ 24 h; v) TFA/ DCM/ 30 \u25e6 C/ 24 h; vi) DMF/ K2CO3 /120 \u25e6 C/ 48 h.\n\n 2\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\n\n\nFig. 1. The structures of the trisamine anchored trimeric 2-pyridyl (9) and 2-quinolylbenzimidazole-based (10) ligands and the corresponding monomeric 2-pyri\u00ad\ndylbenzimidazole congener (13).\n\n\n\n\nFig. 2. The structures of the mononuclear 2-pyridylbenzimidazole-based ruthenium(II) polypyridyl complex (Mono) and the corresponding triazine-anchored (Ru-\n1) and trisamine anchored trinuclear complexes (Ru-2 and Ru-3).\n\n\n As previously observed for the emission quantum yields, the trinu\u00ad\nTable 1 clear complexes (Ru-1, Ru-2,\nPhotophysical parameters of the ruthenium(II) polypyridyl complexes.\n Ru-3) have significantly less singlet oxygen quantum yields relative\n Compound \u03bbaex \u03bbaem \u03c6a\u0394 \u03a6bem to the mononuclear complex (Mono). This may in part be attributed to\n Mono 469 664 0.79 0.09 the increased flexibility of the trinuclear complexes, thus increasing the\n Ru-1 472 674 0.26 0.03 probability of non-radiative dissipation of the excited state via vibra\u00ad\n Ru-2 465 653 0.31 0.05 tional relaxation and/or kinetic energy [49\u201351]. The trend in the\n Ru-3 503 759 0.13 0.01\n calculated \u0278\u0394 values is, as for the emission quantum yields, of the\na: Parameter determined in acetonitrile; b: parameter determined in DMF. following order: Mono > Ru-2 > Ru-1 > Ru-3 (Table 1).\n\n 3\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\n\n\n Fig. 3. The degradation of 9,10-dimethylanthracene (DMA) in the presence of either Mono (a) or Ru-1 (b) in DMF over 60 s.\n\n\n With this in mind, the ability of the ruthenium(II) complexes to Upon irradiation of the compounds with a blue LED light at 455 nm\ngenerate 1O2 in aqueous media (~1% DMSO in deionized H2O) was (330 mW.cm\u2212 2), a general concentration-dependent reduction in HeLa\ninvestigated using 9,10-anthracenediyl-bis(methylene)dimalonic acid cell survival was noted (Fig. S22), and the IC50 values of the complexes\n(ADMA) as a singlet oxygen quencher. All the complexes were noted to are summarized in Table 2. Generally, there was no discernible corre\u00ad\nproduce singlet oxygen species (Fig. S20), indicated by the time- lation between the singlet oxygen quantum yields of the trinuclear\ndependent reduction of the absorbance of ADMA at 400 nm (Fig. 4, in complexes and their phototoxicity. This is particularly self-evident for\nwhich ADMA is being degraded in the presence of complex Ru-2). complex Ru-3, which has the lowest singlet oxygen quantum yield (\u03d5\u0394\n However, the singlet oxygen quantum yields of the ruthenium(II) = 0.13) but was one of the most active complexes (IC50 = 34.63 \u03bcM).\npolypyridyl complexes were not calculated in aqueous media. This is This may be attributed to the fact that ruthenium complexes have been\ndue to the interaction of the endoperoxide (formed upon the irreversible reported to indiscriminately exploit both type I and type II PDT mech\u00ad\nreaction of ADMA with singlet oxygen) with the respective complexes, anisms, and this may lead to the observation of no correlation between\nresulting in precipitation of the complexes out of solution (see the singlet oxygen quantum yields and the observed phototoxicity\nFig. S21a). Despite this, all the complexes have demonstrated the ability [56\u201358].\nto generate singlet oxygen in aqueous media and dichlorodihydro\u00ad The phototoxicity of the complexes follows the general trend: Ru-2\nfluorescein diacetate (DCFDA) assays confirm the ability of the com\u00ad \u2248 Ru-3 > Ru-1 > Mono. Structurally, complex Ru-1 consists of the\nplexes to produce ROS in vitro upon irradiation (Fig. S25).\n\n Table 2\n2.3. Phototoxicity studies\n The in vitro cytotoxicity (dark) and phototoxicity of the ruthenium(II) poly\u00ad\n pyridyl complexes on human cervical cancer cells (HeLa).\n To evaluate the potential of the trinuclear ruthenium complexes as\nPDT agents, their toxicity in the dark as well as upon light irradiation in Compound Light IC50 (\u03bcM)b Dark IC50 (\u03bcM) P.I.a\n\nthe human cervical cancer cell line (HeLa) was evaluated using the WST- Mono >45 >300 N/A\n1 assay. The dark toxicity of the complexes was performed at gradient Ru-1 42.53 \u00b1 1.16 >300 >7.05\n Ru-2 34.05 \u00b1 1.09\nconcentrations of 5\u2013300 \u03bcM. Of note, none of the compounds inhibited\n >300 >8.81\n Ru-3 34.63 \u00b1 1.11 >300 >8.66\nHeLa cell survival to \u226450% in the absence of light with IC50 > 300 \u03bcM Cisplatin [59] \u2013 7.6 \u2013\n(Fig. S22). These results bode extremely well for the potential applica\u00ad\n a: Phototoxicity Index: IC50 Dark/ IC50 Light; b: 24 h incubation in HeLa cells in\ntion of these complexes as PSs for PDT and are comparable with that of\n the dark followed by exposure to a 455 nm Thorlabs M455L3 blue LED (330 mW.\nPrajith and co-workers who reported two trinuclear complexes of a\n cm\u2212 2 or 1188 J/cm2) for 60 min.\nsimilar structural motif with IC50 values of 287 \u03bcM and 280 \u03bcM [55].\n\n\n\n\n Fig. 4. The degradation of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ADMA) in the presence of Ru-2 in aqueous media over 1200 s.\n\n 4\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\npharmacologically privileged s-triazine core, which is contained in low nuclearity (one metal center vs. three) and relatively lower charge\nseveral FDA-approved drugs [60]. Additionally, s-triazine derivatives (dicationic vs hexacationic). As such, these factors merit opportunities\nhave been noted to have several protein targets, ranging from cyclin- for the further development and study of trinuclear, highly charged\ndependent kinase (CDK) to dihydrofolate reductase, depending on the metallodrugs as potential PDT agents.\nchemical structure [61]. However, the data generated shows that\nchanging the trisamine core (in complexes Ru-2 and Ru-3) to the s-\ntriazine core (in complex Ru-1) does not significantly improve the 2.4. Clonogenic assays\nphototoxicity, as we had initially envisaged, as the s-triazine-based\ncomplex (Ru-1) is the least active trinuclear complex (IC50 = 42.53 \u03bcM). The treatment of cervical cancer patients with advanced stage or\n To explore the effect of the 2-aryl functionality of the benzimidazole recurrent tumours remains a major hurdle, and this is reflected in the\nscaffold, the trinuclear trisamine anchored 2-pyridyl (Ru-2) and 2-qui\u00ad very low five-year survival rates of <5%, despite intensive therapy\nnolinyl (Ru-3) benzimidazole-based complexes were evaluated for [66\u201368]. Moreover, the development of drug resistance by recurrent\ntheir phototoxicity. The positive effects that an extended pi-system ex\u00ad cervical cancer highlights the need for the identification of potential\nerts on the photophysical properties and the phototoxicity of ruthenium therapeutic agents which exhibit long-term cytotoxicity [69]. To this\ncomplexes are well documented in the literature [62\u201364]. From the data end, we investigated the long-term cytotoxic activity of the ruthenium\ngenerated, there was no statistically significant difference in the (II) polypyridyl complexes (Ru-2 and Ru-3), which showed the most\nphototoxicity of complexes Ru-2 and Ru-3 in the HeLa cell line (with promising activity in the short-term viability assay (WST-1 assay), using\ncomparable IC50 values of 34.05 \u00b1 1.09 \u03bcM and 34.63 \u00b1 1.11 \u03bcM, clonogenic assays. Furthermore, the mononuclear congener (Mono) was\nrespectively). This observation was counter-intuitive, as with previous included in these experiments to corroborate the benefit of having more\nstudies the extension of the pi-system on the 2-position of the benz\u00ad than one ruthenium(II) metal center on the long-term cytotoxicity of the\nimidazole scaffold resulted in the enhancement of the antiproliferative tested trinuclear complexes. The clonogenic assay entails treating cells\nactivity of structurally similar ruthenium(II)-p-cymene complexes [43]. with the selected complexes (Mono, Ru-2 and Ru-3) at various con\u00ad\n A comparison of the phototoxicity of the mononuclear complex centrations (\u00bd IC50, IC50 and 2\u00d7 IC50) for 24 h. Thereafter, a set of cells\n(Mono) to that of the trinuclear complexes reveals that the trinuclear were irradiated with a blue LED light at 455 nm (330 mW.cm\u2212 2) for 60\ncomplexes generally show enhanced phototoxicity in the tested cell line. min, and another set of cells was maintained in the dark. This was fol\u00ad\nThis may be attributed in part to two factors: increased nuclearity and lowed by the replating of the cells at lower densities in drug-free media\noverall charge density. Prajith and co-workers recently reported a direct and the cells were allowed to grow over 12\u201315 days (with drug free\nproportionality of the nuclearity of structurally similar ruthenium(II) growth media being replenished every 2 to 3 days).\npolypyridyl complexes to the cellular uptake [55]. Additionally, Mat\u00ad When the HeLa cells were treated and maintained in the absence of\nshitse and co-workers reported the direct correlation between the light (Fig. 5a and c), all the tested complexes showed a concentration-\noverall charge of metal complexes and their cellular uptake [65]. Thus, dependent reduction of their long-term survival. However, it is worth\nwe attribute the low activity of the mononuclear complex (Mono) to its noting that the dark activity of the Ru-2 and Ru-3 trimetallic ruthenium\n (II) complexes surpassed that of the mononuclear congener (Mono).\n\n\n\n\nFig. 5. Representative images and quantification of clonogenic assays of HeLa cells treated with \u00bd IC50, IC50 and 2\u00d7 IC50 concentrations of the trinuclear complexes\n(Ru-2 and Ru-3) and the mononuclear complex (Mono) for 24 h, and either maintained in the dark (A and C) or irradiated with blue light (455 nm) for 60 min (B and\nD). Cells were replated at lower densities and maintained in drug-free media for 12\u201315 days and crystal violet-stained colonies were imaged and quantified using\nImageJ. The graphs represent the mean colony area \u00b1 SEM for each treatment condition as a percentage of the vehicle control (untreated). Cisplatin was included as\na positive control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n 5\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\nMore noteworthy, complex Ru-3 nearly eradicated HeLa colonies at the values were reported in ppm and Hz, respectively. Infrared spectroscopy\nhighest treated concentration (2\u00d7 IC50). was conducted on a Perkin-Elmer Spectrum 100 FT-IR spectrometer\n Relative to the HeLa cells that were maintained in the dark, upon using Attenuated Total Reflectance (ATR) with bond vibrations\nlight irradiation for 60 min (455 nm, 330 mW.cm\u2212 2), a striking reduc\u00ad measured in reciprocal centimetres (cm\u2212 1). Mass Spectrometry (MS)\ntion in the colony forming ability of HeLa cells was observed when determinations were carried out using Electron Impact (EI) on JEOL\ntreated with all three tested compounds individually at all tested con\u00ad GCmatell instrument or Electrospray Ionization (ESI) on a Waters API\ncentrations (Fig. 5b and d). Indeed, the images show that following light Quattro Micro triple quadrupole mass spectrometer with data recorded\nirradiation, there was an almost complete absence of colonies of HeLa using the positive mode. A B\u00fcchi Melting Point Apparatus B-540 ma\u00ad\ncells treated with complexes Ru-2 and Ru-3 (Fig. 5b). These data are chine was used to obtain the uncorrected melting points of each com\u00ad\nimportant as they further validate the phototoxic effects of the tested pound. UV/ Vis electronic spectra were recorded on a Shimadzu UV-\nruthenium(II) polypyridyl complexes against cervical cancer cells with 2550 spectrophotometer. Ambient temperature fluorescence spectra\nthe trinuclear complexes Ru-2 and Ru-3 being highly effective relative were measured on a Cary Eclipse Fluorescence Spectrophotometer\nto the Mono complex, lending impetus to the use of polymetallic (G9800A). The purity of the complexes was determined using an Agilent\ncomplexes. HPLC 1260 equipped with an Agilent DAD 1260 UV/ Vis detector and an\n Agilent Pursuit 5 C18 column (5 \u03bcM, 150 mm \u00d7 4.6 mm). The com\u00ad\n3. Conclusions pounds were eluted using a mixture of solvent A (0.1% trifluoroacetic\n acid in deionized water) and solvent B (methanol) at a flow rate of 0.5\n A series of trinuclear hexacationic ruthenium(II) benzimidazole- mL/ min. The gradient elution conditions were as follows: 90% solvent A\nbased complexes (Ru-1, Ru-2, Ru-3) and a corresponding mono\u00ad between 0 and 2 min, 90\u201310% solvent A from 2 to 8 min, 10% solvent A\nnuclear congener (Mono) were synthesized and fully characterized. from 8 to 20 min. The compounds, cis-dichlorobis(bipyridine)ruthenium\nInvestigation of the photophysical properties of the complexes revealed (II), tert-butyl (3-aminopropyl)carbamate (1), 3-(2-(pyridin-2-yl)-1H-\ntheir ability to produce singlet oxygen upon irradiation in DMF (\u0278\u0394 = benzo[d]imidazol-1-yl)propan-1-amine (5), N1-(2-nitrophenyl)-N2,N2-\n0.13\u20130.79) and aqueous media. When evaluated for their dark toxicity in bis(2-((2-nitrophenyl)amino)ethyl)ethane-1,2-diamine (7), N1-(2-(bis\nthe human cervical cancer cell line (HeLa), the compounds did not show (2-((2-aminophenyl)amino)ethyl)amino)ethyl)benzene-1,2-diamine\na reduction in cell survival of 50% or below. Upon light irradiation, all (8), tris(2-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)ethyl)amine (9)\nthe trinuclear complexes (Ru-1, Ru-2 and Ru-3) show concentration- and tris(2-(2-(quinolin-2-yl)-1H-benzo[d]imidazol-1-yl)ethyl)amine\ndependent antiproliferative activity, with IC50 values ranging between (10) [43], 2-nitro-N-propylaniline (11), N1-propylbenzene-1,2-diamine\n34.05 \u03bcM and 42.53 \u03bcM. Despite the mild activity of the complexes, it is (12) and 1-propyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole (13), were\nworth noting that all the complexes exhibit enhanced phototoxicity synthesized following published literature methods [28,41,72,73].\nindices (PI in the range: 7.05\u20138.81) relative to those reported for clini\u00ad\ncally administered Photofrin\u00ae (PI: 4.4) [70] and 5-aminolevulinic acid 4.2. Synthesis of the 2-pyridylbenzimidazole s-triazine ligand (6)\n(PI: >1) in the HeLa cell line [71]. Furthermore, the investigation of the\nlong-term activity of selected complexes (Mono, Ru-2 and Ru-3) 4.2.1. Synthesis of tert-butyl (3-((2-nitrophenyl)amino)propyl)carbamate\nrevealed that all the tested complexes show mild dark cytotoxicity. (2)\nHowever, upon light irradiation, the long-term cytotoxicity of com\u00ad Tert-butyl (3-aminopropyl)carbamate (1) (1.25 g, 7.28 mmol) was\nplexes Mono, Ru-2 and Ru-3 is significantly enhanced, in a dose- added to DMF (10 mL) at room temperature. Thereafter, 1-fluoro-2-\ndependent manner. The superior cytotoxicity of the trinuclear com\u00ad nitrobenzene (1.15 mL, 10.9 mmol) was added and the reaction\nplexes (Ru-2 and Ru-3) relative to the mononuclear congener (Mono) is mixture was allowed to stir at 100 \u25e6 C for 24 h. Upon complete reaction of\nevident once more, as complexes Ru-2 and Ru-3 show enhanced long- the starting material, the reaction mixture was diluted with toluene\nterm phototoxicity in HeLa cells in comparison with the mononuclear (250 mL) and the solvent was removed using rotary evaporation. The\ncomplex (Mono). resultant crude residue was re-dissolved in 100 mL of dichloromethane\n Overall, the short- and long-term photocytotoxicity of the trinuclear (DCM) and washed with a saturated brine solution (80 mL) and\ncomplexes (Ru-1, Ru-2, Ru-3), surpasses that of the corresponding extracted with two more aliquots of DCM (2 \u00d7 100 mL). The organic\nmononuclear complex (Mono). Although we are currently investigating extracts were collected and dried over anhydrous sodium sulfate, and\nstrategies to enhance the generally modest biological activity of the excess solvent was reduced in vacuo. The desired product (2) was iso\u00ad\ncomplexes reported in this study, this study lends impetus to the lated by column chromatography as a yellow powder (1.59 g, 5.45\ndevelopment of polynuclear metallodrugs as potential PDT agents. mmol). Rf (3:1 petroleum ether: ethyl acetate): 0.50. Yield: 75%. 1H\n NMR (300 MHz, CDCl3) \u03b4 (ppm): 8.17 (ddd, J = 8.6, 4.2, 1.4 Hz, 1H,\n4. Experimental ArH), 8.06 (s, 1H, ArH), 7.50\u20137.37 (m, 1H, ArH), 6.85 (dd, J = 8.6, 5.3\n Hz, 1H, ArH), 6.72\u20136.58 (m, 1H, ArH), 4.62 (s, 1H, NH), 3.50 (t, J = 6.3\n4.1. General remarks Hz, 1H, CH2), 3.37 (t, J = 7.0 Hz, 2H, CH2), 3.32\u20133.20 (m, 2H, CH2),\n 2.26\u20132.07 (m, 1H, CH2), 2.00\u20131.82 (m, 2H, CH2), 1.45 (s, 9H, CH3). 13C\n All reactions were carried out in an inert argon atmosphere unless NMR (101 MHz, CDCl3) \u03b4 (ppm): 156.09, 145.40, 136.28, 131.95,\nstated otherwise. All reagents were purchased from commercial sources 126.95, 115.34, 113.67, 79.49, 40.50, 38.25, 29.62, 28.39. EI-MS (m/\n(Sigma-Aldrich, Combi-blocks) and used without further purification. z): Observed: 296.2 (25% [M + H]+, Calculated: 296.2. FT-IR (ATR) \u03bd\nSolvents were reduced at 40 \u25e6 C in reduced pressure using a B\u00fcchi (cm\u2212 1): 1510 (N\u2013O stretch). MP (\u25e6 C): 89.7\u201391.2 \u25e6 C.\nRotavapor. The heat to reactions conducted above room temperature\nwas supplied by a hot plate and silicone oil. All aqueous solutions were 4.2.2. Synthesis of tert-butyl (3-((2-aminophenyl)amino)propyl)carbamate\nprepared using deionized water. Reactions were monitored by TLC using (3)\naluminium-backed Merck silica-gel F254 plates, and compounds were Tert-butyl (3-((2-nitrophenyl)amino)propyl)carbamate (2) (1.59 g,\nvisualized under a UV lamp. All column chromatography was carried 5.38 mmol) was dissolved in anhydrous methanol (10 mL) and allowed\nout using Fulka Silica Gel 60, 40\u201363 \u03bcm. Nuclear Magnetic Resonance to stir for 5 min. Ammonium chloride (2.92 g, 54.5 mmol) and zinc\nspectra were recorded on either a Bruker X400 MHz spectrometer (1H at powder (7.13 g, 107 mmol) were added, and the resultant mixture was\n399.95 MHz and 13C at 100.65 MHz) or a Varian Mercury XR300 MHz allowed to stir at room temperature for 1 h. The reaction mixture was\n(1H at 299.95 MHz, 13C at 75.46 MHz) with tetramethylsilane (TMS) as filtered through Celite\u00ae and rinsed with copious methanol. The filtrate\nthe internal standard for chemical shifts. Chemical shifts and J-coupling was subsequently collected, and the excess solvent was reduced by\n\n 6\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\nrotary evaporation. The resultant residue was redissolved in DCM (100 benzimidazole imine C\u2013 \u2013N), 1586 (pyridyl C\u2013 \u2013N). EI-MS (m/z):\nmL) and washed with two aliquots of a saturated brine solution (2 \u00d7 100 Observed: 358.3 (100%, [C18H15N9 + H]+), Calculated: 358.3. MP (\u25e6 C):\nmL) and deionized water (100 mL). The organic extract was collected, 100.2\u2013102.8.\ndried over anhydrous sodium sulfate and excess solvent was reduced in\nvacuo and the resultant crude product was purified using column chro\u00ad 4.3. The general synthesis of the ruthenium(II) polypyridyl complexes\nmatography (100% ethyl acetate). The desired compound (3) was iso\u00ad (Mono, Ru-1, Ru-2, Ru-3)\nlated as a dark brown oil (0.813 g, 3.07 mmol). Rf (ethyl acetate): 0.82.\nYield: 57%. 1H NMR (300 MHz, CDCl3) \u03b4 (ppm): 6.87\u20136.74 (m, 1H, The appropriate trimeric 2-pyridylbenzimidazole ligand (1 eq.)\nArH), 6.76\u20136.57 (m, 3H, ArH), 4.71 (s, 1H, NH), 3.37\u20133.13 (m, 6H, H-g, (either N2,N4,N6-tris(3-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)pro\u00ad\nCH2), 1.93\u20131.74 (m, 2H, CH2), 1.45 (s, J = 17.8 Hz, 9H, CH3). 13C NMR pyl)-1,3,5-triazine-2,4,6-triamine, tris(2-(2-(pyridin-2-yl)-1H-benzo[d]\n(151 MHz, CDCl3) \u03b4 (ppm): 156.19, 137.15, 134.50, 120.52, 118.88, imidazol-1-yl)ethyl)amine or tris(2-(2-(quinolin-2-yl)-1H-benzo[d]imi\u00ad\n116.55, 112.15, 79.24, 41.57, 38.35, 29.70, 28.40. EI-MS (m/z): dazol-1-yl)ethyl)amine) was dissolved in anhydrous ethanol (5 mL). To\nObserved: 266.2 (100% [M + H]+, Calculated: 266.2. FT-IR (ATR) \u03bd this stirring solution, cis-dichlorobis(bipyridine)ruthenium(II) (3 eq.)\n(cm\u2212 1): 1691 cm\u2212 1 (C\u2013\n \u2013O stretch), 3378 cm\u2212 1 (1\u05af NH2 stretch). was added and the reaction mixture was refluxed at 78 \u25e6 C overnight.\n After 24 h, the reaction mixture was cooled to room temperature and a\n4.2.3. Synthesis of tert-butyl (3-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1- solution of ammonium hexafluorophosphate (6 eq.) in anhydrous\nyl)propyl)carbamate (4) ethanol (2 mL) was added, and the reaction mixture was stirred for an\n Tert-butyl (3-((2-aminophenyl)amino)propyl)carbamate (3) (0.100 additional 30 min at room temperature. The contents of the reaction\ng, 0.383 mmol) was dissolved in anhydrous ethanol (10 mL) and allowed flask were subsequently filtered through Celite\u00ae. The solvent was\nto stir under argon for 5 min. Thereafter, 2-pyridinecarboxaldehyde removed under reduced pressure, which resulted in a solid precipitate.\n(0.0550 mL, 0.575 mmol), trifluoracetic acid (TFA) (0.00293 mL, The desired complexes were isolated by suction filtration and washed\n0.0383 mmol) and magnesium sulfate (0.276 g, 2.29 mmol) were with cold ethanol (10 mL).\nsequentially added to the reaction vessel. The reaction mixture was\nallowed to stir at 45 \u25e6 C overnight. The reaction mixture was subse\u00ad 4.3.1. The synthesis of the triazine-based ruthenium(II) polypyridyl\nquently filtered, and the filtrate collected. The excess solvent was complex (Ru-1)\nremoved by rotary evaporation, and the resultant crude mixture was The s-triazine ligand N2,N4,N6-tris(3-(2-(pyridin-2-yl)-1H-benzo[d]\nredissolved in dichloromethane (30 mL) and washed with a saturated imidazol-1-yl)propyl)-1,3,5-triazine-2,4,6-triamine, compound (6)\nsodium bicarbonate solution (30 mL) and a saturated brine solution (30 (0.0301 g, 0.0361 mmol), was reacted with cis-dichlorobis(bipyridine)\nmL). The organic extracts were collected and dried over anhydrous so\u00ad ruthenium(II) (0.0557 g, 0.115 mmol) under reflux at 78 \u25e6 C overnight.\ndium sulfate, and the excess solvent was removed in vacuo. Silica gel Thereafter, NH4PF6 (0.0353 g, 0.217 mmol) was added and this mixture\ncolumn chromatography was used to isolate compound 4 as a dark was allowed to stir for 1 h. The desired complex (Ru-1) was isolated as a\nyellow oil (0.0898 g, 0.255 mmol). Rf (Ethyl Acetate: Petroleum Ether burgundy solid (0.06737 g, 0.0325 mmol) by suction filtration and\n2:1): 0.40. Yield: 68%. 1H NMR (300 MHz, CDCl3) \u03b4 (ppm): 8.69 (dd, washed with cold ethanol (10 mL). Yield: 90%. 1H NMR (600 MHz,\nJ = 2.3, 1.5 Hz, 1H, ArH), 8.39 (d, J = 8.0 Hz, 1H, ArH), 7.87\u20137.69 (m, DMSO) \u03b4 (ppm): 8.84 (d, J = 7.0 Hz, ArH), 8.75 (d, J = 8.0 Hz, 3H,\n2H, ArH), 7.45\u20137.35 (m, 1H, ArH), 7.35\u20137.21 (m, 3H, ArH), 5.87 (s, 1H, ArH), 8.64 (d, J = 8.1 Hz, 3H, ArH), 8.24 (t, J = 7.6 Hz, 4H, ArH),\nNH), 4.79 (t, J = 7.0 Hz, 2H, CH2), 3.12 (dd, J = 11.8, 5.8 Hz, 2H, CH2), 8.22\u20138.11 (m, 10H, ArH), 8.08 (t, J = 7.7 Hz, 3H, ArH), 7.96 (d, J = 8.4\n2.18\u20132.04 (m, 2H, CH2), 1.40 (s, 9H, CH3). 13C NMR (151 MHz, CDCl3) Hz, 4H, ArH), 7.91 (d, J = 5.0 Hz, 3H, ArH), 7.84 (d, J = 5.0 Hz, 3H,\n\u03b4 (ppm): 156.05, 149.54, 149.07, 148.51, 141.47, 137.27, 135.87, ArH), 7.77 (s, 8H, ArH), 7.72 (d, J = 5.1 Hz, 4H, ArH), 7.64\u20137.40 (m,\n125.64, 124.28, 123.94, 123.31, 119.90, 110.44, 78.97, 43.07, 37.17, 22H, ArH), 7.13\u20137.03 (m, 6H, ArH), 5.76\u20135.66 (m, 3H, NH), 4.99\u20134.86\n29.84, 28.45. EI-MS (m/z): Observed: 353.2 (100% [M + H]+, Calcu\u00ad (m, 6H, CH2), 3.03 (dt, J = 18.4, 6.1 Hz, 6H, CH2), 2.08 (bs, 6H, CH2).\nlated: 353.2. FT-IR (ATR) \u03bd (cm\u2212 1): 1678 cm\u2212 1 (C\u2013 \u2013O stretch), 1580 13\n C NMR (151 MHz, DMSO) \u03b4 (ppm): 158.17, 157.95, 157.85, 157.66,\n(pyridyl C\u2013\u2013N). 157.61, 157.09, 156.40, 156.18, 155.64, 153.53, 153.20, 152.47,\n 152.24, 151.91, 151.76, 150.72, 149.46, 148.74, 140.46, 139.07,\n4.2.4. Synthesis of the s-triazine ligand N2,N4,N6-tris(3-(2-(pyridin-2-yl)- 138.99, 138.48, 138.21, 138.14, 138.07, 137.90, 136.76, 128.39,\n1H-benzo[d]imidazol-1-yl)propyl)-1,3,5-triazine-2,4,6-triamine (6) 128.30, 128.23, 128.03, 127.78, 127.31, 127.28, 126.34, 125.96,\n To a stirring solution of 3-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1- 125.49, 125.13, 124.99, 124.85, 124.77, 124.72, 124.47, 124.20,\nyl)propan-1-amine (5) (0.501 g, 1.98 mmol) in DMF (10 mL), cyanuric 123.82, 115.43, 115.43, 113.35, 113.35, 78.31, 78.31, 43.79, 43.73,\nchloride (0.0978 g, 0.528 mmol) and potassium carbonate (0.274 g, 37.61, 29.78, 28.69, 28.69. FT-IR (ATR) \u03bd (cm\u2212 1): 1450 (C=Nimine), 834\n1.982 mmol) were sequentially added. The reaction mixture was stirred (P\u2013F). MP (\u25e6 C): 224.6 (decomp.). MS (HR-ESI, m/z): Observed:\nat 120 \u25e6 C for 72 h. The reaction vessel was allowed to cool to room 325.0881 (60% [C35H30N7Ru]2+), Calculated: 325.0798. Purity:\ntemperature and toluene (300 mL) was added. Excess solvent was 98.45%, by LC (tR = 2.034 min).\nremoved in vacuo and the residue was re-dissolved in DCM (20 mL) and\nwashed with two aliquots of a saturated brine solution (2 \u00d7 20 mL). The 4.3.2. The synthesis of the trisamine-2-pyridylbenzimidazole ruthenium(II)\norganic extracts were collected and dried over sodium sulfate. The polypyridyl complex (Ru-2)\nexcess solvent was removed via rotary evaporation and the crude residue The trisamine-based ligand, tris(2-(2-(pyridin-2-yl)-1H-benzo[d]\nwas purified using column chromatography. Compound 6 was isolated imidazol-1-yl)ethyl)amine (9) (0.0447 g, 0.0588 mmol) was reacted\nas an off-white solid (0.539 g, 0.648 mmol). Rf (2: 1 petroleum ether: with cis-dichlorobis(bipyridine)ruthenium(II) (0.0854 g, 0.1764 mmol)\nethyl acetate): 0.48. Yield: 33%. 1H NMR (400 MHz, DMSO) \u03b4(ppm): under reflux at 78 \u25e6 C overnight. Thereafter, NH4PF6 (0.173 g, 1.06\n8.74 (d, J = 4.4 Hz, 3H, ArH), 8.33 (d, J = 7.9 Hz, 3H, ArH), 8.01 (t, J = mmol) was added and was allowed to stir for a further 1 h. The desired\n7.8 Hz, 3H, ArH), 7.68 (ddd, J = 22.7, 15.5, 7.8 Hz, 7H, ArH), 7.52 (dd, complex (Ru-2) was isolated as a dark red solid (0.0688 g, 0.0241 mmol)\nJ = 7.9, 4.6 Hz, 4H, ArH), 7.37\u20137.17 (m, 7H, ArH), 6.91 (br s, 2H, NH), by suction filtration and washed with cold ethanol (10 mL). Yield: 42%.\n5.75 (s, 1H, NH), 4.82 (t, J = 7.2 Hz, 6H, CH2), 2.97 (dd, J = 12.5, 7.3 1\n H NMR (600 MHz, DMSO) \u03b4 (ppm): 8.82\u20138.71 (m, 12H, ArH), 8.57 (t,\nHz, 6H, CH2), 1.98\u20131.85 (m, 6H, CH2). 13C NMR (151 MHz, CDCl3) \u03b4 J = 9.0 Hz, 3H, ArH), 8.48 (d, J = 9.3 Hz, 3H, ArH), 8.24\u20138.17 (m, 6H,\n(ppm): 156.03, 151.17, 149.66, 142.57, 137.89, 136.84, ArH), 8.17\u20137.93 (m, 16H, ArH), 7.88\u20137.81 (m, 7H, ArH), 7.77 (t, J = 5.7\n124.69,123.69, 122.81, 122.32, 121.94, 120.02, 112.51, 111.23, 43.32, Hz, 3H, ArH), 7.72\u20137.44 (m, 34H, ArH), 7.29\u20136.97 (m, 22H, ArH), 5.77\n38.13, 30.65. FT-IR (ATR) \u03bd (cm\u2212 1): 1680 (triazine C\u2013 \u2013N, and (dd, J = 12.5, 8.4 Hz, 3H, ArH), 5.11\u20134.87 (m, 6H, CH2), 3.50\u20133.34 (m,\n\n 7\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\n7H, CH2). 13C NMR (151 MHz, DMSO) \u03b4 (ppm): 157.76, 157.64, 4.4. Photophysical and photochemical properties\n157.33, 156.96, 156.78, 156.67, 153.14, 152.05, 151.91, 151.50,\n151.45, 151.34, 150.68, 150.53, 149.17, 148.43, 148.38, 140.54, Singlet oxygen quantum yields (\u0278\u0394) and emission quantum yields\n138.59, 138.51, 138.37, 138.28, 138.16, 138.11, 138.01, 137.13, (\u0278em) were determined using the comparative method, reported in the\n137.08, 128.31, 127.96, 127.52, 127.45, 126.42, 126.36, 125.77, literature [44,45] with the necessary equations provided in the Sup\u00ad\n124.84, 124.74, 124.60, 124.05, 123.86, 115.79, 113.25, 112.98, plementary Information. Tris(bipyridine)ruthenium(II) chloride ([Ru\n109.20, 53.55, 8.99. FT-IR (ATR) \u03bd (cm\u2212 1): 1438 (C=Nimine), 832 (bpy)3Cl2]) served as a benchmark with, \u0278\u0394 and \u0278em values of 0.57 and\n(P\u2013F). MP (\u25e6 C): 229.1 (decomp.). MS (HR-ESI, m/z): Observed: 0.018 in DMF and acetonitrile, respectively [46\u201348]. When calculating\n325.0867 (20% [C34H28N8Ru]2+), Calculated: 325.0735. Purity: the singlet oxygen quantum yields (\u0278\u0394) the absorbance of the sample\n99.50%, by LC (tR = 3.724 min). was not corrected for.\n\n4.3.3. The synthesis of the trisamine-2-quinolylbenzimidazole ruthenium 4.5. Cell culture\n(II) polypyridyl complex (Ru-3)\n A mixture of the trimeric 2-quinolylbenzimidazole ligand, tris(2-(2- The HeLa cervical cancer cell line was cultured and maintained in\n(quinolin-2-yl)-1H-benzo[d]imidazol-1-yl)ethyl)amine (10) (0.0520 g, Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma Aldrich,\n0.0602 mmol) and cis-dichlorobis(bipyridine)ruthenium(II) (0.0875 g, USA). The growth medium was supplemented with 10% heat-\n0.181 mmol) was refluxed at 78 \u25e6 C overnight. Thereafter, NH4PF6 inactivated foetal bovine serum, 100 \u03bcg/mL streptomycin, and 100 U/\n(0.0588 g, 0.361 mmol) was added and allowed to stir for 1 h. The mL penicillin (Gibco, Life Technologies, USA). The cells were main\u00ad\ndesired complex (Ru-3) was isolated as a red solid (0.142 g, 0.487 tained in an environment with 5% CO2 at 37 \u25e6 C to maintain physio\u00ad\nmmol) by suction filtration and washed with cold ethanol (10 mL). logical pH and temperature respectively. The culture medium was\nYield: 80%. 1H NMR (600 MHz, DMSO) \u03b4 (ppm): 8.91\u20138.78 (m, 2H, replaced every 48\u201372 h.\nArH), 8.74\u20138.69 (m, 1H, ArH), 8.59\u20138.43 (m, 8H, ArH), 8.40\u20138.20 (m,\n7H, ArH), 8.13\u20137.67 (m, 20H, ArH), 7.62\u20137.36 (m, 14H, ArH), 4.5.1. Cytotoxicity studies\n7.28\u20137.18 (m, 8H, ArH), 7.17\u20136.97 (m, 8H, ArH), 6.87 (d, J = 8.1 Hz, Briefly, HeLa cells were seeded at a density of 4500 cells/ well and\n2H, ArH), 6.82\u20136.61 (m, 2H, ArH), 5,53 (t, J = 8,2 Hz, 3H, consequently incubated at 37 \u25e6 C for 48 h to enable adhesion. Thereafter,\nArH),5.33\u20134.02 (m, 6H, CH2), 3.13\u20132.93 (m, 6H, CH2). 13C NMR (151 the cells were incubated for 48 h with either the vehicle control (0.1%\nMHz, DMSO) \u03b4 (ppm): 158.27, 157.42, 157.07, 156.99, 151.16, DMSO) or various concentrations (5\u201335 \u03bcM) of the tested ruthenium(II)\n150.13, 149.24, 146.61, 142.51, 141.08, 139.47, 139.07, 137.45, complexes. The original medium was removed after 48 h and replaced\n137.24, 136.78, 132.05, 130.14, 129.87, 128.59, 128.42, 127.71, with fresh RPMI 1640 without phenol red. The cells were photo\u00ad\n127.61, 125.89, 125.03, 124.73, 124.04, 123.11, 121.73, 120.42, irradiated with a 455 nm Thorlabs M455L3 LED for 60 min (330 mW.\n115.73, 114.01, 110.53, 55.33, 54.41, 43.66, 40.88. FT-IR (ATR) \u03bd cm\u2212 2). Fresh RPMI was subsequently added, and the cells were incu\u00ad\n(cm\u2212 1): 1458 (C=Nimine), 836 (P\u2013F). MP (\u25e6 C): 234.7 (decomp.). MS bated for an additional 24 h. A separate set of cells were incubated with\n(HR-ESI, m/z): Observed: 415.1322 (100% [C114H91N22Ru3]5+), the tested complexes at concentrations ranging from 5 to 300 \u03bcM in the\nCalculated: 415.0991. Purity: 98%, by LC (tR = 2.009 min). absence of light. Cell proliferation and viability were quantified by\n treating the cells with 4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-tetra\u00ad\n4.3.4. The synthesis of the ruthenium(II) polypyridyl complex (Mono) zol-3-ium-5-yl)benzene-1,3-disulfonate (WST-1) according to a proced\u00ad\n The 1-propyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole ligand (13) ure described by Ishiyama et al. [74] The absorbance at 420 nm was\n(0.0289 g, 0.122 mmol) was dissolved in anhydrous ethanol (5 mL) measured with a Molecular Devices Spectra Max M5 plate reader. The\nunder argon. To this stirring solution, cis-dichlorobis(bipyridine)ruthe\u00ad absorbances recorded were appropriately adjusted for the growth media\nnium(II) (0.0651 g, 0.134 mmol) was added and the reaction mixture (RPMI 1640) and the respective complexes.\nwas refluxed at 78 \u25e6 C overnight. After 24 h, the reaction mixture was\ncooled to room temperature and a solution of ammonium hexa\u00ad 4.5.2. Intracellular ROS (DCFDA Assay)\nfluorophosphate (0.0397 g, 0.244 mmol) in anhydrous ethanol (2 mL) The 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate (DCFDA) assay was\nwas added, and the reaction mixture was stirred for an additional 30 min used to quantify the production of intracellular reactive oxygen species\nat room temperature. The contents of the reaction flask were subse\u00ad (ROS) by the ruthenium(II) complexes [75]. HeLa cells were plated at a\nquently filtered through Celite\u00ae. Excess DCM was removed under density of 2 \u00d7 104 cells/ well and allowed to adhere overnight. After\nreduced pressure, which resulted in a burgundy solid precipitate. The adhesion, the cells were incubated with 35 \u03bcM of each of the ruthenium\ndesired complexes were isolated by suction filtration and washed with (II) complexes for 4 h in the dark. Thereafter, DCFDA (at a concentration\ncold ethanol (10 mL). Yield: 97.1% 1H NMR (600 MHz, DMSO) \u03b4 of 10 \u03bcM) was added and the cells were incubated in the dark for 30 min.\n(ppm): 8.84 (dd, J = 12.7, 5.7 Hz, 3H, ArH), 8.73 (d, J = 8.1 Hz, 1H, The DCFDA solution was removed and the cells were washed three times\nArH), 8.64 (d, J = 8.3 Hz, 1H, ArH), 8.23 (dd, J = 14.9, 7.3 Hz, 2H, ArH), with PBS to remove any extracellular DCFDA and ruthenium(II) com\u00ad\n8.16 (dt, J = 15.9, 7.9 Hz, 2H, ArH), 8.07 (t, J = 7.9 Hz, 1H, ArH), 8.00 plexes. The cells were subjected to irradiation with a ThorLabs 455 L3\n(d, J = 8.5 Hz, 1H, ArH), 7.92 (d, J = 5.5 Hz, 1H, ArH), 7.92 (d, J = 5.5 LED for 15 min. Similarly, another 96-well plate was maintained in the\nHz, 1H, ArH), 7.78 (ddd, J = 17.9, 15.9, 5.5 Hz, 4H, ArH), 7.59 (t, J = dark. Fluorescence was measured using a multi-plate reader with exci\u00ad\n6.6 Hz, 1H, ArH), 7.57\u20137.51 (m, 2H, ArH), 7.51\u20137.43 (m, 3H, ArH), 7.08 tation and emission wavelengths of 485 nm and 353 nm, respectively.\n(t, J = 7.8 Hz, 1H, ArH), 5.71 (d, J = 8.3 Hz, 1H, ArH), 4.98\u20134.80 (m, 2H, H2O2 was used as a positive control and vehicle-treated (0.1% DMSO)\nCH2), 2.00\u20131.85 (m, 2H, CH2), 0.89 (t, J = 7.4 Hz, 3H, CH3). 13C NMR cells were used as a negative control.\n(151 MHz, DMSO) \u03b4 (ppm): 157.96, 157.61, 157.08, 153.09, 152.26,\n151.83, 150.73, 148.78, 140.38, 138.69, 138.47, 138.20, 138.10, 4.5.3. Clonogenic assays\n137.93, 137.00, 128.37, 128.28, 128.21, 128.01, 126.32, 126.03, 125.4, HeLa cells were seeded at a density of 1.5 \u00d7 105 in 35 mm dishes and\n124.99, 124.87, 124.70, 124.43, 115.36, 113.60, 46.99, 23.02, 11.06. were incubated for 24 h at 37 \u25e6 C to allow adhesion. Thereafter, the cells\nFT-IR (ATR) \u03bd (cm\u2212 1): 1446 (C=Nimine), 830 (P\u2013F). MP (\u25e6 C): 297.8 were treated with various concentrations (1/2 IC50, IC50 and 2\u00d7 IC50) of\n(decomp.). MS (HR-ESI, m/z): Observed: 325.5878 (100% [M-2PF6]2+), selected ruthenium(II) polypyridyl complexes (Ru-2, Ru-3 and Mono)\nCalculated: 325.5843. Purity: 97%, by LC (tR = 2.799 min). or 0.1% DMSO (vehicle) for 24 h and were either maintained in the\n absence of light or photoirradiated with a 455 nm Thorlabs M455L3 LED\n for 60 min (330 mW.cm\u2212 2). The cells were then subjected to clonogenic\n\n 8\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n\nassays in drug free media and surviving cells were determined after [11] J. Karges, H. Chao, G. Gasser, Critical discussion of the applications of metal\n complexes for 2-photon photodynamic therapy, J. Biol. Inorg. Chem. 25 (8) (2020)\n12\u201315 days, as previously described by Kimani and co-workers [76].\n 1035\u20131050.\n [12] S. Bonnet, Why develop photoactivated chemotherapy? Dalton Trans. 47 (31)\nCRediT authorship contribution statement (2018) 10330\u201310343.\n [13] H. Kostron, T. Hasan, Photodynamic Medicine: From Bench to Clinic,\n Photodynamic Medicine: From Bench to Clinic, Royal Society of Chemistry,\n Athi Welsh: Writing \u2013 original draft, Investigation, Formal analysis, Cambridge, 2016.\nData curation. Refilwe Matshitse: Writing \u2013 review & editing, Inves\u00ad [14] L.C.-C. Lee, K.K.-W. Lo, Luminescent and Photofunctional Transition Metal\n Complexes: From Molecular Design to Diagnostic and Therapeutic Applications,\ntigation, Data curation. Saif F. Khan: Writing \u2013 review & editing, J. Am. Chem. Soc. 144 (32) (2022) 14420\u201314440.\nInvestigation, Data curation. Tebello Nyokong: Writing \u2013 review & [15] L. Zhang, N. Montesdeoca, J. Karges, H. Xiao, Immunogenic Cell Death Inducing\nediting, Resources. Sharon Prince: Writing \u2013 review & editing, Super\u00ad Metal Complexes for Cancer Therapy, Angew. Chem. Int. Ed. 62 (21) (2023)\n e202300662.\nvision, Resources, Project administration, Funding acquisition. Gregory [16] D.V. Straten, V. Mashayekhi, H.S.D. Bruijn, S. Oliveira, D.J. Robinson, Oncologic\nS. Smith: Writing \u2013 review & editing, Supervision, Resources, Project Photodynamic Therapy : Basic Principles , Current Clinical Status and Future\nadministration, Funding acquisition, Conceptualization. Directions, Cancers (Basel) 9 (2) (2017) 19.\n [17] S. Monro, K.L. Colon, H. Yin, J. Roque, P. Konda, S. Gujar, R.P. Thummel, L. Lilge,\n C.G. Cameron, S.A. McFarland, Transition Metal Complexes and Photodynamic\n Therapy from a Tumor-Centered Approach : Challenges , Opportunities , and\nDeclaration of competing interest Highlights from the Development of TLD1433, Chem. Rev. 119 (2019) 797\u2013828.\n [18] M.H. Keefe, K.D. Benkstein, J.T. Hupp, Luminescent sensor molecules based on\n coordinated metals: a review of recent developments, Coord. Chem. Rev. 205 (1)\n The authors declare that they have no known competing financial (2000) 201\u2013228.\ninterests or personal relationships that could have appeared to influence [19] C.K. Prier, D.A. Rankic, D.W.C. MacMillan, Visible Light Photoredox Catalysis with\n Transition Metal Complexes: Applications in Organic Synthesis, Chem. Rev. 113 (7)\nthe work reported in this paper.\n (2013) 5322\u20135363.\n [20] J. Karges, T. Yempala, M. Tharaud, D. Gibson, G. Gasser, A Multi-action and Multi-\nData availability target RuII\u2013PtIV Conjugate Combining Cancer-Activated Chemotherapy and\n Photodynamic Therapy to Overcome Drug Resistant Cancers, Angew. Chem. Int.\n Ed. 59 (18) (2020) 7069\u20137075.\n Data will be made available on request. [21] J. Karges, F. Heinemann, M. Jakubaszek, F. Maschietto, C. Subecz, M. Dotou,\n R. Vinck, O. Blacque, M. Tharaud, B. Goud, E. Vin\u0303uelas Zah\u0131\u0301nos, B. Spingler,\n I. Ciofini, G. Gasser, Rationally Designed Long-Wavelength Absorbing Ru(II)\nAcknowledgments Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy, J. Am.\n Chem. Soc. 142 (14) (2020) 6578\u20136587.\n We gratefully acknowledge and thank the University of Cape Town, [22] L. Conti, S. Ciambellotti, G.E. Giacomazzo, V. Ghini, L. Cosottini, E. Puliti,\n M. Severi, E. Fratini, F. Cencetti, P. Bruni, B. Valtancoli, C. Giorgi, P. Turano,\nthe National Research Foundation of South Africa (UID:129288, Ferritin nanocomposites for the selective delivery of photosensitizing ruthenium-\n120815), the International Centre for Genetic Engineering and polypyridyl compounds to cancer cells, Inorgan. Chem. Front. 9 (6) (2022)\nBiotechnology (ICGEB), the DSI-CSIR Photonics Centre - African Laser 1070\u20131081.\n [23] F. Li, E.J. Harry, A.L. Bottomley, M.D. Edstein, G.W. Birrell, C.E. Woodward, F.\nCentre and the South African Medical Research Council (SAMRC) under\n R. Keene, J.G. Collins, Dinuclear ruthenium(ii) antimicrobial agents that\na Self-Initiated Research Grant for financial support. The views and selectively target polysomes in vivo, Chem. Sci. 5 (2) (2014) 685\u2013693.\nopinions expressed are those of the author(s) and do not necessarily [24] F. Li, M. Feterl, Y. Mulyana, J.M. Warner, J.G. Collins, F.R. Keene, In vitro\n susceptibility and cellular uptake for a new class of antimicrobial agents: dinuclear\nrepresent the official views of the SAMRC.\n ruthenium(II) complexes, J. Antimicrob. Chemother. 67 (11) (2012) 2686\u20132695.\n [25] F. Li, Y. Mulyana, M. Feterl, J.M. Warner, J.G. Collins, F.R. Keene, The\nAppendix A. Supplementary data antimicrobial activity of inert oligonuclear polypyridylruthenium(ii) complexes\n against pathogenic bacteria, including MRSA, Dalton Trans. 40 (18) (2011)\n 5032\u20135038.\n Supplementary data to this article can be found online at https://doi. [26] F.E. Poynton, S.A. Bright, S. Blasco, D.C. Williams, J.M. Kelly, T. Gunnlaugsson,\norg/10.1016/j.jinorgbio.2024.112545. The development of ruthenium(ii) polypyridyl complexes and conjugates for in\n vitro cellular and in vivo applications, Chem. Soc. Rev. 46 (24) (2017) 7706\u20137756.\n [27] X.Y. Ng, K.W. Fong, L.V. Kiew, P.Y. Chung, Y.K. Liew, N. Delsuc, M. Zulkefeli, M.\nReferences L. Low, Ruthenium(II) polypyridyl complexes as emerging photosensitisers for\n antibacterial photodynamic therapy, J. Inorg. Biochem. 250 (2024) 112425.\n [28] L.-I. Rylands, A. Welsh, K. Maepa, T. Stringer, D. Taylor, K. Chibale, G.S. Smith,\n [1] Y.-P. Ho, S.C.F. Au-Yeung, K.K.W. To, Platinum-based anticancer agents:\n Structure-activity relationship studies of antiplasmodial cyclometallated\n Innovative design strategies and biological perspectives, Med. Res. Rev. 23 (5)\n ruthenium (II), rhodium (III) and iridium (III) complexes of 2-\n (2003) 633\u2013655.\n phenylbenzimidazoles, Eur. J. Med. Chem. 161 (2019) 11\u201321.\n [2] C. Zhang, C. Xu, X. Gao, Q. Yao, Platinum-based drugs for cancer therapy and anti-\n [29] N. Baartzes, A. Jordaan, D.F. Warner, J. Combrinck, D. Taylor, K. Chibale, G.\n tumor strategies, Theranostics 12 (5) (2022) 2115\u20132132.\n S. Smith, Antimicrobial evaluation of neutral and cationic iridium(III) and rhodium\n [3] E.V. Sazonova, G.S. Kopeina, E.N. Imyanitov, B. Zhivotovsky, Platinum drugs and\n (III) aminoquinoline-benzimidazole hybrid complexes, Eur. J. Med. Chem. 206\n taxanes: can we overcome resistance? Cell Death Dis. 7 (1) (2021) 155.\n (2020) 112694.\n [4] S. Jin, Y. Guo, Z. Guo, X. Wang, Monofunctional Platinum(II) Anticancer Agents,\n [30] J. Yellol, S.A. Pe\u0301rez, G. Yellol, J. Zajac, A. Donaire, G. Vigueras, V. Novohradsky,\n Pharmaceuticals 14 (2) (2021) 133\u2013156.\n C. Janiak, V. Brabec, J. Ruiz, Highly potent extranuclear-targeted luminescent\n [5] C.S. Allardyce, P.J. Dyson, Metal-based drugs that break the rules, Dalton Trans. 45\n iridium(iii) antitumor agents containing benzimidazole-based ligands with a\n (2016) 3201\u20133209.\n handle for functionalization, Chem. Commun. 52 (2016) 14165\u201314168.\n [6] G.K. Gransbury, P. Kappen, C.J. Glover, J.N. Hughes, A. Levina, P.A. Lay, I.\n [31] G.S. Yellol, A. Donaire, J.G. Yellol, V. Vasylyeva, C. Janiak, J. Ruiz, On the\n F. Musgrave, H.H. Harris, Comparison of KP1019 and NAMI-A in tumour-mimetic\n antitumor properties of novel cyclometalated benzimidazole Ru(ii), Ir(iii) and Rh\n environments, Metallomics 8 (2016) 762\u2013773.\n (iii) complexes, Chem. Commun. 49 (2013) 11533\u201311535.\n [7] J. Karges, Clinical Development of Metal Complexes as Photosensitizers for\n [32] B. Liu, S. Monro, M.A. Jabed, C.G. Cameron, K.L. Colo\u0301n, W. Xu, S. Kilina, S.\n Photodynamic Therapy of Cancer, Angew. Chem. Int. Ed. 61 (5) (2022)\n A. McFarland, W. Sun, Neutral iridium(iii) complexes bearing BODIPY-substituted\n e202112236.\n N-heterocyclic carbene (NHC) ligands: synthesis, photophysics, in vitro theranostic\n [8] M.A\u0301. Mart\u00ednez, M.P. Carranza, A. Massaguer, L. Santos, J.A. Organero, C. Aliende,\n photodynamic therapy, and antimicrobial activity, Photochem. Photobiol. Sci. 18\n R. de Llorens, I. Ng-Choi, L. Feliu, M. Planas, A.M. Rodr\u00edguez, B.R. Manzano,\n (10) (2019) 2381\u20132396.\n G. Espino, F.A. Jalo\u0301n, Synthesis and Biological Evaluation of Ru(II) and Pt(II)\n [33] M. Lari, M. Mart\u00ednez-Alonso, N. Busto, B.R. Manzano, A.M. Rodr\u00edguez, M.I. Acun\u0303a,\n Complexes Bearing Carboxyl Groups as Potential Anticancer Targeted Drugs, Inorg.\n F. Dom\u00ednguez, J.L. Albasanz, J.M. Leal, G. Espino, B. Garc\u00eda, Strong Influence of\n Chem. 56 (22) (2017) 13679\u201313696.\n Ancillary Ligands Containing Benzothiazole or Benzimidazole Rings on\n [9] K. Singha, P. Laha, F. Chandra, N. Dehury, A.L. Koner, S. Patra, Long-Lived\n Cytotoxicity and Photoactivation of Ru(II) Arene Complexes, Inorg. Chem. 57 (22)\n Polypyridyl Based Mononuclear Ruthenium Complexes: Synthesis, Structure, and\n (2018) 14322\u201314336.\n Azo Dye Decomposition, Inorg. Chem. 56 (11) (2017) 6489\u20136498.\n [34] P. Sen, N. Nwahara, T. Nyokong, Photodynamic antimicrobial activity of\n[10] S.-Q. Zhang, T.-T. Meng, J. Li, F. Hong, J. Liu, Y. Wang, L.-H. Gao, H. Zhao, K.-\n benzimidazole substituted phthalocyanine when conjugated to Nitrogen Doped\n Z. Wang, Near-IR/Visible-Emitting Thiophenyl-Based Ru(II) Complexes: Efficient\n Graphene Quantum Dots against Staphylococcus aureus, Main Group Chem. 20\n Photodynamic Therapy, Cellular Uptake, and DNA Binding, Inorg. Chem. 58 (20)\n (2021) 175\u2013191.\n (2019) 14244\u201314259.\n\n\n 9\n\fA. Welsh et al. Journal of Inorganic Biochemistry 256 (2024) 112545\n\n[35] S. Swavey, A. Wertz, J. Erb, Bichromophoric Properties of Ruthenium(II) [56] L. Conti, E. Macedi, C. Giorgi, B. Valtancoli, V. Fusi, Combination of light and Ru\n Polypyridyl Complexes Bridged by Boron Dipyrromethenes: Synthesis, (II) polypyridyl complexes: Recent advances in the development of new anticancer\n Electrochemical, Spectroscopic, Computational Evaluation, and Plasmid DNA drugs, Coord. Chem. Rev. 469 (2022) 214656.\n Photoreactions, Eur. J. Inorg. Chem. 2019 (32) (2019) 3690\u20133698. [57] J. Liu, C. Zhang, T.W. Rees, L. Ke, L. Ji, H. Chao, Harnessing ruthenium(II) as\n[36] S.A. Archer, A. Raza, F. Dro\u0308ge, C. Robertson, A.J. Auty, D. Chekulaev, J. photodynamic agents: Encouraging advances in cancer therapy, Coord. Chem. Rev.\n A. Weinstein, T. Keane, A.J.H.M. Meijer, J.W. Haycock, S. MacNeil, J.A. Thomas, 363 (2018) 17\u201328.\n A dinuclear ruthenium(ii) phototherapeutic that targets duplex and quadruplex [58] J. Karges, S. Kuang, F. Maschietto, O. Blacque, I. Ciofini, H. Chao, G. Gasser,\n DNA, Chem. Sci. 10 (12) (2019) 3502\u20133513. Rationally designed ruthenium complexes for 1- and 2-photon photodynamic\n[37] L. Zeng, S. Kuang, G. Li, C. Jin, L. Ji, H. Chao, A GSH-activatable ruthenium(ii)-azo therapy, Nat. Commun. 11 (1) (2020) 3262.\n photosensitizer for two-photon photodynamic therapy, Chem. Commun. 53 (12) [59] I. Koyuncu, Y. T\u00fcl\u00fcce, H. Slahaddin Qadir, M. Durgun, C.T. Supuran, Evaluation of\n (2017) 1977\u20131980. the anticancer potential of a sulphonamide carbonic anhydrase IX inhibitor on\n[38] L. Conti, E. Macedi, C. Giorgi, B. Valtancoli, V. Fusi, Combination of light and Ru cervical cancer cells, J. Enzyme Inhib. Med. Chem. 34 (1) (2019) 703\u2013711.\n (II) polypyridyl complexes: Recent advances in the development of new anticancer [60] H.E. Hashem, A.E.-G.E. Amr, E.S. Nossier, M.M. Anwar, E.M. Azmy, New\n drugs, Coord. Chem. Rev. 469 (2022) 214656. Benzimidazole-, 1,2,4-Triazole-, and 1,3,5-Triazine-Based Derivatives as Potential\n[39] Z. Huang, A.P. King, J. Lovett, B. Lai, J.J. Woods, H.H. Harris, J.J. Wilson, EGFRWT and EGFRT790M Inhibitors: Microwave-Assisted Synthesis, Anticancer\n Photochemistry and in vitro anticancer activity of Pt(iv)Re(i) conjugates, Chem. Evaluation, and Molecular Docking Study, ACS Omega 7 (8) (2022) 7155\u20137171.\n Commun. 57 (85) (2021) 11189\u201311192. [61] S. Cascioferro, B. Parrino, V. Spano\u0300, A. Carbone, A. Montalbano, P. Barraja,\n[40] X. Zeng, Y. Wang, J. Han, W. Sun, H.-J. Butt, X.-J. Liang, S. Wu, Fighting against P. Diana, G. Cirrincione, 1,3,5-Triazines: A promising scaffold for anticancer drugs\n Drug-Resistant Tumors using a Dual-Responsive Pt(IV)/Ru(II) Bimetallic Polymer, development, Eur. J. Med. Chem. 142 (2017) 523\u2013549.\n Adv. Mater. 32 (43) (2020) 2004766. [62] R. Satrialdi, V. Munechika, Y. Biju, H. Takano, Y. Harashima, Yamada, The\n[41] A. Welsh, L. Rylands, V.B. Arion, S. Prince, G.S. Smith, Synthesis and optimization of cancer photodynamic therapy by utilization of a pi-extended\n antiproliferative activity of benzimidazole-based, trinuclear neutral porphyrin-type photosensitizer in combination with MITO-Porter, Chem. Commun.\n cyclometallated and cationic, N^N-chelated ruthenium(ii) complexes, Dalton Trans. 56 (7) (2020) 1145\u20131148.\n 49 (2020) 1143\u20131156. [63] L. Wang, H. Yin, M.A. Jabed, M. Hetu, C. Wang, S. Monro, X. Zhu, S. Kilina, S.\n[42] A. Welsh, M. Mbaba, S. Prince, G.S. Smith, Synthesis, molecular modeling and A. McFarland, W. Sun, \u03c0-Expansive Heteroleptic Ruthenium(II) Complexes as\n preliminary anticancer evaluation of 2-ferrocenylbenzimidazole metallofragments, Reverse Saturable Absorbers and Photosensitizers for Photodynamic Therapy,\n J. Mol. Struct. 1246 (2021) 131122. Inorg. Chem. 56 (6) (2017) 3245\u20133259.\n[43] N. Msimango, A. Welsh, S. Prince, G.S. Smith, Synthesis and anticancer evaluation [64] L.K. McKenzie, H.E. Bryant, J.A. Weinstein, Transition metal complexes as\n of trinuclear N^N quinolyl-benzimidazole-based PGM complexes, Inorg. Chem. photosensitisers in one- and two-photon photodynamic therapy, Coord. Chem. Rev.\n Commun. 144 (2022) 109840. 379 (2019) 2\u201329.\n[44] S. Fery-Forgues, D. Lavabre, Are Fluorescence Quantum Yields So Tricky to [65] R. Matshitse, N. Nwaji, M. Mananga, E. Prinsloo, T. Nyokong, Effect of number of\n Measure? A Demonstration Using Familiar Stationery Products, J. Chem. Educ. 76 positive charges on the photophysical and photodynamic therapy activities of\n (9) (1999) 1260. quarternary benzothiazole substituted zinc phthalocyanine, J. Photochem.\n[45] A. Ogunsipe, J.-Y. Chen, T. Nyokong, Photophysical and photochemical studies of Photobiol. A Chem. 367 (2018) 253\u2013260.\n zinc(ii) phthalocyanine derivatives\u2014effects of substituents and solvents, New J. [66] H. Tokunaga, T. Nakanishi, T. Iwata, D. Aoki, T. Saito, S. Nagase, F. Takahashi,\n Chem. 28 (7) (2004) 822\u2013827. N. Yaegashi, Y. Watanabe, Effects of chemotherapy on patients with recurrent\n[46] K. Suzuki, A. Kobayashi, S. Kaneko, K. Takehira, T. Yoshihara, H. Ishida, Y. Shiina, cervical cancer previously treated with concurrent chemoradiotherapy: a\n S. Oishi, S. Tobita, Reevaluation of absolute luminescence quantum yields of retrospective multicenter survey in Japan, Int. J. Clin. Oncol. 20 (3) (2015)\n standard solutions using a spectrometer with an integrating sphere and a back- 561\u2013565.\n thinned CCD detector, Phys. Chem. Chem. Phys. 11 (42) (2009) 9850\u20139860. [67] H.J. Long, Management of Metastatic Cervical Cancer: Review of the Literature,\n[47] W. Spiller, H. Kliesch, D. Wo\u0308hrle, S. Hackbarth, B. Ro\u0308der, G. Schnurpfeil, Singlet J. Clin. Oncol. 25 (20) (2007) 2966\u20132974.\n oxygen quantum yields of different photosensitizers in polar solvents and micellar [68] Y. Zhang, X. Zhang, H. Zhu, Y. Liu, J. Cao, D. Li, B. Ding, W. Yan, H. Jin, S. Wang,\n solutions, J. Porphyrins Phthalocyanines 2 (2) (1998) 145\u2013158. Identification of Potential Prognostic Long Non-Coding RNA Biomarkers for\n[48] A.M. Potocny, J.J. Teesdale, A. Marangoz, G.P.A. Yap, J. Rosenthal, Spectroscopic Predicting Recurrence in Patients with Cervical Cancer, Cancer Manag. Res. 12\n and 1O2 Sensitization Characteristics of a Series of Isomeric Re(bpy)(CO)3Cl (2020) 719\u2013730.\n Complexes Bearing Pendant BODIPY Chromophores, Inorg. Chem. 58 (8) (2019) [69] H. Zhu, H. Luo, W. Zhang, Z. Shen, X. Hu, X. Zhu, Molecular mechanisms of\n 5042\u20135050. cisplatin resistance in cervical cancer, Drug. Des. Devel. Ther. 10 (2016)\n[49] E.T. Papish, O.E. Oladipupo, Factors that influence singlet oxygen formation vs. 1885\u20131895.\n ligand substitution for light-activated ruthenium anticancer compounds, Curr. [70] E. Delaey, F. van Laar, D. De Vos, A. Kamuhabwa, P. Jacobs, P. de Witte,\n Opin. Chem. Biol. 68 (2022) 102143. A comparative study of the photosensitizing characteristics of some cyanine dyes,\n[50] C. Kreitner, K. Heinze, Excited state decay of cyclometalated polypyridine J. Photochem. Photobiol. B Biol. 55 (1) (2000) 27\u201336.\n ruthenium complexes: insight from theory and experiment, Dalton Trans. 45 (35) [71] A. Frei, R. Rubbiani, S. Tubafard, O. Blacque, P. Anstaett, A. Felgentra\u0308ger,\n (2016) 13631\u201313647. T. Maisch, L. Spiccia, G. Gasser, Synthesis, Characterization, and Biological\n[51] L.D. Ramos, H.M. da Cruz, K.P. Morelli Frin, Photophysical properties of rhenium Evaluation of New Ru(II) Polypyridyl Photosensitizers for Photodynamic Therapy,\n (i) complexes and photosensitized generation of singlet oxygen, Photochem. J. Med. Chem. 57 (17) (2014) 7280\u20137292.\n Photobiol. Sci. 16 (4) (2017) 459\u2013466. [72] N.M. Motimani, S. Ngubane, G.S. Smith, Polynuclear heteroleptic ruthenium(II)\n[52] S. Doose, H. Neuweiler, M. Sauer, Fluorescence Quenching by Photoinduced photoredox catalysts: Evaluation in blue-light-mediated, regioselective thiol-ene\n Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules, reactions, Polyhedron 212 (2022) 115616.\n Chem. Phys. Chem. 10 (9-10) (2009) 1389\u20131398. [73] M.D. Hossain, M.-A. Haga, B. Gholamkhass, K. Nozaki, M. Tsushima, N. Ikeda,\n[53] H.N. Akl, D. Salah, H.S. Abdel-Samad, A.A. Abdel Aziz, A.A. Abdel-Shafi, Fractional T. Ohno, Syntheses, Spectroelectrochemistry and Photoinduced Electron-Transfer\n dependence of the free energy of activation on the driving force of charge transfer Processes of Novel Ru and Os Dyad and Triad Complexes with Functionalized\n in the quenching of the excited states of substituted phenanthroline homoleptic Diimide Ligands, Collect. Czechoslov. Chem. Commun. 66 (2) (2001) 307\u2013337.\n ruthenium(ii) complexes in aqueous medium, RSC Adv. 13 (19) (2023) [74] M. Ishiyama, H. Tominaga, M. Shiga, K. Sasamoto, Y. Ohkura, K. Ueno,\n 13314\u201313323. A Combined Assay of Cell Vability and in Vitro Cytotoxicity with a Highly Water-\n[54] S.A. McFarland, A. Mandel, R. Dumoulin-White, G. Gasser, Metal-based Soluble Tetrazolium Salt, Neutral Red and Crystal Violet, Biol. Pharm. Bull. 19 (11)\n photosensitizers for photodynamic therapy: the future of multimodal oncology? (1996) 1518\u20131520.\n Curr. Opin. Chem. Biol. 56 (2020) 23\u201327. [75] M. Oparka, J. Walczak, D. Malinska, L.M.P.E. van Oppen, J. Szczepanowska, W.J.\n[55] N.U. Prajith, P.V. Priyanka, V. Alexander, Synthesis, characterization, H. Koopman, M.R. Wieckowski, Quantifying ROS levels using CM-H2DCFDA and\n photophysical, lipophilicity, and in vitro fluorescence studies of mono-, di-, and HyPer, Methods 109 (2016) 3\u201311.\n trinuclear Ru(II) polypyridyl complexes of pyridinyl benzimidazole derivatives, [76] S. Kimani, S. Chakraborty, I. Irene, J. de la Mare, A. Edkins, A. du Toit, B. Loos,\n J. Biol. Inorg. Chem. 27 (3) (2022) 357\u2013372. A. Blanckenberg, A. Van Niekerk, L.V. Costa-Lotufo, K.N. ArulJothi, S. Mapolie,\n S. Prince, The palladacycle, BTC2, exhibits anti-breast cancer and breast cancer\n stem cell activity, Biochem. Pharmacol. 190 (2021) 114598.\n\n\n\n\n 10\n\f", "pages_extracted": 10, "text_length": 85167}