Discovery of Phosphoric Acid Mono-{2-[(E/Z)-4-(3,3-dimethyl-butyrylamino)-3,5-difluoro-benzoylimino]-thiazol-3-ylmethyl} Ester (Lu AA47070): A Phosphonooxymethylene Prodrug of a Potent and Selective hA_2A Receptor Antagonist

Abstract

The discovery and structure-activity relationship of a series of hA(2A) receptor antagonists is described. Compound 28 was selected from the series as a potent and selective compound and was shown to be efficacious in an in vivo model of Parkinson's disease. It had acceptable ADME properties; however, the low intrinsic solubility of this compound was limiting for its developability, because the oral bioavailability from dosing in suspension was significantly lower than the oral bioavailability from solution dosage. As a consequence, prodrugs of 28 were prepared with dramatically increased aqueous solubility. The prodrugs efficiently delivered 28 into systemic circulation, with no detectable levels of prodrug in plasma samples. From this investigation, we selected 32 (Lu AA47070), a phosphonooxymethylene prodrug of 28, as a drug candidate.

Identification of an Aminothiazole with Antifungal Activity against Intracellular Histoplasma capsulatum

“…Growth was monitored by daily measurements of turbidity (absorbance at 595 nm) and RFP fluorescence (530-nm excitation and 590-nm emission wavelengths) on a Synergy2 plate reader (BioTek). For linear regression and computation of 50% inhibitory concentration (IC 50 ) values, yeast growth at 4 days (93 to 102 h) was used. All tests were performed on three biological replicates, and differences were compared by using Student's t test.…”

“…All tests were performed on three biological replicates, and differences were compared by using Student's t test. For determination of assay efficacy, the known antifungal drug AmB was added at concentrations from 0.003 M to 1.7 M. For library screening, compounds were added to yeast at a concentration of 10 M. Compounds that inhibited yeast growth at this concentration were rescreened against yeast cells at concentrations from 0.0095 M to 10 M to establish an IC 50 for yeast cells.…”

“…Using macrophage replication as an indicator of nontoxicity, 16 of the 28 compounds were not toxic to macrophages at concentrations of 5 M or lower, as determined by RFP fluorescence or by culture turbidity. Determination of the IC 50 against macrophages for these 16 compounds allowed computation of overall yeast selectivity (ratio of the IC 50 for yeasts compared to the IC 50 for host cells). We identified 7 compounds that are at least 5-fold more toxic to yeast cells than to macrophages (Fig.…”

“…This is independent of the promoter used for RFP expression, as RFP driven by the Histoplasma TEF1 promoter instead of the histone 2B promoter also exhibited this trend (data not shown). Since the elevated RFP expression level at subinhibitory concentrations decreases the accuracy of the linear regression (resulting in an underestimation of the compound's efficacy), we used absorbance measurements of culture turbidity for precise IC 50 determinations and determination of the selectivity of the compounds (Fig. 2C).…”

“…2C). Multiple dose-response analyses with biological replicates determined the IC 50 against Histoplasma OSU76 yeast cells to be 0.87 Ϯ 0.16 M (Fig. 4A).…”