Methimazole, an Effective Neutralizing Agent of the Sulfur Mustard Derivative 2-Chloroethyl Ethyl Sulfide.

Sulfur mustard (SM), designated by the military as HD, is a highly toxic and dangerous vesicant that has been utilized as a chemical warfare agent since World War I. Despite SM's extensive history, an effective antidote does not exist. The effects of SM are predominantly based on its ability to alkylate important biomolecules. Also, with the potential for a fraction of SM to remain unreacted up to days after initial contact, a window of opportunity exists for direct neutralization of unreacted SM over the days following exposure. In this study, we evaluated the structure-activity relationship of multiple nucleophilic molecules to neutralize the toxic effects of 2-chloroethyl ethyl sulfide (CEES), a monofunctional analogue of SM, on human keratinocyte (HaCaT) cells. Cell viability, relative loss of extracellular matrix adhesions, and apoptosis caused by CEES were measured via MTT, cell-matrix adhesion (CMA), and apoptosis protein marker assays, respectively. A set of five two-carbon compounds with various functional groups served as a preliminary group of first-generation neutralizing agents to survey the correlation between mitigation of CEES's toxic effects and functional group nucleophilicity. Apart from thioacids, which produced additive toxicity, we generally observed the trend of increasing protection from cytotoxicity with increasing nucleophilicity. We extended this treatment strategy to second-generation agents which contained advantageous structural features identified from the first-generation molecules. Our results show that methimazole (MIZ), a currently FDA-approved drug used to treat hyperthyroidism, effectively reduced cytotoxicity, increased CMA, and decreased apoptosis resulting from CEES toxicity. MIZ selectively reacts with CEES to produce 2-(2-(ethylthio)ethylthio)-1-methyl-1H-imidazole (EEMI) in media and cell lysate treatments resulting in the reduction of toxicity. Based on these results, future development of MIZ as an SM therapeutic may provide a viable approach to reduce both the immediate and long-term toxicity of SM and may also help mitigate slower developing SM toxicity due to residual intact SM.

Deacetylated Sialic Acid Sensitizes Lung and Colon Cancers to Novel Cucurbitacin-Inspired Estrone Epidermal Growth Factor Receptor (EGFR) Inhibitor Analogs.

Cancers utilize sugar residues such as sialic acids (Sia) to improve their ability to survive. Sia presents a variety of functional group alterations, including O-acetylation on the C6 hydroxylated tail. Previously, sialylation has been reported to suppress EGFR activation and increase cancer cell sensitivity to Tyrosine Kinase Inhibitors (TKIs). In this study, we report on the effect of deacetylated Sia on the activity of three novel EGFR-targeting Cucurbitacin-inspired estrone analogs (CIEAs), MMA 294, MMA 321, and MMA 320, in lung and colon cancer cells. Acetylation was modulated by the removal of Sialate O-Acetyltransferase, also known as CAS1 Domain-containing protein (CASD1) gene via CRISPR-Cas9 gene editing. Using a variety of cell-based approaches including MTT cell viability assay, flow cytometry, immunofluorescence assay and in-cell ELISA we observed that deacetylated Sia-expressing knockout cells (1.24-6.49 µM) were highly sensitive to all CIEAs compared with the control cells (8.82-20.97 µM). Apoptosis and varied stage cell cycle arrest (G0/G1 and G2/M) were elucidated as mechanistic modes of action of the CIEAs. Further studies implicated overexpression of CIEAs' cognate protein target, phosphorylated EGFR, in the chemosensitivity of the deacetylated Sia-expressing knockout cells. This observation correlated with significantly decreased levels of key downstream proteins (phosphorylated ERK and mTOR) of the EGFR pathway in knockout cells compared with controls when treated with CIEAs. Collectively, our findings indicate that Sia deacetylation renders lung and colon cancer cells susceptible to EGFR therapeutics and provide insights for future therapeutic interventions.

Early in vitro evidence indicates that deacetylated sialic acids modulate multi-drug resistance in colon and lung cancers via breast cancer resistance protein.

Cancers utilize sugar residues to engage in multidrug resistance. The underlying mechanism of action involving glycans, specifically the glycan sialic acid (Sia) and its various functional group alterations, has not been explored. ATP-binding cassette (ABC) transporter proteins, key proteins utilized by cancers to engage in multidrug resistant (MDR) pathways, contain Sias in their extracellular domains. The core structure of Sia can contain a variety of functional groups, including O-acetylation on the C6 tail. Modulating the expression of acetylated-Sias on Breast Cancer Resistance Protein (BCRP), a significant ABC transporter implicated in MDR, in lung and colon cancer cells directly impacted the ability of cancer cells to either retain or efflux chemotherapeutics. Via CRISPR-Cas-9 gene editing, acetylation was modulated by the removal of CAS1 Domain-containing protein (CASD1) and Sialate O-Acetyl esterase (SIAE) genes. Using western blot, immunofluorescence, gene expression, and drug sensitivity analysis, we confirmed that deacetylated Sias regulated a MDR pathway in colon and lung cancer in early in vitro models. When deacetylated Sias were expressed on BCRP, colon and lung cancer cells were able to export high levels of BCRP to the cell's surface, resulting in an increased BCRP efflux activity, reduced sensitivity to the anticancer drug Mitoxantrone, and high proliferation relative to control cells. These observations correlated with increased levels of cell survival proteins, BcL-2 and PARP1. Further studies also implicated the lysosomal pathway for the observed variation in BCRP levels among the cell variants. RNASeq data analysis of clinical samples revealed higher CASD1 expression as a favorable marker of survival in lung adenocarcinoma. Collectively, our findings indicate that deacetylated Sia is utilized by colon and lung cancers to engage in MDR via overexpression and efflux action of BCRP.

Rapid, One-Step Synthesis of α-Ketoacetals via Electrophilic Etherification.

Herein, we report a rapid, one-step synthesis of α-ketoacetals via electrophilic etherification of α-alkoxy enolates and monoperoxyacetals. Methyl, primary, and secondary α-ketoacetals were obtained in 44-63% yields from tetrahydropyranyl substrates; using methyl tetrahydropyranyl, alkyl tetrahydropyranyl, or methyl tetrahydrofuranyl peroxyacetals, however, methyl and primary products were isolated in 66-90% yields. The present method is applied to C-O bond formation at tertiary carbons, via alkyl and methyl peroxyacetals, with yields of 25-65%. Intermolecular "alkoxyl" transfer, from peroxyacetal to α-alkoxy enolate, relies heavily on decreased steric bulk surrounding the peroxide bond and site of etherification; additionally, we found the α-OCH3 group to be critical in ensuring product formation. α-Ketoacetals demonstrated excellent reactivity, as selective, nucleophilic attack at the unprotected carbonyl furnished α-hydroxy acetals in 80-100% yields; subsequent hydrolysis of the foregoing compounds provided α-hydroxy aldehydes in yields of 58-90%.

Deacetylated sialic acids modulates immune mediated cytotoxicity via the sialic acid-Siglec pathway.

Cancers utilize glycans to evade the immune system via the Sialic acid (Sia)-Siglec (Sialic-acid-binding immunoglobulin-like lectins) pathway. Specifically, atypical structural forms of sialic acid bind to inhibitory Siglec receptors on natural killer (NK) cells resulting in the suppression of immune cell mediated cytotoxicity. The mechanism of action that governs the Sia-Siglec pathway in cancers is not understood. Specifically, how deviations from the typical form of Sia mechanistically contribute. Here, we focused on modulating 9-O and 7, 9-O-acetylation of Neu5Ac, via CRISPR-Cas9 gene editing, a functional group that is absent from Sias on many types of cancer cells. The two genes that are responsible for regulating the level of acetylation on Neu5Ac, are Sialic acid acetylesterase (SIAE) and Sialic acid acetyltransferase (CASD1). These genes modulated Siglec binding in colon, lung and a noncancerous kidney cell line. In the absence of SIAE, Neu5Ac is acetylated, engagement of cancer associated Siglecs is reduced while binding was increased when the ability to acetylate was removed via CASD1 knock out. In the absence of SIAE NK mediated cytotoxicity increased in both colon and lung cancer cells. In addition to modulating Siglec binding, SIAE expression modulates the level of Sias in a cell, and the α2-6-linkage of Sias-which is specifically upregulated and associated with cancers. Uncovering how functional group alterations on Neu5Ac contribute mechanistically to both Siglec receptor binding, the Sia-Siglec immune evasion pathway, and the production of cancer associated glycosidic linkages-offers a promising avenue for targeted cancer immune therapies in the future.

Electrophilic Etherification of α-Heteroaryl Carbanions with Monoperoxyacetals as a Route to Ketene O,O- and N,O-Acetals.

Alkyl ketene acetals are useful reactants in a variety of synthetic processes, and yet, there are limited routes to their formation as isolable products. We now report the successful synthesis and isolation of heteroaryl ketene acetals through intermolecular transfer of alkoxyl (δ+OR) from electrophilic peroxides to lithiated benzofurans, indoles, and pyridines. Primary and secondary peroxyacetals enable selective transfer of the nonanomeric alkoxy group in moderate to high yield; substrates bearing an electron-donating substituent show enhanced reactivity toward electrophilic oxygen. Heteroaryl ketene acetals are remarkably stable throughout traditional purification techniques; the superior stability of ketene N,O-acetals compared to ketene O,O-acetals is presumably due to increased aromaticity of the indole and pyridine structures. The presented method overcomes typical problems associated with alkyl ketene acetal synthesis as reported products withstood workup and flash column chromatography procedures.

Oxacycle synthesis via intramolecular reaction of carbanions and peroxides.

The intramolecular reaction of dialkyl peroxides with carbanions, generated via chemoselective metal-heteroatom exchange or deprotonation, provides a new approach to cyclic ethers. Applied in tandem with C-C bond formation, the strategy enables a one-step annelation to form oxaospirocycles.

Tandem application of C-C bond-forming reactions with reductive ozonolysis.

Several variants of reductive ozonolysis, defined here as the in situ generation of aldehydes or ketones during ozonolytic cleavage of alkenes, are demonstrated to work effectively in tandem with a number of C-C bond-forming reactions. For reactions involving basic nucleophiles (1,2-addition of Grignard reagents, Wittig or Horner-Emmons olefinations, and directed aldol reactions of lithium enolates), the one-pot process offers a rapid and high-yielding alternative to traditional two-step protocols.

Pyridine is an organocatalyst for the reductive ozonolysis of alkenes.

Whereas the cleavage of alkenes by ozone typically generates peroxide intermediates that must be decomposed in an accompanying step, ozonolysis in the presence of pyridine directly generates ketones or aldehydes through a process that neither consumes pyridine nor generates any detectable peroxides. The reaction is hypothesized to involve nucleophile-promoted fragmentation of carbonyl oxides via formation of zwitterionic peroxyacetals.