Electrochemical and EPR Characterization of 1,4-dihydropyridines. Reactivity Towards Alkyl Radicals

This work reports the electrochemical oxidation of a series of three synthesized 4-substituted-1,4-dihydropyridine derivatives in different electrolytic media. Also, an EPR characterization of intermediates and the reactivity of derivatives towards ABAP-derived alkyl radicals are reported. Dynamic, differential pulse and cyclic voltammetry studies on a glassy carbon electrode showed an irreversible single-peak due to the oxidation of the 1,4-dihydropyridine (1,4-DHP) ring via 2-electrons to the corresponding pyridine derivative. Levich plots were linear in different media, indicating that the oxidation process is diffusion-controlled. Calculated diffusion coefficients did not exhibit significant differences between the derivatives in the same medium. The oxidation mechanism follows the general pathway (electron, H + , electron, H + ) with formation of an unstable pyridinium radical. One-electron oxidation intermediate was confirmed with controlled potential electrolysis (CPE) and EPR experiments. On applying N-tert-butyl- &#102 -phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap, these unstable radical intermediates from the oxidation of 1,4-DHP derivatives were intercepted. The final product of the CPE, i.e. pyridine derivative, was identified by GC-MS technique. Direct reactivity of the synthesized compounds towards alkyl radicals was demonstrated by UV-Vis. spectroscopy and GC-MS technique. Results indicate that these derivatives significantly react with the radicals, even compared with a well-known antioxidant drug such as nisoldipine.     

Oxidation of C4-hydroxyphenyl 1,4-dihydropyridines in dimethylsulfoxide and its reactivity towards alkylperoxyl radicals in aqueous medium

This work reports the electrochemical oxidation of three newly synthesized C4-hydroxyphenyl-substituted 1,4-dihydropyridine derivatives in dimethylsulfoxide. The reactivity of the compounds with ABAP-derived alkylperoxyl radicals in aqueous buffer pH 7.4, was also studied. The oxidation mechanism involves the formation of the unstable dihydropyridyl radical, which was confirmed by controlled-potential electrolysis (CPE) and ESR experiments. The final product of the CPE, that is, pyridine derivative, was identified by GC-MS technique for the three derivatives. A direct reactivity of the synthesized compounds toward ABAP-derived alkylperoxyl radicals was found. The pyridine derivative was identified by GC-MS as the final product of the reaction. Results reveal that this type of 1,4-DHPs significantly reacts with the radicals, even compared with commercial 1,4-DHP drugs with a well-known antioxidant ability.     

Structural effects on the reactivity 1,4-dihydropyridines with alkylperoxyl radicals and ABTS radical cation

A series of eight commercial C-4 substituted 1,4-dihydropyridines and other synthesized related compounds were tested for direct potential scavenger effect towards alkylperoxyl radicals and ABTS radical cation in aqueous Britton-Robinson buffer pH7.4. A direct quenching radical species was established. The tested 1,4-dihydropyridines were 8.3-fold more reactive towards alkylperoxyl radicals than ABTS cation radical, expressed by their corresponding kinetic rate constants. Furthermore, NPD a photolyte of nifedipine and the C-4 unsubstituted 1,4-DHP were the most reactive derivatives towards alkylperoxyl radicals. The pyridine derivative was confirmed by GC/MS technique as the final product of reaction. In consequence, the reduction of alkylperoxyl and ABTS radicals by 1,4-dihydropyridines involved an electron transfer process. Also, the participation of the hydrogen of the 1-position appears as relevant on the reactivity. Results of reactivity were compared with Trolox.     

1,4-dihydropyridines: reactivity of nitrosoaryl and nitroaryl derivatives with alkylperoxyl radicals and ABTS radical cation

In the present paper, a direct quenching of radical species by a number of synthesized nitrosoaryl 1,4-dihydropyridines and their parent nitroaryl 1,4-dihydropyridines was determined in aqueous media at pH 7.4. These two series of compounds were compared with the C-4 unsubstituted 1,4-dihydropyridines derivatives and the corresponding C-4 aryl substituted 1,4-dihydropyridines derivatives. Kinetic rate constants were assessed by UV-Vis spectroscopy. Nitrosoaryl derivatives were more reactive than the parent nitroaryl 1,4-dihydropyridines.

Our results strongly support the assumption that the reactivity between the synthesized 1,4-dihydropyridines derivatives with alkylperoxyl radicals involves electron transfer reactions, which is documented by the presence of pyridine as final product of reaction and the complete oxidation of the nitroso group to give rise the nitro group in the case of the nitrosoaryl 1,4-dihydropyridines derivatives.     

1,4-Dihydropyridines: Reactivity of Nitrosoaryl and Nitroaryl Derivatives with Alkylperoxyl Radicals and ABTS Radical Cation

In the present paper, a direct quenching of radical species by a number of synthesized nitrosoaryl 1,4-dihydropyridines and their parent nitroaryl 1,4-dihydropyridines was determined in aqueous media at pH 7.4. These two series of compounds were compared with the C-4 unsubstituted 1,4-dihydropyridines derivatives and the corresponding C-4 aryl substituted 1,4-dihydropyridines derivatives. Kinetic rate constants were assessed by UV-Vis spectroscopy. Nitrosoaryl derivatives were more reactive than the parent nitroaryl 1,4-dihydropyridines.

Our results strongly support the assumption that the reactivity between the synthesized 1,4-dihydropyridines derivatives with alkylperoxyl radicals involves electron transfer reactions, which is documented by the presence of pyridine as final product of reaction and the complete oxidation of the nitroso group to give rise the nitro group in the case of the nitrosoaryl 1,4-dihydropyridines derivatives.     

Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

1,4-Dihydropyridines possess a broad range of biological activities, such as the ability to control the influx of calcium into cells, as well as neuroprotective, antineurodegenerative, cognition and memory enhancing, anti-inflammatory, antiviral and many other properties. Chirality plays an important role in the biological activity of 1,4-dihydropyridines. The chemoenzymatic synthesis of 1,4-dihydropyridine derivatives in enantiopure form as the key intermediates for the synthesis of enantiopure drugs and chiral analogues of symmetrical drugs has become an advantageous alternative to the other synthetic methods. Hydrolytic enzymes, as efficient chemo-, regio- and stereoselective biocatalysts have been successfully applied for the asymmetrisation or kinetic resolution of various 1,4-dihydropyridine derivatives. Several synthetic strategies to overcome the inactivity of hydrolytic enzymes towards 1,4-dihydropyridine carboxylic acids have been developed during the last decade, often based on the introduction of a spacer between an enzymatically labile group and the 1,4-DHP nucleus. Good to excellent enantioselectivities can be obtained by careful optimisation of the reaction temperature and the organic (co)solvent used in the enzymatic transformations.     

Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

1,4-Dihydropyridines possess a broad range of biological activities, such as the ability to control the influx of calcium into cells, as well as neuroprotective, antineurodegenerative, cognition and memory enhancing, anti-inflammatory, antiviral and many other properties. Chirality plays an important role in the biological activity of 1,4-dihydropyridines. The chemoenzymatic synthesis of 1,4-dihydropyridine derivatives in enantiopure form as the key intermediates for the synthesis of enantiopure drugs and chiral analogues of symmetrical drugs has become an advantageous alternative to the other synthetic methods. Hydrolytic enzymes, as efficient chemo-, regio- and stereoselective biocatalysts have been successfully applied for the asymmetrisation or kinetic resolution of various 1,4-dihydropyridine derivatives. Several synthetic strategies to overcome the inactivity of hydrolytic enzymes towards 1,4-dihydropyridine carboxylic acids have been developed during the last decade, often based on the introduction of a spacer between an enzymatically labile group and the 1,4-DHP nucleus. Good to excellent enantioselectivities can be obtained by careful optimisation of the reaction temperature and the organic (co)solvent used in the enzymatic transformations.     

A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.     

Novel cationic amphiphilic 1,4-dihydropyridine derivatives for DNA delivery

In order to find new efficient and safe agents for gene delivery, we have designed and synthesized nine novel single- and double-charged amphiphiles on the base of 1,4-dihydropyridine (1,4-DHP) ring. Some biophysical properties of the amphiphilic dihydropyridines and their complexes with DNA were examined. We investigated the transfer of beta-galactosidase gene into fibroblasts (CV1-P) and retinal pigment epithelial (D 4O7) cell lines in vitro. The structure-property relationships of the compounds were investigated in various ways. The net surface charges of 1,4-DHP liposomes were highly positive (25-49 mV). The double-charged compounds condensed DNA more efficiently than single-charged and the condensation increases with the increasing +/- charge ratio between the carrier and DNA. Double-charged compounds showed also buffering properties at endosomal pH and these compounds were more efficient in transfecting the cells, but transfection efficiency of amphiphiles was cell type-dependent. The length of alkyl chains in double-charged compounds affected the transfection efficacy. The most active amphiphile (compound VI) was double-charged and had two C(12) alkyl chains. At optimal charge ratio (+/- 4), it was 2.5 times more effective than PEI 25 and 10 times better than DOTAP, known efficient polymeric and liposomal transfection agents. Formulation of amphiphiles with DOPE did not change their activities. Our data demonstrate some important effects of amphiphile structure on biophysics and activity. The data also suggest that cationic amphiphilic 1,4-DHP derivatives may find use as DNA delivery system.     

Rapid and efficient oxidation of Hantzsch 1,4-dihydropyridines with sodium periodate catalyzed by manganese (III) Schiff base complexes

Rapid and efficient oxidation of Hantzsch 1,4-dihydropyridine with sodium periodate is reported. The Mn(III)-salophen/NaIO4 catalytic system converts 1,4-dihydropyridines to their corresponding pyridine derivatives at room temperature in a 1:1, CH3CN/H2O mixture. The ability of various Schiff base complexes in the oxidation of 1,4-dihydropyridine was also investigated.     

4-alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines

Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine metabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4-2H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4-2H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms.     

Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines

A variety of different 4-substituted 1,4-dihydropyridine Hantzsch esters are substrates for ring dehydrogenation by a cytochrome P-450 (P-450) enzyme (P-450 UT-A); the substitutent could be varied from a hydrogen to a naphthalenyl, but a pyrenyl derivative was not dehydrogenated. When a 4-alkyl group is present, both the P-450 which oxidizes the substrate and other P-450s can be inactivated (by putative alkyl radicals). P-450s did not discriminate with regard to removal of the 4-H atoms from an enantiomeric pair of dihydropyridines. Losses of the 4-proton and N-methyl from a N-methyl-1,4-dihydropyridine occur at similar rates. The calculated intrinsic kinetic hydrogen isotope effect (Dk) for dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid dimethyl ester was 2.9 in a reconstituted P-450 UT-A enzyme system. No significant kinetic hydrogen isotope effect was observed in microsomal incubations for the dehydrogenation of this compound or 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester in a variety of competitive and noncompetitive experiments. In light of previous studies on the magnitude of kinetic hydrogen isotope effects in P-450 systems (e.g. Miwa et al., 1983 (Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449], the mechanistic proposals of Augusto et al., 1982 (Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Biol. Chem. 257, 11288-11295)) for enzyme inactivation by 4-alkyl-substituted Hantzsch pyridine esters, and other precedents for sequential electron transfer in amine oxidation by P-450s, we interpret these results as being consistent with P-450-mediated 1-electron oxidation of dihydropyridines followed by the facile loss of the 4-proton, with subsequent electron transfer to complete the reaction.     

Enzymatic activation of hydrazine derivatives. A spin-trapping study

The oxidative metabolism of hydralazine, isoniazid, iproniazid, and phenylhydrazine has been studied using spin-trapping techniques. The oxidation of these hydrazine derivatives, catalyzed by horseradish peroxidase and prostaglandin synthetase, produces reactive free radical intermediates. Enzymatic activation of hydralazine produce the nitrogen-centered hydralazyl radical (RNHNH); phenylhydrazine formed only the phenyl radical. Iproniazid, on the other hand, formed both the isopropyl radical and a hydroperoxy radical. The formation of the hydroperoxy radical was not inhibited by superoxide dismutase. The horseradish peroxidase-catalyzed oxidation of isoniazid produced two different carbon-centered radicals. The identity of these radicals is not clear; however, they may arise from an acyl (RCO) radical and an alkyl (R) radical.     

Studies on the antioxidant activity of some chromonylrhodanine derivatives

Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2‐diphenyl‐1‐picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric‐ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin‐trapping technique. The 5,5‐dimethyl‐ 1‐pyrroline‐1‐oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71–94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231–672%. Similarly, these compounds caused 28–58% inhibition in the intensity of the DMPO‐OOH radical EPR signal and the DMPO‐OH radical (from 12–48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC₅₀: 0.51–0.56 µmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance. Copyright © 2014 John Wiley & Sons, Ltd.     

Antioxidant activity of 4-flavonil-1,4-dihydropyridine derivatives

The effect of 4-flavonil-1,4-dihydropyridine derivatives on a chemical system involving a superoxide radical anion was tested using the chemiluminescence and spectrophotometry methods. All tested compounds enhanced the light emission from the system. The obtained results indicated that the tested derivatives may catalyze the conversion of superoxide radicals, thus showing superoxide dismutase activity.     

Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals.

Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.     

Synthesis and study of antiviral and anti-radical properties of aminophenol derivatives

A number of sterically-hindered o-aminophenol derivatives have been synthesized, and their antiviral activity in parallel with reactivity towards commonly encountered free-radical intermediates was investigated. Of the compounds tested, the highest activity in suppressing replication of Herpes simplex type l viruses was displayed by N-acyl and N-aryl derivatives of 4,6-di-tert-butyl-2-aminophenol, which were able to interact with organic free radicals and, at the same time, manifested low reactivity towards reactive oxygen species.     

Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy

We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr (*) EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr (*)] intermediate has an electropositive and pH-dependent microenvironment [g(x) value of 2.0077(0) at pH >or= 8.0 and 2.0066(2) at 4.0 相似文献   

Synthesis,structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthesized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated. The compounds are potential candidates for targeted spin probes in EPR imaging.     

Studies on the antioxidant activities of some new chromone compounds

Recent reviews evidence that the naturally occurring compounds containing the chromone skeleton exhibit antiradical activities, providing protection against oxidative stress. The antioxidant activities of 13 new synthesized chromonyl‐2,4‐thiazolidinediones, chromonyl‐2,4‐imidazolidinediones and chromonyl‐2‐thioxoimidzolidine‐4‐ones were evaluated using in vitro antioxidant assays, including superoxide anion radical (), hydroxyl radical (), 2,2‐diphenyl‐1‐picryl‐hydrazyl free radical (DPPH^?) scavenging capacity and total antioxidant capacity ferric ion reducing activity. Superoxide anion radical was produced using potassium superoxide/18‐crown‐6‐ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe(II) + H₂O₂) was a generator of hydroxyl radicals. Chemiluminescence, spectrophotometry, electron paramagnetic resonance (EPR) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) as the spin trap were the measurement techniques. The results showed that the majority of the chromone derivatives tested showed a strong scavenging effect towards free radicals, similar to the chemiluminescence reaction with superoxide anion radical with a high activity, inhibition of the DMPO‐OOH radical EPR signal (24–58%), the DMPO‐OH radical EPR signal (4–75%) and DPPH radical EPR signal (6–100%) at 1 mmol/L. Several of the examined compounds exhibited the high reduction potentials. The results obtained show that the new synthesized chromone derivatives may directly scavenger reactive oxygen species and thus may play a protective role against oxidative damage. Copyright © 2014 John Wiley & Sons, Ltd.