Thienopyrimidine-based P2Y12 platelet aggregation inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 20, 2009, Pages 5919-5923

  Kortum, Steven W ^a   Lachance, Rhonda M ^a   Schweitzer, Barbara A ^a   Yalamanchili, Gopichand ^a   Rahman, Hayat ^a   Ennis, Michael D ^a   Huff, Rita M ^a   TenBrink, Ruth E ^a  

^a PFIZER GLOBAL RESEARCH AND DEVELOPMENT   (United States)

Author keywords

P2Y12; Platelet aggregation inhibition; Thienopyrimidine

Indexed keywords

ANTITHROMBOCYTIC AGENT; PURINERGIC P2Y12 RECEPTOR; PYRIMIDINE DERIVATIVE; THIENOPYRIMIDINE; TICAGRELOR; UNCLASSIFIED DRUG; FIBRINOLYTIC AGENT; PURINERGIC P2 RECEPTOR;

ARTICLE; DRUG DESIGN; DRUG INHIBITION; DRUG STRUCTURE; DRUG SYNTHESIS; IC 50; PROTEIN BINDING; THROMBOCYTE AGGREGATION; CHEMISTRY; DRUG ANTAGONISM; DRUG EFFECT; HUMAN; METABOLISM; STRUCTURE ACTIVITY RELATION; SYNTHESIS;

FIBRINOLYTIC AGENTS; HUMANS; PLATELET AGGREGATION; PLATELET AGGREGATION INHIBITORS; PROTEIN BINDING; PYRIMIDINES; RECEPTORS, PURINERGIC P2; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 70349207147     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2009.08.059     Document Type: Article

References (19)

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16. 1H NMR (300 MHz, chloroform-d) δ ppm 1.11 - 1.31 (m, 3H) 2.61 (qd, J = 7.52, 1.21 Hz, 2H) 3.76-3.83 (m, 3H) 5.79 (br s., 2H) 6.62 (t, J = 1.31 Hz, 1H).
17. 12 assays: dry compounds are diluted as 10 mM dimethylsulfoxide (DMSO) stocks and are tested in a seven-point, threefold dilution series run in triplicate beginning at 10 μM. A 1 mM DMSO intermediate stock is made in a dilution plate and from this the seven dilutions are made. The highest concentration is diluted into water containing 0.02% Bovine serum albumin (BSA), and the remaining six concentrations are diluted into assay buffer containing 0.02% BSA. To a polypropylene assay plate the following are added: (a) 30 μL of assay buffer containing one protease inhibitor cocktail tablet per 50 mL; (b) 30 μL of 1 nM 33P 2-MeSADP made in assay buffer containing 0.02% BSA and 12.5 mg/mL ascorbic acid; (c) 30 μL of cold 1.5 μM 2-MeSADP for the positive control wells, or assay buffer containing 0.02% BSA and 12.5 mg/mL ascorbic acid for the negative control wells; (d) (Method a) 60 μL of 10 μg/well membranes, (Method b) 60 μL of 1 μg/well membranes, (Method c) 60 μL of 0.3 μg/well membranes.Incubate the plates at room temperature for 1 h. Stop the reaction using a cell harvester to aspirate/transfer the supernatant onto GF/B UniFilter plates, and wash three times with wash buffer, aspirating between each wash. The filter plates are dried for approximately 20 min in an oven at 50 °C. Back seals are adhered to the filter plates and 25 μL of Microscint 20 scintillation fluid is added. The filter plates are sealed, shaken for 30 min, and counted on a Top Count.
18. 12 assay without protein with the following exceptions:(Method a) 60 μL of 1 μg/well membranes made with 5X human serum albumin (HSA) (1.75%) and 5X AGP (0.075%), (Method b) 60 μL of 1 μg/well membranes made with 5X HSA (1.75%) and 5X AGP (0.075%), (Method c) 60 μL of 0.3 μg/well membranes made with 5X HSA (1.75%) and 5X AGP (0.075%).
19. Human platelet-rich plasma (hPRP) preparation:Fifty millilitres polypropylene centrifuge tubes were filled with whole blood and centrifuged at 910g × 10 s and then 200g × 15 min at room temperature to obtain platelet-rich plasma (PRP). PRP was removed using a wide bore transfer pipet avoiding disturbance of the buffy coat (polymorphonuclear leukocytes (PMNLs)) and red blood cells, and placed in a clean polypropylene tube. Remaining plasma, buffy coat and red cells were centrifuged at 2380g × 15 min, again at room temperature, to obtain platelet-poor plasma (PPP). PPP was removed, in the same manner as for the PRP, and placed in a clean polypropylene tube. PRP platelet counts were determined using a Z1 Coulter Particle Counter and individual PRP was diluted to 300,000 platelets/μL with autologous PPP.hPRP aggregation assay: the 96-well hPRP aggregation assay monitors aggregation based on the increase of light transmittance through a stirring suspension of platelets after stimulation by adenosine diphosphate (ADP) as they aggregate into large particles. Assays were performed using SpectroMax 190 Microplate Spectrophotometer with SoftMax Pro software from Molecular Devices. Platelets were tested for their sensitivity to compounds in 180 μL reaction volumes containing 144 μL PRP, 18 μL of 10X compound and 14 μL of 10X ADP in a 96-well polystyrene plate. Reactions were initiated by the addition of 20 μM ADP. Compounds were tested at a final concentration of 30 μM. Each concentration of inhibitor was tested in duplicate.