The results of flexible docking in terms of interaction energies of reference compound and initially selected nine selected alkaloids Furthermore, the interactions of all alkaloids with the crucial residues present at enzyme active site cavities were examined carefully in terms of classical hydrogen bond interaction, non-classical hydrogen bond interaction, hydrophobic interaction, and electrostatic interaction (Table , the 2D interaction diagrams all alkaloids with the protein M Pro were given in Supporting Figure ). All the alkaloids and the reference compound famotidine were found to be involved in multiple points interactions with the residues present in M Pro active sites. Interestingly, the data in Table clearly depicts that alkaloids named CNP0368366, CNP0416047, 3-hydroxy anisotine, and Anisotine showed better or almost equal interaction energies compared to reference famotidine. Although the Anisotine was previously linked to M Pro binding , the other three alkaloids namely CNP0368366, CNP0416047 and 3-hydroxy anisotine are entirely new leads against M Pro . Encouraged by these findings, we have chosen CNP0416047, 3-hydroxy anisotine, and anisotine as hit compounds and further examined their interaction profiles along with reference famotidine in details (Figure and Figure ). Regretfully, we neglected CNP0368366 from further examinations as it showed probable carcinogenicity in ADMET prediction (Supporting Table ). The reference famotidine docked between the domains I and II and spanned the area between subsite S1, S1' and S4 by interacting with Asn142 (2.71Å), His163 (2.25Å), Phe140 (2.29Å), Met165(2.80Å), Arg188 (2.50Å), Glu166 (2.20Å) through conventional hydrogen bond, with Asp187 (2.75Å), Arg188 (2.75Å) via non-classical hydrogen bonds, and with active site residues His41 (4.74Å), Cys145 (5.37Å) through long-distance electrostatic interaction, but failed to provide any strong hydrogen bonding interaction with catalytic residues. The alkaloid hit CNP0416047 positioned similarly inside the catalytic cavity and showed strong conventional hydrogen bond interaction with active site residue CYS145 (2.66Å) and conserved neighbouring residue Asn142 (2.06Å), and displayed non-classical hydrogen bond and hydrophobic interactions with multiple residues interactions with Leu141 (2.58Å), His164 (3.05Å), Glu166 (2.99Å), Met165 (4.93Å), Met165 (4.93Å), Leu141 (4.80Å) along with catalytic residue His41 (4.80Å). In addition to spanning S1, S1' and S4 subsites, alkaloid CNP0416047 also covered S2 subsites through exhibiting hydrophobic interactions via its tyrosine moiety linked to C6 of quinazoline ring. The next alkaloids 3-hydroxy anisotine and anisotine showed similar interactions and covered the S1, S1' and S4 subsites of catalytic cavity. Both 3-hydroxy anisotine and anisotine exhibited strong conventional and non-classical hydrogen bonding interactions with residues present near S1 subsite such as His163, Ser144, Met165, Glu166, Phe140, and showed hydrophobic interactions with catalytic residues Cys145 and His41. Compared to anisotine, the 3-hydroxy anisotine showed one extra conventional hydrogen Therefore, we have identified three natural quinazoline alkaloids namely CNP0416047, 3-hydroxy anisotine, and anisotine as hit compounds against the main protease (M Pro ) of SARS_CoV_2 after carrying out rigid docking, pharmacokinetic analysis and flexible docking.