The introduction of substituents at the para-position of the phenyl ring (compounds S8-S17) resulted in a decrease in antiproliferative activity against all selected cancer cell lines compared to the activity of the unsubstituted A-ring analogue (S1 and S2). The introduction of substituents at the para-position of the phenyl ring (compounds S8-S17) resulted in a decrease in antiproliferative activity against all selected cancer cell lines compared to the activity of the unsubstituted A-ring analogue (S1 and S2). In general, in the second phase of the study, the -N-H analogs (S8-S12) were more potent than the -N-Me analogs (S13-S17).
As mentioned above, inhibition of tubulin polymerization and mitotic spindle formation may be one of the reasons for the formation of polyploid nuclei. As mentioned above, inhibition of tubulin polymerization and mitotic spindle formation may be one of the reasons for the formation of polyploid nuclei. The activity of the tested compounds was compared with paclitaxel (PTX), CA-4 and 0.2% DMSO as reference and control, respectively.
According to Nguyen et al. all 15 structurally diverse CBSIs formed a hydrogen bond with Cys241β (Cys239β in this study) [46]. According to Nguyen et al. all 15 structurally different CBSIs formed a hydrogen bond with Cys241β (Cys239β in this study) [47]. However, the studied compounds S1, S19, and S22 had lower affinity for the colchicine binding site than the reference CA-4, and these findings were also in agreement with the results of the tubulin polymerization study described above.
However, the S1, S19, and S22 compounds examined had lower affinity to colchicine binding site than reference CA-4, and those findings were also in line with the results of the tubulin polymerization study described above.
Conclusions
S19 formed two potential hydrogen bonds between the sulfur atom and the -NH group of the compound and Gln247α and Ala354β (Figure S8). Our data are in agreement with previous studies that pointed to the sulfur atom as an essential factor for the anticancer activity of monastrol and other thio-derivatives (3,4-dihydropyrimidine-2(1H)-thiones). Replacement of sulfur with oxygen resulted in a loss of cytotoxic activity, probably due to the "soft nature" of the sulfur atom, making the compounds more nucleophilic [49].
Namely, we evaluated the ability of compounds S1, S19 and S22 to compete with colchicine, whose intrinsic fluorescence increases upon binding to tubulin. We chose two of the most critical concentrations of tested compounds, selected during the cell-free tubulin polymerization study (10 µM and 50 µM). As shown in Figure 9, the fluorescence of the colchicine-tubulin complex was reduced in the presence of S1, S19, and S22 in a dose-dependent manner, suggesting that these compounds bind at the colchicine site and may belong to CBSIs.
Vinblastine (VBL, used as negative control) did not compete with colchicine because it inhibits tubulin polymerization by binding at different sites, located on β-tubulin chain. Namely, the sulfur atom of S1 showed hydrogen bonding interaction with amino acids of tubulin (Asn101α) (Figure S7). The replacement of sulfur by an oxygen led to the loss of the cytotoxic activity, probably due to the "soft nature" of the sulfur atom that makes compounds more nucleophilic [48].
Namely, we evaluated the ability of compounds S1, S19 and S22 to compete with colchicine, whose intrinsic fluorescence increases upon binding to tubulin. We chose two of the most critical concentrations of tested compounds, selected during the cell-free tubulin polymerization study (10 µM and 50 µM). As shown in Figure 9, the fluorescence of the colchicine-tubulin complex was reduced in the presence of S1, S19, and S22 in a dose-dependent manner, suggesting that these compounds bind at the colchicine site and may belong to CBSIs.
Vinblastine (VBL, used as a negative control) did not compete with colchicine because it inhibits tubulin polymerization by binding to different sites located on the β-tubulin chain. Results are shown as intrinsic fluorescence of the colchicine-tubulin complex normalized to control (0.2% DMSO). Furthermore, we demonstrated that compounds S1, S19, and S22 disrupted mitotic spindle organization and inhibited tubulin polymerization through the colchicine binding site in a dose-dependent manner.
Materials and Methods 1. Experimental Part
The crude product purified twice by column chromatography (the first column: SiO 2 , n-hexane: ethyl acetate, 9:1, the second column: SiO 2 , chloroform) gave a yellow solid in 96% yield. The crude product, purified twice by column chromatography (the first column: SiO 2 , n-hexane: ethyl acetate, 10:1, the second column SiO 2 , chloroform), gave a yellow solid in 84% yield. The crude product purified twice by column chromatography (the first column: SiO 2 , n-hexane: ethyl acetate, 10:1, the second column: SiO 2 , chloroform) gave a yellow solid in 98% yield.
The crude product was purified twice by column chromatography (the first column SiO2, n-hexane:ethyl acetate, 6:1, the second column SiO2, chloroform) to give a yellow solid in 68%. The crude product purified twice by column chromatography (the first column: SiO 2 , n-hexane: ethyl acetate, 6:1, the second column: SiO 2 , chloroform), gave a yellow solid in 98% yield. 5-(4-Fluorophenyl)-2-methoxypyridine (2): The crude product purified by column chromatography (SiO2, n-hexane:ethyl acetate 11:1) gave a white solid in 86% yield.
5-(3,4-Difluorophenyl)-2-methoxypyridine (3): The crude product purified by column chromatography (SiO2, n-hexane:ethyl acetate 10:1), gave white solid in 96% yield. 5-(4-Fluorophenyl)-2-methoxypyridine (2): The crude product purified by column chromatography (SiO2, n-hexane:ethyl acetate 11:1), gave white solid in 86% yield. 5-(3,4-Difluorophenyl)-2-methoxypyridine (3): The crude product purified by column chromatography (SiO2, n-hexane:ethyl acetate 10:1), gave white solid in 96% yield.
The crude product was purified by column chromatography (SiO2, n-hexane:ethyl acetate 1:1), affording a white solid in 61% yield. The crude product purified by column chromatography (SiO2, n-hexane:ethyl acetate 1:1) gave a colorless solid in 48% yield. The crude product was purified by column chromatography on SiO2 using suitable solvents as eluent.
The crude product purified by column chromatography (SiO 2 , n-hexane:ethyl acetate 1:1) gave a white solid in 90% yield. The crude product was purified by column chromatography on SiO 2 using the appropriate solvent as eluent. The crude product purified by column chromatography (SiO 2 , n-hexane: ethyl acetate 1:1) gave a white solid in 90% yield.
The crude product was purified by column chromatography (SiO2, n-hexane:ethyl acetate) to give a white solid in 96% yield. The crude product purified by column chromatography (SiO2, n-hexane: .ethyl acetate 1:1) gave white solid in 71% yield. The crude product purified by column chromatography (SiO2, n-hexane:ethyl acetate 1:1) gave a pale yellow oil in 96% yield.
The crude product was purified by column chromatography (SiO 2 , n-hexane:ethyl acetate 3:1) to give a white solid in 91% yield.
