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  • Substitutions on the benzene ring of the


    Substitutions on the benzene ring of the phenyl acetic AXL1717 australia moiety were evaluated to study their effect on DP and CRTH2 selectivity (). Compound has strong affinity for CRTH2, but it only has moderate affinity for DP. Small substitutions such as methoxy () and fluorine () next to the aryloxy group improve the compounds’ affinity for the DP receptor (). However, ethoxy derivative has decreased activity on DP compared to methoxy analog , which suggests that the increased bulk may be detrimental. Introduction of methoxy groups on both sides next to the aryloxy group afforded a compound () with high affinity for CRTH2 and dramatically decreased activity on DP. The linker between the carboxylic acid and the phenyl ring was also studied (). A methylene linker () is preferred over an ethylene linker () or direct attachment of carboxylic acid to the phenyl ring (). Further study of the amide moiety () provided additional improvement on DP potency. Longer -alkyl groups, such as -propyl (), -butyl () and -pentyl (), improved the affinity for DP, while maintaining the affinity for the CRTH2 receptor. Investigation of branched alkyl groups, such as isopropyl () or isobutyl (), and cyclic alkyl groups, such as cyclopropyl () or cyclopentyl (), showed that these groups were well tolerated. However, they did not lead to improvements on the affinity towards either receptor. The cyclobutyl analog (), on the other hand, displayed increased affinity for the DP receptor while maintaining the affinity for CRTH2. All comparisons here are made to the parental ethyl amide (). Because of its high affinity towards both receptors, compound (AMG 009) was further investigated in DP and CRTH2 functional assays. AMG 009 inhibited PGD-induced down-modulation of CRTH2 on CD16 negative granulocytes (eosinophils) in human whole blood with a of 1nM. In addition, AMG 009 also inhibited PGD-induced cAMP response mediated by DP in platelets in 80% human whole blood with a of 148nM. The selectivity of AMG 009 was studied in a commercially available GPCR panel and AMG 009 demonstrated excellent selectivity, including its selectivity against prostanoid receptors TP, EP2 and EP4. The pharmacokinetic properties of AMG 009 were evaluated in several species (). AMG 009 has low to moderate clearance across species and good oral absorption. Since guinea pigs have been shown to respond to aerosolized PGD in a similar manner as humans, AMG 009 was investigated in an acute guinea pig model of PGD-induced airflow obstruction. In this model, airway resistance is measured in response to increased concentrations of PGD using a whole body plethysmography. Groups of four Dunkin-Hartley guinea pigs were dosed once with vehicle or AMG 009 at 3, 10 or 30mg/kg by subcutaneous administration 15min prior to PGD challenges. The enhanced pause (Penh), a measure of airway resistance, to inhaled PGD was measured over a 10-min span after the guinea pigs were subjected to 1-min challenges with aerosolized saline or PGD in saline of ascending concentrations at 0.063, 0.125, 0.25, 0.5 and 1.0mg/mL. Treatment with AMG 009 resulted in a dose dependent decrease in airway resistance provoked by PGD aerosol (). The in vivo effect of AMG 009 was most likely due to its DP antagonistic activity, because CRTH2 selective antagonist () did not reduce airway resistance in this guinea pig model. The ability of AMG 009 to block the guinea pig CRTH2 and DP receptors was also evaluated in vitro using a H-PGD displacement assay with cells transfected with the guinea pig CRTH2 receptors (IC=3 nM) and a PGD-induced cAMP response assay with cells expressing the guinea pig DP receptors (=131nM). In summary, we have discovered a novel series of CRTH2 antagonists and optimized their properties to also inhibit the DP receptor. Based in part on the evaluation of CRTH2 and DP activities and the outcomes of pharmacokinetic studies, we selected AMG 009 () as a candidate compound for evaluation in clinical studies. This compound is a potent inhibitor of PGD-induced CRTH2 receptor down-modulation and DP mediated cAMP response in human whole blood. This compound was also efficacious in a guinea pig model of airway resistance.