ABSTRACT
The search for new drugs for the treatment of leishmaniasis is an important strategy for improving the current therapeutic arsenal for the disease. There are several limitations to the available drugs including high toxicity, low efficacy, prolonged parenteral administration, and high costs. Steroids are a diverse group of compounds with various applications in pharmacology. However, the antileishmanial activity of this class of molecules has not yet been explored. Therefore, in the present study, we investigated the antileishmanial activity and cytotoxicity of novel steroids against murine macrophages with a focus on the derivatives of cholesterol (CD), cholic acid (CA), and deoxycholic acid (DA). Furthermore, the mechanism of action of the best compound was assessed, and in silico studies to evaluate the physicochemical and pharmacokinetic properties were also conducted. Among the sixteen derivatives, schiffbase2, CD2 and deoxycholic acid derivatives (DOCADs) were effective against promastigotes of Leishmania species. Despite their low toxicity to macrophages, the majority of DOCADs were active against intracellular amastigotes of L. amazonensis, and DOCAD5 exhibited the best biological effect against these parasitic stages (IC50 = 15.34 µM). Neither the CA derivatives (CAD) nor DA alone inhibited the intracellular parasites. Thus, the absence of hydroxyl in the C-7 position of the steroid nucleus, as well as the modification of the acid group in DOCADs were considered important for antileishmanial activity. The treatment of L. amazonensis promastigote forms with DOCAD5 induced biochemical changes such as depolarization of the mitochondrial membrane potential, increased ROS production and cell cycle arrest. No alterations in parasite plasma membrane integrity were observed. In silico physicochemical and pharmacokinetic studies suggest that DOCAD5 could be a good candidate for an oral drug. The data demonstrate the potential antileishmanial effect of certain steroid derivatives and encourage new in vivo studies.
Subject(s)
Cholesterol/pharmacology , Deoxycholic Acid/pharmacology , Drug Discovery/methods , Leishmania/drug effects , Leishmaniasis/drug therapy , Macrophages, Peritoneal/drug effects , Trypanocidal Agents/pharmacology , Administration, Oral , Animals , Cell Cycle Checkpoints/drug effects , Cholesterol/analogs & derivatives , Cholesterol/chemical synthesis , Cholesterol/pharmacokinetics , Cholic Acid/chemical synthesis , Cholic Acid/pharmacokinetics , Cholic Acid/pharmacology , Deoxycholic Acid/analogs & derivatives , Deoxycholic Acid/chemical synthesis , Deoxycholic Acid/pharmacokinetics , Dose-Response Relationship, Drug , Leishmania/growth & development , Leishmania/metabolism , Leishmaniasis/parasitology , Macrophages, Peritoneal/parasitology , Membrane Potential, Mitochondrial/drug effects , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondria/metabolism , Models, Biological , Molecular Structure , Oxidative Stress/drug effects , Parasitic Sensitivity Tests , Structure-Activity Relationship , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/pharmacokineticsABSTRACT
Lopezia racemosa Cav. is a plant used in Mexican traditional medicine to heal inflammatory diseases. From this plant we isolated the novel compound 6-O-palmitoyl- 3-O-ß-D-glucopyranosylcampesterol (1) and 6-O-palmitoyl-3-O-ß-D-glucopyranosyl-ß-sitosterol (2), previously reported to have cytotoxic activity on several cancer cell lines. We evaluated the anti-inflammatory activity of 1 in vivo by mouse ear edema induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 57.14% inhibition was observed. The aim of our study was to obtain callus cultures derived from this plant species with the ability to produce the compounds of interest. Callus cultures were initiated on MS basal medium amended with variable amounts of naphthaleneacetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D), combined or not with 6-benzylaminopurine (BAP). Ten treatments with these growth regulators were carried out, using in vitro germinated seedlings as source of three different explants: hypocotyl, stem node, and leaf. Highest yield of 1 was observed on callus derived from leaf explants growing in medium containing 1.0 mg/L 2,4-D and 0.5 mg/L BAP. Selected callus lines produced less 1 than wild plants but the in vitro cultured seedlings showed higher production. So we conclude that it could be attractive to further investigate their metabolic potential.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Cholesterol/analogs & derivatives , Inflammation/drug therapy , Onagraceae/metabolism , Phytosterols/pharmacology , Plant Extracts/pharmacology , Animals , Anti-Inflammatory Agents/chemical synthesis , Cells, Cultured , Cholesterol/chemical synthesis , Cholesterol/chemistry , Cholesterol/pharmacology , Ear/pathology , Edema/drug therapy , Germination/physiology , Male , Mice , Phytochemicals/pharmacology , Phytosterols/chemical synthesis , Phytosterols/chemistry , Seeds/physiology , Tetradecanoylphorbol AcetateABSTRACT
7-Hydroperoxycholesterol is considered to be an intermediate compound of the cholesterol oxidation path as the first product formed when cholesterol is oxidized by triplet oxygen. However, there is a limitation on cholesterol mechanism studies because of the lack of 7-hydroperoxycholesterol analytical standard due to its low stability. To verify the formation of hydroperoxides in cholesterol model systems heated at 140, 180, and 220 °C, 7α-hydroperoxycholesterol was synthesized by cholesterol photooxidation followed by rearrangement at room temperature in chloroform. Its structure was confirmed on the basis of 13C NMR and mass spectra obtained by APCI-LC-MS. The synthesized compound was also used as standard for the quantification of 7-hydroperoxycholesterol as the sum of 7α- and 7ß-hydroperoxycholesterol. The results demonstrated that 7-hydroperoxycholesterol is the first compound formed when the temperature is lower (140 °C). However, the concentration of the 7-hydroperoxycholesterol depends on the temperature and time of exposure: the higher the time, the higher the amount of 7-hydroperoxycholesterol at lower temperatures, and the lower the time, the lower the amount of 7-hydroperoxycholesterol at higher temperatures (180 and 220 °C). By the formation of 7-hydroperoxycholesterol, the known cholesterol oxidation mechanism in three phases (initiation, propagation, and termination) could be confirmed; once at lower temperatures, the stage of cholesterol oxidation is at initiation, at which hydroperoxide formation predominates.
Subject(s)
Cholesterol/analogs & derivatives , Hot Temperature , Models, Chemical , Cholesterol/analysis , Cholesterol/chemical synthesis , Cholesterol/chemistry , Cholesterol/isolation & purification , Kinetics , Photochemical ProcessesABSTRACT
Disodium 3beta,21-dihydroxypregn-5-en-20-one disulfate (2), sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3), sodium 3beta,21-dihydroxypregn-5-en-20-one 21-sulfate (4), and disodium 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one disulfate (6) have been synthesized and completely characterized for the first time from readily available materials. Sulfation was performed using triethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. Selective sulfation of 3beta,21-dihydroxypregn-5-en-20-one rendered sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3) as the major compound. The synthetic sulfated steroids as well as natural disulfated polyhydroxysteroids (7-9) isolated by us from the antarctic ophiuroid Astrotoma agassizii and the synthetic derivatives disodium 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene 3-acetate, 2,21-disulfate (7a) and 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene (7b) were comparatively evaluated for their inhibitory effect on the replication of one DNA (HSV-2) and two RNA (PV-3, JV) viruses. In general, steroids with sulfate groups at C-21 and C-2 or C-3 were the most effective in their inhibitory action against HSV-2 and also proved to be active against PV-3 and JV.