Fragment based design of new H4 receptor-ligands with anti-inflammatory properties in vivo

Article abstract of DOI:10.1021/jm7014217

Using a previously reported flexible alignment model we have designed, synthesized, and evaluated a series of compounds at the human histamine H ₄ receptor (H₄R) from which 2-(4-methyl-piperazin-l-yl)- quinoxaline (3) was identified as a new lead structure for H₄R ligands. Exploration of the structure-activity relationship (SAR) of this scaffold led to the identification of 6,7-dichloro 3-(4-methylpiperazin-l-yl) quinoxalin-2(1H)-one (VUF 10214, 57) and 2-benzyl-3-(4-methyl-piperazin-l-yl) quinoxaline (VUF 10148, 20) as potent H₄R ligands with nanomolar affinities. In vivo studies in the rat reveal that compound 57 has significant anti-inflammatory properties in the carrageenan-induced paw-edema model.

Full text of DOI:10.1021/jm7014217

J. Med. Chem. 2008, 51, 2457–2467
2457
Fragment Based Design of New H4 Receptor-Ligands with Anti-inflammatory Properties
in Vivo
Rogier A. Smits,† Herman D. Lim,† Agnes Hanzer,† Obbe P. Zuiderveld,† Elena Guaita,‡ Maristella Adami,‡ Gabriella Coruzzi,‡
Rob Leurs,† and Iwan J. P. de Esch*,†
Leiden/Amsterdam Center for Drug Research (LACDR), DiVision of Medicinal Chemistry, Department of Pharmacochemistry, Faculty of Exact
Sciences, Vrije UniVersiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, Department of Human Anatomy,
Pharmacology, and Forensic Medicine, Section of Pharmacology, UniVersity of Parma, Via Volturno 39, 43100 Parma, Italy
ReceiVed NoVember 11, 2007
Using a previously reported flexible alignment model we have designed, synthesized, and evaluated a series
of compounds at the human histamine H4 receptor (H4R) from which 2-(4-methyl-piperazin-1-yl)-quinoxaline
(3) was identified as a new lead structure for H4R ligands. Exploration of the structure-activity relationship
(SAR) of this scaffold led to the identification of 6,7-dichloro 3-(4-methylpiperazin-1-yl)quinoxalin-2(1H)-
one (VUF 10214, 57) and 2-benzyl-3-(4-methyl-piperazin-1-yl)quinoxaline (VUF 10148, 20) as potent H4R
ligands with nanomolar affinities. In vivo studies in the rat reveal that compound 57 has significant anti-
inflammatory properties in the carrageenan-induced paw-edema model.
Introduction
Histamine is an endogenous amine that exerts a wide array
of physiological activities by a group of four G-protein coupled
receptors (GPCR’s). The histamine H1 receptor (H1R) mediates
inflammation and vasodilatation, and H1R antagonists have long
been used for the treatment of allergic disorders such as hay
fever.1 The H2R is well-known for its regulation of gastric acid
secretion and H2R antagonists have been successfully used for
the treatment of gastric ulcers.2 The H3R is being studied
intensively and has been shown to play an important role in
several cognitive processes as well as in the regulation of food
intake and the regulation of sleep and wakefulness. Currently
H3R antagonists are being evaluated for their clinical efficacy
in ADHDa, dementia and narcolepsy.3
Figure 1. Previously reported H4R ligands 1 and 2.
The H4R is the most recently discovered histamine receptor
subtype. The DNA sequence was discovered in the human
genome by several groups, and subsequent pharmacological
characterization identified it as a novel histamine receptor.4–8
Although the sequence identity with the H1R and H2R is
relatively low, the H4R protein has a considerable sequence
identity with the H3R receptor (31% overall, 54% in the
transmembrane region).9 Nevertheless, the H4R has a distinct
pharmacological profile.9 The H4R plays a role in immunological
and inflammatory processes and is predominantly expressed on
hematopoetic and immune cells such as eosinophils, mast cells,
and macrophages as well as in peripheral tissues such as spleen,
thymus, and bone marrow.10 Currently, the H4R is considered
a promising target for the treatment of various chronic inflam-
matory diseases such as inflammatory bowel disease, asthma,
and rheumatoid arthritis.11–13 Quite recently, its role has also
been postulated in the proliferation of colon carcinoma cells,
in the modulation of angiogenesis, and in mediating pruritis.14,15
Previously we reported the discovery of VUF6884 (1, Figure
1), a tricyclic clozapine analogue with high affinity for the H4R
(pKi 7.6, full agonist R ) 1), and saturation binding analysis
demonstrated that this ligand bound to the orthosteric binding
site of the H4R.16
We also showed that the H4R antagonist JNJ7777120 (2)17,18
(pKi 7.8, antagonist R ) 0) displaces 1 from its H4R binding
site. On the basis of these findings and on the structural
similarities between 1 and 2, we proposed that they had
overlapping binding modes and created a model for the future
design of H4R ligands (Figure 1). In this paper, we will discuss
the design and synthesis, structure–activity relationship, and
pharmacological evaluation of a new class of potent H4R ligands,
which was discovered on the basis of the flexible alignment
model of 1 and 2.16
Chemistry
Compounds 3–7 (Table 1) were prepared in one step from
their commercially available chloro precursors by substitution
with N-methylpiperazine under microwave irradiation. Starting
from 2,4-dichloroquinazoline (8), quinazoline fragment 9 was
synthesized according to patent literature (Scheme 1).19 Starting
from phenylenediamine (10) and 4,5-dichlorophenylenediamine
(11) (Scheme 2), intermediate quinoxalinones 12-17 were
prepared as described in literature,20–22 except for intermediate
* To whom correspondence should be addressed. Phone: +31(0)205987841.
Fax: +31(0)205987610. E-mail: ideesch@few.vu.nl.
†
Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Uni-
versiteit Amsterdam.
‡
Department of Human Anatomy, Pharmacology and Forensic Medicine,
University of Parma.
a Abbreviations: ADHD, attention deficit hyperactivity disorder; SEM,
standard error of the mean; PEI, pectin esterase inhibitor; DIPEA,
diisopropylethylamine, PMB, para-methoxybenzylamine.
10.1021/jm7014217 CCC: $40.75
2008 American Chemical Society
Published on Web 03/22/2008

2458 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Smits et al.
Table 1. Structures and Affinity of Several Heterocyclic Fragments for the H4R
a Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R. pKi’s are calculated from
at least three independent measurements as the mean ( SEM. b Ligand efficiency (∆g) is calculated as the binding energy per non-hydrogen atom (∆g )
∆G/Nnon-hydrogen atoms with ∆G ) -RT ln Ki).
Scheme 1a
a Reagents and conditions: (a) brine with 9% NH4OH, Zn, DCM, reflux; (b) N-methylpiperazine, mw, 140°C.
Scheme 2a
neat phosphorus oxychloride. After removal of excess reagent
and no further work up, microwave-assisted heating was used
to couple the crude chlorides with neat N-methylpiperazine to
yield quinoxalines 18-23 (Scheme 2).
Commercially available 2,3-dichloroquinoxaline (24) and
2,3,6,7-tetrachloroquinoxaline (25) were reacted with N-meth-
ylpiperazine in the presence of diisopropylethylamine in tet-
rahydrofuran under microwave irradiation to give key interme-
diates 26 and 27 in high yields (Scheme 3). Unsubstituted
quinoxaline 26 was then added to solutions of alcohols and
sodium hydride in dimethylformamide to give compounds 28–50
(Table 2).23 Thioether 51 and phenolether 52 were synthesized
by the same procedure from intermediates 26 and 27, respec-
tively. Methoxy analogues 53 and 54 were synthesized from
their corresponding precursors by refluxing in methanol in the
presence of sodium methoxide. Benzylamine 55 was synthesized
from intermediate 26 by microwave-assisted heating in neat
benzylamine. Compound 56 could either be prepared by
hydrolysis of methoxy analogue 53 with aqueous sodium
hydroxide or by substitution of chloride 26 under microwave-
assisted heating with sodium hydroxide in tetrahydrofuran. A
similar microwave procedure was used for the synthesis of
a Reagents and conditions: (a) R2COCO2H, EtOH, reflux; (b) ethylox-
obutyric acid, r.t. (for compound 15); (c) POCl3, reflux; (d) N-methylpip-
erazine, mw, 140°C.
quinoxalinone 57 from 27.
Monosubstitution of the 6 position (the 6 position mentioned
in the text corresponds to the numbering in Figure 2) of the
quinoxaline scaffold was also explored. For this, 2-amino-4-
chloroaniline (58) was converted to 6-chloro-quinoxalin-
2,3(1H,4H)-dione (59) and subsequently to its chlorinated
product 2,3,6-trichloroquinoxaline (60), as has been described
15, which was synthesized from 4,5-dichlorophenylenediamine
(11) and ethyl 2-oxo-4-phenylbutyrate. The different quinoxa-
line-2-ones were converted to their corresponding chlorides with

Anti-inflammatory H4 Receptor-Ligands in ViVo
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2459
Scheme 3a
a Reagents and conditions: (a) N-methylpiperazine, DIPEA, THF, mw, 160°C; (b) NaH, ROH, DMF, r.t.; (c) NaH, thiophenol, DMF, r.t.; (d) NaH,
phenol, DMF, r.t.; (e) NaMeO, MeOH, reflux; (f) benzylamine, mw, 160°C; (g) aq NaOH, THF, or dioxane, mw, 120°C.
in literature (Scheme 4).24 From this precursor, 2,6-dichloro-
3-methoxy quinoxaline (61) was synthesized according to a
modified procedure from literature.24 The original procedure
mentioned above failed to give 61 in sufficiently high purity.
After multiple attempts, purities around 75% were found by
NMR due to formation of two chloro-substituted regioisomers.
However, three subsequent crystallizations of this impure
material yielded desired product (61) in very high purity (>95%
by NMR). Subsequent substitution of 61 with N-methylpipera-
zine resulted in methoxy analogue 62. Attempts to convert 62
to target compound 63 under acidic conditions failed as these
conditions led to regeneration of precursor (59). However,
demethylation of 62 under basic conditions, using sodium
hydroxide in water, proceeded more mildly and gives 63 in good
yields. Although compound 61 has been reported in literature,
no evidence has been given that can confirm its proposed
structure; in this particular synthesis, two regioisomers are
formed of which the structures cannot be assigned unambigu-
ously by NMR.24 Absolute proof with regard to the isolated
regioisomers of the target compound quinoxalinone 63, as
synthesized following this scheme, could also not be obtained
using standard analytical characterization. Because this informa-
tion is relevant for the SAR, we decided to obtain definite proof
of the structure of compound 63 by a fully regioselective
synthesis (Scheme 5).
2,5-Dichloro-nitrobenzene (64) was substituted with p-
methoxybenzylamine (PMB), yielding benzylamine 65, which
was subsequently hydrogenated in THF with Raney nickel and
hydrogen to give aniline 66. Ring closure of 66 to N-protected
quinoxaline 67 was done by heating in neat diethyloxalate.
Compound 67 was chlorinated with POCl3 in the presence of
Hünigs base and DMF to yield 68. Analogous to a procedure
described in literature, removal of the PMB group from
chloroquinoxaline 68 was achieved with concentrated sulfuric
acid to give the desired monochloro-substituted quinoxalinone
(69).25 Quinoxaline 63 was finally obtained regioselectively
through this route by substitution of the 2-chloro position of
69 with N-methylpiperazine. A comparison of the melting point,
NMR, and LCMS data showed that the compounds obtained
with both synthetic routes were indeed identical. Using the same
procedure for the preparation of 63 from 69, quinoxalines 70
and 71 were obtained with N-methylhomopiperazine and boc-
protected 3-aminomethylpyrrolidine, respectively. Removal of
the boc group with HCl/dioxane finally gave compound 72 as
the hydrochloric salt.
When 68 was directly substituted with N-methylpiperazine,
PMB-protected compound 73 was obtained.
Finally, to enable comparison of the SAR of the methylpip-
erazine moiety with other series of H4R ligands, the N-
ethylpiperazine substituted compound 74 was prepared by the
same method as had been used for 20 (Table 1) using
intermediate 14 and N-ethylpiperazine.
Results and Discussion
To test our model, we synthesized a series of compounds in
which two-ring heterocyclic scaffolds would be directly con-
nected to a N-methylpiperazine group. Relatively small com-
pounds such as quinoxaline 3 would be able to fit in the H4R
binding site in such a way that the heterocyclic ring could
occupy the same space as the chloro-substituted phenyl ring of
1 and the indole ring of 2 while the N-methylpiperazine group
would be able to adopt a similar conformation as in 1 and 2
(Figure 3A). Furthermore, because the tricyclic scaffold of 1
(e.g., clozapine) and its analogues are known to be very
promiscuous GPCR ligands, it was also considered necessary
to move away from this particular scaffold. Therefore, the hybrid
scaffolds based on the structures of 1 and 2 were considered a
good starting point for further exploration of the H4R binding
site and evaluation of our flexible alignment model. Previous
SAR studies of a number of H4R ligands showed that modifica-
tion of the N-methylpiperazine moiety is very detrimental for
H4R affinity.16,17 We therefore initially did not alter this moiety
in the newly designed compounds. In the initial series, we varied
the number and the position of the heterocyclic nitrogen atoms
as well as the position of the N-methylpiperazine moiety (Table
1). 6-Chloro-substituted compounds 5 and 6 were immediately
included in our initial compounds because SAR data from the

2460 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Table 2. SAR of Quinoxalines Substituted at the R3 Position
Smits et al.
no.
R1
R2
R3
pKi ( SEMa
histamine
thioperamide
7.92 ( 0.07
7.20 ( 0.06
6.05 ( 0.07
6.70 ( 0.02
4.99 ( 0.06
7.40 ( 0.04
6.44 ( 0.02
5.13 ( 0.04
6.49 ( 0.02
6.53 ( 0.05
6.32 ( 0.03
5.53 ( 0.15
6.33 ( 0.05
5.53 ( 0.03
5.36 ( 0.11
6.15 ( 0.40
5.86 ( 0.16
5.64 ( 0.05
6.57 ( 0.05
5.89 ( 0.03
5.63 ( 0.06
5.77 ( 0.03
5.80 ( 0.04
6.24 ( 0.08
5.66 ( 0.08
5.63 ( 0.20
5.81 ( 0.02
4.88 ( 0.03
5.24 ( 0.03
6.64 ( 0.08
5.40 ( 0.19
6.47 ( 0.02
7.21 ( 0.03
7.58 ( 0.04
7.93 ( 0.05
8.25 ( 0.07
5.40 ( 0.04
7.20 ( 0.09
5.93 ( 0.11
7.24 ( 0.03
5.60 ( 0.02
6.64 ( 0.03
6.32 ( 0.06
6.01 ( 0.07
5.83 ( 0.08
5.57 ( 0.01
3
H
H
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
H
CH3
C6H5
CH2C6H5
SPh
NHCH2Ph
OPh
OCH2Ph
O(CH2)2Ph
O(CH2)4Ph
OCH2-2-Pyridyl
OCH2-3-Pyridyl
OCH2-4-Pyridyl
OCH2-(4-OCH3-Ph)
OCH2-(4-CH3-Ph)
OCH2-(4-Cl-Ph)
OCH2-(3-Cl-Ph)
O(3-pyridyl)
O(4-Cl-Ph)
O(3,4-Cl-Ph)
O(4-F-Ph)
O(3-CH3-Ph)
O(4-CH3-Ph)
O-(4-OCH3-Ph)
O-(3-N,N-dimethylaniline)
O-cyclohexyl
OCH2CH(CH3)2
OCH2CH3
18
19
20
51
55
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
52
56
62
63
57
21
22
52
54
23
26
73
70
72
74
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O(CH2)3-N,N-dimethylamine
OCH3
OH
OCH3
OH
OH
(CH2)2Ph
CH3
OPh
H
6-Cl
6-Cl
6,7-Cl
6,7-Cl
6,7-Cl
6,7-Cl
6,7-Cl
H
OCH3
CF3
Cl
H
structure as depicted in Scheme 5
6-Cl
6-Cl
H
N-methylhomopiperazine
aminomethylpyrrolidine
N-ethylpiperazine
OH
OH
CH2C6H5
a Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R. pKi’s are calculated from
at least three independent measurements as the mean ( SEM.
of H4R ligands. Fragment 3, together with its chloro analogue
6 has the highest ligand efficiency and thus can be considered
the most promising for further optimization.26
The introduction of a second nitrogen at the four position of
quinoline 4 enhanced the affinity 5-fold, as can be seen in
quinoxaline 3. The introduction of a second nitrogen atom at
the three position gave quinazoline 9, which showed slightly
decreased H4R binding properties. As had been proposed before,
the introduction of a chlorine atom at the 6-position was
Figure 2. 2-(4-Methylpiperazin-1-yl)quinoxaline (3) as a new lead
beneficial and resulted in an increase in H4R affinity of
approximately 10-fold in both the quinoline (compare 4 and 5)
and quinoxaline (compare 3 and 6) scaffolds. Isoquinoline 7
was inactive at the H4R because the methylpiperazine moiety
is most likely positioned unfavorably relative to the heterocyclic
ring.
compound for H4R ligands.
reported series of H4R ligands indicated that the introduction
of a chlorine atom on the all-carbon six-membered ring of
several heterocyclic scaffolds could be beneficial for H4R
affinity.16–18
The alignment model suggested that substitution of quinoxa-
line 3 with an additional aromatic ring system could occupy
the aromatic pocket, which is also occupied by one of the
After the synthesis and pharmacological evaluation of the
compounds in Table 1, we identified 2-(4-methylpiperazin-1-
yl)quinoxaline (3) as a new lead compound for the development

Anti-inflammatory H4 Receptor-Ligands in ViVo
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2461
Scheme 4a
a Reagents and conditions: (a) diethyloxalate, reflux; (b) POCl3, reflux; (c) NaOMe, MeOH, 50°C; (d) N-methylpiperazine, DIPEA, THF, mw; (e) 5%
NaOH, 70°C.
Scheme 5a
a Reagents and conditions: (a) p-methoxybenzylamine, 1-propanol, reflux; (b) Raney Nickel/H2, THF, r.t.; (c) diethyloxalate, reflux; (d) POCl3, DIPEA,
toluene, reflux; (e) NHR1R2, DIPEA, EtOAC, microwave, 120°C; (f) H2SO4, r.t.; (g) 2 M HCl in Et2O, r.t.
aromatic rings of 1 when it binds to the H4R (Figure 3B).16
Analogues of quinoxaline 3 could be readily synthesized with
a variety of substituents at the 3-position. To probe the aromatic
pocket, we therefore initially synthesized the methyl (18), phenyl
(19), and benzyl (20) analogues of quinoxaline 3. The methyl
analogue showed an almost 10-fold increase in H4R affinity
compared to 3, whereas phenyl substitution in 19 is not well
tolerated and leads to a drop in H4R affinity. Interestingly,
benzyl-substituted analogue 20 bound to the H4R with high
affinity (33 nM), suggesting that an additional interaction with
an aromatic pocket had indeed been found. Therefore, the
investigation was extended by introducing various substituents
as well as heteroatoms such as oxygen, nitrogen, and sulfur at
the 3-position (Table 2).

2462 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Smits et al.
Figure 3. (A) Superposition of 1 and 2 (both in green) with quinoxaline 3 (in light blue) can be seen. Quinoxaline 3 is able to occupy the shared
aromatic pockets of 1 and 2, while all three N-methylpiperazine moieties are in the same position. (B) Quinoxalines substituted with a phenyl
(compound 19 in blue) and benzyl (compound 20 in red) moiety at the 3-position and their superposition with 1.
The introduction of oxygen (28) or sulfur (51) in the benzyl
moiety of 20 was detrimental for H4R affinity, but both
compounds bind with higher affinity than quinoxaline 3,
suggesting that a large number of substituents at the 3-position
indeed enhance H4R affinity.
To see if the introduction of two chlorine atoms on the
quinoxaline heterocycle would give a linear SAR, we synthe-
sized phenethyl-, methyl-, phenoxy- and methoxy analogues 21,
22, 52, and 54. As can be seen in Table 2, two chlorine
substituents increased the affinity of the methoxy (52) and
methyl (22) substituted quinoxalines but not of the phenylethyl
(21) and phenoxy (22) substituted quinoxalines. In the last two
compounds, this modulation appeared to even be quite detri-
mental for H4R affinity. During the course of these investiga-
tions, a series of substituted quinoxalinones was reported by
Johnson and Johnson scientists in patent literature as new and
very potent H4R antagonists. Although only a small series of
compounds is reported, quinoxalines 63 and 57 are also reported
as H4R antagonists with nanomolar affinities (31 and 32 nM,
respectively).27
The SAR of the methylpiperazine group was studied to a
limited extent because even minor changes such as the replace-
ment of the methyl group by an ethyl group (compare 20 and
74) is very detrimental for H4R affinity. The sensitivity to
chemical modulation of the N-methylpiperazine group was seen
before in benzimidazole, indole, and dibenzo-oxazepine series
that were reported as H4R ligands.17,16,28 However, patent
literature reports that in a series of aminopyrimidines and
benzofuropyrimidines, the N-methylpiperazine moiety can be
successfully replaced with 3-aminomethylpyrollidine and several
other bioisosteres.29,30 In the quinoxaline series, attempts to find
a suitable bioisostere were unsuccesfull because homopiperazine
70 and aminomethylpyrollidine 72 were found to have affinities
that were about 100-fold lower than N-methylpiperazine 63.
For further pharmacological characterization, quinoxalines 20
and 57 were selected from Table 2. The affinity for the other
histaminergic receptors was determined in order to assess the
selectivity of these ligands for the H4R over the other histamine
receptor subtypes (Table 3). Although compound 57 showed
limited inhibition of radioligand binding to the other histamin-
ergic receptors, at a concentration of 10 µM, compound 20
effectively displaces [3H]mepyramine binding to the H1R.
Compound 20 was designed with a working model that made
use of compound 1 (Figure 3), which had been demonstrated
to have a high affinity (pKi ) 8.11 ( 0.10) for the H1R.16
Therefore, we were prompted to determine the pKi at the H1R,
which was found to be 6.13 ( 0.1 and showed that 20 also
possessed some affinity for the H1R. As was already pointed
Benzylamine analogue 55 did not improve binding, whereas
the benzyl alcohol substituted analogue 29 showed about a
5-fold enhancement of affinity. The introduction of extra carbon
atoms in the alkoxy spacer of 29 showed that two carbons
(compare 29 and 30) were tolerated but four carbons (compare
29 and 31) were detrimental for H4R binding. Because
compound 29 had quite reasonable H4R affinity, we investigated
whether the properties of the aromatic ring of the benzyl alcohol
moiety could be optimized for H4R interaction. 2-Pyridyl
analogue 32 had similar affinity to 29, whereas a nitrogen in
the 3- or 4-position of the pyridine ring (compounds 33 and
34) decreased the H4R affinity. Analogues 35-37 with -OCH3,
-CH3, or chlorine substituents did not show improved H4R
affinity.
Parallel to the synthesis of the benzyl alcohols, we synthesized
a series of pyridinol and phenol-substituted compounds based
on 28 in which we also introduced a number of different
substituents. In these series (compounds 39-46), none of the
synthesized compounds showed an affinity that was higher than
28.
Several quinoxalines were synthesized with aliphatic ethers
that enhanced the binding affinity of 3 only slightly when
substituted with an ethoxy (49) or methoxy (52) group.
However, when the size of these substituents was increased to
isobutoxy (48) or cyclohexyloxy (47), the affinity dropped up
to 10-fold for compound 47. The introduction of a flexible
aliphatic side chain with a basic amine (compound 50) at the
3-position of quinoxaline 3 was not tolerated.
More interesting, quinoxalinone 56 had an affinity almost
comparable to benzylquinoxaline 20. It was anticipated that the
introduction of a chlorine atom on the heterocyclic core would
improve the H4R affinity after having optimized the R2 position
as had been the case in other series.16,17 By this rationale,
compounds 62 and 63 were synthesized. The 6-chloro substitu-
ent gave an enhancement in H4R afiinity of 14- and 5-fold in
compounds 62 and 63, respectively. We also synthesized 6,7-
dichloro-substituted quinoxaline 57 and found that it was even
more potent, showing an H4R affinity of 6 nM.

Anti-inflammatory H4 Receptor-Ligands in ViVo
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2463
Table 3. Pharmacological Evaluation of Selected Quinoxalines at the
Four Human Histaminergic Receptor Subtypes
evaluated in vivo and displayed significant anti-inflammatory
activity in the carrageenan induced paw edema model in rats.
a
b
H1
H2
H3
H4
compound (% inhibition) (% inhibition) (% inhibition) (pKi ( SEM)
Experimental Section
20
57
86c
7
62
42
26
64
7.40 ( 0.04
8.25 ( 0.07
In Vivo Pharmacology. Carrageenan-Induced Edema
Model. Animals. Male Wistar rats (180–200 g; Harlan-Italy, Milan)
were housed under controlled standard conditions (23 °C temper-
ature, 12 h light/dark cycle, and 65% humidity). Food and water
were provided ad libitum. The experiments received the approval
of the local Animal Ethics Committee of the University of Parma,
Italy.
a % Inhibition of radioligand binding to the human H1-H3 receptors
was determined in duplo at a concentration of 10 µM of ligand. b Values
are taken from Table 2. c The pKi of 20 at the human H1R was determined
to be 6.13 ( 0.1 (n ) 3).
Induction of Acute Inflammation. Inflammation was induced
in fasted rats by subplantar injection of carrageenan (0.1 mL of
1% suspension in carboxymethycellulose) into the left hind paw.
As previously described, carrageenan-induced edema was measured
with a plethysmometer (Basile, Comerio, Italy) immediately prior
to the injection of carrageenan and thereafter at 2, 4, and 6 h.33
Edema was expressed for each animal as % increase in paw volume
after carrageenan injection relative to the preinjection value,
considered as 100.
Compound Administration. Equivalent volumes (0.1 mL per
100 g) of the test compounds or vehicle were administered
subcutaneously (s.c.) in separate groups of rats immediately prior
to carrageenan injection. Before use solutions of the histamine H4
receptor–ligands in DMSO were freshly prepared.
Data Analysis. Data are expressed as mean ( SEM. A value of
P < 0.05 was considered statistically significant. The software
package Prism GraphPad 3.0 (GraphPad Software Inc., San Diego,
CA) was used to process data.
In Vitro Pharmacology. Radioligand Displacement Studies
at the Human H1, H2, and H3 Receptors. Single-point radioligand
displacement studies at the human H1, H2, and H3 receptors were
preformed by Cerep (Le Bois l’Evêque, France) at a concentration
of 10 µM of ligand. These measurements were performed in duplo.
The pKi at the H1R was determined according to a procedure
described in literature.34
Figure 4. Anti-inflammatory effect of compounds 20 and 57 and
inactive control 47 on paw edema induced by subplantar injection of
carrageenan (1% in CMC) in rats. Data are expressed as mean ( SEM
n ) 6 rats per group. Comparisons between multiple groups were made
by using one-way analysis of variance (ANOVA), followed by
Dunnett’s test. *P < 0.05 compared with vehicle-treated animals
(Student t test for grouped data).
out earlier, it can be envisioned that dual action H1/H4 ligands
might have great potential in the treatment of several histamine-
mediated allergic and inflammatory disorders.31
Compound 20 and 57 were selected to study the anti-
inflammatory effect on the new series of H4R ligands in vivo.
After 2 h both compounds, respectively, coadministered at 10
mg/kg and 30 mg/kg with carrageenan, caused a significant
inhibition of carrageenan-induced edema when compared to the
vehicle (Figure 4). The effect of 57 was still found to be
significant 6 h after administration. Compound 47 (pKi ) 4.88
( 0.03), which was selected for this study as an inactive control,
displayed no significant inhibition of the carrageenan-induced
edema.
Radioligand Displacement Studies at the Human H4
Receptor. Cell Culture and Transfection. HEK 293T cells were
maintained in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 50 IU/mL
penicillin, and 50 µg/mL streptomycin in 5% CO2 humidified
atmosphere at 37 °C. Approximately 4 million cells were seeded
in a 10 cm dish and cultured overnight before transfection. For
transfection of each dish of cells, the transfection mixture was
prepared in 1 mL serum-free DMEM and contained 5 µg of human
H4R receptor plasmid and 15 µL of 1 mg/mL 25 kDa linear
polyethyleneimine (Polyscience, Inc., USA). The mixture was
incubated for 10–15 min at room temperature before it was added
into the monoloyer cell culture loaded with 5 mL of fresh cell
culture medium. Two days after transfection, the cells were washed
with PBS containing 1 mM EDTA, collected as pellet by centrifug-
ing, and stored at –20 °C until use.
Conclusion
Albeit limited by the available GPCR protein structural
information and the complications when applying biophysical
screening approaches to these targets, the growing of a micro-
molar affinity fragment into nanomolar ligands can be consid-
ered a fragment based design approach that has been success-
fully used in many GPCR projects before.2,33 In this work, we
have described the discovery of a series of new histamine H4R
ligands starting from an in silico flexible alignment model of
the known H4R agonist 1 and antagonist 2. It was used to design
a series of small heterocyclic fragments as H4 receptor–ligands,
of which 2-(4-methylpiperazin-1-yl)quinoxaline (3) binds to the
H4R with a Ki in the submicromolar range. The growing of
fragments into more druglike compounds by introducing a
variety of substituents onto this new scaffold was guided by
the in silico model, thereby strengthening the rational design.
This approach led to a series of potent H4R ligands with affinities
in the low nanomolar range. Two of these compounds, 6,7-
dichloro-3-(4-methylpiperazin-1-yl)quinoxalin-2(1H)-one (57)
and 2-benzyl-3-(4-methyl-piperazin-1-yl)quinoxaline (20) were
[3H] Histamine Binding Assay. For the radioligand binding
study, pellets of transfected cells were homogenized in H4R binding
buffer (100 mM Tris-HCl, pH 7.4). The saturation binding assay
was performed using different concentrations of [3H]histamine
(Perkin-Elmer Life Science, Inc., USA), while nonspecific binding
was determined by incubation in the presence of 3–10 µM of
compound 2 in a total assay volume of 200 µL. For the displacement
binding assay, the membranes were typically incubated with
10-4-10-11 M of ligands (stock concentration was 10 mM 1
DMSO) in the presence of [3H]histamine in a total volume of 200
µL. The reaction mixtures were incubated for 1 h at room
temperature (22 °C), and harvested on 96-well glass fiber C plates
that were pretreated with 0.3% 750 kDa PEI. The binding assay
data were analyzed using Prism 4.0 (Graphpad Software Inc., USA).
Flexible Alignment. The alignment model was created with
Molecular Operating Environment 2006.08 (MOE) from Chemical
Computing Group (Montreal, Canada). The compounds in Figure
3 were aligned with the flexible alignment module of MOE using

2464 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Smits et al.
default parameters and similarity term “partial charge” added with
a weight factor of 1. The alignment with highest mutual similarity
score F (F ) 32.1926, S ) 159.991, U ) 127.7983) was selected
and refined further using the “refine existing alignment” option with
an energy cutoff of 7.0 kcal/mol.
13%) of a light-orange solid; mp 74.4–76.0 °C. 1H NMR (CDCl3)
δ (ppm): 8.81 (s, 1H), 7.52–7.40 (m, 3H), 7.04–6.96 (m, 1H), 3.85
(t, J ) 5.0 Hz, 4H), 2.34 (t, J ) 5.0 Hz, 4H), 2.17 (s, 3H). 13C
NMR (CDCl3) δ (ppm): 161.05, 158.86, 151.98, 133.68, 127.06,
125.41, 122.13, 119.32, 54.78, 46.01, 43.69; MS (ESI) m/z 229
(M + H)+.
General Remarks. Carrageenan was purchased from Sigma-
Aldrich (St. Louis, MO). Other chemicals and reagents were
obtained from commercial suppliers and were used without further
purification. Yields given are isolated yields unless mentioned
otherwise. Flash column chromatography was typically carried out
on an Argonaut Flashmaster II flash chromatography system, using
prepacked Isolute Flash Si II columns with the UV detector
operating at 254 nm. All melting points are uncorrected and were
measured on an Optimelt automated melting point system from
2-Benzyl-3-(4-methylpiperazin-1-yl)quinoxaline (20). 3-Ben-
zylquinoxalin-2(1H)-one (14) (998 mg, 4.2 mmol) and POCl3 (6.0
mL) were added to a round-bottom flask and heated at reflux. After
5 h, the mixture was allowed to cool to r.t. and was poured onto
crushed ice. Concentrated ammonia was then used to adjust the
mixture to pH 6, and the obtained suspension was left to stir
vigorously overnight at r.t. After filtration of the suspension and
washing of the obtained solids with water, the product was dried
in vacuo yielding 1.06 g (41.6 mmol, 99%) of 2-benzyl-3-
chloroquinoxaline as a white solid. This was used directly in the
next step without further purification. A microwave tube charged
with 2-benzyl-3-chloroquinoxaline (400 mg, 1.57 mmol) and
N-methylpiperazine (3.0 mL) was heated at 120 °C for 5 min. Then
water (25 mL) was added and the aqueous layer was extracted with
EtOAc. The combined organic extracts were dried with brine and
Na2SO4. Evaporation of the organic solvents and subsequent
purification over SiO2 (EtOAc 90%, Et3N 5%, MeOH 5%) yielded
446 mg (1.40 mmol, 89%) of a yellow solid; mp 89.0–90.0 °C. 1H
NMR (CDCl3) δ (ppm): 7.82 (dd, J ) 1.4 Hz, J ) 8.3 Hz, 1H),
7.62–7.46 (m, 2H), 7.34–7.14 (m, 5H), 4.35 (s, 2H), 3.32 (t, J )
4.8 Hz, 4H), 2.57 (t, J ) 4.8 Hz), 2.35 (s, 3H). 13C NMR (DMSO-
d6) δ (ppm): 155.89, 151.33, 140,04, 138.80, 138.24, 128.97,
128.63, 128.29, 128.16, 127.18, 126.69, 126.31, 54.75, 49.59, 45.98,
40.71; MS (ESI) m/z 319 (M + H)+.
1
Stanford Research Systems. All H NMR and 13C NMR spectra
were measured on a Brüker 200 or Brüker 400.
Analytical HPLC-MS analyses were conducted using a Shimadzu
LC-8A preparative liquid chromatograph pump system with a
Shimadzu SPD-10AV UV–vis detector with the MS detection
performed with a Shimadzu LCMS-2010 liquid chromatograph–
mass spectrometer. The buffer mentioned under conditions I and
II is a 0.4% (w/v) NH4CO3 solution in water, adjusted to pH 8.0
with NH4OH. The analyses were performed using the following
two conditions:
Condition I: an Xbridge (C18)5µm column (100 mm × 4.6 mm)
with the following two solvents: solvent A, 90% MeCN-10%;
solvent B, 90% water-10% buffer; flow rate ) 2.0 mL/min; start:
5% A, linear gradient to 90% A in 10 min, then 10 min at 90% A,
then 10 min at 5% A. Total run time: 30 min.
Condition II: Xbridge (C18)5µm column (100 mm × 4.6 mm)
with the following two solvents: solvent A, MeCN with 0.1% formic
acid; solvent B, water with 0.1% formic acid; flow rate ) 2.0 mL/
min; start: 5% A, linear gradient to 90% A in 10 min, then 5 min
at 90% A, then 5 min at 5% A. Total run time: 20 min. Compound
purities under both conditions were calculated as the percentage
peak area of the analyzed compound by UV detection at 254 nm.
Analytical HPLC-MS analyses for condition III were carried out
on Agilent 1100 Series HPLC-MS system including an Agilent
G1315B DAD. Condition III: XBridge (C18)3.5µm column (2.1
mm × 50 mm) with the following two solvents: solvent A, a 5
mM solution of NH4HCO3 in water set to pH 9.0 using 19 mM
NH3; solvent B, 100% MeCN. The MS detection was performed
with an Agilent G1956B LC/MSD SL using a multimode ion
source. Flow rate ) 1.2 mL/min; start: 95% A, linear gradient to
5% A in 1.25 min, then 0.75 min at 5% A, followed by 1.0 min
95% A, 5% B. Total run time: 3 min.
2-Chloro-3-(4-methylpiperazin-1-yl)quinoxaline (26). To a
solution of 2,3-dichloroquinoxaline (6.60 g, 33.2 mmol) in toluene
(45 mL) was added triethylamine (4.6 mL, 33.0 mmol) and the
solution was heated at reflux. N-methylpiperazine (3.7 mL, 33.4
mmol) was added and heating was continued for 2 h. The resulting
mixture was diluted with saturated NaHCO3 solution. The organic
layer was separated, and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
over MgSO4, and concentrated in vacuo. Purification of the residue
over SiO2 (90% EtOAc, 5% Et3N, 5% MeOH) (EtOAc) yielded
1
7.70 g (29.3 mmol, 88%) of a yellow solid; mp 67.4–68.6 °C. H
NMR (CDCl3) δ (ppm): 7.87–7.78 (m, 2H), 7.65–7.47 (m, 2H),
3.60 (t, J ) 4.9 Hz, 4H), 2.63 (t, J ) 4.9 Hz, 4H), 2.37 (s, 3H).
13C NMR (CDCl3) δ (ppm): 152.38, 141.46, 139.99, 138.03,
129.94, 127.54, 127.13, 126.87, 54.55, 48.77, 45.96. MS (ESI) m/z
263 (M + H)+.
2,6,7-Trichloro-3-(4-methylpiperazin-1-yl)quinoxaline (27). A
microwave tube charged with 2,3,6,7-tetrachloro-quinoxaline (500
mg, 1.87 mmol), N-methylpiperazine (0.21 mL, 1.89 mmol), DIPEA
(0.37 mL, 2.12), and THF (3.0 mL) was heated at 140 °C. After 5
min, the reaction mixture was diluted with water and extracted with
EtOAc. The combined organic layers were washed with brine and
dried over Na2SO4. The extracts were concentrated and filtered over
a short column of SiO2 (EtOAc as eluent) Evaporation of the solvent
yielded 523 mg (1.58 mmol, 84%) of the title compound. 1H NMR
(CDCl3) δ (ppm): 7.90 (s, 1H), 7.87 (s, 1H), 3.61 (t, J ) 4.8 Hz,
1H), 2.62 (t, J ) 4.8 Hz, 1H), 2.36 (s, 1H). 13C NMR (CDCl3) δ
(ppm): 152.66, 142.30, 138.93, 136.46, 134.28, 130.89, 128.05,
127.42, 54.22, 48.60, 45.89.
2-(4-Methylpiperazin-1-yl)-3-phenoxyquinoxaline (28). To a
round-bottom flask were added 2-chloro-3-(4-methylpiperazin-1-
yl)quinoxaline (26) (1.0 g, 3.81 mmol), phenol (0.43 g, 4.57 mmol),
DMF (3.0 mL), and NaOH (0.23 g, 5.75 mmol). The resulting
suspension was stirred at r.t. for 2 h. After completion, the mixture
was diluted with water and the aqueous layer was extracted with
EtOAc. The combined organic extracts were thoroughly washed
with water and brine and then dried over Na2SO4. Evaporation of
the solvent gave an oil that was purified over SiO2 (90% EtOAc,
5% Et3N, 5% MeOH) yielding 0.89 g (2.78 mmol, 73%) of a yellow
solid; mp 96.8–97.6 °C. 1H NMR (CDCl3) δ (ppm): 7.73 (dd, J )
8.1 Hz, J ) 1.2 Hz, 1H), 7.56–7.20 (m, 8H), 3.82 (t, J ) 4.9 Hz,
Synthetic Methods. 2-(4-Methylpiperazin-1-yl)quinoxaline
(3). 2-Chloroquinoxaline (309 mg, 1.88 mmol) and N-methylpip-
erazine (2.0 mL) were added to a microwave tube and heated at
160 °C for 5 min. The resulting mixture was diluted with water
and extracted with EtOAc. Drying over Na2SO4 and evaporation
of the solvent yielded 402 mg (1.76 mmol, 94%) of a brown solid.
Mp 108.6–110.9 °C; 1H NMR (CDCl3): δ (ppm) 8.56 (s, 1H), 7.85
(dd, J ) 8.2 Hz, J ) 1.3 Hz, 1H), 7.66 (dd, J ) 8.4 Hz, J ) 1.3
Hz, 1H), 7.59–7.50 (m, 1H), 7.41–7.24 (m, 1H), 3.79 (t, J ) 5.1
Hz, 4H), 2.54 (t, J ) 5.1 Hz, 4H), 2.34 (s, 3H); 13C NMR (CDCl3):
δ (ppm) 152.09, 141.46, 136.66, 135.55, 129.92, 128.50, 126.32,
124.60, 54.61, 46.07, 44.48.
2-(4-Methylpiperazin-1-yl)quinazoline (9). A solution of 2,4-
dichloroquinazoline (1.2 g, 6.03 mmol) in DCM (24 mL) covered
with brine (24 mL) containing 9% NH4OH was treated with
powdered zinc (1.2 g), and the resulting biphasic mixture was then
refluxed for 3.5 h. After cooling to r.t., it was filtered through celite
and the organic layer was removed under reduced pressure. The
residue was diluted with EtOAc and washed with 1 N HCl solution,
after which it was dried and concentrated to dryness. Without
purification, N-methylpiperazine (4.0 mL) was added, causing a
crystalline solid to separate almost immediately. The product was
diluted with ethyl acetate and washed with NaHCO3 and water.
Drying over Na2SO4 and rotary evaporation yielded the crude title
compound. Purification over SiO2 yielded 180 mg (0.79 mmol,

Anti-inflammatory H4 Receptor-Ligands in ViVo
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2465
4H), 2.61 (t, J ) 4.9 Hz, 4H), 2.36 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 152.61, 149.95, 147.02, 138.95, 135.79, 129.37, 127.18,
126.49, 126.00, 125.62, 125.03, 121.60, 54.88, 47.67, 46.04; MS
(ESI) m/z 321 (M + H)+.
150.96, 132.27, 128.87, 125.20, 125.20, 124.43, 114.87, 54.32,
45.70, 45.42; MS (ESI) 313 m/z (M + H)+.
2,6-Dichloro-3-methoxyquinoxaline (61). A 5.4 M solution of
NaOMe in methanol (2.9 mL, 15.7 mmol) was diluted with
methanol (36 mL) and added over a 6 h period to a slurry of 2,3,6-
trichloroquinoxaline (3.0 g, 12.8 mmol) in methanol (36 mL) at
50 °C. After addition, the obtained solution was refluxed for 16 h
and the solvent was evaporated. The residue was taken up in CHCl3
(100 mL) and washed with water and brine. After drying over
Na2SO4, the organic layer was removed and the residue was purified
over SiO2 (toluene) to yield a white solid. After three crystalliza-
tions, 369 mg (1.61 mmol, 13%) of the title compound was obtained
as white needles (purity >95% by NMR); mp 110.9–111.4 °C. 1H
NMR (CDCl3) δ (ppm): 7.86–7.82 (m, 2H), 7.51 (dd, J ) 8.8 Hz,
J ) 2.2 Hz, 1H), 4.14 (s, 3H).
2-Ethoxy-3-(4-methylpiperazin-1-yl)quinoxaline (49). To 2-
chloro-3-(4-methylpiperazin-1-yl)quinoxaline (26) (355 mg, 1.28
mmol) in ethanol (5.0 mL) was added NaOH (54 mg, 1.35 mmol,
in 1.0 mL water) and the obtained solution was heated at reflux
for 2 h. After cooling to room temperature, the solution was added
to a separatory funnel containing EtOAc and water and the aqueous
phase was extracted with EtOAc. The combined organic phases
were washed with water and brine and dried over MgSO4.
Purification over SiO2 (EtOAc 90%, MeOH 5%, Et3N 5%) yielded
214 mg (0.79 mmol, 61%) of the title compound as a yellow oil.
As a side-product, 30 mg (0.12 mmol, 9%) of compound 56 was
1
also isolated. Analytical data for 49: H NMR (CDCl3) δ (ppm):
6-Chloro-3-methoxy-2-(4-methylpiperazin-1-yl)quinoxaline
(62). To a microwave tube were added 2,6-dichloro-3-methoxy-
quinoxaline (61) (200 mg, 0.87 mmol), N-methylpiperazine (0.2
mL, 1.80 mmol), and THF (3.0 mL). After heating the solution for
5 min at 140 °C, the obtained suspension was diluted with EtOAc
(30 mL) and washed with saturated NaHCO3, water, and brine.
Drying with Na2SO4 and evaporation of the organic layer yielded
an off-white solid. Purification over SiO2 (EtOAc 90%, MeOH 5%,
Et3N 5%) yielded 168 mg (0.57 mmol, 66%) of a white-yellow
solid; mp 99.9–101.6 °C. 1H NMR (CDCl3) δ (ppm): 7.66 (d, J )
2.3 Hz, 1H), 7.59 (d, J ) 8.7 Hz, 1H), 7.34 (dd, J ) 8.8 Hz, J )
2.4 Hz, 1H), 4.01 (s, 3H), 3.71 (t, J ) 5.0 Hz, 4H), 2.56 (t, J ) 5.0
Hz, 4H), 2.34 (s, 3H). 13C NMR (CDCl3) δ (ppm): 151.24, 146.83,
136.80, 136.73, 130.49, 127.02, 126.85, 125.09, 54.85, 53.72, 47.42,
46.03; MS (ESI) m/z 293 (M + H)+.
7.70–7.62 (m, 2H), 7.44–7.30 (m, 2H), 4.54 (q, J ) 7.1 Hz, 2H),
3.73 (t, J ) 4.9 Hz, 4H), 2.57 (t, J ) 5.0 Hz, 4H), 2.34 (s, 3H),
1.46 (t, J ) 7.0 Hz, 3H). 13C NMR (CDCl3) δ (ppm): 150.34,
146.87, 138.04, 136.36, 126.13, 126.01, 125.81, 125.38, 62.25,
54.81, 47.46, 46.01, 14.23; MS (ESI) m/z 273 (M + H)+.
2-Methoxy-3-(4-methylpiperazin-1-yl)quinoxaline (52). 2-
Chloro-3-(4-methylpiperazin-1-yl)quinoxaline (26) (1.0 g, 3.81
mmol) and a 5.4 M solution of NaOMe in MeOH (0.9 mL, 4.86
mmol) were dissolved in MeOH (20 mL) and heated at reflux. After
3 h, the obtained mixture was cooled to r.t. and diluted with EtOAc
(60 mL). The organic layer was then washed with water and brine
and dried over Na2SO4. Evaporation of the organics yielded 932
mg (3.61 mmol, 95%) of a light-brown solid; mp 74.0–76.3 °C.
1H NMR (CDCl3) δ (ppm): 7.71–7.65 (m, 2H), 7.45–7.33 (m, 2H),
4.09 (s, 3H), 3.71 (t, J ) 4.9 Hz, 4H), 2.57 (t, J ) 5.0 Hz, 4H),
2.34 (s, 3H). 13C NMR (CDCl3) δ (ppm): 150.77, 146.95, 138.14,
136.36, 126.30, 126.07, 125.85, 125.51, 54.87, 53.52, 47.50, 46.05;
MS (ESI) m/z 259 (M + H)+.
3-(4-Methylpiperazin-1-yl)quinoxalin-2(1H)-one (56). Method
1: Synthesis from 2-Chloro-3-(4-methylpiperazin-1-yl)quinoxa-
line. A microwave tube containing 2-chloro-3-(4-methylpiperazin-
1-yl)quinoxaline (26) (150 mg, 0.57 mmol), NaOH (60 mg, 1.5
mmol), THF (2.0 mL), and water (0.5 mL) was heated at 120 °C
for 20 min. After completion, the solution was partitioned between
EtOAc and water and the aqueous layer was extracted with EtOAc.
After drying over Na2SO4, the solvent was evaporated and the
residue was purified over SiO2 (EtOAc 90%, Et3N 5%, MeOH 5%)
yielding 100 mg (0.41 mmol, 72%) of a white solid. 1H NMR
(CDCl3) δ (ppm): 11.29 (bs, 1H), 7.53–7.48 (m, 1H), 7.24–7.08
(m, 3H), 4.03 (t, J ) 4.9 Hz, 4H), 2.58 (t, J ) 5.0– Hz, 4H), 2.34
(s, 3H). 13C NMR (CDCl3) δ (ppm): 153.41, 150.74, 133.05,
128.32, 125.75, 124.91, 124.02, 114.21, 54.95, 46.50, 45.99; MS
(ESI) m/z 245 (M + H)+.
Method 2: Synthesis from 2-Methoxy-3-(4-methylpiperazin-
1-yl)quinoxaline. 2-Methoxy-3-(4-methylpiperazin-1-yl)-quinoxa-
line (52) (100 mg, 0.39 mmol) and 5% NaOH in water (5.0 mL)
were heated at reflux for 20 h. After cooling to room temperature,
the pH was adjusted to 7.0 with diluted HCl. The aqueous solution
was extracted with DCM, and the combined extracts were washed
with brine. Drying over Na2SO4 and evaporation of the solvent
yielded 84 mg (0.34 mmol, 88%) of a white solid. An analytical
sample was recrystallized from EtOAc to yield white needles; mp
206.4–207.8 °C.
6,7-Dichloro-3-(4-methylpiperazin-1-yl)quinoxalin-2(1H)-
one (57). To a microwave tube were added 2,6,7-trichloro-3-(4-
methylpiperazin-1-yl)-quinoxaline (26) (212 mg, 0.64 mmol),
dioxane (3.0 mL), NaOH (52 mg, 1.30 mmol), and water (1.0 mL).
The mixture was heated in the microwave for 5 min at 120 °C and
diluted with water. The obtained suspension was thoroughly
extracted with DCM, and the organic layers were dried with
Na2SO4. The residue that was obtained after evaporation of the
solvent was triturated with EtOAc to yield 114 mg (0.36 mmol,
57%) of an off-white solid; mp 262.4–263.6 °C. 1H NMR (DMSO-
d6) δ (ppm): 7,52 (s, 1H), 7.26 (s, 1H), 3.92 (m, 4H), 2.41 (t, J )
4.9 Hz, 4H), 2.19 (s, 3H). 13C NMR (CDCl3) δ (ppm): 151.51,
6-Chloro-3-(4-methylpiperazin-1-yl)quinoxalin-2(1H)-one (63).
Method 1: Synthesis from 6-Chloro-3-methoxy-3-(4-methylpip-
erazin-1-yl)quinoxaline (62). 6-Chloro-3-methoxy-3-(4-methylpip-
erazin-1-yl)quinoxaline (62) (135 mg, 0.46 mmol) and 5% aqueous
NaOH (10 mL) were heated at 70 °C. After 4 h, the clear solution
was neutralized (pH 7) with diluted HCl and extracted with DCM.
The combined extracts were then washed with brine. After drying
over Na2SO4 and evaporation of the organics, the residue was
purified over SiO2 (EtOAc 90%, MeOH 5%, Et3N 5%) to yield 35
mg (0.13 mmol, 28%) of the title compound as a white solid; mp
1
228.5–230.0 °C. H NMR (CDCl3) δ (ppm): 11.23 (bs, 1H), 7.40
(d, J ) 8.5 Hz, 1H), 7.14 (dd, J ) 8.6 Hz, J ) 2.2 Hz, 1H), 7.03
(d, J ) 2.2 Hz, 1H), 4.03 (t, J ) 4.8 Hz, 4H), 2.58 (t, J ) 5.0 Hz,
4H), 2.35 (s, 3H). 13C NMR (CDCl3) δ (ppm): 153.32, 150.36,
131.74, 129.94, 128.91, 126.74, 124.40, 113.91, 54.89, 46.47, 45.93;
MS (ESI) m/z 279 (M + H)+.
Method 2: Synthesis from 3,7-Dichloroquinoxalin-2(1H)-
one (69). N-Methylpiperazine (1.0 mL), 3,7-dichloro-quinoxalin-
2(1H)-one (69) (300 mg, 1.40 mmol), and EtOAc (2.0 mL) were
added to a microwave tube and were heated at 120 °C for 10 min.
After cooling to r.t., the mixture was diluted with EtOAc and
washed with water and brine. Drying of the organic phase over
Na2SO4 and evaporation of the solvent gave the crude product that
was recrystallized from EtOAc to yield 227 mg (0.81 mmol, 58%)
of off-white crystals; mp 230.0–232.0 °C. 1H NMR (CDCl3) δ
(ppm): 11.01 (bs, 1H), 7.36 (d, J ) 8.6 Hz, 1H), 7.10 (dd, J ) 8.6
Hz, J ) 2.2 Hz, 1H), 7.03 (d, J ) 2.1 Hz, 1H), 3.98 (t, J ) 4.9 Hz,
4H), 2.52 (t, J ) 5.0 Hz, 4H), 2.30 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 153.26, 150.38, 131.75, 129.91, 128.91, 126.73, 124.38,
113.88, 54.90, 46.49, 45.94; MS (ESI) m/z 279 (M + H)+.
5-Chloro-N-(4-methoxybenzyl)-2-nitroaniline (65). A solution
of 3,4-dichloro-nitrobenzene (37.0 g, 0.19 mol) and 4-methoxy-
benzylamine (50 mL, 0.38 mol) in n-propanol (200 mL) was heated
at reflux for 16 h, after which the solvent was removed under
reduced pressure. The residue was taken up in EtOAc and washed
with water and brine. Drying of the organic phase over Na2SO4
and removal of the solvent yielded a yellow solid that was
recrystallized from abs EtOH to yield 42.6 g (0.15 mol, 76%) of
yellow crystals; mp 70.0–72.0 °C. 1H NMR (CDCl3) δ (ppm): 8.33
(br s, 1H), 8.11 (d, J ) 9.1 Hz, 1H), 7.27–7.23 (m, 2H), 6.89 (d,

2466 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Smits et al.
J ) 8.7 Hz, 2H), 6.82 (d, J ) 2.1 Hz, 1H), 6.61 (dd, J ) 9.1 Hz,
J ) 2.1 Hz, 1H), 4.41 (d, J ) 5.4 Hz, 2H), 3.80 (s, 3H).
5-Chloro-N′-(4-methoxybenzyl)benzene-1,2-diamine (65). To
a solution of 64 (8.7 g, 29.7 mmol) in THF (150 mL) in a round-
bottom flask was added Raney nickel (2.0 g of a 50% suspension
in water) and the resulting mixture was stirred vigorously. Hydrogen
gas was added with a balloon until TLC indicated complete
conversion of the starting material (16 h). The catalyst was removed
by filtration, and the reaction mixture was evaporated to dryness,
yielding 7.74 g (29.5 mmol, 98%) of a dark-brown solid that was
used in the next step without further purification. 1H NMR (DMSO-
d6) δ (ppm): 7.27 (d, J ) 8.7 Hz, 2H), 6.89 (d, J ) 8.7 Hz, 2H),
6.50 (d, J ) 8.2 Hz, 1H), 6.37 (dd, J ) 8.1 Hz, J ) 2.2 Hz, 1H),
6.26 (d, J ) 2.3 Hz, 1H), 5.33 (m, 1H), 4.71 (s, 2H), 4.21 (d, J )
6.1 Hz, 2H), 3.73 (s, 3H).
4.79 (dd, J ) 14.2 Hz, J ) 5.0 Hz, 1H), 4.59–4.54 (m, 1H),
4.50–4.41 (m, 2H), 3.18 (s, 3H), 3.00–2.92 (m, 1H), 2.75–2.66 (m,
1H). 13C NMR (D2O) δ (ppm): 153.49, 147.74, 132.71, 128.45,
126.48, 125.11, 121.49, 116.65, 59.01, 54.58, 51.10, 33.14, 28.02;
MS (ESI) m/z 279 (M + H)+.
Acknowledgment. Thanks goes out to Jelmer Koole for
technical assistance. The fruitful discussions with Maikel
Wijtmans are highly appreciated. The assistance of Kamoncha-
nok Sansuk for performing the H1R binding assay is also highly
appreciated.
Supporting Information Available: LCMS purity data for
compounds 5, 7, 9, 18-23, 26, 28-57, 62, 63, 70, and 72-74 and
synthetic procedures for compounds 4-7, 18, 19, 21-32, 29-48,
50-52, 54, 55, 70, 73,and 74. This material is available free of
charge via the Internet at http://pubs.acs.org.
7-Chloro-1-(4-methoxybenzyl)quinoxaline-2,3(1H,4H)-dione
(67). Crude 66 (7.7 g, 0.029 mol) and diethyloxalate (16.0 mL,
154 mmol) were heated at 140 °C for 16 h. After completion, the
reaction mixture was carefully cooled to r.t. upon which the desired
product crystallized from the reaction flask. The product was filtered
off and washed with cold EtOH to yield 8.35 g (26.4 mmol, 91%)
References
(1) Hill, S. J. Distribution, properties, and functional characteristics of
three classes of histamine receptor. Pharmacol. ReV. 1990, 42, 45–
83.
(2) Black, J. W.; Duncan, W. A.; Durant, C. J.; Ganellin, C. R.; Parsons,
E. M. Definition and antagonism of histamine H2-receptors. Nature
1972, 236, 385–390.
(3) Celanire, S.; Wijtmans, M.; Talaga, P.; Leurs, R.; de Esch, I. J. P.
Histamine H3 receptor antagonists reach out for the clinic. Drug
DiscoVery Today. 2005, 23/24, 1613–1627.
1
of the title compound as white crystals; mp 236.0–238.0 °C. H
NMR (DMSO-d6): δ (ppm): 12.18 (br s, 1H), 7.28–7.13 (m, 5H),
6.88 (d, J ) 8.7 Hz, 2H), 5.30 (s, 2H), 3.71 (s, 3H). 13C NMR
(DMSO-d6) δ (ppm): 158.25, 155.39, 153.17, 127.87, 127.27,
126.96, 126.58, 124.84, 123.06, 116.77, 114.89, 113.84, 54.80,
44.74.
3,7-Dichloro-1-N-(4-methoxybenzyl)quinoxalin-2-one (68). Com-
pound 67 (6.46 g, 20.4 mmol) and DIPEA (6.8 mL, 39.0 mmol)
were dissolved in DMF (5.0 mL), after which POCl3 (6.0 mL, 64.4
mmol) was carefully added. The resulting mixture was heated at
95 °C for 60 min and carefully poured onto crushed ice. After
vigorous stirring, the obtained suspension was extracted with DCM,
and the combined organic extracts were washed with water and
brine and dried over Na2SO4. The solvent was reduced to about 20
mL and the solution was filtered over a pad of silica using DCM
with 5% of methanol to elute the compound. Evaporation of the
collected filtrate yielded 5.15 g (15.4 mmol, 75%) of the title
compound as a white solid; mp 182.0–183.0 °C. 1H NMR (CDCl3)
δ (ppm): 7.80 (d, J ) 8.6 Hz, 1H), 7.59 (d, J ) 2.0 Hz, 1H), 7.43
(dd, J ) 8.6 Hz, J ) 2.0 Hz, 1H), 7.27 (d, J ) 8.6 Hz, 2H), 6.89
(d, J ) 8.6 Hz, 2H), 5.45 (s, 2H), 3.71 (s, 3H).
(4) Liu, C.; Ma, X-J.; Jiang, X.; Wilson, S. J.; Hofstra, C. L.; Blevitt, K.;
Li, X.; Chai, W.; Carruthers, N.; Lovenberg, T. W. Cloning and
pharmacological characterization of a fourth histamine receptor (H4)
expressed in bone marrow. Mol. Pharmacol. 2001, 59, 420–426.
(5) Morse, K. L.; Behan, J.; Laz, T. M.; West, R. E., Jr.; Greenfender,
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.;
Green, J.; Bayne, M.; Monsma, F. J., Jr. Cloning and characterization
of a novel human histamine receptor. J. Pharmacol. Exp. Ther. 2001,
296, 1058–1066.
(6) Nguyen, T.; Shapiro, D. A.; George, S. R.; Setola, V.; Lee, D. K.;
Cheng, R.; Rauser, L.; Lee, S. P.; Lynch, K. R.; Roth, B. L.; O’Dowd,
B. F. Discovery of a novel member of the histamine receptor family.
Mol. Pharmacol. 2001, 59, 427–433.
(7) Oda, T.; Morikawa, N.; Saito, Y.; Masuho, Y.; Matsumoto, S.
Molecular cloning and characterization of a novel type of histamine
receptor preferentially expressed in leukocytes. J. Biol. Chem. 2000,
275, 36781–36786.
(8) Zhu, Y.; Michalovich, D.; Wu, H-L.; Tan, K. B.; Dytko, G. M.;
Mannan, I. J.; Boyce, R.; Alston, J.; Tierney, L. A.; Li, X.; Herrity,
N. C.; Vawter, L.; Sarau, H. M.; Ames, R. S.; Davenport, C. M.;
Hieble, J. P.; Wilson, S.; Bergsma, D. J.; Fitzgerald, L. R. Cloning,
expression, and pharmacological characterization of a novel human
histamine receptor. Mol. Pharmacol. 2001, 59, 434–444.
(9) de Esch, I. J. P.; Thurmond, R. L. J. A.; Jongejan, A.; Leurs, R. The
histamine H4 receptor as a new therapeutic target for inflammation.
Trends Pharmacol. Sci. 2005, 26, 462–9.
(10) Gantner, F.; Sakai, K.; Tusche, M. W.; Cruikshank, W. W.; Center,
D. M.; Bacon, K. B. Histamine H4 and H2 receptors control histamine-
induced interleukin-16 release from human CD8+ T cells. J. Phar-
macol. Exp. Ther. 2002, 303, 300–7.
(11) Varga, C.; Horvath, K.; Berko, A.; Thurmond, R. L.; Dunford, P. J.;
Whittle, B. J. Inhibitory effects of histamine H4 receptor antagonists
on experimental colitis in the rat. Eur. J. Pharmacol. 2005, 522, 130–
138.
(12) 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. A potent and selective histamine
H4 receptor antagonist with anti-inflammatory properties. J. Pharmacol.
Exp. Ther. 2004, 309, 404–413.
(13) Ikawa, Y.; Suzuki, M.; Shiono, S.; Ohki, E.; Moriya, H.; Negishi, E.;
Ueno, K. Histamine H4 receptor expression in human synovial cells
obtained from patients suffering from rheumatoid arthritis. Biol. Pharm.
Bull. 2005, 10, 2016–2018.
3,6-Dichloroquinoxalin-2(1H)-one (69). Compound 68 (1.0 g,
2.98 mmol) was dissolved in concentrated H2SO4 (6.0 mL) and
stirred at room temperature. After 20 min, the deep-red reaction
mixture was carefully poured onto a small amount of crushed ice
and neutralized with 15 N sodium hydroxide solution while cooling
at 0 °C. A cloudy suspension was obtained that was extracted
thoroughly with n-butanol. The combined organic layers were
washed with a small amount of brine and dried over Na2SO4.
Removal of the solvent yielded 640 mg (100%) of the crude title
1
compound as a light-yellow solid. H NMR (DMSO-d6) δ (ppm):
7.73 (d, J ) 8.6 Hz, 1H), 7.39–7.30 (m, 2H); 13C NMR (DMSO-
d6) δ (ppm): 150.80, 149.27, 134.57, 133.15, 129.40, 129.29,
123.58, 114.67.
7-Chloro-3-(3-(methylamino)pyrrolidin-1-yl)quinoxalin-2(1H)-
one Hydrochloride (72). To compound 69 (100 mg, 0.47 mmol)
in a microwave tube were added Boc-aminomethylpyrrolidine (103
mg, 0.51 mmol), DIPEA (0.9 mL, 0.51 mmol), and EtOAc (2.0
mL), and the resulting suspension was heated at 120 °C for 10
min. After cooling to room temperature, the mixture was diluted
with EtOAc and washed with water and brine. Drying of the organic
phase over Na2SO4 and rotary evaporation gave the crude product
that was purified over SiO2 (EtOAC:hexanes, 1:4) to yield 124 mg
(64%) of the Boc-protected intermediate (72) as a light-yellow solid.
This compound was then stirred in 2 M HCl ether (2.0 mL) and
dioxane (2.0 mL) until removal of the Boc group was completed
(LCMS). Evaporation of the solvents yielded the hydrochloric salt
as a yellow-white solid. 1H NMR (400 MHz, 341 K, D2O) δ (ppm):
7.85 (d, J ) 8.8 Hz, 1H), 7.62 (dd, J ) 8.8 Hz, J ) 2.2 Hz, 1H),
7.58 (d, J ) 2.1 Hz, 1H), 4.92 (dd, J ) 14.1 Hz, J ) 6.8 Hz, 1H),
(14) Bell, J. K.; McQueen, D. S.; Rees, J. L. Involvement of histamine H4
and H1 receptors in scratching induced by histamine receptor agonists
in BalbC mice. Br. J. Pharmacol. 2004, 142, 374–380.
(15) Cianchi, F.; Cortesini, C.; Schiavone, N.; Perna, F.; Magnelli, L.; Fanti,
E.; Bani, D.; Messerini, L.; Fabbroni, V.; Perigli, G.; Capaccioli, S.;
Masini, E. The role of cyclooxygenase-2 in mediating the effects of
histamine on cell proliferation and vascular endothelial growth factor

Anti-inflammatory H4 Receptor-Ligands in ViVo
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2467
production in colorectal cancer. Clin. Cancer Res. 2005, (19 Pt 1),
6807–6815.
(16) Smits, R. A.; Lim, H. D.; Stegink, B.; Bakker, R. A.; de Esch, I. J. P.;
Leurs, R. Characterization of the histamine H4 receptor binding site:
Part I. Synthesis and pharmacological evaluation of dibenzodiazepine
derivatives. J. Med. Chem. 2006, 49, 4512–4516.
(17) Jablonowski, J. A.; Grice, C. A.; Chai, W.; Dvorak, C. A.; Venable,
J. D.; Kwok, A. K.; Ly, K. S.; Wei, J.; Baker, S. M.; Desai, P. J.;
Jiang, W.; Wilson, S. J.; Thurmond, R. L.; Karlsson, L.; Edwards,
J. P.; Lovenberg, T. W.; Carruthers, N. I. The first potent and selective
non-imidazole human histamine H4 receptor antagonists. J. Med.
Chem. 2003, 46, 3957–3960.
(18) Venable, J. D.; Cai, H.; Chai, W.; Dvorak, C. A.; Grice, C. A.;
Jablonowski, J. A.; Shah, C. R.; Kwok, A. K.; Ly, K. S.; Pio, B.;
Wei, J.; Desai, P. J.; Jiang, W.; Nguyen, S.; Ling, P.; Wilson, S. J.;
Dunford, P. J.; Thurmond, R. L.; Lovenberg, T. W.; Karlsson, L.;
Carruthers, N.; Edwards, J. P. Preparation and biological evaluation
of indole, benzimidazole and thienopyrrole piperazine carboximida-
mides: Potent human histamine H4 antagonists. J. Med. Chem. 2005,
48, 8289–8298.
(19) Greenlee, W.; Gangly, A.; Wasley, J. W. F. Certain 1-(2-naphthyl)
and 1-(2-azanaphthyl)-4-(1-phenylmethyl) piperazines, dopamine re-
ceptor subtype specific ligands. U.S. Patent US 6,040,448, 2000.
(20) Lawrence, D. S.; Copper, J. E.; Smith, C. D. Structure-activity studies
of substituted quinoxalinones as multiple-drug-resistance antagonists.
J. Med. Chem. 2001, 44, 594–601.
(21) Duffy, K. J.; Haltiwanger, R. C.; Huang, Y.; Kanialian-Beck, A.;
Luengo, J. I. A novel synthesis of 2-(2-quinoxalino)-3,5-diarylfurans.
Tetrahedron Lett. 1998, 39, 8017–8020.
(22) Mustafa, M. E.; Takaoka, N.; Ishikawa, N. Trifluoropyruvic acid
hydrate in heterocyclic synthesis. Synthesis of trifluoromethylated five,
six, and seven membered heterocycles with two or more heteroatoms.
Bull. Soc. Chim. Fr. 1986, 944–954.
(23) Hori, M.; Suzuki, K.; Yamamoto, T.; Nakajima, F.; Ozaki, A.; Ohtaka,
H. Design and syntheses of a series of novel serotonin3 antagonists.
Chem. Pharm. Bull. 1993, 41, 1832–1841.
(24) Sarges, R.; Howard, H. R.; Browne, R. G.; Lebel, L. A.; Seymour,
P. A.; Koe, K. 2-Amino[1,2,4]triazolo[4,3-a]quinoxalines. A novel
class of potent adenosine receptor antagonists and potential rapid-
onset antidepressants. J. Med. Chem. 1990, 33, 2240–2254.
(25) Cannizzo, S.; Guerrera, F.; Siracusa, M. A. Synthesis of substituted
[1]benzothieno[2,3-b]pyrazines. J. Heterocycl. Chem. 1990, 27, 2175–
79.
(26) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful
metric for lead selection. Drug DiscoVery Today 2004, 9, 430–431.
(27) Quinoxaline compounds. U. S. Patent US2005/0070527 A1, 2005.
(28) Terzioglu, N.; van Rijn, R. M.; Bakker, R. A.; de Esch, I. J. P.; Leurs,
R. Synthesis and structure-activity relationships of indole- and
benzimidazole piperazines as histamine H4 receptor antagonists.
Bioorg. Med. Chem. Lett. 2004, 14, 5251–5256.
(29) Sato, H.; Tanaka, K.; Shimazaki, M.; Urbahns, K.; Sakai, K.; Gantner,
F.; Bacon, K. 2-Aminopyrimidine derivatives. Patent WO/2005/
054239, 2005.
(30) Dyke, H.; Price, S.; Cramp, S. Pyrimidine compounds for the treatment
of inflammatory disorders. European Patent EP1767537A1, 2007.
(31) Daugherty, B. L. Histamine H4 antagonism: a therapy for chronic
allergy. Br. J. Pharmacol. 2004, 142, 5–7.
(32) Ganellin, C. R.; Hosseini, S. K.; Khalaf, Y. S.; Tertiuk, W.; Arrang,
J. M.; Garbarg, M.; Ligneau, X.; Schwartz, J. C. Design of potent
non-thiourea H3 receptor antagonists. J. Med. Chem. 1995, 38, 3342–
3350.
(33) Coruzzi, G.; Adami, M.; Guaita, E.; de Esch, I. J. P.; Leurs, R.
Antiinflammatory and antinociceptive effects of the selective histamine
H4-receptor antagonists JNJ7777120 and VUF6002 in a rat model of
carrageenan-induced inflammation. Eur. J. Pharmacol. 2007, 563, 240–
244.
(34) Bakker, R. A.; Weiner, D. M.; ter Laak, T.; Beuming, T.; Zuiderveld,
O. P.; Edelbroek, M.; Hacksell, U.; Timmerman, H.; Brann, M. R.;
Leurs, R. 8R-lisuride is a potent stereospecific histamine H1-receptor
partial agonist. Mol. Pharmacol. 2004, 65, 538–549.
JM7014217