Cell stability and cellular localisation of compounds 136, 141 and 75

As the compounds have rapid uptake and good cytotoxicity profiles, some efforts were made in order to understand the mechanism by which the compounds are inducing cell death. As explained in Chapter 3, unnatural acetylated glycosides can be fed into cells and be rapidly internalised. Once inside the cells, cytosolic esterases are able to release the deprotected modified glycosides than are subsequently processed by the cells.212 After deprotection, the compounds could be susceptible to the enzymatic release of Amonafide by endogenous enzymes, as previous results demonstrated. This mechanism is depicted in Scheme 4.4.2 using 136 as an example.

Scheme 4.4.2. Different species that could be found inside the cells due to deprotection of acetyl groups by cytosolic esterases and release of Amonafide by endogenous glycosidases.

An experiment was performed to investigate if these glyconaphthalimide compounds were compatible with deacetylation by cytolosic esterases. HeLa cells were incubated with compounds 136 (50 μM) and 141 (50 μM), respectively for 2 h, followed by the replacement of the cell media by water to cause cell death and membrane disruption due to osmotic pressure. After 30 min, the suspension was taken and centrifuged to remove the cellular material and the supernadant was analysed using MS. Analysis of the sample using

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137 APCI showed that only acetylated derivatives 136 and 141 were found (Figures A.4.11- 12).

To further prove that Amonafide had not been released inside the cells, HeLa cells were incubated for 1 h with 136 (50 μM), 141 (50 μM), and Amonafide (50 μM), respectively, and fluorescence spectra of the compounds inside the cells were recorded using confocal microscopy (Figure 4.4.8).

As it can be observed in Figure 4.4.8, the fluorescence emission spectra of the compounds localised inside the cells possess a band centred at 460 nm, which is ~50 nm blue shifted than the corresponding for Amonafide. Therefore proving that compounds 136 and 141 did not release Amonafide inside the cells.

Figure 4.4.8 Fluorescence emission spectra of 136 (50 μM, _max= 460 nm, black line), 141 (50 μM,  max=

460 nm, red line) and Amonafide (50 μM, 

max= 550 nm, blue line) incubated in HeLa cells for 1 h (exc= 405 nm). Figure representative of three independent experiments.

Because of these very positive results that clearly show that this design principle worked satisfactory, HeLa cells were next incubated with the two compounds at longer time points. However 6 h was the longest incubation time possible to be recorded, as the cells were starting to show high levels of toxicity and after 24 h of incubation no cells were alive. Figure 4.4.9 is a representative image of the incubation of HeLa cells with compound 136 (50 μM), 141 (50 μM), 75 (50 μM) and Amonafide (50 μM) incubated for 6 h.

For compounds 136, 141 and 75 there was no remarkable difference between 1 h and 6 h incubation, however in the case of Amonafide it can be said that there is a higher content

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138

of compound within the nucleus. This difference indicated that compounds 136 and 141 are not releasing Amonafide.

After proving that the toxicity induced by 136 and 141 was not due to the release of Amonafide, a cellular localisation study was carried out to understand the mechanism of toxicity. Figure 4.4.10 represents an enlarged image of the incubation of compounds 136 (50 μM) and 141 (50 μM) respectively, in HeLa cells for 1 h. It can be noted that the compounds were localising mainly in two specific regions.

Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide

139 Figure 4.4.9. Incubation of HeLa cells for 6 h with a) Amonafide (50 μM), b) Compound 136 (50 μM), c) compound 141 (50 μM) and d) 75 (50 μM). Images representative of three independent experiments.

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In one hand, it was postulated that the areas showing fiber-like emission was due to mitochondrial localisation. On the other hand, the were areas showing clusters that possessed higher fluorescence intensity and were located right beside the nucleus, causing it to curve around it adopting a bean shape. It was hypothesised that the later could correspond to lysosomal localisation, as they possess lower pH values (ca. pH 4-5) and as demonstrated in section 4.3.4, the fluorescence intensity of these compounds is higher at lower pH. Interestingly, compound 75 (50 μM) exhibited similar subcellular localisation (Figure 4.4.10c).

Figure 4.4.10. Enlarged image of HeLa cells incubated with a) 136 (50 μM), b) 141 (50 μM) and c) 75 (50

Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide

141 To demonstrate that the compounds were mainly localised in the mitochondria, HeLa cells were transfected with a plasmid that contains a red fluorescent protein (DsRed). DsRed possesses an excitation and emission band of 557 and 592 nm, respectively, making it suitable for its visualisation along with these compounds. The transfection was done using Lipofectamine as transfection reagent.

The successfully transfected HeLa cells were incubated with compounds 136 (50 μM), 141 (50 μM) and 75 (50 μM) for different periods of time (Figure 4.4.11). As can be seen in Figure 4.4.11. The overlapped images with these compounds and DsRed

Figure 4.4.11. Confocal image of HeLa cells transfected with DsRed and incubated for 6 h with a) 136 (50

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matched, proving that these compound localised in the mitochondria. It is feasible that the localisation of the compounds in the mitochondria damage the cells over time, thus causing cell death. However, no detailed experiments on the cell death were carried out.

A transfection carried out with Lysosomes-RFP, lysosomal labelling protein (λexc = 555 nm, λem = 584 nm), was also attempted, to prove the localisation of these compounds in the lysosome. Unfortunately, this transfection did not work, possibly due to an old plasmid batch being used. However, due to time constrains no further transfection was attempted with a new batch.