Medicinal Chemistry

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Medicinal Chemistry

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Small-molecule inhibitors of spleen tyrosine kinase as therapeutic agents for immune disorders: will promise meet expectations?

Following on the heels of the US FDA approval of tofacitinib (Xeljanz, Pfizer, USA), an inhibitor of the JAK family members, and ibrutinib (Imbruvica, Janssen, Belgium), an inhibitor of BTK, for the treatment of rheumatoid arthritis and chronic lymphocytic leukemia, respectively, there is now renewed interest in the biopharmaceutical industry in the development of orally active small-molecule agents targeting key protein kinases implicated in immune regulation. One such ‘immunokinase’ target is SYK, a non-receptor tyrosine protein kinase critical for transducing intracellular signaling cascades for various immune recognition receptors, such as the B-cell receptor and the Fc receptor. Here, we review and discuss the progress and challenges in the development of small-molecule inhibitors of SYK and their potential as a new class of disease-modifying immunosuppressive agents for certain inflammatory and autoimmune disorders.

The clinical utility of biologics targeting TNF-α, the IL-6 receptor, CD20 (B-cell depletion) and CD80/86 (inhibition of T-cell costimulatory signal) in the management of rheumatoid arthritis (RA) supports the notion that several major and redundant pathways are capable of promoting and/or sustaining inflammation and autoimmunity. However, there is still a tremendous unmet clinical need for more efficacious therapeutic agents, as a significant proportion of RA patients either do not respond to these marketed biologics or are unable to achieve a sustained response when treatment is ceased. This is not surprising as many human autoimmune disorders, including RA, are polygenic; therefore, therapeutic perturbation of multiple redundant and distinct mechanisms is required in order to achieve greater disease activity control, if not to ultimately modify the disease to induce a state of disease remission. Therapeutic modulation predicated on targeting key intracellular enzymes essential for integrating signal transduction pathways represents a reasonable, if not attractive, strategy to deliver the much-needed

10.4155/FMC.14.126 © 2014 Future Science Ltd

Matthew C Lucas*,1 & Seng-Lai Tan2 Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA 2 EMD Serono Research & Development Institute, 45A Middlesex Turnpike, Billerica, MA 01821, USA *Author for correspondence: [email protected] 1

breakthrough efficacy. Much of the initial enthusiasm for such an approach, however, has been hampered by the disappointing clinical results of various small-molecule inhibitors targeting the p38 pathway [1–3] . A change of tides may be happening following the recent US FDA approval of tofacitinib (Xeljanz, Pfizer, USA) and ibrutinib (Imbruvica, Janssen, Belgium) for RA and chronic lymphocytic leukemia therapy, respectively. Tofacitinib is an inhibitor of the JAK family members, whereas ibrutinib targets BTK. Interestingly, the potential therapeutic utility of another kinase inhibitor, namely imatinib (Gleevec, Novartis, Switzerland), is being explored for RA therapy. Originally developed to inactivate the BCR–ABL tyrosine kinase for the treatment of certain leukemic cancers, imatinib was found to be efficacious in preclinical models of RA and may exert its effects by selectively inhibiting a variety of signaling pathways central to the development of RA [4] . Perhaps one lesson gleaned from the clinical success of BCR–ABL, JAK and BTK inhibitors is that these enzymes and their functions were first elucidated by the identification of disease-associated

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Key terms Autoimmunity: A condition in which the body’s normal immune response is misdirected towards its own tissues, which can lead to hypersensitivity and various disease states. Immunokinase: A kinase that is part of the immune signaling cascade, for example BTK , IRAK4, JAK1, JAK2, JAK3, NIK, NIK beta subunit, PI3K delta, ZAP70, MYD88 and TYK2. Signalosome: A group of proteins that form a complex that mediates signal transduction.

human mutations in the corresponding genes. In the case of JAK3, a member of the JAK kinase family, and BTK, human mutations have been linked to severe immunodeficiencies, confirming their important role in the immune response and suggesting that pharmacological inhibition of these kinases is likely tolerable in human. In addition, as we have learned in the field of anticancer therapy, exquisite kinase selectivity may be desirable but not the rule of thumb in the development of successful therapeutic kinase inhibitors. Predominantly expressed in hematopoietic cells, JAK3 mediates the signal transduction of the common gamma chain (γc) of the type I cytokine receptor family (e.g., IL-2R, -4R, -7R, -9R, -15R and -21R). Blunting these proinflammatory cytokine signals is expected to halt disease progression of inflammatory and autoimmune disease. While the development of highly selective JAK3 inhibitors is conceptually preferred, it is clear that in the JAK signaling pathway for cytokines, JAK members function as dimers by pairing with each other (e.g., JAK1 and JAK3). Furthermore, JAK1 is a primary signaling pathway for IL-6, so a JAK1 inhibitor could function like an IL-6 inhibitor. Inhibition of JAK2 may also benefit RA patients as this JAK member is important for GM-CSF-dependent signaling and an anti-GM-CSF antibody (mavrilimumab, CAM-3001, MedImmune, USA) has met its clinical endpoint in a recent RA trial with an acceptable safety profile [5] . Thus, targeting different combinations of JAK members may still have specificity while offering greater efficacy, provided an adequate therapeutic window can be attained. Notwithstanding these questions, the biopharmaceutical industry has continued to invest in the development of protein kinase inhibitors as therapeutic agents for immune-related disorders [6] . The biopharmaceutical deals centering on less than a handful of ‘immunokinase’ targets, specifically JAK, BTK, SYK and PI3Kδ, have generated an estimated US$12 billion in value creation for the industry. Among these, SYK was at one point considered to be a ‘hot’

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immunokinase target but has rapidly fallen from its pedestal, with AstraZeneca and Pfizer both returning rights to Rigel’s oral and inhaled SYK inhibitors for RA and asthma therapy, respectively. Here, we review and discuss the challenges and progress in the development of SYK inhibitors for the treatment of inflammatory and autoimmune disorders. SYK as a therapeutic target in immunological disorders SYK is a cytoplasmic tyrosine kinase of 72 kDa that contains a tandem pair of SH2 domains and a kinase domain. Expressed at higher levels in the hematopoietic lineage of the immune system, SYK plays a critical role in mediating signal transduction triggered by the activation of a number of immune recognition receptors, including but not limited to the B-cell receptor (BCR), Fc receptors, CD74 and integrins [7,8] . These immune recognition receptors or ‘immunoreceptors’ activate SYK through a common mechanism involving SYK interaction with the immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic tail of the immunoreceptor. Upon immunoreceptor engagement, the tyrosine residues on ITAMs are rapidly phosphorylated by membraneassociated Src family non-receptor tyrosine kinases, such as LYN and FYN. The SH2 domains of SYK recognize and bind the phosphotyrosines on ITAMs. Thus, phophorylated ITAMs act as docking sites for SYK, where SYK becomes activated as a consequence of protein conformational changes. The functional role of SYK is best delineated in B cells, where upon antigen engagement of the BCR, activated SYK orchestrates the formation of a plasma membrane-associated signaling complex known as the BCR signal­osome. The signalosome, which includes SYK itself, PLCγ2, PI3Kδ, BTK and BLNK, in turn catalyzes the phosphorylation of a number of protein substrates and, thus, facilitates the propagation of downstream signaling events. Depending on the cellular context, including the provision of other extracellular signals, the signal transduction cascades are integrated to generate the variable cellular responses, such as proliferation, survival, differentiation, phagocytosis, migration and secretion of cytokines. Aberrant activation of immunoreceptor signaling can contribute to the initiation and/or maintenance of chronic inflammation and autoimmunity. For example, inappropriate activation of B cells as a result of chronic engagement of the BCR by extracellular autoantigen can lead to B-cell differentiation and secretion of pathogenic autoantibodies and proinflammatory cytokines. Similarly, autoantigen–immuno­ globulin complexes can activate monocytes/macrophages

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Small-molecule inhibitors of spleen tyrosine kinase as therapeutic agents for immune disorders 

and mast cells through FcγR and FceR engagement, respectively. Although no human mutations linking SYK function and immunological defects have been described yet, the importance of B cells in the pathogenesis of autoimmune disease is unequivocally demonstrated by the clinical benefits of B-cell-directed therapies, such as rituximab and belimumab [9] . Autoantibodies, which are a hallmark of many autoimmune diseases, can also contribute to disease by engaging the FcR expressed on many immune effector cells, including macrophages, mast cells and neutrophils. Furthermore, FcR-deficient mice are resistant to the development of autoimmunity in preclinical models [10] . Given the critical role of SYK in BCR and FcR signaling, as well as in other signaling pathways (discussed below), therapeutic approaches targeting SYK are expected to deliver better and broader efficacy for treatment of autoimmune diseases. The relative higher expression of SYK in the hematopoietic tissues also suggests pharmacological inhibition of SYK may have a wider therapeutic window compared with proteins that are more ubiquitously expressed. As expected, inhibition of SYK, either by pharmacological or genetic means, resulted in attenuation of various immunoreceptor-mediated cellular responses in vitro and encouraging therapeutic effects in various preclinical disease models [11] . In the following section, we review progress towards positioning SYK inhibitors in different immune disease indications, as well as discuss some of the shortcomings of these studies. Rheumatoid arthritis

Initial preclinical studies in several murine arthritis models showed administration of SYK inhibitor R406 resulted in a dose-dependent improvement in disease severity, suggesting that SYK inhibition might be beneficial in RA [11] . Subsequent clinical evaluation of fostamatinib (R788; the methylene phosphate pro-drug of R406, Rigel, USA) in RA patients who did not adequately respond to the standard-of-care treatment with methotrexate showed fostamatinib was significantly superior to the placebo group with respect to American College of Rheumatology criteria 50 and 70 at doses of 100 mg twice daily and 150 mg once daily [12] . However, fostamatinib failed to show significant efficacy in another clinical trial with RA patients who displayed inadequate response to the anti-TNF-α biologic, and although post-hoc analyses of the data suggested that a further trial in this population is warranted, this appears unlikely at this time [13] . Adverse events reported in these trials included hypertension, diarrhea and neutropenia. Because fostamatinib displays a relatively poor kinase selectivity profile [14] , it is difficult to ascertain

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if SYK is solely responsible for clinical benefit and/or the side effects. The blood pressure increase associated with fostamatinib use is believed to be an off-target effect, owing to inhibition of other kinases – the VEGFR2 being a prime suspect [12] . Approximately 25% of the treated patients needed antihypertensive therapy or needed to increase their dose of antihypertensive therapy, with the subjects’ blood pressure returning to baseline at the end of the study. Thus, the risk for hypertension associated with fostamatinib use appears to be manageable, although the long-term effects of fostamatinib in RA patients, who are prone to cardiovascular risk, remain unknown. Asthma & allergic rhinitis

Given the role of SYK in mediating FceRI signaling in mast cells and basophils, SYK inhibitors should have a therapeutic niche in allergic diseases, such as asthma and allergic rhinitis (reviewed in [11]). However, there have been some setbacks in the clinical evaluation of SYK inhibitors in these indications. One of the earliest SYK inhibitors to progress into the clinic, R112, was able to improve symptoms of seasonal allergic rhinitis [15] . A follow-up compound, R343, completed several Phase I clinical trials for asthma and although it was safe and well tolerated, it fell short of its primary or secondary endpoints in a Phase II clinical trial and development was subsequently halted. However, both these compounds were derived from a poorly selective scaffold, so it is difficult to determine if the lack of clinical efficacy is due to inadequate coverage of SYK inhibition. More recent studies have further substantiated a role for SYK in mediating allergic airway responses in rat and sheep allergen-induced airway constriction models and ex vivo in non-human primate lungs [16] . Furthermore, a SYK inhibitor, NVP-QAB-205, was effective in reducing airway hyper-responsiveness in a chronic mouse model of asthma [17] . Interestingly, depletion of SYK by topical administration of siRNA via nasal instillation seemed to inhibit recruitment of inflammatory cells to the bronchoalveolar lavage fluid of allergen-sensitized mice [18] . Immune thrombocytopenic purpura

Immune thrombocytopenic purpura (ITP) is a disease associated with autoimmune-driven destruction of platelets, which leads to thrombocytopenia, bruising and bleeding. The mechanism involves formation of anti-platelet antibodies and accelerated phagocytosis of platelets carried out by macrophages expressing FcγR. In a murine model of ITP, mice pretreated with R788 were protected from thrombocytopenia. Following these encouraging results, R788

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Key term T-cell receptor: Found on the surface of T cells, it is responsible for recognizing antigens and, thus, triggering a cascade that leads to downstream events, such as cellular proliferation, differentiation and cytokine production. Aberrant signaling can lead to disease.

was administered to 16 adult human patients with chronic refractory ITP in a small Phase II trial. Half of the ITP patients achieved a sustained response at a median R788 dose of 125 mg twice daily [19] . A Phase III trial was recently announced and results are expected in 2015 [20] . IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is the most common cause of glomerulonephritis and is a significant cause of end-stage renal disease. Current treatment options for IgAN are inadequate, consisting of supportive care to control blood pressure and proteinuria [21] . The disease pathogenesis involves the synthesis and release into the circulation an aberrant form of IgA1 antibodies with characteristics that favor mesangial cell deposition. SYK inhibition may arrest or slow destruction of the glomeruli by reducing the production of the IgA1 antibodies and thus mesangial deposition. Moreover, Kim et al. showed fostamatinib can reduce proinflammatory cytokine production and proliferation of human mesangial cells stimulated with IgA1 immune complex from IgAN patients [22] . Thus, SYK inhibitor may also halt or slow the destructive inflammatory pathways leading to downstream renal tissue damage by blocking the signaling of IgA1 immune complex receptors on mesangial cells. These proposed mechanisms might contribute the therapeutic effects seen with fostamatinib (18) in its ability to reverse the inflammation in the glomeruli and improve kidney function in a mouse model with established glomerulonephritis [23] . Predicated on these encouraging preclinical results and a high unmet medical need, a Phase II study evaluating fostamatinib in IgAN has been announced [20] . Systemic lupus erythematosus

Another immune disorder that might benefit from SYK inhibitor therapy is systemic lupus erythematosus (SLE). A chronic autoimmune disease, SLE is characterized by the presence of autoantibodies, the formation of immune complexes and inflammation in multiple organs. B cells in SLE have multiple abnormalities and are believed to play a central role in these aspects [24] . Preclinical studies evaluating R788 in various lupus models support the development of SYK inhibitors for SLE therapy [25,26] . However, as

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discussed above, these results are tempered by the fact that R788 is not a SYK-selective inhibitor. The best evidence supporting a critical role for B cells in SLE pathogenesis comes from clinical practice of therapeutic agents targeting B cells [9] . Despite not meeting its clinical endpoints in SLE clinical trials, B-cell-depletion therapy using rituximab is being used off-label for refractory cases. Belimumab, a monoclonal antibody that neutralizes the B-cell survival factor BLyS fared better in SLE clinical trials and received FDA approval for SLE therapy. In this regard, it is worth pointing out that optimal BLyS responsiveness in B cells requires SYK, suggesting a potential crosstalk mechanism between the BLyS and BCR pathways in transmitting its survival signal [27] . Abnormal T-cell activation and production of cytokines are also well-known characteristics of SLE and these may also drive the initiation and maintenance of the autoimmune reaction [28] . Intriguingly, higher SYK expression and activity were detected in T cells of SLE patients compared with controls [29] . However, the hyporesponsiveness of the T cells in these SLE patients did not correlate with SYK expression. The mechanism underlying this heightened T-cell receptor (TCR) response is apparently due to the swapping out of one of the TCR subunits, CD3ζ, with the Fcg receptor common gamma chain (FcRγ), which recruits and signals through SYK [30] . If confirmed in a larger SLE patient cohort, these results might suggest a potential strategy for selecting a subset of SLE patients based on dysregulated SYK-dependent T-cell activation who may benefit from treatment with a SYK inhibitor. Post-operative ileus

Post-operative ileus is a gastrointestinal motility disorder caused by physical disturbances to the bowel during abdominal surgery. Inflammation within the intestinal muscularis, characterized by infiltration of neutrophils, mast cells and macrophages, is thought to contribute to the development of post-operative ileus. van Bree et al. evaluated a potent and selective aminopyrimidine SYK inhibitor 25 in a mouse model of post-operative ileus [31] . The investigators found 25 dampened manipulation-induced intestinal muscular inflammation, restored intestinal transit and prevented recruitment of immune effector cells to the muscularis, probably by inhibiting mast cell degranulation and the activation of macrophages in response to intestinal manipulation. Thus, SYK inhibition may exert additional effects through inhibition of migration and recruitment of immune cells to the site of tissue inflammation, either via FcR or integrin signaling.

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Small-molecule inhibitors of spleen tyrosine kinase as therapeutic agents for immune disorders 

SYK inhibitors: recent progress

Review

examples (e.g., 1 & 2, Figure 1), one can speculate that they will have encountered similar challenges to the Gilead (USA) and Roche (Switzerland) investigators (vide infra). The Merck group appears to also have found alternatives (e.g., 3, Figure 1) [38] , which have allowed them to replace the carboxylic acid, although they seem to have returned to the acid functionality in subsequent applications (e.g., 4, Figure 1) [39] . In 2013, a patent application from Boehringer Ingelheim (Germany) disclosed another variation of the aminopyrimidine scaffold that is favored by Merck [40] . Amongst the compounds claimed was compound 5, an exceptionally potent (SYK IC50 = 0.5 nM) inhibitor with only a small shift in the presence of 1% human serum albumin. This suggests that cellular potency might also be reasonable. Although no information on selectivity is provided in the patent, again, one could speculate that the indole ring might interact with Pro455 to achieve some level of selectivity of binding. A series of Merck GMBH patent applications have appeared in the recent literature covering wide structural diversity. Most recently, Burgdorf et al. disclosed a series of pyridopyrimidine derivatives [41] . The medicinal chemistry behind this scaffold is discussed below. This scaffold is clearly distinguished from the set of other scaffolds published by the group,

Patent literature review: 2010–2014

A steady stream of patents and patent applications have been published over the last few years and several reviews of this literature have been prepared [32–34] . A review of the patent literature from 2010–2012 is therefore redundant and is not included here. Rather, selected patent applications published in 2013 and 2014 are discussed either if they complement these reviews or if they are important to highlight and augment the discussion of some of the common scaffolds that have emerged in the peer-reviewed literature discussed below. Merck Sharp & Dohme (USA) have been the source of several new patent applications in 2013 and 2014. Based on some of their most potent compounds, one can speculate that they are seeking selective inhibitors of SYK and that they may be following a strategy to optimize binding interactions with key residues Pro455 and Asn458 in the SYK binding pocket similar to that described by Lucas et al. and Currie et al. (vide infra) [35,36] . Interestingly, the Merck group appears to have found a way to achieve this using a different vector and an aminopyrimidine scaffold [37] . Given the high-molecular weights and the presence of the carboxylic acid in many of their potent

CF3

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Figure 1. Aminopyrimidine SYK inhibitors. SYK IC50 binding potency is shown for comparison, but it should be noted that these values may have been obtained from different assay types and conditions.

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