Methide Precursors Research Articles

Targeted Discovery of a Natural ortho-Quinone Methide Precursor and Green Generation of Its Oligomers.

ortho-Quinone methides (o-QMs) are a class of highly reactive intermediates that serve as important nonisolable building blocks (NBBs) in organic synthesis and small-molecule library construction. Because of their instability and nonisolability, most reported o-QMs are generated through in situ chemical synthesis, and only a few natural o-QMs have been reported due to the lack of directed discovery strategies. Herein, a new natural o-QM precursor (trichophenol A, 2) was identified from the fungal strain of Trichoderma sp. AT0167 through genome mining, which was generated by trilA (nonreducing polyketide synthase) and trilB (2-oxoglutarate dependent dioxygenase). Combinatorial biosynthesis via two other known NRPKS genes with trilA and trilB was performed, leading to the generation of five new trichophenol o-QM oligomers (trichophenols D-H, 5-9). The strategy combining genome mining with combinatorial biosynthesis not only targetedly uncovered a new natural o-QM precursor but also produced various new molecules through oligomerization of the new o-QM and its designated o-QM acceptors without chemical synthesis and isolation of intermediates, which was named NBB genome mining-combinatorial biosynthesis strategy for o-QM molecule library construction. This study provides a new strategy for the targeted discovery of natural o-QMs and small-molecule library construction with natural o-QMs.

Atomistic Origins of Resurrection of Aged Acetylcholinesterase by Quinone Methide Precursors.

Nerve agents are organophosphates (OPs) that act as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine. After inhibition, a dealkylation reaction of the phosphorylated serine, known as the aging of AChE, can occur. When aged, reactivators of OP-inhibited AChE are no longer effective. Therefore, the realkylation of aged AChE may offer a pathway to reverse AChE aging. In this study, molecular modeling was conducted to propose new ligands as realkylators of aged AChE. We applied a methodology involving docking and quantum mechanics/molecular mechanics (QM/MM) calculations to evaluate the resurrection kinetic constants and ligand interactions with OP-aged AChE, comparing them to data found in the literature. The results obtained confirm that this method is suitable for predicting kinetic and thermodynamic parameters of ligands, which can be useful in the design and selection of new and more effective ligands for AChE realkylation.

I Bind It That Way – Bioorthogonal Unmasking of Profluorescent Quinone Methides

Proof‐of‐concept studies for the bioorthogonally controlled generation of pro‐fluorescent quinone‐methides are presented here. The novel concept relies on a click‐to‐release tetrazine unit that chemically cages a pro‐fluorescent quinone methide precursor. On a synthetically readily accessible model compound we demonstrate that IEDDA reaction of the tetrazine with a TCO leads to the liberation of a pro‐fluorescent quinone methide, which is readily captured by nearby nucleophiles to form a fluorescent scaffold. Unlike in previous approaches to access bioorthogonally activatable fluorogenic probes, where the tetrazine unit acted as a quencher of fluorescence, the role of the tetrazine is redefined here. Such repurposed role of tetrazines is foreseen to address the limitations of tetrazine‐quenched red / NIR excitable fluorogenic probes.

4-Amidophenol Quinone Methide Precursors: Effective and Broad-Scope Nonoxime Reactivators of Organophosphorus-Inhibited Cholinesterases and Resurrectors of Organophosphorus-Aged Acetylcholinesterase.

Acetylcholinesterase (AChE) inhibition by organophosphorus (OP) compounds poses a serious health risk to humans. While many therapeutics have been tested for treatment after OP exposure, there is still a need for efficient reactivation against all kinds of OP compounds, and current oxime therapeutics have poor blood-brain barrier penetration into the central nervous system, while offering no recovery in activity from the OP-aged forms of AChE. Herein, we report a novel library of 4-amidophenol quinone methide precursors (QMP) that provide effective reactivation against multiple OP-inhibited forms of AChE in addition to resurrecting the aged form of AChE after exposure to a pesticide or some phosphoramidates. Furthermore, these QMP compounds also reactivate OP-inhibited butyrylcholinesterase (BChE) which is an in vivo, endogenous scavenger of OP compounds. The in vitro efficacies of these QMP compounds were tested for reactivation and resurrection of soluble forms of human AChE and BChE and for reactivation of cholinesterases within human blood as well as blood and brain samples from a humanized mouse model. We identify compound 10c as a lead candidate due to its broad-scope efficacy against multiple OP compounds as well as both cholinesterases. With methylphosphonates, compound 10c (250 μM, 1 h) shows >60% recovered activity from OEt-inhibited AChE in human blood as well as mouse blood and brain, thus highlighting its potential for future in vivo analysis. For 10c, the effective concentration (EC50) is less than 25 μM for reactivation of three different methylphosphonate-inhibited forms of AChE, with a maximum reactivation yield above 80%. Similarly, for OP-inhibited BChE, 10c has EC50 values that are less than 150 μM for two different methylphosphonate compounds. Furthermore, an in vitro kinetic analysis show that 10c has a 2.2- and 92.1-fold superior reactivation efficiency against OEt-inhibited and OiBu-inhibited AChE, respectively, when compared to an oxime control. In addition to 10c being a potent reactivator of AChE and BChE, we also show that 10c is capable of resurrecting (ethyl paraoxon)-aged AChE, which is another current limitation of oximes.

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.

Investigations on Superbase Mediated Reactivity of N‐Tosylhydrazones with Aza‐ortho‐Quinone Methide Precursors

We have encountered a superbase‐mediated chemoselective reaction of N‐tosylhydrazones with aza‐ortho‐quinone methide precursors. When tosylhydrazone was treated with ortho‐aminobenzyl alcohol in super basic conditions (KOH+DMSO), we observed the formation of 2‐substituted quinoline. The reaction was found to be general, and by this method, mono‐, di‐ and tri‐substituted quinolines could be made. We could prove experimentally and theoretically that the reaction proceeded via the formation of an azine from the basic decomposition of N‐tosylhydrazones. Finally, the reaction of tosylhydrazone with N‐(2‐(chloromethyl) phenyl)‐4‐methylbenzenesulfonamide (aza‐ortho‐quinone methide precursor) under super basic conditions afforded hydrazine substituted sulfonamides.

Superoxide-responsive quinone methide precursors (QMP-SOs) to study superoxide biology by proximity labeling and chemoproteomics.

Superoxide is a reactive oxygen species (ROS) with complex roles in biological systems. It can contribute to the development of serious diseases, from aging to cancers and neurodegenerative disorders. However, it can also serve as a signaling molecule for important life processes. Monitoring superoxide levels and identifying proteins regulated by superoxide are crucial to enhancing our understanding of this growing field of redox biology and signaling. Given the high reactivity and very short lifetime of superoxide compared to other ROS in biological systems, proteins redox-modified by superoxide should be in close proximity to where superoxide is generated endogenously, i.e. superoxide hotspots. This inspires us to develop superoxide-specific quinone methide-based precursors, QMP-SOs, for proximity labeling of proteins within/near superoxide hotspots to image superoxide and profile proteins associated with superoxide biology by chemoproteomics. QMP-SOs specifically react with superoxide to generate an electrophilic quinone methide intermediate, which subsequently reacts with nucleophilic amino acids to induce a covalent tag on proteins, as revealed by liquid chromatography-mass spectrometry (LC-MS) and shotgun MS experiments. The alkyne handle on the covalent tag enables installation of fluorophores onto the tagged proteins for fluorescence imaging of superoxide in cells under oxidative stress. By establishing a chemoproteomics platform, QMP-SO-TMT, we identify DJ-1 and DLDH as proteins associated with superoxide biology in liver cancer cells treated with menadione. This work should provide insights into the crosstalk between essential cellular events and superoxide redox biology, as well as the design principles of quinone methide-based probes to study redox biology through proximity labeling and chemoproteomics.

Trapping of thermally generated ortho- and para-quinone methides by imidazoles and pyrazoles: a simple route to green synthesis of benzopyrone-azole hybrids and their evaluation as α-glucosidase inhibitors.

An efficient green approach for the trapping of in situ generated ortho-and para-quinone methide intermediates by imidazoles and pyrazoles has been developed. A wide range of quinone methide precursors based on simple phenols are compatible with the experimental protocol under mild thermal conditions. This methodology was demonstrated to be suitable for the synthesis of methylene-linked benzopyrone-azole hybrids using naturally occurring coumarin and chromone Mannich bases. In most cases, the products were isolated in good to excellent yields without chromatographic purification. In vitro studies showed that some of the synthesized compounds exhibit inhibitory activity towards α-glucosidase.

Synthesis of Functionalized Tetrahydroquinoline Containing Indole Scaffold via Chemoselective Annulation of Aza-ortho-quinone Methide Precursor.

The chemoselective annulation of aza-ortho-quinone methide generated by in situ o-chloromethyl sulfonamide has been achieved with bifunctional acyclic olefin. This efficient approach provides access to the diastereoselective synthesis of functionalized tetrahydroquinoline derivatives containing indole scaffolds through the inverse-electron-demand aza-Diels-Alder reaction under mild reaction conditions with excellent results (up to 93% yield, > 20:1 dr). Moreover, this article realized the cyclization of α-halogeno hydrazone with electron-deficient alkene affording the tetrahydropyridazine derivatives, which had never been reported.

Catalyst-free aza-Michael addition of azoles to 3-hydroxypyridine-based quinone methides

We have studied the reaction of various 1H-azoles with 2-((dimethylamino)methyl)pyridin-3-ol and 2-bromo-6-(hydroxymethyl)pyridin-3-ol as precursors of ortho- and para-quinone methides of 3-hydroxypyridine series, correspondingly. This process proved to be convenient and efficient for the synthesis of hybrid heterocycles containing both pyridine and azole moiety - 2- and 6-[(azolyl)methyl]pyridin-3-ols. The protocol has been successfully extended to 2,6-bis-(hydroxymethyl)pyridine-3-ol, which is simultaneously ortho- and para-quinone methide precursor, involving both functionalities. Finally, two new heterocyclic systems 9Н-pyrazolo[5,1-b]pyrido[2,3-e][1,3]oxazine and 12H-benzo[4,5]imidazo[2,1-b]pyrido[2,3-e][1,3]oxazine were obtained as a result of cascade aza-Michael/intramolecular nucleophilic substitution reactions from 2-((dimethylamino)methyl)pyridin-3-ol and 2-(methylthio)benzimidazole or 3,4,5-tribromopyrazole, correspondingly.

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.

A scalable and diversity-oriented synthesis of (±)-paeoveitols

Gram scale synthesis of (±)-paeoveitol, a nor-diterpene harboring an unusual benzofuran stitched benzopyran 6/5/6/6 tetracyclic ring system, and its analogues, emanating from bench-top reagents is reported. Scandium triflate-mediated inverse electron demand [4 + 2] cycloaddition reaction between pre-synthesized paeoveitol-D analogue (dienophile) and ortho-quinone methide precursor (diene), both resulting from the same starting material delivered (±)-paeoveitol in decent yield. Fine tuning of the synthetic strategy not only helped to procure scalable quantities of natural product but also provided ample scope to look for the diversity-oriented synthesis of its analogues.

Synthesis, photophysical properties, anti-Kasha photochemical reactivity and biological activity of vinyl- and alkynyl-BODIPY derivatives

New photoactivable BODIPY fluorescent dyes, which are quinone methide (QM) precursors, were synthesized. The series of BODIPY dyes has a photoreactive group linked to the BODIPY core by vinyl or ethynyl spacers. Molecules have bathochromic shifted absorption and emission maxima and higher fluorescence quantum yields compared to the previously reported BODIPY QM precursors. They show anti-Kasha photochemical reactivity and undergo photomethanolysis reaction upon photoexcitation at λ < 350 nm. Although UV light is needed for the photochemical generation of QMs and attachment to potential bio-targets, bathochromic shifted absorption and emission has advances in sensing and fluorescence labeling. Antiproliferative investigation was conducted for the vinyl derivative against three human cancer lines with the cells kept in dark or irradiated. Upon excitation with visible light antiproliferative activity was enhanced with IC50 values in low micromolar concentration.

Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents.

Combining the selectivity of G‐quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V‐shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.

Directed Crosslinking of RNA by Glutathione‐Triggered PNA‐Quinone‐Methide‐Conjugates

PNA conjugates of redox-labile quinone methide precursors can be activated by glutathione at concentrations typical for the cytosol. The quinone methide, an electrophilic short-lived intermediate, then reacts with nucleophiles. When the PNA part is hybridized with a complementary strand of RNA, directed alkylation of the nucleobases can form crosslinks in yields up to 71 %.

High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green (MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or non-catalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670 nm by restricting flexibility of dye. Integrating a phosphate-caged quinone methide precursor with MG yielded a covalent labeling fluorogenic probe, allowing real-time imaging of membrane alkaline phosphatase (ALP, a model catalytic protein) activity in live cells with over 100-fold enhancement of fluorescence intensity. Moreover, MG is also applicable to image non-catalytic protein by conjugation with protein-specific ligand. A fluorogenic probe consisted of c-RGDfK peptide and MG proved to be compatible with wash-free and real-time visualization of non-catalytic integrin αvβ3 in live cells with high contrast.

4+n] Annulation Reactions Using ortho-Chloromethyl Anilines as Aza-ortho-Quinone Methide Precursors

AbstractAza-ortho-quinone methides are important reactive intermediates that have found broad applications in synthetic chemistry. Recently, 1,4-elimination of ortho-chloromethyl aniline derivatives has emerged as a novel, powerful and convenient method for aza-ortho-quinone methide generation. This review will highlight the recent applications of aza-ortho-quinone methide precursors in annulation reactions to access various biologically important nitrogen-containing heterocycles. The general mechanisms are briefly discussed as well.1 Introduction2 [4+n] Annulation Reactions Using ortho-Chloromethyl Anilines as Aza-ortho-Quinone Methide Precursors2.1 [4+2] Annulation Reactions2.2 [4+1] Annulation Reactions2.3 [4+3] Annulation Reactions3 Conclusion and Perspective

Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity.

Quinone methide precursors 1a–e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10−2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10−5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105–106 M−1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M−1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a–e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

METHODS OF SYNTHESIS OF 2-AMINOCHROMANES

The review summarizes the existing methods of the synthesis of 2-aminochromanes. N,O-acetals in which the dihydropyran ring is spiroannulated or condensed with any carbo- or heterocyclic fragment at the b or c bond are not considered in the review because of the wide variety of methods for their preparation. The main attention is paid to (4+2) annulation processes with the participation of o-quinone methide precursors, salicylic aldehydes and their imino derivatives, as well as substitution reactions in chroman-2-ols and conjugated addition to electron-deficient 4 H -chromenes.

Methods of synthesis of 2-aminochromanes

The review summarizes the existing methods of the synthesis of 2-aminochromanes. N,O-acetals in which the dihydropyran ring is spiroannulated or condensed with any carbo- or heterocyclic fragment at the b or c bond are not considered in the review because of the wide variety of methods for their preparation. The main attention is paid to (4+2) annulation processes with the participation of o-quinone methide precursors, salicylic aldehydes and their imino derivatives, as well as substitution reactions in chroman-2-ols and conjugated addition to electron-deficient 4H-chromenes.