Novel Epoxides of Soloxolone Methyl: An Effect of the Formation of Oxirane Ring and Stereoisomerism on Cytotoxic Profile, Anti-Metastatic and Anti-Inflammatory Activities In Vitro and In Vivo

Abstract

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and βO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18βH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and β-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC₅₀^(24h) = 0.7-4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and βO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and βO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates.

Keywords: 18βH-glycyrrhetinic acid; ABC transporters; CDDO-Me; anti-inflammatory activity; anti-metastatic activity; apoptosis; epoxide; pharmacophore group; soloxolone methyl; toxicity.

Scheme 1

Synthesis of soloxolone methyl epoxides (αO-SM) and (βO-SM).

Figure 1

Evaluation of the pro-apoptotic activity of αO-SM and βO-SM in B16 melanoma cells. (A) Dose-dependent effect of SM epoxides on the apoptosis induction in B16 melanoma cells. Flow cytometry analysis, double staining using Annexin V-FITC/PI. (B) Dose-dependent effect of SM epoxides on mitochondrial membrane potential in B16 cells. Flow cytometry analysis, staining with JC-1. (C) The ratio of JC-1 aggregates to monomers for the control and triterpenoid incubated cells. The data are expressed as mean ± SD of three independent experiments. (D) Activation of caspase-3/-7 in B16 cells incubated with SM, αO-SM or βO-SM at 1 µM for 24 h. The data are expressed as mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

The effects of SM epoxides on doxorubicin uptake by B16 melanoma cells. (A) The effect of αO-SM, βO-SM or SM on doxorubicin cytotoxicity induced in B16 cells. B16 cells were incubated with SM (0.5 µM) or αO-SM/βO-SM (1 µM) either in the presence or in the absence of doxorubicin (1 µM) for 72 h followed by the assessment of cell viability by MTT assay. Control–untreated cells. The data are represented as the mean ± SD of three independent experiments. (B) Doxorubicin (1 µM) fluorescence intensity in B16 cells exposed to SM (0.5 µM) or αO-SM/βO-SM (1 µM) and doxorubicin for 3 h. (C) Relative fluorescence intensity of B16 cells incubated with doxorubicin, SM and SM-epoxides. The data are expressed as mean ± SD of three independent experiments. (D) The binding modes of αO-SM, βO-SM and SM to the transmembrane domain of P-glycoprotein, MRP1 and MXR1. Molecular docking was performed by Autodock Vina. Green-, blue- and orange-colored molecules represent αO-SM, βO-SM and SM, respectively. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

The effect of αO-SM and βO-SM on the pro-metastatic phenotype of B16 melanoma cells. (A,B) Clonogenic activity of B16 melanoma cells incubated with αO-SM, βO-SM or SM for 14 days. The data are expressed as mean ± SD of six independent experiments. (C,D) Scratch assay showing the effect of SM-epoxides on the motility of B16 cells. Representative images (C) and quantitative data (D) presented as the mean ± SD of three independent experiments. (E) The effect of SM-epoxides on the adhesiveness of B16 cells. The cells were incubated with the evaluated compounds for 24 h, followed by their treatment with TrypLE, washing with PBS and measurement of relative cell numbers by MTT assay. The data are expressed as mean ± SD of five independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Anti-metastatic activity and toxicity of α-O-SM and βO-SM on the B16 melanoma model in vivo. (A) Experimental setup. A metastatic model of B16 melanoma was induced by the intravenous (i.v.) injection of B16 cells (10⁶ cells/mL) in C57Bl6 mice. On day 4 after tumor transplantation, mice were treated with intraperitoneal (i.p.) injections of α-O-SM, βO-SM or SM in 10% Tween-80 (30 mg/kg) or 10% Tween-80 (vehicle) thrice a week, receiving eight injections in total. Dacarbazine (DTIC) in water for injections (40 mg/kg, i.p.) was used as the reference drug. The mice were sacrificed on day 21 after tumor transplantation. (B,C) The number of surface metastases (B) and metastasis inhibition index (MII) (C) in mice with B16 melanoma after administration of α-O-SM, βO-SM, SM or DTIC. D-F. Toxic effects of triterpenoids in mice with B16 melanoma. The dynamics of body weight during the experiment (D), body weight at the end of experiment (E) and organ indexes (F) of mice with B16 melanoma after α-O-SM, βO-SM, SM and DTIC administration. The organ indexes of mice were calculated as (organ weight/body weight) and normalized on the average organ indexes of control mice. (G) Representative histological images of livers and kidneys of healthy mice and mice with B16 melanoma after α-O-SM, βO-SM, SM and DTIC administration. Hematoxylin and eosin staining. Original magnification ×400. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 5

Anti-inflammatory potential of SM epoxides in vitro and in vivo. (A) The effect of SM-epoxides on the viability of murine J774 macrophages. (B) The effect of SM epoxides on NO production by IFNγ/LPS-stimulated J774 cells. (C,D) The effect of SM-epoxides on the motility of J774 cells. Representative images (C) and quantitative data (D) of scratch assay represented the mean ± SD of three independent experiments. (E–H) Effect of SM-epoxides on the development of carrageenan-induced peritonitis in vivo. (E) Experimental setup. Balb/C mice were pretreated intraperitoneally (i.p.) with αO-SM, βO-SM, SM (30 mg/kg in 10% Tween-80) or dexamethasone (1 mg/kg) 1 h prior to peritonitis induction by i.p. injection of 1% carrageenan. Four hours after peritonitis induction, mice were sacrificed, and peritoneal exudates were obtained. (F–H) Total (F) and differential (G) leukocyte counts as well as TNFα levels (H) in the peritoneal exudates of healthy mice and mice with carrageenan-induced peritonitis after αO-SM, βO-SM, SM or dexamethasone administration. * p < 0.05; ** p < 0.01; *** p < 0.001.