Identification of 2-mercaptohexanoic acids as dual inhibitors of 5-lipoxygenase and microsomal prostaglandin E₂ synthase-1

5-Lipoxygenase (5-LO) and microsomal prostaglandin E? synthase (mPGES)-1 are key enzymes in the biosynthesis of leukotrienes and prostaglandin (PG)E?, respectively, and are considered as valuable targets for the treatment of inflammatory diseases. Here, we present the identification of 2-mercaptohexanoic acid derivatives as dual inhibitors of 5-LO and mPGES-1. The lead compound 2(4-(3-biphenyloxypropoxy)phenylthio)hexanoic acid (21) inhibits human 5-LO and mPGES-1 in cell-free assays with an IC?? = 3.5 and 2.2 μM, respectively, and suppresses 5-LO in intact cells with even a higher potency (IC??=0.9 μM). Compound 21 (10 μM) neither significantly inhibited the related 12- or 15-LOs nor cyclooxygenase-1 and -2 or cytosolic phospholipase A?. Based on the selective and potent inhibition of 5-LO and mPGES-1, further assessment of these 2-mercaptohexanoic acids in preclinical models of inflammation are warranted.     

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BackgroundSubcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.MethodsFLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.Results5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca^2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB₄. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.ConclusionThe cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.General significanceWe present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.     

Synthesis and biological evaluation of a class of 5-benzylidene-2-phenyl-thiazolinones as potent 5-lipoxygenase inhibitors

A class of 5-lipoxygenase (5-LO) inhibitors characterized by a central 5-benzylidene-2-phenyl-thiazolinone scaffold was synthesized as a new series of molecular modifications and extensions of a previously reported series. Compounds were tested in a cell-based and a cell-free assay and furthermore evaluated for their influence on cell viability. The presented substituted thiazolinone scaffold turned out to be essential for both the 5-LO inhibitory activity and the non-cytotoxic profile. With (Z)-5-(4-methoxybenzylidene)-2-(naphthalen-2-yl)-5H-thiazol-4-one (2k, ST1237), a potent, direct, non-cytotoxic 5-LO inhibitor with IC(50) of 0.08 μM and 0.12 μM (cell-free assay and intact cells), we present a promising lead optimization and development for further investigations as novel anti-inflammatory drug.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.     

SAR-study on a new class of imidazo[1,2-a]pyridine-based inhibitors of 5-lipoxygenase

A novel class of 5-lipoxygenase (5-LO) inhibitors characterized by a central imidazo[1,2-a]pyridine scaffold, a cyclohexyl moiety and an aromatic system, is presented. This scaffold was identified in a virtual screening study and exhibits promising inhibitory potential on the 5-LO. Here, we investigate the structure-activity relationships of this compound class. With N-cyclohexyl-6-methyl-2-(4-morpholinophenyl)imidazo[1,2-a]pyridine-3-amine (14), we identified a potent 5-LO inhibitor (IC(50)=0.16μM (intact cells) and 0.1μM (cell-free)), which may possess potential as an effective lead compound intervening with inflammatory diseases and certain types of cancer.     

Structure based design and syntheses of amino-1H-pyrazole amide derivatives as selective Raf kinase inhibitors in melanoma cells

The synthesis of a novel series of N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl amide derivatives 6a-o, 7a-s and their antiproliferative activities against A375P melanoma cell line were described. Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among them, N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-(3-(4-chloro-3-(trifluoromethyl)phenyl) ureido)-2-methylbenzamide 7c exhibited potent activities (GI(50)=0.27 μM). Especially, 7c was found to be a potent and selective B-Raf V600E and C-Raf inhibitor (IC(50)=0.26 μM, IC(50)=0.11 μM, respectively), showing a possibility as melanoma therapeutics.     

I. Novel HCV NS5B polymerase inhibitors: discovery of indole 2-carboxylic acids with C3-heterocycles

SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 μM, replicon EC(50)>100 μM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 μM, replicon EC(50)=1.4 μM) and 7r (NS5B IC(50)=0.017 μM, replicon EC(50)=0.3 μM) with improved enzyme and replicon activity.     

Development of 2-substituted-N-(naphth-1-ylmethyl) and N-benzhydrylpyrimidin-4-amines as dual cholinesterase and Aβ-aggregation inhibitors: Synthesis and biological evaluation

A group of 2-substituted N-(naphth-1-ylmethyl)pyrimidin-4-amines (6a-k) and N-benzhydrylpyrimidin-4-amines (7a-k) in conjunction with varying steric and electronic properties at the C-2 position were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. The naphth-1-ylmethyl compound 6f (2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine) exhibited optimum dual ChE (AChE IC(50)=8.0 μM, BuChE IC(50)=3.9 μM) and hAChE-promoted Aβ-aggregation inhibition (30.8% at 100 μM), whereas in the N-benzhydryl series, compound 7f (N-benzhydryl-2-(4-cyclohexylpiperazin-1-yl)pyrimidin-4-amine) exhibited optimum combination of dual ChE (AChE IC(50)=10.0 μM, BuChE IC(50)=7.6μM) and hAChE-promoted Aβ-aggregation inhibition (32% at 100 μM). These results demonstrate that a 2,4-disubstituted pyrimidine ring serves as a suitable template to target multiple pathological routes in AD, with a C-2 cyclohexylpiperazine substituent providing dual ChE inhibition and potency whereas a C-4 diphenylmethane substituent provides Aβ-aggregation inhibition.     

Phenylsulphonyl urenyl chalcone derivatives as dual inhibitors of cyclo-oxygenase-2 and 5-lipoxygenase

Two series of phenylsulphonyl urenyl chalcone derivatives (UCH) with various patterns of substitution were tested for their effects on nitric oxide (NO) and prostaglandin E2 (PGE2) overproduction in RAW 264.7 macrophages. None of the tested compounds reduced NO production more than 50% at 10 microM but most of them inhibited the generation of PGE2 with IC50 values under the micromolar range. Me-UCH 1, Me-UCH 5, Me-UCH 9, Cl-UCH 1, and Cl-UCH 9 were selected to evaluate their influence on human leukocyte functions and eicosanoids generation. These derivatives selectively inhibited cyclo-oxygenase-2 (COX-2) activity in human monocytes being Me-UCH 5 the most potent (IC50 0.06 microM). Selected compounds also reduced leukotriene B4 synthesis in human neutrophils by a direct inhibition of 5-lipoxygenase (5-LO) activity, with IC50 values from 0.5 to 0.8 microM. In addition, lysosomal enzyme secretion, such as elastase or myeloperoxidase as well as superoxide generation in human neutrophils were also reduced in a similar range. Our findings indicate that UCH derivatives exert a dual inhibitory effect on COX-2/5-LO activity. The profile and potency of these compounds may have relevance for the modulation of the inflammatory and nociceptive responses with reduction of undesirable side-effects associated with NSAIDs.     

Natural forms of vitamin E and 13'-carboxychromanol, a long-chain vitamin E metabolite, inhibit leukotriene generation from stimulated neutrophils by blocking calcium influx and suppressing 5-lipoxygenase activity, respectively

Leukotrienes generated by 5-lipoxygenase (5-LOX)-catalyzed reaction are key regulators of inflammation. In ionophore-stimulated (A23187; 1-2.5 μM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited leukotriene B(4) (LTB(4)) with an IC(50) of 5-20 μM for γ-tocopherol, δ-tocopherol (δT), and γ-tocotrienol, but a much higher IC(50) for α-tocopherol. 13'-Carboxychromanol, a long-chain metabolite of δT, suppressed neutrophil- and HL-60 cell-generated LTB(4) with an IC(50) of 4-7 μM and potently inhibited human recombinant 5-LOX activity with an IC(50) of 0.5-1 μM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key event for 5-LOX activation. Further investigation showed that δT suppressed cytosolic Ca(2+) increase and/or LTB(4) formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 13'-carboxychromanol decreased cellular production of LTB(4) regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that δT does not likely affect fMLP receptor-mediated signaling or store depletion-induced calcium entry. Instead, we found that δT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inflammatory effects of vitamin E forms in vivo.     

Design, synthesis and anticancer activity of novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivatives

On the basis of the chemical structures of psorospermin with a xanthone template and acronycine derivatives with an acridone template, rac-1 and rac-2 constructed on an 1,2-dihydrobenzofuro[4,5-b][1,8]naphthyridin-6(11H)-one scaffold were designed and synthesized as potential anticancer agents. Their anticancer activities were evaluated against five human cancer cell lines. Rac-2 showed similar anticancer activity to doxorubicin and rac-1 exhibited even higher anticancer activity against LNCaP (IC(50)=0.14 μM), DU145 (IC(50)=0.15 μM), PC3 (IC(50)=0.30 μM) and MCF-7 (IC(50)=0.26 μM) cancer lines than doxorubicin and rac-2. Also, rac-1 revealed very potent anticancer activity (IC(50)=0.15 μM) against MCF-7/ADR cell (doxorubicin-resistant breast cancer cell) lines and induced G2/M phase arrest of the cell cycle in MCF-7/ADR cells.     

The antiplasmodial activity of norcantharidin analogs

The antiplasmodial activities of sixty norcantharidin analogs were tested in vitro against a chloroquine sensitive (D6, Sierra Leone) and chloroquine resistant (W2) strains of Plasmodium falciparum. Forty analogs returned IC(50) values <500 μM against at least one of the P. falciparum strains examined. The ring open compound 24 ((1S,4R)-3-(allylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid) is the most active aliphatic analog (D6 IC(50)=3.0±0.0 and W2 IC(50)=3.0±0.8 μM) with a 20-fold enhancement relative to norcantharidin. Surprisingly, seven norcantharimides also displayed good antiplasmodial activity with the most potent, 5 returning D6=8.9±0.9 and W2 IC(50)=12.5±2.2 μM, representing a fivefold enhancement over norcantharidin.     

IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils.

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.     

Pharmacology of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid), a potent, orally active leukotriene biosynthesis inhibitor.

MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.     

In situ amplification of 5-lipoxygenase and 5-lipoxygenase-activating protein in allergic airway inflammation and inhibition by leukotriene blockade

Leukotrienes are important mediators of the eosinophilic influx and mucus hypersecretion in the lungs in a murine model of asthma. We used in situ PCR in this model of human asthma to detect lung mRNA for 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP), key proteins necessary for leukotriene synthesis. Lung tissue was obtained on day 28 from mice treated with i.p. (days 0 and 14) and intranasal (days 14, 25, 26, and 27) OVA or saline. After fixation, the tissue sections underwent protease- and RNase-free DNase digestion, before in situ RT-PCR using target-specific cDNA amplification. 5-LO and FLAP-specific mRNA was visualized by a digoxigenin detection system, and positive cells were analyzed by morphometry. 5-LO and FLAP-specific mRNA and protein were associated primarily with eosinophils and alveolar macrophages in the airways and pulmonary blood vessels in OVA-sensitized/challenged mice. 5-LO and FLAP protein expression increased on a per-cell basis in alveolar macrophages of OVA-treated mice compared with saline controls. Pulmonary blood vessel endothelial cells were also positive for 5-LO, FLAP mRNA, and protein. 5-LO inhibition significantly decreased 5-LO and FLAP-specific mRNA and protein expression in the lung inflammatory cells and endothelial cells. These studies demonstrate a marked increase in key 5-LO pathway proteins in the allergic lung inflammatory response and an important immunomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.     

Synthesis of novel inhibitors of β-glucuronidase based on benzothiazole skeleton and study of their binding affinity by molecular docking

Benzothiazole derivatives 1-26 have been synthesized and their in vitro β-glucuronidase potential has been evaluated. Compounds 4 (IC(50)=8.9 ± 0.25 μM), 5 (IC(50)=36.1 ± 1.80 μM), 8 (IC(50)=8.9 ± 0.38 μM), 13 (IC(50)=19.4 ± 1.00 μM), 16 (IC(50)=4.23 ± 0.054 μM), and 18 (IC(50)=2.26 ± 0.06 μM) showed β-glucuronidase activity potent than the standard (d-saccharic acid 1,4-lactone, IC(50)=48.4 ± 1.25 μM). Compound 9 (IC(50)=94.0 ± 4.16 μM) is found to be the least active among the series. All active analogs were also evaluated for cytotoxicity and none of the compounds showed any cytotoxic effect. Furthermore, molecular docking studies were performed using the gold 3.0 program to investigate the binding mode of benzothiazole derivatives. This study identifies a novel class of β-glucuronidase inhibitors.     

6-Nitrobenzimidazole derivatives: potential phosphodiesterase inhibitors: synthesis and structure-activity relationship

6-Nitrobenzimidazole derivatives (1-30) synthesized and their phosphodiesterase inhibitory activities determined. Out of thirty tested compounds, ten showed a varying degrees of phosphodiesterase inhibition with IC(50) values between 1.5±0.043 and 294.0±16.7 μM. Compounds 30 (IC(50)=1.5±0.043 μM), 1 (IC(50)=2.4±0.049 μM), 11 (IC(50)=5.7±0.113 μM), 13 (IC(50)=6.4±0.148 μM), 14 (IC(50)=10.5±0.51 μM), 9 (IC(50)=11.49±0.08 μM), 3 (IC(50)=63.1±1.48 μM), 10 (IC(50)=120.0±4.47 μM), and 6 (IC(50)=153.2±5.6 μM) showed excellent phosphodiesterase inhibitory activity, much superior to the standard EDTA (IC(50)=274±0.007 μM), and thus are potential molecules for the development of a new class of phosphodiesterase inhibitors. A structure-activity relationship is evaluated. All compounds are characterized by spectroscopic parameters.     

A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells.

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).     

Antagonists of 5-HT₆ receptors. Substituted 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines-Synthesis and 'structure-activity' relationship

Synthesis and biological evaluation of a new series of structurally unrestricted and intramolecular hydrogen bond restricted derivatives of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines (angular tricyclics) and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines (linear tricyclics) are described. Structurally restricted derivatives are highly potent and selective blockers of 5-HT(6) receptors with little difference between angular or linear shape of the tricyclic core, the angular species being only slightly more potent. The angular representative of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines, 5, can be considered as more favorable candidate for further development as it shows only weak 5-HT(2B) blocking activity (IC(50)=6.16 μM as compared with IC(50)=1.8 nM for 5-HT(6) receptors) and very low hERG potassium channel blocking potency (IC(50)=54.2 μM). The linear analog, 11, is less favorable as while showing no binding to the 5-HT(2B) receptor at concentrations of up to 10 μM, it exhibits quite a high potency to block the hERG channel (IC(50)=0.5 μM).