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. 2022 Nov 12;27(22):7807.
doi: 10.3390/molecules27227807.

The C30-Modulation of Betulinic Acid Using 1,2,4-Triazole: A Promising Strategy for Increasing Its Antimelanoma Cytotoxic Potential

Gabriela Nistor  1   2 , Marius Mioc  1   2 , Alexandra Mioc  2   3 , Mihaela Balan-Porcarasu  4 , Roxana Racoviceanu  1   2 , Alexandra Prodea  1   2 , Andreea Milan  1   2 , Roxana Ghiulai  2   5 , Alexandra Semenescu  2   6 , Cristina Dehelean  2   6 , Codruța Șoica  2   5
Affiliations

The C30-Modulation of Betulinic Acid Using 1,2,4-Triazole: A Promising Strategy for Increasing Its Antimelanoma Cytotoxic Potential

Gabriela Nistor et al. Molecules. .

Abstract

Cancer, in all its types and manifestations, remains one of the most frequent causes of death worldwide; an important number of anticancer drugs have been developed from plants, fungi and animals, starting with natural compounds that were later derivatized in order to achieve an optimized pharmacokinetic/pharmacological profile. Betulinic acid is a pentacyclic triterpenic compound that was identified as an anticancer agent whose main advantage consists in its selective activity, which ensures the almost total lack of cytotoxic side effects. Conjugates of betulinic acid with substituted triazoles, scaffolds with significant pharmacological properties, were synthesized and tested as anticancer agents in order to achieve new therapeutic alternatives. The current paper aims to obtain a C30-1,2,4-triazole derivative of betulinic acid simultaneously acetylated at C3 whose biological activity was tested against RPMI melanoma cells. The compound revealed significant cytotoxic effects at the tested concentrations (2, 10 and 50 μΜ) by significantly decreasing the cell viability to 88.3%, 54.7% and 24.5%, respectively, as compared to the control. The compound's testing in normal HaCaT cells showed a lack of toxicity, which indicates its selective dose-dependent anticancer activity. The investigation of its underlying molecular mechanism revealed an apoptotic effect induced at the mitochondrial level, which was validated through high-resolution respirometry studies.

Keywords: apoptosis; betulinic acid; cell viability; high-resolution respirometry; triazole; triterpenes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Synthesis pathways used to obtain 3β-O-Acetyl-30-(1H-1,2,4-triazole-3-ylsulfanyl)-betulinic acid (BA-TZ); TSC = thiosemicarbazide, TZ = 1,2,4-triazole-3-thiol, BA = betulinic acid, BrBA = 3β-O-Acetyl-30-bromo-betulinic acid; reaction conditions: a. reflux, 30 min; b. H2O, NaOH, reflux, 1h; c. acetic anhydride, pyridine, DMAP, r.t, 12h; d. NBS, CCl4, r.t, 48h; e. DMF, K2CO3, r.t, 72 h.
Figure 2
Figure 2
Cell viability of HaCaT cells after 24 h treatment with BA-TZ 0.08, 0.4, 2, 10 and 50 μM, determined using the MTT assay. Untreated cells were used as the negative control, whereas 5-FU was used as the positive control. The results are expressed as cell viability percentage (%) normalized to the negative control (100%). The data represent the mean values ± SD of three independent experiments performed in triplicate. The statistical differences vs. the negative control was determined using two-way ANOVA analysis followed by Bonferroni’s multiple comparisons post-test (*** p < 0.0001).
Figure 3
Figure 3
Cell viability of RPMI-7951 cells after 24 h treatment with BA-TZ 0.08, 0.4, 2, 10 and 50 μM, determined using the MTT assay. Untreated cells were used as negative control, while 5-FU was used as positive control. The results are expressed as cell viability percentage (%) normalized to the negative control (100%). The data represent the mean values ± SD of three independent experiments performed in triplicate. The statistical differences vs. the negative control was determined using two-way ANOVA analysis followed by Bonferroni’s multiple comparisons post-test (* p < 0.05 and *** p < 0.0001).
Figure 4
Figure 4
Morphological observation of RPMI-7951 cells using DAPI staining, after treatment with TZ and BA-TZ at 10 and 50 μM for 24 h. The staurosporine solution (5 μM) was used as positive control for apoptotic changes at the nuclear level. The yellow arrows represent signs of apoptosis, such as nuclear shrinkage, condensation, fragmentation and cellular membrane disruption.
Figure 5
Figure 5
Mitochondrial respiratory rates of permeabilized HaCaT cells following 24 h treatment with BA-TZ 10 μM. Data represent the mean ± SD of five individual experiments. The statistical differences vs. control was determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons post-test. Values with p < 0.05 were considered to have a statistically significant difference (p > 0.5 no significant differences).
Figure 6
Figure 6
Mitochondrial respiratory rates of permeabilized RPMI-7951 cells following 24 h treatment with BA-TZ 10 μM. Data represent the mean ± SD of five individual experiments. The statistical differences vs. control was determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons post-test. Values with p < 0.05 were considered to have a statistically significant difference (*p < 0.05, ** p < 0.01 and *** p < 0.001).
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