Design,synthesis, and evaluation of novel N-(4-phenoxybenzyl)aniline derivatives targeting acetylcholinesterase, β-amyloid aggregation and oxidative stress to treat Alzheimer’s disease

Novel hybrids N-(4-phenoxybenzyl)aniline were designed, synthesized, and evaluated for their potential AChE inhibitory activity along with antioxidant potential. The inhibitory potential (IC₅₀) of synthesized analogs was evaluated against human cholinesterases (hAChE and hBChE) using Ellman’s method. Among all the tested compounds, 42 with trimethoxybenzene substituent showed maximum hAChE inhibition with the competitive type of enzyme inhibition (IC₅₀ = 1.32 µM; Ki = 0.879 µM). Further, parallel artificial membrane permeation assay (PAMPA-BBB) showed favorable BBB permeability by most of the synthesized compounds. Meanwhile, compound 42 also inhibited AChE-induced Aβ aggregation (39.5–66.9%) in thioflavin T assay. The in vivo behavioral studies showed dose-dependent improvement in learning and memory by compound 42. The ex vivo studies also affirmed the significant AChE inhibition and antioxidant potential of compound 42 in brain homogenates.     

Some factors affecting root formation onin vitro regenerated pea shoots

The rooting ability of 2 cm long shoots ofPisum sativum L., derived from differentin vitro shoot-tip cultures in two pea cultivars Bohatýr and Kleine Rheinländerin was evaluated. In three mutually independent experiments the full and half-strength Murashige-Skoog medium (containing full or half concentration of macro and microelements), with sucrose concentrations 10–30 g l-¹, and with various NAA and IAA combinations, was tested. The variant with half concentration of macro- and microelements, supplemented with 30 g l¹ sucrose, and with growth regulators in the quantity of 1 μM proved optimum.     

Efficient synthesis of novel dialkyl-3-cyanopropylphosphate derivatives and evaluation of their anticholinesterase activity

Based on the broad spectrum of biological activities associated with organophosphates, a novel type of this class of compounds was synthesized, bearing a nitrile group, from the sodium alkoxide-catalyzed reaction of dialkylphosphites with γ-ketonitriles at 80 °C under solvent-free conditions. A reaction mechanism involving a phospha-Brook type rearrangement is proposed. Eight title compounds were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using Ellman’s spectrophotometric method. The synthesized derivatives exhibited mostly a moderate activity against both cholinesterases. The IC₅₀ values for BChE were in a smaller concentration range (5.96–23.35 µM) compared to those for AChE inhibition (9.61–53.74 µM). The diethyl-3-cyano-1-p-tolylpropylphosphate which displayed the higher dual inhibitory potency towards both cholinesterases could be considered as a potential candidate for developing new drugs to treat Alzheimer’s disease.     

Salicylanilide diethyl phosphates as cholinesterases inhibitors

Based on the presence of dialkyl phosphate moiety, we evaluated twenty-seven salicylanilide diethyl phosphates (diethyl [2-(phenylcarbamoyl)phenyl] phosphates) for the inhibition of acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.) and butyrylcholinesterase (BChE) from equine serum. Ellman’s spectrophotometric method was used. The inhibitory activity (expressed as IC₅₀ values) was compared with that of the established drugs galantamine and rivastigmine. Salicylanilide diethyl phosphates showed significant activity against both cholinesterases with IC₅₀ values from 0.903 to 86.3 μM. IC₅₀s for BChE were comparatively lower than those obtained for AChE. All of the investigated compounds showed higher inhibition of AChE than rivastigmine, and six of them inhibited BChE more effectively than both rivastigmine and galantamine. In general, derivatives of 4-chlorosalicylic acid showed enhanced activity when compared to derivatives of 5-halogenated salicylic acids, especially against BChE. The most effective inhibitor of AChE was O-{5-chloro-2-[(3-bromophenyl)carbamoyl]phenyl} O,O-diethyl phosphate with IC₅₀ of 35.4 μM, which is also one of the most potent inhibitors of BChE. O-{5-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphate exhibited in vitro the strongest inhibition of BChE (0.90 μM). Salicylanilide diethyl phosphates act as pseudo-irreversible cholinesterases inhibitors.     

The effect of 5-azacytidine on the growth and thymidine kinase activity of coleoptile sections ofTriticum

The growth of coleoptile sections ofTriticum and its stimulation by indole-3-acetic acid (IAA) are inhibited by 5-azacytidine added into the cultivation medium. 50 per cent depression of the elongation was observed at 2×10^?3M 5-azacytidine concentration. Thymidine kinase activity in cell-free extracts prepared from coleoptile sections treated with 5-azacytidine, and caleulated per 10 mg of their wet weight, is increased while IAA administration resulted in its depression. The observed changes in thymidine kinase activity can be explained assuming the different uptake of water due to 5-azacytidine and IAA treatment.     

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay

Abstract

Ellman’s method is a standard protocol for the determination of cholinesterases activity. Though the method is ready for laboratory purposes, it has some drawbacks as well. In the current article, 2,6-dichloroindophenol acetate is performed as a chromogenic substrate suitable for acetylcholinesterase (AChE) activity examination. Michaelis constant and maximal velocity for 2,6-dichloroindophenol acetate were determined (38.0?µM and 244 pkat) and compared to the values for acetythiocholine (K_m 0.18?mM; V_max 5.1?nkat). Docking for 2,6-dichloroindophenol acetate and human AChE was done as well. In conclusion, 2,6-dichloroindophenol acetate seems to be suitable chromogenic substrate for AChE and spectrophotometry and based on this it can be easily performed whenever AChE activity should be tested.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^?8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^?7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^?5, 5 × 10^?6, and 5 × 10^?7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^?8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1′-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Synthesis and in vitro evaluation of novel rhodanine derivatives as potential cholinesterase inhibitors

Based on a broad spectrum of biological activities of rhodanines, we synthesized aromatic amides and esters of 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (rhodanine-3-acetic acid) via carbodiimide- or PCl₃-mediated coupling. Both esters and amides were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum using Ellman’s spectrophotometric method. The derivatives exhibited mostly a moderate activity against both cholinesterases. IC₅₀ values for AChE were in a closer concentration range of 24.05–86.85 μM when compared to BChE inhibition (7.92–227.19 μM). The esters caused the more efficient inhibition of AChE than amides and parent acid. The esterification and amidation of the rhodanine-3-acetic acid increased inhibition of BChE, even up to 26 times. Derivatives of 4-nitroaniline/phenol showed the activity superior to other substituents (H, Cl, CH₃, OCH₃, CF₃). Rhodanines produced a balanced inhibition of both cholinesterases. Seven derivatives produced the more potent inhibition of AChE than rivastigmine, a clinically used drug; additional three compounds were comparable. Two amides exceeded inhibitory potency of rivastigmine towards BChE. Importantly, this is the first evidence that rhodanine-based compounds are able to inhibit BChE.     

Investigation of salicylanilide and 4-chlorophenol-based N-monosubstituted carbamates as potential inhibitors of acetyl- and butyrylcholinesterase

Based on the presence of carbamate moiety, twenty salicylanilide N-monosubstituted carbamates concomitantly with their parent salicylanilides and five newly prepared 4-chlorophenyl carbamates obtained from isocyanates were investigated using Ellman’s method for their in vitro inhibitory activity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum. The carbamates and salicylanilides exhibited mostly a moderate inhibition of both cholinesterase enzymes with IC₅₀ values ranging from 5 to 235 µM. IC₅₀ values for AChE were in a narrower concentration range when compared to BChE, but many of the compounds produced a balanced inhibition of both cholinesterases. The derivatives were comparable or superior to rivastigmine for AChE inhibition, but only a few of carbamates also for BChE. Several structure-activity relationships were identified, e.g., N-phenethylcarbamates produce clearly favourable BChE inhibition. The compounds also share convenient physicochemical properties for CNS penetration.     

Production of plant growth regulators by someFusarium species

About 10 species ofFusarium were screened and tested for their growth regulatory activity on oat coleoptile straight growth test. Culture filtrates of four species contained growthstimulating factors while others showed growth-inhibition responses on oat sections. On the whole,F. moniliforme was found to be produce higher stimulatory effects. Chromatographic analysis revealed the presence of several indole auxin and gibberellin-like plant-growth regulators in their culture filtrates. The quantity and biological activity of each indole spot was also measured.     

Comparison of esterase isoenzyme patterns in seeds of someAllium species and in cultivars ofAllium cepa L

Esterase isoenzyme patterns were studied in seeds of 6 cultivars ofAllium cepa L. and of14 species ofAllium, namelyAllium aflatunense B. Fedtsch.,A. altaicum (Pall.) Reyse,A. Cristophii Trautv.,A. fistulosum L.,A. jajlae Vved.,A. Karsianum Fom.,A. nutans L.,A. porrum L. cv. Gigant,A. praemixtum Vved.,A. pskemense Vved.,A. ramosum L.,A.rotundum L.,A. schoenoprasum L.,A. stipitatum Regel. The cultivars differ in their isoenzyme patterns, the cultivar Ka?tická stands apart from all the other cultivars, probably due to the high alkalinity of its seed extract. The examined species, arranged according to their mutual similarity of isoenzyme patterns, form several groups corresponding to individual sections of the genus. Our results corroborate the recognizing of the sectionCepa and the subsectionPhyllodolon. The maintaining ofA. jajlae andA. rotundum as well described species fits better with our results, than degrading them to subspecies ofA. scorodoprason. Our results agree in main features with those obtained by the immunological method. Some few differences appear concerning individual species. It is evident that esterase isoenzyme patterns can be used in chemotaxonomy ofAllium on an infrageneric level, and, at least inA. cepa, also on an infraspecific level.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Design,synthesis and evaluation of new 4-arylthiazole-2-amine derivatives as acetylcholinesterase inhibitors

A series of new 4-arylthiazole-2-amine derivatives as acetylcholinesterase inhibitors (AChEIs) were designed and synthesized, Furthermore, their inhibitory activities against acetylcholinesterase in vitro were tested by Ellman spectrophotometry, and the results of inhibitory activity test showed that most of them had a certain acetylcholinesterase inhibitory activity in vitro. Moreover, the IC₅₀ value of compound 4f was to 0.66 μM, which was higher than that of Rivastigmine and Huperzine-A as reference compounds, and it had a weak inhibitory effect on butyrylcholinesterase. The potential binding mode of compound 4f with AChE was investigated by the molecular docking, and the results showed that 4f was strongly bound up with AChE with the optimal conformation, in addition, their binding energy reached −11.27 Kcal*mol⁻¹. At last, in silico molecular property of the synthesized compounds were predicted by using Molinspiration online servers. It can be concluded that the lead AChEIs compound 4f presented satisfactory drug-like characteristics.     

Role of sucrose-phosphate synthase in partitioning of carbon in leaves

Variations in leaf starch accumulation were observed among four species (wheat [Triticum aestivum L.], soybean [Glycine max L. Merr.], tobacco [Nicotiana tabacum L.], and red beet [Beta vulgaris L.]), nine peanut (Arachis hypogea L.) cultivars, and two specific peanut genotypes grown under different nutritional regimes. Among the genotypes tested, the activity of sucrose phosphate synthase was correlated negatively with leaf sucrose content in seven of the nine peanut cultivars as well as the two peanut cultivars grown with different mineral nutrition. The peanut cultivars differed in the effect of 10 millimolar sucrose on sucrose phosphate synthase activity in leaf extracts. Enzyme activity in crude leaf extracts was inhibited by sucrose (10-42%) in four of the cultivars tested whereas five cultivars were not. Overall, the results suggest that a correlation exists between the activity of sucrose phosphate synthase and starch/sucrose levels in leaves.     

Characterization of cholinesterases from the parasitic nematode Trichinella spiralis

1. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors.2. The 5.3 and 13S forms identified in Trichinella extracts are also found in C. elegans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident.3. The level of cholinesterases in excretory/secretory products is low.4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.     

Design and development of 1,3,4-oxadiazole derivatives as potential inhibitors of acetylcholinesterase to ameliorate scopolamine-induced cognitive dysfunctions

The novel hybrids bearing 4-aminopyridine (4-AP) tethered with substituted 1,3,4-oxadiazole nucleus were designed, synthesized, and evaluated for their potential AChE inhibitory property along with significant antioxidant potential. The inhibitory potential (IC₅₀) of synthesized analogs was evaluated against human cholinesterases (hAChE and hBChE) using Ellman’s method. Among all the compounds, 9 with 4-hydroxyl substituent showed maximum hAChE inhibition with the non-competitive type of enzyme inhibition (IC₅₀ = 1.098 µM; Ki = 0.960 µM). Further, parallel artificial membrane permeation assay (PAMPA-BBB) showed significant BBB permeability in most of the synthesized compounds. Meanwhile, compound 9 also inhibited AChE-induced Aβ aggregation (38.2–65.9%) by thioflavin T assay. The in vivo behavioral studies showed dose-dependent improvement in learning and memory by compound 9. The ex vivo studies also affirmed the significant AChE inhibition and antioxidant potential of compound 9 in brain homogenates.     

Karyological variation in the group ofMyosotis alpestris (boraginacea)

A total of 6 population samples ofMyosotis stenophylla Knaf, a rare species showing great ecological disjunction in its distribution, were examined to clarify the present status of its karyological variation. In order to elucidate relationships between lowland tetraploid populations ofM. stenophylla and diploid and tetraploid montane populations ofM. alpestris F.W. Schmidt, four population samples ofM. alpestris were also examined. The karyotypes of all populations ofM. alpestris s.l. studied were highly asymmetrical and heterogeneous, being composed of metacentric, submetacentric, subtelocentric and satellited acrocentric chromosomes. The karyotype formula for haploid chromosome set was established: n=x=12=6m+2sm+3st+1t^SAT. Multivariate analysis based on chromosome length and shape showed significant differences between diploid and tetraploid forms ofM. alpestris s.l. Four numerical parameters, used to characterize the karyotype ofM. stenophylla, revealed significant differences between populations on serpentine and on non-serpentine substrates. In addition, the noticeable affinity of the karyotype of non-serpentine populations to that ofM. alpestris tetraploids has been shown by means of discriminant analysis. These data suggest that the unique features of serpentine play an important role in the origin of karyotypic differentiation within populations ofM. stenophylla.     

The effect of ferrous ions,tungstate and selenite on the level of formate dehydrogenase inClostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation

In a medium containing a trace element solution and 10^-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10^-4 M tungstate and 10^-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO₂ to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO₂ exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate.     

Intraspecific variation in sensitivity to ultraviolet-B radiation in endogenous hormones and photosynthetic characteristics of 10 wheat cultivars grown under field conditions

Field studies were conducted to determine the potential of altering endogenous hormones and photosynthetic characteristics and intraspecific variation in sensitivity of 10 wheat (Triticum aestivum) cultivars (four tolerant, two middle sensitive and four sensitive) to enhanced ultraviolet-B (UV-B, 280–315 nm) radiation under field conditions. The supplemental UV-B radiation was 5.00 kJ m⁻², simulating a depletion of 20% stratospheric ozone. Responses were cultivar-specific. Out of the 10 tested wheat cultivars, six showed significant decrease in IAA content. UV-B radiation significantly increased ZR content in two wheat cultivars and significantly decreased in five cultivars. ABA content of three wheat cultivars was increased significantly, while that of five cultivars was decreased significantly. UV-B radiation significantly increased the stomatal conductance of three cultivars, and significantly decreased that of four cultivars. Intercellular CO₂ concentrations were significantly increased in five cultivars and significantly decreased in one cultivar (Mianyang 20). Transpiration rate of three cultivars significantly increased, while that of three cultivars significantly decreased. UV-B radiation significantly decreased the net photosynthetic rate of six cultivars. Intraspecific differences were found for the different measured parameters. For seven measured parameters, UV-B radiation had significant effects on five wheat cultivars, while no effect on the others. Significant correlations were observed between net photosynthetic rate and stomatal conductance, intercellular CO₂ concentrations and transpiration rate in eight cultivars. UV-B radiation might change stomatal conductance, intercellular CO₂ concentrations and transpiration rate, thus resulting in changes in net photosynthetic rate.     

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.