Identification of Spiro-Fused Pyrrolo[3,4- a]pyrrolizines and Tryptanthrines as Potential Antitumor Agents: Synthesis and In Vitro Evaluation

Abstract

A series of heterocyclic compounds containing a spiro-fused pyrrolo[3,4-a]pyrrolizine and tryptanthrin framework have been synthesized and studied as potential antitumor agents. Cytotoxicity of products was screened against human erythroleukemia (K562) and human cervical carcinoma (HeLa) cell lines. Among the screened compounds. 4a, 4b and 5a were active against human erythroleukemia (K562) cell line, while 4a and 5a were active against cervical carcinoma (HeLa) cell line. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G2/M phase and induced apoptosis. Using confocal microscopy, we found that with 4a and 5a treatment of HeLa cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 76-91% of cells. We discovered that HeLa cells after treatment with compounds 4a and 5a significantly reduced the number of cells with filopodium-like membrane protrusions (from 63 % in control cells to 29% after treatment) and a decrease in cell motility.

Keywords: 1,3-dipolar cycloaddition; antiproliferative activity; cell cycle; cell death; cell motility; morphological changes (cytoskeleton); one-pot synthesis; pyrrolo[3,4-a]pyrrolizine; tryptanthrin-derived azomethine ylide; tumor cells.

Figure 1

ORTEP representation of the molecular structure of 4c and 5d.

Figure 2

Antiproliferative activity of spiro-fused pyrrolo[3,4-a]pyrrolizines and tryptanthrines against K562 (A) and HeLa (B) cell lines for 72 h.

Figure 3

Annexin V-FITC/Propidium iodide (PI) dual staining assay of K562 (A) and HeLa (B) cells treated with cycloadducts 4a and 5a at concentrations 5, 10 and 20 μg/mL using flow cytometry.

Figure 4

Effect of spiro-fused tryptanthrines and pyrrolo[3,4-a]pyrrolizines 4a and 5a on the distribution of K562 cells in the cell cycle.

Figure 5

Effect of spiro-fused tryptanthrines and pyrrolo[3,4-a]pyrrolizines 4a and 5a on the distribution of HeLa cells in the cell cycle.

Figure 6

Changes in the relative expression levels of p53 and MDM2 proteins in HeLa cells under the treatment with compounds 4a and 5a (at concentration 10 μg/mL).

Figure 7

Microscopic images of the HeLa cells wound area in the scratch assay and wound area (%) in the scratch assay after 24 h incubation post spiro-fused cycloadducts 4a and 5a treatment at concentration 10 μg/mL. * p value < 0.05.

Figure 8

State of actin cytoskeleton of HeLa cells after treatment with spiro-fused tryptanthrines and pyrrolo[3,4-a]pyrrolizines 4a and 5a. (I): Images demonstrate the different stages of cell actin cytoskeleton. (II): Pie charts demonstrate percentage of cells with normal stress fibers (A) and disassembled stress fibers (B). (III): Pie charts demonstrate percentage of cells with filopodia-like deformations (C), and without filopodia-like deformations (D). Inserts: 1—stress fibers; 2—disassembled stress fibers.