Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Molecular Modeling of the Multiple-Substrate Activity of the Human Recombinant Intra-Melanosomal Domain of Tyrosinase and Its OCA1B-Related Mutant Variant P406L.

Tyrosinase serves as the key enzyme in melanin biosynthesis, catalyzing the initial steps of the pathway, the hydroxylation of the amino acid L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), followed by the subsequent oxidation of L-DOPA into dopaquinone (DQ), and it facilitates the conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into 5,6-indolequinone-2-carboxylic acid (IQCA) and 5,6-dihydroxy indole (DHI) into indolequinone (IQ). Despite its versatile substrate capabilities, the precise mechanism underlying tyrosinase's multi-substrate activity remains unclear. Previously, we expressed, purified, and characterized the recombinant intra-melanosomal domain of human tyrosinase (rTyr). Here, we demonstrate that rTyr mimics native human tyrosinase's catalytic activities in vitro and in silico. Molecular docking and molecular dynamics (MD) simulations, based on rTyr's homology model, reveal variable durability and binding preferences among tyrosinase substrates and products. Analysis of root mean square deviation (RMSD) highlights the significance of conserved residues (E203, K334, F347, and V377), which exhibit flexibility during the ligands' binding. Additionally, in silico analysis demonstrated that the OCA1B-related P406L mutation in tyrosinase substantially influences substrate binding, as evidenced by the decreased number of stable ligand conformations. This correlation underscores the mutation's impact on substrate docking, which aligns with the observed reduction in rTyr activity. Our study highlights how rTyr dynamically adjusts its structure to accommodate diverse substrates and suggests a way to modulate rTyr ligand plasticity.

Design, synthesis and bioactivity evaluation of the combination of evodiamine and erlotinib linked by indolequinone.

Compared with single-targeted therapy, the design and synthesis of heterozygous molecules is still a significant challenge for the discovery of antitumor drugs. Quinone oxidoreductase-1 (NQO1) is a potential target for selective cancer therapy due to its overexpression in many cancer cells and its unique bioredox properties. Based on the principle of combinatorial drug design, we successfully synthesized a new hybrid molecules 13 with an indolequinone structure. We found that the synthesized compounds exhibited much higher cytotoxicity against the tested cancer cells than free drugs. Further mechanism studies confirmed that compound 13 induced cell apoptosis was achieved by regulating p53-dependent mitochondrial pathway and cell cycle arrest at the G0/G1 phase.

Decaging-to-labeling: Development and investigation of quinone methide warhead for protein labeling.

Biomolecule labeling in living systems is crucial for understanding biological processes and discovering therapeutic targets. A variety of labeling warheads have been developed for multiple biological applications, including proteomics, bioimaging, sequencing, and drug development. Quinone methides (QMs), a class of highly reactive Michael receptors, have recently emerged as prominent warheads for on-demand biomolecule labeling. Their highly flexible functionality and tunability allow for diverse biological applications, but remain poorly explored at present. In this regard, we designed, synthesized, and evaluated a series of new QM probes with a trifluoromethyl group at the benzyl position and substituents on the aromatic ring to manipulate their chemical properties for biomolecule labeling. The engineered QM warhead efficiently labeled proteins both in vitro and under living cell conditions, with significantly enhanced activity compared to previous QM warheads. We further analyzed the labeling efficacy with the assistance of density functional theory (DFT) calculations, which revealed that the QM generation process, rather than the reactivity of QM, contributes more predominantly to the labeling efficacy. Noteworthy, twelve nucleophilic residues on the BSA were labeled by the probe, including Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp and Tyr. Given their high efficiency and tunability, these new QM warheads may hold great promise for a broad range of applications, especially spatiotemporal proteomic profiling for in-depth biological studies.

To what extent may aminochrome increase the vulnerability of dopaminergic neurons in the context of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that progresses over time and is characterized by preferential reduction of dopaminergic neurons in the substantia nigra. Although the precise mechanisms leading to cell death in neurodegenerative disorders, such as PD, are not fully understood, it is widely accepted that increased oxidative stress may be a prevalent factor contributing to the deterioration of the nigrostriatal dopaminergic fibers in such conditions. Aminochrome, generated from dopamine (DA) metabolism, plays an important role in multiple pathogenic mechanisms associated with PD. Its capacity to induce a gradual reduction in dopaminergic neurons is due to its endogenous neurotoxicity. The formation of aminochrome results in the production of various reactive oxygen species (ROS), including pro-inflammatory factors, superoxide, nitric oxide, and hydroxyl radicals. This, in turn, causes loss of dopaminergic neurons, reducing DA uptake, and reduced numbers and shortened dendrites. Notably, o-quinones, which are more cytotoxic, arise from the oxidation of DA and possess a higher capacity to impede cellular defense mechanisms, thereby resulting in the death of neuronal cells. Aminochrome potentially contributes to the pathophysiology of PD by forming adducts with various proteins. All of the aforementioned effects suggest that aminochrome may play a crucial role in the pathophysiology of PD. Thus, aminochrome may serve as a more relevant preclinical model for PD, facilitating a better understanding of its pathophysiological processes and identification of novel therapeutic strategies aimed at preventing or slowing disease progression.

Synthesis and Photochemical Properties of Fluorescent Metabolites Generated from Fluorinated Benzoylmenadiones in Living Cells.

This work describes the reactivity and properties of fluorinated derivatives (F-PD and F-PDO) of plasmodione (PD) and its metabolite, the plasmodione oxide (PDO). Introduction of a fluorine atom on the 2-methyl group markedly alters the redox properties of the 1,4-naphthoquinone electrophore, making the compound highly oxidizing and particularly photoreactive. A fruitful set of analytical methods (electrochemistry, absorption and emission spectrophotometry, and HRMS-ESI) have been used to highlight the products resulting from UV photoirradiation in the absence or presence of selected nucleophiles. With F-PDO and in the absence of nucleophile, photoreduction generates a highly reactive ortho-quinone methide (o-QM) capable of leading to the formation of a homodimer. In the presence of thiol nucleophiles such as ß-mercaptoethanol, which was used as a model, o-QMs are continuously regenerated in sequential photoredox reactions generating mono- or disulfanylation products as well as various unreported sulfanyl products. Besides, these photoreduced adducts derived from F-PDO are characterized by a bright yellowish emission due to an excited-state intramolecular proton transfer (ESIPT) process between the dihydronapthoquinone and benzoyl units. In order to evidence the possibility of an intramolecular coupling of the o-QM intermediate, a synthetic route to the corresponding anthrones is described. Tautomerization of the targeted anthrones occurs and affords highly fluorescent stable hydroxyl-anthraquinones. Although probable to explain the intense visible fluorescence emission also observed in tobacco BY-2 cells used as a cellular model, these coupling products have never been observed during the photochemical reactions performed in this study. Our data suggest that the observed ESIPT-induced fluorescence most likely corresponds to the generation of alkylated products through reduction species, as demonstrated with the ß-mercaptoethanol model. In conclusion, F-PDO thus acts as a novel (pro)-fluorescent probe for monitoring redox processes and protein alkylation in living cells.

Tacrine First-Phase Biotransformation and Associated Hepatotoxicity: A Possible Way to Avoid Quinone Methide Formation.

Tacrine was withdrawn from clinical use as a drug against Alzheimer's disease in 2013, mainly due to drug-induced liver injury. The culprit of tacrine-associated hepatotoxicity is believed to be the 7-OH-tacrine metabolite, a possible precursor of quinone methide (Qmeth), which binds to intracellular -SH proteins. In our study, several different animal and human models (liver microsomes, primary hepatocytes, and liver slices) were used to investigate the biotransformation and hepatotoxicity of tacrine and its 7-substituted analogues (7-methoxy-, 7-phenoxy-, and 7-OH-tacrine). Our goal was to find the most appropriate in vitro model for studying tacrine hepatotoxicity and, through rational structure modifications, to develop derivatives of tacrine that are less prone to Qmeth formation. Our results show that none of animal models tested accurately mimic human tacrine biotransformation; however, the murine model seems to be more suitable than the rat model. Tacrine metabolism was overall most accurately mimicked in three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs). In this system, tacrine and 7-methoxytacrine were hydroxylated to 7-OH-tacrine, whereas 7-phenoxytacrine formed, as expected, only trace amounts. Surprisingly, however, our study showed that 7-OH-tacrine was the least hepatotoxic (7-OH-tacrine < tacrine < 7-methoxytacrine < 7-phenoxytacrine) even after doses had been adjusted to achieve the same intracellular concentrations. The formation of Qmeth-cysteine and Qmeth-glutathione adducts after human liver microsome incubation was confirmed by all of the studied tacrine derivatives, but these findings were not confirmed after incubation with 3D PHH spheroids. Therefore, the presented data call into question the suggested previously hypothesized mechanism of toxicity, and the results open new avenues for chemical modifications to improve the safety of novel tacrine derivatives.

Heterocyclic Iminoquinones and Quinones from the National Cancer Institute (NCI, USA) COMPARE Analysis.

This review uses the National Cancer Institute (NCI) COMPARE program to establish an extensive list of heterocyclic iminoquinones and quinones with similarities in differential growth inhibition patterns across the 60-cell line panel of the NCI Developmental Therapeutics Program (DTP). Many natural products and synthetic analogues are revealed as potential NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates, through correlations to dipyridoimidazo[5,4-f]benzimidazoleiminoquinone (DPIQ), and as potential thioredoxin reductase (TrxR) inhibitors, through correlations to benzo[1,2,4]triazin-7-ones and pleurotin. The strong correlation to NQO1 infers the enzyme has a major influence on the amount of the active compound with benzo[e]perimidines, phenoxazinones, benz[f]pyrido[1,2-a]indole-6,11-quinones, seriniquinones, kalasinamide, indolequinones, and furano[2,3-b]naphthoquinones, hypothesised as prodrugs. Compounds with very strong correlations to known TrxR inhibitors had inverse correlations to the expression of both reductase enzymes, NQO1 and TrxR, including naphtho[2,3-b][1,4]oxazepane-6,11-diones, benzo[a]carbazole-1,4-diones, pyranonaphthoquinones (including kalafungin, nanaomycin A, and analogues of griseusin A), and discorhabdin C. Quinoline-5,8-dione scaffolds based on streptonigrin and lavendamycin can correlate to either reductase. Inhibitors of TrxR are not necessarily (imino)quinones, e.g., parthenolides, while oxidising moieties are essential for correlations to NQO1, as with the mitosenes. Herein, an overview of synthetic methods and biological activity of each family of heterocyclic imino(quinone) is provided.

Arylcyanomethylenequinone Oximes: An Overview of Synthesis, Chemical Transformations, and Biological Activity.

Quinone methides are a class of biologically active compounds that can be used in medicine as antibacterial, antifungal, antiviral, antioxidant, and anti-inflammatory agents. In addition, quinone methides have the potential to be used as pesticides, dyes, and additives for rubber and plastics. In this paper, we discuss a subclass of quinone methides: methylenequinone oximes. Although the first representatives of the subgroup were synthesized in the distant past, they still need to be additionally studied, while their chemistry, biological properties, and perspective of practical applications require to be comprehensively summarised. Based on the analysis of the literature, it can be concluded that methylenequinone oximes exhibit a diversified profile of properties and outstanding potential as new drug candidates and reagents in organic synthesis, both of electrophilic and nucleophilic nature, worthy of wide-ranging further research.

Synthesis of Oxo-Bridged Dibenzoazocines through Ruthenium-Catalyzed [4 + 3]-Cycloannulation of Aza-ortho-quinone Methides with Carbonyl Ylides.

Oxo-bridged dibenzoazocines are furnished within a single synthetic step at room temperature via ruthenium-catalyzed [4 + 3]-cycloannulation of aza-ortho-quinone methides with carbonyl ylides. Exclusive diastereoselectivity, excellent yield, mild reaction conditions, and broad substrate scope are distinguishing features of this protocol. The product could be prepared on a gram scale and could be further functionalized into diverse substituted dihydroisobenzofuran derivatives and a dibenzoazocine scaffold.

Metal-free and enantioselective synthesis of 1,4-benzoxazepines from para-quinone methide derivatives and α-bromohydroxamates.

A sequential asymmetric conjugate addition/cyclisation of α-bromohydroxamates with para-quinone methide derivatives has been developed, which provides enantioenriched 1,4-benzoxazepines in generally high yields (up to 95%) and good enantioselectivities (up to 97 : 3 er). This protocol not only offers a novel and straightforward strategy for constructing chiral 1,4-benzoxazepines, but also demonstrates the potential of α-bromohydroxamates as three-atom synthons in asymmetric cyclisation reactions.

An ortho-Quinone Methide Mediates Disulfide Migration in the Biosynthesis of Epidithiodiketopiperazines.

The transannular disulfide functions as a key structural element imparting diverse biological activities to epidithiodiketopiperazines (ETPs). Although mechanisms were proposed in previous studies, α,ß'-disulfide formation in ETPs is not well-determined owing to the failure to identify the hypothetical intermediate. Herein, we characterize the key ortho-quinone methide (o-QM) intermediate and prove its involvement in the carbon-sulfur migration from an α,α'- to an α,ß'-disulfide by elucidating pretrichodermamide A biosynthesis, which is catalyzed by a FAD-dependent thioredoxin oxygenase TdaE harboring a noncanonical CXXQ motif. Biochemical investigations of recombinant TdaE and mutants demonstrated that the construction of the α,ß'-disulfide was initiated by Gln140 triggering proton abstraction for generation of the essential o-QM intermediate, accompanied by ß'-acetoxy elimination. Subsequent attack on the α,α'-disulfide by Cys137 led to disulfide migration and spirofuran formation. This study expands the biocatalytic toolbox for transannular disulfide formation and sets the stage for the targeted discovery of bioactive ETPs.

Chemistry of Isoeugenol and Its Oxidation Products: Mechanism and Kinetics of Isoeugenol as a Skin Sensitizer.

Structurally similar phytochemical compounds may elicit markedly different skin sensitization responses. Eugenol and isoeugenol are natural phenylpropanoids found in various essential oils are frequently used as fragrance ingredients in consumer products due to their pleasing aromatic properties. Both compounds are also skin sensitizers with isoeugenol being a stronger sensitizer than eugenol. The most commonly accepted mechanisms for haptenation by eugenol involve formation of a quinone methide or an ortho-quinone intermediate. The mechanism for the increased skin response to isoeugenol remains elusive, although quinone methide intermediates have been proposed. The recent identification of diastereomeric 7,4'-oxyneolignans as electrophilic, thiol-depleting isoeugenol derivatives has revived interest in the possible role of elusive reactive intermediates associated with the isoeugenol's haptenation process. In the present work, integrated non-animal skin sensitization methods were performed to determine the ability of syn-7,4'-oxyneolignan to promote haptenation and activation of further molecular pathways in keratinocytes and dendritic cells, confirming it as a candidate skin sensitizer. Kinetic NMR spectroscopic studies using dansyl cysteamine (DCYA) confirmed the first ordered nature of the nucleophilic addition for the syn-7,4'-oxyneolignan. Computational studies reaffirmed the "syn" stereochemistry of the isolated 7,4'-oxyneolignans along with that of their corresponding DCYA adducts and provided evidence for the preferential stereoselectivity. A plausible rationale for isoeugenol's strong skin sensitization is proposed based on the formation of a hydroxy quinone methide as a reactive intermediate rather than the previously assumed quinone methide.

Sensing of Catecholamine in Human Urine Using a Simple Colorimetric Assay Based on Direct Melanochrome and Indolequinone Formation.

We used the first enzyme-free synthesis and stabilization of soluble melanochrome (MC) and 5,6-indolequinone (IQ) derived from levodopa (LD), dopamine (DA), and norepinephrine (NE) oxidation to develop a simple colorimetric assay for catecholamine detection in human urine, also elucidating the time-dependent formation and molecular weight of MC and IQ using UV-Vis spectroscopy and mass spectrometry. The quantitative detection of LD and DA was achieved in human urine using MC as a selective colorimetric reporter to demonstrate the potential assay applicability in a matrix of interest in therapeutic drug monitoring (TDM) and in clinical chemistry. The assay showed a linear dynamic range between 5.0 mg L-1 and 50.0 mg L-1, covering the concentration range of DA and LD found in urine samples from, e.g., Parkinson's patients undergoing LD-based pharmacological therapy. The data reproducibility in the real matrix was very good within this concentration range (RSDav% 3.7% and 6.1% for DA and LD, respectively), also showing very good analytical performances with the limits of detection of 3.69 ± 0.17 mg L-1 and 2.51 ± 0.08 mg L-1 for DA and LD, respectively, thus paving the way for the effective and non-invasive monitoring of dopamine and levodopa in urine from patients during TDM in Parkinson's disease.

Chemoenzymatic Synthesis of (+)-Xyloketal B.

Xyloketal B is a pentacyclic fungal marine natural product that has shown potential for the treatment of diseases such as Alzheimer's disease and atherosclerosis. Herein, we describe the first asymmetric synthesis of this natural product, which relies on a chemoenzymatic strategy. This approach leverages a biocatalytic benzylic hydroxylation to access to an ortho-quinone methide intermediate which is captured in a [4 + 2] cycloaddition to stereoselectively yield a key cyclic ketal intermediate enroute to (+)-xyloketal B. The relative configuration of this intermediate was rapidly confirmed as the desired stereoisomer using MicroED. To complete the synthesis, a second ortho-quinone methide was accessed through a reductive approach, ultimately leading to the stereoselective synthesis of (+)-xyloketal B.

Light and hydrogen peroxide dual-responsive DNA interstrand crosslink precursors with potent cytotoxicity.

Arylboronic acid/esters and phenyl selenides-based quinone methide (QM) precursors were reported to induce DNA interstrand crosslink (ICL) formation upon reaction with the inherently high concentrations of H2O2 in cancer cells. However, some normal cells (such as macrophages) also contain high-levels of H2O2, which may interfere with precursors' selectivity. In order to enhance the spatiotemporal specificity by the photolysis, we developed photo- and H2O2- dual-responsive DNA ICL precursors 1-3, bearing a photo-responsive coumarin moiety and a H2O2 inducible phenyl selenide group. Precursors 1-3 are efficiently activated by photoirradiation and H2O2 to generate reactive QMs crosslinking DNA. Moreover, the reactivity of precursors can be modulated by the introduction of aromatic substituents (OMe, F), and the electron donating group (OMe) displays a more pronounced promoting effect on DNA ICL formation. A subsequent piperidine heat stability study confirmed that the formed QMs primarily alkylate dAs, dGs and dCs in DNA. Furthermore, 1-3 inhibit lung cancer cell (H1299) growth by inducing DNA damage and producing toxic reactive oxygen species (ROS) upon photolysis of released coumarin. This study illustrates the potent cytotoxicity achieved by novel photo/H2O2 dual-responsive QM precursors 1-3, affording a novel strategy for the development of inducible DNA interstrand cross-linkers.

Ring Expansion of Isatins via 1,2-Phospha-Brook Rearrangement: A Route to the Synthesis of 2-Quinolinone-Derived p-Quinone Methides.

A Lewis acid-mediated one-carbon homologation approach to installing a 2-quinolinone core embedded with para-quinone methides, in a high yield of up to 92%, and with high regioselectivity has been developed. Also, post-synthetic modifications, including C-P, C-S, and C-C bond formations, have been demonstrated by the 1,6-addition of suitable nucleophiles. Further, cyclopropanation of 2-quinolinone-embedded p-QM is also demonstrated affording a contiguous quaternary spiro center.

A Novel Method to Construct 2-Aminobenzofurans via [4 + 1] Cycloaddition Reaction of In Situ Generated Ortho-Quinone Methides with Isocyanides.

A new approach for the synthesis of 2-aminobenzofurans has been described via Sc(OTf)3 mediated formal cycloaddition of isocyanides with the in situ generated ortho-quinone methides (o-QMs) from o-hydroxybenzhydryl alcohol. Notably, as a class of readily available and highly active intermediates, o-QMs were first used in the construction of benzofurans. This [4 + 1] cycloaddition reaction provides a straightforward and efficient methodology for the construction of 2-aminobenzofurans scaffold in good yield (up to 93% yield) under mild conditions.

p-Quinone Methide-Mediated Nonenzymatic Formation of Chalcane-Containing Dimers in Dragon's Blood.

Chalcane-containing dimers are major compounds identified from dragon's blood, the red resin that accumulates in Dracaena trees after injury. The key step for the formation of these dimers was a p-quinone methide (p-QM, 3) mediated nonenzymatic Michael addition. Compound 3 is derived from the spontaneous dehydration of chalcane alcohol-M274 (2). Two dihydroflavonol-4-reductases, discovered in D. cambodiana, reduce dihydrochalcone-M272 (7) to 2. Moreover, the application potential of p-QMs was demonstrated using a 3-like p-QM to synthesize diverse dimeric derivatives.

Reactivity of quinone methides with carbenes generated from α-diazocarbonyl compounds and related compounds.

Over the years, quinone methides have broadly been applied in synthesis and biological systems for synthesizing heterocyclic compounds and biologically active molecules. In this feature article, we have discussed the novel and uncovered reactivity of o-quinone methides, p-quinone methides, aza-o-quinone methides, and indolyl-2-methides with carbenes generated from α-diazocarbonyl compounds and related compounds. Two in situ-generated transient intermediates undergo cycloannulation reactions, metathesis-type reactions, 1,6-conjugate addition reactions, cyclopropanation reactions, and many other transformations to access nitrogen- and oxygen-containing heterocyclic compounds and beyond. The reactivity of quinone methides and carbenes is observed in various metal catalysts, Brønsted-acids, Lewis acids, phase transfer catalysts, additives, and visible-light-induced transformations.