Indolizines as novel potent inhibitors of 15-lipoxygenase

15-Lipoxygenase (15-LO) has been implicated in oxidation of low-density lipoproteins (LDL) and this enzyme may be involved in the development of atherosclerosis. We have examined 1-substituted indolizines as possible inhibitors of 15-LO from soy beans and from rabbit reticulocytes. Most compounds studied were significantly more active as inhibitors of 15-LO from soy beans than quercetin. The indolizines were slightly less potent inhibitors of the mammalian enzyme, but we found good correlation between inhibitory activity against both 15-LO enzymes studied. Several of the compounds were only weak DPPH scavengers and they may therefore be regarded as so-called non antioxidant inhibitors of 15-LO.     

New bis-catechols 5-lipoxygenase inhibitors

Three polyhydroxy-2-phenylnaphthalenes (1-3) and the oxy analogue of tetrahydroxypavinan (4) were prepared and evaluated for their antioxidant properties (inhibition of diphenylpycrylhydrazyl radical (DPPH), reduction of iron (III) ion) and inhibition of 5-lipoxygenase (5-LO) activity. Their three-dimensional structures were established on the basis of spectroscopic data and semiempirical calculations. Compounds 1 and 2 were found as potent 5-LO inhibitors as nordihydroguaiaretic acid (NDGA), whereas 4 is 2.5 times less potent than NDGA. The reliability of the 3-D structures with the 5-LO inhibition properties is discussed. Their antioxidant properties show that tested compounds are expected to act as redox inhibitors.     

Design and synthesis of pyrimido[4,5-b][1,4]benzothiazine derivatives, as potent 15-lipoxygenase inhibitors

A group of 2-substituted pyrimido[4,5-b][1,4]benzothiazines were designed, synthesized, and evaluated as potential inhibitors of 15-lipoxygenase (15-LO). Compounds 4d and 4e showed the best IC50 of 15-LO inhibition (IC50=18 and 34 microM, respectively). All compounds were docked into 15-LO. As a result the sulfur atom was oriented toward the iron atom of the active site of 15-LO. We suggest the interaction of the iron atom is essential for the activity of the inhibitors.     

Indolizine 1-sulfonates as potent inhibitors of 15-lipoxygenase from soybeans

A number of indolizine 1-sulfonates have been prepared by cyclization of cyclopropenones with pyridines followed by trapping of the intermediate 1-indolizinol with a sulfonyl halide, and examined as inhibitors of 15-lipoxygenase (15-LO). The compounds display IC(50) values between 15 and 42 microM; all are more active than the well-known 15-LO inhibitor quercetin (IC(50) 51 microM). A wide variety of substituents are well tolerated. The enzyme inhibition was not affected by preincubation or the presence of a detergent and no significant particle formation was observed. Hence, inhibition from aggregates of indolizines, promiscuous inhibition, is highly unlikely.     

Structural modifications of coumarin derivatives: Determination of antioxidant and lipoxygenase (LOX) inhibitory activity

In the present project, a series of coumarin analogues, were synthesised and evaluated for their antioxidant and soybean lipoxygenase inhibitory activity. A variety of structural modifications on the coumarin scaffold revealed interesting structure–activity relationships concerning the different biological assays. Prenyloxy-coumarins 9 and 10 displayed the best combined inhibition of lipid peroxidation and soybean lipoxygenase. Thiocoumarins 11 and 14 were identified as potent lipoxygenase inhibitors whereas hydrazone analogues 15 and 16 were efficient DPPH radical scavengers.     

Alpha-tocopherol decreases tumor necrosis factor-alpha mRNA and protein from activated human monocytes by inhibition of 5-lipoxygenase

Cardiovascular disease is the leading cause of morbidity in Westernized populations. Low levels of alpha-tocopherol (AT) are associated with increased incidence of atherosclerosis and increased intakes appear to be protective. AT supplementation decreases interleukin 1 and 6 release from human monocytes. Thus, the aim of this study was to examine the effect of AT on an important proinflammatory cytokine, tumor necrosis factor-alpha (TNF) release from human monocytes. AT supplementation (1200 IU/day for 3 months) significantly decreased TNF release from activated human monocytes. Mechanisms that were examined included its effect as a general antioxidant, its inhibitory effect on protein kinase C (PKC), and the cycloxygenase-lipoxygenase pathway. While AT decreased TNF release from activated monocytes, other antioxidants had no effect on TNF release. Specific PKC inhibitors had no effect on TNF release from activated monocytes. The inhibition of TNF release by AT in activated monocytes was reversed by leukotriene B(4) (LTB(4)), a major product of the 5-lipoxygenase (5-LO) pathway. Similar observations were seen with inhibitors of 5-lipoxygenase. Indomethacin, a COX inhibitor, in the presence and absence of AT failed to affect TNF activity. These findings suggest that AT decreases TNF release from activated human monocytes via inhibition of 5-lipoxygenase. Also, AT as well as a 5-LO inhibitor significantly decreased TNF mRNA. Furthermore, AT and the 5-LO inhibitor decreased NFkappab-binding activity. Thus, in activated human monocytes, AT appears to inhibit TNF mRNA and protein by inhibition of 5-LO.     

Tryptamine and homotryptamine-based sulfonamides as potent and selective inhibitors of 15-lipoxygenase

A series of inhibitors of mammalian 15-lipoxygenase based on tryptamine and homotryptamine scaffolds is described. Compounds with aryl substituents at C-2 of the indole core of tryptamine and homotryptamine sulfonamides (e.g., 37a-p) proved to be potent inhibitors of the isolated enzyme. Selected compounds also demonstrated desirable inhibition selectivities over isozymes 5- and P-12-LO.     

Cysteinyl-flavan-3-ol conjugates from grape procyanidins. Antioxidant and antiproliferative properties

New bio-based antioxidant compounds have been obtained by depolymerisation of grape polymeric flavanols in the presence of cysteine. Their preparation and purification, as well as their antiradical/antioxidant and antiproliferative properties are reported. 4beta-(S-cysteinyl)epicatechin 5, 4beta-(S-cysteinyl)catechin 6 and 4beta-(S-cysteinyl)epicatechin 3-O-gallate 7 were efficiently purified from the crude depolymerised mixture by cation-exchange chromatography and preparative reversed-phase chromatography. The new compounds were more efficient than the underivatised (-)-epicatechin 1 as scavengers of the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) and weak growth inhibitors of human colon carcinoma HT29 cells. The order of antiradical and antiproliferative efficiency was 7 >5 approximately 6 >1, the same for both assays.     

Synthesis and biological evaluation of purine-pyrazole hybrids incorporating thiazole,thiazolidinone or rhodanine moiety as 15-LOX inhibitors endowed with anticancer and antioxidant potential

Novel purine-pyrazole hybrids combining thiazoles, thiazolidinones and rhodanines, were designed and tested as 15-LOX inhibitors, potential anticancer and antioxidant agents. All tested compounds were found to be potent 15-LOX inhibitors with IC₅₀ ranging from 1.76 to 6.12 µM. The prepared compounds were evaluated in vitro against five cancer cell lines: A549 (lung), Caco-2 (colon), PC3 (prostate), MCF-7 (breast) and HepG-2 (liver). Compounds 7b and 8b displayed broad spectrum anticancer activity against the five tested cell lines (IC₅₀ = 18.5–95.39 µM). While, compound 7h demonstrated moderate anticancer activity against lung A549 and colon Caco-2 cell lines. Antioxidant screening revealed that six compounds (5a, 5b, 6b, 7b, 7h and 8b) with IC₅₀ ranging from 0.93 to 14.43 µg/ml were found to be more potent scavengers of 2,2- diphenyl-1-picrylhydrazyl (DPPH) than the reference ascorbic acid with IC₅₀ value of 15.34 µg/ml. Compounds 7b, 7h and 8b, when evaluated for their antioxidant activity, where found to be potent DPPH scavengers. Moreover, compound 7b displayed twice the potency of ascorbic acid as NO scavenger. Docking study was performed to elucidate the possible binding mode of the most active compounds with the active site of 15-LOX enzyme. Collectively, the purine-pyrazole hybrids having thiazoline or thizolidinone moieties (7b, 7h and 8b) constitute a promising scaffold in designing more potent 15-LOX inhibitors with anticancer and antioxidant potential.     

Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages.

Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.     

Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO-transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.     

Novel imidazole compounds as a new series of potent,orally active inhibitors of 5-lipoxygenase

Replacement of the dihydroquinolinone pharmacophore of Zeneca's ZD2138 by ionizable imidazolylphenyl moiety has lead to the discovery of a novel series of potent and orally active 5-lipoxygenase (5-LO) inhibitors. The synthesis and structure-activity relationship (SAR) of this series of compounds are described herein.     

Platelet-derived growth factor (PDGF) induces pulmonary vascular remodeling through 15-LO/15-HETE pathway under hypoxic condition

15-lipoxygenase (15-LO) is known to play an important role in chronic pulmonary hypertension. Accumulating evidence for its down-stream participants in the vasoconstriction and remodeling processes of pulmonary arteries, while how hypoxia regulates 15-LO/15-hydroxyeicosatetraenoic acid (15-HETE) to mediate hypoxic pulmonary hypertension is still unknown. Platelet-derived growth factor (PDGF) is an important vascular regulator whose concentration increases under hypoxic condition in the lungs of both humans and mice with pulmonary hypertension. The present study was carried out to determine whether hypoxia advances the pulmonary vascular remodeling through the PDGF/15-LO/15-HETE pathway. We found that pulmonary arterial medial thickening caused by hypoxia was alleviated after a treatment of the hypoxic rats with imatinib, which was associated with down-regulations of 15-LO-2 expression and 15-HETE production. Moreover, the increases in cell proliferation and endogenous 15-HETE content by hypoxia were attenuated by the inhibitors of PDGF-β receptor in pulmonary artery smooth muscle cells (PASMCs). The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of 15-LO inhibitors or 15-LO RNA interference. These results suggest that hypoxia promotes PASMCs proliferation and survival, contributing to pulmonary vascular medial hypertrophy, which is likely to be mediated via the PDGF-BB/15-LO-2/15-HETE pathway.     

Use of a continous assay of oxygen consumption to evaluate the pharmacology of 5-lipoxygenase inhibitors

A variety of assay systems have been utilized to evaluate the inhibition of the key enzyme in leukotriene (LT) biosynthesis, 5-lipoxygenase (5-LO). We have developed an assay utilizing a cytosolic preparation of 5-LO from rat basophilic leukemia (RBL-1) cells. Enzyme activity was monitored by continous measurement of oxygen consumption. High performance liquid chromatography (HPLC) analysis of products showed exclusive generation of 5-LO products. The assay proved useful for the evaluation of a variety of chemical classes of lipoxygenase inhibitors and clearly differentiated those compounds which extended the lag phase (e.g. A-64077) as opposed to the propagation phase of the enzyme activity (e.g. SK & F 105561). The data generated were in reasonable agreement with results from the assay of isolated human monocyte 5-LO and, with the exception of compounds which appear to have a significant effect on 5-LO translocation (e.g. MK-886 and Wy-49 232), inhibition of LT production by intact monocytes. This assay system proved to be a convenient and informative method to analyze inhibition of 5-LO activity.     

5-Lipoxygenase inhibitors: convenient synthesis of 4-[3-(4-heterocyclylphenylthio)phenyl]-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide analogues

A convenient synthetic route to 4-[3-(4-heterocyclylphenylthio)phenyl]-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide analogues as 5-LO inhibitors is described. This methodology enabled rapid development of structure-activity relationships (SARs) leading to improvement of pharmacological properties. Thus, new compounds with higher 5-LO inhibitory potency were discovered. The stereo-chemistry requirements of the tetrahydropyran ring are also discussed.     

Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection

The olive oil phenol hydroxytyrosol (3), as well its metabolite homovanillic alcohol (4), were subjected to chemoselective lipase-catalysed acylations, affording with good yield 10 derivatives (5-14) bearing C(2), C(3), C(4), C(10) and C(18) acyl chains at C-1. Hydroxytyrosol (3) and its lipophilic derivatives showed very good DPPH. radical scavenging activity. Compounds 3, 4 and their lipophilic analogues 5-14 were subjected to the atypical Comet test on whole blood cells: 3 and its analogues 5 and 6, with little hydrophobic character (logP相似文献   

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Pyrazole-based sulfonamide and sulfamides as potent inhibitors of mammalian 15-lipoxygenase

A series of inhibitors of mammalian 15-lipoxygenase (15-LO) based on a 3,4,5-tri-substituted pyrazole scaffold is described. Replacement of a sulfonamide functionality in the lead series with a sulfamide group resulted in improved physicochemical properties generating analogs with enhanced inhibition in cell-based and whole blood assays.     

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.