Synthesis of betulonic aldehyde derivatives

Selective functionalization of betulonic aldehyde (the oxidation product of betulin) was studied with the aim of obtaining new physiologically active substances. We developed a method for the synthesis of azomethine derivatives at the C-28 aldehyde group and benzylidene derivatives at the 2-methylene group of the A ring. The structure of the synthesized products was proved by ¹H NMR.     

Advances in the synthesis and pharmacological activity of lupane-type triterpenoid saponins

Lupeol, betulin and betulinic acid are members of the so-called lupane-type triterpenoids. These natural products found worldwide in quite of lot of vegetables, fruits and plant species exhibit promising pharmacological activities including anti-inflammatory, anti-HIV and antitumor activities. Nevertheless, the poor pharmacokinetic properties of these cholesterol-like triterpenoids hampered further pharmaceutical developments. The synthesis of lupane-type saponins, i.e., sugar-derived lupanes, seems to be a good avenue to improve both their water solubility and pharmacological activity. The aims of this review are twofold: first, to describe the biological activity of naturally occurring lupane-type saponins, and second, report the different methodologies employed for the elaboration of glycosidic linkages at the C-3 and/or C-28 positions on the lupane core. The synthesis of both natural and unnatural lupane-type saponins is discussed with an emphasis on molecules exhibiting relevant biological activities.     

Synthesis and cytotoxicity of lupane-type triterpenoid glyceryl esters

A new series of betulinic acid and betulin derivatives were synthesized by introducing a D-glycerol moiety at the C-3 and/or C-28 positions of the lupane skeleton. The resulting glyceryl esters were evaluated in vitro for their cytotoxic activity against A549, DLD-1 and WS1 human cell lines. The structure-activity relationships study revealed that the incorporation of a glycerol unit at the C-3 or C-28 position of the lupane core resulted in compounds exhibiting potent cytotoxic activity together with decreased liposolubility.     

Synthesis,cytotoxicity, and haemolytic activity of chacotrioside lupane-type neosaponins and their germanicane-type rearrangement products

The concise synthesis, via a stepwise glycosylation approach, of lupeol, betulin and betulinic acid O-glycosides bearing a chacotriosyl moiety at the C-3 position is described. All neosaponins as well as their rearrangement products of the germanicane-type were evaluated in vitro for their anticancer and haemolytic activities. Although betulinic acid and betulin 3β-O-chacotriosides were neither cytotoxic nor haemolytic, their rearrangement products allobetulin and 28-oxoallobetulin 3β-O-chacotriosides (9 and 10) exhibited a cytotoxicity profile up to fourfold superior to betulinic acid against human breast (MCF7) and prostate (PC-3) adenocarcinomas cell lines (IC₅₀ = 10–18 μM). One important result was that only chacotriosides featuring non-polar functions at the C-28 position (6, 9 and 10) exerted a haemolytic activity against red blood cells.     

Cell adaptations of Rhodococcus rhodochrous IEGM 66 to betulin biotransformation

The findings of a comprehensive study on R. rhodochrous IEGM 66 and triterpenoid betulin interactions during its biotransformation were reported. In the presence of betulin, rhodococci were shown to form heterogeneous cell aggregates. The enhanced size of the aggregates from 12–15 μm to 25– 35 μm was consistent with the increase in betulin concentration from 0.5 to 3.0 g/L. The confocal laser scanning microscopy indicated a high (80.0%) level of rhodococcal viability during betulin biotransformation regardless of the betulin concentration. Experiments employing the combined confocal laser scanning and atomic force microscopy system confirmed that interactions between actinobacterial cells and betulin occur by direct contact. Transforming activities of the crude cell extracts from R. rhodochrous IEGM 66 were compared, and localization of enzymes catalyzing betulin oxidation to betulone was determined. Additionally the effects of betulin on fatty acid composition of rhodococci and their morphometric and morphofunctional characteristics during biotransformation were studied. Our findings could be used to develop approaches for enhanced betulin bioavailability, thus leading to improved biotransformation efficiency.     

Lipid Atlas of Keratinocytes and Betulin Effects on its Lipidome Profiled by Comprehensive UHPLC–MS/MS with Data Independent Acquisition Using Targeted Data Processing

Betulin is a pentacyclic triterpene with demonstrated healing properties in mid‐dermal wounds. A few earlier studies have provided insights into the wound healing effects on the molecular level. However, there are still questions left on the molecular targets of betulin. Therefore, a pharmacolipidomics analysis of betulin is undertaken in human immortalized keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. For this purpose, lipid extracts of keratinocytes treated with betulin and untreated controls are comprehensively analyzed by an untargeted UHPLC–ESI–QTOF‐MS/MS lipidomics profiling workflow using data‐independent acquisition. Targeted data processing allows the identification of 611 lipid species from 21 different lipid classes. Statistical analysis of the identified lipids shows significant changes in 440 lipid species that can be described as downregulation of cholesteryl esters and triacylglycerides and upregulation of glycerophospholipids, sphingolipids, and diacylglycerides. Additionally, some other signals corresponding to triterpenes are found in the betulin group and suggested that betulin is incorporated (in the membrane) and metabolized in keratinocytes.     

Study of the betulin molecule in a water environment; ab initio and molecular simulation calculations

Ab initio and molecular simulation methods were used in calculations of the neutral individual betulin molecule, and molecular simulations were used to optimize the betulin molecule immersed in various amounts of water. Individual betulin was optimized in different force fields to find the one exhibiting best agreement with ab initio calculations obtained in the Gaussian03 program. Dihedral torsions of active groups of betulin were determined for both procedures, and related calculated structures were compared successfully. The selected force field was used for subsequent optimization of betulin in a water environment, and a conformational search was performed using quench molecular dynamics. The total energies of betulin and its interactions in water bulk were calculated, and the influence of water on betulin structure was investigated.     

Betulin binds to melanocortin receptors and antagonizes alpha-melanocyte stimulating hormone induced cAMP generation in mouse melanoma cells

Betulin is a principal component of birch bark and is known to possess a broad range of biological activities, including antiinflammatory, antiviral and anticancer actions. The present study was carried out in vitro to clarify the influence of betulin on melanocortin (MC) receptor-ergic signalling by using COS-7 cells transfected with corresponding human MC receptor DNA. The results showed that betulin binds to the human melanocortin MC1, three to five receptors with selectivity to the MC1 subtype (K(i) value 1.022 +/- 0.115 microM). Betulin binds to the MC receptors with the following potency order-MC > MC3 > MC5 > MC4. Betulin itself does not stimulate cAMP generation, however, it slightly antagonizes alpha-melanocyte-stimulating hormone (alpha-MSH)-induced cAMP accumulation in the mouse melanoma cell line B16-F1. As a water-insoluble substance, betulin was dissolved in DMSO therefore DMSO competition with the labelled ligand NDP-MSH for the binding to the MC receptors was tested in the identical experimental set-up. We found that DMSO competes for binding to all the MC receptor subtypes, at 20% concentration and above. Selectivity for one or another receptor subtype was not observed. We have demonstrated for the first time, the ability of the plant compound betulin to bind to the MC receptors. One may suggest MC receptor MC1 subtype as the essential target for the antimelanoma action of betulin and its structurally close molecules such as betulinic acid. Moreover, we have found a new non-peptide small molecule MC mimetic, that is betulin. Thus, we report a new chemical motif for the binding to the MC receptors that could be used as a template for the search of more selective MC mimetics.     

The dose dependent effects of betulin on porcine chondrocytes

The past two decades triterpenes have attracted attention because of their pharmacological potential, especially its anti-oxidant activity. The present study was aimed to evaluate the possible protective effects of the triterpene betulin on porcine chondrocytes. For this, the cells were treated with different doses of betulin (0.02, 0.32 and 5.12 μg/mL) and without betulin. Biochemical measures of necrosis, mitochondrial activity, DNA content and sulphated glycosaminoglycans (sGAG) were reported. In addition, the gene expression of extracellular matrix molecules (ECM), proteases and soluble factors were examined. The abundance of reactive oxygen species (ROS) was also reported. Among the concentrations tried 0.32 μg/mL of betulin was found to be optimum because it effectively promoted the gene expressions of type II collagen, aggrecan and inhibited the gene expression of matrix metalloproteinase 2 (MMP-2). The chemiluminescence (CL) assay indicated that betulin treated chondrocytes had better free radical scavenging activity than the chondrocytes cultured without betulin. Alcian blue staining revealed that the chondrocytes were functionally active and able to synthesis sGAG. The free radical scavenging activity ensures betulin as protectant of chondrocytes and it further maintains the proliferation and basic activities of chondrocytes.     

Biotransformation optimization of betulin into betulinic acid production catalysed by cultured Armillaria luteo‐virens Sacc ZJUQH100‐6 cells

Aims: Betulinic acid has attracted attention in terms of its important biological and pharmacological characteristics. The main objective of this work was to optimize the variables of biotransformation process in order to enhance betulinic acid production from betulin catalysed by fungus Armillaria luteo‐virens Sacc ZJUQH100‐6. Methods and Results: Fractional factorial design and response surface methodology were applied to optimize the main parameters that affect betulinic acid production in the growing‐cells system. Results indicated that the addition of Tween 80 and substrate concentration were identified as the significant factors on betulinic acid formation, and the central composite experimental design was then adopted to derive a statistical model for optimizing biotransformation conditions. The optimum conditions were observed at pH 6·0, 0·57% Tween 80, 15 mg l^?1 betulin and at 3 days of stage of inoculation. Conclusions: Under the optimized conditions, the highest productivity of betulinic acid predicted was 9·32%, which increased by 74·53% in comparison with that of the nonoptimized. The verified experiment revealed that the model can well simulate betulin biotransformation. Moreover, the bioconversion of betulin and betulin‐28‐monooxygenase activities was compared between the optimized and the nonoptimized conditions. Significance and Impact of the Study: Current data imply that betulinic acid production from betulin can be effectively enhanced through biotransformation optimization strategy.     

Betulin and ursolic acid synthetic derivatives as inhibitors of Papilloma virus

The synthesis of new betulin and ursolic acid derivatives and evaluation of their antiviral activity in vitro is reported. Betulin was modified at positions C-3, C-20 and C-28 to afford the derivatives with nicotinoyl-, methoxycynnamoyl-, alkyne and aminopropoxy-2-cyanoethyl-moieties. The two stage conversion of betulin to the new ursane-type triterpenoid by treatment of allobetulin with Ac₂O–HClO₄ is suggested. Cyanoethylation of ursonic acid oxime led to cyanoethyloximinoderivative. According to the results of antiviral screening against human papillomavirus type 11 the selectivity index for tested triterpenoids has a range from 10 to 35 with no cellular cytotoxicite, the most remarkable activity was found for 3β,28-di-O-nicotinoylbetulin. 3β,28-Dihydroxy-29-norlup-20(30)-yne was also active against HCV replicon (EC₅₀ 1.32; EC₉₀ 16.82; IC₅₀ 12.41; IC₉₀ >20; SI₅₀ 9.4; SI₉₀ >1.19). 28-O-Methoxycynnamoylbetulin was active against influenza type A virus (H1N1) (ЕС₅₀ 2; IC₅₀ >200; SI >100).     

Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana

Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC₅₀ values 3.49?±?0.02, 4.17?±?0.03 and 87.52?±?0.03?µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds.     

Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence

The senescence-promoting activities of methyl jasmonate and its related compounds were compared with respect to structure-activity relationships. The activities were assayed by using oat ( A vena saliva L. cv. Victor) leaf segments in the presence of 2 μg/ml kinetin. Dextrorotatory methyl jasmonate prepared from an authentic sample of the racemate mixture was less active than the naturally occurring levorotatory form especially at its low concentrations (0.1 to 2.5 μg/ml). The activity of jasmonic acid, the free acid form of methyl jasmonate, was much less than the methyl ester, and this relationship was true for the other compounds tested. The reduction of the unsaturated bond in the substituent at the C-2 position and the keto group at the C-3 position greatly reduced the activity. The length of the n -alkyl substituents at the C-2 position had also a significant effect on the activity. From these results, it is concluded that the important functional groups for the high senescence-promoting activity of the methyl jasmonate related compounds are the methyl acetate substituent at the C-l position, the 2' cis -pentenyl or n -pentyl group at the C-2, position and the keto group at the C-3 position in methyl jasmonate.     

Glycosidation of lupane-type triterpenoids as potent in vitro cytotoxic agents

The weak hydrosolubility of betulinic acid (3) hampers the clinical development of this natural anticancer agent. In order to circumvent this problem and to enhance the pharmacological properties of betulinic acid (3) and the lupane-type triterpenes lupeol (1), betulin (2), and methyl betulinate (7), glycosides (beta-D-glucosides, alpha-L-rhamnosides, and alpha-D-arabinosides) were synthesized and in vitro tested for cytotoxicity against three cancerous (A-549, DLD-1, and B16-F1) and one healthy (WS1) cell lines. The addition of a sugar moiety at the C-3 or C-28 position of betulin (2) resulted in a loss of cytotoxicity. In contrast, the 3-O-beta-D-glucosidation of lupeol (1) improved the activity by 7- to 12-fold (IC50 14-15.0 microM). Moreover, the results showed that cancer cell lines are 8- to 12-fold more sensitive to the 3-O-alpha-L-rhamnopyranoside derivative of betulinic acid (IC50 2.6-3.9 microM, 22) than the healthy cells (IC50 31 microM). Thus, this study indicates that 3-O-glycosides of lupane-type triterpenoids represent an interesting class of potent in vitro cytotoxic agents.     

Synthesis,biological evaluation and SAR analysis of O-alkylated analogs of quercetin for anticancer

O-Alkylated quercetin analogs were synthesized and their anticancer activities were assessed by a high-throughout screening (HTS) method. The structure–activity relationships (SAR) showed that introduction of long alkyl chain such as propyl group at the C-3 OH position or short alkyl chain such as ethyl group at the C-4′ OH position were very important for keeping inhibitory activities against the 16 cancer cell lines. Furthermore, when the two n-butyl groups were introduced into the C-3, C-7 or C-4′, C-7 positions, the anticancer activity was enhanced.     

Biotransformation of betulin to betulone by growing and resting cells of the actinobacterium Rhodococcus rhodochrous IEGM 66

The ability of Rhodococcus actinobacteria to transform betulin to betulone was proved and reported for the first time. Betulone, the product of regioselective oxidation of a 3β-hydroxyl group of betulin, is a useful intermediate in the synthesis of novel biologically active compounds. Of 56 strains of Rhodococcus tested, Rhodococcus rhodochrous IEGM 66 was selected because it had the highest betulin-transforming ability. It was shown that R. rhodochrous IEGM 66 growing cells transformed 0.5 g/L betulin to betulone with 45% conversion rate within 240 h. A substantial reduction in the time of betulin (0.5 g/L) biotransformation was achieved by using resting cells, which catalyzed the production of 75% betulone after 96 h. At higher initial betulin concentrations (1.0–3.0 g/L), resting cells catalyzed 40–60% betulone production within 24 h.     

Synthesis of anomeric 1,5-anhydrosugars as conformationally locked selective α-mannosidase inhibitors

Anomeric 1,5-anhydrosugar 2 was synthesized from d-glucose derived N-Cbz protected aminodiol 8. The key step involves, acid catalyzed hydrolysis of 1,2-acetonide group in 8 to get hemiacetal that concomitantly undergoes formation of the pyranose ring by attack of C-3 hydroxyethyl group on anomeric C-1, leading to the formation of dioxabicyclo[3.2.1]octane skeleton which on hydrogenolyis gave 2. The glycosidase inhibitory activities of hydroxy- and amino-substituted anomeric 1,5-anhydrosugars 1 and 2, respectively, showed selective inhibition of α-mannosidase. These results were substantiated by molecular docking studies using WHAT IF software and AUTODOCK 4.0 program.     

Synthesis of the betulin dipropionate from the upper birch bark

A one-step synthesis of the betulin dipropionate directly from the birch bark without a separate stage of the betulin preparation is described in this paper. Extracts with different content of the betulin dipropionate were shown to form depending on the conditions of acylation of the upper birch bark with propionic acid. The product with the maximum content of the betulin dipropionate was prepared from the starting fraction of 2?C5 mm of the upper birch bark and the fraction of 10?C20 mm that was preliminarily activated with superheated water vapor. The upper bark extract was analyzed by gas chromatography. The structure of betulin dipropionate was confirmed by element analysis, ¹H NMR, ¹³C NMR, and FTIR spectroscopy.     

Ameliorative effect of betulin from Betula platyphylla bark on scopolamine-induced amnesic mice

Alzheimer’s disease (AD) is a neurodegenerative disease induced by cholinergic neuron damage or amyloid-beta aggregation in the basal forebrain region and resulting in cognitive disorder. We previously reported on the neuroprotective effects of Betula platyphylla bark (BPB) in an amyloid-beta-induced amnesic mouse model. In this study, we obtained a cognitive-enhancing compound by assessing results using a scopolamine-induced amnesic mouse model. Our results show that oral treatment of mice with BPB and betulin significantly ameliorated scopolamine-induced memory deficits in both passive avoidance and Y-maze tests. In the Morris water maze test, administration of BPB and betulin significantly improved memory and cognitive function indicating the formation of working and reference memories in treated mice. Moreover, betulin significantly increased glutathione content in mouse hippocampus, and the increase was greater than that from betulinic acid treatment. We conclude that BPB and its active component betulin have potential as therapeutic, cognitive enhancer in AD.     

Extraction of betulin from birch bark and study of its physico-chemical and pharmacological properties

Betulin-containing products were obtained in a yield of about 40% from absolutely dry birch bark. The content of betulin in the products was 74–75% or 85–89% depending on the presence of sodium or potassium hydroxide, respectively. The one-step method of extraction of high-purity betulin (97.7%) is presented. Betulin was identified by physic-chemical methods, i.e., elemental analysis, IR and NMR spectroscopy, and electron scanning microscopy. The thermal characteristics of betulin were also studied. It was shown that betulin exhibits gastroprotective, hepatoprotective, and capillary-strengthening properties.