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  • Herein we explored by docking studies the


    Herein, we explored by docking studies the effectiveness of replacing the dimethyl substitution at C (6) of dihydrotriazine with a folded azaspiro-containing substituent, along with the introduction of differently substituted phenyl- and benzyl-rings or alkyl chains, linked at the position 1 of the main core of the derivatives (see Fig. 1, Fig. 2, Fig. 3). This kind of decoration was designed with the aim to clarify the relevance of steric properties for dihydrotriazines targeting the hDHFR. In fact, azaspiro derivatives could allow to better fulfil the requirements set forth by the pyridopyrimidine scaffold and the aniline ring of reference compound I. Indeed, the docking results suggest a beneficial role played by the azaspiro group bearing an aromatic substituent, since it aptly superposed on the aniline moiety of the reference compound I, displayed a comparable pattern of hydrophobic contacts with the surrounding residues, which are predicted to stabilize the inhibitor in the enzyme cavity (Table 1S). The concomitant presence of a meta substituted phenyl ring tethering the azaspiro dihydrotriazine core impaired the activity of these derivatives with respect to cycloguanil-like dihydrotriazine derivatives lacking of the azaspiro moiety, especially when decorated with electron-donor groups. On the contrary, the presence of lipophilic and electron-withdrawing groups (e.g., chlorine), at the meta position of the phenyl ring sometimes allows the inhibitor to maintain one of the two key H-bonds with the enzyme, involving I7 (see Fig. 6). Interestingly, the most potent compounds 4 and 6 featuring valuable anti-influenza B activity (mean EC50 ∼ 0.2 μM) displayed additional π−π stacking interactions projecting the aforementioned phenyl ring towards Y121, and also one H-bond between their carbonyl oxygen mCAP and the S59 side-chain. Based on these data, the combination of a carbonyl H-bond acceptor moiety and an aromatic lipophilic group (such as the benzoyl moiety) would be preferred. Accordingly, the predicted energy values of the complexes hDHFR-4 and hDHFR-6 listed in Table 1S were lower than that of the congeners featuring aliphatic groups at piperidine nitrogen (see 2, 3). This seems to be further supported by the lack of activity for all the derivatives being unsubstituted in this position and bearing a variable phenyl ring at N (1) of dihydrotriazine (see 1, 7; EC50 > 100 μM). When derivatives characterized by an alkyl chain at N (1) of dihydrotriazine (12–21, Fig. 3) are considered, only compound 20 (Influenza B virus, EC50 = 3.0 μM) exhibited an effective docking mode within the enzyme cavity. As shown in Fig. 7, its flexible alkyl side chain permitted to the dihydrotriazine portion of the ligand to fill the binding crevice better than reference compound I and previous congeners, which were decorated with conformationally rigid aromatic rings at N (1) of dihydrotriazine. Notably, this allowed to detect one H-bond with I7, while the sulfone moiety displayed H-bonds with S59. More interestingly, the presence of an oxygen atom along with the aliphatic chain placed at N (1) allowed intramolecular hydrogen contacts with the surrounding NH2 group onto the dihydrotriazine core. As a consequence, the overall positioning of the ligand oriented the second NH2 group towards I7, while the azaspiro portion and the sulfone group properly recapitulate the binding mode of the aniline fragment in reference compound I.
    Conclusion This work represents a follow-up of a previous investigation on dihydrotriazine-based derivatives as dual antiviral agents against influenza and RSV viruses, acting on the host factor DHFR. In the new compounds series, the nitrogen of the spiropiperidine nucleus allowed the study of the role of different functionalized side chains, to understand the chemical space for enhanced interactions within the hDHFR enzyme. Whereas the previous series of cycloguanil-like dihydrotriazines emerged as highly potent influenza B and RSV inhibitors displaying nanomolar activity, the novel azaspiro dihydrotriazines, that we presented herein, possessed lower antiviral activity. By enzyme inhibition assays a DHFR-mediated mechanism of action was also confirmed for the new compounds, as we previously demonstrated for cycloguanil-like dihydrotriazines. The two novel compounds with superior activity and selectivity against influenza B and RSV were characterized by the piperidine nitrogen bearing a H-bond acceptor moiety linked to an aromatic lipophilic groups, such as the p-tolylcarbonyl (4) or (4-fluorophenyl)carbamoyl ones (6), joined with the 1-phenyl azaspiro dihydrotriazine. Also compound 20 has been found as promising new hit, combining a methoxyethyl chain at N (1) with an ethylsulfonic group at spiropiperidine nitrogen. Thus, besides the previously studied cycloguanil-like dihydrotriazine derivatives, also the above adequately substituted azaspiro dihydrotriazines may represent valuable starting points deserving further structural optimization. The new SAR insights gathered from this study provide crucial information to further develop this class of host DHFR-directed antiviral agents.