Chemistry of the compounds
The compounds evaluated in this study include azole-acridine molecular hybrids of triazole and pyrazole containing acridines designed rationally for targeted therapy with nucleic acid as one of the molecular targets. Figure 1 shows the molecular triazole and pyrazole hybrids elected for inhibition of main protease SARS-CoV Mpro by molecular docking. The synthesis of the acridine based triazole hybrids has been reported previously with 1,3-dipolar cycloaddition as a key reaction in the triazole heterocycle synthesis.13 A convergent reaction between 3-phenylpyrazole and acridine in the presence of sodium hydride and 2-bromoethylamine hydrobromide salt afforded the ethyl linked acridine based pyrazole hybrid 6 analogous to triazole 2.
Molecular docking studies: To determine the binding affinity, BE, of the azole derivatives
To probe the potential of the synthesized compounds for anti-coronavirus activity, a docking strategy was employed to not only determine their enzymatic binding affinities and preferred conformations on the SARS-CoV-2 main protease (Mpro) and RdRp but also to identify the key interactions involved in their binding sites. Notably, the selected binding site was derived from the liganded crystal structures of SARS-CoV-2 Mpro (PDBID: 6lu7) which co-crystallized with peptide-like compounds as candidate inhibitors.4, 17 Experimentally determined α-ketoamide inhibitor20 13b was used as a reference inhibitor to corroborate the methodology used in this docking study as well as for comparative assessment of the potential of these new compounds as potential inhibitors of SARS-CoV-2 Mpro through their binding affinities. For RdRp, the binding conformations and affinities of the ligands were determined relative to the crystallographic site of remdesivir.6
In addition, other drugs such as chloroquine (CQ),21 hydroxychloroquine (HCQ)22 and remdesivir,6, 23 which are currently being used or under clinical trials were included in the docking study for comparative purposes. Table 1 shows the binding affinities of the most preferred binding poses obtained for each of the compounds evaluated in this study based on the docking scoring function.
All the compounds 1-6 displayed binding affinities (in the range of -4.7 kcal/mol to -6.5 kcal/mol), CQ (-4.9 kcal/mol), HCQ (-5.3 kcal/mol) and remdesivir (-5.5 kcal/mol) comparable to that of α- ketoamide inhibitor 13b (B.E = -5.0 kcal/mol). Hybrids based on triazole exhibited binding affinities, B.E, in the range of -4.7 kcal/mol to -6.5 kcal/mol on docking to the active site of SARS- CoV-2 Mpro protease as defined by co-crystallized peptide-like inhibitors (B.E of 13b = -5.0 kcal/mol).4,17
As observed in Figure 3 and Figure 4, the docking models reveal hydrogen bonds are among the key interactions with residues in the active site of the protease which include Thr24/26/45, His41/164, Gln189, Met49, Gly143 and Cys145 - all of which are located in domain I and II of SARS-CoV-2 Mpro protease.4,20
The presence of acridine framework in the triazole derivatives 1-4 (-5.5 kcal/mol to -6.5 kscal/mol) led to improved binding affinities relative to non-acridine based triazole 5 (-4.7 kcal/mol), possibly due to improved hydrophobic interactions between the acridine moiety and the amino acid residues in the binding pocket of SARS-CoV-2 Mpro.
It is notable that for acridine containing azole hybrids 1-4 and 6, the presence of acridine moiety orients the molecule in such a way that the 9-amino group forms at least two key H-bonds with thiol and imidazole groups of Cys145 and His 41 amino acid residues, respectively. In a similar fashion, the two amino acids (Cys145 & His41) contribute to the interaction of α-ketoamide inhibitor 13b with SARS-CoV-2 Mpro though through reversible covalent bond. Importantly, Cys145 and His41 residues are catalytic dyads responsible for the catalytic actions of SARS-CoV-2 Mpro as well as other SARS-CoV protease.4, 10, 20 Variations of the groups anchored on the azole moiety has some influence on the binding affinity of the azole hybrid and thus allowed preliminary evaluation of the structure activity relationships of these set of azole hybrids.
Hydrophobic interactions are significant in the binding of the azole tail to the Thr24/26 binding pocket, with the phenyl moiety interactions being greater than that of cyclopropyl and hydroxymethyl group (H-bond) in that order. While non-acridine based triazole 5 assumes an orientation in which the triazole moiety behaves as the tail end as in the other azole derivatives, the t-butyl carbarmate (t-boc) moiety forms H-bond with Cys145 but not His41. This reduced hydrogen bonding with the catalytic dyad of SARS-CoV-2 Mpro could account for the observed relatively low binding affinity (-4.7 kcal/mol) of triazole 5. It therefore implies that these azoles may exert their anti-SARS-CoV-2 Mpro inhibition through disruption of Cys145---His41 interactions of the catalytic dyad.
In a similar fashion to the inhibition activity against protease Mpro, the azole compounds 1 - 6 have exhibited good binding affinities to SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) in the range of -5.6 to -7.1 kcal/mol (Table 1). All the azole-acridine hybrids 1-4 and 6 displayed good affinities for RdRp active site comparable to that of remdesivir (-6.6 kcal/mol) with binding energies below -6.3 kcal/mol. Their binding affinities are interestingly better than that of CQ (-5.4 kcal/mol) and HCQ (-6.1 kcal/mol). Also, azole-acridine hybrids exhibit better affinities for RdRp active site than the non-acridine containing triazole 5 (-5.6 kcal/mol) implying that the acridine moiety plays an important role in the activity of the azole derivatives. The presence of the phenyl group on the triazole and pyrazole moieties appears to improve the binding affinities of the azole- acridine hybrids (2, 4 and 6) as evidenced by binding energies below -6.6 kcal/mol (Table 1) in comparison azole-acridines 1 (-6.6 kcal/mol) and 3 (-6.3 kcal/mol) with hydroxymethyl and cyclopropyl groups respectively. Besides, the chloro and methoxy groups on the acridine framework contribute to improved binding affinities of the azole-acridine evidenced by favorable binding of azole-acridine 2 and 6 (-7.1 kcal/mol each) compared to 4 (-6.8 kcal/mol). While the acridine framework in each of the azole-acridine hybrids occupy the same pocket in the RdRp, it is interesting to note that the preferred conformation of azole-acridine 1 adapts an opposite orientation to that of the other azole-acridines 2, 3, 4 and 6 (Figure S1) in the RdRp-ligand complexes. This could be attributed to the polarity of the binding pocket of the triazole moiety in 1 as evidenced by hydrogen bonding of the hydroxyl group with amino acid residues Asp618/760/761 as compared to the hydrophobic binding pocket of the phenyl group of azole- acridines 2, 4, and 6 (Figures S1 and S3). The observed better binding affinities suggest that the azole-acridine hybrids may disrupt the activities of RNA-dependent RNA-polymerase by forming significant local interactions with the active site limiting the replication activity of the viral RNA polymerase.6
The results from these docking strategies provide preliminary evidence to support the rationale for anti-coronavirus assays of these reported azole compounds developed from our laboratory13 and suggest further study on their in vitro and/or in vivo assays are necessary to validate these models.
The cheminformatics described herein imply that these compounds may hold promise for optimization as lead compounds and possibly in the development of drug candidates for development into anti-coronavirus agents.