Investigation of the terminal P4 domain in a series of d-phenylglycinamide-based factor Xa inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 17, Issue 24, 2007, Pages 6910-6913

  Franciskovich, Jeffry B ^a   Masters, John J ^a   Weber, Wayne W ^a   Klimkowski, Valentine J ^a   Chouinard, Michael ^a   Sipes, Philip R ^a   Johnson, Lea M ^a   Snyder, David W ^a   Chastain, Marcia K ^a   Craft, Trelia J ^a   Towner, Richard D ^a   Gifford Moore, Donetta S ^a   Froelich, Larry L ^a   Smallwood, Jeffrey K ^a   Foster, Ronald S ^a   Smith, Gerald F ^a   Liebeschuetz, John W ^b,c   Murray, Christopher W ^b,d   Young, Stephen C ^b,e  

^a ELI LILLY AND COMPANY   (United States)

^b Tularik Ltd   (United Kingdom)

^c CAMBRIDGE CRYSTALLOGRAPHIC DATA CENTRE   (United Kingdom)

^d ASTEX PHARMACEUTICALS   (United Kingdom)

^e VERTEX PHARMACEUTICALS   (United States)

Author keywords

Antithrombotics; d Phenylgylcinamides; Factor Xa inhibitors

Indexed keywords

BLOOD CLOTTING FACTOR 10A; BLOOD CLOTTING FACTOR 10A INHIBITOR; DEXTRO PHENYLGLYCINAMIDE DERIVATIVE; SERINE PROTEINASE; UNCLASSIFIED DRUG;

ANIMAL EXPERIMENT; ANTICOAGULATION; AREA UNDER THE CURVE; ARTICLE; CRYSTAL STRUCTURE; DRUG SYNTHESIS; ENZYME INHIBITOR INTERACTION; MAXIMUM PLASMA CONCENTRATION; NONHUMAN; RAT;

ANTICOAGULANTS; ANTITHROMBIN III; CRYSTALLOGRAPHY, X-RAY; FACTOR XA; GLYCINE; HUMANS; MODELS, MOLECULAR; MOLECULAR STRUCTURE; PROTEIN BINDING; STRUCTURE-ACTIVITY RELATIONSHIP;

RATTUS;

EID: 36048956698     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2007.09.105     Document Type: Article

References (11)

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11. Pharmacodynamic surrogate of Xa inhibitor oral exposure. Fisher rats were fasted overnight, followed by a single administration of test compounds suspended in acacia or CMC/Tween 80, by oral gavage. Blood was collected at specified time-points in citrate for generation of plasma at 2000g for 15 min at 10 °C. Compounds were weighed out to the nearest 0.001 mg, adjusted for salt form and purity, and dissolved in GC-MS or HPLC grade methanol (OD at 350 nm < .001) with brief sonication in a water bath. Compounds were serially diluted in methanol and aliquoted into pooled plasma from vehicle treated animals in triplicate, for use as calibration standards. A solution for human factor Xa was prepared by dissolving 18.8 mg of fatty acid-free bovine serum albumin (Sigma RIA grade A-7888) in 15 ml of 1× TBS. A solution of Xa substrate, S-2222 (MidWest Bio-Tech, South Bend, IN), was diluted to 0.5 mg/ml in 18.2 MΩ purified water. Water (5 μl) was added to all wells reserved for standards. Methanol (5 μl of 50%) was added to wells reserved for samples of unknown compound level. Ex vivo plasma (25 μl) from animals dosed with compounds was thawed at 4 °C. Samples and calibration standards were mixed, incubated in a 37 °C water bath for 15 min, and diluted into individual wells of a 96-well, polystyrene, uncoated assay plate containing 50 μl of 3× TBS. Substrate solution was then added to all wells, the plate was placed on mixer for 1 min, and a pre-read was collected. Human factor Xa (Enzyme Research Labs HFXa1240, titrated) was thawed at ambient temperature immediately before use and aliquoted into vehicle at 0.4 U/ml. The factor Xa solution was then aliquoted (20 μl) using a multi-channel pipette to start the enzyme reaction. Plates were read on a Spectromax 250 plate reader (Molecular Devices, Sunnyvale, CA) at 405 nm kinetically for 10 min at 11 s/cycle, immediately followed by an end-point read. Slopes of OD/min were plotted against the known concentrations of calibration standards and samples of unknown concentration were interpolated from four parameter logistic fits of standard curves. Compound concentrations of ex vivo samples from dosed animals were used in SigmaPlot for the calculation of AUCs using a SigmaPlot rev 8.1 area algorithm.