Design,synthesis, and pharmacological evaluation of 2-amino-5-nitrothiazole derived semicarbazones as dual inhibitors of monoamine oxidase and cholinesterase: effect of the size of aryl binding site

A series of 2-amino-5-nitrothiazole derived semicarbazones were designed, synthesised and investigated for MAO and ChE inhibition properties. Most of the compounds showed preferential inhibition towards MAO-B. Compound 4, (1-(1-(4-Bromophenyl)ethylidene)-4-(5-nitrothiazol-2-yl)semicarbazide) emerged as lead candidate (IC₅₀?=?0.212?µM, SI?=?331.04) against MAO-B; whereas compounds 21 1-(5-Bromo-2-oxoindolin-3-ylidene)-4-(5-nitrothiazol-2-yl)semicarbazide (IC₅₀?=?0.264?µM) and 17 1-((4-Chlorophenyl) (phenyl)methylene)-4-(5-nitrothiazol-2-yl)semicarbazide (IC₅₀?=?0.024?µM) emerged as lead AChE and BuChE inhibitors respectively; with activity of compound 21 almost equivalent to tacrine. Kinetic studies indicated that compound 4 exhibited competitive and reversible MAO-B inhibition while compounds 21 and 17 showed mixed-type of AChE and BuChE inhibition respectively. Docking studies revealed that these compounds were well-accommodated within MAO-B and ChE active sites through stable hydrogen bonding and/or hydrophobic interactions. This study revealed the requirement of small heteroaryl ring at amino terminal of semicarbazone template for preferential inhibition and selectivity towards MAO-B. Our results suggest that 5-nitrothiazole derived semicarbazones could be further exploited for its multi-targeted role in development of anti-neurodegenerative agents.

A library of 2-amino-5-nitrothiazole derived semicarbazones (4–21) was designed, synthesised and evaluated for in vitro MAO and ChE inhibitory activity. Compounds 4, 21 and 17 (shown) have emerged as lead MAO-B (IC₅₀:0.212?µM, competitive and reversible), AChE (IC₅₀:0.264?µM, mixed and reversible) and BuChE (IC₅₀:0.024?µM, mixed and reversible) inhibitor respectively. SAR studies disclosed several structural aspects significant for potency and selectivity and indicated the role of size of aryl binding site in potency and selectivity towards MAO-B. Antioxidant activity and neurotoxicity screening results further suggested their multifunctional potential for the therapy of neurodegenerative diseases.     

Combined molecular modeling and cholinesterase inhibition studies on some natural and semisynthetic O-alkylcoumarin derivatives

Coumarins of synthetic or natural origins are an important chemical class exerting diverse pharmacological activities. In the present study, 26 novel O-alkylcoumarin derivatives were synthesized and have been tested at 100 µM for their in vitro inhibitory potential against acetylcholinesterase (AChE) and butyrlcholinesterase (BChE) targets which are the key enzymes playing role in the pathogenesis of Alzheimer’s disease. Among the tested coumarins, none of them could inhibit AChE, whereas 12 of them exerted a marked and selective inhibition against BChE as compared to the reference (galanthamine, IC₅₀ = 46.58 ± 0.91 µM). In fact, 10 of the active coumarins showed higher inhibition (IC₅₀ = 7.01 ± 0.28 µM – 43.31 ± 3.63 µM) than that of galanthamine. The most active ones were revealed to be 7-styryloxycoumarin (IC₅₀ = 7.01 ± 0.28 µM) and 7-isopentenyloxy-4-methylcoumarin (IC₅₀ = 8.18 ± 0.74 µM). In addition to the in vitro tests, MetaCore/MetaDrug binary QSAR models and docking simulations were applied to evaluate the active compounds by ligand-based and target-driven approaches. The predicted pharmacokinetic profiles of the compounds suggested that the compounds reveal lipophilic character and permeate blood brain barrier (BBB) and the ADME models predict higher human serum protein binding percentages (>50%) for the compounds. The calculated docking scores indicated that the coumarins showing remarkable BChE inhibition possessed favorable free binding energies in interacting with the ligand-binding domain of the target. Therefore, our results disclose that O-alkylcoumarins are promising selective inhibitors of cholinesterase enzymes, particularly BChE in our case, which definitely deserve further studies.     

Enzymatic synthesis of gallic acid from tannic acid with an inducible hydrolase of Enterobacter spp

Gallic acid acts as a precursor molecule to synthesize various tannin molecules. These are plant polyphenols and were proved to be good anti-oxidant, anti-cancerous, anti-inflammatory, anti-microbial compounds. In order to fully exploit prominent biological activities of specific tannins and to develop tannin-based new medicines, it is necessary to obtain their pure preparations with an aim of high yield and specificity. In the present study, gallic acid is synthesized by the hydrolysis of tannic acid using a microbial based transformation process. The microorganism was isolated and identified. The ability of the isolated microorganism to covert tannic acid into gallic acid was determined by HPLC and enzyme production.

• Highlights

• The present investigation signifies the role of Enterobacter spp. in various processes:

• ??To synthesize gallic acid (a precursor for food oxidant such as propyl gallate) and a bacteriostatic antibiotic (trimethoprim).

• ??To protect the environment from tannery’s discharge through the process of biodegradation.

• ??To reduce the toxicity of tannins in animal feed.

     

Embriologia Della “Vinca Difformis„ Pourr. (Apocinaceae)

Riassunto

L'A. ha studiato l'embriogia della Vinea difformis Pourr. ed ha potuto stabilire che:

1. l'archisporio è pluricellulare e possono svilupparis talvolta pi[ugrave] cellule madri;

2. normalmente solo una cellula madre arriva a maturità;

3. delle quattro megaspore solo una è fertile e precisamente la pi[ugrave] calazale;

4. lo sviluppo del gametofito è del tipo Normale cioè Monomegasporiale con oangio emisporiale.

Ha inoltre risontrato una anomalia di sviluppo constituita da un gametofito binucleato abnorme per ritardo delle divisioni nucleari cispetto all'acerescimento che è quello di un gametofito ottonucleato.     

A cascade synthesis, in vitro cholinesterases inhibitory activity and docking studies of novel Tacrine-pyranopyrazole derivatives

In this work, we describe the preparation of some new Tacrine analogues modified with a pyranopyrazole moiety. A one-pot multicomponent reaction of 3-methyl-1H-pyrazol-5(4H)-one, aryl(or hetero)aldehydes, malononitrile and cyclohexanone involving a Friedländer condensation led to the title compounds. The synthesized heterocyclic analogues of this molecule were evaluated in vitro for their AChE and BChE inhibitory activities in search for potent cholinesterase enzyme inhibitors. Most of the synthesized compounds displayed remarkable AChE inhibitory activities with IC₅₀ values ranging from 0.044 to 5.80?µM, wherein compounds 5e and 5j were found to be most active inhibitors against AChE with IC₅₀ values of 0.058 and 0.044?µM respectively. Molecular modeling simulation on AChE and BChE receptors, showed good correlation between IC₅₀ values and binding interaction template of the most active inhibitors docked into the active site of their relevant enzymes.     

Potent inhibition of acetylcholinesterase by sargachromanol I from Sargassum siliquastrum and by selected natural compounds

Six hundred forty natural compounds were tested for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Of those, sargachromanol I (SCI) and G (SCG) isolated from the brown alga Sargassum siliquastrum, dihydroberberine (DB) isolated from Coptis chinensis, and macelignan (ML) isolated from Myristica fragrans, potently and effectively inhibited AChE with IC₅₀ values of 0.79, 1.81, 1.18, and 4.16 µM, respectively. SCI, DB, and ML reversibly inhibited AChE and showed mixed, competitive, and noncompetitive inhibition, respectively, with K_i values of 0.63, 0.77, and 4.46 µM, respectively. Broussonin A most potently inhibited BChE (IC₅₀ = 4.16 µM), followed by ML, SCG, and SCI (9.69, 10.79, and 13.69 µM, respectively). In dual-targeting experiments, ML effectively inhibited monoamine oxidase B with the greatest potency (IC₅₀ = 7.42 µM). Molecular docking simulation suggested the binding affinity of SCI (−8.6 kcal/mol) with AChE was greater than those of SCG (−7.9 kcal/mol) and DB (−8.2 kcal/mol). Docking simulation indicated SCI interacts with AChE at Trp81, and that SCG interacts at Ser119. No hydrogen bond was predicted for the interaction between AChE and DB. This study suggests SCI, SCG, DB, and ML be viewed as new reversible AChE inhibitors and useful lead compounds for the development for the treatment of Alzheimer’s disease.     

Ecological Risk Assessment and Quantitative Consequence Analysis

Ecological risk actually refers to two separate things. First, risk to the environment as a result of human activity. Contaminated sites are an example. Second, risk to the biota—flora, fauna, and people—as a result of environmental hazards. Geophysical risk arising from natural hazards is an example. Risk is a combination of likelihoods and consequences. This article examines methods used to quantify the consequences. At the general level, such methods are linked to the methods used to quantify the likelihoods and thus to quantify the risks. It is possible to use the existing frameworks of risk management, health risk assessment, and ecological risk analysis to develop a risk management framework that is suitable for ecological risk assessment. The framework consists of the following steps:

1. Determine concernsby using risk assessment techniques for various scenarios.

2. Identify the consequences by systematically identifying hazards.

3. Undertake calculations by using relevant models.

4. Evaluate certainties, uncertainties, and probabilities involved in the calculations of the vulnerability and of the exposure.

5. Compare with criteriato assess the need for further action.

6. Determine and act on options to control, mitigate, and adapt to the risk.

7. Communicatethe results to those who need to know.

     

Conformation of Some Hexuronic Acids and d-Galactopyranosiduronic Acid Moiety in the Peracetylated and Permethylated Derivatives of Orange Pectic Acid in Solutions

1. The 1C conformation was estimated for α-d-galactopyranosiduronic acid moiety of pectic acid in the permethylated derivative dissolved in 1 n NaOD-D₂O and in the peracetylated derivative dissolved in dimethyl sulfoxide-d₆, and the C1 conformation was estimated for some derivatives of d-galactopyranuronic acid in chloroform-d by NMR spectroscopy.

2. Random conformation of the whole macromolecule was estimated for pectic acid in water on the basis of no appearance of any induced Cotton effects in the 200 ~ 700 mμ region in the ORD spectra of pectic acid-anionic dye complexes.

3. The conformation was supported by the fact that the rate of periodate oxidation of pectic acid at 5° was slightly decreased in comparison with that of amylase in 7 m urea solution.

     

Staining of Negri Bodies

The following procedure for staining Negri bodies in sections is based on methods previously described by MacNeal, by Haynes, and by Richter:

Fixation:

1. 1. Zenker's solution 4 hours at 37°C or Dominici's 3 hours.

2. 2. 70% alcohol, 12 to 18 hours at room temperature.

3. 3. 80% alcohol, about 5 to 6 hours.

4. 4. 90% alcohol, about 4 to 6 hours.

5. 5. Absolute alcohol about 16 hours.

6. 6. Ether and absolute alcohol aa, about 8 hours.

7. 7. 16 to 24 hours in the following mixture: celloidin 1 g., methyl salycilate 25 cc., abs. alcohol 25 cc., ether 25 cc.

8. 8. Chloroform and paraffin, 2 to 3 hours.

9. 10. Paraffin, 1 to 1 1/2 hours.

10. 11. Embed.

staining:

1. 1. Cut sections 4 to 5 μ.

2. 2. Bring section to water and cover with Lugol's iodine for 10 minutes.

3. 3. Decolorize with a 2% sodium thiosulfate (hypo).

4. 4. Wash thoroly with water.

5. 5. Cover with a mixture of equal parts of 0.5% phloxine and 1% eosin Y (National Aniline brand) and leave for 15 minutes.

6. 6. Wash with water and stain 2 to 5 minutes in 0.1% azure B (National Aniline).

7. 7. Wash with 96% alcohol and decolorize in a mixture of 2 parts absolute alcohol with 1 part clove oil, ordinarily for not more than 1/2 to 1 minute.

8. 8. Dehydrate rapidly, clear, and mount in Yucatan Elemi.

     

Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol

During the course of studies on the oxidative metabolism of d-sorbitol by acetic acid bacteria, it was found that d-sorbitol was almost quantitatively converted to 5-keto-d-fructose via l-sorbose by a certain strain of Gluconobacter suboxydans. In addition to 5-keto-d-fructose, three γ-pyrone compounds, kojic acid, 5-oxymaltol, and 3-oxykojic acid, 2-keto-l-gulonate, and several organic acids such as succinic, glycolic, and glyceric acids were confirmed in the culture filtrate of this bacterium.

• The most suitable carbon source for 5-ketofructose fermentation by Gluconobacter suboxydans Strain 1 was confirmed to be d-sorbitol or l-sorbose using growing and resting cells. d-Fructose had little effect on the formation of this dicarbonylhexose.

• The optimal pH for the formation from l-sorbose by intact cells was found to be at 4.2.

• The activity of the pentose phosphate cycle in the resting cells was calculated as 13~17 μatoms/hr/mg of dry cells by the use of the manometric techniques.

• There was no strain tested so far which could accumulate a large amount of 5- keto-d-fructose from d-sorbitol except this bacterium.

• The experimental results shown in this paper makes the prediction that a certain dehydrogenating system of l-sorbose is functional in the organism, and the metabolic pathways of d-sorbitol via l-sorbose and 5-keto-d-fructose is proposed.

     

Fenomeni di Inattivazione e di Riattivazione Enzimatica in Endospermi di semi di Ricino

Abstract

Inactivation and riactivation of enzymes in endosperms of castor bean seeds. — On the basis of previous results, the possibility has been investigated of the reversible interconversion of active and inactive form of enzymes in castor bean seeds, during their development.

The results described here indicate that:

1. the activity of some glycolytic enzymes increases greatly (81% and 400% increase of, respectively, Gl-6-P-dehydrogenase and aldolase) upon incubation of dry seeds for few hours at 4 °C.

2. The decrease of enzyme activity upon dehydration of seeds and the increase during the subsequent imbibition can be shown reproducibly.

3. This same observation is made for oxygen uptake.

These results are interpreted to indicate the reversible inactivation of enzymes caused by dehydration of seeds.     

Design and synthesis of some new carboxamide and propanamide derivatives bearing phenylpyridazine as a core ring and the investigation of their inhibitory potential on in-vitro acetylcholinesterase and butyrylcholinesterase

A series of new carboxamide and propanamide derivatives bearing phenylpyridazine as a core ring were designed, synthesized and evaluated for their ability to inhibit both cholinesterase enzymes. In addition, a series of carboxamide and propanamide derivatives bearing biphenyl instead of phenylpyridazine were also synthesized to examine the inhibitory effect of pyridazine moiety on both cholinesterase enzymes. The inhibitory activity results revealed that compounds 5b, 5f, 5h, 5j, 5l pyridazine-3-carboxamide derivative, exhibited selective acetylcholinesterase (AChE) inhibition with IC₅₀ values ranging from 0.11 to 2.69 µM. Among them, compound 5h was the most active one (IC₅₀ = 0.11 µM) without cytotoxic effect at its effective concentration against AChE. Additionally, pyridazine-3-carboxamide derivative 5d (IC₅₀ for AChE = 0.16 µM and IC₅₀ for BChE = 9.80 µM) and biphenyl-4-carboxamide derivative 6d (IC₅₀ for AChE = 0.59 µM and IC₅₀ for BChE = 1.48 µM) displayed dual cholinesterase inhibitory activity. Besides, active compounds were also tested for their ability to inhibit Aβ aggregation. Theoretical physicochemical properties of the compounds were calculated by using Molinspiration Program as well. The Lineweaver-Burk plot and docking study showed that compound 5 h targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE.     

Poster Abstracts

DOCSPER—A Synthetic Lipid Fit for In Vivo Application

DOCSPER [1,3-Dioleoyloxy-2-(N⁵-carbamoyl-spermine)-propane] is a cationic amphiphile consisting of a hydrophobic 1,3 dioleylglycerol moiety and threefold positively charged spermine head group (). We optimised the 5-step-synthesis of the lipospermine and after up-scaling we have obtained sufficient amounts to initiate preclinical investigations. DOCSPER was tested for its ability to transfect eukaryotic cells in vitro. It has proven to possess high transfection efficiency in comparison to commercially available liposomal transfection agents. Furthermore, DOCSPER was extensively tested in several in vivo studies (). These studies revealed a high transfection efficiency, whereas very low toxicity levels were detected. Thus, the results clearly indicate that the cationic lipid DOCSPER is a reliable, low-risk system for broad applications in gene therapy.

1. Groth D. et al. Int J Pharm 1998; 162:143–157.

2. Nikol S. et al. Int J Angiol 2000; 9:87–95.

3. Armeanu S. et al. Mol Ther 2000; 1(4):366–375.

     

Induction of Catalase Activity by Hydrocarbons in Candida tropicalis рK 233

1. The catalase activity of Candida tropicalis pK 233 was induced by hydrocarbons but not by glucose, galactose, ethanol, acetate or lauryl alcohol.

2. The induction of the catalase activity depending upon hydrocarbons was sensitive to cycloheximide but not to chloramphenicol.

3. Glucose repressed strongly the induction of the catalase activity by hydrocarbons but galactose did not affect seriously.

4. When C. tropicalis was incubated with hydrocarbons, the appearance of microbodies was observed electronmicroscopicaliy.

     

Some properties of turnip yellow mosaic virus isolated from hybrid Brassica Perko PVH in Yugoslavia

Anhand mikroskopischer Untersuchungen und durch Mittelversuche an A. pisum wurden folgende Kenntnisse zur Endosymbiose gewonnen:

• In L₃‐Stadien von A. pisum sind zwischen 55 und 85 potentielle Bakteriocyten vorhanden, von dene ca. 60–80 % besiedelt sind.

• Eine Reduktion des besiedelten Anteils in der F₁‐Generation auf unter 50% läßt eine deutliche Depression in der F₂‐Generation erwarten.

• Das Kriterium Embryonenlänge ist großen Schwankungen unterworfen und eignet sich nur bedingt als Unterscheidungsmerkmal.

• Die von Fröhlich (1990) vorgeschlagene Methodik zum Symbiontizidscreening bei A. pisum mit dem Standard OTC 2000 ppm und der Auszählung der mit TTC angefärbten Bakteriocyten unter dem Mikroskop läßt eine praktikable Testung von Substanzen auf symbiontizide Wirkung bei A. pisum zu. Es wird jedoch als günstiger angesehen, nicht die Larven mit den Pflanzen zu behandeln, wie von Fröhlich (1990) vorgeschlagen, sondern erst nach dem Antrocknen des Spritzbelages Adulte zur Erzeugung von F₁‐Larven anzusetzen.

• Es konnte eindeutig nachgewiesen werden, daß die von den Prüfsubstanzen hervorgerufenen aphiziden Effekte, insbesondere durch Cycloheximid (100/500 ppm) sowie Neemkernextrakt (50%), nicht auf einem symbiontiziden Wirkungsmechanismus beruhen (Ausnahme Oxytetracyclin 2000 ppm als Standard).

     

Tryptophan reaction with free radicals arisen from carbon tetrachloride in a model system. A mass spectrometric study

SUMMARY

The interaction between free radicals derived from the thermal decomposition of carbon tetrachloride and N-acetyl-d, l-tryptophan ethyl ester (TRPAE) under anaerobic and aerobic conditions was studied. The structure of the reaction products formed was deciphered by the GC/MS analysis of their trimethylsilyl derivatives. Under anaerobic conditions no formation of reaction products was detected. Under aerobic conditions the following products were identified:

• 1. A chloro hydroxy unsaturated adduct of TRPAE (2 isomers).

• 2. A dichloro hydroxy unsaturated adduct of TRPAE.

• 3. 12 products which are different pyrrolo[2,3-b]indol derivatives.

Some of the products appeared to have an hydroxyl group as a substituent, all of them contained chlorine and only one contained carbon from CCl₄. Interestingly, the formation of those adducts not containing CCl₃ would be missed during the regular procedures toxicologists use to determine the so-called ‘covalent binding’ employing ¹⁴CCl₄.

Concerning the potential relevance of these findings, we hypothesize that if interactions similar to those here reported occurred at least in part during CCl₄ poisoning, the resulting critical proteins containing tryptophan, e.g. membrane or other and enzymes containing the amino acid in their active center, might be impaired.     

Interaction and photo–induced cleavage studies of meropenem drug with human serum albumin using spectroscopic and molecular docking investigations

The interaction between Meropenem drug and human serum albumin (HSA) has been studied under physiological condition in Tris–HCl buffer solution at pH 7.4 by various spectroscopic (UV spectra, fluorescence spectra, CD spectra), Photo–induced HSA cleavage, and molecular docking techniques. The results of fluorescence titration revealed that the Meropenem strongly quench the intrinsic fluorescence of HSA through a static quenching procedure. Binding constants (K_b) and the number of binding sites (n ? 1) were calculated using modified Stern–Volmer equations. The thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were calculated which revealed that the electrostatic and hydrogen bonding interactions play a major role in HSA–Meropenem association. The distance r between donor (HSA) and acceptor (Meropenem) was obtained according to fluorescence resonance energy transfer (FRET) and the alterations of HSA secondary structure induced by Meropenem were confirmed by FT–IR and CD measurements. The molecular docking technique was utilized to ascertain the mechanism and mode of action towards the molecular target HSA indicating that Meropenem was located within the subdomain IIA of protein by electrostatic interactions and hydrogen bonds, consistent with the corresponding experimental results. Additionally, Meropenem shows efficient photo–induced HSA cleavage. Our results may provide valuable information to understand the mechanistic pathway of drug delivery and to pharmacological behavior of drug.

• Research Highlights

• The interaction of Meropenem with HSA was studied by spectroscopic, photo-induced cleavage and molecular docking techniques.

• The secondary structure of protein has been changed upon the interaction with Meropenem.

• Subdomain IIA of the HSA is found to be the main binding site for Meropenem.

Communicated by Ramaswamy H. Sarma     

Alkynyl and β-ketophosphonates: Selective and potent butyrylcholinesterase inhibitors

A series of thirty-three alkynyl and β-ketophosphonates were evaluated for their in vitro acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitory activities using Ellman’s spectrophotometric method. None of the examined compounds inhibited AChE activity at tested concentrations while twenty-nine of them showed significant and selective inhibition of BChE with IC₅₀ values between 38.60 µM and 0.04 µM. In addition, structure-activity relationships were discussed. The most effective inhibitors were the dibutyl o-methoxyphenyl alkynylphosphonate 3dc and dibutyl o-methoxyphenyl β-ketophosphonate 4dc. Activities of most potent compounds were also compared with a commercial organophosphorus compound. These results could inspire the design of new inhibitors with stronger activity against BChE.