3.4.3.1 UV-Vis studies
UV-Vis spectroscopy allows the photoreduction of Pt(IV)-diazido compounds to be monitored as a decrease of the N3→Pt charge transfer band (at ca. 300 nm) is observed due to the loss of an azide. Irradiation of compound 3 results only in a decrease in intensity of this LMCT band, whereas for compounds 4 and 5, there is an apparent increase in intensity of the band at ca. 250 nm (refer to Figure 3.15 and Figure 3.16, section 3.3.6). In case of compound 3, similarly to 6, no increase of intensity occurs for the band at 250 nm but a slight shift of the maximum wavelength is observed. This result could be explained in two ways: either the electronic transitions of the attached ligand are not mixing with the transitions of the platinum centre therefore absorbance of the former is similar to the free ligand or the release of the ligand is very slow in comparison to the release of the azide. Most reduction profiles of Pt(IV) complexes,65 as found for compounds 3-6, follow an exponential decay profile. This is illustrated in Figure 3.18 which shows plots of the absorbance of the N3→Pt band, as a function of time. Mechanistic
134 studies of the chemical reduction of other Pt(IV) prodrugs in the presence of reducing agents such as GSH and L-ascorbate have shown that the reduction is second-order and proportional to the concentration of the Pt(IV) complex as well as the reducing agent.66 In the case of the photoreductions carried out on 3-6, it was observed a decrease in the reduction rate when the concentration of the sample was increased, upon the same light exposure (Figure 3.44).
0 100 200 300 400 500 20 30 40 50 60 70 80 90 100 110 Complex 6 40 Complex 6 60 N o r m a li z e d a b s o r b a n c e Time (min)
Figure 3.44: Time dependent photoreduction of complex 6 at 40 μM (t1/2= 192 min) and 60 μM (214 min) concentration in 5% DMSO/95% H2O. The photoirradiation was carried out with green light (517 nm).
The photoreactions proceed at different rates depending on the complex (t1/2= 634 min for 3,220 min for 4, 169 min for 5 and 214 min for 6). Evidently, the half times of the compounds decrease by up to ~4 times less (e.g. compound 5) as compared to the dihydroxo compound 3. This suggests that the carboxylate functionality enhances the photoreactivity of these complexes. All three show
135 much faster kinetics in comparison to compound 3. Furthermore compound 5, the fastest complex in photodegradation, shows the longest Pt–O (carboxylate) bond, which photodissociates upon light exposure. Observing an effect on the reduction rate of Pt(IV) prodrugs with an increasing chain length of the functionality on the carboxylate ligand is not unusual (although the chain is distant from the Pt centre). Ravera et al showed that the reduction potential decreased for a longer carbon chain, in cis,trans,cis-[PtCl2(mpy)(NH3)(RCOO)2], where R= CH3(CH2)n, n=0-4.8 The increased rate of photodegradation in the case of compound 4 versus compound 3 correlates with the extinction coefficients, higher for 4 at the longer wavelengths (Figure 3.13). Compound 4 underwent very little activation with yellow light (550 nm, 2.5 mW/cm2), as judged by the change in the absorbance of the band at ca. 300 nm.
3.4.3.2 EPR studies
The formation of azidyl radicals when Pt-diazido compounds are irradiated may play a role in the mechanism of action of these prodrugs.52 Therefore the amount of azidyl radicals produced on photoactivation of the complexes studied here was determined.
The first experiment was carried out with 1 mM solutions of compounds 3, 4, and 5 in PBS in the presence of DMPO (spin-trap). The results of this study showed that no radicals were trapped in the case of 3 whereas 4 and 5 yielded radicals up to 68 M and 55 M, respectively (Figure 3.21). The identity of the trapped radical was confirmed to be the DMPO-N3 by matching the hyperfine splitting constants (HFSC) to the theoretical data reported previously (Figure 3.45).43, 41
136 Hydroxyl or carboxylate radicals, which could potentially be released from the Pt(IV) complex, would give a 1:2:2:1 pattern when trapped by DMPO. This signal however, was not detected in the spectrum. Even though compound 4 was found to have a slightly lower photodissociation rate than compound 5, the azidyl radical yield after 14 minutes of irradiation was in fact slightly higher for the former. However the initial production of radicals (e.g. after 7 min of irradiation) was lower for 4 than for 5.
Figure 3.45: (A) The structure of the spin-adduct DMPO.-N3 adduct formed by the addition of N3. to DMPO. (B) The origin of the hyperfine splitting pattern from the coupling of the unpaired electron to nitrogen (atom 1), hydrogen (atom 2) and nitrogen (atom 3). (C) EPR signal of an irradiated sample of compound 4 in DMF/H2O (75 %, 25%, v/v) in the presence of DMPO (2 mol eq) at 20 oC.
137 A similar experiment was carried out using DMF as the solvent. Compounds 3, 4, and 5 in DMF gave similar results as in PBS, with compound 4 giving the highest and compound 3 the lowest azidyl radical yield. Surprisingly, compound 6 produced a similar amount of radicals as 3 although the rate of photodegradation is ~3-fold higher. A plausible explanation for this fact is that the released N- methylisatoate ligand (axially bound in 6) could act as a radical scavenger. In order to confirm this hypothesis, complex 3, trans, trans, trans- [Pt(N3)2(OH)2(pyr)2], was irradiated (λ= 463 nm, 64 mW/cm2) in the presence of N-methylisatoic acid (1 or 2 mol eq). The production of azidyl radicals was entirely quenched by N-MIA suggesting that upon release, this ligand can readily react with N3·. The reactivity of similar molecules with reactive oxygen species has been established recently where it was shown that methyl anthranilate, structurally related to N-methylisatoate ligand, can quench singlet oxygen due to the presence of the anilinic moiety.67