Barreiro Eliezer JLaboratorio de Avaliacoo e Sintese de Substancias Bioativas (LASSBio), Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, C. P. 68.006, 21944-910, Rio de Janeiro, RJ BrazilPhone : Fax :
Sulfonylhydrazones are privileged structures with multifaceted pharmacological activity. Exploring the hypoglycemic properties of these organic compounds, we previously revealed a new series of N-sulfonylhydrazones (NSH) as antidiabetic drug candidates. Here, we evaluated the microsomal metabolism, chemical stability, and permeability profile of these NSH prototypes, focusing on the pharmacokinetic differences in N-methylated and non-N-methylated analogs. Our results demonstrated that the N-methylated analogs (LASSBio-1772 and LASSBio-1774) were metabolized by CYP, forming three and one metabolites, respectively. These prototypes exhibited chemical stability at pH 2.0 and 7.4 and brain penetration ability. On the other hand, non-N-methylated analogs (LASSBio-1771 and LASSBio-1773) were hydrolyzed in acid pH and could not cross the artificial blood-brain barrier. The cyano group in LASSBio-1771 was postulated as a possible site of interaction with the heme group, potentially inhibiting CYP enzymes. Moreover, prototypes with the methyl ester group were metabolized by carboxylesterase, and non-N-methylated analogs did not show oxidative metabolism. The prototypes (except LASSBio-1774) showed excellent gastrointestinal absorption. Altogether, our data support the idea that the methyl effect on NSH strongly alters their pharmacokinetic profile, enhances the recognition by CYP enzymes, promotes brain penetration, and plays a protective effect upon acid hydrolysis.
Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.
BACKGROUND AND PURPOSE: Compound LASSBio-881 is an orally effective antinociceptive that binds to cannabinoid receptors and is active mainly on the neurogenic component of pain models. We investigated whether transient receptor potential vanilloid subfamily type 1 (TRPV1) channels are involved in the effects of LASSBio-881. EXPERIMENTAL APPROACH: Modulation of capsaicin (CAP)- and low pH-induced currents was evaluated in TRPV1-expressing Xenopus oocytes. In vivo effects were evaluated in CAP-induced acute and inflammatory changes in nociception, as well as in partial sciatic ligation-induced thermal hypernociception. KEY RESULTS: LASSBio-881 inhibited TRPV1 currents elicited by CAP with an IC(50) of 14 microM, and inhibited proton-gated currents by 70% at 20 microM. Functional interaction with CAP was surmountable. Locally applied LASSBio-881 decreased time spent in CAP-elicited nocifensive behaviour by 30%, and given orally it reduced measures of CAP- or carrageenan-evoked thermal hypernociception by 60 and 40% respectively. In addition, LASSBio-881 decreased the paw withdrawal responses to thermal stimuli of animals with sciatic neuropathy 7-11 days after nerve ligation, at a dose of 300 micromol*kg(-1)*day(-1) p.o. At this dose, hyperthermia was not observed within 4 h following oral administration. CONCLUSIONS AND IMPLICATIONS: LASSBio-881 is a TRPV1 antagonist that apparently competes with CAP. Accordingly, LASSBio- 881 inhibited nociception in models of acute, inflammatory and neuropathic pain presumed to involve TRPV1 signalling. These in vivo actions were not hindered by hyperthermia, a common side effect of other TRPV1 antagonists. We propose that the antinociceptive properties of LASSBio-881 are due to TRPV1 antagonism, although other molecular interactions may contribute to the effects of this multi-target drug candidate.
LASSBio-767 [(-)-3-O-acetyl-spectaline] and LASSBio-822 [(-)-3-O-tert-Boc-spectaline] were recently described as cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline, obtained from the flowers of Senna spectabilis (Fabaceae). We investigated their mechanism of inhibition of acetylcholinesterase and their efficacy in reversing scopolamine-induced amnesia. Competition assays with the substrate acetylthiocholine showed a concentration-dependent reduction in rat brain cholinesterase Vmax without changes in apparent Km. The kinetic data for LASSBio-767 and LASSBio-822 were best fit by a model of simple linear noncompetitive inhibition with Ki of 6.1 microM and 7.5 microM, respectively. A dilution assay showed a fast and complete reversal of inhibition, independent of incubation time. Simulated docking of the compounds into the catalytic gorge of Torpedo acetylcholinesterase showed interactions with the peripheral anionic site, but not with the catalytic triad. Anti-amnestic effects in mice were assessed in a step-down passive avoidance test and in the Morris water maze 30 min after injection of scopolamine (1 mg/kg i.p.). Saline, LASSBio-767, or LASSBio-822 was administered 15 min before scopolamine. Both compounds reversed the scopolamine-induced reduction in step-down latency at 0.1 mg/kg i.p. LASSBio-767 reversed scopolamine-induced changes in water maze escape latency at 1 mg/kg i.p. or p.o., while its cholinergic side effects were absent or mild up to 30 mg/kg i.p. (LD50 above 100 mg/kg i.p.). Thus, the (-)-spectaline derivatives are potent cholinergic agents in vivo, with a unique profile combining noncompetitive cholinesterase inhibition and CNS selectivity, with few peripheral side effects.
The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro- 2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2- (1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1, 3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1, 3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated Na2CO3 solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 UH/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 UH/mL solvent) and 5.0 mL of Na2CO3(sat) at 40 degrees C for 6 h.
Five new piperidine alkaloids were designed from natural (-)-3-O-acetyl-spectaline and (-)-spectaline that were obtained from the flowers of Senna spectabilis (sin. Cassia spectabilis, Leguminosae). Two semi-synthetic analogues (7 and 9) inhibited rat brain acetylcholinesterase, showing IC50 of 7.32 and 15.1 microM, and were 21 and 9.5 times less potent against rat brain butyrylcholinesterase, respectively. Compound 9 (1mg/kg, i.p.) was fully efficacious in reverting scopolamine-induced amnesia in mice. The two active compounds (7 and 9) did not show overt toxic effects at the doses tested in vivo.
Alzheimer's disease (AD) is a progressive neurodegenerative pathology with severe economic and social impact. There is currently no cure, although cholinesterase inhibitors provide effective temporary relief of symptoms in some patients. Nowadays, drug research and development are based on the cholinergic hypothesis that supports the cognition improvement by regulation of the synthesis and release of acetylcholine in the brain. There are only four commercial medicines approved for treatment of AD, and natural products have played an important alternative role in the research for new acetylcholinesterase inhibitors, as exemplified through the discovery of galantamine. This profile conducts us to give in this paper an overview relating the several classes of natural products with anti-cholinesterasic activity as potential templates to the design of new selective and powerful anti-Alzheimer drugs.
The flowers of Cassia spectabilis yielded three new piperidine alkaloids, (-)-3-O-acetylspectaline (1), (-)-7-hydroxyspectaline (2), and iso-6-spectaline (3), together with the known (-)-spectaline (4). The green fruits of this plant were also investigated, resulting in the isolation of 1 and 4. Their structures were elucidated using a combination of multidimensional NMR and MS techniques, and relative stereochemistries were established by NOESY correlations and analysis of coupling constants. The DNA-damaging activity of these compounds was evaluated using a mutant yeast, Saccharomyces cerevisiae, assay.
Title: Design, synthesis, and pharmacological profile of novel fused pyrazolo[4,3-d]pyridine and pyrazolo[3,4-b][1,8]naphthyridine isosteres: a new class of potent and selective acetylcholinesterase inhibitors Barreiro EJ, Camara CA, Verli H, Brazil-Mas L, Castro NG, Cintra WM, Aracava Y, Rodrigues CR, Fraga CA Ref: Journal of Medicinal Chemistry, 46:1144, 2003 : PubMed
A new family of tacrine (THA) analogues (7-9, 12), containing the azaheterocyclic pyrazolo[4,3-d]pyridine or pyrazolo[3,4-b][1,8]naphthyridine systems as isosteres of the quinoline ring of THA, has been synthesized. The compounds were tested in rat brain cholinesterases using Ellman's method, and all were fully efficacious in inhibiting the enzymes. Compounds 9 and 12b were the most potent against acetylcholinesterase (AChE), showing IC(50) of 6.0 and 6.4 microM, and were less active against rat brain butyrylcholinesterase, showing selectivity indexes of 5.3 and 20.9, respectively. Compounds 7-9 and 12 were also tested for their acute neurotoxicity in vitro, using cultured rat cortical cells. Compounds 7 and 8 were not significantly toxic; 9 was toxic at 500 microM, but not at 100 microM. The naphthyridine derivatives 12a and 12b showed a significant concentration-dependent neurotoxicity, being able to kill most cells at 500 microM. Molecular dynamic simulation using the X-ray crystal structure of AChE from Torpedo californica was used to explain the possible binding mode of these new THA isosteres.
The present study describes the synthesis and pharmacological profile of three novel heterocyclic compounds originally designed, on the basis of bioisosterism, as dopamine D2 receptor ligands: 1-[1-(4-chlorophenyl)-1H-pyrazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-579), 1-phenyl-4-(1-phenyl-1H-[1,2,3]triazol-4-ylmethyl)-piperazine (LASSBio-580) and 1-[1-(4-chlorophenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-581). Binding studies performed on brain homogenate indicated that all three compounds bind selectively to D2 receptors. In addition, electrophysiological studies carried out in cultured hippocampal neurons suggested that LASSBio-579 and 581 act as D2 agonists, whereas LASSBio-580 acts as a D2 antagonist.
This study was designed to investigate the effects on single skeletal muscle fibers of a novel thienylhydrazone, referred to as LASSBio-294, which is a bioisoster of pyridazinone compounds that inhibit the cyclic AMP-specific phosphodiesterase (PDE) 4. Twitch and fatigue were analyzed in single skeletal muscle fibers isolated from either the semitendinous or the tibialis anterior muscles dissected from the frog Rana pipiens. LASSBio-294 (12.5-100 microM) increased twitch tension, accelerated the maximal rate of tension decay during relaxation, and had very little effect in the maximal rate of tension development of muscle fibers directly stimulated at < or =30 Hz. The positive inotropic effect of LASSBio-294 developed slowly, reaching its maximum at 40 min and was inversely proportional to the frequency of stimulation, becoming negligible at 60 and 90 Hz. The concentration-response relationship for LASSBio-294-induced potentiation of twitch tension was bell-shaped, with maximal effect occurring at 25 microM. In addition, LASSBio-294 reduced development of fatigue induced by tetanic stimulation of the muscle fibers and reduced the time needed for 80% prefatigue tension recovery after fatigue had developed to 50% of the maximal pretetanic force. These effects of LASSBio-294 can be fully explained by stimulation of the sarcoplasmic reticulum Ca2+ pump and could be ascribed to an increase in cellular levels of cyclic AMP due to PDE inhibition. The novel thienylhydrazone LASSBio-294 may be useful for treatment of patients suffering from conditions in which muscle fatigue is a debilitating symptom (e.g., chronic heart failure).
Title: Pyrazole, an alcohol dehydrogenase inhibitor, has dual effects on N-methyl-D-aspartate receptors of hippocampal pyramidal cells: agonist and noncompetitive antagonist Pereira EF, Aracava Y, Aronstam RS, Barreiro EJ, Albuquerque EX Ref: Journal of Pharmacology & Experimental Therapeutics, 261:331, 1992 : PubMed
Electrophysiological and biochemical studies demonstrated that pyrazole, an inhibitor of alcohol dehydrogenase and a proposed therapeutic agent for treatment of alcoholic intoxication, activated and blocked the N-methyl-D-aspartate (NMDA) receptor and did not interact significantly with the end-plate nicotinic acetylcholine receptor (AChR). Pyrazole, at concentrations as low as 0.5 microM, applied to outside-out patches excised from the membrane of cultured rat hippocampal neurons, elicited single-channel currents of 48 pS which were blocked by DL-2-amino-5-phosphorovaleric acid, a competitive antagonist of NMDA. In addition, binding studies showed that pyrazole displaced 1-(cis-2-carboxypiperidine-4-yl)methyl-1-phosphoric acid from the agonist recognition site of the NMDA receptor in a concentration-dependent manner and enhanced the binding of (+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine to this complex. These data indicate that pyrazole is an agonist at NMDA receptors. However, at higher concentrations, open and burst times as well as the frequency of single-channel currents activated by pyrazole were reduced significantly, a finding which suggests that this compound is also an open channel blocker. In agreement with these results, it was shown biochemically that pyrazole was able to stimulate influx of Ca++ into rat brain microsomes via NMDA receptors and on the other hand to block the influx of Ca++ induced by NMDA. Pyrazole was unable to affect the neuromuscular transmission of frog sartorius muscle-sciatic nerve preparations. Additionally, pyrazole did not interact either with the agonist recognition site or with noncompetitive sites of the AChR. However, this drug had a very weak agonist-like action on the AChR of the Torpedo electric organ, most likely via binding sites different from those described previously for acetylcholine. Therefore, the therapeutic efficacy of pyrazole may be related at least in part to its effects on the NMDA receptor. Furthermore, this compound, because of the small size and rigidity of its molecular structure, becomes a promising drug for the study of the NMDA receptor. Indeed its use may allow a better understanding of the physiological and pathological processes involving this receptor.
