Small Molecule CXCR3 Antagonists - Journal of Medicinal Chemistry

Perspective pubs.acs.org/jmc

Small Molecule CXCR3 Antagonists Stephen P. Andrews*,† and Rhona J. Cox‡ †

Heptares Therapeutics, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, United Kingdom Respiratory, Inflammation & Autoimmunity iMed, AstraZeneca, Respiratory, Inflammation & Autoimmunity IMED, Pepparedsleden, 431 83 Mölndal, Sweden

J. Med. Chem. 2016.59:2894-2917. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/28/19. For personal use only.

‡

ABSTRACT: Chemokines and their receptors are known to play important roles in disease. More than 40 chemokine ligands and 20 chemokine receptors have been identified, but, to date, only two small molecule chemokine receptor antagonists have been approved by the FDA. The chemokine receptor CXCR3 was identified in 1996, and nearly 20 years later, new areas of CXCR3 disease biology continue to emerge. Several classes of small molecule CXCR3 antagonists have been developed, and two have shown efficacy in preclinical models of inflammatory disease. However, only one CXCR3 antagonist has been evaluated in clinical trials, and there remain many opportunities to further investigate known classes of CXCR3 antagonists and to identify new chemotypes. This Perspective reviews the known CXCR3 antagonists and considers future opportunities for the development of small molecules for clinical evaluation.

■

INTRODUCTION The chemokine receptor CXCR3 was first identified in 1996 and has since been shown to play a role in a wide range of physiological and pathological processes.1 It has been the target of many drug discovery programs within biopharmaceutical companies and academic research groups, which have focused mainly on the search for small molecule antagonists capable of blocking the action of the three chemokines that are known to bind to and activate this receptor (CXCL9, CXCL10, and CXCL11).2−4 CXCR3 antagonists were originally proposed for use in inflammatory and autoimmune diseases, but more recent research suggests that they may be of therapeutic use in a broader range of disorders.5 More than 15 classes of CXCR3 antagonists have been identified, mainly from medium- to high-throughput screening, as well as from focused and virtual screening approaches. These include not only basic molecules, which are typical of chemokine receptor antagonists, but also acidic and neutral ligands. Two classes that have been extensively optimized are the piperazinyl-piperidines, e.g., 1 (SCH 546738),6 which have shown efficacy in several preclinical disease models,6,7 and the 8-azaquinazolinones, e.g., 2 (AMG 487; Figure 1).8 Compound 2 is the only small molecule CXCR3 antagonist that has been reported to have been evaluated in clinical trials. It reached Phase IIa trials for psoriasis but failed to show efficacy.8 This has been postulated to be due to variable exposure caused by time-dependent pharmacokinetics, and further development of the compound has been terminated.9,10 This leaves its mechanism untested in humans, but a human monoclonal © 2015 American Chemical Society

antibody that targets CXCL10 (eldelumab, BMS-936557) has been evaluated in Phase II clinical trials for the treatment of ulcerative colitis and Crohn’s disease,11,12 which gives confidence in the clinical value of modifying the CXCR3− CXCL10 axis. To date, more than 40 chemokine ligands and 20 chemokine receptors have been identified,13 but, despite extensive efforts, only two drugs targeting chemokine receptors have been approved by the FDA.14,15 There are clear challenges associated with developing chemokine receptor antagonists,16−18 but the success of the CCR5 inverse agonist maraviroc (3) and the CXCR4 antagonist plerixafor (4) inspires further efforts to develop small molecule chemokine antagonists (Figure 1). Compound 3 binds to CCR5 with high affinity and represents a novel class of antiretrovirals for the treatment of HIV-1 infection.19 Compound 4 is a small molecule that mobilizes hematopoietic stem cells and is used in the treatment of multiple myeloma and for bone marrow transplants in nonHodgkin lymphoma patients.20 As our understanding of the roles of CXCR3 has matured, the opportunities for drug development have expanded. However, Figure 2 shows that although the total number of CXCR3 publications continues to grow, many of these are associated with emerging biology, and the search for small molecule CXCR3 antagonists appears to have slowed over the past decade. The aim of this Perspective is to comprehensively Received: August 28, 2015 Published: November 4, 2015 2894

DOI: 10.1021/acs.jmedchem.5b01337 J. Med. Chem. 2016, 59, 2894−2917

Journal of Medicinal Chemistry

■

Perspective

CXCR3 BIOLOGY

Chemokines comprise a large family of soluble proteins that are approximately 8−10 kDa in size and that have similar threedimensional structures. They are classified by subfamily according to the number and spacing of cysteine residues at their N-termini. The two major families are the CXC chemokines, which have two N-terminal cysteine residues separated by one amino acid, and the CC chemokines, in which the N-terminal cysteines are adjacent.21 There are two minor families of CX3C and C chemokines, as well as a family of atypical chemokines.13 Chemokine receptors are expressed on the surface of leukocytes. They belong to the G protein-coupled receptor (GPCR) superfamily and are classified according to the type of chemokine ligand they bind. For example, CXC receptors (CXCR) bind to CXC ligands (CXCL). Many chemokine receptors are able to recognize and bind several different chemokines, and many chemokines are able to bind to several different receptors, but in nearly all cases, chemokines and their receptors interact within the same structural subfamily.22 The chemokine receptor CXCR3 is activated by the chemokines CXCL9 (previously known as MIG, monokine induced by γ-interferon), CXCL10 (IP-10, interferon-γ-induced protein 10), and CXCL11 (I-TAC, interferon-inducible T-cell α chemoattractant).1,23−25 Following activation, the receptor couples to signaling molecules, including Gαi protein and βarrestin, to initiate intracellular signaling events. These signals cause cell migration (chemotaxis) of the leukocyte along a gradient toward the source of the chemokine.26 After activation, the receptor is desensitized, internalized, and then degraded, followed by synthesis of a new receptor.27 CXCL9, CXCL10, and CXCL11 are predominantly induced by interferon-γ, although they are differentially regulated, and are produced at sites of inflammation. Some studies suggest that these chemokines have overlapping functions in vivo, although others indicate collaboration or competition among them.28

Figure 1. Two most extensively investigated classes of CXCR3 antagonists are the piperazinyl-piperidines (exemplified by 1) and the (aza)quinazolinones (exemplified by 2). Compound 1 is efficacious in preclinical models of inflammation. Compound 2 is the only CXCR3 antagonist that has been evaluated clinically. Only two small molecule chemokine receptor antagonists have been approved by the FDA (compounds 3 and 4).

review the known CXCR3 antagonist chemotypes and to consider the progress that has been made with medicinal chemistry programs. With the recent developments in our understanding of the structure and function of chemokine receptors, attention is also given to the future role of structurebased design in the development of therapeutically relevant small molecule CXCR3 antagonists. Many opportunities remain to develop such molecules to rigorously test animal models of disease and to treat unmet medical needs in human patients. We hope that this review stimulates further interest in the field.

Figure 2. (A) Total number of CXCR3 publications made each year since 1997; (B) number of patents published each year since 2001 and that claim to identify new small molecule CXCR3 antagonists. Analyses were performed in June 2015, in SciFinder, which searches bibliographic metadata in MEDLINE and CAPLUS, including article titles and abstracts. (A) The search term CXCR3 resulted in >4000 hits, which were plotted by publication year (similarly, searching the titles of journal articles resulted in >800 hits). (B) The hits from (A) were refined by document type = patent. Only patents that claimed to identify new small molecules (not peptides) primarily as CXCR3 antagonists were included (e.g., process patents for previously claimed molecules were discarded, as were patents that claimed to identify CXCR1 antagonists that showed some CXCR3 activity in selected cases). 2895



Perspective

Figure 3. Schematic representation of a GPCR and the two-site activation mechanism of a chemokine receptor. In step 1, the chemokine (blue) approaches the GPCR (A) and then binds to the N-terminus of the receptor via its C-terminus (B). In step 2, the N-terminus of the chemokine engages the TM binding site of the GPCR (i.e., within the helical bundle) (C) to trigger an intracellular signaling cascade.

number of diseases not generally considered to be inflammatory or autoimmune in nature, such as Alzheimer’s disease,58 Guillain-Barré syndrome,59 acute respiratory distress syndrome (ARDS),60 chronic prostatitis,61 and others. CXCR3 antagonists have been shown to prevent tumor metastasis in animal models of disease,62−64 although the links between CXCR3 and cancer are complex, depending on the isoform of CXCR3, the cell type, and the tumor microenvironment.33,65 Antagonism of CXCR3, therefore, offers the potential to modulate a variety of chronic diseases.

In any case, spatial and temporal regulation of these ligands appears to direct the generation and migration of T cells to inflamed tissue. CXCL9, CXCL10, and CXCL11 show slightly different binding modes to CXCR3 and have differing efficacies, which may lead to differences in the downstream signaling cascade and chemotaxis.29 CXCR3 is predominantly expressed on helper Th1 CD4+ T cells, effector CD8+ cytotoxic T cells, and some natural killer ̈ T cells and natural killer T cells, but it is absent from naive cells.28 It is additionally expressed on dendritic cells30 and certain B cells.31 A number of nonimmune cells also express CXCR3, including astrocytes,32 fibroblasts, endothelial, epithelial, and smooth muscle cells.33 CXCR3 exists as a number of splice variants containing slightly different amino acid sequences: CXCR3-A, CXCR3-B, and CXCR3-alt. CXCR3-A is the most common and is the variant most frequently expressed on immune cells. Other cell types also express CXCR3-B, which can bind CXCL4 in addition to CXCL9, CXCL10, and CXCL11, and appears to mediate angiogenesis.34 CXCR3-alt is a significantly truncated variant containing only four transmembrane helices and is activated only by CXCL11.35 Strong immune responses are important during acute inflammation, but the immune system can overreact, leading to chronic inflammation. CXCR3 has been linked to a variety of inflammatory and immune diseases.5 In some cases, patient tissue samples show upregulation of CXCR3 or its ligands, and the degree of expression may correlate with disease pathogenesis, progression, or prognosis. In other cases, animal models suggest a link between the disease and CXCR3 or its ligands, either because CXCR3-deficient mice are protected from developing a phenotype associated with the disease or because a CXCR3 antagonist shows beneficial effects in an animal disease model.36 Some diseases are more strongly associated with one of the ligands (CXCL9, CXCL10, or CXCL11) over the other two. CXCR3 antagonism has been associated with inflammatory and autoimmune diseases such as rheumatoid arthritis,37−40 multiple sclerosis,41−43 inflammatory bowel disease,44,45 systemic lupus erythematosus (SLE),46−48 chronic obstructive pulmonary disease (COPD),49,50 psoriasis,48,51 organ transplant rejection52−54 and graft-vs-host disease,55 asthma,56 and type 1 diabetes.57 More recently, CXCR3 has also been suggested to be a therapeutic target for a

■

CHEMOKINE RECEPTOR STRUCTURE AND FUNCTION The chemokine receptors belong to class A of the GPCR superfamily of receptors. GPCRs are cell surface signaling proteins that are often referred to as seven transmembrane receptors (7TMs) owing to their topology, which comprises seven helices arranged in a parallel array within the cell’s lipid bilayer and tethered to one another with intra- and extracellular loops (as shown schematically in Figure 3A). Since the turn of the millennium, GPCR crystallography and structure determination have been revolutionized by advances with lipidic cubicphase technologies66 and protein fusion strategies to improve GPCR crystallization,67 as well as by receptor engineering methods,68 which enable the stabilization, purification, and crystallization of GPCR constructs. Indeed, the 3D coordinates of nearly 30 receptors from GPCR classes A, B, C, and F are now available,69 and these are greatly impacting our understanding of the structure and function of these proteins,70,71 as well as influencing GPCR drug design.72,73 In the majority of these structures, the ligands are found to bind to a similar region of the receptors, in the top of the TM domains, where they often overlap with one another (including small molecule chemokine receptor antagonists).74 This binding cavity will be termed the “TM binding site” throughout this Perspective, and its approximate position is shown schematically in Figure 3A. Furthermore, this TM binding site has been described as comprising major and minor subpockets, and these are shown schematically in Figure 4A.75 Targeting these different subpockets with small molecule antagonists may lead to probe-dependent signal bias and varying pharmacological profiles, but this is still poorly understood (vide infra). 2896



Perspective

Figure 4. Helix box diagrams of the human chemokine receptors CXCR4 (A) and CCR5 (B) that have been solved by X-ray crystallography (CXCR4 and CCR5 share 36% sequence identity). The residues with a role in ligand binding for these receptors are colored in green where they are the same in the CXCR3 sequence and in red where they are different in the CXCR3 sequence. (C) Sequence alignments for CXCR4, CCR5, and CXCR3, where * indicates the highly conserved residues in each TM domain and the colored residues from (A) and (B) are shown in bold (sequence identities: CXCR3/CXCR4 = 40%, CXCR3/CCR5 = 36%).

vMIP-II.81,82 The peptide forms a number of hydrogenbonding interactions and salt bridges with CXCR4, including from three aspartate residues (Asp171 4.60, Asp187 ECL2 , Asp2626.58) and resides mainly in the major site. Compound 5 was found to partially overlap with the peptide, but it binds mainly to the minor site. The structure of CXCR4 in complex with vMIP-II was resolved to 3.1 Å and was enabled by introducing a disulfide link between D187C of the receptor and W5C of the ligand. The binding site was open and negatively charged, and, in contrast to previously solved structures, the N-terminus of the receptor was almost perpendicular to the membrane. The structure revealed a continuous surface contact of 1330 Å2 between the ligand and receptor, as well as electron density for the chemokine’s entire N-terminus, which was located mainly in the minor site of the receptor, where it made polar contacts with D972.63 and E2887.39. The structure of CCR5 has been solved in cocomplex with the inverse agonist 3 (Figures 1 and 4).83 Once again, the ligand was observed to bind in the TM binding site, and this time the small molecule spanned across both the major and minor subpockets. As was observed with the CXCR4 ligands, 3 makes salt bridging interactions with the receptor and very efficiently occupies lipophilic pockets with its alkyl groups (3 is known to have a very high binding efficiency84). Furthermore, the phenyl group of the ligand interacts with Trp2486.48, preventing helical movement, which has been implicated in GPCR activation.

Before chemokine receptor crystal structures were available, it was proposed that they were activated by extracellular chemokine ligands in a two-site mechanism to generate an intracellular response (Figure 3).76 Chemokines are much larger than the majority of other GPCR ligands and bind not only to the TM binding site but also to the extracellular domain of the receptor. It was postulated that in the first stage of binding (recognition) the N-terminus and extracellular loops of the receptor recognize and bind the C-terminus of the chemokine with high affinity, and in the second stage (activation), the N-terminus of the ligand interacts with the TM binding site to trigger a conformational change, which results in intracellular signaling.77 As such, N-terminally truncated chemokines can retain high levels of affinity for their receptors but show decreased levels of functional activity.78 NMR and circular dichroism studies of the Nterminus of CXCR3,79 and of CXCL11,80 provide further support for the two-site mechanism. In solution, the first 48 residues of the N-terminus of CXCR3 were found to access multiple conformations in which stretches of negatively charged residues formed dynamic hydrogen bonds.79 In 2015, a crystal structure was solved with CXCR4 bound to a viral chemokine, showing unequivocally that the ligand binds to both sites of the receptor.81 The structures of two chemokine receptors have been solved by X-ray crystallography, revealing TM binding sites with open, negatively charged cavities that are larger than those in other GPCR structures (Figure 4). CXCR4 has been solved in complex with the small molecule antagonist 5 (IT1t; Figure 5), the peptide antagonist CVX15, as well as the viral chemokine 2897



Perspective

Figure 5. Tool ligands for structure and function studies. Compound 5 has been crystallized in co-complex with CXCR4. CXCR3 ligands 6 and 7 were found to occupy different parts of the large CXCR3 TM binding site, and 8 may also bind in a different site from that of the (aza)quinazolinones. Compounds 9 and 10 were found to have different biases toward G protein signaling and β-arrestin recruitment. Compound 11 has been developed as a radioligand.

■

CXCR3 STRUCTURE-BASED DRUG DESIGN As is the case with other GPCRs, X-ray crystallography with chemokine receptors is greatly enhancing our understanding of the structure and function of these proteins and is highly enabling for the construction of CXCR3 homology models.85,86 Figure 4 shows a comparison of the residues involved in small molecule antagonist binding contacts within the CXCR4 and CCR5 crystal structures, as well as the sequences of these receptors alongside CXCR3. Several studies have recently been reported in which CXCR3 homology models have been used for in silico ligand screening. For example, a CXCR3 model has been built from published CXCR4 X-ray structures, used for the screening of known small molecule CXCR3 antagonists, and then compared with a pharmacophore-based model.87 The homology model was trained with 16 known bioactive molecules (largely from the (aza)quinazolinone and piperazinyl-piperidine classes of ligand, which are described further below), which covered 5 log orders of binding affinity. A further 286 known CXCR3-actives were then screened with the model, from which 246 were correctly predicted to be CXCR3 antagonists. This compared well with a hit rate of 243 actives from the same set using a pharmacophore-based model. Another CXCR3 homology model has been used to prospectively identify new CXCR3/CXCR4 antagonists.88 This model was also constructed from published CXCR4 structural data, and 2 million compounds from the ZINC database were screened in silico. From this set, 17 compounds were purchased and screened in biological assays with CXCR3 and CXCR4. It was predicted that 6 compounds would be CXCR4-selective, 7 would be CXCR3-selective, and 4 would be dual antagonists. Excellent hit rates were obtained, with 4 of the compounds showing the expected CXCR3-selective profile, 3 showing the predicted CXCR4-selective activity, and 2 correctly showing dual antagonism.

A further study has used a CXCR3 homology model built from the CXCR4 crystal structure in combination with sitedirected mutagenesis to map the binding sites of ligands from the piperazinyl-piperidine and 8-azaquinazolinone classes of CXCR3 antagonist: 6 (VUF11211), which contains two basic centers, and the neutral compound 7 (NBI-74330; Figure 5).89 In this elegant study, mutations were made within the CXCR3 binding site that were predicted to affect salt bridges, hydrogen bonds, and π-stacking interactions with the small molecules. The results showed that 6 also bound to the TM binding site, where it spanned across both the major and minor subpockets and was anchored to D1864.60 via a salt bridging interaction. In contrast, compound 7 was found to occupy only the minor site (around TMs 2, 3, and 7), where it partially overlapped with 6. Surprisingly, the neutral 8-azaquinazolinone was also significantly affected by the mutation of a charged residue (D112N2.63). Another closely related 8-azaquinazolinone (2) has also been evaluated in CXCR3 mutagenesis studies. In CXCR3, the mutation D112N2.63 was found to affect the ability of 2 to displace 125I-CXCL11 or inhibit CXCL11-mediated chemotaxis (by 13- and 6-fold, respectively), which is in line with the results observed with 7, above.90 A quaternary ammonium derivative 8 (TAK-779; Figure 5)91 was also evaluated in this study. This compound is known to be a promiscuous antagonist with high affinity for CCR2b and CCR5, as well as CXCR3, and displays a partial inverse agonism mechanism, which differs from the full inverse agonist mechanism of the 8azaquinazolinones.91 Its binding was generally not affected by the mutations that affected the 8-azaquinazolinone, and the authors postulated that 8 may bind at a different site. However, there is currently no data to positively associate this compound with an alternative binding site. The 8-azaquinazolinones have also been shown to exhibit bias in their blocking of G protein activation versus β-arrestin 2898



Perspective

Figure 6. (Aza)quinazolinones and related derivatives developed as CXCR3 antagonists by ChemoCentryx, Amgen, and TaiGen.

chemical classes, and, where possible, series from within these classes have been presented alongside one another to allow the reader to draw comparisons. A limited number of CXCR3 small molecule agonists have also been reported, but they will not be described in detail here.3,95 Research groups have optimized their chemical series with varying assay types (e.g., binding, calcium flux, or chemotaxis), and no single standard assay has been used. To aid comparison between different drug discovery programs, the most commonly used CXCR3 potency assays are described briefly here. Note that the activities of the three natural ligands varies, so some variation is expected between assays using different ligands. Most research groups have optimized against just one chemokine, with additional profiling against the two other chemokines at the start and/or end of the optimization. To our knowledge, there are no examples of small molecule antagonists that are reported to be completely selective for just one or two of the natural chemokines. Radioligand Binding Assay. This is used to study direct binding to CXCR3 by competition with a radiolabeled

recruitment and are able to modulate the activity of chemokines in a probe-dependent manner.92 Up to 187-fold selectivity was observed in CXCL11-dependent G protein activation vs βarrestin recruitment for 9, whereas structurally related boronic acids such as 10 showed bias for β-arrestin recruitment.93 Compound 11 was found to show no signal bias and in its tritiated form is known as RAMX3, a radioligand that has been used to study competitive binding with CXCL11 (Figure 5).94 Further investment for solving X-ray structures of CXCR3 in co-complex with antagonists from different chemical classes and with different signal biases is expected to greatly enhance the field of rational CXCR3 drug design.

■

PROGRESS WITH CXCR3 ANTAGONISTS This section describes the progress that has been made with the discovery and development of small molecule CXCR3 antagonists. Where possible, details of the SAR within chemical series are provided in figures and attention is given to the optimization of drug-like properties, such as solubility, off-target toxicity, and stability. In some cases, there is overlap between 2899



Perspective

Figure 7. Aryl sulfonamides developed as CXCR3 antagonists by TaiGen and Merck Serono.

Chemotaxis. This assay measures the effects on cell migration of CXCR3 antagonism. Cells expressing CXCR3 are incubated with the antagonist. The chemokine is added to the lower wells of a microchemotaxis plate. A membrane is put in place over these wells, and the pretreated cells are added to the top of the membrane. After incubation, the number of cells that have migrated from the upper to the lower chamber is counted using flow cytometry. A similar assay can be carried out using PBMCs instead of transfected cells.96 Internalization. Once CXCR3 has been activated, the receptor is internalized. This internalization assay measures the effects of CXCR3 antagonism on this process. For example, activated mouse T cells are incubated with plasma from mice that have been dosed with the antagonist. The samples are stimulated with the chemokine; then, the level of surface CXCR3 is measured by flow cytometry.98 Amgen and ChemoCentryx. The first patents describing small molecule CXCR3 antagonists were published by ChemoCentryx in 200199 and Tularik (now part of the Amgen group) in 2002.100 These patents described closely related 3-phenyl-3H-quinazolin-4-ones, and this class of CXCR3 antagonists is now one of the most intensely studied. It has led to tool ligands, including probe-dependent modulators such as 9 and 10 and radioligand 11 (all of which are discussed earlier), as well as the clinically evaluated compound 2. An article from 2007 describes the origins of this series.101 High-throughput screening led to the identification of 12 as a CXCR3 antagonist with submicromolar potency in both a radioligand binding assay with 125I-CXCL10 and a calcium flux

chemokine. Membranes from cells expressing CXCR3 are incubated with 125I-labeled chemokine and varying concentrations of antagonist. Binding of the radiolabeled chemokine is measured using scintillation proximity assay (SPA) technology or, after filtration, by liquid scintillation.96 A similar assay in human cells can be carried out using isolated human peripheral blood mononuclear cells (PBMCs), and a tritiated small molecule has also been reported for use as a CXCR3 radioligand.94 GTPγ35S Binding Assay. This allows measurement of antagonism of CXCR3 by inhibition of receptor activation rather than by competitive binding. Membranes from cells expressing CXCR3 are incubated with antagonist before stimulation with a chemokine. GTPγ35S (a hydrolytically stable, labeled analogue of GTP, which binds to a G protein as the first step of the signaling cascade) is added; then, after further incubation, radioactivity associated with CXCR3 is determined.97 A reduction in radioactivity is a measure of blockade of CXCR3 activation by the antagonist. Intracellular Calcium Flux Assay. This is a functional assay measuring changes in cellular downstream effects of CXCR3 activation. Cells expressing CXCR3 are loaded with a Ca2+-sensitive fluorescent dye. Antagonist is added at varying concentrations, followed by the appropriate chemokine at a fixed concentration. The increase in intracellular calcium produced by the agonist chemokine is reflected in changes in fluorescence, which are read using a fluorometric imaging plate reader (FLIPR).96 Reduction in this signal is a measure of activity of the antagonist. 2900



Perspective

Figure 8. Arylpiperazines and derivatives developed as CXCR3 antagonists by Ligand Pharmaceuticals, Boehringer Ingelheim, Actelion, Sanofi, and Merck & Co. This class of compound typically contains a benzamide or aryl group at the position ortho to the (homo)piperazine. 2901



Perspective

CXCR3 (typical examples are shown in Figure 7). This series was built upon in a second patent.112 Merck Serono. Merck Serono has also disclosed a series of aryl sulfonamides as CXCR3 antagonists. A screen of 90 000 compounds using a CXCR3 calcium flux assay enabled the identification of acyl hydrazone 15 as a submicromolar hit.113,114 Subsequent testing of in-house and commercially available analogues that lacked the acyl hydrazone established that it could be replaced by an amide. Further optimization was performed using a chemotaxis assay, and potencies as high as pIC50 = 7.9 were achieved, but the compounds were typically highly unstable in microsomal preparations. A synthetic intermediate acid was found to be much more stable, possibly owing to lower log D, and retained some CXCR3 activity. Exploration of acid isosteres (e.g., tetrazoles, acyl sulfonamides, and hydroxyoxadiazoles) allowed moderate gains in potency over the carboxylic acid and generally retained in vitro stability. Tetrazoles showed the added advantage of improved solubility over amides. The pharmacokinetic properties of 16 have been evaluated in the mouse (t1/2 = 1 h, F = 83%), and the authors suggest that the series is suitable for optimization, but no further work has been reported. Ligand Pharmaceuticals. Several groups have developed arylpiperazines and related derivatives as CXCR3 antagonists, typically containing an amide or aryl group ortho to the (homo)piperazine (Figure 8). Ligand Pharmaceuticals (previously Pharmacopeia) was the first group to publish on this class of CXCR3 antagonist,115 which has also been evaluated with QSAR models.116 High-throughput screening of >4 million compounds identified homopiperazines such as 17 as CXCR3 antagonists in calcium flux assays with CXCL11 (Figure 8).115,117 Within the Ligand Pharmaceuticals’ SAR, the homopiperazine appeared to be required for activity, as contraction to the corresponding piperazine or replacement with acyclic isosteres ablated antagonism, but Sanofi has reported that ring contraction was possible with a very closely related set of compounds (vide infra). Parallel chemistry approaches were used to investigate the SAR of the series at three peripheral positions. Combining this SAR led to compound 18, which showed pIC50 = 7.2 in the calcium flux assay and no significant activity when screened against a panel of 14 other GPCRs, including other chemokine receptors. ADME and pharmacokinetic parameters of this series have not been reported, and Ligand Pharmaceuticals is thought to have discontinued this work. The company has also developed piperazinyl-piperidines in collaboration with Merck & Co. (vide infra). Boehringer Ingelheim. Boehringer Ingelheim reported aryl piperazine 19 as a singleton hit with submicromolar activity in a calcium flux assay against an unknown chemokine.118 The compound showed low hERG activity (pIC50 < 4.5), high permeability in a PAMPA screen, low stability in HLM, and low solubility at pH 7.4. The SAR of 19 has been extensively explored, and the findings are summarized in Figure 8. Boehringer Ingelheim has also filed patents that describe potent examples in this series where the terminal aromatic ring has been replaced with a substituted benzamide group, and these compounds are reminiscent of the homopiperazine analogues reported by Ligand Pharmaceuticals, e.g., 20.119−121 The central phenyl ring can tolerate a third substituent (R3), such as an amide group (akin to those explored by Ligand

assay (Figure 6). The hit compound was found to have low oral bioavailability in the rat (F = 1.5%), and further compounds were designed to improve potency and pharmacokinetic properties. After several rounds of optimization and demonstration of efficacy in a mouse model of bleomycin-induced cellular recruitment to the lung, 8-azaquinazolinone 2 was selected as a clinical candidate.96,102 Compound 2 was evaluated in Phase I clinical trials. Despite no evidence of compound accumulation in preclinical species, accumulation was observed in healthy human subjects. This was particularly marked at higher doses and on multiple dosing, indicating a reduction in clearance of parent drug. Investigation of the metabolic pathway led to the identification of two major human metabolites: the pyridine N-oxide and the O-deethylated phenol. Metabolism of 2 was found to be dependent on CYP3A4, and the phenol metabolite was identified as a time-dependent inhibitor of CYP3A4, causing nonlinear pharmacokinetics.9 The mechanism by which the phenol causes time-dependent CYP inhibition was found to be due to formation of a reactive quinone metabolite that covalently modified CYP3A4.10 Additionally, evaluation of 2 was complicated by the CXCR3 activity of the pyridine N-oxide, which is equivalent to the parent compound. The compound was taken to a Phase II trial in patients with moderate-to-severe psoriasis,8 but it has been reported that further development of the drug has been discontinued due to lack of exposure.17 Several further articles describe the search for a back-up compound to 2 with reduced metabolic liabilities and lack of time-dependent CYP3A inhibition. Several replacements for the 8-azaquinazolinone were identified with comparable potencies and pharmacokinetic properties (Figure 6).103,104 It was hypothesized that the main function of the heterocycle is to hold the peripheral groups in the correct orientation, and further substituents can be used to modulate the formation of metabolites.105 Many compounds contain a 4-cyanophenyl in place of the original 4-ethoxyphenyl to mitigate dealkylation as a metabolic pathway, although these nitriles were later found to often show activity in an in vitro chromosomal aberration assay.106 Reoptimization of the phenylacetamide group led to enhanced activity by the addition of extra fluorine atoms on the phenyl ring. The effect of F and CF3 seems to be particular to fluorine rather than being a general electron-withdrawing effect. Replacement of the pyridine with a variety of sulfones led to compounds with improved metabolic stability and increased activity.107 Ultimately, core changes were abandoned in favor of the original 8-azaquinazolinone core, and the peripheral substituents were optimized to give a small group of well-characterized compounds, of which 13 is the most well described.106 However, the isomer 14 is one of two compounds described in a patent from 2009,108 and a process chemistry article describes synthesis of 2.5 kg of this compound to support toxicological studies.109 It seems likely that 14 advanced the furthest, but its current status is unknown. TaiGen Biotechnology. A patent from TaiGen Biotechnology claimed a series of compounds related to the 8azaquinazolinones.110 Typical examples are shown in Figure 6. Most compounds were described as having a pIC50 > 6.0 in a fluorometric assay based on GDP−GTP exchange. An unrelated series of arylsulfonamides was described in a patent from 2004.111 No specific biological data was given, but most examples were reported to have micromolar activity at 2902



Perspective

Figure 9. Carboxylic acids developed as CXCR3 antagonists by Sanofi.

Figure 10. Natural products identified as CXCR3 antagonists by Merck & Co.

assay with CXCL10 (e.g., compound 23, pIC50 = 6.4). Compound 24 was found to have low intrinsic clearance in HLM and was progressed to rat pharmacokinetic analysis. It showed significantly higher exposure in rat than a trifluoromethylphenyl derivative (22), claimed by Boehringer Ingelheim (AUC = 14 000 and 271 ng·h/mL for 24 and 22, respectively, when dosed orally at 2 mg/kg in rat). The most recent patent on this class of compounds was published in 2015, but the development status of this series is unknown. Sanofi. CXCR3 antagonists have been described in several patents from Sanofi and broadly cover two chemical classes, including arylpiperazines, which are closely related to those described above. In a 2009 patent,124 compounds were claimed as having CXCR3 activities of “less than 200 nM” in a calcium flux assay, but no specific data is given. The compounds are structurally related to the homopiperazines from Ligand Pharmaceuticals, but they are distinguished by the presence of a piperazine (e.g., 25, Figure 8). Two related patents from 2013 disclosed chemical series that are distinct from those claimed in 2009. The first patent claimed a series of cycloalkyl carboxylic acids, of which 26 is a typical example (Figure 9).125 Data from a 125I-CXCL10 radioligand binding assay was provided for all 160 examples, and the SAR is summarized in Figure 9. The most potent example has a reported pIC50 = 7.9, and no further biological data was reported. The second patent claimed related carboxylic acids, but in this case, the acidic group was moved to a different position on

Pharmaceuticals), or a halogen atom and can also be modified to a pyridine. These aryl piperazines were reported to generally exhibit low to moderate stability in HLM, but some examples such as 21 were suitable for in vivo dosing and were orally bioavailable in the mouse. This compound has significantly improved solubility over 19 (>53 μg/mL) and good potency in both calcium flux and chemotaxis assays (pIC50 = 8.0 and 7.7, respectively). Compound 22 was used as a benchmark by Actelion during optimization of their own series of piperazinyl derivatives (Figure 8). The development status of the Boehringer Ingelheim series remains unknown, and there have been no further publications since 2012. Actelion. Actelion has filed two patents on 4-aryl-5piperazinylthiazoles, which are potent antagonists of CXCR3 in calcium flux assays with CXCL10.122,123 In the first, the claims are limited to compounds in which the aryl group is 2benzamidazoyl, and in the second, the claimed compounds generally contain pyrimidines as the aryl group (or 4-methoxyl phenyl, which is less potent; Figure 8). The compounds are reminiscent of the Boehringer Ingelheim series described above, and the SAR trends are similar, but the major structural difference is the replacement of the phenyl core for a thiazole ring, as has similarly been reported by Merck & Co. (Figure 8). Several examples showed subnanomolar potency in a calcium flux assay, and low to moderate hERG inhibition has been reported for the pyrimidine class. The benzimidazole class was reported to be active in a whole blood CXCR3 internalization 2903



Perspective

Figure 11. Piperazinyl-piperidine derivatives developed as CXCR3 antagonists by Merck & Co and Daiichi Sankyo. The pyridine subseries was developed into potent antagonists such as 35 but generally showed limitations with hERG inhibition and PK properties. The pyridzaine subseries was optimized into compounds such as 1 and 31, which have shown efficacy in vivo.

the core structure.126 This is unusual in that the SAR between the two different acid classes appears to be broadly similar, suggesting that the two series may have similar binding modes in a site offering the possibility of accommodating an acid in two different positions. Again, potencies in the radioligand binding assay are reported for all examples, with the most potent example 27 having a reported pIC50 = 8.0.

Merck & Co. Merck & Co. has reported various series of CXCR3 antagonists, including arylpiperazines, natural products, and piperazinyl-piperidines. Three patents have been filed on compounds that are related to the arylpiperazines described above, and these include both phenylpiperazines, which are related to the Ligand Pharmaceuticals series, as well as thiazolylpiperazines.127−129 In radioligand binding assays with 2904



Perspective

found to give higher affinity than (R). Compounds in this subclass were typically highly active in vitro, e.g., 34, which had subnanomolar affinity for CXCR3 despite truncation of the benzylamide to ethyl (pKi = 9.5), but this compound showed no significant improvement in exposure from oral dosing when compared to that of 33 (AUC = 0.95 μM·h from 10 mg/kg po dose). The amide at position R1 was found to be metabolically unstable and was prone to hydrolysis followed by glucoronidation. In some cases, replacement of the amide with heterocyclic isosteres led to high-affinity CXCR3 antagonists with moderately improved oral bioavailability (e.g., 35: pKi = 8.9; AUC = 1.8 μM·h from 10 mg/kg po dose); however, the pyridyl-piperazinyl-piperidine subclass generally exhibited high levels of hERG inhibition and further optimization of in vivo pharmacokinetics was also required. Pyrazine cores were investigated as potential replacements for the pyridine, and some components of the SAR could be transposed from the pyridyl to the pyrazinyl series, such as adding small alkyl groups to the piperazine ring to increase affinity (Figure 11). The pyrazine could tolerate a variety of small substituents, including a primary amine at position R7 and a small lipophilic group at R9. Polar groups were extensively investigated at each end of the molecule (R6 and R8) in order to identify compounds with reduced hERG inhibition. At position R6, benzoyl groups were well-tolerated and the aromatic ring could be modified to a heterocycle. Compound 36 showed good affinity for the CXCR3 receptor, no hERG inhibition at 10 μM, and a considerably enhanced oral bioavailability in rat when compared to those for examples with a pyridine core (pKi = 8.4; AUC = 30.2 μM·h from 10 mg/kg po dose). Polar substituents were tolerated on the amide at R8, such as alkyl sulfonamides and hydroxyls; however, significant improvements in the pharmacokinetic profiles were observed when the amide was replaced by small heterocycles, and hERG activity was also generally low for these derivatives. In particular, 1,2,4-oxadiazol3-yl was favored, and substitution at the 5-position of this heterocycle was explored with several groups such as methyl (unstable in acid), cyclopropyl, hydroxyl, and primary amino (poor pharmacokinetics); methylsulfonamide and dimethylamine (loss of affinity); and ethylamino (good balance of hERG, pharmacokinetics, and affinity). Combining this SAR led to compounds such as 31, which shows a favorable profile with good exposure in the rat from oral dosing (AUC = 30.2 μM·h from 10 mg/kg dose), low hERG inhibition (25% at 10 μM), and high affinity for the CXCR3 receptor (pKi = 8.4). Compound 1 was also identified as an advanced lead compound during this work and has shown efficacy in autoimmune encepahalomyelitis, cardiac allograft, and collagen-induced arthritis animal models.6 When evaluated in vivo, compound 31 delayed disease onset and reduced disease severity in collagen-induced arthritis, experimental autoimmune encephalitis, and dermal inflammation. However, despite little or no adverse findings in 3 month rat toxicity studies at doses up to 60 mg/kg and in cynomolgus monkeys up to 30 mg/kg, unexpected thrombocytopenia and regenerative hemolytic anemia with acanthocytosis were observed at 75−150 mg/kg doses.7 In 2015, Leurs et al. reported the characterization of a tritium-labeled derivative of compound 6 from this series and showed that it was, in fact, an inverse agonist of CXCR3.138 There is no evidence that Merck & Co. or Ligand

human CXCR3, the arylpiperazines’ affinities were in the pIC50 range 5.7−9.0 against 125I-CXCL11 and pIC50 range 5.4−9.0 against 125I-CXCL10. The compounds inhibited chemotaxis with potencies in the pIC50 range 5.7−8.5 against CXCL11 and pIC50 range 6.1−9.3 against CXCL10. Specific activities were not reported for the compounds; however, many claimed examples are 3,5-disubstituted with lipophilic groups on the top phenyl ring, particularly tert-butyl, and contain a heterocycle on the right-hand side, which is a derivative of pyrazole or imidazole, including fusions with phenyl or pyridine (Figure 8). Merck & Co has also screened 51 000 microbial, plant, and marine extracts in a radioligand binding assay using 125ICXCL10 and CXCR3 expressed in RBL cells, which led to six hits. Bioassay-guided fractionation of these hits led to the identification of three classes of naturally occurring small molecule CXCR3 antagonists with micromolar activity (Figure 10).130 Three roselipins were identified from the screen, including 28. These compounds were originally identified as inhibitors of diacylglycerol acyltransferase.131 Compound 29 is the most potent of three diosgenin glycosides found to be active at CXCR3 in this study. Bioactive steroidal glycosides are well-known, although there are limited reports of their interactions with other GPCRs.132 Compound 30 belongs to a class of cyclic 3-alkylopyridinium natural products with a variety of biological activities.133 There have been no reports of further development of these leads. Merck & Co. with Ligand Pharmaceuticals. Piperazinylpiperidine-based ligands were claimed to be CXCR3 antagonists in a series of patents that were published between 2006 and 2009 and originated from Pharmacopeia and ScheringPlough (now part of the Ligand Pharmaceuticals and Merck & Co. groups, respectively). This work has subsequently been described in a series of four medicinal chemistry articles,134−137 leading to advanced lead compounds 1 and 31 (SCH 900875),7 which have shown efficacy in preclinical models of disease (Figure 11). Piperazinyl-piperidine 32 was identified by HTS in a radioligand binding format with 125I-CXCL10 (pKi = 7.0) and was subsequently confirmed as an antagonist of CXCL9, CXCL10, and CXCL11 in hCXCR3-mediated chemotaxis assays (pIC50 = 7.3, 7.7, 7.1, respectively; Figure 11). The hit originated from a combinatorial chemistry library, and its SAR was evaluated extensively by parallel synthesis on solid phase and in solution. Variation of the benzyl group revealed a distinct preference for chlorobenzyl, a motif that has also been seen in other classes of CXCR3 antagonist. Halogenated benzylamides at position R1 also increased affinity (e.g., 33 pKi = 7.4), but they suffered from low to moderate exposure on oral dosing (33 AUC = 0.92 μM·h from 10 mg/kg po dose). Truncation of the N-benzylamide to significantly smaller alkyl groups such as N-methylamide was accompanied by an affinity loss of approximately 1 order of magnitude. The “reversed” amides showed a similar level of affinity, but the resulting aminopyridine motif was considered to be undesirable. Substitution of the piperazinyl-piperidine motif was extensively investigated and was generally well-tolerated with small alkyl groups; however, introduction of a carbonyl group at position R3 or R5 modulated the basicity of the ring nitrogens and caused significant loss of affinity. Alkylation of the piperidine introduced more stereochemical complexity than alkylation of the piperazine, owing to its prochiral carbon at piperidine position 4. R3 was, therefore, the preferred site for introducing a methyl or ethyl group, and (S) substitution was 2905



Perspective

Figure 12. Benzylpiperidines developed as CXCR3 antagonists by Johnson & Johnson and Ono.

Figure 13. Quaternary ammonium derivatives developed as CXCR3 antagonists by VU University Amsterdam, Kyowa Hakko Kogyo with Millenium Pharmaceuticals, Takeda, and GlaxoSmithKline.

does not explicitly claim CXCR3 antagonism. 139 The compounds were shown to be active in rodent models of arthritis, multiple sclerosis, and colitis. Johnson & Johnson. The screening of 256 000 compounds in a CXCR3 calcium flux assay provided a team from

Pharmaceuticals is continuing to progress these compounds, although a reason for program termination is not clear. Daiichi Sankyo. A patent from Daiichi Sankyo describes a series of compounds that show a close resemblance to the Merck & Co. structures (e.g., 37, Figure 11), but the patent 2906



Perspective

Figure 14. Camphor sulfonamides developed as CXCR3 antagonists by GlaxoSmithKline.

43 with an o-bromo or o-iodo substituent converted these compounds from antagonists to full agonists.146 Takeda. One of the earliest CXCR3 antagonists to be reported was 8 (Figure 13). This compound was first described as a CCR5 antagonist with additional but ∼20-fold lower potency at CCR2b.147 Activity in a mouse arthritis model led to the identification of additional CXCR3 activity.148 To our knowledge, no description of CXCR3 SAR in this series has been published, but compound 8 has been used in a variety of animal models of other diseases. Kyowa Hakko Kogyo with Millennium Pharmaceuticals. Three patents from Kyowa Hakko Kogyo and Millennium Pharmaceuticals claim imidazolidines as CXCR3 antagonists.149−151 These compounds have their origins in a series of acetylcholine esterase inhibitors.152−154 Data has been reported from screening at a single concentration in a radioligand binding assay with 125I-CXCL10, which indicates that most compounds claimed in the patents have a pIC50 of greater than 5.0 and provides some limited SAR. There is no conclusive evidence that either company still has an interest in the area, and there has been no further appearance of the compounds in the literature. GlaxoSmithKline. SmithKline Beecham (now part of the GlaxoSmithKline (GSK) group) claimed imidazolium derivatives such as 44 and 45 to be CXCR3 antagonists in 2003.155 This filing describes a calcium flux assay with CXCL10, but potencies are not disclosed for the compounds claimed. The compounds are typically polysubstituted with Cl and/or F atoms and may contain a small substituent on the imidazole, such as a methyl group (Figure 13). No further work has been published on this series. GSK also identified camphor sulfonamide 46 as a CXCR3 antagonist in a high-throughput screen with a calcium flux assay in CHO cells expressing hCXCR3 (Figure 14).156 SAR expansion was carried out by preparing focused libraries of compounds that sequentially altered the pyridine ring, the piperidine ring, and the camphor bicycle and then combining the best groups identified. This led to compounds such as 47, which showed moderate CXCR3 antagonism, good selectivity in a secondary pharmacology screen against 50 targets, and low hERG activity (pIC50 4.2). However, the compound showed inhibition of some P450 isoforms, as well as low solubility (4 μg/mL) and a poor pharmacokinetic profile in rat (Cl = 108 mL/min/kg; t1/2 = 0.5 h, F = 8%). The authors alluded to obtaining improvements in the pharmacokinetic profile through

Johnson & Johnson with a cluster of benzetimide derivatives with CXCR3 activity, including 38, which was the most active (Figure 12).140−142 This activity was confirmed, and new compounds were evaluated using a GTPγ35S binding assay. Despite the similarity to known muscarinic receptor binders, it was established that, while CXCR3 activity resided in both enantiomers of 38, muscarinic activity was limited to a single enantiomer. As the series developed, more advanced compounds tended to show CXCR3 activity only in the enantiomer without muscarinic activity. Exploration of CXCR3 SAR established optimal substitution of the two aryl rings, whereas the imide ring was shown to be tolerant of removal of either carbonyl, or both of them, provided that the nitrogen was then substituted with an acyl or sulfonyl group (e.g., 39). No characterization of the compounds has been disclosed beyond CXCR3 activity and selectivity over a number of muscarinic and other chemokine receptors. Ono. In 2006, Ono Pharmaceutical Company filed a patent claiming spiropiperidines to be CXCR3 antagonists.143 Piperidines were spiroannulated with rings including diketopiperazines, cyclohexanes, and tetrahydropyrimidin-2-ones. Limited amounts of biological data have been published; compounds appear to have potencies in the low micromolar to high nanomolar range in calcium flux assays with hCXCR3. Compound 40 is the most potent compound reported (Figure 12; pIC50 = 6.9). VU University Amsterdam. VU University Amsterdam has investigated a series of compounds containing structural units from both the piperazinyl-piperidine and UCB urea (vide supra) series, hypothesizing that CXCR3 contains a binding pocket accommodating polycycloaliphatic groups.144 The highest activities were achieved with 2-adamantyl and isobornyl derivatives (e.g., 41), although the myrtenyl group that was successful in the urea series showed no advantage in this series (Figure 13). A structurally related series was arrived at by the same group from a screen of a library of 3360 pharmacologically active compounds.145 Compound 42 was identified as a lead, and replacement of the central guanidine with a quaternary ammonium group gave an increase in CXCR3 activity together with reduced lipophilicity. Optimization of the polycyclic aliphatic group, quaternary ammonium, and benzyl led to tool compound 43. Replacement of the m-chloro substituent in 2907



Perspective

Figure 15. Benzimidazoles and benzoxazoles developed as CXCR3 antagonists by Abbott Laboratories and Seoul National University.

Figure 16. Urea derivatives developed as CXCR3 antagonists by UCB.

camphor replacements, but the details of these studies have not been published. Abbott Laboratories. High-throughput screening of the corporate collection at Abbott Laboratories identified benzimi-

dazole 48 as a CXCR3 antagonist with affinity in the low micromolar range in a radioligand binding assay with 125ICXCL10 (Figure 15).157 SAR expansion led to the discovery of compounds with improved affinities and a greater under2908



Perspective

Figure 17. Ergolines developed as CXCR3 antagonists by Novartis.

A third paper in the series describes efforts to replace the problematic lipophilic cyclooctenyl or myrtenyl groups.165 A set of aliphatic heterocycles was prepared, comparable in size and shape to myrtenyl. Acyl piperidines were shown to have good CXCR3 activity and significantly improved physical properties. Molecular modeling suggested that bridged piperidines should more efficiently mimic the shape of myrtenyl group, and, indeed, acylated homotropenes proved to be more active at CXCR3 than the corresponding piperidines. A further bridge was added to the central piperidine to improve metabolic stability (55). This compound had promising in vitro properties, with sufficiently good pharmacokinetics to show an antagonistic effect on CXCR3 receptor internalization in vivo. In the most recent paper in the series, efforts to combine the heterocycle with the homotropene are described.166 Investigation of a range of alternative aromatic heterocycles led to identification of substituted quinolines as potent replacements for the aryl urea found in the original hit. Compound 56 was selected for further profiling and was shown to have good in vivo pharmacokinetic properties in the mouse, with doseproportional exposure up to 100 mg/kg and a sufficient half-life for exposure at 24 h after dosing. In a mouse CXCR3 receptor internalization assay, complete inhibition of CXCR3 internalization ex vivo was observed 9 h postdosing at 30 mg/kg and at 24 h postdosing at 100 mg/kg. No further progression of this series has been reported. Novartis. High-throughput screening of the Novartis compound collection identified lysergic acid derivative 57 with binding pIC50 = 7.3 at hCXCR3 (Figure 17).167,168 Despite its close structural similarity to LSD, this compound was shown to have unexpectedly high selectivity over other GPCRs.169 The different biological profile is explained in terms of lack of a basic center and an alternative conformation of the tetracyclic core. The human microsomal Clint of 57 was reported as 60 μL/min/mg with no evidence for cleavage of the urea, which would have been expected to lead to metabolites with undesirable safety profiles. Limited compounds were made to explore SAR around the central core and urea with no improvements found over the original hit. Replacement of the N,N-diethyl amide with a pyrrolidine amide gave a 10-fold improvement in potency. Substitution of the indole nitrogen with lipophilic substituents

standing of the physicochemical properties of the series. Examples containing an acyl group at the 2-position of the benzimidazole core were generally found to have low solubility (e.g., 48 solubility = 4.5 μM), and reduction of this carbonyl group to the carbinol, or replacement by sulfoxide, resulted in reduced affinity. However, replacement by an imino group afforded both improved activity and good solubility. Very large substituents were well-tolerated at R3, such as alkylamidetethered bicyclic aromatics.158 However, these larger groups were prone to rapid metabolism in mouse liver microsomes and compounds with N-methyl substitution generally showed superior ligand efficiencies. Compound 49 showed good solubility and a favorable pharmacokinetic profile in the mouse (F = 57%; t1/2 = 4.9 h). Further progression of the series has not been reported. Seoul National University. A patent from Seoul National University claims compounds 50 and 51 to be antagonists of CXCR3/CXCL10 for the treatment of osteolytic bone metastasis (Figure 15).159 No specific data for interaction with CXCR3 is given, but it seems likely that this is the therapeutic target.160 UCB. UCB screened 15 000 compounds in a CXCR3 calcium flux assay and confirmed hits with a GTPγ35S binding assay. Compound 52 was selected for further progression based on its molecular properties and similarity to known GPCR ligands (Figure 16).161,162 Despite its high lipophilicity, 52 was deemed to be a good starting point due to the possibility of rapid analogue synthesis. Initial attempts to improve properties gave some improvement in potency and reduction in lipophilicity and consequently led to improvements in solubility and Clint, e.g., 53. Modification or replacement of the urea generally led to loss of activity, whereas quaternary ammonium derivatives were potent in chemotaxis assays but showed poor permeability. A serendipitous finding from an unexpected reaction product led to the identification of cyclic urea replacements.163,164 These heterocycles showed modest CXCR3 activity and high lipophilicity but good in vitro stability and low CYP inhibition, and an advanced compound 54 was shown to have promising mouse in vivo pharmacokinetics (Cl = 2.8 mL/min/kg, AUC = 10 800 ng·h/mL, t1/2 = 5.4 h from a 10 mg/kg po dose in the mouse). 2909



Perspective

Figure 18. Miscellaneous chemotypes developed as CXCR3 antagonists by Galderma Research and Development, Pharmadesign, and University of Zurich. Compound 59 was developed as a CXCR1/CXCR2 antagonist by Schering Plough.

■

led to a loss in potency, but polar substituents not only retained potency in the binding and cellular assays but also offered improved potency in a whole blood assay.170 Compound 58 showed favorable pharmacokinetic properties in vivo (Cl = 13 mL/min/kg, t1/2 = 8.9 h, AUC = 2700 nM·h, F = 97% from a 3 mg/kg po dose in the rat) and is described as a promising tool compound with which to explore the role of CXCR3 in disease models. Galderma Research and Development. In 2013, Galderma Research and Development filed two patents claiming a series of diaminocyclobutenediones to be CXCR1 and CXCR2 antagonists.171,172 These compounds are structurally related to the clinical CXCR1/CXCR2 antagonist 59 (SCH 527123),173 but compound 60 was found to have a high functional potency against CXCR3 (pIC50 = 8.9) as well as submicromolar potency against CXCR1, CXCR2, CCR4, CCR6, and CCR7 (Figure 18). Compound 60 was also found to be moderately stable in rat hepatocytes (t1/2 = 300 min vs 900 min for the clinical CXCR1/CXCR2 antagonist). Polypharmacology involving CXCR3 may be beneficial in some settings,17 but it is unclear whether compounds such as 60, which potently block so many receptors, may be of therapeutic benefit or whether the diaminocyclobutenediones could be developed into more selective CXCR3 ligands. Compound 60 was the only example that showed higher potency at CXCR3 than at CXCR1 and CXCR2, and the majority of other compounds claimed in these patents generally have considerably lower activity at CXCR3. Pharmadesign. Pharmadesign have claimed a small set of compounds as dual antagonists of CXCR3 and CCR5.174 Compound 61 appears to show dose-related chemotaxis in a CXCR3 assay using CXCL10 with 50% inhibition between 50 and 20 μM (Figure 18). University of Zurich. Luedtke et al. reported inhibition of metastasis in a mouse model using a zinc phthalocyanine 62 (Figure 18).175 This is speculated to be caused by inhibiting the binding of CXCL10 to CXCR3, and it was noted that 62 appears to cause CXCR3 internalization without receptor activation. Follow-up studies have not been reported.

CONCLUSIONS

It has been 14 years since the first patent claiming small molecule CXCR3 antagonists was published. Since that time, many classes of CXCR3 antagonist have been identified, and the pace of emerging CXCR3 biology continues to increase. Despite this, there are few publications that describe the evaluation of CXCR3 antagonists in models of disease in vivo. Further investigations into target validation are warranted, and this will require additional investment in the characterization of small molecule antagonists and the identification of new compounds with improved profiles. The challenges associated with developing chemokine receptor antagonists have been well-documented, and, to date, only one small molecule CXCR3 antagonist has entered clinical trials. Compound 2 was evaluated for the treatment of psoriasis but exhibited low efficacy, possibly owing to variable exposure. To our knowledge, there is no evidence that suggests that the lack of efficacy was mechanism-based, and the two successful examples of drugging chemokine receptors with small molecule antagonists (the marketed small molecules 3 and 4, which selectively target CCR5 and CXCR4, respectively) inspire further attempts to develop CXCR3 antagonists for clinical evaluation. Further investigations with emerging noninflammatory indications are of particular interest, given that modulation of inflammatory disease with chemokine receptor antagonists has historically been unsuccessful. The piperazinyl-piperidines and (aza)quinazolinones have been the most extensively evaluated classes of CXCR3 antagonists, and both have demonstrated efficacy in preclinical models of inflammation (compounds 1 and 2, respectively). Efficacy data for antagonists from other chemical classes has generally not been reported, but examples from several series have shown clear SAR in vitro and at least some level of oral exposure when dosed in rodents. These may be suitable for further development and include the aryl sulfonamides (e.g., 16), aryl piperazines (e.g., 21 and 23), camphor sulfonamides (e.g., 47), benzimidazoles (e.g., 49), ureas (e.g., 56, which has been evaluated in vivo for CXCR3 internalization), and ergolines (e.g., 58). However, these classes may require significant improvements in both efficacy and in vivo 2910



Perspective

pharmacokinetics in order to definitively test translation in in vivo models of disease. The hits for the majority of these series were identified by HTS of large compound collections, but complementary techniques, such as virtual and fragment screening, may enable the identification of new compound classes with developable properties. Structure-based drug design with GPCRs, including chemokine receptors, is a rapidly maturing field and may offer rational approaches to ligand development. Indeed, initial examples of virtual screening against models of CXCR3 have been successful in both identifying new chemotypes and correctly predicting their selectivity against other chemokine receptors. Molecular modeling has also been used in combination with site-directed mutagenesis to demonstrate that the 8-azaquinazolinone (7) occupies a part of the large TM binding site that is largely distinct from the piperazinylpiperidine (6) binding site. The range of available binding modes may be the origin of the biased signaling profiles that CXCR3 antagonists exhibit. Further advances with CXCR3 crystallography and improvements in the quality of CXCR3 models may enable small molecules to be designed with defined pharmacological properties and superior safety and efficacy profiles. Furthermore, there is increasing evidence from X-ray crystallography to show that GPCR antagonists can bind in unexpected locations that are remote from the “TM binding site”. For example, BPTU has been observed to bind on the outside of the helices of the class A receptor, P2Y1,176 and an unusually deep pocket has been identified the TM domain of the class B receptor, CRF1,177 suggesting that further studies with CXCR3 antagonists may also identify new druggable binding sites. In short, there remain many opportunities to further develop small molecule CXCR3 antagonists from known chemical classes and to identify new chemotypes through complementary screening approaches. The development of such compounds has slowed in recent years, but further investment in the rational design of new molecules is now required for establishing models in emerging areas of disease and for determining proof of concept in the clinic.

■

working in the respiratory, inflammation, and autoimmunity research area. Prior to this, she worked at AstraZeneca in the UK for eight years. She obtained a DPhil in synthetic organic chemistry at the University of Oxford with Professor Sir Jack Baldwin and followed this with postdoctoral research in the group of Professor Robert M. Williams at Colorado State University.

■

ACKNOWLEDGMENTS This work was funded by Heptares Therapeutics and AstraZeneca. The authors wish to thank Francesca Deflorian for creating the helix box diagrams and sequence similarity chart, and Tomas Drmota, Mark Furber, Magnus Munck af Rosenschöld, Matthew Perry, Peter Sjö, Fiona Marshall, Rob Cooke, Rebecca Nonoo, Dahlia Weiss, and Francesca Deflorian for critically reading the manuscript and providing helpful comments.

■

ABBREVIATIONS USED ARDS, acute respiratory distress syndrome; ECL, extracellular loop; HLM, human liver microsomes; IP-10, interferon-γinduced protein 10; I-TAC, interferon-inducible T-cell α chemoattractant; MIG, monokine induced by γ-interferon; PBMC, peripheral blood mononuclear cells; RBL, red basophilic leukemia; SPA, scintillation proximity assay; TM, transmembrane or transmembrane helix

■

REFERENCES

(1) Loetscher, M.; Gerber, B.; Loetscher, P.; Jones, S. A.; Piali, L.; Clark-Lewis, I.; Baggiolini, M.; Moser, B. Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated Tlymphocytes. J. Exp. Med. 1996, 184, 963−969. (2) Wijtmans, M.; de Esch, I. P. J.; Leurs, R. Therapeutic targeting of the CXCR3 receptor. In Chemokine Receptors as Drug Targets; Smit, M. J.; Lira, S. A.; Leurs, R., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2011; pp 301−322. (3) Wijtmans, M.; Scholten, D.; Mooij, W.; Smit, M. J.; de Esch, I. J. P.; de Graaf, C.; Leurs, R. Exploring the CXCR3 chemokine receptor with small-molecule antagonists and agonists. Top. Med. Chem. 2014, 14, 119−185. (4) Wijtmans, M.; Verzijl, D.; Leurs, R.; de Esch, I. P. J.; Smit, M. J. Towards small-molecule CXCR3 ligands with clinical potential. ChemMedChem 2008, 3, 861−872. (5) Van Raemdonck, K.; Van den Steen, P. E.; Liekens, S.; Van Damme, J.; Struyf, S. CXCR3 ligands in disease and therapy. Cytokine Growth Factor Rev. 2015, 26, 311−327. (6) Jenh, C.-H.; Cox, M. A.; Cui, L.; Reich, E.-P.; Sullivan, L.; Chen, S.-C.; Kinsley, D.; Qian, S.; Kim, H. K.; Rosenblum, S.; Kozlowski, J.; Fine, J. S.; Zavodny, P. J.; Lundell, D. A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection. BMC Immunol. 2012, 13, 2. (7) Poulet, F. M.; Penraat, K.; Collins, N.; Evans, E.; Thackaberry, E.; Manfra, D.; Engstrom, L.; Geissler, R.; Geraci-Erck, M.; Frugone, C.; Abutarif, M.; Fine, J. S.; Peterson, B. L.; Cummings, B. S.; Johnson, R. Drug-induced hemolytic anemia and thrombocytopenia associated with alterations of cell membrane lipids and acanthocyte formation. Toxicol. Pathol. 2010, 38, 907−922. (8) Berry, K.; Friedrich, M.; Kersey, K.; Stempien, M. J.; Wagner, F.; van Lier, J. J.; Sabat, R.; Wolk, K. Evaluation of T0906487, a CXCR3 antagonist, in a phase 2a psoriasis trial. Inflamm. Res. 2004, 53 (Suppl. 3), S222. (9) Tonn, G. R.; Wong, S. G.; Wong, S. C.; Johnson, M. G.; Ma, J.; Cho, R.; Floren, L. C.; Kersey, K.; Berry, K.; Marcus, A. P.; Wang, X.; Van Lengerich, B.; Medina, J. C.; Pearson, P. G.; Wong, B. K. An inhibitory metabolite leads to dose- and time-dependent pharmacokinetics of (R)-N-{1-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluorome-

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +44 (0) 1707 358 693. Notes

The authors declare no competing financial interest. Biographies Stephen P. Andrews is a Principal Scientist in the Medicinal Chemistry group at Heptares Therapeutics. Since 2008, he has applied Heptares’ structure-based design platform to drug discovery with “intractable” GPCRs and has developed hits and leads for various peptide, orphan, purine, and chemokine receptors. Steve coinvented Heptares’ first clinical candidate, the adenosine A2A receptor antagonist HTL-1071. This work became the first published example of structurebased design using GPCR X-ray structures during lead optimization. Steve obtained his Ph.D. from Cambridge University, where he completed the first total synthesis of thapsigargin with Professor Steve Ley CBE FRS, and undertook his postdoctoral studies at ETH-Zürich with Professor Erick Carreira. Rhona J. Cox has been Associate Principal Scientist in Medicinal Chemistry at AstraZeneca in Gothenburg, Sweden, since 2011, 2911



Perspective

thoxy-phenyl)-acetamide (AMG 487) in human subjects after multiple dosing. Drug. Metab. Dispos. 2009, 37, 502−513. (10) Henne, K. R.; Tran, T. B.; VandenBrink, B. M.; Rock, D. A.; Aidasani, D. K.; Subramanian, R.; Mason, A. K.; Stresser, D. M.; Teffera, Y.; Wong, S. G.; Johnson, M. G.; Chen, X.; Tonn, G. R.; Wong, B. K. Sequential metabolism of AMG 487, a novel CXCR3 antagonist, results in formation of quinone reactive metabolites that covalently modify CYP3A4 Cys239 and cause time-dependent inhibition of the enzyme. Drug Metab. Dispos. 2012, 40, 1429−1440. (11) Mayer, L.; Sandborn, W. J.; Stepanov, Y.; Geboes, K.; Hardi, R.; Yellin, M.; Tao, X.; Xu, L. A.; Salter-Cid, L.; Gujrathi, S.; Aranda, R.; Luo, A. Y. Anti-IP-10 antibody (BMS-936557) for ulcerative colitis: a phase II randomised study. Gut 2014, 63, 442−450. (12) Yellin, M.; Paliienko, I.; Balanescu, A.; Ter-Vartanian, S.; Tseluyko, V.; Xu, L.-A.; Tao, X.; Cardarelli, P. M.; LeBlanc, H.; Nichol, G.; Ancuta, C.; Chirieac, R.; Luo, A. A phase II, randomized, doubleblind, placebo- controlled study evaluating the efficacy and safety of MDX- 1100, a fully human anti- CXCL10 monoclonal antibody, in combination with methotrexate in patients with rheumatoid arthritis. Arthritis Rheum. 2012, 64, 1730−1739. (13) Bachelerie, F.; Ben-Baruch, A.; Burkhardt, A. M.; Combadiere, C.; Farber, J. M.; Graham, G. J.; Horuk, R.; Sparre-Ulrich, A. H.; Locati, M.; Luster, A. D.; Mantovani, A.; Matsushima, K.; Murphy, P. M.; Nibbs, R.; Nomiyama, H.; Power, C. A.; Proudfoot, A. E. I.; Rosenkilde, M. M.; Rot, A.; Sozzani, S.; Thelen, M.; Yoshie, O.; Zlotnik, A. International Union of Basic and Clinical Pharmacology. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. Pharmacol. Rev. 2014, 66, 1−79. (14) Pease, J.; Horuk, R. Chemokine receptor antagonists. J. Med. Chem. 2012, 55, 9363−9392. (15) Wijtmans, M.; Scholten, D. J.; de Esch, I. J. P.; Smit, M. J.; Leurs, R. Therapeutic targeting of chemokine receptors by small molecules. Drug Discovery Today: Technol. 2012, 9, e229−e236. (16) Schall, T. J.; Proudfoot, A. E. I. Overcoming hurdles in developing successful drugs targeting chemokine receptors. Nat. Rev. Immunol. 2011, 11, 355−363. (17) Horuk, R. Chemokine receptor antagonists: overcoming developmental hurdles. Nat. Rev. Drug Discovery 2009, 8, 23−33. (18) Solari, R.; Pease, J.; Begg, M. Chemokine Receptors as Therapeutic Targets: Why Aren’t There More Drugs? Eur. J. Pharmacol. 2015, 746, 363−367. (19) Wood, A.; Armour, D. The discovery of the CCR5 receptor antagonist, UK-427,857, a new agent for the treatment of HIV infection and AIDS. Prog. Med. Chem. 2005, 43, 239−271. (20) Micallef, I. N.; Stiff, P. J.; DiPersio, J. F.; Maziarz, R. T.; McCarty, J. M.; Bridger, G.; Calandra, G. Successful stem cell remobilization using plerixafor (Mozobil) plus granulocyte colonystimulating factor in patients with non-Hodgkin lymphoma: results from the plerixafor NHL phase 3 study rescue protocol. Biol. Blood Marrow Transplant. 2009, 15, 1578−1586. (21) Luster, A. D. Chemokines − chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 1998, 338, 436−445. (22) Charo, I. F.; Ransohoff, R. M. The many roles of chemokines and chemokine receptors in inflammation. N. Engl. J. Med. 2006, 354, 610−621. (23) Farber, J. M. Mig and IP-10: CXC chemokines that target lymphocytes. J. Leukocyte Biol. 1997, 61, 246−257. (24) Cole, K. E.; Strick, C. A.; Paradis, T. J.; Ogborne, K. T.; Loetscher, M.; Gladue, R. P.; Lin, W.; Boyd, J. G.; Moser, B.; Wood, D. E.; Sahagan, B. G.; Neote, K. Interferon-inducible T cell alpha chemoattractant (I-TAC): A novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 1998, 187, 2009−2021. (25) Murphy, P. J.; Baggiolini, M.; Charo, I. F.; Hébert, C. A.; Horuk, R.; Matsushima, K.; Miller, L. H.; Oppenheim, J. J.; Power, C. A. International Union of Pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 2000, 52, 145−176.

(26) Pease, J. E.; Williams, T. J. The attraction of chemokines as a target for specific anti-inflammatory therapy. Br. J. Pharmacol. 2006, 147, S212−S221. (27) Meiser, A.; Mueller, A.; Wise, E. L.; McDonagh, E. M.; Petit, S. J.; Saran, N.; Clark, P. C.; Williams, T. J.; Pease, J. E. The chemokine receptor CXCR3 is degraded following internalization and is replenished at the cell surface by de novo synthesis of receptor. J. Immunol. 2008, 180, 6713−6724. (28) Groom, J. R.; Luster, A. D. CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol. Cell Biol. 2011, 89, 207−215. (29) Watts, A. O.; Scholten, D. J.; Heitman, L. H.; Vischer, H. F.; Leurs, R. Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with Gi-protein pathway activation. Biochem. Biophys. Res. Commun. 2012, 419, 412− 418. (30) García-López, M. Á .; Sánchez-Madrid, F.; Rodríguez-Frade, J. M.; Mellado, M.; Acevedo, A.; García, M. I.; Albar, J. P.; Martínez-A, C.; Marazuela, M. CXCR3 chemokine receptor distribution in normal and inflamed tissues: expression on activated lymphocytes, endothelial cells, and dendritic cells. Lab. Invest. 2001, 81, 409−418. (31) Muehlinghaus, G.; Cigliano, L.; Huehn, S.; Peddinghaus, A.; Leyendeckers, H.; Hauser, A. E.; Hiepe, F.; Radbruch, A.; Arce, S.; Manz, R. A. Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cells. Blood 2005, 105, 3965−3971. (32) Goldberg, S. H.; van der Meer, P.; Hesselgesser, J.; Jaffer, S.; Kolson, D. L.; Albright, A. V.; González-Scarano, F.; Lavi, E. CXCR3 expression in human central nervous system diseases. Neuropathol. Appl. Neurobiol. 2001, 27, 127−138. (33) Billottet, C.; Quemener, C.; Bikfalvi, A. CXCR3, a double-edged sword in tumor progression and angiogenesis. Biochim. Biophys. Acta, Rev. Cancer 2013, 1836, 287−295. (34) Lasagni, L.; Francalanci, M.; Annunziato, F.; Lazzeri, E.; Giannini, S.; Cosmi, L.; Sagrinati, C.; Mazzinghi, B.; Orlando, C.; Maggi, E.; Marra, F.; Romagnani, S.; Serio, M.; Romagnani, P. An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4. J. Exp. Med. 2003, 197, 1537− 1549. (35) Ehlert, J. E.; Addison, C. A.; Burdick, M. D.; Kunkel, S. L.; Strieter, R. M. Identification and partial characterization of a variant of human CXCR3 generated by posttranscriptional exon skipping. J. Immunol. 2004, 173, 6234−6240. (36) Lacotte, S.; Brun, S.; Muller, S.; Dumortier, H. CXCR3, inflammation, and autoimmune diseases. Ann. N. Y. Acad. Sci. 2009, 1173, 310−317. (37) Mohan, K.; Issekutz, T. B. Blockade of chemokine receptor CXCR3 inhibits T cell recruitment to inflamed joints and decreases the severity of adjuvant arthritis. J. Immunol. 2007, 179, 8463−8469. (38) Ruth, J. H.; Rottman, J. B.; Katschke, K. J., Jr.; Qin, S.; Wu, L.; LaRosa, G.; Ponath, P.; Pope, R. M.; Koch, A. E. Selective lymphocyte chemokine receptor expression in the rheumatoid joint. Arthritis Rheum. 2001, 44, 2750−2760. (39) Patel, D. D.; Zachariah, J. P.; Whichard, L. P. CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin. Immunol. 2001, 98, 39−45. (40) Qin, S.; Rottman, J. B.; Myers, P.; Kassam, N.; Weinblatt, M.; Loetscher, M.; Koch, A. E.; Moser, B.; Mackay, C. R. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 1998, 101, 746−754. (41) Sporici, R.; Issekutz, T. B. CXCR3 blockade inhibits T-cell migration into the CNS during EAE and prevents development of adoptively transferred, but not actively induced, disease. Eur. J. Immunol. 2010, 40, 2751−2761. (42) Sørensen, T. L.; Trebst, C.; Kivisäkk, P.; Klaege, K. L.; Majmudar, A.; Ravid, R.; Lassmann, H.; Olsen, D. B.; Strieter, R. M.; Ransohoff, R. M.; Sellebjerg, F. Multiple sclerosis: a study of CXCL10 2912



Perspective

and CXCR3 co-localization in the inflamed central nervous system. J. Neuroimmunol. 2002, 127, 59−68. (43) Balashov, K. E.; Rottman, J. B.; Weiner, H. L.; Hancock, W. W. CCR5+ and CXCR3+ T cells are increased in multiple sclerosis and their ligands MIP-1α and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 6873−6878. (44) Chami, B.; Yeung, A. W. S.; van Vreden, C.; King, N. J. C.; Bao, S. The role of CXCR3 in DSS-induced colitis. PLoS One 2014, 9, e101622. (45) Singh, U. P.; Singh, S.; Taub, D. D.; Lillard, J. W., Jr. Inhibition of IFN-γ-inducible protein-10 abrogates colitis in IL-10−/− mice. J. Immunol. 2003, 171, 1401−1406. (46) Moser, K.; Kalies, K.; Szyska, M.; Humrich, J. Y.; Amann, K.; Manz, R. A. CXCR3 promotes the production of IgG1 autoantibodies but is not essential for the development of lupus nephritis in NZB/ NZW mice. Arthritis Rheum. 2012, 64, 1237−1246. (47) Enghard, P.; Humrich, J. Y.; Rudolph, B.; Rosenberger, S.; Biesen, R.; Kuhn, A.; Manz, R.; Hiepe, F.; Radbruch, A.; Burmester, G.-R.; Riemekasten, G. CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients. Arthritis Rheum. 2009, 60, 199−206. (48) Flier, J.; Boorsma, D. M.; van Beek, P. J.; Nieboer, C.; Stoof, T. J.; Willemze, R.; Tensen, C. P. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J. Pathol. 2001, 194, 398−405. (49) Costa, C.; Rufino, R.; Traves, S. L.; Lapa e Silva, J. R.; Barnes, P. J.; Donnelly, L. E. CXCR3 and CCR5 chemokines in induced sputum from patients with COPD. Chest 2008, 133, 26−33. (50) Saetta, M.; Mariani, M.; Panina-Bordignon, P.; Turato, G.; Buonsanti, C.; Baraldo, S.; Bellettato, C. M.; Papi, A.; Corbetta, L.; Zuin, R.; Sinigaglia, F.; Fabbri, L. M. Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 2002, 165, 1404−1409. (51) Rottman, J. B.; Smith, T. L.; Ganley, K. G.; Kikuchi, T.; Krueger, J. G. Potential role of the chemokine receptors CXCR3, CCR4, and the integrin αEβ7 in the pathogenesis of psoriasis vulgaris. Lab. Invest. 2001, 81, 335−347. (52) Belperio, J. A.; Keane, M. P.; Burdick, M. D.; Lynch, J. P., III; Zisman, D. A.; Xue, Y. Y.; Li, K.; Ardehali, A.; Ross, D. J.; Strieter, R. M. Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection. J. Immunol. 2003, 171, 4844−4852. (53) Melter, M.; Exeni, A.; Reinders, M. E. J.; Fang, J. C.; McMahon, G.; Ganz, P.; Hancock, W. W.; Briscoe, D. M. Expression of the chemokine receptor CXCR3 and its ligand IP-10 during human cardiac allograft rejection. Circulation 2001, 104, 2558−2564. (54) Hancock, W. W.; Lu, B.; Gao, W.; Csizmadia, V.; Faia, K.; King, J. A.; Smiley, S. T.; Ling, M.; Gerard, N. P.; Gerard, C. Requirement of the chemokine receptor CXCR3 for acute allograft rejection. J. Exp. Med. 2000, 192, 1515−1519. (55) Duffner, U.; Lu, B.; Hildebrandt, G. C.; Teshima, T.; Williams, D. L.; Reddy, P.; Ordemann, R.; Clouthier, S. G.; Lowler, K.; Liu, C.; Gerard, C.; Cooke, K. R.; Ferrara, J. L. M. Role of CXCR3-induced donor T-cell migration in acute GVHD. Exp. Hematol. 2003, 31, 897− 902. (56) Brightling, C. E.; Ammit, A. J.; Kaur, D.; Black, J. L.; Wardlaw, A. J.; Hughes, J. M.; Bradding, P. The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am. J. Respir. Crit. Care Med. 2005, 171, 1103−1108. (57) Frigerio, S.; Junt, T.; Lu, B.; Gerard, C.; Zumsteg, U.; Holländer, G. A.; Piali, L. β cells are responsible for CXCR3-mediated T-cell infiltration in insulitis. Nat. Med. 2002, 8, 1414−1420. (58) Krauthausen, M.; Kummer, M. P.; Zimmermann, J.; ReyesIrisarri, R.; Terwel, D.; Bulic, B.; Heneka, M. T.; Müller, M. CXCR3 promotes plaque formation and behavioral deficits in an Alzheimer’s disease model. J. Clin. Invest. 2015, 125, 365−378. (59) Chiang, S.; Ubogu, E. E. The role of chemokines in GuillainBarré syndrome. Muscle Nerve 2013, 48, 320−330.

(60) Ichikawa, A.; Kuba, K.; Morita, M.; Chida, S.; Tezuka, H.; Hara, H.; Sasaki, T.; Ohteki, T.; Ranieri, V. M.; dos Santos, C. C.; Kawaoka, Y.; Akira, S.; Luster, A. D.; Lu, B.; Penninger, J. M.; Uhlig, S.; Slutsky, A. S.; Imai, Y. CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin. Am. J. Respir. Crit. Care Med. 2013, 187, 65−77. (61) Breser, M. L.; Motrich, R. D.; Sanchez, L. R.; Mackern-Oberti, J. P.; Rivero, V. E. Expression of CXCR3 on specific T cells is essential for homing to the prostate gland in an experimental model of chronic prostatitis/chronic pelvic pain syndrome. J. Immunol. 2013, 190, 3121−3133. (62) Pradelli, E.; Karimdjee-Soilihi, B.; Michiels, J.-F.; Ricci, J.-E.; Millet, M.-A.; Vandenbos, F.; Sullivan, T. J.; Collins, T. L.; Johnson, M. G.; Medina, J. C.; Kleinerman, E. S.; Schmid-Alliana, A.; SchmidAntomarchi, H. Antagonism of chemokine receptor CXCR3 inhibits osteosarcoma metastasis to lungs. Int. J. Cancer 2009, 125, 2586−2594. (63) Cambien, B.; Karimdjee, B. F.; Richard-Fiardo, P.; Bziouech, H.; Barthel, R.; Millet, M. A.; Martini, V.; Birnbaum, D.; Scoazec, J. Y.; Abello, J.; Al Saati, T.; Johnson, M. G.; Sullivan, T. J.; Medina, J. C.; Collins, T. L.; Schmid-Alliana, A.; Schmid-Antomarchi, H. Organspecific inhibition of metastatic colon carcinoma by CXCR3 antagonism. Br. J. Cancer 2009, 100, 1755−1764. (64) Walser, T. C.; Rifat, S.; Ma, X.; Kundu, N.; Ward, C.; Goloubeva, O.; Johnson, M. G.; Medina, J. C.; Collins, T. L.; Fulton, A. M. Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer. Cancer Res. 2006, 66, 7701−7707. (65) Vandercappellen, J.; Van Damme, J.; Struyf, S. The role of CXC chemokines and their receptors in cancer. Cancer Lett. 2008, 267, 226−244. (66) Cherezov, V. Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin. Struct. Biol. 2011, 21, 559−566. (67) Chun, E.; Thompson, A. A.; Liu, W.; Roth, C. B.; Griffith, M. T.; Katritch, V.; Kunken, J.; Xu, F.; Cherezov, V.; Hanson, M. A.; Stevens, R. C. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 2012, 20, 967−976. (68) Robertson, N.; Jazayeri, A.; Errey, J.; Baig, A.; Hurrell, E.; Zhukov, A.; Langmead, C. J.; Weir, M.; Marshall, F. H. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 2011, 60, 36−44. (69) Shonberg, J.; Kling, R. C.; Gmeiner, P.; Löber, S. GPCR crystal structures: medicinal chemistry in the pocket. Bioorg. Med. Chem. 2015, 23, 3880−3906. (70) Kobilka, B. The structural basis of G protein-coupled receptor signaling (Nobel lecture). Angew. Chem., Int. Ed. 2013, 52, 6380−6388. (71) Lefkowitz, R. J. A brief history of G protein coupled receptors (Nobel lecture). Angew. Chem., Int. Ed. 2013, 52, 6366−6378. (72) Andrews, S. P.; Brown, G. A.; Christopher, J. A. Structure-based and fragment-based GPCR drug discovery. ChemMedChem 2014, 9, 256−275. (73) Congreve, M. C.; Dias, J. M.; Marshall, F. H. Structure based drug design for G protein-coupled receptors. Prog. Med. Chem. 2014, 53, 1−63. (74) Bortolato, A.; Doré, A. S.; Hollenstein, K.; Tehan, B. G.; Mason, J. S.; Marshall, F. H. Structure of class B GPCRs: new horizons for drug discovery. Br. J. Pharmacol. 2014, 171, 3132−3145. (75) Rosenkilde, M. M.; Benned-Jensen, T.; Frimurer, T. M.; Schwartz, T. W. The minor binding pocket: a major player in 7TM receptor activation. Trends Pharmacol. Sci. 2010, 31, 567−574. (76) Rajagopalan, L.; Rajarathnam, K. Structural basis of chemokine receptor function−a model for binding affinity and ligand selectivity. Biosci. Rep. 2006, 26, 325−339. (77) Tehan, B. G.; Bortolato, A.; Blaney, F. E.; Weir, M. P.; Mason, J. S. Unifying family A GPCR theories of activation. Pharmacol. Ther. 2014, 143, 51−60. (78) Scholten, D. J.; Canals, M.; Maussang, D.; Roumen, I.; Smit, M. J.; Wijtmans, M.; de Graaf, C.; Vischer, H. F.; Leurs, R. Pharmacological modulation of chemokine receptor function. Br. J. Pharmacol. 2012, 165, 1617−1643. 2913



Perspective

(79) Raucci, R.; Colonna, G.; Giovane, A.; Castello, G.; Costantini, S. N-terminal region of human chemokine receptor CXCR3: structural analysis of CXCR3(1−48) by experimental and computational studies. Biochim. Biophys. Acta, Proteins Proteomics 2014, 1844, 1868−1880. (80) Palladino, P.; Portella, L.; Colonna, G.; Raucci, R.; Saviano, G.; Rossi, F.; Napolitano, M.; Scala, S.; Castello, G.; Costantini, S. The Nterminal region of CXCL11 as structural template for CXCR3 molecular recognition: synthesis, conformational analysis, and binding studies. Chem. Biol. Drug Des. 2012, 80, 254−265. (81) Qin, L.; Kufareva, I.; Holden, L. G.; Wang, C.; Zheng, Y.; Zhao, C.; Fenalti, G.; Wu, H.; Han, G. W.; Cherezov, V.; Abagyan, R.; Stevens, R. C.; Handel, T. M. Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine. Science 2015, 347, 1117−1122. (82) Wu, B.; Chien, E. Y. T.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. J.; Kuhn, P.; Handel, T. M.; Cherezov, V.; Stevens, R. C. Structures of the CXCR4 chemokine receptor in complex with small molecule and cyclic peptide antagonists. Science 2010, 330, 1066−1071. (83) Tan, Q.; Zhu, Y.; Li, J.; Chen, Z.; Han, G. W.; Kufareva, I.; Li, T.; Ma, L.; Fenalti, G.; Li, J.; Zhang, W.; Xie, X.; Yang, H.; Jiang, H.; Cherezov, V.; Liu, H.; Stevens, R. C.; Zhao, Q.; Wu, B. Structure of the CCR5 chemokine receptor−HIV entry inhibitor maraviroc complex. Science 2013, 341, 1387−1390. (84) Hopkins, A. L.; Keserü, G. M.; Leeson, P. D.; Rees, D. C.; Reynolds, C. H. The role of ligand efficiency metrics in drug discovery. Nat. Rev. Drug Discovery 2014, 13, 105−121. (85) Fricker, S. P.; Metz, M. Chemokine receptor modeling: an interdisciplinary approach to drug design. Future Med. Chem. 2014, 6, 91−114. (86) Kufareva, I.; Abagyan, R.; Handel, T. M. Role of 3D structures in understanding, predicting and designing molecular interactions in the chemokine receptor family. Top. Med. Chem. 2014, 14, 41−85. (87) Huang, D.; Gu, Q.; Ge, H.; Ye, J.; Salam, N. K.; Hagler, A.; Chen, H.; Xu, J. On the value of homology models for virtual screening: discovering hCXCR3 antagonists by pharmacophore-based and structure-based approaches. J. Chem. Inf. Model. 2012, 52, 1356− 1366. (88) Schmidt, D.; Bernat, V.; Brox, R.; Tschammer, N.; Kolb, P. Identifying modulators of CXC receptors 3 and 4 with tailored selectivity using multi-target docking. ACS Chem. Biol. 2015, 10, 715− 724. (89) Scholten, D. J.; Roumen, L.; Wijtmans, M.; Verkade-Vreeker, M. C. A.; Custers, H.; Lai, M.; de Hooge, D.; Canals, M.; de Esch, I. J. P.; Smit, M. J.; de Graaf, C.; Leurs, R. Identification of overlapping but differential binding sites for the high-affinity CXCR3 antagonists NBI74330 and VUF11211. Mol. Pharmacol. 2014, 85, 116−126. (90) Nedjai, B.; Viney, J. M.; Li, H.; Hull, C.; Anderson, C. A.; Horie, T.; Horuk, R.; Vaidehi, N.; Pease, J. E. CXCR3 antagonist VUF10085 binds to an intrahelical site distinct from that of the broad spectrum antagonist TAK-779. Br. J. Pharmacol. 2015, 172, 1822−1833. (91) Verzijl, D.; Storelli, S.; Scholten, D. J.; Bosch, L.; Reinhart, T. A.; Streblow, D. N.; Tensen, C. P.; Fitzsimons, C. P.; Zaman, G. J. R.; Pease, J. E.; de Esch, I. J. P.; Smit, M. J.; Leurs, R. Noncompetitive antagonism and inverse agonism as mechanism of action of nonpeptidergic antagonists at primate and rodent CXCR3 chemokine receptors. J. Pharmacol. Exp. Ther. 2008, 325, 544−555. (92) Bernat, V.; Brox, R.; Heinrich, M. R.; Auberson, Y. P.; Tschammer, N. Ligand-biased and probe-dependent modulation of chemokine receptor CXCR3 signaling by negative allosteric modulators. ChemMedChem 2015, 10, 566−574. (93) Bernat, V.; Admas, T. H.; Brox, R.; Heinemann, F. W.; Tschammer, N. Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3. ACS Chem. Biol. 2014, 9, 2664−2677. (94) Bernat, V.; Heinrich, M. R.; Baumeister, P.; Buschauer, A.; Tschammer, N. Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3. ChemMedChem 2012, 7, 1481−1489.

(95) Stroke, I. L.; Cole, A. G.; Simhadri, S.; Brescia, M.-R.; Desai, M.; Zhang, J. J.; Merritt, R.; Appell, K. C.; Henderson, I.; Webb, M. L. Identification of CXCR3 receptor agonists in combinatorial smallmolecule libraries. Biochem. Biophys. Res. Commun. 2006, 349, 221− 228. (96) Heise, C. E.; Pahuja, A.; Hudson, S. C.; Mistry, M. S.; Putnam, A. L.; Gross, M. M.; Gottlieb, P. A.; Wade, W. S.; Kiankarimi, M.; Schwarz, D.; Crowe, P.; Zlotnik, A.; Alleva, D. G. Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist. J. Pharmacol. Exp. Ther. 2005, 313, 1263−1271. (97) Gonsiorek, W.; Zavodny, P.; Hipkin, R. W. The study of CXCR3 and CCR7 pharmacology using [35S]GTPγS exchange assays in cell membranes and permeabilized peripheral blood lymphocytes. J. Immunol. Methods 2003, 273, 15−27. (98) Jopling, L. A.; Watt, G. F.; Fisher, S.; Birch, H.; Coggon, S.; Christie, M. I. Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI-74330, using a murine CXCR3 internalization assay. Br. J. Pharmacol. 2007, 152, 1260−1271. (99) Schall, T. J.; Dairaghi, D. J.; McMaster, B. E. Compounds and methods for modulating CXCR3 function. WO2001/016114, March 8, 2001. (100) Medina, J. C.; Johnson, M. G.; Li, A.-R.; Liu, J.; Huang, A. X.; Zhu, L.; Marcus, A. P. CXCR3 antagonists. WO2002/083143, October 24, 2002. (101) Johnson, M.; Li, A.-R.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.; Wang, X.; Xu, Q.; Huang, A.; Marcus, A.; Xu, F.; Ebsworth, K.; Sablan, E.; Danao, J.; Kumer, J.; Dairaghi, D.; Lawrence, C.; Sullivan, T.; Tonn, G.; Schall, T.; Collins, T.; Medina, J. Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3. Bioorg. Med. Chem. Lett. 2007, 17, 3339−3343. (102) Storelli, S.; Verdijk, P.; Verzijl, D.; Timmerman, H.; van de Stolpe, A. C.; Tensen, C. P.; Smit, M. J.; De Esch, I. J. P.; Leurs, R. Synthesis and structure-activity relationship of 3-phenyl-3H-quinazolin-4-one derivatives as CXCR3 chemokine receptors antagonists. Bioorg. Med. Chem. Lett. 2005, 15, 2910−2913. (103) Du, X.; Chen, X.; Mihalic, J. T.; Deignan, J.; Duquette, J.; Li, A.-R.; Lemon, B.; Ma, J.; Miao, S.; Ebsworth, K.; Sullivan, T. J.; Tonn, G.; Collins, T. L.; Medina, J. C. Design and optimization of imidazole derivatives as potent CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 608−613. (104) Li, A.-R.; Johnson, M. G.; Liu, J.; Chen, X.; Du, X.; Mihalic, J. T.; Deignan, J.; Gustin, D. J.; Duquette, J.; Fu, Z.; Zhu, L.; Marcus, A. P.; Bergeron, P.; McGee, L. R.; Danao, J.; Lemon, B.; Carabeo, T.; Sullivan, T.; Ma, J.; Tang, L.; Tonn, G.; Collins, T. L.; Medina, J. C. Optimization of the heterocyclic core of the quinazolinone-derived CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 688−693. (105) Du, X.; Gustin, D. J.; Chen, X.; Duquette, J.; McGee, L. R.; Wang, Z.; Ebsworth, K.; Henne, K.; Lemon, B.; Ma, J.; Miao, S.; Sabalan, E.; Sullivan, T. J.; Tonn, G.; Collins, T. L.; Medina, J. C. Imidazo-pyrazine derivatives as potent CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2009, 19, 5200−5204. (106) Chen, X.; Mihalic, J.; Deignan, J.; Gustin, D. J.; Duquette, J.; Du, X.; Chan, J.; Fu, Z.; Johnson, M.; Li, A.-R.; Henne, K.; Sullivan, T.; Lemon, B.; Ma, J.; Miao, S.; Tonn, G.; Collins, T.; Medina, J. C. Discovery of potent and specific CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2012, 22, 357−362. (107) Liu, J.; Fu, Z.; Li, A.-R.; Johnson, M.; Zhu, L.; Marcus, A.; Danao, J.; Sullivan, T.; Tonn, G.; Collins, T.; Medina, J. Optimization of a series of quinazolinone-derived antagonists of CXCR3. Bioorg. Med. Chem. Lett. 2009, 19, 5114−5118. (108) Chen, X.; Henne, K.; Medina, J. C. CXCR3 antagonists. WO2009/094168, July 30, 2009. (109) Chan, J.; Burke, B. J.; Baucom, K.; Hansen, K.; Bio, M. M.; DiVirgilio, E.; Faul, M.; Murry, J. Practical syntheses of a CXCR3 antagonist. J. Org. Chem. 2011, 76, 1767−1774. (110) Lin, C.-C.; Chen, H.-C.; Lee, K.-Y.; Huang, Y.-H.; Fan, Y.-P.; Xiang, Y. Pyrimidinone compounds. US2006/0036093, February 16, 2006. 2914



Perspective

(111) Lin, C.-C.; Liu, J.-F.; Chang, C.-W.; Chen, S.-J.; Xiang, Y.; Cheng, P.-C.; Jan, J. J. Aminoquinoline compounds. US2004/0209902, October 21, 2004. (112) Lin, C.-C.; Liu, J.-F.; Chang, C.-W.; Chen, S.-J.; Xiang, Y.; Cheng, P.-C.; Jan, J. J. Aminoquinoline compounds. US2005/0070573, March 31, 2005. (113) Crosignani, S.; Missotten, M.; Cleva, C.; Dondi, R.; Ratinaud, Y.; Humbert, Y.; Mandal, A. B.; Bombrun, A.; Power, C.; Chollet, A.; Proudfoot, A. Discovery of a novel series of CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2010, 20, 3614−3617. (114) Crosignani, S.; Cleva, C.; Tsaklakidis, C.; Burgdorf, L. Sulfonamides. WO2009/124962, October 15, 2009. (115) Cole, A. G.; Stroke, I. L.; Brescia, M.-R.; Simhadri, S.; Zhang, J. J.; Hussain, Z.; Snider, M.; Haskell, C.; Ribeiro, S.; Appell, K. C.; Henderson, I.; Webb, M. L. Identification and initial evaluation of 4-Naryl-[1,4]diazepane ureas as potent CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2006, 16, 200−203. (116) Afantitis, A.; Melagraki, G.; Sarimveis, H.; IgglessiMarkopoulou, O.; Kollias, G. A novel QSAR model for predicting the inhibition of CXCR3 receptor by 4-N-aryl-[1,4] diazepane ureas. Eur. J. Med. Chem. 2009, 44, 877−884. (117) Cole, A. G.; Brescia, M.-R.; Henderson, I. Preparation of substituted [1,4]-diazepanes as CXCR3 antagonists. WO2007/002742, January 4, 2007. (118) Prokopowicz, A.; Wu, D.; Wu, F.; Swinamer, A.; Kowalski, J.; Ruppel, S.; Young, E.; Skow, D.; Nagaraja, R.; Schreyer, S. Optimization of a biaryl series of CXCR3 antagonists, 244th ACS National Meeting & Exposition, Philadelphia, PA, August 19−23, 2012; MEDI-333. (119) Ginn, J. D.; Sorcek, R. J.; Turner, M. R.; Wu, D.; Wu, F. Preparation of piperidino and piperazino benzamides as therapeutic CXCR3 receptor antagonists. WO2011/084985, July 14, 2011. (120) Kowalski, J. A.; Marshall, D. R.; Prokopowicz, A. S., III; Schlyer, S.; Sibley, R.; Sorcek, R. J.; Wu, D.; Wu, F.; Young, E. R. R. Preparation of (hetero)arylpiperidines and -piperazines as chemokine CXCR3 receptor antagonists. WO2010/126811, November 4, 2010. (121) Ginn, J. D.; Marshall, D. R.; Prokopowicz, A. S.; Schyler, S.; Sibley, R.; Turner, M. R.; Wu, D.; Wu, F. Preparation of piperazinobenzamides as CXCR3 receptor antagonists. US2010/0273781, October 28, 2010. (122) Caroff, E.; Keller, M.; Kimmerlin, Y.; Meyer, E. Preparation of 4-(benzoimidazol-2-yl) thiazole compounds and related aza derivatives as modulators of the CXCR3 receptor. WO2013/114332, August 8, 2013. (123) Caroff, E.; Meyer, E. Preparation of piperazinyltriazolylethanone derivatives for use as CXCR3 receptor modulators. WO2015/ 011099, January 29, 2015. (124) Thorpe, D. S.; Smrcina, M.; Cabel, D. D. Inhibitors of the chemokine receptor CXCR3. WO2009/105435, August 27, 2009. (125) Bata, I.; Budzer-Lantos, P.; Vasas, A.; Bartáné Bodor, V.; Ferenczy, G.; Tömösközi, Z.; Szeleczky, G.; Bátori, S.; Smrcina, M.; Patek, M.; Weichsel, A.; Thorpe, D. S. Cycloalkane carboxylic acid derivatives as CXCR3 receptor antagonists. WO2013/084013, June 13, 2013. (126) Bata, I.; Bartáné Bodor, V.; Vasas, A.; Buzder-Lantos, P.; Ferenczy, G.; Tö mö skö zi, Z.; Szeleczki, G.; Szamosvö lgyi, Z. Substituted β-amino acid derivatives as CXCR3 receptor antagonists. WO2013/174485, November 28, 2013. (127) Samuel, R. G.; Santini, C. Preparation of 2-arylthiazoles as CXCR3 chemokine receptor modulators. WO2007/070433, June 21, 2007. (128) Adams, A. D.; Santini, C. Preparation of pyridine, pyrimidine and pyrazine derivatives as CXCR3 receptor modulators. WO2007/ 100610, September 7, 2007. (129) Adams, A. D.; Green, A. I.; Szewczyk, J. W. Preparation of (hetero)arylthiazolylpiperidinyloxoethylazoles as CXCR3 chemokine receptor antagonists. WO2007/064553, June 7, 2007. (130) Ondeyka, J. G.; Herath, K. B.; Jayasuriya, H.; Polishook, J. D.; Bills, G. F.; Dombrowski, A. W.; Mojena, M.; Koch, G.; DiSalvo, J.;

DeMartino, J.; Guan, Z.; Nanakorn, W.; Morenberg, C. M.; Balick, M. J.; Stevenson, D. W.; Slattery, M.; Borris, R. P.; Singh, S. B. Discovery of structurally diverse natural product antagonists of chemokine receptor CXCR3. Mol. Diversity 2005, 9, 123−129 Also identified was a small peptide with CXCR3 activity.. (131) Tomoda, H.; Tabata, N.; Ohyama, Y.; Omura, S. Core structure in roselipins essential for eliciting inhibitory activity against diacylglycerol acyltransferase. J. Antibiot. 2003, 56, 24−29. (132) For a recent example, see Zhang, S.; Ma, Y.; Li, J.; Ma, J.; Yu, B.; Xie, X. Molecular matchmaking between the popular weight-loss herb Hoodia gordonii and GPR119, a potential target for metabolic disorder. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 14571−14576. (133) Turk, T.; Sepčić, K.; Mancini, I.; Guella, G. 3-Alkylpyridinium and 3-alkylpyridine compounds from marine sponges, their synthesis, biological activities and potential use. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier Science: Amsterdam, 2008; Vol. 35, pp 355−397. (134) McGuinness, B. F.; Carroll, C. D.; Zawacki, L. G.; Dong, G.; Yang, C.; Hobbs, D. W.; Jacob-Samuel, B.; Hall, J. W., III; Jenh, C.-H.; Kozlowski, J. A.; Anilkumar, G. N.; Rosenblum, S. B. Novel CXCR3 antagonists with a piperazinyl-piperidine core. Bioorg. Med. Chem. Lett. 2009, 19, 5205−5208. (135) Shao, Y.; Anilkumar, G. N.; Carroll, C. D.; Dong, G.; Hall, J. W., III; Hobbs, D. W.; Jiang, Y.; Jenh, C.-H.; Kim, S. H.; Kozlowski, J. A.; McGuinness, B. F.; Rosenblum, S. B.; Schulman, I.; Shih, N.-Y.; Shu, Y.; Wong, M. K. C.; Yu, W.; Zawacki, L. G.; Zeng, Q., II. SAR studies of pyridyl-piperazinyl-piperidine derivatives as CXCR3 chemokine antagonists. Bioorg. Med. Chem. Lett. 2011, 21, 1527−1531. (136) Kim, S. H.; Anilkumar, G. N.; Zawacki, L. G.; Zeng, Q.; Yang, D.-Y.; Shao, Y.; Dong, G.; Xu, X.; Yu, W.; Jiang, Y.; Jenh, C.-H.; Hall, J. W., III; Carroll, C. D.; Hobbs, D. W.; Baldwin, J. J.; McGuinness, B. F.; Rosenblum, S. B.; Kozlowski, J. A.; Shankar, B. B.; Shih, N.-Y., III. Identification of novel CXCR3 chemokine receptor antagonists with a pyrazinyl-piperazinyl-piperidine scaffold. Bioorg. Med. Chem. Lett. 2011, 21, 6982−6986. (137) Nair, A. G.; Wong, M. K. C.; Shu, Y.; Jiang, Y.; Jenh, C.-H.; Kim, S. H.; Yang, D.-Y.; Zeng, Q.; Shao, Y.; Zawacki, L. G.; Duo, J.; McGuinness, B. F.; Carroll, C. D.; Hobbs, D. W.; Shih, N.-Y.; Rosenblum, S. B.; Kozlowski, J. A., IV. Discovery of CXCR3 antagonists substituted with heterocycles as amide surrogates: improved PK, hERG and metabolic profiles. Bioorg. Med. Chem. Lett. 2014, 24, 1085−1088. (138) Scholten, D. J.; Wijtmans, M.; van Senten, J. R.; Custers, H.; Stunnenberg, A.; de Esch, I. J. P.; Smit, M. J.; Leurs, R. Pharmacological characterization of [3H]VUF11211, a novel radiolabeled small-molecule inverse agonist for the chemokine receptor CXCR3. Mol. Pharmacol. 2015, 87, 639−648. (139) Hayakawa, I.; Watanabe, J.; Momose, T.; Tomisato, W. Bicyclic nitrogen-containing saturated heterocyclic derivatives. WO2011/ 068171, June 9, 2011. (140) Bongartz, J.-P.; Buntinx, M.; Coesemans, E.; Hermans, B.; Van Lommen, G.; Van Wauwe, J. Synthesis and structure-activity relationship of benzetimide derivatives as human CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 5819−5823. (141) Coesemans, E.; Bongartz, J.-P. A. M.; Van Lommen, G. R. E.; Van Wauwe, J. P. F.; Buntinx, M. Piperidine derivatives as CXCR3 receptor antagonists. WO2007/090826, August 16, 2007. (142) Coesemans, E.; Bongartz, J.-P. A. M.; Van Lommen, G. R. E. Piperidine derivatives as CXCR3 receptor antagonists. WO2007/ 090836, August 16, 2007. (143) Habashita, H.; Shibayama, S. Spiropiperidine compounds as CXCR3 antagonists, and medicinal use thereof. WO2006/129679, December 7, 2006. (144) Wijtmans, M.; Verzijl, D.; van Dam, C. M. E.; Bosch, L.; Smit, M. J.; Leurs, R.; de Esch, I. J. P. Exploring a pocket for polycycloaliphatic groups in the CXCR3 receptor with the aid of a modular synthetic strategy. Bioorg. Med. Chem. Lett. 2009, 19, 2252− 2257. 2915



Perspective

(145) Wijtmans, M.; Verzijl, D.; Bergmans, S.; Lai, M.; Bosch, L.; Smit, M. J.; de Esch, I. J. P.; Leurs, R. CXCR3 antagonists: quaternary ammonium salts equipped with biphenyl- and polycycloaliphaticanchors. Bioorg. Med. Chem. 2011, 19, 3384−3393. (146) Wijtmans, M.; Scholten, D. J.; Roumen, L.; Canals, M.; Custers, H.; Glas, M.; Vreeker, M. C. A.; de Kanter, F. J. J.; de Graaf, C.; Smit, M. J.; de Esch, I. J. P.; Leurs, R. Chemical subtleties in smallmolecule modulation of peptide receptor function: the case of CXCR3 biaryl-type ligands. J. Med. Chem. 2012, 55, 10572−10583. (147) Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Iizawa, Y.; Shiraishi, M.; Aramaki, Y.; Okonogi, K.; Ogawa, Y.; Meguro, K.; Fujino, M. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 5698−5703. (148) Gao, P.; Zhou, X.-Y.; Yashiro-Ohtani, Y.; Yang, Y.-F.; Sugimoto, N.; Ono, S.; Nakanishi, T.; Obika, S.; Imanishi, T.; Egawa, T.; Nagasawa, T.; Fujiwara, H.; Hamaoka, T. The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3. J. Leukocyte Biol. 2003, 73, 273−280. (149) Ohshima, E.; Sone, H.; Kotera, O.; Komatsu, R.; LaRosa, G. J.; Luly, J. R. Preparation of imidazolidinylidenepropanedinitriles and related compounds as CXCR3 chemokine receptor antagonists useful as anti-inflammatory agents. WO2002/085861, October 31, 2002. (150) Ohshima, E.; Sone, H.; Kotera, O.; Komatsu, R.; LaRosa, G. J.; Luly, J. R. Imidazolidine derivatives as chemokine receptor modulators. WO2002/085862, October 31, 2002. (151) Ohshima, E.; Sone, H.; Kotera, O.; Luly, J. R.; LaRosa, G. J. Imidazolidine compounds as chemokine receptor modulators. WO2003/087063, October 23, 2003. (152) Sasho, S.; Obase, H.; Ichikawa, S.; Yoshizaki, R.; Ishii, A.; Shuto, K. Synthesis and in vitro gastrointestinal motility enhancing activity of 3-aryl-2-imidazolidinylidene propanedinitrile derivatives. Bioorg. Med. Chem. Lett. 1994, 4, 615−618. (153) Sasho, S.; Obase, H.; Harakawa, H.; Ichikawa, S.; Kitazawa, T.; Kishibayashi, N.; Yokoyama, T.; Nonaka, H.; Yoshizaki, R.; Ishii, A.; Shuto, K. Synthesis of 2-imidazolidinylidene propanedinitrile derivatives as stimulators of gastrointestinal motility − II. Bioorg. Med. Chem. 1994, 2, 1107−1117. (154) Sasho, S.; Obase, H.; Ichikawa, S.; Kitazawa, T.; Nonaka, H.; Yoshizaki, R.; Ishii, A.; Shuto, K. Synthesis of 2-imidazolidinylidene propanedinitrile derivatives as stimulators of gastrointestinal motility − III. Bioorg. Med. Chem. 1995, 3, 279−287. (155) Axten, J. M.; Foley, J. J.; Kingsbury, W. D.; Sarau, H. M. Preparation of imidazolium chemokine CXCR3 inhibitors. WO2003/ 101970, December 11, 2003. (156) Wang, Y.; Busch-Petersen, J.; Wang, F.; Kiesow, T. J.; Graybill, T. L.; Jin, J.; Yang, Z.; Foley, J. J.; Hunsberger, G. E.; Schmidt, D. B.; Sarau, H. M.; Capper-Spudich, E. A.; Wu, Z.; Fisher, L. S.; McQueney, M. S.; Rivero, R. A.; Widdowson, K. L. Camphor sulfonamide derivatives as novel, potent and selective CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2009, 19, 114−118. (157) Hayes, M. E.; Wallace, G. A.; Grongsaard, P.; Bischoff, A.; George, D. M.; Miao, W.; McPherson, M. J.; Stoffel, R. H.; Green, D. W.; Roth, G. P. Discovery of small molecule benzimidazole antagonists of the chemokine receptor CXCR3. Bioorg. Med. Chem. Lett. 2008, 18, 1573−1576. (158) Hayes, M. E.; Breinlinger, E. C.; Wallace, G. A.; Grongsaard, P.; Miao, W.; McPherson, M. J.; Stoffel, R. H.; Green, D. W.; Roth, G. P. Lead identification of 2-iminobenzimidazole antagonists of the chemokine receptor CXCR3. Bioorg. Med. Chem. Lett. 2008, 18, 2414− 2419. (159) Lee, J. H.; Park, H. Y. Preparation of benzoxazole derivatives as CXCR3/CXCL10 antagonists. KR1446301, October 6, 2014. (160) Lee, J.-H.; Kim, H.-N.; Kim, K.-O.; Jin, W. J.; Lee, S.; Kim, H.H.; Ha, H.; Lee, Z. H. CXCL10 promotes osteolytic bone metastasis by enhancing cancer outgrowth and osteoclastogenesis. Cancer Res. 2012, 72, 3175−3186.

(161) Allen, D. R.; Bolt, A.; Chapman, G. A.; Knight, R. L.; Meissner, J. W. G.; Owen, D. A.; Watson, R. J. Identification and structureactivity relationships of 1-aryl-3-piperidin-4-yl-urea derivatives as CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2007, 17, 697−701. (162) Watson, R. J.; Meissner, J. W. G.; Christie, M. I.; Owen, D. A. Piperidin-4-yl urea derivatives and related compounds as chemokine receptor inhibitors for the treatment of inflammatory diseases. WO2003/070242, August 28, 2003. (163) Watson, R. J.; Allen, D. R.; Birch, H. L.; Chapman, G. A.; Hannah, D. R.; Knight, R. L.; Meissner, J. W. G.; Owen, D. A.; Thomas, E. J. Development of CXCR3 antagonists. Part 2: identification of 2-amino(4-piperidinyl)azoles as potent CXCR3 antagonists. Bioorg. Med. Chem. Lett. 2007, 17, 6806−6810. (164) Owen, D. A.; Watson, R. J.; Meissner, J. W. G.; Allen, D. R. Bicyclic heteroaromatic derivatives as modulators of CXCR3 function. WO2005/003127, January 13, 2005. (165) Watson, R. J.; Allen, D. R.; Birch, H. L.; Chapman, G. A.; Galvin, F. C.; Jopling, L. A.; Knight, R. L.; Meier, D.; Oliver, K.; Meissner, J. W. G.; Owen, D. A.; Thomas, E. J.; Tremayne, N.; Williams, S. C. Development of CXCR3 antagonists. Part 3: tropenyl and homotropenyl-piperidine urea derivatives. Bioorg. Med. Chem. Lett. 2008, 18, 147−151. (166) Knight, R. L.; Allen, D. R.; Birch, H. L.; Chapman, G. A.; Galvin, F. C.; Jopling, L. A.; Lock, C. J.; Meissner, J. W. G.; Owen, D. A.; Raphy, G.; Watson, R. J.; Williams, S. C. Development of CXCR3 antagonists. Part 4: discovery of 2-amino-(4-tropinyl)quinolines. Bioorg. Med. Chem. Lett. 2008, 18, 629−633. (167) Thoma, G.; Baenteli, R.; Lewis, I.; Wagner, T.; Oberer, L.; Blum, W.; Glickman, F.; Streiff, M. B.; Zerwes, H.-G. Special ergolines are highly selective, potent antagonists of the chemokine receptor CXCR3: discovery, characterization and preliminary SAR of a promising lead. Bioorg. Med. Chem. Lett. 2009, 19, 6185−6188. (168) Baenteli, R.; Glickman, F.; Kovarik, J.; Lewis, I.; Streiff, M.; Thoma, G.; Zerwes, H.-G. Ergoline derivatives and their use as chemokine receptor ligands. WO2006/128658, December 7, 2006. (169) However, a recent publication by the same group gives their most advanced CXCR3 antagonist as a potent lead in a H3 agonism project: Auberson, Y. P.; Troxler, T.; Zhang, X.; Yang, C. R.; Fendt, M.; Feuerbach, D.; Liu, Y.-C.; Lagu, B.; Lerchner, A.; Perrone, M.; Lei, L.; Zhang, C.; Wang, C.; Wang, T.-L.; Bock, M. G. Ergoline-derived inverse agonists of the human H3 receptor for the treatment of narcolepsy. ChemMedChem 2014, 9, 1683−1696. (170) Thoma, G.; Baenteli, R.; Lewis, I.; Jones, D.; Kovarik, J.; Streiff, M. B.; Zerwes, H.-G. Special ergolines efficiently inhibit the chemokine receptor CXCR3 in blood. Bioorg. Med. Chem. Lett. 2011, 21, 4745− 4749. (171) Musicki, B.; Aubert, J.; Boiteaux, J.-G.; Clary, L.; Rossio, P.; Schuppli-Nollet, M. Preparation of novel disubstituted 3,4-diamino-3cyclobutene-1,2-dione compounds for use in the treatment of chemokine-mediated diseases. WO2013/061004, May 2, 2013. (172) Musicki, B.; Aubert, J.; Boiteau, J.-G.; Clary, L.; Rossio, P.; Schuppli-Nollet, M. Preparation of novel disubstituted 3,4-diamino-3cyclobutene-1,2-dione compounds for use in the treatment of chemokine-mediated diseases. WO2013/061005, May 2, 2013. (173) Ning, Y.; Labonte, M. J.; Zhang, W.; Bohanes, P. O.; Gerger, A.; Yang, D.; Benhaim, L.; Paez, D.; Rosenberg, D. O.; Nagulapalli Venkata, K. C.; Louie, S. G.; Petasis, N. A.; Ladner, R. D.; Lenz, H.-J. The CXCR2 antagonist, SCH-527123, shows antitumor activity and sensitizes cells to oxaliplatin in preclinical colon cancer models. Mol. Cancer Ther. 2012, 11, 1353−1364. (174) Inoue, N.; Takahashi, O.; Furuya, T.; Fujiwara, H. Chemokine receptor antagonistic medicinal composition. WO2005/123065, December 29, 2005. (175) Vummidi, B. R.; Noreen, F.; Alzeer, J.; Moelling, K.; Luedtke, N. W. Photodynamic agents with anti-metastatic activities. ACS Chem. Biol. 2013, 8, 1737−1746. (176) Zhang, D.; Gao, Z.-G.; Zhang, K.; Kiselev, E.; Crane, S.; Wang, J.; Paoletta, S.; Yi, C.; Ma, L.; Zhang, W.; Han, G. W.; Liu, H.; Cherezov, V.; Katritch, V.; Jiang, H.; Stevens, R. C.; Jacobson, K. A.; 2916



Perspective

Zhao, Q.; Wu, B. Two disparate ligand-binding sites in the human P2Y1 receptor. Nature 2015, 520, 317−321. (177) Hollenstein, K.; Kean, J.; Bortolato, A.; Cheng, R. K. Y.; Doré, A. S.; Jazayeri, A.; Cooke, R. M.; Weir, M.; Marshall, F. H. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 2013, 499, 438−443.

2917


Small Molecule CXCR3 Antagonists - Journal of Medicinal Chemistry

Recommend Documents