Examination of novel 4-aminoquinoline derivatives designed and synthesized by a hybrid pharmacophore approach to enhance their anticancer activities

Abstract

In an attempt to develop effective and potentially safe anticancer agents, thirty-six 4-aminoquinoline derived sulfonyl analogs were designed and synthesized using a hybrid pharmacophore approach. The cytotoxicity of these compounds was determined using three breast tumor cell lines (MDA-MB231, MDA-MB468 and MCF7) and two matching non-cancer breast epithelial cell lines (184B5 and MCF10A). Although most of the compounds were quite effective on the breast cancer cells, the compound 7-chloro-4-(4-(2,4-dinitrophenylsulfonyl)piperazin-1-yl)quinoline (13; VR23) emerged as potentially the most desirable one in this series of compounds. Data from the NCI-60 cancer panel screening show that compound 13 is effective on a wide range of different cancers. Importantly, compound 13 is needed up to 17.6-fold less doses to achieve the same IC₅₀ against cancer than non-cancer cells (MDA-MB468 vs MCF10A), suggesting that it can potentially be less toxic to normal cells. Cancer cells formed multiple centrosomes in the presence of compound 13, resulting in the cell cycle arrest at prometa-meta phase. This abnormality leads to eventual cell demise with sub-G1 DNA content typically shown with apoptotic cells. In addition, compound 13 also causes an increase in lysosomal volume in cancer but not in non-cancer cells, which may contribute at least in part to its preferential cancer cell-killing. The cancer cell-killing effect of compound 13 is highly potentiated when combined with either bortezomib or monastrol.

Figure 1

Design of hybrid compounds. (a) Chemical structures that we previously reported to have anticancer activity (see text). (b) Some sulfonyl analogs with anticancer activity. (c) The design of hybrid compounds.

Figure 2

Schematic presentation of the synthesis of 4- aminoquinoline derived analogs. (a) Piperazine, Triethylamine, 120–130 °C for 6 h; (b) 1,3-Diaminopropane, 120–130 °C for 6 h; and (c) R₁-sulfonyl chloride, Triethylamine, THF, RT, 4 h.

Figure 3

Summary of SAR analysis.

Figure 4

Compound 13 (VR23) causes a cell-cycle arrest at M phase. (a) Cell cycle profile of asynchronous HeLa S3 cells (a control profile). (b) HeLa cells were synchronized at the beginning of S phase by double thymidine (DT) block as described previously. (c) Cells synchronized by DT treatment were released into drug-free complete medium for 6 h, at which time most cells were at late-S to G2/M. (d) Cells were continued to incubate in drug-free medium for additional 6 h, at which time most cells were already in G1 of the next cell cycle. (e) The same as the sample in panel d, but cells were incubated in the presence of 10 µM compound 13.

Figure 5

Compound 13-treated cells are arrested at prometa-metaphase through the inactivation of Cdk1. HeLa S3 cells were synchronized at G2/M phase by incubating in 50 ng/ml nocodazole (noco) for 18 h. The cells were then released into complete medium at time 0 h in the absence (Sham) or presence of 10 µM compound 13. Samples were then taken at the indicated times post-nocodazole. (a) Phosphorylation of Cdk1 on Tyr15 was reduced at 1 h, but dramatically increased at 2–3 h post-nocodazole arrest point. (b) Cdk1 may be inactivated by the combination of a high level of Wee1 kinase and inactivation of Cdc25C. The relative intensity of band signals are quantified by densitometry and presented the results in Supplementary Fig. S3.

Figure 6

Compound 13 causes the formation of multiple spindle poles and uneven segregation. (a) HeLa S3 cells were synchronized at G2/M by nocodazole, followed by incubation for 6 h in drug-free medium in the absence (Sham control) or presence of 5 μM of compound 13. Note the presence of uneven cell sizes (arrows) which were likely generated as a result of nondisjuctional chromosome segregation during mitosis. (b) Multiple spindle phenotypes were seen in 44.4% of compound 13-treated cells. In contrast, the untreated control sample contains very few cells with abnormal spindles.

Figure 7

Lysosomal volumes are increased in MCF7 cells treated with compound 13. Lysomes were visualized by treating MCF7 cells with lysoTracker Red for 48 h in the absence (Sham) or presence of 5 μM compound 13. The nuclei of cells in rows a and c were visualized by staining with DRAQ5. Note that rows a, c and e are enlarged images.

Figure 8

Combination of compound 13 and BTZ is highly effective. (a) 2.7–8.0 μM of compound 13 effectively killed cancer cells by 48 h. Asynchronous MCF7 cells were incubated for 48 h in the absence (Sham control) or presence of compound 13 at 2.7 or 8.0 µM. (b) The combination of low doses of compound 13 (0.75 μM) and BTZ (6 nM) shows synergistic effects on Jurkat cells. Asynchronously growing Jurkat lymphoma cells were incubated for 48 h in the absence (sham control) or presence of BTZ ± VR23, followed by cell analysis by cytometry.