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The aggregation of beta-amyloid (1-40) (Abeta) induced by human recombinant acetylcholinesterase (HuAChE) was studied by means of circular dichroism (CD) and by thioflavin T fluorescence spectroscopy. Abeta was incubated alone and with HuAChE. The kinetic of fibrils formation was followed for 48 hr. The increasing beta-conformation content induced by HuAChE, preliminary to the formation of Abeta fibrils, was determined by circular dichroism. This phenomenon was found to be related to the thioflavin T emission of fluorescence at 490 nm. Incubation experiments were performed in the presence of known AChE inhibitors (physostigmine, edrophonium, decamethonium, propidium) and drugs used for Alzheimer's disease (AD) (tacrine, donepezil), to test their capability of preventing the HuAChE-induced Abeta aggregation. The non-competitive or mixed mode of AChE inhibition was confirmed to be an essential feature. At 100 microM propidium, decamethonium, donepezil and physostigmine were found to inhibit the HuAChE-induced Abeta aggregation by 82, 25, 22 and 30%, respectively.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been extensively studied as a target for new drugs to be used in the treatment of various parasitic diseases. The standard approach to the determination of GAPDH activity utilizes solubilized free enzyme and is limited by the enzyme's low stability. In the current study the stability of GAPDH was significantly increased through the covalent immobilization of the enzyme on a wide-pore silica support containing glutaraldehyde (Glut-P). The optimal conditions for the immobilization were: 100 mg Glut-P stationary phase, approximately 150 microg of enzyme dissolved in pyrophosphate buffer (15 mM, pH 8.5). The mixture was gently agitated for 6 h at 4 degrees C. Under these conditions 91.3% of protein was immobilized on 100 mg of Glut-P support with retention of 2.97% of the initial enzymatic activity. The activity of the immobilized GAPDH was stable for over 30 days. The GAPDH-Glut-P stationary phase was packed into a glass column to produce a GAPDH immobilized enzyme reactor (GAPDH-IMER). The activity and kinetic parameters of the GAPDH-IMER were investigated and the results demonstrated that the enzyme retained its activity and sensitivity to the competitive inhibitor agaric acid.

Surfaces that modulate the behaviour of surface-immobilised biomolecules have been of critical interest in the last decades, with applications in biomedical microdevices. Recently, the advances in nanofabrication also allowed the contemplation of a leap-through in the form of the design and fabrication of biomimetic surfaces that replicate the molecular surfaces of large biomolecular structures such as cytoskeleton aggregates. The process of formation of these long-range ordered nanostructures have enormous biological interest, but increasingly they are good examples of ‘fabrication’ processes leading to large nanostructured areas with the design embedded in their smaller components. To this end, we report here an atomic force microscopy (AFM) study of the high order self assembly of F-actin on mica. Actin is one of the principal structural proteins in eukaryotic cells and is an ideal biopolymer for investigations of new modes of higher order self-assembly. G-actin monomer can be polymerized into long right-handed double helical filaments (F-actin) whose formation is induced by Mg2+, K+, Na+, and ATP. F-actin can be considered as a semiflexible and highly charged polyelectrolyte, with diameter DA ~ 8 nm and persistence length of 10 μm [1]. Atomic force microscopy (AFM) is a useful tool for elucidating the topography of biomolecules-covered surfaces, including proteins, and mica is commonly used as a substrate for AFM imaging at molecular resolution due to its atomically-flat surface. In our previous [2] and present work, we visualised different morphologies of assemblies of actin filaments adsorbed on mica and silicon surfaces with different geometries and physical-chemical properties. However, it is notoriously difficult to immobilize negatively charged samples as actin (theoretical pI 5.23) because the electrostatic repulsion between the negatively charged mica surface and the actin assembly results is a weak immobilisation of the filaments, thus rendering the AFM scanning very difficult and prone to artefacts. On silicon surfaces large, flat and long F-actin fibre bundles were visualised, which could indicate a substrate repulsion that favours the assembly of fibres in the bulk and/or the vicinity of the surface, followed by deposition of pre-‘fabricated’ F-actin rafts on the silicon surface. To counter-act the technological problem described above, mica was chemically treated with Mg2+ to increase the affinity towards F-actin. Interestingly, this treatment rendered the mica surface more hydrophobic. On this treated surface we could visualise with great reproducibility F-actin patterns by both contact and tapping mode AFM approaches (Figures 1 and 2). The most interesting aspect was the capability of fabrication ordered patterns formed by F-actin filaments, through the balanced interplay between F-actin self-assembly forces and forces applied by the AFM tip in a contact mode. More specifically, increasing the force applied by the AFM tip we could observe the shift from the visualisation of individual actin filaments (Figure 3, “e” surface) to parallel actin filaments ‘rafts’ (Figure 1 and Figure 3, surfaces “a” and “c”). In ‘visualisation-only’ contact mode, the average height F-actin filaments above the mica surface was 5.5 nm (Figure 2), consistent with the theoretical value. The spacing between rows was found to be a function of the applied tip force, as can be seen in Figure 2 where three different regions were scanned at forces ranging from 7 to 20 nN. Further increasing the force applied by the AFM tip to 50 nN we could clean the mica surface and ‘dragged’ the filaments, still in an ordered manner, i.e., in a direction perpendicular to their axis (Figure 3, transition from surface “d” to surface “b”). Future work will study the parallel characterization of nanostructured solid surfaces which can modulate, either enhancing or retarding, the oligomerisation and fibrillization of actin filaments, w...

Herein we describe the design, multicomponent synthesis, and biological, molecular modeling and ADMET studies, as well as in vitro PAMPA-blood-brain barrier (BBB) analysis of new tacrine-ferulic acid hybrids (TFAHs). We identified (E)-3-(hydroxy-3-methoxyphenyl)-N-{8[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-N-[2-(naphthalen-2-ylamino)2-oxoethyl]acrylamide (TFAH 10n) as a particularly interesting multipotent compound that shows moderate and completely selective inhibition of human butyrylcholinesterase (IC50 = 68.2 nm ), strong antioxidant activity (4.29 equiv trolox in an oxygen radical absorbance capacity (ORAC) assay), and good β-amyloid (Aβ) anti-aggregation properties (65.6% at 1:1 ratio); moreover, it is able to permeate central nervous system (CNS) tissues, as determined by PAMPA-BBB assay. Notably, even when tested at very high concentrations, TFAH 10n easily surpasses the other TFAHs in hepatotoxicity profiling (59.4% cell viability at 1000 μm), affording good neuroprotection against toxic insults such as Aβ1-40, Aβ1-42, H2O2, and oligomycin A/rotenone on SH-SY5Y cells, at 1 μm. The results reported herein support the development of new multipotent TFAH derivatives as potential drugs for the treatment of Alzheimer's disease.

72 p.-7 fig.-2 tab.-4 sch. Prati, Federica et al., One of the main obstacles toward the discovery of effective anti-Alzheimer drugs is the multifactorial nature of its etiopathology. Therefore, the use of multitarget-directed ligands has emerged as particularly suitable. Such ligands, able to modulate different neurodegenerative pathways, for example, amyloid and tau cascades, as well as cognitive and neurogenic functions, are fostered to come. In this respect, we report herein on the first class of BACE-1/GSK-3β dual inhibitors based on a 3,4-dihydro-1,3,5-triazin-2(1H)-one skeleton, whose hit compound 1 showed interesting properties in a preliminary investigation. Notably, compound 2, endowed with well-balanced potencies against the two isolated enzymes (IC50 of 16 and 7 μM against BACE-1 and GSK-3β, respectively), displayed effective neuroprotective and neurogenic activities and no neurotoxicity in cell-based assays. It also showed good brain permeability in a pharmacokinetic assessment in mice. Overall, triazinone derivatives, thanks to the simultaneous modulation of multiple points of the diseased network, might emerge as suitable candidates to be tested in in vivo Alzheimer’s disease models., This work was supported by IIT, UNIBO, PRIN (20103W4779), MINECO (SAF2012-37979-C03-01), Ministerio de Ciencia y Tecnologia (SAF2010-16365), Instituto de Salud Carlos III, UniRimini and CIRI (PORFESR project).

et al., Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimer's disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3, 4-dihydro-1, 3, 5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3ß. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC50 of (18.03 ±0.01) ¿M and (14.67±0.78)¿UM for BACE-1 and GSK-3ß, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential., This work was supported by IIT, UNIBO, PRIN (20103W4779), MINECO (SAF2012-37979-C03-01), Ministerio de Ciencia y Tecnologia (SAF2010-16365), Instituto de Salud Carlos III, UniRimini, and CIRI (PORFESR project).

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.

The goal of the present work is to establish a correlation between the degree of histone post-translational modifications and the effects caused by treatment of HT29 colon cancer cells with class I-selective (MS-275 and MC1855), class II-selective (MC1568), and non-selective (suberoylanilide hydroxamic acid (SAHA) histone deacetylase inhibitors (HDACi). This correlation could afford a mean to better understand the mechanism of action of new, more potent, and selective HDACi directly on the cells. To this end, LC coupled to MS was applied in studies of time and concentration-dependent treatment with HDACi in HT29 cells. The results were correlated to their potency of histone deacetylase inhibition and to their effects on the cell cycle. The results indicate that the four tested inhibitors show a different pattern of time- and concentration-dependent modification after treatment of HT29 cells. At the selected concentrations, they cause different histone hyperacetylation and different cell cycle effects. In particular, SAHA (non-selective HDACi) affected hyperacetylation of all histones and caused massive cell death. MC1855 (class I-selective HDACi, hydroxamate) proved to be more potent and less toxic (cell arrest in G2/M phase) than SAHA. MS-275 (class I-selective HDACi, benzamide) exhibited a higher degree of hyperacetylation of H4 and a lower degree of H2A, H2B, and H3 acetylation, causing a cell arrest in G0/G1 phase. On the contrary, MC1568 (class II-selective HDACi) produced only a modest hyperacetylation of H4, was ineffective on the other histones, and showed no effect on cell cycle in HT29 cells.

In this work, we further investigated a class of carbamic cholinesterase inhibitors introduced in a previous paper (Rampa et al. J. Med. Chem. 1998, 41, 3976). Some new omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl analogues were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The structure of the lead compound (xanthostigmine) was systematically varied with the aim to optimize the different parts of the molecule. Moreover, such a structure-activity relationships (SAR) study was integrated with a kinetic analysis of the mechanism of AChE inhibition for two representative compounds. The structural modifications lead to a compound (12b) showing an IC(50) value for the AChE inhibition of 0.32 +/- 0.09 nM and to a group of BuChE inhibitors also active at the nanomolar level, the most potent of which (15d) was characterized by an IC(50) value of 3.3 +/- 0.4 nM. The kinetic analysis allowed for clarification of the role played by different molecular moieties with regard to the rate of AChE carbamoylation and the duration of inhibition. On the basis of the results presented here, it was concluded that the cholinesterase inhibitors of this class possess promising characteristics in view of a potential development as drugs for the treatment of Alzheimer's disease.

A capillary electrophoretic method was developed for the separation and quantification of prostaglandin E 1 (PGE 1 , alprostadil) and its major impurities prostaglandin E 2 (PGE 2 , dinoprostone), prostaglandin A 1 (PGA 1 ), and prostaglandin B 1 (PGB 1 ). The documented good stability of the studied analytes in organic solvents led us to try CE separation in non-aqueous media. In mixed solvents (methanol-acetonitrile), good analyte peak shapes were obtained using bile acid salts as buffer electrolytes; in particular, sodium cholate and sodium deoxycholate were found to be suitable additives when used at sub-micellar concentrations. Interestingly, the addition of water to the organic running buffer permitted simultaneous improvement of the separation selectivity and the sample compatibility with the CE system. Optimisation of the experimental conditions was carried out considering the requirements for a method devoted to analysis of impurities: precision, selectivity, and sensitivity (quantitation limits of 0.1%). In a fused-silica extended path capillary of 32.5 cm total length, the complete prostaglandin separation was obtained in about 10 min, using 15 mM sodium deoxycholate in a mixed aqueous-organic solvent. The best separation was attained in the presence of 20% water and 80% of methanol-acetonitrile 75:25 (v/v). The method was then validated for linearity, LOD and LOQ, precision, selectivity, accuracy, and robustness; finally, it was applied to real samples, demonstrating its ability to quantify prostaglandin-related impurities.

The photostability of diltiazem was studied in aqueous solutions at different pH values. Firstly, the hydrolysis of the drug to desacetyldiltiazem in alkaline medium was evaluated and then the drug photodegradation under exposure to UVA-UVB radiation (solar simulator) was monitored by HPLC methods. The main photoproduct was isolated and characterized as diltiazem-S-oxide on the basis of the NMR and mass spectra. The HPLC method was also applied to the selective analysis of diltiazem in commercial formulations. Tests on mutagenicity and photomutagenicity of the drug were also carried out using Salmonella typhimurium TA 102 strain. In this testing the drug neither was mutagenic nor toxic.

Capillary electrophoretic methods, under MEKC conditions, were developed for thesimultaneous rapid analysis of neutral (guaifenesin) and basic (salbutamol and dex-tromethorphan) active ingredients of commercially available cough syrups. The run-ning buffer nature played a key role on the migration of the studied analytes: using0.04 M sodium dodecyl sulfate (SDS) as surfactant, sodium tetraborate and 3-(cyclo-hexylamino)-2-hydroxy-1-propane-sulfonic acid sodium salt (CAPSO) were chosenas running buffer allowing the separation of active ingredients and excipients of differ-ent cough syrups. Qualitative and quantitative information on the composition of theanalyzed formulations were simultaneously obtained for both active ingredients andexcipients by the proposed MEKC methods; when syrups containing volatile UV-non-absorbing components (flavors) were analyzed a GC-MS approach was chosen. Theaqueous matrices of the formulations allowed direct sample injection in CE, whileextraction procedures were applied in order to perform GC analyses. The obtaineddata support the suitability of MEKC and GC-MS as effective, complementary techni-ques for the selective and sensitive quality control of cough syrups.Key Words: Cough syrups; Capillary electrophoresis; GC-MS; Guaifenesin; Salbuta-mol;DextromethorphanMs received: January 10, 2001; revised Ms received: February 16, 2001; accepted:February17,2001

The analysis of donepezil, a centrally acting acetylcholine esterase inhibitor, is described by a CZE method suitable for applications in pharmaceutical field. A rapid migration of the analyte was obtained under acidic conditions (pH 3.0); with detection wavelength of 320 nm a LOD of 0.8 x 10(-3) mg/ml was provided. Applications on real sample (pharmaceuticals) were carried out using two different instruments with comparable results in terms of reproducibility and accuracy. The use of chiral selectors in the running buffer allowed the enantioseparation of donepezil; charged cyclodextrins (carboxymethyl-beta-cyclodextrin and sulfated-beta-cyclodextrin) were suitable for the chiral resolution of the analyte. Interesting results were also obtained using human serum albumin. The protein-based CE enantioseparation was carried out at pH 7.4 avoiding the partial filling technique due to the good absorptivity of donepezil at 320 nm. Interestingly, the use of bicine as BGE provided a significative improvement in the enantioresolution compared to that obtained by phosphate buffer.

The purpose of this study was to obtain information on the photochemical and phototoxic properties of Labetalol, a beta-blocker drug. Preliminary information on the drug photoreactivity was achieved using a flow system with a photochemical reactor on-line with a diode array detection system. Photophysical and photochemical investigations on the drug were performed in aqueous solutions at different pH values using spectrophotometric and fluorimetric methods; the photodegradation quantum yield was found to be 2.7 x 10(-3) at pH 5.8 and 1.5 x 10(-2) at pH 11.5. Forced photodegradation of labetalol solutions under exposure to UVA--UVB radiations (xenon arc lamp) was monitored by reversed-phase liquid chromatography. The main photodegradation products were isolated and characterized by NMR and mass spectrometry; labetalol was found to give 3-amino-1-phenylbutane and salicylamide-4-carboxaldehyde as the main photoproducts. Preliminary phototoxic testings on human keratinocyte cultures were performed evaluating the viability of the cells by the neutral-red uptake assay; mutagenic and photomutagenicity tests were also carried out based on Salmonella typhimurium strains. As a result, labetalol was found to be photolabile,mainly in alkaline medium, but evidences of significant phototoxic and photomutagenic effects by the drug were not observed.

The photostability of melatonin, a hormone used as supplementary drug in the alleviation of jet-lag and other sleep disorders, was studied. The drug photodegradation at different pH values was monitored by HPLC methods. The main photoproduct was isolated and characterised on the basis of the NMR, FTIR, and mass spectra. A HPLC method, in combination with a post-column on-line photochemical derivatisation was developed for the selective and reliable quality control of commercially available melatonin containing products.

Capillary electrophoresis (CE) was applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in pharmaceuticals (tablets). Since a free solution CE system failed to reach a complete separation of closely related compounds (lisinopril, ramipril, benazepril, quinapril), alkylsulfonic additives (sodium heptansulfonate and (+)-10-camphorsulphonic acid) were added to the running buffer: improved separations were obtained suggesting a favourable effect of ion-pairing interactions between analytes and additives. The separations were carried out in acidic medium and a systematic investigation of electrophoretic parameters was made to evaluate the performance of the selected additives. Under the optimized conditions, ramipril and benazepril in their commercial dosage forms were determined confirming the applicability of the developed CE approach to the analysis of pharmaceutical samples; the results were also compared with those obtained applying a previously described and validated HPLC method.

Statistical experimental design was used for the optimization and for robustness evaluation of a capillary electrophoretic method developed for the enantioresolution of salbutamol. Dermatan sulfate was used as chiral selector. The goal of the study was to obtain an efficient and fast separation. An eight-run Plackett-Burman matrix was used during the optimization process for the screening of the factors and to adjust the experimental domain under study. Response surface methodology was adopted after the screening phase to obtain information about how the factors percentage of chiral selector, pH and voltage affected the considered responses resolution and analysis time. The Derringer desirability function, which makes it possible to combine results obtained for properties measured on different scales, was used to simultaneously optimize the two responses. Robustness testing was carried out using a Plackett-Burman matrix. The method was found robust as regards the response resolution while voltage and chiral selector were found to be critical factors for the robustness of analysis time response. The proposed CE method permitted the complete enantioseparation of racemic salbutamol and was applied to its chiral resolution in spiked urine samples.

A series of derivatives of the known M 1 selective muscarinic receptor agonist McN-A-343 ( 1 Download high-res image (66KB) Download full-size image Figure 1 . Design strategy for the synthesis of carbamates 3 – 18 by replacing the butynyl chain of McN-A-343 ( 1 ) by a benzyl moiety. ) was designed with the aim of investigating the effects of structural variations on both the butynyl chain and the phenyl ring of 1 . The butynyl chain was replaced with an aromatic spacer, and the effects of such a modification on the stereoelectronic properties of the molecules were theoretically studied and considered compatible with muscarinic receptor affinity. Substituents on the phenyl ring of 1 were selected so as to vary their electronic and hydrophobic properties. This design strategy did not produce muscarinic M 1 receptor agonists more potent than the prototype 1 , even if some analogues displayed functional selectivity for different muscarinic receptor subtypes. Compounds 3 and 7 Download high-res image (110KB) Download full-size image Scheme 1 . were selective agonists towards muscarinic M 3 receptors, while compounds 14 , 16 and 18 were selective muscarinic M 2 receptor agonists. The most interesting derivative was 8 , a full agonist at muscarinic M 3 receptors devoid of activity at both muscarinic M 1 and M 2 subtypes. The pharmacological profile of the series was further characterized by studying the anticholinesterase and miotic activities of some representative compounds. Compounds 3 – 8 turned out to be weak acetylcholinesterase inhibitors, while derivatives 4 , 6 , 8 and 11 were able to significantly reduce the pupillary diameter in rabbit, indicating 8 as an effective miotic agent.

Continuing our work on tetracyclic tacrine analogues, we synthesized a series of acetylcholinesterase (AChE) inhibitors of 11H-indeno-[1,2-b]-quinolin-10-ylaminic structure. Selected substituents were placed in synthetically accessible positions of the tetracyclic nucleus, in order to explore the structure–activity relationships (SAR) and the mode of action of this class of anticholinesterases. A molecular modeling investigation of the binding interaction of the lead compound ( 1a) with the AChE active site was performed, from which it resulted that, despite the rather wide and rigid structure of 1a, there may still be the possibility to introduce some small substituent in some positions of the tetracycle. However, from the examination of the experimental IC50 values, it derived that the indenoquinoline nucleus probably represents the maximum allowable molecular size for rigid compounds binding to AChE. In fact, only a fluorine atom in position 2 maintains the AChE inhibitory potency of the parent compound, and, actually, increases the AChE-selectivity with respect to the butyrylcholinesterase inhibition. By studying the kinetics of AChE inhibition for two representative compounds of the series, it resulted that the lead compound (1a) shows an inhibition of mixed type, binding to both the active and the peripheral sites, while the more sterically hindered analogue 2n Scheme 1. Reagents: (a) ZnCl2 reflux; (b) (1) benzaldehyde reflux, (2) NaBH4. Figure options Download full-size image Download high-quality image (69 K) Download as PowerPoint slide seems to interact only at the external binding site of the enzyme. This finding seems particularly important in the context of Alzheimer's disease research in the light of recent observations showing that peripheral AChE inhibitors might decrease the aggregating effects of the enzyme on the β-amyloid peptide (βA).

The photostability of the beta-blocker drug Atenolol was evaluated at pH 9, 7.4 and 4.0. The drug was exposed to UVA-UVB radiations and the photoproducts were detected by reversed phase LC methods. The photodegradation was found to increase with the pH value decreasing. The major photodegradation product at pH 7.4 was identified as 2-(4-hydroxyphenyl)acetamide. The LC method developed for routine analyses (column: C-18 Alltima; mobile phase: TEA acetate (pH 4; 0.01 M)-acetonitrile 96:4) was found to be suitable for the stability indicating determination of Atenolol in pharmaceutical dosage forms.

Chemically oversulfated galactosaminoglycans with potential as therapeutic agents (inhibitors of human leukocyte elastase) were tested as chiral selectors in capillary electrophoresis of basic racemates. The high anionic character of these compounds provides them with anodic mobility in acidic buffer; using uncoated capillaries, the enantioresolution of racemic basic drugs was obtained at pH 2.5. Dimethindene, chloroquine and chlorpheniramine were enantioresolved applying negative voltage (-15 kV) while the other analytes (propranolol, pindolol, tetrahydrozoline and cloperastine) exhibited catodic migration. The addition of organic solvents to the running buffer was evaluated in order to increase the resolution; methanol provides the best results and in general, baseline separation of the analytes was reached. The studied oversulfated mucopolysaccharide, shows the same ionic character of heparin but presents different stereochemistry and sites of sulfation. A comparison with heparin, used in the same acidic conditions, may underline the role of ionic, spatial and steric features of glycosaminoglycans in the enantiorecognition.

In an effort to learn more about the general structure-activity relationships of cocaine with the aim to elucidate those structural features that might confer antagonistic properties to such analogues, we describe herein our synthetic efforts to prepare two-carbon bridge functionalized (methoxylated and hydroxylated) analogues. Our approach makes use of a modification of the classical Willstatter synthesis of cocaine: Mannich type cyclization of acetonedicarboxylic acid monomethyl ester with methylamine hydrochloride and 2-methoxysuccindialdehyde in a citrate buffer solution afforded the 6- and 7-substituted 2-carbomethoxy-3-tropinones 3a,b and 4a,b in approximate yields of 64%. Reduction of the (+/-)-tropinone derivatives was performed with sodium amalgam in a sulfuric acid solution to afford a mixture of (+/-)-methoxyecgonine and (+/-)-methoxypseudoecgonine derivatives 5, 11 and 6, 7, 12, 13. Benzoylation of these alcohols yielded the desired cocaine and pseudococaine-like compounds 8, 14 and 9, 10, 15, 16. Additionally, we show that enzymatic hydrolysis of these cocaine analogues using pig liver esterase (PLE) affords a practical means for achieving their chemical resolution. The enantiomers of the methoxycocaine analogues were also prepared starting from chiral (+)- and (-)-6-methoxytropinone. All new analogues were examined for their ability to displace [3H]mazindol binding and to inhibit high-affinity uptake of [3H]dopamine into striatal nerve ending (synaptosomes). It appeared evident that methoxylation of the cocaine two-carbon bridge provides compounds of particular interest: the Ki for the binding of the methoxypseudococaines is about two to four times smaller than the Ki for inhibition of dopamine uptake, thus enabling these compounds capable of countering the effects of cocaine to some extent.

An LC-ESI-MS method was developed for the identification and quantification of fructose-1,6-biphosphate (F1,6BP) and fructose-6-phosphate (F6P), respectively the substrate and the product of the enzymatic reaction catalysed by fructose-1,6-bisphosphatase (F1,6BPase). F1,6BPase, expressed predominantly in liver and kidney, is one of the rate-limiting enzymes of hepatic gluconeogenesis and has become a target for the development of new drugs for type 2 diabetes. The two sugar phosphates were separated on a Phenomenex Luna NH2 column (150 mm x 2.0 mm id) using the following mobile phase: 5 mM triethylamine acetate buffer/ACN (80:20) v/v in a linear pH gradient (from pH = 9 to 10 in 15 min) at the flow rate of 0.3 mL/min. The detection was performed with an IT mass spectrometer in negative polarity (full scan 100-450 m/z) and in SIM mode on the generated anions at m/z = 339 (F1,6BP) and m/z = 259 (F6P). Under the optimised final conditions, the method was validated for accuracy, specificity, precision (inter- and intradays RSD comprised between 1.0 and 6.3% over the range of concentrations used), linearity (50-400 microM), LODs (0.44 microM) and LOQs (1.47 microM), and the method was applied to F6P determination in the F1,6BPase catalysed hydrolysis of F1,6BP.

The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver-Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters (Km(app) and vmax(app)) were derived as well as inhibitory constants (Ki(app)) of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.

New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting the beta-amyloid (A beta) peptide aggregation through binding to both catalytic and peripheral sites of the enzyme. Particularly, compounds 5 and 6 emerged as the most potent heterodimers reported so far, displaying IC50 values for AChE inhibition of 20 and 60 pM, respectively. More importantly, these dual AChE inhibitors inhibit the AChE-induced A beta peptide aggregation with IC50 values 1 order of magnitude lower than that of propidium, thus being the most potent derivatives with this activity reported up to date. We therefore conclude that these compounds are very promising disease-modifying agents for the treatment of Alzheimer's disease (AD).

Vasoactive cocktails are widely used in diagnosing and treating erectile dysfunction, especially in poor responders to prostaglandin E1 (PGE1). However, very little information as to their chemical interactions and stability is available, despite the huge amount of published work regarding their clinical efficacy. Obviously, medical and legal problems are involved.We analyzed four kinds of vasoactive cocktails, composed of papaverine, phentolamine and PGE1 in different combinations, using High Performance Liquid Chromatography analysis after 5 to 60 days of storage at temperatures between 2 and 8C. SPSS MANOVA analysis and a t-test for paired samples were used for statistical purposes.Papaverine and phentolamine concentrations showed no significant variations during the 2 month study, ranging from a minimum of 96.75+/-1.20 to a maximum of 103.00+/-0.20% of the starting values. In the same period, PGE1 showed an accelerated degradation profile, reaching concentration values, after 60 days, of 76.00+/-2.28% and 70.20+/-2.02% when added to phentolamine or papaverine respectively and 70.00+/-2.40% with both.Papaverine and phentolamine are characterized by chemical stability when blended together or with PGE1. Papaverine and/or phentolamine increase the naturally occurring degradation of PGE1 in physiological solution. This effect is most evident in the first 10 days. Papaverine has the greatest deteriorating effect on PGE1. A safe and proper use of these cocktails should take into account the variations of PGE1 concentration.

The effect of the chiral diamine sparteine as a cationic mobile phase modifier on the retention and enantioseparation of five hydrophobic basic drugs on an α1-acid glycoprotein column was studied. Sparteine produced good results over the whole range of its concentration (1–15 mM) in the phosphate buffer mobile phase at pH 6.0 for the selected drugs (alprenolol, propranolol, promethazine, chlorpheniramine and disopyramide). When used as a sole modifier, sparteine greatly reduced retention without significant interference in the enantioseparation of the compounds. Sparteine was also applied in combination with 2.5% of the neutral modifier 2-propanol with good results as well. This study shows sparteine's potential both as a substitute for neutral mobile phase modifier and as an auxiliary in reducing amounts of neutral modifier.

Dermatan sulfate (DS), a complex, polydispersed, sulfate polysaccharide was investigated as a useful chiral selector in capillary electrophoresis for the enantioresolution of a variety of drugs. Analysis was carried out in a fused-silica capillary column of 48.5 cm length (40 cm to detector window) x 50 microns I.D., and the separation buffer consisted of citric acid-Tris containing DS; the applied voltage was 15 kV and the detection wavelength was 220 nm. The effects of buffer pH, the dermatan concentration and run temperature on the enantioseparation and migration were examined. The method was applied to the enantioresolution of a representative set of twenty basic drugs. At all pH values used (3.0, 4.5 and 6.5) the addition of DS resulted in an increased migration time due to analyte-DS interaction. Using DS concentration of 2% (w/W), at pH 4.5, enantiomeric separations could be obtained for more than 50% of the examined drugs; resorcinic drugs; resorcinic moiety was found to be a very favourable structural feature for obtaining high enantioresolution values.

Reversed-phase HPLC separations of basic compounds have been optimized by use of mobile phases containing 1,8-diaminooctane (DAO) as amine modifier and heptanesulfonate as counter-ion. Such studies have demonstrated the high resolution and selectivity of this chromatographic system compared with mobile phases prepared with the more widely used primary amines. With the aim of clarifying the retention mechanism involved, three sets of compounds with different structures and properties were analyzed: sulfamidics and trimethoprim, aromatic diamines and aminophenols, and a standard mixture of lipophilic basic drugs. The conditions investigated were: diaminoalkane modifier concentration, diaminoalkane modifier alkyl-chain length, nature and concentration of the counter-ion, pH of the mobile phase, concentration of the organic modifer, and the nature of the stationary phase as characterized by percentage carbon loading. Analysis of chromatographic behavior as a function of the chromatographic conditions has enabled the proposal of a mechanism of retention of the three classes of compounds.

Unusual substitution of amino group occurs by reactions of some 1,4-diamino-2-nitrobenzenes (semipermanent hair dyes) and nucleophiles (NH3, H2O). The reaction is catalyzed by sodium sulfite. The obtained products are suspected of being toxic substances which may be present in cosmetic matrices. Apparently, this reaction is a nucleophilic aromatic substitution but it may be explained by a mechanism involving a tautomeric form of substrate.

Beta-secretase is the key enzyme involved in Alzheimer's disease thus; inhibition of the enzyme can lead to a potential anti-Alzheimer drug. In the search of an effective lead candidate, we have designed non-peptide inhibitor molecules based on amino aromatic heterocyclic motifs specifically, substituted 1,2,4-thiadiazole analogues. In silico modelling was employed to study interaction of the designed ligands in the enzyme active site using molecular docking approach as well as for Absorption, Distribution, Metabolism and Excretion studies. The synthesized analogues were pharmacologically screened using in vitro FRET technique. Overall results indicate that one of the analogues, compound 8 is the most promising one against beta secretase.

We have synthesized a family of rheinhuprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and ()-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and ()-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and ()-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Derivative UV spectroscopy and high performance liquid chromatography (HPLC) were applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in their pharmaceutical dosage forms. For spectrophotometric determinations, the more appropriate derivative order was selected for each drug: ramipril (third-order), benazepril (second-order), enalapril maleate (second-order), lisinopril (first- and second-order) and quinapril (first-order). Reverse phase HPLC procedures (ODS column) were developed able to provide a single, symmetric peak for each drug; mixtures A-B, where A is 20 mM sodium heptansulphonate (pH 2.5) and B is acetonitrile-THF (95:5 v/v), proved to be suitable mobile phases to obtain selective separations of the cited ACE inhibitors. At ambient temperature, a low pH value (2.5) was found to be critical to avoid peak splitting and band broadening.

The development of a selective and sensitive HPLC method for the determination of semipermanent hair colorants in cosmetic formulations is proposed. The separation and identification of N-hydroxyalkyl nitrophenylenediamines and nitroaminophenols were performed by HPLC with on-line, post-column, photochemical derivatisation, using a reversed phase, ion-pair system with 1,8-diaminooctane and heptansulfonate in the mobile phase. Two UV spectra (photoreactor on and off) were obtained for each analyte, adding information for confirming peak identity. Analyte-modified spectral properties were used to increase the method sensitivity. The proposed method was successfully applied to quality control and stability studies of cosmetic formulations.

A series of 12 chiral arylcarboxylic acids were chromatographed on an immobilized human serum albumin chiral stationary phase (HSA-CSP). The effects of solute structure on chromatographic retentions and enantioselective separations were examined by linear regression analysis and the construction of quantitative structure-enantioselective retention relationships. Competitive displacement studies were also conducted using R-ibuprofen as the displacing agent. The results indicate that the enantioselective retention of the solutes takes place at the indole-benzodiazepine site (site II) on the HSA molecule and that chiral recognition is affected by the hydrophobicity and steric volume of the solutes. The displacement studies also identified a cooperative allosteric interaction induced by the binding of R-ibuprofen to site II.

In an effort to develop multipotent agents against β-secretase (BACE-1) and acetylcholinesterase (AChE), able to counteract intracellular ROS formation as well, the structure of the fluorinated benzophenone 3 served as starting point for the synthesis of a small library of 3-fluoro-4-hydroxy- analogues. Among the series, derivatives 5 and 12, carrying chemically different amino functions, showed a balanced micromolar potency against the selected targets. In particular, compound 12, completely devoid of toxic effects, seems to be a promising lead for obtaining effective anti-AD drug candidates.

(E)-6-Methyl-4′-amino-2-styrylquinoline (3) is a small molecule with the proper features to potentially diagnose, deliver therapy and monitor response to therapy in protein misfolding diseases. These features include compound fluorescent emission in the NIR region and its ability to interact with both Aβ and prion fibrils, staining them with high selectivity. Styrylquinoline 3 also inhibits Aβ self-aggregation in vitro and prion replication in the submicromolar range in a cellular context. Furthermore, it is not toxic and is able to cross the blood brain barrier in vitro (PAMPA test).

Amyloid beta 25–35 [Aβ (25–35)], as a peptide model for full-length Aβ in structural and functional investigations, has been chosen for aggregation studies. The complexity of the Aβ (25–35) aggregation process required a multi-methodological analytical approach to obtain reliable and reproducible results. Here, we describe the results obtained by the use of mass spectrometry (MS) for the structural characterization of the self-assembly species during the aggregation process and for the definition of the self-assembly kinetics and myricetin inhibition patterns, comparing the results with those obtained by using the well-established spectroscopic method based on thioflavin T fluorescence. Flow injection electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS) was applied to monitor the disappearance of the monomer specie in the first steps, whereas matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-ToF-MS) was used to follow monomer and small oligomer self-assembly trends in the early stages of the nucleating process.

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2×6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.

Solid-phase extraction (SPE) using C-18, diol and ion-exchange sorbents followed by UV spectrophotometric (conventional and derivative mode) assay was applied to the analysis of basic, acidic and neutral drugs commercially available in creams. A representative set of drugs (promethazine, chlorhexidine, benzydamine, ketoprofen, ibuprofen, fentiazac, piroxicam, fluorouracil, crotamiton and hydrocortisone acetate) was selected, and for each drug the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedures were capable of removing interfering cream components (excipients including preservatives) allowing accurate spectrophotometric analyses to be performed. In some applications, derivative spectrophotometry was advantageous over the conventional absorption mode with respect to higher selectivity and versatility.

9-Hydroxystearic acid (9-HSA) belongs to the endogenous lipid peroxidation by-products that decrease in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. It acts as a histone deacetylase (HDAC, E.C 3.5.1.98) inhibitor, and the interaction of the two enantiomers of 9-HSA with the catalytic site of the enzyme, investigated by using a molecular modelling approach, has been reported to be different. In this work we tested out this prediction by synthesizing the two enantiomers (R)-9-HSA (R-9) and (S)-9-HSA (S-9) starting from the natural source methyl dimorphecolate obtained from Dimorphotheca sinuata seeds and investigating their biological activity in HT29 cells. Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, R-9 being more active; R-9 and S-9 inhibitory effect induces an increase in histone H4 acetylation. We also demonstrate that the antiproliferative effect brought about by R-9 is more pronounced as well as we observe increase of p21 transcription and protein content, while the expression of cyclin D1 is decreased. Starting from these observations it can be hypothesized that the interaction of R-9 with HDAC1 induce conformational changes in the enzyme causing loss of its interaction with other proteins, like cyclin D1 itself.

The multimodal effective compound (I) inhibits the anticholinesterase (AChE) activity in a submicromolar range and shows a significant reduction of AChE-induced anti-aggregating efficacy without a cytotoxicity in neuronal-like cells SH-SY5Y up to the highest concentration of 50 μM.

The synthesis, pharmacological evaluation and molecular modeling of heterocyclic substituted alkyl and cycloalkyl propargyl amines 1 – 7 of type I , and 9 – 12 of type II , designed as multipotent inhibitors able to simultaneously inhibit monoamine oxidases (MAO-A/B) as well as cholinesterase (AChE/BuChE) enzymes, as potential drugs for the treatment of Alzheimer's disease, are described. Indole derivatives 1 – 7 of type I are well known MAO inhibitors whose capacity to inhibit AChE and BuChE was here investigated for the first time. As a result, compound 7 was identified as a MAO-B inhibitor (IC 50 = 31 ± 2 nM) and a moderately selective eqBuChE inhibitor (IC 50 = 4.7 ± 0.2 μM). Conversely, the new and readily available 5-amino-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydropyrido[2,3- b ][1,6]naphthyridine derivatives 9 – 13 of type II are poor MAO inhibitors, but showed AChE selective inhibition, compound 12 being the most attractive as it acts as a non-competitive inhibitor on Ee AChE (IC 50 = 25 ± 3 nM, K i = 65 nM). The ability of this compound to interact with the AChE peripheral binding site was confirmed by kinetic studies and by molecular modeling investigation. Studies on human ChEs confirmed that 12 is a selective AChE inhibitor with inhibitory potency in the submicromolar range. Moreover, in agreement with its mode of action, 12 was shown to be able to inhibit A β aggregation induced by hAChE by 30.6%.

Simulating protein flexibility is a major issue in the docking-based drug-design process for which a single methodological solution does not exist. In our search of new anti-Alzheimer ligands, we were faced with the challenge of including receptor plasticity in a virtual screening campaign aimed at finding new β-secretase inhibitors. To this aim, we incorporated protein flexibility in our simulations by using an ensemble of static X-ray enzyme structures to screen the National Cancer Institute database. A unified description of the protein motion was also generated by computing and combining a set of grid maps using an energy weighting scheme. Such a description was used in an energy-weighted virtual screening experiment on the same molecular database. Assessment of the enrichment factors from these two virtual screening approaches demonstrated comparable predictive powers, with the energy-weighted method being faster than the ensemble method. The in vitro evaluation demonstrated that out of the 32 tested ligands, 17 featured the predicted enzyme inhibiting property. Such an impressive success rate (53.1%) demonstrates the enhanced power of the two methodologies and suggests that energy-weighted virtual screening is a more than valid alternative to ensemble virtual screening given its reduced computational demands and comparable performance. © 2012 American Chemical Society.

In this work, we report a rational structure-based approach aimed at the discovery of new 2-aminoimidazoles as β-secretase inhibitors. Taking advantage of a microwave-assisted synthetic protocol, a small library of derivatives was obtained and biologically evaluated. Two compounds showed promising activities in both enzymatic and cellular assays. Moreover, one of them exhibited the capability to cross the blood-brain barrier as assessed by the parallel artificial membrane permeability assay. © 2011 Elsevier Ltd. All rights reserved.

The complex etiology of Alzheimer's disease prompts scientists to develop multi-target strategies to combat causes and symptoms. In line with this modern paradigm and as a follow-up to our previous studies, we designed and synthesized a focused collection of new 2-arylbenzofurans and evaluated their biological properties towards specific targets involved in AD, namely human AChE and human BuChE, and Aβ fibril formation. Selected compounds were also tested for their ability to inhibit Aβ neurotoxicity in terms of neuronal viability loss, and to prevent Aβ peptide-binding to cell membrane and intracellular reactive oxygen species (ROS) formation. The different modifications introduced in the structure of our lead compound led to an increase in activity towards one or more of the selected targets: the anticholinesterase activity of some compounds was found to be significantly higher than previously obtained related molecules, and the compounds also proved to possess Aβ anti-aggregating properties and neuroprotective effects. The most interesting multi-target compounds were 18, and 1. Interestingly, 1 also showed good selectivity and moderate affinity for CB1 receptor, opening new perspectives in the field of research on AD, since cannabinoid ligands have been widely reported to have neuroprotective properties.

A reversed phase liquid chromatographic method is proposed for the analysis of basic hair dyes (raw materials and colourant formulations). The performance of the method was enhanced by introducing post-column on-line photochemical derivatisation in combination with a Diode Array Detector. On-line photoderivatisation provided an effective way of selectively transforming the analytes to compounds with different spectral properties. For each analyte two characteristic UV-Visible spectra (photoreactor on and off) were obtained with the same mobile phase and this information in combination with the chromatographic data (k' at pH 3.0 and 4.5) enabled the unambiguous identification of both commonly used, approved, and banned basic hair dyes. Additionally, this approach was found useful to improve the method sensitivity, allowing the determination of analytes present in low concentration (0.03%) in complex commercial formulations.