Guanidine and 2-aminoimidazoline aromatic derivatives as α2-adrenoceptor antagonists. 2. Exploring alkyl linkers for new antidepressants

Article abstract of DOI:10.1021/jm800026x

The preparation of a number of (bis)guanidine and (bis)2-aminoimidazoline derivatives as potential α₂-adrenoceptor antagonists for the treatment of depression is presented. Human brain tissue was used to measure their affinity toward the α₂-adrenoceptors in vitro. Compounds 6b, 8b, 9b, 10b, 15b, 17b, 18b, 20b, and 21b displayed a good affinity (pK _i > 7) and were evaluated in in vitro functional [ ³⁵S]GTPγS binding assays in human prefrontal cortex to determine their agonistic or antagonistic activity. Among these compounds, 17b and 20b showed the expected behavior for an antagonist and were subject to in vivo microdialysis experiments in rats. Significantly, these experiments confirmed the antagonistic properties of 17b and 20b, and therefore both compounds can be considered as potential antidepressants.

Full text of DOI:10.1021/jm800026x

3304
J. Med. Chem. 2008, 51, 3304–3312
Guanidine and 2-Aminoimidazoline Aromatic Derivatives as r2-Adrenoceptor Antagonists. 2.
Exploring Alkyl Linkers for New Antidepressants
Fernando Rodriguez,† Isabel Rozas,*,† Jorge E. Ortega,‡,§ Amaia M. Erdozain,‡,§ J. Javier Meana,‡,§ and Luis F. Callado‡,§
Centre for Synthesis and Chemical Biology, School of Chemistry, Trinity College, UniVersity of Dublin, Dublin 2, Ireland,
Department of Pharmacology, Faculty of Medicine, UniVersity of the Basque Country, 48940-Leioa, Bizkaia,
Spain, and CIBER of Mental Health, CIBERSAM, Spain
ReceiVed January 11, 2008
The preparation of a number of (bis)guanidine and (bis)2-aminoimidazoline derivatives as potential R2-
adrenoceptor antagonists for the treatment of depression is presented. Human brain tissue was used to measure
their affinity toward the R2-adrenoceptors in vitro. Compounds 6b, 8b, 9b, 10b, 15b, 17b, 18b, 20b, and
21b displayed a good affinity (pKi > 7) and were evaluated in in vitro functional [35S]GTPγS binding
assays in human prefrontal cortex to determine their agonistic or antagonistic activity. Among these
compounds, 17b and 20b showed the expected behavior for an antagonist and were subject to in vivo
microdialysis experiments in rats. Significantly, these experiments confirmed the antagonistic properties of
17b and 20b, and therefore both compounds can be considered as potential antidepressants.
Introduction
According to the World Health Organization, by 2020,
depression will be the second largest health burden following
only heart diseases.1 Even though the pathophysiological origin
of this disease continues to be unknown, the monoamine theory
is the most widely accepted,2 stating that depression is a result
of a deficiency of brain monoamine (noradrenaline (NA) or
serotonin) activity.
It is well-known that central noradrenergic transmission is
regulated by inhibitory noradrenergic receptors (R2-ARs), which
are expressed on both somatodendritic areas and axon terminals.
Hence, the activation of these R2-ARs induces an inhibition of
NA release in the brain, and thus, it has been proposed that
depression is associated with a selective increase in the high-
affinity conformation of the R2-ARs in the human brain.3 This
enhanced R2-AR activity could be implicated in the deficit in
noradrenergic transmission described in the etiology of depres-
Figure 1. Structures of known R2-noradrenoceptor targeting antide-
sion. Thus, chronic treatment with antidepressants induces an
in vivo desensitization of the R2-ARs regulating the local release
of NA.4 Taking into account this hypothesis, the development
of selective R2-adrenoceptor antagonists can be considered as
a new and effective therapeutical approach to the treatment of
depressive disorders. In this way, it has been demonstrated that
the administration of different R2-AR antagonists either locally
in the locus coeruleus or systemically increases the release of
NA in the prefrontal cortex.5,6 Moreover, R2-AR antagonists
are also able to enhance the increase of NA induced by selective
reuptake inhibitor antidepressant drugs.7
Some of the most recent antidepressants developed include
mianserin and mirtazapine (Figure 1), which show effective
antidepressant activity by blockade of R2-ARs.8 As part of our
interest in the development of new potential antidepressants, in
a recent work9 we described the synthesis and pharmacological
evaluation of several series of phenyl and diphenyl substituted
(bis)guanidine and (bis)2-aminoimidazoline derivatives with
pressants and those of the high R2-AR affinity compound 1 and two
R2-AR antagonists (2 and 3) previously described in our group.
different heteroatoms in the para position with respect to these
groups. On the basis of the high affinity previously shown by
compound 1 (Figure 1),10 this derivative was used as a lead
compound, and thus, several derivatives were prepared obtaining
two new R2-AR antagonists (2 and 3, Figure 1).
However, despite the fact that many of these molecules
showed R2-AR affinities within the range of the well-known
antagonist idazoxan (pKi ) 7.29; see Table 2), none of them
improved that of the original lead compound (pKi ) 8.80).10
Bearing these results in mind, we decided to avoid the presence
of heteroatoms in the linker and substituents and prepare and
pharmacologically test different mono- and dicationic analogues
of 1, keeping alkyl groups in the linker or as substituents in
order to establish some structure–activity relationships (SARs).
We present in this article not only the efficient synthesis of
a number of symmetrical and nonsymmetrical guanidine and
2-aminoimidazoline analogues of the high affinity R2-AR ligand
1 with alkyl substituents/linkers but also, and more importantly,
a complete pharmacological study. Hence, in vitro assays in
human brain tissue to evaluate the R2-AR affinity and functional
* To whom correspondence should be addressed. Phone: +353 1 896
3731. Fax: +353 1 671 2826. E-mail: rozasi@tcd.ie.
†
University of Dublin.
University of the Basque Country.
CIBER of Mental Health.
‡
§
10.1021/jm800026x CCC: $40.75
2008 American Chemical Society
Published on Web 05/07/2008

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3305
Figure 2. General synthetic schemes for the preparation of the guanidinium and 2-aminoimidazolinium derivatives.
studies to determine the agonist or antagonist nature of those
derivatives with pKi > 7 were performed. Furthermore, in vivo
microdialysis experiments in rats were carried out with the
compounds showing antagonistic properties, to test their effect
on NA release in order to establish their potential use as
antidepressants. As mentioned, the R2-AR affinity and potential
receptor antagonism experiments were performed in human
prefrontal cortex (PFC), since there is an important density of
R2-ARs in this tissue.11 Moreover, many studies have reported
changes in PFC activity in the brain of patients with depres-
sion.12 The final objective of our research is the preparation of
antidepressants, and as such, the use of human brain tissue to
directly characterize the pharmacological properties of the new
compounds will be relevant from a therapeutic perspective.
our study. All the compounds have been tested in their
hydrochloride salt forms.
Pharmacology. The affinity toward the R2-ARs in human
brain PFC tissue of all compounds prepared was measured by
competition with the selective radioligand [3H]RX821002 (2-
methoxyidazoxan), which was used at a constant concentration
of 1 nM.
Affinity of the Monocationic Compounds. Following the
strategy of our previous work,9 we decided to synthesize
molecules that can be considered as fragments of the original
lead compound 1. This would allow us not only to understand
the importance of the second cation in the affinity toward the
R2-ARs and its activity but also to explore the effect of keeping
the alkyl groups in the linker/substituents instead of heteroatomic
groups as in the analogues previously reported by us.9 In
addition, the guanidine containing substrates were subject to
study because of the similarities previously found by our group
between the guanidinium and the 2-aminoimidazolinium cat-
ions.29
The affinities toward the R2-ARs (expressed as pKi) of all
the monocationic compounds studied are shown in Table 2.
Three of the better known R2-AR ligands (idazoxan, clonidine,
and RX821002) were used as references. In the 2-aminoimi-
dazoline series, the removal of one of the cations results in loss
of affinity toward the R2-AR receptors. There is a drop of 2
logarithmic units when comparing 4b to 1, but the loss of the
other phenyl ring does not seem to be important, since the pKi
values of 4b and 5b are within the same range and far from the
affinity of the lead compound. However, there is a trend change
for 6b, as size reduction in the alkyl chain produces an increase
in the affinity (pKi greater than those of idazoxan and clonidine).
As for 7b (lowest pKi within the 2-aminoimidazoline series),
the elimination of the methyl group leads to a decrease in
affinity. Compounds 8b, 9b, and 10b (3,4-disubstituted) showed
pKi > 7 within the range of clonidine and idazoxan. In fact,
the affinity found for 9b is the second highest in this study.
As previously observed,9 guanidine derivatives show lower
R2-ARs affinities than their 2-aminoimidazoline analogues. Thus,
compounds 12b, 13b, 14b, and 16b displayed pKi values similar
to those of their dicationic analogue 24 (Table 2), and 11b has
the lowest affinity of the whole set of substrates described in
this article. Remarkably, 15b and 17b, despite being guanidine
derivatives, have an affinity within the range of Idazoxan.
Results and Discussion
Chemistry. The importance of the roles played by different
guanidine and 2-aminoimidazoline containing compounds in
biological processes has led to the development of various
methodologies that allow introducing both these groups into the
backbone of different molecules.9 Among them, on the basis
of the features of our starting materials and the good results
obtained previously,9,13 we decided to use Kim and Qian’s
strategy.14 This approach consists of the treatment of the
corresponding starting amine (or diamine) with 1 (or 2) equiv
of either N,N′-bis(tert-butoxycarbonyl)thiourea or N,N′-di(tert-
butoxycarbonyl)imidazoline-2-thione13 (as the guanidine or
2-aminoimidazoline precursor, respectively) in the presence of
mercury(II) chloride and an excess of triethylamine (Figure 2).
The Boc-protected guanidine intermediates obtained in the first
step of the synthesis were purified by silica gel column
chromatography, whereas in the case of 2-aminoimidazoline
precursors, a quick flash column chromatography over neutral
alumina was run instead. Afterward, some of the substrates
required recrystallization from the appropriate solvent.
Standard Boc deprotection of the intermediates with an excess
of trifluoroacetic acid in dichloromethane and further treatment
with Amberlyte resin in aqueous solution led to the hydrochlo-
ride salts of the target molecules in good overall yields (Table
1). All the starting amines are commercially available either
from Aldrich or Fluka except for the 9,10-dihydroanthracene-
2,6-diamine, which was synthesized according to the procedure
reported in the literature.15
The Boc-protected derivatives 14a16 and 22a17 have been
previously described; however, none of them was prepared by
Kim and Qian’s methodology. Regarding the final substrates,
compounds 4b, 8b, 10b, 17b, 18b, 19b, 20b, and 21b are new
while 5b,18 6b,18,19 7b,20 11b,21 12b,21,22 13b,21–23 14b,21,22,24
15b,25 16b,26 22b,17 and 23b,27 although previously described
as synthetic intermediates or prepared for other purposes, have
not been tested in human brain tissue as possible antidepressants.
It is also important to mention that compound 9b28 is reported
in a patent as a cloned human R2-AR receptor agonist, but since
it had not been tested in human brain PFC, we included it in
Importance of the Methylene Substituent/Linker in
Terms of the Affinity. Considering the similarities between
the compounds prepared in this study and those reported in our
previous work,9 a comparative analysis has been carried out to
understand the difference in receptor affinity based on the steric
and electronic properties of the chemical group in the linker or
substituent. Hence, the R2-AR pKi values of a number of these
substrates, previously reported by our group,9 are displayed in
Table 3 along with the compounds presented in this work
according to their structural similarities. Thus, for the PhXPhIm
set, the affinity showed by 4b is slightly higher than those

3306 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Rodriguez et al.
Table 1. Overall and First and Second Stage Yields (%) Obtained for All Compounds Prepared
displayed by its analogues 25a, 26a, and 27a, suggesting that
the linker does not have an effect in the affinity for the receptor.
As for the PhXPhGu-like compounds, the methylene linker is
a drawback regarding the affinity, since the pKi for 11b is lower
than those of the compounds containing more electron-rich
linkers. The most important difference in affinity (more than 1
logarithmic unit) is for compound 25b.
Regarding the CH3XPhIm-like derivatives, again the presence
of electron-rich groups seems to be an advantage (5b, 28a, and
29a in Table 3), whereas for the CH3XPhGu set there is no
difference at all (12b, 28b, and 29b in Table 3). In the case of
XPhIm and XPhGu analogues, the methyl derivatives showed
pKi values nearly 1 logarithmic unit higher than their amino
counterparts (Table 3), indicating that in this case alkyl groups
increase the R2-AR affinity.
antagonist 3; thus, in this example the presence of the heteroatoms
represents a burden affinitywise. This difference is not so
important in the guanidine set, 5-ringPhGu, since the pKi values
obtained for 16b and 3b are very similar. In the case of the
6-ringPhIm and 6-ringPhGu-like structures, the presence of the
oxygen atoms helps to increase the affinity toward the R2-ARs,
since 31a and 31b showed pKi values higher than their
cycloalkyl counterparts (Table 3).
It can be concluded that the functionality in the linker or
substituents does not seem to make a considerable difference
in the PhXPhIm, CH3XPhGu or 5-ringPhGu-like structures,
whereas the presence of an electron-rich group increases the
affinity toward the R2-ARs for the PhXPhGu, CH3XPhIm,
6-ringPhIm, and 6-ringPhGu sets. For the rest of structures,
XPhIm, XPhGu, 5-ringPhIm, and the ImPhXPhIm twin sub-
strates reported in our previous work,9 the alkyl group has a
Among the five member ring containing derivatives, 5-ringPhIm,
9b displays a pKi almost 1 logarithmic unit higher than the dioxo

Guanidine and 2-Aminoimidazoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3307
Table 2. Monocations Prepared and Their Affinity for the R2-ARs
Expressed as pKia
20b pKi values are within the range of those of idazoxan and
clonidine. Two aspects should be considered: the intracations
distance and the disposition of the cations with respect to the
linkers. Thus, it seems that the optimal distance between the
cations should be around 12.1 Å and anything larger or shorter
decreases the affinity for the receptor. The drop in the pKi of
18b could be a consequence of the increase in the intracations
distance or of the fact that each one of the cations is now in
meta and para positions with respect to each -CH2- bridge.
In the case of 19b, the drop in the affinity is even more
remarkable indicating that to have both cations meta with respect
to the linker, a decrease in the intracation distance is definitely
a drawback. Nevertheless, more derivatives should be studied
to fully evaluate the effect of the conformational restriction.
As for compound 20b, the lengthening of the bridge resulted
in a longer distance between cations and more than 11/2
logarithmic unit drop in the pKi.
The most significant result in the guanidines series is the good
affinity of 21b (Table 4), which for the first time shows better
affinity than its 2-aminoimidazoline analogue. Actually, this is
the first example of a guanidine containing a twin molecule of
our “in-home library” showing pKi > 7. Regarding 22b and
23b, both presented lower affinities than 24. In this set of
compounds and regarding affinity, the optimal distance between
cations seems to be around 12.3 Å, very similar distance
compared with the optimal in the 2-aminoimidazoline series.
[35S]GTPγS Binding Functional Assays. Those compounds
that displayed an affinity within the range of that of idazoxan
or clonidine (pKi > 7) were subject to [35S]GTPγS binding
experiments to determine their activity.
As members of the G-protein-coupled receptors (GPCRs)
superfamily, when the endogenous substrate binds to the R2-
ARs, they interact with a G-protein triggering a cascade of
biochemical events, which results in transmembrane signaling.
This receptor activation alters the conformation of the G-
proteins, leading to the exchange of GDP by GTP on the
R-subunit, promoting their dissociation into R-GTP and ꢀγ
subunits. A direct evaluation of this G-protein activity can be
made by determining the guanine nucleotide exchange using
radiolabeled GTP analogues. The [35S]GTPγS binding assay
constitutes a functional measure of the interaction of the
receptor, and the G-protein and is a useful tool to distinguish
between agonists (increasing the nucleotide binding), inverse
agonists (decreasing the nucleotide binding), and neutral
antagonists (not affecting the nucleotide binding) of GPCRs.30
Experiments were performed in low-affinity receptor conditions
for agonists (presence of guanine nucleotides and sodium in
the medium), and therefore, typical potency values are 2-3
logarithmic units lower than affinity values obtained in radio-
ligand receptor binding experiments.30
Compounds 6b, 8b, 9b, 10b, and 15b stimulated binding of
[35S]GTPγS, showing a typical agonist dose–response plot. The
potencies of all these substrates were in the micromolar range,
with 10b being the most potent agonist. In Table 5, their EC50
values can be found as well as their percentage efficacy relative
to the well-known R2-AR agonist UK14304 (5-bromo-6-[2-
imidazolin-2-ylamino]quinoxaline).
Conversely, compounds 17b, 18b, 20b, and 21b did not
stimulate binding of [35S]GTPγS by themselves and were
subject to new [35S]GTPγS binding experiments and tested
against the R2-AR agonist UK14304.
a The structures of the reference compounds RX821002, idazoxan, and
clonidine are also presented.
positive effect on the R2-AR affinity. No trend could be
identified for the guanidine twin substrates GuPhXPhGu.9
Affinity of the Dicationic Twin Compounds. Despite the
fact that the monocationic compounds 6b, 8b, 9b, 10b, 15b,
and 17b showed interesting pKi values, none of them increased
the R2-AR affinity of the lead compound 1. Thus, we decided
to prepare some twin substrates with different structural features.
All these derivatives are shown in Table 4 alongside the pKi
values obtained in our study.
The conformationally constrained backbones of compounds
18b, 19b, 21b, and 22b (Table 1), even though the lipophilic
properties were kept, reduce dramatically the rotation of the
bonds around the bridge, and therefore, the cationic moieties
spatial location will be restricted to limited areas. Thus, in the
first approach, comparison of their affinity to that of 1 will help
to understand the range of distances between the cations required
for a better interaction with the receptor. Additionally, the
interaction of these conformationally restricted derivatives will
be energetically favored because no energy would be spent in
reaching the optimally oriented conformation. Conversely, 20b
and 23b (see structures in Table 1) have more conformational
freedom, and their study can also help to understand the
dependence of the affinity on the intracations distance and
rotational freedom.
In Table 6, the effect induced in the UK14304 agonist
stimulation of [35S]GTPγS binding by the presence in the
medium of a single concentration (10-5 M) of each of our
In the 2-aminoimidazoline series, none of the new rigid
substrates improved compound 1 affinity, even though 18b and

3308 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Rodriguez et al.
Table 3. Affinity Values (pKi) for the R2-ARs of Different Sets of Comparable Structures with Different Linkers/Substituentsa
a The asterisk (/) indicates that the R2-AR affinity of these compounds was reported in ref 9 by our group.
Table 4. Twin Molecules R2-AR Affinity (pKi) and Approximate
Intracations Distance
Table 5. Affinity for R2-ARs (pKi), EC50 Values, and Percentage
Efficacy Relative to UK14304 Found for Compounds Showing a Typical
Agonist Dose-Response Plot
compd
pKi
d(C+ · · ·C+)a (Å)
compd
pKi
EC50 (µM)
Emax (%)
RX821002
idazoxan
clonidine
1
18b
19b
20b
24
21b
22b
9.04
7.29
7.68
8.80
7.58
6.32
7.02
6.38
7.96
6.12
5.88
UK14304
6b
8b
9b
10b
15b
8.85
7.82
7.68
8.26
7.33
7.12
11.4 ( 0.3
15.2 ( 0.2
62.9 ( 0.7
4.4 ( 0.3
1.1 ( 0.1
35.5 ( 0.8
100
97
89
98
100
84
12.1
12.4
11.8
14.0
12.2
12.3
11.9
14.2
Table 6. EC50 Values Obtained from the Concentration-Response
Curves for UK14304 Stimulation of [35S]GTPγS Binding in the
Absence or Presence of the Different Compounds (10-5 M)
23b
a Structures were built using the unpublished crystal structure of a related
symmetric dication as template and minimized using MM2.
experiment
UK14304
UK14304 + 17b
UK14304 + 18b
UK14304 + 20b
UK14304 + 21b
EC50 (µM)
11.4 ( 0.3
868.3 ( 112.8
28.8 ( 4.2
103.7 ( 10.8
7.8 ( 0.3
compounds can be found. Addition of 18b to the experiment
did not induce a significant rightward shift in the EC50 value
for the UK14304, whereas 21b resulted in a slight leftwards
shift. These facts are in agreement with the lack of R2-ARs
antagonistic properties of both substrates. On the contrary, 17b
and 20b produced a remarkable rightward shift in the UK14304
EC50 value, a result that is expected for antagonists. Hence, after
these in vitro experiments in human brain PFC, two new
antagonists with affinity similar to that of idazoxan were
identified. Remarkably, considering our previous results,9 17b
is the first guanidine derivative that shows antagonistic behavior
while 20b is the first twin molecule of our series acting as an
antagonist.
In Vivo Microdialysis Experiments. Considering the an-
tagonistic properties and relatively good affinity over the R2-
ARs of compounds 17b and 20b, we tested their potential effect

Guanidine and 2-Aminoimidazoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3309
Figure 4. Effects of the systemic administration of 17b, 20b, or saline
on extracellular NA levels, evaluated in the PFC. Data are given as
the mean ( standard error from three separate animals for each group
and are expressed as percentages of the corresponding basal values.
Arrow represents administration of the different compounds.
Figure 3. Effects of local administration (1–100 µM) by reverse
microdialysis in the PFC of 17b, 20b, idazoxan, RX821002, or
cerebrospinal fluid. Concentrations of the compounds were progressively
increased every two fractions (70 min) in tenfold increments (arrows).
Data are given as the mean ( standard error mean values from three
to six separate animals for each group and are expressed as percentages
of the corresponding basal values.
can be deduced by the increase evoked when they were
systemically administered. However the stronger increase on
NA basal values observed for 17b over 20b could indicate
pharmacokinetic differences such as the ability to cross the BBB
or differences in the catabolism of the substrates.
Considering the results of this series of in vivo microdialysis
experiments, the antagonistic properties of compounds 17b and
20b over R2-ARs, as expected from their behavior in
[35S]GTPγS binding experiments, are confirmed.
on noradrenergic transmission in vivo. Intracerebral microdi-
alysis is a neurochemical technique that has been applied
extensively in pharmacological studies aimed at investigating
the effects of different drugs on brain neurotransmission. This
technique allows collection of a representative concentration
of different neurotransmitters of the area where the probe is
implanted while the animals are awake and freely moving.31
Most antidepressant drugs are able to increase NA extracel-
lular concentrations in different brain areas as the PFC, an area
implicated in depression disease. Considering that R2-ARs exert
a tonic inhibitory action on NA release from the noradrenergic
terminals, we assessed the ability of these two new compounds
to increase NA extracellular concentration in this area. First,
we tested the drugs when administered locally in order to
confirm their antagonistic activity over R2-ARs in vivo. A
second step was to study the effect of these compounds
increasing NA extracellular concentrations when administered
systemically.
Local administration of artificial cerebrospinal fluid (aCSF)
did not change NA basal values (F[8,30]) 0.39, P ) 0.91, n )
4; Figure 3). However, reverse dialysis of 17b (1–100 µM) and
20b (1–100 µM) induced a concentration-related increase in
extracellular NA levels (Emax ) 326 ( 113%, F[1,54] ) 8.05,
P ) 0.0064, n ) 10; Emax ) 255 ( 76%, F[1,46] ) 21.07, P
< 0.0001, n ) 7; respectively; Figure 3). The increases were
very similar to those obtained from local administration (1–100
µM) of two well-known R2-AR antagonists, RX821002 (Emax
) 287 ( 49%, F[1,39] ) 66.78, P < 0.0001, n ) 7; Figure 3)
and idazoxan (Emax ) 235 ( 42%, F[1,39] ) 32.07, P < 0.0001,
n ) 7; Figure 3).
Conclusions
In this paper we have reported the quick and efficient synthesis
of a number of guanidine and 2-aminoimidazoline derivatives. The
final compounds 6b, 8b, 9b, 10b, 15b, 17b, 18b, 20b, and 21b
showed affinities toward the R2-ARs in human brain tissue in
in vitro experiments within the range of those of odazoxan and
clonidine. Compounds 18b, 20b, and 21b are twin molecules,
whereas 6b, 8b, 9b, 10b, 15b, and 17b are monocations.
Compounds 6b, 8b, 9b, 10b, 18b, and 20b are 2-aminoimida-
zoline derivatives, while 15b, 17b, and 21b are guanidine
containing substrates. Generally speaking, the 2-aminoimida-
zoline derivatives displayed higher R2-AR affinities than their
guanidine analogues, as expected from the results obtained in
our previous work.9 However, remarkably and for the first time
in our sets of compounds, we have obtained a guanidine twin
compound (21b) with pKi > 7.
In terms of activity, compounds 6b, 8b, 9b, 10b, 15b, 18b,
and 21b are agonists in [35S]GTPγS experiments. The different
R2-AR activities found for 9b and the dioxo derivative 3,9 which
share the same backbone, can only be explained by the presence
of the two oxygen atoms in the latter one, which could establish
some interactions relevant for the antagonism.
Systemic administration of 17b increased NA extracellular
concentration by 373% ( 73% and stayed high over the end of
the experiment (F[1,33] ) 95.70, P < 0.0001, n ) 6; Figure
4), whereas following administration of 20b, a weak increase
of NA basal values, reaching a maximal effect of 156% ( 35%,
was observed (Figure 4).32 This increase was statistically
significant when the group was compared with the respective
control (F[1,30] ) 5.56, P ) 0.02, n ) 6).
The most important result achieved in this work is the
discovery of compounds 17b and 20b, which are antagonists
in both in vitro [35S]GTPγS binding experiments and in vivo
microdialysis experiments. Remarkably, 17b is the first guani-
dine derivative of our compounds with such characteristics. Yet
again, very subtle structural changes led to different activity in
the R2-ARs. Comparing the structures of 17b and 31b,9 one
might expect similar behavior from both; however, the former
one is an antagonist while the dioxo compound 31b9 is an
agonist. This is just the exact opposite of the one mentioned
Thus, 17b and 20b showed R2-AR antagonist properties in
vivo and were able to cross the blood-brain barrier (BBB), as

3310 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Rodriguez et al.
above for the 2-aminoimidazoline derivatives 9b and 3.9 As a
result, no obvious SAR can be stated activitywise.
compounds systemically administered were dissolved in saline and
injected intraperitoneally.
Samples, collected with the microdialysis procedure (every 35
min), were analyzed by HPLC with electrochemical detection. NA
concentrations were monitorizated by an amperometric detector
(Hewlett-Packard model 1049A) at an oxidizing potential of +650
mV. The mobile phase (12 mM citric acid, 1 mM EDTA, 0.7 mM
octylsodium sulfate, pH 5, and 10% methanol) was filtered,
degassed (Hewlett-Packard model 1100 degasser), and delivered
at a flow rate of 0.2 mL/min by a Hewlett-Packard model 1100
pump. Stationary phase was a column of 150 mm × 2.1 mm
(Thermo Electron Corporation). Samples (injection volume 37 µL)
were injected and NA was analyzed in a run time of 10 min.
Solution of standard noradrenaline was injected every working day
to create a new calibration table.
Regarding compound 20b, it is the first twin molecule of our
compounds that acts as an antagonist. After local administration,
20b induced an increase of NA concentrations in the rat brain;
however, after peripheral administration, it produced a weaker
effect than the one induced by 17b. This different behavior could
be due to the fact that 20b is a larger dicationic molecule, and
therefore, it might be more difficult to cross the BBB.
After the series of encouraging results obtained so far, we
are working in the design and synthesis of new analogues in
order to try to understand the characteristics required to improve
the R2-AR affinity and antagonist activity and to be able to
design new potential antidepressants.
The mean values of the first three samples before substrate
administration were considered as 100% basal value. All measures
of extracellular NA concentrations are expressed as a percentage
of the baseline value ( SE of the mean. One way analysis of
variance (ANOVA) for control group or two way ANOVA between
control and each treated group was assessed for statistical analysis.
After the experiments, rats were sacrificed with an overdose of
chloral hydrate and the brains were dissected to check the correct
implantation of the probe.
Experimental Section
Pharmacology: Materials and Methods. [3H]RX821002 Bind-
ing Assays. Specific [3H]RX821002 binding was measured in 0.55
mL aliquots (50 mM Tris-HCl, pH 7.5) of the neural membranes,
which were incubated with [3H]RX821002 (1 nM) for 30 min at
25 °C in the absence or presence of the competing compounds
(10-12 to 10-3 M, 10 concentrations). Incubations were terminated
by diluting the samples with 5 mL of ice-cold Tris incubation buffer
(4 °C). Membrane bound [3H]RX821002 was separated by vacuum
filtration through Whatman GF/C glass fiber filters. Then the filters
were rinsed twice with 5 mL of incubation buffer and transferred
to minivials containing 3 mL of OptiPhase “HiSafe” II cocktail
and counted for radioactivity by liquid scintillation spectrometry.
Specific binding was determined and plotted as a function of the
compound concentration. Nonspecific binding was determined in
the presence of adrenaline (10-5 M).
Chemistry. All the commercial chemicals were obtained from
Sigma-Aldrich or Fluka and were used without further purification.
Deuterated solvents for NMR use were purchased from Apollo.
Dry solvents were prepared using standard procedures, according
to Vogel, with distillation prior to use. Chromatographic columns
were run using silica gel 60 (230–400 mesh ASTM) or aluminum
oxide (activated, Neutral Brockman I STD grade 150 mesh).
Solvents for synthesis purposes were used at GPR grade. Analytical
TLC was performed using Merck Kieselgel 60 F254 silica gel plates
or Polygram Alox N/UV254 aluminum oxide plates. Visualization
was by UV light (254 nm). NMR spectra were recorded in a Bruker
DPX-400 Avance spectrometer, operating at 400.13 and 600.1 MHz
[35S]GTPγS Binding Assays. The incubation buffer for measur-
ing [35S]GTPγS binding to brain membranes contained, in a total
volume of 500 µL, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl,
50 mM GDP, 50 mM Tris-HCl at pH 7.4, and 0.5 nM [35S]GTPγS.
Protein aliquots were thawed and resuspended in the same buffer.
The incubation was started by addition of the membrane suspension
(40 µg of membrane proteins) to the previous mixture and was
performed at 30 °C for 120 min with shaking. In order to evaluate
the influence of the compounds on [35S]GTPγS binding, eight
concentrations (10-10 to 10-3 M) of the different compounds were
added to the assay. Incubations were terminated by adding 3 mL
of ice-cold resuspension buffer followed by rapid filtration through
Whatman GF/C filters presoaked in the same buffer. The filters
were rinsed twice with 3 mL of ice-cold resuspension buffer and
transferred to vials containing 5 mL of OptiPhase HiSafe II cocktail
(Wallac, U.K.). The radioactivity trapped was determined by liquid
scintillation spectrometry (Packard 2200CA). The [35S]GTPγS
bound was about 7–14% of the total [35S]GTPγS added. Nonspe-
cific binding of the radioligand was defined as the remaining
[35S]GTPγS binding in the presence of 10 µM unlabeled GTPγS.
In Vivo Microdialysis Assays. The experiments were carried
out in male Sprague–Dawley rats weighing between 250 and 300 g.
At the beginning of the experiments, animals were anesthetized
with chloral hydrate (400 mg/kg ip) and a microdialysis probe was
implanted by stereotaxic surgery into PFC brain area. The coor-
dinates selected for the PFC were as follows: AP (anterior to
bregma) +2.8 mm, L (lateral from the midsagittal suture) +1 mm,
DV (ventral from the dura surface) -5 mm.33 After 24 h for animal
recovery, perfusion fluid (aCSF) is pumped through the probe at a
flow rate of 1 µL/min. In the semipermeable membrane, which is
the critical side of the probe and is placed on the selected area,
molecules flow into and out of the cannulae by diffusion. Therefore,
microdialysis technique allows local administration of substrates
dissolved in the perfusion fluid.
1
for H NMR and 100.6 and 150.9 MHz for 13C NMR. Shifts are
referenced to the internal solvent signals. NMR data were processed
using Bruker Win-NMR 5.0 software. Electrospray mass spectra
were recorded on a Mass Lynx NT V 3.4 on a Waters 600 controller
connected to a 996 photodiode array detector with methanol, water,
or ethanol as carrier solvents. Melting points were determined using
an Electrothermal IA9000 digital melting point apparatus and are
uncorrected. Infrared spectra were recorded on a Mattson Genesis
II FTIR spectrometer equipped with a Gateway 2000 4DX2-66
workstation and on a Perkin-Elmer Spectrum One FT-IR spec-
trometer equipped with Universal ATR sampling accessory. Sample
analysis was carried out in Nujol using NaCl plates. Elemental
analysis was carried out at the Microanalysis Laboratory, School
of Chemistry and Chemical Biology, University College Dublin.
General Procedure for the Synthesis of the Hydrochloride
Salts. Each of the corresponding Boc-protected precursors (0.5
mmol) was treated with 15 mL of a 50% solution of trifluoroacetic
acid in DCM for 3 h. After that time, the solvent was eliminated
under vacuum to generate the trifluoroacetate salt. This salt was
dissolved in 20 mL of water and treated for 24 h with IRA400
Amberlyte resin in its Cl- form. Then the resin was removed by
filtration and the aqueous solution washed with DCM (2 × 10 mL).
Evaporation of the water afforded the pure hydrochloride salt.
Absence of the trifluoroacetate salt was checked by 19F NMR.
Hydrochloride Salt of 1-(2-Imidazolidinyliminio)-4-benzylben-
1
zene (4b). Yellowish oil (96%); H NMR (D2O) δ 3.55 (s, 4H),
3.57 (s, 2H), 6.87–7.04 (m, 9H); 13C NMR (D2O) δ 40.2, 42.1,
122.9, 125.5, 128.0, 128.2, 129.4, 132.6, 139.5, 140.6, 157.4; MS
(ESI+) m/z 252.0944 [M + H]+. Anal. (C16H18ClN3 ·1.4H2O) C,
H, N.
Hydrochloride Salt of 1-(2-Imidazolidinylimino)-3,4-dimethyl-
benzene (8b). White solid (96%); mp 83–85 °C; 1H NMR (D2O) δ
2.08 (s, 6H), 3.58 (s, 4H), 6.80 (d, 1H, J ) 8.0 Hz), 6.83 (s, 1H),
7.06 (d, 1H, J ) 8.0 Hz); 13C NMR (D2O) δ 17.9, 18.4, 42.1,
When drugs were locally administered, they were dissolved in a
CSF and applied during 70 min via dialysis probe implanted in the
PFC in increasing concentrations of 1, 10, and 100 µM. The

Guanidine and 2-Aminoimidazoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3311
120.1, 123.7, 130.0, 131.9, 135.4, 138.1, 157.5; MS (ESI+) m/z
190.1163 [M + H]+. Anal. (C11H16ClN3 ·0.2H2O) C, H, N.
Hydrochloride Salt of Imidazolidin-2-ylidene-(5,6,7,8-tetrahy-
dronaphthalen-2-yl)amine (10b). White solid (94%); mp 87–89 °C;
1H NMR (D2O) δ 1.65–1.74 (m, 4H), 2.63–2.75 (m, 4H), 3.70 (s,
4H), 6.88–6.99 (m, 2H), 7.13 (d, 1H, J ) 8.0 Hz); 13C NMR (D2O)
δ 21.8, 22.0, 27.9, 28.3, 42.2, 120.7, 123.9, 129.8, 131.7, 136.4,
138.6, 158.1; MS (ESI+) m/z 216.1380 [M + H]+. Anal.
(C11H16ClN3 ·1.3H2O) C, H, N.
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Hydrochloride Salt of N-(5,6,7,8-Tetrahydronaphthalen-2-
1
yl)guanidine (17b). White solid (95%); mp 39–41 °C; H NMR
(D2O) δ 1.58–1.72 (m, 4H), 2.59–2.72 (m, 4H), 6.81–6.93 (m, 2H),
7.07 (d, 1H, J ) 8.0 Hz); 13C NMR (D2O) δ 21.9, 22.0, 28.0,
28.2, 122.1, 125.4, 129.9, 130.6, 136.7, 138.6, 155.6; MS (ESI+)
m/z 190.1248 [M + H]+. Anal. (C11H16ClN3 ·0.8H2O) C, H, N.
Dihydrochloride Salt of 2,6-Di(2-imidazolidinylimino)-9,10-di-
hydroanthracene (18b). Brown solid (94%); mp, decomposes over
1
220 °C; H NMR (D2O) δ 3.66 (s, 8H), 3.73 (s, 4H), 6.92–7.06
(m, 4H), 7.24 (d, 2H, J ) 8.0 Hz); 13C NMR (D2O) δ 34.1, 42.2,
120.9, 121.8, 128.0, 132.4, 134.6, 137.5, 157.8; MS (ESI+) m/z
347.1572 [M + H]+. Anal. (C20H24Cl2N6 ·2.0H2O) C, H, N.
Dihydrochloride Salt of 2,7-Di(2-imidazolidinylimino)-9H-fluo-
rene (19b). Light-brown solid (95%); mp, decomposes over 240
°C; 1H NMR (D2O) δ 3.64 (s, 8H), 3.68 (s, 2H), 7.06 (d, 2H, J )
8.0 Hz), 7.22 (s, 2H), 7.64 (d, 2H, J ) 8.0 Hz); 13C NMR (D2O)
δ 35.9, 42.1, 119.1, 120.3, 121.2, 133.2, 138.3, 144.5, 157.3; MS
(ESI+) m/z 333.1827 [M + H]+. Anal. (C19H22Cl2N6 ·1.8H2O) C,
H, N.
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Dihydrochloride Salt of 4,4′-Di(2-imidazolidinylimino)-1,2-
diphenylethane (20b). Yellowish solid (96%); mp, decomposes over
210 °C; 1H NMR (D2O) δ 2.91 (s, 4H), 3.70 (s, 8H), 7.13 (d, 4H,
J ) 8.0 Hz), 7.25 (d, 4H, J ) 8.0 Hz); 13C NMR (D2O) δ 35.5,
42.2, 123.6, 129.4, 132.3, 140.4, 158.1; MS (ESI+) m/z 349.1840
[M + H]+. Anal. (C20H26Cl2N6 ·1.3H2O) C, H, N.
Dihydrochloride Salt of 2,6-Diguanidino-9,10-dihydroanthracene
(21b). Brown solid (95%); mp, decomposes over 215 °C; 1H NMR
(D2O) δ 3.81 (s, 4H), 7.06 (d, 2H, J ) 8.0 Hz), 7.10 (s, 2H), 7.31
(d, 2H, J ) 8.0 Hz); 13C NMR (D2O) δ 34.2, 122.9, 123.8, 128.1,
131.5, 135.5, 137.7, 155.7; MS (ESI+) m/z 295.1659 [M + H]+.
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Acknowledgment. F.R. thanks the Consejeria de Educacion
Cultura y Deporte de la Comunidad Autonoma de La Rioja for
his Grant. This research was also supported by a Cycle III HEA
PRTLI grant (F.R.), by Bizkaiko Foru Aldundia (Ekinberri 7/12/
EK/2005/65 and DIPE 06/04), the Basque Government
(SAIOTEK), and Spanish Ministry of Health, Instituto de Salud
Carlos III (REM-TAP). J.E.O. and A.M.E were recipients of
predoctoral fellowships from the MEC and the Basque Govern-
ment, respectively.
Supporting Information Available: Preparation and IR, 1H
NMR, 13C NMR, and MS data for the compounds already described
in the literature (5b–7b, 9b, 11b–13b, 14a, 14b–16b, 22a, 22b,
and 23b) and all new Boc-protected derivatives prepared (4a–13a,
15a–21a, and 23a); a table containing the combustion analysis data
for the new final compounds (4b, 8b, 10b, and 17b–21b); and
preparation of membranes, analysis of binding data, and drugs used
in the pharmacology experiments. This material is available free
of charge via the Internet at http://pubs.acs.org.
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