Anti-Nociceptive Effects of Sphingomyelinase and Methyl-Beta-Cyclodextrin in the Icilin-Induced Mouse Pain Model.

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.

Catalyst- and excess reagent recycling in aza-Michael additions.

16α-Azolyl-pregnenolone derivatives were prepared via 2-butyl-1,1,3,3-tetramethylguanidine (n-Bu-TMG) catalysed aza-Michael addition of 16-dehydropregnenolone (16-DHP) carried out in [bmim][BF4]. The application of the guanidine base and the imidazolium ionic liquid made it possible to recycle not only the catalyst/solvent mixture but also the excess of the N-heterocyclic reagent. By the introduction of CO2 at the end of the reaction, both the guanidine base and the unreacted (excess) reagent could be converted into ionic species that remained dissolved in the ionic liquid phase, while the steroid components were extracted with an apolar solvent. After the removal of CO2, the experiment could be repeated by the addition of the steroid substrate and only an equimolar amount of the N-heterocycle. The methodology was successfully applied to a number of N-heterocycles, such as imidazole, pyrazole, 1,2,3- and 1,2,4-triazoles, and benzimidazole. Indazole and indole could also be converted into the corresponding products, but a stronger base had to be used to obtain a recyclable system.

Urea-Functionalized Heterocycles: Structure, Hydrogen Bonding and Applications.

Ureido-heterocycles exhibiting different triple- and quadruple H-bonding patterns are useful building blocks in the construction of supramolecular polymers, self-healing materials, stimuli-responsive devices, catalysts and sensors. The heterocyclic group may provide hydrogen bond donor/acceptor sites to supplement those in the urea core, and they can also bind metals and can be modified by pH, redox reactions or irradiation. In the present review, the main structural features of these derivatives are discussed, including the effect of tautomerization and conformational isomerism on self-assembly and complex formation. Some examples of their use as building blocks in different molecular architectures and supramolecular polymers, with special emphasis on biomedical applications, are presented. The role of the heterocyclic functionality in catalytic and sensory applications is also outlined.

A Temperature-Controlled Switch between Fürst-Plattner Rule and Anti-Fürst-Plattner Rule Ring Opening of 2,3-Epoxy-steroids with Various Halide Sources in the Presence of Imidazolium Ionic Liquids.

The ring opening of 2α,3α- and 2ß,3ß-epoxy-5α-androstan-17-one with halide reagents (AlCl3, TMSCl, LiCl, and LiBr) was investigated using imidazolium ionic liquids in the dual role of solvent and catalyst. The application of the ionic liquid was shown to result in an increase in the amount of the unusual diequatorial halohydrins especially at temperatures above 100 °C. With a careful choice of reaction conditions, the latter derivatives could be produced with 43-96% selectivity depending on the nature of the halide ion. Moreover, the usual diaxial products could also be isolated in 70-85% yields by a proper change in the reaction conditions. The reusability of the ionic liquid was demonstrated in both types of reactions. The structures of the products were proved unequivocally by nuclear magnetic resonance (NMR) measurements including two-dimensional (2D) techniques as well as high-resolution mass spectrometry (HRMS). Based on quantum chemical calculations, the effect of the ionic liquid could be explained by the stabilization of the transition state leading to the diequatorial product.

Antinociceptive Effects of Lipid Raft Disruptors, a Novel Carboxamido-Steroid and Methyl ß-Cyclodextrin, in Mice by Inhibiting Transient Receptor Potential Vanilloid 1 and Ankyrin 1 Channel Activation.

Transient Receptor Potential Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons, and play an integrative role in pain processing and inflammatory functions. Lipid rafts are liquid-ordered plasma membrane microdomains rich in cholesterol, sphingomyelin, and gangliosides. We earlier proved that lipid raft disintegration by cholesterol depletion using a novel carboxamido-steroid compound (C1) and methyl ß-cyclodextrin (MCD) significantly and concentration-dependently inhibit TRPV1 and TRPA1 activation in primary sensory neurons and receptor-expressing cell lines. Here we investigated the effects of C1 compared to MCD in mouse pain models of different mechanisms. Both C1 and MCD significantly decreased the number of the TRPV1 activation (capsaicin)-induced nocifensive eye-wiping movements in the first hour by 45% and 32%, respectively, and C1 also in the second hour by 26%. Furthermore, C1 significantly decreased the TRPV1 stimulation (resiniferatoxin)-evoked mechanical hyperalgesia involving central sensitization processes, while its inhibitory effect on thermal allodynia was not statistically significant. In contrast, MCD did not affect these resiniferatoxin-evoked nocifensive responses. Both C1 and MCD had inhibitory action on TRPA1 activation (formalin)-induced acute nocifensive reactions (paw liftings, lickings, holdings, and shakings) in the second, neurogenic inflammatory phase by 36% and 51%, respectively. These are the first in vivo data showing that our novel lipid raft disruptor carboxamido-steroid compound exerts antinociceptive and antihyperalgesic effects by inhibiting TRPV1 and TRPA1 ion channel activation similarly to MCD, but in 150-fold lower concentrations. It is concluded that C1 is a useful experimental tool to investigate the effects of cholesterol depletion in animal models, and it also might open novel analgesic drug developmental perspectives.

Palladium nanoparticles on a pyridinium supported ionic liquid phase: a recyclable and low-leaching palladium catalyst for aminocarbonylation reactions.

A new SILP (Supported Ionic Liquid Phase) palladium catalyst was prepared and characterized by 13C and 29Si CP MAS NMR, DTG, FTIR and TEM. The presence of the grafted pyridinium cations on the surface of the support was found to result in the formation of highly dispersed Pd nanoparticles with their diameter in the range of 1-2 nm. The catalyst was proved to be active not only in the aminocarbonylation of some model compounds but also in the synthesis of active pharmaceutical ingredients. Catalyst recycling and palladium leaching studies were carried out for the first time in aminocarbonylations leading to CX-546(1-(1,4-benzodioxan-6-ylcarbonyl)piperidine), Moclobemide, Nikethamide and a precursor of Finasteride. The latter reaction proves that not only aryl iodides but also an iodoalkene can be converted into the products with the help of the heterogeneous catalyst. The results show that the conditions should be always fine-tuned in the reactions of different substrates to achieve optimal results. Palladium loss was also observed to depend considerably on the nature of the reaction partners.

Steroidal ferrocenes as potential enzyme inhibitors of the estrogen biosynthesis.

The potential inhibitory effect of diverse triazolyl-ferrocene steroids on key enzymes of the estrogen biosynthesis was investigated. Test compounds were synthesized via copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes using our efficient methodology published previously. Inhibition of human aromatase, steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities was investigated with in vitro radiosubstrate incubations. Some of the test compounds were found to be potent inhibitors of the STS. A compound bearing ferrocenyl side chain on the C-2 displayed a reversible inhibition, whereas C-16 and C-17 derivatives displayed competitive irreversible binding mechanism toward the enzyme. 17α-Triazolyl-ferrocene derivatives of 17ß-estradiol exerted outstanding inhibitory effect and experiments demonstrated a key role of the ferrocenyl moiety in the enhanced binding affinity. Submicromolar IC50 and Ki parameters enroll these compounds to the group of the most effective STS inhibitors published so far. STS inhibitory potential of the steroidal ferrocenes may lead to the development of novel compounds able to suppress in situ biosynthesis of 17ß-estradiol in target tissues.

Carboxamido steroids inhibit the opening properties of transient receptor potential ion channels by lipid raft modulation.

Transient Receptor Potential (TRP) cation channels, like the TRP Vanilloid 1 (TRPV1) and TRP Ankyrin 1 (TRPA1), are expressed on primary sensory neurons. These thermosensor channels play a role in pain processing. We have provided evidence previously that lipid raft disruption influenced the TRP channel activation, and a carboxamido-steroid compound (C1) inhibited TRPV1 activation. Therefore, our aim was to investigate whether this compound exerts its effect through lipid raft disruption and the steroid backbone (C3) or whether altered position of the carboxamido group (C2) influences the inhibitory action by measuring Ca2+ transients on isolated neurons and calcium-uptake on receptor-expressing CHO cells. Membrane cholesterol content was measured by filipin staining and membrane polarization by fluorescence spectroscopy. Both the percentage of responsive cells and the magnitude of the intracellular Ca2+ enhancement evoked by the TRPV1 agonist capsaicin were significantly inhibited after C1 and C2 incubation, but not after C3 administration. C1 was able to reduce other TRP channel activation as well. The compounds induced cholesterol depletion in CHO cells, but only C1 induced changes in membrane polarization. The inhibitory action of the compounds on TRP channel activation develops by lipid raft disruption, and the presence and the position of the carboxamido group is essential.

Synthesis of 16α-amino-pregnenolone derivatives via ionic liquid-catalyzed aza-Michael addition and their evaluation as C17,20-lyase inhibitors.

Aza-Michael addition of 16-dehydropregnenolone was studied in the presence of a basic ionic liquid, [DBU][OAc] as catalyst and solvent. The reaction was carried out using different primary and secondary amines as N-nucleophiles. The products were obtained in moderate to good yields and were characterized by 1H and 13C NMR, MS and IR. The ionic liquid was found to be an efficient and recyclable catalyst that was reused five times. The products were investigated for the inhibition of in vitro C17,20-lyase activity and displayed moderate inhibitory effect.

Synthesis of novel 13α-18-norandrostane-ferrocene conjugates via homogeneous catalytic methods and their investigation on TRPV1 receptor activation.

13α-Steroid-ferrocene derivatives were synthesized via two reaction pathways starting from an unnatural 16-keto-18-nor-13α-steroid. The unnatural steroid was converted to ferrocene derivatives via copper-catalyzed azide-alkyne cycloaddition or palladium-catalyzed aminocarbonylation. 16-Azido- and 16-N-(prop-2-ynyl)-carboxamido-steroids were synthesized as starting materials for azide-alkyne cycloaddition with the appropriate ferrocene derivatives. Based on our earlier work, aminocarbonylation of 16-iodo-16-ene and 16-iodo-15-ene derivatives was studied with ferrocenylmethylamine. The new products were obtained in moderate to good yields and were characterized by (1)H and (13)C NMR, IR and MS. The solid state structure of the starting material 13α-18-norandrostan-16-one and two carboxamide products were determined by X-ray crystallography. Evidences were provided that the N-propargyl-carboxamide compound as well as its ferrocenylmethyltriazole derivative are able to decrease the activation of TRPV1 receptor on TRG neurons.

The use of supported acidic ionic liquids in organic synthesis.

Catalysts obtained by the immobilisation of acidic ionic liquids (ILs) on solid supports offer several advantages compared to the use of catalytically active ILs themselves. Immobilisation may result in an increase in the number of accessible active sites of the catalyst and a reduction of the amount of the IL required. The ionic liquid films on the carrier surfaces provide a homogeneous environment for catalytic reactions but the catalyst appears macroscopically as a dry solid, so it can simply be separated from the reaction mixture. As another advantage, it can easily be applied in a continuous fixed bed reactor. In the present review the main synthetic strategies towards the preparation of supported Lewis acidic and Brønsted acidic ILs are summarised. The most important characterisation methods and structural features of the supported ionic liquids are presented. Their efficiency in catalytic reactions is discussed with special emphasis on their recyclability.

Synthesis of novel 13α-18-nor-16-carboxamido steroids via a palladium-catalyzed aminocarbonylation reaction.

13α-18-nor-16-Carboxamido steroids were synthesized via a palladium-catalyzed aminocarbonylation reaction of the corresponding iodoalkenes. The starting material was an unnatural 13α-16-keto steroid, obtained by a Wagner-Meerwein rearrangement of a 16α,17α-epoxide in the presence of [BMIM][BF4]. The 13α-16-keto steroid was converted to a mixture of 16-iodo-16-ene and 16-iodo-15-ene derivatives in two steps by Barton's methodology. Aminocarbonylation of the steroidal alkenyl iodides was carried out using different primary and secondary amines as nucleophiles. The products, 16-carboxamido-16-ene and 16-carboxamido-15-ene derivatives, were obtained in good yields and were characterized by (1)H and (13)C NMR, IR and MS. The reduction of the above two unsaturated carboxamides resulted in the same product, 17α-methyl-16α-carboxamido-androstane.

Synthesis of ferrocene-labeled steroids via copper-catalyzed azide-alkyne cycloaddition. Reactivity difference between 2ß-, 6ß- and 16ß-azido-androstanes.

Copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes were found to be an efficient methodology for the synthesis of ferrocene-labeled steroids. At the same time, a great difference between the reactivity of 2ß- or 16ß-azido-androstanes and a sterically hindered 6ß-azido steroid toward both ferrocenyl-alkynes and simple alkynes, such as phenylacetylene, 1-octyne, propargyl acetate and methyl propiolate, was observed.

Synthesis of steroid-ferrocene conjugates of steroidal 17-carboxamides via a palladium-catalyzed aminocarbonylation--copper-catalyzed azide-alkyne cycloaddition reaction sequence.

Steroids with the 17-iodo-16-ene functionality were converted to ferrocene labeled steroidal 17-carboxamides via a two step reaction sequence. The first step involved the palladium-catalyzed aminocarbonylation of the alkenyl iodides with prop-2-yn-1-amine as the nucleophile in the presence of the Pd(OAc)(2)/PPh(3) catalyst system. In the second step, the product N-(prop-2-ynyl)-carboxamides underwent a facile azide-alkyne cycloaddition with ferrocenyl azides in the presence of CuSO(4)/sodium ascorbate to produce the steroid-ferrocene conjugates. The new compounds were obtained in good yield and were characterized by (1)H and (13)C NMR, IR, MS and elemental analysis.

Ionic liquid-promoted Wagner-Meerwein rearrangement of 16α,17α-epoxyandrostanes and 16α,17α-epoxyestranes.

Ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim](+)[PF(6)](-)) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim](+)[BF(4)](-)) were found to promote an unusual Wagner-Meerwein rearrangement of steroidal 16α,17α-epoxides leading to unnatural 13-epi-18-nor-16-one derivatives as the main products. These compounds were isolated in good to excellent yields. 16α-Hydroxy-Δ(13)-18-norsteroids, the results of the usual rearrangement, were obtained as minor components of the reaction mixtures. The ionic liquid [bmim](+)[PF(6)](-) was shown to induce C-ring aromatization of 16α,17α-epoxyestranes due to the formation of HF, the hydrolysis product of [PF(6)](-). Increasing amounts of HF and [PO(2)F(2)](-) were detected by (19)F and (31)P NMR when the ionic liquid was reused. The structures of the steroidal products, 16-oxo-18-nor-13α-steroid derivatives, 16α-hydroxy-Δ(13)-18-norsteroids, and C-aromatic compounds were determined by two-dimensional NMR techniques and high-resolution mass spectrometry (HRMS). The ionic liquids were recirculated efficiently.

Facile ring opening of 2,3-epoxy-steroids with aromatic amines in ionic liquids.

Efficient ring opening of steroidal 2,3-epoxides with stoichiometric amount of aromatic amines has been carried out using an ionic liquid ([bmim](+)[BF(4)](-)) both as solvent and catalyst. The reactions were completely regio- and stereoselective in each case. The aminoalcohol products have chair conformations in ring A. The ionic liquid-mediated ring opening can efficiently be carried out with aliphatic amines like morpholine as well.

Unexpected reactivity difference between iodo-alkene moieties of steroids possessing remote lactame or cycloalkane structural units: a theoretical approach.

The steroidal ring A possessing cyclohexane vs. delta-valerolactame structures shows a 'long-range effect' on the reactivity of the 'iodovinyl' functionality of the ring D. The strikingly different reactivity of 17-iodo-16-ene functionality of steroids is explained on the basis of the redistribution of kinetic energy of the system. The vibrational energy localized on the 'iodovinyl' moiety proved to be determinant in the CI bond-breaking process. The appearance of the lactame moiety in the ring A supports a redistribution of the kinetic energy on the molecule. As a result, a drastic decrease in C-I bond stretching has been obtained, which could diminish the C-I bond breaking, and therefore, results in decreased reactivity of the iodovinyl moiety in agreement with experimental findings.

Microwave-assisted Stille-coupling of steroidal substrates.

Steroidal dienes were synthesised by Stille-coupling of the corresponding alkenyl iodides with vinyltributyltin under microwave irradiation in a domestic microwave oven in drastically reduced reaction times. Rate acceleration was observed also in the one-pot Stille-coupling-Diels-Alder reaction of 17-iodo-5alpha-androst-16-ene. Stereoselectivity of cycloaddition was slightly improved with diethyl maleate as the dienophile, compared to that achieved with thermal heating.

Synthesis of steroidal diacyl hydrazines and their 1,3,4-oxadiazole derivatives.

Homogeneous catalytic hydrazinocarbonylation of some steroid derivatives possessing iodo-alkenyl moiety (17-iodo-androst-16-ene 1, 17-iodo-3-methoxy-estra-1,3,5(10),16-tetraene 2, 17-iodo-4-aza-4-methyl-androst-16-en-3-one 3 and 17-iodo-6beta-hydroxy-3alpha,5alpha-cycloandrost-16-ene 4) were carried out in the presence of a palladium catalyst, a base and acetic or benzoic hydrazide as the nucleophilic reagent. The corresponding N-acetamido-carbamoyl 1a-4a or N-benzamido-carbamoyl derivatives 1b-4b were obtained in high yields. Some of these derivatives served as starting materials for the synthesis of new steroidal 1,3,4-oxadiazole compounds.