Charge Transfer Complex of Neostigmine with 2,3-Dichloro-5,6-Dicyano-1,4-Benzoquinone: Synthesis, Spectroscopic Characterization, Antimicrobial Activity, and Theoretical Study.

BACKGROUND: Electron donor-acceptor interactions are important molecular reactions for the activity of pharmacological compounds. The aim of the study is to develop a charge transfer (CT) complex: synthesis, characterization, antimicrobial activity, and theoretical study. METHOD AND RESULTS: A solid CT complex of neostigmine (NSG) with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was synthesized and characterized by infrared spectra, NMR, and UV-visible spectroscopy. The results confirm the formation of a CT complex. The stability of the CT complex between NSG and DDQ in acetonitrile was determined in solution via spectrophotometric measurement, ie, by calculating the formation constant, molar extinction coefficient, and different spectroscopic parameters. The stoichiometry of the formed NSG-DDQ complex was determined using Job's method. The absorption band of the NSG-DDQ complex can be used for the quantification of NSG. CONCLUSION: The DFT geometry optimization of NSG, DDQ, and the CT complex and the UV comparative study of both theoretical and experimental structures are presented. The experimental results confirm the charge transfer structure. The bacterial study shows that the NSG-DDQ complex has good antibacterial activity against both Gram-positive and Gram-negative bacteria as well as antifungal activity against Candida albicans.

A ratiometric fluorescence probe based on graphene quantum dots and o-phenylenediamine for highly sensitive detection of acetylcholinesterase activity.

By using graphene quantum dots (GQDs) and o-phenylenediamine (OPD), a ratiometric fluorescence probe was designed for the highly sensitive and selective detection of AChE. GQDs with strong fluorescence were synthesized by the one-step hydrothermal method. The optimal emission wavelength of GQDs was 450 nm at the excitation wavelength of 375 nm. MnO2 nanosheets with a wide absorption band of 300-600 nm were prepared at room temperature. Because of the extensive overlap between the absorption spectrum of MnO2 nanosheets and the excitation and emission spectra of GQDs, the fluorescence of GQDs at 450 nm was efficiently quenched by the inner-filter effect. Meanwhile, due to the peroxidase-like activity of MnO2 nanosheets, OPD was catalytically oxidized to 2,3-diaminophenazine (oxOPD), a yellow fluorescent substance with a new emission peak at 572 nm. When AChE was present, the substrate acetylthiocholine (ATCh) was hydrolyzed to thiocholine (TCh) that is capable of decomposing MnO2 nanosheets. Therefore, the quench of GQDs and the oxidation of OPD by MnO2 nanosheets were suppressed, resulting in the fluorescence recovery of GQDs at 450 nm, while the fluorescence decrease of oxOPD at 572 nm. Utilizing the fluorescence intensity ratio F450/F572 as the signal readout, the ratiometric fluorescence method was established to detect AChE activity. The ratio F450/F572 against the AChE concentration demonstrated two linear relationships in the range 0.1-2.0 and 2.0-4.5 mU mL-1 with a detection limit of 0.09 mU mL-1. The method was applied to the detection of positive human serum samples and the analysis of the inhibitor neostigmine. Due to the advantages of high sensitivity, favorable selectivity, and strong anti-interference, the method possesses an application prospect in clinical diagnosis of AChE and the screening of inhibitors. Graphical abstract Schematic presentation of a ratiometric fluorescence method for the detection of acetylcholinesterase (AChE). The fluorescence of graphene quantum dots (GQDs) is quenched and o-phenylenediamine (OPD) is oxidized to generate fluorescent product 2,3-diaminophenazine (oxOPD) by MnO2 nanosheets. When AChE is present, acetylthiocholine iodide (ATCh) is hydrolyzed to thiocholine (TCh) with reducibility for decomposing MnO2 nanosheets. Due to the decomposition of MnO2 nanosheets, the quenching of GQDs and oxidation of OPD are suppressed. The fluorescence of GQDs at 450 nm is enhanced, while the fluorescence of oxOPD at 572 nm is reduced. The fluorescence intensity ratio F450/F572 is used to establish the ratiometric fluorescence method for AChE activity.

Synthesis, Characterization and Cholinesterase Inhibition Studies of New Arylidene Aminothiazolylethanone Derivatives.

BACKGROUND: Alzheimer's disease is caused by the destruction or loss of cholinergic cells that produce or use ACh in the brain, thereby reducing the availability of enzyme to other cells. The major treatment strategy for AD is to decrease the level of cholinesterase in the brain. OBJECTIVE: The aim of this study was to describe the effect of novel series of thiazole derivatives i.e. arylidene aminothiazolylethanones (3a-h) as cholinesterase inhibitors (CEIs). METHOD: A novel series of thiazole derivatives i.e. arylidene aminothiazolylethanones (3a-h) was synthesized by treating 3-chloropentane-2,4-dione (1) with urea followed by reaction with suitably substituted benzaldehydes. Structural confirmation of all the synthesized compounds was carried out by spectroscopic techniques (FTIR, 1H and 13CNMR) and elemental analysis. Furthermore, these derivatives were subjected to biological evaluation as potential inhibitors of cholinesterases i.e. acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). RESULTS: In all synthesized compounds except two compounds i.e. 3a and 3f, all compounds were identified as selective inhibitors of AChE. Compound 3a exhibited potent inhibitory values against AChE (IC50± SEM = 1.78±0.11 µM), exhibiting ≈7 times greater selectivity for AChE over BChE. Kinetics studies were performed to find out the mechanism of inhibition against respective enzyme. In addition, molecular docking studies of most potent inhibitors were also carried out to determine the binding interactions with AChE and BChE, respectively. CONCLUSION: In this study, novel thiazole derivatives i.e. arylidene aminothiazolylethanones were successfully synthesized, characterized and further screened for threir potential as cholinesterase inhibitors. All compounds were found as potent selective inhibitors of AChE except two compounds which exhibited dual inhibitory activities but both of these compounds were highly selective toward AChE as compared to BChE.

Epidural administration of neostigmine-loaded nanofibers provides extended analgesia in rats.

BACKGROUND: In this study, neostigmine-loaded electrospun nanofibers were prepared and then their efficacy and duration of analgesic action were studied after epidural administration in rats by repeated tail flick and formalin tests. METHODS: The neostigmine poly vinyl alcohol (PVA) nanofibers were fabricated by electrospinning methods. The nanofibers (1 mg) were injected into the lumbar epidural space (L5-L6) of rats (n = 6). Cerebrospinal fluid samples of rats were collected 1, 5 and 24 hours after injection and then were sampled once weekly for 4 weeks. Free-neostigmine concentration was measured in the samples spectrophotometrically. Rat nociceptive responses were evaluated by repeated tail-flick and formalin tests for 5 weeks after the nanofibers (1 mg) injection. Locomotor activity of rats was measured in the open-field at the same period. RESULTS: The cerebrospinal fluid concentration of free neostigmine reached 5 µg/ml five hours after injection and remained constant until the end of the experiments. The tail-flick latency of treated rats was significantly (p < 0.01) increased and remained constant up to 4 weeks. Pain scores of the rats in both phases of formalin test were significantly (p < 0.01) reduced during the same periods, Epidural injection of the nanofibers had no effect on locomotor activity of rats in an open-field. CONCLUSIONS: Our results indicate that the neostigmine nanofibers can provide sustained release of neostigmine for induction of prolonged analgesia after epidural administration. High tissue distribution and penetration of the nanofibers in dorsal horn can increase thermal and chemical analgesia duration without altering locomotor activity in rats for 4 weeks.

Development and validation of a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry assay for the determination of neostigmine in small-volume beagle dog plasma and its application to a pharmacokinetic study.

A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 µL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs.

Long-acting anticholinesterases for myasthenia gravis: synthesis and activities of quaternary phenylcarbamates of neostigmine, pyridostigmine and physostigmine.

The N-monophenylcarbamate analogues of neostigmine methyl sulfate (6) and pyridostigmine bromide (8) together with their precursors (5), (7), and the N(1)-methylammonium analogues of (-)-phenserine (12), (-)-tolserine (14), (-)-cymserine (16) and (-)-phenethylcymserine (18) were synthesized to produce long-acting peripheral inhibitors of acetylcholinesterase or butyrylcholinesterase. Evaluation of their cholinesterase inhibition against human enzyme ex vivo demonstrated that, whereas compounds 5-8 possessed only marginal activity, 12, 14, 16 and 18 proved to be potent anticholinesterases. An extended duration of cholinesterase inhibition was determined in rodent, making them of potential interest as long-acting agents for myasthenia gravis.

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 microL/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 microM to 10 mM using this method and Z' values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method.

Molecular modelling and QSAR of reversible acetylcholines-terase inhibitors.

Acetylcholinesterase (AChE) inhibitors are an important class of medicinal agents useful for the treatment of Alzheimer s disease, glaucoma, myasthenia gravis and for the recovery of neuromuscular block in surgery. To rationalize the structural requirements of AChE inhibitors we attempt to derive a coherent AChE-inhibitor recognition pattern based on literature data of molecular modelling and quantitative structure-activity relationship (QSAR) analyses. These data are summarised from nearly all therapeutically important chemical classes of reversible AChE inhibitors, e.g., derivatives of physostigmine, tacrine, donepezil and huperzine A. Interactions observed from X-ray crystallography between these inhibitors and AChE have also been incorporated and compared with modelling and QSAR results. It is concluded that hydrophobicity and the presence of an ionizable nitrogen are the pre-requisites for the inhibitors to interact with AChE. However the mode of interaction i.e., the 3-dimensional (3D) positioning of the inhibitor in the active site of the enzyme varies among different chemical classes. It is also recognised that water molecules play crucial roles in defining these different 3D positioning. The information on AChE-inhibitor interactions provided should be useful for future discovery of new chemical classes of AChE inhibitors, especially from De Novo design and hybrid construction.

Application of the WATR technique for water suppression in 1H NMR spectroscopy in determination of the kinetics of hydrolysis of neostigmine bromide in aqueous solution.

Both ammonium chloride and guanidinium chloride were used to secure water suppression in 1H NMR spectra using the 'Water Attenuation by T2 Relaxation' (WATR) technique. The effect of phosphate buffer in the suppression was investigated over a range of pH values at 80 MHz. The spin-spin relaxation time of water protons at 80 MHz was found to reach a minimum at pH 7.3 in the presence of 0.1 M phosphate buffer and 1 M guanidinium chloride; these conditions were therefore chosen for subsequent use of the WATR technique in a study of the kinetics of hydrolysis of neostigmine bromide. The method was found to be very convenient for studies of the hydrolysis of this representative amide.