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Thuy Anh Tran, Bryan Kramer, Young Jun Shin, Pureza Vallar, P. Douglas Boatman, Ning Zou,
Carleton R. Sage, Tawfik Gharbaoui, Ashwin Krishnan, Biman Pal, Sagar Shakya, Antonio Garrido
Montalban, John W. Adams, Juan Ramirez, Dominic P. Behan, Anna Shifrina, Anthony Blackburn, Tina
Leakakos, Yunqing Shi, Michael Morgan, Abu Sadeque, Weichao Chen, David J. Unett, Steve Chang,
Hsin Hui Shu, Shiu Feng Tung and Graeme Semple*
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S2: Additional In Vitro Assays
S3: Additional data from the in vitro and vivo evaluation of (
S4: PK Profile of ( (sodium salt) in cynomolgus monkey
S5: SC TGA and Adsorption Desorption profiles of ( and (
S7: Broad screening (binding) profile of (
S1
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To determine the potency and efficacy of compounds in cAMP assays in the absence of receptor reserve
effects, the cDNA sequence of the human IP receptor was sub cloned into an expression plasmid
containing a ubiquitin promoter in place of the more commonly used CMV promoter. The ubiquitin
promoter yields a greatly reduced mRNA copy number than the CMV promoter and helps reduce the
level of receptor expression after transfection into a suitable cell line. This plasmid was transiently
transfected into CHO cells using lipofectamine, following standard protocols. The quantity of transfected
plasmid DNA was titrated down from 16 g to 1 ng per transfection using empty vector as carrier DNA.
Transfected cells were used in standard HTRF cAMP assays, as described in the Experimental section, 48
hours post transfection. Data were collected from cells that provided the minimum detectable cAMP
response (typically cells transfected with 1 – 10 ng of plasmid).
Compounds were also evaluated in HTRF cAMP assays using primary human pulmonary arterial smooth
muscle cells (Cell Applications, Inc., #352K 05a, or ThermoFisher/Gibco, #C0095C) using the same
basic assay protocol outlined in the Experimental section. Cells were cultured as recommended by the
suppliers, used at low passage and seeded into 384 well assay plates at a density of 700 900 cell per well
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: Functional evaluation of ( and comparator compounds in recombinant IP receptor cAMP
assays following elimination of receptor reserve and in primary human pulmonary arterial smooth muscle
cells.
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Recombinant IP receptor cAMP
3.3 [1.7, 6.4] (9)
100
Primary human PASMC cAMP
2.9 [1.6, 5.3] (8)
100
Recombinant IP receptor cAMP
151 [112, 203] (9)
48 [43, 53]
Primary human PASMC cAMP
184 [136, 248] (8)
41 [33, 49]
Recombinant IP receptor cAMP
24 [20, 29] (9)
67 [64, 71]
Primary human PASMC cAMP
24 [31, 40] (8)
65 [55, 75]
Iloprost
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Data with some comparator compounds in functional assays.
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0.0013 (36)
n = 36
0.52 (28)
n = 28
0.0008 (98)
n = 26
0.147 (42)
n=3
0.154 (80)
n=2
0.850 (52)
n = 52
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0.051 (92)
n=4
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0.047 (48)
n=2
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0.0055 (84)
n = 39
0.79 (73)
n = 13
0.144 (110 )
n=8
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0.28 (68)
n=6
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0.41 (96 )
n=6
> 10
0.230 (98)
n = 51
> 10
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> 10
All assays were cAMP HTRF agonist assays (Gs) except for EP3v6 which was a melanophore dispersion
assay (Gi).
n.d. = not determined
n ≥ 3 for all compounds with EC50 > 10 M
(
Effect of high dose ( increase in pulmonary arterial wall thickness. As this was a time consuming assay,
only 5 animals were selected at random from each of group for these measurements. As the 10mg/kg
group did not show a statistically significant effect on hypertrophy, no tissue from that group was tested.
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Discovery of 2-(((1r,4r)-4-(((4-Chlorophenyl)(phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetate (Ralinepag): An Orally Active Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension
Journal of medicinal chemistry, 2017
The design and synthesis of a new series of potent non-prostanoid IP receptor agonists that showed oral efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) are described. Detailed profiling of a number of analogues resulted in the identification of 5c (ralinepag) that has good selectivity in both binding and functional assays with respect to most members of the prostanoid receptor family and a more modest 30- to 50-fold selectivity over the EP3 receptor. In our hands, its potency and efficacy are comparable or superior to MRE269 (the active metabolite of the clinical compound NS-304) with respect to in vitro IP receptor dependent cAMP accumulation assays. 5c had an excellent PK profile across species. Enterohepatic recirculation most probably contributes to a concentration-time profile after oral administration in the cynomolgus monkey that showed a very low peak-to-trough ratio. Following the identification of an acceptable solid form, 5c was selected f......Read more
