Molecular modelling studies were performed on the hDHFR inhi

Molecular modelling studies were performed on the hDHFR inhibitors identified (1, 11, 13, 14, 16, 25 and 32) to explore the structural basis of the interaction between the mentioned compounds and the human enzyme. The docking studies were performed using the X-ray crystallographic structure of the hDHFR, in complex with a pyridopyrimidine-based inhibitor (I) (pdb code = 4QHV; resolution = 1.61 Å) [24]. The human DHFR inhibitor, RP 67580 mg I, was considered as positive control (Fig. 1). The main issues to be addressed were to clarify, through docking studies of 1, 11, 13, 14, 16 and 25, the role played by the 4,6-diamino-1,2-dihydrotriazine nucleus and of the hydrophobic framework, including the phenyl ring and the R1-R3 substituents, with respect to the 2,4-diaminopyrimidine core and to the phenyl-substituted pyridine-3-amine moiety of I. In addition, pyrimethamine (32) was also submitted to docking calculations, having shown a similar antiviral behaviour to cycloguanil. As shown in Fig. 4A, I was engaged in H-bonds between the two NH2 substituents placed onto the pyrimidine core and the I7 and E30 carbonyl group and side-chain, respectively. The whole, planar bicyclic ring was involved in π−π stacking with Y33 and F34, while the isopropyl phenyl ring was projected towards F31, I60, P61, displaying Van der Waals contacts. As consequence, these kinds of contacts efficiently stabilized I within the hDHFR binding site, leading the complex to a favourable value of the estimated binding affinity (hDHFR-I ΔG = −28.0 kJ/mol), in harmony with the high compound potency profile (Ki = 11 nM, Table 5). Based on our docking calculations (Table 6), when small bulky alkyl groups are chosen for R1 and R2, the presence of a meta substituent placed in R3, rather than at the ortho and para positions of the phenyl ring, proved to be preferred. Accordingly, cycloguanil (1) was characterized by weak H-bonds with I7 and E30, while the 4-chlorophenyl ring moved towards T56 and Y121, on the opposite side of the cavity occupied by the isopropyl phenyl ring of the reference inhibitor (Fig. 4B). A comparable docking mode was observed for pyrimethamine (32). As shown in Table 6, the related complexes displayed quite adequate and comparable values of predicted binding affinity energies (hDHFR-1 ΔG = −7 kJ/mol; hDHFR-32 ΔG = −8 kJ/mol), turning in moderate affinity toward the human enzyme (1 Ki = 0.41 μM; 32 Ki = 0.47 μM). Interestingly, the most promising derivatives 11, 13, 14, 16 shared a common docking mode, exhibiting the required H-bonds with I7 and E30, by means of the two NH2 groups of 1,2-dihydrotriazine scaffold (as shown for compound 13 in Fig. 5A). In addition, the dimethyl substitution in R1 and R2 of 13, and also the presence of a 3-Br-phenyl ring, proved to be particularly effective to properly mimic the same positioning displayed by I within the X-ray crystallographic structure of hDHFR. As consequence, all of them were efficiently characterized by the most important and crucial bonds with the biological target, as previously discussed for compound I, and therefore experienced the most favourable predicted binding affinity profiles, being in agreement with the experimental data. In particular, compound 13 (hDHFR-13 ΔG = −19 kJ/mol) proved to be the most promising (Ki = 0.07 μM). Conversely, the presence of bulky group or the introduction of a cycloalkyl ring in R1 and R2, moved the derivative toward a quite turned docking mode (if compared with I), as shown for compound 25 (Fig. 5B). Indeed, only one of the two NH2 groups onto the dihydrotriazine ring was able to mimic the role played by those of I, detecting only one H-bond with E30. In addition, the bioisosteric replacement of the pyridopyrimidine scaffold with the smaller dihydrotriazine one, inevitably impaired the ability of the compound to display the same pattern of hydrophobic contacts, previously mentioned for I. This compound was the only one of the series here proposed to exhibit this kind of docking mode, losing key-contacts such as one H-bond with I7 and several Van der Waals and π-π stacking. Conceivably, this positioning compromises the ability of compound 25 to gain effective and crucial bonds with the human biological target, turning in lower affinity values (25 Ki = 13.17 μM) if compared with the other congeners (1, 11, 13, 14, 16: Ki = 0.07–0.41 μM). A perspective of the predicted binding affinity values for the enzyme in complex with any selected docking pose also supports the experimental data since the hDHFR-25 complex exhibited the worse predicted value (hDHFR-25 ΔG = −1 kJ/mol) with respect to those including 1, 11, 13, 14, 16 (ΔG from −20.0 to −7.0 J/mol).