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.

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. Acylglucuronides are intrinsically reactive metabolites capable of undergoing hydrolysis, intra-molecular rearrangement, and formation of covalent adducts with proteins, which may generate potential toxicity. The purpose of this study is to develop an in vitro screening model to assess degradation kinetics of acylglucuronides. METHOD: Zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were incubated in the presence of rat microsomal protein and uridine 5'-diphosphoglucuronic acid (UDPGA), followed by addition of human plasma to evaluate degradation kinetics of the acylglucuronides. As a comparison, authentic acylglucuronide standards of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were chemically synthesized and were evaluated for degradation kinetics. RESULTS: The results demonstrate that degradation half-life values of acylglucuronides of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D determined by the in vitro formation/degradation model were in the same rank-order with those of the authentic acylglucuronide standards. DISCUSSION: For the seven compounds tested, the model placed the stability of the acylglucuronides formed in vitro in a rank-order consistent with authentic acylglucuronide standards. The method allows for a rapid assessment of the stability of 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 overall process is both fast and accurate and meets the challenges of high throughput screening for drug discovery. A custom designed, user-friendly computer program has been incorporated for large-scale data processing and report generation. Several applications are discussed that implement this strategy for rapid selection of compounds in early drug discovery.

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. Quinolinone at 10 mpk showed comparable oral efficacy to that of dexamethasone at 1 mpk in a murine collagen-induced arthritis model.

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. Diazepam was used as a positive control since it is highly brain penetrant. Compound A was used as a negative control since it had known poor brain penetration. After intravenous dosing with either diazepam or compound A, rats were anesthetized and blood was collected, then the brain was removed following no perfusion or whole-body perfusion with saline. The analytes described (compound A, diazepam, and the internal standard) were recovered from plasma or brain homogenate by use of protein precipitation, and were subsequently analyzed by use of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The B/P values determined by use of LC-MS were not significantly different in perfused vs. non-perfused rats (P > or = 0.05). This approach (whole brain collected from non-perfused male rats) is an attractive alternative over brain penetration studies of perfused rats, since it has markedly reduced the technical time and potential for pain and distress required for generating B/P data due to elimination of the requirement for anesthesia and surgical preparation of animals.

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. Extensive validation has been made to ensure the accuracy and reliability of this method. A two-stage vacuum pressure controller has been incorporated in the program to improve SPE efficiency. This automated SPE sample preparation approach combined with liquid chromatography coupled with the high sensitivity and selectivity of tandem mass spectrometry (LC/MS)/MS has been successfully applied on both individual and cassette dosing for pharmacokinetic screening of a large number of VLA-4 antagonists with a limit of quantitation in the range of 1-5 ng/ml. Consequently, a significant throughput increase has been achieved along with an elimination of tedious labor and its consequential tendency to produce errors.

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. This allowed us to prepare compounds such as 24 and 25 for the first time. A novel Pd-mediated N-dealkylation of alpha-(pyrrolidin-1-yl)acetic acid was also uncovered.

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. Highly lipophilic substituents impart undesirable ion channel activity to these CCR5 antagonists. Alkoxy substituents provide a good balance of antiviral activity, pharmacokinetic parameters, and selectivity. Compounds 42b and 42d, containing a 3,4-dimethoxy substituent, are considered the most promising improvements over parent compounds 9. They demonstrate improved antiviral activity while retaining good pharmacokinetic profile and selectivity.

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. The lower limits of quantification (LLOQ) of the method are 0.04 ng/mL for DZP and 0.1 ng/mL for its metabolites. The calibration range is 0.04-200 ng/mL for DZP and 0.1-200 ng/ml for the metabolites. All intra- and inter-assay coefficients of variation (%CV) and mean percent errors of the method are less than 12%. This method successfully addresses the need to determine low therapeutic drug concentrations in small physiological samples, namely rat CSF. Moreover, it can be used to investigate the distribution of the drug and its metabolites among blood plasma, brain tissue, and CSF in pharmacokinetic and pharmacodynamic studies in a variety of laboratory animals. With respect to animal experiments involving assays in CSF, this method addresses two of the three criteria of Russell and Bruch (Principles of Humane Experimental Techniques, 1959, Methuen and Co., London) for minimizing animal use, namely refinement and reduction.

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.

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=0.58 microM h).

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 0.4% TFA produces retention times of 1 min or less for a wide range of compounds. This is a great advantage in new drug discovery where many compounds are analyzed once and eliminated. No time is consumed developing chromatographic conditions for each new compound. The mass spectrometer can be optimized and the samples can be processed and analyzed, all in the same day. Multiple assays can be run consecutively without changing the column or mobile phase between assays.

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. This can result in false rejection of a compound in a drug discovery screen. Several plasma purification methods and enhanced chromatographic selectivity are examined as ways to minimize or avoid excipient effects, particularly for very polar compounds. The combination of efficient sample purification and selective chromatography provides an effective way to diminish the significant interference effects of PEG400 and Tween 80. When appropriate, using negative ion mode MS or changing a dosing vehicle excipient, such as substituting propylene glycol for PEG400, provides an alternative approach for eliminating signal interference. The mechanism of excipient-related signal interference is discussed in relation to both competition of gas-phase proton-transfer reactions and high viscosity of dosing excipients.