## 4 Conclusions  
The main aim of the present study was to develop a multistep synthetic route for the preparation of novel D-ring-fused 5-aminopyranoside (**6a-h**) in the androstane series involving a MW-assisted heterocyclization of a steroidal \(\beta\)-ketonitrile (3) with different arylhydrazines (**5a-h**) as the key step. Although the cyclization of \(\beta\)-ketointriles with monosubstituted hydrazines usually needs a long reaction time (12-72 h), the applied MW-induced protocol allowed the high yield of the desired products within 10-20 min. The ring-closures were found not to be significantly influenced by the electron demand of the substituent R present on the aromatic ring of the reagents, with the only exception of the strong electron-withdrawing NO\({}_{2}\)-group. Proton dissociation processes of the heterocycles were investigated by UV-visible spectrophotometric titrations in aqueous solution, as pK\({}_{\text{a}}\) of a bioactive compound is a key physicochemical parameter affecting the pharmacokinetic properties. Moreover, based on its value, the actual protonation state can be established at physiological pH. Furthermore, the antiproliferative activity of the 5-aminopyranoside was screened in vitro on different human cancer cell lines, along with a noncancerous cell line. Compound **6g** was selected for further studies as among the tested molecules this was the most potent in inhibiting the proliferation of the tested cancer cells, including cisplatin-resistant PC-3 prostate cancer cells. These results indicate that D-ring-fused 5-amino-1-arylpyrazoles can be considered as promising synthetic platforms for obtaining new active steroidal derivatives. Additionally, their biological significance, in particular those of the  
Figure 3: Primary antiproliferative effect of the steroidal D-ring-fused 5-aminopyranoside on different cancerous cell lines and on MRC-5 fibroblasts represented as heat map {compound = 5 \(\mu\)M; 72 h incubation time}. Control represents the viability of cells receiving no treatment. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Compd.**} & \multicolumn{5}{c}{**IC\({}_{50}\)**} \\ \cline{2-7}  & **MRC-5** & **MCF-7** & **PC-3** & **A549** & **HeLa** & **U2 Os** \\ \hline
**6g** (\(\mu\)M) & \(8.9\pm 1.1\) & \(6.2\pm 1.0\) & \(4.9\pm 1.1\) & \(7.9\pm 1.0\) & \(4.0\pm 1.0\) & \(3.5\pm 1.0\) \\ \hline Cisplatin (nM) & \(37.72\pm 1.2\) & \(338.5\pm 1.1\) & \(902\pm 1.6\) & \(369.8\pm 1.2\) & \(254.5\pm 1.2\) & \(342.8\pm 1.2\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: IC\({}_{50}\) (\(\pm\)SD) values of compound **6g** and of cisplatin assessed on noncancerous MRC-5 cells and on various cancer cell lines. CN-group-containing compounds, can be broadened to novel cancer therapy approaches. It is worth noting that the syntheses of similar derivatives with improved water solubility are in progress and the results will be published in the near future. The following are available online at [[http://www.mdpi.com/2076-3417/10/1/229/s1](http://www.mdpi.com/2076-3417/10/1/229/s1)]([http://www.mdpi.com/2076-3417/10/1/229/s1](http://www.mdpi.com/2076-3417/10/1/229/s1)), \({}^{1}\)H NMR and \({}^{13}\)C NMR spectra of the synthesized compounds, Table S1: Mean \(\pm\) SD values of primary growth inhibitory screen (given as cell viability) used for heat map construction, Figure S1: Representative cell viability curves to determine growth inhibition and IC\({}_{50}\) values following treatments with (a) compounds **6g** or (b) cisplatin on different cell lines. Conceptualization, E.F.; M.K. and E.A.E. ; Chemical synthesis and optimization experiments, G.M. and A.B. ; Pharmacological studies, M.K.G. and D.I.A. ; UV-visible spectrophotometric titrations, M.A.M. ; Structural analysis, G.M. ; Formal analysis and interpretation of data, A.B., D.I.A. and M.A.M. ; Methodology, resources, supervision, E.F.; M.K. and E.A.E. ; Writing--original draft preparation, G.M. and M.K.G. ; Writing--review and editing, E.F.; M.K. and E.A.E.