2,4-Diaminopyrimidines as histamine H4 receptor ligands-Scaffold optimization and pharmacological characterization

Article abstract of DOI:10.1016/j.bmc.2009.08.059

The human histamine H₄ receptor (hH₄R) is a promising new target in the therapy of inflammatory diseases and disorders of the immune system. For the development of new H₄R antagonists a broad ligand-based virtual screening was performed resulting in two hits. The dissection of their common annelated aromatic core into its heteromonocyclic components showed that 2,4-diaminopyrimidine is a potent hH₄R affinity scaffold, which was comprehensively investigated. Structure-activity relationship studies revealed that slight structural changes evoke extensive differences in functional activities and potencies: while o- and p-substituted benzyl amines mainly showed partial agonism, m-substituted and rigidified ones exhibited inverse agonist efficacy.

Full text of DOI:10.1016/j.bmc.2009.08.059

Bioorganic & Medicinal Chemistry 17 (2009) 7186–7196
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2,4-Diaminopyrimidines as histamine H4 receptor ligands—Scaffold
optimization and pharmacological characterization q
Kerstin Sander a, Tim Kottke a, Yusuf Tanrikulu b, Ewgenij Proschak b, Lilia Weizel a, Erich H. Schneider c,
Roland Seifert d, Gisbert Schneider b, Holger Stark a,
*
a Johann Wolfgang Goethe-University, Institute of Pharmaceutical Chemistry, ZAFES/LiFF/CMP, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany
b Johann Wolfgang Goethe-University, Institute of Organic Chemistry and Chemical Biology, ZAFES/LiFF/CMP, Siesmayerstr. 70, 60323 Frankfurt/Main, Germany
c University of Regensburg, Department of Pharmacology and Toxicology, Universitätsstr. 31, 93053 Regensburg, Germany
d Medical School of Hannover, Institute of Pharmacology, Carl-von-Neuberg-Str. 1, 30625 Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2009
Revised 26 August 2009
Accepted 28 August 2009
Available online 2 September 2009
The human histamine H4 receptor (hH4R) is a promising new target in the therapy of inflammatory dis-
eases and disorders of the immune system. For the development of new H4R antagonists a broad ligand-
based virtual screening was performed resulting in two hits. The dissection of their common annelated
aromatic core into its heteromonocyclic components showed that 2,4-diaminopyrimidine is a potent
hH4R affinity scaffold, which was comprehensively investigated. Structure–activity relationship studies
revealed that slight structural changes evoke extensive differences in functional activities and potencies:
while o- and p-substituted benzyl amines mainly showed partial agonism, m-substituted and rigidified
ones exhibited inverse agonist efficacy.
Keywords:
Histamine
H4
Ó 2009 Elsevier Ltd. All rights reserved.
GPCR
Topliss scheme
Lead optimization
1. Introduction
with heterotrimeric Gi/o proteins.4 Receptor activation leads to G
protein
a
-subunit-mediated inhibition of some adenylyl cyclase iso-
The biogenic amine histamine (1), 2-(1H-imidazol-4-yl)ethan-
amine, mainly acts via four distinct histamine receptor subtypes
(H1R–H4R). Antagonists for H1R and H2R are used in the therapy
of allergic diseases and gastrointestinal ulcer diseases, respectively,
and have reached blockbuster status.1,2 The first H3R antagonists
are progressing into clinical phase III to be investigated for the
treatment of sleep-wake and cognition disorders, epilepsy, neuro-
pathic pain or obesity.3 As the H4R is the last reported receptor
subtype researchers are making efforts to find potent ligands with
inverse agonist/antagonist efficacy, and only preclinical studies of a
few structures have been reported.
forms. b
c-Subunits released from the heterotrimer cause phospholi-
pase Cb activation and an increase of the intracellular calcium ion
concentration.5 The H4R shows an extraordinary high constitutive
activity.6 According to the two-state model of receptor activation7
the equilibrium between the active G protein-coupling conforma-
tion (R*) and the uncoupled inactive state (R) is shifted to the active
state, which promotes G protein signalling in the absence of a ligand.
In the model, agonists are thought to stabilize the active state,
whereas inverse agonists change the receptor conformation to stabi-
lize the inactive state. Partial agonists stabilize the active state less
efficiently than full agonists and neutral antagonists have no prefer-
ence for the active or inactive state, yet block the effects of both ago-
nists and inverse agonists. In the case of hH4R, full and inverse
agonists have been reported to stabilize the active state.8
Likeall other histaminereceptors, theH4Rbelongs toclass A (rho-
dopsin-like) of G protein-coupled receptors (GPCRs). It interacts
The H4R is predominantly expressed peripherally on hemato-
poietic mononuclear cells, especially on eosinophils, basophils,
T-lymphocytes, dendritic cells, and mast cells. Hence, it shows
highest concentration levels in tissues involved in immune
responses such as bone marrow, lung, and spleen.6 The H4R is
involved in immune or inflammatory responses because histamine
signalling induces cell shape change and chemotaxis of mast cells
as well as eosinophils, mast cell migration, and upregulation of
adhesion molecules on monocytes, and all these effects can be
q
Main parts of this investigation have recently been presented at international
conferences: Sander, K., et al. Lead Structure Identification by Virtual Screening and
Scaffold Modification for New Histamine H4 Receptor Ligands. 20th International
Symposium on Medicinal Chemistry, Vienna/Austria (August 31–September 4,
2008). Drugs Future 2008, 33, 267–268 (P490)—Sander, K., et al. From Hit to Lead on
Histamine H4 Receptor Ligands—Diaminopyrimidines as Potential Anti-Inflamma-
tory Agents. Frontiers in Medicinal Chemistry 2009, Heidelberg/Germany (March
15–18, 2009), Abstract book, p 66 (INF02).
* Corresponding author. Tel.: +49 69 798 29302; fax: +49 69 798 29258.
E-mail address: h.stark@pharmchem.uni-frankfurt.de (H. Stark).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.059

K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
7187
blocked by H4R antagonists.9–13 From these physiological reac-
tions, one can deduce several potential clinical uses for H4R inverse
agonists/antagonists in the broad field of anti-inflammatory ther-
apy,14 like allergy and asthma,15 pruritus16 associated with al-
lergy17 or autoimmune skin conditions, inflammatory bowel
disease,18 rheumatoid arthritis, and pain.19
H4R affinities and agonist efficacy while showing H1R antagonism.28
scaffold hopping approach resulted in quinazolin-4-amine
A
VUF10497, which was reported as a dual acting H1R/H4R ligand
(pKi = 7.7/8.2).31 The combination of tricyclic structural elements
and a methylpiperazine group can also be found in the patent liter-
ature32 (e.g., compound 5), where its H4R affinity has been claimed.
A group of conformationally constrained aminopyrimidines was re-
ported as potent antagonists on human, rat and mouse H4R, high-
Since the hH4R is structurally most closely related to hH3R (37%
protein sequence identity, 58% in transmembrane domains)20 the
identification of selectively acting hH4R ligands is a major challenge
in current histamine research. Because some imidazole-containing
hH3R ligands turned out to be hH4R ligands as well21 a reclassifica-
tion of frequently used reference structures has taken place.22 Some
of those ligands such as thioperamide (2), which exhibits similar
affinities at both hH3R (pKi = 7.2)23 and hH4R (pKi = 7.3),20 are now
used as standard compounds in hH4R binding assays. In addition,
new radioligands with improved selectivity and specific activity
compared to that of [3H]histamine are under investigation.24 So
far, only a few potent and selectively acting H4R ligands are pub-
lished.25 JNJ 7777120 (3; pKi = 8.4) is the most profiled among a ser-
ies of indolylpiperazines and has become the de facto standard
reference H4R antagonist in literature studies. The compound was
the first reported H4R antagonist with anti-inflammatory proper-
ties.26 Elongation of the spacer between the basic moiety and the
aromatic scaffold led to 2-arylbenzimidazoles like 4. On the one
hand, H4R binding in the low nanomolar concentration range has
been reported for these compounds but, on the other hand, they
show high structural similarity to H3R antagonists.27 The atypical
lighted by A-943931,
a potent (pKi = 8.3) and selective H4R
antagonist.33 Another constrained series led to the tetracyclic H4R
antagonist A-987306, which also showed efficacy in pain models.34
Non-annelated aminopyrimidines like 6 have been discovered
simultaneously and described previously by a few groups and have
been published in broad patent applications. In a way these struc-
tures are built up quite similar. They all consist of an aminopyrimi-
dine substituted with an aliphatic basic chain or heterocycle.
However, almost all structures differ with regard to the specific sub-
stitution patterns and ring systems of the pyrimidine core. Chrono-
logically, other 2,4-diaminopyrimidines were first detailed by
chemists from Palau Pharma35 and UCB36 though without giving
specific description of pharmacological data. Pfizer scientists also
published related pyrimidines as hH4R ligands, substituted with ali-
phatic heterocycles,37 while the Johnson & Johnson group prepared
carbon-analog 2-amino-4-alkyl-pyrimidines.38
The objective of our study was the development of novel hH4R
ligands with inverse agonist/antagonist efficacy as well as selectiv-
ity over other aminergic receptors and off-targets, especially the
hH3R. We initiated our project with a virtual screen search of a
SPECS library using compound 3 as reference structure. The search
identified a five-membered heterocycle [d]annelated to pyrimi-
dine. This fused pyrimidine scaffold was theoretically dissected
into its fragments, which were synthetically prepared and tested
separately to figure out the structural requirements for hH4R bind-
ing. The resulting 2,4-diaminopyrimidine core structure was fur-
neuroleptic clozapine has become
a lead in H4R research
(pKi = 6.8)28 despite exhibiting high affinities to multiple biogenic
amine receptors.29 It is thought that it mainly mediates its antipsy-
chotic effects via dopamine D2-like receptors as well as serotonin
5-HT2 receptorsubtypes though clozapineexhibitssubstantial affin-
ity for other biogenic amine receptors such as histamine H1 and
adrenergic
a
1 receptors.30 Optimized oxazepines possess improved
Histamine H4 receptor ligands
Purine scaffold
Screening hits
O
O
N
N
N
N
H
N
H
N
N
N
N
N
N
N
N
X
N
N
N
F
H3C
H3C
Cl
CH3
CH3
N
N
3
4
7
Cl
Cl
N
N
N
N
H3C
NH2
N
NH2
N
10, 12
N
N
N
N
S
Cl
O
N
N
N
H3C
H3C
H3C
CH3
5
6
8
X
N
N
N
N
N
N
H3C
X
N
N
N
N
N
NH
N
N
N
N
N
H3C
H3C
13
9, 11
X
1
2
3
N
N
6
4 Y
N
N
5
H3C
R
14 - 38
Figure 1. Scaffold modification of virtual screening hits based on known compounds. X = H or NH2; Y = NH, N, O or S; R = aromatic substituents. Elements of the hH4R
blueprint are highlighted as follows: the basic moiety (in most cases methylpiperazine) in dark grey, the hydrogen donor/acceptor system in light grey, and the aromatic
moiety in medium grey.

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K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
ther optimized by variation of the key substituents. Affinities, effi-
cacies and potencies of the resulting compounds were investigated
in in vitro assays.
(7 and 8, respectively). Both hits consist of a cyclopenta[d]pyrimi-
dine scaffold substituted with 4-methylpiperazine. 4-Methylpiper-
azine is frequently used as basic moiety in hH4R ligands31,40 and in
most cases it increases affinity. Observing the hits and some repre-
sentative reference ligands in Figure 1 it becomes clear that the
majority of known hH4R ligands have some similarities sharing
the mentioned basic piperazine, a hydrogen donor/acceptor system
2. Scaffold determination
A variety of pyrimidines have been reported as active hH4R li-
gands. With the aim to find novel compounds, a ligand-based vir-
tual screening39 was performed and resulted in two hits with
affinities in the low micromolar concentration range, namely a
3H-[1,2,3]triazolo[4,5-d]pyrimidine and a thieno[2,3-d]pyrimidine
and an aromatic moiety allowing for p-interactions. For reasons of
comparison, in our study the 4-methylpiperazine moiety was kept
constant, while the heterocyclic core was modified (Fig. 1) to study
the structure–affinity/efficacy relationships at this position.
Table 1
hH4R Affinities of reference 3, screening hits 7 and 8, and structural fragments thereof
Compound
Structure
Formula
Affinity on hH4Ra Ki (
lM)
O
H
N
N
3 (JNJ 7777120)
C14H16ClN3O
0.014 0.007
N
H3C
Cl
N
N
N
N
7
C16H18ClN7
3.5
N
N
N
N
H3C
H3C
Cl
N
N
N
N
8
S
C13H18N4S
6.6
CH3
N
N
N
9
C10H14N6
18.4
14.3
15.6
NH
N
N
N
N
H3C
H3C
N
N
10
11
C17H20N6ÁC4H4O4
N
N
N
NH2
N
C10H15N7
NH
N
N
H3C
NH2
N
N
12
C17H21N7Á2(COOH)2
8.1
N
N
N
N
N
N
H3C
H3C
N
13
14
C15H21N5Á2C4H4O4
35.0
N
N
N
N
C16H21N5Á2C4H4O4
0.417 0.165
N
N
H
N
H3C
NH2
N
N
N
15
16
C9H16N6Á2C4H4O4
0.290 0.050
0.098 0.013
N
N
NH2
N
N
H3C
H3C
NH2
N
C16H22N6Á2C4H4O4
N
H
a
[3H]Histamine displacement assay with membrane preparation of Sf9 cells expressing hH4R, co-expressed with G
a
i2 and Gb1c2 subunits;8 mean values of at least two
independent measurements, each in triplicates; SEM given for Ki values below 1 M.
l

K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
7189
H (16)
similar effect could be seen in the increase of affinities of com-
pounds 9 and 10 versus 11 and 12. Compound 15 showed affinity
in the submicromolar concentration range and partial agonist effi-
cacy in two functional assays (vide infra), and hence its scaffold
was kept as a basic pharmacophore. Since benzyl substitution fur-
ther improved binding strength, structure 16 was defined as lead
structure (Table 2). Compounds 15 and 16 have meanwhile been
previously reported as hH4R ligands in a FLIPR assay (pEC50 of 6.4
and 7.0, respectively), consistent with our findings.40
To investigate the influence of substituents on the benzylic por-
tion of the molecule and to optimize the partial agonist 16 with re-
spect to hH4R affinity and efficacy the latter compound was
modified according to classical Topliss scheme.41 Effects of elec-
tron-withdrawing, electron-donating and/or steric substituents
were studied and led to nanomolar ligands with differing function-
ality (cf. Tables 2–4).
L
E
M
4-Cl (19)
4-Cl (19)
4-Cl (19)
L, E
M
4-CH3 (24)
4-CH3 (24)
4-OCH3 (20)
4-OH (21)
3,4-di-Cl (22)
4-CF3 (23)
L, E
M
3-Cl (25)
3-Cl (25) 4-C(CH3)3 (33)
electron-
releasing,
slightly
3-CH3 (27)
3-NO2 (26)
electron-
withdrawing,
lipophilic
2-Cl (28)
2-CH3 (29)
hydrophobic
4-CN (30)
4-F (31)
intermediate electronic effects,
lipophilic, steric
Figure 2. Topliss Tree on scaffold optimization. L = lower affinity; E = equal affinity;
M = higher affinity. In parenthesis corresponding compound numbers are given.
Due to their convenient synthetic accessibility the first synthe-
sized ligands were purine 10 and its corresponding fragment des-
benzyl 9, which showed lower affinities than hits 7 and 8 (Table 1).
Introduction of the 2-amino group known from other hH4R ligands
in patent literature32 (cf. Fig. 2) led to a slight improvement in
hH4R binding of compounds 11 and 12.
The central aromatic moiety of hit 7 was analyzed by dissection
of the components into a triazole (13) and a pyrimidine (14) scaf-
fold. These fragments evoked pronounced differences in receptor
interaction: the triazole did not show any affinity at all, whereas
the pyrimidine retained affinity in the submicromolar concentra-
tion range. As can be seen from comparing the affinities of com-
pounds 10 and 14 only removal of purine N-7 and C-8 caused a
fortyfold increase in receptor binding. This is consistent with the
pyrimidine as the source of affinity increase.
3. Chemistry
Fragments 9 and 11 were prepared by substitution of a chloro-
purine derivative with N-methylpiperazine under basic conditions.
Further addition of benzyl bromide and caesium carbonate led to
compounds 10 and 12 (Scheme 2). Triazole 7 was prepared in a
one-pot-reaction via click chemistry42 (Scheme 1). The Huisgen
1,3-dipolar cycloaddition is an efficient approach to couple azides
and terminal acetylenes to achieve 1,2,3-triazoles.43 Copper(I) ion
catalysis selectively leads to 1,4-substituted triazoles.44 Azide
13a was prepared from benzyl bromide in the microwave oven
using water as the solvent.45
Pyrimidin-4-amines 14–16 and 19–38 were synthesized by
microwave-assisted aromatic nucleophilic substitution reactions46
of 2-amino-4,6-dichloropyrimidine and an appropriate amine
(Scheme 2). As adjuvant base N,N-diisopropylethylamine was
added, and reactions were conducted in the polar protic solvent
Incorporation of a 2-amino group into compound 14 provided
compound 16 and led to an improvement of receptor binding. A
Table 2
hH4R Affinities of compounds 16–38
compounds 16-36 NH2
NH2 compounds 37,38
N
N
N
N
N
Y
N
N
R
N
N
H3C
H3C
n
Compound
Y
R
n
Formula
Affinity on hH4Ra Ki (
lM)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
NH
O
S
H
H
H
C16H22N6Á2C4H4O4
C16H21N5O
0.098 0.013
2.5
13.7
C16H21N5SÁ2C4H4O4. 0.5 H2O
C16H21ClN6Á2C4H4O4
C17H24N6OÁ2C4H4O4
C16H22N6OÁ2C4H4O4
C16H20Cl2N6
C17H21F3N6Á2C4H4O4
C17H24N6Á2C4H4O4
C16H21ClN6
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
4-Cl
0.071 0.010
0.542 0.260
0.237 0.047
0.150 0.023
1.2
0.205 0.033
0.290 0.044
0.538 0.152
0.282 0.112
0.018 0.007
0.025 0.008
0.341 0.099
0.046 0.020
0.012 0.003
3.4
4-OCH3
4-OH
3,4-Di-Cl
4-CF3
4-CH3
3-Cl
3-NO2
3-CH3
2-Cl
C16H21N7O2Á2C4H4O4
C17H24N6Á2C4H4O4
C
16H21ClN6Á2C4H4O4
2-CH3
4-CN
4-F
C17H24N6Á2C4H4O4
C18H22N7Á2C4H4O4
C16H21FN6Á2C4H4O4
C16H20Cl2N6Á2C4H4O4
C20H30N6Á2C4H4O4
C19H28N6Á2C4H4O4
2,6-Di-Cl
4-tert-Butyl
4-iso-Propyl
4-Phenyl
2.2
C
22H26N6
0.716 0.203
376.6
0.146 0.073
0.248 0.071
4-COOH
C17H22N6O2
1
0
C18H24N6Á2C4H4O4
C17H22N6Á2C4H4O4
a
[3H]Histamine displacement assay with membrane preparation of Sf9 cells expressing hH4R, co-expressed with G
ai2 and Gb1c
2 subunits;8 mean values SEM of at least
three independent measurements, each in triplicates; SEM given for Ki values below 1 M.
l

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K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
Table 3
Efficacy values for partial agonists determined in two functional assays
Compound
Codea
Efficacy and potency on hH4R
Functionality
GTP
c
Sb
GTPasec
d
e
d
e
Emax (%)
p EC50
Emax (%)
p EC50
1 (Histamine)
28
29
31
100.0
32.5
23.7
28.5
28.4
7.86 0.20
8.22 0.18
8.07 0.16
7.68 0.16
8.95 0.29
100.0
30.5
19.2
26.6
17.5
7.93 0.06
7.71 0.25
7.93 0.15
7.04 0.27
8.85 0.39
7.05 0.11
Agonist
ST-997
ST-998
ST-999
ST-1006
Partial agonist
Partial agonist
Partial agonist
Partial agonist
Inverse agonist
32
2 (Thioperamide)
À100.0
7.15
0.18
À100.0
a
b
c
Laboratory code for later identification of these compounds.
Functional binding assay with [35S]GTP
S on membrane preparation of Sf9 cells expressing hH4R, co-expressed with G
c
a
i2 and Gb1c2 subunits.8
Functional binding assay with GTPase on membrane preparation of Sf9 cells expressing hH4R, co-expressed with Ga
i2 and Gb1c2 subunits.8
d
e
Values relating to maximal response of 1 as reference full agonist (100%).
Mean values SEM of at least three independent experiments, each at least in duplicates and six point measurements.
Table 4
Functional characterization of partial and inverse agonists
Compound
Substitution of benzyl amine
GTPc
Sa
Functionality
Efficacy
Emax (%)
Potency
b
d
pEC50c/pKb
1 (Histamine)
21
16
30
19
24
20
22
3 (JNJ 7777120)
25
27
26
100.0
37.7
34.5
24.3
24.0
7.86 0.20
6.99 0.14
7.67 0.29
6.22 0.05
7.50 0.27
6.98 0.07
6.79 0.07
7.25 0.22
7.80 0.21
6.70 0.25
6.57 0.24
6.32 0.19
7.36 0.07
7.15 0.18
7.43 0.16
Full agonist
p-OH
Partial agonist
Partial agonist
Partial agonist
Partial agonist
Antagonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
p-CN
p-Cl
p-CH3
p-OCH3
m,p-Di-Cl
—
À32.3
À52.9
À58.7
À66.4
À79.0
À85.3
À95.6
À100.0
À110.7
m-Cl
m-CH3
m-NO2
Tetrahydroisoquinolin-2-yl
37
2 (Thioperamide)
38
Isoindoline-2-yl
a
b
c
Functional binding assay with [35S]GTP i2 and Gb1c2 subunits.8
cS on membrane preparation of Sf9 cells expressing hH4R, co-expressed with Ga
Values relating to maximal response of 1 as reference full agonist (100%), or to maximal inhibition of constitutive activity by 2 as reference inverse agonist (À100%).
pEC50 for full, partial, and inverse agonists determined in agonist mode; mean values SEM of at least three independent experiments, each at least in triplicates and
seven point measurements.
d
pKb for neutral agonist 24 determined in antagonist mode (after prestimulation by 1); mean value SEM of three independent experiments, each in triplicates and seven
point measurements.
iso-propanol. These conditions make it suitable for microwave heat-
ing because the irradiation is well absorbed. Under common condi-
tions, which is heating the reagents in solvents like ethanol,
transformation to the product hardly took place, even after three
days of reflux. Hence, microwave irradiation was necessary to obtain
high yields in a reasonable time (30–60 min). Typically, due to the
higher basicity of secondary amines, higher overall yields were ob-
tained when, in a sequential synthesis, first the benzyl amine and
second 1-methylpiperazine was coupled. Ether 17 and thioether
18 resulted from the reaction of 4-chloro-6-(4-methylpiperazin-1-
yl)pyrimidin-2-amine (17a) with benzyl alcohol,47 and benzyl mer-
captane,48 respectively. Sodium hydride was needed to deprotonate
the alcohol, whereas for thiol activation sodium hydroxide was suf-
ficient. Rigidified structures 37 and 38 resulted from the reaction of
17a with tetrahydroisochinoline and isoindoline, respectively.
All final compounds were re-crystallized from ethanol, a few as
free bases, but most as salts of maleic acid due to ease of handling.
According to NMR spectra, diprotonation takes place on N4-piper-
idine and pyrimidine-4-amine, whereas the latter might be pre-
ferred due to its mesomeric stabilization.
tion binding assay (cf. Tables 1 and 2). For this purpose a mem-
brane preparation from Sf9 cells expressing hH4R, co-expressed
with G protein Gai2 and Gb1c
2 subunits, was made.8 Similar exper-
iments were performed to measure affinities of the most potent
compounds 28, 29, 31, and 32 on the remaining three histamine
receptor subtypes, in each case using an appropriate radioligand,
a
namely [3H]pyrilamine, [3H]tiotidine, and [3H]N -methylhistamine
for hH1R,49 hH2R,50 and hH3R51 binding, respectively (cf. Table 5).
Two functional assays8 were used to determine the efficacy of
compounds 28, 29, 31, and 32on hH4R(cf. Table 3). Bothwere carried
out with the Sf9 cell membrane preparation containing hH4R, co-ex-
pressed with G protein G
based on the exchange of GDP to [35S]GTP
to the receptor. As the complex of the G
hardly dissociates, agonist-induced [35S]GTP
sured. The second assay determines the steady-state GDP/GTP ex-
change by measuring hydrolysis of [
32P]GTP catalysed by the
a
i2 and Gb1c2 subunits. The first assay is
S after a ligand has bound
i2 subunit and [35S]GTP
S binding can be mea-
c
a
cS
c
c-
enzyme GTPase. Its activity is determined by counting the signals
of the released 32Pi. Reference structures in both assays are the
endogenous hH4R ligand 1 as agonist and the imidazole-containing
H3R/H4Rligand2asinverseagonist.In thesemembranesystems, rel-
ative to the baseline (equating with the basal constitutive activity)
compound 1 evokes a stimulation of ꢀ60% (63% and 60% in
4. Pharmacology
[
35S]GTP
cS and GTPase assay, respectively). Inverse agonist efficacy
To determine hH4R affinities all compounds (7–38) as well as
reference structures were examined in a [3H]histamine competi-
of 2 also shows comparable values (À38% and À40%, respectively).

K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
7191
X
X
Compounds 16, 19–22, 24–27, 30, 37, and 38 were additionally
analyzed in the [35S]GTP
S binding assay (cf. Table 4). Partial ago-
X
(i)
(ii)
c
N
N
N
N
N
N
nists and inverse agonists were characterized in the agonist mode,
compound 24 additionally in the antagonist mode (prestimulation
with 1).
N
N
Cl
NH
N
NH
N
N
N
N
N
H3C
H3C
9
11
10
12
: X = H
: X = NH2
: X = H
: X = NH2
NH2
NH2
N
NH2
N
5. Results and discussion
(iii)
(iv)
N
N
N
N
N
Y
N
Y
N
Purines 10 and 12 exhibited hH4R affinity in the same range as
hit 7 did. Fragmentation of the annelated heterocycle of 7 led to tri-
azole 13 and pyrimidine 14. Triazoles can be used as bioisosters of
amides since nitrogen atoms two and three replace the carbonyl’s
weak acceptor function.43 Hence, this heterocycle in compound 13
was thought to take the place of the amide moiety in compound 3.
However, the donor function of the indole nitrogen in compound 3
is missing in compound 13 explaining the loss of affinity. In con-
trast, the pyrimidine in compound 14 includes both, hydrogen
bond donor and acceptor. Hence it is able to replace the car-
bonyl-indole moiety of 3. Because 2-amino substitution of the
pyrimidine led to a fivefold increase of affinity (14?16) this bene-
ficial moiety was kept constant. Structure–activity relationships of
compound 16 are shown in Table 2. The exocyclic 4-amino group is
incorporated in the aromatic system of the pyrimidine and might
act as a hydrogen donor important for receptor interactions.
Changing this heteroatom (Y) to O (17) or S (18) led to a 25- and
140-fold loss of affinity, respectively. These results reinforce previ-
ous descriptions of 2,4-diaminopyrimidines as a potent hH4R affin-
ity scaffold.35,36,40 Replacement of the 4-amino moiety by a
methylene group considerably improved receptor binding to the
low nanomolar concentration range.38 This indicates that differ-
ences in the electrostatic charge distribution also influence the
affinity of these compounds.
Cl
Y
N
N
H3C
H3C
17a
15
17
: Y = Cl
: Y = NH2
: Y = O
18: Y = S
NH2
NH2
NH2
N
(iii)
(vi)
(v)
(iii)
N
N
N
N
N
N
N
Cl
N
Cl
Cl
Cl
N
N
H3C
H3C
n
17a
37
: n = 1
38: n = 0
X
X
X
N
N
N
N
N
Cl
N
N
N
N
H
Cl
R
R
H
H3C
14a
14
: X = H
16, 19-36
: X = H
16a, 19a-36a
: X = NH2
: X = NH2
Scheme 2. Synthesis of compounds 9–12 and 14–38 via substitution reactions.
R = aromatic substituents. Reagents and conditions: (i) 1-methylpiperazine, TEA,
DMF, 12 h, rt; (ii) benzyl bromide, Cs2CO3, DMF, 12 h, 110 °C; (iii) 1-methylpiper-
azine, DIPEA, 2-propanol,
THF, 24 h, 0 °C?rt; Y = S: benzyl mercaptane, NaOH, H2O, ethanol, 12 h, 80 °C; (v)
tetrahydroisoquinoline (n = 1) or isoindoline (n = 0), DIPEA, 2-propanol, W,
lW, 30–60 min, 160 °C; (iv) Y = O: benzyl alcohol, NaH,
l
60 min, 160 °C; (vi) benzyl amine, DIPEA, 2-propanol,
l
W, 30–60 min, 130 °C.
Initially, optimization was carried out for hH4R affinity. The 4-
chloro derivative 19 is nearly equipotent to non-substituted com-
pound 16. When referring to Topliss’ scheme (Fig. 2) the equality
of hydrogen and chlorine at this site already gave a hint, that steric
rests with lipophilic and intermediate electronic effects improve
hH4R binding. This classical approach was used to systematically
examine the ability of various substituents to enhance affinities.41
Indeed, 2-chloro (28), 2-methyl (29), and 4-fluoro (31) derivatives
showed affinities in the low nanomolar concentration range. Com-
pared to these substituents, electron-releasing but hydrophilic
groups like 4-methoxy (20) or 4-hydroxy (21) and electron-with-
drawing rests like 3,4-di-Cl (22) or 4-trifluoromethyl (23) decrease
binding potency (pKi <7). Nevertheless, all exhibit stronger affini-
ties than hits 7 and 8. Additional acidic moieties as in compound
36 seem to be detrimental to hH4R binding (pKi ꢀ3.5).
(i 1.)
(i 2.)
NH
N
N
Br
N
N
N
N
H3C
H3C
13
N3
13a
Scheme 1. Synthesis of compound 13 via click chemistry. Reagents and conditions:
(i) (1) TEA, H2O, 1 h, rt; (2) CuI, 12 h, rt.
Table 5
Histamine receptor subtype affinities and hH3R/hH4R selectivity ratio of selected
To evaluate the effect of the 2-Cl substitution and to investigate
the steric interactions and the lipophilic properties of compound
28 a 2,6-di-Cl-substituted compound (32) was prepared. It shows
a similar binding behaviour to that of compound 28. Most likely,
only one chlorine is required to interact with a lipophilic area with-
in the receptor’s binding pocket. No additional fixation is achieved
by inserting additional steric or lipophilic properties. This
assumption was supported with a series of ligands with steric
substituents: compounds with bulky rests like 4-tert-butyl (33)
and 4-iso-propyl (34) possess affinities in the micromolar concen-
tration range, whereas biphenyl 35 evokes submicromolar receptor
binding. The planar phenyl moiety seemed to allow stronger recep-
tor interactions than iso-alkyl rests. Rigidification may be a useful
concept to increase binding potency and/or selectivity of ligands.52
This is not found with tetrahydroisoquinoline 37 and isoindoline
38. They show a slightly reduced affinity when compared to that
of parent structure 16, which might be caused by conformational
change of the molecule.
compounds
Compound hH1Ra
pKi
hH2Rb
pKi
hH3Rc
pKi
hH4Rd
pKi
hH3R/hH4R Ki/
Ki
28
29
31
32
<6.0
<6.0
<6.0
<6.0
<6.0
<6.0
<6.0
<6.0
6.46
6.35
6.49
6.29
7.77
7.63
7.37
7.94
19.2
17.8
7.1
42.5
a
[3H]Pyrilamine displacement on Sf9 cell membranes co-expressing hH1R and
RGS4;49 mean values of three independent experiments, each in triplicates and one
point measurements.
b
[3H]Tiotidine displacement on Sf9 cell membranes with hH2R-Gs
as fusion
protein;50 mean values of three independent experiments, each in triplicates and
one point measurements.
c
a
[3H]N -Methylhistamine displacement on Sf9 cell membranes co-expressing
hH3R, Gai2, Gb1c
2 and RGS4;51 mean values of three independent experiments,
each at least in duplicates and five point measurements.
d
[3H]Histamine displacement on Sf9 cell membranes co-expressing hH4R, G
ai2
and Gb1c2
seven point measurements.
;
8 mean values of three independent experiments, each in triplicates and

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K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
125
100
75
100
75
50
25
0
1 (Histamine)
2 (Thioperamide)
19
22
25
28
1 (Histamine)
3 (JNJ 7777120)
50
24 (antagonist)
24 (agonist)
25
0
-25
-50
-75
-100
-25
-12 -11 -10 -9
-8
-7
-6
-5
-4
-125
-11
log (compound) [mol/l]
-10
-9
-8
-7
-6
-5
Figure 3. Determination of efficacy and potency of compound 24 (black triangles)
in the agonist and antagonist mode of [35S]GTP
S binding assay. Functional binding
assay with [35S]GTP
S on membrane preparation of Sf9 cells expressing hH4R, co-
expressed with G i2 and Gb1
2 subunits;8 values relating to maximal response of 1
log (compound) [mol/l]
c
c
Figure 4. Structure–efficacy relationships of compounds 19, 22, 25, 28, and
reference structures. Functional binding assay with
a
c
[
35S]GTP
c
G
S
on membrane
ai2 and Gb1c2
as reference full agonist (100%); mean values SEM of three independent exper-
iments, each in triplicates and seven point measurements.
preparation of Sf9 cells expressing hH4R, co-expressed with
subunits;8 values relating to maximal response of 1 as reference full agonist (100%),
or to maximal inhibition of constitutive activity by 2 (À100%); mean values SEM
of at least three independent experiments, each in triplicates and seven point
measurements.
Selected compounds with high affinities, that is pKi values >7.3,
were further characterized with respect to their potency and effi-
cacy on hH4R (Table 3) and their selectivity relative to other hista-
mine receptor subtypes (Table 5). A functional [35S]GTP
cS binding
Functional performance of reference structures and the differ-
entially chloro-substituted derivatives (results of [35S]GTP
cS as-
assay provided evidence that these pyrimidine-2,4-diamines can
show partial agonist efficacy: o-substituted compounds 28, 29,
and 32 cause partial receptor activation. Among all tested struc-
tures, 2-chloro derivative 28 exhibits the highest agonist efficacy
of ꢀ30%, relative to maximal response of 1 as reference full agonist
(100%). With addition of one more chlorine (32) agonist efficacy
somewhat decreases, while potency increases. Compared to func-
tional characteristics of 28, 2-methyl derivative 29 shows reduced
efficacy and similar potency, whereas 4-fluoro substituted com-
pound 31 evokes a similar efficacy but decreased potency. To con-
firm these results the compounds were reassessed by a different
functional assay, which is based on GTPase activity. Profiles were
say) are shown graphically in Figure 4 displaying a range of
partial to inverse agonist efficacy, depending on the substitution
site. Although the number of compounds and structural variation
within the described scaffold is too low to draw a definite conclu-
sion, our results provide first hints to structure–efficacy relation-
ships. Further investigations will be needed to elucidate the
structure–efficacy relationships for agonism/partial agonism/
antagonism/inverse agonism in this series of differentially substi-
tuted diaminopyrimidines. Especially 27 is an interesting com-
pound for follow-up development as it exhibits an inverse
agonistic constitutive activity of about -80%, relative to reference
2. Cyclic structures 37 and 38 possess increased inverse agonist
efficacy similar to that of 2 but more limited possibilities for struc-
tural modifications. However, taking into consideration that the
reference structure is not a full inverse agonist8 it might be possi-
ble to enhance inverse agonist efficacy towards the hH4R beyond 2.
To investigate the selectivity of the most potent hH4R ligands,
affinities on the three other histamine receptor subtypes, hH1R–
hH3R, were determined for selected compounds (Table 5). These
structures possess good selectivity over hH1R and hH2R because
pKi values are below 6. For compound 32 at least a 80-fold selectiv-
ity for hH4R was seen. With regard to hH3R the ligands do show sig-
nificant affinities but like other pyrimidine-containing
compounds34,40 they preferentially bind to hH4R. Compound 19 is
the least selective of compounds tested, the other three ligands fa-
vour the hH4R: affinities of 28 and 29 differ to one order of magni-
tude, whereas the best selectivity (more than 40-fold) is found in
compound 32. Although ligands are at least monoprotonated under
physiological conditions and calculated Slog P values are below 1.5
it can be assumed that these compounds are able to penetrate the
blood/brain-barrier. Structurally related aminopyrimidines of sim-
ilar physicochemical properties are reported to achieve brain/plas-
ma ratios ranging up to 20.33,40 Therefore, some of the compounds
reported here might also be able to penetrate the central nervous
system, though this was not tested. All ligands discussed in this
context are partial agonists and unlikely to be developed as drug
candidates. However, the structures are active on both hH3R and
hH4R and potentially occurring hH3R effects have to be considered
in further investigations. In the periphery dual acting ligands might
be useful in the treatment of pain or cancer. For example, the
endogenous ligand 1 influences proliferation of cancer cells via
consistent with the ones obtained from [35S]GTP
Compounds with pKi values <7.3 were functionally character-
ized in the [35S]GTP
S binding assay. 4-Hydroxy, 4-cyano and 4-
cS assay.
c
chloro derivatives 21, 30 and 19 exhibit receptor activation in
the same range as partial agonist 31 does, whereas 4-methyl deriv-
ative 24 does not show receptor activation nor reduction of its con-
stitutive activity indicating that it possesses neutral antagonist
properties in the respective in vitro system. Hence, this compound
was reinvestigated in the antagonist mode of the [35S]GTP
cS bind-
ing assay and showed the inhibition of receptor activation induced
by the endogenous ligand 1 (Fig. 3).
Among the p-substituted compounds only the 4-methoxy
derivative 20 exhibits weak inverse agonist efficacy. Possibly steric
substituents in p-position evoke inverse agonism but reduced
affinity. In contrast to o- and p-substituted partial agonists, m-
substituted compounds 25–27 more clearly are characterized as
inverse agonists showing a reduction of constitutive activity be-
tween 65% and 85%, compound 2 being the reference. When com-
paring the meta-chlorinated inverse agonist 25 with its
corresponding para-substituted analogue and partial agonist 19
and the dichlorinated compound 22 it is found that the 3,4-di-Cl-
substitution still allows for inverse agonism. In this series of
aminopyrimidines the meta-site seems to have a major influence
on the overall efficacy of the compound.
The conformationally restrained structure 37 possesses a simi-
lar and 38 even a superior potency compared to that of 2 (Table
4). Compared to reference structure 3, which is characterized as in-
verse agonist in both functional assays (cf. Table 4 and Ref. 8), the
meta-substituted and rigidified compounds exhibit stronger in-
verse agonist efficacy.

K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
7193
hH3R and hH4R.53 To probe such applications the diaminopyrimi-
dines’ efficacy towards both receptors will be further investigated
in future studies.
atmosphere for 12 h. The solvent was evaporated, the residue puri-
fied by column chromatography (DCM/ammoniacal MeOH = 9/1)
and the product re-crystallized from acetonitrile.
7.1.2. Example compound 6-(4-methylpiperazin-1-yl)-9H-
purine (9)
6. Conclusions
Yield: 60%. 1H NMR (DMSO-d6): d 13.01 (s, 1H, pur-NH), 8.19 (s,
1H, pur-2H), 8.10 (s, 1H, pur-8H), 4.19 (br s, 4H, prz-2,6H2), 2.39 (t,
J = 4.9, 4H, prz-3,5H2), 2.19 (s, 3H, prz-CH3). 13C NMR (DMSO-d6): d
153.14 (pur-4C), 151.73 (pur-2C), 151.44 (pur-6C), 138.06 (pur-
8C), 118.74 (pur-5C), 54.58 (prz-3,5C), 45.75 (prz-CH3), 44.43
(prz-2,6C). ESI MS: 218.9 (100). Mp: 270 °C. Anal. (C10H14N6): C,
H, N.
New hH4R lead structures were successfully identified by struc-
tural modifications and structure–affinity relationships of the hits
obtained from a virtual screening. By employing classical medicinal
chemical methods optimization was carried out to investigate hH4R
affinity with the outcome that hH4R binding in the nanomolar con-
centration range was achieved with the best compounds 28, 29,
and 32. Functional profiling for receptor activation was determined
for the compounds with pKi values >6.2. The described series of dia-
minopyrimidines covers a broad range of functional properties. Sub-
stitution in o- and p-position obviously led to partial agonist efficacy,
whereas m-substituted and conformationally restrained com-
pounds exhibited inverse agonism. The latter conform to the goal
of designing hH4R ligands with inverse agonist functionality. Such
property is aimed at enhancing efficacy as anti-inflammatory or im-
mune modulating agents, compared to partial agonists and neutral
antagonists.54 Hence, compound 27 represents a new lead structure
for further investigations on expanded structure–efficacy and struc-
ture–affinity relationships in this direction.
7.1.3. Preparation of benzyl purines 10 and 12
Purines 9 or 11 (1.8–3 mmol), benzyl bromide (1.2 equiv),
Cs2CO3 (2 equiv) and DMF (5 ml) were stirred overnight at
110 °C. After cooling and evaporating the solvent the mixture
was purified by column chromatography (DCM/ammoniacal
MeOH = 9.5/0.5). The oily products were crystallized as salts.
7.1.4. Example compound 9-benzyl-6-(4-methylpiperazin-1-yl)-
9H-purine hydrogenmaleate (10)
Yield: 10%. 1H NMR (DMSO-d6): d 12.16 (s, 1H, pur-NH), 7.65 (s,
1H, pur-8H), 5.71 (s, 2H, NH2), 4.10 (br s, 4H, prz-2,6H2), 2.35 (t,
J = 4.9, 4H, prz-3,5H2), 2.19 (s, 3H, prz-CH3). 13C NMR (DMSO-d6):
d 159.50 (pur-2C), 153.80 (pur-4C), 153.50 (pur-6C), 134.67 (pur-
8C), 113.16 (pur-5C), 54.69 (prz-3,5C), 45.85 (prz-CH3), 44.14
(prz-2,6C). ESI MS: 233.9 (100). Mp: 255–258 °C. Anal.
(C10H15N7): C, H, N.
7. Experimental section
7.1. General chemical procedures
Melting points were determined on a Büchi 510 melting point
apparatus (Büchi, Switzerland) and are uncorrected. 1H NMR spectra
were recorded on a Bruker AMX 300 (300 MHz) spectrometer (Bru-
ker, Germany). 1H NMR data are reported in the following order:
chemical shift (d) in ppm downfield from tetramethylsilane as inter-
nal reference; multiplicity (br, broad; s, singlet; d, doublet; dd, dou-
ble doublet; t, triplet; m, multiplet); approximate coupling
constants (J) in hertz (Hz); number and assignment of protons
(mal, maleate; ox, oxalate; prz, piperazine; pur, purine; pyrim,
pyrimidine; ph, phenyl). 13C NMR spectra were recorded on a Bruker
AC 200 (50 MHz) spectrometer (Bruker, Germany). Depending on
scanning time of 13C NMR spectra, signal intensity of pyrim-4C and
pyrim-6C sometimes was too low to be detected. ESI MS was per-
formed on a Fisons Instruments VG Platform II (Manchester, Great
Britain) in positive polarity. Data are listed as mass number
([M+H]+) and relativeintensity (%). Elemental analyses (C, H, N) were
measured on CHN-Rapid (Heraeus, Germany) and were within 0.4%
of the theoretical values for all final compounds. Preparative column
7.2. General procedure for preparation of 14a, 16a, 17a, 19a–36a,
14–16, and 19–38
Monosubstitution of dichloropyrimidines was carried out in a
microwave oven using 4,6-dichloropyrimidines (3–12 mmol), the
designated amine (0.9 equiv of either 1-methylpiperazine or a
substituted benzyl amine), DIPEA (2 equiv), and 2-propanol (5–
15 ml). In sealed vials reactants were stirred at 160 °C in the case
of 1-methylpiperazine and at 130 °C in the case of benzyl amines.
Reaction termination was indicated by TLC; usually 30–60 min were
required, depending on scale and substitution pattern of benzyl
amines. After cooling the solvent was evaporated and the crude
product was purified by column chromatography (DCM/ammonia-
cal MeOH = 9.5/0.5). The second substitution step was accomplished
in the same manner but by adding an excess (2 equiv) of the amine
(in most cases 1-methylpiperazine). Final compounds, either as base
or as hydrogenmaleate, were re-crystallized from ethanol.
chromatography was performed on silica gel 63–200 lM (Merck,
Germany). Hydrogenations were carried out on an autoclave model
IV, 500 mL (Roth, Germany). The microwave oven used is a Biotage
Initiator 2.0 (Biotage, Sweden). Educts (6-chloro-9H-purine, 4,6-
dichloropyrimidine, and the corresponding 2-amines, 1-methylpi-
perazine, propargyl bromide, benzylbromide, benzylalcohol, benzyl
mercaptane, and benzyl amines) and all other reactants (DIPEA, TEA,
Cs2CO3, NaH) were commercially obtained from Sigma–Aldrich,
ABCR, and Acros Organics and were used without further purifica-
tion unless otherwise stated.
Preparation of parent compounds and most affine hH4R ligands
as well as detailed pharmacological assay procedures are described
in the following experimental part, whereas complete analytical
data of final compounds can be found in the Supplementary data.
7.2.1. Example compound N-benzyl-6-(4-methylpiperazin-1-yl)-
pyrimidin-4-amine dihydrogenmaleate (14)
In a sealed vial 4,6-dichloropyrimidine (0.50 g, 3.4 mmol), ben-
zyl amine (0.32 g, 3.1 mmol), DIPEA (0.87 g, 6.8 mmol) were sus-
pended in 2-propanol (4 ml) and heated in the microwave oven
at 130 °C for 30 min. After cooling the solvent was evaporated
and the residue purified by column chromatography (DCM/ammo-
niacal MeOH = 9.5/0.5) to obtain N-benzyl-6-chloropyrimidin-4-
amine (14a) as light yellow crystals (yield: 75%). 14a (0.55 g,
2.5 mmol), together with 1-methylpiperazine (0.50 g, 5 mmol), DI-
PEA (0.65 g, 5 mmol) and 2-propanol (4 ml), was retreated in the
microwave oven at 160 °C for 30 min. Purification by column chro-
matography led to a beige solid, which was crystallized as hydrog-
enmaleate salt and re-crystallized in ethanol (overall yield: 30%).
1H NMR (DMSO-d6): d 8.07 (s, 1H, pyrim-2H), 7.48 (t, J = 6.1, 1H,
NH), 7.33–7.22 (m, 5H, ph-2,3,4,5,6H), 6.13 (s, 4H, mal-(CH)2),
5.77 (s, 1H, pyrim-5H), 4.46 (d, J = 5.0, 2H, CH2), 3.71 (br s, 4H,
7.1.1. Preparation of purines 9 and 11
6-Chloro-9H-purines (4-5 mmol), 1-methylpiperazine (2 equiv)
and TEA (4 equiv) were stirred in DMF (50 ml) at rt under argon

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K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
prz-2,6H2), 3.22 (s, 4H, prz-3,5H2), 2.81 (s, 3H, prz-CH3). 13C NMR
(DMSO-d6): d 166.87 (mal-COOH), 162.83 (pyrim-6C), 161.28 (pyr-
im-4C), 156.91 (pyrim-2C), 139.75 (ph-1C), 133.17 (mal-CH),
128.22 (ph-2,6C), 127.13 (ph-3,5C), 126.68 (ph-4C), 82.11 (pyrim-
5C), 51.94 (prz-3,5C), 43.65 (NH–CH2), 42.22 (prz-CH3), 40.92
(prz-2,6C). ESI MS: 284.1 (100). Mp: 168 °C. Anal. (C16H21N5. 2
C4H4O4): C, H, N.
fluxed for 12 h. After cooling to rt, ethanol was evaporated and
water added leading to the crystallization of the product, which
was washed with small amounts of diethyl ether. It was crystal-
lized as dihydrogenmaleate and re-crystallized twice from ethanol
to give a white solid (yield: 3%). 1H NMR (DMSO-d6): d 7.41–7.21
(m, 5H, ph-2,3,4,5,6H), 6.36 (s, 2H, NH2), 6.14 (s, 4H, mal-(CH)2),
6.04 (s, 1H, pyrim-5H), 4.33 (s, 2H, CH2), 3.72 (br s, 4H, prz-
2,6H2), 3.19 (s, 4H, prz-3,5H2), 2.80 (s, 3H, prz-CH3). 13C NMR
(DMSO-d6): d 167.35 (pyrim-4C), 166.91 (mal-COOH), 161.76 (pyr-
im-6C), 161.60 (pyrim-2C), 138.43 (ph-1C), 133.12 (mal-CH),
128.95 (ph-3,5C), 128.33 (ph-2,6C), 126.91 (ph-4C), 88.92 (pyrim-
5C), 51.92 (prz-3,5C), 42.19 (prz-CH3), 40.73 (prz-2,6C), 32.06 (S–CH2).
ESI MS: 316.0 (100). Mp: 179 °C. Anal. (C16H21N5SÁ2C4H4O4Á0.5H2O): C,
H, N.
7.2.2. Example compound N4-benzyl-6-(4-methylpiperazin-1-
yl)pyrimidine-2,4-diamine dihydrogenmaleate (16)
2-Amino-4,6-dichloropyrimidine (1.00 g, 6.0 mmol), benzyl
amine (0.56 g, 5.4 mmol), DIPEA (1.54 g, 12.0 mmol), and 2-propa-
nol (10 ml) were heated under microwave irradiation at 130 °C for
60 min. Purification was accomplished by column chromatography
(DCM/ammoniacal methanol = 9.5/0.5). The resulting yellowish
powder (yield: 87%), N4-benzyl-6-chloropyrimidin-2,4-diamine
(16a, 1.10 g, 4.7 mmol), was suspended in 2-propanol (10 ml),
and 1-methylpiperazine (0.94 g, 9.4 mmol) and DIPEA (1.22 g,
9.4 mmol) were added. The mixture was stirred in the microwave
oven at 160 °C for 30 min. After purification by column chromatog-
raphy the resulting oil was crystallized by adding maleic acid to
obtain a white solid (overall yield: 34%). 1H NMR (DMSO-d6): d
7.75 (s, 1H, NH), 7.38–7.25 (m, 5H, ph-2,3,4,5,6H), 7.05 (br s, 2H,
NH2), 6.06 (s, 4H, mal-(CH)2), 5.42 (s, 1H, pyrim-5H), 4.45 (d,
J = 5.9, 2H, CH2), 3.74 (br s, 4H, prz-2,6H2), 3.17 (s, 4H, prz-
3,5H2), 2.76 (s, 3H, prz-CH3). 13C NMR (DMSO-d6): d 167.10 (mal-
COOH), 161.56 (pyrim-2C), 157.40 (pyrim-4C), 155.95 (pyrim-6C),
138.20 (ph-1C), 134.94 (mal-CH), 128.44 (ph-3,5C), 127.27 (ph-
2,6C), 127.19 (ph-4C), 72.15 (pyrim-5C), 52.04 (prz-3,5C), 44.25
(NH–CH2), 42.49 (prz-CH3), 40.35 (prz-2,6C). ESI MS: 299.0 (100).
Mp: 169 °C. Anal. (C16H22N6Á2C4H4O4): C, H, N.
7.2.5. Preparation of 1-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-
4-methylpiperazine dihydrogenmaleate (13)
Benzyl bromide (0.34 g, 2 mmol) and NaN3 (0.33 g, 5 mmol) were
mixedinwater(4 ml)andheatedinthemicrowave oven at 120 °C for
30 min. The product was extracted in diethyl ether and the solvent
evaporated to give (azidomethyl)benzene (13a) as a colourless oil
(yield: 100%). Propargyl bromide (0.29 g, 2.4 mmol), 1-methylpiper-
azine (0.24 g, 2.4 mmol), and TEA (0.40 g, 4 mmol) were stirred in
water (4 ml) at rt for 1 h. CuI (0.046 g, 0.2 mmol) and 13a (0.26 g,
2 mmol) were added and the mixture was stirred for further 12 h.
After addition of water and extraction with methylene chloride the
organic layer was dried (Na2SO4) and concentrated to dryness. The
product was purified by column chromatography (DCM:MeOH/
NH3 = 9:1) and crystallized as dihydrogenmaleate (yield: 17%). 1H
NMR (DMSO-d6): d 7.85 (s, 1H, trz-5H), 7.17–7.05 (m, 5H, ph-
2,3,4,5,6H), 5.92 (mal-(CH)2), 5.35 (s, 2H, ph-CH2), 3.46 (s, 2H, prz-
CH2), 2.91 (br s, 4H, prz-2,6H2), 2.48 (s, 4H, prz-3,5H2), 2.26 (s, 3H,
prz-CH3). 13C NMR (DMSO-d6): d 166.92 (mal-COOH), 142.26 (trz-
4C), 136.04 (ph-1C), 133.11 (mal-CH), 128.75 (ph-3,5C), 128.12
(ph-4C), 127.87 (ph-2,6C), 124.35 (trz-5C), 52.72 (ph-CH2), 52.55
(prz-3,5C), 51.11 (prz-CH2), 48.89 (prz-2,6C), 42.25 (prz-CH3). ESI
MS:272.1 (100). Mp: 186–189 °C. Anal. (C15H21N5. 2 C4H4O4): C, H, N.
7.2.3. Example compound 4-(benzyloxy)-6-(4-methylpiperazin-
1-yl)pyrimidin-2-amine (17)
4,6-Dichloropyrimidin-2-amine (2.00 g, 12.2 mmol), 1-meth-
ylpiperazin (1.10 g, 10.9 mmol), DIPEA (3.20 g, 24.4 mmol), and 2-
propanol (15 ml) were heated under microwave irradiation at
160 °C for 60 min. After cooling the product crystallized and was fil-
tered out. 4-Chloro-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine
(17a), a white solid (yield: 68%), was used without further purifica-
tion. Under argon atmosphere, NaH (60%, 0.23 g, 5.7 mmol) was sus-
pended in THF (15 ml) and benzyl alcohol (0.48 g, 4.4 mmol) was
added dropwise. The suspension was stirred at rt for 15 min, subse-
quently cooled to 0 °C and treated with a solution of 17a (0.5 g,
2.2 mmol) in THF (5 ml). The solution was allowed to come to rt
and was stirred for 24 h. The reaction was quenched with a saturated
solution of NH4Cl. THF was reduced under vacuum and the crude
product extracted into ethyl acetate (2 Â 20 ml). The organic layers
were combined, washed with 2 N-NaOH and brine, dried over
Na2SO4 and concentrated to dryness. Further purification was
accomplished by column chromatography (DCM/ammoniacal
methanol = 9.5/0.5) and the product re-crystallized from ethanol
(white solid; yield: 20%). 1H NMR (DMSO-d6): d 7.40–7.27 (m, 5H,
ph-2,3,4,5,6H), 6.07 (s, 2H, NH2), 5.38 (s, 1H, pyrim-5H), 5.23 (s,
2H, CH2), 3.43 (t, J = 4.5, 4H, prz-2,6H2), 2.28 (t, J = 4.7, 4H, prz-
3,5H2), 2.17 (s, 3H, prz-CH3). 13C NMR (DMSO-d6): d 170.55 (pyr-
im-4C), 164.70 (pyrim-6C), 162.34 (pyrim-2C), 137.45 (ph-1C),
128.24 (ph-3,5C), 127.78 (ph-2.6C), 127.58 (ph-4C), 75.64 (pyrim-
5C), 66.00 (O–CH2), 54.28 (prz-3,5C), 45.74 (prz-CH3), 43.54 (prz-
2,6C). ESI MS: 299.9 (100). Mp: 161 °C. Anal. (C16H21N5O): C, H, N.
7.3. Virtual screening
The Specs database55 containing 199,272 compounds was virtu-
ally screened. After in silico washing of the virtual molecules, that
is, protonating basic and deprotonating acidic compounds, the
database was prefiltered with MOE56 to select cationic compounds.
The remaining 9867 molecules were straightforwardly resorted by
their 2-dimensional topological pharmacophore similarity towards
structural features of 3 by the software package CATS.39 A 150-
dimensional correlation vector-based molecular descriptor was
compiled for the reference molecule and all residual molecules in
the database. Next, each screening substance was ranked according
to its pairwise Euclidean distance to the reference. Twenty seven
compounds from the top percentiles of the ranked remnant data-
base were selected by experienced medicinal chemists under con-
sideration of the synthetic accessibility. Another selection criterion
was the frequency of occurrence of certain scaffolds. These hand-
picked molecules were tested in a competition binding assay. Nine
compounds yielded an affinity <30 lM, which implied a hit rate of
33%, and two of these compounds, 3-(4-chlorobenzyl)-7-(4-meth-
ylpiperazin-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (7) and 6-
ethyl-4-(4-methylpiperazin-1-yl)thieno[2,3-d]pyrimidine (8) showed an
affinity in the low micromolar concentration range.
7.2.4. Example compound 4-(benzylthio)-6-(4-methylpiperazin-
1-yl)pyrimidin-2-amine dihydrogenmaleate (18)
7.4. Binding assay on hH1R, hH2R, and hH3R
Compound 17a (0.86 g, 3.8 mmol) and NaOH (0.18 g, 4.4 mmol)
were dissolved in water (10 ml) and ethanol (15 ml). Benzyl mer-
captane (0.71 g, 5.7 mmol) was added and the solution was re-
Prior to the experiments, membranes were sedimented by a
10 min centrifugation at 4 °C and 16,000g and resuspended in

K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
7195
binding buffer (12.5 mM MgCl2, 1 mM EDTA and 75 mM Tris/HCl,
pH 7.4). For determination of Kd and Bmax values, Sf9 membranes
potency of receptor activation (EC50) was performed by GraphPad
Prism 3.02.
(30–70
l
g of protein per tube, depending on the specific expres-
l of binding buffer containing
sion level) were suspended in 250
l
7.7. Steady-state GTPase assay
the radioligand and 0.2% (mass/vol) of bovine serum albumin.
For competition binding studies on hH1R, Sf9 cell membranes co-
expressing the hH1R with RGS4 were incubated with [3H]pyrilamine
(5 nM, 954.6 GBq/mmol). Binding studies on hH2R were performed
Sf9 membranes were thawed, sedimented and resuspended in
10 mM Tris/HCl, pH 7.4. Each assay tube contained 80
Tris/HCl (pH 7.4) with 5.0 mM MgCl2, 100 M EDTA, 100
100 nM GTP, 100 M adenylyl imidodiphosphate, 1.2 mM creatine
phosphate, 1 g of creatine kinase, 0.2% (w/v) BSA, 100 mM NaCl
and H4R ligands at various concentrations. Additionally, each tube
contained 10–20 g of Sf9 membranes co-expressing the hH4R with
i2 and Gb1 2. The tubes were incubated for 2 min at 25 °C before
the addition of 20 l of [ Ci per tube (final vol-
32P]GTP to yield 0.1
ume 100 l). The [
32P]GTP work solutions were prepared in
ll of 50 mM
l
lM ATP,
usingthehH2R-Gs
(10 nM, 2.8564 TBq/mmol). Binding studies on hH3R were per-
formed by incubating membranes co-expressing hH3R, i2,
2 and RGS4 with [3H]N -methylhistamine (1 nM, 2.6048 TBq/
mmol). Non-specific binding was determined in the presence of
10 M of diphenhydramine (hH1R), famotidine (hH2R) or thio-
a
sfusionproteinandtheradioligand[3H]tiotidine
l
l
Ga
a
Gb1c
l
Ga
c
l
l
c-
l
peramide (hH3R).
l
c-
Incubations were performed for 60 min at 25 °C and shaking at
250 rpm. Bound radioligand was separated from free radioligand
by filtration through GF/C filters (for binding on hH2R and hH3R
pretreated with 0.3% (mass/vol) polyethyleneimine) and washed
three times with 2 ml of ice-cold binding buffer (4 °C). Filter-bound
radioactivity was determined by liquid scintillation counting.
20 mM Tris/HCl, pH 7.4.
Reactions were conducted for 20 min at 25 °C and terminated
by the addition of 900 ll of slurry consisting of 5% (w/v) activated
charcoal in 50 mM NaH2PO4 (pH 2.0). Charcoal adsorbs nucleotides
but not 32Pi. Charcoal-quenched reaction mixtures were centri-
fuged for 15 min at room temperature at 15,000 Â g. Supernatant
fluid of the reaction mixtures was removed (600 l
l) and 32Pi was
ˇ
7.5. Binding assay on hH4R
determined in water by detecting the Cerenkov radiation. Enzyme
activities were corrected for spontaneous degradation of
Prior to the experiments, cell membranes were sedimented by a
10 min centrifugation at 4 °C and 16,000g. and resuspended in
binding buffer (12.5 mM MgCl2, 1 mM EDTA and 75 mM Tris/HCl,
pH 7.4). Competition binding experiments were carried out by
[c-
32P]GTP, which was determined by adding a very high concen-
tration of unlabelled GTP (1 mM) to tubes containing all of the
above described components. Competing with [
32P]GTP, the
unlabeled GTP prevents hydrolysis of [
32P]GTP by the enzymatic
32P]GTP, which was
c-
c
-
incubating membranes, 35
lg/well (prepared from Sf9 cells
activities present in Sf9 membranes. The [c-
expressing hH4R, co-expressed with G protein G
a
i2 and Gb1 2 sub-
c
prepared by labelling of GDP with 32P, was highly stable, showing
a spontaneous degradation of <1% in 20 mM Tris/HCl, pH 7.4. The
experimental conditions chosen ensured that not more than 10%
units) in a final volume of 0.2 ml containing binding buffer and
[3H]histamine (10 nM, 566.1 GBq/mmol). Assays were run in trip-
licates with seven appropriate concentrations between 0.1 nM
of the total amount of [c-
32P]GTP added was converted to 32P.
and 100
for 60 min at 25 °C and shaking at 250 rpm. Non-specific binding
was determined in the presence of 100 M unlabelled 1. Bound
lM of the test compound. Incubations were performed
Acknowledgements
l
radioligand was separated from free radioligand by filtration
through GF/B filters pretreated with 0.3% (mass/vol) polyethylene-
imine and washed three times with 0.5 ml of ice-cold binding buf-
fer (4 °C). The amount of radioactivity collected on the filter was
determined by liquid scintillation counting. Determination of affin-
ity values (Ki) were calculated by Graph Pad Prism 3.02.
This work was featured with the second prize of the ‘Art A Han-
cock Young Investigator Award’ at the XXXVIIIth annual meeting of
the European Histamine Research Society.57 It was partly sup-
ported by a grant of the DAAD Germany (PPP Polen D/06/25529),
the Research Training Program (Graduiertenkolleg, GRK) 760
‘Medicinal Chemistry: Molecular Recognition—Ligand-Receptor
Interactions’ of the Deutsche Forschungsgemeinschaft, the
‘Beilstein-Institut zur Förderung der chemischen Wissenschaften’
(Frankfurt/Main), the COST Action BM0806 and by the ‘LOEWE
Lipid Signalling Forschungszentrum’ (LiFF, Frankfurt/Main).
7.6. hH4R [35S]GTP
cS binding assay
Prior to the experiments, cell membranes were sedimented by a
10 min centrifugation at 4 °C and 16,000g and resuspended in
binding buffer (1
75 mM Tris/HCl, pH 7.4). [35S]GTP
mode were performed with binding buffer in the presence of
0.6 nM [35S]GTP
S in a final volume of 0.2 ml. In the antagonist
mode hH4R was stimulated with 100 nM 1 resulting in a final vol-
ume of 0.25 ml. Membranes, 10 g/well (prepared from Sf9 cells
expressing hH4R, co-expressed with G-protein G i2 and Gb1 2 sub-
units) and ligands were preincubated without [35S]GTP
S for
l
M GDP, 12.5 mM MgCl2, 100 mM NaCl and
Supplementary data
cS binding studies in the agonist
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.059.
c
l
References and notes
a
c
c
1. Baroody, F. M.; Naclerio, R. M. Allergy 2000, 55, 17.
30 min to obtain steady-state receptor occupation. Assays were
run in triplicates with seven appropriate concentrations between
2. Dove, S.; Elz, S.; Seifert, R.; Buschauer, A. Mini-Rev. Med. Chem. 2004, 4, 941.
3. Sander, K.; Kottke, T.; Stark, H. Biol. Pharm. Bull. 2008, 31, 2163.
4. Oda, T.; Morikawa, N.; Saito, Y.; Masuho, Y.; Matsumoto, S. J. Biol. Chem. 2000,
275, 36781.
5. Hofstra, C. L.; Desai, P. J.; Thurmond, R. L.; Fung-Leung, W. P. J. Pharmacol. Exp.
Ther. 2003, 305, 1212.
6. Morse, K. L.; Behan, J.; Laz, T. M.; West, R. E., Jr.; Greenfeder, S. A.; Anthes, J. C.;
Umland, S.; Wan, Y.; Hipkin, R. W.; Gonsiorek, W.; Shin, N.; Gustafson, E. L.;
Qiao, X.; Wang, S.; Hedrick, J. A.; Greene, J.; Bayne, M.; Monsma, F. J., Jr. J.
Pharmacol. Exp. Ther. 2001, 296, 1058.
0.01 nM and 100
35S]GTP
S, membranes were further incubated for 120 min at
25 °C and shaking at 250 rpm. Basal [35S]GTP
S binding was mea-
lM of test compound. After addition of
[
c
c
sured in the absence of ligands. The reaction was terminated by ra-
pid filtration through GF/B filters soaked in binding buffer followed
by three washings with 0.5 ml of ice-cold binding buffer (4 °C). The
amount of radioactivity collected on the filter was determined by
liquid scintillation counting. The compounds were tested in three
independent experiments (n P 3, mean SEM). Determination of
7. Leff, P. Trends Pharmacol. Sci. 1995, 16, 89.
8. Schneider, E. H.; Schnell, D.; Papa, D.; Seifert, R. Biochemistry 2009, 48, 1424.
9. Ling, P.; Ngo, K.; Nguyen, S.; Thurmond, R. L.; Edwards, J. P.; Karlsson, L.; Fung-
Leung, W. P. Br. J. Pharmacol. 2004, 142, 161.

7196
K. Sander et al. / Bioorg. Med. Chem. 17 (2009) 7186–7196
10. Buckland, K. F.; Williams, T. J.; Conroy, D. M. Br. J. Pharmacol. 2003, 140, 1117.
11. Gutzmer, R.; Diestel, C.; Mommert, S.; Kother, B.; Stark, H.; Wittmann, M.;
Werfel, T. J. Immunol. 2005, 174, 5224.
12. Tanaka, S.; Ichikawa, A. J. Pharmacol. Sci. 2006, 101, 19.
13. Bäumer, W.; Wendorff, S.; Gutzmer, R.; Werfel, T.; Dijkstra, D.; Chazot, P.;
Stark, H.; Kietzmann, M. Allergy 2008, 63, 1387.
T. A.; Wakefield, B. D.; Wetter, J. M.; Witte, D. G.; Honore, P.; Esbenshade, T. A.;
Brioni, J. D.; Cowart, M. D. J. Med. Chem. 2008, 51, 7094.
35. Carceller González, E.; Salas Solana, J.; Soliva Soliva, R.; Medina Fuentes, E. M.;
Martí Via, J. PCT Intern. Appl., 2007, WO 2007/031529 A1.
36. Raphy, G.; Watson, R. J.; Hannah, D.; Pegurier, C.; Ortmans, I.; Lock, C. J.; Knight,
R. L.; Owen, D. A. PCT Intern. Appl., 2008, WO 2008/031556 A2.
37. Bell, A. S.; Lane, C. A. L.; Mowbray, C. E.; Selby, M. D.; Swain, N. A.; Williams, D.
H. PCT Intern. Appl., 2007, WO 2007/072163 A2.
14. Zampeli, E.; Tiligada, E. Br. J. Pharmacol. 2009, 157, 24.
15. Daugherty, B. L. Br. J. Pharmacol. 2004, 142, 5.
16. Rossbach, K.; Wendorff, S.; Sander, K.; Stark, H.; Gutzmer, R.; Werfel, T.;
Kietzmann, M.; Baumer, W. Exp. Dermatol. 2009, 18, 57.
17. Dunford, P. J.; Williams, K. N.; Desai, P. J.; Karlsson, L.; McQueen, D.; Thurmond,
R. L. J. Allergy Clin. Immunol. 2007, 119, 176.
18. Varga, C.; Horvath, K.; Berko, A.; Thurmond, R. L.; Dunford, P. J.; Whittle, B. J.
Eur. J. Pharmacol. 2005, 522, 130.
19. Cowart, M. D.; Altenbach, R. J.; Liu, H.; Drizin, I.; Wishart, N.; Babinski, D. J.;
Gregg, R. J.; Esbenshade, T. A.; Hsieh, G. C.; Brioni, J. D.; Honore, M. P.; Black, L.
A.; Zhao, C.; Wakefield, B. D. PCT Intern. Appl., 2008, WO 2008/060766 A2.
20. Liu, C.; Ma, X.; Jiang, X.; Wilson, S. J.; Hofstra, C. L.; Blevitt, J.; Pyati, J.; Li, X.;
Chai, W.; Carruthers, N.; Lovenberg, T. W. Mol. Pharmacol. 2001, 59, 420.
21. Lim, H. D.; van Rijn, R. M.; Ling, P.; Bakker, R. A.; Thurmond, R. L.; Leurs, R. J.
Pharmacol. Exp. Ther. 2005, 314, 1310.
38. Cai, H.; Chavez, F.; Edwards, J. P.; Fitzgerald, A. E.; Liu, J.; Mani, N. S.; Neff, D. K.;
Rizzolio, M. C.; Savall, B. M.; Smith, D. M.; Venable, J. D.; Wei, J.; Wollin, R. L.
PCT Intern. Appl., 2008, US 2008/0194577 A1.
39. Schneider, G.; Neidhart, W.; Giller, T.; Schmid, G. Angew. Chem., Int. Ed. 1999,
38, 2894.
40. Altenbach, R. J.; Adair, R. M.; Bettencourt, B. M.; Black, L. A.; Fix-Stenzel, S. R.;
Gopalakrishnan, S. M.; Hsieh, G. C.; Liu, H.; Marsh, K. C.; McPherson, M. J.;
Milicic, I.; Miller, T. R.; Vortherms, T. A.; Warrior, U.; Wetter, J. M.; Wishart, N.;
Witte, D. G.; Honore, P.; Esbenshade, T. A.; Hancock, A. A.; Brioni, J. D.; Cowart,
M. D. J. Med. Chem. 2008, 51, 6571.
41. Topliss, J. G. J. Med. Chem. 1972, 15, 1006.
42. Yan, Z.-Y.; Zhao, Y.-B.; Fan, M.-J.; Liu, W.-M.; Liang, Y.-M. Tetrahedron 2005, 61,
9331.
22. Gbahou, F.; Vincent, L.; Humbert-Claude, M.; Tardivel-Lacombe, J.; Chabret, C.;
Arrang, J. M. Br. J. Pharmacol. 2006, 147, 744.
23. Arrang, J. M.; Garbarg, M.; Lancelot, J. C.; Lecomte, J. M.; Pollard, H.; Robba, M.;
Schunack, W.; Schwartz, J. C. Nature 1987, 327, 117.
24. Igel, P.; Schnell, D.; Bernhardt, G.; Seifert, R.; Buschauer, A. ChemMedChem
2009, 4, 225.
43. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
44. Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Angew. Chem., Int. Ed. 2005, 44, 2210.
45. Ju, Y.; Kumar, D.; Varma, R. S. J. Org. Chem. 2006, 71, 6697.
46. Luo, G.; Chen, L.; Poindexter, G. S. Tetrahedron Lett. 2002, 43, 5739.
47. Beingessner, R. L.; Deng, B. L.; Fanwick, P. E.; Fenniri, H. J. Org. Chem. 2008, 73,
931.
25. Kiss, R.; Keserü, G. M. Exp. Opin. Ther. Patent 2009, 19, 119.
26. Thurmond, R. L.; Desai, P. J.; Dunford, P. J.; Fung-Leung, W. P.; Hofstra, C. L.;
Jiang, W.; Nguyen, S.; Riley, J. P.; Sun, S.; Williams, K. N.; Edwards, J. P.;
Karlsson, L. J. Pharmacol. Exp. Ther. 2004, 309, 404.
27. Lee-Dutra, A.; Arienti, K. L.; Buzard, D. J.; Hack, M. D.; Khatuya, H.; Desai, P. J.;
Nguyen, S.; Thurmond, R. L.; Karlsson, L.; Edwards, J. P.; Breitenbucher, J. G.
Bioorg. Med. Chem. Lett. 2006, 16, 6043.
48. Gibson, C. L.; la Rosa, S.; Ohta, K.; Boyle, P. H.; Leurquin, F.; Lemaçon, A.;
Suckling, C. J. Tetrahedron 2004, 60, 943.
49. Seifert, R.; Wenzel-Seifert, K.; Burckstummer, T.; Pertz, H. H.; Schunack, W.;
Dove, S.; Buschauer, A.; Elz, S. J. Pharmacol. Exp. Ther. 2003, 305, 1104.
50. Wenzel-Seifert, K.; Kelley, M. T.; Buschauer, A.; Seifert, R. J. Pharmacol. Exp.
Ther. 2001, 299, 1013.
51. Schlegel, B.; Laggner, C.; Meier, R.; Langer, T.; Schnell, D.; Seifert, R.; Stark, H.;
Holtje, H. D.; Sippl, W. J. Comput. Aided Mol. Des. 2007, 21, 437.
52. Martin, S. F. Pure Appl. Chem. 2007, 79, 193.
28. Smits, R. A.; Lim, H. D.; Stegink, B.; Bakker, R. A.; de Esch, I. J.; Leurs, R. J. Med.
Chem. 2006, 49, 4512.
29. Ashby, C. R., Jr.; Wang, R. Y. Synapse 1996, 24, 349.
30. Bishara, D.; Taylor, D. Drugs 2008, 68, 2269.
53. Medina, V.; Croci, M.; Crescenti, E.; Mohamad, N.; Sanchez-Jimenez, F.;
Massari, N.; Nunez, M.; Cricco, G.; Martin, G.; Bergoc, R.; Rivera, E. Cancer
Biol. Ther. 2008, 7, 28.
54. Tiligada, E.; Zampeli, E.; Sander, K.; Stark, H. Expert Opin. Investig. Drugs 2009,
in press.
31. Smits, R. A.; Lim, H. D.; Hanzer, A.; Zuiderveld, O. P.; Guaita, E.; Adami, M.;
Coruzzi, G.; Leurs, R.; de Esch, I. J. J. Med. Chem. 2008, 51, 2457.
32. Chavez, F.; Curtis, M. P.; Edwards, J. P.; Gomez, L.; Grice, C. A.; Kearney, A. M.;
Savall, B. M.; Fitzgerald, A. E.; Liu, J.; Mani, N. S. PCT Intern. Appl., 2008, WO
2008/008359.
55. v2007.10, Delft, the Netherlands, www.specs.net.
56. v2007.09,
Chemical
Computing
Group,
Montreal,
Canada;
33. 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. J. Med. Chem. 2008, 51, 6547.
34. Liu, H.; Altenbach, R. J.; Carr, T. L.; Chandran, P.; Hsieh, G. C.; Lewis, L. G.;
Manelli, A. M.; Milicic, I.; Marsh, K. C.; Miller, T. R.; Strakhova, M. I.; Vortherms,
www.chemcomp.com.
57. Sander, K.; Kottke, T.; Proschak, E.; Tanrikulu, Y.; Schneider, E.; Seifert, R.;
Schneider, G.; Stark, H. Development of diaminopyrimidines as histamine H4
receptor ligands—virtual screening and scaffold optimization. XXXVIIIth
Meeting of the European Histamine Research Society, Fulda, Germany, 2009; O27.