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.     

Synthesis and cytotoxic activity of 3-O-acyl/3-hydrazine /2-bromo/20,29-dibromo betulinic acid derivatives

A series of 3-O-acyl, 3-hydrazine, 2-bromo, and 20,29-dibromo betulinic acid derivatives (1-27) have been synthesized and screened for in vitro cytotoxic activity on human cancer cell lines MOLT-4, JurkatE6.1, CEM.CM3, BRISTOL8, U937, DU145, PA-1, A549, and L132. A number of compounds have shown ED(50)<1 microg/mL against the cancer cell lines tested and have shown better cytotoxicity than betulinic acid. Compounds 13, 19, 20, 23, and 27 were the best derivatives and were selected as lead molecules for further development. The structure-activity relationship has been described.     

白桦脂酸的研究进展

白桦脂酸是一种五环三萜酸,存在于多种天然植物,特别是白桦树皮中,毒性低,安全指数高。近年来发现白桦脂酸具有抗肿瘤、抗HIV、抗炎、抗菌、抗疟疾等多种生物活性,特别是在抗黑色素瘤与抗HIV方面具有突出的表现,从而引起了人们极大的研究兴趣。对白桦脂酸的来源和生物活性进行了简要综述。     

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5?mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000?μg/mL respectively. The compound (50 and 100?mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90?min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100?mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4?h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.     

23-Hydroxybetulinic acid-mediated apoptosis is accompanied by decreases in bcl-2 expression and telomerase activity in HL-60 Cells

23-Hydroxybetulinic acid, a derivative of betulinic acid, was investigated for its apoptotic effect and the associated telomerase activity in human leukemia HL-60 cells. Apoptosis and bcl-2 were determined by flow cytometry analysis. A PCR-based telomeric repeat amplification protocol assay was used to detect telomerase activity. Results showed that 23-hydroxybetulinic acid induced growth arrest and apoptotic cell death in HL-60 cells. The apoptotic events were associated with concurrent down-regulation of bcl-2 and the telomerase activity. Our data suggest that 23-hydroxybetulinic acid may be a potential cytotoxic agent for treatment of cancer.     

Design, synthesis and cytotoxic evaluation of keto-C-glycoside fatty acid conjugates.

Keto C-glycoside-fatty acid conjugates were synthesized from 6-hydroxy 2- and 4-keto unsaturated D-C-glycosides. These compounds were tested for cytotoxic activity against LFCl₂A cells (Rat hepatocarcinoma cells). The introduction of a lipid chain to 2-keto C-glycosides induced a drop in the cyctotoxic activity of these compounds. On the other hand 4-keto unsaturated C-glycoside-fatty acid conjugates possessed IC₅₀ values of 0.7–0.001 μM with 21 being the most potent.     

Synthesis and structure-activity relationship study of cytotoxic germanicane- and lupane-type 3beta-O-monodesmosidic saponins starting from betulin

Germanicane-type triterpenes allobetulin (3) and 28-oxoallobetulin (4) can be obtained by the Wagner-Meerwein rearrangement of the more available lupane-type triterpenes betulin (1) and betulinic acid (2), respectively. The medical uses of betulinic acid (2) and its derivatives are limited because of their poor hydrosolubility and pharmacokinetics properties. In order to overcome this major problem, we synthesized and studied the in vitro anticancer activity of a series of 3beta-O-monodesmosidic saponins derived from betulin (14-16), betulinic acid (20-22), allobetulin (23-28) and 28-oxoallobetulin (29-34) based on six different natural sugar residues (d-glucose, l-rhamnose, d-arabinose, d-galactose, d-mannose and d-xylose). This structure-activity relationship study confirmed that betulinic acid saponins are generally better in vitro anticancer agents than those derived from betulin with the exception of betulin 3beta-O-alpha-d-mannopyranoside (15) which exerted a potent cytotoxic activity against lung carcinoma (A-549) and colorectal adenocarcinoma (DLD-1) human cell lines with IC(50) ranging from 7.3 to 10.1mumol/L. Furthermore, although the synthesis of novel germanicane-type saponins was carried out with success, the bioactivity measured for these glycosides was not as high as we anticipated since only the 3beta-O-beta-d-glucopyranoside and 3beta-O-beta-d-galactopyranoside of allobetulin (23,24) showed moderate anticancer activity (IC(50) 30-40 micromol/L).     

Design and synthesis of 4-HPR derivatives for rhabdoid tumors

Rhabdoid tumors (RTs) are aggressive pediatric malignancies with poor prognosis that arise due to loss of the hSNF5/INI1 tumor suppressor. Molecular studies indicate that cyclin D1, a downstream effector of INI1 is up regulated in RT, and is essential for this tumor formation. Previously we demonstrated that 4-HPR, a synthetic retinoid that targets Cyclin D1, is a potential chemotherapeutic agent for RT. To facilitate further chemical development of this retinoid, and to determine its active moiety, we synthesized small chemical libraries of 4-HPR and tested their cytotoxic effect on RT cells. We synthesized 4-HPR (1) and the derivatives (5a-5n) starting from retinoic acid. First, retinoic acid was converted to acid chloride derivatives, then in the presence of DMF, base, and aniline derivatives, we synthesized the corresponding 4-hydroxy phenyl amine derivatives (5a-5n). This procedure gave 70-90% yield. Then, the 4-HPR derivatives were tested for their ability to inhibit RT cells using an in vitro cell survival assay. We found that the 4-hydroxy group at para-position is essential for cytotoxic activity against RT cells. Furthermore, we identified a few derivatives of 4-HPR with higher cytotoxic potencies than 4-HPR. In addition, we demonstrate that either chloro, fluoro or iodo derivatives at meta-position of phenyl ring retain the cytotoxic activity. Interestingly, substitution of iodo-moiety at meta-position (5j) substantially increased the efficacy (IC(50) approximately 3muM, Fig. 1D). These results indicate that chemical modification of 4-HPR may result in derivatives with increased therapeutic potential for RTs and that halogen substituted 4-HPR that retain the activity can be synthesized for further therapeutic and diagnostic use.     

Sulfamic and succinic acid derivatives of 25-OH-PPD and their activities to MCF-7, A-549, HCT-116, and BGC-823 cell lines

In the search for new anti-tumor agents with higher potency than our previously identified compound 1 (25-OH-PPD, 25-hydroxyprotopanaxadiol), 12 novel sulfamic and succinic acid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 1, compounds 2, 3, and 7 exhibited higher cytotoxic activity on A-549 and BGC-823 cell lines, together with lower toxicity in the normal cell. In particular, compound 2 exhibited the best anti-tumor activity in the in vitro assays, which may provide valuable data for the research and development of new anti-tumor agents.     

New ursolic and betulinic derivatives as potential cytotoxic agents

Fifteen new ursolic and betulinic triterpenoids, bearing various functionalities at C-3 and C-28 were synthesized as potential cytotoxic agents. All compounds were obtained by a hemisynthetic route via ursolic and betulinic acids. Preliminary screening of these compounds on human HT 29 colon cancer cells revealed inhibitory activity for three of them. Beta-D-Glucopyranosyl-3beta-hydroxyurs-12(13)-en-28-oate 1c, 3beta-3-(3-pyridyl)-prop-2-enoyloxyurs-12(13)-en-28-oic acid 1i and the potassium salt of 3beta-cinnamoyloxylup-20(29)-en-28-oic acid 2d demonstrated cytotoxic activity in the micromolar range: 8.0, 45.0 and 8.0 microM, respectively.     

Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors

Betulinic acid is a natural product possessing abundant and favourable biological activity, including anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties, while causing minimal toxicity to unaffected cells. The full biological potency of betulinic acid cannot be fully unlocked, however, for a number of reasons, a primary one being its limited solubility in aqueous and biologically pertinent organic media. Aiming to improve the water solubility of betulinic acid without disrupting its structurally related bioactivity, we have prepared different ionic derivatives of betulinic acid. Inhibition bioassays on HIV-1 protease-catalysed peptide hydrolysis indicate significantly improved performance resulting from converting the betulinic acid to organic salt form. Indeed, for one particular cholinium-based derivative, its water solubility is improved more than 100 times and the half maximal inhibitory concentration (IC(50)) value (22 μg mL(-1)) was one-third that of wide-type betulinic acid (60 μg mL(-1)). These encouraging results advise that additional studies of ionic betulinic acid derivatives as a therapeutic solution against HIV-1 infection are warranted.     

Synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates

Beta-carboline represents a class of compounds with potent anti-tumor activity by intercalating with DNA. To further enhance the cytotoxic potency and bioavailability of beta-carboline, a series of novel beta-carboline amino acid ester conjugates were designed and synthesized, and the cytotoxic activities of these compounds were tested using a panel of human tumor cell lines. In addition, the membrane permeability of these compounds was evaluated in vitro using a Caco-2 cell monolayer model. The beta-carboline amino acid ester conjugates demonstrated improved cytotoxic activity compared to the parental beta-carbolines. In particular, the Lys/Arg conjugates were the most potent analogs with an IC(50) value of 4 and 1 microM against human cervical carcinoma cells. The low interaction energy of Arg conjugate based on molecular modeling may contribute to its enhanced cytotoxicity. Taken together, this study provided new insights into structure-activity relationships in the beta-carboline amino acid ester conjugates and identified the beta-carboline Lys/Arg conjugates as promising lead compounds for further in vivo biological and molecular evaluation.     

Identification and egg hatching activity of monohydroxy fatty acid eicosanoids in the barnacle Balanus balanoides.

Monohydroxy fatty acids (MHFAs) were isolated from homogenates of the barnacle Balanus balanoides and identified by gas chromatography-mass spectrometry (GC-MS) as 14- and 17-hydroxy docosahexaenoic acids, 8-, 11-, 12-, 15- and 18-hydroxy eicosapentaenoic acids, 13- and 16-hydroxyoctadecatrienoic acids and 9-, 13- and 15-hydroxyoctadecadienoic acids. Each monohydroxy fatty acid was tested for egg hatching activity in a bioassay using Elminius modestus egg masses, but 8-hydroxy-5, 9, 11, 14, 17-eicosapentaenoic acid (8-HEPE) was the only MHFA with barnacle egg hatching activity. Studies on the egg hatching activity of MHFAs prepared from the oxidation of polyunsaturated fatty acids showed that activity was confined to the 8-hydroxy isomer of eicosapentaenoic acid and arachidonic acid, and that unsaturation at C5 and C14, but not C17, was essential for activity. In addition, the 8(R) conformation is necessary for activity, as 8(R)-HEPE caused egg hatching at 10(-7) M whereas the enantiomer 8(S)-HEPE was inactive.     

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 and antiviral activity of hydrazides and substituted benzalhydrazides of betulinic acid and its derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhydrazides, were synthesized. Their antiviral activities toward of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

Synthesis of 3-O-acyl/3-benzylidene/3-hydrazone/3-hydrazine/17-carboxyacryloyl ester derivatives of betulinic acid as anti-angiogenic agents

New 3-O-acyl, 3-benzylidene, 3-hydrazone, 3-hydrazine, 17-carboxyacryloyl ester derivatives of betulinic acid (2-6, 8-11, 13, 17, 18, 21, and 22) were synthesized and evaluated in vitro for anti-angiogenic activity on endothelial cell cytotoxicity, specificity, and tube-formation ability. All derivatives reported here showed IC(50)<4 microg/mL. Compounds 3, 9, 10, 17, 21, and 22 have shown better cytotoxicity (IC(50)<1.2 microg/mL) than betulinic acid (1) and improved endothelial cell specificity (ECS>10) in some cases. Compounds 10, 17, and 18 have shown 20%, 32%, and 48% reduction in TLS, respectively, and were found better than betulinic acid (1). We have shown that 20,29-dihydrobetulinic acid derivatives have better anti-angiogenic activity as compared to betulinic acid or its other derivatives.     

New betulinic acid derivatives as potent proteasome inhibitors

In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC(50) values of 1.42, 1.56, and 1.80 μM, respectively, which are three to fourfold more potent than the proteasome inhibition controls LLM-F and lactacystin.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Cytotoxic heterocyclic triterpenoids derived from betulin and betulinic acid

The aim of this work was to synthesize a set of heterocyclic derivatives of lupane, lup-20(29)-ene, and 18α-oleanane, and to investigate their cytotoxic activities. Some of those heterocycles were previously known in the oleanane (allobetulin) group; however, to our knowledge the syntheses and biological activities of lupane heterocycles have not been reported before. Starting from betulin (1) and betulinic acid (2), we prepared 3-oxo compounds and 2-bromo-3-oxo compounds 3-10, 2-hydroxymethylene-3-oxo compounds 11-13 and β-oxo esters 14-16. Condensation of these intermediates with hydrazine, phenylhydrazine, hydroxylamine, or thiourea yielded the pyrazole and phenylpyrazole derivatives 17-22, pyrazolones 23-25, isoxazoles 26 and 27, and thiazoles 28-31. Fifteen compounds (14-16, 18-25, and 29-32) have not been reported before. The cytotoxicity was measured using panel of seven cancer cell lines with/without MDR phenotype and non tumor MRC-5 and BJ fibroblasts. The preferential cytotoxicity to cancer cell lines, particularly to hematological tumors was observed, the bromo acids 5, 6 showed highest activity and selectivity against tumor cells.