Use Of Pyrazole Research Articles

Pyrazole-directed, fluoroalcohol-free arylation of C-H bonds

Directed arylation of sp2 C−H bonds is often considered one of the most convenient methods to rapidly increase the complexity of organic molecules. However, most of the available methods utilized fluoroalcohols such as hexafluoroisopropanol (HFIP) as the solvent. Herein we report a simple method for palladium-catalyzed, directed arylation of arene C−H bond using acetic acid solvent. The successful functionalization relies on the use of pyrazole directing group.

Physical Properties, Synthetic Approach and Pharmaceutical Use of Pyrazole

Physical Properties, Synthetic Approach and Pharmaceutical Use of Pyrazole

Molecular cytotoxicity mechanisms of allyl alcohol (acrolein) in budding yeast.

Allyl alcohol (AA) is one of the environmental pollutants used as a herbicide and industrial chemical. AA undergoes enzymatic oxidation in vivo to form Acrolein (Acr), a highly reactive and ubiquitous environmental toxicant. The exposure to AA/Acr has detrimental effects on cells and is highly fatal. In corroboration to the current literature describing AA/Acr toxicity, this study aimed to investigate the molecular cytotoxicity mechanisms of AA/Acr using budding yeast as a eukaryotic model organism. Genome-wide transcriptome analysis of cells treated with a sublethal dose of AA (0.4 mM) showed differential regulation of approximately 30% of the yeast genome. Functional enrichment analysis of the AA transcriptome revealed that genes belong to diverse cellular processes including the cell cycle, DNA damage repair, metal homeostasis, stress response genes, ribosomal biogenesis, metabolism, meiosis, ubiquitination, cell morphogenesis, and transport. Moreover, we have identified novel molecular targets of AA/Acr through genetic screening, which belongs to oxidative stress, DNA damage repair, iron homeostasis, and cell wall integrity. This study also demonstrated the epigenetic basis of AA/Acr toxicity mediated through histone tails and chromatin modifiers. Interestingly, our study disclosed the use of pyrazole and ethanol as probable antidotes for AA intoxication. For the first time, this study also demonstrated the reproductive toxicity of AA/Acr using the yeast gametogenesis (spermatogenesis) model. Altogether, this study unravels the molecular mechanisms of AA/Acr cytotoxicity and facilitates the prediction of biomarkers for toxicity assessment and therapeutic approaches.

Chronic intermittent ethanol exposure in mice leads to an up-regulation of CRH/CRHR1 signaling.

One of the most commonly used approaches to induce ethanol (EtOH) dependence in rodents is EtOH vapor inhalation. This procedure requires the co-administration of pyrazole-an inhibitor of the alcohol dehydrogenase-to obtain stable blood EtOH concentrations (BECs) during the entire induction course. However, pyrazole can produce unwanted side effects. Our goal was to obtain EtOH-dependent mice without pyrazole and to study their behavioral and molecular postdependent phenotype. In particular, we were interested in alterations in the corticotrophin-releasing hormone (CRH) and receptor (CRHR1 and CRHR2) system as a prominent role of CRH in driving the postdependent state via actions in the central extended amygdala (CeA) has been demonstrated in rats but not in postdependent mice. We established an alternative model of chronic intermittent EtOH (CIE) inhalation without the use of pyrazole in C57BL/6N mice. Our CIE exposure protocol involved 8 cycles. One cycle consisted of 8hours with EtOH inhalation and 8hours without EtOH. We then examined withdrawal symptoms. After 2weeks of abstinence, we studied relapse, reinstatement of EtOH-seeking, and stress-induced EtOH self-administration. We also did transcriptional analysis of components of the CRH system during CIE, protracted abstinence, and after stress-induced EtOH self-administration. CIE exposure without pyrazole resulted in reproducible BECs during the induction procedure. Mice showed strong withdrawal scores during 4 to 12hours after the last CIE cycle and enhanced stress-induced EtOH self-administration. This postdependent phenotype during abstinence was accompanied by enhanced Crh and Crhr1 transcripts but no change in Crhr2 transcripts in the CeA. Cue-induced EtOH-seeking behavior and relapse (alcohol deprivation effect) were not affected by the inhalation procedure. We have established a CIE inhalation protocol without pyrazole in mice and showed excessive EtOH self-administration under mild stress and enhanced CRH/CRHR1 signaling in the CeA.

Oxidation of ethanol to acetaldehyde in brain and the possible behavioral consequences.

One of the oldest theories of ethanol’s action is that the first metabolite, acetaldehyde, is responsible for some of the actions in the central nervous system (Hunt, 1996; Hashimoto et a1. 1989; Tan et a1. 1993; Bergamaschi et a1. 1988; Zimatkin and Deitrich, 1997; Thadani and Truitt, 1977; Collins et al. 1988; Heap et al. 1995). This hypothesis fell into disfavor for a number of reasons. The first was that following ethanol ingestion, the blood levels of acetaldehyde are extremely low, provided that the artifactual formation of acetaldehyde is accounted for (Sippel, 1974; Westcott et a1. 1980; Sippel and Eriksson, 1975; Tabakoff et a1. 1976; Zimatkin and Pronko, 1995). The levels in normal humans are nearly undetectable in the blood, of the order of 1 μM. The second problem was that even if the blood acetaldehyde levels were significant, the molecule does not seem to be able to penetrate the blood brain barrier because of the presence of aldehyde dehydrogenase in blood vessels. Substantial blood levels were required before acetaldehyde appeared in the brain (Westcott et a1. 1980; Tabakoff et al. 1976; Sippel and Eriksson, 1975; Sippel, 1974; Sippel, 1974). A third issue was that one could inhibit the oxidation of ethanol to acetaldehyde with pyrazole but intoxication still ensued. Indeed the use of pyrazole was critical in the vapor chamber method of Goldstein where ethanol metabolism was slowed in order to physically addict mice to ethanol (Goldstein and Pal, 1971).

Use of a competitive inhibitor in a kinetic enzymatic method for measuring ethanol in serum.

We describe a kinetic enzymatic method for ethanol in serum, based on the use of pyrazole, a competitive inhibitor for alcohol dehydrogenase (EC 1.1.1.1). The method is rapid and is easily automated for the Cobas Bio centrifugal analyzer. No specimen pretreatment is necessary and the total reaction time is 120 s. The standard curve is linear up to 140 mmol/L. The within-run CV was between 1.9% and 3.5%; the between-run CV ranged from 2.6% to 4.1%. Mean analytical recovery of ethanol added to serum was 100.2%. We compared results of the kinetic enzymatic method with those from a gas-liquid chromatographic (GLC) method and another commercial alcohol dehydrogenase method (TDx REA Ethanol; Abbott Diagnostics). Linear regression analysis gave the following equations: kinetic = 0.991GLC - 0.354 mmol/L (r = 0.992, n = 110) and kinetic = 0.998TDx - 1.741 mmol/L (r = 0.993, n = 70). No interference from methanol or isopropanol was seen.

Inhibition of microsomal oxidation of ethanol by pyrazole and 4-methylpyrazole in vitro. Increased effectiveness after induction by pyrazole and 4-methylpyrazole.

Pyrazole and 4-methylpyrazole, which are inhibitors of alcohol dehydrogenase, were also found to be effective inhibitors of the oxidation of ethanol by liver microsomes (microsomal fractions) in vitro. Ethanol oxidation by microsomes from rats previously treated for 2 or 3 days with either pyrazole or 4-methylpyrazole appeared to be especially sensitive to inhibition in vitro by pyrazole or 4-methylpyrazole. The kinetics of inhibition by pyrazole or 4-methylpyrazole in all microsomal preparations were mixed, as the Km for ethanol was elevated while Vmax was lowered. However, Ki values for pyrazole (about 0.35 mM) and especially 4-methylpyrazole (about 0.03-0.10 mM) were much lower than those found with the saline controls (about 0.7-1.1 mM). In contrast, Ki values for dimethyl sulphoxide as an inhibitor of microsomal ethanol oxidation were similar in all microsomal preparations. Pyrazole and 4-methylpyrazole reacted with microsomes to produce type II spectral changes whose magnitude increased after treatment with either pyrazole or 4-methylpyrazole. Thus the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole appears to be associated with increased interactions with the cytochrome P-450 isoenzyme(s) induced by these compounds. These isoenzymes have properties similar to those of the isoenzyme induced by chronic ethanol treatment. Therefore, caution is needed in the use of pyrazole or 4-methylpyrazole to assess pathways of ethanol metabolism, especially after chronic ethanol treatment, since these agents, besides inhibiting alcohol dehydrogenase, are also effective inhibitors of microsomal ethanol oxidation.

Selective oxidation of steroidal allylic alcohols using pyrazole and pyridinium chlorochoromate

ABASTRACT: This paper presents a modified method for the selective oxidation of allylic alchols. Pyrazole, when used with pyridinium chlorochromate, is a mild and useful reagent system for the rapid and selective oxidation of steroidal allylic alcohols to the corresponding α, β-unsaturated ketones. The reaction of each substrate was carried out by adding the oxidant to a dry methylene chloride solution containing pyrazole and an allylic alchol. This report is the first on the use of pyrazole to augment selective oxidation by a chronium (VI) reagent.

Substituent Effects on the Ionization Step Regulating Desorption and Catalytic Oxidation of Alcohols Bound to Liver Alcohol Dehydrogenase

1. Transient-state kinetic methods, based on the use of pyrazole as a displacing reagent and reporter ligand, have been applied to examine the pH dependence of rate and equilibrium constants for 2-chloroethanol and 2,2-dichloroethanol binding to the binary complex formed between liver alcohol dehydrogenase and NAD+. 2. The apparent affinity of the enzyme . NAD+ complex for the examined alcohols is dependent on two proton dissociation equilibria. One of these equilibria affects the rate of alcohol association to the binary complex with a ligand-independent pKa value of 7.6, attributable to ionization of zinc-bound water in the enzyme . NAD+ complex. The second proton dissociation equilibrium regulates the rate of alcohol desorption from the enzyme . NAD+ . alcohol complex and exhibits a pKa value of 5.4 and 4.5, respectively, with 2-chloroethanol and 2,2'-dichloroethanol as the ligand. Steady-state kinetic data are reported which indicate that the pKa-5.4-equilibrium controls also the apparent rate of enzymic hydride transfer from 2-chloroethanol to NAD+. 3. These results lend strong support to the mechanism of enzyme action proposed by Kvassman and Pettersson according to which the enzyme . NAD+ . alcohol complex participates in an obligatory proton dissociation step which regulates both desorption and catalytic oxidation of the alcohol ligand. The corresponding pKa value is shown to be lineary dependent on the pKa of the free alcohol ligand with a regression coefficient (Brönstedt alpha value) of about 0.6. The latter observation provides direct evidence that the effect of pH on the reactivity of the enzyme . NAD+ . alcohol complex reflects an alcohol/alcoholate ion equilibration of the enzyme-bound substrate. 4. The particular mode of binding and properties of the active-site zinc ion in liver alcohol dehydrogenase suggest that the catalytic function of the metal ion can be related primarily to facilitation of the process of alcohol oxidation through facilitation of the alcoholate ion formation step now established to precede hydride transfer to NAD+.

A study on diazepam and postwithdrawal drinking of ethanol solution in rats

A 10-day ethanol inhalation procedure without the use of pyrazole was used to produce physical dependence on ethanol in Sprague-Dawley rats. During this 10-day period, weight changes in the control animals reflected the weight changes in the ethanoltreated animals. At the end of the inhalation procedure the rate of ethanol clearance from the blood was 0.44 ± 0.2 mg/ml/hr in the dependent animals; also, signs of withdrawal were manifested in all ethanol vapor-treated animals, but not in control animals. Twenty-four hours after removal from the chamber, a 5-day drinking study was started. Control animals and physically dependent animals were each divided into two groups, one for diazepam (5 mg/ kg, orally) administration and one for vehicle administration. The results from the free-choice drinking study (water and ethanol solution, 7%) indicated that diazepam did not enhance ethanol consumption in animals which were physically dependent on ethanol but significantly suppressed ethanol drinking in nondependent animals. Also when examining the effect of physical dependence alone on postwithdrawal drinking, it appeared that no increase in ethanol preference was observed with animals under our experimental design.

Physical dependence on ethanol: Rate of ethanol clearance from the blood and effect of ethanol on body temperature in rats

An inhalation procedure for the administration of ethanol without the use of pyrazole was employed to produce physical dependence on ethanol in rats. A reduction in body temperature was noted at the latter part of the induction phase and in the early hours of withdrawal. In the withdrawal phase the animals experienced convulsions on handling and sponataneous signs of withdrawal. To investigate the rates of ethanol clearance from the blood po and ip routes of ethanol administration were used. A significant enhancement of the rate of ethanol disappearance from the blood was measured in the animals at 48 hr following 10 days of ethanol exposure. Also at this 48-hr time period the usual hypothermic response to ethanol administration was lost in these animals as compared to a control group and to animals receiving ethanol ip, at 7 days after exposure to ethanol vapor. The data indicated that the loss of hypothermic effect from ethanol might be a factor in the increased rate of ethanol clearance. However, in studying the rate of ethanol disappearance from the blood in nondependent animals at 34°C which prevented hypothermia, there was no difference in the rates of ethanol clearance as compared to controls at 23.5°C where the hypothermic effect from ethanol was present. It appears that body temperature does not play a role in the observed acceleration of ethanol clearance from the blood in animals which have undergone withdrawal.

Induction of physical dependence in rats by ethanol inhalation without the use of pyrazole

Rats were exposed to increasing air ethanol concentrations (14 to 28 mg/liter) over a 10-day period without the use of pyrazole. There was a positive correlation between the rising air ethanol concentrations in the chamber and the concentrations of ethanol in the blood. On Day 10 the animals were removed from the chamber with a mean blood ethanol concentration of 3.13 mg/ml and observed for signs of withdrawal over a 24-hr period. During this time all animals manifested spontaneous signs of withdrawal and convulsions on handling. There was a positive correlation between the spontaneous signs and convulsions on handling. The disappearance of ethanol from the blood followed zero-order kinetics over the first 7 hr. In the withdrawal phase of the experiments a definite negative correlation existed between the decreasing blood ethanol concentrations and the rise in the scores for convulsions on handling. This inhalation procedure is a simple, reproducible approach for causing physical dependence on ethanol in rats without the use of pyrazole and with the maintenance of original body weight.

Isotopic probes into pathways of ethanol metabolism

The relative extent of tritium labeling in glucose and water was determined when l-[2- 3H]lactate or [(1 R)1- 3H]ethanol were the labeled substrates for rat liver parenchymal cells, incubated with 20 m m ethanol and 10 m ml-lactate. From the relatively lower specific yield in glucose from the tritiated ethanol one can calculate a percentage contribution of a non-alcohol dehydrogenase-mediated pathway to total ethanol metabolism. This calculated value (about 35%) is somewhat higher than that determined by the use of pyrazole at 5 m m to inhibit alcohol dehydrogenase. The utilization of [(1 R)1- 3H]ethanol is slower than that of unlabeled ethanol, both in the absence and presence of 5 m m pyrazole, indicating isotope discrimination against tritium in both the alcohol dehydrogenase and non-alcohol dehydrogenase pathways. There was only a slight difference in the rate of utilization of normal and fully deuterated ethanol by rat liver cells in the absence of pyrazole. However, in the presence of 5 m m pyrazole, where essentially only the non-alcohol dehydrogenase pathway operates, deuterated ethanol was utilized at only about half the rate of nondeuterated ethanol. These findings are difficult to reconcile with a catalase-mediated pathway of ethanol metabolism in which the rate-limiting factor is the rate of H 2O 2 generation.

Effects of pyrazole, 4-bromopyrazole and 4-methylpyrazole on mitochondrial function

Pyrazole, an inhibitor of alcohol dehydrogenase, has been widely used in studies of ethanol metabolism. Since its specificity has recently been questioned, we studied the effects of pyrazole, methylpyrazole and bromopyrazole on mitochondrial function. These compounds inhibited oxidative phosphorylation, the ATP −32P exchange reaction, and energy dependent and independent calcium uptake. With α-ketoglutarate as substrate, state 3 (coupled) respiration was inhibited, whereas state 4 (resting) respiration was not affected. By contrast, state 4 respiration was stimulated when succinate or ascorbate served as the substrate, while state 3 respiration was slightly inhibited. Regardless of the substrate, the respiratory control ratio was depressed. The activities of succinic dehydrogenase and cytochrome oxidase were stimulated by pyrazole and its derivatives, which may explain the stimulation of succinate and ascorbate oxidation. The inhibitory effects of these compounds were reversed by washing the mitochondria, indicating that no permanent damage to mitochondria had occurred. This is supported by the lack of stimulation of latent ATPase activity and the unchanged barrier to the penetration of NADH. Pyrazole and its derivatives decreased the uptake of citrate and glutamate, but stimulated that of phosphate and malate. Methylpyrazole and bromopyrazole inhibited the transport of reducing equivalents into the mitochondria, as catalyzed by the malate-aspartate, fatty acid and α-glycerophosphate shuttles. The data mandate caution in advocating the therapeutic use of pyrazole or its derivatives in man, and suggest that the use of pyrazole to assess ethanol metabolism and its sequelae in vivo may have limitations.