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. 2022 Sep 15;23(18):10759.
doi: 10.3390/ijms231810759.

Biological Evaluation of 3-Azaspiro[Bicyclo[3.1.0]Hexane-2,5'-Pyrimidines] as Potential Antitumor Agents

Stanislav V Shmakov  1 , Diana K Latypova  1 , Tatiana V Shmakova  2 , Artem A Rubinshtein  3 , Mark V Chukin  3 , Sergei G Zhuravskii  2   3 , Nickolay A Knyazev  4   5 , Alexander V Stepakov  6   7 , Michael M Galagudza  2 , Vitali M Boitsov  1
Affiliations

Biological Evaluation of 3-Azaspiro[Bicyclo[3.1.0]Hexane-2,5'-Pyrimidines] as Potential Antitumor Agents

Stanislav V Shmakov et al. Int J Mol Sci. .

Abstract

A series of heterocyclic compounds containing spirofused barbiturate and 3-azabicyclo[3.1.0]hexane frameworks have been studied as potential antitumor agents. Antiproliferative activity of products was screened in human erythroleukemia (K562), T lymphocyte (Jurkat), and cervical carcinoma (HeLa) as well as mouse colon carcinoma (CT26) and African green monkey kidney epithelial (Vero) cell lines. The most effective among the screened compounds show IC50 in the range from 4.2 to 24.1 μM for all tested cell lines. The screened compounds have demonstrated a significant effect of the distribution of HeLa and CT26 cells across the cell cycle stage, with accumulation of cells in SubG1 phase and induced apoptosis. It was found, using a confocal microscopy, that actin filaments disappeared and granular actin was distributed diffusely in the cytoplasm of up to 90% of HeLa cells and up to 64% of CT26 cells after treatment with tested 3-azaspiro[bicyclo [3.1.0]hexane-2,5'-pyrimidines]. We discovered that the number of HeLa cells with filopodium-like membrane protrusions was reduced significantly (from 91% in control cells to 35%) after treatment with the most active compounds. A decrease in cell motility was also noticed. Preliminary in vivo experiments on the impact of the studied compounds on the dynamics of CT26 tumor growth in Balb/C mice were also performed.

Keywords: 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines]; CT26; HeLa; Jurkat; Vero); alloxan-derived azomethine ylide; antiproliferative activity; cancer cell lines (K-562; cell cycle; cell death; cell motility; cyclopropenes; in vitro and in vivo activity; morphological changes (cytoskeleton).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Synthesis of 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4 via one-pot three-component reactions of alloxane 1, primaryα-amino acids 2, and cyclopropenes 3 1.
Figure 1
Figure 1
Cytotoxicity of selected 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4 against K562 cell line for 24 h (A) and 72 h (B).
Figure 2
Figure 2
Cytotoxicity of selected 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4 against HeLa cell line for 24 h (A) and 72 h (B).
Figure 3
Figure 3
Cytotoxicity of selected 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4 against CT26 cell line for 24 h (A) and 72 h (B).
Figure 4
Figure 4
Cytotoxicity of 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4a,c–i against Jurkat cell line for 24 h (A) and 72 h (B).
Figure 5
Figure 5
Cytotoxicity of 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4a–i against Vero cell line for 24 h (A) and 72 h (B).
Figure 6
Figure 6
Apoptotic activity of HeLa cells treated with spiro-fused cycloadducts 4c–i at concentration 10 μg/mL for 72 h using flow cytometry.
Figure 7
Figure 7
Apoptotic activity of CT26 cells treated with spiro-fused cycloadducts 4c–i at concentration 10 μg/mL for 72 h using flow cytometry.
Figure 8
Figure 8
Effect of cycloadducts 4c–i at concentration 10 μg/mL on the distribution of HeLa cells in the cell cycle.
Figure 9
Figure 9
Effect of cycloadducts 4c–i at concentration 10 μg/mL on the distribution of CT26 cells in the cell cycle.
Figure 10
Figure 10
Effect of cycloadducts 4d, 4e, 4g, and 4h at concentrations 2, 5, and 10 μg/mL on the distribution of HeLa (A) and CT26 (B) cells in the cell cycle.
Figure 11
Figure 11
Microscopic images of the HeLa cells wound area in the scratch assay and wound area (%) in the scratch assay after 24 h incubation post azaspiro[bicycle[3.1.0]hexane-2,5′-pyrimidines] 4 treatment. Bars indicate ± SD; * p value < 0.05.
Figure 12
Figure 12
Microscopic images of the CT26 cells wound area in the scratch assay and wound area (%) in the scratch assay after 24 h incubation post azaspiro[bicycle[3.1.0]hexane-2,5′-pyrimidines] 4 treatment. Bars indicate ± SD; * p value < 0.05.
Figure 13
Figure 13
Microscopic images of the Vero cells wound area in the scratch assay and wound area (%) in the scratch assay after 24 h incubation post azaspiro[bicycle[3.1.0]hexane-2,5′-pyrimidines] 4 treatment. Bars indicate ± SD.
Figure 14
Figure 14
State of actin cytoskeleton of HeLa cells after treatment with 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4d, 4e, 4f, 4h, and 4i. I: Images demonstrate the different stages of cell actin cytoskeleton. II: Pie charts demonstrate percentage of cells with filopodia-like deformations (A) and without filopodia-like deformations (B). III: Pie charts demonstrate percentage of cells with normal stress fibers (C) and disassembled stress fibers (D).
Figure 14
Figure 14
State of actin cytoskeleton of HeLa cells after treatment with 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4d, 4e, 4f, 4h, and 4i. I: Images demonstrate the different stages of cell actin cytoskeleton. II: Pie charts demonstrate percentage of cells with filopodia-like deformations (A) and without filopodia-like deformations (B). III: Pie charts demonstrate percentage of cells with normal stress fibers (C) and disassembled stress fibers (D).
Figure 15
Figure 15
State of actin cytoskeleton of CT26 cells after treatment with 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4d, 4e, 4f, 4h, and 4i. I: Images demonstrate the different stages of cell actin cytoskeleton. II: Pie charts demonstrate percentage of cells with filopodia-like deformations (A) and without filopodia-like deformations (B). III: Pie charts demonstrate percentage of cells with normal stress fibers (C) and disassembled stress fibers (D).
Figure 15
Figure 15
State of actin cytoskeleton of CT26 cells after treatment with 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4d, 4e, 4f, 4h, and 4i. I: Images demonstrate the different stages of cell actin cytoskeleton. II: Pie charts demonstrate percentage of cells with filopodia-like deformations (A) and without filopodia-like deformations (B). III: Pie charts demonstrate percentage of cells with normal stress fibers (C) and disassembled stress fibers (D).
Figure 16
Figure 16
State of actin cytoskeleton of Vero cells after treatment with 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4d, 4e, 4f, 4h, and 4i. I: Images demonstrate the different stages of cell actin cytoskeleton. II: Pie charts demonstrate percentage of cells with filopodia-like deformations (A) and without filopodia-like deformations (B). III: Pie charts demonstrate percentage of cells with normal stress fibers (C) and disassembled stress fibers (D).
Figure 17
Figure 17
Dynamics of tumor growth with a single intraperitoneal administration of drugs based on 3-azaspiro[bicyclo[3.1.0]hexane-2,5′-pyrimidines] 4d, 4e, 4f, and 4i. Bars indicate ±SD.
Figure 18
Figure 18
Organs of the mouse abdominal cavity during autopsy. Animal #12 (group 1, 4e). Macropreparation. There are no signs of local inflammation, adhesion, or sufficient development of adipose tissue. (A)—x 2. No signs of acute intoxication. The arrows indicate the agglomerates of the injected compound observed on the surface of the liver. (B)—x 4. The area of the gastrointestinal angle and the left kidney. The arrows indicate the agglomerates of the injected compound observed on the surface of the spleen.
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