The design of MB327, a bispyridinium compound that ameliorates the nicotinic effects of acute organophosphorus nerve agent (NA) intoxication, followed an observation made by the German pharmacologist Klaus Schoene in the 1970s, who noted therapeutic activity in bispyridinium molecules missing the usual oxime group, CHNOH. Some of these compounds protected mice against soman. One structurally related to obidoxime called HY10 had this action. Its oxime moieties were capped by tert-butyl groups: CH=NOtBu. We modified HY10 by changing the bridge between the pyridinium units from a dimethylene ether to a trimethylene group (CH2OCH2 → CH2CH2CH2) and prepared a novel relative of trimedoxime, called LB1, whose synthesis and stereochemistry are described. Unlike obidoxime or trimedoxime, LB1 because of its capped oxime groups, cannot directly reactivate NA inhibited acetylcholinesterase. Its antidotal activity in mice is now reported. The therapeutic efficacy of LB1, atropine alone, atropine with LB1, atropine with an oxime (HI-6, obidoxime or trimedoxime), and atropine with an oxime and LB1, was studied by determining the LD50 values of the NAs soman, sarin, or tabun in mice treated with these compounds or mixtures. LB1 exceeded MB327 in toxicity and its activity was insufficient for a useful addition to the current standard antidotal treatment (protective ratio data are compared to those of MB327). Although this study produced largely negative biological results, the therapeutically beneficial mechanism of the effective bispyridinium non-oxime analogues is unclear, and has been demonstrated only in vivo. The present study points out directions in structural optimisation unlikely to yield the desired therapeutic outcomes and provides a literature review that could promote creative thinking for the design of widely-desirable non-oxime therapeutics for anticholinesterase inhibitors.
- Klíčová slova
- Bispyridinium, LB1, Mechanism, Mice, Nerve agent, Pyridinecarboxaldoxime,
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota * chemická syntéza chemie farmakologie terapeutické užití MeSH
- atropin terapeutické užití farmakologie MeSH
- cholinesterasové inhibitory toxicita MeSH
- myši MeSH
- nervová bojová látka * toxicita MeSH
- organofosforové sloučeniny * toxicita MeSH
- oximy chemie MeSH
- pyridinové sloučeniny * chemická syntéza chemie terapeutické užití farmakologie MeSH
- soman toxicita MeSH
- trimedoxim chemie chemická syntéza farmakologie terapeutické užití MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- antidota * MeSH
- atropin MeSH
- cholinesterasové inhibitory MeSH
- MB327 MeSH Prohlížeč
- nervová bojová látka * MeSH
- organofosforové sloučeniny * MeSH
- oximy MeSH
- pyridinové sloučeniny * MeSH
- soman MeSH
- trimedoxim MeSH
Recent events involving nerve agents of the A-Series, a once elusive class of chemical warfare agents, have provoked a great concern in the international community. In this paper, continuing our research efforts in Medicinal Chemistry at the Brazilian Institute of Chemical, Biological, Radiological and Nuclear Defense (IDQBRN) (an OPCW-designated Laboratory for environmental samples), we explore ANMP, an A-230 surrogate, in the search for new treatment options for intoxications caused by these chemicals. Five isatin-pyridine oxime hybrids were evaluated as acetylcholinesterase (AChE) reactivators using a modified Ellman's assay. Our results indicate that monocationic hybrids with five methylene units, as well as its oxa-analog, are promising compounds for the design of new AChE reactivators.
- Klíčová slova
- Acetylcholinesterase, Antidotes, Chemical Weapons Convention, Isatin hybrids, Nerve agents,
- MeSH
- acetylcholinesterasa metabolismus MeSH
- chemické bojové látky * toxicita chemie MeSH
- cholinesterasové inhibitory * toxicita chemie MeSH
- isatin * chemie farmakologie analogy a deriváty MeSH
- oximy * chemie farmakologie MeSH
- počítačová simulace MeSH
- pyridiny * chemie farmakologie MeSH
- reaktivátory cholinesterasy * farmakologie chemie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- chemické bojové látky * MeSH
- cholinesterasové inhibitory * MeSH
- isatin * MeSH
- oximy * MeSH
- pyridiny * MeSH
- reaktivátory cholinesterasy * MeSH
Mono-quaternary pyridinium oximes derived from K-oximes K027, K048 and K203 were designed, synthesized and evaluated for the reactivation of organophosphate-inhibited cholinesterases. The incorporation of the halogen atoms to the structure decreased the pKa value of the oxime group resulting in an increased formation of oximate necessary for reactivation. The stability and pKa values were found to be similar to analogous bis-quaternary compounds. Some mono-quaternary oximes resulted as relatively strong inhibitors of human acetylcholinesterase. Nevertheless, the reactivation ability of mono-quaternary oximes for organophosphate-inhibited cholinesterases was lower compared to their bis-quaternary analogues. These results were further confirmed by the determination of reactivation kinetics, when in some cases novel compounds showed improvement reactivation compared to the tested standards, but no improvement to bis-quaternary K-oximes. A computational study investigated reactivation process for K027, and its two analogues for VX-inhibited AChE. This study revealed slight differences between reactivation of mono-quaternary and bis-quaternary oximes. Abbreviations: 2-PAM, pralidoxime; AChE, acetylcholinesterase; ACN, acetonitrile; ATCI, acetylcholine iodide; BChE, butyrylcholinesterase; BTCI, butyrylcholine iodide; Bu3SnSnBu3, bis(tributyltin) Et2O, diethyl ether; ChEs, cholinesterases; CNS, central nervous system; DAD, diode array detector; DIBAL-H, diisobutylaluminium hydride; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTNB, 5,5́-dithiobis-2-nitrobenzoic acid; Et3N, triethylamine; EtOAc, ethyl acetate; EWG, electron withdrawing group; HI-6, asoxime; hrAChE, human recombinant acetylcholinesterase; hrBChE, human recombinant butyrylcholinesterase; hrChEs, human recombinant cholinesterases; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; KD, dissociation constant; kr, first-order reactivation rate constant; kr2, second-order reactivation rate constant; LüH-6, obidoxime; MeOH, methanol; MM, molecular mechanics; MMC-4, methoxime; m.p., melting point; NCIs, non-covalent interactions; NEDPA, 4-nitrophenyl ethyl dimethylphosphoramidate; NEMP, 4-nitrophenyl ethyl methylphosphonate; NIMP, isopropyl methylphosphonate; NMR, nuclear magnetic resonance spectroscopy; OPs, organophosphates; PBS, phosphate-buffered saline; Pd(dppf)Cl2.CH2Cl2, [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) in complex with dichloromethane; pKa, negative decimal logarithm of the dissociation constant; POX, paraoxon; PPh3, triphenylphosphine; QM, quantum mechanics; rt, room temperature; SN2, bimolecular nucleophilic substitution; SNAc, nucleophilic acyl substitution; THF, tetrahydrofuran; TMC-4, trimedoxime; TNB, 5-thio-2-nitrobenzoic acid; UHPLC, ultra high-performance liquid chromatography; UV, ultraviolet; UV-VIS, ultraviolet-visible.
- Klíčová slova
- Cholinesterase, Inhibition, Organophosphate, Oxime, Reactivation,
- MeSH
- acetylcholinesterasa * metabolismus MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory * farmakologie chemie chemická syntéza MeSH
- halogenace MeSH
- kinetika MeSH
- lidé MeSH
- molekulární struktura MeSH
- oximy * chemie farmakologie chemická syntéza MeSH
- pyridinové sloučeniny chemie farmakologie chemická syntéza MeSH
- reaktivátory cholinesterasy farmakologie chemie chemická syntéza MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa * MeSH
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory * MeSH
- oximy * MeSH
- pyridinové sloučeniny MeSH
- reaktivátory cholinesterasy MeSH
The synthesis of nitriles is of utmost importance for preparative organic chemistry. The classical routes are often associated with disadvantages such as toxicity of the reagents and drastic conditions. The uses of enzymes like aldoxime dehydratases (Oxds) and hydroxynitrile lyases constitute attractive benign alternatives. In this review, we summarize the recent trends regarding Oxds. Thousands of oxd genes were sequenced but less than thirty Oxds were investigated on protein level. We give an overview of these Oxds, their sequence analysis, conditions required for their overexpression, and their purification and assays. We then focus on the use of Oxds especially in multistep reactions combining the chemical or chemoenzymatic synthesis of aldoximes from different starting materials with the enzymatic dehydration of aldoximes to nitriles, possibly followed by the hydration of nitriles to amides. Progress in Oxd immobilization is also highlighted. Based on data published mainly in the last 5 years, we evaluate the industrial prospects of these enzyme processes in comparison with some other innovations in nitrile synthesis. KEY POINTS: • Aldoxime dehydratases (Oxds) are promising for cyanide-free routes to nitriles • A comprehensive overview of wet-lab explored Oxds is provided • Recent trends include combining Oxds with other enzymes or chemical catalysts.
- Klíčová slova
- Aldoxime dehydratase, Biocatalyst, Immobilization, Multistep reaction, Nitrile synthesis,
- MeSH
- dehydratasy * metabolismus genetika MeSH
- enzymy imobilizované * metabolismus genetika MeSH
- nitrily * metabolismus MeSH
- oximy metabolismus chemie MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- aldoxime dehydratase MeSH Prohlížeč
- dehydratasy * MeSH
- enzymy imobilizované * MeSH
- nitrily * MeSH
- oximy MeSH
Six novel brominated bis-pyridinium oximes were designed and synthesized to increase their nucleophilicity and reactivation ability of phosphorylated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Their pKa was valuably found lower to parent non-halogenated oximes. Stability tests showed that novel brominated oximes were stable in water, but the stability of di-brominated oximes was decreased in buffer solution and their degradation products were prepared and characterized. The reactivation screening of brominated oximes was tested on AChE and BChE inhibited by organophosphorus surrogates. Two mono-brominated oximes reactivated AChE comparably to non-halogenated analogues, which was further confirmed by reactivation kinetics. The acute toxicity of two selected brominated oximes was similar to commercially available oxime reactivators and the most promising brominated oxime was tested in vivo on sarin- and VX-poisoned rats. This brominated oxime showed interesting CNS distribution and significant reactivation effectiveness in blood. The same oxime resulted with the best protective index for VX-poisoned rats.
- Klíčová slova
- Cholinesterase, Nerve agent, Nucleophile, Organophosphate, Oxime, Reactivation,
- MeSH
- acetylcholinesterasa * metabolismus účinky léků MeSH
- butyrylcholinesterasa * metabolismus MeSH
- chemické bojové látky toxicita MeSH
- cholinesterasové inhibitory * toxicita farmakologie MeSH
- halogenace MeSH
- krysa rodu Rattus MeSH
- nervová bojová látka * toxicita MeSH
- organothiofosforové sloučeniny * toxicita MeSH
- oximy * farmakologie chemie MeSH
- potkani Wistar MeSH
- pyridinové sloučeniny farmakologie MeSH
- reaktivátory cholinesterasy * farmakologie chemie MeSH
- sarin * toxicita MeSH
- stabilita léku MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa * MeSH
- butyrylcholinesterasa * MeSH
- chemické bojové látky MeSH
- cholinesterasové inhibitory * MeSH
- nervová bojová látka * MeSH
- organothiofosforové sloučeniny * MeSH
- oximy * MeSH
- pyridinové sloučeniny MeSH
- reaktivátory cholinesterasy * MeSH
- sarin * MeSH
- VX MeSH Prohlížeč
The current study imposes a new class of organophosphorus (OP)-inhibited cholinesterase reactivators by conceptualizing a family of asymmetric bisoximes with various reactivating scaffolds. Several novel nucleophilic warheads were investigated, putting forward 29 novel reactivating options, by evaluating their nucleophilicity and ability to directly decompose OP compounds. Adopting the so-called zwitterionic strategy, 17 mono-oxime and nine bisoxime reactivators were discovered with major emphasis on the bifunctional-moiety approach. Compounds were compared with clinically used standards and other known experimentally highlighted reactivators. Our results clearly favor the concept of asymmetric bisoximes as leading reactivators in terms of efficacy and versatility. These top-ranked compounds were characterized in detail by reactivation kinetics parameters and evaluated for potential CNS availability. The highlighted molecules 55, 57, and 58 with various reactivating warheads, surpassed the reactivating potency of pralidoxime and several notable uncharged reactivators. The versatility of lead drug candidate 55 was also inspected on OP-inhibited butyrylcholinesterase, revealing a much higher rate compared to existing clinical antidotes.
- Klíčová slova
- Acetylcholinesterase, Butyrylcholinesterase, Nerve agents, Oxime, Pesticides, Reactivator,
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota chemie farmakologie MeSH
- butyrylcholinesterasa * metabolismus chemie MeSH
- cholinesterasové inhibitory chemie farmakologie MeSH
- kinetika MeSH
- lidé MeSH
- organofosforové sloučeniny chemie MeSH
- otrava organofosfáty * farmakoterapie MeSH
- oximy * chemie farmakologie MeSH
- reaktivátory cholinesterasy * chemie farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- antidota MeSH
- butyrylcholinesterasa * MeSH
- cholinesterasové inhibitory MeSH
- organofosforové sloučeniny MeSH
- oximy * MeSH
- reaktivátory cholinesterasy * MeSH
Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.
- Klíčová slova
- Aldoxime dehydratase, Cinnamonitrile, E-Cinnamaldoxime (N-[(2E)−3-phenylprop-2-en-1-ylidene]hydroxylamine), E-Cinnamonitrile (E-3-phenylprop-2-enenitrile), Fragrance nitriles, Immobilization, Metal affinity resin, Phenylacetaldoxime (N-hydroxy-2-phenylethanimine), Phenylacetonitrile,
- MeSH
- dehydratasy * metabolismus MeSH
- enzymy imobilizované MeSH
- nitrily metabolismus MeSH
- odoranty * MeSH
- oximy chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aldoxime dehydratase MeSH Prohlížeč
- dehydratasy * MeSH
- enzymy imobilizované MeSH
- nitrily MeSH
- oximy MeSH
Oxime reactivators of acetylcholinesterase (AChE) are used as causal antidotes for intended and unintended poisoning by organophosphate nerve agents and pesticides. Despite all efforts to develop new AChE reactivators, none of these drug candidates replaced conventional clinically used oximes. In addition to the therapeutic efficacy, determining the safety profile is crucial in preclinical drug evaluation. The exact mechanism of oxime toxicity and the structure-toxicity relationship are subjects of ongoing research, with oxidative stress proposed as a possible mechanism. In the present study, we investigated four promising bispyridinium oxime AChE reactivators, K048, K074, K075, and K203, and their ability to induce oxidative stress in vitro. Cultured human hepatoma cells were exposed to oximes at concentrations corresponding to their IC50 values determined by the MTT assay after 24 h. Their potency to generate reactive oxygen species, interfere with the thiol antioxidant system, and induce lipid peroxidation was evaluated at 1, 4, and 24 h of exposure. Reactivators without a double bond in the four-carbon linker, K048 and K074, showed a greater potential to induce oxidative stress compared with K075 and K203, which contain a double bond. Unlike oximes with a three-carbon-long linker, the number of aldoxime groups attached to the pyridinium moieties does not determine the oxidative stress induction for K048, K074, K075, and K203 oximes. In conclusion, our results emphasize that the structure of oximes plays a critical role in inducing oxidative stress, and this relationship does not correlate with their cytotoxicity expressed as the IC50 value. However, it is important to note that oxidative stress cannot be disregarded as a potential contributor to the side effects associated with oximes.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota farmakologie MeSH
- buňky Hep G2 MeSH
- cholinesterasové inhibitory toxicita MeSH
- lidé MeSH
- organofosfáty toxicita MeSH
- oxidační stres MeSH
- oximy farmakologie chemie MeSH
- pyridinové sloučeniny farmakologie chemie MeSH
- reaktivátory cholinesterasy * farmakologie chemie MeSH
- uhlík MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- antidota MeSH
- cholinesterasové inhibitory MeSH
- organofosfáty MeSH
- oximy MeSH
- pyridinové sloučeniny MeSH
- reaktivátory cholinesterasy * MeSH
- uhlík MeSH
We report a green chemistry approach for preparation of oxime-functionalized ILs as AChE reactivators: amide/ester linked IL, l-alanine, and l-phenylalanine derived salts bearing pyridinium aldoxime moiety. The reactivation capacities of the novel oximes were evaluated towards AChE inhibited by typical toxic organophosphates, sarin (GB), VX, and paraoxon (PON). The studied compounds are mostly non-toxic up to the highest concentrations screened (2 mM) towards Gram-negative and Gram-positive bacteria cell lines and both filamentous fungi and yeasts in the in vitro screening experiments as well as towards the eukaryotic cell (CHO-K1 cell line). Introduction of the oxime moiety in initially biodegradable structure decreases its ability to biodegradation. The compound 3d was shown to reveal remarkable activity against the AChE inhibited by VX, exceeding conventional reactivators 2-PAM and obidoxime. The regularities on antidotal activity, cell viability, plasma stability, biodegradability as well as molecular docking study of the newly synthesized oximes will be used for further improvement of their structures.
- Klíčová slova
- Acetylcholinesterase reactivators, Functionalized ionic liquids, Green chemistry, Oximes, Sustainability,
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota MeSH
- cholinesterasové inhibitory farmakologie chemie MeSH
- iontové kapaliny * MeSH
- oximy farmakologie chemie MeSH
- pyridinové sloučeniny farmakologie chemie MeSH
- reaktivátory cholinesterasy * farmakologie chemie MeSH
- simulace molekulového dockingu MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- antidota MeSH
- cholinesterasové inhibitory MeSH
- iontové kapaliny * MeSH
- oximy MeSH
- pyridinové sloučeniny MeSH
- reaktivátory cholinesterasy * MeSH
- VX MeSH Prohlížeč
Acetylcholinesterase (AChE, EC 3.1.1.7) reactivators (2-PAM, trimedoxime, obidoxime, asoxime) have become an integral part of antidotal treatment in cases of nerve agent and organophosphorus (OP) pesticide poisonings. They are often referred to as specific antidotes due to their ability to restore AChE function when it has been covalently inhibited by an OP compound. Currently available commercial reactivators exhibit limited ability to penetrate the blood-brain barrier, where reactivation of inhibited AChE is crucial. Consequently, there have been numerous efforts to discover more brain-penetrating AChE reactivators. In this study, we examined a derivative of 2-PAM designed to possess increased lipophilicity. This enhanced lipophilicity was achieved through the incorporation of a benzyl group into its molecular structure. Initially, a molecular modeling study was conducted, followed by a comparison of its reactivation efficacy with that of 2-PAM against 10 different AChE inhibitors in vitro. Unfortunately, this relatively significant structural modification of 2-PAM resulted in a decrease in its reactivation potency. Consequently, this derivative cannot be considered as a broad-spectrum AChE reactivator.
- Klíčová slova
- 2-PAM, Antidote, Nerve agent, Pesticide, Reactivator,
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota farmakologie MeSH
- cholinesterasové inhibitory farmakologie metabolismus MeSH
- lidé MeSH
- otrava organofosfáty * MeSH
- oximy farmakologie chemie MeSH
- pralidoximové sloučeniny farmakologie MeSH
- reaktivátory cholinesterasy * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- antidota MeSH
- cholinesterasové inhibitory MeSH
- oximy MeSH
- pralidoxime MeSH Prohlížeč
- pralidoximové sloučeniny MeSH
- reaktivátory cholinesterasy * MeSH
