Antiproliferative and Cytotoxic Properties of Propynoyl Betulin Derivatives against Human Ovarian Cancer Cells: In Vitro Studies.

Due to the incidence of ovarian cancer (OC) and the limitations of available therapeutic strategies, it is necessary to search for novel therapeutic solutions. The aim of this study was to evaluate the cytotoxic effect of betulin 1 and its propynoyl derivatives 2-6 against ovarian cancer cells (SK-OV-3, OVCAR-3) and normal myofibroblasts (18Co). Paclitaxel was used as the reference compound. The propynoyl derivatives 2-6 exhibited stronger antiproliferative and cytotoxic activities compared to betulin 1. In both ovarian cancer cell lines, the most potent compound was 28-propynoylbetulin 2. In the case of compound 2, the calculated IC50 values were 0.2 µM for the SK-OV-3 cells and 0.19 µM for the OVCAR-3 cells. Under the same culture conditions, the calculated IC50 values for compound 6 were 0.26 µM and 0.59 µM, respectively. It was observed that cells treated with compounds 2 and 6 caused a decrease in the potential of the mitochondrial membrane and a significant change in cell morphology. Betulin 1, a diol from the group of pentacyclic triterpenes, has a confirmed wide spectrum of biological effects, including a significant anticancer effect. It is characterized by low bioavailability, which can be improved by introducing changes to its structure. The results showed that chemical modifications of betulin 1 only at position C-28 with the propynoyl group (compound 2) and additionally at position C-3 with the phosphate group (compound 3) or at C-29 with the phosphonate group (compound 6) allowed us to obtain compounds with greater cytotoxic activity than their parent compounds, which could be used to develop novel therapeutic systems effective in the treatment of ovarian cancer.

Design, Synthesis and Biological Evaluation of Quinoline-8-Sulfonamides as Inhibitors of the Tumor Cell-Specific M2 Isoform of Pyruvate Kinase: Preliminary Study.

Cancer cells need to carefully regulate their metabolism to keep them growing and dividing under the influence of different nutrients and oxygen levels. Muscle isoform 2 of pyruvate kinase (PKM2) is a key glycolytic enzyme involved in the generation of ATP and is critical for cancer metabolism. PKM2 is expressed in many human tumors and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various modulators regulate PKM2, shifting it between highly active and less active states. In the presented work, a series of 8-quinolinesulfonamide derivatives of PKM2 modulators were designed using molecular docking and molecular dynamics techniques. New compounds were synthesized using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Compound 9a was identified in in silico studies as a potent modulator of muscle isoform 2 of pyruvate kinase. The results obtained from in vitro experiments confirmed the ability of compound 9a to reduce the intracellular pyruvate level in A549 lung cancer cells with simultaneous impact on cancer cell viability and cell-cycle phase distribution. Moreover, compound 9a exhibited more cytotoxicity on cancer cells than normal cells, pointing to high selectivity in the mode of action. These findings indicate that the introduction of another quinolinyl fragment to the modulator molecule may have a significant impact on pyruvate levels in cancer cells and provides further directions for future research to find novel analogs suitable for clinical applications in cancer treatment.

The Influence of Betulin and Its Derivatives EB5 and ECH147 on the Antioxidant Status of Human Renal Proximal Tubule Epithelial Cells.

Betulin and its derivatives, 28-propyne derivative EB5 and 29-diethyl phosphonate analog ECH147, are promising compounds in anti-tumor activity studies. However, their effect on kidney cells has not yet been studied. The study aimed to determine whether betulin and its derivatives-EB5 and ECH147-influence the viability and oxidative status of human renal proximal tubule epithelial cells (RPTECs). The total antioxidant capacity of cells (TEAC), lipid peroxidation product malondialdehyde (MDA) level, and activity of antioxidant enzymes (SOD, CAT, and GPX) were evaluated. Additionally, the mRNA level of genes encoding antioxidant enzymes was assessed. Cisplatin and 5-fluorouracil were used as reference substances. Betulin and its derivatives affected the viability and antioxidant systems of RPTECs. Betulin strongly reduced TEAC in a concentration-dependent manner. All tested compounds caused an increase in MDA levels. The activity of SOD, CAT, and GPX, and the mRNA profiles of genes encoding antioxidant enzymes depended on the tested compound and its concentration. Betulin showed an cisplatin-like effect, indicating its nephrotoxic potential. Betulin derivatives EB5 and ECH147 showed different impacts on the antioxidant system, which gives hope that these compounds will not cause severe consequences for the kidneys in vivo.

Design, synthesis and biological activity of 1,4-quinone moiety attached to betulin derivatives as potent DT-diaphorase substrate.

In this research, betulin derivatives were bonded to the 1,4-quinone fragment by triazole linker. Furthermore, the enzymatic assay used has shown that these compounds are a good DT-diaphorase (NQO1) substrates as evidenced by increasing enzymatic conversion rates relative to that of streptonigrin. The anticancer activities of the hybrids were tested against a panel of human cell lines, like: melanoma, ovarian, breast, colon, and lung cancers. The structure-activity relationship showed that the activity depends on the type of 1,4-quinone moiety and the tumor cell lines used. It was also found that the anticancer effects were increasing against the cell line with higher NQO1 protein level, like: breast (T47D, MCF-7), colon (Caco-2), and lung (A549) cancers. The transcriptional activity of the gene encoding a proliferation marker (H3 histone), cell cycle regulators (p53 and p21) and apoptosis pathway (BCL-2 and BAX) for selected compounds were determined. The molecular docking study was carried out to examine the interaction between the hybrids and NQO1 enzyme. The computational simulation showed that the type of the 1,4-quinone moiety influences location of the compound in the active site of the enzyme. It is worth noting that the study of new hybrids of betulin as substrate for NQO1 protein may lead to new medical therapeutic applications in the future.

Acetylenic Synthetic Betulin Derivatives Inhibit Akt and Erk Kinases Activity, Trigger Apoptosis and Suppress Proliferation of Neuroblastoma and Rhabdomyosarcoma Cell Lines.

Neuroblastoma (NB) and rhabdomyosarcoma (RMS), the most common pediatric extracranial solid tumors, still represent an important clinical challenge since no effective treatment is available for metastatic and recurrent disease. Hence, there is an urgent need for the development of new chemotherapeutics to improve the outcome of patients. Betulin (Bet), a triterpenoid from the bark of birches, demonstrated interesting anti-cancer potential. The modification of natural phytochemicals with evidenced anti-tumor activity, including Bet, is one of the methods of receiving new compounds for potential implementation in oncological treatment. Here, we showed that two acetylenic synthetic Bet derivatives (ASBDs), EB5 and EB25/1, reduced the viability and proliferation of SK-N-AS and TE671 cells, as measured by MTT and BrdU tests, respectively. Moreover, ASBDs were also more cytotoxic than temozolomide (TMZ) and cisplatin (cis-diaminedichloroplatinum [II], CDDP) in vitro, and the combination of EB5 with CDDP enhanced anti-cancer effects. We also showed the slowdown of cell cycle progression at S/G2 phases mediated by EB5 using FACS flow cytometry. The decreased viability and proliferation of pediatric cancers cells after treatment with ASBDs was linked to the reduced activity of kinases Akt, Erk1/2 and p38 and the induction of apoptosis, as investigated using Western blotting and FACS. In addition, in silico analyses of the ADMET profile found EB5 to be a promising anti-cancer drug candidate that would benefit from further investigation.

Anticancer Activity of the Acetylenic Derivative of Betulin Phosphate Involves Induction of Necrotic-Like Death in Breast Cancer Cells In Vitro.

Betulin (BT) is a natural pentacyclic lupane-type triterpene exhibiting anticancer activity. Betulin derivatives bearing propynoyloxy and phosphate groups were prepared in an effort to improve the availability and efficacy of the drug. In this study, a comparative assessment of the in vitro anticancer activity of betulin and its four derivatives was carried out using two human breast cancer cell lines: SK-BR-3 and MCF-7. In both studied cell lines, 30-diethoxyphosphoryl-28-propynoylbetulin (compound 4) turned out to be the most powerful inhibitor of growth and inducer of cellular death. Detailed examination of that derivative pertained to the mechanisms underlying its anticancer action. Treatment with compound 4 decreased DNA synthesis and up-regulated p21WAF1/Cip1 mRNA and protein levels in both cell lines. On the other hand, that derivative caused a significant increase in cell death, as evidenced by increased lactate dehydrogenase (LDH) release and ethidium homodimer uptake. Shortly after the compound addition, an increased generation of reactive oxygen species and loss of mitochondrial membrane potential were detected. The activation of caspase-3 and fragmentation of genomic DNA suggested an apoptotic type of cell death. However, analysis of cellular morphology did not reveal any nuclear features typical of apoptosis. Despite necrosis-like morphology, dead cells exhibited activation of the cascade of caspases. These observations have led to the conclusion that compound 4 pushed cells to undergo a form of necrotic-like regulated cell demise.

Molecular Structure, In Vitro Anticancer Study and Molecular Docking of New Phosphate Derivatives of Betulin.

A series of 30-diethylphosphate derivatives of betulin were synthesized and evaluated for their in vitro cytotoxic activity against human cancer cell lines, such as amelanotic melanoma (C-32), glioblastoma (SNB-19), and two lines of breast cancer (T47D, MDA-MB-231). The molecular structure and activities of the new compounds were also compared with their 29-phosphonate analogs. Compounds 7a and 7b showed the highest activity against C-32 and SNB-19 cell lines. The IC50 values for 7a were 2.15 and 0.91 µM, and, for 7b, they were 0.76 and 0.8 µM for the C-32 and SNB-19 lines, respectively. The most potent compounds, 7a and 7b, were tested for their effects on markers of apoptosis, such as H3, TP53, BAX, and BCL-2. For the whole series of phosphate derivatives, a lipophilicity study was performed, and the ADME parameters were calculated. The most active products were docked to the active site of the EGFR protein. The relative binding affinity of selected phosphate betulin derivatives toward EGFR was compared with standard erlotinib on the basis of ChemScore and KDEEP score. Positively, all derivatives docked inside the cavity and showed significant interactions. Moreover, a molecular dynamics study also reveals that ligands 7a,b form stable complexes and the plateau phase started after 7 ns.

New phosphate derivatives of betulin as anticancer agents: Synthesis, crystal structure, and molecular docking study.

Betulin derivatives exhibit an antiproliferative activity and have been tested for many cancer cell lines. This paper describes a new series of 3-phosphate derivatives of betulin bearing different substituents at C28 position. The synthesized compounds were tested in vitro for their antiproliferative effect against human leukemia (MV-4-11 and CCRF/CEM), lung carcinoma (A549), prostate cancer (DU 145), melanoma (Hs 294T) cell lines, and murine leukemia P388. To explore the possible mechanism of anticancer activity for the most in vitro active compounds (4, 5, 7 and 8) and betulin, molecular docking was performed to the binding sites of potential anticancer targets, described for the various triterpene derivatives, including topoisomerase I and II, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGFR), transcription factor NF-κB, anti-apoptotic protein Bcl-2 and peroxisome proliferator-activated receptor (PPARγ). According to the results of the docking, the best fit to the binding pocket of PPARγ was shown by compound 4.

Biological Activity and In Silico Study of 3-Modified Derivatives of Betulin and Betulinic Aldehyde.

A series of 3-substituted derivatives of betulin and betulinic aldehyde were synthesized as promising anticancer agents. The newly triterpenes were tested against five human cancer cell lines like biphenotypic B myelomonocytic leukaemia (MV-4-11), adenocarcinoma (A549), prostate (Du-145), melanoma (Hs294T), breast adenocarcinoma (MCF-7) and normal human mammary gland (MCF-10A). The compound 9 showed towards Du-145, MCF-7 and Hs294T cells significant antiproliferative activity with IC50 ranging from 7.3 to 10.6 µM. The evaluation of ADME properties of all compounds also includes their pharmacokinetic profile. The calculated TPSA values for synthetized derivatives are in the range between 43.38 Ų and 55.77 Ų suggesting high oral bioavailability. The molecular docking calculations showed that triterpene 9 fits the active site of the serine/threonine protein kinase Akt.

New Phosphorus Analogs of Bevirimat: Synthesis, Evaluation of Anti-HIV-1 Activity and Molecular Docking Study.

Since the beginning of the human immunodeficiency virus (HIV) epidemic, many groups of drugs characterized by diverse mechanisms of action have been developed, which can suppress HIV viremia. 3-O-(3',3'-Dimethylsuccinyl) betulinic acid, known as bevirimat (BVM), was the first compound in the class of HIV maturation inhibitors. In the present work, phosphate and phosphonate derivatives of 3-carboxyacylbetulinic acid were synthesized and evaluated for anti-HIV-1 activity. In vitro studies showed that 30-diethylphosphonate analog of BVM (compound 14a) has comparable effects to BVM (half maximal inhibitory concentrations (IC50) equal to 0.02 µM and 0.03 µM, respectively) and is also more selective (selectivity indices: 3450 and 967, respectively). To investigate the possible mechanism of antiviral effect of 14a, molecular docking was carried out on the C-terminal domain (CTD) of HIV-1 capsid (CA)-spacer peptide 1 (SP1) fragment of Gag protein, designated as CTD-SP1, which was described as a molecular target for maturation inhibitors. Compared with interactions between BVM and the protein, an increased number of strong interactions between ligand 14a and protein, generated by the phosphonate group, was observed.

5,8-Quinolinedione Scaffold as a Promising Moiety of Bioactive Agents.

Natural 5,8-quinolinedione antibiotics exhibit a broad spectrum of activities including anticancer, antibacterial, antifungal, and antimalarial activities. The structure-activity research showed that the 5,8-quinolinedione scaffold is responsible for its biological effect. The subject of this review report is a presentation of the pharmacological activity of synthetic 5,8-quinolinedione compounds containing different groups at C-6 and/or C-7 positions. The relationship between the activity and the mechanism of action is included if these data have been included in the original literature. The review mostly covers the period between 2000 and 2019. Previously published literature data were used to present historical points.

Novel triazoles of 3-acetylbetulin and betulone as anticancer agents.

The CuAAC reaction of azides and acetylenic triterpenes was used for synthesis of new triazoles of 3-acetylbetulin and betulone. The triazole derivatives were evaluated for their anticancer activity in vitro against amelanotic melanoma C-32, ductal carcinoma T47D and glioblastoma SNB-19 cell lines. 28-[1-(3'-Deoxythymidine-5'-yl)-1H-1,2,3-triazol-4-yl]carbonylbetulone 6e exhibited a significant IC50 value (0.17 µM) against the human glioblastoma SNB-19 cell line, an almost 5-fold higher potency while compared with reference cisplatin.

Alkoxy and Enediyne Derivatives Containing 1,4-Benzoquinone Subunits-Synthesis and Antitumor Activity.

The compounds produced by a living organism are most commonly as medicinal agents and starting materials for the preparation of new semi-synthetic derivatives. One of the largest groups of natural compounds consists of products containing a 1,4-benzoquinone subunit. This fragment occurs in three enediyne antibiotics, dynemicin A, deoxydynemicin A, and uncilamicin, which exhibit high biological activity. A series of alkoxy derivatives containing 1,4-naphthoquinone, 5,8-quinolinedione, and 2-methyl-5,8-quinolinedione moieties was synthesized. Moreover, the 1,4-benzoquinone subunit was contacted with an enediyne fragment. All obtained compounds were characterized by spectroscopy and spectrometry methods. The resulting alkane, alkene, alkyne and enediyne derivatives were tested as antitumor agents. They showed high cytotoxic activity depending on the type of 1,4-benzoquinone subunit and the employed tumor cell lines. The synthesized derivatives fulfill the Lipinski Rule of Five and have low permeability through the blood-brain barrier.

Synthesis, Anti-Breast Cancer Activity, and Molecular Docking Study of a New Group of Acetylenic Quinolinesulfonamide Derivatives.

In this study, a series of regioisomeric acetylenic sulfamoylquinolines are designed, synthesized, and tested in vitro for their antiproliferative activity against three human breast cacer cell lines (T47D, MCF-7, and MDA-MB-231) and a human normal fibroblast (HFF-1) by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. The antiproliferative activity of the tested acetylenic quinolinesulfonamides is comparable to that of cisplatin. The bioassay results demonstrate that most of the tested compounds show potent antitumor activities, and that some compounds exhibit better effects than the positive control cisplatin against various cancer cell lines. Among these compounds, 4-(3-propynylthio)-7-[N-methyl-N-(3-propynyl)sulfamoyl]quinoline shows significant antiprolierative activity against T47D cells with IC50 values of 0.07 µM. In addition, 2-(3-Propynylthio)-6-[N-methyl-N-(3-propynyl)sulfa-moyl]quinoline and 2-(3-propynylseleno)-6-[N-methyl-N-(3-propynyl)sulfamoyl]quinoline display highly effective atitumor activity against MDA-MB-231 cells, with IC50 values of 0.09 and 0.50 µM, respectively. Furthermore, most of the tested compounds show a weak cytotoxic effect against the normal HFF-1 cell line. Additionally, in order to suggest a mechanism of action for their activity, all compounds are docked into the binding site of two human cytochrome P450 (CYP) isoenzymes. These data indicate that some of the title compounds display significant cytotoxic activity, possibly targeting the CYPs pathways.

Novel Triazole Hybrids of Betulin: Synthesis and Biological Activity Profile.

Betulin derivatives containing a 1,2,3-triazole ring possess a wide spectrum of biological activities, including antiviral, anticancer, and antibacterial activity. A series of novel triazoles were prepared by the 1,3-dipolar cycloaddition reaction between the alkyne derivatives of betulin and organic azides. The chemical structures of the obtained compounds were defined by ¹H and 13C NMR, IR, and high-resolution mass spectrometry (HR-MS) analysis. The target triazoles were screened for their antiviral activity against DNA and RNA viruses. The cytotoxic activity of the obtained compounds 5a-k and 6a-h was determined using five human cancer cell lines (T47D, MCF-7, SNB-19, Colo-829, and C-32) by a WST-1 assay. The bistriazole 6b displayed a promising IC50 value (0.05 µM) against the human ductal carcinoma T47D (500-fold higher potency than cisplatin). The microdilution method was applied for an evaluation of the antimicrobial activity of all of the compounds. The triazole 5e containing a 3'-deoxythymidine-5'-yl moiety exhibited antibacterial activity against two gram-negative bacteria vz. Klebsiellapneumoniae and Escherichia coli (minimal inhibitory concentration (MIC) range of 0.95-1.95 µM).

New acetylenic derivatives of betulin and betulone, synthesis and cytotoxic activity.

Betulin 1 and its semisynthetic derivatives exhibit a cytotoxic activity toward various cancer cell lines. These compounds are a promising and potential anticancer candidates. A series of betulin derivatives was prepared and tested for the antiproliferative activity in vitro against T47D breast cancer, CCRF/CEM leukemia, HL-60 promyelocytic leukemia, SW707 colorectal, murine P388 leukemia, as well as BALB3T3 normal fibroblasts cell lines. Cisplatin and betulin 1 were used as a reference compounds. Some derivatives of betulin showed a higher cytotoxic activity than the parent compound 1. Two derivatives (5 and 17) were 24-fold potent than betulin 1 against the human promyelocytic leukemia cell line (HL-60), with an IC50 value of 0.3 µg/mL.

Synthesis, Structure and Cytotoxic Activity of Mono- and Dialkoxy Derivatives of 5,8-Quinolinedione.

A series of 5,8-quinolinedione derivatives containing one or two alkoxy groups was synthesized and characterized by ¹H- and (13)C-NMR, IR and MS spectra. X-ray diffraction was used to investigate the crystal structures of 6-chloro-7-(2-cyjanoethoxy)-5,8-quinolinedione and 6,7-di(2,2,2-trifloroethoxy)-5,8-quinolinedione. All studied compounds were tested in vitro for their antiproliferative activity against three human cancer cell lines and human normal fibroblasts. Most of the compounds showed higher cytotoxicity than the starting compound, 6,7-dichloro-5,8-quinolinedione, and cisplatin, which was used as a reference agent.

Betulin Phosphonates; Synthesis, Structure, and Cytotoxic Activity.

Betulin derivatives are a widely studied group of compounds of natural origin due to their wide spectrum of biological activities. This paper describes new betulin derivatives, containing a phosphonate group. The allyl-vinyl isomerization and synthesis of acetylenic derivatives have been reported. Structural identification of products as E and Z isomers has been carried out using ¹H-, (13)C-, (31)P-NMR, and crystallographic analysis. The crystal structure in the orthorhombic space group and analysis of crystal packing contacts for 29-diethoxyphosphoryl-28-cyclopropylpropynoyloxy-lup-20E(29)-en-3ß-ol 8a are reported. All new compounds were tested in vitro for their antiproliferative activity against human T47D (breast cancer), SNB-19 (glioblastoma), and C32 (melanoma) cell lines.

Influence of 28-O-propynoylbetulin on proliferation and apoptosis of melanotic and amelanotic human melanoma cells.

INTRODUCTION: A relatively new approach in treatment of malignant melanoma is the use of betulin and its synthetic derivatives that have anticancer properties. The aim of the study was to determine the effect of an acetylenic derivative of betulin, 28-O-propynoylbetulin, on cell growth and apoptosis induction in human melanotic and amelanotic melanoma cells. MATERIALS AND METHODS: The A2058 and C32 cell lines were incubated with 28-O-propynoylbetulin (working solutions from 0.1 to 10 µg/ml). To evaluate cell proliferation, a sulforhodamine B based assay was conducted. In order to elucidate the early stages of apoptosis in both melanoma cell lines, caspase-3 activity was evaluated. RESULTS: The administration of 28-O-propynoylbetulin at a concentration equal to or less than 1 µg/ml did not cause a statistically significant change in the cell proliferation in either melanoma cell line (compared to control, p>0.05). Higher concentrations of the compound (3 and 10 µg/ml) inhibited the cell growth (in comparison to control, p<0.05). These results corresponded with caspase-3 activity results that revealed an increase of enzyme activity after 24-hour incubation with 3 and 10 µg/ml of the compound (compared to control, p<0.05). DISCUSSION: The study revealed that 28-O-propynoylbetulin may have diverse effects on melanoma cells and could be a strong inhibitor of cell growth (C32 cells) or exert a more potent proapoptotic effect (A2058 cells). These findings support the possibility of the use of EB5 in different antimelanoma approaches.

ANTIPROLIFERATIVE ACTIVITY OF NOVEL ACETYLENIC DERIVATIVES OF BETULIN AGAINST G-361 HUMAN MELANOMA CELLS.

Acetylenic derivatives of betulin were tested in vitro for their antiproliferative activity against G-361 human melanoma cells. Two types of betulin derivatives were studied: monoesters, obtained by modification of the hydroxyl group at C-28 position, and diesters modified at both C-28 and C-3 positions. To assess cell proliferation, a colorimetric sulforhodamine B based method was used. All the tested monoesters inhibited cellular growth and 28-O-propynoylbetulin showed the strongest cytotoxic effect. Esterification of the C-3 hydroxyl group of the molecule abolished its growth inhibitory activity.