Synthesis and evaluation of novel ligands for the histamine H4 receptor based on a pyrrolo[2,3-d]pyrimidine scaffold

Article abstract of DOI:10.1016/j.bmcl.2012.10.139

Starting from a known H₄R ligand based on a pyrimidine skeleton, a series of novel analogues based on a pyrrolo[2,3-d]pyrimidine scaffold have been prepared. Whereas the original pyrimidine congener shows good affinity at hH₄R (K_i = 0.5 μM), its lacks selectivity with a K _i value for the hH₃R of 1 μM. Within the newly synthesized pyrrolo[2,3-d]pyrimidines, several congeners show K_i values of less than 1 μM at the hH₄R and show a much improved selectivity profile. Therefore, these series represent an interesting starting point for the discovery of novel hH₄R ligands.

Full text of DOI:10.1016/j.bmcl.2012.10.139

Bioorganic & Medicinal Chemistry Letters 23 (2013) 132–137
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel ligands for the histamine H4 receptor
based on a pyrrolo[2,3-d]pyrimidine scaffold
Ling-Jie Gao a, J. Stephan Schwed b, Lilia Weizel b, Steven De Jonghe a, Holger Stark b, Piet Herdewijn a,
⇑
a KU Leuven, Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Minderbroedersstraat 10, 3000 Leuven, Belgium
b Johann Wolfgang Goethe University, Institut fuer Pharmazeutische Chemie, ZAFES/CMP, Biozentrum, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from a known H4R ligand based on a pyrimidine skeleton, a series of novel analogues based on a
Received 14 September 2012
Revised 29 October 2012
Accepted 31 October 2012
Available online 15 November 2012
pyrrolo[2,3-d]pyrimidine scaffold have been prepared. Whereas the original pyrimidine congener shows
good affinity at hH4R (Ki = 0.5
l
M), its lacks selectivity with a Ki value for the hH3R of 1
lM. Within the
newly synthesized pyrrolo[2,3-d]pyrimidines, several congeners show Ki values of less than 1
l
M at the
hH4R and show a much improved selectivity profile. Therefore, these series represent an interesting start-
ing point for the discovery of novel hH4R ligands.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Histamine 4 receptor
Pyrrolo[2,3-d]pyrimidine
Pyrimidine
The human histamine 4 (hH4R) receptor is the newest member
of the histamine receptor family. It has been discovered in 2000,
independently by several research groups,1–5 and since then, has
attracted much attention from the medicinal chemistry commu-
nity as promising drug target. The hH4R is mainly expressed on
cells of the immune system (e.g., mast cells, eosinophils, mono-
cytes, dendritic cells, T-cells), although recent reports also show
evidence for expression of the hH4R on non-hematopoietic cells
and in the brain. At the cellular level, activation of the hH4R in-
duces chemotaxis of mast cells and eosinophils and triggers cal-
cium mobilization in mast cells, monocytes and eosinophils.
Moreover, the hH4R modulates the release of various inflammatory
mediators.6 Therefore, the hH4R is currently exploited as potential
drug target for the treatment of various chronic inflammatory and
allergic diseases such as inflammatory bowel disease, asthma,
rheumatoid arthritis and pruritis.7,8
In search for novel hH4R antagonists, a wide variety of different
structural ligands have been discovered. The first non-imidazole
H4R antagonist was JNJ7777120 (Fig. 1), a (piperazinylacyl)indole
analogue that displays strong affinity for the hH4R (Ki = 4 nM).9 Up
to now, it is still considered as the reference antagonist, and this
compound has been used in animal models of different inflamma-
tory disorders (mouse allergic airway inflammation, a mouse perio-
tonitis and a rat colitis model).10 However, the short half-life of this
compound prevents its further development. In search for other ser-
ies of compounds that act as hH4R antagonists, the 2-amino-pyrim-
idine scaffold turned out to be a privileged structure. The first
patents disclosing 2-amino-pyrimidines as hH4R antagonists be-
came public in 2005.11,12 Since then, a wide variety of analogues
have been prepared. Overall, it seems that 5- and 6-positions of
the pyrimidine scaffold tolerate a wide variety of substituents, while
the2-amino group is mostly essential for hH4R binding. The N-meth-
ylpiperazine substituent provides usually one of the most potent
analogues, but the N-methylpiperazine moiety can be successfully
replaced with several analogues. In order to broaden structural vari-
ety, a second and third ring has been appended to the pyrimidine
ring giving rise to bicyclic and tricyclic analogues, respectively
(Fig. 1). Examples of tricyclic analogues include benzothieno[2,3-
d]pyrimidines, benzofuro[2,3-d]pyrimidines13 and benzo[6,7]cyclo-
hepta[1,2-d]pyrimidines.14 Bicyclic congeners that have been
synthesized as hH4R antagonists are quinazolines,15 furo[3,2-
d]pyrimidines,16 pyrazolo[4,3-d]pyrimidines17 and purines.18
However, much to our surprise, the structurally highly related
pyrrolo[2,3-d]pyrimidine scaffold has not been explored in the
hH4R field (or at least not reported in literature). In this letter,
we report our efforts towards the design of novel hH4R ligands
based on a pyrrolo[2,3-d]pyrimidine core structure.
The chemistry started off with the synthesis of 2,6-diamino-4-
(N-methyl-piperazin-1-yl)pyrimidine 2 from 6-chloropyrimidin-
2,6-diamine by
a simple nucleophilic aromatic substitution
reaction (Scheme 1). Although the synthesis of this compound has
been described in literature,19 switching the solvent from 2-
methoxyethanol (as originally described) to water greatly increased
the yield of the reaction and shortened the reaction time from 16 to
4 h.
In order to build up the pyrrolo[2,3-d]pyrimidine scaffold, the
construction of the pyrrole moiety on the preformed pyrimidine
⇑
Corresponding author. Tel.: +32 (0)16 33 73 87.
E-mail address: piet.herdewijn@rega.kuleuven.be (P. Herdewijn).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.139

L.-J. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 132–137
133
Figure 1. Polycyclic structures of hH4R ligands.
manner, yielding the 6-substituted pyrrolo[2,3-d]pyrimidine ana-
logues (Scheme 2). A standard way for derivatisation of the lactam
group involves activation of the carbonyl group by halogenation
using phosphorus oxychloride. Unfortunately, these harsh condi-
tions only resulted in complex reaction mixtures. Recently, we re-
ported on the use of a convenient phosphonium mediated SNAr
reaction for the derivatisation of a lactam functionality.21 Treat-
ment of 4a–h with benzotriazol-1-yloxytris(dimethylamino)-
phosphoniumhexafluorophosphate (BOP), DBU and a nitrogen
nucleophile (piperazine, N-alkyl-piperazine, pyrrolidine) leads to
the formation of the 4-N-substituted pyrrolo[2,3-d]pyrimidine
analogues 5a–s. Although the yield of these reactions was low to
moderate (30–70%), its shortness makes it an attractive route.
When Boc-protected pyrrolidines were used as nucleophile, a final
acidic deprotection step was necessary in order to deliver the de-
sired compounds. Methylation of the exocyclic amino group of
compound 5g was performed by a reductive alkylation reaction
using sodium cyanoborohydride and formaldehyde, affording the
N,N-dimethylated analogue 6.
Scheme 1. Synthesis of 2,6-diamino-4-(N-methyl-piperazin-1-yl)pyrimidine.
Reagents and conditions: (a) N-methylpiperazine, H2O, 4 h, 83%.
ring from 2 was unsuccessful, as only starting material could be
recovered. Therefore, the sequence of reactions was changed, as
we first constructed the pyrolo[2,3-d]pyrimidine scaffold, followed
by decoration of the skeleton. Hereto, 2,6-diamino-4-hydroxy-
pyrimidine 3 was selected as commercially available starting
material. Reaction with a range of
a-halo-carbonyl derivatives
yielded the desired 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine ana-
logues 4a–h.20 All these reactions proceeded in a regiospecific
Scheme 2. Synthesis of 6-H and 6-phenyl substituted pyrrolo[2,3-d]pyrimidines. Reagents and conditions: (a) R6COCH2X (X = Cl or Br), NaOAc, H2O, dioxane; (b) BOP, DBU,
amine, DMF, rt; (c) NaCNBH3, 30% aq formaldehyde, CH3OH, rt.

134
L.-J. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 132–137
Scheme 3. Synthesis of 2-amino-4-N-piperazinyl-6-methyl-pyrrolo[2,3-d]pyrimidine. Reagents and conditions: (a) chloroacetone, DMF, 60 °C; (b) BOP, DBU, piperazine,
DMF, rt.
In contrast to the regiospecific reactions mentioned in Scheme
2, the reaction of 3 with chloroacetone yielded a mixture of two
compounds, that is 2-amino-4-hydroxy-6-methyl-7H-pyrrolo[2,3-
d]pyrimidine 7 and 2,4-diamino-5-methyl-furo[2,3-d]pyrimidine
8 (Scheme 3).22 Separation by flash chromatography on silicagel
yielded the pure furo[2,3-d]pyrimidine derivative 8, whereas the
desired pyrrolo[2,3-d]pyrimidine derivative 7 could not be isolated
in pure form, but was always contaminated with the furo[2,3-
d]pyrimidine analogue 8. Therefore, the mixture was further
treated in a one-step BOP-mediated amination reaction, using
piperazine as nucleophile, yielding a mixture of 2,4-diamino-
5-methyl-furo[2,3-d]pyrimidine 8 (which remained unchanged in
this reaction) and the desired pyrrolo[2,3-d]pyrimidine derivative
9. At that stage, both compounds could be easily separated afford-
ing pure 9.
For alkylation reactions at the pyrrole nitrogen, compounds 5a
and 5g were selected as starting materials (Scheme 4). As treat-
ment of 5a and 5g with alkyl halides led predominantly to alkyl-
ation of the terminal nitrogen atom of the piperazine moiety,
protection prior to alkylation was necessary. The Boc-protected
intermediate 10a was easily obtained by treatment of the 4-N-pip-
erazinyl intermediate 5a with Boc anhydride and 10b was obtained
by treatment of the 4-oxo-pyrrolo[2,3-d]pyrimidine 4c with N-Boc-
piperazine. Although alkylation reactions of these Boc-protected
intermediates with a suitable alkyl or benzyl halide, predomi-
nantly gave the mono-alkylated compound (with an alkyl moiety
on the pyrrole nitrogen), the reaction mixture was always contam-
inated with substantial amounts of the dialkylated compound (al-
kyl on pyrrole nitrogen and exocyclic amino group). They were,
however, easily separated on column, and finally, acidic cleavage
Scheme 4. Synthesis of alkylated pyrrolo[2,3-d]pyrimidine analogues. Reagents and conditions: (a) (Boc)2O, Et3N, DMF, rt; (b) BOP, DBU, N-Boc-piperazine, DMF, rt; (c) R7X
(X = I or Br), NaH, THF, rt; (d) TFA/CH2Cl2, rt; (e) NaCNBH3, 30% aq formaldehyde, CH3OH, rt.

L.-J. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 132–137
135
Table 1
of the Boc group afforded a series of 7-N-substituted pyrrolo[2,3-
SAR of 6-substituted pyrrolo[2,3-d]pyrimidines at hH4R
d]pyrimidine analogues 12a–d. Reductive amination of compound
12d with formaldehyde led to the N-methyl-piperazinyl product
13a as major compound, whereas the double methylated product
13b (i.e. methylation of the exocyclic amino group and the pipera-
zine nitrogen) was isolated as a minor side product.
R4
N
R6
N
H
H2N
R6
N
All compounds synthesized were evaluated for their affinities at
hH4R and in selected cases for the human histamine H3 receptor
(hH3R). For these in vitro determinations radioligand competition
binding assays were used as previously described in Tomasch
et al.23 In brief, determination of hH4R affinity was performed with
crude membrane preparations of Sf9 cells transiently expressed
hH4R in full length and co-expressed with G-Protein subunits
Compd.
R4
hH4R Ki (
lM)
cLogP
LE
H
N
5a
H
45.92
0.58
0.37
0.45
0.36
N
H
N
G
a
and Gb1 2. [3H]Histamine was used as radioligand and
i/o
c
9
CH3
Phenyl
2.67
1.41
1.08
2.68
JNJ7777120 (10 lM concentration) for the determination of non
specific binding. For the determination of human H3 receptor affin-
N
H
N
ity crude membrane preparations of HEK293 cells stably expressing
a
the hH3R in full length were used. [3H]N -Methylhistamine was
5b
N
used as radioligand and 10
nation of non specific binding. All determinations were performed
in four different concentrations ranging from 0.1 to 100
lM Pitolisant was used for the determi-
CH3
N
lM
5c
Phenyl
5.40
3.25
0.31
in triplicates in at least two independent experiments. All
binding data analysis was performed with GraphPad Prism™
(Version 3.02).
N
Compound 2 was previously evaluated for its functional activity
in hH4R transfected cell lines with a Ca2+ Influx FLIPR Assay.17 As
starting point for our internal drug discovery program, this com-
pound was evaluated for its binding affinity towards the hH4R in
a radioligand competition binding assay, whereby compound 2
Table 2
SAR of 6-phenyl pyrrolo[2,3-d]pyrimidines at hH4R
CH3
N
displays a Ki value of 0.53 lM. The appendage of a pyrrole ring to
the pyrimidine ring afforded the pyrrolo[2,3-d]pyrimidine ana-
logue 5a, which lost all affinity for the hH4R, displaying a Ki value
of 45.92 lM (Table 1). In order to restore activity, additional sub-
N
N
R
stituents were introduced on the pyrrole moiety. The presence of
a small methyl group at position 6 of the pyrrolo[2,3-d]pyrimidine
scaffold (compound 9) led to a greatly improved binding affinity
N
H
H2N
N
Compd.
R
hH4R Ki (
l
M)
cLogP
LE
(Ki = 2.67 lM). The insertion of a phenyl at position 6 yielded com-
5d
5e
5f
5g
5h
5i
4-F
4-Cl
4-OCH3
4-CH3
4-CN
4-Br
2.64
3.28
3.32
3.73
7.32
8.99
36.12
3.40
3.97
3.05
3.75
2.70
4.12
4.56
0.32
0.31
0.30
0.31
0.28
0.29
0.24
pound 5b with comparable activity as the 6-methyl analogue 9.
The presence of a piperazinyl (5b) or 4-N-piperazinyl (5c) moiety
at 4-position of the skeleton led only to marginal differences in
binding affinity.
In order to probe into the optimal substitution pattern on the
aromatic ring, a series of analogues was prepared, carrying halo-
gens, electron-withdrawing and electron-donating substituents
on the phenyl ring (Table 2, compounds 5d–5j). Most of these ana-
logues do show activity in the hH4R binding assay, displaying Ki
5j
3,4-di-Cl
Table 3
SAR of 6-(4-fluorophenyl)pyrrolo[2,3-d]pyrimidines at hH4R
values between 2.64 and 8.99
3,4-dichlorophenyl ring (compound 5j) leading to a drastic de-
crease in affinity (Ki = 36.12 M).
lM, with only the presence of a
R
N
l
A series of analogues was prepared to study the effect of the size
of the alkyl group on the piperazinyl moiety (Table 3). Whereas
hydrogen (5k), methyl (5d), and ethyl groups (5l) all have compa-
rable hH4R binding affinities, increasing the size towards an isopro-
N
N
F
N
H
H2N
N
pyl group (compound 5m) led
a drastic loss of affinity
(Ki = 48.01 M). It confirms the trend observed in other series of
l
Compd.
R
hH4R Ki (lM)
cLogP
LE
hH4R antagonists, that sterically demanding substituents are not
tolerated.
5k
5d
5l
H
0.39
2.64
4.07
2.82
3.40
3.93
4.24
0.38
0.32
0.29
0.28
Methyl
Ethyl
Isopropyl
Structurally related diamines frequently used in other H4R drug
design programs were also incorporated onto the pyrrolo[2,3-
d]pyrimidine scaffold (Table 4). It seems that for the 6-unsubstituted
pyrrolo[2,3-d]pyrimidine analogues, the N-methyl-homopiperazi-
nyl substituent (analogue 5n) shows better binding affinity than
its 4-N-piperazinyl counterpart (compound 5a). In the 6-phenyl, as
well as in the 6-(4-fluorophenyl) series, the 4-N-Me-homopiperazi-
nyl (compounds 5o and 5p) substituent show comparable affinity at
hH4R, when compared to their 4-N-Me-piperazinyl congeners
5m
48.01
(compounds 5c and 5d). Both optically pure enantiomers of 3-ami-
no-pyrrolidine were coupled to the pyrrolo[2,3-d]pyrimidine scaf-
fold. Both isomers show good affinity in the hH4R binding assay,
with the (S)-isomer 5r (Ki = 0.98 lM) being slightly higher affine

136
L.-J. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 132–137
Table 4
Table 5
Replacements for the piperazinyl moiety
SAR of alkylated pyrolo[2,3-d]pyrimidines at hH4R
R4
R4
N
N
R6
R6
H2N
N
R2
N
N
H
N
R7
Compd.
R4
CH3
R6
hH4R Ki (
lM)
cLogP
LE
Compd. R2
R4
R6
R7
hH4R Ki
cLogP LE
(lM)
N
CH3
N
5n
H
8.72
1.41
2.93
1.14
0.38
4-F-
phenyl
6
N(CH3)2
H
10.45
4.32
0.26
N
N
CH3
N
H
N
5o
5p
Phenyl
3.23
3.38
0.33
0.30
12a
12b
12c
12d
NH2
NH2
NH2
NH2
H
CH3
36.35
8.13
0.53
2.12
2.30
2.78
0.36
0.35
0.28
0.25
N
N
CH3
N
H
N
n-
butyl
4-F-phenyl
H
H
N
N
N
N
H
N
NH2
NH2
benzyl
25.22
44.15
5q
5r
5s
4-F-phenyl
4-F-phenyl
4-F-phenyl
1.71
0.98
5.95
2.41
2.41
3.15
0.34
0.36
0.31
N
H
N
4-F-
phenyl
CH3
CH3
N
HN
CH3
N
NH
4-F-
phenyl
13a
13b
NH2
67.87
3.35
4.06
0.23
0.21
N
CH3
N
4-F-
phenyl
than the corresponding (R)-distomer 5s (Ki = 1.71 lM). When the 3-
amino-pyrrolidine moiety was coupled via its exocyclic amino
group to the pyrrolo[2,3-d]pyrimidine skeleton (yielding compound
NHCH3
CH3
>100
N
5s), a fivefold decrease in binding affinity (Ki = 5.95 lM) was
observed.
In another round of optimization, structural variation was intro-
duced on the pyrrole nitrogen (Table 5). For the 6-unsubstituted
pyrrolo[2,3-d]pyrimidine analogues, the introduction of a small
Table 6
Affinity profile of selected compounds
Compd.
hH4R Ki (
lM)
hH3R Ki (
lM)
cLogP
LE
methyl group (compound 12a; Ki = 36.35
effect on the binding affinity, when compared to the pyrrolo[2,3-
d]pyrimidines, bearing free NH group (compound 5a;
Ki = 45.92 M). However, insertion of bulkier substituents such as
lM) did not have any
2
5k
0.53
0.39
1.08
84.66
0.42
2.82
0.57
0.38
a
l
a n-butyl (affording analogue 12b) or benzyl (furnishing derivative
12c), increased the affinity for the hH4R (Ki values of 8.13 and
demonstrated the essential contribution of the exocyclic amino
group on hH4R binding.
25.22
(Ki = 45.92
l
M, respectively), when compared to the analogue 5a
An important way of assessing the ‘drug-likeness’ of compound
series is the determination of their lipophilicity. The cLogP values
of all analogues were calculated. Although there are a number of
cases where binding affinity increases with additional lipophilic
substituents (e.g. compare compounds 5a and 5b), this is not
always the case (e.g. analogues 5k/5m, 5d/5j, 5n/5o). It indicates
that the increased binding affinity within this series is not due to
aspecific lipophilic effects.
In order to assess the effectiveness of a compound optimization
process, ligand efficiency (LE) is a valuable concept.24 Throughout
this paper, the LE values (in kcal/mol) have been calculated for
all derivatives made. It has been stated that a minimum LE of 0.3
is required in a hit or lead compound to be useful. The original
hit 2 had an LE value of 0.57, whereas. compound 5k, the result
of this optimization process, is endowed with a LE index of 0.38,
and therefore still suitable as lead compound.
lM). On the other hand, for the 6-(4-fluorophenyl) ana-
logues, methylation of the pyrrole nitrogen led to a drastic
decrease in binding affinity. This is clear when the binding affinity
data are compared between compounds 5k (Ki = 0.39
corresponding methylated analogue 12d, which is endowed with
a Ki value of 44.15 M. Similarly, N-methylation of pyrrolo[2,3-
d]pyrimidine analogue 5d (Ki = 2.64 M) led to a 30-fold loss in
binding affinity (13a; Ki = 67.87 M). It indicates that for this ser-
ies, having a hydrogen bond donor at that position may be impor-
tant for affinity.
Methylation of the 2-amino group (6; Ki = 10.45
fivefold decrease in activity, when compared to its 2-amino coun-
terpart (5d; Ki = 2.64 M). A more pronounced effect is observed
lM) and its
l
l
l
lM), leads to a
l
when the exocyclic amino group, as well as the pyrrole NH moiety,
are both methylated as in compound 13b, giving rise to a complete
loss in activity, displaying a Ki value of more than 100
lM. These
In conclusion, starting from a known H4R ligand based on
observations are in agreement with previous SAR studies that
a
pyrimidine skeleton, a series of analogues based on a

L.-J. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 132–137
137
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(Ki = 0.53
(Table 6). Within the newly synthesized pyrrolo[2,3-
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We grateful thank Professor J.C. Schwartz (Bioproject, Saint-
Gregoire, France) for providing hH3R-HEK293 cells and Professor
R. Seifert (Hannover, Germany) for Sf9 cells and the baculovirus
stock solutions encoding for hH4R and the G-Proteins G
and
a
i/o
Gb1 2.This work was supported by a Grant from the Fund of Scien-
c
tific Research (to P.H.; FWO research project G.0614.09N). Further
support by the Hessian LOEWE programs OSF, NeFF, AFA as well as
by the EU COST Actions BM0806, BM1007, and CM1103 is
acknowledged (to J.S.S., L.W. and H.S.).
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17. Carceller Gonzalez, E.; Virgili Bernado, M. WO2010043633.
18. Sander, K.; Kottke, T.; Tanrikulu, Y.; Proschak, E.; Weizel, L.; Schneider, E. H.;
Seifert, R.; Schneider, G.; Stark, H. Bioorg. Med. Chem. 2009, 17, 7186.
19. 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.; Brioni, J. D.; Cowart, M.
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