## 3 Results and Discussion
### Molecular Docking  
In order to investigate the molecular interactions between compounds 4, 7, 8 and 10 and the active binding site of \(\beta\)1-adrenergic receptor, molecular docking studies were performed on a crystallographic structure of the target protein obtained from the Protein Data Bank (PDB code 7 BVQ) [22]. In this structure the receptor is complexed with carazol, a previously identified inverse agonist endowed with a carbazole based chemical structure [22]. In the PDB is reported another interesting crystallographic structure of \(\beta\)1 receptor (PDB code 7 JJO), very recently deposited, in which the protein is complexed with ISO, a known \(\beta\)1-adrenergic receptor ligand. Even though this structure presents a more complete aminoacidic sequence, it does not belong to _Homo sapiens_. In spite of this shortcoming, it was compared after alignment with 7 BVQ. From this comparison a sequence similarity of 86% was found between the two proteins and that an Asp residue in the protein binding site plays a key role during ligand-receptor interaction. In fact, in 7 JJO ISO interacts with Asp121 forming a hydrogen bond and an ionic interaction through its nitrogen group, and with Val122 by a hydrophobic interaction through the aromatic moiety, whereas in 7 BVQ the crystallographic ligand carazolol interacts by two hydrogen bonds with Asp1138 (corresponding to Asp121 of 7 JJO). Beside this interaction, carazolol is anchored to Asn1363 of \(\beta\)1 receptor through its hydroxyl group by an additional hydrogen bond. The binding energy value obtained after redocking of carazolol was -7.7 kcal/mol. As shown in Figure 3, in our experiments, although the four molecules might adopt different orientations in the complex, all of them accommodate in the binding site and are able to interact with the key residues of the active site, including Asp1138 (Table 1).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Compound** & **Binding Energy** \\
**(kal/mol)** & \\ \hline
**4** & \(-8.0\) \\ \hline
**7** & \(-7.7\) \\ \hline
**8** & \(-7.5\) \\ \hline
**10** & \(-7.8\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Binding Energy for compounds 4, 7, 8 and 10 in the \(\beta\)1-adrenergic receptor (PDB code 7 BVQ).  
In particular, compound 4 interacts with the protein active site forming two hydrogen bonds: a first with Asp1138 through its carbazole nitrogen, with a distance of 3.15 A, and the second with Ser1228 through its carbonyl group, with a distance of 2.84 A, whereas compound 7 interacts by a hydrogen bond through its carbonyl group with Asp1138 with a distance of 3.43 A. Compounds 8 and 10 although allocated in the same binding pocket interact through their side chain with Val1139 by hydrophobic interactions that stabilize the complexes. Overall, these results suggest that our compounds are able to accommodate in the active site of the \(\beta\)1 receptor with an affinity similar to that of the crystallographic ligands (Table 1).  
Figure 3: Ligand-binding pocket of the active site of the \(\beta\)1 adrenergic receptor. The protein backbone is represented in background as ribbons, and the key residues for ligand interaction (Asp1138, Asn1363, Ser1128, Val1139) are also indicated. (**A**) Superimposed binding modes of all the five ligands: carazol (yellow), **4** (cyan), **7** (grey), **8** (orange) and **10** (green). Specific binding mode of carazol (**B**), **4** (**C**), **7** (**D**), **8** (**E**) and **10** (**F**).