Synthesis of furanotriterpenoids from betulin and evaluation of Tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitory properties of new semi-synthetic triterpenoids.

For the first time, a synthetic route for preparing lupane and oleanane derivatives with a hydrogenated furan ring as a cycle A of triterpene scaffold is described. Most of the synthesized compounds, furanoterpenoids and their synthetic intermediates, were non-toxic against the tested cancer and non-cancerous cell lines, and evinced significant inhibitory activity with IC50 1.0-9.0 µM in the tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibition test. Lupane derivatives - 1-oxime 7, 1,10-seco-hydroxynitrile 11 and furanoterpenoid 14 - were selected as those expected to be the most promising compounds. The results of molecular modeling evinced the strongest binding of compound 11 to the active site of Tdp1 compared to the reference drug. Simultaneously, only compound 11 at subtoxic concentration (10 µM) produced a synergetic effect on the topotecan activity against HeLa-V cells.

Synthesis and Anticancer Activity of A-Ring-Modified Derivatives of Dihydrobetulin.

Multidrug resistance (MDR) is a common phenomenon in clinical oncology, whereby cancer cells become resistant to chemotherapeutic drugs. A common MDR mechanism is the overexpression of ATP-binding cassette efflux transporters in cancer cells, with P-glycoprotein (P-gp) being one of them. New 3,4-seco-lupane triterpenoids, and the products of their intramolecular cyclization with the removed 4,4-gem-dimethyl group, were synthesized by the selective transformations of the A-ring of dihydrobetulin. Among the semi-synthetic derivatives, the MT-assay-enabled methyl ketone 31 (MK), exhibiting the highest cytotoxicity (0.7-16.6 µM) against nine human cancer cell lines, including P-gp overexpressing subclone HBL-100/Dox, is identified. In silico, MK has been classified as a potential P-gp-inhibitor; however, the Rhodamine 123 efflux test, and the combined use of P-gp-inhibitor verapamil with MK in vitro, showed the latter to be neither an inhibitor nor a substrate of P-gp. As the studies have shown, the cytotoxic effect of MK against HBL-100/Dox cells is, arguably, induced through the activation of the ROS-mediated mitochondrial pathway, as evidenced by the positive Annexin V-FITC staining of apoptotic cells, the cell cycle arrest in the G0/G1 phase, mitochondrial dysfunction, cytochrome c release, and the activation of caspase-9 and -3.

The Molecular Mechanisms of Oleanane Aldehyde-ß-enone Cytotoxicity against Doxorubicin-Resistant Cancer Cells.

Oleanane aldehyde-ß-enone (OA), being the semi-synthetic derivative of the triterpenoid betulin, effectively inhibits the proliferation of HBL-100 and K562 cancer cells (IC50 0.47-0.53 µM), as well as the proliferation of their resistant subclones with high P-gp expression HBL-100/Dox, K562/i-S9 and K562/i-S9_Dox (IC50 0.45-1.24 µM). A molecular docking study, rhodamine efflux test, synergistic test with Dox, and ABC transporter gene expression were used to investigate the ability of OA to act as a P-gp substrate or inhibitor against Dox-resistant cells. We noted a trend toward a decrease in ABCB1, ABCC1 and ABCG2 expression in HBL-100 cells treated with OA. The in silico and in vitro methods suggested that OA is neither a direct inhibitor nor a competitive substrate of P-gp in overexpressing P-gp cancer cells. Thus, OA is able to overcome cellular resistance and can accumulate in Dox-resistant cells to realize toxic effects. The set of experiments suggested that OA toxic action can be attributed to activating intrinsic/extrinsic or only intrinsic apoptosis pathways in Dox-sensitive and Dox-resistant cancer cells, respectively. The cytotoxicity of OA in resistant cells is likely mediated by a mitochondrial cell death pathway, as demonstrated by positive staining with Annexin V-FITC, an increasing number of cells in the subG0/G1 phase, reactive oxygen species generation, mitochondrial dysfunction, cytochrome c migration and caspases-9,-6 activation.

Synthesis, cytotoxic evaluation, and molecular docking studies of the semi-synthetic "triterpenoid-steroid" hybrids.

Synthetic transformations of steroids for drug discovery and improvement of drug effectiveness have been an important part of modern medicinal chemistry and pharmaceutical sciences. Pentacyclic triterpenoids, being represented in the nature by various structures and biogenetically related to steroids, can largely expand the spectrum of biologically active steroidal agents via synthesis of the so-called "triterpenoid-steroid" hybrids. In the presented work, the nitrile anion cyclizations of 3,4-secolupane and 3,4-seco-oleanane nitriles and follow-up synthetic transformations of the cyclized products with formation of the gemm-dimethyl-free A ring "triterpenoid-steroid" hybrids were studied. Furthermore, the resulting cyclic compounds were modified at C3, C4, and/or C5 positions of ring A, as well as at C20, C28, and C30 positions of the isopropylidene moiety in the case of lupane triterpenoids. The cytotoxic effect of the synthesized compounds against seven cancer cell lines HEp-2, HCT 116, MS, RD TE32, A549, MCF7, and PC3 was evaluated. The in silico identification of potential anticancer protein targets with regard to the compounds, which were active at micromolar concentrations against tested cell lines, was carried out. The molecular docking studies showed that compound 19, which demonstrated most pronounced cytotoxicity (IC50 0.64-3.17 µM) against all tested cell lines, fits well the active sites of CDK6 and HER2/neu.

Synthesis of cytotoxically active derivatives based on alkylated 2,3-seco-triterpenoids.

Extremely low content of biologically active triterpenoids with the fragmented or contracted ring A extractable from plants is the main disadvantage of their use in drug discovery and practical pharmacology. Development of new methods for synthesis of these compounds and their structural analogs from bioavailable triterpene precursors gives an opportunity to obtain promising agents for pharmacology with excellent yields. A new approach to synthesis of alkylated A-seco-triterpenoids, including the Beckmann fragmentation of 3-methyl-substituted allobetulin or betulinic acid methyl ester with 2-hydroxyimino group in the ring A was proposed. These compounds were used to prepare a series of 2,3-seco- and five-membered ring A lupane and oleanane derivatives, cytotoxicity of which was screened in vitro against the cancer (HEp-2, HCT 116, A549, RD TE32, MS) and non-cancerous (HEK 293) cell lines. Methyl 3-bromomethyl-1-cyano-3-oxo-2,3-seco-2-norlup-20(29)-en-30-al-28-oate was selected as the most active compound (IC50 3.4-10.4 µM for HEp-2, HCT 116, RD TE32, MS cells) capable of triggering caspase-8-mediated apoptosis in HCT 116 cells accompanied by typical apoptotic chromatin condensation, without any loss of mitochondrial membrane permeability.

Preparation of novel ring-A fused azole derivatives of betulin and evaluation of their cytotoxicity.

An efficient scheme to synthesize novel ring-A fused heterocyclic derivatives of betulin was developed. The starting reaction of this synthesis was one-pot selective bacterial oxidation of betulin to betulone used as the key compound to synthesize the substituted azoles such as C(2)-C(3)-fused 1,2,3-triazoles, oxazoles and 1,2,4-triazine, as well as C(1)-C(2)-fused isoxazoles. The semi-synthetic compounds were screened for their cytotoxic activity against human cancer cell lines A549, HCT 116, HEp-2, MS and RD TE32 with use of the photometric MTT assays. Among the tested compounds, N-acetyltriazole of betulin (10) displayed impressive cytotoxic activity with IC50 2.3-7.5 µM against HCT 116, HEp-2, MS and RD TE32 cell lines as well as 3-methyl-4-oxido-1,2,4-triazine-derivative of betulonic acid (12) that was active against HCT 116 and HEp-2 cell lines with IC50 1.4 and 1.5 µM, respectively. Comparative experiments showed triazole (10) to have a lower cytotoxicity to normal epithelial cells, in comparison with compound (12). In accord with the in vivo acute toxicity test, the LD50 of triazole (10) exceeded 600 mg/kg. The ability of the most potent active triazole (10) to trigger apoptotic cell death was explored in the Annexin V-FITC test and by analyzing of caspase activity and morphological alterations in mitochondria and nuclei of HCT 116 cells.

Functionalization, cyclization and antiviral activity of A-secotriterpenoids.

Triterpene derivatives with an α,ß-alkenenitrile moiety in the five-membered ring A have been synthesized by nitrile anion cyclizations of 1-cyano-2,3-secotriterpenoids. Oxime-containing precursors, 2,3-secointermediates and five-membered ring A products of cyclizations were screened for in vitro antiviral activity against enveloped viruses - influenza A virus and human immunodeficiency virus type I (HIV-1). Lupane ketoxime and the 2,3-secolupane C-3 aldoxime which possess antiviral activities against both influenza A virus (EC50 12.9-18.2 µM) and HIV-1 (EC50 0.06 µM) were the most promising compounds.

Synthesis, transformation and biological evaluation of 2,3-secotriterpene acetylhydrazones and their derivatives.

It has been previously shown that semi-synthetic A-secotriterpene acetylhydrazones of 1-cyano-28-methoxy-28-oxo-2,3-seco-2-norlup-20(29)-en-3-al and 1-cyano-2,3-seco-2-nor-19ß,28-epoxy-18αH-olean-3-al (1, 2) inhibit the vesicular stomatitis virus (VSV) replication. To improve the antiviral activity against VSV, structural modifications of compounds 1 and 2 were performed, and new A-secoderivatives containing the acetylhydrazone fragment were obtained from betulonic acid and its methyl ester, allobetulone, and 3-oxo-18ßH-glycyrrhetinic acid methyl ester. The inhibitory effects of these compounds on VSV replication in porcine embryo kidney (PEK) cells were determined after infection. It was shown that introduction of the 3'-acetyl-5'-methyl-1',3',4'-oxadiazoline fragment into lupane triterpene structures lead to an increase in the antiviral activity of A-secotriterpene derivatives. However, the presence of a heterocyclic moiety enhanced toxic activity and reduced the therapeutic indices of these agents. Investigation in the anti-proliferative activity of the heterocyclic derivatives has shown high sensitivity of A-549, MS and RD tumor cell lines to lupane (R)-oxadiazoline 11a. The pro-apoptotic effect of 11a was confirmed by the AnnexinV/PI analysis.