Computational and Spectroscopic Studies on the Formation of Halogen-Bonded Complexes Between Tertiary Amines and CBr4 and Application in the Light-mediated Amino Acid Coupling.

In recent years, halogen-bonded complexes (XBCs), in solution, have played a pivotal role in inducing photochemical organic reactions. In this work, we explore the ability of various tertiary amines to act as XB acceptors in the presence of the XB donor CBr4 by computational and spectroscopic studies. DFT studies clearly showcase the formation of XBCs between the studied tertiary amines and CBr4. Simultaneously, computational and experimental UV-Vis studies display intense red shifts that are consistent with charge transfer observed from tertiary amines to CBr4. A detailed NMR study revealed a clear chemical shift of the carbon carrying the bromine atoms upon mixing the XB acceptor with the donor, suggesting that this spectroscopic technique is indeed an experimental tool to identify the generation of XBCs. An application of the ability of such XBCs to activate a carboxylic acid under UVA irradiation or sunlight is presented for amino acid coupling. Among the various tertiary amines studied, the pair DABCO-CBr4 was found to work well for the photochemical amide bond formation. Direct infusion-HRMS studies allowed us to propose a general mechanism for the photochemical amino acid coupling in the presence of a tertiary amine and CBr4, initiated by the photoactivation of an XBC.

Free Fatty Acid Determination in Broccoli Tissues Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Broccoli (Brassica oleracea L. var. italica Plenck) is a widely consumed vegetable, very popular due to its various nutritional and bioactive components. Since studies on the lipid components of broccoli have been limited so far, the aim of the present work was the study of free fatty acids (FFAs) present in different broccoli parts, aerial and underground. The direct determination of twenty-four FFAs in broccoli tissues (roots, leaves, and florets) was carried out, using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method in a 10 min single run. Linolenic acid was found to be the most abundant FFA in all different broccoli parts in quantities ranging from 0.76 to 1.46 mg/g, followed by palmitic acid (0.17-0.22 mg/g) and linoleic acid (0.06-0.08 mg/g). To extend our knowledge on broccoli's bioactive components, for the first time, the existence of bioactive oxidized fatty acids, namely hydroxy and oxo fatty acids, was explored in broccoli tissues adopting an HRMS-based lipidomics approach. 16- and 2-hydroxypalmitic acids were detected in all parts of broccoli studied, while ricinoleic acid was detected for the first time as a component of broccoli.

Asymmetric Synthesis of Saturated and Unsaturated Hydroxy Fatty Acids (HFAs) and Study of Their Antiproliferative Activity.

Hydroxy fatty acids (HFAs) constitute a class of lipids, distinguished by the presence of a hydroxyl on a long aliphatic chain. This study aims to expand our insights into HFA bioactivities, while also introducing new methods for asymmetrically synthesizing unsaturated and saturated HFAs. Simultaneously, a procedure previously established by us was adapted to generate new HFA regioisomers. An organocatalytic step was employed for the synthesis of chiral terminal epoxides, which either by alkynylation or by Grignard reagents resulted in unsaturated or saturated chiral secondary alcohols and, ultimately, HFAs. 7-(S)-Hydroxyoleic acid (7SHOA), 7-(S)-hydroxypalmitoleic acid (7SHPOA) and 7-(R)- and (S)-hydroxymargaric acids (7HMAs) were synthesized for the first time and, together with regioisomers of (R)- and (S)-hydroxypalmitic acids (HPAs) and hydroxystearic acids (HSAs), whose biological activity has not been tested so far, were studied for their antiproliferative activities. The unsaturation of the long chain, as well as an odd-numbered (C17) fatty acid chain, led to reduced activity, while the new 6-(S)-HPA regioisomer was identified as exhibiting potent antiproliferative activity in A549 cells. 6SHPA induced acetylation of histone 3 in A549 cells, without affecting acetylated α-tubulin levels, suggesting the selective inhibition of histone deacetylase (HDAC) class I enzymes, and was found to inhibit signal transducer and activator of transcription 3 (STAT3) expression.

N-(2-Aminophenyl)-benzamide Inhibitors of Class I HDAC Enzymes with Antiproliferative and Antifibrotic Activity.

Inhibitors of histone deacetylases (HDACs) have received special attention as novel anticancer agents. Among various types of synthetic inhibitors, benzamides constitute an important class, and one is an approved drug (chidamide). Here, we present a novel class of HDAC inhibitors containing the N-(2-aminophenyl)-benzamide functionality as the zinc-binding group linked to various cap groups, including the amino acids pyroglutamic acid and proline. We have identified benzamides that inhibit HADC1 and HDAC2 at nanomolar concentrations, with antiproliferative activity at micromolar concentrations against A549 and SF268 cancer cell lines. Docking studies shed light on the mode of binding of benzamide inhibitors to HDAC1, whereas cellular analysis revealed downregulated expression of EGFR mRNA and protein. Two benzamides were investigated in a mouse model of bleomycin-induced pulmonary fibrosis, and both showed efficacy on a preventative dosing schedule. N-(2-Aminophenyl)-benzamide inhibitors of class I HDACs might lead to new approaches for treating fibrotic disorders.

An Efficient Light-Mediated Protocol for the Direct Amide Bond Formation via a Novel Carboxylic Acid Photoactivation Mode by Pyridine-CBr4.

The direct amide bond formation between a carboxylic acid and an amine still constitutes a challenging reaction for both academia and industry. We demonstrate herein that several pairs of amines (halogen bond acceptors) and organohalogen sources may be used for the photochemical amidation reaction under either UVA or sunlight irradiation. Our studies led to the identification of pyridine-CBr4 as an efficient agent to perform amide synthesis under LED 370 nm irradiation, avoiding super-stoichiometric quantities. An extended substrate scope was demonstrated, showing that the widely used amino and carboxyl protecting groups are compatible with this photochemical protocol, while a number of industrially interesting products and bioactive compounds were synthesized. Direct infusion-high resolution mass spectrometry studies suggest an unprecedented type of carboxylic acid activation mode upon irradiation, involving the generation of a symmetric anhydride, an active ester with pyridine N-oxide and a mixed anhydride with hypobromous acid.

Targeted and Suspect Fatty Acid Profiling of Royal Jelly by Liquid Chromatography-High Resolution Mass Spectrometry.

Royal jelly (RJ) is a bee product produced by the mandibular and hypopharyngeal glands of worker honeybees which has attracted special attention because of its numerous pharmacological activities and its applications to dermatology and cosmetics. In 2020, we demonstrated a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the determination of seven medium-chain FFAs in RJ samples. The aim of the present work was to extend our studies on FA profiling of RJ, exploring the presence of common long-chain saturated, mono-unsaturated and poly-unsaturated free FAs in RJ samples using this LC-HRMS method. Among twenty common FAs studied by a targeted approach, palmitic acid, stearic acid and oleic acid were found at concentrations higher than the rest of the FAs (the concentrations of these three acids ranged from 37.4 to 48.0, from 17.7 to 24.0 and from 9.4 to 11.1 mg/100 g of fresh RJ, respectively). The high mass accuracy of LC-HRMS allowed the application of a suspect approach, which enabled the exploration of various C9 and C11 FAs, as well as hydroxylated C12 FAs. Nonenoic acid was indicated as the most abundant among these acids. In addition, for the first time, the presence of a variety of regio-isomers of hydroxymyristic, hydroxypalmitic and hydroxystearic acids was demonstrated in RJ samples.

Free fatty acid profiling of Greek yogurt by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis.

Yogurt is a fermented dairy product of high nutritional value, very popular in many parts of the world. Free fatty acids (FFAs), which are formed during fermentation, may cause changes in organoleptic properties of yogurt, and thus, the determination of FFAs is of importance. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of a large set of common and uncommon FFAs in yogurt samples, avoiding any derivatization step. Twenty-five common saturated and unsaturated FAs, together with 21 saturated hydroxy fatty acids (SHFAs) and 17 saturated oxo fatty acids (SOFAs), were analyzed in 26 cow and 7 sheep Greek yogurt samples. A detailed analysis of bioactive SHFAs and SOFAs was carried out in yogurt samples for the first time. Differences at the concentrations of six common FAs and five oxidized FAs between the cow and sheep samples were observed. Based on these FAs, Principal Component Analysis (PCA) allows the discrimination of cow from sheep yogurt samples.

Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples.

Fatty acids (FAs) play pleiotropic roles in living organisms, acting as signaling molecules and gene regulators. They are present in plants and foods and may affect human health by food ingestion. As a consequence, analytical methods for their determination in biological fluids, plants and foods have attracted high interest. Undoubtedly, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs. Due to the inherent poor ionization efficiency of FAs, their chemical derivatization prior to analysis is often employed. Usually, the derivatization of the FA carboxyl group aims to charge reversal, allowing detection and quantification in positive ion mode, thus, resulting in an increase in sensitivity in determination. Another approach is the derivatization of the double bond of unsaturated FAs, which aims to identify the double bond location. The present review summarizes the various classes of reagents developed for FA derivatization and discusses their applications in the liquid chromatography-MS (LC-MS) analysis of FAs in various matrices, including plasma and feces. In addition, applications for the determination of eicosanoids and fatty acid esters of hydroxy fatty acids (FAHFAs) are discussed.

Lipidomics Analysis of Free Fatty Acids in Human Plasma of Healthy and Diabetic Subjects by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS).

Targeted analytical methods for the determination of free fatty acids (FFAs) in human plasma are of high interest because they may help in identifying biomarkers for diseases and in monitoring the progress of a disease. The determination of FFAs is of particular importance in the case of metabolic disorders because FFAs have been associated with diabetes. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of 74 FFAs in human plasma. The method is fast (10-min run) and straightforward, avoiding any derivatization step and tedious sample preparation. A total of 35 standard saturated and unsaturated FFAs, as well as 39 oxygenated (either hydroxy or oxo) saturated FFAs, were simultaneously detected and quantified in plasma samples from 29 subjects with type 2 diabetes mellitus (T2D), 14 with type 1 diabetes mellitus (T1D), and 28 healthy subjects. Alterations in the levels of medium-chain FFAs (C6:0 to C10:0) were observed between the control group and T2D and T1D patients.

The discovery and development of transmembrane serine protease 2 (TMPRSS2) inhibitors as candidate drugs for the treatment of COVID-19.

INTRODUCTION: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused the devastating pandemic named coronavirus disease 2019 (COVID-19). Unfortunately, the discovery of antiviral agents to combat COVID-19 is still an unmet need. Transmembrane serine protease 2 (TMPRSS2) is an important mediator in viral infection and thus, TMPRRS2 inhibitors may be attractive agents for COVID-19 treatment. AREAS COVERED: This review article discusses the role of TMPRSS2 in SARS-CoV-2 cell entry and summarizes the inhibitors of TMPRSS2 and their potential anti-SARS activity. Two known TMPRSS2 inhibitors, namely camostat and nafamostat, approved drugs for the treatment of pancreatitis, are under clinical trials as potential drugs against COVID-19. EXPERT OPINION: Due to the lack of the crystal structure of TMPRSS2, homology models have been developed to study the interactions of known inhibitors, including repurposed drugs, with the enzyme. However, novel TMPRSS2 inhibitors have been identified through high-throughput screening, and appropriate assays studying their in vitro activity have been set up. The discovery of TMPRSS2's crystal structure will facilitate the rational design of novel inhibitors and in vivo studies and clinical trials will give a clear answer if TMPRSS2 inhibitors could be a new weapon against COVID-19.

Synthesis of benzoxazole-based vorinostat analogs and their antiproliferative activity.

Hydroxamic acid derivatives constitute an interesting novel class of antitumor agents. Three of them, including vorinostat, are approved drugs for the treatment of malignancies, while several others are currently under clinical trials. In this work, we present new vorinostat analogs containing the benzoxazole ring as the cap group and various linkers. The benzoxazole-based analogs were synthesized starting either from 2-aminobenzoxazole, through conventional coupling, or from benzoxazole, through a metal-free oxidative amination. All the synthesized compounds were evaluated for their antiproliferative activity on three diverse human cancer cell lines (A549, Caco-2 and SF268), in comparison to vorinostat. Compound 12 (GK601), carrying a benzoxazole ring replacement for the phenyl ring of vorinostat, was the most potent inhibitor of the growth of three cell lines (IC50 1.2-2.1 µΜ), similar in potency to vorinostat. Compound 12 also inhibited human HDAC1, HDAC2 and HDAC6 like vorinostat. This new analog also showed antiproliferative activity against two colon cancer cell lines genetically resembling pseudomyxoma peritonei (PMP), namely HCT116 GNAS R201C/+ and LS174T (IC50 0.6 and 1.4 µΜ, respectively) with potency comparable to vorinostat (IC50 1.1 and 2.1 µΜ, respectively).

The design and discovery of phospholipase A2 inhibitors for the treatment of inflammatory diseases.

AREAS COVERED: This review article summarizes the most important synthetic PLA2 inhibitors developed to target each one of the four major types of human PLA2 (cytosolic cPLA2, calcium-independent iPLA2, secreted sPLA2, and lipoprotein-associated Lp-PLA2), discussing their in vitro and in vivo activities as well as their recent applications and therapeutic properties. Recent findings on the role of PLA2 in the pathobiology of COVID-19 are also discussed. EXPERT OPINION: Although a number of PLA2 inhibitors have entered clinical trials, none has reached the market yet. Lipoprotein-associated PLA2 is now considered a biomarker of vascular inflammation rather than a therapeutic target for inhibitors like darapladib. Inhibitors of cytosolic PLA2 may find topical applications for diseases like atopic dermatitis and psoriasis. Inhibitors of secreted PLA2, varespladib and varespladib methyl, are under investigation for repositioning in snakebite envenoming. A deeper understanding of PLA2 enzymes is needed for the development of novel selective inhibitors. Lipidomic technologies combined with medicinal chemistry approaches may be useful tools toward this goal.

Saturated Oxo Fatty Acids (SOFAs): A Previously Unrecognized Class of Endogenous Bioactive Lipids Exhibiting a Cell Growth Inhibitory Activity.

The discovery of novel bioactive lipids that promote human health is of great importance. Combining "suspect" and targeted lipidomic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) approaches, a previously unrecognized class of oxidized fatty acids, the saturated oxo fatty acids (SOFAs), which carry the oxo functionality at various positions of the long chain, was identified in human plasma. A library of SOFAs was constructed, applying a simple green photochemical hydroacylation reaction as the key synthetic step. The synthesized SOFAs were studied for their ability to inhibit in vitro the cell growth of three human cancer cell lines. Four oxostearic acids (OSAs) were identified to inhibit the cell growth of human lung carcinoma A549 cells. 6OSA and 7OSA exhibited the highest cell growth inhibitory potency, suppressing the expression of both STAT3 and c-myc, which are critical regulators of cell growth and proliferation. Thus, naturally occurring SOFAs may play a role in the protection of human health.

α-Ketoheterocycles Able to Inhibit the Generation of Prostaglandin E2 (PGE2) in Rat Mesangial Cells.

Prostaglandin E2 (PGE2) is a key mediator of inflammation, and consequently huge efforts have been devoted to the development of novel agents able to regulate its formation. In this work, we present the synthesis of various α-ketoheterocycles and a study of their ability to inhibit the formation of PGE2 at a cellular level. A series of α-ketobenzothiazoles, α-ketobenzoxazoles, α-ketobenzimidazoles, and α-keto-1,2,4-oxadiazoles were synthesized and chemically characterized. Evaluation of their ability to suppress the generation of PGE2 in interleukin-1ß plus forskolin-stimulated mesangial cells led to the identification of one α-ketobenzothiazole (GK181) and one α-ketobenzoxazole (GK491), which are able to suppress the PGE2 generation at a nanomolar level.

Free Saturated Oxo Fatty Acids (SOFAs) and Ricinoleic Acid in Milk Determined by a Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS) Method.

Oxidized saturated fatty acids, containing a hydroxyl or an oxo functionality, have attracted little attention so far. Recent studies have shown that saturated hydroxy fatty acids, which exhibit cancer cell growth inhibition and may suppress ß-cell apoptosis, are present in milk. Herein, we present the application of a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the detection and quantification of various saturated oxo fatty acids (SOFAs) previously unrecognized in milk. This robust and rapid analytical method, which involves simple sample preparation and a single 10-min run, revealed the presence of families of oxostearic acids (OSAs) and oxopalmitic acids (OPAs) in milk. 8OSA, 9OSA, 7OSA, 10OSA and 10OPA were found to be the most abundant SOFAs in both cow and goat milk. Higher contents of SOFAs were found in cow milk in comparison to goat milk. Together with SOFAs, ricinoleic acid, which is isobaric to OSA, was detected and quantified in all milk samples, following a "suspect" HRMS analysis approach. This unique natural fatty acid, which is the main component (>90%) of castor oil triglycerides, was estimated at mean content values of 534.3 ± 6.0 µg/mL and 460 ± 8.1 µg/mL in cow and goat milk samples, respectively.

Saturated Hydroxy Fatty Acids Exhibit a Cell Growth Inhibitory Activity and Suppress the Cytokine-Induced ß-Cell Apoptosis.

The field of bioactive lipids is ever expanding with discoveries of novel lipid molecules that promote human health. Adopting a lipidomic-assisted approach, two new families of previously unrecognized saturated hydroxy fatty acids (SHFAs), namely, hydroxystearic and hydroxypalmitic acids, consisting of isomers with the hydroxyl group at different positions, were identified in milk. Among the various regio-isomers synthesized, those carrying the hydroxyl at the 7- and 9-positions presented growth inhibitory activities against various human cancer cell lines, including A549, Caco-2, and SF268 cells. In addition, 7- and 9-hydroxystearic acids were able to suppress ß-cell apoptosis induced by proinflammatory cytokines, increasing the possibility that they can be beneficial in countering autoimmune diseases, such as type 1 diabetes. 7-(R)-Hydroxystearic acid exhibited the highest potency both in cell growth inhibition and in suppressing ß-cell death. We propose that such naturally occurring SHFAs may play a role in the promotion and protection of human health.

A Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of Free Hydroxy Fatty Acids in Cow and Goat Milk.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Method for the Determination of Free Fatty Acids in Milk.

The determination of free fatty acids (FFAs) in milk is of importance for quality control, legislative purposes, authentication and product development. We present herein a liquid chromatography-high resolution mass spectrometry method for the direct determination of FFAs in milk. The method involves mild sample preparation, avoids time-consuming derivatization and allows the direct quantification of twenty-two FFAs in a 10-min single run. It was validated and applied in thirteen cow milk and seven goat milk samples. Saturated fatty acids C16:0, C18:0 and unsaturated C18:1 (n-9) were found to be the major components of milk FFAs at concentrations of 33.1 ± 8.2 µg/mL, 16.5 ± 5.3 µg/mL and 14.8 ± 3.8 µg/mL, respectively, in cow milk and at concentrations of 22.8 ± 1.8 µg/mL, 12.7 ± 2.8 µg/mL and 13.3 ± 0.3 µg/mL, respectively, in goat milk. Other saturated and unsaturated FFAs were found in significantly lower quantities. Saturated fatty acids C6:0, C8:0 and C10:0 were found in higher quantities in goat milk than in cow milk. The levels of the important (for human health) odd-chain FFAs C15:0 and C17:0 were estimated in cow and goat milk.

Study of the Royal Jelly Free Fatty Acids by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS).

The lipidome of royal jelly (RJ) consists of medium-chained (8-12 carbon atoms) free fatty acids. We present herein a liquid chromatography-high resolution mass spectrometry (HRMS) method that permits the determination of RJ fatty acids and at the same time the detection of suspect fatty acids. The method allows for the direct quantification of seven free fatty acids of RJ, avoiding any derivatization step. It was validated and applied in seven RJ samples, where the major RJ fatty acid trans-10-hydroxy-2-decenoic acid (10-HDA) was found to vary from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. Four additional suspect fatty acids were simultaneously detected taking advantage of the HRMS detection.