Synthesis and biological evaluation of 2-aryliminopyrrolidines as selective ligands for I1 imidazoline receptors: Discovery of new sympatho-inhibitory hypotensive agents with potential beneficial effects in metabolic syndrome

Article abstract of DOI:10.1021/jm501456p

New 2-aryliminopyrrolidines (1-18) were synthesized and tested for their binding properties on I₁ imidazoline receptors vs α₂-adrenergic receptors and their blood pressure effects after both systemic and intracerebral administrations

Full text of DOI:10.1021/jm501456p

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 2‑Aryliminopyrrolidines as
Selective Ligands for I1 Imidazoline Receptors: Discovery of New
Sympatho-Inhibitory Hypotensive Agents with Potential Beneficial
Effects in Metabolic Syndrome
Vincent Gasparik,‡ Hugues Greney,‡ Stephan Schann,† Josiane Feldman, Lyne Fellmann,
Jean-Daniel Ehrhardt, and Pascal Bousquet*
Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Faculte
́
de Med
́ ́ ́ ́
ecine, EA 7296, Federation de Medecine
Translationnelle, Universite
́
de Strasbourg, 11 rue Humann, 67000 Strasbourg, France
S
* Supporting Information
ABSTRACT: New 2-aryliminopyrrolidines (1−18) were synthe-
sized and tested for their binding properties on I1 imidazoline
receptors vs α2-adrenergic receptors and their blood pressure
effects after both systemic and intracerebral administrations. The
purposes of this study were: (i) to analyze structure−activity and
affinity relationships on I1 imdazoline receptors and (ii) to
propose some leader compounds for the development of new
sympatho-inhibitory drugs with potential applications in hyper-
tension and/or metabolic syndrome, i.e., a cluster of cardiovas-
cular (hypertension) and metabolic disorders. Our study high-
lights decisive arguments of SAR concerning both the affinity for
I1Rs and the hypotensive activity of 2-aryliminopyrrolidines.
Binding assays showed high affinity and selectivity of some compounds for I1 imidazoline receptors over α2-adreergic receptors.
Compound 13 (laboratory reference LNP599; Ki = 3.2 nM on I1imidazoline receptors) is the prototype for the development of
new centrally acting agents targeting specifically I1imidazoline receptors to be used in the management of hypertension and/or
metabolic syndrome.
INTRODUCTION
■
imidazoline receptors (IRs) are involved in several biological
regulatory systems. The major one is the central regulation of
blood pressure (BP).1−3 They are also involved in other
physiological functions such as insulin release,4,5 intraocular
pressure regulation,6 and cardiac rhythm control.7,8 IRs are
classified in three different subtypes. The first subtype of
imidazoline receptors, I1Rs, is located in the rostro-ventrolateral
medulla of the brainstem and was shown to be involved in the
central regulation of the cardiovascular function.9 The
hypothesis of their existence and their role was first proposed
by Bousquet and co-workers in the mid 1980s.10,11 I1Rs are
sensitive to clonidine and other imidazoline-like drugs but not
to catecholamines. Therefore, they are different from α2AARs,
which is the subtype of adrenoceptors involved in the central
regulation of BP.12 Recently, we demonstrated that I1Rs are
present in adipose tissue and that selective activation of I1Rs
not only reduces BP but also improves metabolic disorders
which are associated with hypertension in the so-called MetS.13
Clonidine (Figure 1) was the leader compound of the first
generation of centrally acting antihypertensive drugs. It was
shown to bind to both I1Rs and α2ARs. Its side effects, sedation
in particular, were clearly attributed to the activation of
α2ARs.14 Drugs such as moxonidine and rilmenidine display
Figure 1. Structures of imidazolines and related compounds.
some selectivity for I1Rs over α2ARs, as compared to clonidine,
they have a lower affinity for α2ARs.15 Consequently, they also
have fewer side effects in hypertensive patients.16,17
Over the last two decades, very active research on
imidazoline derivatives and IRs led to the discovery of
compounds either selective for α2ARs or new hybrid drugs
Received: September 22, 2014
© XXXX American Chemical Society
A
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Article
partially selective for α2ARs over I1Rs.18−20 Interesting
hypotheses were suggested to explain the selectivity of some
drugs for I1Rs by physicochemical and biophysical parame-
ters.21,22
a
Scheme 1. Synthesis of the 2-Aryliminopyrrolidines 1−18
Our group is particularly interested in the cardiovascular
effects of potential therapeutical interest of drugs selective for
I1Rs. Bruban et al. showed that drugs binding and acting
exclusively on I1Rs are able to decrease BP. Thus, S23515
(Figure 1) has a significant selectivity for I1Rs over α2ARs,23
whereas 30 (LNP509, Figure 1) was the first compound
binding to I1Rs, but devoid of any adrenergic property, able to
lower the BP after ic injection.24−26 30 even lowered the BP of
genetically engineered animals as D79N mice,27 whose α2AARs
were no longer functional. IRC with nanomolar affinities for
I1Rs and great selectivities over α2ARs were synthesized. One of
these IRC, 31 (LNP630, Figure 1), decreases BP efficiently.28
Other compounds selective for I1Rs, 32 and 33 (LNP911 and
LNP906, Figure 1) which exhibited a subnanomolar affinity,
were developed as a radioligand29 and a photoaffinity−
radioligand,30 respectively, but they had no hypotensive activity.
32 behaves rather as an antagonist (personal observation),
whereas 33, as a photoactivable ligand, is unusable in in vivo
experiments. These two molecules were used as tools to study
and characterize I1Rs. They are iminopyrrolidines by contrast
with the historical leader compounds that are either imidazo-
lines (S23757)23 or amino-imidazolines (clonidine, 31) or
amino-oxazolines (rilmenidine).
a
Reaction conditions: (a) lactam 19a−b, R3-Ph-NH2, POCl3, 1,2-
dichloroethane, 60 °C, 6 h. For R groups definitions, see Table 1.
presence of catalytic amount of tetramethylguanidine.36 The
nitroester 20 was then hydrogenated and cyclized, affording the
lactam in good yield (Scheme 2).37 The choice of only alkyl
a
Scheme 2. Preparation of Lactams 19b
a
Reaction conditions: (a) nitromethane, TMG, rt, 12 h; (b) (i) H2,
10% Pd/C, AcOH, rt, 12 h, (ii) TEA, EtOH, reflux, 20 h.
and halogen as substituents is based on the physicochemical
properties of such groups that serve the lipophilicity of central
acting drugs.38 Most of the anilines used in the study were
obtained from commercial sources, but anilines 21−29 were
synthesized. The starting point for the synthesis is the
commercial mixture of 4- and 5-nitroindan as described in
Scheme 3. After catalytic hydrogenation of the former mixture,
the two anilines 21 and 22 were separated by column
chromatography. Aniline 25 was prepared from 21,39 which
was acylated with Ac2O to give 23 in 90% yield and treated in
acetic acid with chlorine to give compound 24 in a nearly
quantitative yield. The latter was then hydrolyzed in 4 M HCl
to afford 25 in quantitative yield. The way to 28 was somewhat
different. Aniline 21 was acylated as described above, but the
product 23 was halogenated with bromine to afford 26 in 92%
yield. Treatment of 26 with trimethylboroxine using the
standard Suzuki coupling conditions, in dioxan, gave the
methylated derivative 27 in 80% yield.40 Aniline 28 was
obtained quantitatively after the hydrolysis of the acetamido
group in acidic condition. Aniline 29 was obtained from 22
following the procedure described for the preparation of 25.
Because of the high affinity of 32 for I1Rs,29 the aim of the
present work was to further analyze structure−activity relation-
ships of 2-aryliminopyrrolidines. Requirements for their
selectivity and affinity for I1Rs and their sympatho-inhibitory
properties, mainly their BP effects, were studied. As a matter of
fact, drugs suitable for use in MetS need to reduce BP as
hypertension takes part of MetS. That is the reason why we
screened our compounds for their BP effects first. In addition,
according to our previous studies,13 we can assume that drugs
highly selective for I1Rs, which reduce BP through a centrally
mediated sympathetic inhibition, will most likely also improve
the metabolic abnormalities of MetS.31 We have especially
focused our work on compounds that are substituted on the
position-2 of the phenyl ring, since previous studies have shown
that this kind of substitution was required for their biological
activity.32−35 The choice of the substituents on the aromatic
ring is based on a classical rational strategy: starting from a
substitution on the ortho position, we have made variations on
the other positions to state about the influence of the region
that is substituted on the biological activity. Cycles were
introduced to determine the limits of the steric hindrance on
the considered positions. We have also carried out some
variations on the pyrrolidinyl ring to determine the influence of
such substitutions on both the selectivity and the hypotensive
activity of 2-aryliminopyrrolidines derivatives.
PHARMACOLOGY
■
Hemodynamic Effects. The 18 compounds 1−18 were
evaluated in vivo for their effects on mean arterial blood
pressure (MBP) and heart rate (HR). On the basis of our
experience in this field, results were defined as functionally
significant for variations greater than 15% of the basal values.
Cardiovascular parameters were recorded after intravenous
injections (iv, 3 and 10 mg/kg when the 3 mg/kg dose was
inactive) and ic injections (0.3 mg/kg) in normotensive
anaesthetized Wistar rats (Table 1). The ic route of
administration allowed us to confirm the central origin of the
observed effects. These two routes of administration are
commonly used by all the groups studying centrally acting
drugs in different mammal species.27,41−43 The first series of
compounds tested included those bearing a methyl group on
position-5 of the pyrrolidinyl ring (1−11). Compounds whose
phenyl ring is disubstituted on positions-2 and -6 (1), on
positions-2 and -5 (2, 3), and on positions-2 and -4 (4) never
decreased BP after the iv injection more than 10% of the initial
CHEMISTRY
■
Compounds 1−18 were synthesized by coupling the
appropriate lactam with a chosen aniline derivative and using
a dehydrating agent, i.e., phosphorus oxychloride, in 1,2-
dichloroethane at 60 °C under inert atmosphere (Scheme 1).32
To determine the influence of the substitutions on the
pyrrolidinyl ring, we chose two starting lactams differently
methylated. The lactam 19a was commercially available. The
lactam 19b was prepared according to standard procedures.
First, nitromethane is reacted with methyl crotonate in the
B
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a
Scheme 3. Synthesis of Indanamine Derivatives 21−29
a
Reaction conditions: (a) H2, 10% Pd/C, MeOH, rt, 12 h, separation of isomers; (b) Ac2O, 0 °C to rt; (c) X2, AcOH, 0 °C to rt, 1 h; (d)
trimethylboroxine, 10%mol Pd(PPh3)4, 2 N Na2CO3, dioxan, reflux, 5 h; (e) 4 N HCl, reflux, 2 h.
Table 1. Pharmacological Data of Compounds 1−18
a
cardiovascular effects
binding affinities Ki
intravenous injection
intracisternal injection (0.3 mg/kg)
(nM)
compd
R1
R2
R3
2,6-Me2
max ΔMBP (% var) max ΔHR (% var) max ΔMBP (% var) max ΔHR (% var)
I1R
α2AR
1
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
−5*
−9*
−4*
−6*
na
na
na
na
nd
nd
nd
nd
nd
nd
2
H
2-F, 5-Me
3
H
2-Cl, 5-Me
2-Me, 4-F
−7*
−7*
na
na
nd
4
H
−6*
−8*
na
na
nd
5
H
2,3-Me2
−25*
−28*
−37*
−37*
−8*
−18*
−20*
−20*
−26*
−4*
−20
−25
−32
−29
na
−25
−30
−29
−10
na
70
5
6
8
6
>104
>104
>104
>104
nd
6
H
2-Me, 3-Cl
2,3-(CH2)3
2,3-(CH2)4
3,4-(CH2)3
2-Cl, 4,5-(CH2)3
2,3-(CH2)3, 4-Cl
2,3-Me2
27
19
73
7
H
8
H
9
H
nd
nd
2.8 0.3
42
3.2 0.5
19
10
11
12
13
14
15
16
17
18
H
−9*
−3*
na
na
nd
H
−6*
−7*
na
na
>104
= 104
>104
>104
>104
>104
nd
Me
Me
Me
Me
Me
Me
Me
−26#
−30#
−35#
−10*
−26*
−8*
−20#
−18#
−22#
−6*
−25
−30
−30
na
−31
−33
−30
na
5
H
2-Me, 3-Cl
2,3-(CH2)3
2,3-(CH2)3, 4-Cl
2,3-(CH2)3, 4-Me
3,4-(CH2)3
2,3-(CH2)4
H
6
H
0.22 0.02
2.8 0.2
nd
H
−22*
−4*
−32
na
−15
na
H
H
−5*
−3*
na
na
nd
nd
a
Effects on mean arterial blood pressure (MBP) and heart rate (HR) in normotensive anesthetized rats. Results presented are means of at least three
independent experiments: for iv injections, # = 3 mg/kg and * = 10 mg/kg. Results were defined as functionally significant for variations greater than
15% of the basal value. Affinities for I1Rs were measured with [125I] PIC on PC12 cells; affinities for α2ARs were measured with [3H] RX 821002 on
CHO transfected cells. Data are presented as mean SEM. na, non active; nd, not determined.
MBP value, even at the high dose of 10 mg/kg. The same
compounds, administered via the ic route did not significantly
modify MBP. On the opposite, compound 5, which is 2,3-
dimethylated, lowered MBP by 25% and HR by 18% at 10 mg/
kg iv. Compound 6, which has a methyl on position-2 and a
chlorine on position-3, also decreased MBP but its hypotensive
effect lasted much longer than that of 5. For both compounds,
hypotensive effect after ic administration and effects on HR
were similar. Compound 7, which is constituted of an indan-4-
yl moiety, was more potent than 5 and 6 because MBP and HR
were decreased by 37% and 20%, respectively, after iv at the
same dose. After ic injection, it produced marked effects on
these parameters, which decreased by 32% and 29%,
respectively. Changing from an indan-4-yl to a tetrahydro-
naphth-5-yl scaffold, compound 8, did not change the efficiency
on both MBP and HR, which was quite as important as for 7,
either after iv or ic administrations. On the opposite, compound
9, which is an isomer of position of compound 7 having an
indan-5-yl moiety, was unable to affect MBP nor HR, after iv
and ic administrations. Compound 10, with a chlorine atom on
position-7 of the indan-5-yl moiety reduced neither arterial
pressure nor HR after both administration ways. Compound
11, which is a derivative of 7 with a chlorine atom in position-4
of the indan-4-yl moiety, was inactive after both iv and ic
administrations.
The compounds of the second series bear a methyl group on
position-4 of the pyrrolidinyl ring (Table 1, entry 12−18).
Compounds 12 and 13, which are position isomers of
compounds 5 and 6, respectively, displayed interesting activities
because MBP was decreased by 26% and 30%, respectively, at
the dose of 3 mg/kg iv, whereas HR was decreased by 20%.
This was also the case for compound 14, which lowered MBP
by 35% at the same dose. Hypotensive and bradycardic
activities of these three compounds were marked after ic
injections, ranging from −25 to −30%. Compound 15, which
bears a chlorine atom on position-4 of the indan-4-yl moiety,
did not significantly modify MBP and HR, after iv and ic
administrations. Compound 16, which is substituted by a
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methyl group instead of a chlorine atom on position-4 of the
indan-4-yl moiety, had a marked activity on both MBP and HR
at the dose of 10 mg/kg iv and was also active after ic injection.
Compound 17, a position isomer of compound 14 having an
indan-5-yl moiety was inactive, after both administration ways.
The tetrahydronapht-5-yl analogue 18 was also unable to
decrease both MBP and HR.
Binding Assays. Compounds that were active on MBP and
HR were evaluated for their binding properties on I1Rs and
α2ARs (Table 1). The first important statement is that none of
the compounds evaluated exhibited significant affinity on α2ARs
(Ki > 10−5 M).
Compounds 5 and 8 had good affinity for I1Rs, with Ki values
of 70 and 73 nM, respectively. Compounds 6, 7, 12, and 14
showed even better affinities, with Ki values ranging from 19 to
42 nM. Finally, compounds 13 and 16 had the highest affinities
among the active compounds, with Ki values of 3.2 and 2.8 nM,
respectively. We also evaluated two inactive compounds, 11
and 15, with Ki values of 2.8 and 0.22 nM, respectively.
Discussion. One of the goals of the present work was to
obtain compounds selective for I1Rs over α2ARs with
hypotensive activity. None of the tested compounds caused
initial vasoconstriction after systemic injection, opposite to
what happens with hybrid drugs, such as clonidine.44 This fact
indicates that our pyrrolidine ligands are devoid of adrenergic
properties because the vasoconstrictive effect of clonidine-like
compounds is known to be caused by the stimulation of
vascular αARs.45 This was confirmed by the binding assays
performed on hypotensive compounds, because no significant
affinities for α2AARs were measured (Ki values ≥104 nM)
(Table 1). Our experiments also showed that all hypotensive
compounds bound specifically to I1Rs (Table 1); they can
therefore be considered as compounds selective for I1Rs over
α2ARs.
targeting both I1Rs and α2ARs23,27 or even drugs selective for
α2ARs such as dexmedetomidine and marsanidine,20,51,52 have
greater potencies than I1 selective drugs as they act at lower
doses. α2ARs and I1Rs are differently involved in BP blood
pressure regulation. We showed previously that both types of
receptors can synergistically interact to cause hypotensive
effects at very low doses.27 Therefore, I1Rs selective drugs
which cannot trigger such a synergism act at higher doses.
Nevertheless, I1Rs selectivity has important advantages, such
as the lack of the side effects clearly associated with α2ARs, i.e.,
sedation and mucosal dryness in particular. In addition, the
more selective the drugs are for I1Rs, the more they have
beneficial metabolic effects (cholesterol reduction and improve-
ment of glucose tolerance). Thus, as far as metabolic effects and
MetS management are concerned, activation of α2ARs goes
rather in the unfavorable direction.53,54
Concerning the structure−activity relationships, the binding
selectivity seems to be due to the presence of the methyl group
on the pyrrolidinyl ring, wherever it is located in position-4 or
-5; position-3 was not considered since we demonstrated in a
previous work that it was not favorable for the biological
activity.24 This steric hindrance may disfavor the linking
interactions between ligands and α2ARs because nonmethylated
2-aryliminopyrrolidines still bind to α2ARs, but to a lesser
extent than imidazoline compounds.55 The same observation
was supported by a study dealing with derivatives of
brimonidine, an amino-imidazoline compound: the one which
was methylated on the imidazolinyl ring had lost its adrenergic
properties.56 This assumption is also supported by our recent
work on IRC demonstrating that methylated rilmenidine and
methylated clonidine (31) are devoid of adrenergic properties,
the later still being able to induce hypotensive response.28
However, it is noticeable that, in the case of aliphatic
aminopyrrolines, to which belongs 30, the methylation on the
pyrrolinyl ring is not necessary for I1/α2 selectivity.
It has repeatedly been reported that many ligands selective
for I1Rs exhibited a biphasic binding to the receptors,
suggesting that I1Rs is a G protein-coupled receptor as assessed
by the effect of nonhydrolyzable GTP on the binding
curves.29,46,47 The high affinity sites are the ones involved in
the biological activities of the I1Rs.24,28,48,49 These are the
reasons why only the high affinity sites are taken into account
here. However, no direct correlation between the hypotensive
effect of the 2-aryliminopyrrolidines and their affinities for I1Rs
has been sought because the hypotensive potential of I1
selective drugs depends not only on their affinity for I1Rs but
also on a combination of many different parameters such as the
capability of crossing the BBB after systemic delivery, their
potential metabolism even after central administration, and
their lipophilicity. Besides several arguments suggest that the
signaling pathways which have been associated with I1Rs so far,
such as cAMP48 and DAG,50 are probably not the only ones to
be involved. Thus, so far, our knowledge in this matter is not
sufficient to correlate the hypotensive effects of I1 selective
drugs to their effects on second messengers. In all these
domains, our series of 2-aryliminopyrrolidines is not distin-
guishable from amino-imidazolines or amino-oxazolines.
Some active 2-aryliminopyrrolidines described in the present
study can decrease BP by about 30%; as the tested animals were
initially normotensive, a 30% reduction of BP is important,
close to the maximal possible effect (maximal efficacy).
Nonetheless, the doses which produce these effects are quite
large compared to the ones of amino-imidazolines, such as
clonidine, which cause similar effects. Thus, hybrid drugs
Furthermore, the substitution on the pyrrolidinyl ring
appears to influence the affinity of the ligands. While
compounds 7 and 14 had the same Ki values for I1Rs, the Ki
value of 12 is lower than that of 5. Also, the Ki values of 13 and
15 are lower than those of 6 and 11, respectively. This suggests
that methylation on position-4 of the pyrrolidinyl ring is more
appropriate than in position-5 to improve the affinity of ligands
toward I1Rs.
How the aromatic ring is substituted leads to some
interesting observations. As shown previously, a sole substituent
on position-2 did not lead to hypotensive compounds, whatever
its nature, alkyl group, or halogen atom,28,57 opposite to what
was observed with imidazoline compounds and nonselective
pyrrolidines.32,33,35 In the first series, in which compounds 1−
11 bear a methyl group on position-5 of the pyrrolidinyl ring,
addition of a second substituent on the phenyl ring had
different influences on the hypotensive activity, according to its
position. When it was placed in position-6, in position-5, or in
position-4 of the phenyl ring (compounds 1−4), compounds
were unable to decrease MBP, even after ic injection. But when
the additional substituent was placed on position-3 (5), the
compound was very active. We synthesized other compounds
with this kind of substitutions, and all of them were able to
decrease MBP, whatever the route of administration (6−8). In
the second series of compounds, the methyl group is on
position-4 of the pyrrolidinyl ring (12−18). Compounds 12−
14, substituted on positions-2 and -3 of the phenyl, were very
efficient hypotensive agents. Other combinations of substitu-
D
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tions in both series, either on positions-3 and -4 (9, 17), or
positions-2, -4, and -5 (10) of the phenyl, did not lead to active
compounds. On the basis of these observations, we can
conclude that the cardiovascular effects of such selective 2-
aryliminopyrrolidines compounds were strongly dependent on
the nature of the aniline moiety. The presence of substituents
on both positions-2 and -3 of the aromatic ring was required for
the hypotensive activity. These 2-aryliminopyrrolidines are the
first to be selective for I1Rs and to be hypotensive after systemic
administration.
The influence of the nature of the substituent on position-3
on the pharmacological activity was not clear because changing
from a methyl group to a chlorine atom did not modify the
hypotensive activity (see 5 vs 6 and 12 vs 13). Nevertheless, it
seems to influence, in some extent, the binding properties.
Affinity of compound 6 was higher than that of 5 (3-fold). This
influence is noteworthy in the second series, as the chlorinated
compound 13 had a rather higher affinity for I1Rs than its alkyl
analogues 12 and 14, with a significant gain on the Ki value (13-
and 6-fold, respectively).
Addition of a third substituent on position-4 of a phenyl ring
already substituted on positions-2 and -3 clearly increased the
affinity of the ligands for I1Rs. This is the case for 11 vs 7 (7-
fold in Ki gain) and for compounds 15 and 16 vs 14 (90- and 7-
fold in Ki gain, respectively). Such an observation is in
agreement with previous observations when we compare the
affinities of clonidine and its para-substituted analogues58,59 and
also when 32 was compared with its non-iodinated
analogues.28,29 This increase in affinity seems also to be
dependent on the nature of this substituent. The chlorine atom
was more efficient than the methyl group to improve the
binding properties (see 15 vs 16 with 12-fold in Ki gain). The
2-aryliminopyrrolidines we have synthesized display higher
affinities for I1Rs than 30, whose Ki = 537 nM. Some of them
have affinities as high as our reference I1Rs selective ligand 32
(Ki = 0.22 nM) or clonidine and its methylated analogue 31 (Ki
= 14.1 and 0.93 nM, respectively) and even higher than that of
rilmenidine and its methylated analogue (Ki = 83.1 and 363
nM, respectively).28 But although such additional substitutions
increased the affinity of the ligands for I1Rs, they did not
necessarily increase their hypotensive activity (see 11 vs 7 and
15 vs 14). Indeed, compound 16 is the sole of the series
substituted in position-4 of the phenyl exhibiting a marked
hypotensive activity. This difference of behavior between 16
and the chlorinated analogues 11 and 15 might not be due to
differences of steric hindrance, because the van der Waals
volumes of chlorine atom and methyl group are very close, with
values of 22.4 and 21.6 Å3, respectively.60,61 The cause may be
the differences of their intrinsic electronic natures. The
chlorinated compounds may interact via halogen bonding62,63
with residues of the active site of the receptor protein in a
manner that favor the binding interactions but that does not
allow the shaping of the active conformation of the protein. As
a consequence, there will be no functional response. Another
possible reason for the discrepancy of activity could be the lack
of stability of this chlorinated molecule in vivo. The para
position of the chlorine atom toward the amidine function may
favor the rapid metabolism of the molecule.64 This kind of
electronic activation might not occur with the methyl analogue.
Of course, this assumption deserves experimental confirmation.
Investigations are in progress to define the nature of the
interactions involved in the binding of the ligands, their
metabolic pathway, and even to look at the possible antagonist
activity of such nonhypotensive ligands.
One can notice that all the compounds that decreased BP
after systemic administration also reduced BP after ic injection.
It strongly suggests that the hypotensive activity of such
compounds is due to their central sympatho-inhibitory action.
This assumption is strengthened by the fact that all hypotensive
compounds were able to decrease HR, which is centrally
triggered. As a matter of fact, a drug which simultaneously
reduces BP and HR after both systemic and central
administration necessarily inhibits the sympathetic nervous
system activity. This claim was recently confirmed when we
showed that compound 13 decreased the sympathetic activity
directly measured on the renal nerve.13
Compounds 1−4, 9−11, 15, 17, and 18, which were inactive
after iv injection, were also inactive after direct central
administration. Indeed, their lack of activity on BP was not
due to their inability to cross the blood−brain barrier (BBB).
We calculated pKa and Log P of each molecule of the set (see
Supporting Information).65 These parameters (pKa ranging
7.9−9.3 units, Log P ranging 2.3−3.5 units) are in accordance
with those required for a central action,66 which are to be
compared with those of rilmenidine (pKa = 9.22; Log P = 1.63)
and clonidine (pKa = 8.11; Log P = 1.59).67 These assumptions
were completed by in silico estimations of the BBB crossing
capacity of compounds 1−18 and reference compounds
clonidine and rilmenidine (see Supporting Information). BBB
transport parameters were calculated with ACD-iLab software
and a correlation was established between LogBB, which is the
logarithm of the distribution ratio of a compound between
brain tissue and plasma, and Log(PS·fu, brain), which is the
logarithm of the brain/plasma equilibration rate, in order to
predict if a compound is able to reach the CNS.68 As a result, all
compounds were estimated able to cross the BBB.
Our new active 2-aryliminopyrrolidines selective for I1Rs are
more promising hypotensive agents than any other pyrrolidine
derivatives tested so far because they are active after systemic
administration. This statement is even strengthened by our
recent work dealing with the treatment of experimental MetS
by a sympatho-inhibitory drug. Our leader compound (13) was
fully tested in a rodent model of MetS. Compound 13, which is
orally bioavailable, not only reduces BP but also weight gain
and total cholesterol; moreover, it improves glucose tolerance
and insulin resistance, all relevant biological parameters related
to MetS.13,69 Other compounds belonging to our series of 2-
aryliminopyrrolidines, highly selective for I1Rs and shown to
reduce BP, are currently tested for their metabolic effects.
CONCLUSION
■
As a logical continuation of our previous structure−activity
relationship studies on 2-aryliminopyrrolidines, additional
compounds (1−18) were prepared and tested for their binding
properties on I1Rs and their cardiovascular in vivo effects after
both systemic and direct intracerebral administrations. Some
compounds have high affinity for I1Rs and are selective for I1Rs
vs α2ARs. They were totally devoid of any α2-adrenergic
property. The selectivity of such ligands for I1Rs over α2ARs
was the consequence of the presence of the methyl group on
position-4 or -5 of the pyrrolidinyl ring. The position-4 isomere
seems more relevant, because the affinity to I1Rs of 4-
substituted compounds is higher. These 2-aryliminopyrroli-
dines must be substituted on positions-2 and -3 of the aromatic
ring to cause hypotension and bradycardia, whatever the way of
E
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Journal of Medicinal Chemistry
Article
homogenizer. After centrifugation at 75000g for 20 min, the pellet was
washed twice in cold Tris-HEPES buffer and centrifuged. Pellets were
resuspended in the same buffer at a concentration of 1−2 mg of
protein/mL. Membrane preparations were stored at −80 °C until use.
Binding Experiments. Competition binding assays were performed
using 0.5 nM [125I]PIC (2200 Ci/mmol (PerkinElmer, USA)) and
seven different concentrations of the unlabeled ligand under
investigation, ranging from 10−10 to 10−4 M. Nonspecific binding
was defined as [125I]PIC binding in the presence of 10 μM PIC. Each
point is the mean of 3−5 experiments performed in triplicate using
different membrane preparations.
Incubation was initiated by the addition of PC12 cell membranes
(10−30 μg of protein) in a final volume of 250 μL of Tris−HEPES
buffer (5 mM Tris−HEPES, pH 7.7, 0.5 mM EDTA, 0.5 mM EGTA,
and 0.5 mM MgCl2) and was carried out at 25 °C for 45 min. The
reaction was stopped by rapid vacuum filtration through GF/B glass
fiber filters treated with 0.3% PEI with a Brandel harvester, followed by
three rapid washes of the filters with 3 mL of ice-cold 50 mM Tris−
HCl buffer, pH 7.4. Radioactivity retained on the dried filters was
determined in a Minaxi gamma counter (Packard, Meriden, CT, USA).
α2-Adrenoreceptors Binding Assays. Membranes were pre-
pared as described by Newman-Tancredi et al.70 These membranes
(30 μg protein/mL for CHO-hα2A) were incubated for 60 min at
room temperature in binding buffer (33 mM Tris HCl, 1 mM EDTA,
pH 7.5) in a final volume of 500 μL containing 1 nM [3H]RX821002.
The incubation was stopped by rapid filtration under vacuum through
GF/B glass fiber filters followed by three successive washes with ice-
cold binding buffer. Nonspecific binding was defined with 100 μM
(−)epinephrine.
administration. The affinity is also strongly governed by the
presence of a substituent on position-4 of the aromatic ring, a
halogen atom being very efficient in improving the affinity for
I1Rs; the lack of hypotensive effect of some of such 4-
substituted compounds might be due to unexpected additional
pharmacological properties. It is interesting to note that the
methylated derivative in position-4 causes hypotension.
The compounds tested in this study which decreased BP
after iv administration as well as after ic injection, i.e., those
which were able to cross the BBB, had variables potencies but
had efficacies similar to those of reference drugs, such as
clonidine, moxonidine, and rilmenidine.27,41−43
Although affinity for I1Rs is a necessary but not a sufficient
condition to cause hypotension, the present study shows
further structure−activity relationship information on 2-
aryliminopyrrolidines and provides the first examples of
pyrrolidine ligands highly selective and with high affinity for
I1Rs, which are hypotensive after systemic injection. Their lack
of adrenergic properties is expected to avoid the adverse effects
usually caused by centrally active antihypertensive drugs of the
first generation, such as sedation. Selective ligands such as 13
are valuable tools to further study the pharmacology of I1Rs and
to further explore the field of central regulation of the
cardiovascular and metabolic functions. The results of this
study, strengthened by those we recently obtained in the
treatment of the MetS by 13 (LNP599),13,69 support the
therapeutic potential of such 2-aryliminopyrrolidines.
Data Analysis. Competition experiments were analyzed using the
iterative nonlinear least-squares curve-fitting program GraphPad. Ki
were determined using the method of Cheng and Prussof.71 Data are
presented as mean SEM.
EXPERIMENTAL SECTION
■
Pharmacology. In Vivo Studies. All animal care and experimental
procedures complied with the rules of the European communities
Council Directive of 24 November 1986 (86/609/EEC) and the
French Department of Agriculture (license no. 67-337 to P.
Bousquet).
Chemistry. All solvents were purified according to standard
procedures before use. Thin-layer chromatography was performed on
precoated plate of silica gel 60F254 (Merk). Flash chromatography
was conducted with silica gel Supporting Information 60 (40−63 μm)
as a stationary phase. Melting points were determined in open
capillaries on a Gallenkamp apparatus and are uncorrected. NMR
spectra were recorded in CDCl3 or D2O on a Bruker AV300
spectrometer. Chemical shifts (δ) are expressed in parts per million
(ppm), and coupling constants (J) are expressed in hertz (Hz). The
abbreviations used for the multiplicities are as follow: m, unspecified
multiplet, s, singlet, d, doublet, t, triplet, q, quartet. All target
compounds were tested as their hydrochloride salts. Elemental
analyses were performed at the Service de Microanalyses, University
of Strasbourg, Strasbourg, France. Analytical results obtained for C, H,
and N were within 0.4% of the calculated theoretical values (see
Supporting Information). Lactam 19a and anilines used to prepare
compounds 1−6, 8, 12, 13, and 18 were obtained from commercial
sources. Procedure and data concerning compounds 19b and 21−29
are described in Supporting Information. Calculations of the physical
properties of compounds and estimations of their BBB crossing ability
are reported in Supporting Information. The 2-aryliminopyrrolidines
1−18 described here were patented recently by the University of
Strasbourg.69
General Procedure for the Preparation of 2-Aryliminopyr-
rolidines 1−18.32 A solution of the appropriate lactam 19a/b (5
mmol) and the chosen aniline derivative (1 equiv) was prepared in
dichloroethane (10 mL), under an argon atmosphere. POCl3 (1 equiv)
was added dropwise to the vigorously stirred mixture, which was then
heated at 60 °C for 6 h. The cooled mixture was hydrolyzed with 5 mL
of a saturated aqueous solution of Na2CO3. The aqueous layer was
extracted twice with dichloromethane. The combined organic layer
was dried over anhydrous Na2SO4, and the solvent was distilled under
reduced pressure. The product was purified by flash chromatography
(3% TEA in AcOEt) to give the 2-aryliminopyrrolidines 1−18. The
hydrochloride salts were prepared by the addition of an anhydrous
ethanolic HCl solution of known concentration to an anhydrous
Arterial Blood Pressure and Heart Rate Measurements in
Anaesthetized Wistar Rats. Adult male Wistar rats were
anesthetized (sodium pentobarbital 50 mg/kg, ip), and physiological
temperature was maintained. The animals were then tracheotomized,
and the left carotid vein was catheterized to allow iv injections. The
animals were immobilized with pancuronium bromide (Pavulon,
Organon) (1 mg/kg, (iv) and artificially ventilated with room air
(Hugo Sachs electronic model 7025). A catheter was introduced into
the left femoral artery and connected to a pressure processor and
recorder (Gould Electronics model BS-272). Heart rate (HR), systolic
(SBP), diastolic (DBP), and mean (MBP) arterial pressures (MBP =
DBP + 1/3(SBP − DBP)) were continuously monitored from the
pressure signal (Gould Biotach amplifier model 13-4615-66). BP and
HR were measured over a 90 min experimental period. Results were
expressed as the maximal variation of mean arterial pressure (mmHg)
and heart rate (bpm) compared to the basal values before treatment.
The corresponding percentages of variation were also determined.
Results were defined as significant for variation greater than 15% of the
basal value. In experiments during which the drug was injected
intracisternally, a volume of 0.2 mL of drug solution was injected after
removal of the same volume of cerebrospinal fluid.
Radioligand Binding Assays. I1−Receptors Binding Assays.
Cell Culture. PC-12 cells obtained from Dr. G. Rebel (IRCAD,
Strasbourg, France) were cultured in 75 cm2 flasks in DMEM (1 g/L
glucose) supplemented with 10% heat-inactivated fetal bovine serum,
100 U/mL penicillin, and 100 μg/mL streptomycin. When the cells
reached confluence (3−4 days after plating), they were harvested by a
2 min exposure to 0.25% trypsin at 37 °C. For binding assays, after
removing the medium, cells at confluence were frozen in the flasks at
−20 °C until used to prepare membranes.
Cell Extracts. Frozen PC-12 cells were scraped into cold Tris-
HEPES buffer (5 mM Tris-HEPES, pH 7.7, 0.5 mM EDTA, 0.5 mM
EGTA, and 0.5 mM MgCl2) and homogenized and lyzed with a Potter
F
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Journal of Medicinal Chemistry
Article
ethanolic solution of the base (1 equiv). The salts were recrystallized
in iPrOH−Et2O. All of them decompose over 200 °C.
× CH2, J = 7.5), 2.32 (m, 1H, 1/2 CH2), 1.97 (qn, 2H, CH2, J = 7.5),
1.75 (m, 1H, 1/2 CH2), 1.16 (d, 3H, CH3, J = 6.4). 13C NMR (D2O) δ
167.7, 145.7, 145.1, 132.7, 125.5, 121.9, 120.1, 56.9, 32.3, 32.0, 30.4,
28.0, 25.3, 19.8. Anal. (C14H18N2·HCl) C, H, N.
N-(2,6-Dimethylphenyl)-5-methylpyrrolidin-2-imine Hydrochlor-
1
ide (1). Yield 32%, white solid. H NMR (D2O) δ 7.24 (d, 1H, Har,
J = 7.6), 7.17 (t, 1H, Har, J = 7.7), 7.06 (d, 1H, Har, J = 7.6), 4.02 (h,
1H, CH, J = 6.4), 3.05 (m, 2H, CH2), 2.34 (m, 1H, 1/2 CH2), 2.24 (s,
3H, CH3), 2.08 (s, 3H, CH3), 1.81 (m, 1H, 1/2 CH2), 1.13 (d, 3H,
CH3, J = 6.4). 13C NMR (D2O) δ 168.2, 139.2, 133.5, 133.1, 131.2,
126.6, 124.0, 56.2, 43.8, 37.0, 28.6, 24.9, 17.2. Anal. (C13H18N2·HCl)
C, H, N.
N-(6-Chloro-2,3-dihydro-1H-inden-5-yl)-5-methylpyrrolidin-2-
1
imine Hydrochloride (10). Yield 50%, white solid. H NMR (D2O) δ
7.18 (s, 1H, Har), 6.91 (s, 1H, Har), 4.07 (h, 1H, CH, J = 6.6), 2.93
(m, 2H, CH2), 2.81 (t, 4H, 2 × CH2, J = 7.5), 2.32 (m, 1H, 1/2 CH2),
1.97 (qn, 2H, CH2, J = 7.5), 1.75 (m, 1H, 1/2 CH2), 1.63 (d, 3H, CH3,
J = 6.4). 13C NMR (D2O) δ 168.5, 145.3, 145.9, 133.4, 127.5, 122.7,
121.6, 57.2, 32.2, 31.8, 30.6, 27.9, 25.5, 21.1. Anal. (C14H17ClN2·HCl)
C, H, N.
N-(2-Fluoro-5-methylphenyl)-5-methylpyrrolidin-2-imine Hydro-
1
chloride (2). Yield 4, white solid. H NMR (D2O) δ 7.20−7.09 (m,
3H, Har), 4.11 (hex, 1H, CH, J = 6.8), 3.05 (m, 2H, CH2), 2.39 (m,
1H, 1/2 CH2), 2.25 (s, 3H, CH3), 1.80 (m, 1H, 1/2 CH2), 1.08 (d,
3H, CH3, J = 6.5). 13C NMR (D2O) δ 168.4, 154.3 (d, J1C−F = 244.5),
N-(7-Chloro-2,3-dihydro-1H-inden-4-yl)-5-methylpyrrolidin-2-
1
imine Hydrochloride (11). Yield 69%, white solid. H NMR (D2O) δ
7.22 (d, 1H, Har, J = 8.3), 7.01 (d, 1H, Har, J = 8.4), 4.05 (h, 1H, CH,
J = 6.4), 3.02 (m, 2H, CH2), 2.93 (t, 2H, CH2, J = 7.6), 2.81 (t, 2H,
CH2, J = 7.5), 2.36 (m, 1H, 1/2 CH2), 2.03 (qn, 2H, CH2, J = 7.6),
1.79 (m, 1H, 1/2 CH2), 1.17 (d, 3H, CH3, J = 6.4). 13C NMR (D2O) δ
168.4, 145.1, 142.5, 130.5, 129.2, 127.8, 124.4, 56.8, 32.6, 30.1, 29.9,
28.2, 24.8, 19.9. Anal. (C14H18ClN2·HCl) C, H, N.
135.7, 130.9, 126.9, 121.6 (d, J2
= 13.2), 116.4 (d, J2C−F= 19.5),
57.2, 30.5, 28.1, 21.3, 19.7. 19CF−F NMR (D2O) δ −128.3. Anal.
(C12H15FN2·HCl) C, H, N.
N-(2-Chloro-5-methylphenyl)-5-methylpyrrolidin-2-imine Hydro-
chloride (3). Yield 47%, white solid. 1H NMR (D2O) δ 7.38 (dd, 1H,
Har, J = 3.1, J = 7.8), 7.18 (d, 1H, Har, J = 2.9), 7.16 (dd, 1H, Har, J =
3.0, J = 7.7), 4.05 (m, 1H, CH), 2.99 (m, 2H, CH2), 2.37 (m, 1H, 1/2
CH2), 2.24 (s, 3H, CH3), 1.77 (m, 1H, 1/2 CH2), 1.16 (d, 3H, CH3, J
= 6.3). 13C NMR (D2O) δ 168.5, 139.4, 131.2, 130.2, 128.0, 126.8,
124.4, 57.1, 30.2, 28.1, 19.8, 19.7. Anal. (C12H15ClN2.·HCl) C, H, N
N-(4-Fluoro-2-methylphenyl)-5-methylpyrrolidin-2-imine Hydro-
chloride (4). Yield 35%, white solid. 1H NMR (D2O) δ 7.22 (dd, 1H,
Har, J = 5.4, J = 8.7), 7.09 (dd, 1H, Har, J = 2.9, J = 8.7), 7.0 (dt, 1H,
Har, J = 2.9, J = 8.8), 4.14 (hex, 1H, CH, J = 6.7), 3.07 (m, 2H, CH2),
2.41 (m, 1H, 1/2 CH2), 2.17 (s, 3H, CH3), 1.77 (m, 1H, 1/2 CH2),
N-(2,3-Dimethylphenyl)-4-methylpyrrolidin-2-imine Hydrochlor-
1
ide (12). Yield 40%, white solid. H NMR (D2O) δ 7.21 (d, 1H,
Har, J = 7.7), 7.16 (t, 1H, Har, J = 7.7), 7.03 (d, 1H, Har, J = 7.6), 3.68
(m, 1H, 1/2 CH2), 3.20 (m, 2H, 1/2 CH2 + CH), 2.72 (m, 2H, CH2),
2.30 (s, 3H, CH3), 2.11 (s, 3H, CH3), 1.12 (s, 6H, 2 × CH3). 13C
NMR (D2O) δ 168.7, 139.5, 133.4, 132.8, 130.5, 126.8, 123.7, 54.5,
43.7, 37.0, 25.6, 19.4, 12.3. Anal. (C13H18N2.·HCl) C, H, N.
N-(3-Chloro-2-methylphenyl)-4-methylpyrrolidin-2-imine Hydro-
chloride (13). Yield 51%, white solid. 1H NMR (D2O) δ 7.45 (d, 1H,
Har, J = 7.9), 7.25 (d, 1H, Har, J = 7.9), 7.20 (t, 1H, Har, J = 7.8), 3.70
(m, 1H, 1/2 CH2), 3.19 (m, 2H, 1/2 CH2 + CH), 2.74 (m, 2H, CH2),
2.22 (s, 3H, CH3), 1.10 (d, 3H, CH3, J = 6.5). 13C NMR (D2O) δ
169.1, 135.5, 133.4, 132.3, 130.1, 128.1, 125.3, 54.2, 37.9, 29.5, 18.1,
14.2. Anal. (C12H15ClN2·HCl) C, H, N.
1.11 (d, 3H, CH3, J = 6.4). 13C NMR (D2O) δ 169.8, 161.9 (d, J1
=
=
248.8), 137.8 (d, J3C−F = 9.2), 129.3 (d, J4C−F = 2.8), 128.3 (d, J3C−F
C−F
9.6), 117.5 (d, J2
= 22.7), 114.2 (d, J2
= 23.0), 55.6, 31.5, 28.8,
C−F
C−F
19.7, 19.5. 19F NMR (D2O) δ −113.4. Anal. (C12H15FN2·HCl) C, H,
N-(2,3-Dihydro-1H-inden-4-yl)-4-methylpyrrolidin-2-imine Hy-
N.
1
drochloride (14). Yield 33%, white solid. H NMR (D2O) δ 7.28
N-(2,3-Dimethylphenyl)-5-methylpyrrolidin-2-imine Hydrochlor-
1
(d, 1H, Har, J = 7.5), 7.21 (t, 1H, Har, J = 7.6), 7.03 (d, 1H, Har, J =
7.5), 3.70 (dd, 1H, 1/2 CH2, J = 10.8, J = 7.8), 3.19 (dd, 1H, 1/2 CH2,
J = 11, J = 6), 3.14 (m, 1H, 1/2 CH2), 2.89 (t, 2H, CH2, J = 7.4), 2.74
(t, 2H, CH2, J = 7.4), 2.65 (m, 2H, CH + 1/2 CH2), 2.01 (qn, 2H,
CH2, J = 7.5), 1.10 (d, 3H, CH3, J = 7). 13C NMR (D2O) δ 168.3,
147.6, 140.2, 130.5, 127.9, 124.9, 122.5, 54.1, 37.8, 32.6, 29.9, 29.5,
24.7, 17.6. Anal. (C14H18N2.·HCl) C, H, N.
ide (5). Yield 43%, white solid. H NMR (D2O) δ 7.24 (d, 1H, Har,
J = 7.6), 7.17 (t, 1H, Har, J = 7.7), 7.06 (d, 1H, Har, J = 7.6), 4.02 (h,
1H, CH, J = 6.4), 3.05 (m, 2H, CH2), 2.34 (m, 1H, 1/2 CH2), 2.24 (s,
3H, CH3), 2.08 (s, 3H, CH3), 1.81 (m, 1H, 1/2 CH2), 1.13 (d, 3H,
CH3, J = 6.4). 13C NMR (D2O) δ 168.2, 139.2, 133.5, 133.1, 131.2,
126.6, 124.0, 56.2, 43.8, 37.0, 28.6, 24.9, 17.2. Anal. (C13H18N2·HCl)
C, H, N.
N-(3-Chloro-2-methylphenyl)-5-methylpyrrolidin-2-imine Hydro-
chloride (6). Yield 44%, white solid. 1H NMR (CDCl3) δ 7.41 (d, 1H,
Har, J = 7.8), 7.23 (d, 1H, Har, J = 7.9), 7.18 (t, 1H, Har, J = 7.8), 4.02
(h, 1H, CH, J = 6.4), 3.08 (m, 2H, CH2), 2.34 (m, 1H, 1/2 CH2), 2.25
(s, 3H, CH3), 2.11 (s, 3H, CH3), 1.83 (m, 1H, 1/2 CH2), 1.13 (d, 3H,
CH3, J = 6.4). 13C NMR (D2O) δ 168.7, 139.5, 133.4, 132.8, 130.5,
126.8, 123.7, 56.2, 32.3, 30.5, 29.7, 24.9, 20.2. Anal. (C12H15ClN2·HCl)
C, H, N.
N-(7-Chloro-2,3-dihydro-1H-inden-4-yl)-4-methylpyrrolidin-2-
1
imine Hydrochloride (15). Yield 43%, white solid. H NMR (D2O) δ
7.22 (d, 1H, Har, J = 8.3), 7.01 (d, 1H, Har, J = 8.4), 3.70 (dd, 1H, 1/2
CH2, J = 10.9, J = 7.7), 3.20 (dd, 1H, 1/2 CH2, J = 10.9, J = 7.2), 3.12
(m, 1H, 1/2 CH2), 2.93 (t, 2H, CH2, J = 7.6), 2.82 (t, 2H, CH2, J =
7.5), 2.65 (m, 2H, CH + 1/2 CH2), 2.04 (qn, 2H, CH2, J = 7.6), 1.08
(d, 3H, CH3, J = 6.8). 13C NMR (D2O) δ 168.5, 145.2, 142.4, 130.4,
129.1, 127.8, 124.5, 54.2, 37.9, 32.2, 30.9, 29.5, 23.8, 17.7. Anal.
(C14H17ClN2.·HCl) C, H, N.
N-(2,3-Dihydro-1H-inden-4-yl)-5-methylpyrrolidin-2-imine Hy-
1
drochloride (7). Yield 42%, white solid. H NMR (D2O) δ 7.28 (d,
N-(7-Methyl-2,3-dihydro-1H-inden-4-yl)-4-methylpyrrolidin-2-
1
imine Hydrochloride (16). Yield 51%, white solid. H NMR (D2O) δ
1H, Har, J = 7.5), 7.21 (t, 1H, Har, J = 7.6), 7.02 (d, 1H, Har, J = 7.6),
4.07 (h, 1H, CH, J = 6.4), 3.03 (m, 2H, CH2), 2.88 (t, 2H, CH2, J =
7.5), 2.74 (t, 2H, CH2, J = 7.4), 2.37 (m, 1H, 1/2 CH2), 2.00 (qn, 2H,
7.06 (d, 1H, Har, J = 8), 6.95 (d,1H, Har, J = 8), 3.7 (dd, 1H, 1/2
CH2, J = 10.8, J = 7.7), 3.21−3.09 (m, 2H, 1/2 CH2 + CH), 2.82 (t,
2H, CH2, J = 7.5), 2.75 (t, 2H, CH2, J = 7.5), 2.67 (m, 2H, CH2), 2.19
(s, 3H, CH3), 2.01 (qn, 2H, CH2, J = 7.5), 1.09 (d, 3H, CH3, J = 6.7).
13C NMR (D2O) δ 168.4, 145.9, 139.8, 135.2, 132.4, 128.5, 128.1,
122.8, 54.0, 37.8, 31.3, 30.1, 29.6, 24.2, 18.1, 17.7. Anal. (C15H20N2·
HCl) C, H, N.
CH2, J = 7.3), 1.79 (m, 1H, 1/2 CH2), 1.17 (d, 3H, CH3, J = 6.4). 13
C
NMR (D2O) δ 168.0, 147.6, 140.3, 130.7, 127.9, 125.0, 122.6, 56.8,
32.6, 30.1, 29.9, 28.2, 24.8, 19.9. Anal. (C14H18N2·HCl) C, H, N.
N-(5,6,7,8-Tetrahydronaphthalen-1-yl)-5-methylpyrrolidin-2-
1
imine Hydrochloride (8). Yield 85%, white solid. H NMR (D2O) δ
7.17 (d, 2H, Har, J = 7), 7.03 (t, 1H, Har, J = 7.1), 4.03 (m, 1H, CH),
3.00 (m, 2H, CH2), 2.72 (t, 2H, CH2, J = 5.7), 2.51 (t, 2H, CH2, J =
5.9), 2.36 (m, 1H, 1/2 CH2), 1.81−1.61 (m, 5H, 1/2 CH2 + 2 ×
CH2), 1.15 (d, 3H, CH3, J = 6.4). 13C NMR (D2O) δ 168.6, 140.1,
133.9, 133.0, 130.2, 126.6, 123.6, 56.6, 29.9, 28.9, 28.2, 24.1, 22.1, 22.0,
19.8. Anal. (C15H20N2·HCl) C, H, N.
N-(2,3-Dihydro-1H-inden-5-yl)-4-methylpyrrolidin-2-imine Hy-
1
drochloride (17). Yield 55%, white solid. H NMR (D2O) δ 7.26
(d, 1H, Har, J = 8.0), 7.08 (d, 1H, Har, J = 1.5), 6.96 (dd, 1H, Har, J =
1.9, J = 7.9), 4.03 (h, 1H, CH, J = 6.6), 2.95 (m, 2H, CH2), 2.80 (t,
4H, 2 × CH2, J = 7.5), 2.32 (m, 1H, 1/2 CH2), 1.97 (qn, 2H, CH2, J =
7.5), 1.75 (m, 1H, 1/2 CH2), 1.16 (d, 3H, CH3, J = 6.4). 13C NMR
(D2O) δ 167.7, 145.7, 145.1, 132.7, 125.5, 121.9, 120.1, 56.9, 32.3,
32.0, 30.4, 28.0, 25.3, 19.8. Anal. (C14H18N2·HCl) C, H, N.
N-(2,3-Dihydro-1H-inden-5-yl)-5-methylpyrrolidin-2-imine Hy-
1
drochloride (9). Yield 31%, white solid. H NMR (D2O) δ 7.26 (d,
1H, Har, J = 8.0), 7.08 (d, 1H, Har, J = 1.5), 6.96 (dd, 1H, Har, J = 1.9,
J = 7.9), 4.03 (h, 1H, CH, J = 6.6), 2.95 (m, 2H, CH2), 2.80 (t, 4H, 2
N-(5,6,7,8-Tetrahydronaphthalen-1-yl)-4-methylpyrrolidin-2-
1
imine Hydrochloride (18). Yield 60%, white solid. H NMR (D2O) δ
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7.17 (d, 2H, Har, J = 7), 7.03 (t, 1H, Har, J = 7.1), 4.03 (m, 1H, CH),
3.00 (m, 2H, CH2), 2.72 (t, 2H, CH2, J = 5.7), 2.51 (t, 2H, CH2, J =
5.9), 2.36 (m, 1H, 1/2 CH2), 1.81−1.61 (m, 5H, 1/2 CH2 + 2 ×
CH2), 1.15 (d, 3H, CH3, J = 6.4). 13C NMR (D2O) δ 168.6, 140.1,
133.9, 133.0, 130.2, 126.6, 123.6, 56.6, 29.9, 28.9, 28.2, 24.1, 22.1, 22.0,
19.8. Anal. (C15H20N2.·HCl) C, H, N.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental part for the syntheses and characterizations of
compounds 19b−29, data of elemental analysis of compounds
1−18, and calculations of their physical properties. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +33 (0) 3 68 85 33 90. Fax: +33 (0)3 68 85 33 88. E-
mail: pascal.bousquet@unistra.fr.
■
(13) Fellmann, L.; Regnault, V.; Greney, H.; Gasparik, V.; Muscat, A.;
Max, J.-P.; Gigou, L.; Orea, V.; Chetrite, G.; Pizard, A.; Niederhoffer,
N.; Julien, C.; Lacolley, P.; Feve, B.; Bousquet, P. A New Pyrroline
Compound Selective for I1-Imidazoline Receptors Improves Metabolic
Syndrome in Rats. J. Pharmacol. Exp. Ther. 2013, 346, 370−380.
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Evidence That Locus Coeruleus Is the Site Where Clonidine and
Drugs Acting at Alpha 1- and Alpha 2-Adrenoceptors Affect Sleep and
Arousal Mechanisms. Br. J. Pharmacol. 1987, 90, 675−685.
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Pharmacological Properties of the Central Antihypertensive Agent,
Moxonidine. Cardiovasc. Ther. 2012, 30, 199−208.
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Agonists: Do They Have a Place in the Management of Hypertension?
Am. J. Cardiovasc. Drugs 2001, 1, 321−326.
Present Address
†For S.S.: Domain Therapeutics, Quai Seb
́
astien Brandt, 67400
Illkirch, France.
Author Contributions
‡V.G. and H.G. contributed equally to this study.
Notes
The authors declare the following competing financial
interest(s): Reseach support from and Institut de Recherche
International Servier (IRIS, Courbevoie, France).
ACKNOWLEDGMENTS
We are grateful to the Institut de Recherche International
Servier (IRIS, Courbevoie, France), for financial support.
■
ABBREVIATIONS USED
■
αARs, alpha-adrenergic receptors; BP, blood pressure; bpm,
beats per minute; DAG, diacylglycerol; DMEM, Dulbecco’s
Modified Eagle Medium; EGTA, ethylene glycol-bis(2-amino-
ethyl ether)-N,N,N′,N′-tetraacetic acid; HEPES, 2-[4-(2-
hydroxyethyl)piperazin-1-yl] ethanesulfonic acid; IRs, imidazo-
line receptors; ic, intracisternal; IRC, imidazoline related
compounds; MetS, metabolic syndrome; PEI, polyethyleni-
mine; PIC, para-iodoclonidine; RX821002, 2-(2-methoxy-1,4-
benzodioxan-2-yl)-2-imidazoline
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Ligands: An Overview of the Different Chemical Structures. Med. Res.
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