Bispyrimidines as Potent Histamine H4 Receptor ... - ACS Publications

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Bispyrimidines as Potent Histamine H4 Receptor Ligands: Delineation of Structure−Activity Relationships and Detailed H4 Receptor Binding Mode Harald Engelhardt,†,‡ Sabine Schultes,†,‡ Chris de Graaf,‡ Saskia Nijmeijer,‡ Henry F. Vischer,‡ Obbe P. Zuiderveld,‡ Julia Dobler,† Katharina Stachurski,† Moriz Mayer,† Heribert Arnhof,† Dirk Scharn,† Eric E. J. Haaksma,†,‡ Iwan J. P. de Esch,‡ and Rob Leurs*,‡ †

Boehringer Ingelheim RCV GmbH & Co KG, Department of Medicinal Chemistry, Dr. Boehringergasse 5-11, 1121 Vienna, Austria Amsterdam Institute of Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Department of Pharmacochemistry, Faculty of Exact Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

‡

S Supporting Information *

ABSTRACT: The basic methylpiperazine moiety is considered a necessary substructure for high histamine H4 receptor (H4R) affinity. This moiety is however also the metabolic hot spot for various classes of H4R ligands (e.g., indolcarboxamides and pyrimidines). We set out to investigate whether mildly basic 2-aminopyrimidines in combination with the appropriate linker can serve as a replacement for the methylpiperazine moiety. In the series of 2-aminopyrimidines, the introduction of an additional 2-aminopyrimidine moiety in combination with the appropriate linker lead to bispyrimidines displaying pKi values for binding the human H4R up to 8.2. Furthermore, the methylpiperazine replacement results in compounds with improved metabolic properties. The attempt to transfer the knowledge generated in the class of bispyrimidines to the indolecarboxamides failed. Combining the derived structure−activity relationships with homology modeling leads to new detailed insights in the molecular aspects of ligand−H4R binding in general and the binding mode of the described bispyrimidines in specific.

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INTRODUCTION Histamine receptors belong to the GPCR gene family and currently four histamine receptor subtypes are known, namely the histamine H1, H2, H3, and H4 receptors. The construction of the human genome database enabled the discovery of the H4 receptor (H4R) in the year 2001.1−3 Initially, the expression of the H4R was thought to be restricted to the periphery on dendritic cells, mast cells, eosinophils, monocytes, basophils, natural killer cells, and T cells.4,5 Recent studies however, revealed its presence in several regions of the CNS.6,7 Meanwhile, there is clear evidence that the H4R plays a role in immune and inflammatory responses4 and modulates itch responses as well.8−10 It has been reported that antagonists of the H4R are able to block the shape change and chemotaxis of eosinophils and mast cells, which are involved in modulating many inflammatory processes, whereas such compounds are also active in a variety of animal models of inflammation and itch11−15 This finding suggests that H4R antagonists could play a role in, e.g., the treatment of asthma, rheumatoid arthritis, and pruritus.16 The first reported selective H 4 R antagonist is the indolecarboxamide, 1a (JNJ7777120; Figure 1),17−19 which is highly potent and selective for H4R.17,18 Several other H4R © 2013 American Chemical Society

ligands have been published, many containing a pyrimidine moiety as aromatic scaffold (see examples in Figure 1).13−15,19−27 Many of the reference H4R ligands, i.e., the compounds frequently used in pharmacological studies and preclinical animal models of diseases, contain a methylpiperazine basic group that has long been considered an essential substructure for ligands with high H4R activity. However, a methylpiperazine moiety is a known metabolic hotspot, severely limiting the half-life of the ligands. In particular, for compound 1a and 1b, extensive studies have shown that the metabolic liability in mouse is linked to the 1-methylpiperazine residue.27 Mouse PK experiments revealed that the Nmethylated piperazine moiety undergoes rapid demethylation, thereby confounding in vivo experiments.27 It is already known that, in the class of indolecarboxamides and pyrimidines, the 1methylpiperazine moiety can be replaced by N-methylpyrrolidin-3-amine or by N-methylazetidin-3-amine,27 yet these replacements still contain a N-methylamine moiety, which is a potential metabolic weak spot.28 Received: December 21, 2012 Published: May 13, 2013 4264

dx.doi.org/10.1021/jm301886t | J. Med. Chem. 2013, 56, 4264−4276

Journal of Medicinal Chemistry

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Figure 1. (a) Indolecarboxamide and pyrimidine-based H4R ligands. (b) Previous published binding modes of reference ligands 2 (magenta carbon atoms, (A)) and 1b (green, (B)) in H1R crystal structure-based H4R homology models. Important residues in the binding pockets are displayed as gray ball-and-sticks. Nitrogen and oxygen are colored blue and red, respectively. H-bonds between polar hydrogen atoms in the ligand (pale cyan) and hydrogen acceptors in H4R residue side chains are depicted as black dotted lines. TM helices 5, 6, and 7 are presented as light-yellow cartoons, while the part of helix 3 containing the residue D943.32 and W903.28 and part of the loop between helix 4 and 5 containing the residue E16345.49 are presented as ribbons.

Scheme 1a

Reagents and conditions: (i) DCM, DIPEA, 20 °C, 24 h; (ii) (a) R2# = H: NMP, 7 bar NH3 atmosphere, 150 °C, 3 d, (b) R2 = isobutyl :isobutylamine, 130 °C, 24 h; (iii) aqueous 3M hydrochloric acid, 20 °C, 16 h; (iv) NMP, DIPEA, 80 °C, 3 d. a

only five examples bearing two pyrimidine subunits appear in the patent application,25 and therefore structure−activity relationships cannot be established based on these data. Furthermore, it was not directly obvious which part of bispyrimidine 5 serves as methylpiperazine replacement. To provide a detailed understanding of the receptor−ligand

Recently, pyrimidine-containing H4R ligands with the core structure of compound 5 were published in a patent application that was filed by Abbott Laboratories.25 In contrast to the previously reported pyrimidine-containing H4R ligands 2−4, this new derivative 5 does not contain a strongly basic amine but instead has a second pyrimidine subunit. Unfortunately, 4265



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Scheme 2a

Reagents and conditions: (i) CuI, Pd(DPPF)Cl2, K2CO3, DME, water, 90 °C, 3 h; (ii) methanol, TEA, Pd on carbon, 20 °C, 4 bar H2 atmosphere, 2 h; (iii) Pd(DPPF)Cl2, Cs2CO3, THF, NMP, water, 100 °C, 16 h. a

Scheme 3a

a

Reagents and conditions: (i) methanol, TEA, Pd on carbon, PdOH, 20 °C, 4 bar H2 atmosphere, 3 h; (ii) isobutylamine, 130 °C, 24 h.

Scheme 4a

Reagents and conditions: (i) NMP, DIPEA, 80 °C, 24 h; (iia) Pd(DPPF)Cl2, Cs2CO3, THF, NMP, water, 100 °C, 16 h; (iib) CuI, Pd(DPPF)Cl2, K2CO3, DME, water, 90 °C, 3 h, followed by methanol, TEA, Pd on carbon, 20 °C, 4 bar H2 atmosphere, 2 h; (iii) aqueous 3M hydrochloric acid, 20 °C, 16 h; (iv,v) NMP, DIPEA, 80 °C, 24 h.

a

Previous combined ligand SAR, H4R mutagenesis, and H4− ligand modeling studies indicate that the piperazine moieties of 1a−b and 2a−b form H-bond interactions with D943.32, while the indole moieties of 1a−b and the aminopyrimidines of 2a−b form H-bond interactions with E1825.46 (see Figure 1b).30 Interestingly, the monkey H4R mimicking41 L1755.39V mutation affects binding of the chlorine substituted compounds 1a and 2a but does not significantly affect the binding affinity of the unsubstituted compounds 1b and 2b, indicating that the aromatic ring moieties of 1a−b and 2a−b are directed toward

interactions at the human H4R and enable comparison with the above-mentioned H4R ligands 1a−4, we set out to generate a comprehensive structure−activity relationships (SAR) for bispyrimidines and try to expand the generated know-how to the series of indolecarboxamides. This information was subsequently used to establish a reliable binding model of compound 5 in the human H4R. In a last step, we have investigated the metabolic stability of the most potent bispyrimidines. 4266



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Scheme 5a

Reagents and conditions: (i) Pd(DPPF)Cl2, Cs2CO3, THF, NMP, water, 100 °C, 16 h; (ii) CuI, Pd(DPPF)Cl2, K2CO3, DME, water, 90 °C, 3 h; (iii) methanol, TEA, Pd on carbon, 20 °C, 4 bar H2 atmosphere, 2 h; (iv) isobutylamine, 130 °C, 24 h.

a

Scheme 6a

a Reagents and conditions: (i) DCM, DIPEA, 0 °C, 16 h; (ii) Pd(DPPF)Cl2, Cs2CO3, THF, NMP, water, 100 °C, 6 h; (iii) isobutylamine, 130 °C, 24 h; (iv) aqueous 3M hydrochloric acid, 20 °C, 16 h; (v) NMP, DIPEA, 80 °C, 3 d; (vi) methanol, TEA, Pd on carbon, 20 °C, 4 bar H2 atmosphere, 3 h.

this residue.30 Furthermore, the binding affinities of 2a−b were decreased by the E1825.46Q mutation while the binding of 1a−b was not significantly affected by this mutation, indicating that the more basic aminopyrimidine moieties of 2a−b form an ionic interaction with the carboxylate side chain of E1825.46.30 MD simulations that considered the aminopyrimidine in the positively ionized double protonated form could better explain the ligand-dependent effects of the H4R mutation studies than simulations in which the aminopyrimidine ring was in its neutral form.30 SAR studies in combination with quantum mechanical conformation analyses and protein−ligand modeling studies indicated that the flexible side chains of 3 and 4 adopt energetically favorable conformations nearby L1755.39 in the H4R binding pocket.29

step to position 2. After deprotection of the Boc group, intermediates 10a−e for the synthesis of indolecarboxamides (see Scheme 2) were generated. A further nucleophilic aromatic substitution reaction of derivates 10a−e with 4,6-dichloropyrimidine-2-amine 11 led to the intermediates 12a−e (Scheme 1), which were used to synthesize several final compounds shown in Schemes 3−4. Derivatives with an alkyl chain at position 6 (24, 42) were synthesized from the chloro intermediates 12a−e applying a Sonogashira coupling followed by a reduction of the triple bond (Scheme 2). The key step in the synthesis of pyrimidines bearing an aryl moiety at position 6 (e.g., 5) was a Suzuki reaction starting from the intermediates 12a−e (Scheme 2). Compounds 23 and 41, which bear amine linked residues at position 6, were synthesized from the chloro intermediates 12a−b via a nucleophilic aromatic substitution reaction (Scheme 3). Palladium-catalyzed reduction of the carbon− chlorine bond with H2 led to ligand 22 and 36 (Scheme 3). Nucleophilic aromatic substitution reaction of 4,6-dichloropyrimidine-2-amine 11 with amine 7a followed by a Suzuki reaction and Boc-deprotection led to intermediate 16a. Intermediate 16b was synthesized starting from intermediate 15 using a Sonogashira coupling with 3-methylbut-1-yne 13 followed by a reduction of the triple bond and a Boc

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CHEMISTRY Schemes 1−8 illustrate the routes that were used to synthesize the compounds described herein. Using a nucleophilic aromatic substitution reaction, tert-butyloxycarbonyl (Boc) protected diamines 7a−c were introduced at position 4 of 2,4dichloropyrimidine 6a. Regiochemistry of this reaction was determined with ROESY−NMR spectra, which proves the structure of 8a−c. Amines 9a−b were substituted in a second 4267



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affinity. The SAR explorations (see Table 1) focused on three parts of the bispyrimidines (Figure 2). Pyrimidine subunit I consists of a pyrimidine which is optionally substituted at position R2 and R6. Pyrimidine subunit II consists of a 2aminopyrimidine moiety, which is substituted with R2′ and R6′. Both pyrimidine subunits are connected via position 4 and 4′ with a linker subunit, which is an ethylene-diamine moiety optionally substituted with L1 and L2, where L1 is next to the pyrimidine subunit I and L2 is next to pyrimidine subunit II. Y3196.51 and Y953.33 are proposed to bind the aromatic rings of H4R ligands by π−π stacking interactions as indicated in previous in silico guided mutagenesis and SAR studies (e.g., Schulte et al30 and Lim et al41) and divide the H4R binding pocket into two parts (Figure 1, Y953.33 is not shown in Figure 1b for clarity but is located next to D943.32). The first part is located between W903.28, E16345.49, and Y3196.51, while the second part is located between Y3196.51, E1825.46, and L1755.39. The assignment of the two subunits of the bispyrimidines to the above-described areas will be part of the following discussion.

deprotection. Derivatives 29−32 were synthesized by a further nucleophilic aromatic substitution reaction of intermediate 16a or 16b with electrophiles 6b,c or 11 (Scheme 4). Compounds bearing substituents at position R6 and R6′ were synthesized from intermediate 31 applying a Sonogashira coupling with 3-methylbut-1-yne 13 followed by a reduction of the triple bond, a Suzuki reaction with phenylboronic acid 14a, or a nucleophilic aromatic substitution reaction with isobutylamine 9b (Scheme 5). Nucleophilic aromatic substitution reaction of 2,4,6-trichloro-pyrimidine 17 with amine 7a followed by a Suzuki reaction with phenylboronic acid 14a led to 18. Regiochemistry of this reaction was determined with ROESY−NMR spectra, which proves the structure of 18. A second nucleophilic aromatic substitution reaction with isobutylamine 9b and Boc deprotection led to intermediate 19. Derivative 33 was synthesized by a further nucleophilic aromatic substitution reaction of intermediate 19 with electrophile 11 (Scheme 6). Nucleophilic aromatic substitution reaction of 4,6-dichloropyrimidine with amine 10a or 16a followed by an additional nucleophilic aromatic substitution reaction with isobutylamine led to 37 and 38 (Scheme 7). Scheme 7a

Figure 2. Bispyrimidines: nomenclature.

The core scaffold of the bispyrimidines, represented by 22, already displays a pKi on the H4R of 6.1 ± 0.1. The introduction of an aminoisobutyl moiety at position R6′ of the pyrimidine subunit II (bispyrimidine derivate 23) increases the H4R affinity by a factor of 10 (23 compared to 22). The level of H4R affinity of the corresponding 6-aminoalkyl-2aminopyrimidines with basic amines (e.g., represented through 4, pKi = 8.5 ± 0.1) cannot be reached. This indicates that bispyrimidine 23 cannot form all interactions identified for 429 or makes new detrimental interactions. A 30-fold increased H4R affinity compared to 22 can be observed if an isopentyl residue is introduced at the position R6′ of the pyrimidine subunit II (bispyrimidine derivate 24). These data indicate that in case of the 6-alkyl-pyrimidine series (e.g., represented by 3, pKi = 8.5 ± 0.1) the methylpiperazine moiety can be replaced by a N′-pyrimidine-2-ylethane-1,2diamine moieties (linker + subunit I) while losing minor binding affinity on H4R (3−8-fold, bispyrimidine 24 and 42). Introduction of a phenyl moiety at the position R6′ of pyrimidine subunit II (compound 5) leads to a 100-fold increased H4R affinity compared to 22. The level of H4R affinity of the corresponding 6-phenyl-2-aminopyrimidines with basic amines (e.g., represented by 2, pKi = 8.1 ± 0.1) can thus be reached by the replacement of the methylpiperazine with subunit I in combination with the appropriated linker. Interestingly, the introduction of a chlorine atom at the meta position of the phenyl residue of R6′ leads only to marginal changes in H4R affinity (compare 5 vs 25), which is in contrast with the indolcarboxamide series but similar to the series of 6-

Reagents and conditions: (i) NMP, DIPEA, 80 °C, 3 h; (ii) NMP, DIPEA, 100 °C, 3 d. a

Indolcarboxamide derivates 46−48 were synthesized using an amide coupling of the acids 21a−b and amines 10a−b (Scheme 8). Scheme 8a

a

Reagents and conditions: (i) HATU, DIPEA, DMF, 20 °C, 1−16 h.

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BIOCHEMISTRY H4R Radioligand Displacement Assay. The affinity of the various compounds was determined by the displacement of [3H]histamine binding from the human H4R as described previously.38 The given pKi values are mean values ± SEM of at least three independent determinations.

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RESULTS AND DISCUSSION To map the binding site of 5 in the H4R receptor, a series of bispyrimidine compounds were synthesized and tested for H4R 4268



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Table 1. H4R Binding Affinity of Bispyrimidines

aryl-2-aminopyrimidines with basic amines,20 e.g., represented by 2. Bispyrimidine 26, which bears on both pyrimidine subunits (R6 and R6′) a phenyl moiety, displays a 150 lower H4R affinity compared to 5. Compound 27 and 28 demonstrate that also the combination of isopentyl or isobutylamino as R6 for pyrimidine subunit I with phenyl as R6′ for pyrimidine subunit II lead to a drop in H4R affinity compared to 23 and 24. This

shows that only one pyrimidine subunit tolerates such kind of substitution at R6/R6′ and indicates that 6-aryl, 6-alkyl-, and 6alkylamino-2-amino-pyrimidines (representing subunit II) only bind in one defined orientation to the H4R. This finding supports the former binding studies with 2−4, which also reveal only one defined binding pose for such pyrimidine derivatives.29,30 4269



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Removal of the amino-group at position R2 of 5 leads to bispyrimidine 29 (see Figure 2 and Table 1), which displays a 250-fold lower H4R affinity compared to 5. A 40-fold improved affinity compared to 29 is observed if a methyl group is introduced at position R2 (see Figure 2 and Table 1; compound 30). The subunit I of 29, an unsubstituted 4aminopyrimidine, has a pKa value of 5.7.31,32 The pKa value is increased by the introduction of a methyl group at position R2 to 6.533 (subunit I of 30) and is further increased by an amino residue at position R2 to 7.431,34 (subunit I of 5). Thus, there is a high probability that the pyrimidine subunit I of 30 and 5 will be protonated during binding due to their increased basicity. This protonation allows the formation of an additional H-bond or an ionic interaction with the H4R and can therefore explain the increased H4R affinity of 5 and 30 compared to 29. The protonation of the pyrimidine subunit I also increases the acidity of the L1 proton (see Figure 3), thereby enhancing the H-bond ability of this proton.35,36 Compound 31 carrying a chlorine atom at position R6 displays a clearly lower H4R affinity compared to 5. Similar to the phenyl substituted bispyrimidine 31 also in the isopentyl series (32), introduction of a chlorine atom at position R6 of the pyrimidine subunit I leads to a dramatic drop of H4R affinity. The reduced affinity of compounds 31 and 32 is in line with the above proposed hypothesis because the introduction of the chlorine atom will reduce the pKa value of the pyrimidine subunit I to 3.6.34 Subunit I will consequently not be protonated, and there will be one H-bond less available for binding to the H4R. Steric limitations in the H4R binding pocket around the position of the chlorine atom could be an additional reason for the drop in affinity of compounds 31 and 32. Previously it was shown by Altenbach et al. that substituents at the 2-amino residue of 6-aryl-2-aminopyrimidines (e.g., 2) results in a drop of H4R affinity by 2 log units.20 Similar to the observations of Altenbach et al.,20 a more then 100-fold drop in H4R affinity can be seen if an isobutyl residue is introduced at position R2′ of the pyrimidine subunit II (compare compound 5 vs 33). Bispyrimidines 34 and 35 demonstrate that the drop of H4R affinity is clearly less pronounced if an isobutylamino residue is introduced to the pyrimidine subunit I at position R2. Derivatives 34 and 35 display only a 7−10-fold decreased H4R affinity compared to 5, which indicates that the H4R binding site for subunit I has clearly distinct binding properties compared to the H4R binding site of subunit II. These observations further corroborate the suggestion that in the bispyrimidine series subunit I can be seen as a replacement of the methylpiperazine. Interestingly, removal of the phenyl moiety of 34 leads to bispyrimidine derivative 36 and does not affect the H4R affinity (Table 1). In the case of 36, it is not directly obvious which pyrimidine subunit replaces the methylpiperazine moiety. Compound 33 clearly shows that at position R2′ of the pyrimidine subunit II no substituent is tolerated, whereas compound 34 and 35 demonstrate that at position R2 of the pyrimidine subunit I alkylamino substituents are tolerated with respect to H4R affinity. These data strongly suggest that the 2isobutylamino-pyrimidine moiety of 36 binds to an area of the H4R where usually the pyrimidine subunit I is located (see Figure 3) and therefore can be considered as the methylpiperazine replacement.

Figure 3. Assignment of pyrimidine subunits for 34, 36, 37, and 38.

Omitting the 2-amino moiety at one subunit and introducing an aminobutyl residue at position R6 of the same subunit (in that case equivalent to R6′) leads to compound 37. Also in the case of 37, it is not directly obvious which pyrimidine subunit replaces the methylpiperazine moiety. However, compared to 37, a slight gain in H4R affinity is observed for 38, which carries at position 6 of one pyrimidine core an aminoisobutyl group and at the other pyrimidine moiety at position 6 a phenyl residue. This indicates that the 6-isobutylamino-pyrimidine core and the 6-phenyl-2-aminopyrimidine moiety can bind to different areas in the H4R because if they would preferably bind to the same region a clear drop in affinity of the H4R would be expected for bispyrimidine 38. It was already shown that a phenyl moiety is not tolerated at position R6 of the pyrimidine subunit I (compare 5 and 26). These SAR considerations suggest that the 6-isobutylamino-pyrimidine moiety of 37 binds to an area of the H4R where usually the pyrimidine subunit I is located (see Figure 3) and therefore can be considered as the methylpiperazine replacement. Next step in the SAR investigation of bispyrimidines was to determine the importance of the H-bond donors at L1 and L2 of the linker subunit (see Figure 2), which connects both pyrimidine subunits. Compounds 39 and 40, which have at position L2 a methyl group instead of a proton and therefore display no longer a H-bond donor function at this position, show a similar affinity on the H4R compared to 5 and 35, indicating that this H-bond donor is not important for binding to the H4R. Introduction of a methyl group to the linker at position L2 of 23, 24, and 34 led to bispyrimidines 41, 42, and 43, which display even a slightly increased H4R affinity compared to 23, 24, and 34. The relevance of a proton at position L1 for the binding of bispyrimidines to the H4R is illustrated by comparison of compound 5 and 44. The absence of the H-bond donor in compounds 44 leads to a 10-fold lower H4R affinity. Figure 4 summarizes the SAR investigation for bispyrimidines. Essential for high H4R affinity are phenyl, alkyl, or aminoalkyl moieties for R6′ and a NH2 group at position 2′ of the pyrimidine subunit II. L2 can accept both a proton or a methyl group, whereas for L1 the proton is necessary for good affinity. At R6 of the pyrimidine subunit I, no substituent is tolerated for optimal H4R affinity. Methyl or aminoalkyl substitution for R2 leads to compounds with pKi values up to 7.5 for H4R binding and with an unsubstituted amino function pKi values higher than 8 are achievable. Next step in our investigation was to test, whether the methylpiperazine replacements identified during the SAR 4270



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Table 2. Binding Affinity of Indolecarboxamide Analogues

Figure 4. Summary of SAR investigation for bispyrimidines. Subunit I shown in blue, subunit II shown in orange, linker unit shown in green.

Figure 5. Proposed binding modes of compounds 5 (purple carbon atoms, (A)) and 46 (green, (B)) in H1R crystal structure-based H4R homology models. Rendering and color coding is the same as in Figure 1. The binding pocket is depicted by a gray surface. Subpocket I (blue part in Figure 4) is located between W903.28, E16345.49, and Y3196.51, while subpocket II (orange part in Figure 4) is located between Y3196.51, E1825.46, and L1755.39. (B) The “loss” of the H-bond between the indole group and E1825.46 (compared to the conserved H-bond interaction in Figure 1b) is indicated by a solid blue arrow, while the distance between the indole C3 atom (indicated by an asterisk) and amino group and of 46 is indicated by a blue dotted arrow.

indolecarboxamides with basic amines,17,18,26 the introduction of a chlorine atom at position R5 of the indolecarboxamide scaffold (compound 46) leads to a 3−4-fold increase of the H4R affinity compared to 45. Compounds 47 and 48 show that introduction of a methyl group to the amide function causes a 10-fold drop in H4R affinity, indicating that a NH-donor containing amide is important for binding to the H4R.

exploration of bispyrimidines can be also used in the series of indolecarboxamides. Table 2 shows replacements of the 1-methylpiperazine moiety by 2-aminopyrimidines for the class of indolecontaining H4R ligands. Replacing 1-methylpiperazine of 1b with the 4-N-(2-aminoethyl)pyrimidine-2,4-diamine fragment leads to compound 45, which displays a 50-fold lower H4R affinity compared tothat of 1b. Similar to the series of 4271



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Table 3. In Vitro Metabolic Stability in Human Liver Microsomes (HLM) and Mouse Liver Microsomes (MLM) and Aqueous Solubility at pH 6.8 compd

half life in HLM [min]

half life in MLM [min]

1a 2 3 4

>45 43 61 >120

2 4 62 >64

The most potent indolecarboxamide derivatives of this series (45−48) still display a 10−50-fold lower H4R affinity than the classical indolecarboxamides that contain basic amines like the methylpiperazine moiety (e.g., 1a and 1b).17,18 Binding of Bispyrimidine Analyzed Using a Human H4R Homology Model. On the basis of our SAR analysis and previous in silico guided mutagenesis studies,30,37,41 we propose that the at N1′ protonated subunit II of bispyrimidine 5 forms an ionic interaction with the negatively ionizable side chains of E1825.46 (Figure 5A) in the same way as in the previously validated binding mode of pyrimidine 2b (Figure 5B).30 In addition, we suggest that a H-bond is formed between the L1 proton with D943.32, which is known from previous studies to be crucial for binding to the H4R.30 The similar distance between the pharmacophoric reference point in the pyrimidine core of 2 (N1 nitrogen) to the positive charge carried by the methylpiperazine (7 Ǻ ) and the distance between the pharmacophoric reference point in the pyrimidine subunit II (N1′ nitrogen) of 5 to the L1 proton of the pyrimidine subunit I (8 Ǻ ) suggests a comparable binding, which is shown in Figures 1a and 5a. Finally, an additional ionic interaction between the at N1 protonated subunit I of 5 and the negatively ionizable side chains of E16345.49 is proposed. The binding mode model of bispyrmidine 5 (Figure 5A) in our previously validated H4R homology model30,37,41 explains: (i) (i)The important role of the acidic L1 proton, which forms an H-bond with D943.32 (compare 5 to 44). (ii) That substitution of L2 is allowed as it is not involved in essential H-bond interactions (compare 5 to 39). (iii) That R6 substituents of subunit I (see Figure 4: blue part) different than hydrogen are not tolerated, as these groups do not fit the limited space between D943.32 and W903.28 (compare 5 to 26, 27, 28, 31, and 24 to 32). (iv) That large R6′ substituents of subunit II (see Figure 4: orange part) are allowed, as these groups can bind in the extracellular region of the H4R binding site close to L1755.39 (compare 22 to 5, 23, 24, and 25).30 (v) That the increased basicity of the pyrimidine subunit I (see Figure 4: blue part) of compound 5 compared to 29 and 3031−34 leads to a higher affinity as a result of the proposed stronger ionic interactions with the negatively ionizable carboxylate group E16345.49 in the second extracellular loop (compare 5 to 29 and 30). (vi) The important role of the basic 2-aminopyrimidine moiety of subunit II (see Figure 4: orange part) that is proposed to interact with E1825.46 in the same way as ligand 2 in Figure 5B.30 (vii) That no substituents are tolerated at R2′ as these residues do not fit the limited space around E1825.46. (viii) That the presence of aminoisobutyl at R2 and phenyl at R6′ (compound 34 and 43) will lead to steric clashes of this moieties, explaining the loss of H4R affinity if the R2 moiety is increased from an unsubstituted amino group

compd

half life in HLM [min]

half life in MLM [min]

solubility @ pH 6.8 [μg/mL]

24 25 41 42

>120 51 101 82

18 9 23 8

>79 >76 >84 >83

to the aminoisobutyl moiety (compare compound 5 and 34 or 39 and 43). (ix) Why no significant increase in H4R affinity is observed when a phenyl moiety is introduced at position R6′ in the presence of the aminoisobuthyl moiety at R2 (compare 36 with 34). Previously, it was shown that substituents larger than methyl at the piperazine moiety of 2 lead to a dramatic drop in H4R affinity.20 Yet, large substituents like pyrimidine moieties are tolerated by the H4R if such moieties are connected via an ethylenediamine linker to the 6-aryl-2amino-pyrimidine scaffold (e.g., 5, 25, and 39). This perceived discrepancy can also be explained by the model presented in Figure 5. It can be clearly shown that the piperazine residue offers a completely different exit vector for substituents than the ethylenediamine. The exit vector in the case of the piperazine substituted pyrimidines points to a lipophilic subpocket which is mainly occupied by tryptophane W903.28 and is only large enough to accommodate a methyl group. In contrast, the ethylenediamine moiety points into a subpocket (between tryptophane W903.28, glutamate E16345.49, and tyrosine Y3196.51) which is big enough to bind a pyrimidine substituted at position 2. The SAR data and binding mode models suggest that although the binding modes of pyrimidines and indole carboxamides overlap,30 the molecular determinants of H4R binding by pyrimidines and indolecarboxamides are not identical.30 This is in line with previous mutation studies that indicated that ionic interactions with the negatively charged carboxylate group of E1825.46 are more important for binding of the basic aminopyrimidine moiety of compound 2 (Figure 1a) than for the binding of the neutral indole carboxamide group in compounds 1b (Figure 1b).30,37,41 Apparently the strong ionic interactions of the two basic moieties in the aminopyrimidines series allow more variation in the geometry/type of the basic groups (and linker between these groups). The SAR of the indole carboxamide series on the other hand suggests that the relative orientation of the single basic group and the aromatic moiety is more restricted for this series as the optimal orientation of the aromatic moiety in the H4R pocket is more important. Comparison of the proposed binding modes of bispyrimidines 5 (Figure 5A) and 2-aminopyrimidine 2 (Figure 1a) and the binding modes of indolecarboxamides 1b (Figure 1b) and 46 (Figure 5B) suggests that the pyrimidine subunit I (see Figure 4: blue part) does not allow the indole ring of 46 to adopt an optimal orientation in the H4R binding pocket. The two aminopyrimidine rings of 5 can form H-bonds with D943.32 and E1825.46 simultaneously (Figure 5A). The indole ring of 46 however has to adopt a conformation perpendicular to the aminopyrimidine group (subunit II of 5) to avoid a steric clash between the 2-amino group and the (C3 atom of the) indole ring. In this binding orientation, 46 cannot form a H-bond interaction with E1825.46 (like in the experimentally vali4272



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dated29,37 binding mode of 1b presented in Figure 1b). This could be an additional explanation why the binding conformation of compounds 45, 46, 47, and 48 are unfavorable. This binding mode hypothesis is in line with point (viii), that the aminoisobutyl at R2 of compound 34, 39, or 43 leads to a steric clash with the R6′ phenyl moiety, which can also explain the observed affinity drop (compare compound 5 and 34 or 39 and 43). Investigation of in Vitro Metabolic Stability. The indolecarboxamide 1a and the pyrimidines 2−4 display a very short half-life in mouse liver microsomes (Table 3 and see also27). This compromises in vivo experiments with these compounds. The bispyrimidine 25 and 42 show a slightly improved microsomal stability in mice. The microsomal stability can be further improved by removing the methyl group of the linker moiety (L2) of compound 42, which is shown by 24. A further option for improvement of metabolic stability in mouse liver microsomes is the replacement of the R6′ alkyl moiety of 42 by an alkylamino group, which leads to compound 41. Compound 24 and 41 display a 5−10-fold increased half-life on mice liver microsomes compared to the starting points 1−4 and show still a reasonable affinity on the human H4R. Moreover, also on the mouse H4R, both compounds still show submicromolar affinity (unpublished observations Nijmeijer). All investigated bispyrimidines display a comparable long half-life on human liver microsomes and the same excellent solubility at pH 6.8 as the starting points 1−4. Interestingly, the ratio between half-life in HLM and MLM is in general greater for the reference compounds 1−4 then the ratio for the bispyrimidines 24, 25, 41, and 42 (1−4, 10−30; 24, 25, 41, and 42, 5−10). It is known that the total amount of cytochrome P450 and/or abundances of individual cytochromes (i.e., 2C19) is different across species.46 A possible explanation for the differences depicted in Table 3 could be that the metabolic responsible cytochrome(s) for 1−4 are different to those for 24, 25, 41, and 42. A similar observation for lack of proportionality between compound half-lives in human and in mouse is shown by Miyata J. et al. for amino substituted pyrimidines.47

amides. The new insights in H4R−ligand interaction will aid the future rational design of new H4R ligands.

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EXPERIMENTAL SECTION

General Remarks. Chemicals and reagents were obtained from commercial suppliers and were used without further purification. All moisture sensitive reactions were carried out under a nitrogen atmosphere in commercially available anhydrous solvents. Proton and carbon NMR spectra were obtained on a Bruker Advance 400 FTNMR or Bruker Advance 500 FT-NMR instrument with chemical shifts (δ) reported relative to tetramethylsilane as an internal standard. High resolution mass spectrometry data were obtained on a LTQ Orbitrap XL (Thermo Scientific) equipped with a NSI Source (Advion Nanomate) in ESI positive mode. Analytical HPLC-MS analyses were conducted using an Agilent 1100 series LC/MSD system. The analytic methods A1 and A2 are defined in the Supporting Information Table S1. Compound purities were calculated as the percentage peak area of the analyzed compound by UV detection at 254 nm. If purity data is not explicitly mentioned, the compound displays a purity >95%. Flash column chromatography was carried out using hand packed silica gel 60 (230−400 mesh) or prepacked silica gel columns from Biotage, and product was eluted under medium pressure liquid chromatography. Preparative high performance chromatography was carried out on a Gilson system (pump system, 333 and 334 prep-scale HPLC pump; fraction collector, 215 liquid handler; detector, Gilson UV/vis 155) using prepacked reversed phase silica gel columns from waters. The methods for preparative high performance chromatography P1−P3 are defined in the Supporting Information Table S1. 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]pyrimidine-2,4diamine (22). 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-6chloropyrimidine-2,4-diamine 12a (20 mg, 0.08 mmol) TEA (11 mg, 0.11 mmol), 5 mg of palladium hydroxide, and 5 mg of palladium on carbon (5%) were suspended in 10 mL of methanol. The mixture was stirred for 3 h at 4 bar hydrogen atmosphere and 20 °C. Afterward, the suspension was filtered. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 10 mg (51%, 0.04 mmol) of the title compound as a pale-yellow solid. Purity by method A1, >95%; RT = 0.31 min. MS (ESI+) m/z 247 (M + H)+. HRMS (ESI+) m/z found 247.1412 [M + H]+, C10 H15 N8 requires M+ 247.142. 1H NMR (400 MHz, DMSO) δ (ppm) 7.60 (d, J = 5.7 Hz, 2H), 6.84 (br, 2H), 5.82 (s, 4H), 5.73 (d, J = 5.9 Hz, 2H), 3.23−3.19 (m, 4H). Using the same method 36 was synthesized. 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-6-N-(2methylpropyl)pyrimidine-2,4,6-triamine (23). 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-6-chloropyrimidine-2,4-diamine 12a (44 mg, 0.16 mmol) was dissolved in 3 mL of isobutylamine, and the mixture was stirred for 24 h at 130 °C. Then 50 mL of DCM and 50 mL of an aqueous sodium hydrogen carbonate solution were added to the reaction mixture, and the organic layer was separated and dried over magnesium sulfate. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 13 mg (24%, 0.04 mmol) of the title compound as a gray solid. Purity by method A1, >95%; RT = 0.99 min. MS (ESI+) m/z 318 (M + H)+. HRMS (ESI+) m/z found 318.2153 [M + H]+, C14 H24 N9 requires M+ 318.2155. 1H NMR (500 MHz, DMSO) δ (ppm) 7.61 (d, J = 5.7 Hz, 1H), 6.89 (br, 1H), 6.03 (br, 2H), 6.97 (br, 1H), 5.83 (s, 2H), 5.73 (d, J = 5.7 Hz, 1H), 5.33 (s, 2H), 3.27−3.22 (m, 2H), 2.93−2.88 (m, 4H), 1.81−1.70 (m, 1H), 0.87 (d, J = 6.7 Hz, 6H). Using the same method, 41 was syntheisized. 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-6-(3methylbutyl)pyrimidine-2,4-diamine (24). Compound 12a (4-N[2-[(2-aminopyrimidin-4-yl)amino]ethyl]-6-chloropyrimidine-2,4-diamine (48 mg, 0.17 mmol), 3-methylbut-1-yne (58 mg, 0.85 mmol), potassium carbonate (82 mg, 0.59 mmol), copper iodide (6 mg, 0.03 mmol), and Pd(DPPF)Cl2 (28 mg, 0.03 mmol) were suspended in 1.5 mL of a 1:1 mixture of DME and water. The reaction mixture was flushed with argon and stirred for 3 h at 90 °C. Afterward, water and DCM was added to the mixture and the organic layer was separated

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CONCLUSION Following the initial description of 5 bispyrimidines as H4R ligands in a patent by Abbott laboratories, we have developed a detailed SAR, leading to bispyrimidines with pKi values up to 8 for binding the human H4R. The comprehensive SAR analysis in combination with homology modeling enabled us to establish a detailed binding model of bispyrimidine 5 in the human H4R and allowed us to identify the pyrimidine subunit that is replacing the N-methylpiperazine moiety found in most H4R ligands. Furthermore, it is shown that the successful replacement of the metabolically labile N-methylpiperazine leads to, e.g., the bispyrimidines (24 and 41), displaying a clearly improved in vitro metabolic stability (mouse) in combination with a reasonable affinity on the human H4R. However, pharmacokinetic and pharmacology studies are necessary to prove whether the improved in vitro metabolic stability in mouse translates into improved in vivo efficacy. Our attempts to identify similar pyrimidine alternatives for the Nmethylpiperazine moiety in the indolecarboxamide series leads to only moderately active derivatives, suggesting distinct binding modes for the indolcarboxamides and bispyrimidines or an unfavorable binding conformation of the indolecarbox4273



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and dried, and the solvent was removed under reduced pressure. The crude product was purified using method P3, yielding 17 mg (31%, 0.05 mmol) of 4-N-[2-[(2-aminopyrimidin-4-yl)amino]ethyl]-6-(3methylbut-1-ynyl)pyrimidine-2,4-diamine. This intermediate, TEA (11 mg, 0.11 mmol), and 5 mg palladium on carbon (5%) were dissolved in 20 mL of methanol. The mixture was stirred for 2 h at 4 bar hydrogen atmosphere and 20 °C. Afterward, the suspension was filtered. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 15 mg (87%, 0.04 mmol) of the title compound as an off-white solid. Purity by method A1, >95%; RT = 1.08 min. MS (ESI+) m/z 317 [M + H]+. HRMS (ESI+) m/z found 317.2211 [M + H]+, C15 H25 N8 requires M+ 317.2202. 1H NMR (500 MHz, DMSO) δ (ppm) 7,61 (d, J = 4.7 Hz, 1H), 6.83 (br, 1H), 6.67 (br, 1H), 5.82 (s, 2H), 5.76 (s, 2H), 5.72 (d, J = 4.7 Hz, 1H), 5.59 (s, 1H), 3.28−3.23 (m, 4H), 2.28−2.21 (m, 2H), 1.57−1.47 (m, 1H), 1.46−1.38 (m, 2H), 0.88 (d, J = 6.6 Hz, 6H). 13C NMR (125 MHz, DMSO) δ (ppm) 163.58, 163.17, 163.09, 162.95, 162.90, 155.87 (not visible in 1D 13C NMR; determined with HSQC), 96.82 (not visible in 1D 13C NMR; determined with HSQC), 94.19 (not visible in 1D 13C NMR; determined with HSQC), ∼40 (both ethyl carbons under the DMSO peak; determined with HSQC), 37.47, 34.91, 27.27, 22.41. Using the same method, 42 was synthesized. 4-N-[2-[(2-Amino-6-chloropyrimidin-4-yl)amino]ethyl]-6phenylpyrimidine-2,4-diamine (31). 4,6-Dichloro-pyrimidine-2amine 11 (100 mg, 0.61 mmol), DIPEA (467 mg, 3.62 mmol), and 4-N-(2-aminoethyl)-6-phenylpyrimidine-2,4-diamine 16a (82 mg, 0.36 mmol) were suspended in 3 mL of NMP and stirred for 24 h at 80 °C. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 90 mg (41%, 0.25 mmol) of the title compound as a pale-yellow solid. Purity by method A1, >95%; RT = 1.11 min. MS (ESI+) m/z 357/359 (M + H)+, Cl distribution. HRMS (ESI+) m/z found 357.1352 [M + H]+, C16 H18 Cl N8 requires M+ 357.1343. 1H NMR (500 MHz, DMSO) δ (ppm) 7.91−7.86 (m, 2H), 7.44−7.41 (m, 3H), 7.26 (br, 1H), 6.97 (br, 1H), 6.41 (s, 2H), 6.23 (s, 1H), 5.99 (s, 2H), 5.77 (br, 1H), 3.45−3.39 (m, 4H). Using the same method 29, 30 and 32 were synthesized. 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-2-N-(2-methylpropyl)-6-phenylpyrimidine-2,4-diamine (33). 4,6-Dichloro-pyrimidine-2-amine 11 (40 mg, 0.24 mmol), DIPEA (467 mg, 1.45 mmol), and 4-N-(2-aminoethyl)-2-N-(2-methylpropyl)-6-phenylpyrimidine-2,4-diamine 19 (49 mg, 0.17 mmol) were suspended in 2 mL of NMP and stirred for 3 days at 80 °C. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 20 mg (20%, 0.05 mmol) of tert-butyl N-[2-[[2(2-methylpropylamino)-6-phenylpyrimidin-4-yl]amino]ethyl]carbamate. This intermediate, TEA (50 mg, 0.50 mmol), 5 mg of palladium hydroxide, and 5 mg of palladium on carbon (5%) were suspended in 10 mL of methanol. The mixture was stirred for 3 h at 4 bar hydrogen atmosphere and 20 °C. Afterward, the suspension was filtered. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 16 mg (84%, 0.04 mmol) of the title compound as a pale-yellow solid. Purity by method A1, >95%; RT = 1.29 min. MS (ESI+) m/z 379 (M + H)+. HRMS (ESI+) m/z found 379.2352 [M + H]+, C20 H27 N8 requires M+ 379.2359. 1H NMR (500 MHz, DMSO) δ (ppm) 8.89−8.85 (m, 1H), 8.25−8.22 (m, 2H), 6.64−6.55 (m, 2H), 7.59 (d, J = 7.2 Hz, 1H), 7.42 (s, 3H), 6.70 (br, 2H), 6.03 (d, J = 7.2 Hz, 1H), 5.38 (s, 1H), 3.58−3.51 (m, 4H), 3.11−3.02 (m, 2H), 1.91−1.81 (m, 1H), 0.92 (d, J = 6.6 Hz, 6H). 4-N-[2-[[6-(2-Methylpropylamino)pyrimidin-4-yl]amino]ethyl]-6-phenylpyrimidine-2,4-diamine (38). 4,6-Dichloro-pyrimidine 20 (120 mg, 0.81 mmol), DIPEA (624 mg, 4.83 mmol), and 4N-(2-aminoethyl)-6-phenylpyrimidine-2,4-diamine 16a (110 mg, 0.48 mmol) were suspended in 3 mL of NMP and stirred for 24 h at 80 °C. Afterward, isobutylamine (2.19 g, 29,94 mmol) was added and the reaction mixture was stirred for 3 days at 100 °C. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 74 mg (24%, 0.20 mmol) of the title

compound as a gray solid. Purity by method A1, >95%; RT = 1.15 min. MS (ESI+) m/z 379 (M + H)+. HRMS (ESI+) m/z found 379.2353 [M + H]+, C20 H27 N8 requires M+ 379.2359. 1H NMR (500 MHz, DMSO) δ (ppm) 7.93−7.87 (m, 3H), 7.46−7.40 (m, 3H), 6.97 (br, 1H), 6.64−6.55 (m, 2H), 6.27 (br, 1H), 5.98 (s, 2H), 5.37 (s, 1H), 3.46−3.37 (m, 2H), 3.31−3.26 (m, 2H), 2.98−2.89 (m, 2H), 1.84−1.72 (m, 1H), 0.87 (d, J = 6.8 Hz, 6H). Using the same method, 37 was synthesized. 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-4-N-methyl-6N-(2-methylpropyl) pyrimidine-2,4,6-triamine (41). Derivative was synthesized according to compound 23 as a pale-yellow solid. Purity by method A1, >95%; RT = 1.07 min. MS (ESI+) m/z 332 [M + H]+. HRMS (ESI+) m/z found 332.2302 [M + H]+, C15 H26 N9 requires M+ 332.2311. 1H NMR (500 MHz, DMSO) δ (ppm) 7.60 (s, 1H), 6.83 (br, 1H), 6.11−6.04 (m, 1H), 5.81 (s, 2H), 5.73−5.68 (m, 1H), 5.36 (s, 2H), 4.90 (s, 1H), 3.58−3.49 (m, 2H), 3.29−3.21 (m, 2H), 2.97−2.90 (m, 2H), 2.88 (s, 3H), 1.81−1.69 (m, 1H), 0.86 (d, J = 6.5 Hz, 6H). 13C NMR (125 MHz, DMSO) δ (ppm) 164.34, 163.21, 162.96, 162.87, 162.27, 155.66 (not visible in 1D 13C NMR; determined with HSQC), 96.82 (not visible in 1D 13C NMR; determined with HSQC), 73.16 (not visible in 1D 13C NMR; determined with HSQC), 48.04, 47.53, ∼40 (signal of one carbon atom under the DMSO peak; determined with HSQC), 35.73, 27.93, 20.33. 4-N-[2-[(2-Aminopyrimidin-4-yl)-methylamino]ethyl]-6-phenylpyrimidine-2,4-diamine (44). 4-N-[2-[(2-Aminopyrimidin-4yl)-methylamino]ethyl]-6-chloropyrimidine-2,4-diamine 12c (60 mg, 0.21 mmol), phenylboronic acid (56 mg, 0.45 mmol), cesium carbonate (283 mg, 0.81 mmol), and Pd(DPPF)Cl2 (17 mg, 0.02 mmol) were suspended in 1 mL of a 3:1:1 mixture of THF, NMP, and water. The reaction mixture was flushed with argon and stirred for 16 h at 100 °C. The crude product was purified using method P3, yielding 40 mg (57%, 0.12 mmol) of the title compound as an off-white solid. Purity by method A1, >95%; RT = 0.97 min. MS (ESI+) m/z 337 (M + H)+. HRMS (ESI+) m/z found 337.1894 [M + H]+, C17 H21 N8 requires M+ 337.1889. 1H NMR (500 MHz, DMSO) δ (ppm) 7.89− 7.86 (m, 2H), 7.74 (d, J = 4.7 Hz, 1H), 7.45−7.40 (m, 3H), 6.97 (br, 1H), 6.21 (s, 1H), 5.98 (s, 2H), 5.94 (d, J = 4.7 Hz, 1H), 5.89 (s, 2H), 3.63−3.56 (m, 2H), 3.37−3.41 (m, 2H), 2.99 (s, 3H). Using the same method, 5, 25, 34, 35, 39, 40, and 43 were synthesized. N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-1H-indole-2-carboxamide (45). Indole-2-carboxylic acid 21a (20 mg, 0.12 mmol), DIPEA (97 mg, 0.75 mmol), and HATU (52 mg, 0.14 mmol) were suspended in 1.5 mL of DMF and stirred for 10 min. Afterward, 4-N(2-aminoethyl)pyrimidine-2,4-diamine 10a (21 mg, 0.14 mmol) was added and the reaction mixture was stirred for a further 2 h at 20 °C. Then 50 mL of DCM and 50 mL of an aqueous sodium hydrogen carbonate solution were added to the reaction mixture, and the organic layer was separated and dried over magnesia sulfate. The solvent was removed under reduced pressure, and the crude product was purified using method P3, yielding 29 mg (81%, 0.10 mmol) of the title compound as a gray solid. Purity by method A1, >95%; RT = 1.11 min. MS (ESI+) m/z 297 (M + H)+. HRMS (ESI+) m/z found 297.1458 [M + H]+, C15 H17 N6 O requires M+ 297.1464. 1H NMR (500 MHz, DMSO) δ (ppm) 11.55 (s, 1H), 8.56−8.51 (m, 1H), 7.62 (br, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.19−7.14 (m, 1H), 7.09 (d, J = 1.7 Hz, 1H), 7.05−7.00 (m, 1H), 6.93 (br, 1H), 5.87 (s, 2H), 5.75 (d, J = 4.5 Hz, 1H), 3.44−3.39 (m, 4H). Using the same method, 46−48 were synthesized. H4R Radioligand Displacement Assay. The affinity of the various compounds was determined by the displacement of [3H]histamine binding from the human H4R as described previously.38 The given pKi values are mean values ± SEM of at least three independent determinations. Determination of Solubility. Solubility measurements were performed by DMSO solution precipitation. This methodology is in line with the method described by K. Sugano et al.39 Microsomal Stability Assay. Liver microsomes were purchased from Xenotech, and the stability assay for the determination of t1/2 was performed as described in a publication from S. M. Skaggs et al.40 4274



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Computational Methods. The H4R homology model and the binding pose of compound 2 and 1b was derived as previously described.30,37,41 Compound 5 was docked in the receptor model using the PLANT42,43 docking algorithm without imposing any restraints. The first ranked binding pose that was overlaying with compound 2 and showing adjacent interactions with the second pyrimidine ring was selected for further energy minimization in MOE44 version 2011.10.27 with fixed backbone atoms. The only Hbond restraints that needed to be applied to keep the interaction was between one of the oxygens of E1825.46 and the adjacent NH2 group of the pyrimidine. Additional dihedral energy constraints were applied to keep the amino groups of both pyrimidine rings in plane with the respective pyrimidine ring.

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(4) Zhang, M.; Thurmond, R. L.; Dunford, P. J. The histamine H4 receptor: a novel modulator of inflammatory and immune disorders. Pharmacol. Ther. 2007, 113, 594−606. (5) Damaj, B. B.; Becerra, C. B.; Esber, H. J.; Wen, Y.; Maghazachi, A. A. Functional expression of H4 histamine receptor in human natural killer cells, monocytes, and dendritic cells. J. Immunol. 2007, 179, 7907−7915. (6) Strakhova, M. I.; Nikkel, A. L.; Manelli, A. M.; Hsieh, G. C.; Esbenshade, T. A.; Brioni, J. D.; Bitner, R. S. Localization of histamine H4 receptors in the central nervous system of human and rat. Brain Res. 2009, 1250, 41−48. (7) Connelly, W. M.; Shenton, F. C.; Lethbridge, N.; Leurs, R.; Waldvolgel, H. J.; Faull, R. L.; Lees, G.; Chazot, P. L. The histamine H4 receptor is functionally expressed on neurons in the mammalian CNS. Br. J. Pharmacol. 2009, 157, 55−63. (8) Dunford, P. J.; Williams, K. N.; Desai, P. J.; McQueen, D.; Karlsson, L.; Thurmond, R. Histamine H4 receptor antagonists are superior to traditional antihistamines in the attenuation of experimental pruritus. J. Allergy Clin. Immunol. 2007, 119, 176−183. (9) Rees, J.; Murray, C. S. Itching for progress. Clin. Exp. Dermatol. 2005, 30, 471−473. (10) Thurmond, R. L.; Gelfand, E. W.; Dunford, P. J. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nature Rev. Drug Discovery 2008, 7, 41−53. (11) Ling, P.; Ngo, K.; Nguyen, S.; Thurmond, R. L.; Edwards, J. P.; Karlsson, L.; Fung-Leung, W. P. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation. Br. J. Pharmacol. 2004, 142, 161−171. (12) Buckland, K. F.; Williams, T. J.; Conroy, D. M. Histamine induces cytoskeletal changes in human eosinophils via the H4 receptor. Br. J. Pharmacol. 2003, 140, 1117−1127. (13) Cowart, M. D.; Altenbach, R. J.; Liu, H.; Hsieh, G. C.; Drizin, I.; Milicic, I.; Miller, T. R.; Witte, D. G.; Wishart, N.; Fix-Stenzel, S. R.; McPherson, M. J.; Adair, R. M.; Wetter, J. M.; Bettencourt, B. M.; Marsh, K. C.; Sullivan, J. P.; Honore, P.; Esbenshade, T. A.; Brioni, J. D. Rotationally constrained 2,4-diamino-5,6-disubstituted pyrimidines: a new class of histamine H4 receptor antagonists with improved druglikeness and in vivo efficacy in pain and inflammation models. J. Med. Chem. 2008, 51, 6547−6557. (14) Mowbray, C. E.; Bell, A. S.; Clarke, N. P.; Collins, M.; Jones, R. M.; Lane, C. A.; Liu, W. L.; Newman, S. D.; Paradowski, M.; Schenck, E. J.; Selby, M. D.; Swain, N. A.; Williams, D. H. Challenges of drug discovery in novel target space. The discovery and evaluation of PF3893787: a novel histamine H4 receptor antagonist. Bioorg. Med. Chem. Lett. 2011, 21, 6596−6602. (15) Sander, K.; Kottke, T.; Tanrikulu, Y.; Proschak, E.; Weizel, L.; Schneider, E. H.; Seifert, R.; Schneider, G.; Stark, H. 2,4Diaminopyrimidines as histamine H4 receptor ligandsscaffold optimization and pharmacological characterization. Bioorg. Med. Chem. 2009, 17, 7186−7196. (16) Akdis, C. A.; Simons, F.; Estelle, R. Histamine receptors are hot in immunopharmacology. Eur. J. Pharmacol. 2006, 533, 69−76. (17) Jablonowski, J. A.; Grice, C. A.; Chai, W.; Dvorak, C. A.; Venable, J. D.; Kwok, A. K.; Ly, K. S.; Wei, J.; Baker, S. M.; Desai, P. J.; Jiang, W.; Wilson, S. J.; Thurmond, R. L.; Karlsson, L.; Edwards, J. P.; Lovenberg, T. W.; Carruthers, N. I. The first potent and selective nonimidazole human histamine H4 receptor antagonists. J. Med. Chem. 2003, 46, 3957−3960. (18) Venable, J. D; Cai, H.; Chai, W.; Dvorak, C. A.; Grice, C. A.; Jablonowski, J. A.; Shah, C. R.; Kwok, A. K.; Ly, K. S.; Pio, B.; Wie, J.; Desai, P. J.; Jiang, W.; Nguyen, S.; Ling, P.; Wilson, S. J.; Dunford, P. J.; Thurmond, R. L.; Lovenberg, T. W.; Karlsson, L.; Carruthers, N. I.; Edwards, J. P. Preparation and Biological Evaluation of Indole, Benzimidazole, and Thienopyrrole Piperazine Carboxamides: Potent Human Histamine H4 Antagonists. J. Med. Chem. 2005, 48, 8289− 8298. (19) Engelhardt, H.; de Esch, I. J. P.; Kuhn, D.; Smith, R. A.; Zuiderveld, O. P.; Dobler, J.; Mayer, M.; Lips, S.; Arnhof, H.; Scharn, D.; Haaksma, E. E. J.; Leurs, R. Detailed structure−activity relationship

ASSOCIATED CONTENT

S Supporting Information *

Description of the analytic and preparative HPLC methods and analytic data (RT, MS, HRMS, and 1H NMR) of compounds 1a, 1b, 2a, 2b, 3−5, 8a−8c, 10a−10e, 12a−12e, 15, 16a, 16b, 18, 19, 25−30, 32, 34−37, 39, 40, 42, 43, and 46−48. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +31(0)205987600. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sandra Dö bel, Jochen Schö n, and Christian Engelhardt for support during the synthesis of the compounds. In addition, Darryl McConnel, Jens Quant, and Heinz Stadtmüller are thanked for the allocation of the necessary resources and infrastructure for synthesis, analysis and DMPK profiling of the compounds. This work was supported by The Netherlands Organization for Scientific Research (NWO) through a VENI grant (grant 700.59.408 to C.d.G.).

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ABBREVIATIONS USED H4R, human histamine H4 receptor; DIPEA, diisopropyl-Nethylamine; TEA, triethylamine; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; Pd(DPPF)Cl 2 , dichloro[1,1′-bis(diphenylphosphino)ferrocene] palladium(II) dichloromethane adduct

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