The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.Trial Registration ISRCTN registry with trial ID ISRCTN13564773.

The classical antifolate N-{4- [(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-L-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl L-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC 50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.

We report, for the first time, the biological activities of four-carbon-atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpolyglutamate synthetase (FPGS) as well as antitumor activity. Extension of the bridge homologation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2), afforded two four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity and inhibitory activity against tumor cells in culture (EC(50) < or = 10(-7) M) compared with the two-carbon bridged analogues. These results support our original hypothesis that the distance and orientation of the side chain p-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine ring are a crucial determinant of biological activity. In addition, this study demonstrates that, for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated target enzymes is not necessarily a predictor of a lack of antitumor activity.

A tandem Diels-Alder/azido Schmidt reaction sequence provides rapid access to the core skeleton shared by several Stemona alkaloids including stenine, neostenine, tuberstemonine, and neotubererostemonine. The discovery and evolution of inter-and intramolecular variations of this process and their applications to total syntheses of (±)-stenine and (±)-neostenine is described. The stereochemical outcome of the reaction depends on both substrate type and reaction condition, enabling the preparation of both (±)-stenine and (±)-neostenine from the same diene/dienophile combination. KeywordsTotal synthesis; Stemona alkaloids; Diels-Alder; azides; Schmidt reaction Chinese and Japanese traditional medicines have for centuries utilized extracts of stemonaceous plants as remedies for the treatment of respiratory ailments. These extracts and the isolated Stemona alkaloids have been associated with insecticidal, anthelmintic, antitussive and various neurochemical effects, although mechanisms have rarely been identified. 1 Recently, interest in these alkaloids was further piqued by the demonstration of effective in vivo activity of two skeletally-related Stemona alkaloids, neostenine 3 and neotuberostemonine 4, against citric acid-induced cough in guinea pig animal models. 2 In addition, the Stemona alkaloid tuberstemonine 2 has demonstrated inhibitory activity on excitatory transmission at the crayfish neuromuscular junction. 3 The Stemona alkaloids have attracted substantial interest from synthetic chemists partly because of these links to biological activity and partly from their challenging structural complexity. Stenine has been the focus of several successful synthetic efforts 4 and has inspired a number of synthetic approaches. 5 In addition, tuberostemonine 2 was synthesized by Wipf. 6 However the stenine isomer, neostenine 3, had not yet been prepared via total synthesis at the outset of this project. 7A noteworthy challenge in any stenine synthesis is the construction of the B ring, which is fused to three additional rings. In addition, each of its carbon atoms is a stereogenic center. This issue was addressed using an intramolecular Diels-Alder cyclization in three out of the four first-published syntheses of this target (Scheme 1; the stenine numbering system used E-mail: jaube@ku.edu. Supporting Information Available. Experimental details and characterization data for all new compounds, including X-ray structures (CIF files) of 3 and 28. This material is available free of charge via the internet at http://pubs.acs.org. throughout is that presented in a recent review 1f ). The first synthesis of stenine by Hart in 1990 not only set the precedent for utilizing a Diels-Alder approach to this target, but also established an iodolactonization/Keck allylation sequence as a solution to the problem of stereoselective ethyl group installation. 4a,b Morimoto utilized a chiral oxazoline-based intramolecular DielsAlder cyclization of 5 to synthesize the naturally occurring enantiomer of stenine. 4c-e Padwa applied a...

The chemokine CCL3/MIP-1␣ is a risk factor in the outcome of multiple myeloma (MM), particularly in the development of osteolytic bone disease. This chemokine, highly overexpressed by MM cells, can signal mainly through 2 receptors, CCR1 and CCR5, only 1 of which (CCR1) is responsive to CCL3 in human and mouse osteoclast precursors. CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. Here we show that formation of mature osteoclasts is blocked by the highly potent and selective CCR1 antagonist CCX721, an analog of the clinical compound CCX354. We also show that doses of CCX721 selected to completely inhibit CCR1 produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease. Similar effects were observed when the antagonist was used prophylactically or therapeutically, with comparable efficacy to that of zoledronic acid. 5TGM1 cells were shown to express minimal levels of CCR1 while secreting high levels of CCL3, suggesting that the therapeutic effects of CCX721 result from CCR1 inhibition on non-MM cells, most likely osteoclasts and osteoclast precursors. These results provide a strong rationale for further development of CCR1 antagonists for the treatment of MM and associated osteolytic bone disease. (Blood. 2012;120(7):1449-1457) IntroductionEstablishment of multiple myeloma (MM) in the bone marrow niche is highly dependent on bone resorption and proximity to active osteoclasts (OCs). 1,2 OC and MM cells support and nourish each other in vitro and in vivo. 1,3,4 MM-associated osteolytic bone disease (OBD), which affects Ͼ 80% of patients, results from heightened bone catabolism and decreased bone formation and is characterized by severe bone pain and high rates of fractures, greatly impacting their quality and length of life. 5,6 The chemokine CCL3/MIP-1␣ is one of the most important OC-activating factors produced by MM cells and is generally thought to contribute significantly to MM-associated OBD. 7 In cell culture, CCL3 is among the most consistently identified OC-activating factors produced by primary and immortalized MM cells. 8 The extent of CCL3 secretion by MM cells has been correlated with the extent of lytic bone lesions in patients. 9 Serum levels of CCL3 are elevated in newly diagnosed MM patients and correlate with the extent of bone disease, bone resorption, and disease prognosis. 10 High levels of CCL3 in bone marrow also correlate with MM disease stage and activity. [11][12][13] Other chemokines that have been implicated in the pathogenesis of MM include CCL5/RANTES, which, like CCL3, is a potent activator of chemokine CCR1 and CCR5 receptors. 14 We and others have shown that the pathogenic interplay between MM cells and the bone marrow environment is mediated, in part, by a paracrine mechanism whereby CCL3, secreted by MM cells, stimulates OC activity. 15 At the same time, CCL3 also inhibits osteoblast (OB) formation, further contributing to the imbalance between bone resorption and bone formation. 16 On th...

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The safety and pharmacokinetic (PK)/pharmacodynamic (PD) profile of the novel CCR1 antagonist CCX354 was evaluated in double-blind, placebo-controlled, single- and multiple-dose phase I studies (1-300 mg/day oral doses). CCX354 was well tolerated and displayed a linear dose-exposure profile, with half-life approaching 7 h at the 300-mg dose. The extent of CCR1 receptor blockade on blood monocytes, which correlated well with plasma concentrations of the drug, was assessed using fluorescently labeled CCL3 binding in whole blood from phase I subjects. High levels of receptor coverage at the 12-h time point were achieved after a single dose of 100 mg CCX354. Preclinical studies indicate that effective blockade of inflammatory cell infiltration into tissues requires ≥90% CCR1 inhibition on blood leukocytes at all times. The comparison of the properties of CCX354 with those published for other CCR1 antagonists has informed the dose selection for ongoing clinical development of CCX354 in rheumatoid arthritis (RA).

N-[4-[1-methyl-2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (5) and its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized in order to investigate the effect of incorporating a methyl group at the C9 position and of conformational restriction at the C8-C9 bridge of N-[4-[2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (1) with respect to dihydrofolate reductase (DHFR) inhibitory activity as well as antitumor activity. The compounds were synthesized by a Wittig reaction of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with ethyl 4-acetylbenzoate followed by catalytic reduction, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The biological results indicated that the addition of a 9-methyl group to the C8-C9 bridge, as in 5, increased recombinant human (rh) DHFR inhibitory potency (IC(50) = 0.42 microM) as well as the potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC(50) = 29 nM, A253 EC(50) = 28.5 nM, and FaDu EC(50) = 17.5 nM) compared with the 9-desmethyl analogue 1. However, the conformationally restricted 4:1 Z/E mixture of 7 and 6 was less potent than 5 in both assays, and the pure E-isomer 6 was essentially inactive. These three classical analogues were also evaluated as inhibitors of Lactobacillus casei, Escherichia coli, and rat and rh thymidylate synthase (TS) and were found to be weak inhibitors. All three analogues 5-7 were good substrates for human folylpolyglutamate synthetase (FPGS). These data suggested that FPGS is relatively tolerant to different conformations in the bridge region. Further evaluation of the cytotoxicity of 5 and 7 in methotrexate (MTX)-resistant CCRF-CEM cell sublines suggested that polyglutamylation was crucial for their mechanism of action. Metabolite protection studies of 5 implicated DHFR as the primary intracellular target. Compound 5 showed GI(50) values in 10(-9)-10(-7) M range against more than 30 tumor cell lines in culture.