Dual-acting drugs: An in vitro study of nonimidazole histamine H 3 receptor antagonists combining anticholinesterase activity

Article abstract of DOI:10.1002/cmdc.201000008

Dual-acting compounds that combine H₃ antagonism with anticholinesterase properties are currently emerging as a novel and promising therapeutic approach in the treatment of multi-factorial disorders primarily characterized by cholinergic deficits such as Alzheimer's disease. A series of novel nonimidazole H₃ ligands was developed from the chemical manipulation of 1,1′-octa-, -nona-, and -decamethylene-bis-piperidines - H₃ antagonists that had been the subject of previous investigations. These compounds were evaluated for in vitro binding affinity, antagonistic potency, and selectivity at rodent and human histamine H₃ receptors, inhibitory activity at rat brain cholinesterase, and in vivo CNS access and cholinomimetic effects. Within the present series, the tetrahydroaminoacridine hybrid 18 stands out as one of the most attractive molecules, synergistically combining nanomolar and selective H₃ antagonism with remarkable anticholinesterase activity. From this original starting point, it is hoped that future investigations will lead to dual-acting compounds that can selectively enhance central cholinergic neurotransmission and thus facilitate the treatment of cognitive disorders.

Full text of DOI:10.1002/cmdc.201000008

DOI: 10.1002/cmdc.201000008
Dual-Acting Drugs: an in vitro Study of Nonimidazole
Histamine H3 Receptor Antagonists Combining
Anticholinesterase Activity
Matteo Incerti,*[a] Lisa Flammini,[b] Francesca Saccani,[b] Giovanni Morini,[a] Mara Comini,[a]
Massimo Coruzzi,[a] Elisabetta Barocelli,[b] Vigilio Ballabeni,[b] and Simona Bertoni[b]
Dual-acting compounds that combine H3 antagonism with an-
ticholinesterase properties are currently emerging as a novel
and promising therapeutic approach in the treatment of multi-
factorial disorders primarily characterized by cholinergic defi-
cits such as Alzheimer’s disease. A series of novel nonimidazole
H3 ligands was developed from the chemical manipulation of
1,1’-octa-, -nona-, and -decamethylene-bis-piperidines—H3 an-
tagonists that had been the subject of previous investigations.
These compounds were evaluated for in vitro binding affinity,
antagonistic potency, and selectivity at rodent and human his-
tamine H3 receptors, inhibitory activity at rat brain cholinester-
ase, and in vivo CNS access and cholinomimetic effects. Within
the present series, the tetrahydroaminoacridine hybrid 18
stands out as one of the most attractive molecules, synergisti-
cally combining nanomolar and selective H3 antagonism with
remarkable anticholinesterase activity. From this original start-
ing point, it is hoped that future investigations will lead to
dual-acting compounds that can selectively enhance central
cholinergic neurotransmission and thus facilitate the treatment
of cognitive disorders.
Introduction
The histamine H3 receptor (H3R) is a Gi-coupled presynaptic
auto- and heteroreceptor that negatively modulates the syn-
thesis and release of histamine and several other neurotrans-
mitters in various brain areas such as the cerebral cortex, hip-
pocampus, basal ganglia, and striatum.[1] It is well known that
these regions play a key role in learning and memory process-
es, in the regulation of the sleep–wake cycle, and in homeo-
static functions such as food and water intake. Therefore, H3
receptor antagonists have long been suggested as potentially
effective therapeutics for various central nervous system (CNS)
pathologies characterized by neurotransmitter deficits, such as
cognitive disorders in Alzheimer’s disease and attention-deficit
hyperactivity disorder (ADHD), and for the regulation of body
weight in obesity.[2] Although generally endowed with good in
vitro potency, first-generation imidazole-based H3 antagonists
exhibited a poor pharmacokinetic profile: their inhibitory
action on cytochrome P450 activity[3] and their low brain pene-
tration,[4] both of which are due to the presence of the imida-
zole ring, accounted for their limited in vivo applicability. As a
consequence, in the last decades the pharmaceutical industry
and academia have undertaken several efforts to synthesize
new nonimidazole H3 antagonists: the most promising com-
pounds, such as GSK189254 and BF2.649, are now being
tested at various phases of clinical trials (http://www.clinical-
trials.gov). However, the most recent and attractive approach
in the field of the treatment of CNS disorders lies in the devel-
opment of multitarget molecules that combine H3 antagonism
with inhibition of histamine Nt-methyltransferase (HTM),[5,6]
acetylcholinesterase (AChE),[7] HTM/AChE,[8] M2 antagonism,[9]
NO-releasing properties,[10,11] or neuroleptic activity.[12] In partic-
ular, the synergic interaction between central H3 blockade and
cholinesterase inhibition could ameliorate cognitive perform-
ances in vivo, by improving cholinergic neurotransmission
without the undesired peripheral effects typical of anticholi-
nesterase agents. Unfortunately, only in vitro data are available
concerning these molecules while no information on their
in vivo activity has been provided to date.
Herein we report the preliminary pharmacological investiga-
tion of a series of nonimidazole H3 ligands developed from the
flexible structures of 1,1’-octa-, -nona-, and -decamethylene-
bis-piperidine derivatives (com-
pounds M35, M41, and M42), al-
ready proven to possess moderate
affinity and potency toward rodent
H3Rs and good antagonistic prop-
erties at human H3Rs.[13,14] The gen-
eral formula of the new com-
pounds 5–19 and of the previously
studied bis-piperidine derivatives
are shown.
[a] Dr. M. Incerti, Prof. G. Morini, Dr. M. Comini, Dr. M. Coruzzi
Dipartimento Farmaceutico, Universitꢀ degli Studi di Parma
V.le G.P. Usberti 27A, 43100 Parma (Italy)
Fax: (+39)0521-905006
E-mail: matteo.incerti@unipr.it
[b] Dr. L. Flammini, Dr. F. Saccani, Prof. E. Barocelli, Prof. V. Ballabeni,
Dr. S. Bertoni
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate
Universitꢀ degli Studi di Parma
V.le G.P. Usberti 27A, 43100 Parma (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000008.
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M. Incerti et al.
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The chemical manipulation of one of the terminal piperidine
rings and its substitution with secondary or tertiary amines of
different steric hindrance and basicity or with the tetrahydroa-
minoacridine nucleus of the AChE inhibitor (AChEI) tacrine led
to the synthesis of a novel class of compounds. They were
evaluated for their binding affinity and antagonistic potency at
rodent and human H3Rs and their possible interactions with
guinea-pig H1 and H2 and human H4 histamine receptor sub-
types. Furthermore, as the molecular structure of these com-
pounds either contains the scaffold of tacrine or shows clear
similarities with that of dibasic cyclic amines endowed with an-
ticholinesterase action,[15] their ability to inhibit rat brain choli-
nesterase activity in vitro was assessed as well. Finally, in vivo
experiments were performed to test the CNS access of the
most promising compound 18 and to assess the occurrence of
peripheral/central cholinomimetic effects. The results obtained
for the newly synthesized derivatives, compared with those
previously collected for prototypical compounds M35, M41,
and M42, will allow a deeper insight into the optimal structural
requirements for potent and selective AChEI–H3 antagonists as
dual-acting molecules.
Results and Discussion
In vitro pharmacology
The binding and functional profile of the novel compounds
toward human and rodent H3 receptors, the data concerning
their possible interactions with the other histamine receptor
subtypes, and their inhibitory activity against rat brain cho-
linesterase are reported in Table 1. The new results are com-
pared with those of M35, M41, and M42.[14]
As indicated in Table 1, all the derivatives tested were able
to displace the radiolabeled ligand [3H](R)-a-methylhistamine
from H3Rs in the rat cerebral cortex membrane assay with simi-
lar affinity to M35, M41, and M42; only when considering the
decamethylene analogues (compounds 7, 10, 13, 16), the pKi
values estimated were definitely lower. This negative trend is
especially true regarding the cyclohexylamine derivatives 5–7,
for which the progressive lengthening of the flexible chain
from eight to ten methylene units causes a decrease of 1.5 log
units of rat H3Rs pKi values (Table 1). Regarding human H3Rs ex-
pressed in cultured SK-N-MC cells, a substantial loss of affinity
is common for all the compounds with respect to the bis-pi-
peridine reference molecules, with the exception of tacrine de-
Table 1. Affinity and antagonistic potency of the compounds under study at various histamine receptors[a] and rat brain cholinesterase inhibitory potency.
Histamine receptors
Cholinesterase
pIC50
Imax [%][i]
[d]
[e]
[f]
[g]
Compd
n
R
rH3 pKi[b]
hH3 pKi[c]
hH3 pKB
gpH3 pKB
gpH1 pKB
gpH2 pKB
hH4 pKi[h]
M35[13,14]
M41[13,14]
M42[13,14]
6
7
8
7.31Æ0.01 8.56Æ0.06 8.28Æ0.07 8.01Æ0.06
7.80Æ0.09 8.35Æ0.09 8.22Æ0.09 7.65Æ0.12
7.53Æ0.08 8.40Æ0.07 7.69Æ0.06 8.12Æ0.20
#
#
#
#
#
#
#
#
#
#
#
5.19Æ0.05
5.68Æ0.17
81Æ5
91Æ2
5
6
7
6
7
8
7.79Æ0.04 7.90Æ0.02 8.18Æ0.17 8.65Æ0.15
7.18Æ0.09 7.18Æ0.08 7.64Æ0.08 7.67Æ0.12
6.28Æ0.17 7.00Æ0.03 7.52Æ0.09 6.76Æ0.12
#
#
#
#
#
§
#
#
#
#
#
5.03Æ0.13
5.46Æ0.05
85Æ1
93Æ3
8
9
10
6
7
8
7.45Æ0.07 7.53Æ0.04 7.94Æ0.07 7.99Æ0.11
7.31Æ0.04 7.58Æ0.04 7.47Æ0.10 7.57Æ0.21
#
#
§
#
#
#
#
#
#
#
5.8Æ0.13
5.93Æ0.08
96Æ4
96Æ1
7.03Æ0.05 7.89Æ0.08 7.77Æ0.05 6.91Æ0.24 5.38Æ0.03
11
12
13
6
7
8
7.44Æ0.09 7.61Æ0.10 8.07Æ0.12 7.49Æ0.17
7.51Æ0.10 7.68Æ0.09 7.72Æ0.11 7.82Æ0.17
#
#
5.26
#
§
#
#
#
#
#
5.05Æ0.08
5.31Æ0.02
90
7.15Æ0.03 8.05Æ0.12 7.39Æ0.13 7.21Æ0.11 4.75Æ0.05[j]
93Æ1
14
15
16
6
7
8
7.56Æ0.15 7.66Æ0.12 8.20Æ0.15 7.86Æ0.07 5.90Æ0.05
7.74Æ0.06 7.89Æ0.07 7.86Æ0.10 7.36Æ0.08 6.30Æ0.10
7.04Æ0.07 7.73Æ0.10 7.89Æ0.14 7.27Æ0.06 4.85Æ0.22[j]
§
#
§
#
#
#
5.21Æ0.03
5.80
5.69Æ0.11
90Æ1
95
93Æ1
17
18
19
6
7
8
7.54Æ0.10 7.94Æ0.06 8.23Æ0.11
7.94Æ0.03 8.73Æ0.06 8.93Æ0.18
7.81Æ0.08 8.66Æ0.01 8.67Æ0.21
ND
ND
ND
ND
ND
ND
§
§
§
#
#
#
7.88Æ0.06
7.57Æ0.04
7.69Æ0.05
94Æ3
95Æ2
95Æ1
[a] Histamine H3 receptors: rat (rH3), human (hH3), guinea pig (gpH3); guinea pig histamine H1 (gpH1) and H2 (gpH2) receptors; human histamine H4 recep-
tors (hH4). [b] Inhibition of [3H]RAMH binding to rat brain membranes. [c] Inhibition of [3H]RAMH binding to SK-N-MC cells stably expressing the human his-
tamine H3 receptor. [d] Antagonist potency at human histamine H3 receptors expressed in SK-N-MC cells. [e] Antagonist potency at H3 receptors expressed
in guinea pig ileum. [f] Antagonist potency at H1 receptors expressed in guinea pig ileum. [g] Antagonist potency at H2 receptors expressed in guinea pig
atrium. [h] Inhibition of [3H]histamine binding to SK-N-MC cells stably expressing the human histamine H4 receptor. [i] Maximum percent inhibition of rat
brain cholinesterase. [j] pD2’ value. #: Inactive up to 10 mm; §: inactive up to 1 mm; ND: not determined.
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ChemMedChem 2010, 5, 1143 – 1149

Nonimidazole H3 Receptor Antagonists
rivatives 18 and 19, which displayed the highest pKi values in
the entire series (Table 1). These data suggest that the chemi-
cal manipulation of one of the terminal heterocyclic rings of
the bis-piperidine derivatives does not lead, in general, to a
significant change in the binding affinity toward rat H3Rs as
long as the flexible bridge contains eight or nine methylene
groups. On the other hand, independent of the chain length,
the replacement of a piperidine ring with a non-tacrine moiety
negatively affects the affinity to human H3Rs. Therefore, we
can speculate that the length of the flexible linker is a critical
feature to drive the species preference of this series of dibasic
asymmetrical H3 antagonists.
ed ileal contractions by the longest N-methylcyclohexylamine
compound 10, that is, behaving like its rigid biphenyl ana-
logue,[15] and by p-chlorophenyl derivatives 14 and 15, where-
as the homologue 11 slightly affected dimaprit H2-induced
positive chronotropic responses of spontaneously beating atria
(Table 1). As concerns tacrine derivatives, the enhancement of
the cholinergic tone, due to their potent anticholinesterase ac-
tivity, prevented the functional evaluation of their selectivity
profile on guinea pig tissues at concentrations higher than
1 mm. Moreover, none of the compounds tested displayed any
cytotoxic activity in the MTT test on SK-N-MC cell culture up to
10 mm (data not shown).
The results of the functional studies confirm the affinity
data: the compounds antagonized (R)-a-methylhistamine ef-
fects showing surmountable and concentration-dependent an-
tagonism at both human and guinea pig H3Rs, although with
variable potency (Table 1). In detail, the progressive separation
of the two basic centers produced a gradual loss of antagonis-
tic potency, analogous to that observed for rat H3 receptor af-
finity and especially evident for cyclohexylamine compounds
toward guinea pig H3Rs. Among these derivatives it is worth
noting that the passage from the octamethylene analogue 5,
exhibiting the highest pKB value in the entire series, to the
least potent decamethylene 7 brings about a decrease in H3
antagonistic potency of nearly two log units (Table 1). As re-
gards the tacrine derivatives, the longest molecules 18 and 19
combined the highest affinity with the most potent antago-
nism toward human H3 receptors within all the compounds as-
sayed. Their blocking potency on guinea pig H3 receptors
could not be determined because of their ability to enhance
basal cholinergic tone and to interfere with electrically induced
cholinergic contractions of the guinea pig ileum, probably be-
cause of their marked anticholinesterase activity. In fact, as ex-
pected, the molecules containing the tetrahydroaminoacridine
scaffold displayed the most potent anticholinesterase activity
in the entire series, exhibiting pIC50 values ranging from 7.57
to 7.88, even higher than that shown by the simple molecule
of tacrine on rat brain cholinesterase (pIC50 =6.54Æ0.01; maxi-
mum percent inhibition: 95Æ2).
In vivo pharmacology
On the basis of its remarkable in vitro H3 antagonistic potency
and selectivity, and of its anticholinesterase activity, compound
18 was administered in vivo to rats to evaluate its CNS access
and side effects profile. As reported in Table 2, when adminis-
Table 2. Ex vivo H3 receptor binding, tremor, salivation, and chewing fol-
lowing i.p. administration of compound 18 and tacrine.
Compd
Dose
[mgkgÀ1
Ex vivo
binding [%][a]
Tremor[b] Salivation[b] Chewing[b]
]
18
18
18
5
10
20
10
95Æ19
85Æ7
86Æ10
ND
0/2
0/2
6/7
7/7
0/2
0/2
1/7
3/7
0/2
0/2
0/7
3/7
Tacrine
[a] Percentage of [3H]RAMH-specific binding in rat cerebral cortex 60 min
after i.p. administration of compound 18; ND: not determined. [b] Choli-
nergic side effects registered 60 min after i.p. administration of com-
pound 18 and tacrine and expressed as a fraction of total animals treated
that showed the symptoms.
tered intraperitoneally (i.p.) from 5 to 20 mgkgÀ1, compound
18 showed a very low displacement of the H3-specific ligand
[3H]RAMH from rat cerebral cortex 60 min later, suggesting lim-
ited access to the CNS compared with the parent molecule
M41.[14] However, it dose-dependently induced a pronounced
hypothermia (DT=1.7Æ0.338C at 20 mgkgÀ1 i.p. **P<0.01 vs.
vehicle-treated rats) (Figure 1). At the highest dose, tremor was
detected in six of seven treated animals, whereas salivation
was present in only one animal (Table 2). As regards tacrine, at
the dose of 10 mgkgÀ1 i.p., roughly equimolar to 20 mgkgÀ1
of compound 18, it lowered the body temperature by 1.26Æ
0.258C after 60 min (*P<0.05 vs. vehicle-treated rats)
(Figure 1), producing a clear tremorogenic effect in all treated
rats and sialogogic and chewing effects in three of seven ani-
mals (Table 2). Taken together, the data collected herein seem
to indicate that, despite the apparently disappointing results
of the ex vivo H3 binding assay, the blood–brain barrier is not
impermeable to the tacrine derivative. Indeed, both hypother-
mia and especially tremor are described as central muscarinic
effects,[16] also overtly displayed by the CNS-penetrating anti-
cholinesterase reference compound in accordance with previ-
ous findings.[17] It is therefore appealing to hypothesize that, al-
It is also interesting to note that, among the remaining com-
pounds, whereas the octamethylene derivatives displayed no
or only weak inhibitory activity on brain cholinesterase up to a
concentration of 10 mm, the elongation of the flexible core and
the presence of a second tertiary nitrogen atom proportionally
increased the anticholinesterase activity (Table 1). These results
are consistent with our previous findings on the pivotal role
played by the length of a rigid linker connecting two basic
centers in the anticholinesterase potency of the compound.[15]
In particular, as nona- and decamethylene derivatives M41,
M42, 6–7, and 9–10 displayed similar activity to their already
described corresponding biphenyl analogues,[15] we can specu-
late that the flexible molecules block the enzyme in their fully
extended conformation.
As regards the interactions with the other histamine recep-
tor subtypes, the newly synthesized compounds generally ex-
hibited a good selectivity profile. Weak effects and only at mi-
cromolar concentrations were shown on histamine H1-mediat-
ChemMedChem 2010, 5, 1143 – 1149
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M. Incerti et al.
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Scheme 1. Reagents and conditions: a) SOCl2, reflux, 4 h; b) primary or sec-
ondary amine, DMF, K2CO3, KI, 808C, 8 h; c) phthalimide, PPh3, DIAD, THF,
08C, 1 h, then room temperature, overnight; d) NH2NH2·H2O, reflux, 1 h; e) 9-
chloro-1,2,3,4-tetrahydroacridine, pentanol, DIPEA, reflux, 3 days.
tion with NH2NH2·H2O of 3a–c to afford the intermediates 4a–c.
The amines 4a–c were then condensed with 9-chloro-1,2,3,4-tetra-
hydroacridine to give the final compounds.
Figure 1. Hypothermic effect induced by vehicle (white column), compound
18 (gray columns), and tacrine (black column), 60 min after intraperitoneal
administration (compound concentrations given in mgkgÀ1); *P<0.05 and
**P<0.01 versus vehicle (one-way ANOVA followed by Dunnett’s post-test).
General methods
Melting points were not corrected and were determined with a
Gallenkamp melting point apparatus. The final compounds were
analyzed on a ThermoQuest Flash EA 1112 elemental analyzer for
C, H, and N. The percentages we found were within Æ0.4% of the
though limited, the degree of central H3 receptor occupancy
by compound 18 may be adequate to potentiate its anticholi-
nesterase action in the CNS without evoking unwanted periph-
eral cholinergic side effects such as sialorrhea. Such a favorable
pharmacological profile could be achieved by a molecule that,
dually acting as a H3 blocker/AChEI, selectively boosts CNS
cholinergic neurotransmission by blunting the tonic inhibition
exerted by histamine, through H3 receptors, primarily on cen-
tral ACh release.[18]
1
theoretical values. The H NMR spectra were recorded on a Bruker
300 spectrometer (300 MHz); chemical shifts (d) are reported in
1
parts per million (ppm). H NMR spectra are reported in the follow-
ing order: multiplicity, approximate coupling constants (J value) in
hertz (Hz) and number of protons; signals were characterized as s
(singlet), d (doublet), dd (doublet of doublets), t (triplet), m (mul-
tiplet), brs (broad signal). Mass spectra were recorded using an
API-150 EX instrument with APCI interface (Applied Biosystems,
MDS Sciex, Toronto, Canada). Reactions were monitored by TLC on
Kieselgel 60 F254 (DC-Alufolien, Merck). Final compounds and inter-
mediates were purified by preparative flash chromatography on
Bꢁchi Sepacoreꢂ, using SiO2 column (Prepacked Cartridges, SiO2
60, 40–63 mm, Bꢁchi); the eluents were mixtures of CH2Cl2/CH3OH
at various volume ratios. When indicated, gaseous NH3 was added
to the methanolic phase to obtain a 5% w/w solution. Abbrevia-
tions are the following: THF: tetrahydrofuran, DMSO: dimethyl sulf-
oxide, DMF: N,N-dimethylformamide, DIAD: diisopropylazodicar-
boxylate, DIPEA: N,N-diisopropylethylamine.
Conclusions
The results collected in the present study widen the conclu-
sions drawn by our previous investigations and allowed further
exploration of the steric requirements for an optimal interac-
tion with and inhibition of two distinct molecular targets: the
histamine H3 receptor and cholinesterase enzyme. Further-
more, the original in vivo data reveal that of the novel deriva-
tives, the hybrid molecule 18 was able to combine a potent H3
antagonism with a remarkable anticholinesterase activity. Thus
18 may represent a promising starting point toward dual-
acting compounds specifically enhancing central cholinergic
neurotransmission. Future studies in preclinical animal models
of learning and/or memory deficits will help to better elucidate
its potential impact on the therapy of cognitive disorders.
General procedure for the synthesis of piperidinoalkyl deriva-
tives (5–16): We synthesized the derivatives 1a–c and 2a–c using
the condition described in the literature[5] by condensation of the
appropriate commercially available a,w-hydroxyhalogenoalkane
with piperidine followed by treatment with SOCl2.
A stirred suspension of w-chloroalkyl intermediate 2a–c (1 mmol),
the appropriate amine (1 mmol), K2CO3 (0.138 g, 1 mmol), and KI
(33.2 mg, 0.2 mmol) in DMF (4 mL) was heated at 808C for 8 h. The
solvent was removed in vacuo to give the crude products, which
were purified by flash chromatography [SiO2, CH2Cl2/CH3OH (NH3)
9:1].
Experimental Section
Chemistry
N-(8-(Piperidin-1-yl)octyl)cyclohexylamine
(5·2HCl·0.5H2O).
The synthesis of the studied compounds was accomplished as
shown in Scheme 1. The target compounds 5–16 were prepared
from w-hydroxyalkylpiperidine derivatives a–c, previously synthe-
sized in our laboratory,[5,13] by treatment with SOCl2 followed by re-
action with the appropriate amine.
White solid (244 mg, 65% yield); mp: 245–2478C (EtOH/Et2O);
1H NMR (D2O): d=1.11–1.57 (m, 14H, (CH2)2-(CH2)4-(CH2)2, cHex,
pip), 1.57–1.88 (m, 10H, cHex, pip), 1.88–2.02 (m, 2H, CH2-CH2-pip),
2.02–2.15 (m, 2H, CH2-CH2-NH), 2.85–2.97 (m, 2H, CH2-pip), 2.97–
3.16 (m, 5H, cHex, pip, CH2-NH), 3.44–3.58 (m, 2H, pip); MS (APCI)
m/z: 294 [M+1]+; Anal. calcd for C19H38N2·2HCl·0.5H2O: C 60.62, H
10.98, N 7.44, found: C 60.49, H 10.96, N 7.50.
For the synthesis of 17–19 a different route was used. The starting
products 1a–c were treated with phthalimide followed by reduc-
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Nonimidazole H3 Receptor Antagonists
General procedure for the synthesis of piperidinoalkylphthali-
mides 3a–3c.[19] DIAD (0.202 g, 1 mmol) was added to a mixture of
(DL 116/92) and with the local Ethical Committee Guidelines for
Animal Research.
w-piperidinoalkylalcohol
(0.87 mmol), phthalimide
(0.147 g,
Rat histamine H3 receptor binding assay. Rat brain membranes,
prepared according to the method of Kilpatrick and Michel,[20] were
incubated for 30 min with [3H](R)-a-methylhistamine (RAMH)
0.5 nm and the compounds under study (1 nm–10 mm), in Tris-HCl
50 mm, pH 7.4, NaCl 50 mm, EDTA 0.5 mm, then rapidly filtered
(AAWP Millipore filters 0.8 mm) under vacuum and rinsed twice
with ice-cold buffer (50 mm Tris-HCl/5 mm EDTA). Nonspecific bind-
ing was defined with 10 mm thioperamide as competing ligand.
1 mmol), and PPh3 (0.262 g, 1 mmol) in dry THF (2.5 mL), stirred at
08C and kept under N2 atmosphere. The reaction mixture was then
allowed to warm to room temperature and stirred overnight. Sol-
vent was evaporated under reduced pressure, and the residue was
purified by flash chromatography [SiO2, CH2Cl2/CH3OH (NH3) 95:5]
to give the piperidinoalkylphthalimide derivative.
2-(8-(Piperidin-1-yl)octyl)isoindoline-1,3-dione (3a). White solid
(179 mg, 60% yield); mp: 56–578C (PE); H NMR HCl (DMSO): d=
1
Human histamine H3 and H4 receptor binding assay. Homoge-
nates of SK-N-MC cells, a human neuroblastoma cell line stably ex-
pressing the human histamine H3 or H4 receptors, were used in ra-
dioligand displacement studies according to the method of Loven-
berg et al. for H3 receptors[21] or Liu et al. for H4 receptors.[22] Mem-
branes were incubated for 60 min at room temperature with
0.5 nm [3H]RAMH (30.0 CimmolÀ1, Amersham Bioscience) or with
10 nm [3H] histamine (18.1 CimmolÀ1, PerkinElmer) in the absence
or presence of competing ligands (0.01 nm–10 mm). Incubation was
terminated by rapid filtration over Millipore AAWPO2500 filters fol-
lowed by two washes with ice-cold buffer (50 mm Tris-HCl/5 mm
EDTA). Nonspecific binding was defined by 10 or 100 mm histamine
as competing ligand for H3 and H4 receptors, respectively.
1.25–1.29 (m,(CH2)6-, pip), 1.55–1.88 (m, 6H, pip), 2.73–2.95 (m, 4H,
pip), 3.32–3.44 (m, 2H, CH2-phth), 3.52–3.57 (m, 2H, CH2-NH), 7.68–
7.88 (m, 4H, Ph), 10.65 (s, 1H, NH+-pip); MS (APCI) m/z: 343
[M+1]+.
General procedure for the synthesis of piperidinoalkylamines
4a–4c.[5] The phthalimide derivative (0.47 mmol) was dissolved in
CH3OH (6 mL) and NH2NH2·H2O (0.070 g, 1.4 mmol) was added to
this solution. The mixture was held at reflux for 1 h. The suspen-
sion was cooled, acidified with concentrated HCl, and filtered. The
filtrate was evaporated under reduced pressure, and the residue
was purified by flash chromatography [SiO2, CH2Cl2/CH3OH (NH3)
95:5] to give the piperidinoalkylamine derivative.
8-(Piperidin-1-yl)octan-1-amine·2HCl (4a). Oil (74.8 mg, 75%
yield); 1H NMR 2HCl (DMSO): d=1.22–1.55 (m, 12H, (CH2)6-, pip),
1.67–1.88 (m, 6H, pip), 2.67–2.85 (m, 4H, pip), 2.69–2.88 (m, 2H,
CH2-pip), 3.37–3.55 (m, 2H, CH2-NH3+), 8.11 (s, 3H, NH3+), 10.63 (s,
1H, NH+-pip); MS (APCI) m/z: 213 [M+1]+.
Human histamine H3 receptor functional assay. Compounds were
added directly to the media containing SK-N-MC cells expressing
the human histamine H3 receptor and the construct gene (b-galac-
tosidase), followed 5 min later by addition of forskolin (5 mm). The
compounds (1 nm–10 mm) were added 10 min prior to RAMH (0.1–
100 nm). After a 6 h incubation at 378C, the medium was aspirated
and the cells were lysed with 25 mL 0.1ꢃ assay buffer (mm compo-
sition: NaH2PO4 10; Na2HPO4 10; pH 8, MgSO4 0.2; MnCl2 0.01) and
after 10 min with 100 mL 1ꢃ assay buffer (NaH2PO4 100; Na2HPO4
100; pH 8; MgSO4 2; MnCl2 0.1) containing 0.5% Triton and 40 mm
b-mercaptoethanol. Color was developed using 25 mL 1 mgmLÀ1
substrate solution (chlorophenol red b-d-galactopyranoside; Roche
Molecular Biochemicals, Indianapolis, IN, USA) and quantified with
a microplate reader by measuring the absorbance at l 570 nm
(Bio-Rad microplate reader 550, Segrate, MI, Italy).[23]
General procedure for the synthesis of piperidinoalkyl deriva-
tives (17–19). A solution of 9-chloro-1,2,3,4-tetrahydroacridine
(0.218 g, 1 mmol) in n-pentanol (6 mL) was treated with the appro-
priate amine 4a–c (1 mmol) and DIPEA (0.517 g, 4 mmol) and held
at reflux for 3 days. The solvent was evaporated under reduced
pressure; the residue was purified by flash chromatography [SiO2,
CH2Cl2/CH3OH (NH3) 95:5].
1,2,3,4-Tetrahydro-N-(8-(piperidin-1-yl)octyl)acridin-9-amine
(17·C2H2O4·2H2O). White solid (234 mg, 45% yield); mp: 128–
1298C (EtOH/Et2O); 1H NMR 2HCl (DMSO): d=1.14–1.38 (m, 10H,
(CH2)2-(CH2)4-(CH2)2, pip), 1.57–1.86 (m, 12H, pip, CH2-CH2-pip, CH2-
CH2-NH, acr), 2.61–2.79 (m, 2H, acr), 2.72–2.96 (m, 4H, CH2-pip,
pip), 2.96–3.08 (m, 2H, acr), 3.28–3.38 (m, 2H, pip), 3.80–3.84 (m,
2H, CH2-NH), 7.55 (t, J=7.8 Hz, 1H, Ph), 7.83 (t, J=7.8 Hz, 1H, Ph),
7.95 (brs, 1H, NH), 8.05 (d, J=8.4 Hz, 1H, Ph), 8.43 (d, J=8.4 Hz,
1H, Ph), 10.77 (s, 1H, NH+-pip), 14.31 (s, 1H, NH+-acr); MS (APCI)
m/z: 394 [M+1]+; Anal. calcd for C26H39N3·C2H2O4·2H2O: C 64.71, H
8.73, N 8.06, found: C 64.95, H 8.68, N 7.99.
Cell viability. Cell viability was determined through colorimetric
quantification of formazan derived from 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic reduction.[24]
SK-N-MC cells were incubated with the compounds under study
(1–10 mm) or with the vehicle for 6 h. At the end of the period of
incubation, 10 mL 5 mgmLÀ1 MTT solution were added to each
well. After 3 h, the culture medium was removed, the cells washed
with phosphate-buffered saline (PBS), and 200 mL formazan solubi-
lization solution (0.1n HCl in anhydrous iPrOH) was added. Culture
medium absorbance was spectrophotometrically read at l 570 nm
(Bio-Rad microplate reader 550, Segrate, MI, Italy). Cell viability was
expressed as viability relative to control.
Pharmacology
Drugs used were purchased from Sigma (St. Louis, MO, USA). Cul-
tured SK-N-MC cells stably expressing human histamine H3 or H4
receptors and the reporter gene b-galactosidase (Johnson & John-
son R&D, San Diego, CA, USA) were used for binding and function-
al studies. Functional experiments were also performed on isolated
organs excised from guinea pigs (250–350 g) whereas in vitro and
ex vivo binding, colorimetric, and behavioral assays were carried
out in male Wistar rats (150–200 g) (Charles River, Italy). Animals
were housed, handled, and cared for according to the European
Community Council Directive 86 (609) EEC, and the experimental
protocols were carried out in compliance with Italian regulations
Functional studies on isolated tissues
Field stimulated guinea pig ileum. Portions of guinea pig ileum
were longitudinally mounted (1 g load) in organ chambers, filled
with Krebs–Henseleit solution (mm composition: NaCl 118.9; KCl
4.6; CaCl2 2.5; KH2PO4 1.2; NaHCO3 25; MgSO4·7H2O 1.2; glucose
11.1) and gassed with 95:5 O2/CO2 at 378C. The tissues were elec-
trically stimulated (0.1 Hz, 1 ms, submax voltage) (LACE, Ospedalet-
to, PI, Italy). The H3 antagonistic activity of the tested compounds
(1 nm–1 mm) was functionally determined on twitch contraction in-
ChemMedChem 2010, 5, 1143 – 1149
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1147

M. Incerti et al.
MED
hibition induced by RAMH cumulatively administered (1 nm–1 mm)
in the presence of 1 mm mepyramine.[25]
as present on the basis of the intensity of tremor provoked by han-
dling during the temperature measurement.[16]
Guinea pig isolated ileum and atria. Guinea pig terminal ileum
portions (H1 receptors) and atria (H2 receptors) were isometrically
suspended in organ baths filled with Krebs–Henseleit (378C) and
Ringer–Locke solution (318C) (mm composition: NaCl 154.0; KCl
5.6; CaCl2 1.08; NaHCO3 5.95; glucose 11.1), respectively, and aerat-
ed with 95:5 O2/CO2. Atrial responses were determined as changes
in the rate of spontaneous beating by means of a connected cardi-
otachograph. Cumulative dose–response curves of histamine in
ileum (1 nm–1 mm) or of H2 agonist dimaprit in atria (0.1–100 mm)
were obtained in the absence and in the presence of the com-
pounds tested up to a 10 mm concentration.[25]
Data analysis
Data are presented as mean ÆSEM of 4–6 independent experi-
ments. The antagonistic potencies were estimated by determining
pKB (“apparent pA2”) as described by Furchgott’s equation.[28] When
insurmountable antagonism was detected, the antagonistic poten-
cy of the drugs was expressed by pD2’ values, determined accord-
ing to Van Rossum’s equation.[29]
In the in vitro binding assays, pIC50 values, estimated from the dis-
placement curves of the tested compounds versus [3H](R)-a-meth-
ylhistamine or [3H]histamine, were converted into pKi values ac-
cording to the Cheng–Prusoff equation.[30]
Rat brain cholinesterase inhibition. The inhibition of brain choli-
nesterase was determined spectrophotometrically using acetylthio-
choline as substrate according to the method of Ellman et al.[26] Ali-
quots of rat brain homogenates were incubated in phosphate
buffer 0.1m (pH 8.0) with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB)
(5 mm) and test compounds or tacrine at appropriate concentra-
tions (10 nm–100 mm). The reaction was started at 378C by adding
20 mL acetylthiocholine (75 mm). The reaction was stopped after
15 min by adding formalin (4%). The hydrolysis of acetylthiocho-
line catalyzed by the enzyme was determined by monitoring the
formation of the yellow 5-thio-2-nitrobenzoate anion at a l 412 nm
(Bio-Rad microplate reader 550, Segrate, MI, Italy). The percent in-
hibition of cholinesterase was calculated as: [(AcontrolÀAsample)/
Acontrol]ꢃ100. Results are provided as pIC50 (ÀlogIC50, where IC50 is
the concentration causing half-maximal inhibition of cholinesterase
activity) and maximum percent inhibition of rat brain cholinester-
ase.
Temperature differences were measured and compared in the
drug-treated and vehicle-treated animals. One-way ANOVA fol-
lowed by Dunnett’s post-test using Prism 5.0 (GraphPad Software,
San Diego, CA, USA) was performed. Tremor, salivation, and chew-
ing were reported in terms of the fraction of total treated animals
that showed the symptoms.
Acknowledgements
This work was supported by a grant from the University of
Parma (FIL 2008). The authors thank Dr. Lovenberg and Dr. Thur-
mond (Johnson & Johnson Pharmaceutical R&D, San Diego, CA,
USA) for kindly providing the cells expressing the human H3R and
the construct of human histamine H4 receptor.
Ex vivo binding study. Ex vivo binding studies were performed by
measuring the displacement of [3H]RAMH from cerebral cortical
membranes of rats intraperitoneally (i.p.) treated with compound
18 (doses ranging from 5 to 20 mgkgÀ1) or vehicle saline 60 min
before the binding assay. Animals were killed by CO2 asphyxiation.
Cerebral cortical membranes were prepared according to Taylor’s
method,[27] by homogenizing cerebral tissues isolated after rat
transcardial perfusion with ice-cold buffer at pH 7.4 (Tris-HCl
50 mm, pH 7.6, NaCl 50 mm, EDTA 0.5 mm). Cerebral cortex homo-
genates were incubated with [3H]RAMH (0.5 nm) for 45 min at
room temperature, then rapidly washed twice with ice-cold buffer
and separated by centrifugation (12800 g at 48C for 5 min). Pellets
were resuspended in buffer solution, mixed with liquid scintillation
(Ultima Gold, PerkinElmer), and bound radiolabel was determined
by liquid scintillation counting. Thioperamide 10 mm was used to
determine nonspecific binding. Specific [3H]RAMH binding in drug-
treated animals (2–7 rats for each dose) was expressed as percent-
age of the specific [3H]RAMH binding measured in vehicle-treated
animals.
Keywords: cholinesterase inhibitors · dual-acting compounds ·
histamine H3 receptor · neurological agents · tacrine
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Published online on May 28, 2010
ChemMedChem 2010, 5, 1143 – 1149
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1149