## 3 Results and Discussion
### In Vitro Findings  
The interesting results obtained by in silico studies prompted us to investigate the biological activity exerted by compounds 4, 7, 8 and 10. Preliminary analyses indicated that compounds 4 and 7 exerted marked antiadrenergic effects on the Langendorff perfused heart model, reflecting a reduction of the cardiac performance associated with a weak cardiac recovery after washing with Krebs-Henseleit solution (unpublished data). Therefore, we aimed to deeper examine the biological activity induced by compounds 8 and 10. These molecules were tested on H9c2 cells alone or in the presence of ISO, widely used as inducer of myocardial stress and hypertrophy, mimicking the activation of the adrenergic and neurohumoral systems [46]. Effect of Compounds 8 and 10 on Cell Proliferation and ISO-Dependent Hypertrophy in H9c2 Cardiomyocytes  
It has widely ascertained that the activation of \(\beta\)1- and \(\beta\)2-AR represents a main mechanism responsible for the progression of cardiac remodeling [47]. Although it has been shown that the expression of \(\beta\)2-ARs is higher compared to that of \(\beta\)1-ARs in both neonatal rat cardiomyocytes (\(\beta\)1, 36% vs. \(\beta\)2, 64%) [48] and in embryonic rat cardiomyocytes H9c2 cell lines [48; 49], significant findings elucidated the selective contribution of \(\beta\)1 and \(\beta\)2-AR into the complex mechanisms of \(\beta\)-ARs-mediated adverse remodeling. Several studies also support the fact that, among the \(\beta\)-ARs, the \(\beta\)1 subtype is mainly responsible for catecholamine effects and represents the main promoter of cardiomyocyte hypertrophy, interstitial fibrosis and heart failure [50; 51]. For instance, it has been reported that isoproterenol binds to \(\beta\)1-AR with higher affinity compared to \(\beta\)2-AR [52; 53]. Furthermore, the isoproterenol-mediated cardiac responses were inhibited by betaxolol, a \(\beta\)1-AR antagonist, but not by ICI 118551, a \(\beta\)2-AR antagonist. These effects were observed both in vitro (i.e., primary cultures of cardiac ventricular myocytes stimulated with isoproterenol) and in vivo (i.e., rats exposed to continuous infusion of isoproterenol) [48]. Overall, these observations strongly suggest that the \(\beta\)1-subtype predominantly mediates isoproterenol-dependent hypertrophy in the heart. Accordingly, to assess the effects of compounds 8 and 10 on cell viability, we first exposed H9c2 cells to increasing concentrations (1 to 100 nM) of each compound and their cytotoxic potential was evaluated by MTT assay. As shown in Figure 4A, compound 8 did not exert significant effects on cell proliferation at any tested concentration compared to the control cells, while compound 10 induced a significant increase in cardiac cell viability at 10 nM and 50 nM (Figure 4B). These data indicate that both compounds do not negatively affect cell viability and do not show direct cytotoxic effects. Taking into consideration these results, we evaluated the in vitro effects of compounds 8 and 10 against cardiomyocytes hypertrophy. Therefore, we exposed H9c2 cells to ISO at 100 \(\mu\)M for 24 h [36], in the presence or absence of compound 8 or compound 10. Morphological staining analysis revealed that ISO treatment induced a significant increase in cardiac cell size with respect to control cells, indicating that the model of in vitro hypertrophy was successfully established (Figure 5A). Notably, compound 8 resulted was effective in significantly reducing cell size compared to the ISO group only at concentrations of 25 nM and 100 nM. Cells treated with compound 8 alone did not show significant increase in cell size (Figure 5A). The surface area analysis of H9c2 cells reflected the same trend (Figure 5B). The same experiment was carried out for testing the activity of compound 10. In this case, as evinced by morphological staining and cell surface area analysis shown in Figure 6, ISO-treated cells exhibited a significant cardiac cell size increase compared to the control cells.