Requisitos para la inscripción de la Pymes como emisores en el Registro

Early efforts studying the photophysical and photochemical properties of 1 involved the use of electronic absorption spectroscopy supported by Density Functional Theory (DFT), nuclear magnetic resonance (NMR) spectroscopy and

mass spectrometry (MS) methods.[37v]

The UV-Vis spectrum of 1 (Figure 1.10) revealed two absorption maximum at

~260 nm and ~295 nm which were assigned as dissociative LMCT (N3,

OH−>Pt) and mixed 1LMCT/1IL (OH−>Pt,N3 ; IL = interligand) transitions

respectively, by time-dependent DFT.[37v] _{Furthermore, a weak shoulder in the}

visible region at ~414 nm can be gleaned, with a similar mixed 1_LMCT/1_IL

character. The photodecomposition of 1 by UV-Vis features the loss of 294 nm

absorption at different rates, depending on the excitation wavelength. Interestingly, evolution of gas bubbles was observed throughout irradiation of 1, although this was not further discussed at the time.[37v] Further studies on a close derivative of 1, trans,trans,trans-[Pt(N3)2(OH)2(py)(methylamine)],

revealed a gas evolution consisting of N2 (likely recombination of two !N3) and

O2 (from the complex, rather than solvent, verified by 18O-labeled water) by gas

Figure 1.10. The experimental (black) and predicted (red) UV-Vis spectra of 1 in water. Inset: expansion of the 350 – 450 nm region. Blue bars indicate the calculated excited states, with heights equal to extinction coefficients. Reprinted with permission from Farrer et al.[ 3 7 v ] Copyright 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The photoinduced binding of 1 (9 mM) to a DNA-base model, 5’-guanosine monophosphate (5’-GMP, 2 mol equiv.), was studied by 195_{Pt NMR in D}

2O

under 420 nm irradiation.[37v] Complete removal of 1 was observed within 45 min of irradiation and led to two major Pt(II) NMR signals. Synthesis allowed for the elucidation of one of the formed compounds: trans-[Pt(py)2(5-GMP)2]2+ (A),

whilst the other species was reasonably assigned to

trans-[Pt(N3)(py)2(5-GMP)2]+ (B) (Figure 1.11). A third minor (PtIV) species was

observed, which, after irradiation, increased in magnitude over time together with A, whilst B decreased. Subsequent monitoring of these species by electron spray ionization (ESI) MS matched this minor species to a molecular formula of: [Pt(N3)(OH)2(py)2(5’-GMP)]+ (C). 15N labelling of pyridine and azide allowed for

further elucidation of the NMR spectra of 1, although, no photodecomposition studies by 14N or 15N NMR were reported.

Figure 1.11. Proposed molecular structures of the formed species by photoinduced binding of 1 with 5’-GMP under 420 nm irradiation, as observed by 1 9 5_{Pt NMR and ESI-MS.}[ 3 7 v ]

A combination of electron paramagnetic resonance (EPR) and 14_{N NMR studies}

carried out by Butler et al. captured several important characteristic events of the azido ligands, upon irradiation of 1.[37y] Firstly, by using a spin trap, 5,5- dimethylpyrroline N-oxide (DMPO), azidyl radicals (from 14N and 15N azido ligands) were captured in water, phosphate-buffered saline (PBS) and growth medium for cell culture (RPMI-1640). However, the azidyl radical binding to DMPO competes with the formation of N2 following radical dimerisation,

depending on concentration and solvent, confirmed by 14N NMR. Whilst PBS yielded slightly higher concentrations of trapped radicals, RPMI-1640 yielded lower concentrations. The inclusion of various amino acids, vitamins, inorganic salts, and other growth-promoting substances in RPMI-1640 is likely to compete or influence the reaction of azidyl radicals with DMPO. Furthermore, the addition of a two molar equivalent of the DNA-base model, 5’-GMP, had little effect on the capture of azidyl radicals, but did prevent the precipitation of resulting platinum species otherwise formed at such high concentrations (4 mM), likely due to binding of 1 to 5’-GMP.

Introducing known electron transfer mediators, present on the side chains of proteins, tyrosine (Tyr) and tryptophan (Trp), into the photoreactions was carried out to further investigate the possible effect of amino acids on the photocytotoxicity of 1. Tyr! _{and Trp}! _{are weaker oxidising agents than} !_N

3, thus

Tyr and Trp could potentially quench the !_N

3 radical to N3-.

The presence of L-Tyr, had little influence on the trapping of azidyl radicals,

whilst L-Trp completely suppressed the capture of azidyl radicals by DMPO at

Pt OH OH N N N3 N3 5’-GMP Pt N N N3 N3 5’-GMP Pt N N N3 N3 5’-GMP 5’-GMP

A

B

C

0.25 mol equiv., without substantially affecting the photodecompostion of 1. This interestingly translated similarly to the photocytotoxic effect of 1 on the A2780 cancer cell line. Co-incubation of varying concentrations of L-Trp with 1 allowed for an increase in viability. At 500 µM L-Trp, the IC50 values (half maximal

inhibitory concentrations) of 1 were increased by ~7 fold. Even at equal concentrations (~50 µM, L-Trp and 1), viable cells were observed after 24

hours, whereas 50 µM of 1 alone resulted in no viable cells.

This allowed for the first in vitro proof of the dual mechanism of 1, in which the released ligands upon irradiation are responsible for a part of the observed photocytotoxicity. Additionally, the relative depletion of Trp levels in the serum of some cancer patients (breast, lung and ovarian) could be an underlying factor of the effectiveness of 1 as a chemotherapeutic agent.[26,69]

Significant advances have been made in effort to elucidate the mechanism of action, suggesting that the cleaved ligands and platinum centres following photoactivation, act in concert to elicit the observed multi-targeted biological activity. The potential use of NMR spectroscopy to monitor the azido release of 1 was reported. However, it remains challenging to deduct significant information due to the broad resonances for quadrupolar I=1 14_{N and poorly}

sensitive I=1/2 15_{N resonances (due to lack the enhancement by polarisation}

transfer via coupled protons).[57]

Further insights into the release of azide and, yet to be studied, hydroxido ligands as well as the changes around the platinum centre are of utmost importance to improve our understanding of these potential anticancer prodrugs in the field of chemotherapy.