α2-adrenoceptor antagonists: Synthesis, pharmacological evaluation, and molecular modeling investigation of pyridinoguanidine, pyridino-2-aminoimidazoline and their derivatives

Article abstract of DOI:10.1021/jm501635e

We have previously identified phenylguanidine and phenyl-2-aminoimidazoline compounds as high affinity ligands with conflicting functional activity at the α₂-adrenoceptor, a G-protein-coupled receptor with relevance in several neuropsychiatric conditions. In this paper we describe the design, synthesis, and pharmacological evaluation of a new series of pyridine derivatives [para substituted 2- and 3-guanidino and 2- and 3-(2-aminoimidazolino)pyridines, disubstituted 2-guanidinopyridines and N-substituted-2-amino-1,4-dihydroquinazolines] that were found to be antagonists/inverse agonists of the α₂-adrenoceptor. Furthermore, the compounds exert their effects at the α₂-adrenoceptor both in vitro in human prefrontal cortex tissue and in vivo in rat brain as shown by microdialysis experiments. We also provide a docking study at the α_2A- and α_2C-adrenoceptor subtypes demonstrating the structural features required for high affinity binding to the receptor.

Full text of DOI:10.1021/jm501635e

Article
pubs.acs.org/jmc
α2‑Adrenoceptor Antagonists: Synthesis, Pharmacological
Evaluation, and Molecular Modeling Investigation of
Pyridinoguanidine, Pyridino-2-aminoimidazoline and Their
Derivatives
Brendan Kelly,† Michela McMullan,† Carolina Muguruza,‡ Jorge E. Ortega,‡ J. Javier Meana,‡
Luis F. Callado,‡ and Isabel Rozas*,†
†School of Chemistry, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland
‡Department of Pharmacology, University of the Basque Country UPV/EHU, and Centro de Investigacion
́ ́
Biomedica en Red de
Salud Mental, CIBERSAM, 28029 Madrid, Spain
S
* Supporting Information
ABSTRACT: We have previously identified phenylguanidine
and phenyl-2-aminoimidazoline compounds as high affinity
ligands with conflicting functional activity at the α2-
adrenoceptor, a G-protein-coupled receptor with relevance in
several neuropsychiatric conditions. In this paper we describe
the design, synthesis, and pharmacological evaluation of a new
series of pyridine derivatives [para substituted 2- and 3-
guanidino and 2- and 3-(2-aminoimidazolino)pyridines,
disubstituted 2-guanidinopyridines and N-substituted-2-
amino-1,4-dihydroquinazolines] that were found to be
antagonists/inverse agonists of the α2-adrenoceptor. Further-
more, the compounds exert their effects at the α2-adrenoceptor
both in vitro in human prefrontal cortex tissue and in vivo in
rat brain as shown by microdialysis experiments. We also provide a docking study at the α2A- and α2C-adrenoceptor subtypes
demonstrating the structural features required for high affinity binding to the receptor.
Therefore, α2-AR antagonists, which increase the release of
NA and also potentiate dopamine release in the prefrontal
cortex (PFC),3,4 seem useful pharmacological tools for the
treatment of depression (α2-AR antagonists) and schizophrenia
(selective α2C-AR antagonists). Thus, the development of
selective α2-AR antagonists suitable for clinical use can be
considered as an effective approach to the treatment of
neuropsychiatric disorders. For example, one of the most recent
antidepressants marketed is mirtazapine, which shows effective
activity by blockade of α2A- and α2C-ARs but also blocks 5-
HT1A, 5-HT2A, 5-HT2C, and histamine H1 receptors.5
As part of our interest in the development of new α2-AR’s
antagonists,6−10 we have previously described the synthesis and
pharmacological evaluation of several series of diphenyl and
phenyl substituted bis- and monoguanidine (e.g., 1 and 2,
Figure 1) as well as bis- and mono-2-aminoimidazoline
derivatives (e.g., 3, Figure 1), some of which have shown
high affinity and antagonist activity at the α2-AR. Moreover,
compounds 1 and 2, which increase levels of NA in vivo and
INTRODUCTION
■
The burden of mental health conditions is on the rise globally,
and they are the second leading cause of disability worldwide,
surpassing all cancers combined and being outranked only by
cardiovascular diseases.1 For many, the symptoms of
depression, schizophrenia, or bipolar disorder can be treated
with current medications; however, a substantial proportion of
patients obtain no improvements with these treatments.
Two of the most important neuropsychiatric disorders,
depression and schizophrenia, have been linked to the
noradrenergic function in the brain. Pathophysiologically,
depression is characterized by reduced monoamine activity
(particularly noradrenaline (NA) and serotonin) in the brain,
and correcting this imbalance has been the approach of all
currently marketed antidepressants. Depression is associated
with increased density of the α2-adrenoceptors (α2-ARs) in the
brain as well as a selective increase in the high-affinity
conformation of the α2-ARs.2 Moreover, the dorsolateral
prefrontal cortex (DLPFC) is the main anatomical pathway
for the cognitive symptoms of schizophrenia, and the
postsynaptic α2C-ARs located on the DLPFC cells control the
noradrenergic tone that maintains the basal cognitive activity.
Either too little or too much NA impairs DLPFC function.
Received: October 23, 2014
© XXXX American Chemical Society
A
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states existing under physiological conditions. Hence, we
propose to synthesize molecules that can be considered as
conformationally controlled analogues of our lead compounds
1−4 (Figure 1), allowing investigation of the importance of
molecular conformation and rigidity on binding to, and
antagonist activity at, the α2-AR. The pyridine ring and
guanidinium/2-aminoimidazolinium groups can adopt either a
coplanar or out of plane arrangement relative to each other
(Figure 2).
Figure 1. Structures of some high affinity α2-AR antagonists previously
described in our group showing antidepressant activity (1 and 2), a 2-
aminoimidazoline (3), and a disubstituted guanidine (4).
present antidepressant properties in animal models,11 have
become our lead compounds.
Despite these good results, highly similar structures have
often led to opposing functional activity at the receptor, with
prediction of the features necessary for antagonist activity
proving particularly difficult. However, we have recently
described the design (by means of a pharmacophore),
synthesis, and pharmacological evaluation of new phenyl-
guanidinium and 2-aminoimidazolinium derivatives with a
second substitution in the guanidine moiety (e.g., 4, Figure
1). This added structural modification affords exclusively α2-AR
antagonists (in contrast with the analogous monosubstituted
compounds which had previously exhibited a mixture of
antagonist/agonist activities) but has a detrimental effect on
receptor affinity.12
Hence, we present in this article the synthesis of a series of
guanidine and 2-aminoimidazoline derivatives of pyridine as
well as 1,4-dihydroquinazolin-2-amines (emanating from the
promising pharmacological profiles of initial derivatives), the
evaluation of their α2-AR binding affinity using in vitro assays in
human PFC brain tissue, functional studies to determine the
agonist or antagonist nature of a range of these molecules in
human PFC brain tissue, and microdialysis experiments to
assess the target engagement of our compounds in vivo. The
primary aim of this work is to obtain compounds displaying
solely antagonist activity at the α2-AR; thus, EC50 values against
the known agonist UK14304 have also been measured
providing several structure−activity relationships (SARs)
related to both α2-AR affinity and activity.
Figure 2. Conformational preferences of proposed compounds:
pyridinylguanidines (left, up and down), 2-aminoimidazolines (middle,
up and down), and constrained dihydroquinazoline/ dihydropyrido-
pyrimidin-2-amines (right, up and down).
For pyridin-2-yl derivatives, an intramolecular hydrogen
bond (IMHB) between the pyridine nitrogen lone-pair of
electrons and a proton from either the guanidinium or 2-
aminoimidazolinium confers a coplanar arrangement. On the
contrary, pyridin-3-yl derivatives are forced into an out of plane
arrangement due to avoidance of repulsion between aromatic
protons from the pyridine ring and NH protons from the
cationic groups. We have previously examined these con-
formations in a theoretical and spectroscopic study and
predicted that these preferences will be maintained in the
aqueous environment of the body.16
Previous studies6−11 have instructed that substitution of the
aryl ring para to the cationic group is favored for α2-AR affinity
and activity and that steric bulk in that position is not well
tolerated. Thus, the para-substituents found in lead compounds
1 (-NHEt) and 2 [-(CH2)4-] were prepared for all families
(pyridin-2-yl and pyridin-3-yl; guanidines and 2-aminoimidazo-
lines) because of their promising target engagement proved by
microdialysis experiments6,7 and their pharmacological profiles
as antidepressants in animal models.12 Additionally, a series of
substituents with diverse electronic effects were examined in an
effort to obtain further SARs information.
RESULTS AND DISCUSSION
■
Design Strategy. As part of a ligand-based drug design
strategy based on our previously developed pharmacophore,
which contains two contiguous hydrophobic cores connected
through a protonated N-containing fragment,12 we propose to
substitute one of the hydrophobic moieties by a pyridine as a
bioisostere of the phenyl ring present in our previous
derivatives.6−11 The introduction of a pyridine core will result
in a number of beneficial characteristics. On the one hand, the
pyridine ring is more metabolically stable because of its reduced
lipophilicity relative to the phenyl ring.13 On the other hand,
the high basicity of the phenylguanidine functionality (pKaH of
phenylguanidine is 10.88)14 will be substantially reduced
(calculated pKaH of 2-pyridinoguanidine is 9.54).15
Our previous pharmacophore12 as well as the pharmaco-
logical results obtained from this new series of pyridinyl
derivatives directed the choice of subsequent compounds to
explore: substitution of the molecules beyond the cationic
group (N-pyridinyl-N′-substituted guanidines) and further
restraint of molecular conformation (1,4-dihydroquinazolin-2-
amines). Furthermore, 1,4-dihydroquinazolin-2-amines and 1,4-
dihydropyrido[2,3-d]pyrimidin-2-amines maintain many of the
features of the first series of derivatives prepared while
improving the pharmacokinetic profile of the compounds.17
However, it must also be stated that our arylguanidines have
previously demonstrated an ability to cross the blood−brain
barrier in animal models,6,7 probably via amino acid trans-
porters.
Taking into account the mentioned basicity of these
compounds, their conformation must be considered in terms
of the guanidinium and 2-aminoimidazolinium protonation
B
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a
Scheme 1. General Synthetic Scheme for the Preparation of Pyridino(guanidine/-2-aminoimidazoline) Hydrochlorides
a
Reagents: (a) HgCl2, NEt3, CH2Cl2, rt; (b) HCl (4 M in 1,4-dioxane).
a
Scheme 2. General Synthetic Scheme for the Preparation of Disubstituted Guanidine Hydrochlorides
a
Reagents: (a) HgCl2, NEt3, CH2Cl2, rt; (b) HCl (4 M in 1,4-dioxane); (c) HCl (4 M in methanol) for compounds 5f, 8f, and 9f.
Chemistry. Boc-protected intermediates 5a−9a and hydro-
chloride salts 5c−9c (see Scheme 1) have been previously
described by our group,16 while all remaining compounds are
new, to the best of our knowledge, and none of the compounds
have previously been tested for action at the α2-AR. The
synthesis and characterization of all starting compounds and
Boc-protected and neutral intermediates are presented in the
Supporting Information. All compounds were tested pharma-
cologically as the hydrochloride salts because of their high
water solubility.
Our standard synthetic approach for the guanidylation or 2-
aminoimidazolidylation of poorly nucleophilic amines consists
of the treatment of the corresponding starting amine with an
activated N-Boc-protected thiourea18 or imidazolidine-2-
thione19 in the presence of mercury(II) chloride and an excess
of triethylamine to yield the corresponding Boc-protected
intermediates, which can be converted to their hydrochloride
salts on removal of the Boc group with hydrochloric acid
solutions (Scheme 1).
7c,d,l, 8c,d,i−l, 9c,d,i−k, 10c,d, 11c,d, 12c, 13c, 14c,d, and 15d
(see Schemes 1 and 2) in good to excellent overall yields as
presented in Table S1 (Supporting Information). These salts
were purified by reverse phase silica C-8 chromatography, as
well as recrystallization when required. Compounds 5e−g, 7h,
8e−h, and 9e−g were obtained as a mixture of two isomers as
detected by NMR spectroscopy: the major product (E) in
which the imine is in conjugation with the Boc group and the
minor product (Z) in which the imine is in conjugation with
the pyridine ring. The mixture of isomers was carried forward,
as removal of the Boc group generated the same product
regardless. This phenomenon has been extensively studied in
our group computationally and by low temperature NMR.20
It should be noted that compounds 5f, 8f, and 9f show an
acetate protection of the terminal OH group in the R′
substituent; this was required to avoid formation of the cyclic
4,5-dihydrooxazole byproduct during mercury(II) chloride
coupling. The acetate and Boc groups were simultaneously
cleaved using methanolic hydrochloric acid solution to generate
the corresponding guanidine hydrochlorides.
Because of problems with regioselectivity in the guanidyla-
tion of 2-amino-5-ethylaminopyridine, this methodology was
unsuited to prepare compound 10c and an alternative route
was utilized. Nucleophilic aromatic substitution of 2-chloro-5-
nitropyridine with the free base of guanidine hydrochloride
(Scheme 3) proceeded smoothly yielding the neutral guanidine
intermediate (16) that could be isolated after alkaline workup.
This intermediate provided access to three hydrochloride salts:
by treatment with HCl/1,4-dioxane, it yielded 17c; palladium
catalyzed hydrogenation followed by immediate protonation in
Accordingly, Boc-protected derivatives 5a−9a and 11a−15a
were prepared from commercially available N,N′-di(tert-
butoxycarbonyl)-S-methylisothiourea and compounds 5b,
7b−11b, 14b, and 15b from N,N′-di(tert-butoxycarbonyl)-
imidazolidine-2-thione prepared from imidazolidine-2-thione
(Scheme 1). Similarly, disubstituted Boc-protected guanidines
5e,f,g, 7h, 8e,f,g,h, and 9e,f,g were obtained from appropriate
N-Boc-N′-substituted thioureas (Scheme 2).
All these Boc-protected intermediates were then deprotected
using a solution of hydrochloric acid in dry 1,4-dioxane (4 M)
to generate the corresponding hydrochloride salts 5c,d,i−k, 6c,
C
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a
Scheme 3. Synthesis of Guanidine Hydrochlorides 10c, 17c, and 18c
a
t
Reagents: (a) NaOH, BuOH, 82 °C, 8 h; (b) HCl (4 M in 1,4-dioxane); (c) H2 (3 atm.), Pd/C (10%), CH3OH; (d) NaBH(OAc)3, CH3CN/
CH3OH.
a
Scheme 4. Synthesis of Compounds 19i−l and 20j,k,m and Percentage Yields for Each Step
a
Reagents: (a) CS2, NEt3, CH2Cl2, 0 °C, 15 min; (b) CH3I, 0 °C to rt, 2 h; (c) K2CO3 (2.0 equiv), Cu(II)O (0.2 equiv), DMF, 60 °C, 2 h; (d) HCl
(4 M in 1,4-dioxane or 1.25 M in CH3OH).
HCl/1,4-dioxane gave 18c; reductive alkylation with acetalde-
hyde and NaBH(OAc)3 followed by protonation in HCl/1,4-
dioxane afforded 10c. The free-base compounds of the amino
and N-ethylamino derivatives were unstable and needed to be
converted to their corresponding hydrochloride salts in situ. All
salts were purified by reverse-phase chromatography.
hydrochloric acid (1.25 M), generating the desired hydro-
chloride salts (19j and 20j).
Computational Physicochemical and Toxicity Param-
eters. Several physicochemical parameters such as molecular
weight (MW), ClogP, ClogD (at pH 7.4), polar surface area
(PSA), and pKaH, which are associated with the absorption and
distribution steps in the pharmacokinetic phase of a drug, were
calculated using the Marvin package,15 and the results obtained
are gathered in Table S3 (Supporting Information). According
to different authors and different databases,23 CNS-acting drugs
already in the market show the following physicochemical
characteristics: MW less than 400 with a mean value of 310 g
mol−1, (C)logP less than 5 with a preferred value around 1.5−
2.7, 2.5, or 3.43 (depending on the authors), (C)logD greater
than 0 and less than 3 (optimum value around 2.1), total PSA
in the range 40−90 Å2 and pKaH between 7.5 and 10.5
(compounds that are positively charged at pH 7−8 favor brain
penetration). The vast majority of our compounds fall within
the desired ranges of these properties (see Table S3). These
predicted values agree with our observations in animal studies
that compounds administered peripherally and centrally led to
increased levels of NA in rat brain (see microdialysis results and
Informed by the pharmacological results from these initial
sets of compounds, we proceeded to prepare two new series,
the 2-amino-1,4-dihydroquinazoline and the 2-amino-1,4-
dihydropyrido[2,3-d]pyrimidine derivatives 19i−l and
20j,k,m, which were synthesized using the method described
by Das et al.21 This method involves the preparation of S-
methyl-N-substituted dithiocarbamates from the corresponding
amines, carbon disulfide, and iodomethane22 and coupling
them with either 2-aminobenzylamine or 2-amino-3-amino-
ethylpyridine in the presence of copper(II) oxide, followed by
protonation in hydrochloric acid/1,4-dioxane (Scheme 4).
The yields obtained for the preparation of these derivatives
are presented in Table S2 (Supporting Information). Similar to
the disubstituted guanidine family, ethyl alcohol derivatives
were protected as acetates to avoid formation of oxazolidin-2-
thione. These intermediates were hydrolyzed in methanolic
D
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a
Table 1. Affinity for the α2-AR (Expressed as pKi) of All the Compounds Prepared
a
Data represent the mean SEM of three to five separate experiments. The pKi values of idazoxan and RX821002 (2-methoxyidazoxan) are 7.29
and 9.04, respectively.
E
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ref 6). Taken together, it is reasonable to suggest that these
compounds will have favorable pharmacokinetic profiles.
A very important cause of toxicity for many therapeutic
agents is hERG channel blocking that results in cardiotoxicity
and many drugs have been withdrawn from the market for that
reason. Recently, a ligand-based pharmacophore for blocking
hERG has been developed (and experimentally validated) using
the Catalyst software;24 in order to evaluate if our compounds
fulfill such a pharmacophore, and hence produce cardiopathies,
we have computationally optimized (DFT level: B3LYP/6-
31+G*, PCM-water) four of them. These compounds (8l, 9c,
9i and 14d) have been chosen as a sample of all the structures
prepared.
The pharmacophoric elements identified in these four
compounds as well as the distances among them compared
to those of the Catalyst pharmacophore are gathered in Table
S4 (Supporting Information). None of our compounds fulfill
the pharmacophoric requirements, therefore, based on this
model would not be able to block the hERG channels and, in
principle, would have a lower probability to show cardiotoxicity.
Pharmacology and Structure−Activity Relationships.
Affinity for the α2-Adrenoceptors. The α2-AR affinity of all
compounds was measured in human brain PFC tissue by
competition assays with the selective radioligand [3H]-
RX821002 (2-methoxyidazoxan), which was used at a constant
concentration of 1 nM. The results obtained, expressed as pKi
values, are presented in Table 1.
On evaluation of the compounds binding affinity, it became
clear that placement of the cationic group in the 2-position of
the pyridine ring led to higher pKi values (5c−10c, 17c−18c,
5d, 7d−10d, 5i−k, 8i−k, 9i−k, 7l−8l, Table 1). For every
para-substituent (R in Schemes 1 and 2), except for
ethylamino, 2-guanidinopyridines had higher pKi values than
their 3-guanidinopyridine analogues (11c−15c). While this was
also generally true for 2-aminoimidazolines (series d), the
difference was less pronounced and pyridin-3-yl 2-amino-
imidazolinium compounds 14d and 15d gave the highest pKi
values of all. Comparing the differing cationic groups, it can be
said that 2-aminoimidazoliniums (series d) gave slightly higher
pKi values than the corresponding guanidiniums (series c), as
has been seen for previous families of this type of compounds.
For derivatives with the cationic group at the 2-position of
the pyridine ring, the most favorable aryl system was 5,6,7,8-
tetrahydroquinoline (9), followed by unsubstituted pyridine
(5) and then 5-methylpyridine (8). This trend is conserved for
all guanidines (series c), 2-aminoimidazolines (series d) and
disubstituted guanidines (series i, j, k and l). Pyridines with
more inductively electron-withdrawing substituents, such as 5-
Cl (7), -Br (6), -NO2 (17) and -NH2 (18), had poor affinity,
while the 5-NHEt (10) substituted pyridines were the worst of
all. On the contrary, the NHEt substituent gave the highest
affinity molecules for pyridines with the cation at the 3-position
of the pyridine ring (15) as shown in Table 1.
Except for compound 19j, 1,4-dihydroquinazoline and
dihydropyridopyrimidin-2-amines systems (19i,k,l and
20j,k,m) showed disappointingly low pKi values at the α2-AR
in this assay (Table 1). It is unclear why this is the case, though
it could be due to the strain introduced to the system by the
conformationally restricted six-membered ring. Maybe these
restricted guanidines are too constrained and orient the
substituents in the wrong direction; possibly a certain degree
of flexibility, as that present in the hydrogen bonded 2-
guanidinopyridines, is necessary for effective binding to the α2-
AR. Curiously, in the 1,4-dihydroquinazoline series, the ethanol
substituted compound (19j) had a considerably higher pKi
value than the phenyl (19i), 2-furanyl (19k), and n-propyl
(19l) substituted systems, the reverse of that seen for
disubstituted guanidines 5i−k, 8i−k, and 9i−k (Table 1).
This supports our hypothesis that the substituents in the
conformationally restricted guanidines are in the wrong
orientation.
[35S]GTPγS Binding Functional Assays. Compounds with a
pKi > 6.0, as well as some SAR-relevant compounds with lower
pKi values, were subjected to [35S]GTPγS functional experi-
ments to determine their activity profile as agonist or antagonist
of the α2-AR. As members of the GPCR superfamily, receptor
activation leads to the exchange of GDP by GTP on the α-
subunit. A direct evaluation of this G-protein activation can be
made using radiolabeled GTP analogues. The [35S]GTPγS
binding assay is a useful tool to distinguish between agonists
(increasing the nucleotide binding), inverse agonists (decreas-
ing the nucleotide binding), and neutral antagonists (not
affecting the nucleotide binding) of GPCRs. The [35S]GTPγS
nucleotide is not hydrolyzable, and hence once exchange occurs
between GDP and [35S]GTPγS, the α-[35S]GTPγS subunit will
not return to its GDP-bound state, meaning that activation of
the receptor by ligands can be directly monitored using
scintillation counting. Significantly, all compounds tested for
activity (5c−9c, 12c, 15c, 5d, 8d, 9d, 14d, 15d, 5i, 9i, 9j, 5k,
8k, 9k, 7l, 8l, 19j) at the α2-AR proved to be either antagonists
or inverse agonists, with no compounds activating the receptor.
The corresponding dose−response curves for [35S]GTPγS
binding versus ligand concentration can be found in Figure S1
(Supporting Information).
A subset of the compounds that did not stimulate receptor
activation (9c, 9d, 9k, 14d, 15d, and 19j) were tested in new
[35S]GTPγS binding experiments against the known α2-AR
agonist UK14304 to confirm that their inverse agonist/
antagonist activity was mediated through α2-AR. In Table 2,
the antagonist effect induced by the presence of a single
concentration (10−5 M) of our compounds in the medium on
UK14304 agonist stimulation of [35S]GTPγS binding can be
found.
Addition of 9c, 9d, 14d, and 15d to the experiment induced
a small (<10-fold) rightward shift in the EC50 value for
UK14304, suggesting that they possess only weak antagonistic
effects at the α2-AR. On the contrary, 9k and 19j caused a
substantial shift to the EC50 of the standard agonist UK14304,
characteristic of potent antagonists. The results obtained for 9k
and 19j, which have slightly lower binding affinity than the
monosubstituted guanidinium and 2-aminoimidazolinium
compounds tested, indicate improved α2-AR antagonistic
features. It is reasonable to suggest that 9k and 19j have
access to a pocket in the active site not available to 9c, 9d, 14d,
or 15d, which allows them to block activation of the receptor
by UK14304. The conformational restriction introduced in 19j,
Extension into the region beyond the cationic moiety in
disubstituted guanidines 5i,k, and 8i,k,l, led to higher values of
binding affinity relative to analogous mono-substituted deriva-
tives; however, this was not the case for compound 7l which
gave similar results to mono-substituted 7c, and was the
opposite for compounds 9i,j,k, whose affinity was smaller or
similar to the mono-substituted analogue (Table 1). The pKi
values varied with the group attached to the guanidine group,
though the phenyl and 2-furanyl substituents (series i and k)
consistently outperformed the ethanol substituent (series j).
F
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effect of derivatives 14d and 15d on extracellular NA levels by
systemic administration. Control rats were administered with
the vehicle (saline), and intraperitoneal administration of
compounds 14d (10 mg/kg ip) and 15d (10 mg/kg ip)
significantly increased PFC NA extracellular concentration by
138 10% (Ftr[1,44] = 6.84, p < 0.05; Ft[10,44] = 2.47, p =
0.012; Fi[10,44] = 1.10, p = 0.38, n = 6; vs saline) and 186
20% (Ftr[1,44] = 67.00, p < 0.0001; Ft[10,44] = 6.79, p <
0.0001; Fi[10,44] = 6.30, p < 0.0001, n = 6; vs saline),
respectively (see Figure 4).
Table 2. EC50 Values Obtained from the Concentration−
Response Curves for UK14304 Stimulation of [35S]GTPγS
Binding (10−12−10−3 M, 10 Concentrations) in the Absence
or Presence of the Different Compounds (10−5 M)
which may have induced a loss of receptor affinity, seems
however to be ideal in order to orient the ethanol group in the
proposed pocket and enhance antagonist activity.
Figure 4. Effects of systemic 14d, 15d, or saline administration on
extracellular NA concentrations evaluated in PFC. Data are mean
SEM values from three to four experiments and are expressed as
percentages of the corresponding basal values. The arrow represents
administration of the different compounds or saline (1 mL kg−1).
In Vivo Microdialysis Experiments. It has been demon-
strated that the administration of α2-AR antagonists increases
the in vivo release of NA in the PFC.25 Considering their
antagonistic effect and relatively good affinities over the α2-ARs,
14d and 15d compounds were evaluated by microdialysis
experiments. Local administration of 14d by reverse dialysis
through the probe (1−100 μM) induced a significant increase
of extracellular NA levels in the PFC (Emax = 148 4%, P <
0.001; Ftr[1,50] = 25.48, p < 0.0001; Ft[9,50] = 1.28, p = 0.26;
Fi[9,50] = 4.70, p < 0.001, n = 7; vs saline). When 15d was
perfused in the PFC an even larger increase of NA extracellular
In summary, pyridine derivatives 14d and 15d, which are
bioisosteric analogues of some of our previous phenyl
derivatives that had been shown to be agonists,6 not only are
α2-AR antagonists in vivo but also keep that antagonistic
activity when administered systemically, suggesting that they
can cross the blood−brain barrier.
Structure−Activity Relationships and Docking Study.
A docking study was undertaken in order to understand the
SAR observed in the α2-AR competitive binding assays, keeping
in mind that no crystal structure of any of the α2-AR subtypes is
known and that, therefore, the outcomes of this study could
only be considered as hypotheses and guides for the design of
future compounds. Hence, we used the Modeller 9.12
software26 to construct homology models of the human α2A-
AR and α2C-AR subtypes (the most prevalent subtypes in the
human CNS27) using sequences from www.uniprot.org (α2A-
AR, P08913; α2C-AR, P18825), based on three template
structures of the homologous β2-adrenoceptor (β2-AR; 2R4S,28
3D4S,29 and 3SN630), which has 38.9% homology with the α2A-
AR and 35.2% homology with the α2C-AR. Detailed
information on the stepwise minimization process of homology
model refinement carried out in explicit solvation using the
AMBER software, binding site identification based on cocrystal
structures of the β2-adrenoceptor, and induced-fit docking in
the MOE 2010 software can be found in the Supporting
Information.
concentrations was observed (Emax = 429
7%, P < 0.001;
Ftr[1,50] = 182.7, p < 0.0001; Ft[9,50] = 45.97, p < 0.0001;
Fi[9,50] = 54.64, p < 0.0001, n = 7; vs saline). These results are
presented in Figure 3.
Considering the positive results obtained by local admin-
istration of the compounds directly in the brain, we tested the
As expected, the smaller monosubstituted guanidines and 2-
aminoimidazolines have lower docking scores (see Table S5,
Supporting Information) than both disubstituted guanidines
and 1,4-dihydroquinazolin-2-amines. Considering the molecular
interactions and their boundaries as described by Stahl,31 we
observed that these compounds could establish further
interactions within the binding site, which is abundant in
hydrophobic amino acids, favoring the binding of electron-rich
Figure 3. Effects of local administration of compounds 14d and 15d
(1−100 μM) or aCSF in the PFC. Concentration of the compounds
was progressively increased (arrows). Compounds 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 three animals for each group and are expressed as
percentages of the corresponding basal values.
G
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Figure 5. A 2D view of the interactions between 9k and the α2A-AR (left) and a 3D view from the extracellular side with important residues
highlighted (right).
aryl systems. Strikingly, analysis of the top-ranked poses for all
compounds revealed two main types of complex with both
subtypes of the α2-AR.
The pharmacophoric elements for binding of these
compounds to the α2-AR can be summarized as follows: an
aryl ring for hydrophobic interactions with the space of the
receptor adjacent to Ser200/Ser204 (α2A-AR) or Ser214/
Ser218 (α2C-AR), a cationic group for electrostatic binding to
Asp113 (α2A-AR) or Asp131 (α2C-AR), and a substituent for
hydrophobic interactions with the region formed by Tyr and
Phe residues between TM-3 and TM-7. Therefore, the
pharmacophore previously observed by us for α2-AR
antagonists11 has also been detected in these α2-AR docking
experiments, which have provided extra information building
on the previous pharmacophore. Thus, these α2-AR docking
experiments have allowed us to see the residues in the region
occupied by the second guanidinium substituent and have
supported that “electron-rich aromatics” are suitable. The
results also suggest that interaction with Ser200/Ser204
(Ser214/Ser218 of the α2C-AR) should be circumvented in
order to avoid receptor activation. This extra information can
now be used to guide the design of new compounds.
Considering that the membrane preparation used in the
experimental competitive binding assay is from human brain
prefrontal cortex, a tissue rich in the α2C-AR subtype, and given
that compounds in our simulation seem to bind most favorably
to the α2C-AR model, it is possible that there could be a degree
of subtype selectivity. It will be interesting to investigate this
possibility, especially as there is a lack of α2C-AR subtype-
selective ligands. In this sense, the pharmacological modulation
of the α2C-AR subtype could contribute to attenuating some
unwanted effects of adrenergic targeting drugs also mediated
through α2A-AR,32 and also, it has been hypothesized that α2C-
AR subtype antagonism may contribute to improve cognitive
function.33
First, the cationic group of monosubstituted derivatives
generally interacts with Ser200 and Ser204 of the α2A-AR
(Ser214 and Ser218 of the α2C-AR), the two residues
implicated in catechol ligand-binding and receptor activation
(cf. 3SN6 crystal structure of activated β2-AR). The only
exceptions to this are 5,6,7,8-tetrahydroquinoline and ethyl-
amino derivatives which preferentially fill the Ser200/Ser204
region of the receptor with their aryl core and orient the
cationic group in proximity to Asp113 (Asp131 of the α2C-AR),
also heavily implicated in ligand binding.
Second, disubstituted guanidines and 1,4-dihydroquinazo-
lines necessarily adopt a similar pose (Figure 5) in which the
cationic group interacts with Asp113 of the α2A-AR (Asp131 of
the α2C-AR), being unable to place the cationic group in the
Ser200/Ser204 region. It was found that all such compounds
bind in this manner and that furthermore the second
substituent on the cationic moiety extends toward aromatic
residues Tyr109, Phe412, and Tyr416 of TM-3 and TM-7
(Tyr127, Phe423, Tyr427 of the α2C-AR). This helps to explain
the enhanced and exclusive antagonist activity obtained for
these compounds compared to monosubstituted derivatives,
not only in the current series but also in analogous phenyl
guanidine derivatives.
The exact docking scores are not instructive because of the
inherent error associated with docking into homology models
(scores are presented in Supporting Information Table S5);
however, the information gained on the binding mode of
compounds to the α2-AR and the implications this has on
functional activity are highly valuable. Nonetheless, it is
interesting that compounds 9i, 9j, and 9k had the highest
scores and are among the compounds with highest pKi values.
By comparison of docking into the α2A-AR and α2C-AR, the
main ligand-binding residues described above are exactly
homologous in both subtypes, and thus, the same types of
pose are replicated. The main difference is in Tyr109 (α2A-AR)
or Tyr 127 (α2C-AR), which is closer to ligands in the α2C-AR
because of a change in the position of extracellular loop 1
(ECL1) between TM-2 and TM-3. This leads to higher
docking scores on average at the α2C-AR; however, the trends
among ligands are the same as in the α2A-AR.
CONCLUSIONS
■
In this paper we have reported the design, synthesis, and
pharmacological evaluation of a series of 2- and 3-guanidino-
and -2-aminoimidazolinopyridines, disubstituted 2-guanidino-
pyridines, and N-substituted-2-amino-1,4-dihydroquinazolines
with activity as antagonists of the α2-AR both in vitro and in
vivo, with potential for the treatment of depression and
schizophrenia.
All compounds have been prepared following different
synthetic approaches developed in our laboratory. They have
been evaluated for binding affinity and functional activity at the
H
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times with 300 μL of incubation buffer and air-dried (60 min). The
radioactivity trapped was determined by liquid scintillation spectrom-
etry (MicroBeta TriLux counter, PerkinElmer). The [35S]GTPγS
bound was about 7−14% of the total [35S]GTPγS added. Nonspecific
binding of the radioligand was defined as the remaining [35S]GTPγS
binding in the presence of 10 μM unlabeled GTPγS. The
pharmacological parameters of the stimulation curves of the
[35S]GTPγS binding were obtained by nonlinear analysis using
GraphPad Prism software version 5.0.
In Vivo Microdialysis Experiments. Animals. Experiments were
performed on male Sprague−Dawley rats (University of the Basque
Country, Spain). Animals were housed 4/5 per cage in a 12 h light−
dark cycle at room temperature (22 °C) with food and water ad
libitum. All the experimental protocols were approved by the
Committee for Animal Experimentation at the University of the
Basque Country (UPV/EHU). All procedures relating to animal care
and use conformed to European Ethical Standards (6106/10-EEC)
and Spanish Law (RD 53/2013).
Microdialysis Experiments. Male Sprague−Dawley rats (250−300
g) were implanted with a probe in a stereotaxic apparatus under 1.5%
isoflurane anesthesia. The probe was located in the prefrontal cortex
(PFC) according to the coordinates of the atlas of Paxinos and
Watson34 (AP (anterior to bregma), +2.8 mm; L (lateral from the
mid-sagittal suture), +1 mm, DV (ventral from the dura surface), −5
mm).
Experiments were performed 18−24 h after the probe implantation,
and artificial cerebrospinal fluid (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−1 (CMA/microdialysis infusion pump). Drugs, when
locally administered, 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. In each experimental group, animals
received either drugs or their appropriate vehicle.
Samples were collected every 35 min and NA concentrations
analyzed by HPLC apparatus with amperometric detection (VT-03
cell, Decade II, Antec Leyden, Holland) at an oxidizing potential of
0.300 mV. The mobile phase (50 mM fosforic acid, 0.1 mM EDTA, 8
mM sodium chloride, 500 mg L−1 sodium octyl sulfate, pH 6, and 16%
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 C18 column (ALF-
205, 150 mm × 2.1 mm, 3 μm; Antec Leyden, Holland). Samples
(injection volume 30 μL) were injected, and NA was analyzed in a run
time of 18 min.
The mean values of the first three/four samples before drug
administration were considered as 100% basal value. All measures of
extracellular NA concentrations are expressed as percentage of the
baseline value SEM. Two-way analysis of variance (ANOVA) was
followed by Bonferroni’s test between control, and each treated group
was assessed for statistical analysis. F values were expressed as Ftr
(treatment, between groups), Ft (time, within groups), or Fi
(treatment × time, interaction). In these analyses all the experimental
points, including basal values, were considered. All statistical
procedures were performed using GraphPad Prism (GraphPad
Sofware, San Diego, CA, USA).
α2-AR in human brain prefrontal cortex tissue, using
competitive binding assays and [35S]GTPγS experiments,
respectively. Furthermore, compounds with pKi > 7 (14d and
15d) were shown to increase NA levels in vivo by means of
microdialysis experiments in rats, proving target engagement
and showing antagonistic activity not only when they were
locally administered but also when they were given systemically.
Several SARs, particularly in relation to binding affinity, have
been obtained from a combination of our in vitro assays and
computational docking models. Thus, it can be concluded that
2-guanidinopyridines have higher α2-AR affinity than analogous
3-guanidinopyridines; however, the opposite is the case for 2-
aminoimidazolino derivatives. Generally speaking, 2-amino-
imidazolino derivatives displayed higher α2-AR affinities than
their monosubstituted guanidine analogues, as was expected
from the results obtained in our previous work. In the present
work, extension into the space beyond the cationic moiety, as in
disubstituted guanidines, led to small increases in binding
affinity, while such compounds are also conferred with
enhanced antagonistic activity at the α2-AR as shown by their
effects on UK14304 activation of the α2-AR.
Furthermore, for all arylguanidines and aryl-2-aminoimidazo-
lines prepared exclusively, antagonist or inverse agonist activity
was obtained, with no agonists found in [35S]GTPγS
experiments. This work has identified six antagonists (9c, 9d,
9k, 14d, 15d, and 19j) of the α2-AR which significantly reduce
the ability of UK14304 to activate the receptor.
We have identified α2-AR antagonists that are water-soluble
and exert their activity in vivo in rat brain with both central and
peripheral administration. The potential for optimizing these
compounds to obtain orally bioavailable agents that antagonize
the α2-AR is huge, given the relevance of this receptor to the
treatment of depression and schizophrenia.
EXPERIMENTAL SECTION
■
Pharmacology. [3H]RX821002 Binding Assays. Specific [3H]-
RX821002 binding was measured in 0.25 mL aliquots (50 mM Tris-
HCl, 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 (10−12−10−3 M, 10
concentrations). Incubations were terminated by separating free ligand
from bound ligand by rapid filtration under vacuum (1450 Filter Mate
Harvester, PerkinElmer) through GF/C glass fiber filters. The filters
were then rinsed three times with 300 μL of binding buffer, air-dried
(60 min), and counted for radioactivity by liquid scintillation
spectrometry using a MicroBeta TriLux counter (PerkinElmer).
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). Analyses 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
At the end of the experiments, rats were killed by an overdose of
anesthetic and the brains were dissected to check the correct
implantation of the probe.
[35S]GTPγS Binding Assays. The incubation buffer for measuring
[35S]GTPγS binding to brain membranes contained, in a total volume
of 250 μL, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, 50 mM GDP,
50 mM Tris-HCl at pH 7.4 and 0.5 nM [35S]GTPγS. Protein aliquots
were thawed and resuspended in the same buffer. The incubation was
started by addition of the membrane suspension (40 μg of membrane
proteins) to the previous mixture and was performed at 30 °C for 120
min, with shaking. In order to evaluate the influence of the compounds
on [35S]GTPγS binding, 10 concentrations (10−12−10−3 M) of the
different compounds were added to the assay. Incubations were
terminated by separating free ligand from bound ligand by rapid
filtration under vacuum (1450 Filter Mate Harvester, PerkinElmer)
through GF/C glass fiber filters. The filters were then rinsed three
Chemistry. General. All commercial chemicals were obtained
from either Sigma-Aldrich or Fluka and used without further
purification. Deuterated solvents for NMR spectroscopy use were
purchased from Apollo. Dry solvents were prepared using standard
procedures, according to Vogel,35 with distillation prior to use.
Chromatographic columns were run using a Biotage SP4 flash
purification system with Biotage SNAP silica cartridges. Solvents for
synthesis purposes were used at GPR grade. Analytical TLC was
performed using either 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 on Bruker DPX-400
I
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1
Avance spectrometers, operating at 400.13 and 600.1 MHz for H
NMR; 100.6 and 150.9 MHz for 13C NMR. Shifts are referenced to
the internal solvent signals.36 NMR data were processed using Bruker
TOPSPIN software. HRMS spectra were measured on a Micromass
LCT electrospray TOF instrument with a WATERS 2690 autosampler
and methanol/acetonitrile as carrier solvent. Melting points were
determined using a Stuart Scientific melting point SMP1 apparatus and
are uncorrected. Infrared spectra were recorded on a PerkinElmer
Spectrum One FT-IR spectrometer equipped with a Universal ATR
sampling accessory.
1-(Pyridin-2-yl)-3-(phenyl)guanidine Hydrochloride (5i):
Procedure B. Colorless gum (90%). δH (400 MHz, DMSO-d6):
7.19 (d, 1H, J = 8.2, Ar), 7.25 (dd, 1H, J = 7.0, 5.2, Ar), 7.40 (m, 3H,
Ar), 7.53 (app t, 2H, J = 8.1, 7.4, Ar), 7.94 (app td, 1H, 7.0, 1.6, Ar),
8.38 (d, 1H, J = 5.2, Ar), 8.92 (br s, 2H, NH), 11.11 (br s, 1H, NH),
11.60 (br s, 1H, NH). δC (100 MHz, DMSO-d6): 113.6 (CH Ar),
119.7 (CH Ar), 125.3 (CH Ar), 127.4 (CH Ar), 129.9 (CH Ar), 134.1
(q Ar), 139.8 (CH Ar), 146.6 (CH Ar), 151.9 (q Ar), 153.5 (q, CN).
νmax (ATR)/cm−1: 3061 (NH), 2953, 1668 (CN), 1639, 1594,
1569, 1485, 1423, 1373, 1231, 1152, 1075, 779, 758, 696. HRMS
(ESI+) m/z: [M + H]+ calcd for C12H13N4 213.1140. Found:
213.1145. Purity by HPLC: 97.6% (tR = 24.21 min).
HPLC purity analysis was carried out using a Varian ProStar system
equipped with a Varian Prostar 335 diode array detector and a manual
injector (20 μL). For purity assessment, UV detection was performed
at 245 nm, and peak purity was confirmed using a purity channel. The
stationary phase consisted of an ACE 5 C18-AR column (150 mm ×
4.6 mm), and the mobile phase used the following gradient system,
eluting at 1 mL/min: aqueous formate buffer (30 mM, pH 3.0) for 10
min, linear ramp to 85% methanol buffered with the same system over
25 min, hold at 85% buffered methanol for 10 min. Minimum
requirement for purity was set at 95.0%.
Preparation of Hydrochloride Salts from Boc-Protected
Derivatives: Procedure B. To either the 1-(pyridinyl)-2,3-di(tert-
butoxycarbonyl)guanidine, 1-(pyridinyl)-2,3-di(tert-butoxycarbonyl)-
2-iminoimidazolidine, or 1-(pyridinyl)-2-(tert-butoxycarbonyl)-3-sub-
stituted guanidine precursor (1.0 equiv, 0.5 mmol) were added 4 M
HCl/1,4-dioxane (6.0 equiv per Boc group) and a 1:1 solution of
iPrOH/CH2Cl2, such as to maintain a reaction concentration of 0.2 M.
The mixture was stirred at 55 °C until completion, as judged by
disappearance of starting material in TLC. Solvent and excess HCl
were then removed under vacuum, and the crude salt was dissolved in
a minimum volume of water. It was washed with CH2Cl2 (2 × 5 mL)
and then purified using reverse phase chromatography (C-8 silica)
with 100% H2O as mobile phase. Removal of solvent yielded the pure
guanidine/2-aminoimidazoline hydrochloride, which was recrystallized
as described if deemed necessary.
General Procedure for the Protonation of Neutral Guani-
dines: Procedure D. To a solution of the corresponding neutral
guanidine or N-substituted-1,4-dihydroquinazolin-2-amine (1.0 equiv,
0.5 mmol) in CH2Cl2 (0.5 mL) was added excess 4 M HCl/dioxane
(3.0 equiv, 1.5 mmol, 375 μL). Where necessary CH3OH (1 mL) was
added dropwise as needed to aid solubility. Stirring was continued for
1 h after which solvent and excess HCl were removed under vacuum.
The crude salt was then dissolved in a minimum volume of water,
washed with CH2Cl2 (2 × 5 mL), and purified using reverse phase
chromatography (C-8 silica) with 100% H2O as mobile phase. Where
necessary, further purification was carried out.
1-(Pyridin-2-yl)guanidine Hydrochloride (5c): Procedure B.
Colorless crystals (71%) after recrystallization from CH3OH and Et2O.
Mp 77−78 °C, clean melt. δH (600 MHz, DMSO-d6): 7.07 (d, 1H, J =
8.2, Ar), 7.18 (dd, 1H, J = 6.9, 5.4, Ar), 7.88 (m, 1 H, Ar), 8.30 (br s,
4H, NH), 8.32 (d, 1H, J = 5.4, Ar), 11.39 (br s, 1H, NH). δC (150
MHz, DMSO-d6): 113.2 (CH Ar), 119.3 (CH Ar), 139.5 (CH Ar),
146.7 (CH Ar), 151.9 (q Ar), 155.3 (q, Gua). νmax (ATR)/cm−1: 3312
(NH), 3180 (NH), 3130 (NH), 1679 (CN), 1622, 1596, 1561,
1462, 1416, 1319, 1274, 1244, 1154, 1054, 1020, 998, 874, 775. HRMS
(ESI+) m/z: [M + H]+ calcd for C6H9N4 137.0827. Found: 137.0827.
Purity by HPLC: 97.2% (tR = 16.81 min).
1-(Pyridin-2′-yl)-2-iminoimidazolidine Hydrochloride (5d):
Procedure B. White solid (92%). Mp 155−160 °C, decomposition.
δH (600 MHz, DMSO-d6): 3.73 (s, 4H, CH3), 7.12 (d, 1H, J = 8.4,
Ar), 7.20 (dd, 1H, J = 7.0, 5.3, Ar), 7.87 (dt, 1H, J = 7.0, 2.3, Ar), 8.33
(d, 1H, J = 5.3, Ar), 8.90 (br s, 2H, NH), 11.60 (br s, 1H, NH). δC
(150 MHz, DMSO-d6): 42.4 (2 CH2), 112.7 (CH Ar), 119.6 (CH Ar),
139.4 (CH Ar), 147.1 (CH Ar), 150.7 (q Ar), 155.8 (q, Imi). νmax
(ATR)/cm−1: 3451, 3401, 3159 (NH), 3059 (NH), 2957, 2906, 2526,
2384, 2270, 1632 (CN), 1594, 1476, 1448, 1426, 1366, 1344, 1279,
1247, 1199, 1158, 1102, 1053, 1017, 952, 932, 869, 772, 724, 693.
HRMS (ESI+) m/z: [M + H]+ calcd for C8H11N4 163.0984. Found:
163.0979. Purity by HPLC: 97.2% (tR = 18.80 min).
1-(Pyridin-2-yl)-3-(ethoxy)guanidine Hydrochloride (5j):
Procedure B. White solid (83%). Mp 140−143 °C, decomposition.
δH (400 MHz, DMSO-d6): 3.44 (t, 2H, J = 5.2, CH2), 3.61 (t, 2H, J =
5.2, CH2), 5.19 (br s, 1H, OH), 7.09 (d, 1H, J = 8.3, Ar), 7.18 (m, 1H,
Ar), 7.88 (ddd, 1H, J = 8.3, 7.3, 1.1, Ar), 8.31 (dd, 1H, J = 5.0, 1.1, Ar),
8.56 (br s, 2H, NH), 9.65 (br s, 1H, NH), 11.33 (br s, 1H, NH). δC
(100 MHz, DMSO-d6): 44.1 (CH2), 59.5 (CH2), 113.6 (CH Ar),
119.5 (CH Ar), 140.0 (CH Ar), 146.8 (CH Ar), 152.8 (q Ar), 155.0
(q, CN). νmax (ATR)/cm−1: 3449 (OH), 3026 (NH), 1600, 1518,
1492, 1445, 1406, 1358, 1278, 1250, 1185, 1107, 1040, 899, 873, 797,
754, 709, 683. HRMS (ESI+) m/z: [M + H]+ calcd for C8H13N4O
181.1089. Found: 181.1094. Purity by HPLC: 96.4% (tR = 18.11 min).
1-(Pyridin-2-yl)-3-(2-furanylmethyl)guanidine Hydrochlor-
ide (5k): Procedure B. White solid (82%). Mp 142−144 °C,
decomposition. δH (400 MHz, DMSO-d6): 4.67 (d, 2H, J = 5.0, CH2),
6.46−6.51 (m, 2H, Ar), 7.11 (d, 1H, J = 8.2, Ar), 7.21 (dd, 1H, J = 6.9,
5.2, Ar), 7.70 (dd, 1H, 1.6, 0.7, Ar), 7.90 (ddd, 1H, J = 8.2, 6.9, 1.3,
Ar), 8.33 (dd, 1H, J = 5.2, 1.3, Ar), 8.86 (br s, 2H, NH), 9.71 (br s, 1H,
NH), 11.32 (br s, 1H, NH). δC (100 MHz, DMSO-d6): 38.2 (CH2),
108.7 (CH Ar), 111.1 (CH Ar), 113.8 (CH Ar), 119.9 (CH Ar), 140.2
(CH Ar), 143.7 (CH Ar), 146.9 (CH Ar), 150.0 (q Ar), 152.5 (q Ar),
154.6 (q, CN). νmax (ATR)/cm−1: 3092 (NH), 2990, 1674 (CN),
1656, 1628, 1598, 1567, 1499, 1474, 1428, 1416, 1380, 1357 1343,
1323, 1285, 1265, 1200, 1154, 1143, 1132, 1074, 1046, 1018, 997, 929,
915, 902, 875, 834, 784, 773, 748, 715. HRMS (ESI+) m/z: [M + H]+
calcd for C11H13N4O 217.1089. Found: 217.1090. Purity by HPLC:
95.5% (tR = 24.81 min).
1-(5-Bromopyridin-2-yl) guanidine Hydrochloride (6c): Pro-
cedure B. White solid (74%). Mp 76−77 °C, clean melt. δH (600
MHz, DMSO-d6): 7.06 (d, 1H, J = 8.8, Ar), 8.09 (dd, 1H, J = 8.8, 2.5,
Ar), 8.23 (br s, 4H, NH), 8.43 (d, 1H, J = 2.5, Ar), 11.53 (br s, 1H,
NH). δC (150 MHz, DMSO-d6): 114.7 (q Ar), 116.1 (CH, Ar), 142.9
(CH, Ar), 148.1 (CH, Ar), 151.7 (q, Ar), 155.9 (q, Gua). νmax (ATR)/
cm−1: 3313 (NH), 3218 (NH), 3011 (NH), 1684.0 (CN), 1618,
1582, 1551, 1465, 1360, 1308, 1275, 1234, 1137, 1094, 1006, 925, 874,
825, 732. HRMS (ESI+) m/z: [M + H]+ calcd for C6H879BrN4
214.9932. Found: 214.9926. Purity by HPLC: 99.1% (tR = 23.88 min).
1-(5-Chloropyridin-2-yl)guanidine Hydrochloride (7c): Pro-
cedure B. White solid (85%). Mp 162−164 °C, clean melt. δH (600
MHz, DMSO-d6): 7.13 (d, 1H, J = 8.8, Ar), 7.98 (dd, 1H, J = 8.8, 2.6,
Ar), 8.28 (br s, 4H, NH), 8.35 (d, 1H, J = 2.6, Ar), 11.70 (br s, 1H,
NH). δC (150 MHz, DMSO-d6): 115.0 (CH Ar), 126.6 (q Ar), 139.2
(CH Ar), 144.7 (CH Ar), 151.0 (q Ar), 155.7 (q, Gua). νmax (ATR)/
cm−1: 3313 (NH), 3178 (NH), 2953, 1685 (CN), 1617, 1587,
1551, 1465, 1364, 1310, 1274, 1236, 1136, 1113, 1022, 1009, 924, 876,
828, 757, 734, 718. HRMS (ESI+) m/z: [M + H]+ calcd for
C6H835ClN4 171.0437. Found: 171.0433. Purity by HPLC: 99.0% (tR
= 22.68 min).
1-(5-Chloropyridin-2′-yl)-2-iminoimidazolidine Hydrochlor-
ide (7d): Procedure B. White solid (96%). Mp 95−96 °C, clean
melt. δH (600 MHz, DMSO-d6): 3.70 (s, 4H, CH2), 7.23 (d, 1H, J =
8.8, Ar), 7.96 (dd, 1H, J = 8.8, 2.7, Ar), 8.30 (d, 1H, J = 2.7, Ar), 8.86
(br s, 2H, NH), 12.31 (br s, 1H, NH). δC (150 MHz, DMSO-d6): 42.5
(2 CH2), 114.3 (CH Ar), 125.7 (q Ar), 139.1 (CH Ar), 145.2 (CH
Ar), 149.5 (q Ar), 155.5 (q, Imi). νmax (ATR)/cm−1: 3295, 3230
(NH), 3165 (NH), 3112 (NH), 3035, 2951, 1694, 1640 (CN),
1628, 1588, 1569, 1501, 1468, 1442, 1389, 1365, 1301, 1276, 1231,
1180, 1137, 1111, 1047, 1017, 959, 930, 875, 832, 757, 687. HRMS
J
DOI: 10.1021/jm501635e
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(ESI+) m/z: [M + H]+ calcd for C8H10N435Cl 197.0594. Found:
197.0596. Purity by HPLC: 98.7% (tR = 2.36 min).
δC (100 MHz, DMSO-d6): 17.7 (CH3), 38.2 (CH2), 108.7 (CH Ar),
111.1 (CH Ar), 113.4 (CH Ar), 129.0 (q Ar), 140.8 (CH Ar), 143.6
(CH Ar), 146.2 (CH Ar), 150.0 (q Ar), 150.5 (q Ar), 154.6 (q, CN).
νmax (ATR)/cm−1: 3102 (NH), 1664 (CN), 1623, 1609, 1493,
1407, 1381, 1355, 1274, 1247, 1225, 1210, 1158, 1083, 1077, 1018,
925, 912, 882, 821, 771, 748, 669. HRMS (ESI+) m/z: [M + H]+ calcd
for C12H15N4O 231.1246. Found: 231.1241. Purity by HPLC: 98.4%
(tR = 26.96 min).
1-(5-Methylpyridin-2-yl)-3-(propyl)guanidine Hydrochloride
(8l): Procedure B. Yellow solid (90%). Mp 100−102 °C, clean melt.
δH (600 MHz, DMSO-d6): 0.94 (t, 3H, J = 7.5, CH3), 1.58 (app sex,
2H, J = 7.5, 7.1, CH2), 2.25 (s, 3H, CH3), 3.29 (m, 2H, CH2), 6.99 (br
s, 1H, Ar), 7.71 (dd, 1H, J = 8.4, 1.9, Ar), 8.14 (d, 1H, J = 1.9, Ar),
8.59 (br s, 3H, NH), 9.43 (br s, 2H, NH), 11.34 (br s, 2H, NH). δC
(150 MHz, DMSO-d6): 11.0 (CH3), 17.2 (CH3), 21.6 (CH2), 42.5
(CH2), 112.6 (CH Ar), 128.3 (q Ar), 140.3 (CH Ar), 145.8 (CH Ar),
150.0 (q Ar), 154.1 (q, CN). νmax (ATR)/cm−1: 2965, 1674 (CN),
1632, 1608, 1572, 1484, 1343, 1283, 1244, 1136, 1079, 1029, 968, 831,
743. HRMS (ESI+) m/z: [M + H]+ calcd for C10H17N4 193.1453.
Found: 193.1461. Purity by HPLC: 96.9% (tR = 24.57 min).
1-(5,6,7,8-Tetrahydroquinolin-2-yl)guanidine Hydrochloride
(9c): Procedure B. Colorless crystals (87%) after recrystallization
from CH3OH and Et2O. Mp 224−228 °C, decomposition. δH (600
MHz, DMSO-d6): 1.73 (m, 2H, CH2), 1.80 (m, 2H, CH2), 2.68 (app t,
2H, J = 6.1, 6.0, CH2), 2.78 (app t, 2H, J = 6.1, 6.0, CH2), 6.80 (d, 1H,
J = 8.2, Ar), 7.55 (d, 1H, J = 8.2, Ar), 8.22 (br s, 4H, NH), 11.08 (br s,
1H, NH). δC (150 MHz, DMSO-d6): 22.1 (CH2), 22.2 (CH2), 27.2
(CH2), 31.5 (CH2), 110.5 (CH Ar), 127.3 (q Ar), 140.1 (CH Ar),
149.2 (q Ar), 154.0 (q Ar), 155.2 (q, Gua). νmax (ATR)/cm−1: 3323
(NH), 3149 (NH), 2961, 1679 (CN), 1634, 1597, 1566, 1465,
1031, 813. HRMS (ESI+) m/z: [M + H]+ calcd for C10H15N4
191.1297. Found: 191.1293. Purity by HPLC: 98.1% (tR = 27.00 min).
1-(5,6,7,8-Tetrahydroquinolin-2′-yl)-2-iminoimidazolidine
Hydrochloride (9d): Procedure B. White solid (93%). Mp 208−
212 °C, decomposition. δH (600 MHz, DMSO-d6): 1.72−1.76 (m, 2H,
CH2), 1.79−1.83 (m, 2H, CH2), 2.69 (app t, 2H, J = 5.9, 6.3, CH2),
2.84 (app t, 2H, J = 6.1, 6.4, CH2), 3.73 (s, 4H, CH2), 6.92 (d, 1H, J =
8.2, Ar), 7.54 (d, 1H, J = 8.2, Ar), 8.79 (br s, 2H, NH), 11.87 (br s, 1H,
NH). δC (150 MHz, DMSO-d6): 22.2 (CH2), 22.2 (CH2), 27.3
(CH2), 31.5 (CH2), 42.5 (2 CH2), 110.1 (CH Ar), 127.5 (q Ar), 139.9
(CH Ar), 148.1 (q Ar), 154.8 (q Ar), 155.9 (q, Imi). νmax (ATR)/
cm−1: 3344, 3296, 3169 (NH), 3114 (NH), 3058 (NH), 2934, 2848,
1642 (CN), 1595, 1583, 1474, 1464, 1415, 1377, 1351, 1309, 1286,
1264, 1204, 1156, 1137, 1111, 1089, 1066, 1018, 949, 938, 927, 896,
864, 835, 817, 759, 736. HRMS (ESI+) m/z: [M + H]+ calcd for
C12H17N4 217.1453. Found: 217.1458. Purity by HPLC: 97.0% (tR =
26.65 min).
1-(5,6,7,8-Tetrahydroquinolin-2-yl)-3-(phenyl)guanidine Hy-
drochloride (9i): Procedure B. White solid (86%). Mp 180−183
°C, decomposed. δH (600 MHz, DMSO-d6): 1.75 (app t, 2H, J = 5.6,
5.1, CH2), 1.81 (app t, 2H, J = 5.8, 5.1, CH2), 2.71 (app t, 2H, J = 5.6,
CH2), 2.79 (d, 2H, J = 5.8, CH2), 6.92 (d, 1H, J = 8.2, Ar), 7.35 (t, 1H,
J = 7.3, Ar), 7.39 (d, 2H, J = 7.6, Ar), 7.50 (app t, 2H, J = 7.6, 7.3, Ar),
7.60 (d, 1H, J = 8.2, Ar), 9.03 (br s, 2H, NH), 11.40 (br s, 1H, NH),
11.63 (br s, 1H, NH). δC (150 MHz, DMSO-d6): 22.4 (CH2), 22.5
(CH2), 27.5 (CH2), 31.8 (CH2), 111.6 (CH Ar), 124.8 (CH Ar),
127.1 (CH Ar), 127.7 (q Ar), 130.1 (CH Ar), 140.6 (CH Ar), 135.3 (q
Ar), 150.3 (q Ar), 153.6 (q Ar), 154.0 (q, CN). νmax (ATR)/cm−1:
3235 (NH), 2978, 2937, 1672, 1642 (CN), 1595, 1578, 1499, 1467,
1376, 1319, 1282, 1234, 1121, 1079, 1047, 951, 835, 818, 755, 697,
664. HRMS (ESI+) m/z: [M + H]+ calcd for C16H19N4 267.1610.
Found: 267.1605. Purity by HPLC: 95.6% (tR = 31.11 min).
1-(5-Chloropyridin-2-yl)-3-(propyl)guanidine Hydrochloride
(7l): Procedure B. Yellow solid (93%). Mp 102−104 °C, clean melt.
δH (600 MHz, DMSO-d6): 0.94 (t, 3H, J = 7.4, CH3), 1.59 (app sex,
2H, J = 7.4, 7.2, CH2), 3.29 (m, 2H, CH2), 7.12 (s, 1H, Ar), 7.99 (dd,
1H, J = 8.8, 2.5, Ar), 8.37 (d, 1H, J = 2.5, Ar), 8.58 (br s, 3H, NH),
9.17 (br s, 2H, NH), 11.47 (br s, 2H, NH). δC (150 MHz, DMSO-d6):
11.3 (CH3), 21.9 (CH2), 42.9 (CH2), 115.0 (CH Ar), 125.7 (q Ar),
139.7 (CH Ar), 145.1 (CH Ar), 151.0 (q Ar), 154.1 (q, CN). νmax
(ATR)/cm−1: 3268 (NH), 3097 (NH), 3059, 2958, 2931, 2875, 1673,
1646 (CN), 1629 (CN), 1591, 1558, 1469, 1385, 1371, 1342,
1315, 1271, 1243, 1145, 1109, 1075, 1044, 1012, 966, 903, 869, 828,
772, 737, 658. HRMS (ESI+) m/z: [M + H]+ calcd for C9H12N435Cl
211.0750. Found: 211.0745. Purity by HPLC: 99.3% (tR = 26.40 min).
1-(5-Methylpyridin-2-yl)guanidine Hydrochloride (8c): Pro-
cedure B. Colorless crystals (84%) after recrystallization from iPrOH
and Et2O. Mp 188−192 °C, clean melt. δH (600 MHz, DMSO-d6):
2.27 (s, 3H, CH3), 6.98 (d, 1H, J = 8.3, Ar), 7.71 (dd, 1H, J = 8.3, 1.9,
Ar), 8.15 (d, 1H, J = 1.9, Ar), 8.21 (br s, 4H, NH), 11.17 (br s, 1H,
NH). δC (150 MHz, DMSO-d6): 17.1 (CH3), 112.7 (CH Ar), 128.5 (q
Ar), 140.1 (CH Ar), 146.1 (CH Ar), 149.7 (q Ar), 155.2 (q, Gua). νmax
(ATR)/cm−1: 3268 (NH), 2889, 1677 (CN), 1621, 1601, 1563,
1489, 1376, 1315, 1285, 1242, 1086, 1035, 1023, 1002, 909, 873, 832,
798, 738, 718. HRMS (ESI+) m/z: [M + H]+ calcd for C7H11N4
151.0984. Found: 151.0979. Purity by HPLC: 98.3% (tR = 21.57 min).
1-(5-Methylpyridin-2′-yl)-2-iminoimidazolidine Hydrochlor-
ide (8d): Procedure B. White solid (93%). Mp 140−145 °C,
decomposition. δH (600 MHz, DMSO-d6): 2.27 (s, 3H, CH3), 3.71 (s,
4H, CH2), 7.06 (d, 1H, J = 8.3, Ar), 7.70 (dd, 1H, J = 8.3, 2.0, Ar),
8.15 (d, 1H, J = 2.0, Ar), 8.84 (br s, 2H, NH), 11.76 (br s, 2H, NH).
δC (150 MHz, DMSO-d6): 17.2 (CH3), 42.4 (2 CH2), 112.3 (CH Ar),
128.7 (q Ar), 139.9 (CH Ar), 146.6 (CH Ar), 148.6 (q Ar), 155.8 (q,
Imi). νmax (ATR)/cm−1: 3287 (NH), 3123 (NH), 2909, 2702, 2473,
2373, 2073, 1642 (CN), 1599, 1578, 1486, 1454, 1373, 1297, 1280,
1257, 1238, 1222, 1141, 1077, 1032, 989, 935, 918, 875, 860, 831, 790,
741, 721. HRMS (ESI+) m/z: [M + H]+ calcd for C9H13N4 177.1140.
Found: 177.1145. Purity by HPLC: 96.4% (tR = 22.31 min).
1-(5-Methylpyridin-2-yl)-3-(phenyl)guanidine Hydrochlor-
ide (8i): Procedure B. Colorless gum (86%). δH (400 MHz,
DMSO-d6): 2.30 (s, 3H, CH3), 7.10 (d, 1H, J = 8.4, Ar), 7.37−7.41
(m, 3H, Ar), 7.52 (app t, 2H, J = 8.0, 7.5, Ar), 7.78 (dd, 1H, J = 8.4,
1.8, Ar), 8.21 (d, 1H, J = 1.8, Ar), 8.93 (br s, 2H, NH), 11.43 (br s, 2H,
NH). δC (100 MHz, DMSO-d6): 17.7 (CH3), 113.7 (CH Ar), 125.6
(CH Ar), 127.7 (CH Ar), 129.3 (q Ar), 130.3 (CH Ar), 134.8 (q Ar),
140.9 (CH Ar), 146.4 (CH Ar), 150.3 (q Ar), 153.9 (q, CN). νmax
(ATR)/cm−1: 2921, 1637 (CN), 1608, 1595, 1572, 1483, 1369,
1281, 1233, 1140, 1029, 900, 895, 827, 752. HRMS (ESI+) m/z: [M +
H]+ calcd for C13H15N4 227.1297. Found: 227.1297. Purity by HPLC:
98.9% (tR = 27.09 min).
1-(5-Methylpyridin-2-yl)-3-(ethoxy)guanidine Hydrochlor-
ide (8j): Procedure B. White solid (87%). Mp 150−154 °C,
decomposition. δH (400 MHz, DMSO-d6): 2.27(s, 3H, CH3), 3.42 (t,
2H, J = 5.0, CH2), 3.60 (t, 2H, J = 5.0, CH2), 5.20 (br s, 1H, OH),
7.01 (d, 1H, J = 8.3, Ar), 7.72 (dd, 1H, J = 8.3, 2.0, Ar), 8.14 (d, 1H, J
= 2.0, Ar), 8.53 (br s, 2H, NH), 9.59 (br s, 1H, NH), 11.41 (br s, 1H,
NH). δC (100 MHz, DMSO-d6): 17.7 (CH3), 44.1 (CH2), 59.5
(CH2), 113.1 (CH Ar), 128.7 (q Ar), 140.7 (CH Ar), 146.2 (CH Ar),
150.6 (q Ar), 154.9 (q, CN). νmax (ATR)/cm−1: 3352 (OH), 3234
(OH), 3169 (NH), 3001 (NH), 2917, 1670 (CN), 1652, 1633,
1608, 1492, 1459, 1380, 1354, 1312, 1278, 1246, 1200, 1183, 1150,
1084, 1027, 996, 923, 887, 818, 762, 732, 718, 665. HRMS (ESI+) m/
z: [M + H]+ calcd for C9H15N4O 195.1246. Found: 195.1245. Purity
by HPLC: 98.2% (tR = 21.83 min).
1-(5,6,7,8-Tetrahydroquinolin-2-yl)-3-(ethoxy)guanidine Hy-
drochloride (9j): Procedure B. White solid (81%). Mp 74−76 °C,
clean melt. δH (400 MHz, DMSO-d6): 1.70−1.82 (m, 4H, 2 CH2),
2.67 (app t, 2H, J = 6.3, 5.9, CH2), 2.76 (app t, 2H, J = 6.3, 6.0, CH2),
3.43 (app t, 2H, J = 5.2, 4.9, CH2), 3.61 (app t, 2H, J = 5.2, 4.9, CH2),
5.21 (br s, 1H, OH), 6.82 (d, 1H, J = 8.2, Ar), 7.54 (d, 1H, J = 8.2, Ar),
8.45 (br s, 2H, NH), 10.10 (br s, 1H, NH), 11.15 (br s, 1H, NH). δC
(100 MHz, DMSO-d6): 22.6 (CH2), 22.6 (CH2), 27.6 (CH2), 31.9
1-(5-Methylpyridin-2-yl)-3-(2-furanylmethyl)guanidine Hy-
drochloride (8k): Procedure B. White solid (95%). Mp 130−135
°C, clean melt. δH (400 MHz, DMSO-d6): 2.27 (s, 3H, CH3), 4.66 (s,
2H, CH2), 6.46−6.49 (m, 2H, Ar), 7.02 (d, 1H, J = 8.3, Ar), 7.69 (d,
1H, J = 0.6, Ar), 7.73 (dd, 1H, J = 8.3, 2.0, Ar), 8.15 (d, 1H, J = 2.0,
Ar), 8.81 (br s, 2H, NH), 9.66 (br s, 1H, NH), 11.35 (br s, 1H, NH).
K
DOI: 10.1021/jm501635e
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(CH2), 44.1 (CH2), 59.4 (CH2), 110.7 (CH Ar), 127.5 (q Ar), 140.6
(CH Ar), 150.0 (q Ar), 154.0 (q Ar), 154.9 (q, CN). νmax (ATR)/
cm−1: 3405 (OH), 3317 (OH), 2937, 1673 (CN), 1593, 1646,
1493, 1253, 1052, 815, 749. HRMS (ESI+) m/z: [M + H]+ calcd for
C12H19N4O 235.1559. Found: 235.1559. Purity by HPLC: 96.4% (tR =
26.89 min).
1-(5,6,7,8-Tetrahydroquinolin-2-yl)-3-(2-furanylmethyl)-
guanidine Hydrochloride (9k): Procedure B. White solid (86%)
was obtained as a white solid. Mp 182−186 °C, decomposition. δH
(400 MHz, DMSO-d6): 1.69−1.82 (m, 4H, 2 CH2), 2.67 (app t, 2H, J
= 6.0, 5.8, CH2), 2.73 (app t, 2H, J = 6.3, 5.8, CH2), 4.66 (s, 2H, CH2),
6.46−6.49 (m, 1H, Ar), 6.50 (d, 1H, J = 2.8, Ar), 6.84 (d, 1H, J = 8.2,
Ar), 7.55 (d, 1H, J = 8.2, Ar), 7.69 (s, 1H, Ar), 8.87 (br s, 2H, NH),
9.97 (br s, 1H, NH), 11.22 (br s, 1H, NH). δC (100 MHz, DMSO-d6):
22.5 (CH2), 22.6 (CH2), 27.6 (CH2), 31.8 (CH2), 38.3 (CH2), 108.7
(CH Ar), 111.0 (CH Ar), 111.1 (CH Ar), 127.8 (q Ar), 140.74 (CH
Ar), 143.7 (CH Ar), 149.9 (q Ar), 150.0 (q Ar), 154.0 (q Ar), 154.6
(q, CN). νmax (ATR)/cm−1: 3150 (NH), 2938, 1643 (CN), 1603,
1472, 1281, 1179, 1062, 1012, 802, 734. HRMS (ESI+) m/z: [M + H]+
calcd for C15H19N4O 271.1559. Found: 271.1563. Purity by HPLC:
98.4% (tR = 30.64 min).
Ar), 144.4 (CH Ar), 156.2 (q, Gua). νmax (ATR)/cm−1: 3237 (NH),
3115 (NH), 3057 (NH), 2587, 2390, 2331, 2164, 2032, 1658 (CN),
1570, 1507, 1478, 1424, 1385, 1261, 1179, 1131, 1047, 1013, 933, 838,
810, 721, 687. HRMS (ESI+) m/z: [M + H]+ calcd for C6H9N4
137.0827. Found: 137.0829. Purity by HPLC: 99.2% (tR = 2.19 min).
1-(Pyridin-3′-yl)-2-iminoimidazolidine Hydrochloride (11d):
Procedure B. Clear, crystalline solid (98%). Mp 112−115 °C,
decomposition. δH (400 MHz, DMSO-d6): 3.69 (s, 4H, CH2), 7.67
(app t, 1H, J = 6.6, Ar), 7.94 (d, 1H, J = 8.3, Ar), 8.59 (d, 1H, J = 4.4,
Ar), 8.66 (s, 1H, Ar), 8.69 (br s, 2H, NH), 11.08 (br s, 1H, NH). δC
(150 MHz, DMSO-d6): 42.7 (2 CH2), 127.1 (CH Ar), 137.4 (CH Ar),
138.1 (CH Ar), 140.1 (CH Ar), 135.7 (q Ar), 157.5 (q, Imi). νmax
(ATR)/cm−1: 3343 (NH), 3070 (NH), 3040 (NH), 2975, 2864, 2744,
2444, 2067, 1639 (CN), 1608, 1556, 1514, 1468, 1420, 1366, 1339,
1328, 1284, 1252, 1190, 1089, 1046, 1021, 928, 885, 859, 815, 748,
671. HRMS (ESI+) m/z: [M + H]+ calcd for C8H11N4 163.0984.
Found: 163.0987. Purity by HPLC: 99.2% (tR = 4.00 min).
1-(6-Chloropyridin-3-yl)guanidine Hydrochloride (12c): Pro-
cedure B. White solid (92%). Mp 190−196 °C, clean melt. δH (600
MHz, DMSO-d6): 7.59 (d, 1H, J = 8.5, Ar), 7.73 (br s, 4H, NH), 7.76
(dd, 1H, J = 8.5, 2.5, Ar), 8.33 (d, 1H, J = 2.5, Ar), 10.16 (br s, 1H,
NH). δC (150 MHz, DMSO-d6): 125.0 (CH Ar), 132.0 (q Ar), 136.4
(CH Ar), 146.3 (CH Ar), 147.4 (q Ar), 156.3 (q, Gua). νmax (ATR)/
cm−1: 3278 (NH), 3092 (NH), 3050 (NH), 2839, 2163, 2012, 1910,
1667 (CN), 1632, 1603, 1584, 1454, 1417, 1362, 1287, 1266, 1132,
1106, 1029, 1010, 934, 873, 833, 745, 707. HRMS (ESI+) m/z: [M +
H]+ calcd for C6H8N435Cl 171.0437. Found: 171.0433. Purity by
HPLC: 96.0% (tR = 7.43 min).
1-(6-Methylpyridin-3-yl)guanidine Hydrochloride (13c): Pro-
cedure B. White solid (82%). Mp 171−172 °C, clean melt. δH (600
MHz, DMSO-d6): 2.73 (s, 3H, CH3), 7.86 (d, 1H, J = 8.7, Ar), 8.00
(br s, 4H, NH), 8.23 (dd, 1H, J = 8.7, 2.2, Ar), 8.72 (d, 1H, J = 2.2,
Ar), 10.69 (br s, 1H, NH). δC (150 MHz, DMSO-d6): 19.3 (CH3),
127.6 (CH Ar), 132.8 (q Ar), 138.6 (CH Ar), 140.1 (CH Ar), 151.5 (q
Ar), 156.3 (q, Gua). νmax (ATR)/cm−1: 3275 (NH), 2894, 1677 (C
N), 1622, 1603, 1565, 1491, 1456, 1376, 1288, 1244, 1107, 1025, 875,
832, 798, 739, 716. HRMS (ESI+) m/z: [M + H]+ calcd for C7H11N4
151.0984. Found: 151.0980. Purity by HPLC: 99.7% (tR = 3.16 min).
1-(5,6,7,8-Tetrahydroquinolin-3-yl)guanidine Hydrochloride
(14c): Procedure B. White solid (87%). Mp 85−88 °C, clean melt.
δH (600 MHz, DMSO-d6): 1.78 (app t, 2H, J = 5.9, 5.5, CH2), 1.86
(app t, 2H, J = 5.9, 5.5, CH2), 2.85 (t, 2H, J = 6.1, CH2), 2.97 (app t,
2H, J = 6.1, CH2), 7.76 (br s, 4H, NH), 7.85 (s, 1H, Ar), 8.48 (s, 1H,
Ar), 10.25 (br s, 1H, NH). δC (150 MHz, DMSO-d6): 21.5 (CH2),
21.7 (CH2), 27.8 (CH2), 29.1 (CH2), 131.6 (q Ar), 135.9 (q Ar),
138.3 (CH Ar), 139.3 (CH Ar), 152.4 (q Ar), 155.6 (q, Gua). νmax
(ATR)/cm−1: 3274 (NH), 2893, 1676 (CN), 1620, 1598, 1563,
1490, 1376, 1325, 1289, 1244, 1083, 1025, 904, 876, 831, 737, 716,
653. HRMS (ESI+) m/z: [M + H]+ calcd for C10H15N4 191.1297.
Found: 191.1292. Purity by HPLC: 96.4% (tR = 10.11 min).
1-(5,6,7,8-Tetrahydroquinolin-3′-yl)-2-iminoimidazolidine
Hydrochloride (14d): Procedure B. White, crystalline solid (91%).
Mp 122−126 °C, clean melt. δH (600 MHz, DMSO-d6): 1.79 (app p,
2H, CH2), 1.86 (app p, 2H, CH2), 2.87 (app t, 2H, J = 6.1, CH2), 3.01
(app t, 2H, J = 6.2, CH2), 3.70 (br s, 4H, CH2), 7.99 (s, 1H, Ar), 8.54
(s, 1H, Ar), 8.81 (br s, 2H, NH), 11.35 (br s, 1H, NH). δC (150 MHz,
DMSO-d6): 21.3 (CH2), 21.4 (CH2), 27.7 (CH2), 28.3 (CH2), 43.0 (2
CH2), 132.7 (q Ar), 136.7 (CH Ar), 138.0 (CH Ar), 151.3 (q Ar),
158.1 (q, Imi), 164.6 (q Ar). νmax (ATR)/cm−1: 3418 (NH), 3108
(NH), 3020 (NH), 2955, 2932, 2898, 2868, 2812, 2418, 2035, 1622
(CN), 1560, 1504, 1445, 1418, 1322, 1262, 1215, 1087, 1027, 1000,
947, 932, 898, 826, 779, 729, 688. HRMS (ESI+) m/z: [M + H]+ calcd
for C12H17N4 217.1453. Found: 217.1446. Purity by HPLC: 96.1% (tR
= 17.20 min).
1-[5-(Ethylamino)pyridin-2-yl]guanidine Hydrochloride
(10c). To a solution of neutral 1-(5-aminopyridin-2-yl)guanidine
(111 mg, 0.73 mmol) in a mixture of CH3CN/CH3OH (5:1, 3.0 mL)
was added acetaldehyde (1.0 equiv, 0.73 mmol, 41 μL). The solution
was stirred under an argon atmosphere, and sodium triacetoxybor-
ohydride (2.0 equiv, 311 mg, 1.47 mmol) was added. Reaction was
continued for 6 h, after which excess solvent and acetaldehyde were
removed under reduced pressure. The resulting residue was dissolved
in a solution of iPrOH/CH2Cl2 (20%, 10 mL), washed with NaHCO3
(sat., 5 mL) and water (3 mL), dried over MgSO4, filtered, and
concentrated under vacuum. The residue was stirred in a 1.25 M
methanolic HCl solution (4.0 equiv, 2.4 mL, 2.94 mmol) for 3 h.
Excess solvent and HCl were removed under reduced pressure, and
the residue was dissolved in a minimum volume of H2O. The aqueous
layer was washed with CH2Cl2 and concentrated under vacuum to
yield a red solid, which was purified by reverse phase chromatography
(C-8 silica) with 100% H2O as mobile phase to yield a dark red solid
1
identified by H NMR spectroscopy as a mixture of salts. Normal
phase chromatography, eluting with a gradient of CH2Cl2/CMA,
isolated neutral guanidine (32%) as a red solid after restirring in 1.25
M methanolic HCl (2.4 mL). Mp 154−157 °C, clean melt. δH (400
MHz, DMSO-d6): 1.22 (t, 3H, J = 7.2, CH3), 3.43 (q, 2H, J = 7.2,
CH2), 7.12 (d, 1H, J = 9.4, Ar), 7.66 (br s, 4H, NH), 7.75 (d, 1H, J =
9.4, Ar), 7.92 (s, 1H, Ar), 9.78 (br s, 1H, NH). δC (100 MHz, DMSO-
d6): 13.7 (CH3), 37.0 (CH2), 114.3 (CH Ar), 120.7 (q Ar), 135.0 (CH
Ar), 142.0 (CH Ar), 142.0 (q Ar), 157.2 (q, Gua). νmax (ATR)/cm−1:
3313, 3181 (NH), 3130 (NH), 3037 (NH), 2973, 2810, 1702, 1666
(CN), 1608, 1584, 1498, 1469, 1456, 1432, 1337, 1302, 1272, 1230,
1183, 1123, 1102, 1057, 1037, 1003, 942, 909, 875, 857, 821, 781, 730,
694, 672. HRMS (ESI+) m/z: [M + H]+ calcd for C8H14N5 180.1249.
Found: 180.1244. Purity by HPLC: 99.5% (tR = 3.88 min).
1-[5-(Ethylamino)pyridin-2′-yl]2-iminoimidazolidine Hydro-
chloride (10d): Procedure B. Colorless solid (93%). Mp above 220
°C, decomposition. δH (400 MHz, DMSO-d6): 1.22 (m, 3H, CH3),
3.44 (m, 2H, CH2), 3.64 (s, 4H, CH2), 7.16 (d, 1H, J = 8.0, Ar), 7.77
(d, 1H, J = 8.0, Ar), 7.91 (s, 1H, Ar), 8.58 (br s, 2H, NH), 9.32 (s, 1H,
NH, NHEt), 10.65 (br s, 1H, NH). δC (100 MHz, DMSO-d6): 14.1
(CH3), 37.6 (CH2), 43.2 (2 CH2), 114.6 (CH Ar), 122.0 (q Ar), 134.1
(CH Ar), 141.6 (CH Ar), 152.2 (q Ar), 159.4 (q, Imi). νmax (ATR)/
cm−1: 3375 (NH), 3198 (NH), 3057 (NH), 2860, 1675, 1619 (C
N), 1590, 1482, 1458, 1407, 1375, 1321, 1280, 1213, 1154, 1128,
1110, 1085, 1003, 938, 935, 785, 746, 717, 658. HRMS (ESI+) m/z:
[M + H]+ calcd for C10H16N5 206.1406. Found: 206.1401. Purity by
HPLC: 100.0% (tR = 5.07 min).
1-[6-(N-Ethylamino)pyridin-3-yl]guanidine Hydrochloride
(15c): Procedure B. White, crystalline solid (100%). Mp 120−124
°C, decomposition. δH (600 MHz, DMSO-d6): 1.22 (t, 3H, J = 7.1,
CH3), 3.43 (q, 2H, J = 7.1, CH2), 7.13 (d, 1H, J = 9.5, Ar), 7.67 (br s,
4H, NH), 7.75 (dd, 1H, J = 1.9, 9.5, Ar), 7.91 (d, 1H, J = 1.9, Ar), 9.17
(br s, 1H, NH, NHEt), 9.81 (br s, 1H, NH). δC (150 MHz, DMSO-
1-(Pyridin-3-yl)guanidine Hydrochloride (11c): Procedure B.
White solid (85%). Mp 65−67 °C, clean melt. δH (600 MHz, DMSO-
d6): 7.70 (m, 1H, Ar), 7.80 (br s, 4H, NH), 7.95 (d, 1H, J = 7.8, Ar),
8.62 (s, 1H, Ar), 8.68 (s, 1H, Ar), 10.33 (br s, 1H, NH). δC (150 MHz,
DMSO-d6): 125.5 (CH Ar), 133.6 (q Ar), 135.2 (CH Ar), 143.2 (CH
L
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d6): 14.0 (CH3), 37.3 (CH2), 114.6 (CH Ar), 121.0 (q Ar), 135.3 (CH
Ar), 142.4 (CH Ar), 152.3 (q Ar), 157.5 (q, Gua). νmax (ATR)/cm−1:
3310, 3250 (NH), 3179 (NH), 3129 (NH), 3040 (NH), 2973, 2848,
2808, 2770, 1663 (CN), 1605, 1582, 1502, 1439, 1376, 1336, 1302,
1273, 1230, 1166, 1103, 1055, 1021, 1005, 875, 858, 821, 781, 684.
HRMS (ESI+) m/z: [M + H]+ calcd for C8H14N5 180.1249. Found:
180.1246. Purity by HPLC: 96.3% (tR = 2.41 min).
1-(6-(Ethylamino)pyridin-3′-yl)-2-iminoimidazolidine Hy-
drochloride (15d): Procedure B. White, crystalline solid (95%).
Mp above 210 °C, decomposition. δH (600 MHz, DMSO-d6): 1.22 (t,
3H, J = 7.1, CH3), 3.45 (q, 2H, J = 7.1, CH2), 3.65 (s, 4H, CH2), 7.15
(d, 1H, J = 9.4, CH2), 7.76 (d, 1H, J = 9.4, Ar), 7.92 (s, 1H, Ar), 8.55
(br s, 2H, NH), 10.63 (br s, 1H, NH). δC (150 MHz, DMSO-d6): 13.7
(CH3), 37.0 (CH2), 42.7 (2 CH2), 113.9 (CH Ar), 121.5 (q Ar), 133.8
(CH Ar), 141.0 (CH Ar), 151.9 (q Ar), 159.0 (q, Imi). νmax (ATR)/
cm−1: 3199 (NH), 3057 (NH), 2861, 1674, 1619 (CN), 1589,
1482, 1458, 1407, 1375, 1320, 1279, 1213, 1154, 1128, 1108, 1085,
1066, 1020, 1002, 989, 934, 835, 786, 745, 717, 657. HRMS (ESI+) m/
z: [M + H]+ calcd for C10H16N5 206.1406. Found: 206.1401. Purity by
HPLC: 97.9% (tR = 3.21 min).
7.1, Ar), 7.30−7.33 (m, 1H, Ar), 7.34 (t, 2H, J = 7.3, Ar), 7.46 (d, 2H,
J = 7.3, Ar), 8.21 (br s, 1H, NH), 9.84 (br s, 1H, NH). δC (150 MHz,
DMSO-d6): 42.9 (CH2), 123.7 (q Ar), 123.8 (CH Ar), 124.8 (CH Ar),
128.3 (CH Ar), 128.6 (CH Ar), 129.0 (CH Ar), 129.5 (q Ar), 130.6 (q
Ar), 133.1 (CH Ar), 139.7 (CH Ar), 181.5 (q, CN). νmax (ATR)/
cm−1: 3221 (NH), 2810, 2554, 1980, 1744, 1594 (CN), 1534, 1494,
1452, 1380, 1343, 1313, 1265, 1121, 1093, 1029, 939, 835, 748, 694.
HRMS (ESI+) m/z: [M + H]+ calcd for C14H14N3 224.1188. Found:
224.1194. Purity by HPLC: 96.4% (tR = 28.55 min).
N-(Ethoxy)-1,4-dihydroquinazolin-2-amine Hydrochloride
(19j): Procedure D. White solid (95%). Mp 139−140 °C, clean
melt. δH (400 MHz, DMSO-d6): 3.57 (t, 2H, J = 7.5, CH2), 3.90 (t,
2H, J = 7.5, CH2), 3.90 (br s, 1H, OH), 4.84 (d, 2H, J = 5.2, CH2),
7.34−7.50 (m, 4H, Ar), 8.45 (br s, 1H, NH), 9.99 (br s, 1H, NH),
10.96 (br s, 1H, NH). δC (100 MHz, DMSO-d6): 44.0 (CH2), 30.9
(CH2), 48.8 (CH2), 123.6 (CH Ar), 127.8 (CH Ar), 128.8 (CH Ar),
129.1 (q Ar), 129.4 (CH Ar), 131.3 (q Ar), 170.1 (q, CN). νmax
(ATR)/cm−1: 2793, 2561, 2020, 1634 (CN), 1580, 1494, 1455,
1343, 1292, 1244, 1127, 1071, 940, 873, 755, 679. HRMS (ESI+) m/z:
[M + H]+ calcd for C10H14N3O 192.1137. Found: 192.1143. Purity by
HPLC: 97.3% (tR = 19.96 min).
N-(2-Furanylmethyl)-1,4-dihydroquinazolin-2-amine Hydro-
chloride (19k): Procedure D. Tan solid (30%). Mp 50−52 °C, clean
melt. δH (400 MHz, DMSO-d6): 4.66 (s, 2H, CH2), 4.84 (d, 2H, J =
4.6, CH2), 6.29 (br s, 1 H, Ar), 6.40 (s, 1H, Ar), 7.37 (m, 1H, Ar),
7.38−7.40 (m, 2H, Ar), 7.47 (s, 1H, Ar), 7.59 (s, 1H, Ar), 8.31 (br s,
1H, NH), 8.39 (br s, 1H, NH). δC (100 MHz, DMSO-d6): 38.4
(CH2), 41.3 (CH2), 108.8 (CH Ar), 111.1 (CH Ar), 116.0 (CH Ar),
124.8 (CH Ar), 126.7 (CH Ar), 129.0 (CH Ar), 133.8 (q Ar), 140.3 (q
Ar), 143.6 (CH Ar), 152.4 (q Ar), 174.2 (q, CN). νmax (ATR)/cm−1:
2834, 2574, 1623 (CN), 1590, 1560, 1496, 1456, 1299, 1226, 1070,
1011, 939, 883, 753. HRMS (ESI+) m/z: [M + H]+ calcd for
C13H14N3O 228.1137. Found: 228.1146. Purity by HPLC: 97.6% (tR =
27.95 min).
N-(n-Propyl)-1,4-dihydroquinazolin-2-amine Hydrochloride
(19l): Procedure D. White solid (90%). Mp 80−82 °C, clean melt.
δH (400 MHz, DMSO-d6): 0.92 (t, 3H, J = 7.1, CH3), 1.54 (m, 2H,
CH2), 3.27 (m, 2H, CH2), 4.46 (s, 2H, CH2), 7.02−7.09 (m, 2H, Ar),
7.19 (d, 1H, J = 7.2, Ar), 7.24 (t, 1H, J = 7.2, Ar), 8.32 (br s, 1H, NH),
8.79 (br s, 1H, NH), 10.82 (br s, 1H, NH). δC (100 MHz, DMSO-d6):
11.0 (CH3), 21.9 (CH2), 40.8 (CH2), 42.7 (CH2), 123.9 (q Ar), 126.2
(CH Ar), 128.4 (CH Ar), 133.5 (CH Ar), 145.5 (q Ar), 151.8 (CH
Ar), 166.9 (q, CN). νmax (ATR)/cm−1: 3233 (NH), 2872, 2558, 1992,
1618 (CN), 1555, 1496, 1456, 1382, 1333, 1291, 1213, 1179, 1064,
935, 837, 750. HRMS (ESI+) m/z: [M + H]+ calcd for C11H16N3
190.1344. Found: 190.1347. Purity by HPLC: 96.7% (tR = 26.23 min).
N-(Ethoxy)-1,4-dihydropyrido[2,3-d]αin-2-amine Hydro-
chloride (20j): Procedure D. Yellow solid (98%). Mp 235 °C,
decomposed. δH (400 MHz, DMSO-d6): 3.52 (br s, 2H, CH2), 3.86
(br s, 2H, CH2), 4.49 (br s, 2H, CH2), 6.86 (s, 1H, Ar), 7.88 (s, 1H, J
= 8.0, Ar), 7.94 (d, 1H, J = 4.0, Ar), 8.18 (br s, 1H, NH), 8.25 (br s,
1H, NH), 10.61 (br s, 1H, NH). δC (100 MHz, DMSO-d6): 31.1
(CH2), 43.5 (CH2), 49.2 (CH2), 112.9 (CH Ar), 134.0 (q Ar), 135.4
(CH Ar), 142.6 (CH Ar), 151.8 (q Ar), 182.4 (q, CN). HRMS (ESI+)
m/z: [M + H]+ calcd for C9H13N4O 193.1089. Found: 193.1083.
Purity by HPLC: 97.7% (tR = 2.56 min).
1-(5-Nitropyridin-2-yl)guanidine Hydrochloride (17c). To
guanidine hydrochloride (1.1 equiv, 133 mg, 1.39 mmol) and
tBuOH (12 mL) was added finely ground NaOH (3.0 equiv, 151
mg, 3.79 mmol). The mixture was stirred for 30 min at rt after which
2-nitro-5-chloropyridine (1.0 equiv, 200 mg, 1.26 mmol) was added.
Reflux at 120 °C was continued for 6 h. The resulting solution was
i
extracted with PrOH/CH2Cl2 (20%, 4 × 30 mL) and washed with 2
M NaOH solution (30 mL). The organic extracts were combined,
dried over MgSO4, and concentrated in vacuo to give the neutral
guanidine (153 mg, 67%). This was stirred at rt in a 1.25 M solution of
HCl in CH3OH (6.0 equiv, 5.98 mL, 7.47 mmol) for 3 h. Removal of
solvent and purification by reverse phase chromatography (C-8 silica)
with 100% H2O as mobile phase yielded 16c as yellow crystals (85%).
Mp 93−95 °C, clean melt. δH (600 MHz, DMSO-d6): 7.27 (d, 1H, J =
9.1, Ar), 8.49 (br s, 4H, NH), 8.62 (dd, 1H, J = 9.1, 2.7, Ar), 9.12 (d,
1H, J = 2.7, Ar), 12.17 (br s, 1H, NH). δC (150 MHz, DMSO-d6):
113.7 (CH Ar), 135.0 (CH Ar), 140.3 (q Ar), 144.1 (CH Ar), 155.2 (q
Ar), 156.0 (q, Gua). νmax (ATR)/cm−1: 3426 (NH), 3318 (NH),
3185, 2924, 1687 (CN), 1620, 1601, 1552, 1516, 1474, 1340, 1246,
1121, 1079, 1013, 984, 947, 864, 840, 758, 708. HRMS (ESI+) m/z:
[M + H]+ calcd for C6H8N5O2 182.0678. Found: 182.0679. Purity by
HPLC: 96.8% (tR = 18.97 min).
1-(5-Aminopyridin-2-yl)guanidine Hydrochloride (18c). To a
solution of neutral 1-(5-nitropyridin-2-yl)guanidine (100 mg, 0.55
mmol) in CH3OH (6 mL) was added 10% Pd/C (29 mg, 0.028 mmol,
5 mol %). The mixture was stirred under a hydrogen atmosphere (3
atm) for 7 h. It was then diluted with CH3OH (30 mL), filtered
through filter paper, and concentrated under reduced pressure. The
unstable neutral guanidine was immediately converted to the
hydrochloride salt by stirring the residue in a 1.25 M methanolic
HCl solution (6.0 equiv, 2.6 mL, 3.30 mmol) for 3 h. Excess solvent
and HCl were removed under reduced pressure, and the residue was
dissolved in a minimum volume of H2O. The aqueous layer was
washed with CH2Cl2 (2 × 2 mL) and concentrated under reduced
pressure to yield a red solid, which was purified by reverse phase
chromatography (C-8 silica) with 100% H2O as mobile phase.
Recrystallization from CH3OH/Et2O afforded 18c as a red solid
(69%). Mp 200−202 °C, burned. δH (600 MHz, DMSO-d6): 3.82 (br
s, 2H, NH2), 7.03 (d, 1H, J = 8.8, Ar), 7.56 (d, 1H, J = 8.8, Ar), 8.08 (s,
1H, Ar), 8.20 (br s, 4H, NH), 11.39 (br s, 1H, NH). δC (150 MHz,
DMSO-d6): 113.9 (CH Ar), 127.2 (CH Ar), 133.8 (q Ar), 137.5 (CH
Ar), 143.7 (q Ar), 154.7 (q, Gua). νmax (ATR)/cm−1: 3308 (NH),
2795, 2544, 2025, 1682 (CN), 1627, 1602, 1560, 1488, 1396, 1287,
1252, 1226, 1137, 1099, 1033, 877, 825, 807, 731. HRMS (ESI+) m/z:
[M + H]+ calcd for C6H10N5 152.0936. Found: 152.0934. Purity by
HPLC: 97.4% (tR = 4.84 min).
N-(Furan-2-ylmethyl)-1,4-dihydropyrido[2,3-d]pyrimidin-2-
amine Hydrochloride (20k): Procedure D. White powder (86%).
Mp 181 °C, decomposed. δH (400 MHz, DMSO-d6): 4.53 (s, 2H,
CH2), 4.59 (s, 2H, CH2), 6.25 (s, 1H, Ar), 6.34 (s, 1H, Ar), 6.85 (app
t, 1H, J = 8.0, 4.0, Ar), 7.53 (s, 1H, Ar), 7.79 (d, 1H, J = 8.0, Ar), 7.87
(d, 1H, J = 4.0, Ar), 8.11 (br s, 1H, NH), 8.39 (br s, 1H, NH), 8.48 (br
s, 1H, NH). δC (100 MHz, DMSO-d6): 41.1 (CH2), 42.9 (CH2), 107.8
(CH Ar), 110.9 (CH Ar), 112.5 (CH Ar), 123.5 (q Ar), 134.5 (CH
Ar), 141.4 (CH Ar), 142.7 (CH Ar), 151.7 (q Ar), 152.5 (q Ar), 172.5
(q CN). HRMS (ESI+) m/z: [M + H]+ calcd for C12H13N4O
229.1098. Found: 229.1098. Purity by HPLC: 96.2% (tR = 23.27 min).
N-(1,3-Benzodioxol-5-ylmethyl)-1,4-dihydropyrido[2,3-d]-
pyrimidin-2-amine Hydrochloride (20m): Procedure D. White
powder (26%). Mp 196 °C. δH (400 MHz, DMSO-d6): 4.59 (br s, 2H,
N-Phenyl-1,4-dihydroquinazolin-2-amine Hydrochloride
(19i): Procedure D. White solid (93%). Mp 122−124 °C, clean
melt. δH (600 MHz, DMSO-d6): 4.75 (d, 2H, J = 5.2, CH2), 7.04 (br s,
1H, Ar), 7.09 (br s, 1H, Ar), 7.14 (t, 1H, J = 7.3, Ar), 7.22 (t, 1H, J =
M
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CH2), 4.62 (s, 2H, CH2), 5.98 (s, 2H, CH2), 6.76 (app t, 1H, J = 4.0,
Ar), 6.87 (m, 2H, Ar), 6.87 (s, 1H, Ar), 7.74 (d, 1H, J = 4.0, Ar), 7.92
(d, 1H, J = 4.0, Ar), 8.07 (br s, 1H, NH), 8.33 (br s, 1H, NH), 8.49 (br
s, 1H, NH). δC (100 MHz, CDCl3): 42.5 (CH2), 42.7 (CH2), 101.1
(CH2), 112.2 (CH Ar), 108.5 (CH Ar), 115.3 (q Ar), 120.8 (CH Ar),
131.3 (q Ar), 134.3 (CH Ar), 147.1 (CH Ar), 146.9 (q Ar), 152.6 (q
Ar), 181.3 (q CN). HRMS (ESI+) m/z: [M + H]+ calcd for
C15H15N4O2 283.1195. Found: 283.1190. Purity by HPLC: 99.8% (tR
= 24.40 min).
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ASSOCIATED CONTENT
■
S
* Supporting Information
Preparation and characterization of all starting amines and Boc-
protected or neutral guanidine intermediates; spectroscopic and
HPLC data of final hydrochloride salts; further details of
pharmacological assays, calculated pharmacokinetic parameters,
and molecular modeling. This material is available free of
charge via the Internet at http://pubs.acs.org.
̌
́
(8) Goonan, A.; Kahvedzic, A.; Rodriguez, F.; Nagle, P.; McCabe, T.;
AUTHOR INFORMATION
■
Rozas, I.; Erdozain, A. M.; Meana, J. J.; Callado, L. F. Novel Synthesis
and Pharmacological Evaluation as α2-Adrenoceptor Ligands of O-
Phenylisouronium Salts. Bioorg. Med. Chem. 2008, 16, 8210−8217.
(9) Rozas, I. Improving Antidepressant Drugs: Update on Recently
Patented Compounds. Expert Opin. Ther. Pat. 2009, 19, 827−845.
(10) Rodriguez, F.; Rozas, I.; Ortega, J. E.; Erdozain, A. M.; Meana, J.
J.; Callado, L. F. Guanidine and 2-Aminoimidazoline Aromatic
Derivatives as α2-Adrenoceptor Ligands: Searching for Structure−
Activity Relationships. J. Med. Chem. 2009, 52, 601−609.
(11) Muguruza, C.; Rodriguez, F.; Rozas, I.; Meana, J. J.; Uriguen, L.;
Callado, L. F. Antidepressant-like Properties of Three New α2-
Adrenoceptor Antagonists. Neuropharmacology 2013, 65, 13−19.
(12) O’ Donovan, D. H.; Muguruza, C.; Callado, L. F.; Rozas, I.
Guanidine-based α2-Adrenoceptor Ligands: Towards Selective Antag-
onist Activity. Eur. J. Med. Chem. 2014, 82, 242−254.
Corresponding Author
*E-mail: rozasi@tcd.ie. Phone: +353 1 896 3731.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
B.K. is grateful to the HEA-PRTLI-4 (Ireland) for financial
support and to Trinity College Dublin for a travel grant. C.M. is
recipient of a predoctoral fellowship from the University of the
Basque Country (UPV/EHU), Spain. This work was also
supported by grants from the Basque Government (Grant
IT616-13; SAIOTEK S-PE12UN033), the University of the
Basque Country (Grant UFI 11/35), Ministry of Economy and
Competitiveness (Grant SAF2013-48586-R), and the Instituto
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Decision-Making Process in the Structural Modification of Drug
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ABBREVIATIONS USED
■
α2-AR, α2-adrenoceptor; α2C-AR, subtype C α2-adrenoceptor;
α2A-AR, subtype A α2-adrenoceptor; NA, noradrenaline;
[35S]GTPγS, sulfur labeled guanosine 5′-O-[γ-thio]-
triphosphate; PFC, prefrontal cortex; BDNF, brain-derived
neurotrophic factor; EC50, E half maximal effective concen-
tration; UK14304, brimonidine; RX821002, 2-methoxyidazox-
an; aCFS, artificial cerebrospinal fluid; Emax, maximum possible
effect; P or p, probability; Ftr, F value, treatment, between
groups; Ft, F value, time, within groups; Fi, F value, treatment ×
time, interaction; SAR, structure−activity relationship; pKaH,
minus logarithm of the acid constant of a base; IMHB,
intramolecular hydrogen bond; log D, logarithm of the
distribution coefficient; CNS, central nervous system; NMR,
nuclear magnetic resonance; Boc, di-tert-butyl dicarbonate;
DFT, density functional theory; pKi, minus logarithm of the
dissociation constant; GPCR, G-protein-coupled receptor;
GDP, guanine diphosphate; β2-AR, β2-adrenoceptor; TM,
transmembrane; hERG, human ether-a-go-go-related gene
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