Functional Profiling of 2-Aminopyrimidine Histamine H4 Receptor

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Functional Profiling of 2‑Aminopyrimidine Histamine H4 Receptor Modulators Miniperspective Mark S. Tichenor, Robin L. Thurmond, Jennifer D. Venable, and Brad M. Savall* Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, California 92121, United States

ABSTRACT: Histamine is an important endogenous signaling molecule that is involved in a number of physiological processes including allergic reactions, gastric acid secretion, neurotransmitter release, and inflammation. The biological effects of histamine are mediated by four histamine receptors with distinct functions and distribution profiles (H1−H4). The most recently discovered histamine receptor (H4) has emerged as a promising drug target for treating inflammatory diseases. A detailed understanding of the role of the H4 receptor in human disease remains elusive, in part because low sequence similarity between the human and rodent H4 receptors complicates the translation of preclinical pharmacology to humans. This review provides an overview of H4 drug discovery programs that have studied cross-species structure−activity relationships, with a focus on the functional profiling of the 2-aminopyrimidine chemotype that has advanced to the clinic for allergy, atopic dermatitis, asthma, and rheumatoid arthritis.

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H2 (5000 nM) receptors.6 Similarly, synthetic H4 ligands typically have high selectivity margins versus H1 and H2 but sometimes demonstrate cross-reactivity with H3. Preclinical disease models continue to play an important role in validating new disease-modifying mechanisms for therapeutic intervention. Animal disease models are designed to mimic human disease by having an analogous underlying biochemical mechanism. Ideally, when a GPCR modulator has a comparable binding affinity and an identical functional profile at the human and rodent receptors, the efficacy profile in preclinical disease models can strengthen the rationale that the mechanism of action will have therapeutic value in human patients. However, when a molecule signals differently at the human and rodent receptors, disease models cannot be expected to predict human efficacy. Similarly, preclinical toleration studies are used to identify and de-risk toxicities that are related to the mechanism of action. Predicting the human safety profile from an animal toleration study requires the selection of preclinical species having the same functional response as the human receptor. Thus, it is important to track the structure−activity and

BACKGROUND The histamine receptors have been a productive class of pharmaceutical targets, yielding a number of successful drugs for the treatment of allergy (H1)1 and ulcers (H2).2 More recently, H3 antagonists have advanced to late phase clinical trials as promising treatments for narcolepsy, cognitive disorders, and pain.3 The fourth and most recently discovered histamine receptor (H4)4 is expressed primarily on immune cells including eosinophils, mast cells, and dendritic cells and has been shown to participate in inflammatory signaling processes by mediating chemotaxis and cytokine release. H4 antagonists are currently being evaluated as potential treatments for a variety of immunological diseases including allergy, asthma, atopic dermatitis, and rheumatoid arthritis. The H4 receptor is structurally differentiated from other members of the histamine receptor family, having only 35% sequence homology with its nearest neighbor, the H3 receptor, and 30% homology with the H1 receptor.5 The relatively large differences in protein sequence are evident in the divergent selectivity profiles of histamine receptor ligands. The endogenous ligand histamine has nanomolar affinity for the H3 (16 nM) and H4 (8 nM) receptors, but the affinity is roughly 3 orders of magnitude lower for the H1 (6000 nM) and © XXXX American Chemical Society

Received: April 1, 2015

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DOI: 10.1021/acs.jmedchem.5b00516 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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agonism has been detected in primary cells, leaving open the question as to whether the findings in transfected cell systems have any physiological relevance. In addition, no data have been reported on the effects of 1 or other H4 ligands on β-arrestin activation in mouse systems, making it difficult to interpret whether the β-arrestin pathway contributes to any of the efficacy observed with H4 ligands in mouse disease models. Nevertheless, the fact that for several disease models, similar effects are observed in H4 receptor deficient mice and with H4 antagonist treatment supports the conclusion that biased agonism is not contributing to these effects, since it cannot occur in receptor deficient mice. Compound 1 has been used to characterize the biological effects of antagonizing the H4 receptor in animal disease models of asthma, dermatitis, and allergy. The utility of 1 for interrogating H4 pharmacology in vivo is limited by its short plasma half-life due to rapid demethylation of the piperazine Nmethyl group and N-oxidation. Oral dosing of 1 achieves adequate target coverage in acute disease models, but chronic disease models that may require plasma concentrations in excess of the H4 Ki for an extended period require larger doses, which can confound the pharmacology because of potential offtarget effects. H4 antagonist drug discovery programs have focused on identifying new H4 chemotypes with improved pharmacokinetic profiles that can further elucidate the role of H4 preclinically and can be advanced into human clinical trials. Researchers at Pfizer initiated their H4 program with a focus on validating the H4 receptor as a safe and effective mechanism for treating immune-mediated diseases. Initially, 1 was the only selective H4 antagonist reported, and the Pfizer team recognized the need to have multiple H4 ligands from diverse chemotypes to draw conclusions about the safety of H4 modulators. Pfizer employed two lead identification strategies in parallel: evolving new chemotypes using 1 as chemical starting point and employing a high-throughput screen of their compound collection to discover entirely new leads.15 Systematic modification of the indole and the diamine subunits was pursued with a goal of improving metabolic stability, leading to the discovery of a new benzimidazole lead 2 (Figure 2). During

structure−function relationships for multiple species in parallel to inform the selection of animal species for in vivo studies. Speciation is often an issue for developing drugs targeting Gprotein-coupled receptors.7 Human and monkey H4 receptors have the highest sequence homology (98%), followed by the dog (71%), rat (69%), mouse (68%), and guinea pig (65%) receptors.5 Consequently, H4 ligands can have significantly different binding affinities across H4 species orthologs. Histamine itself has high affinity for the human H4 (4.8 nM) and the guinea pig H4 (6 nM) receptors but much lower affinity for the mouse H4 (42 nM) and rat H4 (136 nM) receptors. Large species differences in the binding and functional profiles of synthetic H4 ligands have also been reported.5 In contrast, the protein sequence of H3 is more uniform, each species isoform having greater than 92% sequence homology between the human, mouse, rat, and guinea pig receptors. The larger differences in protein sequence and ligand binding profiles for the H 4 receptor complicate the interpretation of H 4 pharmacology and its translation to the clinic. H4 chemotypes and their structure−activity relationships have been previously reviewed,8−10 focusing on H4 binding affinity. This review provides an overview of H4 medicinal chemistry programs that have studied structure−activity relationships, with a focus on the functional profiling of the 2-aminopyrimidine chemotype that has advanced into the clinic. The H4 antagonist 1 (JNJ 7777120, Figure 1)11 has been frequently used for pharmacological profiling of the H4 receptor

Figure 1. H4 reference compound.

since its disclosure in 2003. Compound 1 is highly selective for H4 and has comparable potency at the human and mouse H4 receptors. Compound 1 is reported to act as an antagonist at the human, rat, and mouse receptors in multiple functional assays, but recently it has been described as a partial agonist under certain assay conditions.12 H4 functional assays typically use recombinant cell lines overexpressing H4, and the functional readouts are sensitive to the receptor density, complicating the comparison of functional results between laboratories. For this reason, validation of antagonism in primary cell systems is important for correlating in vitro functional behavior with in vivo disease model profiling. Characterizing the functional behavior of H4 antagonists has become considerably more complicated following the recent discovery of G-protein-independent signaling through the βarrestin pathway.13 Compound 1 behaves as an antagonist with respect to G-protein signaling and a partial agonist in mediating β-arrestin recruitment to the H4 receptor. A systematic analysis of the G-protein and β-arrestin signaling of indole carboxamide analogs of 1 has identified ligands that are biased for the βarrestin pathway and an unbiased ligand that is an agonist in both pathways.14 However, to date, no evidence of biased

Figure 2. New benzimidazole H4 ligands.

the synthesis of 2, synthetic byproduct 3 was isolated and found to also be a potent H4 ligand. Both compounds were characterized as antagonists using a functional assay consisting of a cell-based β-lactamase reporter assay using HEK-293T cells that were transiently transfected with the human H4 receptor. Compounds 2 and 3 were profiled in a rodent toleration study for an early safety assessment of H4 antagonists in vivo.15a The study design consisted of oral administration to rats for 4 days, at an undisclosed dose that produced free compound concentrations in excess of the Ki’s. Both compounds caused serious adverse effects including dose-dependent lymphoid depletion from spleen, thymus, and gut associated lymphoid tissues, decreased reticulocyte counts, and decreased erythB



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reported to completely block the imetit-induced eosinophil shape change at 30 nM in human whole blood, behaving as a functional antagonist in a native cell type. In addition to demonstrating H4 antagonism in human primary cells, this assay is directly translatable as a human clinical biomarker. Researchers at Abbott initiated a high-throughput screen of their compound collection to identify new H4 chemotypes. The screen used a fluorimetric imaging plate reader (FLIPR) assay that measured changes in intracellular calcium to detect H4 stimulation in a HEK-293 cell line stably expressing the human or rat H4 receptor.18 Screening hit 6 (Figure 4) was selected as

ropoiesis, raising concerns about H4 antagonism as a safe therapeutic mechanism. However, the subsequent development of an analogous rat functional assay identified 2 and 3 as full agonists at the rat H4 receptor. Therefore, the toxicity could not be attributed to H4 antagonism. Additional structurally diverse H4 agonists must be profiled in vivo before the toxicity that was reported in this study can be conclusively attributed to an ontarget H4 agonist effect versus off-target pharmacology. The 2-aminopyrimidine chemotype, which has dominated the H4 chemical landscape,16 was evolved from pyrimdine HTS hits that were independently discovered in multiple research groups. The Pfizer group conducted a high-throughput screen of their internal compound collection using an H4 functional assay, which led to the identification of pyrimidine 4, having moderate binding affinity for H4 (Figure 3). Subsequent SAR

Figure 4. Structure and in vitro potencies of HTS hit 6 and 2aminopyrimidine derivative 7.

a lead on the basis of its structural novelty and moderately potent human H4 binding affinity. The low rat affinity of 6 was identified as an essential parameter for optimization to support profiling in rodent disease models. Early SAR showed that replacing the pyrrole in 6 with an amino substituent (7) caused a large increase in affinity for both the human and rat homologs of H4. Both 6 and 7 were human H4 antagonists and rat partial agonists in the corresponding functional assays. A series of analogs having modifications to the 2-position of the pyrimidine was evaluated for binding and functional activity (Table 2). Removal of the 2-amino substituent (8) and N-

Figure 3. Pfizer HTS hit (4) and clinical compound (5).

led to truncation of the diamine and addition of a 2-amino substituent to the central pyrimidine to give 5 (PF-3893787), demonstrating a 1000-fold improvement in affinity for H4. The functional behavior of 5 was evaluated by transfecting HEK-293T cells with the H4 receptors from a variety of preclinical species (Table 1). Compound 5 behaved as an Table 1. Binding Potency and Functional Behavior of 5.15a species

5, pKi a

5, functional effectb

human macaque dog guinea pig rat mouse

8.21 7.81 5.79 6.91 7.91 7.68

inverse agonist inverse agonist antagonist partial agonist partial agonist partial agonist

Table 2. Modification of the 2-Position of the Pyrimidine18

a

The displacement binding was performed using [3H]histamine and homogenate of HEK 293T cells transiently transfected with the cDNA of corresponding H4 variants. bFunctional activity at recombinant H4 receptors was determined using HEK-293T cells transiently transfected with the cDNA of corresponding H4 variants and a CRE-βlactamase reporter gene. The reversal of histamine inhibition of forskolin-stimulated cAMP was the functional end point.

compd

R

human H4 pKb or pEC50 a

8 7 9 10

-H -NH2 -NHMe -NMe2

7.07 8.35 6.08 4.94

αb

rat H4 pKb or pEC50 a

αb

1

6.03 7.17 5.29 4.74

0.79 0.64 0.46 0.88

When an α value is reported, the compound is an agonist or partial agonist and the potency is reported as pEC50 using a FLIPR assay. If no α value is reported, the antagonist response (pKb) in a FLIPR assay is measured by inhibition of a reference response to histamine in a cell line expressing human or rat H4. bAgonist efficacy, normalized relative to histamine α = 1. a

inverse agonist at the human and monkey H4 receptors, an antagonist at the dog H4 receptor, and a partial agonist at the guinea pig, rat, and mouse receptors. The authors do not report running 5 in animal disease models in part because of the inherent difficulty in interpreting the results in the context of the divergent cross-species functional activity. The rationale for advancing 5 into the clinic was built upon a combination of asthma model efficacy data for reference H4 antagonists and the development of a human primary cell functional assay that could be used as a clinical biomarker.17 Human eosinophils, isolated or in whole blood, are treated with H4 agonists, such as imetit, to induce shape change that is detected by gated autofluorescence forward scatter (GAFS). Compound 5 is

methylation (9 and 10) caused a >10-fold loss in potency relative to 7 for both the human and rat H4 receptors, highlighting the importance of the 2-amino substituent in the binding interactions with H4. Changes to the 2-amino substituent also had a dramatic influence on human and rat H4 functional activity. The pyrimidine 2-H (8) and the 2C



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assay using a guanosine 5′-O-[γ-thio]triphosphate (GTPγS) assay in the HEK-293 cell line. Recognizing the limitations of the FLIPR and the GTPγS assays as artificial systems using overexpressed H4 that may amplify agonist signaling, the Abbott group developed a mouse primary cell functional assay to predict functional behavior in vivo. Despite its agonist or partial agonist behavior in rodent recombinant functional assays, 13 acted as an antagonist in primary cells, blocking histamine-induced shape change in mouse bone marrow mast cells. The H4 agonists histamine and clozapine have been shown to induce itch when administered by intraperitoneal (ip) injection, serving as a direct measure of H4 functional activity in vivo. Following administration of the H4 agonist clobenproprit, 13 caused a dose-dependent reduction in itch. When tested in agonist mode, intraperitoneal administration of 13 did not cause any itch response in mice. In combination, these observations support the antagonist behavior of 13 in vivo, which correlates with the primary cell data as the most predictive in vitro functional assay. These observations point out the risks in relying on overexpressed systems to deduce functional behavior. Researchers at Abbott subsequently designed a new 2,4diaminopyrimidine subseries by restricting the rotation of the substituent in the 6-position by constraining it in a fused ring (Table 4).19 Analogs having fused six-membered (21) and fivemembered (22) rings were potent human H4 antagonists but behaved as partial (21) or full (22) agonists at the rat H4 receptors. Compound 23, having a fused seven-membered ring, had somewhat lower human H4 affinity but behaved as an antagonist at both the human and the rat receptors. This desirable characteristic for in vivo profiling was uncommon in this chemotype, so a focused set of analogs was prepared having modifications to the diamine moiety. Diamine replacements 23−26 recovered the H4 affinity, and the functional antagonism was maintained for both the human and the rodent receptors. Unlike the preceding examples with unpredictable human and rodent functional activities, the fused seven-membered tricycles demonstrate that functional antagonism can be maintained across the human and rodent receptors. Researchers at Janssen Research and Development, LLC (Janssen), conducted a high-throughput screen using a radioligand displacement (3H-histamine) binding assay to identify pyrimidine hit 27 (Figure 5).20 Following the initial binding screen, compounds were profiled in a cellular functional assay, using a β-galactosidase reporter gene in a human neuroblastoma SK-N-MC cell line stably transfected with H4. Introduction of an amino substituent to the 2-position of the pyrimidine core (28) increased binding affinity over 100fold and allowed greater diversity in the diamine component to be incorporated while maintaining binding affinity. From this tricyclic series, 29 was identified as a potent and orally available antagonist at the human H4 receptor.21 Although the in vitro functional data for the mouse H4 receptor were not reported, 29 demonstrated H4 antagonist behavior in vivo. In a mouse antigen asthma model, treatment with 29 significantly reduced the number of inflammatory eosinophils found in lung lavage after antigen challenge. Aligning the N-methylpiperazine rings of HTS hit 27 and 1 places the phenyl rings of both compounds in a similar location (Figure 5). Furthermore, convergent SAR for phenyl ring substituents of 27 and 1 indicated that the two structures bound to the H4 protein in an analogous manner. An analog of

NHMe (9) analogs were human H4 antagonists, but the 2-N,Ndimethyl analog (10) behaved as a full agonist at human H4. Both the binding affinity and functional activity were very sensitive to small modifications to the substituent at the 2position of the pyrimidine in this series. Structurally diverse analogs having modifications to the 6position of the pyrimidine generally maintained potent human and rat H4 binding and covered a broad range of human and rat functional activities (Table 3). Removal of the substituent in Table 3. Modification of the 6-Position of the Pyrimidine18

compd

R

human H4 pKb or pEC50 a

11 7 12 13 14 15 16 17 18 19 20

H t-Bu Ph 4-CN-Ph NHMe Ph-CH2-NHPh-CH2-NMeimidazole-1-yl benzimidazol-1-yl imidazole-4-yl 1-Me-imidazol-4-yl

7.36 8.53 8.39 8.53 8.28 7.19 6.77 5.6 6.75 7.79 8.05

αb 0.42

0.56 0.31 0.82 0.41

rat H4 pKb or pEC50 a 6.94 7.17 7.40 7.23 6.31 6.31 6.47 5.1 6.46 6.41 6.99

αb 0.87 0.64 0.79 0.64 0.93 0.87 0.56 0.77 0.91

When an α value is reported, the compound is an agonist or partial agonist and the potency is reported as pEC50 using a FLIPR assay. If no α value is reported, the antagonist response (pKb) in a FLIPR assay is measured by inhibition of a reference response to histamine in a cell line expressing human or rat H4. bAgonist efficacy, normalized relative to histamine α = 1. a

the pyrimidine 6-position (11) gave a human H4 partial agonist, while phenyl derivatives (12 and 13) were human H 4 antagonists. Nitrogen-linked analogs (14−18) did not have a consistent functional trend at either species, with small structural differences having large functional consequences. For example, an N-methyl substituent on the nitrogen linker changes a partial agonist (15) to an antagonist (16). The 6substituent scan included compounds having the full spectrum of H4 functional activities, including benzimidazole-containing agonist 18. Compounds in this series demonstrate a greater propensity for activation of the rat receptor relative to the human receptor, but the rat and human functional activity varied independently. For example, compound 18 is a full agonist at only the human receptor and compound 20 is an agonist at only the rat receptor. Thus, no clear SAR trend for designing dual human/rat H4 antagonists is apparent from an extensive set of modifications to the 2- and 6-positions of the pyrimidine. The rodent H4 functional activity of compound 13 was studied more thoroughly in preparation for in vivo profiling. The mouse H4 functional profile (pEC50 = 6.91, α = 0.54) correlates closely with the rat H4 functional profile (pEC50 = 7.23, α = 0.64), consistent with the relatively high homology between the mouse and rat H4 receptor sequences (84%). Compound 13 behaved as a full agonist in a second functional D



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Table 4. Modification of the 6-Position of the Pyrimidine19

1 having a cyclohexyl ring (30) in place of the fused phenyl maintained binding affinity for H4, suggesting that saturated analogs of 27 might have high affinity for H4. The 2-amino substituent that was beneficial for potency across multiple subseries was incorporated to give the 2,4-diamino-6-alkylpyrimidine series exemplified by 31. Modification of the diamine (31−34) and the 6-alkyl substituent (34−37) gave a series with consistently high H4 binding affinity, and all of the compounds behaved as antagonists at the human receptor (Table 5). The Janssen group also reported human H4 pA2 values using a Schild analysis, demonstrating potent competitive antagonism with histamine. Among the compounds in Table 5, 32 and 36 (JNJ 39758979) were selected for additional profiling.23 Compound 32, with a piperazine as the diamine fragment at the 4-position, was a functional antagonist at the human receptor and was a partial agonist at the mouse receptor (EC50 = 7.3 nM, α = 0.57), which would complicate the interpretation of results in preclinical disease models. Compound 36 was a functional antagonist at the human, mouse, and monkey H4 receptors (Table 6). The affinity for the rat and guinea pig H4 receptors was moderate, and there was essentially no affinity for the dog H4 receptor. Compound 36 demonstrated dose-dependent activity in models of asthma and dermatitis, which was consistent with antagonism of the H4 receptor. On the basis of its preclinical efficacy and safety profile, 36 was selected as a candidate for human clinical trials. Several additional subseries related to pyrimidine screening hit 27 were discovered, each having unique functional SAR trends. An extensive series of analogs was prepared having sixmembered heterocyclic modifications to the central pyrimidine core, including alternative pyrimidine isomers, pyridine, and pyridazine as heterocyclic replacements.24 The 2-amino substituent on the central heterocycle consistently increased binding affinity, but functional activity was dependent on the identity of the diamine (Table 7). All of the compounds having N-methylpiperazine as the diamine component behaved as human H4 partial agonists (α = 0.63−0.80), independent of the core heterocycle (38−42). Analogs having the (R)-N-methylpyrrolidine as the diamine component had a greater tendency to behave as H4 antagonists in the functional assay compared to N-methylpiperazine

When an α value is reported, the compound is an agonist or partial agonist and the potency is reported as pEC50 using a FLIPR assay. If no α value is reported, the antagonist response (pKb) in a FLIPR assay is measured by inhibition of a reference response to histamine in a cell line expressing human, rat, or mouse H4. bAgonist efficacy, normalized relative to histamine α = 1. a

Figure 5. Identification of the 2,4-diamino-6-alkylpyrimidine series. E



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Table 7. Functional Activity of the N-Methylpiperazine Compounds21,24

Table 5. SAR of 2-Aminopyrimidines22

a

Displacement of [3H]histamine from the recombinant histamine H4 receptor. bFunctional activity at recombinant H4 receptors was determined using a forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing human H4. cAntagonism of histamine inhibition of forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing the human histamine H4 receptor. nd = not determined; hH4 = human H4.

Table 6. Cross-Species H4 Binding Potency and Functional Behavior species

36, Ki (nM)a

36, functional effectb

human mouse monkey dog guinea pig rat

12 5.3 25 >10000 306 188

antagonist antagonist antagonist nd nd antagonist

a

Displacement of [3H]histamine from the recombinant histamine H4 receptor. bFunctional activity at recombinant H4 receptors was determined using a forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing human H4. nd = not determined.

analogs (Table 7). Although pyrimidine analog 43 was a partial agonist (α = 0.53), the corresponding analog having an aminosubstituted core 44 behaved as an antagonist. (R)-NMethylpyrrolidine analogs having pyridine as the central heterocycle 45 and 46 were both antagonists, whereas the direct N-methylpiperazine-containing analogs 40 and 41 were partial agonists. Compounds having a pyridazine core were unique, since the N-methylpiperazine derivative 42 did not have appreciable affinity for the H4 receptor. However, the (R)N-methylpyrrolidine derivative 47 was a potent H4 antagonist. These results above indicate a role for the position and nature of hydrogen bond donor/acceptor interactions in not only the affinity but also the functional activity of ligands for the H4 receptor.25

a

Displacement of [3H]histamine from the recombinant human histamine H4 receptor. bFunctional activity at recombinant H4 receptors was determined using a forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing human H4. c Agonist efficacy, normalized relative to histamine α = 1. dAntagonism of histamine inhibition of forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing the human histamine H4 receptor. F



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CLINICAL STATUS Clinical development has been reported for a few H 4 antagonists including 5, UR-63325 (structure not disclosed), 36, and toreforant (5-(5,7-dimethyl-1H-benzimidazol-2-yl)-4methyl-N-[3-(1-methyl-4-piperidinyl)propyl]-2-pyrimidinamine),26 although limited data are available outside of company web sites and public registries of clinical studies. The only compound with results available in peer reviewed journals is 36. Preclinical toxicology studies with 36 of up to a 6-month duration in rats and 9 months in monkeys have been reported with only minimal findings in rats and no meaningful findings in monkeys.23 In particular, there was no evidence of significant hematologic, bone marrow, or lymphoid organ changes in either species. The lack of toxicity is notable especially given the findings with the previous Pfizer compounds as noted above15a and points to these effects being related to the chemical class or H4 agonism rather than H4 antagonism. In a phase 1 study in healthy volunteers, inhibition of histamine-induced eosinophil shape change was observed upon a single dose of 36 and after daily dosing for 14 days.23 The plasma concentrations at which inhibition was observed (>20 ng/mL) were consistent with the concentrations needed in vitro to inhibit eosinophil shape change.23 Chronic pruritus in diseases such as atopic dermatitis remains an unmet medical need, since few drugs are proven to be effective. H4 antagonists have been shown to block histamineinduced scratching in mice, and such scratching is absent in H4deficient mice.27 Consistent with these findings, 36 was able to inhibit histamine-induced scratching in mice.22 This preclinical observation coupled with the compound’s selectivity for the H4 receptor (greater than 80-fold more potent at the H4 receptor compared to other histamine receptors) makes it a good candidate for exploring the role of the H4 receptor in mediating pruritus in humans. To this end, a clinical study was performed testing the efficacy of 36 in blocking histamine-induced itch in humans.28 Itch induced by intradermal injection of histamine was assessed in healthy male volunteers before and after dosing with 36. Compared to placebo, 36 was able to significantly inhibit the itch response. These data provide evidence that the H4 receptor is involved in the sensation of itch in humans and suggests that antagonists of the receptor, like 36, could be effective treatments for pruritus in a variety of diseases. One disease where pruritus in not well controlled by current therapies is atopic dermatitis. H4 antagonists, including 36, have been shown to be efficacious on both inflammation and pruritus in preclinical dermatitis models.23,29 To test for efficacy in humans, 36 was studied in adult Japanese patients with moderate atopic dermatitis.30 Patients received 100 mg of 36, 300 mg of 36, or matching placebo once a day for 6 weeks. The primary end points in this study were the Eczema Area and Severity Index (EASI) scores, and although the changes in the primary end points were not statistically significant, numerical improvements were noted for both doses of 36 compared to placebo. In addition to this, nominally statistically significant improvement in pruritus was noted across a number of different measures. In general, efficacy was noted for both doses of 36. The data from the preclinical dermatitis model suggested that trough concentrations needed to be in the range of 10−40 ng/ mL for efficacy, which was achieved for both doses.30,23 Given the caveats with the early termination of the study, it is not possible to make firm conclusions about the potential efficacy

of H4 antagonists for the treatment of atopic dermatitis, but the results do provide a strong rationale for further study.

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PERSPECTIVE The discovery of the fourth histamine receptor introduced new opportunities and unique challenges in the field of histamine research. Fourteen years after the discovery of the H4 receptor, a detailed understanding of H4 pharmacology remains elusive, although there is compelling evidence that modulation of H4 function could be beneficial in treating a number of diseases. The large sequence differences between the human and rodent H4 receptors, and the corresponding species differences in functional activity observed for H4 ligands from various structural classes, complicate the interpretation of H 4 pharmacology and its translation to the clinic. Since the disclosure of 1 in 2003, new antagonists with high selectivity for H4 have been discovered. Using high throughput screening strategies, several research groups independently discovered chemical starting points having a central pyrimidine and extensively evaluated the structure−activity relationships in this chemical series. 2-Aminopyrimidines have been the focus of the drug discovery programs of Pfizer, Abbott, and Janssen, in part because of the excellent druglike properties and high selectivity for the H4 receptor. The 2-aminopyrimidine chemical series has produced the clinical compounds 5 and 36. Compound 36 is a potent antagonist at the human and rodent H4 receptors, has excellent subtype selectivity for H4, and has a mouse pharmacokinetic profile that is superior to 1. Thus, its profile compares favorably to 1 as a reference H4 antagonist for interrogating H4 pharmacology in vivo. The three drug discovery programs described in this review encountered species differences in H4 functional activity that complicated the profiling of compounds in preclinical disease models. In all three programs, the rat H4 receptor was found to be more susceptible to agonist/partial agonist activation by 2aminopyrimidine H4 modulators than the human H4 receptor. The investigators took different approaches to addressing these functional challenges and advancing compounds into the clinic. The Pfizer group’s early toleration study highlighted the importance of having the appropriate rodent assays set up early in the program to enable correct correlation of the toxicity to the mechanism of action. The subsequent identification of the rat agonistic behavior of the test compounds leaves an open question about the safety profile of H4 agonists, which have not been studied as extensively as H4 antagonists. Additional toleration studies with structurally diverse H4 agonists are required to provide context for the findings in the Pfizer toleration study. Having identified a human vs rat H4 functional disconnect for the lead molecule 5, the Pfizer group chose not to use preclinical disease model profiling because the results would not have been instructive for assessing the human clinical dose. Instead, the rationale for the effectiveness of the mechanism was based on the results of reference H4 antagonists that were used as surrogates in rodent disease model studies. A human whole blood biomarker that measured the eosinophil shape change by gated autofluorescence forward scatter (GAFS) was utilized to select the clinical dose and to verify target engagement in the clinic. However, this assay only measures the degree of target engagement in the blood ex vivo and may over- or underpredict the doses needed for clinical efficacy depending on the site of action, tissue distribution, and other factors. In addition, the GAFS assay lacks a direct rodent G



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correlation because rodent eosinophils do not exhibit autofluorescence, and therefore, it is not possible to correlate the level of inhibition in this assay with efficacy on disease parameters. The Abbott group emphasized the optimization of rat H4 affinity from the outset of their discovery program, which suggests that they considered efficacy in animal disease models essential for advancing a compound to the clinic. As in the Pfizer work, a majority of the Abbott compounds behaved as rat partial agonists or full agonists in recombinant assays. A mouse bone marrow mast cell assay was used to demonstrate that their lead compound was an antagonist in primary cells, despite behaving as an agonist or partial agonist in two different recombinant receptor assays. The lead compound behaved as an antagonist in an H4 agonist-induced itch study, correlating the in vivo functional result with the primary cell functional assay. The Janssen research group identified several variations on the 2-aminopyrimidine core structure, some having a strong propensity for rodent H4 agonism and others with a bias toward antagonism at both human and rat H4 receptors. Focusing on 6-alkyl-2,4-diaminopyrimidines, the human, mouse, and rat H4 antagonist 36 enabled the use of rodent disease models to study H4 pharmacology and to inform clinical trial design, including disease selection and human dose prediction. Compound 36 was able to inhibit the pruritic effect of histamine in mice and was later shown to also inhibit the pruritic effect of histamine in humans. This ability to carry out the same experiment in humans and mice makes the assessment of histamine-induced pruritus a direct translation of H4 antagonism in vivo from mice to humans. There has been recent progress in identifying new H4 antagonists, but the majority of these compounds are structurally related to two chemical series: 2-aminopyrimidine derivatives and indole carboxamides. Ongoing efforts to expand the chemical diversity of H4 modulators will continue to be important for understanding H4 pharmacology. Selectivity panels sample a relatively small set of receptors with known biological activities, leaving it difficult to rule out the possibility that biological effects of compounds are caused by unknown off targets. Furthermore, selectivity is usually assessed against a panel of human receptors, while the selectivity against rodent off targets remains unknown. With a chemically diverse set of reference compounds, the likelihood of the compounds sharing a common off target is reduced, and conclusions about the pharmacology can be made with more confidence. Thus, continued investigation into the discovery of new, selective H4 antagonists will contribute to future advances in the field.

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Robin L. Thurmond received his B.A. in Chemistry from the University of Virginia. In 1993 he earned his Ph.D. in Biology from the University of Arizona. After postdoctoral work at the Massachusetts Institute of Technology, he joined Janssen Research & Development, LLC, in 1996 and has worked in both Immunology discovery and clinical development. Jennifer D. Venable received her B.Sc. in Chemistry from the University of California, Los Angeles in 1997. In 2001 she earned her Ph.D. from the University of Texas at Austin under the direction of Professor Philip D. Magnus in the area of natural product total synthesis. Since 2002 she has worked in the Immunology therapeutic area at Janssen Research & Development, LLC, as a medicinal chemist. Brad M. Savall received his B.Sc. in Chemistry from the University of WisconsinStevens Point and his Ph.D. from the University of Michigan (2002) under the direction of Prof. William Roush. In 2002 he joined Janssen Research & Development, LLC, in the Immunology therapeutic area, where he contributed to the development of several clinical candidates. In 2009, he transitioned to the Neuroscience therapeutic area where he is involved in the research of several CNS diseases.

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ABBREVIATIONS USED FLIPR, fluorimetric imaging plate reader; CRE, cAMP response element; GTPγS, guanosine 5′-O-[γ-thio]triphosphate; SK-NMC, a neuroblastoma cell line; GAFS, gated autofluorescence forward scatter

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AUTHOR INFORMATION

Corresponding Author

*Phone: 858-320-3393. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Mark S. Tichenor received his B.Sc. in Chemistry from the University of California, San Diego in 2002. In 2007 he earned his Ph.D. from the Scripps Research Institute in the research group of Professor Dale Boger in natural product synthesis and medicinal chemistry. Since 2007, he has worked as a medicinal chemist at Janssen Research & Development, LLC, in Pain & Related Disorders and Immunology therapeutic areas. H



Perspective

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Functional Profiling of 2-Aminopyrimidine Histamine H4 Receptor

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