CCR3 antagonists: a potential new therapy for the treatment of asthma. Discovery and structure-activity relationships

CCR3 antagonist leads with IC(50) values in the microM range were converted into low nM binding compounds that displayed in vitro inhibition of human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-1-yl-n-propylureas and erythro-3-(4-benzyl-2-(alpha-hydroxyalkyl)piperidin-1-yl)-n-propylureas (obtained via Beak reaction of N-BOC-4-benzylpiperidine) exhibited single digit nanomolar IC(50) values for CCR3.     

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.     

From rigid cyclic templates to conformationally stabilized acyclic scaffolds. Part II: Acyclic replacements for the (3S)-3-benzylpiperidine in a series of potent CCR3 antagonists

Conformational analysis of the 3-benzylpiperidine in CCR3 antagonist clinical candidate 1 (BMS-639623) predicts that the benzylpiperidine may be replaced by acyclic, conformationally stabilized, anti-1,2-disubstituted phenethyl- and phenpropylamines. Ab initio calculations, enantioselective syntheses, and evaluation in CCR3 binding and chemotaxis assays of anti-1-methyl-2-hydroxyphenethyl- and phenpropylamine-containing CCR3 antagonists support this conformational correlation.     

CCR5 antagonists as anti-HIV-1 agents. Part 2: Synthesis and biological evaluation of N-[3-(4-benzylpiperidin-1-yl)propyl]-N,N'-diphenylureas

We have previously reported the novel lead compound 1a as a CCR5 antagonist for treatment of HIV-1 infection. SAR studies on incorporating various acyl groups as a replacement for the 5-oxopyrrolidine-3-carbonyl group of the lead structure resulted in the discovery of N-[3-(4-benzylpiperidin-1-yl)propyl]-N,N'-diphenylurea (4a) with significantly improved CCR5 binding affinity. Substitutions (4-Cl, 4e,f; 4-Me, 4i) on the N'-phenyl ring further increased the binding affinity. Introduction of polar substituents on the phenyl ring of the 4-benzylpiperidine moiety enhanced the inhibitory activity of the HIV-1 envelope-mediated membrane fusion (4v,w), suggesting that polar substituents at this position can interfere effectively with HIV-1 cell entry.     

2,4-Disubstituted piperidines as selective CC chemokine receptor 3 (CCR3) antagonists: synthesis and selectivity

Linear unselective CCR3 antagonist leads with IC(50) values in the 200 nM range were converted into low nM binding compounds selective at CCR3 by moving the piperidine nitrogen substituent to the carbon at the 2-position of the ring. Substitution of the piperidine nitrogen with simple alkyl and acyl groups was found to improve the selectivity of this new compound class. In particular, N-{3-[(2S, 4R)-1-(propyl)-4-(4-fluorobenzyl)piperidinyl]propyl}-N'-(3-acetylphenyl)urea exhibited single digit nanomolar IC(50) values for CCR3 with >100-fold selectivity against an extensive counter screen panel.     

In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031

Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC(50)=3.0nM), ligand-induced Ca(2+) flux (IC(50)=5.4nM), and the chemotaxis of human CCR3-expressing cells (IC(50)=19.9nM). YM-344031 (1-10mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.     

CP-481,715, a potent and selective CCR1 antagonist with potential therapeutic implications for inflammatory diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5'-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.     

Discovery and structure-activity relationships of urea derivatives as potent and novel CCR3 antagonists

The synthesis and structure-activity relationships of ureas as CCR3 antagonists are described. Optimization starting with lead compound 2 (IC(50)=190 nM) derived from initial screening hit compound 1 (IC(50)=600 nM) led to the identification of (S)-N-((1R,3S,5S)-8-((6-fluoronaphthalen-2-yl)methyl)-8-azabicyclo[3.2.1]octan-3-yl)-N-(2-nitrophenyl)pyrrolidine-1,2-dicarboxamide 27 (IC(50)=4.9 nM) as a potent CCR3 antagonist.     

Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC(50) 3.2 nM; MCP-1-Induced Chemotaxis IC(50) 0.83 nM; Ca(2+) Flux IC(50) 7.5 nM].     

CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines

Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP-2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern.     

Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other beta-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC(50)) ranged from 0.04 nM with HIV-1(A-92RW009) to 1.3 nM with HIV-1(B-BaL). Experiments performed on the same isolate (HIV-1(B-BaL)) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC(50) values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC(50) values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.     

Identification of a potent and nonpeptidyl ccr3 antagonist

CCR3 is expressed in a variety of leukocyte subsets, especially eosinophils, and may be involved in allergic disorders such as atopic asthma. To clarify the pathophysiological roles of CCR3 in allergic disorders, we developed a nonpeptidyl CCR3 antagonist. This antagonist, which is referred to as "Compound X," that inhibited the binding of [(125)I]Eotaxin to CHO cells transfected with human CCR3 with an IC(50) value of 2.3 nM. In human eosinophils, Compound X also inhibited Eotaxin-induced increases in intracellular Ca(2+) concentrations and chemotaxis. Thus, Compound X appears to be a highly potent CCR3 antagonist. These findings suggest that Compound X may be a useful tool for elucidating the pathophysiological roles of CCR3 in a variety of allergic disorders.     

New insights into the mechanisms whereby low molecular weight CCR5 ligands inhibit HIV-1 infection

CC chemokine receptor 5 (CCR5) is a G-protein-coupled receptor for the chemokines CCL3, -4, and -5 and a coreceptor for entry of R5-tropic strains of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T-cells. We investigated the mechanisms whereby nonpeptidic, low molecular weight CCR5 ligands block HIV-1 entry and infection. Displacement binding assays and dissociation kinetics demonstrated that two of these molecules, i.e. TAK779 and maraviroc (MVC), inhibit CCL3 and the HIV-1 envelope glycoprotein gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that they bind to regions of CCR5 distinct from the gp120- and CCL3-binding sites. We observed that TAK779 and MVC are full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins. Dissociation of [(125)I]CCL3 from CCR5 was accelerated by TAK779, to a lesser extent by MVC, and by GTP analogs, suggesting that inverse agonism contributes to allosteric inhibition of the chemokine binding to CCR5. TAK779 and MVC also promote dissociation of [(35)S]gp120 from CCR5 with an efficiency that correlates with their ability to act as inverse agonists. Displacement experiments revealed that affinities of MVC and TAK779 for the [(35)S]gp120-binding receptors are in the same range (IC(50) ～6.4 versus 22 nm), although we found that MVC is 100-fold more potent than TAK779 for inhibiting HIV infection. This suggests that allosteric CCR5 inhibitors not only act by blocking gp120 binding but also alter distinct steps of CCR5 usage in the course of HIV infection.     

Synthesis and evaluation of cis-3,4-disubstituted piperidines as potent CC chemokine receptor 2 (CCR2) antagonists

A series of cis-3,4-disubstituted piperidines was synthesized and evaluated as CC chemokine receptor 2 (CCR2) antagonists. Compound 24 emerged with an attractive profile, possessing excellent binding (CCR2 IC(50)=3.4 nM) and functional antagonism (calcium flux IC(50)=2.0 nM and chemotaxis IC(50)=5.4 nM). Studies to explore the binding of these piperidine analogs utilized a key CCR2 receptor mutant (E291A) with compound 14 and revealed a significant reliance on Glu291 for binding.     

Human alpha-fetoprotein binds to primary macrophages

We have previously reported that alpha-fetoprotein (AFP) inhibits infection of human monocyte-derived macrophages (MDM) by R5-HIV-1 strains and that a peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds to CCR5. We demonstrate here that AFP binds high- and low-affinity binding sites of MDM, characterized, respectively, by 5.15 and 100nM K(d) values. Heat denaturation or neuraminidase treatment of AFP inhibits this binding, suggesting the involvement of protein-protein and lectin-carbohydrate interactions. Moreover, AFP displaces V3Cs binding to MDM. In addition, MIP-1beta, the most specific CCR5 ligand, displaces AFP binding to MDM (IC(50)=4.3nM). Finally, we demonstrate that AFP binds to a ligand of HIV-gp120 V3Cs domain, CCR5, expressed by MDM and by HeLa cells expressing CCR5. Such binding is not observed in the presence of HeLa cells lacking CCR5. The present results provide strong evidence that AFP directly binds to CCR5 expressed by human primary macrophages and by transfected CCR5+ HeLa cells.     

A small molecule antagonist of chemokine receptors CCR1 and CCR3. Potent inhibition of eosinophil function and CCR3-mediated HIV-1 entry

We describe a small molecule chemokine receptor antagonist, UCB35625 (the trans-isomer J113863 published by Banyu Pharmaceutical Co., patent WO98/04554), which is a potent, selective inhibitor of CCR1 and CCR3. Nanomolar concentrations of UCB35625 were sufficient to inhibit eosinophil shape change responses to MIP-1alpha, MCP-4, and eotaxin, while greater concentrations could inhibit the chemokine-induced internalization of both CCR1 and CCR3. UCB35625 also inhibited the CCR3-mediated entry of the human immunodeficiency virus-1 primary isolate 89.6 into the glial cell line, NP-2 (IC(50) = 57 nm). Chemotaxis of transfected cells expressing either CCR1 or CCR3 was inhibited by nanomolar concentrations of the compound (IC(50) values of CCR1-MIP-1alpha = 9.6 nm, CCR3-eotaxin = 93.7 nm). However, competitive ligand binding assays on the same transfectants revealed that considerably larger concentrations of UCB35625 were needed for effective ligand displacement than were needed for the inhibition of receptor function. Thus, it appears that the compound may interact with a region present in both receptors that inhibits the conformational change necessary to initiate intracellular signaling. By virtue of its potency at the two major eosinophil chemokine receptors, UCB35625 is a prototypic therapy for the treatment of eosinophil-mediated inflammatory disorders, such as asthma and as an inhibitor of CCR3-mediated human immunodeficiency virus-1 entry.     

Design and synthesis of novel CCR3 antagonists

As part of our investigation into the development of potent CCR3 antagonists, a series of piperidine analogues was designed and prepared. Exploration of the piperidine core examined both the basicity and the location of a nitrogen, as well as conformational variants. The bicyclo-piperidine 24c was found to be the most potent inhibitor of CCR3 with an IC(50) of 0.0082 microM in the binding assay and 0.0024 microM in the chemotaxis assay.     

Orally active CCR5 antagonists as anti-HIV-1 agents. Part 3: Synthesis and biological activities of 1-benzazepine derivatives containing a sulfoxide moiety

In order to develop orally active CCR5 antagonists, 1-propyl- or 1-isobutyl-1-benzazepine derivatives containing a sulfoxide moiety have been designed, synthesized, and evaluated for their biological activities. Sulfoxide compounds containing a 2-pyridyl group were first investigated, which led to discovering that the presence of a methylene group between the sulfoxide moiety and 2-pyridyl group was necessary for increased inhibitory activity in a binding assay. After further chemical modification, it was found that replacement of the pyridyl group with an imidazolyl or 1,2,4-triazolyl group enhanced activity in the binding assay and that S-sulfoxide compounds were more active than R-isomers. Particularly, compounds (S)-4r, (S)-4s, and (S)-4w exhibited highly potent CCR5 antagonistic activities (IC50=1.9, 1.7, 1.6 nM, respectively) and inhibitory effects (IC50=1.0, 2.8, 7.7 nM, respectively) in the HIV-1 envelope mediated membrane fusion assay, together with good pharmacokinetic properties in rats. In addition, we established the synthesis of (S)-4r and (S)-4w by asymmetric oxidation with titanium-(S)-(-)-1,1'-bi-2-naphthol complex.     

Synthesis and monoamine transporter binding properties of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes

A series of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2beta-[3'-(substituted benzyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (3a-h) showed high binding affinities for the dopamine transporter (DAT). The IC(50) values ranged from 5.9 to 22nM. On the other hand, the 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (4a-h), with IC(50) values ranging from 65 to 173nM, were approximately 3- to 25-fold less potent than the corresponding 2beta-[3'-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3Beta-(4-Methylphenyl)-2beta-[3'-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC(50) of 5.9nM at the DAT and K(i)s of 454 and 113nM at the NET and 5-HTT, respectively, was the most potent and DAT-selective analog. Molecular modeling studies suggested that the rigid conformation of the isoxazole side chain in 4a-h might play an important role on their low DAT binding affinities.