A chemical switch for the modulation of the functional activity of higher homologues of histamine on the human histamine H3 receptor: Effect of various substitutions at the primary amino function

Article abstract of DOI:10.1021/jm0504353

In an effort to establish the structural requirements for agonism, neutral antagonism, and inverse agonism at the human histamine H₃ receptor (H₃R) we have prepared a series of higher homologues of histamine in which the terminal nitrogen of the side chain has been either mono- or disubstituted with several aliphatic, alicyclic, and aromatic moieties or incorporated in cyclic systems. The novel ligands have been pharmacologically investigated in vitro for their affinities on the human H₃R and H₄R subtypes by radioligand displacement experiments and for their intrinsic H₃R activities via a CRE-mediated β-galactosidase reporter gene assay. Subtle changes of the substitution pattern at the side chain nitrogen alter enormously the pharmacological activity of the ligands, resulting in a series of compounds with a wide spectrum of pharmacological activities. Among the several neutral H₃R antagonists identified within this series, compounds 2b and 2h display an H₃R affinity in the low nanomolar concentration range (pK_i values of 8.1 and 8.4, respectively). A very potent and selective H₃R agonist (11, pEC ₅₀ = 8.9, α = 0.94) and a very potent, though not highly selective, H₃R inverse agonist (2k, pIC₅₀ = 8.9, α = -0.97) have been identified as well.

Full text of DOI:10.1021/jm0504353

J. Med. Chem. 2006, 49, 2549-2557
2549
A Chemical Switch for the Modulation of the Functional Activity of Higher Homologues of
Histamine on the Human Histamine H3 Receptor: Effect of Various Substitutions at the
Primary Amino Function
Marinella Govoni, Herman D. Lim, Dris El-Atmioui, Wiro M. P. B. Menge, Henk Timmerman, Remko A. Bakker,
Rob Leurs, and Iwan J. P. De Esch*
Leiden/Amsterdam Center for Drug Research, Department of Pharmacochemistry, Vrije UniVersiteit Amsterdam, De Boelelaan 1083,
1081 HV Amsterdam, The Netherlands
ReceiVed May 8, 2005
In an effort to establish the structural requirements for agonism, neutral antagonism, and inverse agonism
at the human histamine H3 receptor (H3R) we have prepared a series of higher homologues of histamine in
which the terminal nitrogen of the side chain has been either mono- or disubstituted with several aliphatic,
alicyclic, and aromatic moieties or incorporated in cyclic systems. The novel ligands have been
pharmacologically investigated in vitro for their affinities on the human H3R and H4R subtypes by radioligand
displacement experiments and for their intrinsic H3R activities via a CRE-mediated â-galactosidase reporter
gene assay. Subtle changes of the substitution pattern at the side chain nitrogen alter enormously the
pharmacological activity of the ligands, resulting in a series of compounds with a wide spectrum of
pharmacological activities. Among the several neutral H3R antagonists identified within this series, compounds
2b and 2h display an H3R affinity in the low nanomolar concentration range (pKi values of 8.1 and 8.4,
respectively). A very potent and selective H3R agonist (1l, pEC50 ) 8.9, R ) 0.94) and a very potent,
though not highly selective, H3R inverse agonist (2k, pIC50 ) 8.9, R ) -0.97) have been identified as well.
Introduction
The biogenic amine histamine exerts its multiple biological
activities through the activation of at least four distinct histamine
receptors, i.e., H1, H2, H3, and H4 receptors,1-4 all belonging to
the superfamily of heptahelical (7TM) G-protein coupled
receptors.
The histamine H3 receptor (H3R) was discovered in 1983 by
Arrang and co-workers as a presynaptic autoreceptor,5 and the
gene was successively cloned in 1999 by Lovenberg and
colleagues.3 Tissue distribution analysis indicated that the
expression of the receptor is predominantly restricted to the
brain.6 The H3R does not only mediate the inhibition of synthesis
and release of histamine from histaminergic neurons via a
negative feedback loop,7,8 but also exerts modulatory effects
on other neurotransmitter systems, e.g. the cholinergic,9,10
dopaminergic,11 noradrenergic,12 and serotoninergic13 systems,
in both the central and peripheral nervous systems. It has been
recognized that the H3R is a potential therapeutic target.14-16
H3R antagonists have been proposed to be potential drugs for
the treatment of several CNS disorders, such as attention-deficit
hyperactivity disorder (ADHD),17,18 Alzheimer’s disease,19
epilepsy,20-22 schizophrenia,22,23 and obesity,24-26 whereas the
therapeutic potential of H3R agonists has been shown for
myocardial ischemia,27 inflammatory28 and gastric acid related
diseases,29 migraine, and sleep disorders.30
The constitutive activity of several 7TM receptor systems has
been investigated within the past decade.31-36 For a long time
it was not known whether this phenomenon existed only in cells
overexpressing (mutant) GPCRs or also occurred in vivo.
Constitutive activity has been shown recently for both the
histamine H1 and H2 receptors.37,38 Our group reported that the
therapeutically important H1 and H2 receptor antagonists act,
Figure 1. Structures of 4-[1H-imidazol-4-yl]butylamine (imbutamine,
1a), 5-[1H-imidazol-4-yl]pentylamine (impentamine, 2a), [5-(1H-
imidazol-4-yl)pentyl]isopropylamine (2d), clobenpropit, GT-2331 (cipral-
isant, Perceptin), and proxyfan.
in fact, as inverse agonists, but we also identified neutral
antagonists for both receptors.37,39 Histaminergic neurotrans-
mission in rodent brain was shown to be regulated by consti-
tutively active H3Rs in vitro as well as in vivo,40 and our group
also demonstrated that the human and rat H3Rs stably expressed
in SK-N-MC cells3,6 show a high level of constitutive activ-
ity.41,42 This led to the identification of several standard H3R
antagonists (thioperamide and clobenpropit) as inverse agonists
in this cell system. Moreover, burimamide and impentamine
(Figure 1), previously identified as H3R antagonists,5,43,44 behave
as H3R agonists at both recombinant H3Rs (human and rat).
We also showed that in a small series of impentamine analogues
we were able to manipulate the intrinsic activity (R) by
substitution of the amino group.41
In an attempt to establish a correlation between the structure
of the ligands and their functional activity at the human H3R,
* Author for correspondence. Tel: 31-20-597600. Fax: 31-20-
5987610. E-mail: i.de.esch@few.vu.nl.
10.1021/jm0504353 CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/22/2006

2550 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Scheme 1. Synthesis of Compounds 1c,m and 2c,d,i,ma
GoVoni et al.
sodium triacetoxyborohydride and cleavage of the dimethyl-
sulfamoyl protecting group to give compounds 1c,m and 2c,m
in moderate to good yield (44-70%). Alternatively, the ω-chloro
group of 944 was converted either into an isopropylamino group
or into a cyclohexylamino group via heating with isopropyl-
amine and cyclohexylamine, respectively, to give compounds
11d,i. Deprotection yielded compounds 2d,i.
Aminoalkylimidazoles 1b,d-l,n-p and 2b,e-h,j-l,n-p
were prepared according to the synthetic pathway shown in
Scheme 2. Methanolysis of δ-valerolactone and ꢀ-caprolactone
followed by Swern oxidation of esters 12 and 13 afforded
aldehydes 14 and 15, which were readily converted into
imidazoles using TosMIC chemistry.49 The intermediate 4-to-
syloxazolines 16 and 17 were treated with ammonia in ethanol
under pressure, affording a mixture of amide and methyl and
ethyl ester which were converted into the carboxylic acid via
acidic hydrolysis and then re-esterified to give esters 18 and
19 in good yields (82% and 51%, respectively). Protection of
the imidazole ring with a trityl group, followed by reduction of
esters 20 and 21 with lithium aluminum hydride and Swern
oxidation of the alcohols 22 and 23, yielded the key intermedi-
ates 24 and 25 (49% and 70% yield). Reductive amination with
a suitable amine and subsequent deprotection afforded the final
compounds 1b,d-l,n-p and 2b,e-h,j-l,n-p, in variable
isolated yields (17-95%).
a Reagents: (a) n-BuLi, THF, -70 °C; (b) 2-(ω-iodoalkyloxy)tetrahy-
dropyran; (c) HCl, rt; (d) Swern oxidation; (e) R1R2-amine, AcOH,
NaBH(OAc)3, DCE, rt (method B, reductive amination); (f) R1R2-amine,
100 °C (method A); (g) 30% HBr or 37% HCl, reflux (method D). R1R2-
amines are given in Tables 1 and 2.
we report here the synthesis, affinity, and intrinsic H3R activity
of a variety of analogues of imbutamine and impentamine (1a
and 2a, Figure 1) in which the primary amino function has been
mono- or disubstituted with several aliphatic and aromatic
moieties. All the ligands were additionally screened for their
affinities at the human histamine H4 receptor (H4R) subtype in
order to evaluate H3 versus H4 receptor selectivity of the
compounds.
Pharmacology
Radioligand Displacement Studies. Homogenates of SK-
N-MC cells, stably expressing either the human H3R3 or the
human H4R,50 were used for determining ligand affinities for
the H3R and H4R, respectively, as previously described.51 Cell
homogenates of H3R-expressing cells (475 ( 32 fmol/mg of
protein) were incubated for 40 min at 25 °C with 0.9-1.1 nM
[3H]-NR-methylhistamine (82 Ci/mmol) in 50 mM sodium
phosphate buffer (pH 7.4) with or without competing ligands;
cell homogenates of H4R-expressing cells (620 ( 44 fmol/mg
of protein) were incubated for 60 min at 37 °C with 9-11 nM
[3H]histamine (23.2 Ci/mmol) in 50 mM Tris HCl (pH 7.4),
Chemistry
Aminoalkylimidazoles 1c,m and 2c,m were prepared by
lithiation of a suitable 1,2-diprotected imidazole 445 and
subsequent treatment with 2-(ω-iodoalkyloxy)tetrahydropyrans
to give compounds 5 and 6 in moderate yields (35-40%,
Scheme 1). The ω-hydroxy group of 5 and 6 was converted
into an aldehyde via a Swern oxidation46,47 followed by reductive
amination48 with the appropriate amine in the presence of
Scheme 2. Synthesis of Compounds 1b,d-l,n-p and 2b,e-h,j-l,n-pa
a Reagents: (a) MeOH, H2SO4, reflux; (b) Swern oxidation; (c) TosMIC, NaCN, abs EtOH; (d) NH3/EtOH, 120-140 °C, 10 atm; (e) HCl, reflux; (f)
EtOH, H2SO4, reflux; (g) triphenylmethyl chloride, triethylamine, DCM, rt; (h) LiAlH4, THF, rt; (i) Swern oxidation; (j) R1R2-amine, AcOH, NaBH(OAc)3,
DCE, rt (method B, reductive amination); (k) 1 M HCl or 1 M HBr, reflux (method C). R1R2-amines are given in Tables 1 and 2.

Modulation of the ActiVity of Histamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2551
Table 1. Chemical Structures and Pharmacological Properties of
with or without competing ligands. Incubations were terminated
by rapid dilution and subsequent filtration over Whatman GF/C
filters pretreated with 0.3% polyethyleneimine and the residue
was subsequently washed using ice-cold wash buffer (H3R: 25
mM Tris HCl, 145 mM NaCl, pH 7.4 at 4 °C; H4R: 50 mM
Tris HCl, pH 7.4 at 4 °C). The radioactivity retained on the
filters was measured by liquid scintillation counting. Nonspecific
binding was determined with 1 µM clobenpropit as competing
ligand. Competition isotherms were evaluated by a nonlinear,
least squares curve-fitting using GraphPad Prism (GraphPad
Software, Inc., San Diego, CA).
Colorimetric cAMP Assay. Colorimetric cAMP assays were
performed as previously described.51 Briefly, SK-N-MC cells
stably expressing the human H3R3 and a cyclic AMP responsive
element (CRE)-â-galactosidase reporter gene were grown
overnight in 96-well plates before the assay. To start the assay,
the cells were incubated for 6 h with 1 µM forskolin and
respective ligands in a humidified incubator at 37 °C. Thereafter,
the medium was aspirated and cells were incubated overnight
at room temperature with 100 µL of assay buffer [100 mM
NaH2PO4, 100 mM Na2HPO4, pH 8, 2 mM MgSO4, 0.1 mM
MnCl2, 0.5% Triton, 40 mM â-mercaptoethanol, and 4 mM
o-nitrophenyl-â-D-galactopyranoside (ONPG)]. The absorbance
at 405 nm was determined by using a Victor2 plate reader
(Perkin-Elmer).
Compounds 1a-p for the Human H3 (hH3R) and H4 (hH4R) Receptorsa
hH3R
hH4R:
pKi
b
compd R1
R2
pKib
pEC50
Rb,c
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
H
H
H
H
H
H
H
H
H
H
H
Et
8.4 ( 0.1 8.3 ( 0.1 0.98 ( 0.02
7.6 ( 0.1 7.6 ( 0.1 0.73 ( 0.10
8.0 ( 0.1
7.0 ( 0.1
7.3 ( 0.1
6.7 ( 0.1
7.2 ( 0.1
n-Pr
i-Pr
n-Bu
i-Bu
t-Bu
8.1 ( 0.1 7.8 ( 0.1 0.45 ( 0.07
d
7.1 ( 0.1
-
0.10 ( 0.08
8.2 ( 0.1 8.3 ( 0.1 0.55 ( 0.09
8.5 ( 0.1 8.1 ( 0.1e -0.14 ( 0.10 7.1 ( 0.1
d
6.2 ( 0.1
-
-
-
-
-
0.01 ( 0.06
6.1 ( 0.1
7.4 ( 0.1
7.0 ( 0.1
7.3 ( 0.0
7.7 ( 0.1
6.1 ( 0.1
6.5 ( 0.1
6.3 ( 0.1
6.4 ( 0.2
6.3 ( 0.1
f
d
cyclopentyl 7.4 ( 0.1
cyclohexyl 7.5 ( 0.1
cycloheptyl 7.4 ( 0.1
p-Cl-benzyl 7.8 ( 0.1
-
-
f
d
d
f
0.00 ( 0.05
d
-
Me Me
Et Et
-(CH2)4-
-(CH2)5-
-(CH2)6-
8.5 ( 0.1 8.9 ( 0.1 0.94 ( 0.04
d
7.0 ( 0.1
-
0.07 ( 0.08
7.4 ( 0.1 7.4 ( 0.1 0.47 ( 0.11
7.7 ( 0.1 7.5 ( 0.1 0.64 ( 0.04
7.4 ( 0.1 7.3 ( 0.2 0.35 ( 0.07
a The values are expressed as means ( SEM of separate experiments,
each performed in triplicate, unless indicated otherwise. b N > 3. c R )
intrinsic activity value. d Could not be estimated. e pIC50 value. f Biphasic
behavior (partial agonism up to 1 µM, non-H3R-mediated effects at 10 and
100 µM).
Analytical Methods. Protein levels were determined spec-
trophotometrically by a Packard Argus 400 microplate reader
according to the method of Bradford,52 using bovine serum
albumin as a standard. All data shown are expressed as a mean
( SEM.
Table 2. Chemical Structures and Pharmacological Properties of
Compounds 2a-p for the Human H3 (hH3R) and H4 (hH4R) Receptorsa
hH3R
hH4R:
pKi
Results and Discussion
compd R1
R2
pKib
pEC50
Rb,c
b
A previous study from our group identified compound 2d
(Figure 1 and Table 2), an impentamine analogue with an
isopropyl group at the amino group of the side chain, as a neutral
H3R antagonist41 in SK-N-MC cells stably transfected with the
human H3R.3 In the same study it was also shown that the
modification of the primary amino group of the H3R agonist
impentamine, via substitution or incorporation in a piperidine
ring, dramatically affected the pharmacological activity of the
ligands, resulting in a series of compounds spanning the whole
spectrum of pharmacological activities.
The amine function of impentamine is thought to interact with
the aspartate residue Asp114 in transmembrane domain 3, which
is highly conserved in the family of biogenic amine recep-
tors.53,54
In view of the fact that this series of ligands may be of great
help to understand the mechanism of receptor (in)activation,
we have prepared a series of imbutamine and impentamine
analogues in which the primary amino function has been mono-
or disubstituted with either a variety of aliphatic (1b-g,l,m,
2b-g,l,m), alicyclic (1h-j, 2h-j), or aromatic moieties (1k,
2k) or incorporated in cyclic systems (1n-p, 2n-p).
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
H
H
H
H
H
H
H
H
H
H
H
Et
8.3 ( 0.1 8.3 ( 0.1
0.71 ( 0.05 6.6 ( 0.1
0.11 ( 0.09 6.5 ( 0.1
0.44 ( 0.11 6.8 ( 0.1
d
8.1 ( 0.1
-
n-Pr
i-Pr
n-Bu
i-Bu
t-Bu
8.3 ( 0.1 8.0 ( 0.1
7.9 ( 0.1 7.4 ( 0.1e -0.55 ( 0.11 6.9 ( 0.1
8.4 ( 0.1 8.3 ( 0.1
0.55 ( 0.09 6.7 ( 0.1
0.35 ( 0.07 6.9 ( 0.1
0.04 ( 0.05 7.2 ( 0.1
0.02 ( 0.08 7.4 ( 0.1
8.8 ( 0.1 8.1 ( 0.2
d
7.4 ( 0.1
-
-
d
cyclopentyl 8.4 ( 0.1
cyclohexyl 8.0 ( 0.1 7.9 ( 0.1e -0.51 ( 0.12 7.4 ( 0.1
cycloheptyl 8.5 ( 0.1 8.0 ( 0.1e -0.65 ( 0.03 7.3 ( 0.1
p-Cl-benzyl 8.6 ( 0.1 8.9 ( 0.2e -0.97 ( 0.01 7.2 ( 0.2
Me Me
Et Et
-(CH2)4-
-(CH2)5-
-(CH2)6-
7.8 ( 0.2 7.5 ( 0.2
0.68 ( 0.08 6.3 ( 0.1
0.06 ( 0.02 6.2 ( 0.1
0.32 ( 0.07 6.1 ( 0.1
0.46 ( 0.08 6.5 ( 0.1
0.22 ( 0.07 6.8 ( 0.1
d
7.2 ( 0.1 -
7.5 ( 0.1 7.5 ( 0.1
8.1 ( 0.1 7.7 ( 0.1
7.8 ( 0.1 7.3 ( 0.2
a The values are expressed as means ( SEM of separate experiments,
each performed in triplicate, unless indicated otherwise. b N > 3. c R )
intrinsic activity value. d Could not be estimated. e pIC50 value.
of impentamine, 2l (Table 2), shows instead reduced affinity
and potency, with a pKi value of 7.8 and a pEC50 value of 7.5,
as well as a diminished intrinsic H3R activity, resulting in partial
H3R agonism (R ) 0.68). It must be noted, however, that the
parent compound 2a, opposite to what was observed in our
previous study, where it behaved as a nearly full agonist (R )
0.9),41 is now found to be also a partial agonist (R ) 0.71),
with almost the same intrinsic activity as 2l. A discussion of
these differences is described below. A further increase of the
steric hindrance on the primary amine, as achieved via introduc-
tion of a diethylamino group, is generally not well-tolerated, as
shown by the decreased H3R affinities of compounds 1m and
2m. Parallel to the decrease in the H3R affinity, a diminished
H3R intrinsic activity is observed; in fact, both ligands are
neutral H3R antagonists.
As shown in Table 1, substitution of the NR-position of
imbutamine (1a) caused in general a decrease in the H3R
affinity, with the exception of compounds 1f, bearing an isobutyl
substituent, and NR,NR-dimethylimbutamine (1l), in which the
terminal amino function is dimethylated. With a pEC50 of 8.9,
1l is the most potent ligand of this series and it displays a nearly
full agonistic intrinsic activity (R ) 0.94) on the human H3R.
This behavior has already been observed in the case of
histamine, where mono- or dimethylation of the amino function
of the side chain results in agonists that are more active than
histamine at the H3R.5,55 Surprisingly, the dimethylated analogue

2552 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
GoVoni et al.
Figure 2. The effects of several selected compounds on the basal
signaling of the H3R, as measured by CRE-mediated â-galactosidase
reporter gene assay in forskolin-stimulated (1 µM) SK-N-MC cells.
(A) Dose-response curves of the full H3R agonist 1a (9, imbutamine),
the partial H3R agonists 1b (2) and 1c (1), the neutral H3R antagonist
1g ([), and the partial inverse H3R agonists 1f (b) (see also Table 1).
(B) Dose-response curves of the partial H3R agonists 2a (9, impen-
tamine) and 2c (2), the neutral H3R antagonist 2h ([), the partial
inverse H3R agonist 2d (1), and the full inverse H3R agonist 2k (b)
(see also Table 2). Representative dose-response curves of the full
H3R agonist histamine (4) and of the partial inverse H3R agonist
thioperamide (0) are shown (A and B). Data are normalized to the
basal signaling observed in the assay (set to 100%).
Figure 3. (A) Rightward shift of concentration-response curves of
imbutamine (1a) in the presence of increasing concentrations of NR-
cyclopentylimpentamine (2h) determined by the inhibition of the CRE-
stimulated â-galactosidase transcription in SK-N-MC cells stably
expressing the human H3R. (B) The log of the (dose ratio - 1) at each
concentration of antagonist was plotted versus the log of 2h concentra-
tion. The pA2 value was determined from the x-intercept on the linear
Schild regression plot and was in this particular experiment 8.1. The
data points represent means ( SEM of triplicate determinations from
one representative experiment.
In the impentamine series shown in Table 2, compounds 2c,
2e, 2f, 2h, 2j, and 2k display an H3R affinity comparable to
that of the parent compound 2a or slightly increased.
exhibits the highest H3R affinity among this series of ligands.
Unfortunately, no definite correlation between the alkyl residue
and the intrinsic H3R activity could be observed. In fact, 1f
shows partial inverse agonism (R ) -0.14, Table 1), whereas
2f is a partial agonist (R ) 0.35, Table 2).
Upon comparing imbutamine and impentamine derivatives,
it can be appreciated that, with the exception of the parent
compounds (1a, 2a) and those bearing a dimethyl substitution
(1l, 2l), the H3R affinities are higher for the impentamine
analogues. Alkylation in the NR-position of 1a and 2a with
longer alkyl groups, such as ethyl, n-propyl, or n-butyl, has only
limited effect on the H3R affinity in both series (1b,c,e and
2b,c,e), while ramification to isopropyl (1d, 2d) or tert-butyl
(1g, 2g) is detrimental for H3R affinity (see Tables 1 and 2).
Alkylation of the histamine side-chain terminal nitrogen with
bulky groups leads to a loss of H3R intrinsic activity, generating
partial agonists56 or antagonists.57 Similarly, alkylation of
imbutamine and impentamine with the above-mentioned resi-
dues, e.g. ethyl, n-propyl, isopropyl, n-butyl, and tert-butyl,
causes a decrease in the intrinsic activity of the compounds
(Figure 2), therefore generating partial agonists (1b,c,e and 2c,e)
or neutral antagonists (1d,g and 2b,g, Tables 1 and 2).
Remarkably, compound 2d (Table 2), also bearing an isopropyl
moiety, which was identified as a neutral antagonist (R ) -0.10
( 0.10) in our previous study,41 in the present research behaves
as a partial inverse agonist (R ) -0.55). However, a different
assay system has been used in the current study that is more
sensitive than the classical cAMP measurements used in the
previous study.41 Thus, enhancement of the van der Waals
volume of the NR-position diminishes agonist properties and
increases antagonistic activity, but no definite relation between
the substituting alkyl residue and the intrinsic H3R activity of
the compound can be derived. Surprisingly, ramification to
isobutyl (1f and 2f) generates compounds with higher H3R
affinity; in fact, compound 2f (Table 2), with a pKi of 8.8,
Substitution with alicyclic residues (cyclopentyl, cyclohexyl
or cycloheptyl) exerts a different behavior for imbutamine and
impentamine derivatives; in the former, alkylation with either
of the moieties consistently produces a 10-fold decrease in the
H3R affinity (1h-j, Table 1), whereas in the latter H3R affinities
are not significantly altered (2h-j, Table 2). As already seen
in case of alkylation with aliphatic residues, insertion of alicyclic
residues results in a loss of agonistic activity compared to the
unsubstituted compounds; in fact, 1j and 2h are neutral
antagonists, whereas 2i and 2j are partial inverse agonists.
In view of its nanomolar affinity for the H3R (pKi ) 8.4)
and lack of intrinsic H3R activity (R ) 0.02 ( 0.08), NR-
cyclopentylimpentamine (2h, Table 2) can be considered an
interesting pharmacological tool. As depicted in Figure 3, ligand
2h effectively antagonizes the response to the full agonist
imbutamine (1a, Figure 3), yielding a pA2 value of 8.1.
Ligands 1h and 1i (Table 1), respectively alkylated with a
cyclopentyl and a cyclohexyl moiety, are also showing a
diminished H3R agonistic activity; in fact, they behave as partial
agonists at concentrations up to 1 µM, but they turn into apparent
(partial) inverse agonists at higher concentrations (10 and 100
µM). This “biphasic” behavior is probably due to non-H3R-
mediated effects at high concentrations that interfere with an
accurate estimation of the intrinsic H3R activity of the afore-
mentioned compounds in our cell-based assay. Further experi-
ments showed indeed that responses to 1h and 1i in SK-N-MC
cells transfected with the human H3R could not be antagonized
by the neutral H3R antagonist 2h (data not shown).

Modulation of the ActiVity of Histamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2553
Introduction of para-substituted phenyl rings has proven to
be a successful strategy to improve affinity and/or potency of
the compounds, as seen with the very potent H3R antagonist
clobenpropit (Figure 1).1,58 However, introduction of a p-
chlorobenzyl moiety on the primary amino function of 1a and
2a, yielding ligands 1k and 2k (Tables 1 and 2), results in an
increased affinity only in the case of the impentamine derivative
2k (about 4-fold more active than the parent compound 2a,
(about 40%) to the H3R cDNA,67 and it shares about 60%
sequence identity with the H3R in the transmembrane regions,50
suggesting that the two receptors might have a similar mode of
ligand recognition. Although the development of selective H3R
ligands is not the primary goal of this study, the synthesized
compounds were additionally screened for their affinity for the
human H4R in order to investigate the H3/H4 receptor selectivity
of the synthesized compounds. The affinity of most of the
compounds is lower for the human H4R than for the human
H3R (Tables 1 and 2). Impentamine (2a) and its ethyl (2b),
n-butyl (2e), and isobutyl (2f) derivatives show a high selectivity
for the H3R over H4R, the difference in affinity between the
two receptors ranging between 1.6 and 1.9 log units (Table 2),
but the best H3R selectivity among the synthesized compounds
is displayed by NR,NR-dimethylimbutamine 1l (Table 1), with
2.4 log units difference between the two receptors’ affinities
(pKi ) 8.5 on H3R versus 6.1 on H4R). Excluding ligands
1a,d,g,h,j,k (Table 1) and 2g (Table 2), which have almost the
same affinity for both receptors and therefore lack H3/H4
receptor selectivity, all the other compounds of the series bear
a selectivity that ranges between 0.5 and 1.5 log units for the
H3 over the H4 receptor (Tables 1 and 2).
Table 2). The potency of ligand 2k is also enhanced (pEC50
)
8.9), but remarkably a huge decrease in the intrinsic H3R activity
of the compound can be observed as well, leading to the only
full inverse agonist (R ) -0.97) identified among the ligands
investigated in this study.
Incorporation of the NR-amino group of histamine into a
pyrrolidine nucleus led to a compound with reduced potency
and a maximal response that was only 56% with respect to
histamine, implying that this compound is a partial H3R
agonist.59 Similarly, incorporation of the amino group of
imbutamine and impentamine in a pyrrolidine (1n, 2n), piperi-
dine (1o, 2o), or azepane (1p, 2p) ring generates ligands with
reduced potency and intrinsic H3R activity (Tables 1 and 2),
all the compounds being partial agonists with R values ranging
from 0.22 (2p) to 0.64 (1o). Ligand 2o (Table 2), a partial
agonist in this study, displaying an R value of 0.46, was
previously found to behave as a full agonist (R ) 1.00).41
Parallel to a diminished H3R potency and intrinsic activity, these
ligands also show H3R affinities that are decreased by 4-10-
fold, excluding compound 2o, for which the decrease in affinity
was less marked (pKi of 8.1 vs a pKi value of 8.3 for 2a). The
only apparent relation between the structures of the compounds
and their activities is that affinity and potency values are slightly
higher for the impentamine derivatives 2n-p (Table 2) than
for ligands 1n-p (Table 1), while the opposite behavior could
be observed for the intrinsic activity, compounds 1n-p display-
ing higher partial agonistic activity over 2n-p.
When compared to our previous study,41 three of the six
compounds analyzed in that research showed a different intrinsic
H3R activity. More precisely, impentamine (2a) and NR-
pyrrolidine-impentamine (2o) behaved in the present study as
partial agonists (R values respectively 0.78 and 0.46, Table 2)
whereas they were identified as full agonists (R ) 0.9 and 1.00)
in the previous one. NR-isopropylimpentamine (2d), earlier
recognized as a neutral antagonist, now behaved as a partial
inverse agonist (R ) -0.55, Table 2). The other three (2i,k,l,
Table 2) showed instead values comparable to those formerly
measured.
Conclusions
The synthesis of a series of imbutamine and impentamine
analogues in which the primary amino function has been mono-
or disubstituted with several aliphatic, alicyclic, and aromatic
moieties, as well as incorporated in cyclic systems, has been
reported in this study. The novel ligands were pharmacologically
investigated in vitro for their affinities on the human H3R and
H4R subtypes and for their intrinsic H3R activities.
Modification of the primary amine function of 1a and 2a
dramatically affects the H3R binding and functional properties
of the compounds. Subtle changes in the substitution of the side
chain nitrogen have great influence on the H3R pharmacological
activities of the ligands: agonists, antagonists, and inverse
agonists are in fact present in this series of compounds.
Previously, we had indeed proposed that the side chain amine
function of impentamine (2a) interacts with the aspartate residue
Asp114 in transmembrane domain 3.53 We hypothesized that
rotameric changes of the side chain of this amino acid residue
could influence the agonistic properties of the interacting
ligands.41
However, substitution with the same moiety on the two parent
compounds (1a and 2a) yields ligands with different structure-
activity relationships (SAR) at the H3R, depending on the length
of the side chain. This is possibly due to the existence of diverse
binding pockets and a different set of receptor site points
available for interaction with derivatives of 1a and 2a that are
very much dependent on the distance between the basic nitrogen
of the side chain and the imidazole nucleus, as well as on the
kind of substituting moiety present on the aforementioned basic
nitrogen. Our group already proposed that the differences in
SAR observed for the lipophilic tails of different classes of H3R
antagonists could be explained by the existence of two distinct
lipophilic pockets available for antagonist binding.53 The
obtained qualitative model for the H3R revealed a molecular
determinant explaining intrinsic activity, as the conformation
of the aspartic acid residue differs according to whether it is
binding to agonists or antagonists.53
However, it is important to point out that impentamine was
initially recognized as an H3R antagonist44 and subsequently
proved to act also as a partial agonist, depending on the test
system.60
More recently, Fox and co-workers61 have reported the partial
agonistic behavior of GT-2331 (cipralisant, Perceptin, Figure
1), so far presumed to be an H3R antagonist.62 Furthermore,
Gbahou et al.63 identified proxyfan (Figure 1) as a protean H3
receptor ligand with the ability to display agonism, neutral
antagonism, and inverse agonism, depending on the test system.
Thus, the complex pharmacodynamic profile of the discussed
compounds may be caused by different assay and species
differences. The distribution of distinct receptor active states
or alternative coupling routes in different cells or tissue may
also play a role.
Several neutral H3R antagonists (1d,g,j,m and 2b,g,h,m) have
been identified within this series, but also a very potent and
selective H3R agonist (1l) and a very potent, though not highly
selective, H3R inverse agonist (2k). It has proven very difficult
Recently, another histamine receptor subtype, the H4R, was
cloned by several groups and identified as a G-protein coupled
receptor.4,50,64-68 The H4R has significant sequence homology

2554 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
GoVoni et al.
dichloromethane (DCM) (50 mL) was cooled to -60 °C. A solution
of DMSO (2.79 g, 0.036 mol) in anhydrous dichloromethane (10
mL) was added dropwise. After stirring for 10 min, a solution of
5 (2.94 g, 0.012 mol) in dichloromethane (15 mL) was added
dropwise. After additional stirring for 15 min, triethylamine (9.03
g, 0.089 mol) was added. The mixture was allowed to warm to
room temperature and poured into water. The aqueous layer was
extracted with dichloromethane, and the combined organic layers
were washed successively with dilute sodium carbonate (5%), water,
and brine and then dried (MgSO4) and concentrated in vacuo to
yield 2.72 g (93%) of product as a mixture of the 4- and 5-isomers
to draw SAR among these series of ligands, since there does
not seem to be a clear correlation between the modifications
applied to the side chain nitrogen and the different H3R affinities
and functional activities. Very small modifications can lead to
dramatic changes in the affinity as well as in the functional
activity of the generated ligands. In this perspective, the side
chain nitrogen can be seen as a very subtle chemical switch for
the modulation of the functional activity of higher homologues
of histamine on the human H3R.
The target compounds were additionally investigated for their
affinities on the human H4R subtype in order to check
selectivity, which led to the identification of NR,NR-dimeth-
ylimbutamine (1l), showing an H3R/H4R affinity ratio of 2.4
log units, as a potent and selective agonist for the human H3R.
The H3R is one of the few GPCRs that has been shown to
modulate important physiological processes by means of its
constitutive activity.40 This underlines not only the impact of
assay setup and species differences when evaluating the behavior
of ligands on the histamine H3R but also the usefulness of the
compounds presented in this study as pharmacological tools.
In view of the many potential therapeutic applications of H3R
antagonists,16,73,74 the availability of high-affinity neutral H3R
antagonists such as NR-ethylimpentamine 2b and NR-cyclopen-
tylimpentamine 2h as well as of the potent inverse H3R agonist
NR-p-chlorobenzylimpentamine 2k may help in unraveling the
effects and clinical applications of inverse H3R agonists or
neutral H3R antagonists.
1
(12.5:87.5). H NMR (CDCl3): δ 1.95 (m, 4H, central CH2’s, 4-
+ 5-isomer), 2.50 (m, 2H, CH2CHO 4- + 5-isomer), 2.70 (t, 2H,
imidazole-5(4)-CH2, 4- + 5-isomer), 2.78 + 2.82 (s, 6H, N(CH3)2,
4- + 5-isomer), 6.78 + 6.90 (s, 1H, imidazole-4(5)H, 4- +
5-isomer), 7.75 + 7.80 (s, 1H, imidazole-2H, 4- + 5-isomer), 9.70
+ 9.75 (t, 1H, CHO 4- + 5-isomer).
5-Hydroxypentanoic Acid Methyl Ester (12). δ-Valerolactone
(47.17 g, 0.47 mol) in methanol (300 mL) and concentrated H2-
SO4 (4 mL) was heated to reflux for 16 h. The mixture was cooled
to room temperature and the solvent was removed in vacuo. The
residue was dissolved in dichloromethane, washed three times with
a saturated solution of NaHCO3, dried over MgSO4, and evaporated
to dryness, yielding 46.91 g (76%) of product. 1H NMR (CDCl3):
δ 1.48-1.80 (m, 4H, central CH2’s), 2.30 (m, 2H, CH2COOCH3),
3.58 (m, 3H, CH2OH + CH3OCO).
4-[4-(Toluene-4-sulfonyl)-4,5-dihydrooxazol-5-yl]butyric Acid
Methyl Ester (16). To a stirred suspension of tosyl methyl
isocyanide (TosMIC) (49.68 g, 0.25 mol) and 14 (33.15 g, 0.25
mol) in absolute ethanol (800 mL) was added finely powdered
sodium cyanide (1.24 g, 0.025 mol). The yellow-orange suspension
was stirred for 30 min, filtered, and washed with hexane/ether (1:
Experimental Section
1
1), to give 40.06 g (49%) of product. H NMR (CDCl3): δ 1.75
General Procedures. Reagents were obtained from commercial
suppliers and used without further purification. Solvents used were
either AR or HPLC grade. Dry THF and dichloromethane were
freshly distilled from LiAlH4 and CaH2, respectively. Compounds
1a and 2a were obtained from our own laboratory stock.44 The
2-(ω-iodoalkyloxy)tetrahydropyrans were prepared by refluxing the
corresponding 2-(ω-chloroalkyloxy)tetrahydropyrans69 with 1.1
equiv of sodium iodide in acetone and purified by distillation.70,71
Melting points were measured on an Electrothermal IA 9200
(m, 4H, 2 × CH2), 2.36 (m, 2H, 1 × CH2), 2.46 (s, 3H, p-CH3),
3.55 (s, 3H, CH3OCO), 4.79 (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).
4-(1H-Imidazol-4-yl)butyric Acid Ethyl Ester (18). In a
resealable pressure tube, a solution of 16 in absolute ethanol
saturated with ammonia was heated between 120 and 140 °C. After
20 h the reaction mixture was cooled to room temperature and the
solvent was removed in vacuo. The residue (45.67 g) was dissolved
in HCl (37%, 300 mL) and heated to reflux for 16 h. The mixture
was cooled to room temperature, evaporated to dryness, dissolved
in water, washed with ether, then concentrated in vacuo. The residue
in ethanol (300 mL) and concentrated H2SO4 (ca. 3 mL) was heated
to reflux. After 16 h the mixture was cooled to room temperature
and evaporated to dryness. The residue was dissolved in water and
washed with ether, and then the water layer was basified with
NaHCO3 and extracted with dichloromethane. The combined
organic extracts were dried over MgSO4 and evaporated in vacuo,
1
apparatus. H NMR spectra were recorded on a Bruker AC-200
(200 MHz) spectrometer. Chemical shifts are given in ppm using
the residual undeuterated solvent as reference. Elemental analyses
were performed by the Mikroanalytisches Labor Pascher, Remagen-
Bandorf, Germany. Flash chromatography was performed on J.T.
Baker Kieselgel 60. All reactions were performed under an
atmosphere of dry nitrogen.
5(4)-(4-Hydroxybutyl)imidazole-1-sulfonic Acid Dimethyl-
amide (5). 2-(tert-Butyldimethylsilanyl)imidazole-1-sulfonic acid
dimethylamide 445 (8.59 g, 0.030 mol) was dissolved in THF (100
mL) and cooled to -70 °C. n-Butyllithium in hexane (20.35 mL,
0.033 mol) was added dropwise at -65 °C. After 30 min, a solution
of 2-(4-iodobutyloxy)tetrahydropyran (8.42 g, 0.030 mol) in THF
was added dropwise and the mixture was gradually allowed to warm
to room temperature overnight. The solution was acidified with
HCl (6 M) and stirred for 3 h, and then the solvents were removed
in vacuo. The residue was diluted with water, basified with K2-
CO3, and extracted with dichloromethane. The organic layers were
combined, washed with brine, dried (MgSO4), and concentrated in
vacuo. The product was purified by flash chromatography by
gradually switching the eluent from ethyl acetate to ethyl acetate/
methanol (9/1) to afford 2.94 g (40%) of product as a mixture of
1
yielding 18.46 g (82%) of product. H NMR (CDCl3): δ 1.20 (t,
3H, CH3CH2OCO), 1.95 (m, 2H, central CH2), 2.32 (t, 2H, CH2-
COO), 2.62 (t, 2H, imidazole-CH2), 4.05 (q, 2H, CH3CH2OCO),
6.78 (s, 1H, imidazole-5H), 7.65 (s, 1H, imidazole-2H), 10.08 (s,
1H, imidazole-NH).
4-(1-Trityl-1H-imidazol-4-yl)butyric Acid Ethyl Ester (20).
Compound 18 (15.20 g, 0.083 mol) was dissolved in dichlo-
romethane (200 mL). Triphenylmethyl chloride (23.03 g, 0.083 mol)
in dichloromethane (200 mL) was added dropwise, followed by
triethylamine (17.18 mL, 0.12 mol). The reaction mixture was
stirred at room temperature overnight, and then it was washed
successively with an aqueous solution of K2CO3 (1 M) and water.
The organic layer was dried over MgSO4 and then concentrated
under reduced pressure to give 34.22 g (97%) of product. 1H NMR
(CDCl3): δ 1.20 (t, 3H, CH3CH2OCO), 1.95 (m, 2H, central CH2),
2.30 (t, 2H, CH2COO), 2.55 (t, 2H, imidazole-CH2), 4.05 (q, 2H,
CH3CH2OCO), 6.50 (s, 1H, imidazole-5H), 7.00-7.38 (m, 16H,
imidazole-2H + Ph3C).
1
the 4- and 5-isomers (10:90). H NMR (CDCl3): δ 1.70 (m, 4H,
central CH2’s 4- + 5-isomer), 1.93 (s, 1H, OH, 4- + 5-isomer),
2.60 + 2.75 (t, 2H, imidazole-5(4)-CH2, 4- + 5-isomer), 2.80 +
2.85 (s, 6H, N(CH3)2, 4- + 5-isomer), 3.65 (t, 2H, CH2OH 4- +
5-isomer), 6.80 + 6.92 (s, 1H, imidazole-4(5)H, 4- + 5-isomer),
7.80 + 7.85 (s, 1H, imidazole-2H, 4- + 5-isomer).
5(4)-(4-Oxobutyl)imidazole-1-sulfonic Acid Dimethylamide
(7). A solution of oxalyl chloride (2.27 g, 0.018 mol) in anhydrous
4-(1-Trityl-1H-imidazol-4-yl)butan-1-ol (22). A suspension of
20 (34.22 g, 0.081 mol) in THF (200 mL) was added, with efficient

Modulation of the ActiVity of Histamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2555
stirring, to an ice-cold suspension of LiAlH4 (3.06 g, 0.081 mol)
in THF (50 mL). The reaction mixture was stirred for 15 min at
room temperature. Next, water (5 mL), followed by saturated K2-
CO3 solution (5 mL), was added dropwise. The suspension was
filtered and concentrated in vacuo to afford 25.81 g (84%) of
4H, central CH2’s), 2.70 (m, 2H, imidazole-CH2), 2.90-3.18 (m,
4H, CH2NHCH2), 7.20 (s, 1H, imidazole-5H), 8.55 (s, imidazole-
2H). Anal. (C9H17N3‚2C2H2O4) C, H, N.
[4-(1H-Imidazol-4-yl)butyl]propylamine Oxalic Acid Salt (1c).
Method D yielded 0.46 g (77%) of a white powder. Mp: 156-
158 °C. 1H NMR (D2O): δ 0.90 (t, 3H, CH3CH2CH2), 1.50-1.80
(m, 6H, CH3CH2CH2 + central CH2’s), 2.72 (t, 2H, imidazole-
CH2), 2.85-3.15 (m, 4H, CH2NHCH2), 7.19 (s, 1H, imidazole-
5H), 8.50 (s, imidazole-2H). Anal. (C10H19N3‚1.5C2H2O4‚1.5H2O)
C, H, N.
1
product. H NMR (CDCl3): δ 1.50-1.78 (m, 4H, central CH2’s),
2.50 (t, 2H, imidazole-CH2), 3.58 (t, 2H, CH2OH), 6.50 (s, 1H,
imidazole-5H), 7.00-7.40 (m, 16H, imidazole-2H + Ph3C).
General Procedures for the Synthesis of the Protected
Aminoalkylimidazoles 10c,m, 11c,d,i,m, 26b,d-l,n-p, and 27b,e-
h,j-l,n-p. Method A. One equivalent of 972 was mixed with 3
equiv of the appropriate amine and heated at 100 °C. After 48 h,
the excess amine was evaporated under reduced pressure and the
residue purified by flash chromatography (starting with ethyl
acetate/methanol (9/1) as eluent and gradually switching to methanol
and methanol/ammonia (95/5) for elution of the product).
Method B (Reductive Amination). One equivalent of the
required aldehyde (7, 8, 24, or 25) was stirred for 10 min with the
appropriate amine (3 equiv in the case of primary amines, 1 equiv
in the case of secondary amines) and 1 equiv of acetic acid in
dichloroethane (DCE) at room temperature. Then 1.5 equiv of
sodium triacetoxyborohydryde was added and the mixture stirred
overnight. Water was added and the reaction stirred for 15 min.
Dichloroethane was removed in vacuo, and the residue was basified
with K2CO3 and extracted with chloroform. The combined organic
layers were washed with brine, dried (MgSO4), and concentrated
in vacuo. The residue was then purified by flash chromatography
as described in method A.
Acknowledgment. The authors thank Dr. T. Lovenberg,
Johnson & Johnson Pharmaceutical Research and Development,
San Diego, CA, for the cells stably expressing the human
histamine H3 and H4 receptors.
Supporting Information Available: Elemental analyses for all
compounds, experimental details, data for compounds 6, 8, 13-
15, 17, 19, 21, 23-25, 10m, 11c,i,m, 26b,d-l, 26n-p, 27b,e-h,
27j-l,n-p, 1d-p, 2b-p, and 1H NMR and LC-MS spectra from
1f and 2d. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Leurs, R.; Smit, M. J.; Timmerman, H. Molecular Pharmacological
Aspects of Histamine Receptors. Pharmacol. Ther. 1995, 66, 413-
463.
(2) Hill, S. J.; Ganellin, C. R.; Timmerman, H.; Schwartz, J. C.; Shankley,
N. P.; Young, J. M.; Schunack, W.; Levi, R.; Haas, H. L. International
Union of Pharmacology. XIII. Classification of Histamine Receptors.
Pharmacol. ReV. 1997, 49, 253-278.
5(4)-(4-Propylaminobutyl)imidazole-1-sulfonic Acid Dimethyl-
amide (10c). Method B with aldehyde 7 and n-propylamine yielded
1
0.49 g (30%) as a mixture of 4- and 5-isomers (9:91). H NMR
(CDCl3): δ 0.82 (t, 3H, CH3 4- + 5-isomer), 1.30-1.72 (m, 6H,
CH2CH2CH3 4- and 5-isomer + central CH2’s, 4- and 5-isomer),
2.40-2.60 (m, 4H, CH2NHCH2 4- + 5-isomer), 2.68 (t, 2H,
imidazole-5(4)-CH2, 4- + 5-isomer), 2.78 + 2.82 (s, 6H, N(CH3)2,
4- + 5-isomer), 6.78 + 6.88 (s, 1H, imidazole-4(5)H, 4- +
5-isomer), 7.72 + 7.78 (s, 1H, imidazole-2H, 4- + 5-isomer).
5(4)-(5-Isopropylaminopentyl)imidazole-1-sulfonic Acid Di-
methylamide (11d). Method A with compound 9 and isopropy-
lamine yielded 1.50 g (36%). 1H NMR (CDCl3): δ 1.44-1.53 (m,
10H, (CH3)2CH + 2 × CH2), 1.57-1.75 (m, 2H, 1 × CH2), 1.81-
2.01 (m, 2H, 1 × CH2), 2.68 (t, 2H, imidazole-5(4)-CH2), 2.82-
2.92 (m, 9H, CH2NH + N(CH3)2 + CH(CH3)2), 6.79 (s, 1H,
imidazole-4(5)H), 7.82 (s, 1H, imidazole-2H).
General Procedures for the Deprotection of the Final Ami-
noalkylimidazoles 1b-p and 2b-p. Method C (Removal of the
Trityl Group). One equivalent of the protected aminoalkylimida-
zole was mixed with 10 equiv of hydrochloric acid (1 N) (1b,d-
l,n-p and 2b,e-h,k,l,n-p) or hydrobromic acid (1 N) (2j) and
the mixture was heated under reflux. After 2 h the mixture was
cooled, washed with diethyl ether, and concentrated in vacuo. The
salt was either washed with diethyl ether, recrystallized from
ethanol/ethyl acetate or ethanol/diethyl ether, or converted into the
oxalic acid or L-(+)-tartaric acid salt according to the following
procedure. The residue was dissolved in water, basified with K2-
CO3, and extracted with CHCl3. The combined organic layers were
dried (MgSO4) and concentrated in vacuo. The free base was
dissolved in ethyl acetate with a few drops of methanol and then
added dropwise to a solution of 2 equiv of oxalic acid dihydrate or
L-(+)-tartaric acid in ethyl acetate/methanol. The precipitate was
collected by filtration.
(3) Lovenberg, T. W.; Roland, B. L.; Wilson, S. J.; Jiang, X.; Pyati, J.;
Huvar, A.; Jackson, M. R.; Erlander, M. G. Cloning and Functional
Expression of the Human Histamine H3 Receptor. Mol. Pharmacol.
1999, 55, 1101-1107.
(4) Oda, T.; Morikawa, N.; Saito, Y.; Masuho, Y.; Matsumoto, S. I.
Molecular Cloning and Characterization of a Novel Type of
Histamine Receptor Preferentially Expressed in Leukocytes. J. Biol.
Chem. 2000, 275, 36781-36786.
(5) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Auto-Inhibition of Brain
Histamine Release Mediated by a Novel Class (H3) of Histamine
Receptors. Nature London 1983, 302, 832-837.
(6) Lovenberg, T. W.; Pyati, J.; Chang, H.; Wilson, S. J.; Erlander, M.
G. Cloning of Rat Histamine H3 Receptor Reveals Distinct Species
Pharmacological Profiles. J. Pharmacol. Exp. Ther. 2000, 293, 771-
778.
(7) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Autoregulation of
Histamine Release in Brain by Presynaptic H3 Receptors. Neuro-
science 1985, 15, 553-562.
(8) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Autoinhibition of
Histamine Synthesis Mediated by Presynaptic H3 Receptors. Neu-
roscience 1987, 23, 149-157.
(9) Clapham, J.; Kilpatrick, G. J. Histamine H3 Receptors Modulate the
Release of [3H]-Acetylcholine from Slices of Rat Entorhinal Cor-
tex: Evidence for the Possible Existence of H3 Receptor Subtypes.
Br. J. Pharmacol. 1992, 107, 919-923.
(10) Yokotani, K.; Murakami, Y.; Okada, S.; Wang, M.; Nakamura, K.
Histamine H3 Receptor-mediated Inhibition of Endogenous Acetyl-
choline Release from the Isolated, Vascularly Perfused Rat Stomach.
Eur. J. Pharmacol. 2000, 392, 23-29.
(11) Schlicker, E.; Fink, K.; Detzner, M.; Gothert, M. Histamine Inhibits
Dopamine Release in the Mouse Striatum via Presynaptic H3
Receptors. J. Neural Transm. Gen. Sect. 1993, 93, 1-10.
(12) Schlicker, E.; Schunack, W.; Gothert, M. Histamine H3 Receptor-
mediated Inhibition of Noradrenaline Release in Pig Retina Discs.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 1990, 342, 497-501.
(13) Schlicker, E.; Betz, R.; Gothert, M. Histamine H3 Receptor-mediated
Inhibition of Serotonin Release in the Rat Brain Cortex. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1988, 337, 588-590.
(14) Krause, M.; Stark, H.; Schunack, W. Medicinal Chemistry of
Histamine H3 Receptor Agonists. The Histamine H3 Receptor, A
Target for New Drugs; Elsevier: Amsterdam, 1998.
Method D (Removal of the Dimethylsulfamoyl Group). The
same procedure as described in method A was adopted, but 30%
HBr (1c,m and 2c,d,i,m) was used and the mixture was refluxed
for 16 h. The dihydrobromic acid salt was either washed with diethyl
ether, recrystallized from ethanol/ethyl acetate or ethanol/ diethyl
ether, or converted into the oxalic acid or tartaric acid salt as
described above.
Ethyl[4-(1H-imidazol-4-yl)butyl]amine Oxalic Acid Salt (1b).
Method C yielded 0.011 g (20%) of a white powder. Mp: 135-
137 °C. 1H NMR (D2O): δ 1.20 (t, 3H, CH3CH2), 1.58-1.85 (m,
(15) Onodera, K.; Watanabe, T. Histamine H3 Antagonists as Potential
Therapeutics in the CNS. The Histamine H3 Receptor, A Target for
New Drugs; Elsevier: Amsterdam, 1998.
(16) Leurs, R.; Blandina, P.; Tedford, C.; Timmerman, H. Therapeutic
Potential of Histamine H3 Receptor Agonists and Antagonists. Trends
Pharm. Sci. 1998, 19, 177-183.

2556 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
GoVoni et al.
(17) Miyazaki, S.; Imaizumi, M.; Onodera, K. Effects of Thioperamide,
a Histamine H3 Antagonist, on a Scopolamine-Induced Learning
Deficit Using an Elevated Plus-Maze Test in Mice. Life Sci. 1995,
57, 2137-2144.
(18) Onodera, K.; Miyazaki, S.; Imaizumi, M.; Stark, H.; Schunack, W.
Improvement by FUB 181, a Novel Histamine H3-Receptor
Antagonist, of Learning and Memory in the Elevated Plus-maze Test
in Mice. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1998, 357, 508-
513.
(19) Passani, M. B.; Cangioli, I.; Bacciottini, L.; Mannaioni, P. F.
Thioperamide and Cimetidine Modulate Acetylcholine Release from
the Amygdala of Freely Moving Rats. Inflamm. Res. 2000, 49, S43-
44.
(20) Yokoyama, H.; Onodera, K.; Iinuma, K.; Watanabe, T. Effect of
Thioperamide, a Histamine H3 Receptor Antagonist, on Electrically
Induced Convulsions in Mice. Eur. J. Pharmacol. 1993, 234, 129-
133.
(21) Yokoyama, H.; Onodera, K.; Maeyama, K.; Sakurai, E.; Iinuma, K.;
Leurs, R.; Timmerman, H.; Watanabe, T. Clobenpropit (VUF 9153),
a New Histamine H3 Receptor Antagonists, Inhibits Electrically
Induced Convulsions in Mice. Eur. J. Pharmacol. 1994, 260, 23-
28.
(22) Schlicker, E.; Marr, I. The Moderate Affinity of Clozapine at H3
Receptors Is Not Shared by Its Two Major Metabolites and by
Structurally Related and Unrelated Atypical Neuroleptics. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1996, 353, 290-294.
(23) Pillot, C.; Ortiz, J.; Heron, A.; Ridray, S.; Schwartz, J.-C.; Arrang,
J. M. Cyproxifan, a Histamine H3 Antagonist/Inverse Agonist,
Potentiates Neurochemical and Behavioral Effects of Haloperidol in
the Rat. J. Neurosci. 2002, 22, 7272-7280.
(24) Sakata, T.; Yoshimatsu, H.; Kurokawa, M. Hypothalamic Neuronal
Histamine: Implications of Its Homeostatic Control of Energy
Metabolism. Nutrition 1997, 13, 403-411.
(25) Takahashi, K.; Suwa, H.; Ishikawa, T.; Kotani, H. Targeted Disruption
of H3 Receptors Results in Changes in Brain Histamine Tone Leading
to an Obese Phenotype. J. Clin. InVest. 2002, 110, 1791-1799.
(26) Bray, G. A.; Tartaglia, L. A. Medicinal Strategies in the Treatment
of Obesity. Nature 2000, 404, 672-677.
(27) Levi, R.; Smith, N. C. Histamine H3 Receptors: A New Frontier in
Myocardial Ischemia. J. Pharmacol. Exp. Ther. 2000, 292, 825-
830.
(28) McLeod, R. L.; Aslanian, R.; Del Prado, M.; Duffy, R.; Egan, R.
W.; Kreutner, W.; McQuade, R.; Hey, J. A. Sch 50971, an Orally
Active Histamine H3 Receptor Agonist, Inhibits Central Neurogenic
Vascular Inflammation and Produces Sedation in the Guinea Pig. J.
Pharmacol. Exp. Ther. 1998, 287, 43-50.
(29) Bertaccini, G.; Coruzzi, G.; Poli, E. The Histamine H3 Receptor: A
Novel Prejunctional Receptor Regulating Gastro-Intestinal Function.
Aliment Pharmacol. Ther. 1991, 5, 585-591.
(30) Mignot, E.; Taheri, S.; Nishino, S. Emerging Therapeutic Targets
for Sleep Disorders. Nature Neurosci. 2002, 5, 1071-1075.
(31) Costa, T.; Ogino, Y.; Munson, P. J.; Onaran, O.; Rodbard, D. Drug
Efficacy at Guanine Nucleotide-Binding Regulatory Protein-Linked
Receptors: Thermodynamics Interpretation of Negative Antagonism
and of Receptor Activity in the Absence of Ligand. Mol. Pharmacol.
1992, 41, 549-560.
(32) Lefkowitz, R. L.; Cotecchia, S.; Samama, P.; Costa, T. Constitutive
Activity of Receptors Coupled to Guanine Nucleotide Regulatory
Proteins. Trends Pharmacol. Sci. 1993, 14, 303-307.
(33) Milligan, G.; Bond, R. A.; Lee, M. Inverse Agonism: Pharmacologi-
cal Curiosity or Potential Therapeutic Strategy? Trends Pharmacol.
Sci. 1995, 16, 10-13.
(34) 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.
(39) Govoni, M.; Bakker, R. A.; van de Wetering, I.; Smit, M. J.; Menge,
W. M. B. P.; Timmerman, H.; Elz, S.; Schunack, W.; Leurs, R.
Synthesis and Pharmacological Identification of Neutral Histamine
H1-Receptor Antagonists. J. Med. Chem. 2003, 46, 5812-5824.
(40) 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 2000, 408, 860-864.
(41) Wieland, K.; Bongers, G.; Yamamoto, Y.; Hashimoto, T.; Yama-
todani, A.; Menge, W. M. P. B.; 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.
(42) Drutel, G.; Peitsaro, N.; Karlstedt, K.; Wieland, K.; Smit, M. J.;
Timmerman, H.; Panula, P.; Leurs, R. Identification of Rat H3
Receptor Isoforms with Different Brain Expression and Signaling
Properties. Mol. Pharmacol. 2001, 1, 1-8.
(43) Vollinga, R. C.; Menge, W. M. P. B.; Leurs, R.; Timmerman, H.
New Analogues of Burimamide as Potent and Selective Histamine
H3 Receptor Antagonists: The Effect of Chain Length Variation of
the Alkyl Spacer and Modifications of the N-Thiourea Substituent.
J. Med. Chem. 1995, 38, 2244-2250.
(44) Vollinga, R. C.; Menge, W. M. P. B.; Leurs, R.; Timmerman, H.
Homologues of Histamine as Histamine H3 Receptor Antagonists:
A New Potent and Selective H3 Antagonist, 4(5)-(5-Aminopentyl)-
1H-imidazole. J. Med. Chem. 1995, 38, 266-271.
(45) Ngochindo, R. I. 2-Protecting Groups for 5-Lithiation in the Syntheses
of Imidazoles. J. Chem. Soc., Perkin Trans. 1 1990, 6, 1645-1648.
(46) Mancuso, A. J.; Swern, D. Activated Dimethyl Sulfoxide: Useful
Reagents for Synthesis. Synthesis 1981, 165-185.
(47) Omura, K.; Swern, D. Oxidation of Alcohols by “Activated” Dimethyl
Sulfoxide. A Preparative, Steric and Mechanistic Study. Tetrahedron
1978, 34, 1651-1660.
(48) Abdel-Magid, A. F.; Maryanoff, C. A.; Carson, K. G. Reductive
Amination of Aldehydes and Ketones by Using Sodium Triacetoxy-
borohydride. Tetrahedron Lett. 1990, 31, 5595-5598.
(49) Horne, D. A.; Yakushijin, K.; Bu¨chi, G. A Two-Step Synthesis of
Imidazoles from Aldehydes Via 4-Tosyloxazolines. Heterocycles
1994, 39, 139-153.
(50) Liu, C.; Ma, X. J.; Jiang, 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.
(51) Kitbunnadaj, R.; Zuiderveld, O. P.; De Esch, I. J. P.; Vollinga, R.
C.; Bakker, R. A.; Lutz, M.; Spek, A. L.; Cavoy, E.; Deltent, M.-F.;
Menge, W. M. P. B.; Timmerman, H.; Leurs, R. Synthesis and
Structure-Activity Relationships of Conformationally Constrained
Histamine H3 Receptor Agonists. J. Med. Chem. 2003, 46, 5445-
5457.
(52) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation
of Microgram Quantities of Protein Utilizing the Principle of Protein-
Dye Binding. Anal. Biochem. 1976, 72, 248-254.
(53) De Esch, I. J. P.; Timmerman, H.; Menge, W. M. P. B.; Nederkoorn,
P. H. J. A Qualitative Model for the Histamine H3 Receptor
Explaining Agonistic and Antagonistic Activity Simultaneously. Arch.
Pharm. Pharm. Med. Chem. 2000, 333, 254-260.
(54) Uveges, A. J.; Kowal, D.; Zhang, Y.; Spangler, T. B.; Dunlop, J.;
Semus, S.; Jones, P. G. The Role of Transmembrane Helix 5 in
Agonist Binding to the Human H3 Receptor. J. Pharmacol. Exp. Ther.
2002, 301, 451-458.
(55) Van der Werf, J. F.; Bast, A.; Bijloo, G. J.; Van der Vliet, A.;
Timmerman, H. HA Autoreceptor Assay with Superfused Slices of
Rat Brain Cortex and Electrical Stimulation. Eur. J. Pharmacol. 1987,
138, 199-206.
(35) Leurs, R.; Rodriguez Pena, M. S.; Bakker, R. A.; Alewijnse, A. E.;
Timmerman, H. Constitutive Activity of G-protein Coupled Receptors
and Drug Action. Pharm. Acta HelV. 2000, 74, 327-331.
(36) Milligan, G. Constitutive Activity and Inverse Agonists of G Protein-
Coupled Receptors: A Current Perspective. Mol. Pharmacol. 2003,
64, 1271-1276.
(37) 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 Antagonists Accounts for Up-
regulation of Spontaneously Active Histamine H2 Receptors. Proc.
Natl. Acad. Sci. 1996, 93, 6802-6807.
(56) Schwartz, J.-C.; Arrang, J.-M.; Garbarg, M.; Schunack, W. InnoVatiVe
Approaches in Drug Research; Elsevier: Amsterdam, 1986; pp 73-
89.
(57) Stark, H.; Lipp, R.; Arrang, J.-M.; Garbarg, M.; Schwartz, J.-C.;
Schunack, W. Acylated and Alkylated Histamine Derivatives as New
Histamine H3-receptor Antagonists. Eur. J. Med. Chem. 1994, 29,
695-700.
(58) Van der Goot, H.; Schepers, M. J. P.; Sterk, G. J.; Timmerman, H.
Isothiourea Analogues of Histamine as Potent Agonists or Antagonists
of the Histamine H3-Receptor. Eur. J. Med. Chem. 1992, 27, 511-
517.
(38) Bakker, R. A.; Wieland, K.; Timmerman, H.; Leurs, R. Constitutive
Activity of the Histamine H1 Receptor Reveals Inverse Agonism of
Histamine H1 Receptor Antagonists. Eur. J. Pharmacol. 2000, 381,
R5-R7.
(59) Arrang, J. M.; Garbarg, M.; Lancelot, J. C.; Lecomte, J. M.; Pollard,
H.; Robba, M.; Schunack, W.; Schwartz, J. C. Highly Potent and
Selective Ligands for Histamine H3-Receptors. Nature 1987, 327,
117-123.

Modulation of the ActiVity of Histamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2557
(60) Leurs, R.; Kathmann, M.; Vollinga, R. C.; Menge, W. M. B. P.;
Schlicker, E.; Timmerman, H. Histamine Homologues Discriminating
between Two Functional H3 Receptor Assay. Evidence for H3
Receptor Heterogeneity? J. Pharm. Exp. Ther. 1996, 276, 1009-
1015.
(61) Fox, G. B.; Bao Pan, J.; Esbenshade, T. A.; Bennani, Y. L.; Black,
L. A.; Faghih, R.; Hancock, A. A.; Decker, M. W. Effects of
Histamine H3 Receptor Ligands GT-2331 and Cyproxifan in a
Repeated Acquisition Avoidance Response in the Spontaneously
Hypertensive Rat Pup. BehaV. Brain Res. 2002, 131, 151-161.
(62) Tedford, C. E.; Phillips, J. G.; Gregory, R.; Pawlowski, G. P.; Fadnis,
L.; Khan, A.; Ali, S. M.; Handley, M. K.; Yates, S. L. Development
of trans-2-[1H-Imidazol-4-yl] Cyclopropane Derivatives as New
High-Affinity Histamine H3 Receptor Ligands. J. Pharm. Exp. Ther.
1999, 289, 1160-1168.
(63) Gbahou, F.; Rouleau, A.; Morisset, S.; Parmentier, R.; Crochet, S.;
Lin, J.-S.; Ligneau, X.; Tardivel-Lacombe, J.; Stark, H.; Schunack,
W.; Ganellin, C. R.; Schwartz, J.-C.; Arrang, J.-M. Protean Agonism
at Histamine H3 Receptors in Vitro and in Vivo. Proc. Natl. Acad.
Sci. 2003, 100, 11086-11091.
(64) Nakamura, T.; Itadani, H.; Hikada, Y.; Ohta, M.; Tanaka, K.
Molecular Cloning and Characterization of a New Histamine Recep-
tor, H4R. Biochem. Biophys. Res. Commun. 2000, 279, 615-620.
(65) Morse, K. L.; Behan, J.; Laz, T. M.; West, R. E. J.; 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. J. Cloning and Characterization
of a Novel Human Histamine Receptor. J. Pharm. Exp. Ther. 2001,
296, 1058-1066.
(67) Zhu, Y.; Michalovich, D.; Wu, H. L.; Tan, K. B.; Dytko, G. M.;
Mannan, I. J.; Boyce, R.; Altson, 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.
(68) Hough, L. B. Genomics Meets Histamine Receptor: New Subtypes,
New Receptors. Mol. Pharmacol. 2001, 59, 415-419.
(69) Guan, J.; Kyle, D. E.; Gerena, L.; Zhang, Q.; Milhous, W. K.; Lin,
A. J. Design, Synthesis, and Evaluation of New Chemosensitizers
in Multi-Drug-Resistant Plasmodium falciparum. J. Med. Chem.
2002, 45, 2741-2748.
(70) Ames, D. E.; Islip, P. J. Syntheses of Long-chain Acids. Part IV.
Synthesis of the Octadeca-5,12-dienoic Acids. J. Chem. Soc. 1963,
4363-4368.
(71) Rumbero, A.; Borreguero, I.; Sinisterra, J. V.; Alcantara, A. R.
Acyclic Phenylalkanediols as Substrates for the Study of Enzyme
Recognition: Synthesis of Substrates and Enzymatic Resolution via
Hydrolysis and Transesterification. Tetrahedron 1999, 55, 14947-
14960.
(72) Vollinga, R. C.; Menge, W. M. B. P.; Timmerman, H. A New
Convenient Route for the Synthesis of 4(5)-(ω-Aminoalkyl)-1H-
Imidazoles. Recl. TraV. Chim. Pays-Bas 1993, 112, 123-125.
(73) Hancok, A. A.; Fox, G. B. Perspectives on cognitive domains, H3
receptor ligands and neurological disease. Expert Opin. InVestig.
Drugs 2004, 13, 1237-1248.
(74) Celanire, S.; Wijtmans, M.; Talaga, P.; Leurs, R.; De Esch, I. J. P.
Keynote Review: Histamine H(3) Receptor Antagonists Reach Out
for the Clinic. Drug DiscoVery Today 2005, 23-24, 1613-27.
(66) Nguyen, T.; Shapiro, D. A.; George, S. R.; Setola, V.; Lee, D. K.;
Cheng, R.; Rauser, L.; Lee, S. P.; Lynch, K. R.; Roth, B. L.; O’Dowd,
B. F. Discovery of a Novel Member of the Histamine Receptor
Family. Mol. Pharmacol. 2001, 59, 427-433.
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