Recent studies from the literature have identified
Recent studies from the literature have identified new cysteine protease inhibitor of antiviral agents able to target diverse host factors indispensable for efficient virus replication: as an example, the human DEAD-box polypeptide 3 (DDX3) inhibitor, 1-[4-(4-methyl-1H-1,2,3-triazol-1-yl)phenyl]-3-o-tolylurea (16d), has elicited interest for its broad spectrum of antiviral activity against HIV, Hepatitis C virus (HCV), Dengue virus (DENV) and West Nile virus (WNV) with EC50 values ranging from 0.97 to 16.5 μM and its favorable safety profile (mean SI∼ 162) . Also the (imidazo-pyridazinyl) benzenesulfonamide derivative (10) has displayed a significant antiviral profile (mean EC50∼ 54 nM and SI∼ 339) against three single stranded +RNA viruses, such as human rhinovirus 1 (HRV1), coxsackie virus B3 (CVB3) and HCV (1 b-genotype), impairing the activity of phosphatidylinositol 4-kinase IIIβ (PI4KB) . By comparison with the aforementioned host-factor directed compounds, also the azaspiro dihydrotriazines 4 and 6 may represent valid hits, worthy of further assessment to develop more efficient antiviral agents.
3.2. Inhibition of hDHFR enzyme. In order to verify the host factor DHFR inhibition as mechanism of action by which the novel azaspiro dihydrotriazines exploit their antiviral effect, the most potent compounds (4, 6 and 20) were assayed against the recombinant hDHFR enzyme, displaying sub-micromolar or low micromolar Ki values (Table 2). Interestingly, these compounds exhibited an inhibitory behaviour (Ki) on hDHFR comparable to the antiviral activity (EC50) showed against Influenza B virus: in fact, the most potent compound 6 (EC50 = 0.19 μM) provided the lowest Ki value equal to 0.53 μM, and the less effective compound 20 (EC50 = 3.0 μM) possessed the highest inhibition constant (Ki = 1.40 μM). Therefore, as previously demonstrated for cycloguanil-like dihydrotriazines , the host (human) DHFR was found to be the molecular target of the novel azaspiro dihydrotriazine derivatives.
Molecular modeling In our previous work towards the identification of novel and safe antiviral agents, we explored the molecular docking poses of cycloguanil-like dihydrotriazine derivatives, featuring a promising antiinfluenza activity (11a, 13a, 14a, 16a) at the X-ray crystallographic structure of the human DHFR. The chosen X-ray data included the host enzyme in complex with a pyridopyrimidine-based inhibitor (I) (pdb code = 4QHV; resolution = 1.61 Å) (Fig. 4) . In virtue of the related structural similarity between the dihydrotriazine scaffold and the co-crystallized ligand, our previous docking calculations allowed to gain a better understanding of the mechanism of action of our first series of derivatives . As shown in Fig. 4, the co-crystallized inhibitor I displayed two H-bonds between the two amine groups linked to the pyrimidine core and the I7 and E30 carbonyl groups and side-chain, respectively. Notably, this kind of contacts were thought to be the most relevant in the DHFR inhibitor activity. Based on our previous findings, the introduction of bulky group or of a cycloalkyl ring at C (6) of the 1-phenyl substituted dihydrotriazine core caused a different positioning of the derivative with respect to the reference compound I. In fact, this kind of substituents forced the central ring to establish only a H-bond between one of its NH2 groups and E30. Conversely, smaller substituents were better tolerated, especially when combined with a meta substituted phenyl ring at N (1) of dihydrotriazine ring . Accordingly, the most promising cycloguanil-like dihydrotriazine derivatives 11a, 13a, 14a, 16a (hDHFR Ki = 0.07–0.13 μM) shared a common docking mode, featuring two H-bonds with I7 and E30, thanks to the two NH2 groups onto the dihydrotriazine scaffold (in Fig. 5 the docking pose of compound 14a is depicted). In particular, the dimethyl substitution on the dihydrotriazine core together with lipophilic substituents at the meta position of the phenyl ring, as shown for 14a featuring a 3-CF3-phenyl moiety, proved to be particularly effective.