Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton

Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes.     

Molecular and crystal structures of 5,7-dichloro-4-nitro-2,1,3-benzoxadiazole and products of its reactions with secondary amines

The molecular and crystal structures of 5,7-dichloro-4-nitro-2,1,3-benzoxadiazole and products of its reactions with hexamethyleneimine and dimethylamine were established by X-ray diffraction analysis. The molecular structures and the systems of hydrogen bonds in the crystals of these compounds are discussed.     

Capillary electrophoresis with laser induced-fluorescence detection of profens derivatized with the water-soluble fluorogenic reagent 4-N-(4-N'-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole

Profens, including pranoprofen, fenoprofen, flurbiprofen, ketoprofen and ibuprofen (Ib), were derivatized by a water-soluble benzofurazan fluorescent reagent, 4-N-(4-N'-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole and then were run on capillary electrophoresis in a NH4Ac-HAc buffer of pH 3.1 containing 2.4 mM beta-cyclodextrin. At room temperature, the derivatization reaction was catalyzed by triphenyl phosphine and diphenyl disulfide in acetonitrile medium, and the derivatives fluoresce around 530 nm when excited at 488 nm. With the CE running on a 50 cm x 50 microm I.D. length fused-silica capillary of by using Ar+ laser induced-fluorescence detection, the detection limits attained were in the range of 0.16 to 0.3 fmol.     

Energetic and structural properties of 4-nitro-2,1,3-benzothiadiazole

The energetic study of 4-nitro-2,1,3-benzothiadiazole has been developed using experimental techniques together with computational approaches. The standard (p^° = 0.1 MPa) molar enthalpy of formation of crystalline 4-nitro-2,1,3-benzothiadiazole (181.9 ± 2.3 kJ · mol⁻¹) was determined from the experimental standard molar energy of combustion −(3574.3 ± 1.3) kJ · mol⁻¹, in oxygen, measured by rotating-bomb combustion calorimetry at T = 298.15 K. The standard (p^° = 0.1 MPa) molar enthalpy of sublimation, at T = 298.15 K, (101.8 ± 4.3) kJ · mol⁻¹, was determined by a direct method, using the vacuum drop microcalorimetric technique. From the latter value and from the enthalpy of formation of the solid, it was calculated the standard (p^° = 0.1 MPa) enthalpy of formation of gaseous 4-nitro-2,1,3-benzothiadiazole as (283.7 ± 4.9) kJ · mol⁻¹. Standard ab initio molecular orbital calculations were performed using the G3(MP2)//B3LYP composite procedure and several working reactions in order to derive the standard molar enthalpy of formation 4-nitro-2,1,3-benzothiadiazole. The ab initio results are in good agreement with the experimental data.     

Use of 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the beta-blocker metoprolol in serum by high-performance liquid chromatography.

Fluorogenic reagents having a benzofurazan moiety, viz., 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 7-fluoro-4-nitro-2,1,3-benzoxadiazole and 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole, were compared for the sensitive analysis of their derivatives by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection. Of the proline derivatives, DBD-proline was the most sensitive with a detection limit of 2 fmol. The optimum concentrations of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and H2O2 for the post-column reaction were 0.5 and 75 mmol dm-3 respectively and amino acids and beta-blockers derivatized with DBD-F were detected in the range 0.2-40 fmol (signal-to-noise ratio = 3) using the proposed method. The lower detection limit of metoprolol (a beta-blocker having an isopropylamino group) spiked in serum was 0.8 ng ml-1 using 20 microl of serum (signal-to-noise ratio = 5).     

Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.     

Syntheses and characterizations of 4-(3,17beta-dihydroxyestra-1,3,5(10)-trien-6alpha- and 6beta-yl)amino-7-nitro-2,1,3-benzoxadiazoles as fluorescent probes.

4-(3,17Beta-dihydroxyestra-1,3,5(10)-trien-6alpha- and 6beta-yl)amino-7-nitro-2,1,3-benzoxadiazoles have been synthesized and characterized as fluorescent probes for use in a receptor assay and/or a homogeneous immunoassay for estradiol. The fluorescence intensities are strongly dependent upon the solvent polarity used. The intensities in water were reduced to less than 1% of those in ethyl acetate, and a blue shift was also observed in polar solvents. The quenched fluorescence in aqueous solution was recovered by adding bovine serum albumin or an anti-estradiol antibody. Adding intact estradiol inhibited the fluorescence recovered by the antibody.     

Utilization of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) for kinetic spectrophotometric assay of befunolol hydrochloride in its pharmaceutical formulation

A simple, accurate, precise, and sensitive kinetic spectrophotometric method for determination of befunolol hydrochloride is described. The method is based on the formation of a colored product with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in methanol at 70 degrees C for 45 min. The red-colored product was measured at 523 nm. The optimization of various experimental conditions is described; Beer's law was obeyed in the range 15.25 x 10(-6) to 122.04 x 10(-6) M. The results obtained showed good recoveries (100.1 +/- 0.80%). Application of the proposed method to a pharmaceutical formulation was successfully achieved. The determination of befunolol hydrochloride by fixed time, fixed concentration, and rate constant methods was feasible with the calibration equation obtained. However, the fixed time method proved to be more applicable.     

Synthesis and structure of 6- and 7-nitro-1-oxoisochroman-4-spirooxiranes

The reaction of diazomethane with 4-nitrophthalic acid anhydride gave 4- and 5-nitro-2-carbomethoxy--diazoacetophenones and 6- and 7-nitro-1-oxoisochroman-4-spirooxiranes. The structure of 7-nitro-1-oxoisochroman-4-spirooxirane was investigated by x-ray diffraction analysis.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1322–1325, October, 1989.     

Vibrational spectra of 2-fluoro-5-nitro-, 2-fluoro-4-nitro-, 4-fluoro-2-nitro- and 5-fluoro-2-nitrotoluene

The mid- and far-IR absorption spectra of four substituted toluenes, namely 2-fluoro-5-nitro-, 2-fluoro-4-nitro-, 4-fluoro-2-nitro- and 5-fluoro-2-nitrotoluene were recorded. Vibrational assignments are proposed assuming C_s, symmetry for the molecules.     

Heterocyclic bioantioxidants 4. Chlorolysis of 3-nitro-4-s-benzylcoumarin

3-Nitro-4-S-benzylcoumarin reacts with sulfuryl chloride to give an unstable compound, which in turn reacts with p-toluidine to give 3-nitro-4-p-tolylaminocoumarin.For Communication 3, see [1].Translated from Khimiya Geterotsiklicheskikh Soednenii, No. 7, 888–889, July, 1992.     

LC-electron capture-APCI(-)-MS determination of nitrobenzoxadiazole derivatives

Nitrobenzoxadiazole (NBD) derivatives are determined with limits of detection ranging down to 20 nmol l(-1) using liquid chromatography-mass spectrometry (LC-MS) with electron capture (EC) ionisation. An atmospheric pressure chemical ionisation (APCI) interface operated in the negative ion mode is used as ionisation source. Amine derivatives of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBDCl) as well as the isocyanate derivatives of 4-nitro-7-piperazino-2,1,3-nitrobenzoxadiazole (NBDPZ) have been analysed using this technique. The parameters favouring electron capture mechanisms have been investigated thoroughly under consideration of the competing mechanism of deprotonation to allow a better understanding of the electron capture process and to improve selectivity of the analysis.     

Determination of telmisartan in human blood plasma: Part I: Immunoassay development

Telmisartan is an angiotensin II receptor antagonist and a known drug against high blood pressure. In this report, the development of a new and rapid analytical technique, an enzyme-linked immunosorbent assay (ELISA) for the determination of telmisartan in human blood plasma is described. The immunoassay is based on a conversion of 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) to 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA), which is detected by fluorescence spectroscopy. The limit of detection was 0.1 ng/mL, the limit of quantification was 0.3 ng/mL and the working range extended from 0.3 ng/mL to 300 ng/mL.     

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.     

Nucleophilic substitution and ring closure reactions of 4-chloro-3-nitro-2-quinolones

4-Chloro-3-nitro-2-quinolones 3 obtained from the 4-hydroxy quinolones 1 by nitration and chlorination, reacted with sodium azide to the 4-azido derivatives 4 which cyclized on thermolysis to yield the furoxanes 5 . Nucleophilic substitution reactions of 3 led to the 4-amino-, 4-fluoro- and 4-alkoxy-3-nitroquinolones 7, 8 and 9 , respectively. With thiols either 4-thio-3-nitro- 10 or 3,4-dithioquinolones 11 were obtained depending on the basic catalyst.     

Synthesis of 3-nitro-4-amino- and 3,4-diaminothiocoumarins

Nitration of 4-hydroxythiocoumarin gave 3-nitro-4-hydroxythiocoumarin, which was converted to 3-nitro-4-chlorothiocoumarin by the action of phosphorus oxychloride in dimethylformamide. The corresponding 3-nitro-4-aminothiocoumarins were synthesized by reaction of 3-nitro-4-chlorothiocoumarin with ammonia or amines. The 3-nitro-4-aminothiocoumarins were hydrogenated in alcohol over Raney nickel to give 3,4-diaminothiocoumarins. The-aminovinylcarbonyl form of the 4-substituted 3-aminothiocoumarins was established on the basis of the UV, IR, and PMR spectra.See [1] for our preliminary communication.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 1, pp. 43–47, January, 1978.     

Design and synthesis of a hydrophilic fluorescent derivatization reagent for carboxylic acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH2), and its application to capillary electrophoresis with laser-induced fluorescence detection

A hydrophilic fluorescent derivatization reagent for fatty acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH(2)), was designed and synthesized. NBD-PZ-NH(2) possesses not only a fluorophore and a reacting group but also a positive charge group and, thus, was hydrophilic and suitable for application to capillary electrophoresis. NBD-PZ-NH(2) reacted with fatty acids in the presence of triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (DPDS) at room temperature within 10 min. The derivatives were strongly fluoresced and were positively charged at pH below 3. The derivatives of C4-C20 fatty acids were separated within 10 min in 50% acetonitrile in water containing 30 mM ammonium acetate and 1.0 M acetic acid by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The detection limits attained were 6.5 nM (signal-to-noise ratio of 3). It is proposed that NBD-PZ-NH(2) is a prominent derivatization reagent for fatty acids which is suitable for CE-LIF application.     

Tautomerism of the Thioamide Group in 4-Methyl-7-nitro-2,3,4,5-tetrahydro-1,5-benzodiazepine-2-thione

When 4-methyl-7-nitro-2,3,4,5-tetrahydro-1H-1,5-benzodiazepin-2-one were reacted with phosphorus pentasulfide, the corresponding benzodiazepine-2-thione and its thiol tautomer were formed, which via the 2-methylmercapto derivative were converted to 4-(2-acetylhydrazino)-2-methyl-8-nitro-2,3-dihydro-1H-1,5-benzodiazepine.     

Synthesis of 5(and 6)-nitro-4-methyl-2,3-dihydro-1h-1,5-benzo-2-diazepinones

The reaction of 4-nitro-o-phenylenediamine (I) with acetoacetic ester at room temperature under acid catalysis gives ethyl 3-(2-amino-5-nitrophenylamino)crotonate (II), which is readily cyclized to 7-nitro-4-methyl-2,3-dihydro-1H-1,5-benzo-2-diazepinone (III) on heating with alkaline agents. The reaction of I with acetoacetic ester in refluxing xylene gives isomeric 8-nitro-4-methyl-2,5-dihydro-1H-1,5-benzo-2-diazepinone (IVa) or 8-nitro-4-methyl-2,3-dihydro-1H-1,5-benzo-2-diazepinone (IVb), which are readily interconverted. The synthesis of IV is complicated by the side formation of 5-nitro-2-methylbenzimidazole (V) and thermal rearrangement of IVa and IVb to 5-nitro-1-isopropenylbenzimidazolone (VI). 6-Nitro-1-isopropenylbenzimidazolone (VII) is similarly obtained on heating III.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 696–699, May, 1972.     

Methylation of 4-nitro-3(5)-pyrazolecarboxylic acid

Reactions of 4-nitro-3(5)-pyrazolecarboxylic acid dipotassium salt with different methylating agents in various solvents have been investigated to improve the synthesis of isomeric 1-methyl-4-nitro-3- and -5-pyrazolecarboxylic acids.