Study of Cytotoxicity of 3-Azabicyclo[3.1.0]hexanes and Cyclopropa[ a]pyrrolizidines Spiro-Fused to Acenaphthylene-1(2 H)-one and Aceanthrylene-1(2 H)-one Fragments Against Tumor Cell Lines

Abstract

A series of 3-azabicyclo[3.1.0]hexanes and cyclopropa[a]pyrrolizidines spiro-fused to acenaphthylene-1(2H)-one and aceanthrylene-1(2H)-one frameworks have been studied for their in vitro antiproliferative activity against human erythroleukemia (K562), cervical carcinoma (HeLa), melanoma (Sk-mel-2), osteosarcoma (U2OS), as well as murine melanoma (B16) cell lines. Using confocal microscopy, it was found that cultivation with the tested spiro-fused compounds led to the disappearance of stress fibers (granular actin was distributed diffusely in the cytoplasm in up to 56% of treated cells) and decrease in filopodia-like deformations (up to 69% after cultivation), which indirectly suggests a decrease in cell motility. The human melanoma cell line scratch test showed that these cells lose their ability to move after cultivation with the tested spiro-fused compounds and do not fill the scratched strip. This was also supported by docking simulations with actin-related targets (PDB ID: 8DNH, 2Q1N). Using flow cytometry, the impact on the mitochondrial membrane potential showed that the tested compounds led to a significant increase in the number of cells with decreased mitochondrial membrane potential from 10% for the control up to 55-80% for the cyclopropa[a]pyrrolizidine adducts. The obtained results support the antitumor effect of the tested spiro-compounds and encourage the extension of the study in order to improve their anticancer activity as well as reduce their toxicological risks.

Keywords: 1,3-dipolar cycloaddition; 3-spiro[3-azabicyclo[3.1.0]-hexanes]; antiproliferative activity; azomethine ylides; cell motility; cyclopropa[a]pyrrolizidines; cyclopropenes; morphological changes (cytoskeleton); spiro-acenaphthylene-1,2′-bicyclo[3.1.0]hexanes; spiro-acenaphthylene-1,2′-cyclopropa[a]pyrrolizines; tumor cell lines.

Figure 1

Selected examples of biologically active acenaphthylenes, azabicyclo[3.1.0]hexanes, and 1-azabicyclo[3.3.0]octanes (pyrrolizines or pyrrolizidines) [14,15,16,17,25,27,29,34,41,42,43].

Figure 2

Structures of acenaphthylene-1,2′-cyclopropa[a]pyrrolizines and their aceanthrylene analogues 1a–k.

Figure 3

Structures of acenaphthylene-1,2′-bicyclo[3.1.0]hexanes and their aceanthrylene analogues 2a–i.

Figure 4

Cytotoxicity of racemic spiro-fused cyclopropa[a]pyrrolizines 1a–k (A) and 3-azabicyclo[3.1.0]hexanes 2a–I (B) against the K562 cell line for 72 h.

Figure 5

Cytotoxicity of racemic spiro-fused cyclopropa[a]pyrrolizines 1a–k (A) and 3-azabicyclo[3.1.0]hexanes 2a–i (B) against the HeLa cell line for 72 h.

Figure 6

Cytotoxicity of selected racemic spiro-fused cyclopropa[a]pyrrolizines 1a–k (A) and 3-azabicyclo[3.1.0]hexanes 2a–i (B) against the Sk-mel-2 cell line for 72 h.

Figure 7

Cytotoxicity of racemic spiro-fused cyclopropa[a]pyrrolizines 1a–k (A) and 3-azabicyclo[3.1.0]hexanes 2a–i (B) against the U2OS cell line for 72 h.

Figure 8

Cytotoxicity of selected racemic spiro-fused cyclopropa[a]pyrrolizines 1a–k (A) and 3-azabicyclo [3.1.0]hexanes 2a–i (B) against the B16 cell line for 72 h.

Figure 9

Microscopic images of treated cells and state of actin cytoskeleton of HeLa cells after cultivation with compounds 1e, 1f, 1h, 1i, 2b, 2c, and 2d (10 μg/mL).

Figure 10

Microscopic images of treated cells and state of actin cytoskeleton of Sk-mel-2 cells after cultivation with compounds 1e, 1f, 1h, 1i, 2b, 2c, and 2d (10 μg/mL).

Figure 11

Microscopic images of treated cells and state of actin cytoskeleton of B16 cells after cultivation with compounds 1e, 1f, 1h, 1i, 2b, 2c, and 2d (10 μg/mL).

Figure 12

Microscopic images of the Sk-mel-2 cell wound area in the scratch assay and wound area (%) in the scratch assay after 36 h incubation with spiro-fused cyclopropa[a]pyrrolizidines 1c, 1e, 1f, 1h, 1i, and 3-azabicyclo[3.1.0]hexanes 2b, 2c, 2d, and 2g. p value < 0.05 (*), 0.01 (**), 0.001 (***).

Figure 12

Microscopic images of the Sk-mel-2 cell wound area in the scratch assay and wound area (%) in the scratch assay after 36 h incubation with spiro-fused cyclopropa[a]pyrrolizidines 1c, 1e, 1f, 1h, 1i, and 3-azabicyclo[3.1.0]hexanes 2b, 2c, 2d, and 2g. p value < 0.05 (*), 0.01 (**), 0.001 (***).

Figure 13

Changes in the mitochondrial membrane potential (∆Ψm) of K562 cells treated with the studied compounds.

Figure 14

Docked view of 1c, 1f, 2b, and 2c with the target protein (PDB ID: 8DNH).

Figure 15

Predicted binding models of compounds 1b and 2b within the target-binding cleft of MDM2 protein (for PDB ID: 7BJ6).