Characterization of the histamine H4 receptor binding site. Part 1. Synthesis and pharmacological evaluation of dibenzodiazepine derivatives

Article abstract of DOI:10.1021/jm051008s

A series of dibenzodiazepine derivatives was synthesized to probe the binding site of the recently discovered histamine H₄ receptor (H ₄R). Optimization of the lead structure clozapine (2) resulted in (E)-7-chloro-11(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine (7j), a potent H₄R agonist (H₄R, pK, = 7.6). Pharmacological data suggests that the series of nonimidazole compounds can be used to describe the orthosteric binding site of the H₄R because both 2 and 7j displace [³H]histamine in a competitive manner. Furthermore, it is demonstrated that the effects of 7j are competitively antagonized by the selective H₄R antagonist JNJ 7777120 (1), indicating considerable overlap of their binding sites. On the basis of the derived structure-activity relationships and additional pharmacological results, a pharmacophore model was constructed, which will be the premise for the design of novel H₄R ligands.

Full text of DOI:10.1021/jm051008s

4512
J. Med. Chem. 2006, 49, 4512-4516
Characterization of the Histamine H4 Receptor Binding Site. Part 1. Synthesis and
Pharmacological Evaluation of Dibenzodiazepine Derivatives
Rogier A. Smits,† Herman D. Lim,† Bart Stegink, Remko A. Bakker, Iwan J. P. de Esch, and Rob Leurs*
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
ReceiVed October 7, 2005
A series of dibenzodiazepine derivatives was synthesized to probe the binding site of the recently discovered
histamine H4 receptor (H4R). Optimization of the lead structure clozapine (2) resulted in (E)-7-chloro-11-
(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine (7j), a potent H4R agonist (H4R, pKi ) 7.6). Pharma-
cological data suggests that the series of nonimidazole compounds can be used to describe the orthosteric
binding site of the H4R because both 2 and 7j displace [3H]histamine in a competitive manner. Furthermore,
it is demonstrated that the effects of 7j are competitively antagonized by the selective H4R antagonist JNJ
7777120 (1), indicating considerable overlap of their binding sites. On the basis of the derived structure-
activity relationships and additional pharmacological results, a pharmacophore model was constructed, which
will be the premise for the design of novel H4R ligands.
Introduction
Histamine is an extensively studied biogenic amine that is
present in a variety of tissues. Its physiological role is mediated
through the activation of four G-protein coupled receptors
(GPCRs) that significantly differ in location, (patho)physiology
and ligand binding. The histamine H4 receptor (H4R) has only
Figure 1. JNJ 7777120 (1).
recently been discovered independently by several research
groups.1-5 This receptor is mainly expressed in eosinophils, T
cells, dendritic cells, basophils, and mast cells. The administra-
tion of selective H4R antagonist 1 (Figure 1) inhibits the H4R-
mediated calcium influx and the chemotaxis of mast cells.6
Furthermore, it has been shown that zymosan-induced neutrophil
Figure 2. Clozapine (2).
recruitment from the bone marrow can be blocked by thio-
peramide, a potent inverse agonist at both the H4R and H3R
subtypes.7 Together with the reports on the role of histamine
as a leukocyte chemoattractant, these findings suggest an
important role for the H4R for the treatment of inflammatory
diseases such as asthma, inflammatory bowel disease, and
several dermatological disorders.8-13 It has been suggested that
the H4R is involved in the histamine-induced itch, and H4R
antagonists could potentially lead to the first effective treatment
of pruritis.14,15
The human H4R is most closely related to the human H3R.
The two proteins have a sequence identity of 31% overall and
54% in the transmembrane domains where the orthosteric
binding sites of the class A aminergic GPCRs are located. The
orthosteric binding site of the H4R is the site where its natural
ligand histamine binds. It is not surprising, therefore, that most
imidazole-containing ligands (agonists and antagonists) that have
been developed for the H3R also display considerable affinity
for the H4R.13,16 Remarkably, the more recently developed
nonimidazole H3R inverse agonists and antagonists, compounds
that have a high clincial potential in H3R-mediated diseases,
all lack H4R affinity.17
been extensively studied, and many receptors tolerate its tricyclic
dibenzodiazepine structure to some extent.18,19 Because 2 acts
as an antagonist on most GPCRs we were intrigued to find that
it acts as a full agonist at the human H4R (R ) 1, pEC50 ) 6.7;
pKi ) 6.8).16 Taking into account the novelty of the target and
the size and rigidity of tricyclic ligand 2, we consider this
compound to be an interesting starting point to explore the H4R
binding site. Therefore, a series of dibenzodiazepine analogues
were synthesized to explore the structure-activity relationship
(SAR) of this class of compounds at the human H4R.
Chemistry. We synthesized several different clozapine
analogues using a modified method described by Nagarajan et
al. (1974) (Scheme 1).20 First, substituted chloro or fluoroben-
zoic acids were converted to their corresponding acid chlorides
by SOCl2. These acid chlorides were then added to a solution
of different substituted o-aminophenols and triethylamine in
THF to obtain the respective amide intermediates. Ring closure
of the amides was achieved with NaOH in DMF. POCl3 was
used to convert the benzoxazepines in situ to iminochlorides
that could then react with different cyclic amines. Compound
14 was synthesized according to a literature procedure.21 Most
of the compounds obtained were then converted to fumaric acid
salts to improve handling of the compounds.
A nonimidazole reference compound that has considerable
affinity for the H4R is the widely used antipsychotic drug
clozapine (2) (Figure 2).1 The binding of 2 to other GPCRs has
Pharmacology. Radioligand Displacement Studies. Ho-
mogenates of SK-N-MC cells stably expressing human H3Rs
or H4Rs were used to determine ligand affinities for the H3R or
H4R. These methods have been described previously.16 An
* Corresponding author. Phone: +31(0)205987579. Fax: +31(0)-
205987610. E-mail: leurs@few.vu.nl.
† These authors contributed equally to this work.
10.1021/jm051008s CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/01/2006

Dibenzodiazepine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4513
Scheme 1. General Synthesis of Clozapine Analoguesa
Table 2. H4 Receptor Affinity of Clozapine Analogues with Alterations
in the Piperazine Side Chain as Determined by the Displacement of [3H]
Histamine
compd
X1
X2
pKi ( SEM
7c
7a
7d
7e
13
2
O
O
O
O
NH
NH
NH
NH
NH
N-CH3
N-CH2CH3
N-i-Propyl
N-CH2CH2OH
N-CH3
6.49 ( 0.1
7.37 ( 0.1
6.54 ( 0.1
5.97 ( 0.1
5.33 ( 0.2
6.75 ( 0.1
<5
a
Reagents and Conditions: (a) SOCl2, Reflux; (b) Et3N, THF, 0 °C-
RT; (c) NaOH, DMF, 120 °C; (d) POCl3, Reflux (e) N-methylpiperazine,
Toluene, Reflux
Table 1. H4 Receptor Affinity of Clozapine Analogues with Alterations
in the Tricyclic Framework as Determined by the Displacement of [3H]
Histamine
14
7f
CH2
O
<5
(Table 2). The distal nitrogen in unsubstituted compound 7c is
less basic than its methylated analogue 7a. The same holds true
for ethylated analogue 7d, yet no increase in H4R affinity can
be observed compared to 7c. A steric component might,
therefore, also play a role in the binding of these compounds.
A further increase in the size of this methyl substituent decreases
the H4R affinity even more. The absence of the distal nitrogen
atom in the piperazine side chain, as can be seen in the
piperidine (14) and morpholine (7f) analogues, even leads to a
dramatic loss of H4R affinity.
Changing the R1 chlorine from the 8- to the 7-position
(compounds 7a and 7j) gave a slight increase in affinity.
Changing this substituent at the 7-position to a methyl group
as in analogue 7k made the affinity drop slightly. The unsub-
stituted compound 7b also showed a decrease in affinity
compared to that of 7a, indicating the importance of a lipophilic
substituent on the left aromatic ring. An investigation of the
SAR of the right aromatic ring showed that a chlorine substituent
at the 2- (compare 7b and 11) and 3-positions (compare 7h
and 7a) was detrimental to affinity, whereas a chlorine sub-
stituent at the 4-position was tolerated (7l). Fluorine substitution
at the 2-position also decreased H4R affinity (compare 7a and
7g). Fluorine substitution at the 3- and 4-positions was allowed,
but these compounds were no more potent than, for example,
the unsubstituted 7-chloro analogue 7j. The functional profile
of the compounds in Table 3 was also studied to see if they
would retain the full agonistic behavior that was found for
clozapine. Interestingly, all of the compounds in Table 3 were
full agonists except for 11, which acted as a partial agonist.
Chlorine substitution at the 2-position appeared to be detrimental
for H4R affinity as well as for the full agonistic profile of the
clozapine analogues.
compd
X1
NH
X2
X3
X4
pKi ( SEM
2
8
9
7a
10
7b
11
12
1a
N
N
N
N
N
N
N
CH2
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
6.75 ( 0.1
5.69 ( 0.1
5.85 ( 0.2
7.37 ( 0.1
5.32 ( 0.2
6.88 ( 0.1
5.34 ( 0.0
<5
S
N-CH3
O
CH2
O
O
O
7.80 ( 0.1
a Structure as depicted in Figure 1.
analysis of ligand binding to the H1R and H2R was performed
according to a literature procedure.22
Colorimetric cAMP Assay. SK-N-MC cells stably express-
ing the H3R or the H4R with a CRE-â-galactosidase reporter
gene were employed to determine the functional acivity of either
the H3R or the H4R. This assay has been described previously.16
NFKB-Luciferase Reporter Assay. COS-7 cells transiently
transfected with the H1R were used for the determination of
the functional activity at the H1R according to literature.22
Results and Discussion
Initially, we optimized the tricyclic carbon framework of
clozapine for H4R affinity.
As can be seen in Table 1, the replacement of the X1 nitrogen
of clozapine (2) by a sulfur atom or methylamine group
(compare 2, 8, and 9 with 7a) or a carbon atom (compare 10
and 7b), is detrimental to H4R affinity. In contrast, the
substitution of the X1 position with oxygen increases H4R
affinity 4-fold (7a). Lie´gois and co-workers have recently
reported crystallographic data showing that the nature of the
atom that fuses the phenyl rings determines the dihedral angle
between the planes of both aromatic rings.23 This angle,
therefore, strongly determines the 3D structure of the molecules.
Although an electronic or steric effect might also explain the
increased receptor binding, conformational differences of the
minimized structures can also be seen in the clozapine analogue
series (data not shown).
Table 3 contains several tricyclic structures that bind to the
H4R with nanomolar affinity, and of these, compound 7j has
been taken as a model compound to study the affinity and
functional behavior at the other histamine receptors.
These data show that 7j has about a 5-fold higher affinity
for the H1R than that for the H4R and is 330 times more selective
for the H4R over the H3R. These data suggest that a compound
can be designed that is relatively inactive at the H2R and the
H3R, while binding in the lipophilic domains of the H1R and
H4R active sites. In fact, dual action ligands acting as both H1R
and H4R antagonists or inverse agonists have been suggested
to be a potential new way to treat inflammatory diseases.10
Although one group reported known H1R ligands (doxepine,
cinnarazine, and promethazine) to be active at the H4R3 we were
Changing the piperazine substituent in 7a from methyl to any
other substituent decreased affinity for the H4R in all cases

4514 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Smits et al.
The Bmax value for [3H]histamine did not change significantly
when increasing concentrations of the selected compounds were
used. In contrast, the Kd value for [3H]histamine increased upon
increasing concentrations 2 and 7j. These data indicate that 2
and 7j compete for the same binding site as [3H]histamine in
the H4R. Compounds 2 and 7j, therefore, do not act as allosteric
modulators on the recombinant H4R system but as orthosteric
ligands.
Table 3. H4 Receptor Affinity of Clozapine Analogues with Varying
Aromatic Substituents as Determined by the Displacement of [3H]
Histamine
We could also show that known H4R antagonist 1 competi-
tively antagonized the agonistic effect of 7j (Figure 4). Schild
plot analysis resulted in a pA2 value of 7.8 for 1, which nicely
correlates with the H4R affinity of pKi 7.8.16
compd
R1
R2
pEC50
R
pKi ( SEM
7b
11
g
7h
7i
7a
7j
7k
7l
H
H
H
6.52 ( 0.19
6.66 ( 0.04
7.09 ( 0.11
6.99 ( 0.11
7.83 ( 0.11
7.55 ( 0.12
7.70 ( 0.10
6.98 ( 0.08
7.83 ( 0.07
7.69 ( 0.08
7.41 ( 0.02
1
0.5
1
1
1
1
1
1
1
6.88 ( 0.1
5.34 ( 0.0
6.72 ( 0.1
6.51 ( 0.1
7.56 ( 0.1
7.37 ( 0.1
7.55 ( 0.1
7.10 ( 0.1
7.34 ( 0.1
7.51 ( 0.1
7.41 ( 0.0
When the structure of 1 is compared to that of 7j, several
similarities can be noticed. Most strikingly, both ligands have
a piperazine moiety linked to an aromatic ring system. For both
classes of compounds, it has been shown that the basic nitrogen
of this piperazine is ideally substituted by a methyl group,
whereas other substituents lead to a drop in affinity.24,25
Furthermore, halogenation of the aromatic rings lead to similar
subtle differences in affinity. On the basis of these similarities
in structure and SAR and because the pharmacological results
suggest that 7j and 1 compete for the same H4R binding site,
we were tempted to perform preliminary modeling studies to
investigate the structural similarities of these compounds, even
though 1 is an (neutral) H4R antagonist and 7j is a full H4R
agonist.16
2-Cl
2-F
3-Cl
4-F
H
H
H
4-Cl
4-F
3-F
8-Cl
8-Cl
8-Cl
8-Cl
7-Cl
7-CH3
7-Cl
7-Cl
7-Cl
7m
7n
1
1
Table 4: Affinity and Functional Profile of 7j at the Different
Histamine Receptors
compd 7j
receptor
pKi ( SEM
pEC50
R
For these similarity studies, the procedure as described by
Labute and Williams was used.26 Calculations were carried out
using the flexible alignment module of Molecular Operating
Environment (MOE), employing the MMFF94 force field. The
best scoring fit (both in terms of similarity and objective
function; similarity F ) 104.6195, objective function S )
187.6832, dU ) 0.7498 kcal/mol) is shown in Figure 5. In this
superposition, the key structural elements are perfectly super-
posed. The chlorinated aromatic ring of 7j is superimposed on
the phenyl ring of 1 with the chlorine atoms pointing in the
same direction. The carbonyl group in 1 is flexible enough to
give a good overlay of the protonated nitrogens in both
compounds. The methyl substituents also overlap perfectly,
whereas the bulkier piperazine moiety in compound 1 and 7j
occupy approximately the same space. Obviously, this qualita-
tive pharmacophore model is unable to explain the mode of
action of the ligands (agonism for 7j, neutral antagonism for
1). With the limited number of structurally diverse classes of
H1
H2
H3
H4
8.11 ( 0.10
5.06 ( 0.05
5.04 ( 0.14
7.55 ( 0.09
8.17 ( 0.14
n.d.a
-1
n.d.a
n.d.a
1
n.d.a
7.70 ( 0.10
a Not determined.
unable to reproduce those results to combine them with the SAR
found for the clozapine analogues at the H4R.16
Clozapine (2) and its metabolites have been reported to have
affinity for the D1-D4 receptors, several 5-HT subreceptors,
the R1-adrenoreceptor, the H1R, and the muscarinic receptor
subtypes. Compound 2 and one of its metabolites have also been
reported to act as allosteric modulators of GPCRs, although
evidence for this is sometimes contradictory.18,19 Therefore, we
investigated whether 2 and 7j bind to the H4R as orthosteric or
allosteric ligands by looking at the Bmax value of the H4R in
the presence of increasing concentrations of 2 and 7j (Figure
3).
Figure 3. Potential orthosteric binding of 2 and 7j by saturation binding analysis. Binding of [3H]histamine to the H4R. The presence of 2 (A) and
7j (B) dose-dependently increases the Kd value without affecting the Bmax of [3H]histamine binding, suggesting that 2 and 7j displace [3H]histamine
in a competitive manner.

Dibenzodiazepine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4515
its orthosteric binding site in the H4R. Given the structural
similarities of 7j and 1, we have conducted preliminary modeling
studies and propose a model that gives a description of the H4R
binding site of 7j. The results from these modeling studies are
currently being used to design new chemical entities that will
fit the H4R binding site. In the search for new antipsychotic
drugs, many analogues of 2 have been made in the past that
also seem to fit the H4R pharmacophore. The fact that many of
these analogues can modulate the H4R and cause a possible
clinically relevant but yet unknown response mediated by this
histamine receptor should, therefore, be taken into consideration.
Experimental Section
General Remarks. Compounds 2 and 11 were purchased from
Sigma RBI (U.S.A.). Compounds 8, 9, 10, and 13 were donated
by Dr. Aebischer from the Sandoz research institute in Bern.
Clozapine analogue 12 was purchased from Tocris Cookson, Ltd.
(U.K.).
The THF and toluene used in the reactions were freshly distilled
from lithium aluminum hydride and calcium hydride, respectively.
Other chemicals and reagents were obtained from commercial
suppliers and were used without further purification. The yields
given are isolated yields unless otherwise mentioned and were
calculated on the free base after purification by flash chromatog-
raphy. Flash column chromatography was 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 IA9200 electrothermal meltingpoint apparatus. All
1H NMR and 13C NMR spectra were measured on a Bru¨ker 200.
Analytical HPLC-MS analyses were conducted using a Shimadzu
LC-8A preparative liquid chromatograph pump system with a
Shimadzu SPD-10AV UV-Vis detector set at 254 nm, with the
MS detection performed with a Shimadzu LCMS-2010 liquid
chromatograph mass spectrometer. The analyses were performed
using the following two conditions. Condition I: an Alltima(C18)
5 µm column (150 mm × 4.6 mm) with the following two
solvents: solvent A, 10% MeOH- 90% H2O- 0.1% formic acid;
solvent B, 95% MeOH- 5% H2O- 0.1% formic acid; flow rate )
1.0 mL/min; start: 100% A, linear gradient time is 15 min, then
10 min at 100% B, then 15 min at 100% A. The total run time was
40 min. Condition II: Xbridge(C18) 5 µm column (100 mm × 6
mm), isocratic elution with varying percentages of acetonitrile-
H2O containing 0.1% formic acid; flow rate ) 1.0 mL/min. The
total run time was 40 min. Compounds that were isolated as fumaric
acid salts all showed an extra peak around 4.1 min under condition
I (fumaric acid was found to have tR) 4.1 min in this system).
Under condition II, fumaric acid blancs were used to determine
the tR of fumaric acid. Purities calculated are based on RP HPLC-
UV peak surface area of the compounds, taking the fumaric acid
peaks into account.
Methods. General Procedure. The synthesis of 7b from
precursors 6b and 5b described below is a general procedure that
has been used for the synthesis of all tricyclic oxazepines.
2-Fluoro-N-(2-hydroxyphenyl)-Benzamide (5b). To 2-fluo-
robenzoic acid (5.0 g, 36 mmol) was added thionyl chloride (10
mL). The solution was refluxed for 2 h; after which the excess of
thionyl chloride was removed under reduced pressure, and the
residual traces were then removed by coevaporation with dry
toluene. The acid chloride was then added dropwise to a solution
of 2-aminophenol (3.9 g, 36 mmol) and Et3N (10 mL, 72 mmol)
in dry THF (75 mL) at 0 °C. After the addition of the acid chloride,
the mixture was allowed to warm up to room temperature overnight
while stirring under a nitrogen atmosphere. After stirring overnight,
the mixture was diluted with water (400 mL) and neutralized with
4% HCl to pH 7. The precipitated amide was then filtered off over
a glass filter and washed with 4% HCl and copious amounts of
water. The washed product was dried in vacuo overnight in the
presence of P2O5 and was used in the next step without further
Figure 4. Selective histamine H4 antagonist 1 competitively antago-
nizes the H4R effects of 7j activity. (A) Compound 1 shifts the dose-
response curve of 7j to the right in a dose-dependent manner. (B) Schild
plot analysis resulted in a pA2 value of 7.8 for JNJ 7777120 (1).
Figure 5. Alignment of 1 and 7j. The superpostion shown was selected
on the basis of the highest similarity score (F) and the highest objective
function score (S).
H4R ligands reported to date and without taking into account
the receptor protein environment, it will be difficult to address
the functional activity of the ligands. It has already been shown
that for the histamine H3R subtype, subtle changes in the
chemical structures of the H3R ligands can have a major impact
on functional activity. Even within a single class of ligands,
close homologues can vary in H3R activity from agonist to
neutral antagonist and inverse agonist.27 Nevertheless, we
consider this preliminary pharmacophore a useful working
model for H4R affinity.
In conclusion, probing the H4R pharmacophore, we have
optimized the dibenzodiazepine structure of clozapine (2),
resulting in the identification of 7j as a high-affinity full H4R
agonist. Compound 7j was found to displace [3H]histamine from

4516 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Smits et al.
purification. Yield 6.19 g (76%). Mp 144-147 °C. 1H NMR
(DMSO-d6) δ (ppm) 10.10 (s, 1H), 9.48 and 9.43 (2s, 1H, rotamers),
8.17-8.05 (m, 1H), 7.94-7.87 (m, 1H), 7.72-7.52 (m, 1H), 7.45-
7.24, (m, 3H), 7.03-6.80 (m, 2H).
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(7) Takeshita, K.; Bacon, K. B.; Ganter, F. Critical role of L-selectin
and histamine H4 receptor in Zymosan-induced neutrophil recruitment
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(8) Buckland, K. F.; Williams, T. J.; Conroy, D. M. Histamine induces
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Histamine H3 receptor antagonists reach out for the clinic. Drug
DiscoVery Today 2005, 10, 1613-1627.
Dibenzo[b,f][1,4]oxazepin-11(10H)-one (6b). 2-Fluoro-N-(2-
hydroxyphenyl)-benzamide (5b) (500 mg, 2.16 mmol) was dis-
solved in DMF (10 mL), and one equivalent of freshly powdered
NaOH was added. The resulting mixture was heated at reflux for
5 h After cooling to room temperature, the mixture was diluted
with water (100 mL), and the precipitated product was filtered over
a glass filter and washed with 5% NaOH and then water to remove
the unreacted starting material. The solid residue was dried
overnight under vacuum and was used in the next step without
1
further purification. Yield 320 mg (70%). Mp 210-212 °C. H
NMR (DMSO-d6) δ (ppm) 10.55 (s, 1H), 7.78 (dd, J ) 1.6 Hz, J
) 7.7 Hz, 1H), 7.66-7.58 (m, 1H,), 7.37-7.28 (m, 3H), 7.17-
7.11 (m, 3H).
11-(4-Methylpiperazin-1-yl)-dibenzo[b,f][1,4]oxazepine (7b).
10H-Dibenzo[b,f][1,4]oxazepin-11-one (6b) (1.0 g, 4.7 mmol) was
added to POCl3 (5 mL) and heated at reflux overnight. Excess
POCl3 was evaporated under reduced pressure yielding the crude
iminochloride. Dry toluene (10 mL) and N-methylpiperazine (5.3
mL, 47 mmol) were added to the iminochloride, and the mixture
was heated at reflux. After 2 h, the solvent was allowed to cool to
room temperature, and the mixture was added to EtOAc and water.
The aqueous layer was then extracted with EtOAc, and the
combined organic layers were dried over Na2SO4. Evaporation of
the solvent yielded the crude product, which was purified by flash
chromatography (100% EtOAc) to give the free base as a light
yellow oil. Yield 472 mg (34%); 1H NMR (CDCl3) δ (ppm) 7.45-
6.99 (m, 8H), 3.65 (m, 4H), 2.52 (m, 4H), 2.34 (s, 3H); 13C NMR
(DMSO-d6) δ (ppm) 160.82, 160.20, 152.00, 140.44, 132.45,
129.42, 126,78, 125.36, 124.63, 124.00, 123.48, 121.11, 120.03,
54.84, 47.13, 46.02; MS (ESI) m/z 294 (M + H)+.
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Acknowledgment. We greatly acknowledge Dr. T. Loven-
berg (Johnson & Johnson Pharmaceutical Research and Devel-
opment, L.L.C., San Diego, CA) for the gift of the cells
expressing the human H4R and H3R. Compounds 3, 5, 7, and
13 were generously donated by Dr. Aebischer from the Sandoz
research institute in Bern. Thanks also goes out to Andrea van
de Stolpe for expert technical assistance.
(20) Nagarajan, K.; Kulkarni, L.; Venkateswarlu, A.; Shah, R. Condensed
heterotricycles: Dibenz[b,f][1,4]oxazepin-11(10H)-thiones, 11-
substituted dibenz[b,f][1,4]oxazepines & dibenz[b,f][1,4]thiazepines
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Supporting Information Available: LCMS purity data for
compounds 7a-n and 14 and experimental details for compounds
5-7a, 7c-f, 14, 5g-n, 6g-n, and 7g-n. This material is available
free of charge via the Internet at http://pubs.acs.org.
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O. P.; Edelbroek, M.; Hacksell, U.; Timmerman, H.; Brann, M. R.;
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