Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([(11)C]POX) and profiling in live rats is reported. Naive rats intravenously injected with [(11)C]POX showed a rapid decrease in parent tracer to ~ 1% with an increase in radiolabeled serum proteins to 87%, and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 min reflecting covalent modification of proteins by [(11)C]POX. Ex vivo biodistribution and imaging profiles in naive rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection, and persisted over the 60 min experiment. This showed for the first-time that even low POX exposures (~200 ng of tracer) can rapidly enter brain. Rats given an LD(50) dose of non-radioactive paraoxon at the LD(50) 20 or 60 min prior to [(11)C]POX tracer revealed protein pools were blocked. Blood radioactivity at 20 min were markedly lower than naive levels due to rapid protein modification by non-radioactive POX, however by 60 min, the blood radioactivity returned to near naive levels. Live rat tissue imaging-derived radioactivity values were 10-37% of naive levels in non-radioactive POX pretreated rats at 20 min, but by 60 min the AUC values had recovered to 25-80% of naive. The live rat imaging supported blockade by non-radioactive POX pretreatment at 20 min and recovery of proteins by 60-min. Significance Statement Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX etc. To study the distribution and penetration of POX into the CNS and other tissues, a positron emission tomography (PET) tracer analog, [(11)C]POX, was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.
Organophosphorus esters (OPs) were originally developed as pesticides but were repurposed as easily manufactured, inexpensive, and highly toxic chemical warfare agents. Acute OP toxicity is primarily due to inhibition of acetylcholinesterase (AChE), an enzyme in the central and peripheral nervous system. OP inhibition of AChE can be reversed using oxime reactivators but many show poor CNS penetration, indicating a need for new clinically viable reactivators. However, challenges exist on how to best measure restored AChE activity in vivo and assess the reactivating agent efficacy. This work reports the development of molecular imaging tools using radiolabeled OP analog tracers that are less toxic to handle in the laboratory, yet inhibit AChE in a similar fashion to the actual OPs. Carbon-11 and fluorine-18 radiolabeled analog tracers of VX and sarin OP agents were prepared. Following intravenous injection in normal Sprague-Dawley rats (n = 3-4/tracer), the tracers were evaluated and compared using noninvasive microPET/CT imaging, biodistribution assay, and arterial blood analyses. All showed rapid uptake and stable retention in brain, heart, liver, and kidney tissues determined by imaging and biodistribution. Lung uptake of the sarin analog tracers was elevated, 2-fold and 4-fold higher uptake at 5 and 30 min, respectively, compared to that for the VX analog tracers. All tracers rapidly bound to red blood cells (RBC) and blood proteins as measured in the biodistribution and arterial blood samples. Analysis of the plasma soluble activity (nonprotein/cell bound activity) showed only 1-6% parent tracer and 88-95% of the activity in the combined solid fractions (RBC and protein bound) as early as 0.5 min post injection. Multivariate analysis of tracer production yield, molar activity, brain uptake, brain area under the curve over 0-15 min, and the amount of parent tracer in the plasma at 5 min revealed the [(18)F]VX analog tracer had the most favorable values for each metric. This tracer was considered the more optimal tracer relative to the other tracers studied and suitable for future in vivo OP exposure and reactivation studies.
Oxime antidotes regenerate organophosphate-inhibited acetylcholinesterase (AChE). Although they share a common mechanism of AChE reactivation, the rate and amount of oxime that enters the brain are critical to the efficacy, a process linked to the oxime structure and charge. Using a platform based on the organophosphate [(18) F]-VXS as a positron emission tomography tracer for active AChE, the in vivo distribution of [(18) F]-VXS was evaluated after an LD50 dose (250 mug/kg) of the organophosphate paraoxon (POX) and following oximes as antidotes. Rats given [(18) F]-VXS tracer alone had significantly higher radioactivity (two- to threefold) in the heart and lung than rats given LD50 POX at 20 or 60 min prior to [(18) F]-VXS. When rats were given LD50 POX followed by 2-PAM (cationic), RS194b (ionizable), or monoisonitrosoacetone (MINA) (neutral), central nervous system (CNS) radioactivity returned to levels at or above untreated naive rats (no POX), whereas CNS radioactivity did not increase in rats given the dication oximes HI-6 or MMB-4. MINA showed a significant, pairwise increase in CNS brain radioactivity compared with POX-treated rats. This new in vivo dynamic platform using [(18) F]-VXS tracer measures and quantifies peripheral and CNS relative changes in AChE availability after POX exposure and is suitable for comparing oxime delivery and AChE reactivation in rats.
Title: Positron emission tomography studies of organophosphate chemical threats and oxime countermeasures Thompson CM, Gerdes JM, VanBrocklin HF Ref: Neurobiol Dis, 133:104455, 2020 : PubMed
There is a unique in vivo interplay involving the mechanism of inactivation of acetylcholinesterase (AChE) by toxic organophosphorus (OP) compounds and the restoration of AChE activity by oxime antidotes. OP compounds form covalent adducts to this critical enzyme target and oximes are introduced to directly displace the OP from AChE. For the most part, the in vivo inactivation of AChE leading to neurotoxicity and antidote-based therapeutic reversal of this mechanism are well understood, however, these molecular-level events have not been evaluated by dynamic imaging in living systems at millimeter resolution. A deeper understanding of these critically, time-dependent mechanisms is needed to develop new countermeasures. To address this void and to help accelerate the development of new countermeasures, positron-emission tomography (PET) has been investigated as a unique opportunity to create platform technologies to directly examine the interdependent toxicokinetic/pharmacokinetic and toxicodynamic/pharmacodynamic features of OPs and oximes in real time within live animals. This review will cover two first-in-class PET tracers representing an OP and an oxime antidote, including their preparation, requisite pharmacologic investigations, mechanistic interpretations, biodistribution and imaging.
Title: Divergent synthesis of organophosphate [(11)C]VX- and [(11)C]Sarin-surrogates from a common set of starting materials Hayes TR, Blecha JE, Thompson CM, Gerdes JM, VanBrocklin HF Ref: Appl Radiat Isot, 151:182, 2019 : PubMed
Radiolabeled 1-[(11)C]ethyl, 4-nitrophenyl methylphosphonate (VX surrogate) and 2-[(11)C]-propanyl, 4-nitrophenyl methylphosphonate (sarin surrogate) were developed as organophosphate (OP) tracers. The [(11)C]ethyl- and [(11)C]isopropyl-iodide radiolabeled synthons were obtained by temperature controlled, in loop reactions of [(11)C]CO2 with MeMgBr followed by reduction with LiAlH4, then reaction with HI. Distillation of the [(11)C]alkyl iodides into a solution of hydrogen (4-nitrophenyl)methylphosphonate and cesium carbonate afforded the desired tracers in >95% radiochemical purity, yields from [(11)C]CO2 of 1-3% and 1.7-15.1 GBq/mmol molar activities.
Title: The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE Chao CK, Balasubramanian N, Gerdes JM, Thompson CM Ref: Chemico-Biological Interactions, 291:220, 2018 : PubMed
In this study, the mechanisms of HuAChE and HuBChE inhibition by Me-P(O) (OPNP) (OR) [PNP = p-nitrophenyl; R = CH(2)CH(3), CH(2)CH(2)F, OCH(CH(3))(2), OCH(CH(3)) (CH(2)F)] representing surrogates and fluoro-surrogates of VX and sarin were studied by in vitro kinetics and mass spectrometry. The in vitro measures showed that the VX- and fluoro-VX surrogates were relatively strong inhibitors of HuAChE and HuBChE (k(i) - 10(5)-10(6) M(-1)min(-1)) and underwent spontaneous and 2-PAM-mediated reactivation within 30 min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (k(i) - 10(4)-10(5) M(-1)min(-1)), and in general did not undergo spontaneous reactivation, although HuAChE adducts were partially reactivatable at 18 h using 2-PAM. The mechanism of HuAChE and HuBChE inhibition by the surrogates was determined by Q-TOF and MALDI-TOF mass spectral analyses. The surrogate-adducted proteins were trypsin digested and the active site-containing peptide bearing the OP-modified serine identified by Q-TOF as triply- and quadruply-charged ions representing the respective increase in mass of the attached OP moiety. Correspondingly, monoisotopic ions of the tryptic peptides representing the mass increase of the OP-adducted peptide was identified by MALDI-TOF. The mass spectrometry analyses validated the identity of the OP moiety attached to HuAChE or HuBChE as MeP(O) (OR)-O-serine peptides (loss of the PNP leaving group) via mechanisms consistent with those found with chemical warfare agents. MALDI-TOF MS analyses of the VX-modified peptides versus time showed a steady reduction in adduct versus parent peptide (reactivation), whereas the sarin-surrogate-modified peptides remained largely intact over the course of the experiment (24 h). Overall, the presence of a fluorine atom on the surrogate modestly altered the rate constants of inhibition and reactivation, however, the mechanism of inhibition (ejection of PNP group) did not change.
Title: Radiosynthesis of O-(1-[(18) F]fluoropropan-2-yl)-O-(4-nitrophenyl)methylphosphonate: A novel PET tracer surrogate of sarin Hayes TR, Thompson CM, Blecha JE, Gerdes JM, VanBrocklin HF Ref: J Labelled Comp Radiopharm, 61:1089, 2018 : PubMed
O-(1-Fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate is a reactive organophosphate ester (OP) developed as a surrogate of the chemical warfare agent sarin that forms a similar covalent adduct at the active site serine of acetylcholinesterase. The radiolabeled O-(1-[(18) F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate ([(18) F] fluorosarin surrogate) has not been previously prepared. In this paper, we report the first radiosynthesis of this tracer from the reaction of bis-(4-nitrophenyl) methylphosphonate with 1-[(18) F]fluoro-2-propanol in the presence of DBU. The 1-[(18) F]fluoro-2-propanol was prepared by reaction of propylene sulfite with Kryptofix 2.2.2 and [(18) F] fluoride ion. The desired tracer O-(1-[(18) F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate was obtained in a >98% radiochemical purity with a 2.4% +/- 0.6% yield (n = 5, 65 minutes from start of synthesis) based on starting [(18) F] fluoride ion and a molar activity of 49.9 GBq/mumol (1.349 +/- 0.329 Ci/mumol, n = 3). This new facile radiosynthesis routinely affords sufficient quantities of [(18) F] fluorosarin surrogate in high radiochemical purity, which will further enable the tracer development as a novel radiolabeled OP acetylcholinesterase inhibitor for assessment of OP modes of action with PET imaging in vivo.
2-Pyridinealdoxime methiodide (2-PAM) is a widely used antidote for the treatment of organophosphorus (OP) exposure that reactivates the target protein acetylcholinesterase. Carbon-11 2-PAM was prepared to more fully understand the in vivo mode of action, distribution, and dynamic qualities of this important countermeasure. Alkylation of 2-pyridinealdoxime with [(11)C]CH3I provided the first-in-class [(11)C]2-PAM tracer in 3.5% decay corrected radiochemical yield from [(11)C]CH3I, >99% radiochemical purity, and 4831 Ci/mmol molar activity. [(11)C]2-PAM tracer distribution was evaluated by ex vivo biodistribution and in vivo dynamic positron emission tomography (PET) imaging in naive (OP exposure deficient) rats. Tracer alone and tracer coinjected with a body mass-scaled human therapeutic dose of 30 mg/kg nonradioactive 2-PAM demonstrated statistically similar tissue and blood distribution profiles with the greatest uptake in kidney and significantly lower levels in liver, heart, and lung with lesser amounts in blood and brain. The imaging and biodistribution data show that radioactivity uptake in brain and peripheral organs is rapid and characterized by differential tissue radioactivity washout profiles. Analysis of arterial blood samples taken 5 min after injection showed approximately 82% parent [(11)C]2-PAM tracer. The imaging and biodistribution data are now established, enabling future comparisons to outcomes acquired in OP intoxicated rodent models.
Title: An improved radiosynthesis of O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer Neumann KD, Thompson CM, Blecha JE, Gerdes JM, VanBrocklin HF Ref: J Labelled Comp Radiopharm, 60:337, 2017 : PubMed
O-(2-Fluoroethyl)-O-(p-nitrophenyl) methylphosphonate 1 is an organophosphate cholinesterase inhibitor that creates a phosphonyl-serine covalent adduct at the enzyme active site blocking cholinesterase activity in vivo. The corresponding radiolabeled O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, [18 F]1, has been previously prepared and found to be an excellent positron emission tomography imaging tracer for assessment of cholinesterases in live brain, peripheral tissues, and blood. However, the previously reported [18 F]1 tracer synthesis was slow even with microwave acceleration, required high-performance liquid chromatography separation of the tracer from impurities, and gave less optimal radiochemical yields. In this paper, we report a new synthetic approach to circumvent these shortcomings that is reliant on the facile reactivity of bis-(O,O-p-nitrophenyl) methylphosphonate, 2, with 2-fluoroethanol in the presence of DBU. The cold synthesis was successfully translated to provide a more robust radiosynthesis. Using this new strategy, the desired tracer, [18 F]1, was obtained in a non-decay-corrected radiochemical yield of 8 +/- 2% (n = 7) in >99% radiochemical and >95% chemical purity with a specific activity of 3174 +/- 345 Ci/mmol (EOS). This new facile radiosynthesis routinely affords highly pure quantities of [18 F]1, which will further enable tracer development of OP cholinesterase inhibitors and their evaluation in vivo.
Title: Novel Organophosphate Ligand O-(2-Fluoroethyl)-O-(p-Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability and Analysis of the Inhibition and Reactivation of Cholinesterases Chao CK, Ahmed SK, Gerdes JM, Thompson CM Ref: Chemical Research in Toxicology, 29:1810, 2016 : PubMed
The organophosphate O-(2-fluoroethyl)-O-(p-nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-18 radiolabeled organophosphate inhibitor ([18F]1) of acetylcholinesterase (AChE). In rats, [18F]1 localizes in AChE rich regions of the brain and other tissues where it likely exists as the (CH3)(18FCH2CH2O)P(O)-AChE adduct (ChE-1). Characterization of this adduct would define the inhibition mechanism and subsequent postinhibitory pathways and reactivation rates. To validate this adduct, the stability (hydrolysis) of 1 and ChE-1 reactivation rates were determined. Base hydrolysis of 1 yields p-nitrophenol and (CH3) (FCH2CH2O)P(O)OH with pseudo first order rate constants (kobsd) at pH 7.4 (PBS) of 3.25 x 10-4 min-1 (t1/2 = 35.5 h) at 25 degrees C and 8.70 x 10-4 min-1 (t1/2 = 13.3 h) at 37 degrees C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; ki = 7.5 x 105 M-1 min-1), electric eel acetylcholinesterase (EEAChE) (ki = 3.0 x 106 M-1 min-1), and human serum butyrylcholinesterase (HuBChE; 1.95 x 105 M-1 min-1). Spontaneous and oxime-mediated reactivation rates for the (CH3) (FCH2CH2O)P(O)-serine ChE adducts using 2-PAM (10 muM) were (a) HuAChE 8.8 x 10-5 min-1 (t1/2 = 131.2 h) and 2.41 x 10-2 min-1 (t1/2 = 0.48 h), (b) EEAChE 9.32 x 10-3 min-1 (t1/2 = 1.24 h) and 3.33 x 10-2 min-1 (t1/2 = 0.35 h), and (c) HuBChE 1.16 x 10-4 min-1 (t1/2 = 99.6 h) and 4.19 x 10-2 min-1 (t1/2 = 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo is the formation of (CH3)(FCH2CH2O)P(O)-serine adducts and monoacid (CH3)(FCH2CH2O)P(O)OH from hydrolysis and reactivation.
Radiosynthesis of a fluorine-18 labeled organophosphate (OP) inhibitor of acetylcholinesterase (AChE) and subsequent positron emission tomography (PET) imaging using the tracer in the rat central nervous system are reported. The tracer structure, which contains a novel beta-fluoroethoxy phosphoester moiety, was designed as an insecticide-chemical nerve agent hybrid to optimize handling and the desired target reactivity. Radiosynthesis of the beta-fluoroethoxy tracer is described that utilizes a [(18)F]prosthetic group coupling approach. The imaging utility of the [(18)F]tracer is demonstrated in vivo within rats by the evaluation of its brain penetration and cerebral distribution qualities in the absence and presence of a challenge agent. The tracer effectively penetrates brain and localizes to cerebral regions known to correlate with the expression of the AChE target. Brain pharmacokinetic properties of the tracer are consistent with the formation of an OP-adducted acetylcholinesterase containing the fluoroethoxy tracer group. Based on the initial favorable in vivo qualities found in rat, additional [(18)F]tracer studies are ongoing to exploit the technology to dynamically probe organophosphate mechanisms of action in mammalian live tissues.
Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena occurs. In this report, we prepared a panel of beta-substituted ethoxy and gamma-substituted propoxy phosphonoesters of the type p-NO(2)PhO-P(X)(R)[(O(CH(2))(n)Z] (R=Me, Et; X=O, S; n=2, 3; Z=halogen, OTs) and examined the inhibition of three AChEs by select structures in the panel. The beta-fluoroethoxy methylphosphonate analog (R=Me, Z=F, n=2) was the most potent anti-AChE compound comparable (ki approximately 6 x 10(6)M(-1)min(-1)) to paraoxon against EEAChE. Analogs with Z=Br, I, or OTs were weak inhibitors of the AChEs, and methyl phosphonates (R=Me) were more potent than the corresponding ethyl phosphonates (R=Et). As expected, analogs with a thionate linkage (PS) were poor inhibitors of the AChEs.
Title: Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates Guo L, Suarez AI, Braden MR, Gerdes JM, Thompson CM Ref: Bioorganic & Medicinal Chemistry Lett, 20:1194, 2010 : PubMed
Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (k(i)) ranging from 0.3 x 10(5)M(-1)min(-1) to 10.4 x 10(5)M(-1)min(-1). When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE's ( congruent with 1 x 10(7)M(-1)min(-1)) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling.
