Novel 3'-Substituted-1',2',4'-Oxadiazole Derivatives of 18βH-Glycyrrhetinic Acid and Their O-Acylated Amidoximes: Synthesis and Evaluation of Antitumor and Anti-Inflammatory Potential In Vitro and In Vivo

Abstract

A series of novel 18βH-glycyrrhetinic acid (GA) derivatives containing 3'-(alkyl/phenyl/pyridin(-2″, -3″, and -4″)-yl)-1',2',4'-oxadiazole moieties at the C-30 position were synthesized by condensation of triterpenoid's carboxyl group with corresponding amidoximes and further cyclization. Screening of the cytotoxicity of novel GA derivatives on a panel of tumor cell lines showed that the 3-acetoxy triterpenoid intermediates-O-acylated amidoxime 3a-h-display better solubility under bioassay conditions and more pronounced cytotoxicity compared to their 1',2',4'-oxadiazole analogs 4f-h (median IC₅₀ = 7.0 and 49.7 µM, respectively). Subsequent replacement of the 3-acetoxy group by the hydroxyl group of pyridin(-2″, 3″, and -4″)-yl-1',2',4'-oxadiazole-bearing GA derivatives produced compounds 5f-h, showing the most pronounced selective toxicity toward tumor cells (median selectivity index (SI) > 12.1). Further detailed analysis of the antitumor activity of hit derivative 5f revealed its marked proapoptotic activity and inhibitory effects on clonogenicity and motility of HeLa cervical carcinoma cells in vitro, and the metastatic growth of B16 melanoma in vivo. Additionally, the comprehensive in silico study revealed intermediate 3d, bearing the tert-butyl moiety in O-acylated amidoxime, as a potent anti-inflammatory candidate, which was able to effectively inhibit inflammatory response induced by IFNγ in macrophages in vitro and carrageenan in murine models in vivo, probably by primary interactions with active sites of MMP9, neutrophil elastase, and thrombin. Taken together, our findings provide a basis for a better understanding of the structure-activity relationship of 1',2',4'-oxadiazole-containing triterpenoids and reveal two hit molecules with pronounced antitumor (5f) and anti-inflammatory (3d) activities.

Keywords: 18βH-glycyrrhetinic acid; anti-inflammatory activity; antitumor activity; apoptosis; derivatives; heterocyclic moiety; metastasis; molecular docking; oxadiazole; target prediction.

Figure 1

Structures of semisynthetic derivatives of natural compounds bearing oxadiazole (A,B,D,E,G–J) and amidoxime (C,F) moieties that display promising antitumor activity.

Scheme 1

Synthesis of compounds 3a-h–5a-h. Reagents and conditions: (a) N,N′-carbonyldiimidazole (CDI), CH₂Cl₂, r.t., 2 h; then corresponding amidoxime R-C(=NOH)NH₂ 2a-h, r.t., overnight; (b) tetrabutylammonium fluoride (TBAF), tetrahydrofuran (THF), reflux, 1–2 h; (c) KOH, MeOH, reflux, 1–2 h.

Figure 2

Cytotoxic profiles of novel GA derivatives. (A) Hierarchical clustering of IC₅₀ values of investigated compounds using Euclidean distance. (B) Heatmap illustrating the antitumor selectivity of action of the investigated derivatives. Selectivity index (SI) was calculated as the ratio of IC₅₀ values in normal hFF3 fibroblasts to the IC₅₀ values in corresponding malignant cells.

Figure 3

Compound 5f induces mitochondrial caspase-dependent apoptosis in HeLa cells. (A) Compound 5f causes externalization of phosphatidylserine. HeLa cells were treated by 5f at indicated concentrations for 48 h, stained by annexin V-fluorescein isothiocyanate (annexin V FITC)/ propidium iodide (PI), and analyzed by flow cytometry. (B) Compound 5f stimulates the dissipation of mitochondrial membrane potential in HeLa cells. Tumor cells were treated by 5f as in (A), followed by staining of cells with JC-1 and flow cytometry analysis. (C) Compound 5f activates the caspase cascade. HeLa cells were treated by 5f similarly to (A), followed by labeling of active caspases by fluorescein isothiocyanate (FITC) conjugate of the cell-permeable pancaspase inhibitor VAD-FMK (FITC-VAD-FMK) and flow cytometry analysis. (D) Compound 5f increases the activity of executioner caspases-3/7. HeLa cells were treated by 5f at indicated concentrations for 48 h followed, by measurement of the levels of activity for caspases-3/7 using a Caspase-Glo ^® 3/7 Assay kit (Promega, Madison, WI, USA).

Figure 4

Compound 5f displays significant antimetastatic potential. (A) Compound 5f inhibits the clonogenic activity of HeLa cells. HeLa cells were seeded at a low density and treated by 5f (0.25, 0.5 µM) for 14 days followed by fixation of the cells and their staining with crystal violet. The assessment of the colony formation level was carried out using a ColonyArea ImageJ plugin. (B) Compound 5f inhibits the motility of HeLa cells. HeLa cells were seeded into the upper chamber of the cell invasion/migration (CIM)-Plate and treated by 5f at 0.5 µM for 48 h. FBS-mediated migration of the cells to the lower chamber was analyzed by impedance measurement in real time regimen using the xCELLigence Real-Time Cell Analyzer Dual Plates (RTCA DP) system. (C) Administration of 5f significantly suppresses the metastatic growth of B16 melanoma in vivo. Compound 5f (50 mg/kg) was intraperitoneally injected in B16 melanoma-bearing mice five times. At day 14 after tumor cell transplantation, mice were sacrificed and a number of surface metastases in the lungs were counted. Then, 10% Tween-80 in physiological salt solution was used as the vehicle. (D) Metastatic inhibition indexes (MII) of mice treated by a vehicle or compound 5f. MIIs were calculated relatively to control B16 melanoma-bearing untreated mice. (E) Compound 5f does not cause toxic effects in mice. Liver, kidney, spleen, and thymus indexes of mice in tested groups were calculated relative to the organ indexes of healthy mice.

Figure 5

Compound 3d displays significant dose-dependent anti-inflammatory activity in vitro. (A) The heatmap showed the probability of the anti-inflammatory potential of novel GA derivatives. The bioactivities of compounds were predicted using the PASS Online platform. Inflammation-related terms “anti-inflammatory” (anti-Inflam) and “nitric oxide antagonist” (anti-NO) were showed. Pa and Pi represent the probability of tested compounds “to be active” or “to be inactive”, respectively. (B) Compound 3d is nontoxic to HeLa cells. HeLa cells were incubated in the presence of 3d (10-50 µM) for 24 h followed by analysis of cell viability by MTT assay. (C) Compound 3d dose-dependently inhibits production of TNF-α by IFNγ-stimulated RAW264.7 cells. The cells were co-treated by IFN-γ (20 nM) and 3d (10-50 µM) for 24 h, after which the concentration of TNF-α in culture medium was measured using ELISA. (D) Compound 3d dose-dependently suppresses production of NO through inflamed macrophages. RAW264.7 cells were co-treated by IFN-γ (20 nM) and 3d (10-50 µM) for 24 h, followed by the measurement of nitrite concentration in culture medium by Griess reaction.

Figure 6

Compound 3d effectively inhibits carrageenan-induced inflammation in vivo. (A) Compound 3d suppresses carrageenan-induced paw edema in mice. Compound 3d (50 mg/kg) was administered in sesame oil (vehicle) to mice via gastric gavage 1 h prior to carrageenan (CRG) injection, followed by the measurement of edema weight 5 h after induction of paw inflammation. Indomethacin (IM, 20 mg/kg) was used as a reference drug. (B) Histology of healthy footpads and footpads with carrageenan-induced paw edema after administration of 3d, IM, and vehicle. Hematoxylin and eosin staining. Original magnification of ×100 (top panel) and ×400 (bottom panel). The arrows indicate inflammatory infiltration of paw tissues. Black boxes show areas that were examined further at a higher magnification. (C) Compound 3d inhibits carrageenan-induced peritonitis in mice. Compound 3d (50 mg/kg) in 10% Tween-80 (vehicle) was intraperitoneally injected into mice 1 h prior to peritonitis induction, followed by the counting of leukocytes in peritoneal fluid. Control–uninflamed mice injected only by saline. Dexamethasone (DEX, 1 mg/kg) was used as a reference drug. (D) Compound 3d blocks neutrophil migration into carrageenan-inflamed peritoneum. The distribution of leukocyte subpopulations was measured by microscopy after staining of peritoneal cells with azur–eosin by Romanovsky–Giemsa.

Figure 7

Probable primary protein targets of compound 3d and their association with inflammatory response in mice. Probable targets of 3d listed in a table were predicted using Naïve–Bayes machine learning together with nearest neighbor (NN) search in Polypharmacology Browser 2.0. The protein–protein interaction network was reconstructed with rodent inflammatome-associated genes and revealed probable targets of 3d by using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (confidence score ≥0.7) in Cytoscape.

Figure 8

MMP9, neutrophil elastase (ELANE), and F2 are probable primary targets of compound 3d. (A) Binding energies of 3d and known inhibitors, with the top 5 probable targets of 3d being the most associated with rodent inflammatome. The binding energies were calculated by Autodock Vina. (B) The mode of binding of 3d to MMP9, neutrophil elastase (ELANE), and thrombin (F2). Stereo presentations of the docked pose of 3d in the mentioned proteins superimposed on corresponding inhibitor-bound structures were created using BIOVIA Discovery Studio. Structures of 3d and inhibitors are depicted in blue and yellow sticks, respectively. The 2D representations of docked poses of 3d in MMP9, ELANE, and F2 were depicted using LigPlot+. The green lines and combs represent hydrogen bonds and hydrophobic interactions, respectively. Common amino acid residues, interacting with both 3d and corresponding inhibitors, are highlighted in red circles.