Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC₅₀ <100 μM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K_i value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.     

Design, synthesis, and anti-inflammatory activity both in vitro and in vivo of new betulinic acid analogues having an enone functionality in ring A

Fifteen new betulinic acid analogues were designed, synthesized, and tested for anti-inflammatory activity. Many of these analogues effectively suppress nitric oxide (NO) production in RAW cells stimulated with interferon-γ. Analogue 10 is highly and orally active in vivo for induction of the anti-inflammatory and cytoprotective enzyme, heme oxygenase-1.     

Synthesis of phthalates of betulinic acid and betulin with cytotoxic activity

Synthesis of 3beta-O-phthalic esters from betulinic acid and its esters and synthesis of phthalic esters from betulin and its monoacetates using classical acylation procedure with phthalic anhydride. The evaluation of cytotoxicity of the prepared compounds was using numbers of tumor cell lines in MTT test. It was discovered that hemiphthalic esters had better cytotoxicity than starting compounds as betulinic acid or quite inactive betulin.     

New 30-norlupane derivatives through chemical-microbial semi-synthesis of betulinic acid and their neuroprotective effect

In this study, seven 30-norlupane derivatives (2–8) was obtained from the chemical oxidation of betulinic acid followed by biotransformation via Bacillus megaterium CGMCC 1.1741. And metabolites 2–4 and 6–8 were newly identified products. In the first step, betulinic acid was chemically oxidized to platanic acid (1). Following the chemical oxidation, B. megaterium catalyzed the hydroxylation at C-7, C-11, C-15 and C-23 of platanic acid (1) as well as the oxidation of C-3 hydroxyl group. Compared to the labor-intensive isolation from natural plants, this chemical-microbial semi-synthesis is more capable to provide increased structural diversity of oxygenated 30-norlupane. Finally, the potential neuroprotective effect of the derivatives was assessed on neuron-like PC12 cells induced by cobalt chloride (CoCl₂). Metabolite 6 showed a potent neuroprotective activity.     

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment

The efficacy of praziquantel against a Puerto Rican strain of Schistosoma mansoni was assessed using both in vivo and in vitro approach. The drug effective dose (50%) in the infected mouse model was about 30 times higher when determined against 28-day-old infections than against 7-week-old parasites. Single-sex female infections were also largely refractory to treatment and single-sex male infections moderately refractory, in comparison with bisexual infections. The in vitro approach consisted of overnight exposure of parasite cultures to various drug concentrations, followed by several days of culture in drug-free medium. In vitro results confirmed in vivo data and allowed for the observation of schistosome morphological phenomena after praziquantel exposure. Early worm contraction was observed in all cases, even after exposure to sub-lethal concentrations of praziquantel or upon exposure of the largely refractory 28-day-old schistosomes. In these instances, however, worms resumed movements and normal shape upon drug removal and were able to survive. The inference of these observations on the clinical use of praziquantel and on its mechanism of action is discussed.     

New betulinic acid derivatives induce potent and selective antiproliferative activity through cell cycle arrest at the S phase and caspase dependent apoptosis in human cancer cells

New semisynthetic derivatives of betulinic acid (BA) RS01, RS02 and RS03 with 18-45 times improved cytotoxic activity against HepG2 cells, were tested for their ability to induce apoptosis and cell cycle arrest in HepG2, HeLa and Jurkat cells. All the compounds induced significant increase in the population at the S phase more effectively than BA. RS01, RS02 and RS03 were also found to be potent inducers of apoptosis with RS01 being markedly more potent than BA, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell cycle arrest. The mechanism of apoptosis induction has been studied in HepG2 cells and found to be mediated by activation of the postmitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8. These facts were corroborated by detection of mitochondrial cytochrome c release and DNA fragmentation. Because RS01, RS02 and RS03 exhibited significant improved antitumor activity with respect to BA, they may be promising new agents for the treatment of cancer. In particular, RS01 is the most promising compound with an IC₅₀ value 45 times lower than BA on HepG2 cells and 61 times lower than the one found for the non-tumoral Chang liver cells.     

桦木酸体内抗肿瘤作用的初步研究

目的研究桦木酸对H22、S180小鼠肿瘤细胞药效学作用及对肿瘤细胞膜Ca^2＋、Mg^2＋-ATP酶活性的影响。方法通过限量实验确定桦木酸的给药剂量。动物随机分为6组,分别为桦木酸500、250、125mg／kg剂量组,阳性对照组给予环磷酰胺（20mg／kg）,模型对照组分别给予相同体积的生理盐水和配药溶剂,灌胃给药。观察生命延长时间与瘤重抑制率;紫外分光光度法测定Ca^2＋、Mg^2＋-ATP酶活性,结果桦木酸使S180荷瘤小鼠肿瘤细胞膜Ca^2＋、Mg^2＋-ATP酶活性提高（P〈0．05）。结论桦木酸抗肿瘤作用机制与其上调肿瘤细胞膜p53蛋白的表达,改变肿瘤细胞膜Ca^2＋、Mg^2＋-ATP酶活性有关。     

Synthesis and anti-HIV activity of bi-functional betulinic acid derivatives

Betulinic acid (BA) derivatives with a side chain at C-3 can inhibit HIV-1 maturation. On the other hand, BA derivatives with a side chain at C-28 can block HIV-1 entry. In order to combine the anti-maturation and anti-entry activities in a single molecule, new bi-functional BA derivatives containing side chains at C-3 and C-28 have been synthesized. The most potent compound ([[N-[3beta-O-(3',3'-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]-carbamoyl]methane) inhibited HIV-1 at an EC50 of 0.0026 microM and was at least 20 times more potent than either the anti-maturation lead compound DSB or the anti-entry lead compound IC9564. This bi-functional BA derivative was active against both HIV entry and maturation. These results suggest that bi-functional BA derivatives with dual mechanisms of action have the potential to become clinically useful for AIDS therapy.     

Synthesis of new betulinic acid/betulin-derived dimers and hybrids with potent antimalarial and antiviral activities

Severe malaria and viral infections cause life-threatening diseases in millions of people worldwide every year. In search for effective bioactive hybrid molecules, which may possess improved properties compared to their parent compounds, a series of betulinic acid/betulin based dimer and hybrid compounds carrying ferrocene and/or artesunic acid moieties, was designed and, synthesized de novo. Furthermore, they were analyzed in vitro against malaria parasites (growth inhibition of 3D7-strain P. falciparum-infected erythrocytes) and human cytomegalovirus (HCMV). From this series of hybrids/dimers, the betulinic acid/betulin and artesunic acid hybrids 11 and 12 showed the most potent activities against P. falciparum and HCMV. On the strength of results, additive and/or synergistic effects between the natural or semisynthetic products, such as betulinic acid-/betulin- and artesunic acid-derived compounds, are suggested on the basis of putatively complex modes of antimicrobial action. This advantage may be taken into account in future drug development.     

Synthesis of lupane-type saponins bearing mannosyl and 3,6-branched trimannosyl residues and their evaluation as anticancer agents

The saponins modified with mono- or trimannosyl residues can provide a convenient means of delivering drugs to certain human cells via interactions with mannose receptors. In the study reported therein, we developed a convenient approach for the synthesis of 3-O-mannoside and branched trimannoside derivatives of the saponin lupeol and of C-28 acyl esters of 3-O-acetyl-betulinic acid bearing the same mannosyl entities. Lupeol and 3-O-acetyl-betulinic acid were mannosylated with tetra-O-benzoyl- or tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidates. De-esterification followed by regioselective dimannosylation of the unprotected monosaccharide derivatives with 2equiv of tetra-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate selectively yielded O-3,O-6-linked trimannosides. The cytotoxic activity of selected lupane-type saponins (derivatives of lupeol, betulinic acid, and betulin) toward normal human fibroblasts and various cancer cell lines was also compared.     

In vitro and in vivo chromosomal aberrations induced by megazol

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.     

Synthesis and cytotoxic activity of A-ring modified betulinic acid derivatives

New A-ring modified betulinic acid derivatives having small steric hindrance were prepared and tested for cytotoxic activity on 3 cancer cell lines: 10 compounds showed stronger cytotoxic activity than betulinic acid. Especially, the compounds bearing 1-ene-3-oxo with electron-withdrawing groups at C2 showed strong cytotoxicity.     

In vitro and in vivo interactions of aluminum on NTPDase and AChE activities in lymphocytes of rats

Al adjuvants are used in vaccines to increase the immune response. NTPDase and AChE play a pivotal role and act in the regulation of the immune system. The effect of Al exposure in vitro and in vivo on NTPDase and AChE activities in the lymphocytes of rats was determined. In vitro, ATP hydrolysis was decreased by 20.4% and 17.3% and ADP hydrolysis was decreased by 36.5% and 34.8%, in groups D and E, respectively, when compared to the control. AChE activity was increased by 157.3%, 152.5%, 74.7% and 90.8% in groups B, C, D, and E, respectively, when compared to the control. In vivo, ATP hydrolysis was increased by 85% and 86% and ADP hydrolysis was increased by 104.2% and 74%, in Al plus citrate and Al groups, respectively, when compared to the control. AChE activity was increased by 50.7% in Al plus citrate and by 28.6% in Al groups, when compared to the control. Our results show that Al exposure both in vitro and in vivo altered NTPDase and AChE activities in lymphocytes. These results may demonstrate the ability of Al to elicit the immune system, where NTPDase and AChE activities can act as purinergic and cholinergic markers in lymphocytes.     

Development of a dual-wavelength fluorescent nanoprobe for in vivo and in vitro cell tracking consecutively

Many imaging probes have been developed for a wide variety of imaging modalities. However, no optical imaging probe could be utilized for both microscopic and whole animal imaging. To fill the gap, the dual-wavelength fluorescent imaging nanoprobe was developed to simultaneously carry both visible-range fluorescent dye and near-infrared (NIR) dye. Emission scan confirms that the nanoprobe exhibits two separate peaks with strong fluorescent intensity in both visible and NIR ranges. Furthermore, the dual-wavelength fluorescent nanoprobe has high photostability and colloidal stability, as well as long shelf-life. In vitro cell culture experiments show that the nanoprobe has the ability to label different types of cells (namely, esophageal, prostate, fibroblast and macrophage cell) for fluorescent microscope imaging. More importantly, cell tracking experiments confirm that cell migration and distribution in various organs can be tracked in real time using in vivo whole-body NIR imaging and in vitro microscopic imaging, respectively.