Molecular Docking Simulation
In docking simulation, these compounds have common H-bond interactions with key residues Lys254 and Lys352. However, the docking poses of compounds 01–06 with small R2 substituent have different docking poses than others. The docking pose of compound 06 illustrated in Fig. 3a-b indicates that the oxygen atoms in 1,4-naphthoquinone moiety and amino fragment of acylamino moiety have H-bonds with key residues Lys254 and Lys352. The docking pose of compound 18 illustrated in Fig. 3c-d indicates that the H-bonds with key residues Lys254 and Lys352 are formed with the oxygen atoms in 1,3-dimethoxybenzene moiety and amino fragment of acylamino moiety. It indicates that compounds with the large alkyl moiety in R2 substituent have different docking poses. In addition, Fig. 3 indicates that compound 06 also has H-bonds with residues Asn249 using its amine group and has hydrophobic contacts with residues Cys241, Leu248, Asn249, Ala250, Lys254, Leu255, Ala316, and Lys352. For compound 18, it also has two π-cation interactions with residues Asn258 and Lys352, and it has hydrophobic contacts with residues Gln247, Leu248, Ala250, Lys254, Leu255, Asn258, Met259, Val315, and Lys352. These interactions and hydrophobic contacts supported each compound to bind stabilized in the binding site of β-tubulin protein.
Pharmacophore model
For compound 06, the oxygen atoms in 1,4-naphthoquinone moiety and amino fragment of acylamino moiety were matched with the two H-bond acceptor features in pharmacophore model. For compound 18, the oxygen atoms in 1,3-dimethoxybenzene moiety and amino fragment of acylamino moiety were matched with the two H-bond acceptor features, phenyl moiety of 1,4-naphthoquinone was matched with the aromatic ring feature, and ethyl group of R2 substituent was matched with the hydrophobic feature in pharmacophore model. Figure 4 indicates that although the docking poses of compound 06 with small R2 substituent were differed from the docking pose of compound 18 with large R2 substituent, they can both fit the pharmacophore features in pharmacophore model and supported the results obtained in docking simulation.
CoMFA and CoMSIA models
In the CoMFA model, Fig. 6a illustrated several favorable steric regions (green) closed to the residues Asn249, Asn258, Val315, Ala354, and several unfavorable steric regions (yellow) closed to the residues Cys241, Ala250, Ala316. Basically, the favorable steric regions are located around the R1 substituent of compound 18, and unfavorable steric regions are closed to the terminal of R2 substituent of compound 18.
In the CoMSIA model, the favorable and unfavorable regions of steric field displayed in Fig. 6b are similar to CoMFA model, which has favorable regions around the R1 substituent of compound 18 and unfavorable regions closed to the terminal of R2 substituent of compound 18. Figure 6c indicates that there are a favored hydrophobic field (blue) closed to the terminal of R2 substituent toward the direction of residues Ala316, Lys352 and a disfavored hydrophobic field (white) closed to the terminal of R1 substituent. Figure 6d introduced the favored (cyan) and disfavored (purple) areas of H-bond donor may further improve the activity.
The results of CoMFA and CoMSIA models indicate that the R1 substituent of compound 18 preferred to have disfavored hydrophobic fields and have favorable space toward the direction of residue Asn258. In addition, the terminal of R2 substituent of compound 18 preferred to have hydrophobic substituent toward the direction of residues Ala316 and Lys352, but a long additional chain of substituent would decrease the efficacy of cytotoxicity.