Research paper
2,8-Diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine potent CCR4 antagonists capable of inducing receptor endocytosis

https://doi.org/10.1016/j.ejmech.2016.02.058Get rights and content

Highlights

  • 20 amide and amine derivatives of spirocyclic pyrimidines were prepared.

  • pKi = 8.8 for (R)-(18a) in the [125I]-TARC binding assay, and pIC50 = 8.1 in the [35S]-GTPγS assay.

  • pA2 = 6.7 in human whole blood actin polymerisation assay.

  • Compounds found to induce CCR4 endocytosis.

  • They internalise 50–70% of the cell surface receptors.

Abstract

A number of potent 2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine CCR4 antagonists binding to the extracellular allosteric site were synthesised. (R)-N-(2,4-Dichlorobenzyl)-2-(2-(pyrrolidin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine (R)-(18a) has high affinity in both the [125I]-TARC binding assay with a pKi of 8.8, and the [35S]-GTPγS functional assay with a pIC50 of 8.1, and high activity in the human whole blood actin polymerisation assay (pA2 = 6.7). The most potent antagonists were also investigated for their ability to induce endocytosis of CCR4 and were found to internalise about 60% of the cell surface receptors, a property which is not commonly shared by small molecule antagonists of chemokine receptors.

Introduction

Chemokines are a group of about 50 small, basic proteins of 8–10 kDa, which together with their receptors mainly regulate the recruitment of leukocytes into inflammatory sites. Chemokines exert their effects through the activation of G protein-coupled receptors situated on the cell surface. Ten CC chemokine receptors have been identified so far named as CC-chemokine receptor 1, 2, 3 etc. Most chemokine receptors recognise more than one chemokine and several chemokines bind to more than one receptor [1]. CC chemokine receptor 4 (CCR4) is the only receptor identified so far for the macrophage-derived chemokine (MDC, CCL22) and thymus and activation-regulated chemokine (TARC, CCL17), and shown to be highly expressed in the thymus. T helper 2 (Th2) cytokines in inflamed tissues lead to eosinophilia, high levels of serum IgE and mast cell activation, all of which contribute to the pathogenesis of allergic diseases [2]. Upon exposure to allergen, dendritic cells within tissue secrete CCL22 and CCL17, which can recruit Th2 cells from the circulation. The T cells can then migrate along this chemokine gradient to the dendritic cells. The latter migrate from the inflamed tissue to local lymph nodes where CCL22 and CCL17 may recruit further T cells. Elevated levels of CCL17 and CCL22 as well as accumulation of CCR4-positive cells were observed in lung biopsy samples from patients with atopic asthma following allergen challenge [3]. CCR4 is also expressed by immune suppressive regulatory T cells [4], [5], and a minor subset of Th17 cells [6], [7]. Hence CCR4 antagonists represent a novel therapeutic intervention in diseases where CCR4 is involved, such as asthma [8], lung disease [4], atopic dermatitis [9], allergic bronchopulmonary aspergillosis [10], leukemia [11], colon cancer [12], inflammatory bowel disease [5], the mosquito-borne tropical diseases, such as Dengue fever [13], and allergic rhinitis [14]. In addition, CCR4 antagonists were used as molecular adjuvants in vaccines [15], [16], [17], [18]. Finally, CCR4 monoclonal antibodies were recently explored for CCR4+ T cell leukemia and one such antibody, Mogamulizumab (20 mg injection), was launched in 2012 in Japan for the treatment of relapsed or refractory adult T cell leukemia/lymphoma [19], [20]. In December 2014 approval for additional indication for chemotherapy-native CCR4-positive adult T-cell leukemia-lymphoma (ATL) of Mogamulizumab was granted in Japan. The launch of the humanised monoclonal antibody Mogamulizumab underlines the value of generating cheaper small molecule CCR4 antagonists in this area. Progress in the discovery of small-molecule CCR4 antagonists was reviewed by Purandare and Somerville in 2006 [21]. A number of other CCR4 antagonists have appeared in the literature since the publication of this review [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. These antagonists appear to belong to two chemotypes. The first chemotype, exemplified by compounds 17, includes lipophilic amines, such as Bristol Myers Squibb (BMS) compounds 1 [22] and 2 [24], Astellas compounds 3 [27] and 4 [29], the Daiichi Sankyo compound 5 [31], 6 [33] and 7 [34]. The second chemotype, exemplified by sulfonamides 810, includes AstraZeneca pyrazine 8 [35], the Ono pyrazine 9 [36], and the GlaxoSmithKline indazole 10 [37], [38] (Fig. 1). Indazole 10 was the first small molecule candidate to be progressed to human studies; however, the compound suffered from low solubility and weak potency [39]. Our group has recently published our efforts to identify novel sulfonamide templates for lead optimisation studies [40], [41].

Moreover, our group has recently reported that antagonism of human CCR4 can be achieved through three distinct binding sites on the receptor [42]. Sulfonamides 810 were shown to bind at an intracellular allosteric binding site, arbitrarily named at GSK as site II, which is different from the binding site (site I), where lipophilic amine antagonists, such as 1, 2 and 4 bind. Glu 290, which is found in Helix VII, is the anchor point for site I – perhaps the only residue strongly believed to interact with the chemokine, and these basic site I antagonists. The two allosteric binding sites I and II are distinct from each other and from the orthosteric binding site where CCL17 and CCL22 bind. Similar findings for a CCR4 and CCR5 intracellular binding site were reported by the AstraZeneca group [43]. Our group has so far reported only on sulfonamide (site II) intracellular CCR4 antagonists [37], [38], [40], [41], herein we report our efforts on identifying basic site I allosteric antagonists.

Section snippets

Chemistry

At the time that this work was initiated X-ray crystal structures of G Protein Coupled Receptors were not reported, however, the structures of antagonists 13 were published, together with a CCR4 homology model based on bovine rhodopsin [27]. Our initial lead, the spirocyclic pyrimidine proline amide (±)-11a, was overlaid on BMS pyrimidine homoproline amide 1 docked in the homology model, and showed that the basic nitrogen atoms of the proline and homoproline moieties of (R)-1 and (R)-11a

Results and discussion

The antagonist activity at human CCR4 of the compounds shown in Table 1 was determined by [125I]-TARC radioligand binding assay [44]. This assay used recombinant CCR4 expressing CHO cell membranes adhered to WGA-coated Leadseeker scintillation proximity assay (SPA) beads. The SPA beads contained scintillants that emit light when stimulated by emitted radiation, for example, during binding of the radiolabelled ligand to CCR4 bringing it in close proximity to the bead, and the output was measured

Receptor endocytosis studies

CCL22 and CCL17 were shown to induce CCR4 receptor endocytosis (internalisation from the cell surface of human TH2 cells), resulting in a loss of functional responsiveness [45]. Internalisation of the receptor following agonist exposure is thought to be a means of receptor desensitisation in GPCRs [46]. Endocytosis is an ATP-dependent process, which is largely inhibited at 4 °C. Hence the antibody staining for the receptor endocytosis assays was performed at 4 °C in all cases in order to

Conclusion

A number of 2,8-(diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine derivatives were synthesized and screened as CCR4 antagonists. Compound (±)-18a has high affinity in the [125I]-TARC binding assay with a pKi of 8.8, and the [35S]-GTPγS functional assay with a pIC50 of 8.1. Furthermore, it has high activity in the human whole blood actin polymerisation assay (pA2 = 6.7). The most potent antagonists were investigated for their ability to induce endocytosis of CCR4 and were found to partially

Experimental section

Organic solutions were dried over anhydrous Na2SO4 or MgSO4. TLC was performed on Merck 0.25 mm Kieselgel 60 F254 plates. Products were visualised under UV light and/or by staining with aqueous KMnO4 solution. LCMS analysis was conducted on either System A an Acquity UPLC BEH C18 column (2.1 mm × 50 mm i.d. 1.7 μm packing diameter) eluting with 0.1% formic acid in water (solvent A), and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0.0–1.5 min 3–100% B,

Acknowledgements

We thank Bill J. Leavens for collecting the HRMS data, the Screening and Compound Profiling Department at GlaxoSmithKline for generating the human CCR4 GTPγS data, Jonathan Goodacre for technical assistance and David Hall for helpful discussions.

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