Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase.

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.

Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T790M) EGFR Mutants for the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers.

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

5-oxo-15-HETE: total synthesis and bioactivity.

The first total synthesis of 6(E),8(Z),11(Z),13(E) 5-oxo-15-HETE 4 was accomplished. The synthetic material was evaluated with calcium mobilization assay and compared with 5-oxo-ETE the natural ligand for the OXE receptor.

Substrate selectivity of 5-hydroxyeicosanoid dehydrogenase and its inhibition by 5-hydroxy-Delta6-long-chain fatty acids.

5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of the 5-lipoxygenase (5-LO) product 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), formed by the microsomal enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH). 5-oxo-ETE is a chemoattractant for neutrophils and eosinophils, both in vitro and in vivo. To examine the substrate selectivity of 5-HEDH and to search for potential inhibitors, we prepared a series of 5S-hydroxy fatty acids (C(12) to C(20) containing zero to four double bonds) by total chemical synthesis and examined their metabolism by microsomes from monocytic U937 cells. Although most of these fatty acids were oxidized to their 5-oxo metabolites by 5-HEDH, 5-HETE seemed to be the best substrate. However, substrates containing less than 16 carbons, a methylated alpha-carboxyl group, or a hydroxyl group at the omega-end of the molecule were not substantially metabolized. Some of the fatty acids tested were fairly potent inhibitors of the formation of 5-oxo-ETE by 5-HEDH, in particular 5-hydroxy-6-octadecenoic acid and 5-hydroxy-6-eicosenoic acid. Both substances selectively inhibited 5-oxo-ETE formation by human peripheral blood mononuclear cells incubated with arachidonic acid and calcium ionophore without affecting the formation of leukotriene B(4), 12-HETE, or 12-hydroxy-5,8,10-heptadecatrienoic acid. We conclude that the requirements for appreciable metabolism by 5-HEDH include a chain length of at least 16 carbons, a free alpha-carboxyl group, and a hydrophobic group at the omega-end of the molecule. 5-Hydroxy-Delta(6) C(18) and C(20) fatty acids selectively inhibit 5-HEDH without inhibiting 5-LO, leukotriene A(4) hydrolase, 12-lipoxygenase, or cyclooxygenase. Such compounds may be useful in defining the role of 5-oxo-ETE and its mechanism of synthesis.

Structural requirements for activation of the 5-oxo-6E,8Z, 11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor: identification of a mead acid metabolite with potent agonist activity.

The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a potent chemoattractant for neutrophils and eosinophils, and its actions are mediated by the oxoeicosanoid (OXE) receptor, a member of the G protein-coupled receptor family. To define the requirements for activation of the OXE receptor, we have synthesized a series of 5-oxo-6E,8Z-dienoic acids with chain lengths between 12 and 20 carbons, as well as a series of 20-carbon 5-oxo fatty acids, either fully saturated or containing between one and five double bonds. The effects of these compounds on neutrophils (calcium mobilization, CD11b expression, and cell migration) and eosinophils (actin polymerization) were compared with those of 5-oxo-ETE. The C12 and C14 analogs were without appreciable activity, whereas the C16 5-oxo-dienoic acid was a weak partial agonist. In contrast, the corresponding C18 analog (5-oxo-18:2) was nearly as potent as 5-oxo-ETE. Among the C20 analogs, the fully saturated compound had virtually no activity, whereas 5-oxo-6E-eicosenoic acid had only weak agonist activity. In contrast, 5-oxo-6E,8Z,11Z-eicosatrienoic acid (5-oxo-20:3) and its 8-trans isomer were approximately equipotent with 5-oxo-ETE in activating granulocytes. Because of the potent effects of 5-oxo-20:3, we investigated its formation from Mead acid (5Z,8Z,11Z-eicosatrienoic acid), which accumulates in dietary essential fatty acid deficiency, by neutrophils. The main Mead acid metabolite identified was 5-hydroxy-6,8,11-eicosatrienoic acid, followed by 5-oxo-20:3 and two 6-trans isomers of leukotriene B(3). We conclude that optimal activation of the OXE receptor is achieved with 5-oxo-ETE, 5-oxo-18:2, and 5-oxo-20:3, and that the latter compound could potentially be formed under conditions of essential fatty acid deficiency.

Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant.

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.

TiCl4 catalyzed tandem construction of C-C and C-O bonds: a simple and one-pot atom-economical stereoselective synthesis of spiro-oxindoles.

An atom-economical stereoselective synthesis of [{1-acetyl-5-methyl-6,8-dioxabicyclo(3.2.1)octane}-7-spiro-3'-(indolin-2'-one)] derivatives, containing both the oxindole and 6,8-dioxabicyclo(3.2.1)octane moieties via TiCl(4) catalyzed coupling of 2-acetyl-6-methyl-2,3-dihydro-4H-pyran with isatin derivatives is described.

Steric factors direct Baylis-Hillman and aldol reactions in titanium tetrachloride mediated coupling between alpha-keto esters and cyclohex-2-enone derivatives.

The titanium tetrachloride mediated reaction of alpha-keto esters with 5,5-dimethylcyclohex-2-enone provides the corresponding Baylis-Hillman adducts exclusively whereas a similar reaction of alpha-keto esters with cyclohex-2-enone furnishes the corresponding aldol adducts (with high syn-diasteroeselectivity) as the major product (along with the Baylis-Hillman adducts as the minor product), thus clearly demonstrating the role of the steric factors in directing the reaction pathway.

First example of electrophile induced Baylis-Hillman reaction: a novel facile one-pot synthesis of indolizine derivatives.

Treatment of pyridine-2-carboxaldehyde with activated alkenes such as alkyl vinyl ketones and cyclic enones in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) provides a novel facile one-pot synthesis of indolizine derivatives, thus for the first time describing an electrophile induced Baylis-Hillman reaction.