## 3 Results and Discussion
### Docking Study
#### 3.4.3 Docking Analysis
For instance, compound 8 has a short bond distance of 2.20 A with  
Figure 10: Native ligand (pranlukast, cyan-colored), reference ligand (caffeine, pink-colored), and new ligands: bioconjugates **4** (green-colored) and **8** (bittersweet-colored) at once in the binding site of a 6 RZ4 protein domain. Figure 9: (**a**) The depiction of possible hydrogen bond formation between bioconjugate **8** and 6 RZ4 protein domain. (**b**) The 2D depiction of interactions between bioconjugate **8** and 6 RZ4 protein domain. Cyan lines with big dots—pi–pi interactions, green solid lines—hydrophobic contacts. GLY 560 B, indicating a strong interaction that could lead to better binding affinity. In comparison, atorvastatinn shows competitive distances, such as 1.91 A with LYS 735 B, highlighting its effectiveness as a ligand. The biological significance of these distances is considerable; interactions with amino acids like ARG and SER are crucial for protein function. When comparing these compounds to reference ligands like atorvastatinn and caffeine--where caffeine has a distance of 2.80 A with SER 155 A--it becomes clear that some analyzed compounds may have advantages in binding efficiency. This detailed look at bond distances not only helps interpret molecular docking results but also improves our understanding of how effective ligands can be, guiding the design of better ligands for specific protein interactions. The possibility of modifying the starting compounds allows the synthesis of a wide range of derivatives with potential biological activity. Using caffeine and gramine derivatives as well as steroid derivatives as starting compounds, we obtained ten new steroid-alkaloid bioconjugates. All compounds were characterized by spectroscopic methods. The lowest HOF values for gramine and caffeine bioconjugates with cholic acid were observed. The preliminary results based on in silico data indicated that the obtained compounds, particularly caffeine bioconjugates, can function as cholesterol antagonists, analeptic and respiratory agents, and antihypercholesterolic compounds. The results of molecular docking to selected protein domains (1 HWK and 6 RZ4) highlight the potential of the newly developed bioconjugates in pharmacology and give hope for future drug discovery. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15020591/s1](https://www.mdpi.com/article/10.3390/app15020591/s1)]([https://www.mdpi.com/article/10.3390/app15020591/s1](https://www.mdpi.com/article/10.3390/app15020591/s1)), Figures S1-10: NMR spectra of compounds **1-10**; Figures S11-S20: FT-IR spectra of compounds **1-10**; Figures S21-S30: ESI-MS spectra of compounds **1-10**. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & **Protein** & **Amino Acid** & **Distance** & **Is Compound** \\  & **Domain** & **Residues** & **[Å]** & **Doctor or** \\  & & & & **Acceptor?** \\ \hline
**1** & 1 HWK & ARG 568 B & 3.33 & Acceptor \\  & & SER 565 B & 2.73 & Donor \\
**3** & 1 HWK & ASN 755 B & 2.91 & Acceptor \\  & & ARG 590 A & 2.85 & Acceptor \\
**4** & 1 HWK & ARG 568 B & 3.11 & Acceptor \\  & & GLY 560 B & 2.20 & Acceptor \\
**8** & 1 HWK & ASN 658 A & 3.48 & Acceptor \\  & & SER 661 A & 2.19 & Acceptor \\  & & ARG 590 A & 2.47 & Acceptor \\
**4** & 6 RZ4 & SER 155 A & 3.44 & Acceptor \\  & & SER 193 A & 1.99 & Acceptor \\  & & THR 154 A & 3.70 & Acceptor \\
**8** & 6 RZ4 & SER 155 A & 2.78 & Acceptor \\  & & SER 193 A & 3.39 & Acceptor \\  & & ARG 22 A & 2.81 & Acceptor \\  & & SER 565 B & 2.21 & Acceptor \\  & & ASN 755 B & 2.05 & Acceptor \\ Atorvastatinn & 1 HWK & ASN 690 A & 2.36 & Donor \\  & & SER 684 A & 2.09 & Acceptor \\  & & LYS 735 B & 1.91 & Acceptor \\ Caffeine & 6 RZ4 & SER 155 A & 2.80 & Acceptor \\ \hline \hline \end{tabular}
\end{table} 
Table 6: The predicted hydrogen bonds between investigated ligands and protein domains. **Author Contributions:** Conceptualization, T.P. and B.J. ; methodology, K.O., K.B., H.K. and D.N. ; software, D.N. and T.P.