Cytotoxic profiling of artesunic and betulinic acids and their synthetic hybrid compound on neurons and gliomas

Abstract

Gliomas are brain-born tumors with devastating impact on their brain microenvironment. Novel approaches employ multiple combinations of chemical compounds in synthetic hybrid molecules to target malignant tumors. Here, we report on the chemical hybridization approach exemplified by artesunic acid (ARTA) and naturally occurring triterpene betulinic acid (BETA). Artemisinin derived semisynthetic compound artesunic acid (ARTA) and naturally occurring triterpene BETA were used to synthetically couple to the hybrid compound termed 212A. We investigated the impact of 212A and its parent compounds on glioma cells, astrocytes and neurons. ARTA and BETA showed cytotoxic effects on glioma cells at micromolar concentrations. ARTA was more effective on rodent glioma cells compared to BETA, whereas BETA exhibited higher toxic effects on human glioma cells compared to ARTA. We investigated these compounds on non-transformed glial cells and neurons as well. Noteworthy, ARTA showed almost no toxic effects on astrocytes and neurons, whereas BETA as well as 212A displayed neurotoxicity at higher concentrations. Hence we compared the efficacy of the hybrid 212A with the combinational treatment of its parent compounds ARTA and BETA. The hybrid 212A was efficient in killing glioma cells compared to single compound treatment strategies. Moreover, ARTA and the hybrid 212A displayed a significant cytotoxic impact on glioma cell migration. Taken together, these results demonstrate that both plant derived compounds ARTA and BETA operate gliomatoxic with minor neurotoxic side effects. Altogether, our proof-of-principle study demonstrates that the chemical hybrid synthesis is a valid approach for generating efficacious anti-cancer drugs out of virtually any given structure. Thus, synthetic hybrid therapeutics emerge as an innovative field for new chemotherapeutic developments with low neurotoxic profile.

Keywords: artesunic acid; betulinic acid; cancer cytotoxicity; cell death; hybrid synthesis.

Figure 1. Structure of bevirimat

Figure 2. Natural products hybridization

Given is a scheme displaying the principle of the chemical hybrid synthesis concept. This chemical hybrid synthesis approach is a valid methodology for generating efficacious anti-cancer drugs out of virtually any given structure. Thus, synthetic hybrid therapeutics emerge as an innovative field for new chemotherapeutic developments.

Figure 3. Synthesis of artesunic and betulinic acids hybrid 212A

Figure 4. NMR and Mass spectra of hybrid compound 212A, BETA and ARTA

(A) ¹H-NMR spectrum (300 MHz, CDCl₃) of 212A. (B) ¹³C-NMR spectrum (75 MHz, CDCl₃) of 212A. (C) ESI-MS spectrum of 212A. (D) ¹H-NMR spectrum (300 MHz, CDCl₃) of ARTA. (E) ¹³C-NMR spectrum (75 MHz, CDCl₃) of ARTA. (F) ¹H-NMR spectrum (300 MHz, CDCl₃) of BETA. (G) ¹³C-NMR spectrum (75 MHz, CDCl₃) of BETA.

Figure 4. NMR and Mass spectra of hybrid compound 212A, BETA and ARTA

(A) ¹H-NMR spectrum (300 MHz, CDCl₃) of 212A. (B) ¹³C-NMR spectrum (75 MHz, CDCl₃) of 212A. (C) ESI-MS spectrum of 212A. (D) ¹H-NMR spectrum (300 MHz, CDCl₃) of ARTA. (E) ¹³C-NMR spectrum (75 MHz, CDCl₃) of ARTA. (F) ¹H-NMR spectrum (300 MHz, CDCl₃) of BETA. (G) ¹³C-NMR spectrum (75 MHz, CDCl₃) of BETA.

Figure 4. NMR and Mass spectra of hybrid compound 212A, BETA and ARTA

(A) ¹H-NMR spectrum (300 MHz, CDCl₃) of 212A. (B) ¹³C-NMR spectrum (75 MHz, CDCl₃) of 212A. (C) ESI-MS spectrum of 212A. (D) ¹H-NMR spectrum (300 MHz, CDCl₃) of ARTA. (E) ¹³C-NMR spectrum (75 MHz, CDCl₃) of ARTA. (F) ¹H-NMR spectrum (300 MHz, CDCl₃) of BETA. (G) ¹³C-NMR spectrum (75 MHz, CDCl₃) of BETA.

Figure 4. NMR and Mass spectra of hybrid compound 212A, BETA and ARTA

(A) ¹H-NMR spectrum (300 MHz, CDCl₃) of 212A. (B) ¹³C-NMR spectrum (75 MHz, CDCl₃) of 212A. (C) ESI-MS spectrum of 212A. (D) ¹H-NMR spectrum (300 MHz, CDCl₃) of ARTA. (E) ¹³C-NMR spectrum (75 MHz, CDCl₃) of ARTA. (F) ¹H-NMR spectrum (300 MHz, CDCl₃) of BETA. (G) ¹³C-NMR spectrum (75 MHz, CDCl₃) of BETA.

Figure 5. Properties of investigated parent compounds and hybrid 212A

R_f values were determined in the solvent mixture of hexane : ethylacetate (4:1). All compound purities were confirmed by EA (elemental analysis). The NMR spectra are provided in Figure 4.

Figure 6. Cytotoxic profile of Artesunic acid, Betulinic acid and the hybrid 212A on rodent glioma cells

(A) Rat glioma cells (F98) were treated with 5 μM of ARTA, BETA, 212A and ARTA combined with BETA. Cell morphology was examined after 72h with light microscopy. Scale bars, 200 μm. (B, C) F98 were treated with different concentrations of ARTA, BETA, 212A and ARTA combined with BETA. Cell viability was measured after 72h. Statistical significance was tested with unpaired two-sided t-test vs. control (n≥ 4; *, p ≤ 0.05). The light blue marking indicates the value of IC₅₀ and the dark blue one stands for IC ₉₀ value. This is passable for the following figures. (D, E) Rodent cells were treated with 10 μM of the compounds. Cell death analysis was performed after 24h with 7 AAD and Annexin V. Statistical significance was tested with unpaired two-sided t-test vs. control (n≥ 4; p ≤ 0.05). (F) F98 were treated with 10 μM of ARTA, BETA, 212A and ARTA combined with BETA. Cells were fixated and stained with actin marker Phalloidin 488 and DNA marker Hoechst after 48h. Scale bars represent 20 μm.

Figure 7. Cytotoxic effects of Artesunic acid, Betulinic acid and the hybrid 212A on human glioma cells

(A, D) U87 MG (human glioblastoma/astrocytoma cells) were treated with 10 μM of ARTA, BETA, 212A and ARTA combined with BETA. After 72h representative images with propidium iodide (PI) and light microscopy were taken. Scale bars, 200 μm. (B, C) U87 were treated with different concentration of ARTA, BETA, 212A and ARTA combined with BETA. Cell viability was measured after 72h. Statistical significance was tested with unpaired two-sided t-test vs. control (n≥3; *, p ≤ 0.05). (E) Cells were treated with 10 μM of ARTA, BETA, 212A and the combinatorial treatment of ARTA and BETA. After 72h, cell death was examined with propidium iodide (PI) staining. The images were quantified and related to the control. Statistical significance was tested with unpaired two-sided t-test vs. control (n≥4; *, p ≤ 0.05). (F) TN22 (human glioma cells) were treated with 10 μM of ARTA, BETA, 212A and ARTA combined with BETA. Cell viability was measured after 72h. Statistical significance was tested with unpaired two-sided t-test vs. control (n≥4; *, p ≤ 0.05).

Figure 8. Neurotoxicity and gliomatoxicity profiling of Artesunic acid, Betulinic acid and the hybrid 212A

(A) Primary neurons and astrocytes were treated with 10 μM ARTA, BETA, 212A and ARTA in combination with BETA. After 72h cells were fixed and immunostained for the neuronal marker beta-III- tubulin, the glial marker GFAP and the DNA marker Hoechst. Scale bars, 100 μm. (B) Primary neurons and astrocytes were treated with 10 μM of ARTA, BETA, 212A and ARTA combined with BETA. Cell death was examined after 72h with propidium iodide (PI) and light microscopy. Scale bars, 200 μm. (C, D) Primary neurons and astrocytes were treated with 5 μM (C) and 10 μM (D) of ARTA, BETA, 212A and ARTA combined with BETA. Cell viability was measured after 72h. Statistical significance was tested with unpaired two-sided t-test vs. control (n≥5; *, p ≤ 0.05).

Figure 9. Extent of inhibition of cell migration by Artesunic acid, Betulinic acid and the hybrid on rodent glioma cells

(A, C) A scratch was made into plated cells which were then treated with 5 μM (A) and 10 μM (C). Results were observed at different times. (B, D) Migration distance of the treatment with 5 μM (B) and 10 μM (D) were quantified and compared to the data from 0 h. Statistical significance was tested with unpaired two-sided t-test vs. control (n=3; *, p ≤ 0.05).