4-Bicyclic heteroaryl-piperidine derivatives as potent,orally bioavailable Stearoyl-CoA desaturase-1 (SCD1) inhibitors. Part 1: Urea-based analogs

A new series of urea-based, 4-bicyclic heteroaryl-piperidine derivatives as potent SCD1 inhibitors is described. The structure–activity relationships focused on bicyclic heteroarenes and aminothiazole–urea portions are discussed. A trend of dose-dependent decrease in body weight gain in diet-induced obese (DIO) mice is also demonstrated.     

Bicyclic heteroaryl inhibitors of stearoyl-CoA desaturase: from systemic to liver-targeting inhibitors

Optimization of a lead thiazole amide MF-152 led to the identification of potent bicyclic heteroaryl SCD1 inhibitors with good mouse pharmacokinetic profiles. In a view to target the liver for efficacy and to avoid SCD1 inhibition in the skin and eyes where adverse effects were previously observed in rodents, representative systemically-distributed SCD1 inhibitors were converted into liver-targeting SCD1 inhibitors.     

Discovery of piperidine-aryl urea-based stearoyl-CoA desaturase 1 inhibitors

A series of structurally novel stearoyl-CoA desaturase1 (SCD1) inhibitors has been identified via molecular scaffold manipulation. Preliminary structure–activity relationship (SAR) studies led to the discovery of potent, and orally bioavailable piperidine-aryl urea-based SCD1 inhibitors. 4-(2-Chlorophenoxy)-N-[3-(methyl carbamoyl)phenyl]piperidine-1-carboxamide 4c exhibited robust in vivo activity with dose-dependent desaturation index lowering effects.     

Accumulation of N-3 Polyunsaturated Fatty Acids by Cultured Human Y79 Retinoblastoma Cells

Abstract: The metabolism of the n-3 class of polyunsaturated fatty acids, which occur in relatively high quantities in neural tissues, was studied in human Y79 retinoblastoma cells. These cells contained low levels of n-3 polyunsaturates when grown in culture media supplemented with fetal bovine serum. The cells readily incorporated preformed docosahexaenoic acid (22:6 n-3) into phospholipids, but human skin fibroblasts did this to a similar extent. When 10 to 30 μmol/ml linolenic acid (18:3 n-3) was added, the cells also accumulated 22:6 in phospholipids. The capacity to convert appreciable amounts of 18:3 to 22:6 appears to be a unique property of the retinoblastoma cells as compared with other continuously cultured cell lines. More 18:3 than linoleic acid (18:2 n-6) was incorporated into phospholipids by the retinoblastoma cultures, and 18:3 was channeled to a larger extent into the ethanolamine glycerophospholipid fraction. These findings indicate that retinoblastoma cells handle n-3 polyunsaturated fatty acids in a manner very similar to neural tissue in vivo . Based on the results obtained with this model system, it appears that three processes may contribute to the accumulation of 22:6 in retina and neural tissue: increased ability to incorporate 18:3, the capacity to convert 18:3 to 22:6, and channeling of 18:3 and its metabolites into ethanolamine glycerophospholipids.     

Diverse effects of essential (n−6 andn−3) fatty acids on cultured cells

Fatty acids (FAs) have long been recognized for their nutritional value in the absence of glucose, and as necessary components of cell membranes. However, FAs have other effects on cells that may be less familiar. Polyunsaturated FAs of dietary origin (n–6 andn–3) cannot be synthesized by mammals, and are termed essential because they are required for the optimal biologic function of specialized cells and tissues. However, they do not appear to be necessary for normal growth and metabolism of a variety of cells in culture. The essential fatty acids (EFAs) have received increased attention in recent years due to their presumed involvement in cardiovascular disorders and in cancers of the breast, pancreas, colon and prostate. Manyin vitro systems have emerged which either examine the role of EFAs in human disease directly, or utilize EFAs to mimic thein vivo cellular environment. The effects of EFAs on cells are both direct and indirect. As components of membrane phospholipids, and due to their varying structural and physical properties, EFAs can alter membrane fluidity, at least in the local environment, and affect any process that is mediated via the membrane. EFAs containing 20 carbons and at least three double bonds can be enzymatically converted to eicosanoid hormones, which play important roles in a variety of physiological and pathological processes. Alternatively, EFAs released into cells from phospholipids can act as second messengers that activate protein kinase C. Furthermore, susceptibility to oxidative damage increases with the degree of unsaturation, a complication that merits consideration because lipid peroxidation can lead to a variety of substances with toxic and mutagenic properties. The effects of EFAs on cultured cells are illustrated using the responses of normal and tumor human mammary epithelial cells. A thorough evaluation of EFA effects on commercially important cells could be used to advantage in the biotechnology industry by identifying EFA supplements that lead to improved cell growth and/or productivity.Abbreviations AA arachidonic acid (20 carbons: 4 double bonds,n–6) - BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - cAMP cyclic adenosine monophosphate - CHO Chinese hamster ovary - DAG diacylglycerol - DGLNA dihomo--linolenic acid (203,n–6) - DHA docosahexaenoic acid (226,n–3) - EFA essential fatty acid - EGF epidermal growth factor - EGFR epidermal growth factor receptor - EPA eicosapentaenoic acid (205,n–3) - FA fatty acid - FBS fetal bovine serum - GLNA -linolenic acid (183,n–6) - LA linoleic acid (182,n–6) - LNA -linolenic acid (183,n–3) - LT leukotriene - MDA malondialdehyde - NAD nicotinamide adenine dinucleotide - NDGA nordihydroguaiaretic acid - OA oleic acid (181,n–9) - PG prostaglandin - PKC protein kinase C - PUFA polyunsaturated fatty acid - SFM serum-free medium - TX thromboxane     

Discovery of thiazolylpyridinone SCD1 inhibitors with preferential liver distribution and reduced mechanism-based adverse effects

We discovered a series of novel and potent thiazolylpyridinone-based SCD1 inhibitors based on a 2-aminothiazole HTS hit by replacing the amide bond with a pyridinone moiety. Compound 19 demonstrated good potency against SCD1 in vitro and in vivo. The mouse liver microsomal SCD1 in vitro potency for 19 was improved by more than 240-fold compared to the original HTS hit. Furthermore, 19 demonstrated a dose-dependent reduction of plasma desaturation index with an ED₅₀ of 6.3 mg/kg. Compound 19 demonstrated high liver to plasma and liver to eyelid exposures, indicating preferential liver distribution. The preliminary toxicology study with compound 19 did not demonstrate adverse effects related to SCD1 inhibition, suggesting a wide safety margin with respect to other known SCD1 inhibitors with wider distribution profiles.     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

Systematic evaluation of amide bioisosteres leading to the discovery of novel and potent thiazolylimidazolidinone inhibitors of SCD1 for the treatment of metabolic diseases

Several five- and six-membered heterocycles were introduced to replace the C2-position amide bond of the original 2-aminothiazole-based hit compound 5. Specifically, replacement of the amide bond with an imidazolidinone moiety yielded a novel and potent thiazolylimidazolidinone series of SCD1 inhibitors. XEN723 (compound 22) was identified after optimization of the thiazolylimidazolidinone series. This compound demonstrated a 560-fold improvement in in vitro potency and reduced plasma desaturation indices in a dose dependent manner, with an EC₅₀ of 4.5 mg/kg.     

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

Plant desaturases comprise two independently evolved classes, a structurally well characterized soluble class responsible for the production of monoenes in the plastids of higher plants and the poorly structurally characterized integral membrane class that has members in the plastid and endoplasmic reticulum that are responsible for producing mono- and polyunsaturated fatty acids. Both require iron and oxygen for activity and are inhibited by azide and cyanide underscoring their common chemical imperatives. We previously showed that the Δ⁹ acyl-CoA integral membrane desaturase Ole1p from Saccharomyces cerevisiae exhibits dimeric organization, like the soluble plastidial acyl-ACP desaturases. Here we use two independent bimolecular complementation assays, i.e. yeast two-hybrid analysis and Arabidopsis leaf protoplast split luciferase assay, to demonstrate that members of the plant integral membrane fatty acid desaturase (FAD) family, FAD2, FAD3, FAD6, FAD7, and FAD8, self-associate. Further, the endoplasmic reticulum-localized desaturase FAD2 can associate with FAD3, as can the plastid-localized FAD6 desaturase with either FAD7 or FAD8. These pairings appear to be specific because pairs such as FAD3 and FAD7 (or FAD8) and FAD2 and FAD6 do not interact despite their high amino acid similarity. These results are consistent also with their known endoplasmic reticulum and plastid subcellular localizations. Chemical cross-linking experiments confirm that FAD2 and FAD3 can form dimers like the yeast Ole1p and, when coexpressed, can form FAD2-FAD3 heterodimers. Metabolic flux analysis of yeast coexpressing FAD2 and FAD3 indicates that heterodimers can form a metabolic channel in which 18:1-PC is converted to 18:3-PC without releasing a free 18:2-PC intermediate.     

Changes in electron transport pathways in endoplasmic reticulum of rapeseed in response to cold

We studied changes induced by cold on electron transfer pathways (linked to NADH or NADPH oxidation) in endoplasmic reticulum of rapeseed hypocotyls (Brassica napus L.) from a freezing-sensitive variety (ISL) and freezing-tolerant variety (Tradition). Plantlets were grown at 22 degrees C then submitted to a cold shock of 13 or 35 days at 4 degrees C. We measured the content in NADH, NADPH, NAD and NADP of the hypocotyls and the redox power was estimated by the reduced versus oxidized nucleotide ratio. The contents in cytochromes b (5) and P-450, electron acceptors of NADH and NADPH respectively, were determined by differential spectrophotometry. Finally, activity of both NADH-cytochrome b (5) reductase (E.C.1.6.2.2) and NADPH cytochrome P-450 reductase (E.C.1.6.2.4) was determined by reduction of exogenous cytochrome c. Results show that during cold shock, along with an increase of linolenic acid content, there was a general activation of the NADPH pathway which was observed more quickly in Tradition plantlets than in ISL ones. Due to transfer of electrons that can occur between NADPH reductase and cytochrome b (5), this could favor fatty acid desaturation in Tradition, explaining why linolenic acid accumulation was more pronounced in this variety. Besides, more cytochrome P-450 accumulated in ISL that could compete for electrons needed by the FAD3 desaturase, resulting in a relative slower enrichment in 18:3 fatty acid in these plantlets.