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Title: Photodecomposition pathways for photoactivatable platinum(IV) diazido anticancer complexes
Author: Butler, Jennifer Suzanne
ISNI:       0000 0004 5357 5334
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2014
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Photo-activatable platinum(IV) diazido complexes with the general formula of trans,trans,trans-[Pt(N3)2(OH)(X)(py)(am1)] (X is a hydroxide or carboxylate and am1 is an aliphatic/aromatic amine) show dark-stability under physiological conditions but can induce a photo-cytotoxic effect in cancer cells after irradiation with UVA, blue and/or green light. These platinum(IV) diazido complexes can platinate DNA and induce different lesions that are distinctly different from those generated by the anticancer drug cis-platin. Through the use of EPR, multinuclear NMR, and UV-visible absorption spectroscopy, as well as mass spectrometry and some cell studies, this thesis aims to investigate the pathways of photochemical decomposition and in particular the release of azidyl ligands and their subsequent involvement in photo-cytotoxicity. Firstly, the irradiation of trans,trans,trans-[Pt(N3)2(OH)2(py)2] (40, py = pyridine) with blue light in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) led to the detection of a characteristic quartet-of-triplets EPR spectrum, assigned to the azidyl radical adduct, DMPO-14N3. Irradiation of 15N-40, led to the detection of a quartet-of-doublets EPR spectrum as assigned to the DMPO-15N3 spin adduct. This confirmed that the ●N3 radicals arose from the platinum(IV)- bound azide. The DMPO-N3 spin adduct was also detected from the photoirradiation of trans,trans,trans-[Pt(N3)2(OH)2(MA)(py)] (44, MA = methylamine) with blue light. A greater yield in the DMPO-N3 spin adduct was formed in PBS/D2O. This effect was attributed to the Brownian motion of the ●N3 radicals. Interestingly, photoirradiation in the cell culture medium-, RPMI-1640 led to a reduction in the DMPO-N3 spin adduct. This reduction was accredited to the variety of components present in the cell culture medium which could behave as radical quenchers. Alternative nitrone spin traps, -4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) also led to the trapping of the azidyl radicals from irradiated 40, forming the 4-POBN-N3 (triplet-of-quartets) and DEPMPO-N3 (octet-of-triplets) spin adducts EPR spectra, respectively. DEPMPO was the most efficient azidyl radical trap; with the DEPMPO-N3 spin adduct possessing the longest lifetime in aqueous media. Extending the wavelength of activation to green light (517 nm), also led to the detection of DMPO-N3 from the photo-irradiation of 40 in RPMI-1640, trans,trans,trans-[Pt(N3)2(OH)(SAD)(py)2] (56,SAD=succinate), trans,trans,trans-[Pt(N3)2(OH)(ethyl-methyl-SAD)(py)2] (57) and trans,trans,trans-[Pt(N3)2(OH)(N-MI)(py)2] (58, N-MI = N-methylisatoate) in H2O/DMF. The DEPMPO-N3 was also detected from the gamma-ray irradiation of 40, which appears to be the first report of the activation of a platinum(IV) diazido anticancer complex with gamma-rays, a procedure which might be useful of clinical use. The azidyl radicals generated from irradiated 40 were unreactive towards both glycine and L-tyrosine. However, in the presence of L-tryptophan the azidyl radicals were quenched. Detection of free azide by 14N NMR spectroscopy confirmed that this quenching mechanism proceeded through a one-electron transfer pathway. The photo-cytotoxicity of 40 was supressed in the presence of low doses of L-tryptophan in A2780 ovarian cancer cells. From this study, it was deduced that the photo-cytotoxicity of 40 is comprised of both an acute (radical) and chronic (DNA platination) based mechanisms. Additionally, certain cancers have reported on the depleted serum levels of L-tryptophan, in particular ovarian cancer cells. This suggests the extent of the photo-cytotoxicity of 40 can be controlled in the presence of L-tryptophan. Photo-irradiation of 40 in the presence of melatonin, an analogue of L-tryptophan was also performed. Despite, the structural similarity between L-tryptophan and melatonin, the presence of the 5-methoxy substituent present in melatonin induced an alternative photo-decomposition pathway of 40. Photo-irradiation of 40 in the presence of melatonin with blue light also led to the detection of the quartet EPR spectrum assigned to the hydroxyl radical adduct, DMPO-OH. Through multinuclear NMR and mass spectrometry the quenching of both the azidyl and hydroxyl radicals by melatonin was determined. Additionally, mass spectrometry also detected a mass adduct attributed to a platinum(II)-melatonin complex. This dual antioxidant and metal-binding ability of melatonin was attributed to the observed photo-protective effect in A2780 ovarian cancer cells. Melatonin regulates circadian rhythms with a maximum concentration during dark hours. Consequently, treatment of antineoplastic tissue with 40 during the hours of melatonin production may be ineffective. Platinum accumulation and absorption has been suggested to be mediated by organic cation transporters (OCTs), in particular OCT2. Irradiation of 40 was monitored in the presence of cimetidine, an OCT2 inhibitor. A new strong absorption band at ca. 354 nm was assigned to an SPtII LMCT band. High resolution mass spectroscopy identified a mass adduct assigned to a novel platinum(II)-cimetidine species. The loss of coordinated pyridine from irradiated 40 as detected by 1H NMR spectroscopy and the quenching of the azidyl radical by cimetidine were correlated with the observed photo-protective effect in HaCaT keratinocytes cells.
Supervisor: Not available Sponsor: European Research Council (ERC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; RC0254 Neoplasms. Tumors. Oncology (including Cancer)