Synthesis and Pharmacological Identification of Neutral Histamine H 1-Receptor Antagonists

Article abstract of DOI:10.1021/jm030936t

In the present study we searched for neutral antagonists for the human histamine H₁-receptor (H₁R) by screening newly synthesized ligands that are structurally related to H₁R agonists for their affinity using radioligand d

Full text of DOI:10.1021/jm030936t

5812
J . Med. Chem. 2003, 46, 5812-5824
Syn th esis a n d P h a r m a cologica l Id en tifica tion of Neu tr a l Hista m in e H1-Recep tor
An ta gon ists
Marinella Govoni,†, Remko A. Bakker,†, Ineke van de Wetering,† Martine J . Smit,† Wiro M. B. P. Menge,†
Henk Timmerman,† Sigurd Elz,‡ Walter Schunack,§ and Rob Leurs*,†
Leiden/ Amsterdam Center for Drug Research, Department of Pharmacochemistry, Vrije Universiteit Amsterdam,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, Institut fu¨r Pharmazie, Universita¨t Regensburg,
93040 Regensburg, Germany, and Institut fu¨r Pharmazie, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 2-4,
14195 Berlin (Dahlem), Germany
Received J une 23, 2003
In the present study we searched for neutral antagonists for the human histamine H1-receptor
(H1R) by screening newly synthesized ligands that are structurally related to H1R agonists for
their affinity using radioligand displacement studies and by assessing their functional activity
via performing a NF-κB driven reporter-gene assay that allows for the detection of both agonistic
and inverse agonistic responses. Starting from the endogenous agonist for the H1R, histamine,
we synthesized and tested various analogues and ultimately identified several compounds with
partial inverse agonistic properties and two neutral H1-receptor antagonists, namely 2-[2-(4,4-
diphenylbutyl)-1H-imidazol-4-yl]ethylamine (histabudifen, 18d ) (pKi ) 5.8, R ) 0.02) and 2-[2-
(5,5-diphenylpentyl)-1H-imidazol-4-yl]ethylamine (histapendifen, 18e) (pKi ) 5.9, R ) -0.09).
In tr od u ction
but various human diseases have been ascribed to
constitutive receptor activity induced by mutations in
genes encoding GPCRs.22-26 It is obvious that for these
genetic disorders inverse agonists are essential for
silencing the mutant GPCRs, as neutral antagonists
would be of no use.
Four human G-protein coupled histamine-receptor
subtypes (H1-4) are currently recognized to mediate the
various actions of the monoamine histamine.1-10 Specific
activation or blockade of these receptor subtypes has
led to a tremendous increase in the knowledge of the
roles of histamine in both physiology and pathology.
Stimulation of the histamine H1-receptor (H1R), which
is found throughout the body, causes contraction of
smooth muscles in, e.g. the airways and intestine.11
Moreover, histamine plays a role in allergic conditions
that have often been treated successfully with H1R
antagonists.12
It is increasingly recognized that G protein-coupled
receptors (GPCRs) activate their associated signal trans-
duction pathways not only upon agonist activation but
also in the absence of agonists, resulting in constitutive
receptor activity. Concomitant with the appreciation of
constitutive receptor activity, the phenomenon of in-
verse agonism has found general acceptance as well.13-18
Many ligands that were previously thought to act as
antagonists actually inhibit the constitutive receptor
signaling, indicating their inverse agonistic behavior.
A third class of ligands, the neutral antagonists, al-
though interacting with the receptor, fail to modulate
receptor activity. Inverse agonists display negative
intrinsic activity (R) between -1 and 0 and are relatively
common, whereas neutral antagonists, ligands without
intrinsic activity (R ) 0), are quite rare but known for
several GPCRs, including histamine H2- and H3-
receptors.19-21 The actual therapeutic importance of
constitutive GPCR activity has not been clarified yet,
We have recently shown the constitutive activity of
the wild-type human H1R and we have identified well-
known therapeutics such as cetirizine (Zyrtec), epinas-
tine (Flurinol), loratidine (Claritin) as inverse agonists,
which led us to reconsider their mechanism of action.27,28
No data on the physiological relevance of constitutive
H1R activity are currently available. Constitutive recep-
tor activity can be achieved by high receptor expression-
levels, but also by other means, i.e., removal of sodium
ions29 and coexpression of their cognate GR proteins30-33
or specific Gâγ subunit combinations.28,34 Moreover,
receptor mutation can result in constitutively active
(CAM) or inactive (CIM) mutant receptors.35-38 There-
fore, constitutive H1R activity may contribute to patho-
physiological conditions where either H1R up-regulation
occurs, such as in patients with allergic rhinitis,39 or
GRq protein levels are found to be elevated, such as in
the guinea pig nasal mucosa in a model of nasal hyper-
responsiveness.40 Although an inverse H1R agonist
would suppress any apparent constitutive H1R activity,
long-term exposure of cells expressing constitutively
active GPCRs to inverse agonists may result in receptor
up-regulation.19,41-44 This may have clinical significance,
since an increased sensitivity, development of tolerance,
and recurrence have been attributed to long-term treat-
ment with for instance inverse H2R agonists.45,46 How-
ever, so far H1R up-regulation or sensitization has not
been reported upon prolonged inverse agonist treat-
ment.
* Author for correspondence. Tel.: 31-20-4447579. Fax: 31-20-
4447610. E-mail: leurs@few.vu.nl.
† Vrije Universiteit Amsterdam.
‡ Universita¨t Regensburg.
Clearly, the development of a neutral H1R antagonist
would give a valuable pharmacological tool to study the
§ Freie Universita¨t Berlin.
These authors have contributed equally.
10.1021/jm030936t CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/20/2003

Neutral Histamine H1-Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5813
Sch em e 1. Synthesis of Compounds 1c,e,f,i-k
Reagents: (a) n-BuLi, THF, -70 °C; (b) tert-butyldimethylsilyl chloride; (c) 1-chloro-ω-iodoalkane; (d) 2-(ω-bromoalkyloxy)tetrahydro-
pyran; (e) HCl, rt; (f) isoindole-1,3-dione, Na2CO3, DMF, 90 °C; (g) triphenylphosphine, isoindole-1,3-dione, diisopropylazodicarboxylate,
THF; (h) hydrazine monohydrate, EtOH, reflux; (i) pyrrolidine, rt; (j) 30% HBr, reflux.
potential physiological role of constitutive H1R activity
which, to date, has been hampered by the lack of neutral
H1R antagonists. In the present study we report the
synthesis of a variety of histamine homologues, their
binding affinities, and their intrinsic H1R activity using
a reporter-gene assay.
Ch em istr y
The target aminoalkylimidazoles 1c,e-k were pre-
pared by lithiation of a suitable 1,2-diprotected imid-
47
azole 3
and subsequent treatment with 1-chloro-ω-
iodoalkanes or 2-(ω-bromoalkyloxy)tetrahydropyrans
to give compounds 4c,e and 4f,i,j (Scheme 1). The
ω-hydroxy group of 4c,e was converted into a phthaloyl
group via a Mitsunobu reaction48 followed by hydrolysis
of the phthalimide group to give the amines 1c,e.
Alternatively, the ω-chloro group of 4f,i,j was converted
either into a pyrrolidine via reaction with pyrrolidine
to give compounds 7i-k or via a Gabriel synthesis49 into
compound 6f. Deprotection yielded compounds 1f,i-k .
For aminoalkylimidazoles 1g,h the synthetic pathway
shown in Scheme 2 was adopted. The key step in this
synthesis is the conversion of aldehyde 10 into 4-tosyl-
oxazoline 11 using TosMIC followed by treatment with
ammonia in methanol under pressure50 to give amide
12. Treatment of amide 12 with [I,I-bis(trifluoroacet-
oxy)iodo]benzene (PIFA)51 gave compound 1g, whereas
reduction with LiAlH4 afforded compound 1h .
F igu r e 1. Structures of 2-[1H-imidazol-4-yl]ethylamine (his-
tamine), 2-(thiazol-4-yl)ethanamine (4-TEA), 2-(pyridine-2-yl)-
ethyl-1-amine (2-PEA), VUF 4734 (1), and histaprodifen.
P h a r m a cologica l Resu lts a n d Discu ssion
The compound 10-(1H-imidazol-4-yl)decylamine (1d ;
VUF 4734, Figure 1) has been reported to exhibit the
highest affinity for the guinea pig H1R among a series
of higher histamine analogues53 and subsequently proved
to be a partial inverse agonist for the human H1R (see
Table 1). Therefore, we have prepared a series of ω-(1H-
imidazol-4-yl)alkylamines (1c,e-h ) and investigated the
effect of the length of the alkyl spacer of the histamine
analogues on the human H1R binding affinity and
intrinsic activity. As shown in Table 1, the H1R binding
affinity for the higher homologues of histamine (1a -h )
increases with the elongation of the side chain, whereas
for the potency a maximum is reached for compound
1e, bearing an 11-carbon-atom side chain. The intrinsic
activity of the compounds decreases as the length of the
side chain increases, meaning that the compounds are
turning from partial inverse agonists to full inverse
agonists.
The aminoalkylpyridines 13a -g were prepared ac-
cording to Scheme 3. Lithiation of methylpyridines
14a ,d ,f52 and coupling with 1,8-dibromooctane or 2-(ω-
bromoalkyloxy)tetrahydropyrans yielded compounds
15a -g. A Gabriel synthesis or a Mitsunobu reaction
gave compounds 16a -g. The free amino group was
obtained by hydrazinolysis resulting in compounds
13a -g.

5814 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Sch em e 2. Synthesis of Compounds 1g,h
Govoni et al.
does not influence the capability of the resulting com-
pound to bind to the H1R, provided that the aminoethyl
chain is placed in the 2-position of the pyridine ring,12,54
as in 2-(pyridine-2-yl)ethanamine (2-PEA, Figure 1). On
this basis we decided to investigate the importance of
the imidazole ring in the binding affinity and intrinsic
H1R activity of the higher homologues of histamine.
Various aminoalkylpyridines have been synthesized in
which a long side chain has been attached in the 2-
(13a -c), 3- (13d ,e), and 4-position (13f,g) of the pyri-
dine ring (Table 2). For the 2-isomers (13a -c), an
increase in the H1R binding affinity with the elongation
of the side chain can be observed, similarly to what
happens for the aminoalkylimidazoles with a primary
amino function (compounds 1c, 1d , and 1f), and the pKi
values are comparable to those of the aminoalkyl-
imidazoles. Although 13b and especially 13c appear to
be lacking intrinsic H1R activity, these compounds
exhibited non-H1R-mediated effects at high concentra-
tions that interfered with an accurate estimation of their
intrinsic H1R activity in our cell based assay (data not
shown). Interestingly, the 3- (13d ,e) and 4-isomers
(13f,g) are still active on the receptor, although no
significant differences are observed for the binding
affinities with elongation of the side chain or shifting
the side chain from the meta to the para position. All
the aminoalkylpyridines are (partial) inverse H1R ago-
nists, with the exception of 2-PEA (Table 2), but there
is no apparent relation between the length or the
position of the side chain and the efficacy of the
compound.
Reagents: (a) MeOH, p-toluenesulfonic acid monohydrate,
reflux; (b) Swern oxidation; (c) TosMIC, NaCN, EtOH abs; (d) NH3/
MeOH, 90-110 °C, 10 atm; (e) PIFA, CH3CN/H2O (1/1); (f) LiAlH4,
THF.
Sch em e 3. Synthesis of Compounds 13a -g
Substitution of the 2-position of histamine has proven
to be a successful strategy to obtain H1R-selective
agonists,55 such as the full agonist histaprodifen (see
Figure 1 and Table 3).56 As shown in Table 3 the
monosubstituted compounds 17a and 17b are agonists
with comparable binding affinities and potencies. In-
terestingly, compound 17a displays an intrinsic activity
(R) that is greater than that of histamine itself (Table
3, Figure 3C). The disubstituted compound (17c) is a
partial agonist, displaying a reduced binding affinity
and potency, suggesting that the accommodation in the
ligand-binding pocket of the H1R57 is hindered due to
steric effects of the chlorine atom in the para-position.
Reagents: (a) n-BuLi (ortho-isomers) or LDA (meta- and para-
isomers), THF, -50 °C; (b) 2-(ω-bromoalkyloxy)tetrahydropyran,
-50 °C; (c) 1,8-dibromooctane, -50 °C; (d) HCl, rt; (e) triphenyl-
phosphine, isoindole-1,3-dione, diisopropylazodicarboxylate, THF;
(f) isoindole-1,3-dione, Na2CO3, DMF, 90 °C; (g) hydrazine mono-
hydrate, EtOH, reflux.
In an attempt to obtain compounds with a higher H1R
affinity, the amino group of some of the higher homo-
logues of histamine was exchanged for a pyrrolidine
moiety (1i-k ), by analogy with the classical H1R
antagonists for which it is known that the substitution
of the primary amino function by a pyrrolidine ring
results in an increase in H1R affinity of an order of
magnitude or more.12 This exchange results in an
increased binding affinity of one log unit only in the case
of compound 1i, whereas in the case of longer side
chains (1j and 1k ) the binding affinities are unchanged
or reduced. Moreover, there is no significant difference
among the binding affinities of the three compounds
bearing a pyrrolidine ring at the end of the side chain
(1i-k ). No definite relation between the length of the
side chain and the potency can be observed. Opposite
to observations made for the series of compounds
bearing a primary amino function (1a -h ), in this series
the intrinsic activity increases as the length of the side
chain increases, meaning that the compounds are turn-
ing from full inverse agonists to partial inverse agonists.
However, no neutral antagonist were identified in this
series.
Replacement of the imidazole ring of histamine for a
thiazole moiety leads to 2-(thiazol-4-yl)ethanamine (4-
TEA,12 Figure 1), with a very low affinity for the
receptor (Table 1). Subsequent substitution of the
2-position of 4-TEA with a (substituted) phenyl ring or
a (substituted) phenylmethyl residue was achieved by
compounds 19a -d , all displaying a similar (and low)
binding affinity for the receptor.
Recently, Elz et al. synthesized a series of compounds
constituting a new class of highly active H1R agonists
that combine a histamine moiety linked at the 2-position
with an ω,ω-diphenylalkyl substituent (18a -e; hista-
prodifens, Figure 1) and examined them for H1-hista-
minergic properties on the guinea pig ileum prepara-
tion.58 In this study the affinities, potencies, and effi-
cacies of these compounds, and other analogues,56,58,59
on the cloned human H1R were determined. The di-
phenylmethyl substituent present in the histaprodifen
series is a common feature of various clinically used H1R
antagonists,12 like cetirizine, and is believed to confer
H1R specific agonists have been developed by replace-
ment of the imidazole ring for a pyridine ring, which

Neutral Histamine H1-Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5815
Ta ble 1. Chemical Structures and Pharmacological Properties of the Aminoalkylimidazoles 1a -k for the Human H1 Receptor
a
compd
name or code
X
n
R1
R2
pKia
pIC50
Ra
histamine
4-TEA
NH
S
2
2
6
8
9
10
11
12
13
14
8
H
H
4.2 ( 0.1
<4
6.8 ( 0.1b
1.00
c
d
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-
-
-
e
e
1a
1b
1c
1d
1e
1f
1 g
1h
1i
VUF 4732
VUF 4733
VUF 5695
VUF 4734
VUF 5696
VUF 5671
VUF 5697
VUF 5673
VUF 5669
VUF 5670
VUF 5672
mepyramine
tripelennamine
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
3.3 ( 0.4
4.4 ( 0.2
4.7 ( 0.2
5.3 ( 0.1
5.6 ( 0.1
6.0 ( 0.1
6.4 ( 0.1
6.6 ( 0.1
5.4 ( 0.1
5.4 ( 0.2
5.7 ( 0.1
8.7 ( 0.1
8.0 ( 0.1
-
5.5 ( 0.8
5.7 ( 0.2
6.3 ( 0.2
6.6 ( 0.1
6.0 ( 0.2
5.9 ( 0.2
5.7 ( 0.1
6.2 ( 0.1
6.4 ( 0.3
5.4 ( 0.1
7.9 ( 0.1
7.4 ( 0.1
-0.54 ( 0.13
-0.72 ( 0.03
-0.54 ( 0.06
-0.61 ( 0.04
-0.62 ( 0.09
-0.84 ( 0.06
-0.90 ( 0.11
-0.99 ( 0.05
-0.61 ( 0.08
-0.46 ( 0.33
-1.00
-(CH2)4-
-(CH2)4-
-(CH2)4-
1j
1k
10
12
-0.84 ( 0.02
a
b
The values are expressed as means ( SEM of separate experiments, each performed in triplicate. N > 3. pEC50 value. c Could not
be estimated. Tested up to 100 µM; no effects up to 10 µM. e Low affinity for the receptor; not tested for the efficacy.
d
Ta ble 2. Chemical Structures and Pharmacological Properties of the Aminoalkylpyridines 13a -g for the Human H1 Receptor
a
compd
name or code
isomer
n
pKia
pIC50
Ra
PEA
ortho
ortho
ortho
ortho
meta
meta
para
2
3.8 ( 0.1
4.5 ( 0.1
4.9 ( 0.1
6.0 ( 0.1
5.1 ( 0.1
5.4 ( 0.1
5.3 ( 0.1
5.4 ( 0.1
5.9 ( 0.1b
5.1 ( 0.2
-1.11 ( 0.21
-0.92 ( 0.08
13a
13b
13c
13d
13e
13f
13g
VUF 5680
VUF 5674
VUF 5677
VUF 5675
VUF 5678
VUF 5676
VUF 5679
9
10
12
10
12
10
12
c
d
-
-
c
d
-
-
5.6 ( 0.1
5.9 ( 0.1
5.6 ( 0.2
6.4 ( 0.2
-0.76 ( 0.01
-0.78 ( 0.13
-1.00 ( 0.06e
-0.67 ( 0.05
para
a
b
The values are expressed as means ( SEM of separate experiments, each performed in triplicate. N > 3. pEC50 value. c Could not
be estimated. Tested up to 10 µM; no effects up to 1 µM. e Estimated (see Figure 3B).
d
Ta ble 3. Chemical Structures and Pharmacological Properties of Compounds 17a -e and 19a -d for the Human H1 Receptor
a
compd
name or code
VUF 5963
n
X
R1
Cl
CF3
Cl
H
H
H
CF3
H
Cl
R2
pKia
pEC50
Ra
17a
17b
17c
17d
17e
19a
19b
19c
19d
0
0
0
4
5
0
0
1
1
NH
NH
NH
NH
NH
S
S
S
S
H
H
Cl
H
H
H
H
H
H
5.2 ( 0.1
5.4 ( 0.1
4.7 ( 0.1
<4
5.7 ( 0.1
6.0 ( 0.1
4.3 ( 0.1
1.28 ( 0.06
0.91 ( 0.08
0.50 ( 0.07
VUF 8811
FUB 113
FUB 114
b
b
-
-
b
b
<4
-
-
c
d
4.5 ( 0.1
4.9 ( 0.1
4.2 ( 0.2
4.5 ( 0.1
-
-
c
d
-
-
c
d
-
-
c
d
-
-
a
b
The values are expressed as means ( SEM of separate experiments, each performed in triplicate. N > 3. Low affinity for the receptor;
not tested for the efficacy. c Could not be estimated. Tested up to 100 µM; no effects up to 10 µM.
d
high receptor affinity on these antihistamines. We
investigated the influence of this substituent by testing
several substituted histaprodifens and substituted thi-
azoles.60,61
Compounds 18a -i have interesting structure-
activity relationships, as shown in Table 4. The
elongation of the spacer results in an increased H1R
binding affinity (compounds 18b-e) together with a
reduction of the intrinsic activity from full agonism (18c)
to neutral antagonism (18d and 18e; see also Figure
4).
Considering the binding data, the replacement of a
phenyl ring with a cyclohexyl ring (18f) leads to a
reduced affinity for the receptor, and removal of one
phenyl ring (17d ,e) led to a dramatic decrease in
binding affinity. However, substitution of one of the
phenyl rings in the para-position either with an electron-
withdrawing substituent (18g) or an electron-releasing
substituent (18h ) does not affect the affinity, suggesting
that the electron density on that phenyl ring as well as
substitution on the para position are not important for
the interaction with the receptor. This striking observa-

5816 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Govoni et al.
F igu r e 2. The effects of the full inverse H1 agonist mepyr-
amine (b) and the partial inverse H1 agonist VUF 4734 (O)
on the basal signaling of the H1R, as measured by the
bioluminescent reporter gene assay (see also Table 1). Data
are normalized to the basal signaling observed in the assay
(set to 100%).
tion is in contrast with the well-known structure-
activity relationships for the classical antihistamines12,62
and with data for agonists.63 Those studies indicated
in fact that para-substitution of the “cis” ring of classical
H1R antagonists with a small lipophilic group (i.e. CH3,
Cl) is favorable and confers the compounds an increased
affinity on the receptor. The aromatic character of this
ring seems to be indispensable for H1R activity. In
contrast, the second aromatic “trans” ring can be
replaced by nonaromatic lipophilic groups (e.g. cyclo-
hexyl) without drastic effects on H1R-blocking activity.62
Unlike the known effects for classical H1R antagonists,
the replacement of the primary amino function with a
pyrrolidine ring in compound 18i does not influence its
binding affinity, similarly to what was observed with
the series of aminoalkylimidazoles (compounds 1j and
1k ).
Previously, we have shown the human histamine H1-
receptor to be constitutively active both by measuring
the inositolphosphate levels in transiently transfected
COS-7 cells and by measuring the luciferase activity in
a reporter-gene assay we set up in which a firefly
luciferase reporter-gene is under the transcriptional
control of five NF-κB enhancer elements. However, in
general, the physiological relevance of inverse agonists
needs to be verified in a more physiological environment
before the biological effect of antagonists can be pri-
marily ascribed to negative intrinsic activity.14,15 There-
fore, we investigated the effects of (partial) inverse
agonists and the identified neutral antagonist for the
human histamine H1-receptors endogenously expressed
by HeLa cells.
F igu r e 3. The effects of several selected compounds on the
basal signaling of the H1R, as measured by the bioluminescent
reporter gene assay. (A) Dose-response curves of the partial
inverse H1 agonists VUF 5695 (b), VUF 5696 (9), and VUF
5673 (0) (see also Table 1). (B) Dose-response curves of the
inverse H1 agonists VUF 5675 (O), VUF 5676 (b), and VUF
5679 (9) (see also Table 2). (C) Dose-response curves of the
H1R agonists histamine (+), histaprodifen (b), 2-(3-trifluoro-
methylphenyl)histamine (0), and VUF 5963 (9) (see also Table
3 and Table 4). Representative dose-response curves of the
full inverse H1 agonist mepyramine (/)27,28 are shown (A, B,
and C). Data are normalized to the basal signaling observed
in the assay (set to 100%).
Long-term exposure of cells expressing constitutively
active GPCRs to inverse agonists may result in receptor
up-regulation.19,41-44 Also, sensitization of receptors by
inverse agonist treatment has been shown in cell lines
stably expressing receptors at relatively low density64
as well as in cell lines in which receptor expression is
inducible.65 Although inverse agonist induced receptor
up-regulation may have clinical significance,45,46 no data
on the physiological relevance of constitutive H1R activ-
ity are currently available, in part due to the absence
of available neutral H1R antagonists. So far a receptor
up-regulation or sensitization has not been reported for
the H1R upon prolonged inverse agonist treatment. We
assessed the long-term effects of prolonged inverse
agonist treatment of HeLa cells endogenously express-
ing the H1R at low expression levels (Bmax ) 55 fmol/
mg of protein; Figure 5A) on the H1R expression levels
and H1R signaling properties. The procedure of measur-
ing the inverse-agonist-induced up-regulation of H1Rs
is greatly dependent on the dissociation rates of the
inverse agonists, as the preparation needs to be free of
the inverse agonist used to pretreat the cells, to deter-
mine either the expression level of the H1R or its
signaling properties. Whereas tripelennamine is one of
the few antihistamines that dissociates rather quickly
from the receptor and allows for the measurement of
H1R up-regulation and the increase in Emax of hista-
mine, many other antihistamines, such as mepyramine
and cetirizine, dissociate more slowly from the H1R.66
As seen in Figure 5, a 24 h treatment of HeLa cells with
the inverse H1R agonist tripelennamine28 results in a
2-fold increase of the total H1R-expression level (Figure

Neutral Histamine H1-Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5817
Ta ble 4. Chemical Structures and Pharmacological Properties of Compounds 18a -i and 20a ,b for the Human H1 Receptor
a
compd
name or code
histamine
n
R1
R2
R3
phenyl
phenyl
phenyl
phenyl
phenyl
cyclohexyl
p-Br-phenyl
p-CH3-phenyl
phenyl
pKia
pIC50
Ra
H
H
H
H
H
H
H
H
H
H
4.2 ( 0.1
<4
6.8 ( 0.1b
1.00
c
c
18a
18b
18c
18d
18e
18f
18g
18h
18i
0
1
2
3
4
2
2
2
2
H
H
H
H
H
H
H
H
-
-
-
d
e
5.1 ( 0.1
5.7 ( 0.1
5.8 ( 0.1
5.9 ( 0.1
5.0 ( 0.1
5.6 ( 0.1
5.5 ( 0.1
5.6 ( 0.1
-
histaprodifen
histabudifen
histapendifen
6.4 ( 0.1b
0.69 ( 0.06
0.02 ( 0.03
-0.09 ( 0.18
d
-
d
-
d
f
-
-
d
f
-
-
d
f
-
-
-
d
e
-(CH 2)4-
-
a
b
The values are expressed as means ( SEM of separate experiments, each performed in triplicate. N > 3. pEC50 value. c Low affinity
for the receptor; not tested for the efficacy. Could not be estimated. e Tested up to 100 µM; no effects up to 10 µM. f Tested up to 10 µM;
no effects up to 1 µM.
d
Pretreatment of HeLa cells with 100 µM of the partial
inverse H1R agonist VUF 4734 (1d ), which corresponds
to 20 times its Ki value, did not result in a significant
increase of the Emax of histamine. The Emax of histamine
after pretreatment with VUF 4734 was 146 ( 26% of
that of control cells. Pretreatment of HeLa cells with
16 µM of the identified neutral H1-antagonist hista-
budifen (18d ), corresponding to 10 times its Ki value,
also did not result in a significant increase in the Emax
of histamine compared to control cells. VUF 4734 and
histabudifen could not be tested at higher concentra-
tions due to non-H1R-mediated effects in our cell-based
assay.
Con clu sion s
In the present study we reported the synthesis of a
variety of histamine homologues and testing of their
binding affinities and intrinsic H1R activities. A NF-κB
F igu r e 4. Histabudifen as a neutral H1R antagonist. The
effects of the endogenous agonist histamine (0), the full inverse
driven reporter-gene assay allowing testing of H1R
H1 agonist mepyramine (O), and histabudifen (]) on the
ligands for a dynamic range of intrinsic activities has
modulation of basal H1R signaling, as measured by the
been used.28 Moreover, we show an up-regulation of
bioluminescent reporter gene assay (see also Table 4). Data
endogenously expressed H1R and an increased effective-
ness of histamine to induce the formation of inositol
phosphates upon inverse H1R agonist treatment, un-
derscoring the potential benefit of neutral antagonists.
Novel ligands for the histamine H1-receptor were
synthesized and tested for their affinity and intrinsic
activity for the human histamine H1-receptor, and
several known H1-receptor ligands were screened in
the same system. Most of the compounds presented in
this research are (partial) inverse agonists. Especially
aminoalkylimidazoles 1a -k and aminoalkylpyridines
13a ,d -g are structurally diverse from classical hista-
mine H1-antagonists, as they possess a structure similar
to histamine and H1-agonists; therefore, they constitute
a novel class of inverse agonist ligands for this receptor.
The substitution of the primary amino function in the
aminoalkylimidazole series with a pyrrolidine ring led
to the expected increase of affinity for the receptor only
in the case of compound 1i, possibly due to a different
binding site for the other ligands compared to classical
H1-antagonists.62,67 Previously we have indeed proposed
that different H1R agonists binding pockets could ex-
ist.57 The position of the nitrogen in the aminoalkyl-
pyridines series does not seem to be important, since
are normalized to the basal signaling observed in the assay
(set to 100%). (Inset) Effects of histabudifen (10 µM, HB),
histamine (1 µM, HA), and mepyramine (0.1 µM, M) on H1R-
mediated signaling, and the inhibition of histamine- and
mepyramine-induced H1R-mediated responses by histabudifen.
5A) that is concomitant with a dose-dependent increase
in the Emax, but not in the EC50 value of histamine-
induced inositolphosphates formation (Figure 5B,C).
Although similar effects were observed when mepyr-
amine was used to preincubate the cells, the EC50 of
histamine after such pretreatment of the cells was
rightward shifted (data not shown), most likely due to
the kinetics of mepyramine binding. The maximum
enhancement of H1R signaling observed upon inverse
H1-agonist treatment is a 2-fold increase. These data
are clearly consistent with the notion of constitutive H1R
activity and suggests that the inverse H1R agonist
tripelennamine may act to stabilize the H1R in the cell
membrane. In contrast to the effects observed with
pretreatment of the cells with the inverse agonist
tripelennamine, pretreatment of the cells with 0.63 mM
histamine (10 times its Ki value28) completely prevented
histamine-stimulated inositolphosphates formation (Fig-
ure 5C).

5818 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Govoni et al.
F igu r e 5. Effects of a 24 h pretreatment with the inverse H1 agonist tripelennamine on H1R expression and H1R-mediated
signaling of HeLa cells endogenously expressing H1Rs. (A) Upregulation of endogenously expressed H1Rs after a 24 h pretreatment
of the cells with 10 µM tripelennamine. (B) Increase in the Emax of histamine-induced inositolphosphates accumulation upon
pretreatment of the cells with either 1 or 10 µM tripelennamine for 24 h. (C) Effect of tripelennamine (1 µM, 24 h, b) or histamine
(0.63 mM, 24 h, 0) pretreatment of HeLa cells on the Emax and pEC50 of histamine-induced inositolphosphates accumulation in
comparison to control cells (O).
tetramethylsilane as internal standard. Elemental analyses
were performed by the Department of Microanalysis, Gronin-
gen University, Groningen, The Netherlands. Chromatography
was performed on J . T.Baker silica gel for flash chromatog-
raphy. THF was freshly distilled from LiAlH4, and DMF was
dried on molecular sieves. All reactions were performed under
an atmosphere of dry nitrogen.
all the compounds bind to the receptor. The elongation
of the side chain from the classical aminoethyl chain
turns the compounds into (partial) inverse agonists,
analogous to the aminoalkylimidazoles series, with the
exception of compound 13b, for which we currently have
no explanation available.
It has proven difficult to identify neutral H1-receptor
antagonists by modification of the agonist histamine, a
strategy previously successfully used to identify neutral
H2- and H3-receptor antagonists.19,20 Nonetheless, two
histaprodifen-like compounds (18d and 18e), which
exhibit hybrid structures composed of the endogenous
agonist histamine combined with the classical H1R
antagonist pharmacophore, have been ultimately identi-
fied as neutral H1-receptor antagonists. Consequently,
the modification of agonist structures has again proven
a successful strategy to obtain neutral histamine recep-
tor antagonists. The benzhydryl group in compounds
18d and 18e seems to be important for their neutral
H1R antagonism. Although at this stage it is not possible
to draw definite conclusions on the structural require-
ments necessary for neutral antagonism on the hista-
mine H1-receptor, the structure-activity relationships
for neutral H1R antagonists seem to differ from the
structure-activity relationships of inverse H1R agonists.
In contrast to the effects of pretreatment with inverse
H1-agonist, pretreatment with weak partial inverse
agonists or neutral antagonists for the H1-receptor had
no effect on the histamine-induced H1-receptor-mediated
responses. These compounds will therefore be useful
pharmacological tools and may serve as a starting point
for the development of high-affinity neutral H1R an-
tagonists, the availability of which will allow the as-
sessment of the importance of constitutive H1R activity
in vivo as well as the requirement of inverse H1R
agonistic properties for the therapeutic value of anti-
histamines.
The 1-chloro-ω-iodoalkanes were prepared by refluxing the
corresponding 1,ω-dichloroalkanes with 0.33 equiv of sodium
68
iodide in acetone and purification by distillation.
The 2-(ω-bromo-alkyloxy)tetrahydropyrans were prepared
69,70
from the corresponding ω-bromoalkan-1-ols.
5-(9-H yd r oxyn on yl)im id a zole-1-su lfon ic Acid Di-
m eth yla m id e (4c). Imidazole-1-sulfonic acid dimethylamide
253 (6.86 g, 0.039 mol) was dissolved in THF (150 mL) and
cooled to -70 °C. n-Butyllithium in hexane (29.25 mL, 0.047
mol) was added dropwise at -65 °C. After 15 min, a solution
of tert-butyldimethylsilyl chloride (7.07 g, 0.047 mol) in THF
was added at -65 °C and the solution was allowed to warm
to room temperature and stirred for an additional 30 min. The
mixture was cooled to -70 °C again, and n-butyllithium in
hexane (24.5 mL, 0.039 mol) was added dropwise. After 30 min,
a solution of 2-(9-bromononyloxy)tetrahydropyran (10.00 g,
0.033 mol) in THF was added gradually and the mixture was
allowed to (slowly) warm to room temperature (rt) overnight.
The reaction mixture was poured into water and the THF was
removed under reduced pressure. The product was extracted
with dichloromethane, washed with water, dried (MgSO4), and
concentrated in vacuo. HCl (37%) was added to the crude
product till pH ) 1, and the mixture stirred for 1 h and was
then diluted with water, basified with K2CO3, and extracted
with dichloromethane; the organic layers were combined, dried
(MgSO4), and concentrated in vacuo. The product was purified
by flash chromatography with ethyl acetate as eluent to afford
1.09 g (10%) of white crystals. 1H NMR (CDCl3): δ 1.20 (m,
10H, central CH2’s), 1.50 (m, 4H, 2 × CH2), 2.60 (t, 2H,
imidazole-5-CH2), 2.78 (s, 6H, N(CH3)2), 3.53 (t, 2H, CH2OH),
6.70 (s, 1H, imidazole-4H), 7.78 (s, 1H, imidazole-2H).
5-(11-H yd r oxyu n d ecyl)im id a zole-1-su lfon ic Acid Di-
m eth yla m id e (4e). 2-(tert-Butyl-dimethylsilanyl)imidazole-
1-sulfonic acid dimethylamide 347 (9.23 g, 0.032 mol) was
dissolved in THF (120 mL) and cooled to -70 °C. n-Butyl-
lithium in hexane (20 mL, 0.032 mol) was added dropwise at
-65 °C. After 30 min, a solution of (11-bromoundecyloxy)-
tetrahydropyran (10.63 g, 0.032 mol) in THF was added
dropwise and the mixture was allowed to (slowly) warm to
room temperature overnight. The solution was acidified with
HCl (2 M) and stirred for 1 h, then the solvents were removed
in vacuo. The residue was diluted with water, basified with
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
1
measured on an Electrothermal IA 9200 apparatus. H NMR
spectra were recorded on a Bruker AC-200 (200 MHz) spec-
trometer. Chemical shifts are given in ppm downfield from

Neutral Histamine H1-Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5819
K2CO3, extracted with dichloromethane, dried (Na2SO4), and
concentrated in vacuo. The crude product was purified by flash
chromatography switching gradually the eluent from dichloro-
methane/ethyl acetate (8/2) into ethyl acetate to afford 1.60 g
of product (mixed with 25% of 2) which was used without
further purification.
stirred at room temperature with pyrrolidine (4.37 g, 0.062
mol). After 48 h the unreacted pyrrolidine was removed under
reduced pressure and the residue was diluted with water,
basified with K2CO3, extracted with dichloromethane, dried
(Na2SO4), and concentrated in vacuo to afford 1.30 g (78%) of
1
brown oil. H NMR (CDCl3): δ 1.25 (m, 10H, central CH2’s),
1.75 (m, 6H, 3 × CH2), 2.35 (m, 6H, 2 × CH2 + CH2-
pyrrolidine), 2.65 (t, 2H, imidazole-5-CH2), 2.83 (s, 6H, N(CH3)2),
6.75 (s, 1H, imidazole-4H), 7.78 (s, 1H, imidazole-2H).
5-(10-P yr r olid in -1-yld ecyl)im id a zole-1-su lfon ic Acid
Dim eth yla m id e (7j). The same procedure as for 7i with 4.43
g ((0.0029 mol) of 4j was used. The product was purified by
flash chromatography switching gradually the eluent from
ethyl acetate into methanol/triethylamine (9/1); 760 mg (67%)
of a brown oil was obtained. 1H NMR (CDCl3): δ 1.25 (m, 14H,
central CH2’s), 1.75 (m, 6H, 3 × CH2), 2.50 (m, 6H, 2 × CH2 +
CH2-pyrrolidine), 2.65 (t, 2H, imidazole-5-CH2), 2.83 (s, 6H,
N(CH3)2), 6.85 (s, 1H, imidazole-4H), 7.90 (s, 1H, imidazole-
2H).
5-(12-Ch lor od od ecyl)im id a zole-1-su lfon ic Acid Di-
m eth yla m id e (4f). Using the same procedure as for 4c with
4.46 g (0.026 mol) of 2 and 5.61 g (0.017 mol) of 1-chloro-12-
iodododecane gave 2.69 g of product (mixed with 45% of 5-(tert-
butyldimethylsilanyl)imidazole-1-sulfonic acid dimethylamide)
which was used without further purification.
5-(8-Ch lor ooctyl)im id a zole-1-su lfon ic Acid Dim eth yl-
a m id e (4i). Using the same approach as for 4e with 9.23 g
(0.032 mol) of 3 and 8.70 g (0.032 mol) of 1-chloro-8-iodooctane
gave 9.59 g (93%) of brown oil, which did not need any further
purification by flash chromatography. 1H NMR (CDCl3): δ 1.25
(m, 8H, central CH2’s), 1.65 (m, 4H, 2 × CH2), 2.63 (t, 2H,
imidazole-5-CH2), 2.78 (s, 6H, N(CH3)2), 3.43 (t, 2H, CH2Cl),
6.73 (s, 1H, imidazole-4H), 7.73 (s, 1H, imidazole-2H).
5-(10-Ch lor od ecyl)im id a zole-1-su lfon ic Acid Dim eth yl-
a m id e (4j). Using the same procedure as for 4c with 7.96 g
(0.046 mol) of 2 and 13.05 g (0.043 mol) of 1-chloro-10-
iododecane gave 5.32 g of product (mixed with 60% of 5-(tert-
butyldimethylsilanyl)imidazole-1-sulfonic acid dimethylamide)
which was used for the next step without further purification.
5-[9-(1,3-Dioxo-1,3-d ih yd r oisoin d ol-2-yl)n on yl]im id -
a zole-1-su lfon ic Acid Dim eth yla m id e (5c). A mixture of
4c (1.09 g, 0.0035 mol), triphenylphosphine (0.91 g, 0.0035
mol), and isoindole-1,3-dione (0.51 g, 0.0035 mol) in dry THF
(5 mL) was stirred vigorously and placed in an ice/water bath.
Diisopropyl azodicarboxylate (0.698 g, 0.00345 mol) in dry THF
(5 mL) was added dropwise and stirred at room temperature
overnight. The solvent was removed in vacuo and the residue
was purified by flash chromatography with ethyl acetate as
5-(12-P yr r olid in -1-yld od ecyl)im id a zole-1-su lfon ic Acid
Dim eth yla m id e (7k ). Using the same procedure as for 7j
starting with 4f (1.00 g, (0.0016 mol) gave 500 mg (74%) of a
1
brown oil. H NMR (CDCl3): δ 1.25 (m, 16H, central CH2’s),
1.75 (m, 8H, 4 × CH2), 2.45 (m, 6H, 2 × CH2 + CH2-
pyrrolidine), 2.70 (t, 2H, imidazole-5-CH2), 2.83 (s, 6H, N(CH3)2),
6.80 (s, 1H, imidazole-4H), 7.80 (s, 1H, imidazole-2H).
14-[4-(Tolu en e-4-su lfon yl)-4,5-d ih yd r ooxa zol-5-yl]tet-
r a d eca n oic Acid Meth yl Ester (11). To a stirred suspension
of tosyl methyl isocyanide (TosMIC) (4.98 g, 0.026 mol) and
15-oxo-pentadecanoic acid methyl ester 10 71 (6.99 g, 0.026 mol)
in absolute ethanol (80 mL) was added finely powdered sodium
cyanide (0.12 g, 0.0026 mol). The yellow-orange suspension was
stirred for 30 min, filtered, and washed with ethanol/ether.
The light yellow solid was immediately used for the next step.
1H NMR (CDCl3): δ 1.30 (m, 18H, central CH2’s), 1.60 (m, 6H,
3 × CH2), 2.25 (t, 2H, CH2COOCH3), 2.46 (s, 3H, p-CH3), 3.65
(s, 3H, CH3OCO), 4.76 (d, 1H, oxazoline-4H), 5.05 (q, 1H,
oxazoline-5H), 6.95 (s, 1H, oxazoline-2H), 7.35 (d, 2H, 2,6-
phenyl-H), 7.80 (d, 2H, 3,5-phenyl-H).
1
eluent to afford 0.96 g (63%) of beige solid. H NMR (CDCl3):
δ 1.28 (m, 10H, central CH2’s), 1.65 (m, 4H, 2 × CH2), 2.67 (t,
2H, imidazole-5-CH2), 2.83 (s, 6H, N(CH3)2), 3.62 (t, 2H, CH2-
isoindole-1,3-dione), 6.78 (s, 1H, imidazole-4H), 7.50-7.78 (m,
5H, isoindole-1,3-dione-H + imidazole-2H).
5-[11-(1,3-Dioxo-1,3-dih ydr oisoin dol-2-yl)u n decyl]im id-
a zole-1-su lfon ic Acid Dim et h yla m id e (5e). The same
procedure as for 5c starting from 1.60 g (( 0.0046 mol) of 4e
was used. The residue was purified by flash chromatography
using ethyl acetate/dichloromethane as eluent to afford 2.11
g of product that was used for the next step without further
purification.
14-(1H-Im id a zol-4-yl)tetr a d eca n oic Acid Am id e (12). In
a resealable pressure tube, a solution of 11 in a saturated
solution of ammonia in dry methanol was heated between 120
and 140 °C. After 48 h the reaction mixture was cooled to rt
and the solvent was removed in vacuo. The residue was
triturated with dichloromethane (150 mL) and filtered. A total
of 4.41 g (58%) of a beige solid was collected. 1H NMR
(DMSO): δ 1.25 (m, 18H, central CH2’s), 1.50 (m, 4H, 3 × CH2),
2.05 (t, 2H, CH2CONH2), 2.50 (t, 2H, imidazole-4(5)-CH2), 6.70
(s, 1H, imidazole-4(5)H), 7.65 (s, 1H, imidazole-2H).
9-(1H-Im id a zol-4-yl)n on yla m in e Dioxa la te (1c). Com-
pound 5c (0.96 g, 0.0022 mol) was dissolved in 30% HBr (12
mL) and heated under reflux. After 16 h the mixture was
cooled and concentrated in vacuo, and the residue was diluted
with water, washed with diethyl ether, basified with K2CO3,
and extracted with CHCl3/EtOH (9/1). The organic layers were
combined, dried (MgSO4), and concentrated in vacuo to afford
0.30 g (65%) of brown solid.
The free base was converted into the dioxalate salt, using
the following procedure: the free base was dissolved in ethyl
acetate with a few drops of methanol and then added dropwise
with a solution of 2 equiv of oxalic acid dihydrate in ethyl
acetate/methanol. The precipitate that formed was collected
by filtration to afford 0.42 g (77%) of a light yellow powder.
Mp: 126-128 °C. 1H NMR (D2O): δ 1.27 (m, 10H, central
CH2’s), 1.62 (m, 4H, 2 × CH2), 2.67 (t, 2H, imidazole-4(5)-CH2),
2.94 (t, 2H, CH2NH2), 7.14 (s, 1H, imidazole-4(5)H), 8.50 (s,
1H, imidazole-2H). Anal. (C16H27N3O8) C, H, N.
11-(1H-Im idazol-4-yl)u n decylam in e (1e). Using the same
procedure as for 1c starting with 2.11 g ((0.0040 mol) of 5e
gave 0.78 g (82%) of light yellow powder. Mp: 87.5-88.5 °C.
1H NMR (CDCl3): δ 1.27 (m, 16H, central CH2’s + NH2), 1.50
(m, 4H, 2 × CH2), 2.60 (m, 4H, imidazole-4(5)-CH2 + CH2NH2),
6.70 (s, 1H, imidazole-4(5)H), 7.50 (s, 1H, imidazole-2H). Anal.
(C14H27N3) C, H, N.
5-[12-(1,3-Dioxo-1,3-d ih ydr oisoin dol-2-yl)d od ecyl]im id-
a zole-1-su lfon ic Acid Dim eth yla m id e (5f). Compound 4f
(1.54 g, (0.0025 mol) was mixed with isoindole-1,3-dione (1.22
g, 0.0083 mol), Na2CO3 (0.87 g, 0.0083 mol), and NaI (0.075 g,
0.50 mmol) in dry DMF (40 mL) and heated at 90 °C. After 20
h the solvent was evaporated under reduced pressure, and the
residue was diluted with water and extracted with CHCl3. The
combined organic extract were washed with water, dried
(Na2SO4), and concentrated in vacuo to give 5f as a brown solid
(2.07 g) which was used without further purification.
5-(12-Am in od od ecyl)im id a zole-1-su lfon ic Acid Di-
m eth yla m id e (6f). Compound 5f (2.07 g) was dissolved in
warm ethanol (60 mL), hydrazine monohydrate (0.50 g, 0.01
mol) was added, and the solution was refluxed for 3 h, cooled
to room temperature, and filtered. The filtrate was concen-
trated in vacuo and the residue was diluted with NaOH (1 M)
and then extracted with CHCl3. The organic extracts were
washed with NaOH (0.5 M), dried (Na2SO4), and concentrated
in vacuo. The crude product was purified by flash chromatog-
raphy switching gradually the eluent from ethyl acetate into
methanol/triethylamine (9/1) (Rf ) 0.5); 340 mg (38%) of a
1
yellow solid was isolated. H NMR (CDCl3): δ 1.25 (m, 16H,
central CH2’s + NH2), 1.65 (m, 6H, 3 × CH2), 2.65 (m, 4H,
imidazole-5-CH2 + CH2NH2), 2.83 (s, 6H, N(CH3)2), 6.75 (s,
1H, imidazole-4H), 7.78 (s, 1H, imidazole-2H).
5-(8-P yr r olid in -1-yloctyl)im id a zole-1-su lfon ic Acid Di-
m eth yla m id e (7i). Compound 4i (1.5 g, 0.0047 mol) was

5820 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Govoni et al.
12-(1H-Im id a zol-4-yl)d od ecyla m in e (1f). The same pro-
cedure as for 1c with 0.34 g (0.95 mmol) of 6f was used. After
basification the water was removed in vacuo and the remain-
ing solid was washed with 10 mL portions of 2-propanol. The
filtrate was concentrated under reduced pressure and washed
with fresh 2-propanol. The filtrates were concentrated in vacuo
solution of n-butyllithium (3.65 mL, 0.0058 mol) in 5 mL of
THF at -50 °C over 10 min, then the cooling bath was removed
and the temperature was allowed to rise to -20 °C. The cold
dark red solution was added dropwise to a cold solution (-50
°C) of dibromooctane (2.44 g, 0.0090 mol) in THF (5 mL) and
the mixture was allowed to warm to room temperature and
stirred for an additional 0.5 h. HCl (37%) was added till pH )
1 and stirred for 1.5 h, then the solvent was removed in vacuo.
The residue was washed with diethyl ether, basified with
K2CO3, extracted with dichloromethane, dried (MgSO4), and
evaporated in vacuo. The residue (0.82 g) was used directly
for the next step.
1
to give 0.16 g (67%) of a beige powder. Mp: 150-152 °C. H
NMR (CDCl3): δ 1.20 (m, 16H, central CH2’s + NH2), 1.55 (m,
6H, 3 × CH2), 2.60 (m, 4H, imidazole-4(5)-CH2 + CH2NH2),
6.70 (s, 1H, imidazole-4(5)H), 7.50 (s, 1H, imidazole-2H). Anal.
(C15H29N3) C, H, N.
13-(1H -Im id a zol-4-yl)t r id ecyla m in e Dioxa la t e (1g).
PIFA51 (1.72 g, 0.0040 mol) was dissolved in 6 mL of aceto-
nitrile, and 6 mL of distilled water was added. To this solution
was added 12 (1.17 g, 0.0040 mol), and the suspension was
stirred at room temperature. After 20 h the reaction mixture
was diluted with water (75 mL) and HCl (37%, 8 mL), stirred
for another 30 min, and washed with diethyl ether. The
combined ether layers were extracted with HCl (10%, 30 mL).
The aqueous layers were combined, basified with K2CO3,
extracted with chloroform/ethanol (10%), dried (MgSO4), and
concentrated in vacuo. The residue was purified by flash
chromatography with methanol and then methanol/triethyl-
amine (9/1) to afford 240 mg (23%) of amine. The free base
was converted into a dioxalate; 335 mg (83%) of a white powder
was obtained. Mp: 118-120 °C. 1H NMR (D2O): δ 1.27 (m,
18H, central CH2’s), 1.62 (m, 4H, 2 × CH2), 2.67 (t, 2H,
imidazole-4(5)-CH2), 2.94 (t, 2H, CH2NH2), 7.14 (s, 1H, imid-
azole-4(5)H), 8.50 (s, 1H, imidazole-2H). Anal. (C20H35N3O8)
C, H, N.
14-(1H-Im id a zol-4-yl)tetr a d ecyla m in e Dioxa la te (1h ).
A suspension of 12 (2.00 g, 0.068 mol) in THF (100 mL) was
added, with efficient stirring, to a suspension of LiAlH4 (1.30
g, 0.034 mol) in THF (30 mL). The reaction mixture was stirred
for 1 h at rt, and then heated under reflux. After 20 h the
suspension was cooled to rt and water (1.5 mL) followed by
NaOH 10% aqueous solution (1.5 mL) was added dropwise,
and the mixture was refluxed. After 30 min the reaction
mixture was cooled to room temperature, filtered, and con-
centrated in vacuo to afford 1.66 g (88%) of a yellow solid that
was converted into the dioxalate salt. The yellow precipitate
collected by filtration was washed (under stirring) with
methanol (10 mL) and filtered again to afford 1.12 g (42%) of
a light-yellow powder. Mp: 122-126 °C. 1H NMR (D2O): δ
1.27 (m, 20H, central CH2’s), 1.62 (m, 4H, 2 × CH2), 2.67 (t,
2H, imidazole-4(5)-CH2), 2.94 (t, 2H, CH2NH2), 7.14 (s, 1H,
imidazole-4(5)H), 8.50 (s, 1H, imidazole-2H). Anal. (C21H37N3O8)
C, H, N.
10-P yr id in -2-yld eca n -1-ol (15b). Compound 14a (5.63 g,
0.061 mol) in THF (30 mL) was added dropwise to a solution
of n-butyllithium (37.8 mL, 0.061 mol) in 50 mL of THF at
-50 °C over 10 min, then the cooling bath was removed and
the temperature allowed to rise to -20 °C. The red solution
was cooled again to -50 °C and 16.77 g (0.055 mol) of 2-(9-
bromononyloxy)tetrahydropyran in THF (30 mL) was added
dropwise, then the mixture was allowed to (slowly) warm to
room temperature overnight. The reaction mixture was poured
into water and the THF was removed under reduced pressure.
The residue was acidified with HCl (37%) and stirred for 30
min, washed with diethyl ether, basified with K2CO3, extracted
with dichloromethane, dried (MgSO4), and evaporated in
vacuo. The product was purified by flash chromatography
switching gradually the eluent from dichloromethane/ethyl
acetate (9/1) into ethyl acetate; 4.49 g (35%) of a white waxy
compound was isolated. 1H NMR (CDCl3): δ 1.15 (m, 12H,
central CH2’s), 1.50 (m, 4H, 2 × CH2), 2.60 (t, 2H, pyridine-
2-CH2), 2.98 (br s*, 1H, OH), 3.45 (t, 2H, CH2OH), 6.90 (m,
2H, pyridine-3H + pyridine-5H), 7.38 (m, 1H, pyridine-4H),
8.30 (d, 1H, pyridine-6H).
12-P yr id in -2-yld od eca n -1-ol (15c). The same procedure
as for 15b with 12.35 g (0.037 mol) of 2-(11-bromoundecyloxy)-
tetrahydropyran was used. After the basification the product
precipitated and was filtered and washed with water to afford
1
5.67 g (58%) of a beige powder. H NMR (CDCl3): δ 1.23 (m,
14H, central CH2’s), 1.60 (m, 6H, 3 × CH2), 2.75 (t, 2H,
pyridine-2-CH2), 3.60 (t, 2H, CH2OH), 7.05 (m, 2H, pyridine-
3H + pyridine-5H), 7.55 (m, 1H, pyridine-4H), 8.45 (d, 1H,
pyridine-6H).
10-P yr id in -3-yld eca n -1-ol (15d ). A solution of 2.89 g
(0.031 mol) of 3-methylpyridine 14d in THF (20 mL) was added
dropwise at -20 °C to a freshly prepared solution of lithium
diisopropylamide (19.38 mL, 0.031 mol of n-butyllithium and
3.74 g, 0.037 mol of diisopropylamine) in 70 mL of THF. The
cooling bath was removed and the temperature allowed to rise
to 10 °C. After stirring for 30 min at this temperature the
reaction mixture was cooled to -40 °C and 8.00 g (0.026 mol)
of 2-(9-bromononyloxy)tetrahydropyran in THF (30 mL) was
added dropwise. The cooling bath was removed and the
mixture was allowed to (slowly) warm to room temperature
overnight. The reaction mixture was poured into water and
the THF was removed under reduced pressure. The crude
product was acidified with HCl (37%), stirred for 30 min,
washed with diethyl ether (3 × 50 mL), basified with K2CO3,
extracted with dichloromethane, dried (MgSO4), and evapo-
rated in vacuo, to afford 5.50 g (90%) of a brown waxy
4-(8-P yr r olid in -1-yloctyl)-1H-im id a zole Dioxa la te (1i).
Using the same procedure as for 1c with 7i (1.25 g, 0.0035
1
mol) gave 800 mg (92%) of product. H NMR (CDCl3): δ 1.25
(m, 8H, central CH2’s), 1.50 (m, 4H, 2 × CH2), 1.75 (m, 4H, 2
× CH2), 2.45 (m, 8H, imidazole-4(5)-CH2 + CH2-pyrrolidine +
2 × CH2), 6.70 (s, 1H, imidazole-4(5)H), 7.45 (s, 1H, imidazole-
2H). The free base was converted into the dioxalate salt and
crystallized from methanol/diethyl ether to afford 150 mg
(11%) of light yellow solid. Anal. (C19H31N3O8) C, H, N.
4-(10-P yr r olid in -1-yld ecyl)-1H-im id a zole (1j). Using the
same procedure as for 1c with 7j (0.76 g, 0.0020 mol), followed
by the treatment of the free base with activated charcoal, gave
1
compound. H NMR (CDCl3): δ 1.25 (m, 12H, central CH2’s),
1
1.52 (m, 4H, 2 × CH2), 2.58 (t, 2H, pyridine-2-CH2), 3.60 (t,
2H, CH2OH), 7.15 (m, 1H, pyridine-5H), 7.45 (d, 1H, pyridine-
4H), 8.38 (m, 2H, pyridine-2H + pyridine-6H).
480 mg (87%) of a brown waxy compound. H NMR (CDCl3):
δ 1.20 (m, 12H, central CH2’s), 1.50 (m, 4H, 2 × CH2), 1.70
(m, 4H, 2 × CH2), 2.45 (m, 8H, imidazole-4(5)-CH2 + CH2-
pyrrolidine + 2 × CH2), 6.70 (s, 1H, imidazole-4(5)H), 7.45 (s,
1H, imidazole-2H). Anal. (C17H31N3) C, H, N.
4-(12-P yr r olid in -1-yld od ecyl)-1H-im id a zole (1k ). Using
the same procedure as for 1j gave 210 mg (57%) of a waxy
yellow solid. 1H NMR (CDCl3): δ 1.20 (m, 16H, central CH2’s),
1.50 (m, 4H, 2 × CH2), 1.70 (m, 4H, 2 × CH2), 2.45 (m, 8H,
imidazole-4(5)-CH2 + CH2-pyrrolidine + 2 × CH2), 6.70 (s, 1H,
imidazole-4(5)H), 7.45 (s, 1H, imidazole-2H). Anal. (C19H35N3)
C, H, N.
12-P yr id in -3-yld od eca n -1-ol (15e). Using the same pro-
cedure as for 15d with 1.78 g (0.019 mol) of 14d and 5.36 g
(0.016 mol) of 2-(11-bromoundecyloxy)tetrahydropyran gave
1
2.56 g (61%) of a brown waxy compound. H NMR (CDCl3): δ
1.25 (m, 16H, central CH2’s), 1.55 (m, 6H, 3 × CH2), 2.55 (t,
2H, pyridine-2-CH2), 3.60 (t, 2H, CH2OH), 7.15 (m, 1H,
pyridine-5H), 7.45 (d, 1H, pyridine-4H), 8.38 (m, 2H, pyridine-
2H + pyridine-6H).
10-P yr id in -4-yld eca n -1-ol (15f). The same procedure as
for the preparation of 15d was employed, using 3.02 g (0.032
mol) of 4-methylpyridine 14f, with the exception that 14f
2-(9-Br om on on yl)p yr id in e (15a ). 2-Methylpyridine 14a
(0.54 g, 0.0058 mol) in THF (5 mL) was added dropwise to a

Neutral Histamine H1-Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5821
compound. 1H NMR (D2O): δ 1.25 (m, 10H, central CH2’s), 1.70
solution was added at -40 °C and the reaction mixture was
stirred for 30 min at 20 °C. Upon addition of HCl (37%) a white
precipitate formed, which was filtered. The solid was dissolved
in water, basified with K2CO3, and filtered to afford 4.89 g
(77%) of a yellow solid. 1H NMR (CDCl3): δ 1.25 (m, 12H,
central CH2’s), 1.55 (m, 4H, 2 × CH2), 1.85 (br s, 1H, OH),
2.55 (t, 2H, pyridine-2-CH2), 3.60 (t, 2H, CH2OH), 7.10 (d, 2H,
pyridine-3H + pyridine-5H), 8.45 (m, 2H, pyridine-2H +
pyridine-6H).
12-P yr id in -4-yld od eca n -1-ol (15g). Using the same pro-
cedure as for 15f with 1.78 g (0.019 mol) of 14f gave 3.67 g
(87%) of a brown oil. 1H NMR (CDCl3): δ 1.25 (m, 14H, central
CH2’s), 1.55 (m, 6H, 3 × CH2), 1.75 (br s, 1H, OH), 2.55 (t, 2H,
pyridine-2-CH2), 3.60 (t, 2H, CH2OH), 7.10 (d, 2H, pyridine-
3H + pyridine-5H), 8.45 (m, 2H, pyridine-2H + pyridine-6H).
2-(10-P yr id in -2-yl-d ecyl)isoin d ole-1,3-d ion e (16b). The
same procedure as for 5c with 4.49 g (0.019 mol) of 15b was
used. After removal of the solvent the residue was dissolved
in ethyl acetate and extracted with HCl (1 M). The aqueous
layer was washed once with ethyl acetate, basified with K2CO3,
extracted with CHCl3, dried (MgSO4), and evaporated to afford
6.25 g (90%) of white powder. 1H NMR (CDCl3): δ 1.23 (m,
12H, central CH2’s), 1.65 (m, 4H, 2 × CH2), 2.75 (t, 2H,
pyridine-2-CH2), 3.62 (t, 2H, CH2-N), 7.05 (m, 2H, pyridine-
3H + pyridine-5H), 7.50 (m, 1H, pyridine-4H), 7.65 (m, 2H,
isoindole-1,3-dione), 7.80 (m, 2H, isoindole-1,3-dione), 8.45 (d,
1H, pyridine-6H).
2-(12-P yr idin -2-yldodecyl)isoin dole-1,3-dion e (16c). The
same procedure as for 16b with 5.67 g (0.022 mol) of 15c was
used. During the extraction with HCl (1 M) the hydrochloric
salt of the compound crystallized and was separated by
filtration. The salt was dissolved in water, basified with K2CO3,
extracted with CHCl3, dried (MgSO4), and concentrated in
vacuo to afford 3.19 g (38%) of white powder. 1H NMR
(CDCl3): δ 1.23 (m, 14H, central CH2’s), 1.60 (m, 6H, 3 × CH2),
2.75 (t, 2H, pyridine-2-CH2), 3.65 (t, 2H, CH2-N), 7.05 (m, 2H,
pyridine-3H + pyridine-5H), 7.55 (m, 1H, pyridine-4H), 7.68
(m, 2H, isoindole-1,3-dione), 7.80 (m, 2H, isoindole-1,3-dione),
8.45 (d, 1H, pyridine-6H).
(m, 6H, 2 × CH2 + NH2), 3.00 (m, 4H, pyridine-2-CH2
+
CH2NH2), 7.85 (m, 2H, pyridine-3H + pyridine-5H), 8.43 (m,
1H, pyridine-4H), 8.55 (d, 1H, pyridine-6H). Anal. (C14H26
Cl2N2) C, H, N.
-
10-P yr id in -2-yld ecyla m in e Dih yd r och lor id e (13b). Us-
ing the same procedure as for 6f with 6.25 g (0.017 mol) of
16b gave 2.99 g (74%) of free base. The free base was converted
into the dihydrochloride, decolorized with activated charcoal,
and crystallized from ethanol/ethyl acetate to afford 2.18 g
1
(56%) of white crystals. Mp: 143.5-146.5 °C. H NMR (D2O):
δ 1.25 (m, 14H, central CH2’s + NH2), 1.70 (m, 4H, 2 × CH2),
3.00 (m, 4H, pyridine-2-CH2 + CH2NH2), 7.85 (m, 2H, pyridine-
3H + pyridine-5H), 8.43 (m, 1H, pyridine-4H), 8.55 (d, 1H,
pyridine-6H). Anal.(C15H28Cl2N2) C, H, N.
12-P yr id in -2-yld od ecyla m in e Dih yd r och lor id e (13c).
Using the same procedure as for 13b with 3.19 g (0.0081 mol)
of 16c gave 1.65 g (66%) of white crystals. Mp: 161-164 °C.
1H NMR (D2O): δ 1.25 (m, 16H, central CH2’s), 1.70 (m, 4H,
2 × CH2), 3.00 (m, 4H, pyridine-2-CH2 + CH2NH2), 7.85 (m,
2H, pyridine-3H + pyridine-5H), 8.48 (m, 1H, pyridine-4H),
8.58 (d, 1H, pyridine-6H). Anal. (C17H32Cl2N2) C, H, N.
10-P yr id in -3-yld ecyla m in e Dih yd r och lor id e (13d ). The
same procedure as for 6f was employed starting with 7.57 g
(0.021 mol) of 16d . The free base obtained after extraction was
converted into the dihydrochloride and crystallized from
ethanol/ethyl acetate to afford 1.78 g (28%) of a beige powder.
Mp: 93.5-96.5 °C. 1H NMR (D2O): δ 1.30 (m, 12H, central
CH2’s), 1.68 (m, 4H, 2 × CH2), 2.82 (t, 2H, pyridine-2-CH2),
2.95 (t, 2H, CH2NH2), 7.95 (m, 1H, pyridine-5H), 8.45 (d, 1H,
pyridine-4H), 8.60 (m, 2H, pyridine-2H + pyridine-6H). Anal.
(C15H28Cl2N2) C, H, N.
12-P yr id in -3-yld od ecyla m in e Dih yd r och lor id e (13e).
The same procedure as for 5c with 2.56 g (0.0098 mol) of 15e
was used. The solvents were removed in vacuo and the
remaining oil was hydrazinolysed using the same procedure
as for 6f.
The free base was converted into the dihydrochloride and
crystallized from ethanol/ethyl acetate to afford 1.32 g (40%)
1
2-(10-P yr id in -3-yld ecyl)isoin d ole-1,3-d ion e (16d ). Using
the same procedure as for 16b with 5.50 g (0.023 mol) of 15d
of a yellow powder. Mp: 110-112 °C. H NMR (D2O): δ 1.30
(m, 18H, central CH2’s + NH2), 1.68 (m, 4H, 2 × CH2), 2.82 (t,
2H, pyridine-2-CH2), 2.95 (t, 2H, CH2NH2), 7.95 (m, 1H,
pyridine-5H), 8.45 (d, 1H, pyridine-4H), 8.60 (m, 2H, pyridine-
2H + pyridine-6H). Anal. (C17H32Cl2N2) C, H, N.
1
gave 7.57 g (90%) of brown oil. H NMR (CDCl3): δ 1.23 (m,
12H, central CH2’s), 1.55 (m, 4H, 2 × CH2), 2.55 (t, 2H,
pyridine-2-CH2), 3.62 (t, 2H, CH2-isoindole-1,3-dione), 7.15 (m,
2H, pyridine-3H + pyridine-5H), 7.15 (m, 1H, pyridine-5H),
7.45 (d, 1H, pyridine-4H), 7.65 (m, 2H, isoindole-1,3-dione),
7.80 (m, 2H, isoindole-1,3-dione), 8.38 (m, 2H, pyridine-2H +
pyridine-6H).
10-P yr id in -4-yld ecyla m in e Dih yd r och lor id e (13f). The
same procedure as for 6f with 6.42 g (0.018 mol) of 16f was
used. The free base was converted into the dihydrochloride,
crystallized from ethanol/ethyl acetate, and decolorized with
activated charcoal to afford 2.65 g (49%) of light yellow powder.
Mp: 143.5-144.5 °C. 1H NMR (D2O): δ 1.25 (m, 14H, central
CH2’s + NH2), 1.65 (m, 4H, 2 × CH2), 2.95 (m, 4H, pyridine-
2-CH2 + CH2NH2), 7.85 (d, 2H, pyridine-3H + pyridine-5H),
8.58 (d, 2H, pyridine-2H + pyridine-6H). Anal. (C15H28Cl2N2)
C, H, N.
2-(10-P yr id in -4-yld ecyl)isoin d ole-1,3-d ion e (16f). Using
the same procedure as for 16b with 4.89 g (0.021 mol) of 15f
1
gave 6.42 g (82%) of a yellow solid. H NMR (CDCl3): δ 1.23
(m, 12H, central CH2’s), 1.65 (m, 4H, 2 × CH2), 2.55 (t, 2H,
pyridine-2-CH2), 3.62 (t, 2H, CH2-isoindole-1,3-dione), 7.05 (d,
2H, pyridine-3H + pyridine-5H), 7.45 (d, 1H, pyridine-4H),
7.65 (m, 2H, isoindole-1,3-dione), 7.80 (m, 2H, isoindole-1,3-
dione), 8.45 (m, 2H, pyridine-2H + pyridine-6H).
12-P yr id in -4-yld od ecyla m in e Dih yd r och lor id e (13g).
The same procedure as for 6f with 4.37 g (0.011 mol) of 16g
was used. The crude product was purified by flash chroma-
tography switching gradually the eluent from ethyl acetate
into methanol/triethylamine (9/1) (Rf ) 0.4) to afford 1.48 g
(50%) of yellow solid. The free base was converted into the
dihydrochloride, crystallized from ethanol/ethyl acetate, and
decolorized with activated charcoal to afford 1.28 g (68%) of
2-(12-P yr idin -4-yldodecyl)isoin dole-1,3-dion e (16g). Us-
ing the same procedure as for 16b with 3.67 g (0.014 mol) of
1
15g gave 4.37 g (79%) of brown oil. H NMR (CDCl3): δ 1.23
(m, 14H, central CH2’s), 1.55 (m, 6H, 3 × CH2), 2.55 (t, 2H,
pyridine-2-CH2), 3.62 (t, 2H, CH2-isoindole-1,3-dione), 7.05 (d,
2H, pyridine-3H + pyridine-5H), 7.65 (m, 2H, isoindole-1,3-
dione), 7.80 (m, 2H, isoindole-1,3-dione), 8.45 (m, 2H, pyridine-
2H + pyridine-6H).
1
light yellow powder. Mp: 137.5-139.5 °C. H NMR (D2O): δ
1.25 (m, 18H, central CH2’s + NH2), 1.65 (m, 4H, 2 × CH2),
2.95 (m, 4H, pyridine-2-CH2 + CH2NH2), 7.85 (d, 2H, pyridine-
3H + pyridine-5H), 8.58 (d, 2H, pyridine-2H + pyridine-6H).
Anal. (C17H32Cl2N2) C, H, N.
9-P yr id in -2-yln on yla m in e Dih yd r och lor id e (13a ). The
same procedure as for 5f with 0.82 g of crude 15a was used.
The residue obtained after extraction was purified by flash
chromatography using ethyl acetate as eluent. The product
fractions were hydrazinolysed as described for 6f to give 0.12
g (59%) of free base.
P h a r m a cology. Ma ter ia ls. pNF-κB-Luc was obtained from
Stratagene (La J olla, CA). Mepyramine (pyrilamine maleate)
and tripelennamine hydrochloride were obtained from RBI.
ATP disodium salt, bovine serum albumin, chloroquine diphos-
phate, DEAE-dextran (chloride form), histamine dihydro-
chloride, polyethyleneimine, and Tween 20 were purchased
from Sigma Chemical Company. D-Luciferin was obtained
The free base was converted into the dihydrochloride, using
the following procedure: the free base was mixed with 2 equiv
of HCl (37%) and stirred for 30 min, then the water was
removed in vacuo to afford 0.15 g (95%) of a brown waxy

5822 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Govoni et al.
from Duchefa Biochemie BV, glycerol from Riedel-de-Hae¨n,
and Triton X-100 from Fluka. Cell culture media, penicillin,
and streptomycin were obtained from Life Technologies
(Merelbeke, Belgium). Fetal calf serum (FCS) was obtained
from Integro B. V. (Dieren, The Netherlands), dialyzed fetal
calf serum was obtained from HyClone Laboratories Inc.
[3H]Mepyramine (30 Ci/mmol) and myo-[2-3H]inositol (17 Ci/
mmol) were from Amersham International.
squares curve-fitting procedure using Graphpad Prism (Graph-
Pad Software, Inc., San Diego, CA).
[3H]In ositol P h osp h a te F or m a tion . HeLa cells were
seeded in 24-well plates (105 cells/well) and after 24 h cells
were washed with PBS, labeled, and pretreated with various
concentrations of inverse H1-agonists in inositol-free culture
medium supplemented with 1 µCi/mL myo-[2-3H]inositol for
24 h. Subsequently, the medium was aspirated and cells were
incubated with histamine for 1 h at 37 °C in DMEM containing
25 mM Hepes (pH 7.4) and 20 mM LiCl. Incubations were
stopped by aspiration of the culture medium and the addition
of cold 10 mM formic acid. After 90 min incubation at 4 °C,
[3H]inositol phosphates were isolated by anion exchange
chromatography and counted by liquid scintillation.
All other compounds, including VUF 4732 (1a ), VUF 4733
(1b), VUF 4734 (1d ),53 VUF 8811 (17c), VUF 5963 (17a ),
2-(pyridine-2-yl)ethyl-1-amine (PEA) dihydrochloride, 2-(thi-
azol-4-yl)ethanamine (4-TEA), 2-(3-trifluoromethylphenyl)-
histamine dihydrogenmaleate (17b),56 histaprodifen dimaleate
(18c), histabudifen dimaleate (18d ), histapendifen dimaleate
(18e), the pyrrolidin analogue of histaprodifen as dioxalate
(18i),58 the ring-substituted analogues of histaprodifen di-
hydrogenmaleate [4-Br-histaprodifen (18g) and 4-methyl-
histaprodifen (18h )],59 the histaprodifen analogues 2-(di-
phenylmethyl)histamine dihydrochloride (18a ) and 2-(di-
phenylethyl)histamine dihydrochloride (18b ), 2-(4-phenyl-
butyl)histamine dihydrogenmaleate (FUB 113, 17d ), 2-(5-
phenylpentyl)histamine dihydrogenmaleate (FUB 114, 17e),
the cyclohexyl analogue of histaprodifen dihydrogenmaleate
(18f),55 the 2-(2-phenyl-4-thiazolyl)ethyl-1-amines (19a ,b), and
2-(2-benzyl-4-thiazolyl)ethyl-1-amines (19c,d ),60 were taken
from our own stock.
An a lytica l Meth od s. Protein levels were determined ac-
cording to the method of Bradford,72 using BSA as a standard.
All data shown are expressed as mean ( SEM, and statistical
analyses were carried out by Student’s t-test. P values < 0.05
were considered to indicate a significant difference.
Refer en ces
(1) De Backer, M. D.; Gommeren, W.; Moereels, H.; Nobels, G.; van
Gompel, P.; Leysen, J . E.; Luyten, W. H. Genomic cloning,
heterologous expression and pharmacological characterization
of a human histamine H1 receptor. Biochem. Biophys. Res.
Commun. 1993, 197, 1601-1608.
(2) Fukui, H.; Fujimoto, K.; Mizuguchi, H.; Sakamoto, K.; Horio,
Y.; Takai, S.; Yamada, K.; Ito, S. Molecular cloning of the human
histamine H1 receptor gene. Biochem. Biophys. Res. Commun.
1994, 201, 894-901.
(3) Moguilevsky, N.; Varsalona, F.; Noyer, M.; Gillard, M.; Guil-
laume, J . P.; Garcia, L.; Szpirer, C.; Szpirer, J .; Bollen, A. Stable
expression of human H1-histamine-receptor cDNA in Chinese
hamster ovary cells. Pharmacological characterisation of the
protein, tissue distribution of messenger RNA and chromosomal
localisation of the gene. Eur. J . Biochem. 1994, 224, 489-495.
(4) Gantz, I.; Munzert, G.; Tashiro, T.; Schaffer, M.; Wang, L.; Del
Valle, J .; Yamada, T. Molecular cloning of the human histamine
H2 receptor. Biochem. Biophys. Res. Commun. 1991, 178, 1386-
1392.
(5) Lovenberg, T. W.; Roland, B. L.; Wilson, S. J .; J iang, X.; Pyati,
J .; Huvar, A.; J ackson, M. R.; Erlander, M. G. Cloning and
functional expression of the human histamine H3 receptor. Mol.
Pharmacol. 1999, 55, 1101-1107.
(6) 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,
434-441.
(7) Liu, C.; Ma, X. J .; J iang, X.; Wilson, S. J .; Hofstra, C. L.; Blevitt,
J .; Pyati, J .; 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.
(8) 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.
(9) Nakamura, T.; Itadani, H.; Hidaka, Y.; Ohta, M.; Tanaka, K.
Molecular cloning and characterization of a new human hista-
mine receptor, HH4R. Biochem. Biophys. Res. Commun. 2000,
279, 615-620.
Gifts of mianserine hydrochloride (Organon NV), pcDEF3
(Dr. J . Langer), and the cDNA encoding the human histamine
H1-receptor (Dr. H. Fukui)2 are greatly acknowledged.
Cell Cu ltu r e a n d Tr a n sfection . COS-7 African green
monkey kidney cells were maintained at 37 °C in an humidi-
fied 5% CO2/95% air atmosphere in Dulbecco’s modified Eagle’s
medium (DMEM) containing 2 mM L-glutamine, 50 IU/mL
penicillin, 50 µg/mL streptomycin, and 5% (v/v) fetal calf serum
(FCS); HeLa cells were maintained in the same manner in
medium containing 10% (v/v) FCS. COS-7 cells were tran-
siently transfected using the DEAE-dextran method as previ-
ously described.28 The expression level of the histamine H1-
receptor in transiently transfected COS-7 cells was 3.2 ( 0.4
pmol/mg of protein as determined by radioligand binding
assays. The total amount of DNA transfected was maintained
constant by addition of either pcDEF3 or pcDNA3.
Rep or ter -Gen e Assa y. Reporter-gene assays were pre-
formed as previously described. 28 Briefly, after cotransfection
with pNFκB-Luc (125 µg/1 × 107 cells) and either pcDEF3 or
pcDEF3hH1 (25 µg/1 × 107 cells) cells were seeded in 96 well
whiteplates in serum free culture medium and incubated with
drugs. After 48 h, cells were assayed for luminescence for 3
s/well in a Victor2 (Wallac) 30 mim after aspiration of the
medium and the addition of 25 µL/well luciferase assay reagent
[0.83 mM ATP, 0.83 mM D-luciferin, 18.7 mM MgCl2, 0.78 µM
Na2H2P2O7, 38.9 mM Tris (pH 7.8), 0.39% (v/v) glycerol, 0.03%
(v/v) Triton-X-100, and 2.6 µM DTT]. To detect potential
nonhuman H1R-mediated effects of the tested compounds, the
compounds were tested in parallel on control cells not express-
ing the human H1R.
Hista m in e H1-Recep tor Bin d in g Stu d ies. HeLa cells or
COS-7 cells (harvested 48 h after transfection) for radioligand
binding studies were harvested and homogenized in ice-cold
H1-binding buffer (50 mM Na2/K-phosphate buffer, pH ) 7.4)
as previously described.28 HeLa cell membranes were further
purified by centrifugation of the cell homogenate for 5 min at
1400g and subsequent centrifugation of the supernatant for
20 min at 15 000g. For radioligand binding studies, COS-7 cell
homogenates or HeLa cell membranes were incubated for
30 min at 30 °C in H1-binding buffer containing 1 nM
[3H]mepyramine. The nonspecific binding was determined in
the presence of 1 µM mianserin. Incubations were terminated
by rapid dilution and subsequent filtration over Whatman
GF/C filters that had been treated with 0.3% polyethylene-
imine using ice-cold H1-binding buffer. The radioactivity
retained on the filters was measured by liquid scintillation
counting. Binding data were evaluated by a nonlinear, least
(10) Morse, K. L.; Behan, J .; Laz, T. M.; West, R. E.; Greenfeder, S.
A.; Anthes, J . C.; Umland, S.; Wan, Y.; Hipkin, R. W.; Gonsiorek,
W.; Shin, N.; Gustafson, E. L.; Qiao, X.; Wang, S.; Hedrick, J .
A.; Greene, J .; Bayne, M.; Monsma, F. J . Cloning and Charac-
terization of a Novel Human Histamine Receptor. J . Pharmacol.
Exp. Ther. 2001, 296, 1058-1066.
(11) Dale, H. H.; Laidlaw, P. P. The Physiological Action of â-imid-
azolylethylamine. J . Physiol. 1910, 41, 418-444.
(12) Zhang, M.-Q.; Leurs, R.; Timmerman, H. Histamine H1-receptor
antagonists. Burger’s Medicinal Chemistry and Drug Discovery,
5th ed.; J ohn Wiley & Sons: New York, 1997; pp 495-559.
(13) Costa, T.; Ogino, Y.; Munson, P. J .; Onaran, H. O.; Rodbard, D.
Drug efficacy at guanine nucleotide-binding regulatory protein-
linked receptors: Thermodynamic interpretation of negative
antagonism and of receptor activity in the absence of ligand. Mol.
Pharmacol. 1992, 41, 549-560.
(14) Schutz, W.; Freissmuth, M. Reverse intrinsic activity of antago-
nists on G protein-coupled receptors. Trends Pharmacol. Sci.
1992, 13, 376-380.

Neutral Histamine H1-Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5823
(15) Lefkowitz, R. J .; Cotecchia, S.; Samama, P.; Costa, T. Constitu-
tive activity of receptors coupled to guanine nucleotide regulatory
proteins. Trends Pharmacol. Sci. 1993, 14, 303-307.
(16) Milligan, G.; Bond, R. A.; Lee, M. Inverse agonism: Pharmaco-
logical curiosity or potential therapeutic strategy? Trends Phar-
macol. Sci. 1995, 16, 10-13.
(17) Kenakin, T. The classification of seven transmembrane receptors
in recombinant expression systems. Pharmacol. Rev. 1996, 48,
413-463.
(18) Leurs, R.; Smit, M. J .; Alewijnse, A. E.; Timmerman, H. Agonist-
independent regulation of constitutively active G-protein-coupled
receptors. Trends Biochem. Sci. 1998, 23, 418-422.
(19) Smit, M. J .; Leurs, R.; Alewijnse, A. E.; Blauw, J .; van Nieuw
Amerongen, G. P.; van De Vrede, Y.; Roovers, E.; Timmerman,
H. Inverse agonism of histamine H2 antagonist accounts for
upregulation of spontaneously active histamine H2 receptors.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6802-6807.
(20) Wieland, K.; Bongers, G.; Yamamoto, Y.; Hashimoto, T.; Yama-
todani, A.; Menge, W. M. B. P.; Timmerman, H.; Lovenberg, T.
W.; Leurs, R. Constitutive Activity of Histamine H3 Receptors
Stably Expressed in SK-N-MC Cells: Display of Agonism and
Inverse Agonism by H3 Antagonists. J . Pharmacol. Exp. Ther.
2001, 299, 908-914.
(38) Chen, S.; Lin, F.; Xu, M.; Hwa, J .; Graham, R. M. Dominant-
negative activity of an R1B-adrenergic receptor signal-inactivat-
ing point mutation. EMBO J . 2000, 19, 4265-4271.
(39) Iriyoshi, N.; Takeuchi, K.; Yuta, A.; Ukai, K.; Sakakura, Y.
Increased expression of histamine H1 receptor mRNA in allergic
rhinitis. Clin. Exp. Allergy 1996, 26, 379-385.
(40) Chiba, Y.; Saitoh, N.; Matsuo, K.; Misawa, M. Elevated nasal
mucosal G protein levels and histamine receptor affinity in a
guinea pig model of nasal hyperresponsiveness. Int. Arch. Allergy
Immunol. 2002, 127, 285-293.
(41) Milligan, G.; Bond, R. A. Inverse agonism and the regulation of
receptor number. Trends Pharmacol. Sci. 1997, 18, 468-474.
(42) Elfellah, M. S.; Reid, J . L. Regulation of â1- and â2-adrenoceptors
following chronic treatment with â-adrenoceptor antagonists.
Eur. J . Pharmacol. 1989, 173, 85-92.
(43) Homcy, C. J .; Vatner, S. F.; Vatner, D. E. Beta-adrenergic
receptor regulation in the heart in pathophysiologic states:
Abnormal adrenergic responsiveness in cardiac disease. Annu.
Rev. Physiol. 1991, 53, 137-159.
(44) Yoburn, B. C.; Duttaroy, A.; Shah, S.; Davis, T. Opioid antagonist-
induced receptor upregulation: Effects of concurrent agonist
administration. Brain Res. Bull. 1994, 33, 237-240.
(45) Nwokolo, C. U.; Smith, J . T.; Sawyerr, A. M.; Pounder, R. E.
Rebound intragastric hyperacidity after abrupt withdrawal of
histamine H2 receptor blockade. Gut 1991, 32, 1455-1460.
(46) Merki, H. S.; Wilder-Smith, C. H. Do continuous infusions of
omeprazole and ranitidine retain their effect with prolonged
dosing? Gastroenterology 1994, 106, 60-64.
(21) Morisset, S.; Rouleau, A.; Ligneau, X.; Gbahou, F.; Tardivel-
Lacombe, J .; Stark, H.; Schunack, W.; Ganellin, C. R.; Schwartz,
J . C.; Arrang, J . M. High constitutive activity of native H3
receptors regulates histamine neurons in brain. Nature (London)
2000, 408, 860-864.
(22) Shenker, A.; Laue, L.; Kosugi, S.; Merendino, J . J ., J r.; Minegishi,
T.; Cutler, G. B., J r. A constitutively activating mutation of the
luteinizing hormone receptor in familial male precocious puberty.
Nature (London) 1993, 365, 652-654.
(23) Robinson, P. R.; Cohen, G. B.; Zhukovsky, E. A.; Oprian, D. D.
Constitutively active mutants of rhodopsin. Neuron 1992, 9,
719-725.
(24) Parma, J .; Duprez, L.; Van Sande, J .; Cochaux, P.; Gervy, C.;
Mockel, J .; Dumont, J .; Vassart, G. Somatic mutations in the
thyrotropin receptor gene cause hyperfunctioning thyroid ad-
enomas. Nature (London) 1993, 365, 649-651.
(47) Ngochindo, R. I. 2-Protecting Groups for 5-Lithiation in the
Syntheses of Imidazoles. J . Chem. Soc., Perkin Trans. 1 1990,
6, 1645-1648.
(48) Mitsunobu, O. The Use of Diethyl Azodicarboxylate and Tri-
phenylphosphine in Synthesis and Transformation of Natural
Products. Synthesis 1981, 1-28.
(49) Gibson, M. S.; Bradshaw, R. W. Gabriel-Synthese Prima¨rer
Amine. Angew. Chem. 1968, 80, 986-996.
(50) Horne, D. A.; Yakushijin, K.; Bu¨chi, G. A Two-Step Synthesis
of Imidazoles From Aldehydes Via 4-Tosyloxazolines. Hetero-
cycles 1994, 39, 139-153.
(25) Robbins, L. S.; Nadeau, J . H.; J ohnson, K. R.; Kelly, M. A.;
Roselli-Rehfuss, L.; Baack, E.; Mountjoy, K. G.; Cone, R. D.
Pigmentation phenotypes of variant extension locus alleles result
from point mutations that alter MSH receptor function. Cell
1993, 72, 827-834.
(26) Spiegel, A. M. Defects in G protein-coupled signal transduction
in human disease. Annu. Rev. Physiol. 1996, 58, 143-170.
(27) Bakker, R. A.; Wieland, K.; Timmerman, H.; Leurs, R. Constitu-
tive activity of the histamine H1 receptor reveals inverse agonism
of histamine H1 receptor antagonists. Eur. J . Pharmacol. 2000,
387, R5-R7.
(28) Bakker, R. A.; Schoonus, S.; Smit, M. J .; Timmerman, H.; Leurs,
R. Histamine H1-receptor activation of NF-κB: Roles for Gâγ
and GRq/11-subunits in constitutive and agonist-mediated signal-
ing. Mol. Pharmacol. 2001, 60, 1133-1142.
(29) Costa, T.; Herz, A. Antagonists with negative intrinsic activity
at delta opioid receptors coupled to GTP-binding proteins. Proc.
Natl. Acad. Sci. U.S.A. 1989, 86, 7321-7325.
(30) Senogles, S. E.; Spiegel, A. M.; Padrell, E.; Iyengar, R.; Caron,
M. G. Specificity of receptor-G protein interactions. Discrimina-
tion of Gi subtypes by the D2 dopamine receptor in a reconsti-
tuted system. J . Biol. Chem. 1990, 265, 4507-4514.
(31) Munshi, R.; Pang, I. H.; Sternweis, P. C.; Linden, J . A1 adenosine
receptors of bovine brain couple to guanine nucleotide- binding
proteins Gi1, Gi2, and Go. J . Biol. Chem. 1991, 266, 22285-22289.
(32) Burstein, E. S.; Spalding, T. A.; Brann, M. R. Pharmacology of
muscarinic receptor subtypes constitutively activated by G
proteins. Mol. Pharmacol. 1997, 51, 312-319.
(33) Burstein, E. S.; Spalding, T. A.; Bra¨uner-Osborne, H.; Brann,
M. R. Constitutive activation of muscarinic receptors by the
G-protein Gq. FEBS Lett. 1995, 363, 261-263.
(34) Casarosa, P.; Bakker, R. A.; Verzijl, D.; Navis, M.; Timmerman,
H.; Leurs, R.; Smit, M. J . Constitutive Signaling of the Human
Cytomegalovirus-encoded Chemokine Receptor US28. J . Biol.
Chem. 2001, 276, 1133-1137.
(51) Loudon, G. M.; Radhakrishna, A. S.; Almond, M. R.; Blodgett,
J . K.; Boutin, R. H. Conversion of Aliphatic Amides into Amines
with [I,I-Bis(trifluoroacetoxy)iodo]benzene. 1. Scope of the Reac-
tion. J . Org. Chem. 1984, 49, 4272-4276.
(52) Brandsma, L. Metalation of Heterocyclic Compounds. Prepara-
tive Polar Organometallic Chemistry; Springer-Verlag: Berlin,
1990; Chapter 6, pp 127-139.
(53) Vollinga, R. C.; Menge, W. M.; Leurs, R.; Timmerman, H.
Homologues of histamine as histamine H3 receptor antago-
nists:
A new potent and selective H3 antagonist, 4(5)-(5-
aminopentyl)-1H-imidazole. J . Med. Chem. 1995, 38, 266-271.
(54) Walter, L. A.; Hunt, W. H.; Fosbinder, R. J . J . Am. Chem. Soc.
1941, 63, 2771.
(55) Zingel, V.; Leschke, C.; Schunack, W. Developments in histamine
H1-receptor agonists. Prog. Drug Res. 1995, 44, 49-85.
(56) Leschke, C.; Elz, S.; Garbarg, M.; Schunack, W. Synthesis and
histamine H1 receptor agonist activity of a series of 2-phenyl-
histamines, 2-heteroarylhistamines, and analogues. J . Med.
Chem. 1995, 38, 1287-1294.
(57) ter Laak, A. M.; Timmerman, H.; Leurs, H.; Nederkoorn, P. H.
J .; Smit, M. J .; Donne-Op den Kelder, G. M. Modelling and
mutation studies of the histamine H1 receptor agonist binding
site reveal different binding modes for H1 agonists: Asp116 (TM3)
has a constitutive role in receptor stimulation. J . Comput. Aid.
Mol. Design. 1995, 9, 319-330.
(58) Elz, S.; Kramer, K.; Pertz, H. H.; Detert, H.; ter Laak, A. M.;
Ku¨hne, R.; Schunack, W. Histaprodifens: Synthesis, pharma-
cological in vitro evaluation, and molecular modeling of a new
class of highly active and selective histamine H1-receptor ago-
nists. J . Med. Chem. 2000, 43, 1071-1084.
(59) Elz, S.; Kramer, K.; Leschke, C.; Schunack, W. Ring-substituted
histaprodifen analogues as partial agonists for histamine H1
receptors: Synthesis and structure-activity relationships. Eur.
J . Med. Chem. 2000, 35, 41-52.
(60) Walczynski, K.; Timmerman, H.; Zuiderveld, O. P.; Zhang, M.
Q.; Glinka, R. Histamine H1 receptor ligands. Part I. Novel
thiazol-4-ylethanamine derivatives: Synthesis and in vitro
pharmacology. Farmaco 1999, 54, 533-541.
(61) Walczynski, K.; Guryn, R.; Zuiderveld, O. P.; Zhang, M. Q.;
Timmerman, H. Histamine H1 receptor ligands: Part II. Syn-
thesis and in vitro pharmacology of 2-[2-(phenylamino)thiazol-
4-yl]ethanamine and 2-(2-benzhydrylthiazol-4-yl)ethanamine
derivatives. Farmaco 2000, 55, 569-574.
(62) Ter Laak, A. M.; Venhorst, J .; Donne´-Op den Kelder, G. A.;
Timmerman, H. The Histamine H1-Receptor Antagonist Bind-
ing Site. A Stereoselective Pharmacophoric Model Based upon
(Semi-)Rigid H1-Antagonists and Including a Known Interaction
Site on the Receptor. J . Med. Chem. 1995, 38, 3351-3360.
(35) Samama, P.; Cotecchia, S.; Costa, T.; Lefkowitz, R. J . A muta-
tion-induced activated state of the â2-adrenergic receptor. Ex-
tending the ternary complex model. J . Biol. Chem. 1993, 268,
4625-4636.
(36) Allen, L. F.; Lefkowitz, R. J .; Caron, M. G.; Cotecchia, S.
G-protein-coupled receptor genes as protooncogenes: Constitu-
tively activating mutation of the R1B-adrenergic receptor en-
hances mitogenesis and tumorigenicity. Proc. Natl. Acad. Sci.
U.S.A. 1991, 88, 11354-11358.
(37) Dosil, M.; Giot, L.; Davis, C.; Konopka, J . B. Dominant-negative
mutations in the G-protein-coupled alpha-factor receptor map
to the extracellular ends of the transmembrane segments. Mol.
Cell. Biol. 1998, 18, 5981-5991.

5824 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Govoni et al.
(63) Elz, S.; Kramer, K.; Leschke, C.; Schunack, W. Ring-substituted
Histaprodifens Analogues as Partial Agonists for Histamine H1
Receptor: Synthesis and Structure-activity Relationships. Eur.
J . Med. Chem. 2000, 35, 41-52.
(68) Ahmad, K.; Strong, F. M. The Synthesis of Unsaturated Fatty
Acids. J . Am. Chem. Soc. 1948, 70, 1699-1700.
(69) Carbelleira, N.; Thompson, J . E.; Ayanoglu, E.; Djerassi, C.
Biosynthetic Studies of Marine Lipids. 5. The Biosynthesis of
Long-Chain Branched Fatty Acids in Marine Sponges. J . Org.
Chem. 1986, 51, 2751-2756.
(70) Heiser, U. F.; Dobner, B. Copper-catalysed Coupling of Undec-
10-enylmagnesium Bromide with ω-Functionalised Halogeno-
alkanes as a Key Reaction for the Synthesis of Novel Bipolar
Phospholipids with Different Headgroups and Chain Length. J .
Chem. Soc., Perkin Trans. 1 1997, 1, 805-815.
(64) Berg, K. A.; Stout, B. D.; Cropper, J . D.; Maayani, S.; Clarke,
W. P. Novel actions of inverse agonists on 5-HT2C receptor
systems. Mol. Pharmacol. 1999, 55, 863-872.
(65) Lavreysen, H.; Le Poul, E.; Van Gompel, P.; Dillen, L.; Leysen,
J . E.; Lesage, A. S. J . Supersensitivity of Human Metabotropic
Glutamate 1a Receptor Signaling in L929sA Cells. Mol. Phar-
macol. 2002, 61, 1244-1254.
(66) Wallace, R. M.; Young, J . M. Temperature dependence of the
binding of [3H]mepyramine and related compounds to the
histamine H1 receptor. Mol. Pharmacol. 1983, 23, 60-66.
(67) Wieland, K.; ter Laak, A. M.; Smit, M. J .; Ku¨hne, R.; Timmer-
man, H.; Leurs, R. Mutational analysis of the antagonist-binding
site of the histamine H1 receptor. J . Biol. Chem. 1999, 274,
29994-30000.
(71) Cundy, D. J .; Gurr, P. A. An Improved Synthesis of (18-methyl-
eicosanoic acid. Org. Prep. Proced. Int. 2000, 32, 461-468.
(72) Bradford, M. M. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
J M030936T