# Simple Thalidomide Analogs in Melanoma: Synthesis and Biological Activity
## 3 Results and Discussion
### Biological Results
#### 3.2.2 Effect of Compound **2k** on Microtubule Dynamics and Cell Death by Apoptosis  
Microtubules represent vital cell components shaped by polymerization of \(\alpha\) and \(\beta\) tubulin heterodimers and play crucial roles in several cellular functions, including cell division [34]. Several classes of naturally occurring, as well as synthetic and semi-synthetic, compounds disrupt microtubule organization, altering the tubulin dynamics. Among them, vinca alkaloids and taxanes are two families of microtubule-binding agents currently used for the treatment of different solid and hematological tumors, and are characterized by strong side effects [35]. Generally, microtubule-binding agents are divided into two main groups: microtubule-destabilizing and microtubule-stabilizing agents, able to suppress microtubule dynamics with different mechanisms. Particularly, microtubule-destabilizing agents, including vinca alkaloids, inhibit the tubulin polymerization and thus destabilize microtubules; instead, taxanes, including paclitaxel, bind to polymerized microtubules, stabilizing tubulin filaments [36]. Recently, agents able to regulate microtubule dynam  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & \multicolumn{4}{c}{**IC\({}_{50}\) (μM)**} \\ \hline
**Compound** & **A2058** & **Sk-Mel28** & **MDA-MB-231** & **MCF-7** & **HeLa** & **Hek-293** \\
**2a** & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 \\
**2b** & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 \\
**2c** & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 & \(>\)200 & \(<\)200 \\ics, hampering tubulin polymerization or blocking microtubule disassembly, and free of adverse effects, have received great interest in anticancer therapy [37; 38; 39]. Given that our most active compound, **2k**, did not exert any cytotoxicity against the normal cells, we evaluated its ability to interfere with tubulin by using both an immunofluorescence assay and an in vitro tubulin-polymerization inhibition assay. Regarding the immunofluorescence assay, melanoma A2058 cells were exposed to compound **2k** or vinblastine, used as a reference molecule, for 24 h and then processed as described in the materials and methods. Figure 2 shows that, in the CTRL cells (vehicle-treated), tubulin resulted widely distributed in the cytoplasm, forming a regular arrangement and organization of the microtubules with normal and well-assembled filaments (Panel B, CTRL). On the contrary, A2058 cells exposed to vinblastine, as well as those treated with compound **2k**, reveled a dramatic disorganization of microtubules in which tubulin accumulated in the form of crystals within the cell cytoplasm (see white arrows, Figure 2, Panel B, vinblastine and **2k**). Thus, compound **2k** is able to perturb tubulin assembly, interfering with normal microtubule organization. Next, we evaluated if the detected microtubule disorder could be linked to the ability of compound **2k** to act as a destabilizing agent, inhibiting tubulin polymerization, or as a stabilizing agent. Thus, we performed an in vitro tubulin-polymerization inhibition assay, using paclitaxel and vinblastine as reference molecules, and compound **2k**, all used at a  
Figure 2: Immunofluorescence studies. Human melanoma A2058 cells were incubated with compound **2k**, with vinblastine (both used at their IC\({}_{50}\) values), or with DMSO (CTRL) for 24 h. Cells were observed under the inverted fluorescence microscope at 40\(\times\) magnification. CTRL cells displayed a normal arrangement and organization of the tubulin cytoskeleton. Instead, A2058 cells treated with compound **2k** and vinblastine exhibited an irregular microtubules organization (see white arrows). (**A**) nuclear stain with DAPI (\(\alpha_{\text{ex}}=350\) nm/\(\lambda_{\text{em}}=460\) nm); (**B**) \(\beta\)-tubulin (Alexa Fluor® 568; \(\lambda_{\text{ex}}=644\) nm/\(\lambda_{\text{em}}=665\) nm); (**C**) overlay channel. Representative fields are shown.