Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase,lactoperoxidase, acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase isoenzymes

Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates. Acetylcholinesterase (AChE, E.C. 3.1.1.7) is intimately associated with the normal neurotransmission by catalysing the hydrolysis of acetylcholine to acetate and choline and acts in combination with butyrylcholinesterase (BChE) to remove acetylcholine from the synaptic cleft. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms, whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and in eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effects of rosmarinic acid on tumour-associated carbonic anhydrase IX and XII isoenzymes, AChE, BChE, LPO and GST enzymes were evaluated. Rosmarinic acid inhibited these enzymes with K_is in the range between micromolar to picomolar. The best inhibitory effect of rosmarinic acid was observed against both AChE and BChE.     

Discovery of potent carbonic anhydrase,acetylcholinesterase, and butyrylcholinesterase enzymes inhibitors: The new amides and thiazolidine‐4‐ones synthesized on an acetophenone base

Compounds containing nitrogen and sulfur atoms can be widely used in various fields, including industry, medicine, biotechnology, and chemical technology. Among them, amides of acids and heterocyclic compounds have an important place. These amides and thiazolidine‐4‐ones showed good inhibitory action against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and human carbonic anhydrase isoforms. AChE exists at high concentrations in the brain and red blood cells. BChE is an important enzyme that is plentiful in the liver, and it is released into the blood in a soluble form. They were demonstrated to have effective inhibition profiles with K_i values of 23.76–102.75 nM against hCA I, 58.92–136.64 nM against hCA II, 1.40–12.86 nM against AChE, and 9.82–52.77 nM against BChE. On the other hand, acetazolamide showed K_i value of 482.63 ± 56.20 nM against hCA I, and 1019.60 ± 163.70 nM against hCA II. Additionally, Tacrine inhibited AChE and BChE, showing K_i values of 397.03 ± 31.66 and 210.21 ± 15.98 nM, respectively.     

Theoretical Conformational Analysis in the Determination of Productive Conformations of Substrates for Acetylcholinesterase and Butyrylcholinesterase

All the equilibrium conformations of 34 analogues of acetylcholine (ACh) with the general formula R-C(O)O-Alk-N⁺(CH₃)₃ are calculated by the method of molecular mechanics. In the series R-C(O)O-(CH₂)₂-N⁺(CH₃)₃, a reliable correlation is found between the molecular volume of the substrate and the rate of its hydrolysis by acetylcholinesterase (AChE); the absence of such a correlation is demonstrated for butyryl-cholinesterase (BChE). Theoretical conformational analysis confirms that the completely extended tt conformation of ACh is productive for the hydrolysis by AChE, which agrees with the results of X-ray analysis of AChE. AChE is shown to hydrolyze only those substrates that form equilibrium conformers compatible in the mutual arrangement of trimethylammonium group, carbonyl carbon, and carbonyl oxygen with the tt conformation of ACh; in this case, the rate of substrate hydrolysis depends on the total population of these conformers. A reliable correlation was found between the population of the semifolded (tg^?) conformation of the choline moiety of substrate molecules and rate of their BChE hydrolysis. In a series of CH₃-C(O)O-Alk-N⁺(CH₃)₃, the rate of BChE hydrolysis is demonstrated to depend on the total population of conformations compatible in the mutual arrangement of functionally important atoms with the tg^? conformation of ACh. The tg^? conformation of ACh is concluded to be productive for BChE hydrolysis. Similar orientations of the substrate molecules relative to the catalytic triads of both AChE and BChE are proven to coincide upon the substrate productive sorption in their active sites. It is hypothesized that the sorption stage is rate-limiting in cholinesterase hydrolysis and the enzyme hydrolyzes the ACh molecule in its energetically favorable conformation.     

Novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes: Synthesis,characterization, acetylcholinesterase,butyrylcholinesterase, and carbonic anhydrase inhibitory properties

The thiolation reaction was carried out in a benzene solution at 80°C and p‐substituted ketones and mercaptoacetic acid in a molar ratio (1:4) of in the presence of a catalytic amount of toluene sulfonic acids. The enzyme inhibition activities of the novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives were investigated. These novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives showed good inhibitory action against acetylcholinesterase (AChE) butyrylcholinesterase (BChE), and human carbonic anhydrase I and II isoforms (hCA I and II). AChE inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. Many clinically established drugs are carbonic anhydrase inhibitors, and it is highly anticipated that many more will eventually find their way into the market. The novel synthesized compounds inhibited AChE and BChE with K_i values in the range of 0.64–1.47 nM and 9.11–48.12 nM, respectively. On the other hand, hCA I and II were effectively inhibited by these compounds, with K_i values between 63.27–132.34 and of 29.63–127.31 nM, respectively.     

Design,Synthesis, and Biological Evaluation of Novel Indanone Derivatives as Cholinesterase Inhibitors for Potential Use in Alzheimer's Disease

Indanone derivatives containing meta/para-substituted aminopropoxy benzyl/benzylidene moieties were designed based on the structures of donepezil and ebselen analogs as the cholinesterase inhibitors. The designed compounds were synthesized and their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were measured. Inhibitory potencies (IC₅₀ values) for the synthesized compounds ranged from 0.12 to 11.92 μM and 0.04 to 24.36 μM against AChE and BChE, respectively. Compound 5 c showed the highest AChE inhibitory potency with IC₅₀ value of 0.12 μM, whereas the highest BChE inhibition was achieved by structure 7 b (IC₅₀=0.04 μM). Structure-activity relationship (SAR) analysis revealed that there is no significant difference between meta and para-substituted derivatives in AChE and BChE inhibition. However, the most potent AChE inhibitor 5 c belongs to meta-substituted compounds, while the most active BChE inhibitor is para-substituted derivative 7 b . The order of enzyme inhibition potency based on the substituted amine group is dimethyl amine>piperidine>morpholine. Compounds containing C=C linkage are more potent AChE inhibitors than the corresponding saturated structures. Molecular docking studies indicated that 5 c interacts with AChE in a very similar way to that observed experimentally for donepezil. The introduced indanone-aminopropoxy benzylidenes could be used in drug-discovery against Alzheimer's disease.     

ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE-PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS

Abstract— In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro-striatal projection, the effects of selective destruction of these neurons by 6-hydroxydopamine (6-OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate-putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6-OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE), further experiments were conducted on tissues from animals with intracerebral 6-OHDA lesions. (1) Substrate specificity. Acetylcholine (ACh) was replaced by either acetyl-β-methyl-choline (AcβMeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6-OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with AcβMeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) Kinetics. The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) Selective inhibitors. In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose-response curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide, inhibited the enzyme in dopaminergic neurons only at concentrations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non-cholinergic neurons in mammalian CNS.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

The impact of hydroquinone on acetylcholine esterase and certain human carbonic anhydrase isoenzymes (hCA I,II, IX,and XII)

Abstract

Carbonic anhydrases (CAs) are widespread and the most studied members of a great family of metalloenzymes in higher vertebrates including humans. CAs were investigated for their inhibition of all of the catalytically active mammalian isozymes of the Zn²⁺-containing CA, (CA, EC 4.2.1.1). On the other hand, acetylcholinesterase (AChE. EC 3.1.1.7), a serine protease, is responsible for ACh hydrolysis and plays a fundamental role in impulse transmission by terminating the action of the neurotransmitter ACh at the cholinergic synapses and neuromuscular junction. In the present study, the inhibition effect of the hydroquinone (benzene-1,4-diol) on AChE activity was evaluated and effectively inhibited AChE with K_i of 1.22?nM. Also, hydroquinone strongly inhibited some human cytosolic CA isoenzymes (hCA I and II) and tumour-associated transmembrane isoforms (hCA IX, and XII), with K_is in the range between micromolar (415.81?μM) and nanomolar (706.79?nM). The best inhibition was observed in cytosolic CA II.     

Synthesis of some tetrahydropyrimidine-5-carboxylates,determination of their metal chelating effects and inhibition profiles against acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase

2-(Methacryloyloxy)ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate, is a cyclic urea derivative synthesized from urea, 2-(methacryloyloxy) ethyl acetoacetate and substituted benzaldehyde, and tested in terms of the inhibition of two physiologically relevant carbonic anhydrase (CA) isozymes I and II. Acetylcholinesterase (AChE) is found in high concentrations in the red blood cells and brain. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Also, they were tested for the inhibition of AChE and BChE enzymes and demonstrated effective inhibition profiles with Ki values in the range of 429.24–530.80?nM against hCA I, 391.86–530.80?nM against hCA II, 68.48–97.19?nM against AChE and 104.70–214.15?nM against BChE. On the other hand, acetazolamide clinically used as CA inhibitor, showed Ki value of 281.33?nM against hCA I, and 202.70?nM against hCA II. Also, Tacrine inhibited AChE and BChE showed Ki values of 396.03 and 209.21?nM, respectively.     

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer's disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE-/- mice). Extracellular ACh levels in the hippocampus were 60-fold elevated in AChE-/- mice compared with wild-type (AChE+/+) animals. In AChE-/- mice, calcium-free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose-dependent, up to 16-fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE-/- mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE-/- mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis-norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.     

Piperazine derivatives with potent drug moiety as efficient acetylcholinesterase,butyrylcholinesterase, and glutathione S-transferase inhibitors

Cholinesterases catalyze the breakdown of the neurotransmitter acetylcholine (ACh), a naturally occurring neurotransmitter, into choline and acetic acid, allowing the nervous system to function properly. In the human body, cholinesterases come in two types, including acetylcholinesterase (AChE; E.C.3.1.1.7) and butyrylcholinesterase (BChE; E.C.3.1.1.8). Both cholinergic enzyme inhibitors are essential in the biochemical processes of the human body, notably in the brain. On the other hand, GSTs are found all across nature and are the principal Phase II detoxifying enzymes in eukaryotes and prokaryotes. Specific isozymes are identified as therapeutic targets because they are overexpressed in various malignancies and may have a role in the genesis of other diseases such as neurological disorders, multiple sclerosis, asthma, and especially cancer cell. Piperazine chemicals have a role in many biological processes and have fascinating pharmacological properties. As a result, therapeutically effective piperazine research is becoming more prominent. Half maximal inhibition concentrations (IC₅₀) of piperazine derivatives were found in ranging of 4.59–6.48 µM for AChE, 4.85–8.35 µM for BChE, and 3.94-8.66 µM for GST. Also, piperazine derivatives exhibited Ki values of 8.04 ± 5.73–61.94 ± 54.56, 0.24 ± 0.03–32.14 ± 16.20, and 7.73 ± 1.13–22.97 ± 9.10 µM toward AChE, BChE, and GST, respectively. Consequently, the inhibitory properties of the AChE/BChE and GST enzymes have been compared to Tacrine (for AChE and BChE) and Etacrynic acid (for GST).     

Synthesis,Characterization, and Inhibition Study of Novel Substituted Phenylureido Sulfaguanidine Derivatives as α-Glycosidase and Cholinesterase Inhibitors

A series of six N-carbamimidoyl-4-(3-substituted phenylureido)benzenesulfonamide derivatives were synthesized by reaction of sulfaguanidine with aromatic isocyanates. In vitro and in silico inhibitory effects of the novel ureido-substituted sulfaguanidine derivatives were investigated by spectrophotometric methods for α-glycosidase (α-GLY), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes associated with diabetes mellitus (DM) and Alzheimer's disease (AD). N-Carbamimidoyl-4-{[(3,4-dichlorophenyl)carbamoyl]amino}benzene-1-sulfonamide ( 2f ) showed AChE and BChE inhibitory effects, with K_I values of 515.98±45.03 nM and 598.47±59.18 nM, respectively, while N-carbamimidoyl-4-{[(3-chlorophenyl)carbamoyl]amino}benzene-1-sulfonamide ( 2e ) showed strong α-GLY inhibitory effect, with K_I values of 103.94±13.06 nM. The antidiabetic effects of the novel synthesized compounds are higher than their anti-Alzheimer's effects, because the inhibition effect of the compounds on the α-GLY with diabetic enzyme is greater than the effect on esterase enzymes. Indeed, inhibition of the metabolic enzymes is important for the treatment of DM and AD.     

Design,synthesis and cholinesterase inhibitory properties of new oxazole benzylamine derivatives

Abstract

The enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are primary targets in attenuating the symptoms of neurodegenerative diseases. Their inhibition results in elevated concentrations of the neurotransmitter acetylcholine which supports communication among nerve cells. It was previously shown for trans-4/5-arylethenyloxazole compounds to have moderate AChE and BChE inhibitory properties. A preliminary docking study showed that elongating oxazole molecules and adding a new NH group could make them more prone to bind to the active site of both enzymes. Therefore, new trans-amino-4-/5-arylethenyl-oxazoles were designed and synthesised by the Buchwald-Hartwig amination of a previously synthesised trans-chloro-arylethenyloxazole derivative. Additionally, naphthoxazole benzylamine photoproducts were obtained by efficient photochemical electrocyclization reaction. Novel compounds were tested as inhibitors of both AChE and BChE. All of the compounds exhibited binding preference for BChE over AChE, especially for trans-amino-4-/5-arylethenyl-oxazole derivatives which inhibited BChE potently (IC₅₀ in µM range) and AChE poorly (IC₅₀?100?µM). Therefore, due to the selectivity of all of the tested compounds for binding to BChE, these compounds could be applied for further development of cholinesterase selective inhibitors.

• HIGHLIGHTS

• Series of oxazole benzylamines were designed and synthesised

• The tested compounds showed binding selectivity for BChE

• Naphthoxazoles were more potent AChE inhibitors

     

Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K_i was 0.01 mM for AChE and 0.06 mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000 min⁻¹ M⁻¹, which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20 h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K_i), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20 h, the reactivation maximum was 60% for 1 mM K074 and K075, and only 20% for 1 mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate.Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate.For both enzymes, progress curves for hydrolysis of PhA at very low concentration (?K_m) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.     

Prenylated xanthones from mangosteen as promising cholinesterase inhibitors and their molecular docking studies

Garcinia mangostana is a well-known tropical plant found mostly in South East Asia. The present study investigated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of G. mangostana extract and its chemical constituents using Ellman's colorimetric method. Cholinesterase inhibitory-guided approach led to identification of six bioactive prenylated xanthones showing moderate to potent cholinesterases inhibition with IC₅₀ values of lower than 20.5 μM. The most potent inhibitor of AChE was garcinone C while γ-mangostin was the most potent inhibitor of BChE with IC₅₀ values of 1.24 and 1.78 μM, respectively. Among the xanthones, mangostanol, 3-isomangostin, garcinone C and α-mangostin are AChE selective inhibitors, 8-deoxygartanin is a BChE selective inhibitor while γ-mangostin is a dual inhibitor. Preliminary structure-activity relationship suggests the importance of the C-8 prenyl and C-7 hydroxy groups for good AChE and BChE inhibitory activities. The enzyme kinetic studies indicate that both α-mangostin and garcinone C are mixed-mode inhibitors, while γ-mangostin is a non-competitive inhibitor of AChE. In contrast, both γ-mangostin and garcinone C are uncompetitive inhibitors, while α-mangostin is a mixed-mode inhibitor of BChE. Molecular docking studies revealed that α-mangostin, γ-mangostin and garcinone C interacts differently with the five important regions of AChE and BChE. The nature of protein–ligand interactions is mainly hydrophobic and hydrogen bonding. These bioactive prenylated xanthones are worthy for further investigations.     

Synthesis and discovery of potent carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase,and α‐glycosidase enzymes inhibitors: The novel N,N′‐bis‐cyanomethylamine and alkoxymethylamine derivatives

During this investigation, N,N′‐bis‐azidomethylamines, N,N′‐bis‐cyanomethylamine, new alkoxymethylamine and chiral derivatives, which are considered to be a new generation of multifunctional compounds, were synthesized, functional properties were investigated, and anticholinergic and antidiabetic properties of those compounds were studied through the laboratory tests, and it was approved that they contain physiologically active compounds rather than analogues. Novel N‐bis‐cyanomethylamine and alkoxymethylamine derivatives were effective inhibitors of the α‐glycosidase, cytosolic carbonic anhydrase I and II isoforms, butyrylcholinesterase (BChE), and acetylcholinesterase (AChE) with K_i values in the range of 0.15–13.31 nM for α‐glycosidase, 2.77–15.30 nM for human carbonic anhydrase isoenzymes I (hCA I), 3.12–21.90 nM for human carbonic anhydrase isoenzymes II (hCA II), 23.33–73.23 nM for AChE, and 3.84–48.41 nM for BChE, respectively. Indeed, the inhibition of these metabolic enzymes has been considered as a promising factor for pharmacologic intervention in a diversity of disturbances.     

Theoretical conformational analysis in the determination of productive conformations of substrates for acetylcholinesterase and butyrylcholinesterase

All the equilibrium conformations of 34 analogues of acetylcholine (ACh) with the general formula R-C(O)O-Alk-N+(CH3)3 are calculated by the method of molecular mechanics. In the series R-C(O)O-(CH2)2-N+(CH3)3, a reliable correlation is found between the molecular volume of the substrate and the rate of its hydrolysis by acetylcholinesterase (AChE); the absence of such a correlation is demonstrated for butyrylcholinesterase (BChE). Theoretical conformational analysis confirms that the completely extended tt conformation of ACh is productive for the hydrolysis by AChE, which agrees with the results of X-ray analysis of AChE. AChE is shown to hydrolyze only those substrates that form equilibrium conformers compatible in the mutual arrangement of trimethylammonium group, carbonyl carbon, and carbonyl oxygen with the tt conformation of ACh; in this case, the rate of substrate hydrolysis depends on the total population of these conformers. A reliable correlation was found between the population of the semifolded (tg-) conformation of the choline moiety of substrate molecules and the rate of their BChE hydrolysis. In a series of CH3-C(O)O-Alk-N+(CH3)3, the rate of BChE hydrolysis is demonstrated to depend on the total population of conformations compatible in the mutual arrangement of functionally important atoms with the tg- conformation of ACh. The tg- conformation of ACh is concluded to be productive for BChE hydrolysis. Similar orientations of the substrate molecules relative to the catalytic triads of both AChE and BChE are proven to coincide upon the substrate productive sorption in their active sites. It is hypothesized that the sorption stage is rate-limiting in cholinesterase hydrolysis and the enzyme hydrolyzes the ACh molecule in its energetically favorable conformation.     

Synthesis,Spectroscopic Analysis,and in Vitro/in Silico Biological Studies of Novel Piperidine Derivatives Heterocyclic Schiff-Mannich Base Compounds

In the present study, 3-substitued-4-(4-hydroxybenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones ( S1-8 ) were synthesized by treating 4-hydroxybenzaldehyde ( B ) with eight different 3-substitued-4-amino-4,5-dihydro-1H-1,2,4-triazole-5-ones ( T1-8 ) in acetic acid medium, separately. The synthesized Schiff bases ( S ) were reacted with formaldehyde and secondary amine such as 4-piperidinecarboxyamide to afford novel heterocyclic bases. 3-Substitued-4-(4-hydroxybenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones ( T ) were treated with 4-piperidinecarboxyamide in the presence of formaldehyde to synthesize eight new 1-(4-piperidinecarboxyamide-1-yl - methyl)-3-substitued-4-(4-hydroxybenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones ( M1-8 ). The structure characterization of compounds was carried out using ¹H-NMR, IR, HR-MS, and ¹³C-NMR spectroscopic methods. The inhibitory properties of the newly synthesized compounds were calculated against the acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and glutathione S-transferase (GST) enzymes. Ki values were calculated in the range of 20.06±3.11–36.86±6.17 μM for GST, 17.87±2.91–30.53±4.25 μM for AChE, 9.08±0.69–20.02±2.88 μM for BChE, respectively, Besides, IC₅₀ values were also calculated. Best binding scores of -inhibitors against used enzymes were calculated as −12.095 kcal/mol, −12.775 kcal/mol, and −9.336 kcal/mol, respectively. While 5-oxo-triazole piperidine-4-carboxamide moieties have a critical role in the inhibition of AChE and GST enzymes, hydroxy benzyl moiety is important for BChE enzyme inhibition.     

Comprehensive Phytochemical Content by LC/MS/MS and Anticholinergic,Antiglaucoma, Antiepilepsy,and Antioxidant Activity of Apilarnil (Drone Larvae)

Apilarnil is 3–7 days old drone larvae. It is an organic bee product known to be rich in protein. In this study, the biological activities of Apilarnil were determined by its antioxidant and enzyme inhibition effects. Antioxidant activities were determined by Fe³⁺, Cu²⁺, Fe³⁺-TPTZ ((2,4,6-tris(2-pyridyl)-s-triazine), reducing ability and 1,1-diphenyl-2-picrylhydrazyl (DPPH⋅) scavenging assays. Also, its enzyme inhibition effects were tested against carbonic anhydrase I and II isoenzymes (hCA I, hCA II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Antioxidant activity of Apilarnil was generally lower than the standard molecules in the applied methods. In DPPH⋅ radical scavenging assay, Apilarnil exhibited higher radical scavenging than some standards. Enzyme inhibition results towards hCA I (IC₅₀: 14.2 μg/mL), hCA II: (IC₅₀: 11.5 μg/mL), AChE (IC₅₀: 22.1 μg/mL), BChE (IC₅₀: 16.1 μg/mL) were calculated. In addition, the quantity of 53 different phytochemical compounds of Apilarnil was determined by a validated method by LC/MS/MS. Compounds with the highest concentrations (mg analyte/g dry extract) were determined as quinic acid (1091.045), fumaric acid (48.714), aconitic acid (47.218), kaempferol (39.946), and quercetin (27.508). As a result, it was determined that Apilarnil had effective antioxidant profile when compared to standard antioxidants.