Bispyrimidines as potent histamine H4 receptor ligands: Delineation of structure-activity relationships and detailed H4 receptor binding mode

Article abstract of DOI:10.1021/jm301886t

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 pK_i 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.

Full text of DOI:10.1021/jm301886t

Article
pubs.acs.org/jmc
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 consid-
ered 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.
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 methylpiper-
azine 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 N-
methylated piperazine moiety undergoes rapid demethylation,
thereby confounding in vivo experiments.27 It is already known
that, in the class of indolecarboxamides and pyrimidines, the 1-
methylpiperazine moiety can be replaced by N-methylpyrroli-
din-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
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 H4R antagonist is the
indolecarboxamide, 1a (JNJ7777120; Figure 1),17−19 which is
highly potent and selective for H4R.17,18 Several other H4R
Received: December 21, 2012
Published: May 13, 2013
© 2013 American Chemical Society
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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.
a
Scheme 1
a
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.
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,
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
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a
Scheme 2
a
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 3
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.
a
Scheme 4
a
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.
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.
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 L1755.39
V
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
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a
Scheme 5
a
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 6
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
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-dichloro-
pyrimidine-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-dichloro-
pyrimidine-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
the more basic aminopyrimidine moieties of 2a−b form an
30
ionic interaction with the carboxylate side chain of E1825.46
.
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 model-
ing studies indicated that the flexible side chains of 3 and 4
adopt energetically favorable conformations nearby L1755.39 in
the H4R binding pocket.29
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 2,4-
dichloropyrimidine 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
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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 isobutyl-
amine 9b (Scheme 5).
Nucleophilic aromatic substitution reaction of 2,4,6-tri-
chloro-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-dichloro-
pyrimidine with amine 10a or 16a followed by an additional
nucleophilic aromatic substitution reaction with isobutylamine
led to 37 and 38 (Scheme 7).
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 2-
aminopyrimidine 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.
a
Scheme 7
Figure 2. Bispyrimidines: nomenclature.
The core scaffold of the bispyrimidines, represented by 22,
a
Reagents and conditions: (i) NMP, DIPEA, 80 °C, 3 h; (ii) NMP,
already displays a pKi on the H4R of 6.1
0.1. The
DIPEA, 100 °C, 3 d.
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-2-
aminopyrimidines with basic amines (e.g., represented through
Indolcarboxamide derivates 46−48 were synthesized using
an amide coupling of the acids 21a−b and amines 10a−b
(Scheme 8).
4, pKi = 8.5
0.1) cannot be reached. This indicates that
a
Scheme 8
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-1,2-
diamine 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-
a
Reagents and conditions: (i) HATU, DIPEA, DMF, 20 °C, 1−16 h.
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.
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
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Table 1. H4R Binding Affinity of Bispyrimidines
aryl-2-aminopyrimidines with basic amines,20 e.g., represented
by 2.
shows that only one pyrimidine subunit tolerates such kind of
substitution at R6/R6′ and indicates that 6-aryl, 6-alkyl-, and 6-
alkylamino-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 deriva-
tives.29,30
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
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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 4-
aminopyrimidine, 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 2-
isobutylamino-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 methylpiper-
azine 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 bispyrimi-
dines. 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
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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.
exploration of bispyrimidines can be also used in the series of
indolecarboxamides.
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.
Table 2 shows replacements of the 1-methylpiperazine
moiety by 2-aminopyrimidines for the class of indole-
containing 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
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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]
solubility @ pH 6.8
compd
half life in HLM
[min]
half life in MLM
[min]
solubility @ pH 6.8
[μg/mL]
1a
2
>45
43
2
4
58
24
25
41
42
>120
51
18
9
>79
>76
>84
>83
61
3
61
<2
4
>62
>64
101
82
23
8
4
>120
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
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).
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
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.
́
methylpiperazine (7 Å) 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 Å) 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
Comparison of the proposed binding modes of bispyrimi-
dines 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 vali-
(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
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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).
amides. The new insights in H4R−ligand interaction will aid the
future rational design of new H4R ligands.
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 FT-
NMR 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-2,4-
diamine (22). 4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-6-
chloropyrimidine-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.
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 cyto-
chromes (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
4-N-[2-[(2-Aminopyrimidin-4-yl)amino]ethyl]-6-N-(2-
methylpropyl)pyrimidine-2,4,6-triamine (23). 4-N-[2-[(2-Ami-
nopyrimidin-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-(3-
methylbutyl)pyrimidine-2,4-diamine (24). Compound 12a (4-N-
[2-[(2-aminopyrimidin-4-yl)amino]ethyl]-6-chloropyrimidine-2,4-dia-
mine (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
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 N-
methylpiperazine 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 indolecarbox-
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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-(3-
methylbut-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.
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
1
379.2353 [M + H]+, C20 H27 N8 requires M+ 379.2359. H 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-6-
N-(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-Amino-6-chloropyrimidin-4-yl)amino]ethyl]-6-
phenylpyrimidine-2,4-diamine (31). 4,6-Dichloro-pyrimidine-2-
amine 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
4-N-[2-[(2-Aminopyrimidin-4-yl)-methylamino]ethyl]-6-phe-
nylpyrimidine-2,4-diamine (44). 4-N-[2-[(2-Aminopyrimidin-4-
yl)-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
1
Cl N8 requires M+ 357.1343. H 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-methyl-
propyl)-6-phenylpyrimidine-2,4-diamine (33). 4,6-Dichloro-pyr-
imidine-2-amine 11 (40 mg, 0.24 mmol), DIPEA (467 mg, 1.45
mmol), and 4-N-(2-aminoethyl)-2-N-(2-methylpropyl)-6-phenylpyri-
midine-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.
1
requires M+ 337.1889. H 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-car-
boxamide (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
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
1
found 379.2352 [M + H]+, C20 H27 N8 requires M+ 379.2359. H
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-pyri-
midine 20 (120 mg, 0.81 mmol), DIPEA (624 mg, 4.83 mmol), and 4-
N-(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
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
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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 H-
bond 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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ASSOCIATED CONTENT
* Supporting Information
Description of the analytic and preparative HPLC methods and
■
S
1
analytic data (RT, MS, HRMS, and H 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.
AUTHOR INFORMATION
Corresponding Author
*Phone: +31(0)205987600. E-mail: r.leurs@vu.nl.
■
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.
ACKNOWLEDGMENTS
■
We thank Sandra Dobel, Jochen Schon, and Christian
̈
̈
Engelhardt for support during the synthesis of the compounds.
In addition, Darryl McConnel, Jens Quant, and Heinz
Stadtmuller 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.).
ABBREVIATIONS USED
■
H4R, human histamine H4 receptor; DIPEA, diisopropyl-N-
ethylamine; TEA, triethylamine; HATU, 2-(7-aza-1H-benzo-
triazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
Pd(DPPF)Cl2, dichloro[1,1′-bis(diphenylphosphino)-
ferrocene] palladium(II) dichloromethane adduct
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