Reactive Michael Acceptor Research Articles

Microwave-Assisted Rearrangement of Costunolide Catalyzed by Palladium(II)

The structure of costunolide was modified before conducting the Cope rearrangement under thermal conditions. This modification was undertaken to block the highly reactive Michael acceptor α-methylene-γ-lactone, thus preventing side reactions at high temperatures. In this work, the reaction was performed under heating or microwave irradiation using costunolide, not its modified version. Rates of Cope reactions catalyzed by palladium(II) assisted by microwave were substantially enhanced. The rearrangements of costunolide to elemanolide, eudesmanolides, and germacranolide were catalyzed by palladium acetate, bis(benzonitrile)palladium dichloride, and a mixture of both organometallics. Rates of microwave promoted rearrangements of costunolide were six times faster than the reactions assisted by heating. The best success rate for elemanolide (7) was obtained using a mixture of Pd(OAc)2 and (NCPh)2PdCl2. In this case, compound 7 was obtained in a yield of 34%, while the other sesquiterpene lactones were formed only in trace amounts. The reaction products were isolated using semi-preparative high performance liquid chromatography and further purified through sequential analytical high performance liquid chromatography. Bioactivity profiles that used etiolated wheat coleoptiles were conducted for compounds 6, 7, and 9. The tested compounds presented very high phytotoxicity, with inhibition up to 97%.

Self-Assembled Lanthanide Antenna Glutathione Sensor for the Study of Immune Cells.

The small molecule 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves as a reactive non-fluorescent Michael acceptor, which after reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after self-assembling with this cation in water. This behavior makes 2b a highly selective GSH biosensor, which has demonstrated high potential for studies in murine and human cells of the immune system (CD4+ T, CD8+ T, and B cells) using flow cytometry. GSH can be monitored by the fluorescence of the product of addition to 2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was able to capture baseline differences in GSH intracellular levels among murine and human CD4+ T, CD8+ T, and B cells. We also successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of CD4+ naïve T cells into regulatory T cells (TREG).

Bioorthogonal Photocatalytic Decaging-Enabled Mitochondrial Proteomics.

Spatiotemporally resolved dissection of subcellular proteome is crucial to our understanding of cellular functions in health and disease. We herein report a bioorthogonal and photocatalytic decaging-enabled proximity labeling strategy (CAT-Prox) for spatiotemporally resolved mitochondrial proteome profiling in living cells. Our systematic survey of the photocatalysts has led to the identification of Ir(ppy)2bpy as a bioorthogonal and mitochondria-targeting catalyst that allowed photocontrolled, rapid rescue of azidobenzyl-caged quinone methide as a highly reactive Michael acceptor for proximity-based protein labeling in mitochondria of live cells. Upon careful validation through in vitro labeling, mitochondria-targeting specificity, in situ catalytic activity as well as protein tagging, we applied CAT-Prox for mitochondria proteome profiling in living Hela cells as well as hard-to-transfect macrophage RAW264.7 cells with approximately 70% mitochondria specificity observed from up to 300 proteins enriched. Finally, CAT-Prox was further applied to the dynamic dissection of mitochondria proteome of macrophage cells upon lipopolysaccharide stimulation. By integrating photocatalytic decaging chemistry with proximity-based protein labeling, CAT-Prox offers a general, catalytic, and nongenetic alternative to the enzyme-based proximity labeling strategies for diverse live cell settings.

Phospholane‐Phosphite Ligands for Rh Catalyzed Enantioselective Conjugate Addition: Unusually Reactive Catalysts for Challenging Couplings

The use of Rh catalysts derived from a phospholane‐phosphite ligand were found to be more productive than the classic rhodium/BINAP system in enantioselective conjugate additions. These catalysts enable the use of lower amounts of aryl boronic acid in an asymmetric arylation reaction that required an impractical excess of nucleophile. This catalyst was also found to enable the coupling of a poorly reactive Michael acceptor, N‐CBz‐2‐3‐dehydro‐4‐piperidone, or the coupling of poorly reactive 2‐furyl boronic acids at ambient or near temperatures.

Lewis Acid Promoted Trapping of Chiral Aza-enolates

We present a study on sequential conjugate addition of ­Grignard reagents to alkenyl-heteroarenes followed by trapping of the resulting enolates, yielding moderate to good diastereoselectivities. Contrary to conventional wisdom, one-pot conjugate addition/trapping using two reactive Michael acceptors in combination with Grignard reagents can proceed via conjugate addition to the least reactive Michael acceptor. This unusual chemoselectivity is triggered by the presence of a Lewis acid, reverting the usual reactivity order of Michael acceptors.

Cope elimination mediated by human FMO1 leads to formation of reactive Michael Acceptor intermediate

Cope elimination mediated by human FMO1 leads to formation of reactive Michael Acceptor intermediate

Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review).

Targeted cancer therapies are used to inhibit the growth, progression, and metastasis of the tumor by interfering with specific molecular targets and are currently the focus of anticancer drug development. Protein kinase B, also known as Akt, plays a central role in many types of cancer and has been validated as a therapeutic target nearly two decades ago. This review summarizes the intracellular functions of Akt as a pivotal point of converging signaling pathways involved in cell growth, proliferation, apoptotis and neo-angiogenesis, and focuses on the drug design strategies to develop potent anticancer agents targeting Akt. The discovery process of Akt inhibitors has evolved from adenosine triphosphate (ATP)-competitive agents to alternative approaches employing allosteric sites in order to overcome the high degree of structural similarity between Akt isoforms in the catalytic domain, and considerable structural analogy to the AGC kinase family. This process has led to the discovery of inhibitors with greater specificity, reduced side-effects and lower toxicity. A second generation of Akt has inhibitors emerged by incorporating a chemically reactive Michael acceptor template to target the nucleophile cysteines in the catalytic activation loop. The review outlines the development of several promising drug candidates emphasizing the importance of each chemical scaffold. We explore the pipeline of Akt inhibitors and their preclinical and clinical examination status, presenting the potential clinical application of these agents as a monotherapy or in combination with ionizing radiation, other targeted therapies, or chemotherapy.

Design and synthesis of new non nucleoside inhibitors of DNMT3A

DNA methylation, an epigenetic modification regulating gene expression, is a promising target in cancer. In an effort to identify new non nucleosidic inhibitors of DNA methyltransferases, the enzymes responsible for DNA methylation, we carried out a high-throughput screening of 66,000 chemical compounds based on an enzymatic assay against catalytic DNMT3A. A family of propiophenone derivatives was identified. After chemical optimization and structure activity relationship studies, a new inhibitor (33) was obtained with an EC50 of 2.1μM against DNMT3A. The mechanism of inhibition of the compound was investigated as it forms a reactive Michael acceptor group in situ. Thereby, the Michael acceptor 20 was identified. This compound was further characterized for its biological activity in cancer cells.

New Monocyclic, Bicyclic, and Tricyclic Ethynylcyanodienones as Activators of the Keap1/Nrf2/ARE Pathway and Inhibitors of Inducible Nitric Oxide Synthase.

A monocyclic compound 3 (3-ethynyl-3-methyl-6-oxocyclohexa-1,4-dienecarbonitrile) is a highly reactive Michael acceptor leading to reversible adducts with nucleophiles, which displays equal or greater potency than the pentacyclic triterpenoid CDDO in inflammation and carcinogenesis related assays. Recently, reversible covalent drugs, which bind with protein targets but not permanently, have been gaining attention because of their unique features. To explore such reversible covalent drugs, we have synthesized monocyclic, bicyclic, and tricyclic compounds containing 3 as an electrophilic fragment and evaluated them as activators of the Keap1/Nrf2/ARE pathway and inhibitors of iNOS. Notably, these compounds maintain the unique features of the chemical reactivity and biological potency of 3. Among them, a monocyclic compound 5 is the most potent in these assays while a tricyclic compound 14 displays a more robust and specific activation profile compared to 5. In conclusion, we demonstrate that 3 is a useful electrophilic fragment for exploring reversible covalent drugs.

Michael acceptor in gambogic acid--Its role and application for potent antitumor agents.

Gambogic acid (GA), a natural product with unique structure, was reported to have broad antiproliferation activities against cancer cell lines. As a reactive Michael acceptor, the 10-position of GA is susceptible to nucleophiles, thus limiting its clinical application as an anticancer agent. Moreover, the 6-OH forms an intramolecular hydrogen bond with 8-CO, which can make the 9, 10 double bond more reactive to nucleophiles. In this essay, two strategies (A and B) were applied to solve the above-mentioned problems. Strategy A was to increase the steric hindrance of C-10 to reduce the activity of GA towards nucleophiles. Strategy B was to replace the hydroxyl of C-6 with other substituents based on the assumption that the intra-molecular hydrogen bond could increase the electrophilicity of C-10. Results showed the electrophilicity of C-10 disappeared as well as the antiproliferation activity against cancer cell lines by introducing a methyl group at C-10. Strategy B showed that the electrophilicity of C-10 was reduced dramatically while maintained the activity by replacement of the hydroxyl of C-6 with neutral or basic groups.

ChemInform Abstract: Cascade Michael Addition/Cycloketalization of Cyclic 1,3‐Dicarbonyl Compounds: Important Role of the Tethered Alcohol of α,β‐Unsaturated Carbonyl Compounds on Reaction Rate and Regioselectivity.

Reactions of α,β-unsaturated aldehydes and cyclic 1,3-dicarbonyl compounds proceed primarily by cascade Knoevenagel condensation/six-π-electron electrocyclization (K6EC, formal [3 + 3] cycloaddition), while α,β-unsaturated ketones usually react with cyclic 1,3-dicarbonyl compounds in a 1,4-addition manner. This paper discloses our findings that under acidic conditions, α,β-unsaturated carbonyl compounds (ketones and aldehydes) with a tethered alcohol react with cyclic 1,3-dicarbonyl compounds in a highly regioselective 1,4-addition fashion via in situ generation of a hypothetical α-methylene cyclic oxonium ion as the reactive Michael acceptor. Our studies uncovered the important effect of the tethered alcohol on the reaction rate and/or efficiency and some new mechanistic aspects of the cascade Michael addition/cycloketalization. Finally, the substrate scope was examined, and 43 analogues of penicipyrone and tenuipyrone were prepared in good to excellent yields.

Electrophilic warhead-based design of compounds preventing NLRP3 inflammasome-dependent pyroptosis.

Pyroptosis is a caspase-1-dependent pro-inflammatory form of programmed cell death implicated in the pathogenesis of autoinflammatory diseases as well as in disorders characterized by excessive cell death and inflammation. Activation of NLRP3 inflammasome is a key event in the pyroptotic cascade. In this study, we describe the synthesis and chemical tuning of α,β-unsaturated electrophilic warheads toward the development of antipyroptotic compounds. Their pharmacological evaluation and structure-activity relationships are also described. Compound 9 was selected as a model of this series, and it proved to be a reactive Michael acceptor, irreversibly trapping thiol nucleophiles, which prevented both ATP- and nigericin-triggered pyroptosis of human THP-1 cells in a time- and concentration-dependent manner. Moreover, 9 and other structurally related compounds, inhibited caspase-1 and NLRP3 ATPase activities. Our findings can contribute to the development of covalent, multitarget antipyroptotic compounds targeting molecular components of the NLRP3 inflammasome regulatory pathway.

Design, synthesis and biological evaluation of FLT3 covalent inhibitors with a resorcylic acid core

A series of simplified ring-opened resorcylic acid lactone (RAL) derivatives were conveniently synthesized to target FLT3 and its mutants either irreversibly or reversibly. Our design of covalent FLT3 inhibitors is based on cis-enone RALs (e.g., L-783,277) that have a β-resorcylic acid as the core structure. The designed compounds contain three types of Michael acceptors (acrylamide, vinylsulfonamide and maleimide) as potential covalent traps of a cysteine residue at the binding site of kinases. A variety of functional substitutions were also introduced to maximize the binding interactions. Biological evaluations revealed that compound 17, despite the presence of a highly reactive maleimide Michael acceptor, is a potent covalent FLT3 inhibitor which shows some specificity in cellular assays. On the other hand, compounds 2 and 6 containing acrylamide or vinylsulfonamide groups are reversible towards FLT3 binding, and are potent and selective inhibitors of mutant FLT3-ITD versus wt-FLT3. They also inhibit cell proliferation in FLT3-ITD expressing cell line MV-4-11 as compared to wt-FLT3 expressing cell line THP-1 and non-FLT3 cell lines (K562, HL60 and Hek-293T).

A simple and widely applicable hit validation strategy for protein–protein interaction inhibitors based on a quantitative ligand displacement assay

Identification of inhibitors for protein–protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.

Cascade Michael addition/cycloketalization of cyclic 1,3-dicarbonyl compounds: important role of the tethered alcohol of α,β-unsaturated carbonyl compounds on reaction rate and regioselectivity.

Reactions of α,β-unsaturated aldehydes and cyclic 1,3-dicarbonyl compounds proceed primarily by cascade Knoevenagel condensation/six-π-electron electrocyclization (K6EC, formal [3 + 3] cycloaddition), while α,β-unsaturated ketones usually react with cyclic 1,3-dicarbonyl compounds in a 1,4-addition manner. This paper discloses our findings that under acidic conditions, α,β-unsaturated carbonyl compounds (ketones and aldehydes) with a tethered alcohol react with cyclic 1,3-dicarbonyl compounds in a highly regioselective 1,4-addition fashion via in situ generation of a hypothetical α-methylene cyclic oxonium ion as the reactive Michael acceptor. Our studies uncovered the important effect of the tethered alcohol on the reaction rate and/or efficiency and some new mechanistic aspects of the cascade Michael addition/cycloketalization. Finally, the substrate scope was examined, and 43 analogues of penicipyrone and tenuipyrone were prepared in good to excellent yields.

Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor

The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

Kinetics and molecular dynamic modeling of a potent and selective marine natural product inhibitor of human cathepsin L (975.3)

Protease inhibitors are an important drug target class due to their wide and varied roles in both physiological and pathological states. In particular, the human cysteine cathepsin proteases have been implicated in the progression and metastasis of cancer, participation in the development of neurodegenerative diseases, and are important processing enzymes for the production of multiple neuropeptides. Past marine natural products screening efforts have successfully yielded potent and selective inhibitors of proteases, hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Gallinamide A, a depsipeptide secondary metabolite isolated from a collection of filamentous marine cyanobacteria, was identified as a hit structure with potent activity. With 30 minutes of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9009 ± 262 M‐1 s‐1. A preincubation‐dilution experiment revealed an irreversible mode of inhibition, and comparative IC50 values display a 28‐ to 331‐ fold greater selectivity toward cathepsin L than closely related human cysteine cathepsins V or B. Gallinamide A shows no inhibitory activity against the exopeptidase cathepsin H, further exemplifying its selectivity. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enone functional group of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L.Grant Funding Source: Supported by NIH T32GM007752, NIH FIC U01 TW006634, and NIH R01MH077305

The Marine Cyanobacterial Metabolite Gallinamide A Is a Potent and Selective Inhibitor of Human Cathepsin L

A number of marine natural products are potent inhibitors of proteases, an important drug target class in human diseases. Hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Herein, we have shown that gallinamide A potently and selectively inhibits the human cysteine protease cathepsin L. With 30 min of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9000 ± 260 M(-1) s(-1). Preincubation-dilution and activity-probe experiments revealed an irreversible mode of inhibition, and comparative IC50 values display a 28- to 320-fold greater selectivity toward cathepsin L than closely related human cysteine cathepsin V or B. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enamide of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L.

Ralfuranone Thioether Production by the Plant Pathogen Ralstonia solanacearum

Ralfuranones are aryl-substituted furanone secondary metabolites of the Gram-negative plant pathogen Ralstonia solanacearum. New sulfur-containing ralfuranone derivatives were identified, including the methyl thioether-containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione.

NHC Organocatalytic Formal LUMO Activation of α,β‐Unsaturated Esters for Reaction with Enamides

An effective wake-up call: Stable α,β-unsaturated esters were activated by the addition of a chiral N-heterocyclic carbene (NHC) organocatalyst, and the resulting reactive Michael acceptor intermediates reacted with enamide nucleophiles to furnish optically pure products (see scheme; Ts=p-toluenesulfonyl). These products can be converted readily into bioactive δ-lactams, piperidines, and their derivatives. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.