Chemoselective Hydrogenation of 6-Alkynyl-3-fluoro-2-pyridinaldoximes: Access to First-in-Class 6-Alkyl-3-Fluoro-2-pyridinaldoxime Scaffolds as New Reactivators of Sarin-Inhibited Human Acetylcholinesterase with Increased Blood-Brain Barrier Permeability.

Novel 6-alkyl- and 6-alkenyl-3-fluoro-2-pyridinaldoximes have been synthesised by using a mild and efficient chemoselective hydrogenation of 6-alkynyl-3-fluoro-2-pyridinaldoxime scaffolds, without altering the reducible, unprotected, sensitive oxime functionality and the C-F bond. These novel 6-alkyl-3-fluoro-2-pyridinaldoximes may find medicinal application as antidotes to organophosphate poisoning. Indeed, one low-molecular-weight compound exhibited increased affinity for sarin-inhibited acetylcholinesterase (hAChE) and greater reactivation efficiency or resurrection for sarin-inhibited hAChE, compared with those of 2-pyridinaldoxime (2-PAM) and 1-({[4-(aminocarbonyl)pyridinio]methoxy}methyl)-2-[(hydroxyimino)methyl]pyridinium chloride (HI-6), two pyridinium salts currently used as antidote by several countries. In addition, the uncharged 3-fluorinated bifunctional hybrid showed increased in vitro blood-brain barrier permeability compared with those of 2-PAM, HI-6 and obidoxime. These promising features of novel low-molecular-weight alkylfluoropyridinaldoxime open up a new era for the design, synthesis and discovery of central non-quaternary broad spectrum reactivators for organophosphate-inhibited cholinesterases.

Potent 3-Hydroxy-2-Pyridine Aldoxime Reactivators of Organophosphate-Inhibited Cholinesterases with Predicted Blood-Brain Barrier Penetration.

A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.

Selective irreversible chemical tagging of cysteine with 3-arylpropiolonitriles.

Exquisite chemoselectivity for cysteine has been found for a novel class of remarkably hydrolytically stable reagents, 3-arylpropiolonitriles (APN). The efficacy of the APN-mediated tagging was benchmarked against other cysteine-selective methodologies in a model study on a series of traceable amino acid derivatives. The selectivity of the methodology was further explored on peptide mixtures obtained by trypsin digestion of lysozyme. Additionally, the superior stability of APN-cysteine conjugates in aqueous media, human plasma, and living cells makes this new thiol-click reaction a promising methodology for applications in bioconjugation.

The risk associated with organophosphorus nerve agents: from their discovery to their unavoidable threat, current medical countermeasures and perspectives.

The first organophosphorus nerve agent was discovered accidently during the development of pesticides, shortly after the first use of chemical weapons (chlorine, phosgene) on the battlefield during World War I. Despite the Chemical Weapons Convention banning these substances, they have still been employed in wars, terrorist attacks or political assassinations. Characterised by their high lethality, they target the nervous system by inhibiting the acetylcholinesterase (AChE) enzyme, preventing neurotransmission, which, if not treated rapidly, inevitably leads to serious injury or the death of the person intoxicated. The limited efficacy of current antidotes, known as AChE reactivators, pushes research towards new treatments. Numerous paths have been explored, from modifying the original pyridinium oximes to developing hybrid reactivators seeking a better affinity for the inhibited AChE. Another crucial approach resides in molecules more prone to cross the blood-brain barrier: uncharged compounds, bio-conjugated reactivators or innovative formulations. Our aim is to raise awareness on the threat and toxicity of organophosphorus nerve agents and to present the main synthetic efforts deployed since the first AChE reactivator, to tackle the task of efficiently treating victims of these chemical warfare agents.

No-observed-adverse-effect-level (NOAEL) assessment as an optimized dose of cholinesterase reactivators for the treatment of exposure to warfare nerve agents in mice.

Despite the international convention on the prohibition of chemical weapons ratified in 1997, the threat of conflicts and terrorist attacks involving such weapons still exists. Among these, organophosphorus-nerve agents (OPs) inhibit cholinesterases (ChE) causing cholinergic syndrome. The reactivation of these enzymes is therefore essential to protect the poisoned people. However, these reactivating molecules, mainly named oximes, have major drawbacks with limited efficacy against some OPs and a non-negligible ChE inhibitor potential if administered at an inadequate dose, an effect that they are precisely supposed to mitigate. As a result, this project focused on assessing therapeutic efficacy, in mice, up to the NOAEL dose, the maximum dose of oxime that does not induce any observable toxic effect. NOAEL doses of HI-6 DMS, a reference oxime, and JDS364. HCl, a candidate reactivator, were assessed using dual-chamber plethysmography, with respiratory ventilation impairment as a toxicity criterion. Time-course modeling parameters and pharmacodynamic profiles, reflecting the interaction between the oxime and circulating ChE, were evaluated for treatments at their NOAEL and higher doses. Finally, the therapeutic potential against OPs poisoning was determined through the assessment of protective indices. For JDS364. HCl, the NOAEL dose corresponds to the smallest dose inducing the most significant therapeutic effect without causing any abnormality in ChE activity. In contrast, for HI-6 DMS, its therapeutic benefit was observed at doses higher than its NOAEL, leading to alterations in respiratory function. These alterations could not be directly correlated with ChE inhibition and had no adverse effects on survival. They are potentially attributed to the stimulation of non-enzymatic cholinergic targets by HI-6 DMS. Thus, the NOAEL appears to be an optimal dose for evaluating the efficacy of oximes, particularly when it can be linked to respiratory alterations effectively resulting from ChE inhibition.

Dynamic micelles of mannoside glycolipids are more efficient than polymers for inhibiting HIV-1 trans-infection.

Mannoside glycolipid conjugates are able to inhibit human immunodeficiency virus type 1 (HIV-1) trans-infection mediated by human dendritic cells (DCs). The conjugates are formed by three building blocks: a linear or branched mannose head, a hydrophilic linker, and a 24-carbon lipid chain. We have shown that, even as single molecules, these compounds efficiently target mannose-binding lectins, such as DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) important for HIV-1 transmission. With the goal to optimize their inhibitory activity by supramolecular structure formation, we have compared saturated and unsaturated conjugates, as single molecules, self-assemblies of dynamic micelles, and photopolymerized cross-linked polymers. Surface plasmon resonance showed that, unexpectedly, polymers of trivalent conjugates did not display a higher binding affinity for DC-SIGN than single molecules. Interactions on a chip or in solution were independent of calcium; however, binding to DCs was inhibited by a calcium chelator. Moreover, HIV-1 trans-infection was mostly inhibited by dynamic micelles and not by rigid polymers. The inhibition data revealed a clear correlation between the structure and molecular assembly of a conjugate and its biological antiviral activity. We present an interaction model between DC-SIGN and conjugates-either single molecules, micelles, or polymers-that highlights that the most effective interactions by dynamic micelles involve both mannose heads and lipid chains. Our data reveal that trivalent glycolipid conjugates display the highest microbicide potential for HIV prophylaxis, as dynamic micelles conjugates and not as rigid polymers.

Reactivators of acetylcholinesterase inhibited by organophosphorus nerve agents.

Since the September 11, 2001, terrorist attacks in the United States, the specter of a chemical threat against civilian populations has renewed research interest in chemical warfare agents, their mechanisms of action, and treatments that reverse their effects. In this Account, we focus specifically on organophosphorus nerve agents (OPNAs). Although some OPNAs are used as pest control, the most toxic chemicals in this class are used as chemical warfare agents in armed conflicts. The acute toxicity of OPNAs results from the irreversible inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) via the formation of a covalent P-O bond at the serine hydroxyl group in the enzyme active site. AChE breaks down the neurotransmitter acetylcholine at neuronal synapses and neuromuscular junctions. The irreversible inhibition of AChE causes the neurotransmitter to accumulate in the synaptic cleft, leading to overstimulation of cholinergic receptors, seizures, respiratory arrest, and death. The current treatment for OPNA poisoning combines an antimuscarinic drug (e.g., atropine), an anticonvulsant drug (e.g., diazepam), and an AChE reactivator of the pyridinium aldoxime family (pralidoxime, trimedoxime, obidoxime, HI-6, HLö-7). Because of their high nucleophilicity, oximes can displace the phosphyl group from the catalytic serine, thus restoring the enzyme's catalytic activity. During 50 years of research in the reactivator field, researchers have synthesized and tested numerous structural modifications of monopyridinium oximes and bispyridinium oximes. In the past decade, medicinal chemists have focused their research on the more efficient bispyridinium reactivators, but all known reactivators have several drawbacks. First, due to their permanent positive charge, they do not cross the blood-brain barrier (BBB) efficiently and do not readily reactivate AChE in the central nervous system. Second, no single oxime is efficient against a wide variety of OPNAs. Third, oximes cannot reactivate "aged" AChE. This Account summarizes recent strategies for the development of AChE reactivators capable of crossing the BBB. The use of nanoparticulate transport and inhibition of P-glycoprotein efflux pumps improves BBB transport of these AChE reactivators. Chemical modifications that increased the lipophilicity of the pyridinium aldoximes, the addition of a fluorine atom and the replacement of a pyridyl ring with a dihydropyridyl moiety, enhances BBB permeability. The glycosylation of pyridine aldoximes facilitates increased BBB penetration via the GLUT-1 transport system. The development of novel uncharged reactivators that can move efficiently across the BBB represents one of the most promising of these new strategies.

Cobalt tris(4-vinylphenyl)corrole: out of the frying pan into the polymer.

A novel cobalt corrole bearing 4-vinylphenyl groups at the 5,10,15-meso-positions of the macrocycle has been synthesized from tris(4-bromophenyl)corrole using a Suzuki coupling reaction. The spectral and electrochemical properties are reported in CH2Cl2 along with its ability to form a highly stable six-coordinate complex and cross-linked corrole-based polymer in a 59% yield.

Synthesis of novel mannoside glycolipid conjugates for inhibition of HIV-1 trans-infection.

Mannose-binding lectins, such as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), are expressed at the surface of human dendritic cells (DCs) that capture and transmit human immunodeficiency virus type-1 (HIV-1) to CD4(+) cells. With the goal of reducing viral trans-infection by targeting DC-SIGN, we have designed a new class of mannoside glycolipid conjugates. We report the synthesis of amphiphiles composed of a mannose head, a hydrophilic linker essential for solubility in aqueous media, and a lipid chain of variable length. These conjugates presented unusual properties based on a cooperation between the mannoside head and the lipid chain, which enhanced the affinity and decreased the need for multivalency. With an optimal lipid length, they exhibited strong binding affinity for DC-SIGN (K(d) in the micromolar range) as assessed by surface plasmon resonance. The most active molecules were branched trimannoside conjugates, able to inhibit the interaction of the HIV-1 envelope with DCs, and to drastically reduce trans-infection of HIV-1 mediated by DCs (IC(50s) in the low micromolar range). This new class of compounds may be of potential use for prevention of HIV-1 dissemination, and also of infection by other DC-SIGN-binding human pathogens.

Long-Term Anxiety-like Behavior and Microbiota Changes Induced in Mice by Sublethal Doses of Acute Sarin Surrogate Exposure.

Anxiety disorder is one of the most reported complications following organophosphorus (OP) nerve agent (NA) exposure. The goal of this study was to characterize the long-term behavioral impact of a single low dose exposure to 4-nitrophenyl isopropyl methylphosphonate (NIMP), a sarin surrogate. We chose two different sublethal doses of NIMP, each corresponding to a fraction of the median lethal dose (one mild and one convulsive), and evaluated behavioral changes over a 6-month period following exposure. Mice exposed to both doses showed anxious behavior which persisted for six-months post-exposure. A longitudinal magnetic resonance imaging examination did not reveal any anatomical changes in the amygdala throughout the 6-month period. While no cholinesterase activity change or neuroinflammation could be observed at the latest timepoint in the amygdala of NIMP-exposed mice, important modifications in white blood cell counts were noted, reflecting a perturbation of the systemic immune system. Furthermore, intestinal inflammation and microbiota changes were observed at 6-months in NIMP-exposed animals regardless of the dose received. This is the first study to identify long-term behavioral impairment, systemic homeostasis disorganization and gut microbiota alterations following OP sublethal exposure. Our findings highlight the importance of long-term care for victims of NA exposure, even in asymptomatic cases.

A New Class of Bi- and Trifunctional Sugar Oximes as Antidotes against Organophosphorus Poisoning.

Recent events demonstrated that organophosphorus nerve agents are a serious threat for civilian and military populations. The current therapy includes a pyridinium aldoxime reactivator to restore the enzymatic activity of acetylcholinesterase located in the central nervous system and neuro-muscular junctions. One major drawback of these charged acetylcholinesterase reactivators is their poor ability to cross the blood-brain barrier. In this study, we propose to evaluate glucoconjugated oximes devoid of permanent charge as potential central nervous system reactivators. We determined their in vitro reactivation efficacy on inhibited human acetylcholinesterase, the crystal structure of two compounds in complex with the enzyme, their protective index on intoxicated mice, and their pharmacokinetics. We then evaluated their endothelial permeability coefficients with a human in vitro model. This study shed light on the structural restrains of new sugar oximes designed to reach the central nervous system through the glucose transporter located at the blood-brain barrier.

Cationic cyclization of 2-alkenyl-1,3-dithiolanes: diastereoselective synthesis of trans-decalins.

An unprecedented and highly diastereoselective 6-endo-trig cyclization of 2-alkenyl-1,3-dithiolanes has been developed yielding trans-decalins, an important scaffold present in numerous di- and triterpenes. The novelty of this 6-endo-trig cyclization stands in the stepwise mechanism involving 2-alkenyl-1,3-dithiolane, acting as a novel latent initiator. It is suggested that the thioketal opens temporarily under the influence of TMSOTf, triggering the cationic 6-endo-trig cyclization, and closes after C-C bond formation and diastereoselective protonation to terminate the process. DFT calculations confirm this mechanistic proposal and provide a rationale for the observed diastereoselectivity. The reaction tolerates a wide range of functionalities and nucleophilic partners within the substrate. We have also shown that the one-pot 6-endo-trig cyclization followed by in situ 1,3-dithiolane deprotection afford directly the corresponding ketone. This improvement allowed the achievement of the shortest total synthesis of triptophenolide and the shortest formal synthesis of triptolide.

An Antidote Screening System for Organophosphorus Poisoning Using Zebrafish Larvae.

Organophosphorus (OP) cholinesterase inhibitors, which include insecticides and chemical warfare nerve agents, are very potent neurotoxicants. Given that the actual treatment has several limitations, the present study provides a general method, called the zebrafish-OP-antidote test (ZOAT), and basic scientific data, to identify new antidotes that are more effective than the reference pyridinium oximes after acute OP poisoning. The reactivation capacity of a chemical compound can be measured using in vivo and ex vivo acetylcholinesterase (AChE) assays. We demonstrated that it is possible to differentiate between chemical compound protective efficacies in the central and peripheral nervous system via the visual motor response and electric field pulse motor response tests, respectively. Moreover, the ability to cross the brain-blood barrier can be estimated in a physiological context by combining an AChE assay on the head and trunk-tail fractions and the cellular and tissue localization of AChE activity in the whole-mount animal. ZOAT is an innovative method suitable for the screening and rapid identification of chemicals and mixtures used as antidote for OP poisoning. The method will make it easier to identify more effective medical countermeasures for chemical threat agents, including combinatorial therapies.

Persistent brainwave disruption and cognitive impairment induced by acute sarin surrogate sub-lethal dose exposure.

Warfare neurotoxicants such as sarin, soman or VX, are organophosphorus compounds which irreversibly inhibit cholinesterase. High-dose exposure with nerve agents (NA) is known to produce seizure activity and related brain damage, while less is known about the effects of acute sub-lethal dose exposure. The aim of this study was to characterize behavioral, brain activity and neuroinflammatory modifications at different time points after exposure to 4-nitrophenyl isopropyl methylphosphonate (NIMP), a sarin surrogate. In order to decipher the impacts of sub-lethal exposure, we chose 4 different doses of NIMP each corresponding to a fraction of the median lethal dose (LD50). First, we conducted a behavioral analysis of symptoms during the first hour following NIMP challenge and established a specific scoring scale for the intoxication severity. The intensity of intoxication signs was dose-dependent and proportional to the cholinesterase activity inhibition evaluated in mice brain. The lowest dose (0.3 LD50) did not induce significant behavioral, electrocorticographic (ECoG) nor cholinesterase activity changes. Animals exposed to one of the other doses (0.5, 0.7 and 0.9 LD50) exhibited substantial changes in behavior, significant cholinesterase activity inhibition, and a disruption of brainwave distribution that persisted in a dose-dependent manner. To evaluate long lasting changes, we conducted ECoG recording for 30 days on mice exposed to 0.5 or 0.9 LD50 of NIMP. Mice in both groups showed long-lasting impairment of theta rhythms, and a lack of restoration in hippocampal ChE activity after 1-month post-exposure. In addition, an increase in neuroinflammatory markers (IBA-1, TNF-α, NF-κB) and edema were transiently observed in mice hippocampus. Furthermore, a novel object recognition test showed an alteration of short-term memory in both groups, 1-month post-NIMP intoxication. Our findings identified both transient and long-term ECoG alterations and some long term cognitive impairments following exposure to sub-lethal doses of NIMP. These may further impact morphopathological alterations in the brain.

A HTS assay for the detection of organophosphorus nerve agent scavengers.

A new pro-fluorescent probe aimed at a HTS assay of scavengers is able to selectively and efficiently cleave the P-S bond of organophosphorus nerve agents and by this provides non-toxic phosphonic acid has been designed and synthesised. The previously described pro-fluorescent probes were based on a conventional activated P-Oaryl bond cleavage, whereas our approach uses a self-immolative linker strategy that allows the detection of phosphonothioase activity with respect to a non-activated P-Salkyl bond. Further, we have also developed and optimised a high-throughput screening assay for the selection of decontaminants (chemical or biochemical scavengers) that could efficiently hydrolyse highly toxic V-type nerve agents. A preliminary screening, realised on a small alpha-nucleophile library, allowed us to identify some preliminary "hits", among which pyridinealdoximes, alpha-oxo oximes, hydroxamic acids and, less active but more original, amidoximes were the most promising. Their selective phosphonothioase activity has been further confirmed by using PhX as the substrate, and thus they offer new perspectives for the synthesis of more potent V nerve agent scavengers.

Theoretical mechanistic study of the TBD-catalyzed intramolecular aldol reaction of ketoaldehydes.

The intramolecular aldol reaction of acyclic ketoaldehydes catalyzed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) is investigated using density functional theory calculations. Compared to the proline-catalyzed aldol reaction, the use of TBD provides a unique and unusual complete switch of product selectivity. Three mechanistic pathways are proposed and evaluated. The calculations provide new insights into the activation mode of bifunctional guanidine catalysts. In the favored mechanism, TBD first catalyzes the enolization of the substrate and then the C-C bond formation through two concerted proton transfers. In addition, the computationally predicted stereochemical outcome of the reaction is in agreement with the experimental findings.

Generation of Nucleophilic Chromium Acetylides from gem-Trichloroalkanes and Chromium Chloride: Synthesis of Propargyl Alcohols.

Nucleophilic mixed chromium(II) and chromium(III) acetylides are generated from the smooth reduction of primary 1,1,1-trichloroalkanes with chromium(II) chloride in the presence of an excess amount of triethylamine at room temperature. These species arise from chromium(III) vinylidene carbenoids. It has been demonstrated that uncommon low-valent Cr(II) acetylides are formed by C-H insertion of Cr(II)Cl(2) into terminal alkynes, formed in situ through the Fritsch- Buttenberg-Wiechell (FBW) rearrangement, whereas Cr(III) acetylides are concomitantly generated by HCl elimination from the chromium(III) vinylidene carbenoid. Both divergent pathways result, overall, in the formation of nucleophilic acetylides. In situ trapping with electrophilic aldehydes afforded propargyl alcohols. Furthermore, deuteration experiments and the use of deuterium labeled 1,1,1-trichloroalkane substrates demonstrated the prevalence of low-valent Cr(II) acetylides, potentially useful, yet highly elusive synthetic intermediates.

Efficacy Assessment of an Uncharged Reactivator of NOP-Inhibited Acetylcholinesterase Based on Tetrahydroacridine Pyridine-Aldoxime Hybrid in Mouse Compared to Pralidoxime.

(1) Background: Human exposure to organophosphorus compounds employed as pesticides or as chemical warfare agents induces deleterious effects due to cholinesterase inhibition. One therapeutic approach is the reactivation of inhibited acetylcholinesterase by oximes. While currently available oximes are unable to reach the central nervous system to reactivate cholinesterases or to display a wide spectrum of action against the variety of organophosphorus compounds, we aim to identify new reactivators without such drawbacks. (2) Methods: This study gathers an exhaustive work to assess in vitro and in vivo efficacy, and toxicity of a hybrid tetrahydroacridine pyridinaldoxime reactivator, KM297, compared to pralidoxime. (3) Results: Blood-brain barrier crossing assay carried out on a human in vitro model established that KM297 has an endothelial permeability coefficient twice that of pralidoxime. It also presents higher cytotoxicity, particularly on bone marrow-derived cells. Its strong cholinesterase inhibition potency seems to be correlated to its low protective efficacy in mice exposed to paraoxon. Ventilatory monitoring of KM297-treated mice by double-chamber plethysmography shows toxic effects at the selected therapeutic dose. This breathing assessment could help define the No Observed Adverse Effect Level (NOAEL) dose of new oximes which would have a maximum therapeutic effect without any toxic side effects.

Insight into the complexation mode of bis(nitrilotriacetic acid) (NTA) ligands with Ni(2+) involved in the labeling of histidine-tagged proteins.

According to literature reports and our own findings, the binding of new Ni(2+)-preloaded bis(nitrilotriacetic acid) (NTA) ligands with polyhistidine-tagged proteins has been found to be accompanied by a one- to two-order-of-magnitude increase in affinity, compared to the binding of a single Ni(2+)-preloaded NTA moiety. In spite of the introduction of a second NTA chelating group, a cooperative effect that is less than the theoretical maximum has been observed. Herein, we present a rational explanation for the observed stability of the ternary complex involving the postulated bis-NTA-(Ni(2+))(2) species and multivalent polyhistidine tags. We have found that prior to the formation of the ternary complex, the Ni(2+)-preloading step of bis-NTA ligands does not form the expected bis-NTA-(Ni(2+))(2) exclusively. Instead of the major formation of bis-NTA-(Ni(2+))(2) species, it appears that cyclic discrete 1:1 and 2:2 entities are predominantly formed. It is proposed that these species interact upon ring-opening with multivalent histidine tags. The occurrence of this phenomena accounts for the overall one- to two-order-of-magnitude increase in affinity of ternary complexes involving bis-NTA ligands.

Novel chiral derivatizing isothiocyanate-based agent for the enantiomeric excess determination of amines.

The expedient synthesis of a novel chiral (1S,2S)-N-[(2-isothiocyanato)cyclohexyl] trifluoromethanesulfonamide and its use as a derivatizing auxiliary for the straightforward determination of optical purity of chiral amines are reported.