Synthesis and preliminary evaluation of new ursolic and oleanolic acids derivatives as antileishmanial agents

A series of new ursolic and oleanolic acids derivatives was synthesized via ursolic or oleanolic acids, previously extracted from South American Ilex species. These new compounds were tested for in vitro antiparasitic activity on Leishmania amazonensis and Leishmania infantum strains. Some of these compounds showed activity against the promastigote forms of L. amazonensis or L. infantum, with IC₅₀ ranging from 5 to 12 μM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC₅₀ = 2-50 μM). From a structure-activity relationships point of view, these pharmacological results enlightened mainly the importance of an acetylation at position 3 of the oleanolic acid skeleton in the activity against the L. amazonensis strain, and of a bis-(3-aminopropyl)piperazine moiety on the carboxylic function of ursolic acid against the L. infantum strain.     

Antileishmanial activity of isolated triterpenoids from Pourouma guianensis.

The inhibiting activity of triterpenoids isolated from the methanolic extract of Pourouma guianensis (Moraceae) leaves is described for promastigotes and intracellular amastigotes of Leishmania amazonensis. Whereas the fractions containing apigenin, friedelin, epi-friedelinol, arjunolic acid, hyptatic acid B, stigmasterol and sitosterol were of no or relatively low inhibitory activity, fractions containing tormentic acid, 2alpha,3beta-dihydroxyursan-12-en-28-oic acid, 2alpha,3beta-dihydroxyolean-12-en-28-oic acid, oleanolic acid and ursolic acid were very potent in inhibiting promastigote growth at 100 microg/ml. Of the eleven isolated compounds, however, only ursolic acid and oleanolic acid showed high activity against intracellular amastigotes (IC50 value = 27 microg/ml and 11 microg/ml, respectively), which was superior to the control drug Glucantime (IC50 value = 83 microg/ml). The antileishmanial activity of oleanolic acid was directed against the parasite and not due to activation of nitric oxide intermediates by macrophages, but this triterpenoid also significantly inhibited the phagocytic capacity of those cells at concentrations above 40 microg/ml, indicating a cytotoxic effect. These results indicate that Pourouma guianensis contains many triterpenoids and some, such as ursolic and oleanolic acids, may serve as lead compounds for new antileishmanial drugs, but chemical modifications may be necessary to avoid unselective cytotoxicity.     

Synthesis of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents by Suzuki-Miyaura cross-coupling reactions

A series of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives, structural analogues of alkaloid chimanine B, was synthesized in good yields using efficient palladium(0)-catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon Leishmania amazonensis and Leishmania infantum strains. Biological results showed activity against the promastigote forms of L. amazonensis and L. infantum with IC50 ranging from 0.5 to 7 microM. From a Structure-Activity Relationships point of view, these pharmacological results mainly enlightened the importance of the 4-lateral C6, C7 or C8 alpha-unsaturated trans-alkenyl chain of unsubstituted pyrrolo[1,2-a]quinoxaline moiety.     

Triterpenic acids and flavonoids from Satureja parvifolia. Evaluation of their antiprotozoal activity

Bioassay-guided fractionation of a Satureja parvifolia MeOH extract led to the isolation of eriodictyol, luteolin and ursolic and oleanolic acids as its active components against Plasmodium falciparum K1. This is the first time these compounds are reported as constituents of S. parvifolia. Ursolic acid showed an IC50 of 4.9 microg/ml, luteolin 6.4 microg/ml, oleanolic acid 9.3 microg/ml and eriodictyol 17.2 microg/ml. Antiplasmodial activity of eriodictyol and luteolin is reported here for the first time. Besides, the four compounds showed activity against P. falciparum 3D7 strain and Trypanosoma brucei rhodesiense. Eriodictyol showed moderate activity on all the parasites but was the most selective compound as a result of its rather low cytotoxicity (IC50 174.2 microg/ml) on the mammalian KB cell line.     

Abietane diterpenoids and triterpenoic acids from Salvia cilicica and their antileishmanial activities

Bioguided-fractionation of an acetone extract of the roots of Salvia cilicica (Lamiaceae) led to isolation of two new diterpenes, 7-hydroxy-12-methoxy-20-nor-abieta-1,5(10),7,9,12-pentaen-6,14-dione and abieta-8,12-dien-11,14-dione (12-deoxy-royleanone), together with oleanolic acid, ursolic acid, ferruginol, inuroyoleanol and cryptanol. Their structures were determined spectroscopically, which included HREIMS and 2D NMR spectroscopic analysis. The new abietane derivatives showed appreciable in vitro antileishmanial activity against intracellular amastigote forms of both Leishmania donovani (IC(50) values of 170 and 120 nM, respectively) and Leishmania major (IC(50) values of 290 and 180 nM, respectively). The triterpenoic acids were found to be potently active against amastigote (IC(50) values of 7-120 nM) and moderately active against promastigote stages (IC(50) values of 51-137 nM) of the two Leishmania species.     

Antibacterial activity of triterpene acids and semi-synthetic derivatives against oral pathogens

Triterpene acids (ursolic, oleanoic, gypsogenic, and sumaresinolic acids) isolated from Miconia species, along with a mixture of ursolic and oleanolic acids and a mixture of maslinic and 2-a-hydroxyursolic acids, as well as ursolic acid derivatives were evaluated against the following microorganisms: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, and Enterococcus faecalis, which are potentially responsible for the formation of dental caries in humans. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) during the evaluation of the antibacterial activity. All the isolated compounds, mixtures, and semi-synthetic derivatives displayed activity against all the tested bacteria, showing that they are promising antiplaque and anticaries agents. Ursolic and oleanolic acids displayed the most intense antibacterial effect, with MIC values ranging from 30 microg/mL to 80 microg/mL. The MIC values of ursolic acid derivatives, as well as those obtained for the mixture of ursolic and oleanolic acids showed that these compounds do not have higher antibacterial activity when compared with the activity observed with either ursolic acid or oleanolic acid alone. With regard to the structure-activity relationship of triterpene acids and derivatives, it is suggested that both hydroxy and carboxy groups present in the triterpenes are important for their antibacterial activity against oral pathogens.     

Antileishmanial activity of lapachol analogues

The antileishmanial activity of lapachol, isolapachol, and dihydrolapachol, along with soluble derivatives (potassium salt) and acetate was obtained. All the compounds were assayed against metacyclic promastigotes of two different species of Leishmania associated to tegumentar leishmaniasis: L. amazonensis and L. braziliensis. All compounds presented significant activity, being isolapachol acetate the most active against promastigotes, with IC50/24h = 1.6 +/- 0.0 microg/ml and 3.4 +/- 0.5 microg/ml for, respectively, L. amazonensis and L. braziliensis. This compound was also assayed in vivo against L. amazonensis and showed to be active. Its toxicity in vitro was also established, and at concentration similar to the IC50, no toxicity was evidenced. In all experiments, pentamidine isethionate was used as a reference drug. The present results reinforce the potential use of substituted hydroxyquinones and derivatives as promising antileishmanial drugs and suggest a continuing study within this class of compounds.     

The synthesis and the in vitro cytotoxicity studies of bisnaphthalimidopropyl polyamine derivatives against colon cancer cells and parasite Leishmania infantum

Bisnaphthalimidopropyl derivatives (BNIPSpd, BNIPDaoct, BNIPDanon, BNIPDadec, BNIPDpta and BNIPDeta) were synthesised in yields ranging from 50% to 70% and their cytotoxicity against colon cancer cells (Caco-2) and the parasite Leishmania infantum determined using the MTT assay. Cytotoxicity within Caco-2 cells was manifested with IC(50) values between 0.3 and 22 microM. Compounds with the central longer alkyl chains exhibited the highest cytotoxicity. Against L. infantum, IC(50) values were encompassed within a narrower concentration range of 0.47-1.54 microM. In the parasites, the presence of nitrogen in the central chain and the length of the central alkyl chains did not especially enhance cytotoxicity. This may be due to the way these compounds are transported in the cells.     

The in vitro leishmanicidal activity of hexadecylphosphocholine (miltefosine) against four medically relevant Leishmania species of Brazil

The in vitro leishmanicidal activity of miltefosine? (Zentaris GmbH) was assessed against four medically relevant Leishmania species of Brazil: Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) chagasi. The activity of miltefosine against these New World species was compared to its activity against the Old World strain, Leishmania (Leishmania) donovani, which is known to be sensitive to the effects of miltefosine. The IC50 and IC90 results suggested the New World species harboured similar in vitro susceptibilities to miltefosine; however, miltefosine was approximately 20 times more active against the Old World L. (L.) donovani than against the New World L. (L.) chagasi species. The selectivity index varied from 17.2-28.9 for the New World Leishmania species and up to 420.0 for L. (L.) donovani. The differences in susceptibility to miltefosine suggest that future clinical trials with this drug should include a laboratory pre-evaluation and a dose-defining step.     

Antileishmanial activity of indole alkaloids from Aspidosperma ramiflorum

The present study was designated to evaluate the antileishmanial activity of acid and basic fractions that were obtained after acid-basic extraction, from ethanolic 70% crude extract and pure compounds from the stem bark of Aspidosperma ramiflorum. The basic alkaloidal fraction presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis (LD(50) value<47 microg/ml). Based on these findings, the basic fraction was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 16.3+/-1.6 microg/ml and 4.9+/-0.9 microg/ml, respectively). Our results are promising, showing that these compounds are biologically active against Gram-positive bacteria.     

Design, synthesis and antileishmanial in vitro activity of new series of chalcones-like compounds: a molecular hybridization approach

The chalcone-like series 1a-1g was efficiently synthesized from Morita-Baylis-Hillman reaction (52-74% yields). Compounds 1a-1g were designed by molecular hybridization based on the anti-inflammatory drug methyl salicylate (3) and the antileishmanial moiety of the Morita-Baylis-Hillman adducts 2a-2g. The 1a-1g compounds were much more actives than precursor series 2a-2g, for example, IC(50)=7.65 μM on Leishmania amazonensis and 10.14 μM on Leishmania chagasi (compound 1c) when compared to IC(50)=50.08 μM on L. amazonensis and 82.29 μM on L. chagasi (compound 2c). The IC(50) values of compound 3 (228.49 μM on L. amazonensis and 261.45 μM on L. chagasi) and acryloyl salicylate 4 (108.50 μM on L. amazonensis and 118.83 μM on L. chagasi) were determined here, by the first time, on Leishmania.     

New clerodane diterpenoids from Laetia procera (Poepp.) Eichler (Flacourtiaceae), with antiplasmodial and antileishmanial activities

Extracts of Laetia procera (Flacourtiaceae) displayed significant in vitro activity against Plasmodium falciparum. P. falciparum bioassay guided fractionation of a trunk bark extract of this plant led to the isolation of six clerodane diterpenoids (1-6) and a butanolide (7). Five of these compounds are new and called Laetiaprocerine A-D (3-6) and Laetianolide A (7). Their structures were established on the basis of 1D and 2D NMR experiments. Absolute configurations of 1 and 2 were determined by a modified Mosher's method and the absolute configuration of 5 by chemical correlation. The clerodane diterpenoids displayed activities against P. falciparum with an IC50 down to 0.5 microM on FCb1 and F32 strains, and also cytotoxicity toward human tumor cell line MCF7. The most active compound showed a selectivity index of 6.8. Some of these compounds also displayed activities against Leishmania amazonensis amastigote axenic stages and promastigote.     

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC₅₀) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC₅₀ and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural^(r) (a furvina-containing antifungal ointment) for the treatment of CL.     

熊果酸衍生物的合成及其对肺癌细胞活性抑制的研究

目的：探讨合成的熊果酸衍生物对肺癌LTEP-α-2肿瘤细胞增殖的抑制作用。方法：熊果酸经氧化、肟化和腙化合成为3-氧代熊果酸、3-肟基熊果酸和3-（2＇,4＇-二硝基）-苯腙熊果酸。采用MTT法检测熊果酸及其衍生物对肺癌LTEP-α-2细胞的体外抗肿瘤活性。结果：熊果酸及其衍生物对肺癌LTEP-α-2细胞有抑制作用,且3-肟基熊果酸对肺癌LTEP-α-2细胞抑制作用高于其他化合物,其抑制率为90.0%,IC50为7.4μg/m L。结论：熊果酸衍生物3-肟基熊果酸抗肺癌LTEP-α-2肿瘤细胞活性高于熊果酸的活性。     

Leishmanicidal activity of primary S-nitrosothiols against Leishmania major and Leishmania amazonensis: implications for the treatment of cutaneous leishmaniasis.

Nitric oxide (NO) is considered a key molecule in the defense against intracellular pathogens, particularly Leishmania. The expression of inducible nitric oxide synthase and consequent production of NO by infected macrophages has been shown to correlate with leishmaniasis resistance in the murine model as well as in human patients. Nitric oxide donors have been used successfully in the treatment of cutaneous leishmaniasis in humans, although their mechanisms of action are not fully understood. In the present work, the dose-dependent cytotoxic effects of the NO-donors S-nitroso-N-acetyl-l-cysteine (SNAC) and S-nitrosoglutathione (GSNO) against Leishmania were evaluated. GSNO inhibited the growth of Leishmania major and Leishmania amazonensis with in vitro 50% inhibitory concentrations (IC(50)) of 68.8+/-22.86 and 68.9+/-7.9 micromol L(-1), respectively. The IC(50) for SNAC against L. major and L. amazonensis were, respectively, 54.6+/-8.3 and 181.6+/-12.5 micromol L(-1). The leishmanicidal activity of GSNO, but not of SNAC, was reversed by ascorbic acid (AA) and dithiothreitol (DTT), suggesting that the mechanism of action of GSNO is related to the transnitrosation of parasite proteins. These results demonstrate that SNAC and GSNO have leishmanicidal activity, and are thus potential therapeutic agents against cutaneous leishmaniasis.     

Carnosic acid, a new class of lipid absorption inhibitor from sage

The methanolic extract from the leaves of Salvia officinalis L. (sage) showed significant inhibitory effect on serum triglyceride elevation in olive oil-loaded mice (500 and 1000 mg/kg, p.o.) and inhibitory activity (IC(50): 94 microg/mL) against pancreatic lipase, which is participated in digestion of lipids. Through bioassay-guided separation using the inhibitory activity against pancreatic lipase activity, 4 abietan-type diterpenes (carnosic acid, carnosol, royleanonic acid, 7-methoxyrosmanol) and a triterpene (oleanolic acid) were isolated from the active fraction. Among these compounds, carnosic acid and carnosol substantially inhibited pancreatic lipase activity with IC(50) values of 12 microg/mL (36 microM) and 4.4 microg/mL (13 microM), respectively. Carnosic acid significantly inhibited triglyceride elevation in olive oil-loaded mice at doses of 5-20 mg/kg (p.o.). However, other constituents (carnosol, royleanonic acid, oleanolic acid) did not show any effects at a dose of 200 mg/kg (p.o.). Furthermore, carnosic acid (20 mg/kg/day, p.o.) reduced the gain of body weight and the accumulation of epididymal fat weight in high fat diet-fed mice after 14 days.     

Leishmanicidal Triterpenes from Lantana camara

Two new natural triterpenes, lantaninilic acid and lantoic acid, along with the known triterpenes lantadene A, and oleanolic, ursolic, betulinic, lantanolic, and camaric acid, were obtained from the aerial parts of Lantana camara through bioassay‐guided isolation, monitoring the in vitro antileishmanial activity against promastigotes of Leishmania major. Oleanolic acid ( 3 ), ursolic acid ( 4 ), lantadene A ( 5 ), and lantanilic acid ( 7 ) showed significant leishmanicidal activities with IC₅₀ values of 53.0, 12.4, 20.4, and 21.3 μM , respectively. The IC₅₀ value of ursolic acid ( 4 ; 12.4 μM ) was found to be comparable with that of the standard drugs, pentamidine (IC₅₀ 15.0 μM ) and amphotericin B (IC₅₀ 0.31 μM ). The in vitro activities of L. camara and its constituents against promastigotes of Leishmania major are reported here for the first time.     

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.     

Biological evaluation of substituted quinolines

Several quinolines were synthesized and evaluated in vitro against several parasites (Trypanosoma brucei, T. cruzi, Leishmania infantum, L. amazonensis, Plasmodium falciparum). Then, they were evaluated in vitro (at 10 microM), against HTLV-1 transformed cells. A few of them displayed interesting activities, comparable to the reference drugs.     

Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.