Potent AChE Inhibitor Research Articles

Selective inhibition of human acetylcholinesterase by xanthine derivatives: In vitro inhibition and molecular modeling investigations

The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC50=6.40μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC50=7.25μM)<pentoxifylline (hAChE IC50=6.60μM)⩽propentofylline (hAChE IC50=6.40μM). These compounds were less potent relative to the reference agent donepezil (hAChE IC50=0.04μM). Moreover, they all exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC50>50μM) relative to the reference agent donepezil (hBuChE IC50=13.60μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.

Carbamate Derivatives of Indolines as Cholinesterase Inhibitors and Antioxidants for the Treatment of Alzheimer’s Disease

The cascade of events that occurs in Alzheimer's disease involving oxidative stress and the reduction in cholinergic transmission can be better addressed by multifunctional drugs than cholinesterase inhibitors alone. For this purpose, we prepared a large number of derivatives of indoline-3-propionic acids and esters. They showed scavenging activity against different radicals in solution and significant protection against cytotoxicity in cardiomyocytes and primary cultures of neuronal cells exposed to H2O2 species and serum deprivation at concentrations ranging from 1 nM to 10 μM depending on the compound. For most of the indoline-3-propionic acid derivatives, introduction of N-methyl-N-ethyl or N-methyl-N-(4-methoxyphenyl) carbamate moieties at positions 4, 6, or 7 conferred both acetyl (AChE) and butyryl (BuChE) cholinesterase inhibitory activities at similar concentrations to those that showed antioxidant activity. The most potent AChE inhibitors were 120 (3-(2-aminoethyl) indolin-4-yl ethyl(methyl)carbamate dihydrochloride) and 94 (3-(3-methoxy-3-oxopropyl)-4-(((4-methoxyphenyl)(methyl) carbamoyl)oxy)indolin-1-ium hydrochloride) with IC50s of 0.4 and 1.2 μM, respectively.

Pyridonepezils, new dual AChE inhibitors as potential drugs for the treatment of Alzheimer's disease: Synthesis, biological assessment, and molecular modeling

The synthesis, biological assessment and molecular modeling of new pyridonepezils1–8, able to inhibit human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE), are described. The new compounds have been designed as hybrids resulting from a conjunctive approach that combines the N-benzylpiperidine moiety, present in donepezil, and the 2-amino-6-chloropyridine heterocyclic ring system, connected by an appropriate polymethylene linker. Compounds 1–8 were prepared by reaction of 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2-amino-6-chloropyridine-3,5-dicarbonitrile (14)] with 2-(1-benzylpiperidin-4-yl)alkylamines (9–12). The biological evaluation of molecules 1–8 showed that compounds 1–6 are potent AChE inhibitors, in the submicromolar, while compounds 7 and 8 are on the nanomolar range, the most potent, 2-amino-6-((3-(1-benzylpiperidin-4-yl)propyl)amino)pyridine-3,5-dicarbonitrile (7), showing a IC50 (hAChE) = 9.4 ± 0.4 nM. Inhibitors 2–8 are permeable as determined in the PAMPA assay. Compared to donepezil, compound 7 is in the same range of inhibitory activity for hAChE, and 703-fold more selective for hAChE than for hBuChE. Molecular modeling investigation on pyridonepezil7 supports its dual AChE inhibitory profile, binding simultaneously at the catalytic active and at peripheral anionic sites of the enzyme. The theoretical ADME analysis of pyridonepezils1–8 has been carried out. Overall, compound 7, a potent and selective dual AChEI, can be considered as a candidate with potential impact for further pharmacological development in Alzheimer's therapy.

Inhibition of cholinesterase activity and amyloid aggregation by berberine-phenyl-benzoheterocyclic and tacrine-phenyl-benzoheterocyclic hybrids

A series of berberine-phenyl-benzoheterocyclic (26–29) and tacrine-phenyl-benzoheterocyclic hybrids (44–46) were synthesised and evaluated as multifunctional anti-Alzheimer’s disease agents. Compound 44b, tacrine linked with phenyl-benzothiazole by 3-carbon spacers, was the most potent AChE inhibitor with an IC50 value of 0.017μM. This compound demonstrated similar Aβ aggregation inhibitory activity with cucurmin (51.8% vs 52.1% at 20μM, respectively), indicating that this hybrid is an excellent multifunctional drug candidate for AD.

Screening of new huprines—Inhibitors of acetylcholinesterases by electrospray ionization ion trap mass spectrometry

Acetylcholinesterase inhibitors (AChEI) are one of the drugs families validated for clinical use in the treatment of Alzheimer's disease (AD). For this reason, finding new more potent and more selective AChEIs is always of interest. Since 1961, the inhibitory activity of AChEI is evaluated through the Ellman's method. Herein, we reported a MS-based evaluation of potential new AChEI with the determination of their inhibitory activity (IC(50) and K(I)). Compared to the Ellman's method, that uses the substrate analog acetylthiocholine, the electrospray ionization ion trap mass spectrometry (ESI-IT-MS) consists in monitoring the conversion ratio of a low concentration of the natural substrate - acetylcholine to choline. We present here the inhibition activity of huprine X and six of its derivates (bearing different functional groups at position 9) towards the recombinant human (rhAChE) and Electrophorus electricus acetylcholinesterase (EelAChE). Mechanisms of action of selected inhibitors were evaluated by means of Lineweaver-Burk plot analysis. The Michaelis-Menten constants (K(M)), inhibitory constants (K(I)) were examined as well as the IC(50) to allow classifying a series of huprine derivatives by inhibition potency by a comparison with a reference (huprine X). Our results demonstrate that these drugs are very potent AChE inhibitors, especially (±)-huprine 6 with an inhibitory activity on recombinant human AChE (rhAChE) in the picomolar range. This study reveals the interest of huprine compounds in the treatment of AD.

Design, synthesis and structure–activity relationship (SAR) studies of 2,4-disubstituted pyrimidine derivatives: Dual activity as cholinesterase and Aβ-aggregation inhibitors

A novel class of 2,4-disubstituted pyrimidines ( 7a– u, 8a– f, 9a– e) that possess substituents with varying steric and electronic properties at the C-2 and C-4 positions, were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. In vitro screening identified N-(naphth-1-ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine ( 9a) as the most potent AChE inhibitor (IC 50 = 5.5 μM). Among this class of compounds, 2-(4-methylpiperidin-1-yl)- N-(naphth-1-ylmethyl)pyrimidin-4-amine ( 9e) was identified as the most potent and selective BuChE inhibitor (IC 50 = 2.2 μM, selectivity index = 11.7) and was about 5.7-fold more potent compared to the commercial, approved reference drug galanthamine (BuChE IC 50 = 12.6 μM). In addition, the selective AChE inhibitor N-benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine ( 7d), exhibited good inhibition of hAChE-induced aggregation of Aβ 1–40 fibrils (59% inhibition). Furthermore, molecular modeling studies indicate that a central pyrimidine ring serves as a suitable template to develop dual inhibitors of cholinesterase and AChE-induced Aβ aggregation thereby targeting multiple pathological routes in AD.

Novel Carbamates as Orally Active Acetylcholinesterase Inhibitors Found to Improve Scopolamine-Induced Cognition Impairment: Pharmacophore-Based Virtual Screening, Synthesis, and Pharmacology

A systematic virtual screening (VS) experiment, consisting of the development of 3D-pharmacophore, screening of virtual library, synthesis, and pharmacology, is reported. The predictive pharmacophore model (correlation = 0.955) with one H-bond donor and three hydrophobic features was developed using HypoGen on a training set of 24 carbamates as AChE inhibitors. The model was validated on a test set of 40 carbamates (correlation = 0.844). The pharmacophore-based VS of virtual library led to the identification of novel carbamates as potent AChE inhibitors. The synthesis and pharmacological evaluation of nine carbamates against three diverse assay systems, namely (i) in vitro Ellman method, (ii) in vivo passive avoidance test, and (iii) aldicarb-sensitivity assay, led to the discovery of orally active novel AChE inhibitors which improved scopolamine-induce cognition impairment in Swiss male mice. Finally, two novel lead compounds 85 and 86 are selected as candidate molecules for further optimization.

New Bisquaternary Isoquinolinium Inhibitors of Brain Cholinesterases - Synthesis and Anticholinesterase Activity

Abstract: Alzheimer´s disease (AD) is one of the most discussed diseases of present time. Because of the aging of our population it is high threat for future. Due to this, new drugs combating AD are still developed. In this study, twelve bisquaternary isoquinolinium de-rivatives differing in the linker between two heteroarenium rings were synthesized and tested for their potency to inhibit brain cho-linesterases. According to the obtained in vitro results, the anticholinesterase activity grew up with the length of the connection chain. Possible binding to the enzyme cavity was described using software Autodock. Moreover, LogP and molecular volume of prepared com-pounds were predicted. As resulted, the highest probability of synthesized compounds to penetrate blood-brain barrier could be expected for the compound with linker having eight methylenes. Keywords: Quinolinium, Quaternary compounds, Inhibitor, Cholinesterase, Alzheimer´s disease, Anticholinergic. INTRODUCTION At present, there is increasing number of people suffering from diseases connecting with the increasing age. Among them, Alz-heimer´s disease (AD) is well discussed by the scientific commu-nity. It belongs along with cancer, AIDS and cardiovascular dis-eases among the highest threats influencing the whole population. Although this disease is known already for more than one hundred years, there is no treatment able to stop its degenerative progress. Currently, there are just few AD treatments (Donepezil, Galan-tamine, Rivastigmine, Tacrine, Memantine) approved by FDA. All of them, except Memantine, are potent inhibitors of acetylcho-linesterase (AChE; EC 3.1.1.7). Although the cholinergic approach is able to have only one- or two-year benefit, economically, one or two more years of the productive life are very important [1,2]. Due to this reason, development of new AChE inhibitors as new AD-drugs candidates is still possible approach how to combat AD [3-5]. During last years, there were published several articles discuss-ing penetration of quaternary compounds into the blood-brain bar-rier (BBB) [6-8]. Thanks to this, also quaternary compounds with increased lipophility could be considered as an alternative for treatment of AD or at least as its supplement. Recently, there were published several articles describing quaternary compounds (expe-cially pyridinium compounds) as potential AD treatment [9-12]. In 2004, we found that more potent inhibitors of AChE than pyridi-nium salts seem to be quaternary compounds with conjugated aro-matic rings (quinolinium, isoquinolinium or acridinium) [13]. Thanks to the added aromatic rings, also their lipophilicity is higher if compared with pyridinium compounds. And with the lipophilic-ity, hand in hand, higher penetration through the BBB can be ex-pected. Because of the fact that affinity of bisquaternary compounds is higher than that of monoquaternary ones, in this article, we turned our attention to bisquaternary isoquinolinium compounds differing in the length of the connection chain between two isoquinolines.

N‐substituted isoquinoline derivatives as potential AChE inhibitors

Abstract magnified imageN‐substituted donepezil‐related isoquinolines have been prepared as potential acetylcholinesterase inhibitors (AChEIs). Microwave assisted procedures and solution‐phase parallel synthesis were chosen to optimize the synthetic approach and improve the yields. All synthesized compounds were tested for their AChE inhibitory activity by colorimetric Ellman method and some of them (10, 13, and 28) displayed low inhibitory effects at μM concentrations. J. Heterocyclic Chem., (2010).

Metabolic profiling of bioactive Pancratium canariense extracts by GC‐MS

Pancratium canariense Ker Gawler is a plant species belonging to family Amaryllidaceae. Plants from this family are known to synthesise a particular type of bioactive compounds, named Amaryllidaceae alkaloids, which have shown AChE inhibitory activity. To perform the metabolite profiling of methanolic extracts from P. canariense in order to identify bioactive compounds. Methanolic extracts from bulbs, leaves and fruits were separated into alkaloid-free apolar and polar fractions, as well as alkaloid fractions, and subjected to AChE assay. Metabolite profiling of extracts and fractions of P. canariense was carried out by GC-EI-MS and LC-ESI-TOF-MS. AChE inhibitory activities of the alkaloid fractions at a concentration of 10 microg/mL were 29.80 +/- 0.91, 40.93 +/- 4.60 and 58.06 +/- 1.18% for the bulbs, leaves and fruits, respectively. Seventy-six metabolites-mono-, di- and trisaccharides, fatty acids, amino acids, sterols as well as several Amaryllidaceae alkaloids-were detected. Further purification of the alkaloids from the methanolic extracts resulted in the detection of 31 compounds including several potent AChE inhibitors such as habranthine and galanthamine, and the structural elucidation of 3-O-acetylhabranthine, a new natural compound with potential AChE inhibitory activity. The described method resulted in effective integration of both GC-EI-MS and LC-ESI-TOF-MS strategies, which permitted the identification of many metabolites, as well as the structural elucidation of new compounds with potential AChE inhibitory activity.

Design, synthesis and evaluation of flavonoid derivatives as potent AChE inhibitors

A new series of flavonoid derivatives have been designed, synthesized and evaluated as potent AChE inhibitors. Most of them showed more potent inhibitory activities to AChE than rivastigmine. The most potent inhibitor isoflavone derivative 10d inhibit AChE with a IC 50 of 4 nM and showed high BChE/AChE inhibition ratio (4575-fold), superior to donepezil (IC 50 = 12 nM, 389-fold). Molecular docking studies were also performed to explore the detailed interaction with AChE.

Tacripyrines, the First Tacrine−Dihydropyridine Hybrids, as Multitarget-Directed Ligands for the Treatment of Alzheimer’s Disease

Tacripyrines (1-14) have been designed by combining an AChE inhibitor (tacrine) with a calcium antagonist such as nimodipine and are targeted to develop a multitarget therapeutic strategy to confront AD. Tacripyrines are selective and potent AChE inhibitors in the nanomolar range. The mixed type inhibition of hAChE activity of compound 11 (IC(50) 105 +/- 15 nM) is associated to a 30.7 +/- 8.6% inhibition of the proaggregating action of AChE on the Abeta and a moderate inhibition of Abeta self-aggregation (34.9 +/- 5.4%). Molecular modeling indicates that binding of compound 11 to the AChE PAS mainly involves the (R)-11 enantiomer, which also agrees with the noncompetitive inhibition mechanism exhibited by p-methoxytacripyrine 11. Tacripyrines are neuroprotective agents, show moderate Ca(2+) channel blocking effect, and cross the blood-brain barrier, emerging as lead candidates for treating AD.

λ-cyhalothrin and cypermethrin induced in vivo alterations in the activity of acetylcholinesterase in a freshwater fish, Channa punctatus (Bloch)

In the present study, the in vivo effects of λ-cyhalothrin and cypermethrin on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) were evaluated for 96 h in brain, muscle and gills of Channa punctatus. Both compounds exhibited tissue specific as well as dose dependent decrease in the activity of AChE. The treated fish showed a significant decrease in the activity of AChE in brain and a lesser inhibition in muscle and gills in response to the increasing concentrations of λ-cyhalothrin as well as cypermethrin. Our results indicated that the brain was the main target organ for both insecticides, followed by muscle and gills, as determined by AChE inhibition study. However, these organs showed variations in the degree of AChE inhibition for separate treatments of both insecticides. The λ-cyhalothrin was a more potent AChE inhibitor as compared to cypermethrin. These findings indicated that apart from the established mechanism of delayed closure of sodium ion channels, these pyrethroids inhibit the activity of AChE in C. punctatus which could further aggravate their neurotoxic effects.

Synthesis of Fenthion Sulfoxide and Fenoxon Sulfoxide Enantiomers: Effect of Sulfur Chirality on Acetylcholinesterase Activity

Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the organophosphate pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were transformed into chiral fenoxon sulfoxides using a two-step protocol. The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (>82%) from the parent sulfide. Single recrystallizations afforded chiral fenthion sulfoxides with >99% ee, measured by chiral HPLC analysis. The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis of the (S)-(-)-antipode. Inhibition of human recombinant (hrAChE) and electric eel (eeAChE) acetylcholinesterase were assayed with fenthion, fenoxon, and the racemates and enantiomers of fenthion sulfoxide and fenoxon sulfoxide. Results revealed stereoselective inhibition with (R)-(+)-fenoxon sulfoxide when compared with that of (S)-(-)-fenoxon sulfoxide (IC50 of 6.9 and 6.5 microM vs 230 and 111 microM in hrAChE and eeAChE, respectively). Fenthion sulfoxide (R or S enantiomers) did not present anti-AChE properties. Although the stereoselective sulfoxidation of fenthion to (R)-(+)-fenthion sulfoxide by FMO represents a detoxification pathway, the results of this study support the notion that subsequent oxidative desulfuration of (R)-(+)-fenthion sulfoxide (in vivo) may represent a critical bioactivation pathway, resulting in the production of (R)-(+)-fenoxon sulfoxide, a potent AChE inhibitor.

Synthesis and biological evaluation as AChE inhibitors of new indanones and thiaindanones related to donepezil

Sixty-four new indanones and thiaindanones related to donepezil were synthesized and evaluated in vitro as potential AChE inhibitors. Among them, 11 derivatives were found to inhibit the enzyme in the submicromolar range; the best compound revealed its inhibitory activity with an IC 50 in the same range (0.06 μM) than the reference compound, donepezil (IC 50 = 0.02 μM).

Acute effects of physostigmine and galantamine on the binding of [18F]fluoro-A-85380: A PET study in monkeys

2-[18F]fluoro-3-[2S-2-azetidinylmethoxy]pyridine ([18F]fluoro-A-85380) is an alpha4beta2 subtype selective nicotinic cholinergic agonist with potential suitability for studying changes in endogenous acetylcholine synaptic concentration. Physostigmine, a potent AChE inhibitor, and galantamine, an allosteric modulator of nAChRs, are widely used for the treatment of Alzheimer's disease. Before studying patients with this neurodegenerative disease, positron emission tomography (PET) studies in monkeys were performed to assess the impact of these two compounds on the radiotracer distribution volumes. Physostigmine was administered i.v. at two dosages: 150 microg/kg/h and 37.5 microg/kg/h for 160 min. Galantamine was administered i.v. at two dosages: 2 or 4 mg over 20 min. For PET data analysis, a model with one tissue (radioactivity of the parent compound in plasma and radioactivity in brain tissue) compartment was chosen because reliable parameter estimates could not be obtained with a more complex model. The higher dose of physostigmine produced a 40%, 23%, and 30% reduction of distribution volumes in the putamen, the temporal, and frontal cortices, respectively. The lower dose of physostigmine produced a reduction of 33%, 31%, and 24% in the same structures, respectively. Galantamine (4 mg or 2 mg) produced no significant change of distribution volumes in the basal ganglia, the temporal and frontal cortex. The effects of physostigmine, a more potent AChE inhibitor than galantamine, could be interpreted as a desensitization of nAChRs, due to a prolonged exposure to high synaptic concentration of acetylcholine or as a competition with acetylcholine.

Structure−Activity Relationships of Acetylcholinesterase Noncovalent Inhibitors Based on a Polyamine Backbone. 3. Effect of Replacing the Inner Polymethylene Chain with Cyclic Moieties

In the present paper we expanded SAR studies of 3, the ethyl analogue of the AChE inhibitor caproctamine (2), by investigating the role of its octamethylene spacer separating the two amide functions through the replacement with dipiperidine and dianiline moieties. Compounds 4 and 8 were the most interesting of the two series of compounds. Compound 4 was the most potent AChE inhibitor with a pIC50 value of 8.48 +/- 0.02, while displaying also significant muscarinic M2 antagonistic activity (pKb value of 6.18 +/- 0.20). The availability of a suitable assay allowed us to verify whether 2, 3, 4, and 8 inhibit AChE-induced Abeta aggregation. Although all four derivatives caused a mixed type of AChE inhibition (active site and PAS), only 4 and 8, which bear an inner constrained spacer, were able to inhibit AChE-induced Abeta aggregation to a greater extent than donepezil. Clearly, the ability of an AChE inhibitor, based on a linear polyamine backbone, to bind both AChE sites may not be a sufficient condition to inhibit also AChE-induced Abeta aggregation. Dipiperidine derivative 4 emerged as a valuable pharmacological tool and a promising lead compound for new ligands to investigate and, hopefully, treat Alzheimer's disease.

Potential neurotoxicity of a novel aminoacridine analogue

1. A class of compounds, 9-aminoacridines, have long been known to be reversible inhibitors of acetylcholinesterase (AChE-EC 3.1.1.7), the most familiar of which is 9-amino-1,2,3,4-tetrahydroacridine (Tacrine). 2. A novel aminoacridine was synthesised: -2-tertiary-butyl-9-amino-1,2,3,4- tetrahydroacridine (2tBuTHA). 3. In vitro comparisons of the acetylcholinesterase inhibitory potential and neurotoxicity compared to Tacrine were performed using a chemically differentiated neuroblastoma cell line (Neuro 2A). 2tBuTHA, but not Tacrine, was cytotoxic to the neural cell following 20 h exposure, despite being the least potent AChE inhibitor (IC80 AChE 12.53 microM +/- 1.14 s.e.m., Neutral Red Uptake IC50 9.53 microM +/- 0.98 s.e.m., MTT Reduction IC80 14.6 microM +/- 1.43 s.e.m.). 4. In vivo studies used a novel application of a five arm radial maze to assess neuropharmacological effects on working memory in control and Scopolamine (1 mg kg-1 i.p.) treated mice. There was an impairment of short term cognitive function with 2tBuTHA (15 mg kg-1 i.p.), but not Tacrine (10 mg kg-1 i.p.) which improved the Scopolamine deficit as expected. 5. This combined in vitro and in vivo data infers a neurotoxic property for the novel compound 2tBuTHA, a close structural analogue of Tacrine.

Patent Evaluation: Novel Benzylpiperidines as Potent AChE Inhibitors

Patent Evaluation: Novel Benzylpiperidines as Potent AChE Inhibitors

Biological Activity and Metabolism of Phosphoramidate Insecticides

The mode of action of phosphoramidothioates was examined with reference to their anti-AChE activity. N-Isopropylphosphoramidothioate, isofenphos, was the most toxic to the adzuki bean weevil among N-alkyl substituted compounds. LD50 values for the insects were not correlated with the in vitro anti-AChE activity of these oxons. It was demonstrated that phosphoramidothioates and these oxons became potent inhibitors of AChE by the incubation with the rat liver microsomal NADPH system and the chemical treatment with peracid. The inhibition of AChE activity was reduced by the addition of SKF 525-A to the microsomal system. The stereospecificity of isofenphos and its oxon on biological activity was obvious. The (+)-isomers of both the compounds were more toxic to insects than the (-)-isomers. Chiral isomers and racemate of isofenphos-oxon were activated with the microsomal NADPH system and the I50 values became 1.47×10-7M, and 6.19×10-5M for the (+)- and (-)-oxon, respectively. The data suggested that the (+)-isomer was easily bioactivated and the I50 values of chiral isomers after the bioactivation were sufficient to explain their insecticidal activity. 14C-Isofenphos metabolism was different between animals and plants. In mammals and insects, isofenphos was rapidly transformed into the water soluble metabolites, and in plant isofenphos and isofenphos-oxon were very persistent. Isofenphos was metabolized by (1) oxidative desulfuration, (2) desphenylation, (3) desN-alkylation and (4) N-desalkylation, and the formation of water soluble metabolites through isofenphos-oxon was proposed to be the main metabolic pathway. In all experiments, the conversion to water soluble metabolites was dominant in the cleavage of P-O-C (aryl) bond and only desphenyl compounds were found in plants. The effect of chirality was seemingly small in the metabolism of isofenphos based on the examination in the rat liver microsomal system and in the housefly. The amount of isofenphos-oxon metabolized from the (-)-isofenphos was always larger than that from the (+)-isomer, but quantitative differences of other metabolites between both the chiral isofenphos was obscure.