We present the synthesis and characterization of six new heteroleptic osmium(II) complexes of the type [Os(C^N)(N^N)2]OTf (N^N = 2,2'-bipyridine and dipyrido[3,2-d:2',3'-f]quinoxaline; C^N = deprotonated methyl 1-butyl-2aryl-benzimidazolecarboxylate) with varying substituents in the R3 position of the phenyl ring of the cyclometalating C^N ligand. The new compounds are highly kinetically inert and absorb a full-wavelength range of visible light. An investigation of the antiproliferative activity of the new compounds has been performed using a panel of human cancer and noncancerous 2D cell monolayer cultures under dark conditions and green light irradiation. The results demonstrate that the new Os(II) complexes are markedly more potent than conventional cisplatin. The promising antiproliferative activity of selected Os(II) complexes was also confirmed using 3D multicellular tumor spheroids, which have the characteristics of solid tumors and can mimic the tumor tissue microenvironment. The mechanism of antiproliferative action of complexes has also been investigated and revealed that the investigated Os(II) complexes activate the endoplasmic reticulum stress pathway in cancer cells and disrupt calcium homeostasis.

Czech Academy of Sciences Institute of Biophysics CZ 61200 Brno Czech Republic

Departamento de Química Inorgánica Universidad de Murcia and Murcia BioHealth Research Institute E 30071 Murcia Spain

Department of Biophysics Faculty of Science Palacky University in Olomouc CZ 78371 Olomouc Czech Republic

SUIC ACTI Universidad de Murcia E 30071 Murcia Spain

See more in PubMed

Sung H.; Ferlay J.; Siegel R. L.; Laversanne M.; Soerjomataram I.; Jemal A.; Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-Cancer J. Clin. 2021, 71, 209–249. 10.3322/caac.21660. PubMed DOI

Rosenberg B. Platinum complexes for the treatment of cancer. Interdiscip. Sci. Rev. 1978, 3, 134–147. 10.1179/030801878791926119. DOI

Ott I.; Gust R. Non platinum metal complexes as anti-cancer drugs. Arch. Pharm. 2007, 340, 117–126. 10.1002/ardp.200600151. PubMed DOI

Medici S.; Peana M.; Nurchi V. M.; Lachowicz J. I.; Crisponi G.; Zoroddu M. A. Noble metals in medicine: Latest advances. Coord. Chem. Rev. 2015, 284, 329–350. 10.1016/j.ccr.2014.08.002. DOI

Luttrell W. E.; Giles C. B. Toxic tips: Osmium tetroxide. J. Chem. Health Saf. 2007, 14, 40–41. 10.1016/j.jchas.2007.07.003. DOI

Kushwaha R.; Kumar A.; Saha S.; Bajpai S.; Yadav A. K.; Banerjee S. Os(II) complexes for catalytic anticancer therapy: recent update. Chem. Commun. 2022, 58, 4825–4836. 10.1039/d2cc00341d. PubMed DOI

Ortega E.; Ballester F. J.; Hernández-García A.; Hernández-García S.; Guerrero-Rubio M. A.; Bautista D.; Santana M. D.; Gandía-Herrero F.; Ruiz J. Novel organo-osmium(II) proteosynthesis inhibitors active against human ovarian cancer cells reduce gonad tumor growth in Caenorhabditis elegans. Inorg. Chem. Front. 2021, 8, 141–155. 10.1039/c9qi01704f. DOI

Peacock A. F. A.; Habtemariam A.; Moggach S. A.; Prescimone A.; Parsons S.; Sadler P. J. Chloro half-sandwich osmium(II) complexes: Influence of chelated N,N-ligands on hydrolysis, guanine binding and cytotoxicity. Inorg. Chem. 2007, 46, 4049–4059. 10.1021/ic062350d. PubMed DOI

Cebrián-Losantos B.; Krokhin A. A.; Stepanenko I. N.; Eichinger R.; Jakupec M. A.; Arion V. B.; Keppler B. K. Osmium NAMI-A analogues: synthesis, structural and spectroscopic characterization, and antiproliferative properties. Inorg. Chem. 2007, 46, 5023–5033. 10.1021/ic700405y. PubMed DOI

Dorcier A.; Ang W. H.; Bolaño S.; Gonsalvi L.; Juillerat-Jeannerat L.; Laurenczy G.; Peruzzini M.; Phillips A. D.; Zanobini F.; Dyson P. J. In vitro evaluation of rhodium and osmium RAPTA analogues: The case for organometallic anticancer drugs not based on ruthenium. Organometallics 2006, 25, 4090–4096. 10.1021/om060394o. DOI

Romero-Canelón I.; Salassa L.; Sadler P. J. The contrasting activity of iodido versus chlorido ruthenium and osmium arene azo- and imino-pyridine anticancer complexes: Control of cell selectivity, cross-resistance, p53 dependence, and apoptosis pathway. J. Med. Chem. 2013, 56, 1291–1300. 10.1021/jm3017442. PubMed DOI

Zhang P.; Huang H. Future potential of osmium complexes as anticancer drug candidates, photosensitizers and organelle-targeted probes. Dalton Trans. 2018, 47, 14841–14854. 10.1039/c8dt03432j. PubMed DOI

King A. P.; Wilson J. J. Endoplasmic reticulum stress: an arising target for metal-based anticancer agents. Chem. Soc. Rev. 2020, 49, 8113–8136. 10.1039/d0cs00259c. PubMed DOI

Licona C.; Delhorme J.-B.; Riegel G.; Vidimar V.; Cerón-Camacho R.; Boff B.; Venkatasamy A.; Tomasetto C.; da Silva Figueiredo Celestino Gomes P.; Rognan D.; Freund J.-N.; Le Lagadec R.; Pfeffer M.; Gross I.; Mellitzer G.; Gaiddon C. Anticancer activity of ruthenium and osmium cyclometalated compounds: identification of ABCB1 and EGFR as resistance mechanisms. Inorg. Chem. Front. 2020, 7, 678–688. 10.1039/c9qi01148j. DOI

Yang Q.-Y.; Ma R.; Gu Y.-Q.; Xu X.-F.; Chen Z.-F.; Liang H. Arene-ruthenium(II)/osmium(II) complexes potentiate the anticancer efficacy of metformin via glucose metabolism reprogramming. Angew. Chem., Int. Ed. 2022, 61, e20220857010.1002/anie.202208570. PubMed DOI

Lazic S.; Kaspler P.; Shi G.; Monro S.; Sainuddin T.; Forward S.; Kasimova K.; Hennigar R.; Mandel A.; McFarland S.; Lilge L. Novel osmium-based coordination complexes as photosensitizers for panchromatic photodynamic therapy. Photochem. Photobiol. 2017, 93, 1248–1258. 10.1111/php.12767. PubMed DOI

Ballester F. J.; Ortega E.; Bautista D.; Santana M. D.; Ruiz J. Ru(II) photosensitizers competent for hypoxic cancers via green light activation. Chem. Commun. 2020, 56, 10301–10304. 10.1039/d0cc02417a. PubMed DOI

Karges J. Clinical development of metal complexes as photosensitizers for photodynamic therapy of cancer. Angew. Chem., Int. Ed. 2022, 61, e20211223610.1002/anie.202112236. PubMed DOI

Lumpkin R. S.; Kober E. M.; Worl L. A.; Murtaza Z.; Meyer T. J. Metal-to-ligand charge-transfer (MLCT) photochemistry: experimental evidence for the participation of a higher lying MLCT state in polypyridyl complexes of ruthenium(II) and osmium(II). J. Phys. Chem. 1990, 94, 239–243. 10.1021/j100364a039. DOI

Lu N.; Deng Z.; Gao J.; Liang C.; Xia H.; Zhang P. An osmium-peroxo complex for photoactive therapy of hypoxic tumors. Nat. Commun. 2022, 13, 2245.10.1038/s41467-022-29969-z. PubMed DOI PMC

Zhang P. Y.; Wang Y.; Qiu K. Q.; Zhao Z. Q.; Hu R. T.; He C. X.; Zhang Q. L.; Chao H. A NIR phosphorescent osmium(II) complex as a lysosome tracking reagent and photodynamic therapeutic agent. Chem. Commun. 2017, 53, 12341–12344. 10.1039/c7cc07776a. PubMed DOI

Bansal Y.; Silakari O. The therapeutic journey of benzimidazoles: A review. Bioorg. Med. Chem. 2012, 20, 6208–6236. 10.1016/j.bmc.2012.09.013. PubMed DOI

Singla P.; Luxami V.; Paul K. Benzimidazole-biologically attractive scaffold for protein kinase inhibitors. RSC Adv. 2014, 4, 12422–12440. 10.1039/c3ra46304d. DOI

Hachey A. C.; Havrylyuk D.; Glazer E. C. Biological activities of polypyridyl-type ligands: implications for bioinorganic chemistry and light-activated metal complexes. Curr. Opin. Chem. Biol. 2021, 61, 191–202. 10.1016/j.cbpa.2021.01.016. PubMed DOI PMC

Yellol J.; Perez S. A.; Buceta A.; Yellol G.; Donaire A.; Szumlas P.; Bednarski P. J.; Makhloufi G.; Janiak C.; Espinosa A.; Ruiz J. Novel C,N-Cyclometalated Benzimidazole Ruthenium(II) and Iridium(III) Complexes as Antitumor and Antiangiogenic Agents: A Structure–Activity Relationship Study. J. Med. Chem. 2015, 58, 7310–7327. 10.1021/acs.jmedchem.5b01194. PubMed DOI

Wang C.; Lystrom L.; Yin H.; Hetu M.; Kilina S.; McFarland S. A.; Sun W. Increasing the triplet lifetime and extending the ground-state absorption of biscyclometalated Ir(iii) complexes for reverse saturable absorption and photodynamic therapy applications. Dalton Trans. 2016, 45, 16366–16378. 10.1039/c6dt02416e. PubMed DOI

Pracharova J.; Vigueras G.; Novohradsky V.; Cutillas N.; Janiak C.; Kostrhunova H.; Kasparkova J.; Ruiz J.; Brabec V. Exploring the effect of polypyridyl ligands on the anticancer activity of phosphorescent iridium(III) complexes: From proteosynthesis inhibitors to photodynamic therapy agents. Chem.—Eur. J. 2018, 24, 4607–4619. 10.1002/chem.201705362. PubMed DOI

Boff B.; Gaiddon C.; Pfeffer M. Cancer cell cytotoxicity of cyclometalated compounds obtained with osmium(II) complexes. Inorg. Chem. 2013, 52, 2705–2715. 10.1021/ic302779q. PubMed DOI

Li J.; Zeng L.; Wang Z.; Chen H.; Fang S.; Wang J.; Cai C.-Y.; Xing E.; Liao X.; Li Z.-W.; Ashby C. R. Jr; Chen Z.-S.; Chao H.; Pan Y. Cycloruthenated self-assembly with metabolic inhibition to efficiently overcome multidrug resistance in cancers. Adv. Mater. 2022, 34, 2100245.10.1002/adma.202100245. PubMed DOI PMC

Irikura M.; Tamaki Y.; Ishitani O. Development of a panchromatic photosensitizer and its application to photocatalytic CO2 reduction. Chem. Sci. 2021, 12, 13888–13896. 10.1039/d1sc04045f. PubMed DOI PMC

Broussard J. A.; Webb D. J.; Kaverina I. Asymmetric focal adhesion disassembly in motile cells. Curr. Opin. Cell Biol. 2008, 20, 85–90. 10.1016/j.ceb.2007.10.009. PubMed DOI

Valastyan S.; Weinberg R. A. Tumor metastasis: Molecular insights and evolving paradigms. Cell 2011, 147, 275–292. 10.1016/j.cell.2011.09.024. PubMed DOI PMC

Brandhagen B. N.; Tieszen C. R.; Ulmer T. M.; Tracy M. S.; Goyeneche A. A.; Telleria C. M. Cytostasis and morphological changes induced by mifepristone in human metastatic cancer cells involve cytoskeletal filamentous actin reorganization and impairment of cell adhesion dynamics. BMC Cancer 2013, 13, 35.10.1186/1471-2407-13-35. PubMed DOI PMC

Zanoni M.; Piccinini F.; Arienti C.; Zamagni A.; Santi S.; Polico R.; Bevilacqua A.; Tesei A. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci. Rep. 2016, 6, 19103.10.1038/srep19103. PubMed DOI PMC

Thoma C. R.; Zimmermann M.; Agarkova I.; Kelm J. M.; Krek W. 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Adv. Drug Delivery Rev. 2014, 69–70, 29–41. 10.1016/j.addr.2014.03.001. PubMed DOI

Baker B. M.; Chen C. S. Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues. J. Cell Sci. 2012, 125, 3015–3024. 10.1242/jcs.079509. PubMed DOI PMC

Kimlin L. C.; Casagrande G.; Virador V. M. In vitro three-dimensional (3D) models in cancer research: An update. Mol. Carcinog. 2013, 52, 167–182. 10.1002/mc.21844. PubMed DOI

Kacsir I.; Sipos A.; Bényei A.; Janka E.; Buglyó P.; Somsák L.; Bai P.; Bokor É. Reactive oxygen species production is responsible for antineoplastic activity of osmium, ruthenium, iridium and rhodium half-sandwich type complexes with bidentate glycosyl heterocyclic ligands in various cancer cell models. Int. J. Mol. Sci. 2022, 23, 813.10.3390/ijms23020813. PubMed DOI PMC

Maillet A.; Yadav S.; Loo Y. L.; Sachaphibulkij K.; Pervaiz S. A novel Osmium-based compound targets the mitochondria and triggers ROS-dependent apoptosis in colon carcinoma. Cell Death Dis. 2013, 4, e65310.1038/cddis.2013.185. PubMed DOI PMC

Kandioller W.; Balsano E.; Meier S. M.; Jungwirth U.; Göschl S.; Roller A.; Jakupec M. A.; Berger W.; Keppler B. K.; Hartinger C. G. Organometallic anticancer complexes of lapachol: metal centre-dependent formation of reactive oxygen species and correlation with cytotoxicity. Chem. Commun. 2013, 49, 3348–3350. 10.1039/c3cc40432c. PubMed DOI

Romero-Canelón I.; Mos M.; Sadler P. J. Enhancement of selectivity of an organometallic anticancer agent by redox modulation. J. Med. Chem. 2015, 58, 7874–7880. 10.1021/acs.jmedchem.5b00655. PubMed DOI PMC

Scalcon V.; Top S.; Lee H. Z. S.; Citta A.; Folda A.; Bindoli A.; Leong W. K.; Salmain M.; Vessières A.; Jaouen G.; Rigobello M. P. Osmocenyl-tamoxifen derivatives target the thioredoxin system leading to a redox imbalance in Jurkat cells. J. Inorg. Biochem. 2016, 160, 296–304. 10.1016/j.jinorgbio.2016.04.005. PubMed DOI

Gaiddon C.; Gross I.; Meng X.; Sidhoum M.; Mellitzer G.; Romain B.; Delhorme J. B.; Venkatasamy A.; Jung A. C.; Pfeffer M. Bypassing the resistance mechanisms of the tumor ecosystem by targeting the endoplasmic reticulum stress pathway using ruthenium- and osmium-based organometallic compounds: An exciting long-term collaboration with Dr. Michel Pfeffer. Molecules 2021, 26, 5386.10.3390/molecules26175386. PubMed DOI PMC

Suntharalingam K.; Johnstone T. C.; Bruno P. M.; Lin W.; Hemann M. T.; Lippard S. J. Bidentate Ligands on Osmium(VI) Nitrido Complexes Control Intracellular Targeting and Cell Death Pathways. J. Am. Chem. Soc. 2013, 135, 14060–14063. 10.1021/ja4075375. PubMed DOI PMC

Chow M. J.; Babak M. V.; Tan K. W.; Cheong M. C.; Pastorin G.; Gaiddon C.; Ang W. H. Induction of the endoplasmic reticulum stress pathway by highly cytotoxic organoruthenium Schiff-base complexes. Mol. Pharmaceutics 2018, 15, 3020–3031. 10.1021/acs.molpharmaceut.8b00003. PubMed DOI

Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 2012, 13, 89–102. 10.1038/nrm3270. PubMed DOI

Deniaud A.; Sharaf el dein O.; Maillier E.; Poncet D.; Kroemer G.; Lemaire C.; Brenner C. Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene 2008, 27, 285–299. 10.1038/sj.onc.1210638. PubMed DOI

Lebeau P. F.; Platko K.; Byun J. H.; Austin R. C. Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells. J. Biol. Chem. 2021, 296, 100779.10.1016/j.jbc.2021.100779. PubMed DOI PMC

Bruker . Bruker; AXS Inc.: Madison, Wisconsin, USA, 2001.

Sheldrick G. Crystal structure refinement with SHELXL. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 3–8. 10.1107/s2053229614024218. PubMed DOI PMC

Spek A. L. PLATON SQUEEZE: a tool for the calculation of the disordered solvent contribution to the calculated structure factors. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 9–18. 10.1107/s2053229614024929. PubMed DOI

Karges J.; Heinemann F.; Jakubaszek M.; Maschietto F.; Subecz C.; Dotou M.; Vinck R.; Blacque O.; Tharaud M.; Goud B.; Viñuelas Zahínos E.; Spingler B.; Ciofini I.; Gasser G. Rationally designed long-wavelength absorbing Ru(II) polypyridyl complexes as photosensitizers for photodynamic therapy. J. Am. Chem. Soc. 2020, 142, 6578–6587. 10.1021/jacs.9b13620. PubMed DOI

Ghosh G.; Yin H.; Monro S. M. A.; Sainuddin T.; Lapoot L.; Greer A.; McFarland S. A. Synthesis and characterization of Ru(II) complexes with π-expansive imidazophen ligands for the photokilling of human melanoma cells. Photochem. Photobiol. 2020, 96, 349–357. 10.1111/php.13177. PubMed DOI PMC