Li-Min Yang

Differential neuropsychopharmacological influences of naturally occurring tropane alkaloids anisodamine versus scopolamine.

Two naturally occurring tropane alkaloids, anisodamine and scopolamine, structurally dissimilar in one OH group, are well established as muscarinic acetylcholine receptor (mAChR) antagonists in clinic and basic research. However, experimental evidence for central effects of anisodamine is limited and conflicting compared with that of scopolamine. In the present study, Morris water maze test, long-term potentiation (LTP) recording and receptor radioligand binding assays were used to explore the disparity in neuropsychopharmacological influences of anisodamine versus scopolamine and possible mechanisms. Anisodamine, at 10-40-fold higher doses than those of scopolamine, did not produce any spatial cognitive deficits as scopolamine, but tended to improve cognition at the repeated high doses. LTP in vivo was then adopted to predict BBB permeability of the muscarinic antagonists following systemic drug administration. Contrary to scopolamine, anisodamine did not influence the formation of LTP in the CA(1) region of rat hippocampus at 40-fold higher dose than that of scopolamine. Additionally, receptor radioligand binding assays (RRLBA) revealed that the binding affinity of anisodamine to mice brain mAChR was much lower than that of scopolamine. The findings suggested that anisodamine did not impair cognition nor depress LTP primarily due to its poor BBB permeability. This work enlarged knowledge of structure-activity relationship among tropane alkaloids, meanwhile providing evidence for more reasonable drug prescription in clinic.

Stereoselectivity of satropane, a novel tropane analog, on iris muscarinic receptor activation and intraocular hypotension

AbstractAim:To study the stereoselectivity of satropane (3-paramethylbenzene sulfonyloxy-6-acetoxy tropane), a novel tropane analog, on iris muscarinic receptor activation and intraocular hypotension.Methods:The assays for radioligand-receptor binding, the contractile responses of isolated iris muscle, the miosis response, and the intraocular hypotension of the enantiomers of satropane were investigated.Results:In the binding analysis, S(-)satropane (lesatropane) completely competed against the [3H]quinuclydinyl benzilate-labeled ligand at muscarinic receptors in the iris muscle, whereas R(+)satropane failed to completely compete. In an isolated iris contractile assay, R,S(±)satropane and S(-)satropane produced a concentration-dependent contractile response with similar efficacy and potency to that of carbachol. R(+)satropane did not induce any contractile response. In the pupil diameter measurement assay in vivo, S(-)satropane induced miosis much more effectively than pilocarpine, while R(+)satropane failed to produce any miosis. In the water loading-induced and methylcellulose-induced ocular hypertensive models, S(-)satropane, but not R(+)satropane, significantly suppressed intraocular pressure at a much lower concentration than pilocarpine.Conclusion:The agonistic and hypotensive properties of satropane on rabbit eyes are stereoselective, with the S(-)isomer being its active form.

Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

Aim:To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells, and the underlying mechanisms.Methods:PC12 cells were treated with different concentrations of glutamate for 24 or 48 h. The cell viability was measured using MTT assay, and the expression and activation of GSK-3β were detected with Western blot. β-Catenin translocation was detected using immunofluorescence. Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin.Results:Glutamate (1, 3, and 10 mmol/L) induced PC12 cell death in a dose-dependent manner. Moreover, treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus. Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation, and markedly enhanced β-catenin transcriptional activity.Conclusion:Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation, suggesting potential benefits of muscarinic agonists for Alzheimers disease.

(1R,3S,5R,6S)-6-Acet­oxy-3-(4-methyl­phenyl­sulfon­yloxy)tropane

In the title compound [systematic name: (1R,3S,5R,6S)-8-methyl-3-(4-methylphenylsulfonyloxy)-8-azabicyclo[3.2.1]octan-6-yl acetate], C17H23NO5S, the fused piperidine ring exists in a chair conformation with the N atom and one C atom displaced by 0.876 (2) and −0.460 (3) Å, respectively, on opposite sides of the mean plane defined by the other four atoms. The fused pyrrolidine ring adopts an envelope conformation with the N atom deviating by 0.644 (3) Å from the mean plane of the other four atoms.

Quantitative determination of a novel enantiomeric tropane analog, (−)-satropane, in biological fluids using liquid chromatography/tandem mass spectrometry

A sensitive, accurate and precise liquid chromatography-tandem mass spectrometry method was developed for the determination of (-)-satropane (3alpha-paramethyl-benzenesulfonyloxy-6beta-acetoxy-tropane) in rabbit aqueous humor. Since (-)-satropane may be absorbed from the aqueous humour with resultant systemic side effects, the LC-MS/MS method was also evaluated for its applicability in analyzing plasma samples containing this compound. (-)-Satropane and phentolamine (the internal standard, represented as IS) were detected by multiple reaction monitoring using the transitions m/z 354-182 and 282-212, respectively. The calibration curve was linear over the ranges 2-500 and 5-1000 ng/mL, and the values of the lower limit of quantification were 2 and 5 ng/mL for the microdialysis dialysate and rat plasma samples, respectively. The intra-day and inter-day precision and accuracy were better than 8.6 and 6.00%, respectively, in both matrices investigated. The absolute recovery of the plasma samples was more than 76.30%. The average matrix effects of (-)-satropane were 91.72 and 83.05% in the microdialysis dialysate and plasma samples, respectively. The validated method was successfully applied to analyze (-)-satropane in microdialysis dialysate and rat plasma samples, and this assay has been used to quantify (-)-satropane in the pharmacokinetic and toxicokinetic studies in our laboratory.

Development and validation of a reversed-phase HPLC method for determination of lesatropane and enantiomeric impurity

Lesatropane is a novel muscarinic receptor agonist and is currently being under preclinical development in China as a single enantiomer drug for the treatment of primary glaucoma. A reversed-phase chiral HPLC method for determination of lesatropane and enantiomeric impurity was developed. Enantiomeric separation of lesatropane from its enantiomer (desatropane) was achieved in normal-phase mode with Chiralpak AD-H and in reversed-phase mode with Chiralpak AS-RH. The conditions using a Chiralpak AS-RH column and mobile phase of K(2) HPO(4) -KH(2) PO(4) (pH 7.0; 0.02 M)-acetonitrile (69:31, v/v) at a flow rate of 0.5 ml/min have been fully validated with satisfactory specificity, linearity, accuracy, and precision. The method was found to be suitable for the simultaneous quantitation of lesatropane and enantiomeric impurity desatropane.

(1R,3S,5R,6S)-6-Acet­oxy-8-methyl-3-(p-tolyl­sulfon­yloxy)-8-azoniabicyclo­[3.2.1]octane (2R,3R)-2,3-bis­(benzo­yloxy)-3-carboxy­propanoate

The title compound, C17H24NO5S+·C18H13O8 −, is the key intermediate during the preparation of lesatropane [systematic name (1R,3S,5R,6S)-6-acetoxy-3-(4-methylphenylsulfonyloxy)tropane], a potential antiglaucoma agent. The tertiary N atom of the tropane ring is involved in intermolecular N—H⋯O hydrogen bonding, and the carboxylate groups are involved in intermolecular O—H⋯O hydrogen bonding.

(1R,3S,5R,6S)-6-Hydr­oxy-3-tosyl­oxy­tropan-8-ium chloride

The title compound, C15H22NO4S+·Cl−, is a hydrolysis product of lesatropane [(1R,3S,5R,6S)-6-acetoxy-3-tosyloxytropane] hydrochloride, a potential antiglaucoma agent. As in lesatropane, the piperidine and pyrrolidine rings in the title compound adopt chair and envelope conformations, respectively. There are two molecules in the unit cell with similar conformations. The crystal structure is stabilized by intermolecular O—H⋯Cl and N—H⋯Cl hydrogen bonds.