According to the docking study results
According to the docking study results, the difference in biological activity of the tested compounds despite of their similar chemical structure could be explained. For the active molecules like 26, the presence of aromatic substituent with its conformational freedom seems to be crucial for the interaction, in addition to the presence of electron withdrawing group which favor MTX spatial arrangements. The inactive molecules like 25 were much more constrained and possibly having different structural attributes related to the aromatic ring.
Ligand-based active site alignment study by docking inside the binding pocket is a well-known technique for structural analysis of ligand complexes . In the present, study flexible alignment comparative modeling experiment was performed (Fig. 5). An alignment is considered to be successful when the molecule strain energy is small, possessing similar shapes and their aromatic atoms overlap to show the similarity between the 3D structures of the most active compound 26 and MTX. Initial approach was applied to employ CharmM /MMFF94 flexible alignment automatically generated superposition with minimal user bias.
There was a good alignment profile between compound 26 (IC50 0.06μM) and MTX (IC50 0.08μM) explaining its activity pattern (Fig. 5a). Fig. 5b clearly indicates a different alignment profiles where carbonyl groups are not on the same side between 25 (IC50>100.0μM) and MTX which is in consistency with the obtained experimental data.
The surface map for the DHFR binding pocket was calculated (Fig. 6). MTX showed to occupy the whole space lying into the groove pocket (Fig. 6a). Compound 26 active site alignment showed a pattern which resemble that of MTX, filling all the area of the active site with its bulkiness and fit into the hydrophobic pocket, forming favorable binding contact (Fig. 6b). Results for 26 showed larger blue hydrophobic areas which are responsible for the interaction with amino u73122 residues inside the enzyme active pocket. On the other hand, compound 25 structure was pointed out toward the surface wall of the active site and deprived of any receptor exposure clashes explaining its poor DHFR inhibitory activity (Fig. 6c). The hydrophobic distributions of the least active compound 25, showed less lipophilic moieties and hence the required lipophilicity for effective binding to DHFR is absent.
Conclusion A new series of 2,4-substituted-1,3-thiazoles and thiazolo[4,5-d]pyridazine both bearing the 2-thioureido function with anticipated antitumor and DHFR inhibitory activity were synthesized. A new method to prepare the thiazolo[4,5-d]pyridazine heterocycle was adopted. Compound 26 proved to be the most active DHFR inhibitor with IC50 of 0.06μM (merely comparable to MTX, IC50 0.08μM); while compounds 22 and 23 were active with IC50 of 0.1 and 2.5μM, respectively. Compound 4 showed antitumor activity against NCI-H522 non-small cell lung, HT29 colon, and T-47D breast cancers with GI values of 40.4, 30.7 and 27.7%, respectively; while compound 20 showed 31.4, 25.2, 37.7, 25.1, 41.0GI% against NCI-H522 non-small cell lung, HT29 colon, SK-OV-3 ovarian, MCF7 breast and T-47D breast cancers respectively. In addition compound 21 showed antitumor activity against NCI-H522 non-small cell lung, HT29 colon and TK-10 renal with GI values of 31.7, 29.4, and 34.7%, respectively. Compound 26 proved lethal to HS 578T breast cancer cell line with IC50 value of 0.8μM, inducing cell cycle arrest and apoptosis through increasing the percentage of cells in Pre-G1 and enhancing G2/M phase. Structure activity correlations of the investigated compounds revealed that the 3-[4-chlorophenyl)thioureido]- series is more active than the 3-[(4-methoxyphenyl)-thioureido]- counterparts; also the type of substituent at positions 7- of the thiazolo[4,5-d]pyridazine affected the DHFR inhibition potency. The order of activity in the 3-[(4-chloro-phenyl)thioureido]- series, was 4-BrPh>4-CH3OPh>pH>4-CH3Ph; while in the 3-[(4-methoxy-phenyl)-thioureido]-series was 4-CH3OPh>pH>4-CH3Ph=4-BrPh. Meanwhile, compound 26 showed high affinity binding energy value of 46.65Kcal/mol toward Phe 31 residue which is linked to the thiazolo[4,5-d]pyridazine ring while Arg 22 residue is linked to 7-Phenyl moiety in addition to a network of π-π interaction and hydrogen bonding. The obtained data could be used as template for further development of new DHFR inhibitors.