Electrophilic Agents Research Articles

An improved, scalable synthesis of Notum inhibitor LP-922056 using 1-chloro-1,2-benziodoxol-3-one as a superior electrophilic chlorinating agent.

Background: The carboxylesterase Notum has been shown to act as a key negative regulator of the Wnt signalling pathway by mediating the depalmitoleoylation of Wnt proteins. LP-922056 (1) is an orally active inhibitor of Notum. We are investigating the role of Notum in modulating Wnt signalling in the central nervous system and wished to establish if 1 would serve as a peripherally restricted control. An accessible and improved synthetic route would allow 1 to become more readily available as a chemical tool to explore the fundamental biology of Notum and build target validation to underpin new drug discovery programs.Results: An improved, scalable synthesis of 1 is reported. Key modifications include: (1) the introduction of the C7-cyclopropyl group was most effectively achieved with a Suzuki–Miyaura cross-coupling reaction with MIDA-boronate 11 (5 → 6), and (2) C6 chlorination was performed with 1-chloro-1,2-benziodoxol-3-one (12) (6 → 7) as a mild and selective electrophilic chlorination agent. This 7-step route from 16 has been reliably performed on large scale to produce multigram quantities of 1 in good efficiency and high purity. Pharmacokinetic studies in mouse showed CNS penetration of 1 is very low with a brain/plasma concentration ratio of just 0.01. A small library of amides 17 were prepared from acid 1 to explore if 1 could be modified to deliver a CNS penetrant tool by capping off the acid as an amide. Although significant Notum inhibition activity could be achieved, none of these amides demonstrated the required combination of metabolic stability along with cell permeability without evidence of P-gp mediated efflux.Conclusion: Mouse pharmacokinetic studies demonstrate that 1 is unsuitable for use in models of disease where brain penetration is an essential requirement of the compound but would be an ideal peripherally restricted control. These data will contribute to the understanding of drug levels of 1 to overlay with appropriate in vivo efficacy endpoints, i.e., the PK-PD relationship. The identification of a suitable analogue of 1 (or 17) which combines Notum inhibition with CNS penetration would be a valuable chemical probe for investigating the role of Notum in disease models.

Ultrasound-assisted fabrication of N-cyano-N-arylbenzenesulfonamides at ambient temperature: improvements with biosynthesized Ag/feldspar nanocomposite

A mild and effective method for ultrasound-assisted N-sulfonylation of arylcyanamides with aryl sulfonyl chloride is introduced at ambient temperature; synthesis of assorted derivatives of N-cyano-N-arylbenzenesulfonamide as electrophilic cyanation agent can be further improved using Ag/feldspar nanocomposite, accessible via an simple, eco-friendly and inexpensive method using Hedera helix leaf extract. The important advantages of this method include clean conditions, high purity and yield, and easy workup procedure with minimum generation of waste. The structure and morphology nanocatalyst were characterized by energy-dispersive X-ray spectroscopy, field emission scanning electron microscope, transmission electron microscopy, X-ray powder diffraction and elemental mapping.

TEMPO-Me: An Electrochemically Activated Methylating Agent.

Bench- and air-stable 1-methoxy-2,2,6,6-tetramethylpiperidine (TEMPO-Me) is relatively unreactive at ambient temperature in the absence of an electrochemical stimulus. In this report, we demonstrate that the one-electron electrochemical oxidation of TEMPO-Me produces a powerful electrophilic methylating agent in situ. Our computational and experimental studies are consistent with methylation proceeding via a SN2 mechanism, with a strength comparable to the trimethyloxonium cation. A protocol is developed for the electrochemical methylation of aromatic acids using TEMPO-Me.

Decarboxylation and electrophilic substitution in 3-tert-butyl-4-oxopyrazolo[5,1-c][1,2,4]triazines

Reactivity of 7-R-3-tert-butyl-4-oxo-4,6-dihydropyrazolo[5,1-c][1,2,4]triazine-8-carboxylic acids (R = NH2, N3, H) and products of their decarboxylation towards different electrophilic agents (chloroacetone, Boc2O/NaN3, DMF/POCl3, TMSBr, N-halosuccinimides, and HNO2) was studied. A selective synthesis of new C(7),C(8)-functionalized and N(1)-tert-butyloxycarbonyl-substituted 4-oxopyrazolo[5,1-c][1,2,4]triazines was performed. The structures of the synthesized compounds were confirmed by IR and NMR spectroscopy, high resolution mass spectrometry and single crystal X-ray diffraction analysis.

A new version of Umemoto’s reagents: A three-step one-pot preparation of 2,3,7,8-tetrafluoro-S-(trifluoromethyl)dibenzothiophenium triflate

A new one-pot method was developed for the practical preparation of powerful electrophilic trifluoromethylating agent, 2,3,7,8-tetrafluoro-S-(trifluoromethyl)dibenzothiophenium triflate 2. This method comprised of three steps; (1) in situ generation of trifluoromethanesulfinyl trifluoroacetate 7, (2) treatment of 3,3′,4,4′-tetrafluorobiphenyl with 7 in the presence of trifluoromethanesulfonic acid and trifluoroacetic anhydride, and (3) treatment of the resulting mixture with hydrogen peroxide. By means of this effective three-step one-pot method, 2 was prepared in a good isolated yield by simple water-washing method. It is thus expected that easy access to 2 may lead to its wider or new applications to many areas, because tetrafluoro reagent 2 is much more powerful than 2,8-difluoro-S-(trifluoromethyl)dibenzothiophenium triflate 1.

A Very Strong Methylation Agent: [Me2Cl][Al(OTeF5)4

A new chloronium-containing salt, [Me2 Cl][Al(OTeF5 )4 ], was synthesized on multigram scale by means of a simple one-pot procedure. The isolated product can be handled at room temperature and used as a strong electrophilic methylation agent. This is demonstrated by the methylation of the very weak bases P(CF3 )3 , PF3 , MeI, and MeBr.

Functionalized Nanomaterial Assembling and Biosynthesis Using the Extremophile Deinococcus radiodurans for Multifunctional Applications.

The development of functionalized nanomaterial biosynthesis processes is important to expand many cutting-edge nanomaterial application areas. However, unclear synthesis mechanisms and low synthesis efficiency under various chemical stresses have limited the use of these biomaterials. Deinococcus radiodurans is an extreme bacterium well known for its exceptional resistance to radiation oxidants and electrophilic agents. This extremophile, which possesses a spontaneous self-assembled surface-layer (S-layer), has been an optimal model organism to study microbial nanomaterial biotemplates and biosynthesis under various stresses. This review summarizes the S-layers from D. radiodurans as an excellent biotemplate for various pre-synthesized nanomaterials and multiple applications, and highlights recent progresses about the biosynthesis of functionalized gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), as well as gold and silver bimetallic nanoparticles using D. radiodurans. Their formation mechanisms, properties, and applications are discussed and summarized to provide significant insights into the design or modification of functionalized nanomaterials via natural materials. Grand challenges and future directions to realize the multifunctional applications of these nanomaterials are highlighted for a better understanding of their biosynthesis mechanisms and functionalized modifications.

Studies on predicting reactive sites of 3,9-diazatetraasteranes by conceptual density functional theory and experiment

Theoretical studies on predicting potential reactive sites of attractive compounds are of significance, which are capable of providing deeper insight into their chemical behaviors as well as foundations for discovering new drug molecules. The 3,9-diazatetraasterane derivatives are a kind of cage compounds equipping with potent biological activities. In this paper, predication of reactive sites of 3,9-diazatetraasteranes bearing phenyl and ester groups (6,12-diphenyl-3,9-diazahexacyclo[6.4.0.02.7.04.11.05.10]-dodecane-1,5,7,11-tetracarboxylate) was carried out by the conceptual density functional theory. Firstly, a conformational search of 3,9-diazatetraasterane was performed by molecular dynamics simulations associating with Quantum Mechanics calculations in order to obtain relative stable conformers. And then the reactive site prediction of 3,9-diazatetraasterane was carried out by the Fukui function and condensed Fukui function. The results exhibited that the potentially chemical reactive site is the N atom of 3,9-diazatetraasterane scaffold, which is readily attacked by electrophilic agent. Meanwhile, the calculated results were validated by experimental outcomes in the derivation of 3,9-diazatetraasteranes.

YpdA, a putative bacillithiol disulfide reductase, contributes to cellular redox homeostasis and virulence in Staphylococcus aureus.

The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.

Technology and Properties of Composite Materials with Modifier of Chloride Aluminum

The paper discusses the important issues concerning recycling of industrial sulfur waste and bottom ash. The process flow diagram was designed to form protective coatings on cement concrete by impregnation with molten sulfur, which featured high strength, performance properties and corrosion resistance. The process parameters were specified. The addition of bottom ash slag to cement concrete not only offers a means for recovering waste from thermal power stations, but also increases the material strength. Besides, the impregnation process provides low thermal conductivity for concrete while giving the material high heat insulation. A higher strength of the samples containing bottom ash slag results from a high dispersity and reactive surface of bottom ash slag. The paper investigates the influence of the electrophilic agent (aluminum chloride) on the properties of sulfur binder. It was shown that addition of aluminum chloride encouraged the formation of a more effective protective coating that gave the samples of cement concrete higher strength, density, water resistance. The electrophilic agent stimulated the synthesis process for inorganic sulfides and composite materials based on them. It was found that addition of the electrophilic agent (aluminum chloride) reduced the viscosity of molten sulfur significantly, which was attributed to the formed short-chain sulfur radicals. Such a melt has a relatively high penetration capability to ensure the formation of a high-quality protective coating.

Self-immolative systems for the disclosure of reactive electrophilic alkylating agents.

In this paper we report the design, synthesis and assessment of the first examples of self-immolative systems triggered by non-acidic electrophilic agents such as methyl, allyl or benzylic halides. These systems provide a visual colorimetric disclosure response upon exposure to these electrophilic reagents under mild, basic conditions without the need for the use of analytical instrumentation.

Convenient Synthesis of Ethyl 5-Oxohomoadamantane-4-carboxylate: A Useful Precursor of Polyfunctional Homoadamantanes

A facile and convenient synthesis of ethyl 5-oxohomoadamantane-4-carboxylate is reported, and its chemical properties as a cage analogue of acetoacetic ester are investigated. Various derivatives of homoadamantane were synthesized through the reaction of 5-oxohomoadamantane-4-carboxylate with electrophilic agents, binucleophiles, and hydrazoic acid. Some new unusual products were obtained by the reaction of that β-keto ester with nitric acid and nitrosyl chloride. Cage compounds synthesized could be used as precursors for the diverse condensed heterocyclic compounds with potential viral ion channel abrogating activity that possess conformationally rigid lipophilic moieties.

Molecular Pathways Associated With Methylmercury-Induced Nrf2 Modulation.

Methylmercury (MeHg) is a potent neurotoxin that affects particularly the developing brain. Since MeHg is a potent electrophilic agent, a wide range of intracellular effects occur in response to its exposure. Yet, the molecular mechanisms associated with MeHg-induced cell toxicity have yet to be fully understood. Activation of cell defense mechanisms in response to metal exposure, including the up-regulation of Nrf2- (nuclear factor erythroid 2-related factor 2)-related genes has been previously shown. Nrf2 is a key regulator of cellular defenses against oxidative, electrophilic and environmental stress, regulating the expression of antioxidant proteins, phase-II xenobiotic detoxifying enzymes as well phase-III xenobiotic transporters. Analogous to other electrophiles, MeHg activates Nrf2 through modification of its repressor Keap1 (Kelch-like ECH-associated protein 1). However, recent findings have also revealed that Keap1-independent signal pathways might contribute to MeHg-induced Nrf2 activation and cytoprotective responses against MeHg exposure. These include, Akt phosphorylation (Akt/GSK-3β/Fyn-mediated Nrf2 activation pathway), activation of the PTEN/Akt/CREB pathway and MAPK-induced autophagy and p62 expression. In this review, we summarize the state-of-the-art knowledge regarding Nrf2 up-regulation in response to MeHg exposure, highlighting the modulation of signaling pathways related to Nrf2 activation. The study of these mechanisms is important in evaluating MeHg toxicity in humans, and can contribute to the identification of the molecular mechanisms associated with MeHg exposure.

An Empirical Understanding of the Glycosylation Reaction.

Reliable glycosylation reactions that allow for the stereo- and regioselective installation of glycosidic linkages are paramount to the chemical synthesis of glycan chains. The stereoselectivity of glycosylations is exceedingly difficult to control due to the reaction's high degree of sensitivity and its shifting, simultaneous mechanistic pathways that are controlled by variables of unknown degree of influence, dominance, or interdependency. An automated platform was devised to quickly, reproducibly, and systematically screen glycosylations and thereby address this fundamental problem. Thirteen variables were investigated in as isolated a manner as possible, to identify and quantify inherent preferences of electrophilic glycosylating agents (glycosyl donors) and nucleophiles (glycosyl acceptors). Ways to enhance, suppress, or even override these preferences using judicious environmental conditions were discovered. Glycosylations involving two specific partners can be tuned to produce either 11:1 selectivity of one stereoisomer or 9:1 of the other by merely changing the reaction conditions.

ELECTROPHYLIC HETEROCYCLIZATION OF 5-METHYL-6-TIOXOPYRAZOLO[3,4-d]PYRIMIDINE-4-ON

Derivatives of pyrazolo[3,4-d]pyrimidine have valuable biological activity. The structural fragment of pyrazolopyrimidines is widely used to develop new effective pharmaceuticals. A new promising direction in the construction of polycyclic systems based on pyrazolopyrimidine is the method of electrophilic intramolecular heterocyclicization.The halogenation of 5-methyl-6-thioxopyrazolo[3,4-d]pyrimidin-4-one was carried out in acetic acid medium at room temperature with twice the excess of halogen. It was found that halogenation (with bromine and iodine) of 5-methyl-6-thioxopirazolo[3,4-d]pyrimidin-4-one results the annelation of the thiazolinic cycle with the formation of linear polynuclear systems of a salt-like structure. 7-Halogenomethyl-7-methyl-1,4,6,7-tetrahydropyrazolo[3,4-d][1,3]thiazolo[3,2-a]pyrimidin-4-one was obtained via the action of sodium sulfite in the DMSO-water medium.To study the effect of the nature of the electrophilic reagent on the direction of the electrophilic intramolecular cyclization of 5-methyl-6-thioxopyrazolo[3,4-d]pyrimidin-4-one the p-methoxyphenyltetrachloride was used (tested). Electrophilic heterocyclization take place under similar conditions (room temperature, glacial acetic acid medium), with the formation of the tricyclic system with an exocyclic aryltellurium moiety – 7-(dichloro-(4-methoxyphenyl) telluromethyl)-7-methyl-1,4,6,7-tetrahydropyrazolo[3,4-d][1,3]thiazolo[3,2-a]pyrimidin-4-one. Thus, the direction of electrophilic intramolecular cyclization of 5-methyl-6-thioxopyrazolo[3,4-d]pyrimidine-4-one using as electrophilic agents of halogens (bromine, iodine) and p-methoxyphenyltetrachloride does not depend on the nature of the electrophile and takes place with the participation of the exocyclic sulfur atom in the second position of the pyrimidine and lead to the formation of a condensed system of linear structure – halogenomethyl or aryl telluromethyl derivatives of pyrazolo[3,4-d][1,3]thiazolo[3,2-a]pyrimidin-4-one. The composition and structure of all synthesized compounds were proved by elemental analysis and 1H-NMR spectra.

Differential effects of lipopolysaccharide on mouse sensory TRP channels

Acute neurogenic inflammation and pain associated to bacterial infection have been traditionally ascribed to sensitization and activation of sensory nerve afferents secondary to immune cell stimulation. However, we recently showed that lipopolysaccharides (LPS) directly activate the Transient Receptor Potential channels TRPA1 in sensory neurons and TRPV4 in airway epithelial cells. Here we investigated whether LPS activates other sensory TRP channels expressed in sensory neurons. Using intracellular Ca2+ imaging and patch-clamp we determined the effects of LPS on recombinant TRPV1, TRPV2, TRPM3 and TRPM8, heterologously expressed in HEK293T cells. We found that LPS activates TRPV1, although with lower potency than for TRPA1. Activation of TRPV1 by LPS was not affected by mutations of residues required for activation by electrophilic agents or by diacylglycerol and capsaicin. On the other hand, LPS weakly activated TRPM3, activated TRPM8 at 25 °C, but not at 35 °C, and was ineffective on TRPV2. Experiments performed in mouse dorsal root ganglion (DRG) neurons revealed that genetic ablation of Trpa1 did not abolish the responses to LPS, but remain detected in 30% of capsaicin-sensitive cells. The population of neurons responding to LPS was dramatically lower in double Trpa1/Trpv1 KO neurons. Our results show that, in addition to TRPA1, other TRP channels in sensory neurons can be targets of LPS, suggesting that they may contribute to trigger and regulate innate defenses against gram-negative bacterial infections.

Evaluation of Starting Materials for PMIs (Potentially Mutagenic Impurities): A Vortioxetine Case Study

The nature of the starting material (SM) and the presence of (potentially) mutagenic impurities (PMIs) can correlate strongly, since many syntheses involve the use of potentially mutagenic electrophilic (alkylating) agents as SMs. Since the regulatory guidelines are far from clear and straightforward, selection of the appropriate SMs is a very challenging task for pharmaceutical companies. Here, the principal criteria for the selection of SMs have been identified based on the existing guidelines. Three SMs in the synthesis of vortioxetine drug substance were selected and justified on the basis of their incorporation into the structure of the API, on whether they have defined chemical properties and structure or not, on the number of synthetic steps between the SM and the API, on commercial availability and formation of impurities, and their control. Determination of a theoretical purge factor (TPF) was successful in evaluating the risk of using potentially mutagenic SM 1 and SM 3, with their related impur...

N6-tert-Butoxycarbonyl derivatives of sydnone imines: Preparation and synthetic use

A highly efficient gram-scale method for the C(4)-functionalization of sydnone imine hydrochloride derivatives was developed. This new approach was realized through consecutive reactions including the preparation of a C(3)-substituted N6-tert-butoxycarbonyl sydnone imine derivative, deprotonation with n-BuLi and reaction of the lithiated-intermediate with an electrophilic agent, and removal of the Boc protecting group.

Abstract LB-B17: Characterization of small molecule inhibitors of the Nrf2-Keap1 interaction using MicroScale Thermophoresis

Abstract The molecular complex between Keap 1 (Kelch-like ECH-associated protein) and Nrf2 (nuclear factor erythroid 2-related factor 2) plays a major role in the regulation of cyto-protective responses to oxidative stress and electrophilic agents. The Keap1-Nrf2 pathway has been established as a therapeutic target for oxidative stress-related conditions including inflammatory, cardiovascular, neurodegenerative diseases and cancer. The European Lead Factory (ELF) is a major European project generating new lead structures for drug discovery programs in the public and private sectors. This is achieved by screening molecular targets against the Joint European Compound Library (JECL) combining compounds contributed by participating pharmaceutical companies or synthesised by chemistry SMEs. Target proposals are submitted to the ELF by European academics and SMEs and selected programs are screened against the library within the European Screening Centre. Hit compounds are provided to the program owner who gains exclusive rights to exploit them for drug discovery or as novel pharmacological tools. The aim of this project was to identify non-electrophilic inhibitors of the Keap1-Nrf2 interaction. Protein-protein interaction (PPI) targets are typically difficult to drug due to large, often quite shallow interaction surfaces between proteins, which along with other factors is associated with PPI screens often returning a high proportion of false positives. Therefore, designing a high-throughput screening (HTS) triage process that confirms target engagement of hits is essential, ideally involving the use of orthogonal biophysical assays. Here we present the successful development of a label-free MicroScale Thermophoresis (MST) assay to identify inhibitors of the Keap1-Nrf2 interaction. 318,132 compounds from the JECL were tested in a fluorescence polarization assay using a fluorescein-labelled Nrf2 peptide mimic. The HTS campaign yielded a hit rate of 0.4% and many false positives compounds were deselected due to fluorescence interference or redox reactivity. Following analytical assessment, visual inspection and legal clearance, 8 compounds were selected for further characterization. Assay development indicated that label-free MST was a suitable platform to confirm target engagement prior to investing in a Chemistry program. Two structurally related hits showed a concentration-dependent binding response to the full-length Keap1 protein in MST, which was abolished after denaturation of the Keap1 protein. These compounds were competitive with one another and with an unlabeled Nrf2 peptide mimic confirming that they were able to disrupt the interaction between Keap1 and Nrf2. Both compounds contain a chiral center and synthesis of the individual enantiomers confirmed that binding was specific to the S-stereoisomer. This data package provided the confidence to initiate a full analogue program with over 110 compounds synthesized and eventually led to ligand-bound crystal structures which helped rationalize the structure-activity relationships. Citation Format: Julie M. Rainard, Angus J. Morrison, Andrew D. Pannifer, Philip S. Jones, Richard J. Mead, Stuart P. McElroy. Characterization of small molecule inhibitors of the Nrf2-Keap1 interaction using MicroScale Thermophoresis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B17.

Regioselective copper-catalyzed aminoborylation of styrenes with bis(pinacolato)diboron and diazo compounds.

A Cu(i)-catalyzed aminoborylation reaction of styrenes is reported. In this transformation, diazo compounds are used as the electrophilic amination agent. The in situ generated benzylcopper species is trapped by the electrophilic nitrogen terminus of the diazo substrate to afford borylated hydrazones in a regioselective manner.