Docking analysis targeted to the whole enzyme: an application to the prediction of inhibition of PTP1B by thiomorpholine and thiazolyl derivatives.

PTP1b is a protein tyrosine phosphatase involved in the inactivation of insulin receptor. Since inhibition of PTP1b may prolong the action of the receptor, PTP1b has become a drug target for the treatment of type II diabetes. In the present study, prediction of inhibition using docking analysis targeted specifically to the active or allosteric site was performed on 87 compounds structurally belonging to 10 different groups. Two groups, consisting of 15 thiomorpholine and 10 thiazolyl derivatives exhibiting the best prediction results, were selected for in vitro evaluation. All thiomorpholines showed inhibitory action (with IC50 = 4-45 µΜ, Ki = 2-23 µM), while only three thiazolyl derivatives showed low inhibition (best IC50 = 18 µΜ, Ki = 9 µΜ). However, free binding energy (E) was in accordance with the IC50 values only for some compounds. Docking analysis targeted to the whole enzyme revealed that the compounds exhibiting IC50 values higher than expected could bind to other peripheral sites with lower free energy, Eo, than when bound to the active/allosteric site. A prediction factor, E- (ΣEo × 0.16), which takes into account lower energy binding to peripheral sites, was proposed and was found to correlate well with the IC50 values following an asymmetrical sigmoidal equation with r2 = 0.9692.

Selected heterocyclic compounds as antioxidants. Synthesis and biological evaluation.

Reactive oxygen species, oxidative stress, and oxidative damage are increasingly assigned important roles as harmful factors in pathological conditions and ageing. ROS are potentially reactive molecules derived from the reduction of molecular oxygen in the course of aerobic metabolism. ROS can also be produced through a variety of enzymes. Under normal circumstances, ROS concentrations are tightly controlled by physiological antioxidants. When excessively produced, or when antioxidants are depleted, ROS can impose oxidative damage to lipids, proteins, sugars and DNA. This reduction-oxidation imbalance, called oxidative stress, can subsequently contribute to the development and progression of tissue damage and play a role in the pathology of various diseases. An antioxidant is defined as "any substance that, when present at low concentrations compared with those of a substrate, significantly delays, prevents or removes oxidative damage to this target molecule". Despite evidence that oxidative damage contributes to a wide range of clinically important conditions, few antioxidants act as effective drugs in vivo. Inter alia, the difficulty of measuring antioxidant efficacy in vivo makes the interpretation of results from clinical trials difficult. A large number of synthetic compounds have been reported to possess antioxidant activity. Several of them derive from natural antioxidants, others have various structures. In this review, some of the most often reported classes of heterocyclic antioxidant compounds, as well as methods for evaluation of their antioxidant activity are discussed.

Medicinal chemistry of 2,2,4-substituted morpholines.

We have designed a number of 2-hydroxy (alkoxy)-2-aryl-4-alkyl-(5,6-alkyl)-morpholines with interesting biological activities, i.e. sympathomimetic, analgesic, drug metabolizing enzyme modulating ability, antioxidant potential, anti-inflammatory and anti-dyslipidemic properties. They are synthesised by reaction of the proper 2-aminoethanol with aryl-bromomethyl-ketone. The intermediate hydroxyaminoketone is spontaneously cyclised to form the tetrahydro-1,4-oxazine ring. The produced 2-hydroxy substituted morpholines form the 2-alkoxy derivatives. The 2-hydroxy or 2-alkoxy-1,4-oxazine derivatives can lose water or alcohol, to give styrene-type products. The conditions for oxazine ring formation and water or alcohol abstraction are investigated. Ionisation constants and partition coefficients have been determined and related to structure and activity. The biological activities of these compounds are quite interesting: The 2-phenyl analogues acquire the structural requirements for sympathomimetic amines, and present a structure related to the pethidine-type analgesics. Indeed, they show central dopaminergic and analgesic, antagonized by naloxone, activities. They also affect drug metabolising enzymes. The 2- phenyl and especially the 2-biphenyl derivatives are good antioxidants, also possessing anti-inflammatory and immunomodulating action. The later class of compounds reduces remarkably plasma triglycerides, total cholesterol and LDL-cholesterol in hyperlipidemic rats. Since NO plays a multiple role in atheromatosis and inflammation, some 2-nitroxy-alkoxy derivatives are synthesized and their NO-liberating ability assessed. These nitric esters are also found to be potent hypolipidemic-hypocholesterolemic agents. In a mechanism elucidation attempt, it is indicated that these derivatives may act as squalene synthase inhibitors.

Effect of some biologically interesting substituted tetrahydro-1,4-oxazines on drug metabolising enzymes and on inflammation.

The effect on hepatic drug metabolising enzymes was evaluated for three representative structures and that were selected from a series of substituted oxazine derivatives designed to possess particular pharmacological properties such as analgesic, antioxidant and hypolipidemic activity. In addition, since xenobiotic metabolism, reactive oxygen and nitrogen species, atherosclerosis and inflammation are interrelated and mutually affected, the effects of and on acute inflammation in vivo and lipoxygenase activity in vitro were also investigated. It was found that treatment of rats with caused induction of cytochrome P450, enhancement of the metabolism of aminopyrine in vitro and of zoxazolamine and hexobarbital in vivo. Compound appeared to induce particularly erythromycin N-demethylation, while, a nitric ester, reduced the catalytically active cytochrome P450, although it increased the metabolism of specific cytochrome P450 substrates, i.e. 4-nitrophenol and erythromycin. Compounds and with strong hypolipidemic and antioxidant properties, reduced acute inflammatory response in two inflammation models and inhibited lipoxygenase activity in vitro. These results are helpful in optimising the biological profile as well as the potential applications of substituted oxazines.

Effect of novel anti-inflammatory ethanolamine derivatives with antioxidant properties on drug metabolising enzymes.

The influence of four ethanolamine derivatives with anti-inflammatory and antioxidant activity on the in vitro aminopyrine N-demethylation was studied. It was found that these compounds inhibit the N-demethylation of aminopyrine. 1-Cyclohexyl-5-(2-hydroxy-ethylamino)-pentan-2-one (compound 4), possessing the highest inhibitory activity and found earlier to be a potent anti-inflammatory agent, is further tested in vivo on zoxazolamine-induced paralysis, after a single administration to rats, and on aminopyrine N-demethylation, rat hepatic total cytochrome P450 and protein (postmitochondrial and microsomal) content, after a prolonged treatment. It was found that the examined compound had no significant influence on the above biotransformations, however, it could decrease the catalytically active hepatic cytochrome P450 content. These results, considered together with some structural and physicochemical properties of the compound, indicate that this compound may act as a CYP2D6 substrate.

Nitric oxide in atherosclerosis.

*NO is produced endogenously from L-arginine by NOSs. Among its multiple activities, the homeostatic control of the vascular endothelium is crucial for atherosclerosis, a pathogenic condition connected with elevated levels of LDL, the main plasma cholesterol carrier. Oxidised LDL is proatheromatic, and toxic peroxidation products contribute to further endothelial damage. *NO controls vascular tone, inhibits LDL oxidation and has hypocholesterolaemic activity. This review is referred to the chemistry, biology and role of *NO on atherosclerosis and its treatment by *NO donors or modulators.

Hypocholesterolemic and hypolipidemic activity of some novel morpholine derivatives with antioxidant activity.

In this investigation, we study the synthesis and the evaluation of antioxidant and hypocholesterolemic activity of a number of 2-biphenylyl morpholine derivatives, which are structurally similar to some substituted morpholines possessing antioxidant activity, as well as to hypocholesterolemic 3-biaryl-quinuclidines. The novel derivatives are found to inhibit the ferrous/ascorbate induced lipid peroxidation of microsomal membrane lipids, the most potent derivative, 2-(4-biphenyl)-4-methyl-octahydro-1,4-benzoxazin-2-ol (compound 7), having an IC(50) value of 250 microM. In addition, these compounds demonstrate hypocholesterolemic and hypolipidemic action. The most active compound (7) decreases total cholesterol, low density lipoprotein, and triglycerides in plasma of Triton WR-1339 induced hyperlipidemic rats by 54%, 51%, and 49%, respectively, at 28 micromol/kg (ip). The above results indicate that the new molecules may be proven useful as leads for the design of novel compounds as potentially antiatherogenic factors.

Cytochrome P450 modification by a new oxazine derivative with hypolipidemic activity.

Plasma lipoprotein levels, related to atheromatosis, are influenced by liver function. Microsomal enzyme inducers are reported to modify serum lipoproteins and triglycerides. In this study, the effects of subchronic and acute treatment of rats with 3-(4-biphenyl)-3-n-propoxy-octahydro-1,4-pyrido[2,1-c]oxazine, a novel compound with hypolipidemic and antioxidant activities, on rat hepatic microsomal protein and total cytochrome P450, as well as on p-nitrophenol hydroxylase (CYP2E) and erythromycin N-demethylase (CYP3A) activities are examined. The subchronic treatment had no significant effect on liver weight, microsomal protein and total cytochrome P450. The acute administration lowered considerably cytochrome P450 content. The metabolic activities of CYP2E1 and CYP3A1/2 were not altered by the subchronic treatment, but were notably decreased after the single administration of 3-(4-biphenyl)-3-n-propoxy-octahydro-1,4-pyrido[2,1-c]oxazine. The inhibition of drug metabolism by 3-(4-biphenyl)-3-n-propoxy-octahydro-1,4-pyrido[2,1-c]oxazine cannot be completely correlated with the modification of plasma cholesterol, triglycerides and LDL cholesterol, although published data connect microsomal enzyme induction with a decrease of these parameters. This discrepancy could be attributed to the different biochemical events involved in enzyme induction and inhibition.

Antioxidant activity of guaiazulene and protection against paracetamol hepatotoxicity in rats.

The effect of guaiazulene, a lipophilic azulene derivative widely found in nature, on radical-mediated processes is examined. The ability of guaizulene to inhibit rat hepatic microsomal membrane lipid peroxidation and to scavenge hydroxyl radicals, as well as to interact with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), was estimated. It was found that guaiazulene can inhibit lipid peroxidation very significantly, having an IC50 value of 9.8 microM. It can also scavenge hydroxyl radicals and interact with DPPH. The protection afforded by guaiazulene to rats with paracetamol-induced liver injury was also investigated. Paracetamol hepatotoxicity is caused by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which causes oxidative stress and glutathione (GSH) depletion. Hepatic cytosolic protein, GSH, glutathione transferase and glutathione reductase levels are determined as indices of hepatic injury with or without the administration of guaiazulene. It was found that all parameters affected by paracetamol are restored to normal by guaiazulene treatment, while the administration of guaiazulene alone has no effect on the performed tests compared with the control values. It was concluded that the significant protection against paracetamol-induced GSH depletion and hepatic damage afforded by guaiazulene is probably connected with its antioxidant activity. A molecular mechanism of action of guaiazulene is suggested.

Synthesis, antioxidant and anti-inflammatory activity of novel substituted ethylenediamines and ethanolamines. A preliminary quantitative structure-activity relationship study.

Six substituted oxo- or hydroxy-aminoethanols and ethylenediamines were synthesized and tested as anti-inflammatory agents. 1-Substituted 4-(2-aminoethylamino)-1-butanones and 1-substituted 4-(2-hydroxy-ethylamino)-1-butanones were prepared by reacting the appropriate 4-chloro-1-butanone with the corresponding aminoalcohol or ethylenediamine. 1-Substituted 4-(2-aminoethylamino)-1-butanols were prepared by the reduction of the ketones with NaBH4 or NaBH3CN. The RM values of the synthesized compounds were determined as an expression of their lipophilicity. The effect of these compounds on in vitro non-enzymatic lipid peroxidation, their hydroxyl radical scavenging activity and their ability to interact with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH) were studied. The effect of the synthesized compounds on inflammation, using the carrageenan induced rat paw edema model was studied. Both anti-inflammatory and antioxidant activities depended on some structural characteristics of the synthesized compounds. It was also attempted to correlate the above mentioned activities with some physicochemical parameters using a quantitative structure-activity relationship approach. The primary amino group appeared to be of importance for antioxidant activity in this series of compounds.

Effect of guaiazulene on some cytochrome P450 activities. Implication in the metabolic activation and hepatotoxicity of paracetamol.

The in vitro and in vivo effect of guaiazulene, a natural azulene derivative, on rat hepatic cytochrome P450 (CYP) is investigated. Furthermore, paracetamol hepatotoxicity is induced in rats and the activity of specific cytochrome P450 forms, involved in the metabolic activation of paracetamol to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) is examined, after the administration of guaiazulene, using diagnostic cytochrome P450 substrates. It is found that guaiazulene inhibited considerably CYP1A2 and CYP2B1 and had a weak effect on CYP1A1 in rat hepatic microsomal fractions. Guaiazulene administered to rats did not produce any macroscopic toxic effect and caused no change of liver weight, microsomal protein and total cytochrome P450 content. Guaiazulene inhibited CYP1A2 activity in rats with or without paracetamol intoxication. Considering that CYP1A2 participates in the formation of NAPQI, as well as in the metabolic activation of several toxic and carcinogenic compounds, these results, in combination with the antioxidant activity of guaiazulene that we have found in previous investigations, indicate potential useful applications of guaiazulene.

Biotransformation and detoxification of insecticidal metyrapone analogues by carbonyl reduction in the human liver.

1. The carbonyl reduction of insecticidal metyrapone analogues to their hydroxyl metabolites by human liver microsomes and cytosol was examined. Metabolite quantification was performed by means of hplc determination and inhibition experiments, using specific carbonyl reductase inhibitors, were conducted. 2. The cytotoxicity of the ketones and their hydroxy metabolites was assessed with the MTT test, using Chang liver cells. 3. It was found that the alcohol derivatives are the major metabolite, both in microsomes and cytosol. The microsomal reductive metabolism, considered to be mediated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) (EC 1.1.1.146), was more extensive than the cytosolic carbonyl reduction. In each case, this metabolism was inhibited significantly by equimolar concentrations of the microsomal 11 beta-HSD inhibitor glycyrrhetinic acid and the cytosolic carbonyl reductase inhibitor quercitrin, respectively. 4. The parent ketones were more cytotoxic than their alcohol metabolites. 5. These results demonstrate that the metyrapone analogues are extensively metabolized by human liver microsomes, presumably by 11 beta-HSD, to the less cytotoxic and readily excretable alcohols. 6. Since the metyrapone analogues can inhibit ecdysone 20-monooxygenase (EC 1.14.99.22), our results indicate potential application of these compounds as insecticides, which would be safer for humans, due to their reductive detoxification, mainly by the hepatic microsomal 11 beta-HSD, to the less toxic hydroxy metabolites.

Investigation of the effect of chamazulene on lipid peroxidation and free radical processes.

Oxygen toxicity and related free radical reactions are implicated in numerous pathophysiological conditions, like atherosclerosis, inflammation, gastric ulceration, neuronal degeneration, tumour promotion. The flowers of Matricaria chamomilla, Asteraceae, have been used therapeutically for conditions in which oxidative stress is supposed to be implicated. We considered interesting to investigate the effect of Chamazulene, the active substance of chamomile, on free radical processes. Membrane lipid peroxidation was induced by Fe2+/ascorbate and assessed as the 2-thiobarbituric acid reactive material. The hydroxyl radical scavenging activity was studied as the competition of Chamazulene with DMSO for HO. generated by Fe3+/ascorbate. Finally, the interaction of Chamazulene with the N-centered stable free radical DPPH was estimated photometrically (517 nm). It was found that Chamazulene inhibited lipid peroxidation in a concentration and time dependent manner presenting an IC50 of 18 microM after 45 min incubation. It could also inhibit the autoxidation of DMSO (33 mM) by 76% at 25 mM, and had a weak capacity to interact with DPPH. In conclusion, Chamazulene presents interesting properties concerning radical processes.

An approach to QSAR of 16-substituted pregnenolones as microsomal enzyme inducers.

In this study, we attempt to correlate quantitatively the structure of eight 16-substituted pregnenolones with microsomal enzyme inducing activity. We also performed some electrostatic potential calculations to get further insight into the properties of these substituents. It was found that pregnenolone-16 alpha-carbonitrile is the most active steroidal inducer among the pregnenolone derivatives tested. The receptor-inducer interaction is facilitated by a favourable electronic effect of the 16 alpha-substituents. The orientation of the electronegative area at position 16 seems to influence activity. Lipophilic and volume effects of the 16 alpha-substituents do not seem to be important for microsomal enzyme induction. However, substituent length has some influence on drug metabolising enzyme activity, probably interfering with receptor-inducer interactions.

Novel piperidine derivatives: inhibitory properties towards cytochrome P450 isoforms, and cytoprotective and cytotoxic characteristics.

The ability of a series of eight piperidine derivatives, substituted at positions 1, 3 and 4, to inhibit P450-dependent metabolism of specific substrates, is reported. Five different P450 isoforms (1A1, 1A2, 2B1, 2E1 and 3A1) in differentially induced rat liver microsomes were used for this purpose. From the results it is concluded that compound 2 was the most potent and moreover, highly selective inhibitor for P4502B1 with an IC(50) of 2.5 µM. Compound 3 appeared to have high selectivity for P4501A1 but not for P4501A2 (IC(50)s 80 and > 1000 µM, respectively). P4502B1 was found to be the most susceptible P450 isoform for inhibition by compounds 2, 3 and 6, while P4502E1 was largely insensitive to the inhibitory properties of all piperidine derivatives. A preliminary SAR study for the cytotoxicity, cytoprotective and P450 inhibitory properties of the piperidine derivatives, was also attempted. Using freshly isolated rat hepatocytes, the toxicity of the compounds was estimated and expressed as lactate dehydrogenase (LDH) leakage, lipid peroxidation (LPO) levels and GSH depletion. Considering the P450 inhibition and cytotoxicity results, compounds 2 and 3 were tested for possible protective activity against paracetamol-induced cytotoxicities. It was found that compound 2 protects completely against LDH leakage and LPO caused by paracetamol in rat hepatocytes isolated from ß-naphthoflavone (ß-NF) pretreated rats. It is concluded that the piperidine structures studied proved to be potentially valuable lead compounds for the design of potent and selective P450 inhibitors and for non-toxic cytoprotective agents as well.

Novel 1,4 substituted piperidine derivatives. Synthesis and correlation of antioxidant activity with structure and lipophilicity.

A series of novel piperidine derivatives was prepared and their lipophilicity was determined (as RM values). These compounds as well as two intermediate alpha-keto-esters were tested for antioxidant activity. It was found that the cysteamine derivatives were efficient antioxidants, i.e. they could inhibit lipid peroxidation, act as hydroxyl radical scavengers and interact with 2,2-diphenyl-1-picrylhydrazyl radicals. This interaction could be attributed to the free SH group and this activity seemed to be favoured by increased lipophilicity. Replacement of SH by NH2 or OH resulted in a decreased antioxidant activity of the compounds. However, the described activities seem not to be connected with any O2-.scavenging ability, at least under the experimental conditions applied. Furthermore, cysteamine derivatives seem to induce O2-.generation, a phenomenon often observed with thiol compounds. The antioxidant activity of the intermediate alpha-keto-esters varied and is probably mediated by different mechanisms.

Influence of mercury and cadmium intoxication on hepatic microsomal CYP2E and CYP3A subfamilies.

The effects of subchronic mercuric acetate and of acute mercuric acetate and cadmium chloride administration to rat hepatic microsomal protein and total cytochrome P450, as well as on p-nitrophenol hydroxylase (CYP2E) and erythromycin N-demethylase (CYP3A) activities were examined. It was found that Hg2+ and Cd2+ intoxication resulted in a significant decrease of total cytochrome P450 content. Acute Hg2+ and Cd2+ exposure decreased microsomal protein level. These metals also reduced CYP2E1 activity, while they did not seem to alter CYP3A1-mediated drug metabolism. This effect on CYP2E1 may be connected to free radical generation during Hg2+ and Cd2+ intoxication. Investigation is in progress using more P450 markers for elucidation of the effect of Hg2+ and Cd2+ on P450 activities.

Effect on active oxygen species of alliin and Allium sativum (garlic) powder.

Considering that oxygen toxicity and the related free radical attack are involved in many pathophysiological conditions, and that Allium sativum (ASP) has been used therapeutically for many of them since antiquity, we examined the intervention of ASP and alliin in free radical processes. It was found that ASP presented antioxidant activity and alliin was a very good hydroxyl radical scavenger. ASP presented good reducing ability, interacting with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH). These results indicate an involvement of the active Alluim sativum ingredients in free radical processes.