Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics.

Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test.

Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERβ, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERβ can regulate ERα activity.

Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation.

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation.

Estrogen receptor ligands. Part 10: Chromanes: old scaffolds for new SERAMs.

The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models.

Identification of neutral 4-O-alkyl quinolone nonpeptide GnRH receptor antagonists.

A series of neutral, nonbasic quinolone GnRH antagonists were prepared via Mitsunobu alkylation of protected and unprotected 4-hydroxy quinolone intermediates. The synthetic route was improved by utilization of unique reactivity and convergency afforded by the use of mono and bis-trimethylsilylethyl protected quinolones. Potent neutral GnRH antagonists were identified, including ether and lactam derivatives, that show similar in vitro binding affinity and functional activity as compared to the earlier basic 4-aminoalkyl quinolone series of nonpeptide GnRH antagonists.

Syntheses and structure-activity relationship studies of piperidine-substituted quinolones as nonpeptide gonadotropin releasing hormone antagonists.

Syntheses and structure-activity relationships of piperidine-substituted quinolones as nonpeptide gonadotropin releasing hormone antagonists are described. Some of substituents on the piperidine ring that were investigated included a fused phenyl group, a (6R)-trifluoromethyl group, (6S) and (6R)-methyl group. This study showed that GnRH binding potency was tolerated by a small group at the 6-position of the piperidine, and blocking the 6-position by a trifluoromethyl group reduced clearance rate and increased oral bioavailability.

Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha.

Estrogen receptor alpha (ER alpha) and ER beta are members of the steroid nuclear receptor family that modulate gene transcription in an estrogen-dependent manner. ER mRNA and protein have been detected both peripherally and in the central nervous system, with most data having come from the rat. Here we report the development of an ER beta-selective antibody that cross-reacts with mouse, rat, and human ER beta protein and its use to determine the distribution of ER beta in the murine brain.

Modification of the pyridine moiety of non-peptidyl indole GnRH receptor antagonists.

The synthesis of a number of indole GnRH antagonists is described. Oxidation of the pyridine ring nitrogen, combined with alkylation at the two position, led to a compound with an excellent in vitro activity profile as well as oral bioavailability in both rats and dogs.

Identification and characterization of a functionally distinct form of human estrogen receptor beta.

Estrogen receptors are important for the development and maintenance of many different tissues in the body including the breast, uterus, brain and bone. There are two known genes encoding estrogen receptors, Estrogen Receptor alpha (ER alpha) and Estrogen Receptor beta (ER beta). These receptors are transcription factors with distinct functional domains involved in DNA binding, ligand binding and transcriptional regulation.

2-Arylindoles as gonadotropin releasing hormone (GnRH) antagonists: optimization of the tryptamine side chain.

A series of 2-arylindoles containing novel heteroaromatic substituents on the tryptamine tether, based on compound 1, was prepared and evaluated for their ability to act as gonadotropin releasing hormone (GnRH) antagonists. Successful modifications of 1 included chain length variation (reduction) and replacement of the pyridine with heteroaromatic groups. These alterations culminated in the discovery of compound 27kk which had excellent in vitro potency and oral efficacy in rodents.

Orally bioavailable, indole-based nonpeptide GnRH receptor antagonists with high potency and functional activity.

Stereospecific introduction of a methyl group to the indole-3-side chain enhanced activity in our tryptamine-derived series of GnRH receptor antagonists. Further improvements were achieved by variation of the bicyclic amino moiety of the tertiary amide and by adjustment of the tether length to a pyridine or pyridone terminus. These modifications culminated in analogue 24, which had oral activity in a rat model and acceptable oral bioavailability and half-life in dogs and monkeys.

Potent nonpeptide GnRH receptor antagonists derived from substituted indole-5-carboxamides and -acetamides bearing a pyridine side-chain terminus.

A pyridine side-chain terminus has been incorporated into the indole-5-carboxamide and indole-5-acetamide series of GnRH antagonists. Potent activity was observed in binding and functional assays. Certain branched or cyclic tertiary amides were identified as preferred in each series.

Substituted indole-5-carboxamides and -acetamides as potent nonpeptide GnRH receptor antagonists.

The 2-aryltryptamine class of GnRH receptor antagonists has been modified to incorporate carboxamide and acetamide substituents at the indole 5-position. With either a phenol or methanesulfonamide terminus on the N-aralkyl side chain, potent binding affinity to the GnRH receptor was achieved. A functional assay for GnRH antagonism was even more sensitive to structural modification and revealed a strong preference for branched tertiary amides.

A potent, nonpeptidyl 1H-quinolone antagonist for the gonadotropin-releasing hormone receptor.

Extensive development of the structure-activity relationships of a screening lead determined three important pharmacophores for gonadotropin-releasing hormone (GnRH) receptor antagonist activity. Incorporation of the 3,4,5-trimethylphenyl group at the 3-position, 2-(2(S)-azetidinyl)ethoxy group at the 4-position, and N-4-pyrimidinylcarboxamide at the 6-position of the quinolone core resulted in the identification of 4-(2-(azetidin-2(S)-yl)ethoxy)-7-chloro-2-oxo-3-(3,4,5-trimethylphenyl)-1,2-dihydroquinoline-6-carboxylic acid pyrimidin-4-ylamide (1) as a potent antagonist of the GnRH receptor. A 10(4)-fold increase in in vitro binding affinity is observed for the GnRH receptor as compared to the initial screening lead.

Quinolones as gonadotropin releasing hormone (GnRH) antagonists: simultaneous optimization of the C(3)-aryl and C(6)-substituents.

A series of 3-arylquinolones was prepared and evaluated for their ability to act as gonadotropin releasing hormone (GnRH) antagonists. A variety of substitution patterns of the 3-aryl substituent are described. The 3,4,5-trimethylphenyl substituent (23h) was found to be optimal.

Potent antagonists of gonadotropin releasing hormone receptors derived from quinolone-6-carboxamides.

SAR studies which focused upon the C-6 position of a recently described series of quinolone gonadotropin releasing hormone antagonists are reported. Synthetic access to diverse quinolone-6-carboxamides was achieved via the palladium-catalyzed amino-carbonylation reactions of iodide 4 with various amines. Amides related to 9y were especially potent, functional antagonists of rat and human GnRH receptors.

Inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase and cholesterol biosynthesis by beta-lactone inhibitors and binding of these inhibitors to the enzyme.

The beta-lactones L-659,699 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2- oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic acid) and its radioactive derivative 3H-L-668,411 (the 2,3-ditritiated methyl ester of L-659,699) inhibited a partially purified preparation of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase with an IC50 of 0.1 microM. These compounds were also found to inhibit the incorporation of [14C]acetate into sterols in cultured Hep G2 cells with an IC50 of 3 microM.

On the etiology of subcapsular lenticular opacities produced in dogs receiving HMG-CoA reductase inhibitors.

The administration of high dosages of various hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors has resulted in the development of subcapsular lenticular opacities in dogs. While dogs receiving cataractogenic doses of HMG-CoA reductase inhibitors experienced profound decreases in circulating serum cholesterol concentrations (40-60% reductions in total serum cholesterol), a causal relationship between serum cholesterol lowering and cataractogenesis was not established. A strong relationship was demonstrated, however, between the systemic exposure to inhibitor (plasma drug levels) and the cataractogenic potential of the various compounds studied.

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631).

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase.

Metabolism of lovastatin by rat and human liver microsomes in vitro.

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry.

Separation and identification of triglycerides, cholesteryl esters, cholesterol, 7-dehydrocholesterol, dolichol, ubiquinone, alpha-tocopherol, and retinol by high performance liquid chromatography with a diode array detector.

A simple isocratic high performance liquid chromatography (HPLC) system is described that allows separation and identification of cholesteryl esters, triglycerides, ubiquinone, alpha-tocopherol, dolichol, cholesterol, 7-dehydrocholesterol, and retinol. This consisted of a normal phase cyanopropyl column with 0.1% isopropanol in heptane as the solvent.

Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699.

A beta-lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylating), EC, 4.1.

Mevinolinic acid biosynthesis by Aspergillus terreus and its relationship to fatty acid biosynthesis.

Mevinolinic acid, the open acid form of mevinolin, which is a metabolite of Aspergillus terreus, has been shown to be a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (Alberts et al., Proc. Natl.

Studies on the in vivo synthesis of triacylglycerol in mouse liver.

The in vivo biosynthesis of hepatic glycerolipids was examined by studying the incorporation of [2-3H] glycerol into triacylglycerol in the mouse. The isotope was administered by rapid injection into the portal vein. The incorporation of glycerol was linear for about 1 min and maximal rates were seen in the presence of additional oleic acid.

A sensitive assay for phosphatidate phosphohydrolase in mouse liver microsomes.

A technique is described for the assay of phosphatidate phosphohydrolase using 1,2-[9,10-3H]dioleoyl-sn-glycero-3-phosphate as a substrate. This substrate was prepared enzymatically using mouse liver microsomes washed with 0.5 M NaCl, which synthesize minimal amounts of neutral lipids at high enzyme concentrations.