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. 2021 Aug 28;9(9):1104.
doi: 10.3390/biomedicines9091104.

Betulinic Acid Decorated with Polar Groups and Blue Emitting BODIPY Dye: Synthesis, Cytotoxicity, Cell-Cycle Analysis and Anti-HIV Profiling

David Kodr  1 , Jarmila Stanková  2 , Michaela Rumlová  3 , Petr Džubák  2 , Jiří Řehulka  2 , Tomáš Zimmermann  1 , Ivana Křížová  3 , Soňa Gurská  2 , Marián Hajdúch  2 , Pavel B Drašar  1 , Michal Jurášek  1
Affiliations

Betulinic Acid Decorated with Polar Groups and Blue Emitting BODIPY Dye: Synthesis, Cytotoxicity, Cell-Cycle Analysis and Anti-HIV Profiling

David Kodr et al. Biomedicines. .

Abstract

Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 μM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 μM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.

Keywords: BODIPY; betulinic acid; bevirimat; cancer; cell-cycle; cytotoxicity; fluorescent microscopy; maturation inhibitor.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Chemical structure of betulinic acid and its derivatives.
Figure 2
Figure 2
Synthesis of functionalized BODIPY dyes. Reagents and conditions: (a) β-Ala, DMSO-H2O, 30 °C, 12 h; (b) 3-azidopropylamine, DCM, 30 min, RT; (c) H2, Pd/C, AcOEt, 2h, RT.
Figure 3
Figure 3
Synthesis of “aminopropyl” derivatives by azide reduction (panel A) and Boc chemistry (panel B). Reagents and conditions: (a) 3-azidopropylamine, 4-DMAP, HOBt, EDC, DMF, 36 h, RT; (b) 2,2-dimethylsuccinic anhydride, 4-DMAP, p-TsOH, THF, 2 h, MW-130 °C; (c) PPh3, THF/H2O, 23 h; (d) BODIPY-NH2, 4-DMAP, DCC, DCM, 12 h, RT; (e) BODIPY-CO2H, DCC, 4-DMAP, DCM, 12 h, RT; (f) N-Boc-1,3-diaminopropane, EDC, HOBt, 4-DMAP, DMF, 48 h, RT; (g) 2M HCl/Et2O, 12 h, RT (under argon); (h) BODIPY-SMe, CHCl3-THF, Et3N, 30 min, RT.
Figure 4
Figure 4
Synthesis of “piperazinyl” derivatives. Reagents and conditions: (a) Ac2O, pyridine, 12 h, RT; (b) i. (COCl)2, DCM, DMF, 2 h, RT, ii. 1-(2-N-boc-aminoethyl)piperazine, Et3N, DCM, 12 h, RT; (c) 4M NaOH, THF-MeOH, 3 h, RT; (d) 2M HCl/Et2O, 12 h, RT (under argon); (e) BODIPY-SMe, CHCl3-THF, Et3N, 30 min, RT; (f) 2,2-dimethyl succinic anhydride, 4-DMAP, p-TsOH, THF, 2 h, MW-130 °C.
Figure 5
Figure 5
Live cell imaging and colocalization experiments of active compounds (4, 6) and BODIPY-SMe (panel A) and visualization of Pearson´s and Mander´s coefficient (panel B).
Figure 6
Figure 6
Effect of selected tested compounds on CA-SP1 processing of HIV-1 Gag polyprotein. HEK 293 cells produced HIV-1 particles pseudotyped with VSV-glycoproteins in the absence (lanes 2 and 3) or presence of selected tested compounds (lanes 4–12). At 48 h post-transfection, VSV-G pseudotyped HIV-1 viruses released from the HEK 293 cells were analysed by Western blot using an anti-HIV-1 CA antibody (duplicate of blot shown in Figure S57).
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