Producción de citas

The idea behind the synthesis of the new SiPc-SubPc triad is similar as that used by Cook and co-workers, in the way that the SubPc units will be introduced at the axial positions of the SiPc.^149c However, instead of parting from a dihydroxy SiPc, we wanted to part from the commercially available SiPcCl2, to be able to follow our previously applied methodology for the axial substitution of the SiPcs. Therefore, the SubPc units must bear a primary alcohol available for conjugation. In this respect, the same linker as that used in Subchapter 1 of this Chapter could be used.

SubPcCl 16 was prepared as before^86c and its axial chloride substituent replaced by 2-hydroxyethyl disulfide following the axial substitution procedure developed in our group (Scheme 15).¹⁴¹ The progress of the substitution reaction was followed by TLC, and in this case the reaction was completed after 6 h. The crude was then subjected to column chromatography on silica gel using toluene/THF with a gradient from 30:1 to 10:1 as the eluent. Subsequent trituration in water yielded the desired SubPc 18 with a 56% yield.

Scheme 15. Synthesis of the axial SubPc units bearing the appropriate linker for incorporation in the SiPcCl2.

0 0.2 0.4 0.6 0.8 1 1.2

0 0.2 0.4 0.6 0.8 1 1.2

350 450 550 650 750

Emission / a.u.

Absorption / a.u.

Wavelength / nm 4

18

3.2.2 Synthesis of the target SiPc-(SubPc)

triad

The next step involved the nucleophilic displacement of the chlorine atoms in SiPcCl2 with the primary alcohol 18. To this end, SiPcCl2 and 5 equivalents of 18 were dissolved in pyridine, and DIPEA was added as a base. After refluxing the reaction mixture overnight, the SiPc-SubPc triad 17 was formed (Scheme 16).

Scheme 16. Incorporation of SubPc 18 at the axial positions of SiPcCl2, following the standard procedure, resulting in formation of SiPc-SubPc triad 17.

The purification of 17 was difficult and tedious because of the inherent instability problems in both standard silica (which is slightly acidic) or silica previously neutralised with Et3N. In both cases, the compound decomposed rapidly, probably because the Pc unit is acid-sensitive while SubPcs are known to be base-sensitive. Part of the crude reaction mixture could finally be purified by SEC with Bio-Beads as the stationary phase and CHCl3

as the eluent, yielding triad 17 as a dark purple solid. However only a fraction of the compound elutes as pure compound, the remaining part eluting together with the starting products SiPcCl2 and SubPc 18, and/or decomposition products, for which an estimated yield of the product is reported. Because of this inherent instability of the molecule, it was not possible to detect the molecular ion by mass spectrometry, yet ¹H-NMR characterization is conclusive about the purity and identity of the purified fraction of the product (see Figure 69).

. . .

. . . . .

. . . .

. .

. .

Figure 69. ¹H-NMR of SiPc-(SubPc)2 triad 17, recorded in CDCl3.

3.2.3 Spectral features and photophysical properties

The absorption and photochemical properties of the SiPc-SubPc triad 17, SubPc 18 and reference SiPc 4 were measured in DMF (Table 8).

Table 8. Electronic absorption and photophysical data for SiPc 4, SubPc 18 and the compound does not contain any SubPc moiety. ^e Not applicable because the compound does not contain any Pc moiety.

The UV-Vis spectra of SubPc 18 and SiPc 4 show their typical broad Soret band (at 300-400 nm) and sharp Q band (at 562 and 675 nm, respectively). Accordingly, the UV-Vis spectrum of the SiPc-SubPc triad 17 shows a broad Soret band at 300-400 nm and two Q-bands, the first one originating from the two axial SubPc units (at 562 nm) and the second one from the SiPc unit (at 675 nm), in an expected 2:1 stoichiometrical ratio (Figure 70).

Figure 70. The sum of spectral features in DMF for the reference compounds 18 and 4, in the right stoichiometry (2:1), matches the spectrum of the SubPc-SiPc triad 17.

φF were measured by exciting the compounds at 520 nm (for SubPcs) or 610 nm (for Pcs).

φΔ, in turn, were again measured by the relative method, using DPBF as a chemical scavenger and excitation with visible light filtered below 455 nm (hν > 455 nm).As expected, compared to the reference SiPc 4 and the SubPc 18, the SiPc-SubPc triad 17 shows a significant deactivation of its fluorescence and ¹O2 generation, probably as a result of an electronic energy transfer process in which the SubPc emission band and the SiPc Q-band partially overlap (Figure 68).

0

3.3 Proposed tumor responsive activation of the SiPc(SubPc)

triad

According to the design of the molecule (Figure 67b), initially the fluorescent and ¹O2 -generating properties of 17 are quenched, most probably by electronic energy transfer between the SubPc units and the Pc core. Upon entering a reducing environment, e.g., like that in tumor cells, the disulfide linker would be cleaved and the quenching would be undone, resulting in a sudden generation of ¹O2 and a strong fluorescence of both the SiPc and SubPc units. In the following subsections, this behavior has been probed in a physiologically relevant medium, by means of using DTT as a strong reducing agent to achieve the PS activation. In this way we hope to gain understanding of how both the fluorescence and ¹O2 generation of 17 would be influenced in a tumor like environment.

3.3.1 Effect of DTT concentration on the fluorescence properties of the SiPc(SubPc)

triad

To investigate the influence of intratumor-like disulfide cleavage on the fluorescence properties of 17, its emission spectrum was monitored for 24 h in PBS buffer solutions with varying DTT concentrations (Figure 71).

Sample preparation was as follows: 17 is dissolved in DMF to give a 1 mM solution, which was diluted to 0.4 μM with PBS (with 0.5% Cremophor EL). DTT was dissolved in deionized water to give a 1 M solution. Mixtures of 17 (0.4 μM) with DTT (2 μM, 5 mM, 10 mM, 20 mM or 50 mM) or without DTT in PBS (25 mL) were prepared and stirred continuously.

Figure 71. Representation of the fluorescence spectra of 17 in PBS (0.5% Cremophor EL) with a) 0 μM DTT, b) 2μM DTT, c) 5 mM DTT, d) 20 mM DTT and e) 50 mM DTT, from t =

0 h to t = 24 h.

Figure 72. Normalized emission maximum of 17 in PBS (0.5% Cremophor EL) in function of time for a) the SubPc units (F571) and b) the phthalocyanine unit (F680).

As can be seen in Figure 72a, the fluorescence intensity of the SiPc unit (at 674 nm - λex = 345 nm) exhibits approximately a 9-, 12- and 18-fold increment in the presence of 5, 20 and 50 mM of DTT respectively, while in the absence of DTT or in the presence of only 2 µM of DTT there is no SiPc fluorescence enhancement. This increase in fluorescence intensity reflects that the SiPc emission is recovered upon disulfide cleavage. On the other hand, the fluorescence intensity of the SubPc unit (at 571 nm - λex = 345 nm) displays a similar initial recovery, but reaches a maximum recovery around 2 hours, after which the fluorescence intensity starts decreasing again (Figure 72b).

To explain this unexpected result, the influence of DTT on the absorption of 17 was studied (Figure 73). From this experiment, it became clear that 17 was unstable to the strong reducing environment, indicated by a decrease of the SubPc absorption at 562 nm in function of time, the extent of the decrease being greater at higher concentrations of DTT.

This instability, although unexpected and in principle undesired, does not necessarily urge to discard the use of 17 as an activatable tumor responsive and theranostic PS for PDT, however, as the outcome caused by the actual decomposition of the SubPc unit or by the foreseen cleavage of the disulfide linker is the same with respect to both its ¹O2 generation and the fluorescence of the SiPc unit. Indeed, both outcomes would be activated once 17 enters the strong reducing environment of a tumor cell, just as they are in solution.

Figure 73. UV-Vis spectra of 17 in PBS (0.5% Cremophor EL) with a) 0 µM DTT, b) 2 µM

3.3.2 Effect of DTT concentration on

O

generation of the SiPc(SubPc)

triad

To investigate the effect of a reducing environment (as in tumor cells) on the capacity of triad 17 for ¹O2 generation in physiologically relevant medium, ¹O2 studies were performed in the same buffer solutions with varying DTT concentrations. The rate of DPBF photodegradation upon irradiation of the SiPc-SubPc triad 17, which is proportional to the amount of ¹O2 formation, is measured in combination with 0 µM, 2 µM, 5 mM, 20 mM and 50 mM of DTT and represented in Figure 74.

Figure 74. Effect of DTT on the capacity of 17 for ¹O2 generation: normalized absorption (A/A0) of DPBF at various concentrations of DTT over time, showing its photodegradation rate sensitized by 17 upon excitation with light above 455 nm.

A clear trend can be deduced from this data, demonstrating that more efficient ¹O2

generation results from samples exposed to a higher concentration of DTT. Thus, the degree of disulfide cleavage seems to be directly correlated to the ¹O2 generation efficiency of 17. In the absence of DTT or in the presence of only 2 µM of DTT (corresponding to the extracellular glutathione concentration) there is no enhancement of the ¹O2 generation, which reflects the need of a strong reducing environment to promote such activation via disulfide cleavage. Moreover, dark control experiments confirm that, under all conditions, compound 17 is unable to produce ¹O2 without hν> 455nm

illumination.

In summary, SiPc-SubPc triad 17 is a PS with the ability to generate ¹O2, the main reactive species in PDT, in a substantially higher amount once entered in a reducing environment simulating that of tumor cells. However, due to the difficulty in the preparation and purification of the SiPc-SubPc triad, and the inherent instability problems, no in vitro tests of PDT efficacy are performed for this PS.

0,25

Norm. Absorbance DPBF (A/A0) / a.u.

Irradiation time / min

0 µM DTT 2 µM DTT

5 mM DTT 10 mM DTT

20 mM DTT 50 mM DTT

0 µM DTT dark control 50 mM DTT dark control

4