Biphenyl-substituted oxazolidinones as cholesteryl ester transfer protein inhibitors: modifications of the oxazolidinone ring leading to the discovery of anacetrapib.

The development of the structure-activity studies leading to the discovery of anacetrapib is described. These studies focused on varying the substitution of the oxazolidinone ring of the 5-aryloxazolidinone system. Specifically, it was found that substitution of the 4-position with a methyl group with the cis-stereochemistry relative to the 5-aryl group afforded compounds with increased cholesteryl ester transfer protein (CETP) inhibition potency and a robust in vivo effect on increasing HDL-C levels in transgenic mice expressing cynomolgus monkey CETP.

2-Arylbenzoxazoles as CETP inhibitors: raising HDL-C in cynoCETP transgenic mice.

We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.

2-Arylbenzoxazoles as CETP inhibitors: substitution and modification of the alpha-alkoxyamide moiety.

The development of a series of 2-arylbenzoxazole alpha-alkoxyamide and beta-alkoxyamine inhibitors of cholesteryl ester transfer protein (CETP) is described. Highly fluorinated alpha-alkoxyamides proved to be potent inhibitors of CETP in vitro, and the highly fluorinated 2-arylbenzoxazole beta-alkoxyamine 4 showed a desirable combination of in vitro potency (IC(50)=151 nM) and oral bioavailability in the mouse.

Discovery of N-{N-[(3-cyanophenyl)sulfonyl]-4(R)-cyclobutylamino-(L)-prolyl}-4-[(3',5'-dichloroisonicotinoyl) amino]-(L)-phenylalanine (MK-0668), an extremely potent and orally active antagonist of very late antigen-4.

Extremely potent very late antigen-4 (VLA-4) antagonists with picomolar, whole blood activity and slow dissociation rates were discovered by incorporating an amino substituent on the proline fragment of the initial lead structure. This level of potency against the unactivated form of VLA-4 was shown to be sufficient to overcome the poor pharmacokinetic profiles typical of this class of VLA-4 antagonists, and sustained activity as measured by receptor occupancy was achieved in preclinical species after oral dosing.

Automated sample preparation and purification of homogenized brain tissues.

A robotic homogenized tissue sample transferring method has been developed by using a Packard MultiProbe II 8-tip system. It enables robotically transferring homogenized tissue samples from individual test tubes into a 96-well format plate for further sample purification and analysis. Extensive validation has been made to establish the accuracy and variability of this method.

A simple in vitro model to study the stability of acylglucuronides.

Introduction: Compounds containing the carboxylic functional group (e.g. non-steroidal anti-inflammatory drugs) can be metabolized to form acylglucuronides.

High throughput metabolic stability screen for lead optimization in drug discovery.

A high throughput approach for the determination of in vitro metabolic stability and metabolic profiles of drug candidates has been developed. This approach comprises the combination of a Biomek FX liquid handling system with 96-channel pipetting capability and a custom-designed 96-well format on-line incubator with efficient thermal conductivity. This combination facilitates automated reagent preparation, sample incubation, and sample purification for microsome stability studies.

The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection.

A potent and selective anthrax LF inhibitor 40, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, was identified through SAR study of a high throughput screen lead. It has an IC50 of 54 nM in the enzyme assay and an IC50 of 210 nM in the macrophage cytotoxicity assay. Compound 40 is also effective in vivo in several animal model studies.

p38 MAP kinase inhibitors: metabolically stabilized piperidine-substituted quinolinones and naphthyridinones.

Quinolinones and naphthyridinones with C7 N-t-butyl piperidine substituents were found to be potent p38 MAP kinase inhibitors. These compounds significantly suppress TNF-alpha release in both cellular and LPS-stimulated whole blood assays. They also displayed excellent PK profiles across three animal species.

Anthrax lethal factor inhibition.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site.

Comparison of the effects of perfusion in determining brain penetration (brain-to-plasma ratios) of small molecules in rats.

In the process of drug discovery, brain and plasma measurements of new chemical entities in rodents are of interest, particularly when the target receptors are in the brain. Brain-to-plasma ratios (B/P) obtained from a rodent pharmacokinetic assay are useful in helping determine which compounds are brain penetrant. The study reported here was performed to determine whether whole-body saline perfusion for complete blood removal was required to accurately measure brain tissue compound concentrations.

High-throughput pharmacokinetics screen of VLA-4 antagonists by LC/MS/MS coupled with automated solid-phase extraction sample preparation.

Automation of plasma sample preparation for pharmacokinetic studies on VLA-4 antagonists has been achieved by using 96-well format solid-phase extraction operated by Beckman Coulter Biomek 2000 liquid handling system. A Biomek 2000 robot is used to perform fully automated plasma sample preparation tasks that include serial dilution of standard solutions, pipetting plasma samples, addition of standard and internal standard solutions, performing solid-phase extraction (SPE) on Waters OASIS 96-well plates. This automated sample preparation process takes less than 2 h for a typical pharmacokinetic study, including 51 samples, 24 standards, 9 quality controls, and 3-6 dose checks with minimal manual intervention.

Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 3: SAR studies on the benzylpyrazole segment.

Extensive SAR studies in our benzylpyrazole series of CCR5 antagonists have shown that both lipophilic and hydrophilic substituents on the phenyl of the benzyl group increase antiviral potency. However, improvements in pharmacokinetic profiles were generally only observed with more lipophilic substitutions. 4-Biphenyl (51) performed the best in this regard.

Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 2: Discovery of potent, selective, and orally bioavailable compounds.

Modifications of the alkyl acetic acid portion and the phenyl on pyrrolidine in our lead pyrazole compound 1 afforded the isopropyl compound 9. This compound is a potent CCR5 antagonist showing good in vitro antiviral activity against HIV-1, an excellent selectivity profile, and good oral bioavailability in three animal species. During this investigation, a new method for the preparation of alpha-(pyrrolidin-1-yl)-alpha,alpha-dialkyl acetic acid from a pyrrolidine and alpha-bromo-alpha,alpha-dialkyl acetic acid using silver triflate was discovered.

Simple and sensitive liquid chromatography/tandem mass spectrometry method for the determination of diazepam and its major metabolites in rat cerebrospinal fluid.

Diazepam (DZP) is one of the most commonly prescribed drugs for treating status epilepticus (SE). A simple, sensitive and selective LC/MS/MS method with a wide linear calibration range was developed to quantify DZP and its major metabolites, N-desmethyldiazepam (DMDZP), temazepam (TZP), and oxazepam (OZP), in rat cerebrospinal fluid (CSF). The method was used to simultaneously determine the concentrations of all analytes in a small sample volume (as little as 25 microL) of rat CSF.

p38 Inhibitors: piperidine- and 4-aminopiperidine-substituted naphthyridinones, quinolinones, and dihydroquinazolinones.

We have synthesized a series of C7-piperidine- and 4-aminopiperidine-substituted naphthyridinones, quinolinones, and dihydroquinazolinones that are highly potent inhibitors of both p38MAP kinase activity and TNF-alpha release. The 4-aminopentamethylpiperidine naphthyridinone 5, which was designed to block metabolism at major 'hot spots', combined excellent inhibitory potency with good oral bioavailability in the rat.

Effect of signal interference from dosing excipients on pharmacokinetic screening of drug candidates by liquid chromatography/mass spectrometry.

The effect of dosing vehicle excipients such as PEG400, propylene glycol, Tween 80, and hydroxypropyl-beta-cyclodextrin on the accuracy of LC/MS measurements used in pharmacokinetic studies is examined. Using PEG400 as a probe compound, the concentration-time profile of the excipient in plasma from rats dosed both orally and intravenously is determined. These excipient plasma concentrations can result in a 2-5-fold increase in calculated plasma clearance values when the excipient interferes with the quantitation of the dosed compound.

Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase.

The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.

Rapid pharmacokinetic screening for the selection of new drug discovery candidates using a generic isocratic liquid chromatography--atmospheric pressure ionization tandem mass spectrometry method.

A generic isocratic HPLC-APCI-MS-MS method has been developed for the determination of plasma concentrations of bioactive compounds for the selection of potential new drug discovery candidates. A 4.6 x 50 mm cyano phase column eluted with an acetonitrile/water mobile phase containing 20 mM ammonium acetate and 0.

Synthesis of nodulisporic acid 2' '-oxazoles and 2' '-thiazoles.

[reaction--see text] The semisynthetic conversion of nodulisporic acid A (1) into a set of three heterocyclic side chain derivatives provided compounds, highlighted by 6, with an improved spectrum of ectoparasiticidal activity and pharmacokinetic profile relative to the natural product.

Systemic efficacy of nodulisporamides against fleas on dogs.

Nodulisporic acid A (NSA) has been shown previously to be safe in dogs and to deliver >90% flea control for 4 days following a single oral administration. Three newly prepared nodulisporamide derivatives were subsequently identified from an artificial membrane flea feeding system as exhibiting potency substantially greater than NSA. To determine if they have superior in vivo activity, these 3 nodulisporamides, as well as NSA, were evaluated in dogs at 15 mg/kg/os.

Characterization of a technique for rapid pharmacokinetic studies of multiple co-eluting compounds by LC/MS/MS.

A method for rapid pharmacokinetic screening of multiple potential drug candidates has been developed. This technique, based on the ability of liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to independently monitor multiple components, enables the quantification of substances which may or may not be chromatographically resolved. Our results indicate that the limit of quantitation and accuracy of this multiple-compound LC/MS/MRM quantitation method are comparable to a single-compound LC/MS/MRM quantitation method.

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.

Potent, orally absorbed glucagon receptor antagonists.

The SAR of 2-pyridyl-3,5-diaryl pyrroles, ligands of the human glucagon receptor and inhibitors of p38 kinase, were investigated. This effort resulted in the identification of 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrr ole 49 (L-168,049), a potent (Kb = 25 nM), selective antagonist of glucagon.

A tacrolimus-related immunosuppressant with reduced toxicity.

Background: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility.