## 3 Results and Discussion  
### Synthesis and Spectroscopic Characterization  
C3-substituted indole compounds have attracted significant attention due to their widespread pharmaceutical application [34; 35; 37]. Among the various C8 caffeine derivatives, 8-mercaptoethanol derivatives are particularly noteworthy because of their considerable pharmaceutical properties [28; 29]. Furthermore, bromosubstituted steroid derivatives serve as valuable substrates in the synthesis of new molecules [39]. In this study, we synthesized a series of bioconjugates derived from thione alkaloids and bromosubstituted steroid derivatives to develop novel bioactive compounds. Since thione compounds can exist as tautomeric forms of thione-thiol, we carried out the reaction of gramine and caffeine thione derivatives with bromoacetate of methyl lithocholate, methyl deoxycholate, methyl cholate, cholesteryl, and dihydrocholesterol (see Scheme 1). Yields ranged from 61% to 90% (for gramine derivatives) and 82% to 97% (for caffeine derivatives). \begin{table}
\begin{tabular}{c c c} \hline \hline
**PDB ID** & **Search Space Center (x, y, z)** & **Size of the Search Space (x, y, z)** \\ \hline
1 HWK & 18, 8, 15 & 42, 41, 39 \\
6 RZ4 & 40, 28, 57 & 36, 44, 45 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The search spaces of the analyzed binding sites of the protein domains. All new compounds were characterized by the spectroscopic method. The \({}^{1}\)H NMR spectra of all new compounds showed signals characteristic of the cholic acid and alkaloid derivatives skeletons. The characteristic signals from axial C3\(\beta\)-H protons were observed for all derivatives at 4.59-4.83 ppm. The presence of signals from C7\(\beta\)-H protons at 4.89 ppm and 4.91 ppm for derivatives **7** and **10**, and signals from C12\(\beta\)-H at 5.08 ppm for compounds **6** and **9** and at 5.09 ppm for derivatives **7** and **10**, provided further confirmation of the structure of the newly synthesized bioconjugates. The signals assigned to CH\({}_{3}\)-18 were present in the range of 0.63-0.73 ppm, those assigned to CH\({}_{3}\)-19 in the range of 0.79-1.01 ppm, and those assigned to CH\({}_{3}\)-21 in the range of 0.80-0.92 ppm. These signals were observed in all compounds studied. For compounds **1**, **2**, and **5-7** (gramine derivatives), aromatic ring signals were present in the range of 6.99-7.65 ppm, while signals from the protons of the C10\({}^{\prime}\) -methylene group were observed at about 5.50 ppm. For the caffeine bioconjugates (compounds **3**, **4**, **8-10**), the three \(N\)-methyl group diagnostic signals were observed at about 3.39, 3.53, and 3.90 ppm. The diagnostic signals for the CH\({}_{2}\)-27 protons in derivatives **5-7** and **8-10** and the CH\({}_{2}\)-29 protons in compounds **1**, **2** and **3**, **4** were observed in the range 4.01-4.22 ppm. In the \({}^{13}\)C NMR spectra, diagnostic signals from the C-18, C-19, and C-21 carbon atoms were present at 11.83-12.37 ppm, 18.26-22.95 ppm, and 17.47-21.16 ppm, respectively, for all derivatives. The signal from the C-3 carbon atom was present in the range of 75.38-76.46 ppm for all derivatives, while the signal from the C-5 carbon atom was observedat 139.43 ppm and 139.11 ppm, and C-6 carbon atom at 122.82 ppm and 123.12 ppm for compounds **1** and **3**, respectively. The signal from the C-7 carbon atom was observed at 70.63 ppm and 70.66 ppm for compounds **7** and **10**, respectively. The indole ring signals for the gramine derivatives (compounds **1**, **2**, **5**-**7**) were observed in the 111-137 ppm range. The carbonyl carbon atoms of caffeine (derivatives **3**, **4**, **8**-**10**) exhibited signals at 150 ppm, whereas the carbonyl group of steroid derivatives displayed signals near 170 ppm. The FT-IR spectra of all new derivatives exhibited absorption bands between 2800 and 3000 cm\({}^{-1}\), originating from the stretching vibration of the C-H bonds in aromatic rings.