The Influence of Betulin Derivatives EB5 and ECH147 on the Expression of Selected TGFß Superfamily Genes, TGFß1, GDF15 and BMP2, in Renal Proximal Tubule Epithelial Cells.

Betulin derivatives are proposed to serve as an alternative to the drugs already established in oncologic treatment. Drug-induced nephrotoxicity leading to acute kidney injury frequently accompanies cancer treatment, and thus there is a need to research the effects of betulin derivatives on renal cells. The objective of our study was to assess the influence of the betulin derivatives 28-propynylobetulin (EB5) and 29-diethoxyphosphoryl-28-propynylobetulin (ECH147) on the expression of TGFß1, BMP2 and GDF15 in renal proximal tubule epithelial cells (RPTECs) cultured in vitro. The changes in mRNA expression and copy numbers were assessed using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and the standard curve method, respectively. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the effect of the betulin derivatives on the protein concentration in the culture media's supernatant. The assessment of the betulin derivatives' influence on gene expression demonstrated that the mRNA level and protein concentration did not always correlate with each other. Each of the tested compounds affected the mRNA expression. The RT-qPCR analyses showed that EB5 and ECH147 induced effects similar to those of betulin or cisplatin and resulted in a decrease in the mRNA copy number of all the analyzed genes. The ELISA demonstrated that EB5 and ECH147 elevated the protein concentration of TGFß1 and GDF15, while the level of BMP2 decreased. The concentration of the derivatives used in the treatment was crucial, but the effects did not always exhibit a simple linear dose-dependent relationship. Betulin and its derivatives, EB5 and ECH147, influenced the gene expression of TGFß1, BMP2 and GDF15 in the renal proximal tubule epithelial cells. The observed effects raise the question of whether treatment with these compounds could promote the development of renal fibrosis.

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.

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.

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.

Design and Synthesis of Novel Betulin Derivatives Containing Thio-/Semicarbazone Moieties as Apoptotic Inducers through Mitochindria-Related Pathways.

Two new series of betulin derivatives with semicarbazone (7a-g) or thiosemicarbazone (8a-g) groups at the C-28 position were synthesized. All compounds were evaluated for their in vitro cytotoxicities in human hepatocellular carcinoma cells (HepG2), human breast carcinoma cells (MCF-7), human lung carcinoma cells (A549), human colorectal cells (HCT-116) and normal human gastric epithelial cells (GES-1). Among these compounds, 8f displayed the most potent cytotoxicity with an IC50 value of 5.86 ± 0.61 µM against MCF-7 cells. Furthermore, the preliminary mechanism studies in MCF-7 cells showed that compound 8f could trigger the intracellular mitochondrial-mediated apoptosis pathway by losing MMP level, which was related with the upregulation of Bax, P53 and cytochrome c expression; the downregulation of Bcl-2 expression; activation of the expression levels of caspase-3, caspase-9, cleaved caspase-3 and cleaved caspase-9; and an increase in the amounts of intracellular reactive oxygen species. These results indicated that compound 8f may be used as a valuable skeleton structure for developing novel antitumor agents.

Enhancement of the Antioxidant and Skin Permeation Properties of Betulin and Its Derivatives.

This study investigated the antioxidant activity DPPH, ABTS, and Folin-Ciocalteu methods of betulin (compound 1) and its derivatives (compounds 2-11). Skin permeability and accumulation associated with compounds 1 and 8 were also examined. Identification of the obtained products (compound 2-11) and betulin isolated from plant material was based on the analysis of 1H- NMR and 13C-NMR spectra. The partition coefficient was calculated to determine the lipophilicity of all compounds. In the next stage, the penetration through pig skin and its accumulation in the skin were evaluated of ethanol vehicles containing compound 8 (at a concentration of 0.226 mmol/dm3), which was characterized by the highest antioxidant activity. For comparison, penetration studies of betulin itself were also carried out. Poor solubility and the bioavailability of pure compounds are major constraints in combination therapy. However, we observed that the ethanol vehicle was an enhancer of skin permeation for both the initial betulin and compound 8. The betulin 8 derivative showed increased permeability through biological membranes compared to the parent betulin. The paper presents the transformation of polycyclic compounds to produce novel derivatives with marked antioxidant activities and as valuable intermediates for the pharmaceutical industry. Moreover, the compounds contained in the vehicles, due to their mechanism of action, can have a beneficial effect on the balance between oxidants and antioxidants in the body, minimizing the effects of oxidative stress. The results of this work may contribute to knowledge regarding vehicles with antioxidant potential. The use of vehicles for this type of research is therefore justified.

Leishmanicidal Activity of Betulin Derivatives in Leishmania amazonensis; Effect on Plasma and Mitochondrial Membrane Potential, and Macrophage Nitric Oxide and Superoxide Production.

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. METHODS: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. RESULTS: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. CONCLUSION: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.

Synthetic Betulin Derivatives Inhibit Growth of Glioma Cells In Vitro.

BACKGROUND/AIM: Glioma is the most malignant tumour of the human brain still lacking effective treatment modalities. Betulin, a pentacyclic triterpene abundantly found in the birch bark, has been shown to demonstrate interesting anti-cancer activity towards many cancer cells. We determined the effects of acetylenic synthetic betulin derivatives (ASBDs) as anti-tumour agents on glioma cells in vitro. MATERIALS AND METHODS: T98G and C6 glioma cell viability and proliferation were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and BrdU (bromo deoxyuridine) test, respectively. Cell-cycle progression and induction of apoptosis were investigated with flow cytometry. RESULTS: ASBDs significantly decreased glioma cell viability/survival and inhibited proliferation in a dose-dependent manner in vitro. Moreover, ASBDs were more cytotoxic than clinically used chemotherapeutics - temozolomide and cisplatin. CONCLUSION: ASBDs may be considered for further study as potent anti-tumour agents in glioma treatment.

Determination of binding constants for strong complexation by affinity capillary electrophoresis: the example of complexes of ester betulin derivatives with (2-hydroxypropyl)-γ-cyclodextrin.

Complexation plays an important role in many biological phenomena, the analysis of different samples, optimization of separation processes, and increasing the pharmacological activity of drugs. This paper discusses the features of using mobility shift affinity capillary electrophoresis for studying strong complexation. Electrophoretic peaks for this case are often triangular. It was shown that the use of electrophoretic mobility obtained from the peak apex time to calculate binding constants leads to significant systematic and random errors, and the parameter a1 of the Haarhoff-Van der Linde function should be used instead of the apex time. Distorted triangular peaks with dips were shown to be observed at too high a ratio of analyte concentration in the sample to ligand concentration in the background electrolyte, and the peaks and parameter a1 significantly shifted. It was found that the permissible excess of analyte concentration over ligand concentration was approximately 10-35, provided that the parameter a1 was used, but the peak shape should be used as a landmark, and only triangular peaks without dips should be fitted with the function. The lowest possible analyte concentration should be utilized, which allows the use of a wider range of ligand concentration leading to higher precision of determining the binding constants values. Kinetically labile 1:1 complexes between (2-hydroxypropyl)-γ-cyclodextrin (HP-γ-CD) and betulin 3,28-diphthalate (DPhB) and betulin 3,28-disuccinate (DScB) were studied as an example. The binding constant logarithms at 25 °C are 7.23 ± 0.03 and 7.13 ± 0.10 for the HP-γ-CD complexes of DPhB and DScB, respectively. Graphical Abstract.

Strong complexation of water-soluble betulin derivatives with (2-hydroxypropyl)-γ-cyclodextrin studied by affinity capillary electrophoresis.

The complexation between (2-hydroxypropyl)-γ-cyclodextrin (HP-γ-CD) and water-soluble betulin derivatives, betulin 3,28-disulfate (DSB) and betulin 3-acetate-28-sulfate (ASB), belonging to the class of pentacyclic lupane triterpenoids, was studied using mobility shift ACE (ms ACE). It was found that the complexation is a high-affinity interaction. In this case, a very low amount of HP-γ-CD should be added to the BGE, and triangular peaks are observed as a result of ligand deficiency in the sample zone. Le Saux et al. showed in 2005 that using the parameter a1 of the Haarhoff-Van der Linde (HVL) function instead of the migration time measured at the peak apex eliminates the effect of ligand deficiency on effective electrophoretic mobility. Therefore, the electrophoretic mobilities of asymmetrical peaks of DSB and ASB were calculated in this way. The obtained experimental data correspond to 1:1 complexes. The calculated values of binding constants logarithms at 25°C are 6.70 ± 0.05 and 7.03 ± 0.10 for the HP-γ-CD complexes of DSB and ASB, respectively.

Predictive classification models and targets identification for betulin derivatives as Leishmania donovani inhibitors.

Betulin derivatives have been proven effective in vitro against Leishmania donovani amastigotes, which cause visceral leishmaniasis. Identifying the molecular targets and molecular mechanisms underlying their action is a currently an unmet challenge. In the present study, we tackle this problem using computational methods to establish properties essential for activity as well as to screen betulin derivatives against potential targets. Recursive partitioning classification methods were explored to develop predictive models for 58 diverse betulin derivatives inhibitors of L. donovani amastigotes. The established models were validated on a testing set, showing excellent performance. Molecular fingerprints FCFP_6 and ALogP were extracted as the physicochemical properties most extensively involved in separating inhibitors from non-inhibitors. The potential targets of betulin derivatives inhibitors were predicted by in silico target fishing using structure-based pharmacophore searching and compound-pharmacophore-target-pathway network analysis, first on PDB and then among L. donovani homologs using a PSI-BLAST search. The essential identified proteins are all related to protein kinase family. Previous research already suggested members of the cyclin-dependent kinase family and MAP kinases as Leishmania potential drug targets. The PSI-BLAST search suggests two L. donovani proteins to be especially attractive as putative betulin target, heat shock protein 83 and membrane transporter D1.

Combination of phase-solubility method and capillary zone electrophoresis to determine binding constants of cyclodextrins with practically water-insoluble compounds.

The combined method based on phase-solubility technique and capillary zone electrophoresis (PS-CZE) was suggested for the determination of binding (stability) constants of cyclodextrins (CD) complexes with water-insoluble organic compounds that have no or weak UV chromophores. In this method, the insoluble compounds are agitated at the desired temperature in CD solutions with different concentration up to the attainment of equilibrium and then CZE is used to determine the concentration of the compounds that have passed into the solutions. To avoid precipitation and complex dissociation, the background electrolyte (BGE) for CZE should contain ethanol and, if necessary, cyclodextrin. The samples should be diluted with the BGE without CD so that the CD concentrations in BGE and samples were equal to preclude a baseline shift. Using the suggested approach, the inclusion complexes between betulinic and betulonic acids, pentacyclic lupane-type triterpenes, and hydroxypropyl-ß- and γ-cyclodextrins (HP-ß-CD and HP-γ-CD) were studied. It was found that solubility of the acids studied in HP-ß-CD solutions did not differ from their solubility in pure water. That is, the HP-ß-CD complexes of the acids studied were not formed in noticeable amount. At the same time, the acids formed inclusion complexes with HP-γ-CD, what possibly was caused by the greater size of the HP-γ-CD molecule as compared to HP-ß-CD. To determine binding constants by Higuchi and Connors method, the acids solubility was determined by CZE after their agitation in the solutions of HP-γ-CD (with 0.6 molar substitution) at 25 °C for 3 days. The dependences of acids solubility on HP-γ-CD concentration deviated from straight line in the range of high concentration (AN mode). This can be explained by a self-association of HP-γ-CD molecules. Using the linear segment of the solubility dependences on CD concentration, the binding constants were determined. Their logarithms for the HP-γ-CD complexes with betulonic and betulinic acids were 3.88 ± 0.14 and 3.82 ± 0.12, respectively.

Anticancer properties of ester derivatives of betulin in human metastatic melanoma cells (Me-45).

BACKGROUND: Betulinic acid and betulin are triterpenes that have anticancer properties in various types of cancer. Unfortunately, the bioavailability and the bio-distribution of betulinic acid and its metabolic precursor, betulin are very low because of poor solubility in aqueous buffers. METHODS: In this study, we examined the anticancer properties of the ester derivatives of betulin compared to their precursors in a malignant melanoma cell line. We assessed five amino acid esters of betulin. The compounds contained four basic amino acids-natural lysine (l-Lys-OH) and three of its derivatives (l-Dap-OH, l-Dab-OH, and l-Orn-OH)-and alanine (l-Ala-OH) as a negative control (amino acid without an amine group in the side chain). The derivatives were more soluble than their precursors (betulin and betulinic acid) in water. The betulin esters were tested in the malignant melanoma cell line Me-45. To evaluate the cytotoxicity, MTT test was performed after 24, 48 and 72 h of incubation with the test compounds at a concentration range of 0.75-100 µM. For analysis of the apoptotic activity, TUNEL assay was performed. Additionally, expression of caspase-3 and PARP-1 was investigated immunocytochemically. RESULTS: The highest biological activity was observed with the lysine ester. The results showed that the highest cytotoxicity and the highest number of positively stained nuclei in metastatic melanoma Me-45 cells were obtained after 72 h of incubation with betulin derivatives containing lysine and ornithine. CONCLUSIONS: The betulin ester derivatives showed enhanced antitumor activity compared to their non-modified precursors. Esters of betulin can be more potent anticancer agents than their precursor as a consequence of the rapid bioavailability and increased concentration in cancer cells.

Synthesis and in vitro antitumor evaluation of betulin acid ester derivatives as novel apoptosis inducers.

Nineteen betulin derivatives modified at the C-3 and C-28 positions were synthesized and assessed for antitumor activities against the MGC-803, PC3, Bcap-37, A375, and MCF-7 human cancer cell lines in vitro by MTT assay. Some derivatives (compounds 3a-3d and 5) displayed strong antitumor properties, with IC50 values between 4 and 18 µM. Compound 3c, containing piperidine group at C-28 position, had IC50 values of 4.3, 4.5, 5.2, 7.5, and 5.2 µM on the five cancer cell lines, respectively. Subsequent fluorescence staining and flow cytometric analysis indicated that compound 3c induced apoptosis in MGC-803 cell line, with an apoptosis ratio of 31.11% after 36 h of treatment at 10 µM 3c.

The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment - In vitro studies.

BACKGROUND: Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications. OBJECTIVE: To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells. METHODS: In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids - natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, -CH2-CH2-CH2-CH2-NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (-CH2-CH2-CH2-NH2)>Dab (-CH2-CH2-NH2)>Dap (-CH2-NH2)>Ala (CH3)>betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72h of incubation with the test compounds at a concentration range of 0.75-100µM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically. RESULTS: The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50=7µM) and ornithine (IC50=10µM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain. CONCLUSIONS: The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.

Betulin derivatives impair Leishmania braziliensis viability and host-parasite interaction.

Leishmaniasis is a public health problem in tropical and subtropical areas of the world, including Venezuela. The incidence of treatment failure and the number of cases with Leishmania-HIV co-infection underscore the importance of developing alternative, economical and effective therapies against this disease. The work presented here analyzed whether terpenoids derived from betulin are active against New World Leishmania parasites. Initially we determined the concentration that inhibits the growth of these parasites by 50% or IC50, and subsequently evaluated the chemotactic effect of four compounds with leishmanicidal activity in the sub-micromolar and micromolar range. That is, we measured the migratory capacity of Leishmania (V.) braziliensis in the presence of increasing concentrations of compounds. Finally, we evaluated their cytotoxicity against the host cell and their effect on the infectivity of L. (V.) braziliensis. The results suggest that (1) compounds 14, 17, 18, 25 and 27 are active at concentrations lower than 10 µM; (2) compound 26 inhibits parasite growth with an IC50 lower than 1 µM; (3) compounds 18, 26 and 27 inhibit parasite migration at pico- to nanomolar concentrations, suggesting that they impair host-parasite interaction. None of the tested compounds was cytotoxic against J774.A1 macrophages thus indicating their potential as starting points to develop compounds that might affect parasite-host cell interaction, as well as being leishmanicidal.