Discovery of small molecule human C5a receptor antagonists Hitesh J. Sanganee a, * , Andrew Baxter a , Simon Barber a , Alastair J. H. Brown b , Denise Grice a , Fraser Hunt a , Sarah King a , David Laughton a , Garry Pairaudeau c , Bob Thong a , Richard Weaver a , John Unitt a a AstraZeneca R&D Charnwood, Medicinal Chemistry, Bakewell Road, Loughborough, LE11 5RH, UK b AstraZeneca R&D Alderley Park, Mereside, Macclesfield, SK10 4TG, UK c AstraZeneca R&D Mölndal, Pepparedsleden 1 431 83 Mölndal, Sweden article info Article history: Received 10 October 2008 Revised 23 December 2008 Accepted 24 December 2008 Available online 31 December 2008 Keywords: C5a antagonist Inverse agonists Small molecule Ugi multicomponent abstract A novel series of small molecule C5a antagonists is reported. In particular, in vitro metabolic studies and solution based combinatorial synthesis are demonstrated as useful tools for the rapid identification of antagonists with low in vitro clearance. Members of this series specifically inhibited the binding of 125 I-labeled C5a to human recombinant C5a receptor (C5aR). In functional cell assays these compounds displayed surmountable antagonism against C5a and did not demonstrate any detectable agonist activity. Ó 2009 Elsevier Ltd. All rights reserved. The complement system is comprised of a cascade of interre- lated proteases that are activated in response to immunoglobins binding to a foreign antigen. Activation of the complement systems leads to a stepwise hierarchy of proteolytic cleavage events ulti- mately leading to the release of bioactive fragments (C3a, C4a and C5a) known as anaphylatoxins. C5a is recognized as a promi- nent mediator of inflammation through recruitment of inflamma- tory cells to the site of injury or infection. The functional effects of C5a are mediated by specific interac- tion with the G-protein coupled receptor C5aR, expressed on a variety of cells including mast cells and neutrophils. 1 Consistent with its proinflammatory properties, C5a has been implicated in the pathology of a wide range of disorders, including rheumatoid arthritis, asthma, respiratory distress syndrome, sepsis and inflam- matory bowel disease. 2 Significant progress has been made in re- cent years on the understanding of the complement system and several non-peptidic C5a antagonists have been recently reported in the literature (Fig. 1). 3 Interest in this area is driven by the potential therapeutic appli- cation of a C5aR antagonist in the treatment of inflammatory dis- eases such as arthritis, asthma and COPD. We report in this paper the discovery of a novel series of small molecule C5aR antagonists. High throughput screening of the AstraZeneca compound col- lection followed by a hit to lead (HtL) program identified a number of chemical series with clear structure activity relationships (SAR). The most promising of these was the bisamide series, as exempli- fied by compounds 1 and 2. The bisamide series was identified following an HTS using a whole cell binding Fluorescent Microvolume Assay Technology (FMAT TM ) assay, 6,10 which measured the inhibition of Alexa-647 la- beled C5a binding to human recombinant C5aR (hrC5aR) expressed in HEK-293 cells with G-protein Ga16. 10 The compounds were subsequently confirmed as potent functional C5aR antagonists by determining their inhibition of C5a-stimulated intracellular cal- cium mobilization in the hrC5aR HEK cells (see Table 1). Activity against the native receptor was also confirmed by measuring com- pound mediated inhibition of C5a-stimulated calcium mobilization in human neutrophils. 16 For example, Compound (2) had a pA 2 va- lue of 7.4 in this neutrophil assay. Activity against the related C5a binding receptor C5L2 was determined using an FMAT binding as- say 10 and the series was found to be selective. For example com- pound (2) tested up to 30 lM displayed no inhibition of Alexa- 647 labeled C5a binding to C5L2. In additional selectivity profiling, compound (2) was inactive at GPCRs FPR, CXCR2, CXCR4 and CCR3, when tested up to 10 lM (data not shown). Species selectivity of the bisamide series was examined in recombinant receptor cell cal- cium mobilization assays and was demonstrated to have apparent selectivity for the human C5aR (see Table 2), similar to previous re- ports on small molecule C5aR antagonists. 7 In order to further characterize the functional antagonism of the bisamide series we measured the ability of exemplars from this series to inhibit C5a stimulated GTPcS binding in HEK cell mem- branes expressing the hrC5aR. 10 Concentration response curves to C5a were performed following pre-treatment of membranes 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.12.104 * Corresponding author. Tel.: +44 01509 644167. E-mail address: Hitesh.sanganee@astrazeneca.com (H.J. Sanganee). Bioorganic & Medicinal Chemistry Letters 19 (2009) 1143–1147 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl