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  • Grapiprant is a selective antagonist for prostaglandin E PGE


    Grapiprant () is a selective antagonist for prostaglandin E (PGE) receptor subtype 4 (EP4) identified as a clinical candidate for the treatment of inflammatory pain associated with osteoarthritis (OA). It is currently under development for use in humans and dogs. The projected dosing regimen of grapiprant for humans is 50–100mg, PO, BID (twice a day) based on the pharmacology and pharmacokinetic (PK) data in clinical studies. In order to identify another clinical candidate with improved efficacy, safety, and pharmacokinetic profiles with potential for QD (once a day) dosing, we embarked on a back-up discovery project to discover the second generation of grapiprant (). The target profiles of this candidate are summarized as follows. The back-up candidate should demonstrate improved potency over grapiprant, i.e.; (1) antagonize PGE-mediated cAMP elevation in transfectants expressing human EP4 receptor-with a p>8.6; (2) exhibit selectivity of >100 over other PG receptor subtypes; (3) clearly demonstrate improved oral activity over grapiprant in key acute and chronic inflammatory pain models; i.e., betamethasone celestone mg induced mechanical hyperalgesia and complete Freund’s adjuvant (CFA) induced weight bearing deficit in the rat; (4) demonstrate a PK profile in animals predictive of QD dosing in human. From a medicinal chemistry perspective, the back-up compound should have improved physicochemical characteristics; lower molecular weight and lower lipophilicity while improving efficacy and PK profile. In order to meet the above criteria, we pursued an alternative core structure different from the sulfonylurea since the structure–activity relationships (SAR) studies around grapiprant revealed compounds consisting of the sulfonylurea core had extremely low volume of distribution and high to moderate clearance in rats and other experimental animals. High throughput screening (HTS) of the Pfizer compound library using a human EP4 functional assay measuring PGE-induced cAMP formation in HEK-293 cells expressing human EP4 receptor and the subsequent verification in a membrane binding assay using [H]PGE resulted in the identification of -acyl sulfonamide as a moderately potent EP4 antagonist (IC: 302 nM) without EP4 selectivity over other subtypes (). HTS hit has high molecular weight (MW: 610.08) and is quite lipophilic (ALogP: 5.70, cLogD: 5.80). Further screening of HTS hit were initiated by using the in-house compound libraries. First, a bioisosteric transformation, of the -acyl phenylsulfonamide moiety to the corresponding carboxy group was well tolerated and resulted in the identification of compound with reduced molecular weight. Compound displayed modest EP4 selective functional antagonism (IC: 370nM) and good stability in human liver microsomes (HLM) (>120min), however it did not have acceptable physicochemical properties (typically for lead compounds, solubility in phosphate-buffered saline (PBS)>10μM; molecular weight <400; ALogP<4) (). Next, with further reduction of MW and lipophilicity (ALogP and cLogD) in mind, a structural similarity search by using a simplified pharmacophore query as shown in led to identify ca. 1000 compounds. The subsequent filtering of these compounds by applying Lipinski’s rule of 5, and eliminating compounds with toxicophore, , narrowed down to 50 hit compounds. These 50 compounds were evaluated in a series of assays of functional EP4 receptor antagonism, binding selectivity against EP receptors, HLM stability, human cytochrome P450 inhibition, aqueous solubility, and membrane permeability. As a result, compound was shown to be superior to the others, demonstrating EP4 selective functional antagonism (IC: 575nM), binding affinity for EP4 receptor (i: 73nM), stability in HLM (>120min), no notable CYP 450 inhibition, high Caco-2 cell permeability (: 25×10cm/s), good solubility in PBS (>10μM), lower MW (MW: 384.33), and acceptable lipophilicity (ALogP: 3.61, cLogD: 2.14). The hit-to-lead efforts resulted in the identification of compound that has a core structure of --acylaminomethylbenzoic acid (). The molecules containing carboxylic acid often have undesirable metabolic instability, limited permeability, and potential toxicities. Despite the drawbacks of the carboxylic acid functional group, exhibited selective EP4 functional antagonism with a suitable metabolic profile and good physicochemical properties. Thus, we envisaged that the optimization efforts around would provide the backup candidate that meets the target profiles.