Guanidine-based α2-adrenoceptor ligands: Towards selective antagonist activity

Article abstract of DOI:10.1016/j.ejmech.2014.05.057

Depression has been linked to a selective increase in the high affinity conformation of the α₂-adrenergic autoreceptors (α2-ARs) in the human brain as well as to an overexpression of α2-ARs in the hippocampus and cerebral cortex. Thus, the development of novel α2-AR antagonists represents an attractive source of new antidepressants. This paper describes the design, synthesis and pharmacological evaluation of 30 new guanidinium and 2-iminoimidazolidinium as potential α2-AR antagonists. In order to design this new series of α2-AR antagonists, a pharmacophore model was developed using the GALAHAD software. This study suggested that increased substitution in the space surrounding the cationic guanidine moiety might lead selectively to antagonist activity. Following the preparation of compounds incorporating this feature and competitive radioligand binding, [ ³⁵S]GTPγS functional assays revealed that this structural modification affords exclusively α2-AR antagonists, in contrast with the analogous unsubstituted compounds in which a mixture of antagonist/agonist activities was previously observed.

Full text of DOI:10.1016/j.ejmech.2014.05.057

European Journal of Medicinal Chemistry 82 (2014) 242e254
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Guanidine-based a2-adrenoceptor ligands: Towards selective
antagonist activity
,
*
Daniel H. O'Donovan a, Carolina Muguruza b, Luis F. Callado b, Isabel Rozas a
a School of Chemistry, TBSI, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland
b
ꢀ
ꢀ
Department of Pharmacology, University of the Basque Country UPV/EHU, and Centro de Investigacion Biomedica en Red de
Salud Mental CIBERSAM, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Depression has been linked to a selective increase in the high affinity conformation of the a2-adrenergic
autoreceptors ( 2-ARs) in the human brain as well as to an overexpression of 2-ARs in the hippocampus
and cerebral cortex. Thus, the development of novel 2-AR antagonists represents an attractive source of
new antidepressants. This paper describes the design, synthesis and pharmacological evaluation of 30
new guanidinium and 2-iminoimidazolidinium as potential 2-AR antagonists. In order to design this
new series of 2-AR antagonists, a pharmacophore model was developed using the GALAHAD software.
Received 5 March 2014
Received in revised form
19 May 2014
Accepted 23 May 2014
Available online 27 May 2014
a
a
a
a
a
This study suggested that increased substitution in the space surrounding the cationic guanidine moiety
might lead selectively to antagonist activity. Following the preparation of compounds incorporating this
Keywords:
Pharmacophore
Alpha 2-adrenoceptor
Antagonists
feature and competitive radioligand binding, [35S]GTP
g
S functional assays revealed that this structural
2-AR antagonists, in contrast with the analogous unsubstituted com-
pounds in which a mixture of antagonist/agonist activities was previously observed.
© 2014 Elsevier Masson SAS. All rights reserved.
modification affords exclusively
a
Human prefrontal cortex tissue
[
35S]GTP
gS
1. Introduction
suggested that not only the presynaptic autoreceptors but also the
postsynaptic 2-ARs may play a role in the activity of some anti-
depressants [6]. It has also been shown that 2-AR activation re-
duces neurogenesis in the hippocampus, an effect now seen as a
fundamental causative factor in depression [7]. As such, 2-AR
a
Depression is a mental disorder characterized by a long-lasting
low mood, behavioural changes, and increased morbidity and
mortality. As a major worldwide health burden, the World Health
Organisation has predicted that by the year 2020, depression will
constitute the single largest contributor to morbidity and disability
in the developed world [1]. Although the complete aetiology re-
mains unclear, depression has been closely linked to changes in
monoaminergic neurotransmission [2], and virtually all clinically
effective antidepressant drugs have been shown to exert activity
upon monoaminergic targets [3].
a
a
blockade results in increased hippocampal neurogenesis [8].
Interestingly, a selective increase in the high-affinity conformation
of
a
2-ARs has been found in the hippocampus and cerebral cortex
2-AR antag-
of patients suffering from depression [9]. Moreover,
a
onists have been shown to induce gene expression of the plasticity-
promoting protein Arc, thereby improving neuroplasticity in
depressed individuals [10,11]. All these theories strongly support
The a2-adrenoceptors (
activation of presynaptic
a
a
2-ARs) represent one such target, as the
2-ARs autoreceptors by endogenous
the application of
a
2-AR antagonists as antidepressants.
2-ARs has been studied
The antidepressant potential of the a
noradrenaline (NA) inhibits the release of neurotransmitters into
the synapse [4], hence, antagonism of 2-ARs results in increased
synaptic concentrations of monoamines [5]. Recent studies have
since the 1960s when researchers identified the tetracyclic anti-
depressants mianserin (1) and mirtazapine (2) [12] and attributed
their activity to their a2-AR antagonism (Fig. 1) [13]. Studies have
a
shown that mirtazapine exhibits a level of clinical efficacy similar to
that of the serotonin transporter inhibitor venlafaxine [14], which is
the best-selling antidepressant at the time of writing. However, as
with other clinically used antidepressants, mirtazapine still pre-
sents unwanted side effects and a long onset of action, prompting
Abbreviations:
a gS, radio labelled gua-
2-AR, Alpha 2-adrenoceptor; [35S]GTP
nosine 50-O-[gamma-thio]triphosphate; pKi, minus logarithm of the binding con-
stant; [3H]RX821002, 2-methoxyidazoxan; UK14304, Brimonidine; EC50
maximal effective concentration; EGFR, Epidermal.
* Corresponding author.
, Half-
the need for improved
a2-AR antagonist antidepressants. It has
also been suggested that co-administration of selective
a
2-AR
E-mail address: rozasi@tcd.ie (I. Rozas).
http://dx.doi.org/10.1016/j.ejmech.2014.05.057
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
243
Fig. 1. Structures of a2-AR antagonists mianserin (1), mirtazapine (2), compounds 3 and 5 and a2-AR agonists 4 and 6.
antagonist drugs might potentiate the effects of noradrenaline and
serotonin reuptake inhibitor antidepressants by enhancing extra-
cellular NA concentrations in the brain [15].
allow us to draw analogies between our own series of arylguani-
dine 2-AR ligands and other, structurally distinct, 2-AR
antagonists.
a
a
Our group has been interested in the development of novel
AR antagonists since the 2000s, during which time we synthesized
and evaluated more than 80 arylguanidinium and aryl-2-
a
2-
All 3D structures were generated using the CONCORD program
[22], followed by geometry optimisation using the Tripos molecular
mechanics force field [23]. In cases where enantiomers are present
(i.e. idazoxan and mianserin), both enantiomers were used in the
study. The GALAHAD program was then run several times modi-
fying the parameters (see experimental section) until a pharma-
cophore model matching all ten antagonists (twelve including both
enantiomers of idazoxan and mianserin) was determined. Although
the program constructed several competing models, we chose this
particular one as it represents the only case in which the phar-
macophoric elements match satisfactorily with every antagonist
molecule in the study. The final optimal model includes four
pharmacophoric elements; namely three hydrophobic regions and
a central protonated nitrogen (Fig. 3).
iminoimidazolidinium a2-AR ligands as potential antidepressants,
including animal studies with several promising in vitro candidate
compounds [16e19]. Although these studies produced a wealth of
pharmacological data, it proved difficult to establish definitive
structureeactivity relationships. For example, guanidine derivative
3
was found to be an a2-AR antagonist, while its 2-
iminoimidazolidinium analogue 4 behaves as an agonist [17];
however, in the case of compounds 5 and 6 this pattern is reversed
[16] (Fig. 1). As such, the question of which structural features
might consistently lead to antagonistic activity was of central
importance to our efforts to develop novel a2-AR blockers incor-
porating the arylguanidinium and aryl-2-iminoimidazolidinium
structural motifs.
It is noteworthy that both enantiomers of idazoxan and com-
pound 7 overlap with only three of the four pharmacophoric el-
ements and neglect to overlap with the hydrophobic region
labelled as HY1 (Fig. 3), while every other compound overlaps
with all four elements. Moreover, many compounds also incor-
porate hydrogen-bonding moieties within the HY1 region of
chemical space. We reasoned that a new generation of arylgua-
Thus, in order to gain further insight into which structural ele-
ments impart
pharmacophore hypothesis using the GALAHAD pharmacophore
perception software [20]. By examining a diverse range of 2-AR
antagonists taken from the literature, we aimed to highlight the
features associated with 2-AR antagonist activity and to incor-
a2-AR antagonism, we sought to develop a 3D
a
a
nidine a2-AR ligands should benefit from the introduction of
porate these structural elements in the design of a new generation
of arylguanidinium/aryl-2-iminoimidazolidinium derivatives.
hydrophobic and/or hydrogen-bonding features which overlap
with the HY1 region of chemical space, thereby hoping to produce
a series of compounds with improved antagonist activity and
avoiding the mixture of agonist/antagonist activities observed in
our previous studies.
2. Results and discussion
2.1. Computational design
This design scheme gives rise to two classes of compounds as
potential antagonists, namely N,N0-disubstituted arylguanidine and
4-substituted-2-aryliminoimidazolidine derivatives, as repre-
sented in Fig. 4. The phenyl ring R1 substituents can be chosen on
In order to develop a pharmacophore model for a2-AR antago-
nism, we employed the GALAHAD program as implemented in the
Sybyl X 1.3 software suite [21]. GALAHAD takes a series of com-
pounds with a common biological activity as input and then uses a
genetic algorithm to flexibly align these compounds on the basis of
shared pharmacophoric features such as hydrogen bond acceptors
or donors, hydrophobic regions, or electronically-charged moieties.
The output is a series of 3D pharmacophore models which describe
the spatial relationships between the structural elements corre-
sponding to biological activity; these models can then assist in the
discovery and design of new compounds which incorporate the
desired bioactivity.
the basis of high-affinity a2-AR ligands previously prepared by our
group [16e18], while the R2 substituents will be chosen to incor-
porate various hydrophobic elements and potential hydrogen-
bonding moieties in an attempt to take advantage of the putative
antagonist binding interactions highlighted by this pharmacophore
study.
Introducing examples of these two classes of compounds
(including the two enantiomers from one of the 4-substituted-2-
iminoimidazolines) into our pharmacophore model using the
GALAHAD template alignment mode provided strong support for
our design scheme. As before, the R1 group overlaps with the HY2
region of the pharmacophore, while the newly-introduced R2
group now occupies the region marked as HY1 (Fig. 5). With this
supporting evidence in hand, we proceeded to prepare these
We began by selecting a diverse series of ten known
a2-AR
antagonists, including one high-affinity antagonist taken from our
previous work [16] (Fig. 2, compound 7), all of which incorporate a
characteristic protonated amine moiety. This is a common motif
found in many
a
2-AR antagonists and we hoped that it would serve
compounds and to evaluate their pharmacological potential as
AR antagonists.
a2-
as a central feature in our pharmacophore model which would

244
D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
Fig. 2. Structures of
a
2-AR antagonists used in the pharmacophore development study:BAM 1303 [26], Dibozane [26], A-80426 [26], Delaquamine [26], Piribedil [26], Mianserin
[26], Idazoxan [26], Yohimbine [26], YNS 15P [26] and compound 7 [16].
2.2. Chemistry
final arylguanidine hydrochloride salts 14e18aed. For the prepa-
ration of compounds with R2 ¼ EtOH, the alcohol was protected at
the thiourea stage as acetate 8b; the acetate group was cleaved
during the Boc deprotection step to afford alcohols 14e18b
(Scheme 1).
The 4-substituted 2-iminoimidazolidine series of compounds
were prepared using a similar sequence starting from substituted
imidazolidine-2-thiones 19a,b which were first bis-Boc protected
under standard conditions (Scheme 2) [25]. Boc protected imi-
dazolidine-2-thiones 20a,b were then treated with the appro-
priate aromatic amine in the presence of HgCl2 and triethylamine
to construct the 2-iminoimidazolidine core 21e25a,b. Finally, the
Boc groups were removed using dry HCl in dioxane to afford the
For the preparation of the N,N0-disubstituted arylguanidine se-
ries of compounds, we developed a concise three-step procedure to
facilitate the rapid preparation of structural analogues (Scheme 1)
[24]. Thiourea is first bis-Boc protected under standard conditions
and then converted in situ to the corresponding N-Boc protected N0-
substituted thioureas 8aed by activation with trifluoroacetic an-
hydride, followed by treatment with the appropriate amine.
The substituted thioureas 8aed are then subjected to HgCl2
promoted guanidylation with an aromatic amine to afford Boc
protected N0-substituted arylguanidines 9e13aed. Finally, the Boc
groups are removed using a methanolic solution of HCl to afford the
Fig. 3. Final pharmacophore model overlaid with (upper left) all molecules in the study, and the representative a2-AR antagonists (upper right) piribedil, (lower left) idazoxan and
(lower right) compound 7 with pharmacophoric elements as labelled spheres (HY ¼ hydrophobic region, in blue; NP ¼ protonated nitrogen, in red). The conformations are shown as
generated by the GALAHAD program. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
245
Table 2 presents the
a2-AR affinity data (expressed as pKi
values) for 4-substituted-2-iminoimidazolidine derivatives
(26e30a,b). As before, the pKi values of the corresponding unsub-
stituted 2-iminoimidazolidines (26c-30c) [16e18] are included for
reference. When compared with the N,N0-disubstituted guanidine
series, the substituted 2-iminoimidazolidines generally display
Fig. 4. Proposed structures of potential a
2-AR antagonists of the N,N0-disubstituted-
aryl-guanidine (left) and N-aryl-4-substituted-2-iminoimidazolidine (right) types.
lower affinities for the a2-AR. This contrasts with previous studies
in our group in which unsubstituted 2-iminoimidazolidines
(R2 ¼ H) exhibited higher affinities than their unsubstituted gua-
nidine analogues.
desired 4-substituted-2-iminoimidazolidine hydrochloride salts
26e30a,b.
The effect of the aryl R1 substituent loosely follows the same
order as observed in the guanidine derivatives: 1,2,3,4-
tetrahydronaphthyl
>
4-dimethylamino
>
3,4-
methylenedioxyl > 4-ethylamino > 4-ethyloxy. Regarding the R2
substituent, furan derivatives 26e30b provide higher affinities than
their 4-methyl analogues, echoing the higher affinities in furan
substituted guanidines 14e18a versus their n-propyl substituted
analogues 14e18d.
2.3. Pharmacology
2.3.1. [3H]RX821002 competitive binding assays
The affinity of N,N0-disubstituted guanidines 14e18aed, plus
that of the mono-substituted guanidines 14e18e (R2 ¼ H) towards
In general, the introduction of an R2 substituent gives rise to
a
2-ARs was determined in human prefrontal cortex brain tissue
slightly lower affinities for the
a2-AR when compared with our
using a competitive binding assay with the a2-AR selective radio-
previous series of compounds with R2 ¼ H; this effect is particularly
pronounced for the 2-iminoimidazolidine derivatives. Nonetheless,
we were eager to determine whether the introduction of the R2
ligand [3H]RX821002 (2-methoxyidazoxan), which was used at a
constant concentration of 2 nM. Affinity data for the guanidine
hydrochloride salts 14e18aee, calculated as pKi values, are pre-
group prevented activation of
of our antagonist pharmacophore model. We therefore proceeded
to study the 2-AR activity of those compounds with the most
promising affinities.
a2-ARs by fulfilling the requirements
sented in Table 1; the pKi values of the clinically-used a2-AR agonist
clonidine (see structure in Table 1) and the reference ligand
RX821002 are included for comparison.
a
These results outline a number of structureeaffinity relation-
ships for arylguanidine a2-AR ligands. Broadly speaking, aromatic
R2 substituents (furan-2-ylmethyl and phenyl; 14e18a and 14e18c)
provide higher affinities on average than non-aromatic groups
(ethoxy and n-propyl substituents; 14e18b and 14e18d). N,N0-
disubstituted guanidine derivatives also provide somewhat
reduced affinities compared with their mono-substituted analogues
(14e18e). Furthermore, the effect of the R1 substituent upon af-
finity loosely follows the order: 1,2,3,4-tetrahydronaphthyl > 4-
2.3.2. [35S]GTP
From among the newly-prepared compounds, a set of eleven
compounds with the highest affinity towards 2-AR (26c, 14e, 15a,
16c, 15c, 16a, 16d, 28b, 28a, 17a, 15d) was subjected to [35S]GTP
binding experiments to determine their effect upon 2-ARs as
either agonists or antagonists. The 2-AR agonist UK14304 (bri-
gS binding functional assays
a
gS
a
a
monidine) and a selection of five representative compounds with
lower affinities (14a, 18a, 15b, 14d, 18b) were also included for
comparison.
dimethylamino
> 3,4-methylenedioxyl > 4-ethylamino > 4-
ethyloxy. The higher affinities of compounds with the lipophilic
R1 ¼ 1,2,3,4-tetrahydronaphthyl group are in good agreement with
our pharmacophore model, in which this moiety overlaps with the
hydrophobic regions labelled as HY2 and HY3 (Fig. 3).
The [35S]GTP
g
S binding assay is a useful tool to distinguish be-
tween agonists, inverse agonists, and antagonists of G-protein
coupled receptors such as the 2-ARs [26]. When an agonist binds
a
Fig. 5. Pharmacophore model overlaid with two newly-designed compounds; (left) N,N0-disubstituted arylguanidine (18c) and (right) N-aryl-4-substituted 2-iminoimidazolidine
(28b). Compounds are numbered as per Schemes 1 and 2 (vide infra).
Scheme 1. Preparation of N,N0-disubstituted arylguanidines. (a) (i) NaH, Boc2O, THF, 0 ꢀC e rt, 1 h, (ii) NaH, trifluoroacetic anhydride, R2-NH2, THF, 0 ꢀC e rt, 16 h, 28e91%; (b)
HgCl2, TEA, ArNH2, DCM, 0 ꢀC e rt, 16 h, 49e91%; (c) 1.25 M HCl/MeOH, 35 ꢀC, 3.5 h, 65e94%. *The acetate group is removed during step (c), producing 14e18b with R2 ¼ EtOH.

246
D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
Scheme 2. Preparation of 4-substituted 2-iminoimidazolidines.(a) NaH, Boc2O, THF, 0 ꢀC e rt, 3 h, 81e84%; (b) HgCl2, TEA, ArNH2, DCM, 0 ꢀC e rt, 16 h 62e88%. (c) 4 M HCl/
dioxane, 55 ꢀC, 3.5 h, 58e76%.
to the
leading to the exchange of GDP for GTP on the
its dissociation into the -GTP and bg subunits. The activity of the
a
2-ARs, the conformation of the coupled G protein is altered,
binding experiments in the presence of the a2-AR selective agonist
a
-subunit, promoting
UK14304 in order to confirm their activity as antagonists. In these
a
experiments, the effect of a set concentration (10ꢁ5 M) of each
G-protein can be evaluated by determining the guanine nucleotide
exchange using radiolabeled GTP analogues. As such, agonists lead
to an increase in radio labelled nucleotide binding; inverse agonists
lead to a decrease in nucleotide binding, while antagonists exhibit
no effect on nucleotide binding. As shown in Fig. 6, all compounds
potential antagonist upon [35S]GTP
gS binding stimulation curves
induced by the agonist UK14304 (10ꢁ13 to 10ꢁ4 M) was evaluated.
The rightwards shift of the concentrationeresponse curve for
UK14304 in the presence of these compounds (Fig. 7) confirmed
their
Thus, the addition of antagonists 14e, 26c, 16c, 15a and 15c to
35S]GTP
S binding experiments induced, in all cases, an increase in
a2-AR antagonist effect.
tested showed no stimulatory effect on the [35S]GTP
result which corresponds to antagonist activity.
gS binding, a
[
g
Five compounds (14e, 15a,16a,16c and 26c), which showed high
the EC50 value for UK14304 (Table 3). In the case of mono-
substituted compounds 14e and 26c (R1 ¼ 4-EtO, R2 ¼ H), although
it remains difficult to rationalise their antagonist effect, it is
affinities for the
a
2-AR in radioligand binding assays and did not
stimulate [35S]GTP
g
S binding, were further subjected to [35S]GTP
gS
Table 1
Affinity values (expressed as pKi) obtained for guanidine derivatives 14e18aee.2
Compound
R1
R2
pKi
RX821002
Clonidine
14a
15a
16a
17a
18a
14b
15b
16b
17b
18b
14c
15c
16c
17c
18c
e
e
8.72
7.68
5.83
6.67
6.29
5.97
5.82
4.72
5.73
5.76
4.73
4.93
5.65
6.41
6.58
5.31
5.36
5.37
5.95
6.17
5.08
4.82
6.85
7.06
7.11
6.58
6.40
e
e
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
eCH2-(fur-2-yl)
eCH2-(fur-2-yl)
eCH2-(fur-2-yl)
eCH2-(fur-2-yl)
eCH2-(fur-2-yl)
e(CH2)2eOH
e(CH2)2eOH
e(CH2)2eOH
e(CH2)2eOH
e(CH2)2eOH
eC6H5
eC6H5
eC6H5
eC6H5
eC6H5
e(CH2)2eCH3
e(CH2)2eCH3
e(CH2)2eCH3
e(CH2)2eCH3
e(CH2)2eCH3
eH
14d
15d
16d
17d
18d
14ea
15e [18]
16e [17]
17e [18]
18e [16]
eH
eH
eH
eH
a
The preparation of compound 14e is described in the supplementary material.

D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
247
Table 2
both in terms of affinity and antagonist potency, there now exists a
clear rationale for which structural features can impart antagonist
activity within this class of compounds.
Affinity values (as pKi) obtained for 2-iminoimidazolidine derivatives 26e30aec.3
3. Conclusions
In our previous studies examining mono-substituted guanidine
and unsubstituted 2-iminoimidazolidine derivatives, it was often
difficult to determine the structureeactivity relationships which
Compound
R1
R2
pKi
26a
27a
28a
29a
30a
26b
27b
28b
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
4-OEt
4-NMe2
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
4-OEt
4-NMe2
eCH3
eCH3
eCH3
eCH3
4.85
5.48
6.00
4.91
5.63
5.58
5.88
6.16
5.66
5.77
7.10
7.42
7.33
6.75
7.33
govern functional activity at the
compounds act as antagonists, whereas a significant portion of
them acted instead as 2-AR agonists.
After a molecular modelling study (using GALAHAD pharma-
cophore analysis) to inform the design of new 2-AR antagonists, a
a2-AR. Few of these unsubstituted
a
eCH3
e(fur-2-yl)
e(fur-2-yl)
e(fur-2-yl)
e(fur-2-yl)
e(fur-2-yl)
eH
eH
eH
eH
eH
a
total of 30 new N,N0-disubstituted guanidine and 4-substituted 2-
iminoimidazolidine derivatives plus 2 new mono-substituted gua-
nidine and 4-unsubstituted 2-iminoimidazolidine derivatives were
prepared following our previously established synthetic methods.
While the incorporation of the R2 substituent seems to lead to a
29b
30b
26ca
27c [18]
28c [17]
29c [18]
30c [16]
3,4-(eCH2e)4
4-NHEt
3,4(eOCH2Oe)
reduction in
assays in human brain tissue, it is clear from [35S]GTP
functional experiments that this modification leads to consistent
antagonist activity at 2-ARs. Clearly, the development of aryl-
guanidine 2-AR antagonists represents a continuing challenge, as
a
2-AR affinity as suggested by competitive binding
gS binding
a
The preparation of compound 26c is described in the supplementary material.
a
a
a balance must be struck between the desired antagonist activity
imparted by N,N0-disubstituted guanidines and the increased re-
ceptor affinities associated with their unsubstituted counterparts.
interesting to note that these compounds exhibit a similar shape as
the previously discovered guanidine-type antagonist 17e (R1 ¼ 4-
EtNH, R2 ¼ H) [18].
In the case of compound 16c, the corresponding mono-
substituted guanidine (compound 3 in Fig. 1) had previously been
found to act also as an antagonist [17]. However, the most inter-
esting results are obtained for compounds 15a and 15c. In these
cases, the corresponding mono-substituted guanidine (compound 6
in Fig. 1) was found to act as an agonist [16] and therefore the
introduction of the R2 substituent (furan-2-ylmethylene for 15a or
4. Experimental
4.1. Synthesis
All commercial chemicals were obtained from SigmaeAldrich or
Fluka and were used without further purification. Deuterated sol-
vents 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 a
Biotage SP4 flash purification system with Biotage SNAP silica
phenyl for 15c) has successfully modified their activity from an a2-
AR agonist to an antagonist in human brain prefrontal cortex.
Although the efficacy of these compounds must still be improved
Fig. 6. Dose-response curves for [35S]GTP
g
S binding versus ligand concentration.

248
D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
was washed twice with methylene chloride (2 ꢂ 15 mL). The
aqueous phase was evaporated to yield the crude hydrochloride
salt. The product was purified by reverse phase chromatography (C-
8 silica, typical elution gradient: 100% H2O to 85:15% H2O:aceto-
nitrile). Solvents were removed in vacuo to afford the product.
4.1.1.2. Method B: general method for the deprotection of N-Boc
protected guanidines using methanolic hydrochloric acid. The Boc
protected guanidine (0.39 mmol) was dissolved in 1.25 M meth-
anolic HCl and stirred under argon until the reaction was adjudged
complete (Note: If the reaction fails to proceed to completion after
3 h by TLC, an additional 0.5 mL conc. HCl may be added to the
mixture). At the reaction endpoint, evaporation of solvents and HCl
was followed by reverse phase chromatography (C-8 silica, typical
elution gradient: 100% H2O to 85:15% H2O:acetonitrile). The puri-
fied fractions were evaporated to dryness to yield the product.
Fig. 7. Dose-response curves for UK14304 stimulation of [35S]GTP S binding (10ꢁ13 to
g
10ꢁ4 M, 10 concentrations) in the absence (vehicle) or in the presence of a set con-
centration (10ꢁ5 M) of ligands 14e, 26c, 15a, 16c and 15c.
4.1.1.3. Method C: general method for the deprotection of N,N0-bis-
Boc protected 2-iminoimidazolidines using hydrochloric acid in
dioxane. The bis-Boc protected 2-iminoimidazolidine (0.26 mmol)
was dissolved in methylene chloride (0.8 mL) under argon, to which
4 M HCl in dioxane (0.765 mL, 3.06 mmol) were added. The mixture
was stirred at 55 ꢀC until the reaction was adjudged complete
(typically 3 h, adjudged by TLC and MS analysis). At the reaction
endpoint, solvents and HCl were evaporated, followed by either of
two chromatographic methods. Method 1 comprised of normal
phase silica gel chromatography using a solvent system consisting
of methylene chloride and 80:20:3 CHCl3:MeOH:NH4OH (CMA),
respectively (typical elution gradient: 100% methylene chloride to
1:1 methylene chloride:CMA). The purified fractions were evapo-
rated to dryness, and the residue was dissolved in 1.25 M meth-
anolic HCl (2.5 mL). Evaporation of solvents and HCl gas afforded
the product. Method 2 comprised of reverse phase chromatography
using C-8 silica, typical elution gradient: 100% H2O to 85:15%
H2O:acetonitrile, respectively. The purified fractions were evapo-
rated to dryness to afford the product.
cartridges. 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 aluminium oxide plates.
Visualization was by UV light (254 nm). NMR spectra were recorded
in
a Bruker DPXe400 Avance spectrometer, operating at
400.13 MHz and 600.1 MHz for 1H NMR; 100.6 MHz 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.
HRMS spectra were measured on a Micromass LCT electrospray TOF
instrument with a WATERS 2690 autosampler with methanol as
carrier solvent. Melting points were determined using a Stuart
Scientific Melting Point SMP1 apparatus and are uncorrected. Pu-
rity was assessed using reverse phase HPLC with a diode-array
detector scanning wavelengths from 200 to 950 nm. HLPC anal-
ysis was carried out using a Varian ProStar system equipped with a
Varian Prostar 335 diode array detector and a manual injector
(20 mL). Integration was performed at 245 nm and peak purity was
confirmed using a purity channel. The stationary phase consisted of
an ACE 5C18-AR column (150 ꢂ 4.6 mm). The method developed for
this type of hydrochloride salts which gave optimum retention
times used a gradient from 100% aqueous formate buffer (30 mM,
pH 3.0) to 85% formate buffered methanol (30 mM, pH 3.0) and 15%
aqueous formate buffer. A minimum purity of 95.0% was set for
compounds to be tested pharmacologically.
4.1.2. N-(4-Ethoxyphenyl)-N0-(furan-2-ylmethyl)guanidine
hydrochloride (14a)
Following Method B and starting from 9a (260 mg, 0.73 mmol)
the title compound was obtained as a pale yellow hygroscopic solid
(110 mg, 51%). Mp: 52e60 ꢀC; 1H NMR (D2O):
d7.48 (app s, 1H,
CHeO Fur.), 7.12 (d, J ¼ 8.6 Hz, 2H, CH Ar.), 6.94 (d, J ¼ 8.6 Hz, 2H, CH
Ar.), 6.40 (app s, 2H, 2CH Fur.), 4.44 (s, 2H, NHCH2), 4.02 (q,
J ¼ 7.0 Hz, 2H, CH2CH3), 1.31 (t, J ¼ 7.0 Hz, 3H, CH2CH3); 13C NMR
4.1.1. General methods
4.1.1.1. Method A: general method for the deprotection of N-Boc
protected guanidines using trifluoroacetic acid. The Boc protected
guanidine (0.73 mmol) was treated with solution of 50% trifluoro-
acetic acid in methylene chloride (10 mL). After 3 h stirring at room
temperature, methylene chloride and trifluoroacetic acid were
removed by evaporation, and the residue was dissolved in H2O
(10 mL). To this solution, 1000 mg Amberlite resin in its Clꢁ form
was added, and the reaction was allowed to proceed overnight.
After 16 h the Amberlite was removed by filtration, and the filtrate
(D2O): d157.2 (Cq Ar.), 155.1 (C]N), 148.7 (Cq Fur.), 142.8 (CHeO,
Fur.), 127.5 (CH Ar.), 126.2 (Cq Ar.), 115.3 (CH Ar.), 110.2 (CH Fur.),
107.8 (CH Fur.), 64.0 (CH2CH3), 37.6 (NHCH2), 13.4 (CH2CH3); IR
(neat, cmꢁ1): n 3126 (NH), 2979 (CH), 2343, 1619, 1509, 1476, 1394,
1238, 1172 (CeO), 1114 (CeO), 1042; HRMS (m/z ES): Found:
260.1393 (Mþ. C14H18N3O2 Requires: 260.1399); Purity by HPLC:
95.1% (tR 25.96 min).
4.1.3. N-[4-(dimethylamino)phenyl]-N0-(furan-2-ylmethyl)
guanidine hydrochloride (15a)
Table 3
EC50 values obtained from the concentrationeresponse curves for
UK14304 stimulation of [35S]GTP S binding (10ꢁ13e10ꢁ4 M, 10
g
Following Method B and starting from 10a (260 mg, 0.72 mmol)
the title compound was obtained as an orange hygroscopic solid
concentrations) in the absence or presence of the different N,N0-
disubstituted guanidines (10ꢁ5 M).
(122 mg, 57%). Mp: 96e104 ꢀC; 1H NMR (D2O):
d
7.66 (d, J ¼ 9.0 Hz,
2H, CH Ar.), 7.46 (app s, 1H, CHeO Fur.), 7.44 (d, J ¼ 9.0 Hz, 2H, CH
Compound
EC50 (mM) SEM
Ar.), 6.41 (app s, 2H, 2CH Fur.), 4.49 (s, 2H, CH2), 3.28 (s, 6H,
UK14304
0.4 0.01
7.0 0.54
2.2 0.17
0.7 0.07
2.8 0.24
1.0 0.10
N(CH3)2); 13C NMR (D2O):
d154.6 (C]N), 148.4 (Cq Fur.), 142.9
UK14304 þ 14e
UK14304 þ 26c
UK14304 þ 16c
UK14304 þ 15a
UK14304 þ 15c
(CHeO Fur.), 139.9 (Cq Ar.), 136.0 (Cq Ar.), 126.4 (CH Ar.), 121.8 (CH
Ar.), 110.2 (CH Fur.), 108.0 (CH Fur.), 45.9 (N(CH3)2), 37.9 (CH2); IR
(neat, cmꢁ1): n 3114 (NH), 2322, 1620, 1584, 1515, 1465, 1374, 1258,
1195, 1133, 1072, 1016; HRMS (m/z ES): Found: 259.1554 (Mþ.

D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
249
C14H19N4O Requires: 259.1559); Purity by HPLC: 95.7% (tR
23.55 min).
(45 mg, 94%). Mp: 42e46 ꢀC; 1H NMR (D2O):
d
7.20 (d, J ¼ 8.5 Hz, 2H,
CH Ar.), 6.99 (d, J ¼ 8.5 Hz, 2H, CH Ar.), 4.05 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 3.69 (t, J ¼ 5.0 Hz, 2H, CH2OH), 3.36 (t, J ¼ 5.0 Hz, 2H,
4.1.4. N-(5,6,7,8-tetrahydronaphth-2-yl)-N0-(furan-2-ylmethyl)
guanidine hydrochloride (16a)
NHCH2), 1.33 (t, J ¼ 7.0 Hz, 3H, CH2CH3); 13C NMR (D2O):
d157.1 (Cq
Ar.), 155.6 (C]N), 127.5 (CH Ar.), 126.4 (Cq Ar.), 115.2 (CH Ar.), 64.0
(CH2CH3), 59.3 (CH2OH), 43.1 (NHCH2), 13.3 (CH2CH3); IR (neat,
cmꢁ1): n 3158 (NH), 2979 (CH), 2368, 1617, 1514, 1476, 1394, 1237,
1171 (CeO), 1114 (CeO), 1042; HRMS (m/z ES): Found: 224.1403
(Mþ. C11H18N3O2 Requires: 224.1399); Purity by HPLC: 96.8% (tR
21.97 min).
Following Method B and starting from 11a (155 mg, 0.42 mmol)
the title compound was obtained as a clear hygroscopic solid
(68 mg, 53%). Mp: 72e78 ꢀC; 1H NMR (D2O):
d7.48 (m, 1H, CHeO
Fur.), 7.13 (d, J ¼ 9.0 Hz, 1H, CH Ar.), 6.92 (m, 2H, 2CH Ar.), 6.41 (dd,
J ¼ 3.5, 2.0 Hz, 1H, CH Fur.), 6.38 (d, J ¼ 3.5 Hz, 1H, CH Fur.), 4.44 (s,
2H, NHCH2), 2.68 (m, 4H, 2CH2), 1.70 (m, 4H, 2CH2); 13C NMR (D2O):
d154.9 (C]N), 148.7 (Cq Fur.), 142.8 (CHeO, Fur.), 138.8 (Cq Ar.),
4.1.8. N-[4-(dimethylamino)phenyl]-N0-(2-hydroxyethyl)guanidine
hydrochloride (15b)
137.0 (Cq Ar.), 130.6 (Cq Ar.), 130.0 (CH Ar.), 125.8 (CH Ar.), 122.5 (CH
Ar.), 110.1 (CH Fur.), 107.7 (CH Fur.), 37.6 (NHCH2), 28.2 (CqCH2CH2),
27.9 (CqCH2CH2), 22.0 (CqCH2CH2), 21.8 (CqCH2CH2); IR (neat,
cmꢁ1): n 3143 (NH), 2929 (CH), 2858, 1631, 1600, 1501, 1436, 1346,
1247, 1148, 1074, 1014; HRMS (m/z ES): Found: 270.1608 (Mþ.
Following Method A and starting from 10b (100 mg, 0.31 mmol),
the title compound was obtained as an off-white hygroscopic solid
(65 mg, 80%). Mp: 54e62 ꢀC; 1H NMR (D2O):
d7.68 (d, J ¼ 9.0 Hz, 2H,
CH Ar.), 7.49 (d, J ¼ 9.0 Hz, 2H, CH Ar.), 3.74 (t, J ¼ 5.0 Hz, 2H,
C
16H20N3O Requires: 270.1606); Purity by HPLC: 95.2% (tR
CH2OH), 3.44 (t, J ¼ 5.0 Hz, 2H, NHCH2), 3.29 (s, 6H, N(CH3)2); 13C
29.12 min).
NMR (D2O): d155.1 (C]N), 139.8 (Cq Ar.), 136.1 (Cq Ar.), 126.3 (CH
Ar.), 121.7 (CH Ar.), 59.4 (CH2OH), 45.9 (N(CH3)2), 43.4 (NHCH2); IR
(neat, cmꢁ1): n 3164 (NH), 2578, 2453, 1662, 1628, 1592, 1516, 1469,
1410, 1260, 1184, 1135, 1063; HRMS (m/z ES): Found: 223.1550 (Mþ.
4.1.5. N-(4-ethylaminophenyl)-N0-(furan-2-ylmethyl)guanidine
hydrochloride (17a)
To a solution of 12a (90 mg, 0.25 mmol) in a 1:1 mixture of
C11H19N4O Requires: 223.1559); Purity by HPLC: 99.3% (tR
isopropyl alcohol and methylene chloride (630
mL) was added a 4 M
15.76 min).
solution of HCl in dioxane (377 mL, 1.51 mmol). After 3.5 h stirring at
35 ꢀC, solvents were evaporated and the residue was purified by
4.1.9. N-(5,6,7,8-tetrahydronaphth-2-yl)-N0-(2-hydroxyethyl)
guanidine hydrochloride (16b)
reverse phase chromatography (C-8 silica using a 100% H2O mobile
phase). Removal of solvents in vacuo at
exceeding 35 ꢀC afforded the title compound as a yellow hygro-
scopic gum (70 mg, 84%). 1H NMR (D2O):
a
temperature not
Following Method A and starting from 11b (230 mg, 0.61 mmol)
the title compound was obtained as a yellow hygroscopic solid
d
7.49 (d, J ¼ 8.5 Hz, 2H, CH
(144 mg, 87%). Mp: 64e70 ꢀC; 1H NMR (D2O):
d
7.04 (d, J ¼ 7.5 Hz,
Ar.), 7.48 (app s, 1H, CHeO Fur.), 7.40 (d, J ¼ 8.5 Hz, 2H, CH Ar.), 6.29
(app d, J ¼ 3.0 Hz, 2H, 2CH Fur.), 4.47 (s, 2H, NHCH2), 3.43 (q,
J ¼ 7.5 Hz, 2H, CH2CH3), 1.27 (t, J ¼ 7.5 Hz, 3H, CH2CH3); 13C NMR
1H, CH Ar.), 6.88 (d, J ¼ 7.5 Hz, 1H, CH Ar.), 6.87 (s, 1H, CH Ar.), 3.68
(t, J ¼ 5.0 Hz, 2H, CH2OH), 3.36 (t, J ¼ 5.0 Hz, 2H, NHCH2), 2.60 (m,
4H, 2CH2), 1.63 (m, 4H, 2CH2); 13C NMR (D2O):
d155.0 (C]N), 138.3
(D2O):
d154.6 (C]N),148.5 (Cq-O Fur.), 142.9 (CHeO Fur.), 135.6 (Cq
(Cq Ar.), 135.9 (Cq Ar.), 130.8 (Cq Ar.), 129.8 (CH Ar.), 125.0 (CH Ar.),
121.8 (CH Ar.), 59.4 (CH2OH), 43.2 (NHCH2), 28.3 (CqCH2CH2), 28.0
(CqCH2CH2), 22.1 (CqCH2CH2), 22.0 (CqCH2CH2); IR (neat, cmꢁ1): n
3265 (OH), 3154 (NH), 2928 (CH), 1628, 1601, 1501, 1435, 1352, 1249,
1136,1062 (CeO); HRMS (m/z ES): Found: 234.1609 (Mþ. C13H20N3O
Requires: 234.1606); Purity by HPLC: 95.9% (tR 26.85 min).
Ar.), 132.6 (Cq Ar.), 126.4 (CH Ar.), 123.9 (CH Ar.), 110.1 (CH Fur.),
107.9 (CH Fur.), 46.9 (CH2CH3), 37.8 (NHCH2), 9.7 (CH2CH3); IR (neat,
cmꢁ1): n 3146 (NH), 2943 (CH), 2477, 1656, 1625, 1590, 1513, 1444,
1148 (CeO), 1105 (CeO), 1018; HRMS (m/z ES): Found: 259.1548
(Mþ. C14H19N4O Requires: 259.1599); Purity by HPLC: 95.7% (tR
22.12 min).
4.1.10. N-(4-ethylaminophenyl)-N0-(2-hydroxyethyl)guanidine
hydrochloride (17b)
4.1.6. N-(3,4-methylenedioxyphenyl)-N0-(furan-2-ylmethyl)
guanidine hydrochloride (18a)
A solution of 12b (87 mg, 0.24 mmol) in methanol (1.8 mL) was
treated with 35 equivalents of conc. HCl (0.70 mL, 8.40 mmol). After
3 h stirring at 60 ꢀC, the reaction was adjudged complete (TLC, MS),
solvents were evaporated, and the residue was purified by reverse
phase chromatography (C-8 silica using 100% H2O mobile phase).
Removal of solvents afforded the title compound as an off-white
To a solution of 13a (165 mg, 0.46 mmol) in methylene chloride
(4.0 mL) was added a 4 M solution of HCl in dioxane (689
mL,
2.76 mmol). After 4.5 h stirring at rt, solvents were evaporated and
the residue was purified by reverse phase chromatography (C-8
silica using a 100% H2O mobile phase). Removal of solvents in vacuo
at a temperature not exceeding 35 ꢀC afforded the title compound
as a yellow hygroscopic solid (88 mg, 65%). Mp: 78e82 ꢀC; 1H NMR
solid (49 mg, 77%). Mp: 172e178 ꢀC; 1H NMR (D2O):
d7.49 (d,
J ¼ 8.5 Hz, 2H, CH Ar.), 7.42 (d, J ¼ 8.5 Hz, 2H, CH Ar.), 3.70 (t,
(D2O):
d
7.46 (app s, 1H, CHeO Fur.), 6.84 (d, J ¼ 8.0 Hz, 1H, CH Ar.),
J ¼ 4.5 Hz, 2H, CH2OH), 3.42 (m, 4H, 2CH2), 1.26 (t, 3H, J ¼ 7.0 Hz,
6.73 (s, 1H, CH Ar.), 6.71 (d, J ¼ 8.0 Hz, 1H, CH Ar.), 6.39 (s, 1H, CH
CH2CH3); 13C NMR (D2O):
d155.1 (C]N), 135.7 (Cq Ar.), 132.4 (Cq
Fur.), 6.37 (s, 1H, CH Fur.), 5.96 (s, 2H, OCH2O), 4.42 (s, 2H, NHCH2);
Ar.), 126.3 (CH Ar.), 123.9 (CH Ar.), 59.3 (CH2OH), 46.9 (CH2CH3),
43.4 (NHCH2), 9.7 (CH2CH3); IR (neat, cmꢁ1): n 3266 (OH), 3161
(NH), 2943 (CH), 2662, 2479, 1660, 1626, 1591, 1513, 1443, 1350,
1252, 1058, 1019; HRMS (m/z ES): Found: 223.1557 (Mþ. C11H19N4O
Requires: 223.1559); Purity by HPLC: 98.5% (tR 7.60 min).
13C NMR (D2O):
d155.2 (C]N), 148.7 (Cq Fur.), 147.5 (Cq Ar.), 146.5
(Cq Ar.), 142.8 (CHeO, Fur.), 126.9 (Cq Ar.), 120.0 (CH Ar.), 110.1 (CH
Fur.), 108.4 (CH Ar.), 107.7 (CH Fur.), 107.2 (CH Ar.), 101.4 (OCH2O),
37.6 (NHCH2); IR (neat, cmꢁ1): n 3121,1596 (C]N), 1501, 1486,1446,
1346, 1246, 1204 (CeO), 1032, 923; HRMS (m/z ES): Found:
260.1032 (Mþ. C13H14N3O3 Requires: 260.1035); Purity by HPLC:
97.0% (tR 23.79 min).
4.1.11. N-(3,4-methylenedioxyphenyl)-N0-(2-hydroxyethyl)
guanidine hydrochloride (18b)
Following Method A and starting from 13b (162 mg, 0.44 mmol)
the title compound was obtained as a yellow hygroscopic gum
4.1.7. N-(4-Ethoxyphenyl)-N0-(2-hydroxyethyl)guanidine
hydrochloride (14b)
Following Method A and starting from 9b (60 mg, 0.19 mmol),
the title compound was obtained as a brown, very hygroscopic solid
(113 mg, 98%). 1H NMR (D2O):
d
6.85 (d, J ¼ 7.5 Hz, 1H, CH Ar.), 6.74
(m, 2H, CH Ar.), 5.96 (s, 1H, OCH2O), 3.70 (t, J ¼ 5.0 Hz, 2H, CH2OH),
3.37 (t, J ¼ 5.0 Hz, 2H, NHCH2); 13C NMR (D2O):
d155.6 (C]N), 147.5

250
D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
(Cq Ar.), 146.4 (Cq Ar.), 127.1 (Cq Ar.), 119.8 (CH Ar.), 108.4 (CH Ar.),
107.1 (CH Ar.) 101.5 (OCH2O), 59.4 (CH2OH), 43.2 (NHCH2); IR (neat,
cmꢁ1): n 3163, 1602 (C]N), 1501, 1486, 1352, 1245, 1203 (CeO),
1062, 1032; HRMS (m/z ES): Found: 224.1030 (Mþ. C10H14N3O3
Requires: 224.1035); Purity by HPLC: 96.0% (tR 18.23 min).
4.1.16. N-(3,4-methylenedioxyphenyl)-N0-phenylguanidine
hydrochloride (18c)
Following Method A and starting from 13c (320 mg, 0.90 mmol)
the title compound was obtained as an off-white hygroscopic solid
(208 mg, 79%). Mp: 96e102 ꢀC; 1H NMR (D2O):
d7.42 (t, J ¼ 7.5 Hz,
2H, CH Ar.), 7.36 (t, J ¼ 7.5 Hz, 1H, CH phenyl), 7.30 (d, J ¼ 7.5 Hz, 2H,
4.1.12. N-(4-ethoxyphenyl)-N0-phenylguanidine hydrochloride
(14c)
CH Ar.), 6.82 (d, J ¼ 9.0 Hz, 1H, CH Ar.), 6.76 (s, 1H, CH Ar.), 6.75 (d,
J ¼ 9.0 Hz, 2H, CH Ar.), 5.91 (s, 1H, OCH2O); 13C NMR (D2O):
d154.7
Following Method A and starting from 9c (138 mg, 0.39 mmol)
the title compound was obtained as an off-white hygroscopic solid
(C]N), 147.5 (Cq Ar.), 146.6 (Cq Ar.), 133.4 (Cq Ar.), 129.5 (CH Ar.),
127.6 (CH Ar.), 126.8 (Cq Ar.), 125.5 (CH Ar.), 119.9 (CH Ar.), 108.4 (CH
Ar.),107.1 (CH Ar.) 101.5 (OCH2O); IR (neat, cmꢁ1): n 3129 (NH), 3105
(NH), 2975 (CH), 1619 (C]N), 1580, 1486, 1449, 1244, 1195 (CeO),
1138, 1104, 1032 (CeO), 925; HRMS (m/z ES): Found: 256.1082 (Mþ.
(98 mg, 87%). Mp: 48e55 ꢀC; 1H NMR (D2O):
d
7.49 (t, J ¼ 8.0 Hz, 2H,
CH Ar.), 7.41 (t, J ¼ 8.0 Hz, 1H, CH Ar.), 7.35 (d, J ¼ 8.0 Hz, 2H, CH Ar.),
7.30 (d, J ¼ 9.0 Hz, 2H, CH Ar.), 7.04 (d, J ¼ 9.0 Hz, 2H, CH Ar.), 4.11 (q,
J ¼ 7.0 Hz, 2H, CH2CH3), 1.37 (t, J ¼ 7.0 Hz, 3H, CH2CH3); 13C NMR
C14H14N3O2 Requires: 256.1086); Purity by HPLC: 99.9% (tR
(D2O):
d157.3 (Cq Ar.), 154.7 (C]N), 133.5 (Cq Ar.), 129.5 (CH Ar.),
23.99 min).
127.6 (Cq Ar.), 127.5 (CH Ar.), 126.2 (CH Ar.), 125.4 (CH Ar.), 115.3 (CH
Ar.), 64.0 (CH2CH3), 13.4 (CH2CH3); IR (neat, cmꢁ1): n 3059 (NH),
2978 (CH), 2929 (CH), 2282, 1738, 1616, 1575 (C]N), 1509 (Aryl),
1498, 1476, 1448, 1393, 1301, 1222, 1172, 1114 (CeO), 1087, 1042;
HRMS (m/z ES): Found: 256.1439 (Mþ. C15H18N3O Requires:
256.1450); Purity by HPLC: 98.8% (tR 25.95 min).
4.1.17. N-(4-ethoxyphenyl)-N0-propylguanidine hydrochloride
(14d)
Following Method A and starting from 9d (108 mg, 0.42 mmol)
the title compound was obtained as a yellow hygroscopic solid
(90 mg, 83%). Mp: 104e106 ꢀC; 1H NMR (D2O):
d
7.22 (d, J ¼ 8.5 Hz,
4.1.13. N-[4-(dimethylamino)phenyl]-N0-phenylguanidine
hydrochloride (15c)
2H, CH Ar.), 7.02 (d, J ¼ 8.5 Hz, 2H, CH Ar.), 4.10 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 3.18 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3), 1.59 (m, 2H,
CH2CH2CH3), 1.36 (t, 3H, J ¼ 7.0 Hz, CH2CH3), 0.91 (t, 3H, J ¼ 7.6 Hz,
Following Method A and starting from 10c (122 mg, 0.35 mmol)
the title compound was obtained as an off-white hygroscopic solid
CH2CH2CH3); 13C NMR (D2O):
d157.1 (Cq Ar.), 155.0 (C]N), 127.6
(69 mg, 70%). Mp: 112e130 ꢀC; 1H NMR (D2O):
d
7.68 (d, J ¼ 8.7 Hz,
(CH Ar.), 126.5 (Cq Ar.), 115.3 (CH Ar.), 64.1 (CH2CH3), 42.6
(CH2CH2CH3), 21.0 (CH2CH2CH3), 13.4 (CH2CH3), 9.9 (CH2CH2CH3).
IR (neat, cmꢁ1): n 3125 (NH), 2964 (CH), 2874, 2367, 1616, 1509,
1474, 1393, 1234, 1172 (CeO), 1112 (CeO), 1047. HRMS (m/z ES):
Found: 222.1608 (Mþ. C12H20N3O Requires: 222.1606). Purity by
HPLC: 97.9% (tR 25.19 min).
2H, CH Ar.), 7.53 (d, J ¼ 8.7 Hz, 2H, CH Ar.), 7.47 (t, J ¼ 7.0 Hz, 2H, CH
Ar.), 7.37 (t, J ¼ 7.0 Hz, 1H, CH Ar.), 7.33 (d, J ¼ 7.0 Hz, 2H, CH Ar.),
3.28 (s, 6H, N(CH3)2); 13C NMR (D2O):
d154.1 (C]N), 140.0 (Cq Ar.),
135.9 (Cq Ar.), 133.4 (Cq Ar.), 129.5 (CH Ar.), 127.6 (CH Ar.), 126.5 (CH
Ar.), 125.1 (CH Ar.), 121.7 (CH Ar.), 45.9 (N(CH3)2); IR (neat, cmꢁ1): n
3017 (NH), 2429, 1624, 1575, 1514, 1494, 1232, 1183, 1132, 1019;
HRMS (m/z ES): Found: 255.1609 (Mþ. C15H19N4 Requires:
255.1610); Purity by HPLC: 99.1% (tR 24.07 min).
4.1.18. N-[4-(dimethylamino)phenyl]-N0-propylguanidine
hydrochloride (15d)
4.1.14. N-(5,6,7,8-tetrahydronaphth-2-yl)-N0-phenylguanidine
hydrochloride (16c)
Following Method A and starting from 10d (125 mg, 0.39 mmol)
the title compound was obtained as a clear hygroscopic solid
Following Method A and starting from 11c (280 mg, 0.77 mmol)
the title compound was obtained as a yellow hygroscopic gum.
Recrystallisation from warm H2O provided the purified product as a
(65 mg, 65%). Mp: 50e55 ꢀC; 1H NMR (D2O):
d
7.68 (d, J ¼ 8.6 Hz, 2H,
CH Ar.), 7.47 (d, J ¼ 8.6 Hz, 2H, CH Ar.), 3.29 (s, 6H, N(CH3)2), 3.23 (t,
J ¼ 6.6 Hz, 2H, CH2CH2CH3), 1.61 (m, 2H, CH2CH2CH3), 0.92 (t,
beige solid (203 mg, 88%). Mp: 154e156 ꢀC; 1H NMR (D2O):
d7.40 (t,
J ¼ 7.5 Hz, 3H, CH2CH2CH3); 13C NMR (100 MHz, D2O):
d154.4 (C]
J ¼ 7.5 Hz, 2H, CH Ar.), 7.35 (t, J ¼ 7.5 Hz, 1H, CH Ar.), 7.25 (d,
J ¼ 7.5 Hz, 2H, CH Ar.), 7.10 (d, J ¼ 8.0 Hz, 1H, CH Ar.), 6.96 (d,
J ¼ 8.0 Hz, 1H, CH Ar.), 6.95 (s, 1H, CH Ar.), 2.65 (br s, 4H, 2CH2), 1.67
N), 136.7 (Cq Ar.), 136.2 (Cq Ar.), 126.3 (CH Ar.), 121.7 (CH Ar.), 46.0
(N(CH3)2), 42.9
(CH2CH2CH3), 20.9
(CH2CH2CH3), 10.0
(CH2CH2CH3); IR (neat, cmꢁ1): n 3149 (NH), 2465, 2449, 1624, 1591,
1515, 1463, 1260, 1183, 1134, 1017, 995; HRMS (m/z ES): Found:
221.1772 (Mþ. C12H21N4 Requires: 221.1766); Purity by HPLC: 99.4%
(tR 21.52 min).
(br s, 4H, 2CH2); 13C NMR (D2O):
d154.3 (C]N), 138.7 (Cq Ar.), 136.9
(Cq Ar.), 133.5 (Cq Ar.), 130.6 (Cq Ar.), 130.0 (CH Ar.), 129.5 (CH Ar.),
127.5 (CH Ar.), 125.5 (CH Ar.), 125.2 (CH Ar.), 122.2 (CH Ar.), 28.3
(CqCH2CH2), 28.0 (CqCH2CH2), 22.0 (CqCH2CH2), 21.9 (CqCH2CH2);
IR (neat, cmꢁ1): n 3170 (NH), 2928 (CH), 1654, 1629, 1593, 1574,
1496, 1371, 1229, 1163; HRMS (m/z ES): Found: 266.1651 (Mþ.
4.1.19. N-(5,6,7,8-tetrahydronaphth-2-yl)-N0-propylguanidine
hydrochloride (16d)
C
17H20N3 Requires: 266.1657); Purity by HPLC: 97.3% (tR 29.43 min).
Following Method A and starting from 11a (230 mg, 0.70 mmol)
the title compound was obtained as a yellow hygroscopic gum
4.1.15. N-(4-ethylaminophenyl)-N0-phenylguanidine hydrochloride
(17c)
(135 mg, 73%); 1H NMR (D2O):
d
7.08 (d, J ¼ 8.0 Hz, 1H, CH Ar.), 6.89
Following Method A and starting from 12c (210 mg, 0.59 mmol)
the title compound was obtained as a clear hygroscopic gum
(d, J ¼ 8.0 Hz, 1H, CH Ar.), 6.88 (s, 1H, CH Ar.), 3.16 (t, J ¼ 7.0 Hz, 2H,
CH2CH2CH3), 2.65 (m, 4H, 2CH2), 1.67 (m, 4H, 2CH2), 1.56 (m, 2H,
CH2CH2CH3), 0.90 (t, J ¼ 7.0 Hz, 3H, CH2CH2CH3); 13C NMR (D2O):
(75 mg, 44%); 1H NMR (D2O):
d7.49e7.30 (m, 9H, 9CH Ar.), 3.43 (q,
2H, J ¼ 7.0 Hz, CH2CH3), 1.26 (t, 3H, J ¼ 7.0 Hz, CH2CH3); 13C NMR
d154.6 (C]N), 138.6 (Cq Ar.), 136.6 (Cq Ar.), 130.8 (Cq Ar.), 129.9 (CH
(D2O):
d154.3 (C]N), 135.5 (Cq Ar.), 133.5 (Cq Ar.), 132.7 (Cq Ar.),
Ar.), 125.5 (CH Ar.), 122.2 (CH Ar.), 42.6 (CH2CH2CH3), 28.3
(CqCH2CH2), 28.0 (CqCH2CH2), 22.0 (CqCH2CH2), 21.9 (CqCH2CH2),
21.0 (CH2CH2CH3), 9.9 (CH2CH2CH3); IR (neat, cmꢁ1): n 3147 (NH),
2930 (CH), 1630, 1602, 1501, 1458, 1436, 1354, 1249, 1135; HRMS (m/
z ES): Found: 232.1814 (Mþ. C14H22N3 Requires: 232.1814); Purity
by HPLC: 95.1% (tR 29.19 min).
129.5 (CH Ar.), 127.6 (CH Ar.), 126.5 (CH Ar.), 125.2 (CH Ar.), 123.8
(CH Ar.), 46.9 (CH2CH3), 9.7 (CH2CH3); IR (neat, cmꢁ1): n 2942 (CH),
2651, 2473, 1655, 1624, 1577, 1513, 1496, 1233,1104, 1021; HRMS (m/
z ES): Found: 255.1597 (Mþ. C15H19N4 Requires: 255.1610); Purity
by HPLC: 97.7% (tR 22.56 min).

D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
251
4.1.20. N-(4-ethylaminophenyl)-N0-propylguanidine hydrochloride
(17d)
A solution of 12d (140 mg, 0.44 mmol) in methanol (3.7 mL) was
4.1.24. 4-Dimethylamino-N-(4-methylimidazolidin-2-ylidene)
aniline hydrochloride (27a)
Following Method C and starting from 22a (201 mg, 0.48 mmol)
treated with 60 equivalents of conc. HCl (2.17 mL, 26.25 mmol).
After 2 h stirring at 45 ꢀC, the reaction was adjudged complete (TLC,
MS), solvents were evaporated, and the residue was purified by
reverse phase chromatography (C-8 silica using 100% H2O mobile
phase). Removal of solvents afforded the title compound as a yellow
the title compound was obtained as an off-white hygroscopic gum
(99 mg, 71%). 1H NMR (D2O):
d
7.66 (d, J ¼ 9.0 Hz, 2H, CH Ar.), 7.45 (d,
J ¼ 9.0 Hz, 2H, CH Ar.), 4.20 (m, 1H, CHCH3), 3.85 (t, J ¼ 10.0 Hz, 1H,
CHCH2), 3.25 (dd, J ¼ 10.0, 7.0 Hz, 1H, CHCH2), 3.27 (s, 6H, N(CH3)2),
1.27 (d, J ¼ 6.0 Hz, 3H, CHCH3); 13C NMR (D2O):
d156.7 (C]N), 139.7
hygroscopic gum (50 mg, 39%). 1H NMR (D2O):
d7.48 (d, J ¼ 8.6 Hz,
(Cq Ar.), 136.4 (Cq Ar.), 125.1 (CH Ar.), 121.7 (CH Ar.), 51.1 (CHCH2),
49.1 (CHCH2), 46.0 (N(CH3)2), 19.3 (CHCH3); IR (neat, cmꢁ1): n 3127
(NH), 2971 (CH), 1643, 1600, 1517 (C]N), 1380, 1134, 1063, 996;
HRMS: (m/z ES): Found: 219.1600 (Mþ. C12H19N4 Requires:
219.1610); Purity by HPLC: 99.0% (tR 18.83 min).
2H, CH Ar.), 7.40 (d, J ¼ 8.6 Hz, 2H, CH Ar.), 3.42 (q, J ¼ 7.6 Hz, 2H,
CH2CH3), 3.18 (t, J ¼ 6.5 Hz, 2H, CH2CH2CH3), 1.56 (m, 2H,
CH2CH2CH3), 1.25 (t, 3H, J ¼ 7.6 Hz, CH2CH3), 0.86 (t, J ¼ 7.5 Hz,
CH2CH2CH3); 13C NMR (D2O):
d154.5 (C]N), 135.8 (Cq Ar.), 132.4
(Cq Ar.), 126.3 (CH Ar.), 123.8 (CH Ar.), 46.9 (CH2CH3), 42.8
(CH2CH2CH3), 20.9 (CH2CH2CH3), 9.9 (CH2CH2CH3), 9.7 (CH2CH3);
IR (neat, cmꢁ1): n 3162 (NH), 2966 (CH), 2359, 1621, 1590, 1514,
1455, 1142 (CeO), 1106 (CeO), 1020; HRMS (m/z ES): Found:
221.1763 (Mþ. C12H21N4 Requires: 221.1766); Purity by HPLC: 96.8%
(tR 19.51 min).
4.1.25. 2-Amino-N-(4-methylimidazolidin-2-ylidene)-5,6,7,8-
tetrahydronaphthalene hydrochloride (28a)
Following Method C and starting from 23a (125 mg, 0.29 mmol)
the title compound was obtained as a white solid (57 mg, 73%). Mp:
78e82 ꢀC; 1H NMR (D2O):
d
7.12 (d, J ¼ 8.5 Hz, 1H, CH Ar.), 6.93 (d,
J ¼ 8.5 Hz, 1H, CH Ar.), 6.92 (s, 1H, CH Ar.), 4.16 (m, 1H, CHCH3), 3.81
(t, J ¼ 9.6 Hz, 1H, CHCH2), 3.29 (dd, J ¼ 9.5 Hz, 7.0 Hz, 1H, CHCH2),
2.69 (s, 4H, 2CH2), 1.71 (s, 4H, 2CH2), 1.27 (d, J ¼ 6.0 Hz, 3H, CHCH3);
4.1.21. N-(3,4-methylenedioxyphenyl)-N0-propylguanidine
hydrochloride (18d)
13C NMR (D2O):
d156.9 (C]N), 138.6 (Cq Ar.), 136.2 (Cq Ar.), 131.6
Following Method A and starting from 13d (250 mg, 0.78 mmol),
the title compound was obtained as an off-white hygroscopic solid
(Cq Ar.), 129.8 (CH Ar.), 123.7 (CH Ar.), 120.5 (CH Ar.), 50.9 (CHCH3),
49.0 (CHCH2), 28.3 (CqCH2CH2), 27.9 (CqCH2CH2), 22.0 (CqCH2CH2),
21.9 (CqCH2CH2), 19.2 (CHCH3); IR (neat, cmꢁ1): n 3148 (NH), 2927
(CH), 1648, 1607, 1581, 1505, 1379, 1338, 1266, 1247, 1135, 1065;
HRMS: (m/z ES): Found: 230.1651 (Mþ. C14H20N3 Requires:
230.1657); Purity by HPLC: 98.3% (tR 26.92 min).
(145 mg, 85%). Mp: 176e178 ꢀC; 1H NMR (D2O):
1H, CH Ar.), 6.67 (m, 2H, 2CH Ar.), 5.91 (s, 1H, OCH2O), 3.11 (t,
d
6.80 (d, J ¼ 9.0 Hz,
J ¼ 7.0 Hz, 2H, CH2CH2CH3), 1.51 (m, 2H, CH2CH2CH3), 0.85 (t,
J ¼ 7.0 Hz, 3H, CH2CH2CH3); 13C NMR (D2O):
d154.9 (C]N), 147.5
(Cq Ar.), 146.3 (Cq Ar.), 127.1 (Cq Ar.), 119.8 (CH Ar.), 108.4 (CH Ar.),
107.1 (CH Ar.) 101.5 (OCH2O), 42.6 (CH2CH2CH3), 21.0 (CH2CH2CH3),
9.9 (CH2CH2CH3); IR (neat, cmꢁ1): n 3136 (NH), 2966 (CH),1638 (C]
N), 1622, 1598, 1501, 1486, 1244, 1200 (CeO), 1124 (CeO), 1032;
HRMS (m/z ES): Found: 222.1239 (Mþ. C11H16N3O2 Requires:
222.1243); Purity by HPLC: 97.1% (tR 22.53 min).
4.1.26. 4-Ethylamino-N-(4-methylimidazolidin-2-ylidene)aniline
hydrochloride (29a)
Following Method C and starting from 24a (258 mg, 0.62 mmol)
the title compound was obtained as a white hygroscopic gum
(161 mg, 90%). 1H NMR (D2O):
d
7.47 (d, J ¼ 8.6 Hz, 2H, CH Ar.), 7.36
(d, J ¼ 8.6 Hz, 2H, CH Ar.), 4.13 (m, 1H, CHCH3), 3.78 (t, J ¼ 9.2 Hz,1H,
CHCH2), 3.39 (q, J ¼ 6.8 Hz, 2H, CH2CH3), 3.25 (dd, J ¼ 9.2, 6.8 Hz,1H,
CHCH2), 1.21 (app t, J ¼ 6.8 Hz, 6H, CHCH3 þ CH2CH3); 13C NMR
4.1.22. N-(4-ethoxyphenyl)guanidine hydrochloride (14e)
Following Method A and starting from 9e (235 mg, 0.62 mmol;
see supporting information) the title compound was obtained
without further purification as hygroscopic, off-white solid
(D2O): d156.6 (Cq Ar.), 136.0 (C]N), 132.2 (Cq Ar.), 125.0 (CH Ar.),
123.9 (CH Ar.), 51.1 (CHCH2), 49.0 (CHCH2), 47.0 (CH2CH3), 19.2
(CHCH3), 9.8 (CH2CH3); IR (neat, cmꢁ1): n 3146 (NH), 2975 (CH),
2891, 2639, 2464, 1639, 1600, 1514 (C]N), 1446, 1391, 1270; HRMS:
(m/z ES): Found: 219.1614 (Mþ. C12H19N4 Requires: 219.1610); Pu-
rity by HPLC: 97.9% (tR 17.71 min).
(120 mg, 90%). Mp: 138e140 ꢀC; 1H NMR (D2O):
d
7.16 (d, J ¼ 7.5 Hz,
2H, CH Ar.), 6.94 (d, J ¼ 7.5 Hz, 2H, CH Ar.), 4.02 (q, J ¼ 6.5 Hz, 2H,
CH2CH3),1.27 (t, J ¼ 6.5 Hz, 3H, CH2CH3); 13C NMR (D2O):
d157.7(C]
N), 156.7 (Cq Ar.), 128.1 (Cq Ar.), 126.9 (CH Ar.), 115.8 (CH Ar.), 64.6
(CH2CH3), 13.9 (CH2CH3); IR (neat, cmꢁ1): n 3126 (NH), 2978 (CH),
1663 (C]N), 1619, 1582 (C]N), 1510 (Aryl), 1238, 1114 (CeO), 1041;
HRMS (m/z ES): Found: 180.1131 (Mþ þ H. C9H14N3O Requires:
180.1137); Purity by HPLC: 97.8% (tR 21.96 min).
4.1.27. 5-Amino-N-(4-methylimidazolidin-2-ylidene)benzo[d][1,3]
dioxole hydrochloride (30a)
Following Method C and starting from 25a (191 mg, 0.46 mmol)
the title compound was obtained as a white solid (90 mg, 78%). Mp:
220e222 ꢀC; 1H NMR (D2O):
d
6.86 (d, J ¼ 8.0 Hz, 1H, CH Ar.), 6.76 (s,
4.1.23. 4-Ethoxy-N-(4-methylimidazolidin-2-ylidene)aniline
hydrochloride (26a)
1H, CH Ar.), 6.74 (d, J ¼ 8.0 Hz, 1H, CH Ar.), 5.98 (s, 2H, OCH2O), 4.16
(m,1H, CHCH3), 3.81 (t, J ¼ 9.5 Hz,1H, CHCH2), 3.29 (t, J ¼ 7.5 Hz,1H,
Following Method C and starting from 21a (170 mg, 0.41 mmol)
the title compound was obtained as a clear hygroscopic gum
CH2CH), 1.27 (d, J ¼ 6.0 Hz, 3H, CHCH3); 13C NMR (D2O):
d157.5 (C]
N), 147.4 (Cq Ar.), 146.1 (Cq Ar.), 128.0 (Cq Ar.), 118.3 (CH Ar.), 108.2
(CH Ar.), 105.9 (CH Ar.), 101.5 (OCH2O), 51.0 (CHCH2), 49.0 (CHCH2),
19.2 (CHCH3); IR (neat, cmꢁ1): n 3248 (NH), 2981 (CH), 2858, 1650,
1605, 1488 (C]N), 1449, 1248, 1198, 1130, 1104, 1032, 925, 809;
HRMS: (m/z ES): Found: 220.1082 (Mþ. C11H14N3O2Requires:
220.1086); Purity by HPLC: 96.7% (tR 20.80 min).
(79 mg, 76%). 1H NMR (D2O):
d
7.20 (d, J ¼ 9.0 Hz, 2H, CH Ar.), 6.99 (d,
J ¼ 9.0 Hz, 2H, CH Ar.), 4.15 (m, 1H, CHCH3), 4.08 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 3.81 (t, J ¼ 9.5 Hz, 1H, CHCH2), 3.28 (dd, J ¼ 9.5, 7.0 Hz, 1H,
CHCH2), 1.34 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 1.27 (d, J ¼ 6.0 Hz, 3H,
CHCH3); 13C NMR (D2O):
d157.6 (Cq Ar.), 156.8 (C]N), 127.4 (Cq Ar.),
126.2 (CH Ar.), 115.1 (CH Ar.), 64.1 (CH2CH3) 51.0 (CHCH2), 49.0
(CHCH2), 19.1 (CHCH3) 13.4 (CH2CH3); IR (neat, cmꢁ1): n 3138 (NH),
2976 (CH), 2931, 1646, 1606, 1586 (C]N), 1511, 1477, 1240, 1175,
1115, 1043, 1011, 922; HRMS: (m/z ES): Found: 220.1443 (Mþ.
4.1.28. 4-Ethoxy-N-[4-(fur-2-yl)imidazolidin-2-ylidene]aniline
hydrochloride (26b)
Following Method C and starting from 21b (140 mg, 0.30 mmol)
the title compound was obtained as a clear hygroscopic gum
C
12H18N3O Requires: 220.1450); Purity by HPLC: 99.3% (tR
23.91 min).
(64 mg, 70%). 1H NMR (D2O):
d7.51 (s, 1H, CHeO Fur.), 7.21 (d,

252
D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
J ¼ 8.5 Hz, 2H, CH Ar.), 6.98 (d, J ¼ 8.5 Hz, 2H, CH Ar.), 6.43 (s, 2H, 2ꢂ
CH Fur.), 5.21 (dd, J ¼ 9.5, 6.5 Hz, 1H, CHCH2), 4.06 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 4.04 (app t, J ¼ 9.5 Hz,1H, CHCH2), 3.81 (dd, J ¼ 9.5, 6.5 Hz,
4.1.32. 5-Amino-N-[4-(fur-2-yl)imidazolidin-2-ylidene]benzo[d]
[1,3]dioxole hydrochloride (30b)
Following Method C and starting from 25b (120 mg, 0.25 mmol)
the title compound was obtained as a white solid (56 mg, 72%). Mp:
1H, CHCH2), 1.33 (t, J ¼ 7.0 Hz, 3H, CH2CH3); 13C NMR (D2O):
d157.6
(Cq Ar.), 157.0 (C]N), 150.7 (Cq Fur.), 143.2 (CHeO Fur.), 127.1 (Cq
Ar.), 126.3 (CH Ar.), 115.2 (CH Ar.), 110.1 (CH Fur.), 107.5 (CH Fur.),
64e68 ꢀC; 1H NMR (D2O):
d
7.52 (s, 1H, CH Fur.), 6.87 (d, J ¼ 8.0 Hz,
1H, CH Ar.), 6.80 (s, 1H, CH Ar.), 6.78 (d, J ¼ 8.0 Hz, 1H, CH Ar.), 6.43
(app d, J ¼ 1.0 Hz, 2H, 2ꢂ CH Fur.), 5.99 (s, 2H, OCH2O), 5.22 (dd,
J ¼ 9.5, 6.5 Hz, 1H, CHCH2), 4.02 (t, J ¼ 9.5 Hz, 1H, CHCH2), 3.82 (dd,
64.0 (CH2CH3), 51.5 (CHCH2), 46.9 (CHCH2
þ
CH2CH3), 13.4
(CH2CH3); IR (neat, cmꢁ1): n 3119 (NH), 2979 (CH), 1651, 1606, 1586
(C]N), 1512, 1477, 1244, 1043, 1010; HRMS (m/z ES): Found:
272.1397 (Mþ. C15H18N3O2 Requires: 272.1399); Purity by HPLC:
97.9% (tR 25.99 min).
J ¼ 9.5, 6.5 Hz, 1H, CHCH2); 13C NMR (D2O):
d157.7 (C]N), 150.7 (Cq
Fur.), 147.5 (Cq Ar.), 146.3 (Cq Ar.), 143.2 (CHeO Fur.), 127.7 (Cq Ar.),
118.7 (CH Ar.), 110.1 (CH Fur.), 108.3 (CH Ar.), 107.5 (CH Fur.), 106.2
(CH Ar.), 101.5 (OCH2O), 51.5 (CHCH2), 46.9 (CHCH2); IR (neat,
cmꢁ1): n 3113 (NH), 2898 (CH), 1654, 1626, 1607 (C]N), 1502, 1485,
1450, 1243, 1196, 1033, 926; HRMS (m/z ES): Found: 272.1029 (Mþ.
4.1.29. 4-Dimethylamino-N-[4-(fur-2-yl)imidazolidin-2-ylidene]
aniline dihydrochloride (27b)
C14H14N3O3 Requires: 272.1035); Purity by HPLC: 99.6% (tR
Following Method C and starting from 22b (80 mg, 0.18 mmol)
the title compound was obtained as a hygroscopic off-white solid
23.57 min).
(33 mg, 58%). Mp: 114e118 ꢀC; 1H NMR (D2O):
d7.67 (d, J ¼ 8.5 Hz,
4.1.33. N-(4-Ethoxyphenyl)-2-iminoimidazolidine hydrochloride
(26c)
2H, CH Ar.), 7.52 (s, 1H, CHeO Fur.), 7.49 (d, J ¼ 8.5 Hz, 2H, CH Ar.),
6.45 (app d, J ¼ 10.0 Hz, 2H, 2ꢂ CH Fur.), 5.28 (dd, J ¼ 10.0, 7.0 Hz,
1H, CHCH2), 4.07 (t, J ¼ 10.0 Hz, 1H, CHCH2), 3.87 (dd, J ¼ 10.0,
Following Method A and starting from 21c (200 mg, 0.49 mmol;
see supporting information) the title compound was obtained
without further purification as a hygroscopic, off-white solid
7.0 Hz, 1H, CHCH2), 3.27 (s, 6H, N(CH3)2); 13C NMR (D2O):
d156.7
(C]N), 150.5 (Cq Fur.), 143.2 (CHeO Fur.), 139.9 (Cq Ar.), 136.2 (Cq
Ar.), 125.4 (CH Ar.), 121.7 (CH Ar.), 110.2 (CH Fur.), 107.7 (CH Fur.),
51.6 (CHCH2), 47.0 (CHCH2), 45.9 (N(CH3)2); IR (neat, cmꢁ1): n 3120
(NH), 2450 (CH), 1645, 1602 (C]N), 1517, 1332, 1247, 1134, 1015,
902; HRMS (m/z ES): Found: 271.1546 (Mþ. C15H19N4O Requires:
271.1559); Purity by HPLC: 98.7% (tR 24.13 min).
(110 mg, 92%). Mp: 142e144 ꢀC; 1H NMR (D2O):
d
7.10 (d, J ¼ 8.7 Hz,
2H, CH Ar.), 6.89 (d, J ¼ 8.7 Hz, 2H, CH Ar.), 3.99 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 3.60 (s, 4H, CH2CH2), 1.26 (t, J ¼ 7.0 Hz, 3H, CH2CH3); 13
C
NMR (D2O): d159.1 (C]N), 157.3 (Cq Ar.), 127.9 (Cq Ar.), 126.6 (CH
Ar.), 115.6 (CH Ar.), 64.5 (CH2CH3), 42.7 (CH2CH2), 13.8 (CH2CH3); IR
(neat, cmꢁ1): n 3134 (NH), 2975 (CH), 2878, 1646, 1621 (C]N), 1512
(Aryl), 1488, 1394, 1293, 1238, 1174, 1116 (CeO), 1083, 1045; HRMS
(m/z ES): Found: 206.1294 (Mþ
þ
H. C11H16N3O Requires:
4.1.30. 2-Amino-N-[4-(fur-2-yl)imidazolidin-2-ylidene]-5,6,7,8-
tetrahydronaphthalene hydrochloride (28b)
206.1293); Purity by HPLC: 98.7% (tR 23.09 min).
Following Method C and starting from 23b (98 mg, 0.20 mmol)
the title compound was obtained as a white solid (39 mg, 60%). Mp:
4.2. Pharmacology
76e78 ꢀC; 1H NMR (D2O):
d7.51 (s, 1H, CHeO Fur.), 7.15 (d,
4.2.1. Preparation of membranes
J ¼ 8.0 Hz, 1H, CH Ar.), 7.00 (s, 1H, CH Ar.), 6.99 (d, J ¼ 8.0 Hz, 1H, CH
Ar.), 6.43 (s, 2H, 2ꢂ CH Fur.), 5.22 (dd, J ¼ 10.0, 6.5 Hz, 1H, CHCH2),
4.02 (t, J ¼ 10.0 Hz, 1H, CHCH2), 3.82 (dd, J ¼ 10.0, 6.5 Hz, 1H,
CHCH2), 2.71 (s, 4H, 2ꢂ CH2), 1.72 (s, 4H, 2ꢂ CH2); 13C NMR (D2O):
Neural membranes (P2 fractions) were prepared from the pre-
frontal cortex of human brain tissue obtained at autopsy in the
Instituto Vasco de Medicina Legal, Bilbao, Spain. Postmortem hu-
man 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 (5 mM TriseHCl, pH 7.4) sup-
plemented with 0.25 M sucrose. The crude homogenate was
centrifuged for 5 min at 1000ꢂ g (4 ꢀC), and the supernatant was
centrifuged again for 10 min at 40,000ꢂ g (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 Bradford method using BSA as standard and was
similar in the different brain samples.
d
157.3 (C]N), 150.7 (Cq Fur.), 143.2 (CHeO Fur.), 138.7 (Cq Ar.),
136.7 (Cq Ar.), 131.4 (Cq Ar.), 129.9 (CH Ar.), 124.3 (CH Ar.), 121.1 (CH
Ar.),110.1 (CH Fur.),107.5 (CH Fur.), 51.5 (CHCH2), 46.9 (CHCH2), 28.3
(CqCH2CH2), 27.9 (CqCH2CH2), 22.0 (CqCH2CH2), 21.8 (CqCH2CH2);
IR (neat, cmꢁ1): n 2928 (CH), 1645, 1608 (C]N), 1504, 1435, 1330,
1247, 1193, 1148, 1076, 1011, 907; HRMS (m/z ES): Found: 282.1599
(Mþ. C17H20N3O Requires: 282.1606); Purity by HPLC: 95.2% (tR
28.83 min).
4.1.31. 4-Ethylamino-N-[4-(fur-2-yl)imidazolidin-2-ylidene]aniline
hydrochloride (29b)
Following Method C and starting from 24b (122 mg, 0.26 mmol)
the title compound was obtained as a peach-coloured solid (78 mg,
4.2.2. [3H]RX821002 binding assays
Specific [3H]RX821002 binding was measured in 0.25 mL ali-
quots (50 mM TriseHCl, pH 7.5) of the neural membranes, which
were incubated in 96-well plates with [3H]RX821002 (2 nM) for
30 min at 25 ꢀC in the absence or presence of the competing
compounds (10ꢁ12 Me10ꢁ3 M, 10 concentrations). Non-specific
77%). Mp: 94e98 ꢀC; 1H NMR (D2O):
d
7.52 (m, 3H, CH Fur. þ CH Ar.),
7.46 (d, J ¼ 8.5 Hz, 2H, CH Ar.), 6.42 (app d, J ¼ 12.5 Hz, 2H, CH Fur.),
5.26 (dd, J ¼ 10.0, 6.5 Hz, 1H, CHCH2), 4.04 (t, J ¼ 10.0 Hz, 1H,
CHCH2), 3.85 (dd, J ¼ 10.0, 6.5 Hz,1H, CHCH2), 3.45 (q, J ¼ 7.5 Hz, 2H,
binding was determined in the presence of 10
mM adrenaline.
CH2CH3), 1.28 (t, J ¼ 7.5 Hz, 3H, CH2CH3); 13C NMR (D2O):
d156.9
Specific binding was determined and plotted as a function of the
compound concentration. Incubations were terminated by sepa-
rating free ligand from bound ligand by rapid filtration under
vacuum (1450 Filter Mate Harvester, Perkin Elmer) through GF/C
glass fibre filters. The filters were then rinsed three times with
(C]N), 150.5 (Cq Fur.), 143.3 (CHeO Fur.), 135.8 (Cq Ar.), 132.6 (Cq
Ar.), 125.4 (CH Ar.), 123.9 (CH Ar.), 110.2 (CH Fur.), 107.7 (CH Fur.),
51.5 (CHCH2), 46.9 (CHCH2 þ CH2CH3), 9.7 (CH2CH3); IR (neat liquid,
cmꢁ1): n 3113 (NH), 2940 (CH), 2653, 1637, 1599 (C]N), 1514, 1335,
1247, 1148, 1075, 1014; HRMS (m/z ES): Found: 271.1557 (Mþ.
300 mL binding buffer, air-dried (60 min), and counted for radio-
C
15H19N4O Requires: 271.1559); Purity by HPLC: 95.4% (tR
activity by liquid scintillation spectrometry using a MicroBeta Tri-
Lux counter (Perkin Elmer).
22.63 min).

D.H. O'Donovan et al. / European Journal of Medicinal Chemistry 82 (2014) 242e254
253
4.2.3. Analysis of binding data
work was also supported by grants from Gobierno Vasco (IT616-13;
SAIOTEK S-PE10UN14), the University of the Basque Country (UFI
Analysis of competition experiments to obtain the inhibition
constant (Ki) were performed by nonlinear regression using the
GraphPad Prism program. All experiments were analysed assuming
a one-site model of radioligand binding. Ki values were normalized
to pKi values.
ꢀ
11/35) and the Instituto de Salud Carlos III, Centro de Investigacion
ꢀ
Biomedica en Red de Salud Mental, CIBERSAM.
Appendix A. Supplementary data
4.2.4. [35S]GTP
The incubation buffer for measuring [35S]GTP
membranes contained, in a total volume of 250
3 mM MgCl2, 100 mM NaCl, 50 mM GDP, 50 mM TriseHCl at pH 7.4,
and 0.5 nM [35S]GTP
S. Protein aliquots were thawed and resus-
pended in the same buffer. The incubation was started by addition
of the membrane suspension (20 g of membrane proteins) to the
previous mixture and was performed at 30 ꢀC for 120 min with
gS binding assays
g
S binding to brain
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.057.
mL, 1 mM EGTA,
g
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