A new family of luminescent iridium complexes: synthesis, optical, and cytotoxic studies.

{"full_text": " Dalton\n Transactions\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n View Article Online\n PAPER View Journal | View Issue\n\n\n\n\n A new family of luminescent iridium complexes:\n Cite this: Dalton Trans., 2023, 52,\n synthesis, optical, and cytotoxic studies\u2020\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n 6360\n Gonzalo Mill\u00e1n,\u2021a Mattia Nieddu, \u2021a,b Ic\u00edar P. L\u00f3pez,c Cintia Ezquerro,a\n Jes\u00fas R. Berenguer, a Ignacio M. Larr\u00e1yoz,d,e Jos\u00e9 G. Pichel *c,f and\n Elena Lalinde *a\n\n By using N,N-dibutyl-2,2\u2019-bipyridine-4,4\u2019-dicarboxamide as a diimine (dbbpy) and distinctive cyclometa-\n lated groups, this work reports a new family of cationic phosphorescent Ir(III) cyclometalated [Ir\n (C^N)2(N^N)]X compounds [C^N = di\ufb02uorophenylpyridine (dfppy) a, 2,6-di\ufb02uoro-3-(pyridin-2-yl)benz-\n aldehyde (CHO-dfppy) b, and 2,6-di\ufb02uoro-3-pyridin-2-yl-benzoic acid (COOH-dfppy) c; X = Cl\u2212 2a,b,c-\n Cl; X = PF6\u2212 2b,c-PF6]. For comparative purposes, the related complex [Ir(dfppy)2(H2dcbpy)]+ (3a-PF6)\n incorporating 3,3\u2019-dicarboxy-2,2\u2019-bipyridine as an auxiliary ligand (N^N = H2dcbpy) is also presented. All\n complexes have been fully characterized and their photophysical properties were investigated in detail.\n The theoretically calculated results obtained by density functional theory (DFT) and time-dependent\n density functional theory (TD-DFT) studies indicate that luminescence is derived from mixed 3ML\u2019CT (Ir \u2192\n N^N)/3LL\u2019CT (C^N \u2192 N^N) excited states with the predominant metal-to-diimine charge transfer charac-\n ter. Their antineoplastic activity against tumour cell lines A549 (lung carcinoma) and HeLa (cervix carci-\n noma), as well as the nontumor BEAS-2B (bronchial epithelium) cell line was assessed and \ufb02uorescence\n microscopy studies were performed for their cellular localization. Among them, 2a-Cl exhibited the most\n potent anticancer activity, being higher than cisplatin. However, 2b-Cl and 2c-Cl,-PF6 were the least\n Received 4th January 2023, toxic, while 2b-PF6 and 3a-PF6 exhibited only moderate activity. Confocal microscopy studies for 2a-Cl\n Accepted 4th April 2023\n suggest that complexes localize preferentially in the lysosomes and to a lesser extent in the cytoplasm,\n DOI: 10.1039/d3dt00028a but ultimately causing damage to the mitochondria. Finally, the potential photodynamic behaviour of\n rsc.li/dalton scarcely toxic complexes 2b-Cl, 2b-PF6, 2c-Cl and 3a-PF6 was also studied.\n\n\n\n Introduction chemotherapeutics in oncologic treatments.1\u201310 The cyto-\n toxicity of these drugs mainly relies on the formation of intras-\n Given their e\ufb03cacy, platinum-based agents (cisplatin, carbo- trand cross-links with guanine residues, triggering the inhi-\n platin, and oxaliplatin) are amongst the most prescribed bition of DNA transcription and apoptosis of cancer cells.11,12\n However, despite their e\ufb00ectiveness in cancer chemotherapy,\n their poor selectivity and the acquired resistance of some\n a\n Departamento de Qu\u00edmica-Centro de S\u00edntesis Qu\u00edmica de La Rioja, (CISQ), tumours together with the lack of e\ufb00ective approaches in the\n Universidad de La Rioja, 26006 Logro\u00f1o, Spain. E-mail: elena.lalinde@unirioja.es\n b\n treatment of aggressive metastatic cancers force the develop-\n Chair of Biogenic Functional Materials, Technical University of Munich, Schulgasse,\n 22, Straubing 94315, Germany\n ment of new drugs. To this end, the design of new cisplatin-\n c\n Lung Cancer and Respiratory Diseases Unit (CIBIR), Fundaci\u00f3n Rioja Salud, 26006 like or Pt(IV) derivatives and prodrugs to target specific recep-\n Logro\u00f1o, Spain. E-mail: jgpichel@riojasalud.es tors or tumour cells,13 including new delivery systems to\n reduce side e\ufb00ects,14,15 is a very active area that has been\n d\n Unidad Predepartamental de Enfermer\u00eda, Universidad de La Rioja, Duquesa de la\n Victoria 88, E\u201326006 Logro\u00f1o, Spain. E-mail: ignacio.larrayoz@unirioja.es\n e\n recently reviewed. Moreover, in recent decades, the search for\n Biomarkers and Molecular Signaling Unit (CIBIR), Fundaci\u00f3n Rioja Salud, 26006\n Logro\u00f1o, Spain\n nonplatinum metal-based drugs capable of combining high\n f\n Biomedical Research Networking Center in Respiratory Diseases (CIBERES), ISCIII, anticancer activity, low cytotoxicity and a mechanism of action\n Spain that di\ufb00ers from those of the cisplatin family has increased\n \u2020 Electronic supplementary information (ESI) available: Experimental section, exponentially.16\u201322 In this context, many transition metals have\n tables and figures giving structural, spectroscopic, photophysical, and theore-\n been tested as antineoplastic agents.19,23 In addition, the\n tical data, and biological studies for the compounds. CCDC 2231729 and\n 2231730. For ESI and crystallographic data in CIF or other electronic format see\n incorporation of new strategies, such as thermo-, chemo- and\n DOI: https://doi.org/10.1039/d3dt00028a photodynamic24 therapies, and also synergistic treatments as a\n \u2021 These authors have contributed equally to this work. combination of several modalities to treat cancer are providing\n\n\n 6360 | Dalton Trans., 2023, 52, 6360\u20136374 This journal is \u00a9 The Royal Society of Chemistry 2023\n\f View Article Online\n\n Dalton Transactions Paper\n\n excellent antitumor e\ufb00ects. In particular, photodynamic bearing an acid in the cyclometalated group show increased\n therapy (PDT) is an emerging cancer treatment strategy that solubility, even in water.41 Their optical properties supported\n shows fewer side e\ufb00ects and higher selectivity than conven- by theoretical calculations are presented. Their antineoplastic\n tional therapies. It involves the excitation of a non-toxic photo- activity against tumour cell lines A549 (lung carcinoma) and\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n sensitizer (PS) with light to produce a long-lasting triplet HeLa (cervix carcinoma) as well as the nontumor BEAS-2B\n excited state that can interact with oxygen (3O2) to produce (bronchial epithelium) cell lines was assessed and fluo-\n reactive oxygen species (ROS) through type I (electron transfer) rescence microscopy studies for their cellular localization were\n or type II (energy transfer) mechanisms. Singlet oxygen that is performed. Finally, the potential photodynamic properties of\n generated via the type II mechanism has been implicated as the scarcely toxic complexes 2b-Cl, 2b-PF6, 2c-Cl and 3a-PF6\n the most important mediator of the anticancer e\ufb00ects of PDT. were also evaluated.\n ROS are produced in a wide range of physiological processes,\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n particularly by mitochondria. Nevertheless, uncontrolled and\n excessive production of ROS, or a decreased ability of cells to Results and discussion\n scavenge ROS, gives rise to oxidative stress and subsequent\n damage to various cellular components, causing apoptosis and Synthesis and characterization\n cell necrosis.25,26 The HC^N compounds 2,6-difluoro-3-( pyridin-2-yl) benz-\n In this field, organometallic iridium complexes are an aldehyde (CHO-dfppyH)42 and 2,6-difluoro-3-pyridin-2-yl-\n attractive class of compounds that have demonstrated great benzoic acid (COOH-dfppyH),43 and the diimine N,N\u2032-dibutyl-\n potential as an alternative to platinum-based metallodrugs.27 2,2\u2032-bipyridine-4,4\u2032-dicarboxamide (dbbpy)44 have been pre-\n Iridium complexes have demonstrated promising antiproli- viously reported. The new organometallic chloride-\n ferative activity in vitro and/or in vivo through various mecha- bridged diiridium precursors [Ir(CHO-dfppy)2(\u00b5-Cl)]2 (1b) and\n nisms of action, such as disturbance of cellular redox homeo- [Ir(COOH-dfppy)2(\u00b5-Cl)]2 (1c) were synthesized following\n stasis, interaction with proteins, or regulation of non-apopto- similar procedures to those previously established for\n tic pathways.28\u201332 Moreover, due to their exceptional photo- [Ir(dfppy)2(\u00b5-Cl)]2 (1a).45 The mononuclear complexes 2-Cl\n physical properties and good cell permeability, phosphores- were prepared by refluxing a mixture of the chelating ligand\n cent cyclometalated iridium(III) complexes have been tested in dbbpy and the corresponding iridium precursor (1) in MeOH/\n cellular imaging as biomolecular probes,33\u201335 anticancer CH2Cl2 for 12 h (Scheme 1). Analogous complexes with PF6 as\n drugs, and photosensitizers (PSs) to produce singlet oxygen the counter anion (2b-PF6 and 2c-PF6) could be prepared as\n (1O2).26,36 As cyclometalated iridium complexes integrate pure complexes following a procedure described by Zhou\n the anticancer e\ufb03cacy and excellent phosphorescence pro- et al.,46 instead of anion exchange reactions starting from 2-Cl\n perties, they have shown great potential as theranostic derivatives. This procedure involves the in situ formation of the\n agents.26,34,35,37 In particular, cationic cyclometalated iridium solvated acetonitrile complexes [Ir(CHO-dfppy)2(NCMe)2]PF6\n (III) complexes with ancillary diimine ligands [Ir(C^N)2(N^N)]+ and [Ir(COOH-dfppy)2(MeCN)2], respectively, and the sub-\n stand out in this field because they display rich emissive-state sequent treatment with the dbbpy ligand (Scheme 1), avoiding\n characteristics, which include high quantum yields, large the presence of mixed counterions in the reaction media.\n Stokes shifts, long-lasting luminescence, and good photo- Finally, the synthesis of [Ir(dfppy)2(H2dcbpy)]PF6 (3a-PF6) was\n stability, which can be tuned depending on the C^N back- carried out following a similar procedure to that reported by\n bone and the diimine ligand. Commonly, the 3MLCT, ligand- Amouri, Barbieri et al.47 for related complexes with the 4,4\u2032-\n to-ligand charge-transfer (3LLCT), and intraligand (3IL) dicarboxy-2,2\u2032-bipyridine ligand (see the ESI\u2020 for details).\n excited states compete for the emission depending on the All of the products have been characterized using high-\n energy levels of frontier orbitals. Moreover, together with resolution mass spectrometry and di\ufb00erent spectroscopic\n their cationic nature, proper incorporation of substituents on means (IR and multinuclear 1H, 13C{1H}, and 19F NMR\n the cyclometalated group or the diimine ligand could result spectra and 1H\u20131H COSY, 1H\u201313C HMBC, HSQC correlation\n in the modification of their cell permeability and their experiments; see the ESI,\u2020 Experimental section). The ESI\n physio-chemical and biological activities.38\u201340 mass spectra of complexes 2 and 3a-PF6 confirmed the pres-\n Here, we report a new family of cationic Ir(III) cyclometa- ence of the molecular peak [M]+ with the expected isotopic\n lated [Ir(C^N)2(N^N)]+ compounds with N,N-dibutyl-2,2\u2032-bipyri- distribution, while the precursor complexes 1b and 1c\n dine-4,4\u2032-dicarboxamide as the diimine (dbbpy) and di\ufb00erent showed peaks at m/z 665.02 (100%) and 702.06 (100%)\n cyclometalated backbones (2a\u2013c) [C^N = difluorophenylpyri- corresponding to the bridge splitting [Ir(CHO-dfppy)2Cl +\n dine (dfppy) a, 2,6-difluoro-3-( pyridin-2-yl) benzaldehyde H]+ and [Ir(COOH-dfppy)2 + Na + H2O]+ species, respectively.\n (CHO-dfppy) b, and 2,6-difluoro-3-pyridin-2-yl-benzoic acid The FTIR spectra of complexes 2 and 3a-PF6 exhibit charac-\n (COOH-dfppy) c]. Due to the low solubility of 2b-Cl and 2c-Cl, teristic vibration bands of CvO (1660 cm\u22121) and N\u2013H of\n the related 2b-PF6 and 2c-PF6 complexes were prepared using amide groups (only for 2, 3350\u20133250 cm\u22121), and the com-\n PF6\u2212 as the counter anion. For comparison, the complex [Ir plexes with PF6 show an intense band at 800 cm\u22121. The\n (dfppy)2(H2dcbpy)]+ (3a-PF6; H2dcbpy = 3,3\u2032-dicarboxy-2,2\u2032- NMR images of the binuclear iridium complexes in CDCl3\n bipyridine) is also included. It is remarkable that complexes for 1b and in D2O/KOH mixture for 1c show the presence of\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 6360\u20136374 | 6361\n\f View Article Online\n\n Paper Dalton Transactions\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n Scheme 1 Synthesis of compounds 1, 2(a\u2013c) and 3a-PF6.\n\n\n\n only one type of chemically equivalent cyclometalated with this counter anion. Furthermore, the structures\n ligands, as expected for the formation of a single isomer of complex 2a-Cl and [2c]+ were determined by X-ray\n (two isomers are possible, a meso form and a racemic pair). di\ufb00raction.\n However, in DMSO solution two sets of cyclometalated Single yellow crystals of 2a-Cl were obtained by di\ufb00usion of\n ligands are generated, indicating the cleavage of the chlor- n-hexane into a saturated solution of the corresponding\n ide bridging system in the presence of the highly coordinat- complex in CH2Cl2 at room temperature. However, slow\n ing DMSO, to form [Ir(CHO-dfppy)2Cl(DMSO)] (1b-DMSO) di\ufb00usion of n-heptane into a solution of complex 2c-PF6 in\n (Fig. S.1.1\u2020) and [Ir(COOH-dfppy)2Cl(DMSO)] (1c-DMSO), acetone a\ufb00orded yellow crystals that were identified as 2c-\n respectively. This behaviour is not unusual and has been PO2F2\u2013acetone due to partial hydrolysis of the counter anion.\n reported before.48\u201350 The 1H and 13C{1H} NMR spectra Both complexes crystallized in the P21/n space group and, as\n (CDCl3 for 2a,b, MeOD for 2c-Cl, and acetone-d6 2c-PF6, 3a- expected for centrosymmetric space groups, both enantiomers\n PF6; see the ESI\u2020) showed the presence of the expected (\u0394 and \u039b) are present in the lattice. A view of the cationic part\n signals for a C2 symmetry with the two cyclometalated with selected bond lengths and angles are presented in Fig. 1\n group equivalents and a symmetrical bipyridine ligand. In and Table S1.\u2020 Both cations exhibited the characteristic octa-\n complexes 2, featuring the dbbpy ligand, the singlet corres- hedral environment around the IrIII center, with a mutual cis-\n ponding to the H3 protons, adjacent to the amide function, disposition of C-metalated and a trans arrangement of the\n appeared as the most deshielded signal (\u223c10.6 2a,b-Cl, 9.2 corresponding nitrogen atoms of the 2-(2,4-difluorophenyl)pyr-\n 2c-Cl, 10.30 2b-PF6, 9.21 2c-PF6); however, the amide CONH idine cyclometalated ligands. The bond distances and\n occurred as a broad resonance, which is slightly downfield angles were comparable to those found in analogous\n shifted in complexes 2-Cl relative to 2-PF6 (9.63 2a-Cl, 9.53 compounds.25,51,52 In both anions, the Ir\u2013C distances were\n 2b-Cl vs. 9.36 2b-PF6, 8.34 2c-PF6), likely due to the inter- within the expected values (\u223c2.01 \u00c5). The Ir\u2013N2 distances to\n action with Cl\u2212 (CONH\u22efCl\u22efHNCO), as observed by X-ray the dbbpy ligand (\u223c2.13 \u00c5) were longer than the corresponding\n di\ufb00raction in the solid state. In 2c-Cl, this resonance is lost, Ir\u2013N1 (\u223c2.05 \u00c5, C^N) distance, in accordance with the strong\n likely due to a fast exchange with MeOD. The 13C{1H} and trans influence of the metalated C atom. The chelating C\u2013Ir\u2013\n 19\n F NMR spectra also confirmed the formation of the com- N(C^N) angles are around 80\u00b0 while the N\u2013Ir\u2013N(C^N) were\n plexes. Thus, two doublet signals for the non-equivalent F6 roughly 174.5\u00b0, similar to those observed in related complexes.\n and F8 fluorine resonances are seen in all complexes, with A close look into the structures revealed the presence of hydro-\n the additional expected doublet due to PF6\u2212 in complexes gen bonding interactions. Thus, in complex 2a-Cl, short hydro-\n\n\n 6362 | Dalton Trans., 2023, 52, 6360\u20136374 This journal is \u00a9 The Royal Society of Chemistry 2023\n\f View Article Online\n\n Dalton Transactions Paper\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n Fig. 1 (a) Molecular structure of complex [Ir(dfppy)2(dbbpy)]Cl (2a-Cl), (b) view of the cation [Ir(COOH-dfppy)2(dbbpy)]+ (from structure of 2c-\n PO2F2), and (c) view of the dimer formed by two cations [2c]+ through hydrogen bonds between the ketonic group of one amide and the carboxylic\n group of one cyclometalated ligand. Selected bond distances (\u00c5) and angles (\u00b0); 2a-Cl: Ir(1)\u2013C(10) 2.008(2), Ir(1)\u2013N(1) 2.049(2), Ir(1)\u2013N(2) 2.125(2), Cl\n (1)\u22efH(16) 2.601(1), Cl(1)\u22efH(3A) 2.458(1). N(1)\u2013Ir(1)\u2013C(10) 80.46(8), N(1)\u2013Ir(1)\u2013C(10\u2019) 95.73(8), N(2)\u2013Ir(1)\u2013N(2\u2019) 77.3(1), N(1)\u2013Ir(1)\u2013N(1\u2019) 174.63(9). [2c]+:\n Ir(1)\u2013C(10) 2.009(4), Ir(1)\u2013N(1) 2.042(3), Ir(1)\u2013N(2) 2.131(3). N(1)\u2013Ir(1)\u2013C(10) 80.5(1), N(1\u2019)\u2013Ir(1)\u2013C(10) 97.3(1), N(2)\u2013Ir(1)\u2013N(2\u2019) 76.7(1), N(1)\u2013Ir(1)\u2013N(1\u2019)\n 174.6(1).\n\n\n\n gen bonding between the Cl\u2212 anion and the H atoms of the\n two carboxyamide groups (CONH) is observed (Cl\u22efH3\u2013N 2.458\n (1) \u00c5). Furthermore, Cl is also close to the adjacent H atom of\n the pyridine rings (Cl\u22efH\u2013C(16) 2.601(1) \u00c5). These distances,\n which were shorter than the sum of van der Waals radii\n (2.95 \u00c5), are comparable to those reported in other\n complexes,53\u201358 thus supporting the formation of an ionic pair\n in which the chloride acts as an acceptor of four hydrogen\n donors of the chelating dbbpy ligand. In its turn, in the crystal\n of 2c-PO2F2 the cations dimerize through short hydrogen\n bonding between the ketonic group of one of the carboxya-\n Fig. 2 NCI plot isosurfaces of the noncovalent interactions of 2a-Cl\n mide arms and the carboxylic group of the cyclometalated generated for s = 0.3.\n ligand. The O\u22efH distance (1.79 \u00c5) and the angle O\u22efH\u2013O\n (164.9) are comparable to those seen in other systems.59\n Noncovalent interactions (NCI analysis) have been carried\n out on the ionic pair 2a-Cl. The interactions were colour coded protons of dbbpy ligand and chloride atom developed, thus\n with blue and green colours, indicating strong and moderate supporting the involvement of donor\u2013acceptor interactions\n attractive forces, respectively, while the red and yellow between the anion Cl and the H\u2013X (X = N, C). In addition,\n ones correspond to strong and weak repulsive forces (Fig. 2). green surfaces were also observed for intramolecular C\u2013X\u22ef\u03c0\n In the NCI plot, clear green surfaces between the NH and CH (dfppy) (X = F, H).\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 6360\u20136374 | 6363\n\f View Article Online\n\n Paper Dalton Transactions\n\n Photophysical properties and theoretical calculations 1\n LL\u2032CT (C^N \u2192 dbbpy) transitions with some 1ML\u2032CT contri-\n Absorption spectra. The UV\u2013vis absorption spectra of all bution. The low energy band extending in the region of \u03bb >\n compounds were recorded in dimethyl sulfoxide, and the 400 nm (447 2a-Cl, 421 2b-Cl, 409 nm 2c-Cl) is associated with\n corresponding data are given in Table 1. Selected spectra for 2- the HOMO \u2192 LUMO transitions, having an ML\u2032CT/LL\u2032CT char-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n Cl and 3a-PF6 are shown in Fig. 3. The counter anion for com- acter. The slightly hypsochromic shift on going from 2a-Cl to\n plexes 2 has negligible influence on their maxima. For com- 2c-Cl is reflected in the calculations (S1, cal. 472 2a-Cl, 427 2b-\n plexes 2-Cl, the absorption spectra were also recorded in Cl; 410 nm 2c), and can be attributed to the stabilization of\n di\ufb00erent solvents showing minor variations in their maxima the HOMO due to the presence of the electron withdrawing\n (Table S7 and Fig. S10\u2020). Moreover, concentration dependence CHO and COOH substituents (Fig. 4). Due to strong spin\u2013orbit\n studies in DMSO (1 \u00d7 10\u22126 to 1 \u00d7 10\u22122 M) have been carried out coupling (SOC) associated with iridium, an overlapping with\n for complexes 2-Cl, showing that the three complexes follow the spin-forbidden singlet\u2013triplet metal-to-ligand and ligand-\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n the Beer\u2013Lambert Law and, thus, no remarkable ground-state to-ligand transitions (3ML\u2032CT/3LL\u2032CT) is expected in this\n aggregation phenomena occur in solution (Fig. S11\u2020). region. In complex 3a-PF6, having the dicarboxy-pyridine (3,3\u2032\n According to TD-DFT calculations on complexes 2-Cl (see H2dcbpy), the low energy feature appears at ca. 450 nm with a\n the ESI\u2020), the intense high energy absorption features in the tail extending to 500 nm, also with an ML\u2032CT/LL\u2032CT character\n UV region (<350 nm) are ascribed to spin allowed \u03c0\u2013\u03c0* tran- according to calculations (see the ESI\u2020).\n sitions of the ligands (1IL C^N, L\u2032 dbbpy) with metal to ligand Emission spectra. The room temperature emission spectra\n contribution (MLCT and ML\u2032CT). In mononuclear complexes of the precursor complex 1c and complexes 2 and 3 were\n 2, the moderately intense band around 365 nm can be mainly recorded in DMSO solution (Fig. 5) and in the solid state\n related to the intense S3 transition, which is associated with (Fig. S12\u2020) and the data are listed in Table 2 and Table S8.\u2020\n H-1 to the LUMO. H-1 resides on the cyclometalated ligands For complexes 2-Cl, the spectra were also examined in\n (91\u201392%) and Ir (7%) while the LUMO is located on the dbbpy di\ufb00erent solvents aiming to check the solvatochromic behav-\n ligand. Therefore, this band mainly arises from spin allowed iour, which is common in this type of complexes (Fig. S13\u2020).\n As an example, the emission spectra in DMSO solution are\n shown in Fig. 5a. The precursor complex 1c was highly soluble\n Table 1 Absorption data in DMSO solution (5 \u00d7 10\u22125 M) of complexes\n in DMSO and, as noted before, generated the solvate 1c-DMSO\n 1\u20133 upon dissolution, which exhibits a slightly structured band\n located at 511 nm which is ascribed to a 3LC with a 3MLCT\n Complex \u03bbabs/nm (\u03b5/\u00d710\u22123/M\u22121 cm\u22121) DMSO character (Fig. 5b). In the solid state the emission of 1c was\n 1b-DMSO 293 (29.96), 333 (9.72), 366 (5.58) red shifted to 580 nm, suggesting that the emission might\n 1c-DMSO 277 (60.2), 292 (52.4), 365 (9.6) have originated from molecular aggregation of the dinuclear\n 2a-Cl 273 (62.6), 310 (33.8), 368 (9.5), 447 (1.3), 469 (0.7) derivative in the rigid media through \u03c0\u03c0 stacking of the cyclo-\n 2b-Cl 306 (23.8), 328 (13.7), 358 (4.4), 421 (1.1)\n 2b-PF6 276 (43.2), 305 (32.5), 332 (19.4), 358 (7.8), 421 (1.0) metalated ligands. A highest energy shoulder is observed at\n 2c-Cl 269 (54.2), 284 (60.4), 300 (52.4), 365 (9.6), 409 (1.4) 540 nm that is likely due to the emission from the 3MLCT/3LC\n 2c-PF6 281 (51.4), 300 (34.2), 363 (8.6), 431 (2.4) contribution on individual molecules (Fig. S13\u2020). In DMSO\n 3a-PF6 265 (41.8), 305h (20.94), 361 (6.48), 450 (0.93)\n solution, the complexes 2-Cl and 2-PF6 were brightly emissive\n and exhibited similar photophysical properties. All complexes\n displayed broad unstructured bands in the range of\n 540\u2013565 nm, with a negligible influence of the counter anion\n likely due to the easy breakdown of the hydrogen bonding\n interactions in complexes 2-Cl. In agreement with theoretical\n calculations and previous works,58,60,61 this emission is attrib-\n uted to a mixed 3ML\u2032CT (Ir \u2192 N^N)/3LL\u2032CT (C^N \u2192 N^N)\n excited state with a predominant ML\u2032CT character. The photo-\n luminescence quantum yields (PLQYs) in deaerated solutions\n (\u03d5) were relatively high (from 44.6% for 2b-PF6 to 54.1 for 2c-\n PF6), with lifetimes in the range 0.58 to 0.81 \u03bcs. As expected,\n compared with the deoxygenated atmosphere, the emission\n intensity under an air atmosphere was notably reduced. In the\n case of complex 3a-PF6, the emission is red shifted (\u03bbmax =\n 605 nm) and the photoluminescence quantum yield drops to\n 4.2%, as does the decay to 0.31 \u03bcs. This result is not unex-\n pected and is in accordance with the energy gap law. The\n Fig. 3 Absorption spectra of complexes 2-Cl and 3a-PF6 in DMSO solu-\n measured lower lifetime of complexes 2 (0.58\u2013081 \u03bcs) and 3a-\n tion (5 \u00d7 10\u22125 M) at 298 K. Inset: expansion of the low energy region PF6 (0.31 \u03bcs) measured in deoxygenated solutions in relation\n (400\u2013500 nm). to 1c can be attributed to a notable higher metal contribution\n\n\n 6364 | Dalton Trans., 2023, 52, 6360\u20136374 This journal is \u00a9 The Royal Society of Chemistry 2023\n\f View Article Online\n\n Dalton Transactions Paper\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n Fig. 4 Schematic representation of selected excitation for complexes 2-Cl and 3a-PF6 in DMSO.\n\n\n\n\n Fig. 5 Normalized emission spectra of complexes (a) complexes 2\u20133 in DMSO solution (5 \u00d7 10\u22124 M), (b) 1c in di\ufb00erent media, and (c) of complex\n 2c-Cl in di\ufb00erent solvents.\n\n\n\n\n in the excited state. Comparing the complexes with the dbbpy lower kr in relation to complexes 2, could be related to the rela-\n ligand, the emission maxima follow the order (540 2b < 560 2c tively strong vibrational quenching e\ufb00ect caused by the pres-\n < 565 2a), which is consistent with the stabilization of the ence of the two carboxylic units on the bipyridine ligand.\n HOMO in complexes featuring the CHO and COOH substitu- The influence of the solvent has been examined for com-\n ents. In complex 3a-PF6, the observed red shift can be attribu- plexes 2-Cl (see Fig. 5c, and Table S8 and Fig. S13 in the ESI\u2020\n ted to a remarkable stabilization of the target 3,3\u2032-H2dcbpy for 2c-Cl). For these complexes, the emission band gradually\n LUMO (Fig. 4 and 5a). The reduced quantum yield of this shifts hypsochromically as the solvent polarity decreases (2a-Cl\n complex (Table 2), which is reflected in the higher knr and 565 DMSO, 520 THF and 495 nm toluene; 2b-Cl 540 DMSO,\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 6360\u20136374 | 6365\n\f View Article Online\n\n Paper Dalton Transactions\n\n Table 2 Photophysical data in DMSO solution (5 \u00d7 10\u22124 M)a. Radiative (Kr) and non-radiative (Knr) constants calculated at room temperature\n\n \u03c4 (\u00b5s) aereated/ \u03c6 aereated/\n Complex \u03bbem (nm) Energy/\u03bbem a deoxygenated deoxygenated Kr b/s\u22121 Knr b/s\u22121\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n 1c 510 \u2014 10.14b \u2014/<0.01 1.5 \u00d7 \u00b7103 9.7 \u00d7 104\n 2a-Cl 568 2.2 eV/556 0.18/0.81 0.15/0.53 6.6 \u00d7 105 5.7 \u00d7 105\n 2b-Cl 540 2.5 eV/494 0.42/0.72 0.35/0.51 7.1 \u00d7 105 6.8 \u00d7 105\n 2b-PF6 542 \u2014 0.28/0.58 0.21/0.45 7.7 \u00d7 105 9.6 \u00d7 105\n 2c-Cl 558 2.4 eV/510 0.43/0.76 \u2014/0.54 7.1 \u00d7 105 6.1 \u00d7 105\n 2c-PF6 558 \u2014 0.41/0.73 0.25/0.54 7.4 \u00d7 105 6.3 \u00d7 105\n 3a-PF6 605 1.7 eV/715 0.14/0.31 0.02/0.04 1.4 \u00d7 105 3.1 \u00d7 106\n a\n Predicted from DFT (B3LYP/LANL2DZ (Ir) 31G(d,p)) calculations in DMSO at 298 K, by estimating the energy di\ufb00erence between the optimized\n T1 and singlet state S0. b Values from deoxygenated solution.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n 507 THF and 503 nm toluene; 2c-Cl 615 H2O, 540 DMSO, and density distributions. In all complexes, the SOMO-1 involves Ir\n 517 nm THF). The observed positive solvatochromism indi- metal and the C^N cyclometalating group, while the SOMO is\n cates that the excited state is more polar than the ground state, primarily localized on the bipyridine. The predicted spin den-\n being stabilized by the solvent polarity and confirms the sities have been localized over the bipyridine ligand and, thus,\n strong charge transfer nature of the excited state. For 2a-Cl, the emissions are mainly characterized by both ML\u2032CT from\n the decay and the PLQY decrease with the polarity of the iridium to bipyridine charge transfer and LL\u2032CT from the\n solvent. This feature is very remarkable for complex 2c-Cl, fea- cyclometalated to the bipyridine ligand. There is good agree-\n turing the COOHdfppy cyclometalated ligand, in H2O wherein ment between the experimental and calculated emission wave-\n the PLQY falls to 3.1% with a decay of 0.06 \u03bcs. In this complex, lengths shown in Table 2 (calculated by estimating the energy\n the exchange of carboxylic and H2O protons opens a new di\ufb00erence between the optimized T1 and singlet state S0 in\n channel for deactivation, as reflected in the high value for knr DMSO). The introduction of CHO (2b) and COOH (2c) substitu-\n (1.7 \u00d7 10\u22127 s\u22121). ents on the cyclometalated group caused a slight hypsochro-\n To ascertain the detailed properties of the excited state, the mic shift by the stabilization of the SOMO-1 in relation to 2a-\n optimized geometries of T1 have been calculated using the S0 Cl, while the incorporation of carboxylic units on the bipyri-\n geometries and the B3LYP approach for complexes 2a\u2013c-Cl and dine provoked a remarkable bathochromic shift, which is in\n 3a-PF6. Fig. 6 shows the optimized excited-state structures of good agreement with the experimental data.\n the highest singly occupied molecular orbitals (HSOMOs), Quantum yield 1O2 quantification. In photodynamic\n lowest singly occupied molecular orbitals (LSOMOs), and spin therapy, the presence of singlet oxygen is one of the most\n\n\n\n\n Fig. 6 Calculated SOMO, SOMO-1 and spin density representations of 2 (a, b and c)-Cl and 3a-PF6 in DMSO solution.\n\n\n\n 6366 | Dalton Trans., 2023, 52, 6360\u20136374 This journal is \u00a9 The Royal Society of Chemistry 2023\n\f View Article Online\n\n Dalton Transactions Paper\n\n Cytotoxic activity and selectivity index. The IC50 values were\n determined against two di\ufb00erent human tumours (A549, lung\n carcinoma and HeLa, cervix carcinoma) and nontumoral\n BEAS-2B (bronchial epithelium) cell lines after cellular\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n exposure to the compounds for 72 h and compared to cisplatin\n as reference (Table 3 and Fig. S22\u2020).\n Complexes 2b-Cl and 2c-Cl compounds displayed IC50\n values \u2265100 \u00b5M towards the A549 cell line (Fig. S22\u2020). The low\n toxicity found with this assay could be attributed to their low\n solubility in the aqueous biological media. For that reason, the\n related complexes with PF6\u2212 as the counter anion were pre-\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n pared to test their biological activity. Complexes 2-PF6 dis-\n played better solubility, with no signs of precipitation at con-\n centrations up to 100 \u00b5M. For 2c-PF6, the IC50 value was also\n \u2265100 \u00b5M towards the A549 cell line (Fig. S22\u2020). Based on the\n low cytotoxicity of 2b-Cl, 2c-Cl and 2c-PF6, these complexes\n Fig. 7 Emission band of the singlet oxygen from fresh solution of 2b-\n PF6 (\u03bbex 425 nm). were not further analysed in other cells. However, it is remark-\n able that the antiproliferative activity improved on going from\n 2b-Cl to 2b-PF6 (IC50 values of 27.17 and 74.60 in A549 and\n important factors for the enhancement of the cytotoxic activity HeLa cells, respectively, for 2b-PF6 vs. the lesser e\ufb00ects of 2b-\n of a complex. Therefore, complexes 2b-Cl, 2b-PF6, 2c-Cl and Cl). This change might be attributed to the close interactions\n 3a-PF6 were selected to assess their ability to generate 1O2. between Cl\u2212 anion and the two NH groups of the butylamide\n These complexes generate singlet oxygen (1O2) at room temp- substituents of the dbbpy ligand in compound 2b-Cl, which\n erature in CH2Cl2 solution (Fig. 7). The 1O2 oxygen emission at decreases its ionic character and solubility, thus preventing\n 1270 nm was directly monitored using a near-infrared detector their optimum cellular uptake. Interestingly, complex 2a-Cl,\n upon excitation at 425 nm. To determine the singlet oxygen o\ufb00ered the lower IC50 values, being even more cytotoxic than\n generation, we use a reference method by UV-Visible spec- cisplatin in both A549 (3.73 vs. 6.45) and HeLa (5.53 vs. 13.60)\n troscopy in acetonitrile. ROS generation was visualized using cells (Table 3). These values are similar to those reported for\n the common ROS-capturing agent 1,3-disphenylisobenzofuran related complexes [Ir(C^N)2(N^N)]PF6 (C^N = ppy, dfppy) fea-\n (DPBF). DPBF reacts with ROS to generate 1,2-dibenzoylben- turing as diimine the dibutyl 2,2\u2032-bipyridine-4,4\u2032-dicarboxylate\n zene, resulting in an absorbance decline at 410 nm.62,63 The against HeLa and A549 tumour cells (IC50 1.7\u20132.3).69 Complex\n activity of a mixture of each complex with DPBF in acetonitrile 3a-PF6 that contains two carboxylic acid units on the diimine\n solution, employing [Ru(bpy)3]2 as a reference (\u03c6S\u0394 = 0.56),64 ligand also showed mild cytotoxic activity, with IC50 values\n was measured under blue light irradiation (\u03bb 460 nm). A clear similar to 2b-PF6 (32.87 and 51.22 in A549 and HeLa cells,\n decrease in the absorbance of the DPBF band at 410 nm respectively) (Table 3 and Fig. S22\u2020). As a rule, all the com-\n (Fig. S14\u2020) demonstrated the generation of 1O2 by the Ir(III) plexes were more active towards the A549 cell line rather than\n complexes. The values of \u03c6 obtained for all four complexes HeLa, in accordance with our previous results.62,66\n (0.32 for 2b-PF6, 0.12 for 2c-Cl, 0.10 for 3a-PF6, and 0.09 for In order to evaluate the selectivity index (SI) of complexes\n 2b-Cl) suggest that they might have future potential as an towards the tumour cell lines, we used normal epithelial lung\n intracellular 1O2 generator for photo-chemotherapeutic virus-transformed BEAS-2B cells as a non-tumor reference cell\n development. line. Cisplatin IC50 value towards this cell line was 1.74 \u03bcM,\n slightly lower than those for A549 and HeLa cells, therefore\n Biological studies rendering low SI values of this anti-cancer drug for these cell\n To start, the stability of complexes 2 and 3a was supported by lines (0.27 and 0.13, respectively) (Table 3). Actually, com-\n 1\n H NMR or UV-Vis spectra in DMSO solution or cellular pounds with SI values of <2 are assumed to give general\n medium, respectively, which revealed that they remained unal- toxicity.70,71 IC50 values of cisplatin on BEAS-2B cells and their\n tered within 74 h (Fig. S15\u2013S18 and S20\u2020). Their photostability SI values for A549 and HeLa cells reported here are in good\n in both media was also assessed. The complexes were found to agreement with previous findings.72\u201374 Among the Ir(III) com-\n be stable upon blue light irradiation with a blue lamp plexes, the better values of the selectivity index (SI) were given\n (396 nm) for at least 10 min (Fig. S19 for 3a-PF6 and S21\u2020 for by complex 2a-Cl in both tumoral A549 (3.30) and HeLa (2.23)\n 2b,c-Cl and 3a-PF6). The cytotoxicity of complexes 2 and 3a was cells, due to its higher cytotoxic activity in these cells (IC50 3.73\n determined in vitro against human cell lines by an MTS-based and 5.53 in A549 and HeLa cells, respectively) compared to\n method. We also evaluated the e\ufb00ect of irradiation with UV that in non-tumoral BEAS-2B cells (IC50 12.32 \u00b5M). SI values\n light on the antiproliferative activity of complexes 2b (Cl and for complex 2b-PF6 and 3a-PF6 were 1.90 and 0.73 (A549) and\n PF6), 2c-Cl and 3a-PF6, their interaction with DNA and lipophi- 0.69 and 0.47 (HeLa), respectively (Table 3), indicating low\n licity, as well as their cellular localization. selective toxicity towards cancer cells. Low SI values (<2) for\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 6360\u20136374 | 6367\n\f View Article Online\n\n Paper Dalton Transactions\n\n Table 3 Cytotoxic IC50 values (\u00b5M)a and selectivity indexb of the complexes 2a-Cl, 2b-PF6 and 3a-PF6 in A549, HeLa and BEAS-2B human cell lines\n compared with cisplatin\n\n IC50a SIb\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n Complex A549 HeLa BEAS-2B A549 HeLa\n\n 2a-Cl 3.73 \u00b1 0.76 5.53 \u00b1 0.41 12.32 \u00b1 0.21 3.30 2.23\n 2b-PF6 27.17 \u00b1 1.44 74.6 \u00b1 0.72 51.6 \u00b1 4.71 1.90 0.69\n 3a-PF6 32.87 \u00b1 3.01 51.22 \u00b1 1.29 24.03 \u00b1 3.82 0.73 0.47\n Cisplatin 6.45 \u00b1 0.47c 13.60 \u00b1 0.99d 1.74 \u00b1 0.16 0.27 0.13\n a\n IC50 values presented as mean \u00b1 standard error of the mean of three di\ufb00erent experiments. b Selectivity index (SI) = IC50 nontumor cell\n (BEAS-2B)/IC50 cancer cell (A549 or HeLa), as described in ref. 65, 66. c As determined in ref. 67. d As determined in ref. 68.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n other organometallic complexes with cytotoxic activities whereas the slightly cytotoxic 2c-Cl showed a lower PI activity,\n toward A549, HeLa, and other cancer cell lines have been pre- giving a moderate e\ufb00ect by lowering the IC50 value to 55.52 \u03bcM\n viously described,66,75\u201378 including iridium organometallic upon 15 min of irradiation. In any case, the complexes are\n complexes.18,79,80 more active under UV light than under non-UV light. This\n Photoinduced cytotoxicity and intracellular ROS generation. could be related to their singlet oxygen generation ability in\n To expand our knowledge on the cytotoxic behaviour of these cancer cells. The generation of ROS after UV irradiation was\n complexes, we examine their potential as photosensitizers measured using the ROS indicator H2DCFDA. As shown in\n (PS). Selective activation of a nontoxic photosensitizer by light Table 4, the four complexes show ROS generation after UV\n is an attractive regimen for therapy, especially if the PS is pre- irradiation. The highest values were given by 3a-PF6 (49 \u03bcM),\n ferentially taken up by cancer cells.81 In recent years, lumines- which were in good agreement with its lower UV-IC50 (Fig. 8d)\n cent metal based drugs, in particular Ru(II) Ir(III) and Pt(II) and despite its shorter UV light exposure time (3 min).\n complexes, are being actively investigated due to their out- Relative lipophilicity. The relative hydrophobicity of the\n standing results in PDT based cancer therapy.40,82\u201387 Due to complexes was studied by RP-UPLC with the aim of establish-\n the ability of these iridium complexes to generate 1O2 upon ing a correlation between their cytotoxicity and a\ufb03nity for a\n irradiation and their photostability, they can be considered as lipid environment, a crucial aspect in their pharmacokinetic\n potential tools in phototheranostics. Therefore, we decided to properties. Complexes were dissolved in acetonitrile (\u22481 ppm),\n evaluate the potential use of complexes 2b-Cl and 2c-Cl in and a mixture of acetonitrile with 0.1% HCOOH (A) and H2O\n photodynamic therapy based on their light cytotoxic e\ufb00ects in with 0.1% HCOOH (B) was employed as a mobile phase. The\n normal cell culture. In addition, we also chose 2b-PF6 in order lipophilicity character is based on the values of their retention\n to evaluate the e\ufb00ect of changing the counter-anion Cl\u2212 with times (tR). These values account for the relative interactions\n PF6\u2212 on the induction of the photocytotoxicity. Finally, we also between the hydrophobic stationary phase (Aquity UPLC BEH\n evaluated the photocytotoxicity of 3a-PF6 to test the e\ufb00ect of C18) and the hydrophilic mobile phase with each complex.\n locating the carboxylic units on the diimine ligand. The photo- The longer the tR, the more lipophilic the character of the\n stability of the complexes under similar conditions of complex.62,88 In case of these Ir(III) complexes, the presence of\n irradiation to that employed for PDT assays was supported by substituents on the cyclometalated groups remarkably\n NMR spectroscopy. A549 cells were initially treated with each decreased the tR (H > CHO > COOH) (Table S9\u2020), with 2a-Cl (tR\n complex in Hank\u2019s balanced salt solution (HBSS) for 1 h at = 3.28) and 3a-PF6 (tR = 1.99) being the most lipophilic ones.\n 37 \u00b0C in order to allow cellular interaction and internalization. The higher lipophilicity of 2a-Cl is in accordance with the\n Then, A549 cells were irradiated with a 396 nm LED lamp good cytotoxicity activity found for this complex, which dis-\n located 91 mm apart (5 Mw cm\u22122) for di\ufb00erent times: 3 min plays the lowest IC50 values (Table 3). Di\ufb00erences between the\n (3a-PF6), 10 min (2b-PF6) and 15 min (2b-Cl and 2c-Cl) (see the tR of the rest of compounds were too small to ensure a clear\n ESI\u2020). After the irradiation, cells were washed and incubated in relationship between the results and their cytotoxic activities.\n complete RPMI medium without the presence of any complex Study of the reaction with NADH. Nicotinamide adenine\n for another 72 hours. Finally, cell viability (IC50) was assessed dinucleotide (NADH), and its oxidized form NAD+, is a coen-\n by the MTS test, as detailed in the ESI.\u2020 The observed IC50 zyme whose function is crucial for the catalysis of redox or iso-\n values were significantly lower in the presence of UV light than merization reactions.89 NADH plays an important role in the\n under non-UV light, reflecting the e\ufb00ective phototoxicity of the mitochondrial electron transport chain and the maintenance\n photosensitizers. Thus, as shown in Fig. 8, upon UV light of the cellular redox balance. Thus, any alteration in the intra-\n irradiation a significant increase of the antiproliferative activity cellular concentration of this species might lead to cell\n was observed for 3a-PF6 (IC50: 5.63 \u03bcM) with a short irradiation death.90 Recent studies have reported that some Ir(III) and Os\n time of only 3 min. UV-light irradiation for 15 min for 2b-Cl or (II) complexes display anticancer activity through a non-con-\n 10 min for 2b-PF6, also enhances their antiproliferative activity ventional redox mediated mechanism of action involving cata-\n leading to IC50 values of 14.39 and 18.57 \u03bcM, respectively, lytic photo-oxidation of NADH to NAD+ through transfer hydro-\n\n\n 6368 | Dalton Trans., 2023, 52, 6360\u20136374 This journal is \u00a9 The Royal Society of Chemistry 2023\n\f View Article Online\n\n Dalton Transactions Paper\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n Fig. 8 Dose\u2013response curves for the A549 cell line treated with complex (a) 2b-Cl; (b) 2b-PF6; (c) 2c-Cl and (d) 3a-PF6 either with (triangles) or\n without (circles) UV light: irradiation with a UV 396 nm LED for 15 min (a and c), 10 min (b) and 3 min (d) and followed by MTS cytotoxic assays per-\n formed after 72 h. Non UV-irradiated cells were manipulated identically to UV-irradiated ones. IC50 values are presented as mean \u00b1 standard error of\n the mean of three di\ufb00erent experiments performed in sextuplicate.\n\n\n\n\n genation reactions leading to H2O2 and reactive oxygen\n species.91,92 Thus, the quantification of changes in NADH/\n NAD+, is a good method to know if these compounds can\n Table 4 ROS production in A549 cell line treated with complexes 2b(Cl produce changes in ROS content by this mechanism. The\n and PF6), 2c-Cl and 3a-PF6, either with or without UV light\n interaction with NADH was performed with the cytotoxic\n complex 2a-Cl, and the results are shown in Fig. 9. The reac-\n ROS (arbitrary units)a\n tion of the complex (1 \u00b5M) with NADH (100 \u00b5M) dissolved in a\n Complex Non UVb UVc solution 20% MeOH in H2O was monitored by UV-vis at 298 K\n 2b-Cl Controld 801.42 \u00b1 20.00 10 635.50 \u00b1 451.51** for 8 hours. Following the continuous decrease with time of\n 1 mM 702.67 \u00b1 47.68 46 391.44 \u00b1 818.24*** the NADH absorption peak at 339 nm, due to its interaction\n 25 mM 2976.67 \u00b1 390.74 71 221.42 \u00b1 1906.88*** with the complex to give its oxidized form NAD+,25,93 allowed\n 2b-PF6 Controld 516.33 \u00b1 16.78 7527.17 \u00b1 922.78*\n 2.5 mM 617.67 \u00b1 24.30 54 776.42 \u00b1 5684.69** us to determine the interaction of NADH (conversion to NAD+)\n 25 mM 1702.83 \u00b1 61.91 75 723.75 \u00b1 2670.39*** with the complex. The turnover number (TONs) obtained by\n 2c-Cl Controld 494.58 \u00b1 34.34 7571.42 \u00b1 1329.36** the di\ufb00erence between NADH concentration at t0 and t =\n 1 mM 468.67 \u00b1 24.83 10 578.25 \u00b1 236.44***\n 50 mM 1127.50 \u00b1 118.51 72 319.33 \u00b1 2846.33*** 8 hours was 14.0, showing that the NADH had been oxidated\n 3a-PF6 Controld 577.58 \u00b1 71.84 2476.25 \u00b1 130.39*** to NAD+. This result indicates that this complex can act as a\n 1 mM 633.00 \u00b1 32.92 4583.75 \u00b1 92.10*** moderate catalyst for endogenous NADH oxidation. Thus, it\n 7 mM 999.25 \u00b1 37.02 17 801.33 \u00b1 690.25***\n 49 mM 1832.75 \u00b1 246.03 82 785.67 \u00b1 1296.15*** could be a mitochondrial target, inducing destabilization of\n the redox homeostasis and, ultimately, causing apoptosis.\n a\n ROS was measured using the ROS-sensitive dye, 2\u2032,7\u2032-dichlorodihydro- Interaction of complexes with DNA. Knowing their cytotoxic\n fluorescein diacetate (H2DCFDA) as an indicator. b Cells were non-irra-\n diated. c Cells were irradiated with a UV 396 nm LED for 15 min (2b-Cl activity, the interaction of iridium 2a-Cl, 2b-PF6 and 3a-PF6\n and 2c-Cl), 10 min (2b-PF6) and 3 min (3a-PF6). d ROS values of A549 complexes with DNA was studied by their ability to modify\n cells without complex. Each value represents the mean \u00b1 standard the electrophoretic mobility of the supercoiled covalently\n error from three di\ufb00erent experiments performed in quadruplicate.\n *p < 0.05; **p < 0.01; ***p < 0.001 (Mann\u2013Whitney U test or Student\u2019s closed circular (CCC) and the open circular (OC) forms of\n t-test for comparing two groups). pBR322 plasmid DNA (Fig. S23\u2020). To provide a basis for com-\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 6360\u20136374 | 6369\n\f View Article Online\n\n Paper Dalton Transactions\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n Fig. 9 UV-Vis spectra for the oxidation of NADH (100 \u00b5M) by complex\n 2a-Cl (1 \u00b5M) in a mixture of H2O/MeOH 80/20 under dark conditions.\n\n\n\n\n parison, the incubation of DNA with cisplatin and the low\n cytotoxic complex 2c-PF6 was also performed at the same Fig. 10 Laser confocal microscopy images of live A549 cells incubated\n concentrations and conditions. The binding of cisplatin to with 16 \u00b5M 2a-Cl complex for 1 hour. Cells were stained with speci\ufb01c\n plasmid DNA, for instance, results in a decrease in the mobi- intracellular markers. A. LysoTracker (lysosomal marker, \u03bbex 543 nm) in\n lity of the CCC form and an increase in the mobility of the red. B. 2a-Cl (\u03bbex 405 nm) in green. C. Hoechst (nuclei marker, \u03bbex\n OC form (Fig. S23,\u2020 upper left panel).53,66,94,95 As shown in 405 nm) in blue. D. Merged image showing colocalization of 2a-Cl with\n LysoTracker in lysosomes (yellow). Scale bar = 10 \u00b5m.\n Fig. S23,\u2020 no electrophoretic mobility changes were observed\n after DNA treatment with any complex under the same con-\n ditions, thus indicating that these compounds were either\n not reacting with the DNA or not altering the DNA mobility\n in agarose gels.\n Intracellular localization. Confocal microscopy was per-\n formed to investigate the intracellular localization. In agree-\n ment with the DNA interaction studies, none of the complexes\n showed intranuclear localization in A549 cells, as shown in\n Fig. 10\u201313. When 16 \u00b5M 2a-Cl complex was incubated with\n A549 cells, it showed fast cellular internalization, localizing\n predominantly in lysosomes (Fig. 10), as has been determined\n by a calculated Pearson correlated coe\ufb03cient of 0.79. 2a-Cl\n also localizes to a lesser extent in the cytoplasm, but not in\n mitochondria (Fig. S24 and S25\u2020). However, although 2a-Cl\n was not detected inside the mitochondria, it ends up causing\n them damage. Thus, one-hour incubation with 16 \u00b5M 2a-Cl\n complex induced mitochondrial swelling and the loss of mito-\n chondrial membrane potential was noticed after 30 min\n (Fig. 11), which has been associated with cell death either by\n apoptosis or necrosis, depending on the particular biological\n setting. Accordingly as shown before, this complex e\ufb00ectively\n alters the NADH/NAD+ pair, which can produce ROS, thus\n Fig. 11 Laser confocal microscopy images of live A549 cells and loss of\n altering mitochondrial functions and cell death. mitochondrial membrane potential (MMP) after incubation with 2a-Cl\n Complexes 2b-PF6 and 2c-PF6 were also internalized by complex. (A and B) Cells were stained with speci\ufb01c intracellular markers:\n A549 cells when they were incubated at 32 \u00b5M for 1 hour red: MitoTracker (mitochondrial marker, \u03bbex 633 nm) and blue: Hoechst\n (Fig. 12A and C) or 24 hours (Fig. 12B and D). In agreement (nuclei marker, \u03bbex 405 nm). (A) Control cells. (B) Cells incubated with\n 16 \u03bcM 2a-Cl complex for 1 hour. Scale bar = 10 \u00b5m. (C) Loss of MMP\n with their cytotoxicity results presented in Table 3 and\n after 30 min of incubation with 16 \u03bcM 2a-Cl complex or 10 \u03bcM FCCP.\n Fig. S22,\u2020 the incubation of A549 cells with complex 2b-PF6 Control indicates untreated cells. MMP values represent the mean \u00b1\n for 24 hours resulted in extended cell death while 2c-PF6 standard error from three di\ufb00erent experiments performed in quadrupli-\n complex showed less toxicity for the same time. cate. *p < 0.05 (Mann\u2013Whitney U test for comparing two groups).\n\n\n\n 6370 | Dalton Trans., 2023, 52, 6360\u20136374 This journal is \u00a9 The Royal Society of Chemistry 2023\n\f View Article Online\n\n Dalton Transactions Paper\n\n Interestingly, when we incubated A549 cells with 45 \u00b5M\n of complex 3a-PF6, and followed the cells by confocal\n microscopy, we observed an unexpected behavior. Cells sub-\n jected to 405 nm laser stimulation showed increased toxicity\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n compared to adjacent cells not subjected to the laser stimu-\n lation (Fig. 13). We did not observe laser-induced toxicity in\n A549 cells incubated with any of the complexes 2-Cl.\n However, 405 nm laser stimulation (from the confocal micro-\n scope) not only increased the toxicity of the compound but\n also increased its fluorescence (central region of pictures in\n Fig. 13). When we incubated the cells with 3a-PF6 and the\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n mitochondrial marker MitoTracker, we observed loss of mito-\n chondrial membrane potential and mitochondrial swelling\n (Fig. 13) only when we stimulated the cells with the 405 and\n the 633 lasers. We found that incubation of cells with 3a-PF6\n and MitoTracker and stimulation with 633 nm laser did not\n damage the mitochondria or induced toxicity. Therefore, we\n conclude that 3a-PF6 can be excited using 405 nm irradiation\n increasing its fluorescence and inducing mitochondrial tox-\n icity, possibly by inducing ROS formation (Table 4), leading\n to cell death. Mitochondria play an important role in many\n Fig. 12 Laser confocal microscopy images of live A549 cells incubated\n with 32 \u00b5M 2b-PF6 and 2c-PF6 complexes for up to 24 hours. (A) Cells cellular operations, such as the generation of energy, main-\n exposed to 32 \u00b5M 2b-PF6 for 1 hour (\u03bbex 405 nm). (B) Cells exposed to taining intracellular redox balance and metabolism and,\n 32 \u00b5M 2b-PF6 for 24 hours (\u03bbex 405 nm). (C) Cells exposed to 32 \u00b5M 2c- reports on cyclometalated iridium complexes causing mito-\n PF6 for 1 hour (\u03bbex 405 nm). (D) Cells exposed to 32 \u00b5M 2c-PF6 for chondria mediated dysfunction and apoptosis by several\n 24 hours (\u03bbex 405 nm). Scale bar = 20 \u00b5m.\n factors including reactive oxygen species (ROS) have been pre-\n viously reported.96\n\n\n\n\n Fig. 13 Laser confocal microscopy images of live A549 cells incubated with 45 \u00b5M 3a-PF6 compound for up to 20 minutes. Top pictures: A549\n cells incubated with 3a-PF6 stimulated with a 405 nm laser. Bottom pictures: A549 cells incubated with 3a-PF6 and labelled with MitoTracker stimu-\n lated with 405 and 633 nm lasers. Green: 3a-PF6; magenta: MitoTracker. Scale bar = 20 \u00b5m.\n\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 6360\u20136374 | 6371\n\f View Article Online\n\n Paper Dalton Transactions\n\n\n Conclusion Acknowledgements\n We present here a series of novel Ir(III) cyclometalated cationic This work was supported by the Spanish Ministerio de Ciencia\n complexes incorporating di\ufb00erent substituents in the position e Innovaci\u00f3n (Project PID2019-109742GB-I00) funded by\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n 3 of the 2,4-diflurophenylpyridinate cyclometalated group (H, MCIN/AIE/10.13039/501100011033, by \u201cERDF A way of making\n dfppy; HCO, CHO-dfppy and COOH, COOH-dfppy) and using Europe\u201d, by the \u201cEuropean Union, and by the ADER (Gobierno\n N,N\u2032-dibutyl-2,2\u2032-bipyridine-4,4\u2032-dicarboxamide (dbbpy) as the de La Rioja; Project 2017-I-IDD-00031). G. M. is grateful to UR\n diimine ligand with chloride (2-Cl) or PF6\u2212 ions (2-PF6) as for a PhD grant. We are grateful to E. Alfaro-Arnedo for her\n counter anions, together with [Ir(dfppy)2(H2dcbpy)]PF6 (3a- technical help.\n PF6), to evaluate the influence of altering the position of the\n substituents on the optical properties and bioactivity of the\nOpen Access Article. Published on 05 aprile 2023. Downloaded on 12/05/2026 12:43:04.\n\n\n\n\n complexes. All complexes have been fully characterized by 1H, References\n 13\n C{1H} and 19F NMR spectroscopy, HMRS, and elemental ana-\n lysis. In addition, the structure of complex 2a-Cl and of the 1 M. Fanelli, M. Formica, V. Fusi, L. Giorgi, M. Micheloni\n cation [Ir(COOH-dfppy)2(dbbpy)]+ (from the structure of 2c- and P. Paoli, Coord. Chem. Rev., 2016, 310, 41.\n PO2F2) were confirmed by X-ray di\ufb00raction studies. Crystals of 2 V. Brabec, O. Hrabina and J. Kasparkova, Coord. 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