A rapid method for detection of tyrosinase activity in electrophoresis

This rapid and sensitive method for localizing tyrosinase in polyacrylamide slab gels is based on the condensation of Bestthorn's hydrazone (3 methyl-2-benzothiazolinone hydrazone hydrochloride) with the quinone obtained by enzymatic oxidation of phenol. Both monophenolase and diphenolase activities are localized by this method.     

A new technique for staining catecholic residues in biological samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 microgram level and the final product is chemically stable.     

Synthesis and properties of new rubomycin derivatives

Condensation of rubomycin (daunorubicin) with respective hydrazides yielded novel substituted hydrazones: 13-cyanoacetyl hydrazone rubomycin, 13-L-phenylalanyl hydrazone rubomycin, 13-BOC-3-(uracilyl-1)-DL-alanyl hydrazone rubomycin and 13-BOC-3-(adenylyl-9)-DL-alanyl hydrazone rubomycin. With successive treatment of rubomycin with hydrazine hydrate and respective ketones novel asymmetric azines were prepared: 13-cyclopentylidene hydrazone rubomycin, 13-alpha,alpha'-dimethyl-cyclopentylidene hydrazone rubomycin and 13-(1-phenylethylidene-1) hydrazone rubomycin. 14-Adenylyl-N9-rubomycin was synthesized by interaction of 14-bromorubomycin with adenine and hydrogenation of its analog, 14-N-imidazolyl rubomycin by sodium borhydrite yielded 13-dihydro-14-N-imidazolyl rubomycin. There was observed correlation between the antimicrobial activity of the derivatives against B. mycoides and their cytostatic effect on the cells of murine leukemia NK/LI. The high in vitro activity of 13-cyclopentylidene hydrazone rubomycin showed satisfactory correlation with the results of the study on the antitumor effect in animals.     

A New Technique for Staining Catecholic Residues in Biological Samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 µg level and the final product is chemically stable.     

Iron chelators of the pyridoxal isonicotinoyl hydrazone class Part I. Ionisation characteristics of the ligands and their relevance to biological properties

The orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), and five analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxal p-methoxybenzoyl hydrazone ((PpMBH), pyridoxal m-fluorobenzoyl hydrazone (PmFBH), 3-hydroxy- isonicotinaldehyde isonicotinoyl hydrazone (IIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) were synthesised and characterised and their acid dissociation constants measured by glass electrode potentiometry and UV—Vis spectrophotometry. Analysis of the data showed that at physiological pH all of the ligands are predominantly (av. 80%) in the form of the neutral molecule, allowing passage through cell membranes and access to intracellular iron pools. The results are discussed in the context of the development of an orally effective iron chelator for clinical use.     

Function and stability of abscisic acid acyl hydrazone conjugates by LC-MS2 of ex vivo samples

We prepare a biotinylated conjugate of the ubiquitous plant hormone (S)-(+)-abscisic acid via an acyl hydrazone linkage at the C4' position and demonstrate in vivo cleavage of the otherwise stable acyl hydrazone linkage using LC-MS2. As part of a wider chemical genomic study, biological activity of the conjugate was assessed using standard epidermal peel and gravimetric transpiration assays, showing significant activity but at a level lower than the unconjugated hormone. When deuterated samples of the conjugate were fed to the plant, however, it was apparent by LC-MS2 experiments that significant levels of hydrolysis of the acyl hydrazone had taken place, contrary to in vitro stability assays in artificial sap. We conclude that abscisic acid is liberated in sufficient quantities to account for the observed physiological response and that LC-MS2 monitoring of conjugates is a simple and practical method by which such events may be assessed, whether in plants or other organisms.     

An intramolecular cyclization reaction is responsible for the in vivo inefficacy and apparent pH insensitive hydrolysis kinetics of hydrazone carboxylate derivatives of doxorubicin

Contradictory reports concerning the pH sensitive hydrolysis kinetics of certain hydrazone carboxylates of doxorubicin have appeared in this journal (Kaneko et al., Bioconjugate Chem. 1991, 2, 133. Padilla De Jesús et al., Bioconjugate Chem. 2002, 13, 453). Since the pH stability of the drug-carrier linkage in macromolecular prodrugs has a significant bearing on pharmacological efficacy, the hydrolysis kinetics of low molecular weight and polymeric doxorubicin hydrazone carboxylates were therefore reinvestigated. As observed previously, the conjugates readily release native doxorubicin at pH 5. Unexpectedly, in neutral buffer the hydrazone carboxylate conjugates do not release native doxorubicin, but instead rapidly release a doxorubicin derivative substituted at C-9 by 3,6-dihydro-1,3,4-oxadiazin-2-one with first-order kinetics (t(1/2) = 2.5 h). The proposed intramolecular cyclization reaction involving doxorubicin's C-14 hydroxyl and the carboxylate-substituted hydrazone rationalizes the seemingly anomalous hydrolysis kinetics seen for hydrazone carboxylate linked doxorubicin, and provides a possible explanation for the poor antitumor activity exhibited by polymer-doxorubicin conjugates utilizing this specific type of linkage.     

A sensitive determination of α-keto acids by gas-liquid chromatography and its application to the assay of l-glutamate dehydrogenase and aminotransferases

A sensitive and selective determination of α-keto acids was established by the use of a gas chromatograph equipped with an electron capture detector. α-Keto acids (pyruvic, oxaloacetic, α-ketobutyric, and α-ketoglutaric acids) were reacted with pentafluorophenylhydrazine, and the derivatives were extracted with ethyl ether, reacted with diazomethane, and were subjected to gas-liquid chromatography with an electron capture detector. In the course of the reaction, oxaloacetic acid was decarboxylated, and yielded pyruvic acid. In the case of pyruvic (oxaloacetic) and α-ketobutyric acids two peaks corresponding to the syn and anti forms of the hydrazone appeared, and in the case of α-ketoglutaric acid, two peaks corresponding to the hydrazone and the cyclization compound produced from the hydrazone. The sum of the two peaks was taken for the determination. The present method was applicable to the assay of l-glutamate dehydrogenase, aspartate: 2-oxoglutarate, and l-alanine: 2-oxoglutarate aminotransferases.     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

根皮素异烟酰基腙的合成、表征及抗氧化活性研究

本文旨在提高根皮素的生物活性,以其作为母体通过与异烟肼结合引入C=NNH活性基团,合成了一种新型酰腙类化合物。采用UV,IR,1H NMR,13C NMR及元素分析等手段对产物进行结构表征,并测定了根皮素及其异烟酰基腙的还原能力,清除DPPH自由基及清除ABTS自由基能力。结果表明根皮素异烟酰基腙的抗氧化活性显著优于根皮素。     

Studies on the pathway of methane formation from procarbazine,a 2-methylbenzylhydrazine derivative,by rat liver microsomes

The oxidative metabolism of procarbazine, its azo, hydrazone, and two azoxy derivatives, and methylhydrazine by hepatic microsomes from phenobarbital-pretreated rats was investigated to elucidate the pathway of metabolism that resulted in methane formation from procarbazine. When incubated with microsomal reaction mixtures fortified with NADPH, all of the compounds, except the azoxy isomers, were metabolized to yield methane. A lag phase in methane formation was noted for procarbazine, but not for the other compounds. Kinetic and inhibition studies utilizing methimazole and ethylhydrazine precluded methylhydrazine as an intermediate in methane formation from procarbazine. When the azo derivative was oxidatively metabolized in the presence of liver microsomes, no hydrazone tautomer was detected. Upon monitoring the production of the azo and hydrazone metabolites formed during microsomal metabolism of procarbazine, the azo derivative was formed in sufficient quantities to account for the majority of the methane produced. In addition, small amounts of hydrazone were also detected. It was concluded that both the azo and hydrazone metabolites of procarbazine contribute to methane formation from the terminal methyl group of the hydrazine with the azo derivative being the predominant source and the hydrazone derivative being a minor source of methane. Consideration of the chemical and enzymatic pathways of procarbazine oxidation and the implication of a methyl radical intermediate in methane formation are discussed.     

A sensitive spectrophotometric assay for guanase activity

A highly sensitive and accurate spectrophotometric method was developed for determination of guanase activity with guanine as substrate. The assay is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone and N,N-diethylaniline. Xanthine formed from guanine by guanase is oxidized to uric acid and hydrogen peroxide by xanthine oxidase, and the hydrogen peroxide produced is determined by an oxidative-coupling reaction with 3-methyl-2-benzothiazolinone hydrazone and N,N-diethylaniline mediated by peroxidase. Formation of the indamine dye is greatly affected by the superoxide radical ion (O2-) and pH value. These problems can be overcome by separating the two reactions of hydrogen peroxide formation and color production and carrying out that color-producing reaction at pH 3.0. This method is very sensitive and accurate because the indamine dye has a very high molar extinction coefficient of 29,800. It can be used with various kinds of automatic analyzers such as a Hitachi, Olympus, or Technicon analyzer. Comparative studies showed that this method is more sensitive and reproducible than other methods. Furthermore, guanase activities determined by this method correlated well with those determined by the improved Ellis-Goldberg method. This method should be useful for measurement of guanase activity in banked blood for preventing transfusion hepatitis and could be valuable as a liver function test.     

A Method for Demonstrating Prophenoloxidase after Electrophoresis

The demonstration of prophenoloxidase after electrophoresis is based on its activation by sodium dodecyl sulfate (SDS) or sodium oleate and staining the activated phenoloxidase with dopamine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). A rapid method is presented for demonstrating the presence of activated phenoloxidase using polyacrylamide gel electrophoresis followed by staining in the presence of SDS or sodium oleate.     

Tuning the antiproliferative activity of biologically active iron chelators: characterization of the coordination chemistry and biological efficacy of 2-acetylpyridine and 2-benzoylpyridine hydrazone ligands

2-Pyridinecarbaldehyde isonicotinoyl hydrazone (HPCIH) and di-2-pyridylketone isonicotinoyl hydrazone (HPKIH) are two Fe chelators with contrasting biological behavior. HPCIH is a well-tolerated Fe chelator with limited antiproliferative activity that has potential applications in the treatment of Fe-overload disease. In contrast, the structurally related HPKIH ligand possesses significant antiproliferative activity against cancer cells. The current work has focused on understanding the mechanisms of the Fe mobilization and antiproliferative activity of these hydrazone chelators by synthesizing new analogs (based on 2-acetylpyridine and 2-benzoylpyridine) that resemble both series and examining their Fe coordination and redox chemistry. The Fe mobilization activity of these compounds is strongly dependent on the hydrophobicity and solution isomeric form of the hydrazone (E or Z). Also, the antiproliferative activity of the hydrazone ligands was shown to be influenced by the redox properties of the Fe complexes. This indicated that toxic Fenton-derived free radicals are important for the antiproliferative activity for some hydrazone chelators. In fact, we show that any substitution of the H atom present at the imine C atom of the parent HPCIH analogs leads to an increase in antiproliferative efficacy owing to an increase in redox activity. These substituents may deactivate the imine R–C=N–Fe (R is Me, Ph, pyridyl) bond relative to when a H atom is present at this position preventing nucleophilic attack of hydroxide anion, leading to a reversible redox couple. This investigation describes novel structure–activity relationships of aroylhydrazone chelators that will be useful in designing new ligands or fine-tuning the activity of others. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A colorimetric method for assay of serotonin deamination by monoamine oxidase

The present method involves conversion of the aldehyde produced, as a result of serotonin deamination by monoamine oxidase, to its 2:4 dinitrophenyl hydrazone derivative which gives a stable, bright yellow colour in alkaline solution and can be measured colorimetrically. The derivative is however unstable in the acidic medium and has to be extracted into an organic solvent immediately. The details of the method and its standardization are discussed.     

Nickel transport in Methanobacterium bryantii.

Methanobacterium bryantii, grown autotrophically on H2-CO2, transported nickel against a concentration gradient by a high-affinity system (Km = 3.1 microM). The system had a pH optimum of 4.9 and a temperature optimum of 49 degrees C with an energy of activation of 7.8 kcal/mol (ca. 32.6 kJ/mol). A headspace of H2-CO2 (4:1, vol/vol) was required for maximum rate of transport. The system was highly specific for nickel and was unaffected by high levels of all monovalent and divalent ions tested (including Mg2+) with the sole exception of Co2+. Kinetic experiments indicated that accumulated nickel became increasingly incorporated into cofactor F430 and protein. Nickel transport was inhibited by nigericin, monensin, and gramicidin but not by carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, N,N'-dicyclohexylcarbodiimide, valinomycin plus potassium, or acetylene. The ineffectiveness of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, and N,N'-dicyclohexylcarbodiimide may be related to difficulties in the penetration of these compounds through the outer cell barriers. Nickel uptake was greatly stimulated by an artificially imposed pH gradient (inside alkaline). The data suggest that nickel transport is not dependent on the membrane potential or on intracellular ATP, but is coupled to proton movement.     

Fluorometric determination of delta4-3-ketosteroids using aluminum salt and isonicotinylhydrazine

We have developed a new method for the fluorometric determination of Δ⁴-3-ketosteroids. In a methanolic solution of aluminum salt, Δ⁴-3-ketosteroids reacted with isonicotinylhydrazine (INH), and the resulting hydrazone fluoresced through complex formation with the aluminum ion in the solution. As little as 0.25 nmol of cortisol can be determined by this method.     

Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes

A model consisting of 59Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of 59Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate 59Fe release from 59Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrazones. Several compounds proved to be very potent in mobilizing 59Fe. These included hydrazones derived from 2-hydroxy-1-naphthylaldehyde and benzoic acid hydrazide, p-hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.     

Micro-determination of L-gulono-gamma-lactone oxidase activity.

Highly sensitive assay method of L-gulono-gamma-lactone oxidase (GLO) was constructed. In this method, L-ascorbic acid formed in the enzymatic reaction was converted to its bis(dinitrophenyl)hydrazone derivative, and the amount of the latter was determined by high-performance liquid chromatography. Twenty picomoles of ascorbic acid was detected, which makes this method 25 times more sensitive than the previously used dipyridyl one. By the present method, a minute activity of GLO in liver microsomes prepared from rats of the Osteogenic Disorder Shionogi strain (ODS-od/od) could be measured.     

Synthesis of novel ribavirin hydrazone derivatives and anti-proliferative activity against A549 lung cancer cells

A series of novel ribavirin hydrazone derivatives were synthesized by the reaction of ribavirin hydrazone with benzaldehyde or acetophenone derivatives. The structures of the compounds were determined by IR, ¹H NMR, and HRESIMS. Preliminary biological evaluation showed that one compound (7h) inhibits the growth of A549 cells at 20 μM.