P-nitrophenyl Methylphosphonate Research Articles

Dose Formulation, Biodistribution and PET Imaging Studies of a First-in-Class Fluorine-18 Organophosphorus Cholinesterase Inhibitor Tracer in Rat

Background:: To investigate dynamic live tissue organophosphorus nerve agent uptake and distribution fates resulting in acetylcholinesterase inhibition, we recently reported the first-in-class fluorine-18 [18F] radiolabeled Positron Emission Tomography (PET) imaging tracer known as [18F]O-(2-fluoroethyl)-O-(p-nitrophenyl)methylphosphonate. This tracer has been initially studied in live rats with PET imaging. Objective.: We sought to evaluate the PET tracer in vivo using a new dose formulation of saline, ethanol and L-ascorbic acid, and compare the influence of this formulation on in vivo tracer performance to previous data collected using a CH3CN:PBS formulation. Methods:: A high molar activity [18F]tracer radiosynthesis was used. Doses were formulated as saline, ethanol (≤ 1%) and L-ascorbic acid (0.1%), pH 4.0-4.5. Stability was evaluated to 6 h. Dose injection (i.v.) into male rats was followed by either ex vivo biodistribution profiling at 5, 30, 90 min, or dynamic 90 min PET imaging. Rat biodistribution and PET imaging data were compared. Results and Discussion:: An optimized radiosynthesis (8 ± 2 % RCY) resulted in stable doses for 6 h (>99%). Arterial blood included a tracer and a single metabolite. The ex vivo biodistribution and live tissue PET imaging data revealed rapid radioactivity uptake and distributed tissue levels: heart and lung, highest; liver, moderate; and brain, lowest. Conclusions:: Imaging and biodistribution data were highly correlated with expected radioactivity tissue uptake and distribution in target organs. Lower brain radioactivity levels by PET imaging were found for the new formulation (saline, 1% L-ascorbic acid, < 1% ethanol) as compared to the established CH3CN:PBS formulation. Overall, we found that the i.v. dose formulation changed the in vivo profile of an organophosphorus PET tracer that is considered an important finding for future organophosphorus PET tracer studies.

New Safe Method for Preparation of Sarin-Exposed Human Erythrocytes Acetylcholinesterase Using Non-Toxic and Stable Sarin Analogue Isopropyl p-Nitrophenyl Methylphosphonate and its Application to Evaluation of Nerve Agent Antidotes

A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.

Organophosphate nerve agent toxicity in Hydra attenuata.

The toxicity for analogues of sarin (GB), soman (GD), and VX was evaluated using Hydra attenuata as a model organism. The organophosphate nerve agent analogue simulants used in this investigation included the following: isopropyl p-nitrophenyl methylphosphonate (for GB); pinacolyl p-nitrophenyl methylphosphonate (for GD); and diisopropyl S-(2-diisopropylaminoethyl)phosphorothioate, diethyl S-(2-diisopropylaminoethyl)phosphorothioate, and diethyl S-(2-trimethylaminoethyl)phosphorothioate (for VX). The toxicity of each organophosphate nerve agent was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There is a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate chemical warfare agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogues was 2 orders of magnitude more toxic than the analogue for GD and 4 orders of magnitude more toxic than the analogue for GB.

Stereoselective Detoxification of Chiral Sarin and Soman Analogues by Phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S P- and R P-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R P-enantiomer of the sarin analogue ( k cat=2600 s −1) was the preferred substrate for the wild-type PTE relative to the corresponding S P-enantiomer ( k cat=290 s −1). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k cat values for the R P- and S P-enantiomers were 410 and 4200 s −1, respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R P R C-stereoisomer of the soman analogue was the preferred substrate for PTE. The k cat values for the soman analogues were measured as follows: R P R C, 48 s −1; R P S C, 4.8 s −1; S P R C, 0.3 s −1, and S P S C, 0.04 s −1. With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k cat values for the soman analogues were found to be as follows: R P R C, 0.3 s −1; R P S C, 0.3 s −1; S P R C, 11 s −1, and S P S C, 2.1 s −1. Prior investigations have demonstrated that the S P-enantiomers of sarin and soman are significantly more toxic than the R P-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.

Cetylpyridinium bromide-based oil-in-water microemulsions as a medium for hydrolysis of esters of phosphorus acids in the presence of primary amines

High-resolution 1H NMR technique with Fourier-transform and pulsed-gradient spin-echo was used to study the structure of oil-in-water microemulsions based on cetylpyridinium bromide. The sizes of microdrops and the distribution of components between the disperse and continuous phases were found. It was shown for the hydrolytic decomposition of O,O-bis-(p-nitrophenyl) methyl phosphonate in the presence of amines that the microemulsion medium can affect both the rate and mechanism of hydrolysis. The reaction rate constants depend on the structure of microdrops.

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.

Metal Cation Micelle Mediated Hydrolysis of Phosphonic Acid Esters

p-Nitrophenyl methylphosphonate (PNPMP, 3) and p-nitrophenyl phenylphosphonate (PNPPP, 4) are rapidly hydrolyzed in acidic solutions (pH 3.5−6.0) at 37 °C by excess Zr4+, Ce4+, or Th4+ cations. Brij-35 micelles are employed in the Ce4+ and Th4+ cases. Second-order rate constants (M-1 s-1) with PNPMP are 115 (Zr4+), 53.9 (Ce4+), and 16.9 (Th4+), while for PNPPP they are, respectively, 76.8, 16.1, and 13.1. Relative to water (k2 = 3.6 × 10-11 M-1 s-1 at 30 °C, pH 7.6), kinetic advantages with Zr4+ or Ce4+ exceed 1012.

Kinetics of alkaline hydrolysis of esters of phosphorus acids in micellar and hexagonal phases in the cetyldimethylethylammonium bromide—NaOH—water system

The influence of micellar (Mi) and hexagonal (E) mesophases of the cetyldimethylethylammonium bromide—NaOH—water system (I) on the rates on alkaline hydrolysis ofO-p-nitrophenyl-O,O-diethyl phosphate (2),O-p-nitrophenyl-O-ethylethyl phosphonate (3), andO,O-di(p-nitrophenyl)methyl phosphonate (4) was studied by UV spectrophotometry. The binding constants of the substrates, critical micelle concentrations, and rate constants of reactions in the micellar phase were determined. In micellar solutions of systemI, a tenfold increase in the rates of alkaline hydrolysis of2–4 was observed. An increase in the degree of medium ordering during the formation of the E-phase results in a twofold acceleration of alkaline hydrolysis of2 and3 and in the inhibition of this process in the case of4.

The influence of detergentless microemulsion on the kinetics of hydrolysis of di(p-nitrophenyl) methylphosphonate catalyzed by amines

The influence of the detergentless water/oil microemulsion formed in then-hexane-water-2-propanol system on the kinetics of the hydrolysis of di(p-nitrophenyl) methylphosphonate under the conditions of general base catalysis by primary, secondary, and tertiary aliphatic amines was investigate. The leveling of the catalytic rate constants and basicities of the amines was found for this reaction in a microemulsion. The catalytic rate constants for the amines containing hydrophilic groups increase in microemulsions as compared to those in an aqueous medium, while the catalytic constants for the amines containing hydrophobic groups decrease.

Stereospecific Grignard-Activated Solid Phase Synthesis of DNA Methylphosphonate Dimers.

Stereoregular R(p) or S(p) DNA methylphosphonate dimers have been synthesized on a solid phase support. A deprotected 5'-hydroxyl-N(2)-isobutanoyldeoxyguanosine 3'-O-succinate coupled to high-loaded polyethylene glycol (PEG) coated polystyrene beads (HLP) was activated with a Grignard reagent, t-BuMgCl. After activation was complete, a pure diastereoisomer of 5'-(dimethoxytrityl) N-benzoyldeoxynucleoside 3'-(p-nitrophenyl methylphosphonate) p-nitrophenyl ester (R(p) or S(p)) was added. Coupling of the activated 5'-hydroxyl to the 3'-methylphosphonate ensued, releasing nitrophenol, yielding the R(p) or S(p) dimer, respectively. The dimers were then cleaved from the solid support, deprotected, and purified, yielding methylphosphonate DNA dimers of defined stereochemistry.

Chemical Inactivation of Lipase in Organic Solvent: A Lipase from Pseudomonas aeruginosa TE3285 is More Like a Typical Serine Enzyme in an Organic Solvent than in Aqueous Media.

A microbial lipase from Pseudomonas aeruginosa TE3285 was treated in anhydrous diisopropyl ether with three kinds of serine-reactive reagents, ethyl p-nitrophenyl methylphosphonate (ENMP), diisopropyl fluorophosphate (DFP), and phenylmethylsulfonyl fluoride (PMSF) to lose its catalytic activity for both transesterification in an organic solvent and ester hydrolysis in aqueous system. In contrast with the facile inactivation in an organic solvent, no or very slow inactivation was observed in an aqueous solution. The lipase was shown to behave more like a typical serine enzyme in an organic solvent than in aqueous solution with regard to the chemical inactivation by serine-reactive reagents. The unique behavior of the lipase in an organic solvent may be associated with inferfacial activation of the lipase, which is one of the most distinct characteristics of the lipase family, and the activiation of lipase could be induced by a hydrophobic interaction with an organic solvent.

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 2. Preparation and in vitro and in vivo evaluation of 1-(alkoxymethyl)-2-[(hydroxyimino)methyl]-3-methylimida zolium halides for reactivation of organophosphorus-inhibited acetylcholinesterases.

A series of structurally related mono- and bis-1,3-disubstituted 2-[(hydroxyimino)methyl]imidazolium halides were evaluated in vitro for their ability to reactivate electric eel, bovine, and human erythrocyte (RBC) acetylcholinesterases (AChE) inhibited by ethyl p-nitrophenyl methylphosphonate (EPMP) and 3,3-dimethyl-2-butyl methyl-phosphonofluoridate (soman, GD). All new compounds were characterized for (hydroxyimino)methyl acid dissociation constant, nucleophilicity, octanol-buffer partition coefficient, reversible AChE inhibition, and kinetics of reactivation of EPMP-inhibited AChEs. For GD-inhibited AChEs, maximal reactivation was used to compare compounds since rapid phosphonyl enzyme dealkylation "aging" complicated interpretation of kinetic constants. For comparison, we also evaluated three known pyridinium therapeutics, 2-PAM, HI-6, and toxogonin. In vivo evaluation in mice revealed that when selected imidazolium compounds were coadministered with atropine sulfate, they were effective in providing lifesaving protection against both GD and EPMP challenges. This was a major accomplishment in the search for effective anticholinesterase therapeutics--the synthesis and preliminary evaluation of the first new monoquaternary soman antidotes with potencies superior to 2-PAM. Significantly, there was an apparent inverse relationship between in vitro and in vivo results; the most potent in vivo compounds proved to be the poorest in vitro reactivators. These results suggested that an alternative and possibly novel antidotal mechanism of protective action may be applicable for the imidazolium aldoximes. Selected compounds were also evaluated for their inhibition of AChE phosphorylation by GD and antimuscarinic and antinicotinic receptor blocking effects.

Molecular imprinting of a transition state analogue leads to a polymer exhibiting esterolytic activity

Molecular imprinting of p-nitrophenyl methylphosphonate, a transition state analogue, in poly[4(5)-vinylimidazole] leads to a polymer which hydrolyses p-nitrophenyl acetate at an increased rate and which can then be inhibited by addition of the p-nitrophenol methylphosphonate.

Nonquaternary cholinesterase reactivators. 3. 3(5)-Substituted 1,2,4-oxadiazol-5(3)-aldoximes and 1,2,4-oxadiazole-5(3)-thiocarbohydroximates as reactivators of organophosphonate-inhibited eel and human acetylcholinesterase in vitro.

As an extension of an earlier investigation (J. Med. Chem. 1984, 27, 1431), we prepared a series of 3-substituted 5-[(hydroxyimino)methyl]-1,2,4-oxadiazoles and the corresponding 5-thiocarbohydroximic acid 2-(N,N-dialkylamino)ethyl S-esters. The compounds were evaluated in vitro as reactivators of phosphonylated electric eel and human erythrocyte (RBC) acetylcholinesterases (AChE). The compounds were characterized with respect to (hydroxyimino)methyl acid dissociation constant, nucleophilicity, octanol/buffer partition coefficient, reversible AChE inhibition, and kinetics of reactivating ethyl methylphosphonylated AChE. One compound was also tested for effectiveness in preventing AChE phosphonylation. All of the tested compounds significantly reactivate ethyl methylphosphonylated AChE: the 3-n-octyl- and 3-(1-naphthyl)-substituted aldoximes are as reactive (within a factor of 5-10) toward the inhibited enzymes as the benchmark pyridinium reactivators, 2-PAM and HI-6. All of the substituted thiocarbohydroximic acid S-esters are powerful reversible inhibitors of AChE's: the 3-n-octyl- and 3-(1-naphthyl)-substituted thiocarbohydroximates inhibit eel AChE to 50% initial activity at concentrations less than 5 microM. When added to an eel AChE solution at concentrations between 5 and 50 microM, the 3-phenyl-substituted thiocarbohydroximate effectively antagonizes AChE inhibition by ethyl p-nitrophenyl methylphosphonate (EPMP), suggesting the potential utility of this compound for preventing anti-AChE-agent poisoning.

Irreversible inhibition of pancreatic lipase by bis- p-nitrophenyl methylphosphonate

The reaction of porcine pancreatic lipase with an organophosphorus compound bis- p-nitrophenyl methylphosphonate (BNMP) resulted in the complete and irreversible inhibition of lipase activity on tributyrin emulsion (25°C, pH 7.5, 40 mM Na-veronal-HCl buffer) whereas the activity of the enzyme on p-nitrophenyl acetate solution remained unchanged. The BNMP-modified enzyme did not bind on hydrophobic interfaces (siliconized glass beads). Tyr 49 was presumed to be the modification site, and the conclusion has been made that this residue is implicated in the interface recognition site of pancreatic lipase.

Structure-activity relationships for reactivators of organophosphorus-inhibited acetylcholinesterase: quaternary salts of 2-[(hydroxyimino)methyl]imidazole.

A series of 1,3-disubstituted-2-[(hydroxyimino)methyl]imidazolium halides were prepared and evaluated in vitro with respect to their ability to reactivate acetylcholinesterase inhibited by ethyl p-nitrophenyl methylphosphonate (EPMP) and 3,3-dimethyl-2-butyl methylphosphonofluoridate (GD). The compounds conform to the general formula N(CH3)C(CHNOH)N(CH2OR)CHCH+ X Cl-, where R = CH3, (CH2)3CH3, (CH2)7CH3, CH2C6H5, CH2C10H7, (CH2)3C6H5, CH(CH3)2, CH2C(CH3)3, and CH(CH3)C(CH3)3. For comparison we also evaluated three known pyridinium reactivators, 2-PAM, HI-6, and toxogonin. The imidazolium aldoximes exhibit oxime acid dissociation constants (pKa) in the range 7.9-8.1, bracketing the value of 8.0, believed to be optimal for acetylcholinesterase reactivation. With imidazolium compound in excess over inhibited enzyme, the kinetics of reactivation are well behaved for EPMP-inhibited AChE and depend on the nature of the alkyl ether group R. For GD-inhibited AChE, maximal reactivation was used to compare compounds because rapid phosphonyl enzyme dealkylation and enzyme reinhibition complicate interpretation of kinetic constants.

Nonquaternary cholinesterase reactivators. 2. Alpha-heteroaromatic aldoximes and thiohydroximates as reactivators of ethyl methylphosphonyl-acetylcholinesterase in vitro.

We prepared six pairs of alpha-heteroaromatic aldoximes, RC(= NOH)H, and thiohydroximates, RC(= NOH)S-(CH2)2N(C2H5)2, where R represents various oxadiazole and thiadiazole rings. Each compound was characterized with respect to the following: structure, (hydroxyimino)methyl acid dissociation constant, nucleophilicity toward trigonal carbon and tetrahedral phosphorus, octanol-buffer partition coefficient, reversible inhibition of eel acetylcholinesterase (AChE), and in vitro reactivation of AChE inhibited by ethyl p-nitrophenyl methylphosphonate. Eight of the twelve compounds significantly reactivate ethyl methylphosphonyl-AChE, but inherent reactivities are moderate to low: the most potent nonquaternary reactivator, 3-phenyl-5-[(hydroxyimino)methyl]-1,2,4-oxadiazole, is 17 times less reactive than the well-known reactivator 2-[(hydroxyimino)methyl]-1-methylpyridinium iodide (2-PAM). One of the nonquaternary compounds, 3-phenyl-1,2,4-oxadiazole-5-thiohydroximic acid 2-(diethylamino)ethyl S-ester, is a powerful reversible inhibitor of AChE (I50 = 7.5 microM). The observed relationships between nonquaternary compound structure, reactivation potency, and anti-AChE activity reveal important molecular requirements for high reactivity toward phosphonylated AChE.

Amine-catalyzed alcoholysis of di(p-nitrophenyl) methylphosphonate

We studied the alcoholysis rate of di(p-nitrophenyl) methylphosphonate in the presence of primary, secondary, and tertiary aliphatic amines. The catalytic constants correlate with their basicity.

A covalent affinity column for the purification of acetylcholinesterase

2-Aminoethyl p-nitrophenyl methylphosphonate, a potential inhibitor of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), was coupled to extended sepharose, thus forming covalent affinity support for purification of acetylcholinesterase. Cholinesterase from electric eel was trapped on the column by forming a covalent bond with the insoluble matrix. The enzyme was recovered from the column in very good quality by using the reactivator 2-(hydroximinomethyl)-1-methylpyridinium iodide (2PAM) or 1,1′-trimethylene bis(4-hydroximinomethyl pyridinium) dibromide (TMB 4) . The column functioned as a covalent affinity column for eel cholinesterase and α-chymotrypsin (EC 3.4.4.5).

Reactions of phosphonic acid esters with nucleophiles. Part IV. Hydrazines and hydroxylamines with p-nitrophenyl methylphosphonate

A steric argument accounts for the orders of reactivity of two parallel series of hydrazines and hydroxylamines with p-nitrophenyl methylphosphonates. The α effect is not especially important. The unusually high reactivity of hydroxylamine may reflect O-attack for this species. In some cases hydrogen bonding to the phosphoryl oxygen atom in the transition state is a possibility. The results agree with prevailing theories on the forces which control nucleophilic substitution at a tetrahedral phosphorus atom.