Practica: Transmisor de FM

Photo-activation of a chemotherapeutic (PACT) differs from conventional PDT as the administered complex remains non-toxic until photo-activation with visible light without the requirement of molecular oxygen (type III mechanism). As mentioned, PtIV complexes are kinetically inert making them ideal candidates as photo-activatable platinum prodrugs.

1.8.1 Photo-activatable PtIV _{diiodo complexes}

Similar to PDT, longer wavelengths of light (> 600 nm) are preferred for PACT, for deep penetration into the cancerous tissue. The wavelength of activation for inorganic complexes can be determined from the exhibited electronic transitions. The first photo-activatable PtIV_{-diiodo complexes were synthesised in the Bednarski}

laboratory. All these PtIV complexes possessed an ethylene-diammine ligand as the non-leaving group, opposed to the ammine ligands present in cis-platin. This was specifically chosen to avoid photo-isomerisation and render the resultant complex more stable. Kratochwil investigated the absorption bands of cis,trans- [Pt(en)(Cl)2(X)2] complexes, (where en = ethylenediammine and X = Cl, Br, I). The

complex cis,trans-[Pt(en)(Cl)2(I)2] (32, Figure 1.26) with the least electronegative

atom absorbed energy at the longest wavelength.127 Therefore, the choice of ligands coordinated to the PtIV centre can directly promote activation at longer wavelengths.128 Despite activation at longer wavelengths, complex 32 exhibited an equivalent dark and light cytotoxicity, attributed to its high reduction potential of ca. 75 E/mV. This large reduction potential rationalised its facile reduction by biological reducing agents (e.g. GSH) to the active PtII species.129

41 To increase the dark stability, the chlorido ligands where substituted by hydroxido- based ligands. The resultant complexes (33-35, Figure 1.26) exhibited lower reduction potentials and possessed a LMCT band at ca. 400 nm.

Figure 1.26 Structures of early photo-activatable PtIV-diiodo complexes.

Nuclear magnetic resonance (NMR) studies revealed the reduction of the PtIV- diiodo complexes (32, 33 and 35) by both glutathione (GSH) and N-acetyl-cysteine (NAC).130 Complex 34, the most potent complex, displayed ca. 65% DNA platination after 6 h, in contrast to cis-platin, which induced ca. 90% DNA platination after an equivalent time period.

The abundance of biological reducing agents throughout the body (refer to Figure 1.11), suggested reduction of PtIV to PtII was not specific to tumour cells. Therefore, effective PtIV photo-activatable prodrugs should exhibit dark-stability in the presence of biological reducing agents. Consequently, alternative ligands to iodides were investigated.

42

1.8.2 Photo-activatable PtIV_{diazido complexes}

Transition metal complexes possessing azido ligands have been reported to be light sensitive, regardless of the transition metal centre. Vogler was the first to report on the photo-chemical nature of platinum diazido complexes. Photo-irradiation of trans-[Pt(CN)4(N3)2]2- at 300 nm UVA gave rise to the loss of two azide ligands, in

the form of azidyl (N3) radicals. This proceeded through a simultaneous two one-

electron reduction of the PtIV centre without formation of a PtIII intermediate species,131 _{as shown below.}

The formation of the N3 radicals were confirmed by electron paramagnetic

resonance (EPR). This technique is specific for the characterisation of paramagnetic species and will be discussed in more detail in Chapter III. Moreover, irradiation of cis-[Pt(PPh3)2(N3)2] led to the formation of hexazabenzene (N6) and [Pt(PPh3)2]2.

Bubbles were observed in the irradiated solution, attributed to the decomposition of N6 into nitrogen gas.132 Interestingly, [Pt(N3)6]2-, initially synthesised and

characterised by Beck et al. was photo-irradiated by Volger et al. with ca. 314 nm UVA and reported to undergo a two photon (four-electron) reduction via a PtII

intermediate generating Pt0 , as shown below. The formation of a black precipitate upon additional irradiation confirmed the generation of Pt0.133

The development of second-generation photo-activatable platinum(IV) complexes was paved by the work performed by Bednarski and Vogler and the concept of PDT.

43 Both cis (36) and trans (37) – photo-activatable diammine platinum(IV) complexes

were investigated (Figure 1.27).134,135 _{Complexes 36 and 37 exhibited dark stability}

in the presence of intracellular reducing agents, as monitored by NMR spectroscopy.136,137 Irradiation of complexes 36 and 37 at 365 nm UVA led to the observation of gas bubbles, presumed to be nitrogen gas.138 Both complexes displayed IC50 values (50% inhibitory concentration) similar to both cis and trans-

platin. Interestingly, photo-irradiation of 37 in the presence of dimethyl-sulfide (DMS) led to the formation of a new carbon-to-carbon bond.139 Furthermore, in a separate study performed by Farrer et al., trans-diam(m)ine diazido PtIV complexes were determined to possess a greater photo-cytotoxic activity than their cis-isomers. The difference in activity was proposed to be due to the trans-based complexes targeting different cellular components or inducing different DNA lesions.140

Despite the dark stability of 36 and 37 and their ability to induce a photo-cytotoxic effect in HaCaT human keratinocytes cells, these complexes were limited to photo- activation at 365 nm UVA.

Figure 1.27 Structures of platinum(IV) diammine diazido complexes synthesised in the Sadler group.

Ways to lower the energy of the LMCT band of the PtIV diazido complexes involved the initial replacement of the ammine (NH3) group by a π-acceptor pyridine ligand.

44 Unfortunately, 38 (Figure 1.28) did not exhibit a LMCT band at lower energy (ca. 289 nm). Nevertheless, substitution of the pyridine ligand (38) for the ammine (NH3, 37) ligand decreased the IC50 value from ca. 99.2 µM to 1.9 µM in A2780

ovarian cancer cells. The stark difference in the photo-cytotoxicity values was attributed to the formation of alternative PtII-DNA adducts by 38.

Figure 1.28 Structures of mono-pyridine (38); mono-piperidine (39) and bis- pyridine (40) platinum(IV) diazido complexes.

Photo-irradiation of 38 at 365 nm UVA in the presence of 5-guanosine monophosphate (5-GMP, model for nucleobase guanine) identified binding to the N7 of 5-GMP and generated both trans mono-[Pt(N3)(NH3)(py)(GMP)]+ and bis-

[Pt(NH3)(py)(GMP)2]2+ adducts141 (Figure 1.29). The formation of these different

platinated-DNA adducts suggested that 38 induced its photo-cytotoxic effect through a novel mechanism of action. The formation of such a complex suggested a method of overcoming current resistance mechanisms of platinum anticancer complexes.

45

Figure 1.29 Structures of (A) mono- and (B) bis-guanine species formed from the photo-activation of complex 38 in the presence of 5'guanosine monophosphate (5'- GMP) (figures from ref 141)

Recently, Westendorf reported on 38 photo-irradiatedin vivo. Photo-irradiation of

38 with UVA in mice bearing xenografted OE19 esophageal carcinoma gave rise