Guanidine and 2-aminoimidazoline aromatic derivatives as α2-adrenoceptor ligands: Searching for structure-activity relationships

Article abstract of DOI:10.1021/jm800838r

In this paper, we report the synthesis of three new 2-aminoimidazoline (compounds 4b, 5b, and 6b) and three new guanidine derivatives (compounds 7b, 8b, and 9b) as potential α₂-adrenoceptor antagonists for the treatment of depression. Their pha

Full text of DOI:10.1021/jm800838r

J. Med. Chem. 2009, 52, 601–609
601
Articles
Guanidine and 2-Aminoimidazoline Aromatic Derivatives as r2-Adrenoceptor Ligands:
Searching for Structure-Activity Relationships
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, UniVersity of Dublin, Trinity College, Dublin 2, Ireland, Department of
Pharmacology, UniVersity of the Basque Country, 48940-Leioa, Bizkaia, Spain, Centro de InVestigacio´n Biome´dica en Red de Salud Mental,
CIBERSAM, Spain
ReceiVed July 9, 2008
In this paper, we report the synthesis of three new 2-aminoimidazoline (compounds 4b, 5b, and 6b) and
three new guanidine derivatives (compounds 7b, 8b, and 9b) as potential R2-adrenoceptor antagonists for
the treatment of depression. Their pharmacological profile was evaluated in vitro in human brain tissue and
compared to the potential antidepressant 1 and the agonists 2 and 3. All new substrates were evaluated by
in vitro functional [35S]GTPγS binding assays in human prefrontal cortex to determine their agonistic or
antagonistic activity. Compound 8b was found to be an antagonist in vitro and was subjected to in vivo
microdialysis experiments in rats. Moreover, a new synthesis of the precursor amines for compounds 4b-9b
is presented.
Introduction
Depression affects more than 300 million people in the world
and by 2020 is expected to be the second largest health burden.1
Despite the fact that the neuronal mechanisms underlying
depression are a complicated mesh of integrated actions and
even though no clear pathophysiological mechanism has been
established to explain why depression occurs, the widely
accepted monoaminergic theory2 states that depression is a
consequence of a deficiency of brain monoamine (noradrenaline,
NAa, or serotonin) activity.
It is well-known the role that R2-adrenergic receptors (R2-
ARs), expressed on both somatodendritic areas and axon
terminals, play in central noradrenergic transmission.3 Activation
of these R2-ARs induces an inhibition of NA release in the brain,
and 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.4 A deficit in noradrenergic transmission
has been described in depression and the mentioned enhanced
R2-AR activity could be implicated in such a deficit; thus,
treatment with antidepressants will help to regulate the local
release of NA by inducing in vivo desensitization of the R2-
ARs.5 Supporting this idea, it has been shown that local (in the
locus coeruleus) or systemic administration of R2-AR antagonists
not only increase the liberation of NA in the prefrontal cortex
(PFC)6,7 but also augment the increase of NA induced by
selective reuptake inhibitor antidepressant drugs.8 Therefore,
Figure 1. Structures of known R2-AR targeting antidepressant Mir-
tazapine and three R2-AR ligands previously described in our group.
research in selective R2-adrenoceptor antagonists represents a
valuable therapeutic approach for the treatment of depression.
Mirtazapine (Remeron, Figure 1) is one of the most recent
developed antidepressants and shows efficacy by blocking the
R2-ARs.9 This blockade of R2-ARs in the brain prevents the
negative feedback that NA has in its own release, resulting in
a more efficient noradrenergic neurotransmission.10
Continuing with our work in the development of new R2-AR
antagonists, in two previous articles11 we described the synthesis
and screening in human PFC of a number of bis- and mono-
guanidine and 2-aminoimidazoline derivatives. Among these
derivatives, compounds 1, 2, and 3 (Figure 1) showed affinities
toward the R2-ARs within the same range as Idazoxan and/or
Clonidine.11a Considering that the three analogues are structur-
ally related, one might expect them to display the same activity
in the receptors. However, after a complete in vitro pharmaco-
logical study, compounds 2 and 3 showed agonistic properties,
whereas only compound 1 resulted to be an antagonist. The
antagonism toward the R2-ARs of compound 1, and its potential
as antidepressant, was later confirmed by in vivo microdialysis
experiments carried out in rats.11a As a consequence, it can be
* To whom correspondence should be addressed. Phone: +353 1 896
3731. Fax: +353 1 671 2826. E-mail: rozasi@tcd.ie.
†
Centre for Synthesis and Chemical Biology, School of Chemistry,
University of Dublin, Trinity College.
‡
Department of Pharmacology, University of the Basque Country.
Centro de Investigacio´n Biome´dica en Red de Salud Mental,
§
CIBERSAM.
|
Present address: Departamento de Quimica, Universidad de La Rioja,
Grupo de Sintesis Quimica de La Rioja, UA-CSIC, E-26006 Logron˜o, Spain.
a Abbreviations: NA, noradrenaline; AR, adrenoceptor; GTPγS, gua-
nosine5′-O-(3-thiotriphosphate);PFC,prefrontalcortex;SAR,structure-activity
relationships; aCSF, artificial cerebrospinal fluid.
10.1021/jm800838r CCC: $40.75
2009 American Chemical Society
Published on Web 01/09/2009

602 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Rodriguez et al.
Scheme 1
Scheme 2a
a Reagents and conditions: (a) MeSO3Me, TEA, CH2Cl2, 50 °C; (b) MeSO3Et, TEA, CH2Cl2, 50 °C; (c) TFA, rt.
deduced that in this kind of molecules, very subtle structural
changes lead to a different behavior in the R2-ARs.
properties of the new compounds is relevant from a therapeutic
point of view.
Considering the structures and activities of compounds 1, 2,
and 3, one could speculate whether or not the antagonism of
derivative 1 remains after the loss of one of the methyl groups,
or what type of response would produce in the receptors an
intermediate structure between compounds 1 and 3 bearing an
ethyl-methyl amine. Hence, the synthesis and pharmacological
evaluation of a new series of guanidine and 2-aminoimidazoline
analogues of the lead compounds 1, 2, and 3 is presented in
this article and the results obtained will provide important
structure-activity relationships (SARs). It is worth mentioning
that the lack of a crystal structure for the R2-ARs makes this
type of studies a valuable tool to establish some chemical
functions required for both the affinity and activity in the R2-
ARs.
Results and Discussion
Chemistry. Among the different options to incorporate the
guanidine and 2-aminoimidazoline groups into a molecule that
based on the reaction of primary aromatic amines with 1 equiv
of either N,N′-di(tert-butoxycarbonyl)thiourea (guanidine pre-
cursor) or N,N′-di(tert-butoxy carbonyl)imidazoline-2-thione14
(2-aminoimidazoline precursor) in the presence of mercury (II)
chloride and an excess of triethylamine (Scheme 1) has given
very good results in our group11,14 and for that reason was the
strategy followed in the present work.
In all cases, the reaction was carried out in dichloromethane
and the Boc-protected precursors obtained in the first step of
the synthesis were purified by a quick neutral alumina flash
column chromatography.
In this article, the preparation of six new guanidine and
2-aminoimidazoline analogues of derivatives 1, 2, and 3 and a
new synthesis for their precursor amines is presented. Moreover,
a complete pharmacological study (consisting of in vitro assays
in human brain tissue to evaluate their R2-AR affinity and
determine their activity and in vivo microdialysis experiments
in rats) was performed. The R2-AR affinity and potential receptor
antagonism experiments were carried out in human PFC because
there is an important density of R2-ARs in this tissue,12 and
many studies have reported changes in PFC activity in the brain
of patients with depression.13 Moreover, the final goal of our
research is to obtain antidepressants and, therefore, the use of
human brain tissue to directly characterize the pharmacological
Standard removal of the Boc groups with an excess of
trifluoroacetic acid in dichloromethane followed by treatment
with Amberlyte resin in water led to the hydrochloride salts of
the target molecules in overall good yields ranging from 48 to
75%. The structures and yields of all the new compounds
prepared are displayed in Table 1.
Compound 10 (N-Boc-p-phenylenediamine) was used, as an
advanced intermediate, for the synthesis of the final compounds
4b-9b as depicted in Scheme 2. Thus, treatment of derivative
10 with 1 equiv of methyl methanesulfonate and triethylamine
in dichloromethane led to the monomethylated derivative 11 in
a 25% yield after column chromatography in silica gel. The
ethylamine compound 12 was obtained in a similar manner via

Guanidine and 2-Aminoimidazolines as R2-AR Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 603
Table 1. Overall, First and Second Stage Yields (in %) Obtained for the Compounds Prepared
ethyl methanesulfonate. In this case, the yield increased to 34%.
In both reactions, formation of dialkylated products was
observed.
Boc-removal of compounds 11 and 12 with trifluoroacetic
acid afforded the starting material amines 13 and 14 in good
yields (87% and 84%, respectively).
Affinity toward the r2-ARs. The affinity of all compounds
prepared toward the R2-ARs in human brain PFC tissue was
measured by competition with the R2-AR selective radioligand
[3H]RX821002 (2-methoxy-idazoxan), which was used at a
constant concentration of 1 nM. The affinities obtained,
expressed as pKi, are displayed in Table 2. Three of the most
common R2-AR ligands (Idazoxan, Clonidine, and RX821002)
were used as references.
For the synthesis of the Boc-protected tertiary amine 15, there
are two possible routes (see Scheme 2). Thus, following route
A, heating of amine 11 in dicloromethane at reflux temperature,
with triethylamine and ethyl methanesulfonate as an electrophile,
led to compound 15 in a 39% after column cromatography in
silica gel. Similarly, starting from the ethylamine 12 (route B)
and using methyl methanesulfonate as the alkylating agent,
derivative 15 was obtained in a 37%. Again, in both cases
formation of the polyalkylated products was observed. Finally,
Boc-removal of compound 15 in trifluoroacetic acid afforded
the ethyl-methyl amine 16 in 89% yield.
All the chemicals used for the synthesis described in Schemes
1 and 2 are commercially available either from Aldrich or Fluka
and, to the best of our knowledge, the Boc-protected amines
12 and 15 are new, whereas the methylamine derivative 11 has
been previously described in the literature15 as the anilines
13,15a,c,16 14,16b,17 and 16.18
Pharmacology. As mentioned above and based on our
previous experience, small structural modifications produce
significant changes in terms of affinity or activity (agonism-
antagonism) in the receptors.11 Thus, the presence or absence
of heteroatoms in very similar backbones or the exchange of
the guanidinium and 2-aminoimidazolinium cations can deter-
mine the antagonism of a substrate.11 With that in mind, and
given the similarities of the lead compounds 1, 2, and 3 (Figure
1), there was a need to explore in more detail the pharmacologi-
cal profile of this class of molecules by evaluating the six new
derivatives synthesized. This study would allow finding impor-
tant SARs required for both the affinity and activity of the
compounds studied.
In the 2-aminoimidazoline series, all new compounds dis-
played a pKi value higher than 7 except for the ethylamino
derivative 5b (pKi ) 6.75, see Table 2). These values are within
the range of the well-known R2-AR ligands Idazoxan and/or
Clonidine, with compound 1 keeping the highest affinity of the
series (pKi ) 7.42). Among the new derivatives, the pKi value
obtained for the analogue 6b is very similar to that of compound
1, while the secondary methylamino derivative 4b showed a
slightly lower affinity. This result seems to indicate that for the
monoalkyl amines, the chain shortening is an advantage
affinitywise although more compounds should be evaluated.
Regarding the guanidine containing substrates, among the new
molecules obtained, the order in affinity toward the R2-ARs is
the same as the one found for their 2-aminoimidazoline
counterparts. Thus, the ethyl-methylamino compound 9b shows
the highest pKi (7.12, see Table 2), whereas the monoethylamino
derivative 8b possesses the lowest affinity with a pKi value of
6.58. In all cases, the affinity of the guanidine substrates is lower
than the one displayed by their 2-aminoimidazoline analogues,
as we have observed before.11
It is worth to mention that the affinity showed by compound
9b is the best of its series, better than the affinities showed by
some of the 2-aminoimidazoline counterparts and within the
range of the R2-AR antagonist Idazoxan. Five out of the six
derivatives shown in Table 2 with a dialkyl amine in para
(compounds 1, 2, 3, 6b, and 9b) showed a pKi larger than 7,
whereas the only monoalkyl amine that presents a pKi > 7 is
derivative 4b. This fact seems to indicate that the presence of

604 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Rodriguez et al.
Table 2. R2-ARs Affinity Values (Expressed as Ki and pKi) Obtained for All Compounds Studieda
a * ) Compounds previously prepared and evaluated by us.11a § ) Affinity was measured by competition assays with the R2-AR selective radioligand
[3H]RX821002 (1 nM) in PFC human tissues. † ) Cortical membranes from human postmortem brains were incubated at 25 °C for 30 min with [3H]RX821002
(1 nM) in the absence or presence of the competing compounds (10-12 M to 10-3 M, 10 concentrations).
a tertiary amine in the para position of these molecules is needed
for a good binding to the R2-AR receptors.
The R2-ARs are G-protein coupled receptors (GPCRs) and,
as such, when the endogenous ligand binds to the receptor, a
change in the conformation of the G-proteins occurs, leading
to the exchange of GDP by GTP on the R-subunit, promoting
their dissociation into R-GTP and ꢀγ subunits and resulting in
transmembrane signaling. A direct evaluation of this G-protein
activity can be made by determining the guanine nucleotide
exchange using radiolabeled GTP analogues. The [35S]GTPγS
[35S]GTPγS Binding Functional Assays: Agonism vs
Antagonism. All six new compounds 4b-9b along with
derivaitve 17 were subjected to [35S]GTPγS binding experiments
to determine their nature as agonists or antagonists, and the
results are shown in Table 3. The affinity of the latter one had
been evaluated,11a but its activity had not been studied.

Guanidine and 2-Aminoimidazolines as R2-AR Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 605
Table 3. EC50 Values and Intrinsic Activity Relative to UK14304 (pKi ) 8.85) Found for Compounds Showing a Typical Agonist Dose-Response
Plota
a * ) Compound previously prepared by us whose EC50 had been evaluated.11a § ) Compound previously prepared by us whose EC50 had not been
evaluated.11a † ) Cortical membranes from human postmortem brains were incubated at 30 °C for 2 h in the presence of the different compounds (10-10
M to 10-3 M, 8 concentrations).
Table 4. EC50 Values Obtained from the Concentration-Response
binding assay constitutes a functional measure of the interaction
Curves for UK14304 Stimulation of [35S]GTPγS Binding in the Absence
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.19
Experiments were performed in low-affinity receptor conditions
for agonists (presence of guanine nucleotides and sodium in
the medium), and hence, typical potency values are between
two and three logarithmic units lower than affinity values
obtained in radioligand receptor binding experiments.19
The new compounds 4b, 5b, 6b, 7b, and 9b as well as
derivatives 3 and 17 stimulated binding of [35S]GTPγS, showing
a typical agonist dose-response plot. The potencies (and
structures) of the 2-aminoimidazoline containing compounds 3
and 6b are very similar and the highest of the whole series
(Table 3), whereas the rest of the substrates show potencies
within the same range and close to the one displayed by the
well-known R2-AR agonist UK14304 (5-bromo-6-[2-imidazolin-
2-ylamino]quinoxaline) used as reference.
or Presence of the Different Compounds (10-5 M Concentration)a
experiment
EC50 (µM)
UK14304
11.4 ( 0.3
355 ( 18
16 ( 2
UK14304 + 1a
UK14304 + 2a
UK14304 + 8b
213 ( 18
a Compounds previously prepared by us11a and already evaluated against
UK14304.
nism. Thus, the effect induced in the UK14304 agonist
stimulation of [35S]GTPγS binding by the presence of a single
concentration (10-5 M) of derivative 8b was evaluated and is
presented in Table 4, along with the effect induced by the known
antagonist 1 and the agonist 2.11a Similarly to compound 1,
addition of derivative 8b to the experiment produced a remark-
able rightwards shift in the EC50 value for the UK14304 (Table
4), indicating that compound 8b behaves as an antagonist in
the R2-ARs in human brain PFC in the experiments carried out
in vitro. It is important to highlight that, within this set of
structurally related molecules, only two out of ten compounds
showed antagonism toward the R2-ARs, which suggests that
more specific interactions are required for the antagonism and
makes the rational design of R2-AR antagonists more challenging.
Considering the chemical structure of the lead compound 1,
it is not an easy task to find an obvious explanation for the
antagonism of derivative 8b. In fact, the structures of compounds
Again, molecules with similar structures showed the closest
EC50 values (see compounds 4b and 5b, Table 3). The intrinsic
activity relative to the UK14304 for all these substrates ranges
from 87 to 103% (Table 3). Only compound 8b did not stimulate
binding of [35S]GTPγS and was subjected to new [35S]GTPγS
experiments and tested against the UK14304.
A significant rightwards shift of the EC50 for UK14304 when
including compound 8b in the assay would confirm its antago-

606 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Rodriguez et al.
Figure 3. Effects of intraperitoneal administration of compound 8b
or vehicle (saline) on extracellular NA levels evaluated in the PFC.
Data are given as mean ( standard error mean values from 4-5 separate
animals for each group and are expressed as percentages of the
corresponding basal values. Arrow represents time administration of
the compound or vehicle.
Figure 2. Effects of local administration of compound 8b (1-100
µM), RX821002 (1-100 µM), or aCSF (control rats) in the PFC.
Concentration of the compounds was progressively increased (arrows).
Compounds 8b and RX821002 were dissolved in aCSF and perfused
via reverse dialysis at the time indicated by the arrows (every 70 min).
Data correspond to the mean ( standard error mean values from 3-5
animals for each group and are expressed as percentages of the
corresponding basal values.
0.0001, n ) 9). These results confirm the antagonistic properties
shown by compound 8b in vitro and the ability of the compound
to cross the blood-brain barrier (BBB). At this point, it is not
possible to discard completely the actions of a metabolite of
the compound 8b after systemic administration. However, the
fast effect observed after compound administration makes it
slightly probably. The quantitative determination of the drug
brain penetration of the drug and/or metabolites should be
determined by [brain]/[plasma] determination via single-dose
pharmacokinetic assessment.
Second, it can be concluded that the compound effect on
different brain circuits at the same time did not abolish the NA
increase observed in the PFC after local administration. Under
these premises, the compound 8b fulfils the basic requirements
that indicate its possible activity as antidepressant.
2, 3, 4b, 6b, or 7b are more similar to that of compound 1, and
yet they all showed agonistic properties in the R2-ARs.
In Vivo Microdialysis Experiments. Given the antagonistic
properties over the R2-ARs showed in vitro by derivative 8b,
we proceeded to test its potential effect on noradrenergic
transmission in vivo by microdialysis experiments. This tech-
nique is a widely accepted method for sampling the extracellular
fluid of the brain, allowing the study of different neurotrans-
mitters in the extracellular area where the probe is implanted.20
This technique has been used to investigate the effect of different
compounds on NA concentrations in the PFC, an area widely
implicated in depression. The increase of NA concentration in
the PFC of freely moving rats after drug administration is
accepted as a good predictor for antidepressant activity. In this
context, many antidepressants, including the R2-AR antagonist
Mirtazapine, are able to increase dialysate levels of NA in the
PFC.21 Considering the antagonist properties of compound 8b
on R2-ARs, we assessed its ability to increase NA extracellular
concentrations in that brain area.
Artificial cerebrospinal fluid (aCSF) administration for more
than 5 h did not change NA basal values (F[8,30] ) 0.58; P )
0.77, n ) 4, Figure 2). When derivative 8b was perfused by
reverse dialysis through the probe (1-100 µM), a significant
increase in extracellular NA levels was observed (Emax) 304
( 54%, F[1,52] ) 13.22, P ) 0.0006 vs control, n ) 9; Figure
2) at 100 M concentration of compound 8b. The maximal effect
was very similar to that obtained from the local administration
(1-100 µM) of the well-known R2-AR antagonist RX821002
(Emax ) 290 ( 35%, F[1,40] ) 65.32, P < 0.0001, n ) 7;
Figure 2).
Conclusions
The conclusions in this article are 4-fold. First, the syntheses
of three new 2-aminoimidazoline compounds (4b-6b) and three
new guanidine containing derivatives (7b-9b), as well as a new
methodology for the preparation of their precursor amines (13,
14, and 16) are reported.
Second, it has been shown that compounds 4b, 6b, and 9b
have affinities toward the R2-ARs within the range of those of
Idazoxan and/or Clonidine (pKi > 7). Remarkably, and for the
first time in this type of derivatives, compound 9b, which
contains a guanidinium cation, shows an affinity larger than that
showed by the 2-aminoimidazolinium derivatives 3 and 5b.
Moreover, five out of the six derivatives with a tertiary amine
in para (1, 2, 3, 6b, and 9b) showed a pKi > 7, indicating that
this motif may play an important role in the interactions
established in the binding to the receptor.
However, in good agreement with the pKi values of the
substrates for the R2-ARs, RX821002 (pKi ) 9.04) exerted a
significant increase in extracellular NA levels at the lowest dose
tested (1 µM), whereas 8b (pKi ) 6.58) did not produce a clear
effect until the 100 µM dose.
On a second step, we tested the effect of derivative 8b on
extracellular NA levels by systemic administration.Control rats
were administered with the vehicle (saline). Intraperitoneal
administration of compound 8b (10 mg/kg) increased NA
extracellular concentration by 161 ( 30% in the PFC (Figure
3).
Third, in terms of noradrenergic activity, it has been found
that the new compounds 4b-7b, and 9b, as well as derivatives
2, 3, and 17, showed agonistic properties in the [35S]GTPγS
experiments performed in vitro in PFC human tissue, whereas
compound 8b is an antagonist.
Finally, in terms of the SAR found, despite the structural
similarity between compound 1 and compounds 4b and 6b
(which were obtained by the removal or the addition of a methyl
group in compound 1), the first is an antagonist whereas the
latter ones stimulate binding of [35S]GTPγS. However, com-
pound 8b, which is structurally different than 1, proved to be
an antagonist in [35S]GTPγS binding experiments. The antago-
nism showed by 8b in vitro was later confirmed by in vivo
This increase was statistically significant when the group was
compared with the respective control (F[1,67] ) 22.64, P <

Guanidine and 2-Aminoimidazolines as R2-AR Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 607
General Procedure for the Alkylation of Primary and Second-
ary Amines: Method C. The alkylating agent (10.0 mmol of methyl
methanesulfonate or ethyl methanesulfonate) and 10.0 mmol of TEA
were added at 0 °C over a solution containing 10.0 mmol of the
corresponding amine in DCM (12 mL). The resulting mixture was
heated at reflux temperature for 15 h, and after cooling it was diluted
with 40 mL of DCM and washed with a 10% NaOH solution (2 ×
15 mL) and water (2 × 15 mL). The organic phase was dried over
anhydrous Na2SO4, filtered, and concentrated under vacuum to give
a residue that was purified by silica gel column chromatography,
eluting with the appropriate hexane:EtOAc mixture.
microdialysis experiments carried out in rats. Local and systemic
administration of 8b increased NA extracellular concentration
in the rat brain.
Hence, it is clear that very subtle structural modifications can
lead to significant changes in the activity toward the R2-ARs
and that these interesting and somehow surprising results
encourage us to continue working in the synthesis of new series
of compounds to try to find more SARs that help to understand
the affinity and activity of this kind of substrates in the R2-
ARs.
General Procedure for the Boc-Deprotection and Preparation
of the Starting Material Amines: Method D. A solution containing
10.0 mmol of the Boc-protected compound (11, 12, or 15) in 15
mL of TFA was stirred at room temperature for 2 h. Then the
solvent was eliminated under vacuum to generate the trifluoroacetate
salt. This salt was redissolved in 20 mL of an aqueous solution of
NaOH (2M) and washed with DCM (3 × 15 mL). The organic
layer was washed with water (2 × 10 mL), dried over anhydrous
Na2SO4, filtered, and concentrated to give the corresponding free
amine as an oil.
Experimental Section
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
for 1H NMR and 100.6 and 150.9 MHz for 13C NMR, respectively.
Shifts are referenced to the internal solvent signals. NMR data were
processed using Bruker Win-NMR 5.0 software. HRMS spectra
were recorded on a Waters (Micromass) LCT-Tof mass spectrom-
eter operating in the positive ion electrospray mode. The source
was operated at 100 °C with a cone voltage of 30 V. The instrument
was operated at a resolution of 5000 fwhm. Spectra were recorder
over a range m/z 100 to m/z 1000. The MS was controlled and
data acquired and mass measured with MassLynx 4.0 software.
Methanol, water, or ethanol were used 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 spectrometer equipped with Universal ATR
sampling accessory. Elemental analysis was carried out at the
Microanalysis Laboratory, School of Chemistry and Chemical
Biology, University College Dublin.
General Procedure for the Synthesis of Boc-Protected 2-Imi-
noimidazolidine and Boc-Protected Guanidine Derivatives: Method
A. Each of the corresponding anilines was treated in DCM at 0 °C
with 1.1 equiv of mercury (II) chloride, 1.0 equiv of N,N′-di(tert-
butoxycarbonyl) imidazolidine-2-thione (for the 2-aminoimidazoline
precursors), or N,N′-di(tert-butoxycarbonyl) thiourea (for the guani-
dine precursors) and 3.1 equiv of TEA. The resulting mixture was
stirred at 0 °C for 1 h and for the appropriate duration at room
temperature. Then, the reaction mixture was diluted with EtOAc
and filtered through a pad of celite to get rid of the mercury sulfide
formed. The filter cake was rinsed with EtOAc. The organic phase
was washed first with water (2 × 30 mL) and then with brine (1 ×
30 mL), dried over anhydrous Na2SO4, and concentrated under
vacuum to give a residue that was purified by neutral alumina
column flash chromatography, eluting with the appropriate hexane:
EtOAc mixture.
Dihydrochloride Salt of N-Imidazolidin-2-ylidene-N′-methyl-
benzene-1,4-diamine (4b): Method B. Red solid (93%); mp
1
230-232 °C. H NMR (D2O) δ 3.07 (s, 3H, CH3), 3.74 (s, 4H,
CH2), 7.44 (d, 2H, J ) 8.5 Hz, Ar), 7.54 (d, 2H, J ) 8.5 Hz, Ar).
13C NMR (D2O) δ 36.4 (CH3), 42.3 (CH2), 123.1, 125.2, 134.1,
136.0 (A.), 158.0 (CN). HRMS (ESI+) m/z calcd [M + H]+
191.1291, found 191.1291. Anal. calcd for (C10H16Cl2N4 ·0.4H2O):
C, H, N.
Dihydrochloride Salt of N-Ethyl-N′-imidazolidin-2-ylidene-
benzene-1,4-diamine (5b): Method B. White solid (93%); mp
198-200 °C. 1H NMR (D2O) δ 1.29 (t, 3H, J ) 7.5 Hz, CH3CH2),
3.47 (q, 2H, J ) 7.5 Hz, CH3CH2), 3.75 (s, 4H, CH2), 7.45 (d, 2H,
J ) 9.0 Hz, Ar), 7.53 (d, 2H, J ) 9.0 Hz, Ar). 13C NMR (D2O) δ
9.7 (CH3CH2), 42.3 (CH2), 46.9 (CH3CH2), 123.7, 125.1, 132.4,
136.0 (Ar), 158.0 (CN). HRMS (ESI+) m/z calcd [M + H]+
205.1448, found 205.1443. Anal. calcd for (C11H18Cl2N4 ·1.5H2O):
C, H, N.
Dihydrochloride Salt of N-Ethyl-N′-imidazolidin-2-ylidene-N-
methyl-benzene-1,4-diamine (6b): Method B. White solid (93%);
mp 48-50 °C. 1H NMR (D2O) δ 1.15 (t, 3H, J ) 7.5 Hz, CH3CH2),
3.26 (s, 3H, CH3), 3.63 (q, 2H, J ) 7.5 Hz, CH3CH2), 3.76 (s, 4H,
CH2), 7.49 (d, 2H, J ) 9.0 Hz, Ar), 7.64 (d, 2H, J ) 9.0 Hz, Ar).
13C NMR (D2O) δ 9.3 (CH3CH2), 42.3 (CH2), 44.1 (CH3), 54.8
(CH3CH2), 122.5, 125.1, 136.6, 137.4 (Ar), 157.9 (CN). HRMS
(ESI+) m/z 219.1604 calcd [M + H]+, found 219.1605. Anal. calcd
for (C12H20Cl2N4 ·1.8H2O): C, H, N.
Dihydrochloride Salt of N-(4-Methylamino-phenyl)-guanidine
1
(7b): Method B. Pinkish solid (95%); mp 74-76 °C. H NMR
(D2O) δ 3.08 (s, 3H, CH3), 7.46 (d, 2H, J ) 8.5 Hz, Ar), 7.56 (d,
2H, J ) 9.0 Hz, Ar). 13C NMR (D2O) δ 36.4 (CH3), 123.3, 126.7,
134.6, 135.3 (Ar), 155.6 (CN). HRMS (ESI+) m/z 165.1135 calcd
[M + H]+, found 165.1138. Anal. calcd for (C8H14Cl2N4 ·1.3H2O):
C, H, N.
Dihydrochloride Salt of N-(4-Ethylamino-phenyl)-guanidine
1
(8b): Method B. White solid (94%); mp 126-128 °C. H NMR
(D2O) δ 1.31 (t, 3H, J ) 7.0 Hz, CH3CH2), 3.48 (q, 2H, J ) 7.0
Hz, CH3CH2), 7.49 (d, 2H, J ) 8.0 Hz, Ar), 7.54 (d, 2H, J ) 8.0
Hz, Ar). 13C NMR (D2O) δ 9.8 (CH3CH2), 46.7 (CH3CH2), 123.6,
126.7, 133.2, 135.0 (Ar), 155.6 (CN). HRMS (ESI+) m/z 179.1297
General Procedure for the Synthesis of the Dihydrochloride
Salts: Method B. Each of the corresponding Boc-protected precur-
sors (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
dihydrochloride salt. Absence of the trifluoroacetate salt was
checked by 19F NMR.
calcd [M
(C9H16Cl2N4 ·1.0H2O): C, H, N.
+
H]+, found 179.1303. Anal. calcd for
Dihydrochloride Salt of N-[4-(Ethyl-methyl-amino)-phenyl]-
guanidine (9b): Method B. White solid (96%); mp decomposes over
150 °C. 1H NMR (D2O) δ 1.16 (t, 3H, J ) 7.0 Hz, CH3CH2), 3.27
(s, 3H, CH3), 3.64 (q, 2H, J ) 7.0 Hz, CH3CH2), 7.52 (d, 2H, J )
8.5 Hz, Ar), 7.65 (d, 2H, J ) 8.5 Hz, Ar). 13C NMR (D2O) δ 9.2
(CH3CH2), 44.1 (CH3), 54.7 (CH3CH2), 122.6, 126.6, 135.9, 137.7
(Ar), 155.5 (CN). HRMS (ESI+) m/z 193.1448 calcd [M + H]+,
found 193.1450. Anal. calcd for (C10H18Cl2N4 ·0.2H2O): C, H, N.

608 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Rodriguez et al.
N-Methyl-benzene-1,4-diamine (13): Method D. Red oil (87%);
added. Nonspecific binding of the radioligand was defined as the
remaining [35S]GTPγS binding in the presence of 10 µM unlabeled
GTPγS.
Microdialysis Experiments. Male Sprague-Dawley rats (250-300
g) were implanted with a probe in a stereotaxic apparatus under
chloral hydrate anesthesia (400 mg/kg ip). The probe was located
in the prefrontal cortex (PFC) according to the coordinates of the
atlas of Paxinos and Watson22 (AP (anterior to bregma) +2.8 mm,
L (lateral from the midsagittal suture) +1 mm, DV (ventral from
the dura surface) -5 mm).
Experiments were performed 20-24 h after the probe implanta-
tion and aCSF (148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2 and
0.85 mM MgCl2; pH 7.4) was pumped at a flow rate of 1 µL/min
(CMA/Microdialysis infusion pump). Drugs, when locally admin-
istered, were dissolved in aCSF and applied during 70 min via
dialysis probe in increasing concentrations of 1, 10, and 100 µM.
Drugs systemically administered were dissolved in saline and
injected intraperitoneally.
1
IR (nujol) ν 3400, 3339, 3222 cm-1. H NMR (CDCl3) δ 2.76 (s,
3H, CH3), 3.29-3.38 (m, 3H, NH2 + CH3NH), 6.52 (d, 2H, J )
9.0 Hz, Ar), 6.62 (d, 2H, J ) 9.0 Hz, Ar). 13C NMR (CDCl3) δ
31.4 (CH3), 113.7, 116.5, 137.4, 142.2 (Ar).
N-Ethyl-benzene-1,4-diamine (14): Method D. Red oil (84%);
1
IR (nujol) ν 3378, 3335, 3217 cm-1. H NMR (CDCl3) δ 1.24 (t,
3H, J ) 7.0 Hz, CH3CH2), 2.85-3.19 (m, 5H, NH2 + NH +
CH3CH2), 6.53 (d, 2H, J ) 8.5 Hz, Ar), 6.62 (d, 2H, J ) 8.5 Hz,
Ar). 13C NMR (CDCl3) δ 15.0 (CH3CH2), 39.6 (CH3CH2), 114.5,
116.8, 137.6, 141.6 (Ar).
N-Ethyl-N-methyl-benzene-1,4-diamine (16): Method D. Brown-
1
ish oil (89%); IR (nujol) ν 3433, 3345 cm-1. H NMR (CDCl3) δ
1.10 (t, 3H, J ) 7.0 Hz, CH3CH2), 2.82 (s, 3H, CH3), 3.21-3.43
(m, 4H, NH2 + CH3CH2), 6.67 (d, 2H, J ) 9.0 Hz, Ar), 6.71 (d,
2H, J ) 9.0 Hz, Ar). 13C NMR (CDCl3) δ 10.9 (CH3CH2), 38.4
(CH3), 48.2 (CH3CH2), 115.8, 116.5, 137.5, 143.1 (Ar).
Pharmacology: Materials and Methods. Preparation of Mem-
branes. Neural membranes (P2 fractions) were prepared from the
PFC of human brains obtained at autopsy in the Instituto Vasco de
Medicina Legal, Bilbao, Spain. Postmortem human brain samples
of each subject (∼1 g) were homogenized using a Teflon-glass
grinder (10 up-and-down strokes at 1500 rpm) in 30 volumes of
homogenization buffer (1 mM MgCl2 and 5 mM Tris-HCl, pH 7.4)
supplemented with 0.25 M sucrose. The crude homogenate was
centrifuged for 5 min at 1000g (4 °C), and the supernatant was
centrifuged again for 10 min at 40000g (4 °C). The resultant pellet
was washed twice in 20 volumes of homogenization buffer and
recentrifuged in similar conditions. Aliquots of 1 mg protein were
stored at -70 °C until assay. Protein content was measured
according to the method Bradford using BSA as standard and was
similar in the different brain samples.
Samples were collected every 35 min and NA concentrations
analyzed by HPLC apparatus with amperometric detection (Hewlett-
Packard model 1049A) at an oxidizing potential of +650 mV. The
mobile phase (12 mM citric acid, 1 mM EDTA, 0.7 mM octylsodio
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,
USA). Samples (injection volume 30 µL) were injected and NA
analyzed in a run time of 10 min.
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 percentage of
the baseline value ( SEM. 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.
[3H]RX821002 Binding 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 What-
man 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 radioactiv-
ity 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).
Drugs. [3H]RX821002 (specific activity 59 Ci/mmol) was
obtained from Amersham International, UK. [35S]GTPγS (1250 Ci/
mmol) was purchased from DuPont NEN (Brussels, Belgium).
Idazoxan HCl was synthesized by Dr. F. Geijo at SA Lasa
Laboratories, Barcelona, Spain. Clonidine HCl, GDP, GTP, GTPγS,
RX821002 HCl, and UK14304 were purchased from Sigma (St.
Louis, MO). All other chemicals were of the highest purity
commercially available.
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 S-PE07UN13 and IT-199/07), and Spanish Ministry
of Health, Instituto de Salud Carlos III, (Ciber-SAM). A.M.E.
was recipient of a predoctoral fellowship from the Basque
Government.
Analysis of Binding Data. Analysis of competition experiments
to obtain the inhibition constant (Ki) were performed by nonlinear
regression using the GraphPad Prism program. All experiments were
analyzed assuming a one-site model of radioligand binding. Ki
values were normalized to pKi values.
[35S]GTPγS Binding Assays. The incubation buffer for measuring
[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. To evaluate the
influence of the compounds on [35S]GTPγS binding, 8 concentra-
tions (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,
transferred to vials containing 5 mL of OptiPhase HiSafe II cocktail
(Wallac, UK), and the radioactivity trapped was determined by
liquid scintillation spectrometry (Packard 2200CA). The
[35S]GTPγS bound was about 7-14% of the total [35S]GTPγS
Supporting Information Available: Preparation, 1H NMR, 13
C
NMR, and MS data of all new Boc-protected derivatives prepared
(4a-9a, 11, 12, and 15), a table containing the combustion analysis
data and the 1H and 13C NMR spectra for the new final compounds
(4b-9b) is presented. This material is available free of charge via
the Internet at http://pubs.acs.org.
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