[Developments of Profluorescent Nitroxide Probes for Highly Sensitive and Selective Detection of Biological Redox Molecules].

Disruption of the redox balance in vivo is closely involved in the development of various diseases associated with oxidative stress. Therefore, methods for the in vivo analysis of antioxidants and free radicals are essential to elucidate the pathogenic mechanisms of such diseases. Although profluorescent nitroxide probes can be used to evaluate redox molecules with high sensitivity, these probes have low selectivity. Recently, we developed two profluorescent nitroxide probes, 15-((9-(ethylimino)-10-methyl-9Hbenzo[a]phenoxazin-5-yl)amino)-3,11-dioxa-7-azadispiro-hexadecan-7-yloxyl (Nile-DiPy) and 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen), which had high sensitivity and selectivity toward ascorbic acid and lipid-derived radicals, respectively. These probes can react sensitively and selectively to each target molecule and can be used in animal experiments. In this paper, we review the design strategies and application of these profluorescent nitroxide probes.

Solid-gas reactions for nitroxyl (HNO) generation in the gas phase.

We present a novel nitroxyl (HNO) generation method, which avoids the need of using a liquid system or extreme experimental conditions. This method consists of the reaction between a gaseous base and an HNO donor (Piloty's acid) in the solid phase, allowing the formation of gaseous HNO in a fast and economical way. Detection of HNO was carried out indirectly, measuring the nitrous oxide (N2O) byproduct of HNO dimerization using infrared spectroscopy, and directly, using mass spectrometry techniques and an electrochemical HNO sensor.

Green Efficient One-Pot Synthesis and Separation of Nitrones in Water Assisted by a Self-Assembled Nanoreactor.

This article reports an alternative method for preparing nitrones using a tetrahedral capsule as a nanoreactor in water. Using the hydrophobic cavity of the capsule allowed us to reduce the reaction times and easily separate the nitrones from the reaction mixture, obtaining reaction yields equal or comparable to those obtained with the methods already reported. Furthermore, at the basis of this methodology, there is an eco-friendly approach carried out that can certainly be extended to other synthesis methods for the preparation of other substrates by exploiting various types of macrocyclic hosts, suitably designed and widely used in supramolecular chemistry.

Effects of Glutathione and Histidine on NO Release from a Dimeric Dinitrosyl Iron Complex (DNIC).

Rates of NO release from synthetic dinitrosyl iron complexes (DNICs) are shown to be responsive to coordination environments about iron. The effect of biologically relevant cellular components, glutathione and histidine, on the rate of NO release from a dimeric, "Roussin's Red Ester", DNIC with bridging µ-S thioglucose ligands, SGlucRRE or [(µ-SGluc)Fe(NO)2]2 (SGluc = 1-thio-ß-d-glucose tetraacetate), was investigated. From the Griess assay and X-band EPR data, decomposition of the product from the histidine-cleaved dimer, [(SGluc)(NHis)Fe(NO)2], generated Fe(III) and increased the NO release rate in aqueous media when compared to the intact SGlucRRE precursor. In contrast, increasing concentrations of exogenous glutathione generated the stable [(SGluc)(GS)Fe(NO)2]- anion and depressed the rate of NO release. Both of the cleaved, monomeric intermediates were characterized with ESI-MS, EPR, and FT-IR spectroscopies. On the basis of the Griess assay coupled with data from an intracellular fluorometric probe, both the monomeric DNICs and dimeric SGlucRRE diffuse into smooth muscle cells, chosen as appropriate archetypes of vascular relaxation, and release their NO payload. Ultimately, this work provides insight into tuning NO release beyond the design of DNICs, through the incubation with safe, accessible biological molecules.

Doxyl Nitroxide Spin Probes Can Modify Toxicity of Doxorubicin towards Fibroblast Cells.

The biological properties of doxyl stearate nitroxides (DSs): 5-DS, Met-12-DS, and 16-DS, commonly used as spin probes, have not been explored in much detail so far. Furthermore, the influence of DSs on the cellular changes induced by the anticancer drug doxorubicin (DOX) has not yet been investigated. Therefore, we examined the cytotoxicity of DSs and their ability to induce cell death and to influence on fluidity and lipid peroxidation (LPO) in the plasma membrane of immortalised B14 fibroblasts, used as a model neoplastic cells, susceptible to DOX-induced changes. The influence of DSs on DOX toxicity was also investigated and compared with that of a natural reference antioxidant α-Tocopherol. By employing the trypan blue exclusion test and double fluorescent staining, we found a significant level of cytotoxicity for DSs and showed that their ability to induce apoptosis and modify plasma membrane fluidity (measured fluorimetrically) is more potent than for α-Tocopherol. The most cytotoxic nitroxide was 5-DS. The electron paramagnetic resonance (EPR) measurements revealed that 5-DS was reduced in B14 cells at the fastest and Met-12-DS at the slowest rate. In the presence of DOX, DSs were reduced slower than alone. The investigated compounds, administered with DOX, enhanced DOX-induced cell death and demonstrated concentration-dependent biphasic influence on membrane fluidity. A-Tocopherol showed weaker effects than DSs, regardless the mode of its application-alone or with DOX. High concentrations of α-Tocopherol and DSs decreased DOX-induced LPO. Substantial cytotoxicity of the DSs suggests that they should be used more carefully in the investigations performed on sensitive cells. Enhancement of DOX toxicity by DSs showed their potential to act as chemosensitizers of cancer cells to anthracycline chemotherapy.

Homo-Tris-Nitrones Derived from α-Phenyl-N-tert-butylnitrone: Synthesis, Neuroprotection and Antioxidant Properties.

Herein we report the synthesis, antioxidant and neuroprotective power of homo-tris-nitrones (HTN) 1-3, designed on the hypothesis that the incorporation of a third nitrone motif into our previously identified homo-bis-nitrone 6 (HBN6) would result in an improved and stronger neuroprotection. The neuroprotection of HTNs1-3, measured against oligomycin A/rotenone, showed that HTN2 was the best neuroprotective agent at a lower dose (EC50 = 51.63 ± 4.32 µM), being similar in EC50 and maximal activity to α-phenyl-N-tert-butylnitrone (PBN) and less potent than any of HBNs 4-6. The results of neuroprotection in an in vitro oxygen glucose deprivation model showed that HTN2 was the most powerful (EC50 = 87.57 ± 3.87 µM), at lower dose, but 50-fold higher than its analogous HBN5, and ≈1.7-fold less potent than PBN. HTN3 had a very good antinecrotic (IC50 = 3.47 ± 0.57 µM), antiapoptotic, and antioxidant (EC50 = 6.77 ± 1.35 µM) profile, very similar to that of its analogous HBN6. In spite of these results, and still being attractive neuroprotective agents, HTNs 2 and 3 do not have better neuroprotective properties than HBN6, but clearly exceed that of PBN.

Recent Advances on Nitrones Design for Stroke Treatment.

The recent advances of tetramethylpyrazine nitrones and quinolylnitrones for the treatment of stroke have been reviewed and compared with other agents, showing promising therapeutic applications. As a result of a functional transformation of natural product ligustrazine, (Z)-N-tert-butyl-1-(3,5,6-trimethylpyrazin-2-yl)methanimine oxide (6) is a multitarget small nitrone showing potent thrombolytic activity and free radicals scavenging power, in addition to nontoxicity and blood-brain barrier permeability. Similarly, antioxidant (Z)-N-tert-butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine oxide (17) is a novel agent for cerebral ischemia therapy as it is able to scavenge different types of free radical species, showing strong neuroprotection and reduced infarct size.

Synthesis, antioxidant properties and neuroprotection of α-phenyl-tert-butylnitrone derived HomoBisNitrones in in vitro and in vivo ischemia models.

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1'Z)-1,1'-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50 = 1.24 ± 0.39 µM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity = 95.8 ± 3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.

Synthesis of Nitroxide Diradical Using a New Approach.

A new synthetic pathway to diradical organic systems is proposed. The effectiveness of this approach was exemplified by the synthesis of a new nitroxide diradical. An interaction of perfluorobiphenyl with lithium tert-butylamide, followed by oxidation of the thusly formed N4,N4'-di-tert-butyl-2,2',3,3',5,5',6,6'-octafluorobiphenyl-4,4'-diamine with meta-chloroperoxybenzoic acid, led to the polyfluorinated nitroxide diradical, N,N'-(perfluorobiphenyl-4,4'-diyl)bis(N-tert-butyl(oxyl)amine), with a good total yield. The polyfluorinated diradical is stable and can be isolated in free form and completely characterized. The structure of the nitroxide diradical was proved by single-crystal X-ray diffraction analysis. According to the X-ray diffraction data, the diradical is considerably twisted: dihedral angles between the planes of the nitroxide groups and aromatic cycles are 65.1° and 69.5°, and between aromatic cycles 52.6°. Quantum chemical calculations predict well-balanced size of both intramolecular and intermolecular exchange interactions with J from -2.65 to -1.14 cm-1.

Syntheses and Reactions of Pyrroline, Piperidine Nitroxide Phosphonates.

Organophosphorus compounds occupy a significant position among the plethora of organic compounds, but a limited number of paramagnetic phosphorus compounds have been reported, including paramagnetic phosphonates. This paper describes the syntheses and further transformations of pyrroline and piperidine nitroxide phosphonates by well-established methods, such as the Pudovik, Arbuzov and Horner-Wadsworth-Emmons (HWE) reactions. The reaction of paramagnetic a-bromoketone produced a vinylphosphonate in the Perkow reaction. Paramagnetic a-hydroxyphosphonates could be subjected to oxidation, elimination and substitution reactions to produce various paramagnetic phosphonates. The synthesized paramagnetic phosphonates proved to be useful synthetic building blocks for carbon-carbon bond-forming reactions in the Horner-Wadsworth-Emmons olefination reactions. The unsaturated compounds achieved could be transformed into various substituted pyrroline nitroxides, proxyl nitroxides and paramagnetic polyaromatics. The Trolox® equivalent antioxidant capacity (TEAC) of new phosphonates was also screened, and tertiary a-hydroxyphosphonatate nitroxides exhibited remarkable antioxidant activity.

The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine.

Azanone (HNO, nitroxyl) is a highly reactive molecule that, in the past few years, has drawn significant interest because of its pharmacological properties. However, the understanding of how, when, and where endogenous HNO is produced remains a matter of discussion. In this study, we examined the ability of myoglobin to produce HNO via the peroxidation of hydroxylamine with H2O2 using both experimental and computational approaches. The production of HNO was confirmed using an azanone selective electrochemical method and by the detection of N2O using FTIR. The catalytic capacity of myoglobin was characterized by the determination of the turnover number. The reaction kinetics of the hydroxylamine peroxidation were studied by both electrochemical and UV-vis methods. Further evidence about the reaction mechanism was obtained by EPR spectroscopy. Additionally, quantum mechanical/molecular mechanics experiments were performed to calculate the energy barrier for HNO production and to gain insight into the reaction mechanism. Our results confirm that myoglobin produces HNO via the peroxidation of hydroxylamine with a great catalytic capacity. In addition, our mechanistic study allows us to state that the Mb ferryl state is the most likely intermediate that reacts with hydroxylamine, yielding important evidence for endogenous HNO generation.

Human Serum Albumin Labelled with Sterically-Hindered Nitroxides as Potential MRI Contrast Agents.

Four albumin-nitroxide conjugates were prepared and tested as metal-free organic radical contrast agents (ORCAs) for magnetic resonance imaging (MRI). Each human serum albumin (HSA) carrier bears multiple nitroxides conjugated via homocysteine thiolactones. These molecular conjugates retain important physical and biological properties of their HSA component, and the resistance of their nitroxide groups to bioreduction was retained or enhanced. The relaxivities are similar for these four conjugates and are much greater than those of their individual components: the HSA or the small nitroxide molecules. This new family of conjugates has excellent prospects for optimization as ORCAs.

(Pyrrole-2,5-Diyl)-Bis(Nitronyl Nitroxide) and-Bis(Iminonitroxide): Specific Features of the Synthesis, Structure, and Magnetic Properties.

In contrast to diradicals connected by alternant hydrocarbons, only a few studies have addressed diradicals connected by nonalternant hydrocarbons and their heteroatom derivatives. Here, the synthesis, structure, and magnetic properties of pyrrole-2,5-diyl-linked bis(nitronyl nitroxide) and bis(iminonitroxide) diradicals are described. The diradicals show characteristic electron spin resonance spectra in dilute glassy solutions, from which conclusions about the presence of distinct conformations, their symmetry, and interspin distance were made. X-ray diffraction analysis of the diradicals revealed that paramagnetic moieties lie in the plane of the pyrrole ring, because of the formation of an intramolecular hydrogen bond, ONO…HN, with O…H distances of 2.15-2.23 Å. The N-O groups participating in the formation of H-bonds have greater bond lengths (~1.29 Å) as compared with nonparticipating groups (~1.27 Å). The nitronyl nitroxide and iminonitroxide diradicals showed an intramolecular antiferromagnetic interaction, with J = -77.3 and -22.2 cm-1, respectively (H = -2JS1S2).

Protective effects of two novel nitronyl nitroxide radicals on heart failure induced by hypobaric hypoxia.

AIMS: Hypobaric hypoxia (HH), linked to oxidative stress, impairs cardiac function. We synthesized a novel nitronyl nitroxide radical, an HPN derivative (HEPN) and investigated the protective effects of HEPN and HPN against HH-induced heart injury in mice and the underlying mechanisms of action. MAIN METHODS: Mice were administered with HPN (200 mg/kg) or HEPN (200 mg/kg) 30 min before exposed to HH. The cardiac function was measured. Serum AST, CK, LDH and cTnI were estimated. Heart tissue oxidase activity, SOD, CAT, GSH-Px, ROS and MDA were estimated. ATP content, Na+/K+-ATPase and Ca2+/Mg2+-ATPase activity was measured. The expression of HIF-1, VEGF, Nrf2, HO-1, Bax, Bcl-2, Caspase-3 was estimated. KEY FINDINGS: Results showed that pretreatment with HEPN or HPN led to a dramatic decrease in the activity of biochemical markers AST, CK, LDH and cTnI in murine serum. They increased the activity of SOD, CAT and GSH-Px and reduced the level of ROS and MDA in the hearts of mice. HEPN and HPN could increase the expression of Nrf2 and OH-1. They could maintain the ATPase activity. The Bax and Caspase-3 expression as well as the ratio of Bax/Bcl-2 were significantly downregulated and the Bcl-2 expression was upregulated by HPN or HEPN compared to the HH group. They may attenuate the HH-induced oxidant stress via free radical scavenging activity. SIGNIFICANCE: The present study showed that the nitronyl nitroxide radical HEPN and HPN may be potential therapeutic agents for treatment of HH-induced cardiac dysfunction.

Synthesis and spin trapping properties of polystyrene supported trifluoromethylated cyclic nitrones.

Polystyrene supported fluorinated cyclic nitrone spin-traps: Resin-2-HFDMPO (2-hydroxymethyl-2-methyl-5-(trifluoromethyl)-3,4-dihydro-2H-pyrrole-1-oxide) and Resin-2-PFDMPO (2-(3-hydroxypropyl)-2-methyl-5-(trifluoromethyl)-3,4-dihydro-2H-pyrrole 1-oxide) containing a trifluoromethyl pyrroline-N-oxide core were developed to detect free radicals under flow conditions. A continuous flow EPR technique was used to evaluate the spin trapping properties of these tethered nitrones. While both resins trapped radicals, polymer supported nitrone Resin-2-PFDMPO with a longer and more flexible linker showed a more information rich spectrum than Resin-2-HFDMPO.

Antioxidant Activity of Tetranitrosyl Iron Complex with Thiosulfate Ligands and Its Effect on Catalytic Activity of Mitochondrial Enzymes In vitro.

The antioxidant and antiradical properties of the tetra nitrosyl iron complex with thiosulfate ligands (TNIC) were studied in vitro in mouse brain homogenates. It was found for the first time that TNIC is an effective antioxidant. The effect of TNIC on the catalytic activity of mitochondrial enzymes cytochrome c oxidase and monoamine oxidase A was studied. It was shown for the first time that TNIC is an inhibitor of the catalytic activity of cytochrome c oxidase and monoamine oxidase A in animal brain mitochondria in vitro.

Reactions of Nitroxides, Part 17. Synthesis, Fungistatic and Bacteriostatic Activity of Novel Five- and Six-Membered Nitroxyl Selenoureas and Selenocarbamates.

The reactions of 3-isoselenocyanato-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 3-isoselenocyanatomethyl-2,2,5,5-tetramethyl-3-pyrrolidine-1-oxyl, and 4-isoselenocyanato-2,2,6,6-tetramethylpiperidine-1-oxyl with selected amines and alcohols give the corresponding novel nitroxyl selenoureas and selenocarbamates, all bearing a nitroxyl moiety. Synthesized selenoureas and selenocarbamates show significant activity against pathogenic fungi and bacteria. In contrast to piperidine nitroxides, pyrrolidine, five-membered nitroxyl selenoureas and selenocarbamates show excellent antifungal and antibacterial activity against pathogenic fungi and bacteria, respectively.

Synthetic methodology for preparation of dinitrosyl iron complexes.

After the first structural characterization of dinuclear dinitrosyl iron complex (DNIC) in 1958 and discovery of natural dinitrosyl iron unit (DNIU) [Fe(NO)2] in 1964-1965, continued investigations on natural and synthetic DNICs explored their ubiquitous functions as (1) a product for nitrosylation of non-heme Fe proteins and chelatable iron pool, (2) a biological vehicle for iron and nitric oxide, (3) a novel redox-active unit for stabilization and activation of small molecules, (4) an electrocatalyst for water splitting, and (5) a precursor for electrodeposition of Fe-containing hybrid material. From a synthetic chemistry perspective, herein, we summarize four synthetic methodologies for preparation of structure-characterized DNICs in the attempt to attract continued development of unexplored DNICs featuring novel functions. As collected from CCDC database, structure-characterized DNICs can be classified into (1) tetrahedral {Fe(NO)2}9 DNICs with C/N/P/O/S/Se/Cl/Br/I ligation modes, (2) five-/six-coordinate {Fe(NO)2}9 DNICs with N/O ligation modes, (3) tetrahedral {Fe(NO)2}10 DNICs with C/Sn/N/P/O/S/H ligation modes, (4) metallothiolate-bound {Fe(NO)2}9/{Fe(NO)2}10 DNICs, and (5) dinuclear {Fe(NO)2}9-{Fe(NO)2}9, {Fe(NO)2}9-{Fe(NO)2}10, and {Fe(NO)2}10-{Fe(NO)2}10 DNICs with thiolate/alkoxide/pyrazolate/CO bridging ligands. After buildup of the DNIU [Fe(NO)2] using NO, NO+, and NO2- as alternative sources of nitrosyl ligands, ligand substitution and modification reaction of DNICs, redox interconversion between {Fe(NO)2}9 and {Fe(NO)2}10 cores, and transformation between mononuclear and dinuclear DNICs establish the comprehensive pathways to bridge alternative types of DNICs in the chemical library of structure-characterized DNICs. This review on the synthetic methodology for preparation of DNICs will facilitate the incorporation of DNIU [Fe(NO)2] into (bio)materials for potential applications of DNICs in chemistry, catalysis, biology, and biomedicine.

Synthesis, neuroprotective and antioxidant capacity of PBN-related indanonitrones.

In this work six PBN-related indanonitrones 1-6 have been designed, synthesized, and their neuroprotection capacity tested in vitro, under OGD conditions, in SH-SY5Y human neuroblastoma cell cultures. As a result, we have identified indanonitrones 1, 3 and 4 (EC50 = 6.64 ±â€¯0.28 µM) as the most neuroprotective agents, and in particular, among them, indanonitrone 4 was also the most potent and balanced nitrone, showing antioxidant activity in three experiments [LOX (100 µM), APPH (51%), DPPH (36.5%)], being clearly more potent antioxidant agent than nitrone PBN. Consequently, we have identified (Z)-5-hydroxy-N-methyl-2,3-dihydro-1H-inden-1-imine oxide (4) as a hit-molecule for further investigation.

Direct formylation of 2-pyridone core of 3-N-methylcytisine via Duff reaction; synthesis of 9-enyl, 9-ynyl and 9-imino derivatives.

The first direct synthesis of 3-N-methyl-9-formylcytisine via electrophylic formylation is described. It is established, that Vilsmeier-Haack and Gatterman variants of this reaction are unsuccessful in the case with 3-substituted (-)-cytisine derivatives, but Duff procedure (with hexamethylenetetramine in trifluoroacetic acid) gives a possibility to obtain the target pseudo aromatic aldehyde with the 69% yield. Convenient precursors for [4 + 2]- or [3 + 2]-cycloaddition reactions are obtained by means of condensation of synthesized 3-N-methyl-9-formylcytisine with acetone, nitromethane and phosphorous ylides with yields from 70 to 87%. Alternative aprroach to alkenyl products and to 9-alkynyl-3-methylcytisine is realized using the Heck and Sonogashira cross-coupling reactions of methyl vinyl ketone, cyclohexenone or trimethylsilylacetylene with 9-bromo-3-methylcytisine (55, 70 and 60% accordingly). It is shown, that interaction of 3-N-methyl-9-formylcytisine with hydroxylamines leads to corresponding nitrone (93%) and oxime (70%). All individual compounds are isolated by column chromatography and completely characterized on the basis of NMR spectroscopy data.