Cytotoxicity determination of 80b and 93b

To confirm and investigate the different effects of the diastereomers 80a and 80b on the viability of cancer cells, different cancer cell lines were exposed to various concentrations of the two diastereomers (racemic) for 24 h followed by cell viability determination via the MTT method.

These cell lines include the afore used HT29 cells, the classical cervical cancer cell line HeLa, and two melanoma cell lines, the metastatic WM983B which inherits a natural BRAF mutation (V600E) and p53 wild-type (wt), and WM1366 with a NRAS mutation (61L).

0,1 1 10

0 20 40 60 80 100 120 140

cell survival rate [%]

concentration 80b [µM]

HeLa HT-29 WM1366 WM983B

0,1 1 10

0 20 40 60 80 100 120 140

HeLa HT-29 WM1366 WM983B

cell survival rate [%]

concentration 1a [µM]

Fig. 32: Cell survival rates of cancer cells after treatment with racemic 80b or racemic 80a for 24 h. The cell viability was determined via the MTT method and calculated as percentage of surviving cells compared to untreated controls.^[213]

Indeed, Fig. 32 reveals again a different behaviour in terms of cytotoxicity for the different diastereomers. Whereas 80b displays quite high cytotoxicities with estimated LD50- or IC50-values in the range of 2.9 μM (HT-29) to 0.4 μM (WM983B), 80a is much less cytotoxic with cell survival rates of 82.8% (HT-29) to 36.4% (WM983B) at a concentration of 30 μM. Generally highest cytotoxicity was seen for both compounds in the p53 wt cell-line WM983B, lowest toxicity in HT-29 cells, confirming the previous mentioned trend of low sensitivity of these cells for our kinase inhibitors.

In addition to these in-depth cytotoxicity studies of 80b and 80a, also 93b and 93a were tested in a concentration dependant MTT assay in HT-29 cells. The results are shown in Fig. 33.

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0,1 1 10

0 20 40 60 80 100 120 140

HT29 cell survival rate [%]

concentration [µM]

93b 93a

Fig. 33: Cell survival rates of HT-29 cells after 24 h treatment with 93a and 93b. The overall cytotoxicity is quiet low in comparison to 80b and the a isomer again shows no significant cytotoxicity.

It becomes clear, that 93b is much less cytotoxic than 80b, and its diastereoisomer 93a again shows no cytotoxicity at all. One should notice however, that during the experiments for both compounds large red crystals were observed in the cell medium, indicating solubility issues for both diastereoisomers as a new parameter which might influence cytotoxicity. This leads to further studies on 80a and 80b, to see whether differences in solubility might be one reason for their different cytotoxic behaviour, since we already experienced different crystallisation behaviour during crystal growth for X-ray determination of the stereochemistry.

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3.1.6.1. Solubility studies in cell medium of 80a and 80b

To investigate possible solubility issues of 80a and 80b, both compounds were tested again for a cell-based cytotoxicity in HeLa cells, since these cells seem to detach later from the solid support in comparison to HT-29 cells when undergoing apoptosis. Throughout the incubation time with a compound concentration of 30 µM-0.1 µM, a crystallisation of 80a and 80b was monitored.

Whereas 80a obviously crystallises in the cell medium like 93a and 93b, 80b seems to mainly enter the cells and to visibly crystallise inside or attached to the cells at concentrations of 30, 10, 7.5 and 5 µM (Fig. 34). Interestingly, in the higher concentration range this crystallisation effect, as well as first signs of apoptosis, could already be observed only 5-6 h after compound addition.

Fig. 34: HeLa cells 24 h after substance addition. A) 30 µM of 80b: the compound seems to mainly enter the cells and crystallises until the cells die. A smaller amount forms crystals in the medium. Most of the cells died and only cell fragments remain. B) Reference: cells were incubated with 1% DMSO and the cells are healthy. C) 30 µM of 80a: the compound forms large and small crystals in the cell medium which seems to have minor effect on the cells. Only a few cells have died, the others look healthy. D) 10 µM of 80b: the cells show signs of apoptosis (rounding, detachment from the ground, blebing) and obvious red spots inside or on top of the cells. E) With the lower concentration (2.5 µM of 80b) some cells look apoptotic as well, most look healthy and no obvious localisation of compound can be seen. Samples A)-C) are prepared in medium without phenolred, while D) and E) are prepared in phenolred containing medium.

10 µM 80b 2.5 µM 80b

D)

30 µM 80b

30 µM 80a

Control E)

A) B) C)

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To verify our assumption that the crystal formation of 80b occurs inside cells and not on their surface, scanning electron microscopy (SEM) experiments were performed by Michael Hellwig at the centre for material sciences at the Philipps-Universität Marburg. Accordingly, HeLa cells were grown on small cover glasses without surface treatment in 6-well plates, incubated for 14-16 h with 5-10 µM of 80b and were afterwards either chemically fixed or freeze dried. When simply washing and then freezing the cells in liquid nitrogen, they almost completely detach instantly. The shortened incubation time was chosen based on the fact, that detachment of the cells is much easier on simple glass in comparison to the treated surface of the well plates used previously. For freeze dried samples, the cells were first washed with PBS and afterwards quickly with ddH2O to avoid the formation of salt crystals on the surface. This however leads to a swelling of the cells due to osmotic effects, and following SEM experiments showed large holes in the cell membrane, as well as partially broken cells, indicating this method of fixation not suitable for our purposes.

Using chemical fixation on the other hand, by protein and lipid cross-linking with glutaraldehyde and osmium tetroxide followed by dehydration with ethanol,^[216] gave good results in cell surface and shape, as can be seen by the SEM images shown in Fig. 35. In both samples, freeze dried cells and chemically fixed cells, no sign of crystals on the cell surface could be found at compound concentrations of 5 µM or 10 µM, which strengthened our assumption that the compound crystallises after entering the cell. However, for chemically fixed cells the compound might have been dissolved during the washing steps with ethanol and for freeze dried cells disturbance in the cell membrane might have released the compound, indicating no final prove for 80b to enter the cells before crystallisation. In addition to that, affected cells might have undergone apoptosis and thereby been detached from the glass surface before or during treatment.

Fig. 35: HeLa cells which were chemically fixed 16 h after addition of 10 µM of 80b. No signs of crystals on the cell surface were found in two independent samples.

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Assuming that 80b indeed enters the cells, a similar crystallisation effect, called crystal-like drug inclusions (CLDIs), is known for the approved leprosy drug clofazimine (trade-name: Lamprene).

The active ingredient accumulates to very high levels in tissue, forming intracellular liquid crystal-like supramolecular organisations, probably membrane-bound, multi-layered, liquid crystal-crystal-like, semi-synthetic cytoplasmic structures.^[217] Possible ways of crystal-formation discussed for clofazimine, including pH-dependant solubility through protonation state, protein-binding and associated complex formation with membranes, or precipitation as aggregates/crystals, which may be actively phagocytosed,^[217,218] are possible explanations for the crystal formation of 80b.

Whether or not 80b crystallises inside or on top of the cells, this effect is a deal-breaker for finding a suitable anticancer drug. Therefore, the classical cytotoxicity-based approach was dropped in favour for an approach investigating the drug like abilities of this compound class.

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