Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cell homeostasis.

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, which is characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMC homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam-cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMC proliferation and migration, impacting atherosclerosis. In summary, this work shows that ChA is sufficient to induce lysosomal dysfunction in VSMCs, that, in ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered, and, finally, that recombinant LAL can be a therapeutic approach for lysosomal dysfunction.

Cholesteryl hemiazelate identified in CVD patients causes in vitro and in vivo inflammation.

Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1ß and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.

Novel Foscan®-derived ring-fused chlorins for photodynamic therapy of cancer.

Photodynamic therapy (PDT) is an established anticancer treatment that combines the use of a photosensitiser (PS) and a light source of a specific wavelength for the generation of reactive oxygen species (ROS) that are toxic to the tumour cells. Foscan® (mTHPC) is a clinically-approved chlorin used for the PDT treatment of advanced head and neck, prostate and pancreatic cancers but is characterized by being photochemically unstable and associated with prolonged skin photosensitivity. Herein, we report the synthesis of new 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, having the meso-tetra(3-hydroxyphenyl)macrocycle core of mTHPC, by exploring the [8π + 2π] cycloaddition of a meso-tetra(3-hydroxyphenyl)porphyrin derivative with diazafulvenium methides. These chlorins have photochemical properties similar to Foscan® but are much more photostable. Among the novel compounds, two chlorins with a hydroxymethyl group and its azide derivative present in the 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused system, are promising photodynamic agents with activity in the 100 nM range against triple-negative breast cancer cells and, in the case of azidomethyl chlorin, a safer phototherapeutic index compared to Foscan®.

The role of solvents and concentrations in the properties of oxime bearing A2B corroles.

A comprehensive study on the electronic spectral, photophysical and acid-base properties of phenyl- and methyl-oxime corrole derivatives and of triphenylcorrole (model corrole) has been performed, aiming to shed light on the existing species in the ground and excited states. Solvents and corrole concentration are found to govern the properties of the studied compounds and are determinants of their applicability in in vivo studies. In THF, the neutral corrole has two tautomeric forms (T1 and T2). In DMSO, the deprotonated form shows a characteristic long-wavelength Q band slightly shifted to blue when compared with the T1 tautomer and a higher fluorescence quantum yield. In ACN, with the increase of the corrole concentration formation of an aggregate due to homoconjugation (with dimer characteristics) is observed, and pioneeringly reported using UV-Vis and fluorescence studies and confirmed by carrying out titrations with TFA. The effect of the oxime group on the pK values of a corrole is found to influence the formation of a homoconjugate, namely by precluding its formation (at higher concentrations) when compared with the model corrole. TDDFT electronic quantum calculations support the experimental observations, namely the existence of tautomers and deprotonated species, with their respective electronic spectral features, further allowed proposing a structure for the homoconjugate complex in ACN. The characteristics of the oxime-corroles, namely a pK of ∼ 5, absorption and emission at ca. 650 nm and solvent dependent properties, make them good candidates for their use in biological systems either as probes, sensors, or as new sensitizers for photodynamic therapy.

Differential Tunneling-Driven and Vibrationally-Induced Reactivity in Isomeric Benzazirines.

Quantum mechanical tunneling of heavy-atoms and vibrational excitation chemistry are unconventional and scarcely explored types of reactivity. Once fully understood, they might bring new avenues to conduct chemical transformations, providing access to a new world of molecules or ways of exquisite reaction control. In this context, we present here the discovery of two isomeric benzazirines exhibiting differential tunneling-driven and vibrationally-induced reactivity, which constitute exceptional results for probing into the nature of these phenomena. The isomeric 6-fluoro- and 2-fluoro-4-hydroxy-2H-benzazirines (3-a and 3'-s) were generated in cryogenic krypton matrices by visible-light irradiation of the corresponding triplet nitrene 3 2-a, which was produced by UV-light irradiation of its azide precursor. The 3'-s was found to be stable under matrix dark conditions, whereas 3-a spontaneously rearranges (τ1/2 ∼64 h at 10 and 20 K) by heavy-atom tunneling to 3 2-a. Near-IR-light irradiation at the first OH stretching overtone frequencies (remote vibrational antenna) of the benzazirines induces the 3'-s ring-expansion reaction to a seven-member cyclic ketenimine, but the 3-a undergoes 2H-azirine ring-opening reaction to triplet nitrene 3 2-a. Computations demonstrate that 3-a and 3'-s have distinct reaction energy profiles, which explain the different experimental results. The spectroscopic direct measurement of the tunneling of 3-a to 3 2-a constitutes a unique example of an observation of a species reacting only by nitrogen tunneling. Moreover, the vibrationally-induced sole activation of the most favorable bond-breaking/bond-forming pathway available for 3-a and 3'-s provides pioneer results regarding the selective nature of such processes.

Insights into the anticancer activity of chiral alkylidene-ß-lactams and alkylidene-γ-lactams: Synthesis and biological investigation.

Chiral alkylidene-ß-lactams and alkylidene-γ-lactams were synthesized and screened for their in vitro activity against four human cancer cell lines (melanoma, esophageal, lung and fibrosarcoma carcinoma). Alkylidene-ß-lactams were synthesized via Wittig reaction of diverse phosphorus ylides with benzhydryl 6-oxopenicillanate, derived from 6-aminopenicillanic acid. Moreover, novel chiral alkylidene-γ-lactams were synthesized through a multistep strategy starting from a chiral substrate (d-penicillamine). The in vitro assays allowed the identification of four compounds with IC50 values < 10 µM for A375 cell line, and three compounds with IC50 values < 10 µM for OE19 cell line. The effect of the most promising compounds on cell death mechanism, reactive oxygen species generation as well as the evaluation of their ability to act as MMP-9 inhibitors were studied. The reported results unveil the potential of alkylidene-ß-lactams as anticancer agents.

High Instantaneous Inhibitory Potential of Bictegravir and the New Spiro-ß-Lactam BSS-730A for HIV-2 Isolates from RAL-Naïve and RAL-Failing Patients.

Integrase inhibitors (INIs) are an important class of drugs for treating HIV-2 infection, given the limited number of drugs active against this virus. While the clinical efficacy of raltegravir and dolutegravir is well established, the clinical efficacy of bictegravir for treating HIV-2 infected patients has not been determined. Little information is available regarding the activity of bictegravir against HIV-2 isolates from patients failing raltegravir-based therapy. In this study, we examined the phenotypic and matched genotypic susceptibility of HIV-2 primary isolates from raltegravir-naïve and raltegravir-failing patients to raltegravir, dolutegravir, and bictegravir, and to the new spiro-ß-lactam BSS-730A. The instantaneous inhibitory potential (IIP) was calculated to help predict the clinical activity of bictegravir and BSS-730A. Isolates from raltegravir-naïve patients were highly sensitive to all INIs and BSS-730A. Combined integrase mutations E92A and Q148K conferred high-level resistance to raltegravir, and E92Q and T97A conferred resistance to raltegravir and dolutegravir. The antiviral activity of bictegravir and BSS-730A was not affected by these mutations. BSS-730A displayed strong antiviral synergism with raltegravir. Mean IIP values at Cmax were similar for all INIs and were not significantly affected by resistance mutations. IIP values were significantly higher for BSS-730A than for INIs. The high IIP values of bictegravir and BSS-730A for raltegravir-naïve and raltegravir-resistant HIV-2 isolates highlight their potential value for treating HIV-2 infection. Overall, the results are consistent with the high clinical efficacy of raltegravir and dolutegravir for HIV-2 infection and suggest a promising clinical profile for bictegravir and BSS-730A.

Diels-Alder Cycloaddition Reactions in Sustainable Media.

Diels-Alder cycloaddition reaction is one of the most powerful strategies for the construction of six-membered carbocyclic and heterocyclic systems, in most cases with high regio- and stereoselectivity. In this review, an insight into the most relevant advances on sustainable Diels-Alder reactions since 2010 is provided. Various environmentally benign solvent systems are discussed, namely bio-based derived solvents (such as glycerol and gluconic acid), polyethylene glycol, deep eutectic solvents, supercritical carbon dioxide, water and water-based aqueous systems. Issues such as method's scope, efficiency, selectivity and reaction mechanism, as well as sustainability, advantages and limitations of these reaction media, are addressed.

Switching on H-Tunneling through Conformational Control.

H-tunneling is a ubiquitous phenomenon, relevant to fields from biochemistry to materials science, but harnessing it for mastering the manipulation of chemical structures still remains nearly illusory. Here, we demonstrate how to switch on H-tunneling by conformational control using external radiation. This is outlined with a triplet 2-hydroxyphenylnitrene generated in an N2 matrix at 10 K by UV-irradiation of an azide precursor. The anti-orientation of the nitrene's OH moiety was converted to syn by selective vibrational excitation at the 2ν(OH) frequency, thereby moving the H atom closer to the vicinal nitrene center. This triggers spontaneous H-tunneling to a singlet 6-imino-2,4-cyclohexadienone. Computations reveal that such fast H-tunneling occurs through crossing the triplet-to-singlet potential energy surfaces. Our experimental realization provides an exciting novel strategy to attain control over tunneling, opening new avenues for directing chemical transformations.

Reactivity of steroidal 1-azadienes toward enamines: an approach to novel chiral penta- and hexacyclic steroids.

The chemical behavior of steroidal N-sulfonyl-1-azadienes toward carbonyl compounds, in the presence of pyrrolidine, is described. With aldehydes, these azadienes participate in hetero-Diels-Alder reactions with the in situ generated enamines. The stereoselectivity results from the approach of the dienophiles from the less hindered α-face of the steroid, with the pyrrolidine moiety endo and retention of the enamine trans geometry. This diastereoselective synthetic methodology led to a new class of chiral pentacyclic steroids. Interestingly, the studied steroidal scaffolds follow a different mechanistic pathway with cyclic ketones. They undergo a diastereoselective annulation reaction, under enamine catalysis, affording chiral hexacyclic steroids.

Synthesis of 5H-chromeno[3,4-b]pyridines via DABCO-catalyzed [3 + 3] annulation of 3-nitro-2H-chromenes and allenoates.

The DABCO-catalyzed [3 + 3] annulation between 3-nitro-2H-chromenes and benzyl 2,3-butadienoate has been developed as a route to 5H-chromeno[3,4-b]pyridine derivatives. Under optimal reaction conditions, 5H-chromeno[3,4-b]pyridines incorporating two allenoate units were obtained in moderate to good yields (30-76%). The same type of transformation could be carried out using butynoates as allene surrogates. Mechanistic studies by mass spectrometry allowed the identification of the key intermediates involved in the reaction mechanism. The reported synthetic methodology represents an entirely new approach for the synthesis of the 5H-chromeno[3,4-b]pyridine core structure based on allene chemistry.

Meso-Substituted Corroles from Nitrosoalkenes and Dipyrromethanes.

The synthesis of bilanes and hexapyrroles containing an oxime functionality, prepared by two and three consecutive hetero-Diels-Alder reactions (or conjugated additions) between nitrosoalkenes and dipyrromethanes, is described. Bilanes underwent oxidative macrocyclization to afford a new class of trans-A2B-corroles. Porphyrins could also be obtained by reacting bilanes with aldehydes in the presence of trifluoroacetic acid, followed by an oxidative step.

Recent Advances in the Chemistry of Conjugated Nitrosoalkenes and Azoalkenes.

This review aims to present the most recent contributions in the chemistry of nitrosoalkenes and azoalkenes, highlighting the chemical behavior that makes them important and versatile building blocks in organic synthesis. These are heterodienes used in the assembly of a variety of heterocyclic systems, spanning from five- to seven-membered heterocycles, as well as for the functionalization of heterocycles.

Corroles and Hexaphyrins: Synthesis and Application in Cancer Photodynamic Therapy.

Corroles and hexaphyrins are porphyrinoids with great potential for diverse applications. Like porphyrins, many of their applications are based on their unique capability to interact with light, i.e., based on their photophysical properties. Corroles have intense absorptions in the low-energy region of the uv-vis, while hexaphyrins have the capability to absorb light in the near-infrared (NIR) region, presenting photophysical features which are complementary to those of porphyrins. Despite the increasing interest in corroles and hexaphyrins in recent years, the full potential of both classes of compounds, regarding biological applications, has been hampered by their challenging synthesis. Herein, recent developments in the synthesis of corroles and hexaphyrins are reviewed, highlighting their potential application in photodynamic therapy.

Current Advances in the Synthesis of Valuable Dipyrromethane Scaffolds: Classic and New Methods.

This review presents the most recent developments on the synthesis of dipyrromethanes, covering classical synthetic strategies, using acid catalyzed condensation of pyrroles and aldehydes or ketones, and recent breakthroughs which allow the synthesis of these type of heterocycles with new substitution patterns.

Cholesteryl hemiesters alter lysosome structure and function and induce proinflammatory cytokine production in macrophages.

RATIONALE: Cholesteryl hemiesters are oxidation products of polyunsaturated fatty acid esters of cholesterol. Their oxo-ester precursors have been identified as important components of the "core aldehydes" of human atheromata and in oxidized lipoproteins (Ox-LDL). We had previously shown, for the first time, that a single compound of this family, cholesteryl hemisuccinate (ChS), is sufficient to cause irreversible lysosomal lipid accumulation (lipidosis), and is toxic to macrophages. These features, coupled to others such as inflammation, are typically seen in atherosclerosis. OBJECTIVE: To obtain insights into the mechanism of cholesteryl hemiester-induced pathological changes in lysosome function and induction of inflammation in vitro and assess their impact in vivo. METHODS AND RESULTS: We have examined the effects of ChS on macrophages (murine cell lines and primary cultures) in detail. Specifically, lysosomal morphology, pH, and proteolytic capacity were examined. Exposure of macrophages to sub-toxic ChS concentrations caused enlargement of the lysosomes, changes in their luminal pH, and accumulation of cargo in them. In primary mouse bone marrow-derived macrophages (BMDM), ChS-exposure increased the secretion of IL-1ß, TNF-α and IL-6. In zebrafish larvae (wild-type AB and PU.1:EGFP), fed with a ChS-enriched diet, we observed lipid accumulation, myeloid cell-infiltration in their vasculature and decrease in larval survival. Under the same conditions the effects of ChS were more profound than the effects of free cholesterol (FC). CONCLUSIONS: Our data strongly suggest that cholesteryl hemiesters are pro-atherogenic lipids able to mimic features of Ox-LDL both in vitro and in vivo.

(1H-Tetrazol-5-yl)-Allenes: Building Blocks for Tetrazolyl Heterocycles.

(1H-Tetrazol-5-yl)-allenes have been prepared for the first time, and their reactivity toward aziridines explored. Reaction of a (1-benzyl-1H-tetrazol-5-yl)-phosphonium chloride and acyl chlorides in the presence of triethylamine afforded the target allenes via Wittig reaction of the in situ generated phosphorus ylide and ketenes. 1-(1-Benzyl-1H-tetrazol-5-yl)propa-1,2-diene and 3-methyl-, 3-ethyl- and 3-benzyl derivatives undergo microwave-induced formal [3 + 2] cycloaddition with cis-N-benzyl-2-benzoyl-3-phenylaziridine, through C-N bond cleavage, to give selectively tetrasubstituted pyrroles. In contrast, with (1H-tetrazol-5-yl)-allenes bearing bulkier substituents at C-3, such as i-propyl or a tert-butyl, 4-methylenepyrrolidines were obtained exclusively via [3 + 2] cycloaddition of the in situ generated azomethine ylide. The latter allenes also gave 4-methylenepyrrolidines on reacting with cis-2-benzoyl-N-cyclohexyl-3-phenylaziridine, whereas with the other allenes, pyrroles were obtained as major products together with the formation of 4-methylenepyrrolidines. All the studied (1H-tetrazol-5-yl)-allenes reacted with N-benzyl-cis-3-phenylaziridine-2-carboxylate to give the corresponding 4-methylenepyrrolidines exclusively.

Synthesis of chiral hexacyclic steroids via [8π + 2π] cycloaddition of diazafulvenium methides.

First examples of [8π + 2π] cycloaddition of 16-dehydropregnenolone (16-DPA) acetate with diazafulvenium methides leading to chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused steroids are reported. These hexacyclic steroids were obtained exclusively or selectively with the approach of the 1,7-dipole by the less hindered α-face of 16-DPA. Quantum chemical calculations at the DFT level were carried out, using the cycloaddition of 1-methyl- and 1-benzyl-diazafulvenium methides with N-phenylmaleimide as model reactions, in order to rationalize the stereochemistry outcome. The results indicate that endo cycloadditions of the more stable dipole conformation, having the 1-substituent pointing outward, are significantly more favorable than the alternative exo cycloaddition.

Synthesis of New 2-Halo-2-(1H-tetrazol-5-yl)-2H-azirines via a Non-Classical Wittig Reaction.

The synthesis and reactivity of tetrazol-5-yl-phosphorus ylides towards N-halosuccinimide/TMSN3 reagent systems was explored, opening the way to new haloazidoalkenes bearing a tetrazol-5-yl substituent. These compounds were obtained as single isomers, except in one case. X-ray crystal structures were determined for three derivatives, establishing that the non-classical Wittig reaction leads to the selective synthesis of haloazidoalkenes with (Z)-configuration. The thermolysis of the haloazidoalkenes afforded new 2-halo-2-(tetrazol-5-yl)-2H-azirines in high yields. Thus, the reported synthetic methodologies gave access to important building blocks in organic synthesis, vinyl tetrazoles and 2-halo-2-(tetrazol-5-yl)-2H-azirine derivatives.