Efficiency and selectivity of ionic liquids in microwave-assisted extraction of major lichen phenolic compounds: a scalable process with recycling of ionic liquids.

INTRODUCTION: Pseudevernia furfuracea, a lichen used classically for cosmetic applications, contains interesting metabolites possessing antimicrobial and anti-inflammatory or antioxidant properties. OBJECTIVES: Ionic liquid combined to microwave-assisted extraction (IL-MAE) was successfully applied for metabolites extraction from Pseudevernia furfuracea. MATERIALS AND METHODS: Three imidazolium and pyridinium-based ionic liquids (ILs): 1,3-dimethylimidazolium methylsulphate [C1 C1 Im][MeSO4 ], 1-ethyl-3-methylimidazolium ethylsulphate [C2 C1 Im][EtSO4 ], and N-ethylpyridinium ethylsulphate [C2 Py][EtSO4 ] were assessed for this process. The efficiency of the extraction method was evaluated using thin-layer chromatography (TLC) coupled to a Camag® spectrophotodensitometer and using high-performance liquid chromatography (HPLC) analysis. RESULTS: ILs under MAE showed extraction time efficiency (15 min vs. 24 h for conventional heating) and high selectivity in extracting the targeted metabolites: atranorin (AT), methyl-ß-orcinol carboxylate (MOC), fumarprotocetraric acid (Fum. Ac.), and physodic acid (Phys. Ac.) despite the increased degradation of AT under MAE. We showed a tunable selectivity of ILs towards extracting metabolites by changing anion or cation due to the modification of the interaction between the IL and the metabolites. While [C2 Py][EtSO4 ] was the most efficient IL and could extract all the targeted metabolites, [C2 C1 Im][EtSO4 ] was the most selective. It fully extracted AT and partially Fum. Ac. Moreover, the lichen prepared by mixing procedure provided AT and Fum. Ac. more than the milled one. A 100 times scale-up extraction was successfully performed on mixed samples with full IL recycling after back extraction. CONCLUSION: IL-MAE is reliable for lichen metabolites extraction. The method is reproducible, scalable, with possible IL recycling, opening the door for potential industrial applications.

From Quinoxaline, Pyrido[2,3-b]pyrazine and Pyrido[3,4-b]pyrazine to Pyrazino-Fused Carbazoles and Carbolines.

2,3-Diphenylated quinoxaline, pyrido[2,3-b]pyrazine and 8-bromopyrido[3,4-b]pyrazine were halogenated in deprotometalation-trapping reactions using mixed 2,2,6,6-tetramethyl piperidino-based lithium-zinc combinations in tetrahydrofuran. The 2,3-diphenylated 5-iodo- quinoxaline, 8-iodopyrido[2,3-b]pyrazine and 8-bromo-7-iodopyrido[3,4-b]pyrazine thus obtained were subjected to palladium-catalyzed couplings with arylboronic acids or anilines, and possible subsequent cyclizations to afford the corresponding pyrazino[2,3-a]carbazole, pyrazino[2',3':5,6] pyrido[4,3-b]indole and pyrazino[2',3':4,5]pyrido[2,3-d]indole, respectively. 8-Iodopyrido[2,3-b] pyrazine was subjected either to a copper-catalyzed C-N bond formation with azoles, or to direct substitution to introduce alkylamino, benzylamino, hydrazine and aryloxy groups at the 8 position. The 8-hydrazino product was converted into aryl hydrazones. Most of the compounds were evaluated for their biological properties (antiproliferative activity in A2058 melanoma cells and disease-relevant kinase inhibition).

New 5-ylidene rhodanine derivatives based on the dispacamide A model.

A practical approach for the preparation of (5Z) 5-ylidene rhodanine derivatives bearing the (4,5-dihalogeno-pyrrol-2-yl)carbamoyl fragment of dispacamide A is reported. The new compounds were obtained in good yields (19-88 %) by Knoevenagel condensation according to a solution-phase microwave dielectric heating protocol in the presence of organic bases (piperidine, TEA, and AcONa) from a set of N-substituted rhodanines 2(a-i). The ten synthetic products 3(a-j) have been synthesized with a Z-geometry about their exocyclic double bond and the structure of one of these compounds (3) was confirmed by a single X-ray diffraction analysis. The new (5Z) 5-ylidene rhodanine derivatives 3(a-j) were tested against eight protein kinases.

Design and Synthesis of Novel N-Benzylidene Derivatives of 3-Amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine under Microwave, In Silico ADME Predictions, In Vitro Antitumoral Activities and In Vivo Toxicity.

The synthesis of a series of new N-benzylidene derivatives of 3-amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine 10(a-l) bearing two points of molecular diversity is reported. These new compounds were synthesized in five steps including two steps under microwave dielectric heating. They were fully characterized using 1H and 13C NMR, FTIR and HRMS. The in silico physicochemical properties of compounds 10(a-l) were determined according to Lipinski's rules of five (RO5) associated with the prediction of their bioavailability. These new compounds 10(a-l) were tested for their antiproliferative activities in fibroblasts and eight representative human tumoral cell lines (Huh7 D12, Caco2, MDA-MB231, MDA-MB468, HCT116, PC3, MCF7 and PANC1). Among them, the compounds 10h and 10i showed sub-micromolar cytotoxic activity on tumor cell lines (0.23 < IC50 < 0.3 µM) and no toxicity on fibroblasts (IC50 > 25 µM). A dose-dependent inhibition of Store-Operated Ca+2 Entry (SOCE) was observed in the HEK293 cell line with 10h. In vitro embryotoxicity and angiogenesis on the mCherry transgenic zebrafish line showed that 10h presented no toxic effect and no angiogenic effect on embryos with a dose of 5 µM at 72 hpf.

An efficient approach to dispacamide A and its derivatives.

Dispacamide A and new analogs of this marine alkaloid were prepared in seven steps with an overall yield ranging from 12 to 33%. The key step of the strategy was a stereocontrolled Knoevenagel condensation under microwave dielectric heating in the last step. In this condensation, the 2-aminoimidazolin-4-one hydrochloride partners 10a-c were synthesized in three steps with good overall yields (33-79%) via the ring closure of N-guanidino acetic acids 9a-c and the aldehydes 5a,b as the two others building-blocks, in 3 steps with 60-66% overall yields. The six synthetic products have been obtained with a Z geometry about their exocyclic bond on the basis of (13)C/(1)H long-range coupling constants using a gHSQMBC experiment.

A practical approach to new (5Z) 2-alkylthio-5-arylmethylene-1-methyl-1,5-dihydro-4H-imidazol-4-one derivatives.

A practical protocol for the preparation of (5Z)-2-alkylthio-5-arylmethylene-1-methyl-1,5-dihydro-4H-imidazol-4-one derivatives is reported. The new compounds were obtained in good yield and stereoselectivity in two steps, namely a solvent-free Knoevenagel condensation under microwave irradiation, followed by an S-alkylation reaction with various halogenoalkanes.

Cold-spray ionization mass spectrometry of the choline chloride-urea deep eutectic solvent (reline).

Cold-Spray Ionization mass spectrometry (CSI-MS) that can be compared to an electrospray ionization (ESI) source acting with a nebulizing gas cooled by liquid nitrogen is used for analyzing reline as Deep Eutectic Solvent (DES). The association of cholinium chloride salts with urea molecules is evidenced in negative CSI-MS through the chloride adduct formation. The structure of the supramolecular assemblies forming the reline ions that are observed on CSI mass spectra is rationalized by chemical quantum calculations. The theoretical studies indicate that the ionic network organization is only supported by a maximization of hydrogen bonds of the chlorides with the hydroxyl and methyl moieties of the cholinium cations and the amino groups of urea. The studies of gas-phase fragmentation of the supra-molecular ionic assemblies detected in CSI-MS are performed using the in-source collision-induced dissociation experiments. The experimental measurements in CSI-MS, interpreted at the light of the molecular modelization results, suggest that the insertion of urea in adducts of chlorides with cholinium cations does not lead to the most stable ions.

Design and Microwave Synthesis of New (5Z) 5-Arylidene-2-thioxo-1,3-thiazolinidin-4-one and (5Z) 2-Amino-5-arylidene-1,3-thiazol-4(5H)-one as New Inhibitors of Protein Kinase DYRK1A.

Here, we report on the synthesis of libraries of new 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones 3 (twenty-two compounds) and new 2-amino-5-arylidene-1,3-thiazol-4(5H)-ones 5 (twenty-four compounds) with stereo controlled Z-geometry under microwave irradiation. The 46 designed final compounds were tested in order to determine their activity against four representative protein kinases (DYR1A, CK1, CDK5/p25, and GSK3α/ß). Among these 1,3-thiazolidin-4-ones, the molecules (5Z) 5-(4-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one 3e (IC50 0.028 µM) and (5Z)-5-benzo[1,3]dioxol-5-ylmethylene-2-(pyridin-2-yl)amino-1,3-thiazol-4(5H)-one 5s (IC50 0.033 µM) were identified as lead compounds and as new nanomolar DYRK1A inhibitors. Some of these compounds in the two libraries have been also evaluated for their in vitro inhibition of cell proliferation (Huh7 D12, Caco2, MDA-MB 231, HCT 116, PC3, and NCI-H2 tumor cell lines). These results will enable us to use the 1,3-thiazolidin-4-one core as pharmacophores to develop potent treatment for neurological or oncological disorders in which DYRK1A is fully involved.

Do Deep Eutectic Solvents Form Uniform Mixtures Beyond Molecular Microheterogeneities?

We have performed small-angle neutron scattering in a momentum transfer range (0.05 < Q < 0.5 Å-1) to study long-range order and concentration fluctuations in deep eutectic solvents (DESs) and their aqueous solutions. Ethaline (choline chloride/ethylene glycol), glycerol/lactic acid, and menthol/decanoic acid mixtures were selected to illustrate individually the case of ionic, nonionic, and hydrophobic mixtures. Carefully designed isotopic labeling was used to emphasize selectively the spatial correlations between the different solvent components. For ethaline DESs and their aqueous solutions, a weak low-Q peak observed only for certain compositions and some partial structure factors revealed the mesoscopic segregation of ethylene glycol molecules that do not participate in the solvation of ionic units, either because they are in excess with respect to the eutectic stoichiometry (1:4 neat ethaline) or substituted by water (4w-ethaline and higher aqueous dilutions). For the nonionic hydrophilic solutions, such a mesoscopic segregation was not observed. This indicates that the better balanced interactions between the three nonionic H-bonded components (water, lactic acid, and glycerol) favor homogeneous mixing. For the hydrophobic DESs, we observed an excess of coherent scattering intensity centered at Q = 0, which could be reproduced by a model of noninteracting spherical domains. Local concentration fluctuations are not excluded either. However, unlike liquid mixtures with a tendency to demix, we have found no evidence of expansion of domains with different compositions to a large scale.

Electron Transfer Kinetics in a Deep Eutectic Solvent.

Electron transfer (ET) kinetic rate constants ks in Ethaline (1:2 choline chloride + ethylene glycol) have been measured for two common redox couples (ferrocene/ferrocenium and ferrocyanide/ferricyanide) on a glassy carbon electrode and compared with ET kinetics in ionic liquids and classical organic solvents in the same conditions (acetonitrile and water). Particular care has been taken to treat ohmic drop in DES. For both couples, we found that ET rate constants are just a little lower than those measured in classical solvents (around 50% or less). These results contrast with ET rates in ionic liquids where electron transfers are considerably slower (100 times lower). Data are discussed as a function of the solvent relaxation time using Marcus Theory for an adiabatic electron transfer.

One-pot synthesis of 5-arylidene-2-imino-4-thiazolidinones under microwave irradiation.

A rapid and easy solvent free one-pot synthesis of 5-arylidene-2-imino-4-thiazolidinones by condensation of the thioureas with chloroacetic acid and an aldehyde under microwave-irradiation is described.

Sample preparation for an optimized extraction of localized metabolites in lichens: Application to Pseudevernia furfuracea.

Lichens are symbiotic organisms known for producing unique secondary metabolites with attractive cosmetic and pharmacological properties. In this paper, we investigated three standard methods of preparation of Pseudevernia furfuracea (blender grinding, ball milling, pestle and mortar). The materials obtained were characterized by electronic microscopy, nitrogen adsorption and compared from the point of view of extraction. Their microscopic structure is related to extraction efficiency. In addition, it is shown using thalline reactions and mass spectrometry mapping (TOF-SIMS) that these metabolites are not evenly distributed throughout the organism. Particularly, atranorin (a secondary metabolite of interest) is mainly present in the cortex of P. furfuracea. Finally, using microwave assisted extraction (MAE) we obtained evidence that an appropriate preparation can increase the extraction efficiency of atranorin by a factor of five.

Synthesis and toxicity evaluation of hydrophobic ionic liquids for volatile organic compounds biodegradation in a two-phase partitioning bioreactor.

Synthesis of several hydrophobic ionic liquids (ILs), which might be selected as good candidates for degradation of hydrophobic volatile organic compounds in a two-phase partitioning bioreactor (TPPB), were carried out. Several bioassays were also realized, such as toxicity evaluation on activated sludge and zebrafish, cytotoxicity, fluoride release in aqueous phase and biodegradability in order to verify their possible effects in case of discharge in the aquatic environment and/or human contact during industrial manipulation. The synthesized compounds consist of alkylimidazoliums, functionalized imidazoliums, isoqinoliniums, triazoliums, sulfoniums, pyrrolidiniums and morpholiniums and various counter-ions such as: PF6(-), NTf2(-) and NfO(-). Toxicity evaluation on activated sludge of each compound (5% v/v of IL) was assessed by using a glucose uptake inhibition test. Toxicity against zebrafish and cytotoxicity were evaluated by the ImPACCell platform of Rennes (France). Fluoride release in water was estimated by regular measurements using ion chromatography equipment. IL biodegradability was determined by measuring BOD28 of aqueous samples (compound concentration,1mM). All ILs tested were not biodegradable; while some of them were toxic toward activated sludge. Isoquinolinium ILs were toxic to human cancerous cell lines. Nevertheless no toxicity was found against zebrafish Danio rerio. Only one IL released fluoride after long-time agitation.

Synthesis of N,N'-bis(5-arylidene-4-oxo-3,5-dihydro-4H-imidazol-2-yl)diamines bearing various linkers and biological evaluation as potential inhibitors of kinases.

The synthesis in 4 steps of new N,N'-bis(5-arylidene-4-oxo-3,5-dihydro-4H-imidazol-2-yl)diamines issued from various symmetric primary diamines as linkers was reported. The key step of our strategy has been the sulphur/nitrogen displacement of (5Z)-5-arylidene-2-ethylsulfanyl-3,5-dihydro-4H-imidazol-4-ones 6 with respectively ethylenediamine 7a, piperazine 7b and N,N'-bis(3-aminopropyl)piperazine 7c using solvent-free reaction conditions under microwave irradiation with retention of configuration. These compounds were tested for their kinase inhibitory potencies toward four kinases (GSK-3α/ß, DYRK1A, CLK1 and CLK3).

Synthesis of New N,N'-Bis(5-arylidene-4-oxo-4,5-dihydrothiazolin-2-yl)piperazine Derivatives Under Microwave Irradiation and Preliminary Biological Evaluation.

New N,N'-bis(5-arylidene-4-oxo-4,5-dihydrothiazoline-2-yl)diamine derivatives 5 were prepared in two steps from rhodanine and piperazine, or 1,4-bis(3-amino-propyl)piperazine, under microwave reaction conditions with retention of configuration. Some of these compounds were tested for in vitro antiproliferative activities and for their kinase inhibitory potencies towards six kinases (CDK5/p25, GSK3α/ß, DYRK1A, DYRK2, CLK1, and CLK2). The compound 5d showed nanomolar activity towards DYRK1A kinase (IC(50) = 0.041 µM).

Ionic liquids based microwave-assisted extraction of lichen compounds with quantitative spectrophotodensitometry analysis.

Ionic liquids based extraction method has been applied to the effective extraction of norstictic acid, a common depsidone isolated from Pertusaria pseudocorallina, a crustose lichen. Five 1-alkyl-3-methylimidazolium ionic liquids (ILs) differing in composition of alkyl chain and anion were investigated for extraction efficiency. The extraction amount of norstictic acid was determined after recovery on HPTLC with a spectrophotodensitometer. The proposed approaches (IL-MAE and IL-heat extraction (IL-HE)) have been evaluated in comparison with usual solvents such as tetrahydrofuran in heat-reflux extraction and microwave-assisted extraction (MAE). The results indicated that both the characteristics of the alkyl chain and anion influenced the extraction of polyphenolic compounds. The sulfate-based ILs [C(1)mim][MSO(4)] and [C(2)mim][ESO(4)] presented the best extraction efficiency of norstictic acid. The reduction of the extraction times between HE and MAE (2 h-5 min) and a non-negligible ratio of norstictic acid in total extract (28%) supports the suitability of the proposed method. This approach was successfully applied to obtain additional compounds from other crustose lichens (Pertusaria amara and Ochrolechia parella).

Synthesis and preliminary biological evaluation of new derivatives of the marine alkaloid leucettamine B as kinase inhibitors.

New derivatives of the marine alkaloid leucettamine B were prepared in five steps with overall yields ranging from 23 to 30%. The key step of our strategy has been the sulfur/nitrogen displacement under solvent-free microwave irradiation of (5Z) 5-benzo[1,3]-dioxo-5-ylmethylene-2-ethylsulfanyl-3,5-dihydroimidazol-4-one 3 with a mono-protected ethylenediamine 2. After deprotection of the N-Boc group, the amino derivative of leucettamine B 5 was subjected to reductive amination in two steps with retention of configuration of the double bond, to lead to eight new analogs of leucettamine B. The effect of these compounds on CK1alpha/beta, CDK5/p25, and GSK-3alpha/beta were investigated.