## 2 Results & Discussion
### In Silico Studies  
#### 2.3.1 Molecular Docking  
The scaffolds similar to the target compounds were reported earlier with respect to their potent anticancer activities [56]. This prompted us to perform a computational molecular docking study to understand the interaction between the most active antiproliferative compound **35** and dihydrofolate reductase (DHFR) by using the GLIDE docking module of Schrodinger suite 2014-3 [57]. The docking study was carried out on compound **35**, where the structure of the complex of DHFR and methotrexate was selected as the docking model (PDB ID: 1U72) [58]. Table 3 depicts the results of docking along with the major interactions for compound **35** and methotrexate with DHFR. More detailed analysis of compound **35** with the DHFR receptor is presented in Figure 5. Compound **35** showed a two-hydrogen bond interaction with the active residues Glu30 and Thr56. The amino part of pyrimidine-2-amine of compound **35** acts as a hydrogen bond donor and established a H-bond interaction with the active site residue Thr56 (d = 3.57 A), and the nitrogen atom of 5-chloropyrazin-2-yl group acts as a hydrogen bond acceptor and formed a H-bond with the active residue Glu30 (d = 2.02 A). Further, the pyrimidine-2-amine group of **35** formed an arene-arene (\(\uppi\)-\(\uppi\)) interaction with the Phe34 residue. Additionally, several hydrophobic interactions were observed between compound **35** and the active site residues of DHFR i.e., Ile7, Val8, Ala9, Leu22, Phe31, Tyr33, Phe34, Ile60, Leu67, Val115, and Tyr121, which stabilized the binding of compound **35** in the active site of DHFR.  
Figure 6 illustrates the superimposition of compound **35** and co-crystal (Methotrexate) at the active site of the human dihydrofolate reductase receptor. Compound **35** overlapped well with the native ligand Methotrexate at the active site of DHFR and formed key interactions with the active site residues of DHFR.  
A bioisosteric 6-membered nitrogenous heterocyclic ring such as 2''-pyridinyl ring containing compound **35** has very good antiproliferative activity and binding affinity compared to compounds of chloropyrazine-tethered pyrimidine hybrids with a simple phenyl ring. This observation can be verified in the case of compound **27** as it has low antiproliferative activity as well as a low docking score and interaction with active site residues compared to compound **35**. Figure 7 represents the receptor-ligand interaction diagram (2D view) of compounds **27** at the active site of the human dihydrofolate reductase receptor.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**S. No.**} & \multirow{2}{*}{**Ligand Name**} & \multirow{2}{*}{**Docking Score**} & \multicolumn{2}{c}{**Interactions**} \\ \cline{3-4}  & & & **H-Bonds** & **\(\pi\)-\(\pi\)** & **Hydrophobic** \\ \hline
1 & **Compound 35** & \(-6.834\) & Glu30, Thr56 & Phe34 & Ile7, Val8, Ala9, Leu22, Phe31, \\  & & & & Tyr33, Phe34, Ile60, Leu67, \\  & & & & Val115, Tyr121 \\
2 & **Compound 27** & \(-5.211\) & Glu30 & Phe34 & Val8, Ala9, Leu22, Phe34, \\  & & & & Ile60, Leu67, Tyr121 \\
3 & **Methotrexate** & \(-11.808\) & Ile7, Glu30, Gln35, & Ile7, Val8, Ala9, Leu22, Phe31, \\  & & & Arg70, Val115 & Tyr33, Phe34, Ile60, Pro61, \\  & & & & Leu67, Val115, Tyr121 \\ \hline \hline \end{tabular}
\end{table}
Table 3: GLIDE docking results for compounds **35**, **27** and methotrexate at the active site of human dihydrofolate reductase.  
Figure 5: Docking model of compound **35** in the active site of the human dihydrofolate reductase receptor. The red dashed line represents a hydrogen bond. The dark green color residue (Phe34) is involved in the \(\pi\)-\(\pi\) interaction.