Dihydroxy-Substituted Coumarins as Fluorescent Probes for Nanomolar-Level Detection of the 4-Amino-TEMPO Spin Label.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM-a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH-the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

Koumine, Humantenine, and Yohimbane Alkaloids from Gelsemium elegans.

Nine new alkaloids of the koumine (1-4), humantenine (5-7), and yohimbane (8, 9) types as well as 12 known analogues were isolated from the leaves and vine stems of Gelsemium elegans. Compound 1 is the first N-4-demethyl alkaloid of the koumine type, compound 7 is the first nor-humantenine alkaloid, and compounds 8 and 9 are the first N-1-oxide and the first seco-E-ring alkaloids, respectively, of the yohimbane type. Compounds 1 and 7 exhibited moderate cytotoxicity against five human tumor cell lines with IC50 values in the range 4.6-9.3 µM.

Tuning the reactivity of TEMPO by coordination to a Lewis acid: isolation and reactivity of MCl3(η1-TEMPO) (M = Fe, Al).

Addition of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) to MCl(3) (M = Fe, Al) results in the formation of MCl(3)(η(1)-TEMPO) [M = Fe (1), Al (2)]. Both 1 and 2 oxidize alcohols to generate ketones or aldehydes along with the reduced complexes MCl(3)(η(1)-TEMPOH) [M = Fe (3), Al (4)]. Complexes 1-4 were fully characterized, including analysis by X-ray crystallography. Additionally, control experiments indicated that neither MCl(3) (M = Al, Fe) nor TEMPO are capable of effecting the oxidation of alcohols independently.

"Marking" the nitrogen atoms of phenyl-(2-pyridyl)-(3-pyridyl)-(4-pyridyl)-methane. Synthesis and absolute configuration of the corresponding tris(pyridine N-oxide).

To "mark" the nitrogen atoms in phenyl-(2-pyridyl)-(3-pyridyl)-(4-pyridyl)methane (1), we have synthesized the corresponding tris(pyridine N-oxide) 2 by oxidation of 1 with m-chloroperbenzoic acid. The nitrogen atoms of 2 are unequivocally determined by the X-ray crystal analysis of a single crystal of rac-2 whereas the nitrogen atoms cannot be assigned at all in the case of rac-1. N-Oxide 2 can be resolved by chiral high-performance liquid chromatography under similar conditions to those used for the resolution of 1. The calculated circular dichroism (CD) curve for (R)-2 on the basis of time-dependent density functional theory reproduces the experimental spectra very well to suggest that the second-eluted fraction ([CD(+)283]-2) is the R isomer, namely (R)-[CD(+)283]-2. The independent absolute configuration determinations for 1 and 2 are in keeping with the chemical correlation between the two compounds by oxidation of (R)-1 into (R)-2.

Biphenyl and biphenyl ether quinolizidine N-oxide alkaloids from Lagerstroemia indica L.

Four new biphenyl and biphenyl ether quinolizidine N-oxide alkaloids, 5- EPI-dihydrolyfoline N-oxide (1), decamine N-oxide (2), lagerstroemine N-oxide (3), and lagerine N-oxide (4), were isolated from the aerial parts of Lagerstroemia indica, and their structures were established by extensive spectroscopic studies. In addition, the inhibitory effects of isolated compounds on rat lens aldose reductase (RLAR) were examined.

Two new morphinane alkaloids from Sinomenium acutum.

Two new morphinane alkaloids, 1-hydroxy-10-oxo-sinomenine (1) and 4,5-epoxy-14-hydroxy sinomenine N-oxide (2), have been isolated from the stems of Sinomenium acutum. Their structures were established by various spectral analyses, especially 2D NMR experiments. The structure of 2 was confirmed by single crystal X-ray diffraction. The absolute configurations of 1 and 2 were deduced by comparison of CD spectra with the known alkaloid sinomenine (3). Compound 1 was tested for DPPH inhibition and gave IC(50) of 27.9 µM. Compound 2 was tested for neuroprotective effect and showed significant activity against ß-amyloid(25-35)-induced oxidative injury (*P < 0.05) at 10 µM in PC-12 cells.

The anti-tumor effects of alkaloids from the seeds of Strychnos nux-vomica on HepG2 cells and its possible mechanism.

To screen the anti-tumor effects of the four alkaloids: brucine, strychnine, brucine N-oxide and isostrychnine from the seed of Strychnos nux-vomica, MTT assay was used to examine the growth inhibitory effects of these alkaloids on human hepatoma cell line (HepG2). Brucine, strychnine and isostrychnine revealed significant inhibitory effects against HepG2 cell proliferation, whereas brucine N-oxide didn't have such an effect. In addition, brucine caused HepG2 cell shrinkage, membrane blebbing, apoptotic body formation, all of which are typical characteristics of apoptotic programmed cell death. The results of flow cytometric analysis demonstrated that brucine caused dose-dependent apoptosis of HepG2 cells through cell cycle arrest at G0/G1 phase, thus preventing cells entering S or G2/M phase. Immunoblot results revealed that brucine significantly decreased the protein expression level of cyclooxygenase-2, whereas increased the expression caspase-3 as well as the caspase-3-like protease activity in HepG2 cells, suggesting the involvement of cyclooxygenase-2 and caspase-3 in the pro-apoptotic effects exerted by brucine. Therefore, this paper indicate that the major alkaloids present in the seed of Strychnos nux-vomica are effective against HepG2 cells proliferation, among which brucine proceed HepG2 cells death via apoptosis, probably through the participation of caspase-3 and cyclooxygenase-2.

Geissoschizine methyl ether N-oxide, a new alkaloid with antiacetylcholinesterase activity from Uncaria rhynchophylla.

Geissoschizine methyl ether N-oxide, a new oxindole alkaloid, along with 14 known alkaloids, was isolated from the aerial part of Uncaria rhynchophylla. Their structures were identified by comprehensive spectral methods, including 2D NMR experiments, and confirmed by comparing with the literature data. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that the new compound exhibited anti-AChE activity with IC50 value of 23.4 µM.

Trace level liquid chromatography tandem mass spectrometry quantification of the mutagenic impurity 2-hydroxypyridine N-oxide as its dansyl derivative.

A derivatization LC-MS/MS method was developed and qualified for the trace level quantification of 2-hydroxypyridine N-oxide (HOPO). HOPO is a coupling reagent used in the syntheses of active pharmaceutical ingredients (APIs) to form amide bonds. HOPO was recently confirmed to generate a positive response in a GLP Ames bacterial-reverse-mutation test, classifying it as a mutagenic impurity and as such requiring its control in APIs to the threshold of toxicological concern (TTC). The derivatization reagent 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) was used in a basic solution to convert HOPO into the corresponding dansyl-derivative. The derivative was separated from different APIs and reagents by liquid chromatography. The detection of the HOPO dansyl-derivative was achieved by mass spectrometry in selected reaction monitoring (SRM) mode. The LC-MS/MS method had a reporting limit of 0.1ng/mL HOPO, which corresponds to 0.1ppm HOPO relative to an API at 1mg/mL, and a linearity range of 0.1-25ng/mL HOPO analyte. Recoveries of HOPO standards spiked into three different API matrices at 0.2, 1.2, and 20ppm levels were all within 90-100%. An SRM-based confirmatory methodology using the ratios of two fragment ions at three CID energies was developed to verify the identity of HOPO when present at ≥0.6ppm. This identity confirmation can be employed to prevent potential false positive detection of mutagenic impurities at trace level. It can be broadly applicable for the confirmation of analytes when the analytes generate at least two major fragments in tandem mass spectrometry experiments.

Separation and identification of DMPO adducts of oxygen-centered radicals formed from organic hydroperoxides by HPLC-ESR, ESI-MS and MS/MS.

Many electron spin resonance (ESR) spectra of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) radical adducts from the reaction of organic hydroperoxides with heme proteins or Fe(2+) were assigned to the adducts of DMPO with peroxyl, alkoxyl, and alkyl radicals. In particular, the controversial assignment of DMPO/peroxyl radical adducts was based on the close similarity of their ESR spectra to that of the DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the peroxyl adducts from the DMPO/superoxide adduct. Although recent reports assigned the spectra suggested to be DMPO/peroxyl radical adducts to the DMPO/methoxyl adduct based on independent synthesis of the adduct and/or (17)O-labeling, (17)O-labeling is extremely expensive, and both of these assignments were still based on hyperfine coupling constants, which have not been confirmed by independent techniques. In this study, we have used online high performance liquid chromatography (HPLC or LC)/ESR, electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) to separate and directly characterize DMPO oxygen-centered radical adducts formed from the reaction of Fe(2+) with t-butyl or cumene hydroperoxide. In each reaction system, two DMPO oxygen-centered radical adducts were separated and detected by online LC/ESR. The first DMPO radical adduct from both systems showed identical chromatographic retention times (t(R) = 9.6 min) and hyperfine coupling constants (a(N) = 14.51 G, a(H)(beta) = 10.71 G, and a(H)(gamma) = 1.32 G). The ESI-MS and MS/MS spectra demonstrated that this radical was the DMPO/methoxyl radical adduct, not the peroxyl radical adduct as was thought at one time, although its ESR spectrum is nearly identical to that of the DMPO/superoxide radical adduct. Similarly, based on their MS/MS spectra, we verified that the second adducts (a(N) = 14.86 G and a(H)(beta) = 16.06 G in the reaction system containing t-butyl hydroperoxide and a(N) = 14.60 G and a(H)(beta) = 15.61 G in the reaction mixture containing cumene hydroperoxide), previously assigned as DMPO adducts of t-butyloxyl and cumyloxyl radical, were indeed from trapping t-butyloxyl and cumyloxyl radicals, respectively.

Microbial transformation of papaveraldine.

Preparative-scale fermentation of papaveraldine (1), the known benzylisoquinoline alkaloid, with Mucor ramannianus 1839 (sih) has resulted in a stereoselective reduction of the ketone group and the isolation of S-papaverinol (2) and S-papaverinol N-oxide (3). The structure elucidation of both metabolites was based primarily on 1D-, 2D-NMR analyses and chemical transformations. The absolute configuration of 2 was determined using Horeau's method of asymmetric esterification. These metabolism results were consistent with the previous plant cell transformation studies on papaverine and isopapaverine.

Synthesis and purification of 5,5-dimethyl-1-pyrroline-N-oxide for biological applications.

General procedures for the synthesis and purification of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and its synthetic intermediates for electron paramagnetic resonance (EPR) spin trapping is described. The synthetic methods are not new per se and are based on Todd's original scheme (1959). In contrast to his account, however, we provide a detailed description of the numerous procedures and precautions necessary to obtain DMPO of very high 'chemical' and (most importantly) 'EPR' purity for use in biological spin trapping.

Isolation and structure determination of novel phosphatidylinositol turnover inhibitors, piericidin B5 and B5 N-oxide, from Streptomyces sp.

In the course of a screening program for inhibitors of epidermal growth factor (EGF)-induced phosphatidylinositol turnover in human epidermoid carcinoma cell line A431, we discovered two novel compounds of the piericidin family from the culture broth and mycelia of a Streptomyces strain MJ288-OF3. We named them piericidin B5 and B5 N-oxide. By NMR and mass spectrometric analyses, the molecular formulas of piericidin B5 and B5 N-oxide were determined to be C27H41NO4 (MW 443) and C27H41NO5 (MW 459), respectively. Piericidin B5 and B5 N-oxide inhibited phosphatidylinositol turnover of A431 cells with IC50S of 10.0 micrograms/ml and 1.1 micrograms/ml, respectively.

Analgesic and anti-inflammatory properties of brucine and brucine N-oxide extracted from seeds of Strychnos nux-vomica.

To further understand the purpose of the traditional processing method of the seeds of Strychnos nux-vomica L. (Loganiaceae) as well as analgesic and anti-inflammatory activities of brucine and brucine N-oxide extracted from this medicinal plant, various pain and inflammatory models were employed in the present study to investigate their pharmacological profiles. Both brucine and brucine N-oxide revealed significant protective effects against thermic and chemical stimuli in hot-plate test and writhing test. However, on different phases they exerted analgesic activities in formalin test. Brucine N-oxide showed stronger inhibitory effect than brucine in carrageenan-induced rat paw edema, both of them significantly inhibited the release of prostaglandin E2 in inflammatory tissue, reduced acetic acid-induced vascular permeability and the content of 6-keto-PGF1a in Freund's complete adjuvant (FCA) induced arthritis rat's blood plasma. In addition, brucine and brucine N-oxide were shown to reduce the content of 5-hydroxytryptamine (5-HT) in FCA-induced arthritis rat's blood plasma, while increase the content of 5-hydroxytryindole-3-acetic acid (5-HIAA) accordingly. These results suggest that central and peripheral mechanism are involved in the pain modulation and anti-inflammation effects of brucine and brucine N-oxide, biochemical mechanisms of brucine and brucine N-oxide are different even though they are similar in chemical structure.

[Preparation and structural elucidation of insularine-N-oxides].

Oxidation of insularine (I) with m-chloroperbenzoic acid yielded four insularine-N-oxides. Two of them are identical in every respect with our insularine-2 beta-N-oxide (II) and insularine-2' beta-N-oxide (III), two rare examples of naturally occurring head-to-tail bisbenzylisoquinoline N-oxides newly isolated from Cyclea sutchuenensis, which confirmed the structures of the two novel natural N-oxides. The other two are new compounds and their structures have been established as insularine-2' alpha-N-oxide (IV) and insularine-2 beta,2' beta-N, N-dioxide (V), on the basis of spectral data (UV, IR, 1HNMR, NOEDS AND MS) analysis.

[A new steroidal alkaloid from the bulbus of Fritillaria wabuensia].

AIM: To investigate the chemical constituents of the bulbs of Fritillaria wabuensia. METHODS: Chromatography techniques were used to isolate the chemical constituents. EI-MS, 1HNMR, 13 CNMR and DEPT were used to determine the structures of the isolated constituents. RESULTS: Three alkaloids were isolated from the bulbs of Fritillaria wabuensia, and were identified as imperialine (I), imperialine-beta-N-oxside (II), isoverticine-beta-N-oxide (III). CONCLUSION: Isoverticine-beta-N-oxide was isolated from the bulbs of Fritillaria wabuensia for the first time. Isoverticine-beta-N-oxide is a new alkaloid.

[Pharmacokinetics of the alkaloids from the processed seeds of Strychnos nux-vomica in rats].

AIM: To study the pharmacokinetic process about the concentration in rat plasma of the alkaloids from processed seeds of Strychnos nux-vomica with RP-HPLC method. METHODS: Hypersil BDS C18 column was used and the mobile phase consisted of acetonitrile-water at the flow rate of 0.8 mL.min-1. The UV detection wave length was 254 nm. RESULTS: The concentration-time data of strychnine, brucine, strychnine N-oxide and brucine N-oxide were all in accordance with an open two-compartment model after i.v. alkaloids. Their parameters were as follows: T1/2 alpha were (8 +/- 5), (4 +/- 3), (6.2 +/- 1.7) and (3.0 +/- 0.8) min, T1/2 beta were (262 +/- 125), (416 +/- 131), (285 +/- 50) and (342 +/- 141) min, CL were (17 +/- 4), (21 +/- 12), (1.9 +/- 1.8) and (2.8 +/- 1.1) mL.min-1, Vc were (1.4 +/- 0.5), (1.7 +/- 1.1), (0.24 +/- 0.16) and (0.23 +/- 0.06) L.kg-1, Vd were (6.0 +/- 1.2), (12 +/- 7), (0.8 +/- 0.6) and (1.5 +/- 0.6) L.kg-1, AUC were (57,578 +/- 25,578), (35,240 +/- 15,616), (93,088 +/- 22,375) and (177,712 +/- 120,110) h.microgram.L-1, respectively. CONCLUSION: The method is a good reference for pharmacokinetics in human bodies.

[Studies on the chemical constituents of alkaloids in seeds of Strychnos nux-vomica L].

Thirteen alkaloids were isolated from the seeds of Strychnos nux-vomica. They were identified as strychnine, beta-colubrine, pseudostrychnine, strychnine N-oxide, brucime, brucine N-oxide, novacine, icajine, vomicine, isostrychnine, isobrucine, isobrucine N-oxide and isostrychnine N-oxide by chemical and spectroscopic analysis.

(+)-Sparteine N-16-oxide, a lupin alkaloid from Lygos raetam var. sarcocarpa.

A new lupin alkaloid, (+)-sparteine N-16-oxide was isolated from Lygos raetam var. sarcocarpa, together with five known alkaloids: (+)-aphylline, (+)-17 oxosparteine, (-)-5,6-dehydrolupanine, (-)-N-formyl cytisine and (+)-ammodenderine. The structure of the new alkaloid including absolute configuration was determined by spectroscopic methods and chemical transformation.