Synthesis and biological evaluation of pyrrolinic isosteres of rilmenidine. Discovery of cis-/trans-dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H-pyrrol-2-yl)- amine (LNP 509), an I1 imidazoline receptor selective ligand with hypotensive activity

Article abstract of DOI:10.1021/jm001111b

To find new compounds selective for purported I₁ imidazoline receptors (I₁Rs), over I₂ imidazoline binding sites (I₂BS) and α2-adrenoceptors (α₂ARs), a series of pyrrolinic isosteres of rilmenidine has been prepared and their biological activity at I₁Rs, I₂BS, and α₂ARs evaluated. This isosteric replacement provided us with compounds which still bound to I₁Rs but not to I₂BS nor to α₂-ARs. A limited structure-affinity relationship was generated around the heterocyclic moiety of these ligands. One compound in this series, LNP 509 (1e) [cis-/trans-dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H-pyrrol-2-yl) -amine], had no detectable affinity at α₂ARs yet was capable of lowering blood pressure after central administration. These pyrrolinic analogues constitute a new chemical class of imidazoline related compounds with high selectivity for the I₁Rs. They could be used as new tools in the study of I₁Rs and in the conception of new centrally acting hypotensive drugs.

Full text of DOI:10.1021/jm001111b

1588
J . Med. Chem. 2001, 44, 1588-1593
Syn th esis a n d Biologica l Eva lu a tion of P yr r olin ic Isoster es of Rilm en id in e.
Discover y of cis-/tr a n s-Dicyclop r op ylm eth yl-(4,5-d im eth yl-4,5-d ih yd r o-3H-
p yr r ol-2-yl)-a m in e (LNP 509), a n I1 Im id a zolin e Recep tor Selective Liga n d
w ith Hyp oten sive Activity
Stephan Schann,† Ve´ronique Bruban,† Kenia Pompermayer,† J osiane Feldman,† Bruno Pfeiffer,‡ Pierre Renard,‡
Elizabeth Scalbert,§ Pascal Bousquet,† and J ean-Daniel Ehrhardt*,†
Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Universite´ Louis Pasteur, Faculte´ de Me´decine, 11,
rue Humann, 67000 Strasbourg, France, ADIR, 1, rue Carle Hebert, 92415 Courbevoie Cedex, France, and IRIS, 6,
place des Ple´iades, 92415 Courbevoie Cedex, France
Received November 3, 2000
To find new compounds selective for purported I1 imidazoline receptors (I1Rs) over I2 imidazoline
binding sites (I2BS) and R2-adrenoceptors (R2ARs), a series of pyrrolinic isosteres of rilmenidine
has been prepared and their biological activity at I1Rs, I2BS, and R2ARs evaluated. This isosteric
replacement provided us with compounds which still bound to I1Rs but not to I2BS nor to R2-
ARs. A limited structure-affinity relationship was generated around the heterocyclic moiety
of these ligands. One compound in this series, LNP 509 (1e) [cis-/trans-dicyclopropylmethyl-
(4,5-dimethyl-4,5-dihydro-3H-pyrrol-2-yl)-amine], had no detectable affinity at R2ARs yet was
capable of lowering blood pressure after central administration. These pyrrolinic analogues
constitute a new chemical class of imidazoline related compounds with high selectivity for the
I1Rs. They could be used as new tools in the study of I1Rs and in the conception of new centrally
acting hypotensive drugs.
In tr od u ction
Clonidine (Figure 1) is an antihypertensive drug
known to act within the central nervous system. This
activity was first attributed to an activation of R2-
adrenoceptors (R2ARs); still, in the mid 1980s, Bousquet
and co-workers showed that clonidine also interacted
with imidazoline binding sites (IBS) of the nucleus
reticularis lateralis of the brainstem. Micro-injection of
clonidine in this region lowered the blood pressure
whereas catecholamines did not.1
Then, radioligand binding studies led to the clas-
sification of IBS in two subtypes.2 On one hand, I1
imidazoline receptors (I1Rs), sensitive to clonidine and
idazoxan, constitute the subtype responsible for the
hypotensive activity of imidazolines and related com-
pounds.3 These receptors are distributed in both pe-
ripheral and central nervous systems. Agmatine,4 CDS
(clonidine displacing substance),5 and more recently
harmane6 have been proposed as endogenous ligands.
Two different signaling pathways associated to these
F igu r e 1.
receptors in PC 12 cells have been discovered: the PC-
PLC (phosphatidylcholine-phospholipase C) and the
c-AMP pathways.7,8 On the other hand, I2 imidazoline
binding sites (I2BS), sensitive to idazoxan and insensi-
tive to clonidine, have been shown to be an allosteric
site on monoamine oxidase (MAO).9 However, this
classification may not be definitive since at least one
additional subtype has already been proposed.10
According to recent studies, these IBS are involved
in different physiological functions such as blood pres-
sure11 and intraocular pressure regulation,12 insulin
release,13,14 protein synthesis in astrocytes,15 gastric acid
secretion,16 neurotransmitter release,17 food intake,18
MAO activity,19 and arrhythmogenesis.20
Although clonidine, which displays nanomolar affini-
ties for both I1Rs and R2ARs, has shown a powerful
antihypertensive activity, its use is limited by side
effects resulting from R2ARs activation. For instance,
sedation is known to be a consequence of R2ARs activa-
tion in another medullary region, the locus coeruleus.21
The second generation of centrally acting antihyperten-
* Corresponding author. Tel: +33 (0)3 90 24 33 92. Fax: +33 (0)3
90 24 33 88. E-mail: J ean-Daniel.Ehrhardt@medecine.u-strasbg.fr.
† Universite´ Louis Pasteur.
‡ ADIR.
§ IRIS.
10.1021/jm001111b CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/24/2001

Synthesis of Pyrrolinic Isosteres of Rilmenidine
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1589
Ta ble 1. Physical and Chemical Data of Compounds 1a -g
compd
R
method
mpa (°C)
solvb
yieldc (%)
formula
anal.d
1a
1b
1c
1d
1e
1f
H
A
A
A
A
B
B
B
223-225
144-146
201-203
198-199
143-145
163-165
204-206
1
2
1
1
1
2
3
65
87
42
75
56
39
49
C11H18N2‚HCl‚1/2H2O
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
5-Me
3-Me
4-Me
C
C
C
C
C
12H20N2‚HCl‚1/4H2O
12H20N2‚HCl‚1/4H2O
12H20N2‚HCl‚1/4H2O
12H22N2‚HCl
cis-/trans-4,5-diMe
5-Et
cis-4,5-(CH2)4
12H22N2‚HCl
1g
C15H24N2‚HCl
a
b
Melting points of the hydrochloride salt. Crystallization solvent of the hydrochloride salt: 1 (2-PrOH-Et2O), 2 (2-PrOH-EtOAc),
and 3 (EtOH-EtOAc). c Yield of coupling reaction with C,C-dicyclopropylmethylamine. All compounds were analyzed within 0.4% of
the theoretical values.
d
Sch em e 1a
a
Reagents: (a) (CH3O)2SO2, 60 °C; (b) (C3H5)2CHNH2‚HCl, MeOH; (c) Lawesson’s reagent, THF, reflux; (d) CH3I, 2-PrOH.
Ta ble 2. Binding Data of Selected Compounds
sive drugs, represented by rilmenidine and moxonidine
(Figure 1), appeared to have greater selectivity for I1Rs
pKi ( sem
over R2ARs and a much improved acceptability;11 al-
though their affinities at R2ARs were reduced as com-
pared to clonidine, these drugs still bound to these
receptors. Substances with virtually no affinity at R2-
ARs but binding to I1Rs, which could be very useful tools
for further biochemical and pharmacological character-
ization of the receptors, were missing. Since then, such
compounds have been discovered. Munk et al. described
the new agent AGN 192403 (Figure 1) with very
interesting binding properties but devoid of any anti-
hypertensive activity.22 Pigini et al.23 reported the
synthesis of benazoline and tracizoline (Figure 1) with
high selectivity for I1Rs over R2ARs.8 However, these
agents were not hypotensive and still had nanomolar
affinities for I2BS. Ligands highly selective for I1Rs,
versus both I2BS and R2ARs, and able to reduce blood
pressure were still missing. In this study, preparation
of such compounds is reported.
compd
I1Rs
I2BS
R2ARs
1a
1b
1c
1d
6.29 ( 0.11
5.80 ( 0.08
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
6.19 ( 0.10
6.27 ( 0.11
6.77 ( 0.09
cis-/trans-1e
1f
1g
<5
rilmenidine
7.13 ( 0.10
7.25 ( 0.08
analogues have been described yet. Moreover, there is
no report on pyrrolinic isosteres of imidazoline com-
pounds.
We describe here the synthesis of a series of ami-
nopyrrolines (1a -g), structurally related to rilmeni-
dine, as well as some of their pharmacological proper-
ties. The structures of these compounds are presented
in Table 1.
Ch em istr y
Aminoimidazolines (also called iminoimidazolidines),
2-alkylimidazolines, aminooxazolines, guanidines, or
primary amines (AGN 192403) were previously reported
as imidazoline related compounds. As we will discuss
below, aminopyrrolines constitute an additional chemi-
cal class of I1Rs ligands. We were particularly interested
in aminopyrrolines derived from rilmenidine, the latter
having high selectivity for I1Rs over I2BS (Table 2).
Numerous clonidine analogues have previously been
synthesized,24-27 but to our knowledge, no rilmenidine
Two different methods were used to couple the dicy-
clopropylmethylamine moiety with lactams 2 (Scheme
1). In method A, lactams were activated by reaction with
dimethyl sulfate to obtain the 2-methoxypyrrolines 3a -
d . These methoxy intermediates reacted easily with
dicyclopropylmethylamine in acidic conditions to form
the desired compounds 1a -d .28 In the other coupling
method (method B), the lactams were first transformed
into thiolactams 4e-g with Lawesson’s reagent.29 In
fact, thiolactams are more convenient to handle on

1590 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Schann et al.
Sch em e 2a
a
Reagents: (a) H2/Rh-Al2O3, t-BuOH, 15 atm, 68%.
small scale than lactams: they are easier to detect in
TLC and to separate in chromatography; they are
solids whereas lactams are liquids. These thiolactams
4e-g were then methylated with MeI to form the
activated intermediates 5 which reacted with dicyclo-
propylmethylamine in acidic conditions to give the
products 1e-g.
F igu r e 2. Effect on MAP of cumulative doses of cis-/trans-1e
(squares) and repeated injections of vehicle (circles) adminis-
tered intracisternally to anaesthetized rabbits. Data are mean
( sem of six experiments.
Compound 1e is a mixture of cis and trans adducts.
These two adducts are present in same amounts as
1
shown by H NMR of 1e.
The starting lactams 2a -c are commercially available
and the others 2d -g have been synthesized according
to published procedures,30,31 except 2g which was
obtained in the cis conformation by reduction of oxindole
with Rh-Al2O3 under pressure (15 atm) (Scheme 2).32
The steric tolerance around the pyrrolinic cycle of 1a
was then explored by the introduction of alkyl groups
on the heterocyclic frame. We observed that methylation
at C-3 decreased the affinity for I1Rs (pKi < 5 for 1c),
whereas methylations at C-4 (1d ) and C-5 (1b) had
weak effects on binding (pKi, respectively, of 6.19 and
5.80 versus 6.29 for the unsubstituted analogue 1a ).
Bulkier alkyl groups were then added at C-4 and C-5
of the pyrroline ring. Compared to the unsubstituted
analogue, 1f (5-ethyl) and 1e (cis-/trans-4,5-dimethyl)
maintained potency on I1Rs binding with a small
increase of affinity for 1f (pKi ) 6.77) and no significant
change for 1e (pKi ) 6.27). When carbons C-4 and C-5
were included in a cyclohexyl moiety with a cis config-
uration, the so obtained compound 1g showed a de-
creased I1Rs affinity (pKi < 5) reflecting the higher limit
of steric hindrance. These results clearly showed a
limited steric bulk tolerance around the pyrrolinic ring.
Moreover, irrespective of their substituents, none of
these pyrrolinic analogues exhibited significant affinity
for I2BS or R2ARs.
The next step was the cardiovascular evaluation of
these new agents compared to the reference compound,
rilmenidine. For this purpose, we studied the blood
pressure effects of cis-/trans-1e. This diastereoisomer
mixture was ineffective after intravenous administra-
tion to aneaesthetized rabbits up to the dose of 5 mg/kg
(data not shown). But after intracisternal administra-
tion, it caused an important dose-dependent reduction
of the mean arterial pressure (MAP) (Figure 2). One
explanation for this difference could be that it was
simply unable to cross the blood brain barrier. In fact,
the basicity of aminopyrrolines is greater than that of
aminooxazolines;34 the proportion of ionized molecules
of 1e is therefore higher than that of rilmenidine at
physiological pH. Still, we cannot rule out other expla-
nations such as metabolism to explain this difference
in activity.
P h a r m a cology
Bin din g Exper im en ts. Affinities of compounds 1a-g
for I1Rs, I2BS, and R2ARs were determined by receptor
binding assays. The radioligands used were [3H]cloni-
dine (I1Rs), [3H]idazoxan (I2BS), and [3H]RX821002 (R2-
ARs). Although I1Rs involved in the hypotensive effect
of imidazoline like-drugs are located in the brainstem,
we used bovine chromaffin cell membrane preparations
for I1Rs binding assay wherein binding has been shown
to be pharmacologically equivalent to that performed
on bovine rostral ventrolateral medullary membranes.3
I2BS binding assay was performed on rabbit renal cortex
and R2ARs on calf frontal cortex membranes.
In Vivo Stu d ies. The effects of compound 1e (cis/
trans mixture) on mean arterial blood pressure were
determined after intravenous and intracisternal admin-
istrations according to methods described elsewhere.33
Resu lts a n d Discu ssion
Binding characteristics of compounds 1a -g are pre-
sented in Table 2. Rilmenidine was considered as the
reference compound.
The isosteric replacement of the oxazoline ring of
rilmenidine by a pyrrolinic ring to give 1a had only weak
influence on I1Rs affinity (pKi ) 7.13 for rilmenidine
and 6.29 for 1a ) and I2BS affinity (pKi < 5 for both
rilmenidine and 1a ). On the contrary, the affinity for
R2ARs was attenuated: 1a had no detectable affinity
for this receptor while rilmenidine had a pKi value of
7.25. These data show the crucial implication of the
oxygen atom of rilmenidine in the binding with R2ARs.
One can imagine that this oxygen takes part in a
H-bond within this receptor. But replacement of this
oxygen atom must also cause an increase of basicity of
the ligands: the basicity of aminopyrrolines is greater
than that of aminooxazolines.34 This difference can also
account for the loss of R2ARs affinity observed for 1a .
The oxygen atom of the oxazoline ring seems to play a
weaker role in the binding with I1Rs. This observation
indicates that I1Rs and R2ARs are sensitive to subtle
structural differences and therefore may accommodate
two distinct pharmacophores.
After intracisternal injection, the maximal effect of
the cis/trans mixture 1e was similar to that of rilmeni-
dine (49 ( 6% versus 41 ( 3%). It has been proved that
the rank order of I1Rs binding affinities is similar to
the rank order of hypotensive activities after intracis-
ternal injection, i.e., clonidine ) moxonidine > rilmeni-
dine, supporting a link between I1Rs and imidazoline-
induced hypotension.3,35 Data we obtained with cis-/
trans-1e is consistent with this rank ordering, and 1e
is therefore the first substance selective for I1Rs over

Synthesis of Pyrrolinic Isosteres of Rilmenidine
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1591
2-Meth oxy-3-m eth yl-4,5-d ih yd r o-3H-p yr r ole (3c): 37%;
bp 60 °C/55 mmHg; MS m/z 82(35), 70(100), 56(11), 41(12).
2-Meth oxy-4-m eth yl-4,5-d ih yd r o-3H-p yr r ole (3d ): 15%;
bp 62 °C/50 mmHg; MS m/z 113(100), 98(34), 56(80), 41(38).
Am in op yr r olin es 1a -d . To a solution of the required
methoxy derivative 3a -d (20 mmol) in 10 mL of methanol was
added C,C-dicyclopropylmethylamine hydrochloride (2.95 g, 20
mmol).40 The mixture was stirred under nitrogen for 48 h.
Solvent was removed in vacuo and then water and 2 N NaOH
were added. This basic aqueous solution was extracted with
ether (3 × 50 mL), and the ether layer was dried and
evaporated to give, after crystallization, the product 1a -d .
Dicyclop r op ylm e t h yl-(4,5-d ih yd r o-3H -p yr r ol-2-yl)-
a m in e h yd r och lor id e (1a ‚HCl): 65%; mp base 133-134 °C
(cyclohexane), mp hydrochloride 223-225 °C (2-PrOH-ether);
1H NMR (D2O) δ 3.67 (t, 2H, H-5, J ) 7.2 Hz), 2.75-2.98 (m,
3H, H-3 + CH), 2.10-2.30 (m, 2H, H-4), 1.00-1.22 (m, 2H, 2
× CH), 0.28-0.72 (m, 8H, 4 × CH2); 13C NMR (D2O) δ 168.4
(C2), 62.1 (CH-NH), 48.0 (C5), 31.1 (C3), 20.8 (C4), 14.3 + 2.9
+ 1.9 (C3H5); MS m/z 178(33), 163(97), 149(68), 137(61), 135-
(32), 122(31), 109(54), 95(52), 67(63), 55(50), 41(75). Anal.
(C11H18N2‚HCl‚1/2H2O) C, H, N.
R2ARs and I2BS. Our results support the hypothesis
that an exclusive action on cerebral I1Rs is sufficient to
induce hypotension. They corroborate the observation
of Ernsberger et al. who showed that moxonidine, a
mixed I1Rs and R2ARs ligand, decreased the MAP of
mice with impaired R2ARs after intracisternal admin-
istration.36
The synthesis of selective I1Rs molecules may help
clarify the respective role of I1Rs and R2ARs in the
hypotensive activity of clonidine related compounds.37
Con clu sion
In this study, a series of pyrrolinic analogues of
rilmenidine was synthesized. Due to the isosteric sub-
stitution described, R2ARs affinity was lost, while I1Rs
affinity was only slightly affected. This result reveals
an interesting difference in the pharmacophores of I1-
Rs and R2ARs: the distinctive importance of the oxygen
atom of rilmenidine for these two receptor bindings.
Moreover, these new compounds did not bind to I2BS.
Thus, new selective ligands for I1Rs over I2BS and R2-
ARs were obtained. It was also shown that there is a
limited steric hindrance around the pyrrolinic ring of
these analogues. cis-/trans-1e, one of these analogues,
was highly selective for I1Rs, and it reduced mean
arterial pressure of rabbits when administrated intra-
cisternally. These results support the fact that I1Rs are
involved in the central regulation of MAP and are
targeted by imidazoline related drugs. cis-/trans-1e
represents a prototype compound with good selectivity
for I1Rs and cardiovascular activity.
Dicyclopr opylm eth yl-(5-m eth yl-4,5-dih ydr o-3H-pyr r ol-
2-yl)-a m in e h yd r och lor id e (1b‚HCl): 87%; mp base 99-
101 °C (cyclohexane), mp hydrochloride 144-146 °C (2-PrOH-
1
EtOAc); H NMR (D2O) δ 3.97-4.15 (m, 1H, H-5), 2.70-2.96
(m, 3H, H-3 + CH), 2.21-2.43 (m, 1H, H-4), 1.63-1.85 (m,
1H, H-4), 1.26 (d, 3H, CH3, J ) 6.6), 0.95-1.19 (m, 2H, 2 ×
CH), 0.20-0.68 (m, 8H, 4 × CH2); 13C NMR (D2O) δ 167.6 (C2),
61.9 (CH-NH), 57.1 (C5), 30.9 (C3), 28.9 (C4), 20.6 (CH3), 14.3
+ 3.0 + 1.9 (C3H5); MS m/z 192(29), 177(100), 164(14), 163-
(50), 151(57), 149(24), 136(16), 123(34), 95(77), 67(61), 55(50),
41(75). Anal. (C12H20N2‚HCl‚1/4H2O) C, H, N.
Dicyclopr opylm eth yl-(3-m eth yl-4,5-dih ydr o-3H-pyr r ol-
2-yl)-a m in e h yd r och lor id e (1c‚HCl): 42%; mp base 102-
104 °C (cyclohexane), mp hydrochloride 201-203 °C (2-PrOH-
ether); 1H NMR (D2O) δ 3.50-3.78 (m, 2H, H-5), 3.08-3.27
(m, 1H, H-3), 2.75 (t, 1H, CH, J ) 7.5 Hz), 2.29-2.50 (m, 1H,
H-4), 1.77-1.97 (m, 1H, H-4), 1.33 (d, 3H, CH3, J ) 7.4), 1.02-
1.22 (m, 2H, 2 × CH), 0.20-0.75 (m, 8H, 4 × CH2); 13C NMR
(D2O) δ 171.6 (C2), 62.2 (CH-NH), 46.2 (C5), 38.4 (C3), 29.3 (C4),
16.8 (CH3), 14.3 + 3.1 + 1.8 (C3H5); MS m/z 192(30), 177(100),
164(19), 163(67), 151(71), 149(32), 136(24), 123(45), 95(47), 67-
(58), 55(56), 41(75). Anal. (C12H20N2‚HCl‚1/4H2O) C, H, N.
Dicyclopr opylm eth yl-(4-m eth yl-4,5-dih ydr o-3H-pyr r ol-
2-yl)-a m in e h yd r och lor id e (1d ‚HCl): 75%; mp base 113-
114 °C (cyclohexane), mp hydrochloride 198-199 °C (2-PrOH-
ether); 1H NMR (D2O) δ 3.73-3.83 (m, 1H, H-5), 3.22-3.31
(m, 1H, H-5), 2.97-3.09 (m, 1H, H-3), 2.47-2.84 (m, 3H, H-3
+ H-4 + CH), 1.15 (d, 3H, CH3, J ) 6.4), 0.90-1.25 (m, 2H, 2
× CH), 0.28-0.70 (m, 8H, 4 × CH2); 13C NMR (D2O) δ 167.9
(C2), 62.0 (CH-NH), 54.5 (C5), 38.6 (C3), 30.1 (C4), 18.3 (CH3),
14.3 + 3.0 + 2.0 (C3H5); MS m/z 192(21), 177(100), 164(10),
163(47), 151(51), 149(23), 136(17), 123(34), 95(39), 67(49), 55-
(31), 41(45). Anal. (C12H20N2‚HCl‚1/4H2O) C, H, N.
Meth od B: Gen er a l P r oced u r e for th e P r ep a r a tion of
Com p ou n d s 1e-g. Th iola cta m s 4e-g. Lawesson’s reagent
(14.88 g, 36.8 mmol) was added to a solution of the required
lactam 2e-g (73.6 mmol) in dry THF (400 mL). The mixture
was stirred at room temperature for 24 h. Solvent was then
removed in vacuo and the product purified by flash chroma-
tography (EtOAc-hexane 3/7) to give the corresponding thi-
olactam 4e-g.
Mixtu r e of cis- a n d tr a n s-4,5-d im eth yl-p yr r olid in e-2-
th ion e (4e): 98%; 1H NMR (CDCl3) δ 8.72 (br, 1H, NH), 4.01
(m, 0.5H, H-5), 3.55 (m, 0.5H, H-5), 2.89-3.16 (m, 1H, H-3),
2.42-2.71 (m, 1.5H, H-3 + H-4), 2.02-2.23 (m, 0.5H, H-4),
1.27 (d, 1.5H, CH3, J ) 6.4), 1.15 (d, 1.5H, CH3, J ) 6.7), 1.10
(d, 1.5H, CH3, J ) 6.7), 0.98 (d, 1.5H, CH3, J ) 6.8); MS m/z
129(100), 114(59), 81(17), 86(17), 71(21), 55(43), 44(30), 42-
(13).
5-Eth yl-p yr r olid in e-2-th ion e (4f): 53%; 1H NMR (CDCl3)
δ 8.50 (br, 1H, NH), 3.79-3.93 (m, 1H, H-5), 2.76-3.05 (m,
2H, H-3), 2.23-2.41 (m, 1H, H-4), 1.45-1.90 (m, 3H, H-4 +
Exp er im en ta l Section
Ch em istr y. All solvents were purified by standard methods
before use. Flash chromatographies38 were performed on
Merck silica gel Si 60 (40-63 µm) and TLC on silica gel 60
F254 (Merck). All organic layers were washed with brine and
then dried with Na2SO4. Melting points were determined in
open capillaries on a Gallenkamp apparatus and are uncor-
1
rected. H and 13C NMR spectra were recorded at 200 and 50
MHz, respectively, on a Bruker AC 200 spectrometer. Solvents
used were CDCl3 (CHCl3 at 7.26 ppm as reference) and D2O
(t-BuOH at 1.28 ppm as reference) for 1H NMR and D2O (t-
BuOH at 70.4 and 30.3 ppm as reference) for 13C NMR. The
signals are described as: s (singlet); d (doublet); t (triplet); m
(multiplet); and br (broad). Chemical shifts (δ) are given in
ppm and coupling constants (J ) in Hz. The mass spectra were
recorded using a LKB 2091 apparatus. The related intensities
of the mass spectrum peaks are listed in parentheses. Elemen-
tal analyses were performed at the Service de microanalyse,
Universite´ Louis Pasteur, Strasbourg.
All target compounds were tested as hydrochloride salts.
These salts were prepared by the addition of ethanolic HCl to
an ethanolic solution of the base.
Meth od A: Gen er a l P r oced u r e for th e P r ep a r a tion of
Com p ou n d s 1a -d . 2-Meth oxyp yr r olin es 3a -d . Lactam
2a -d (0.2 mol) was added to dimethyl sulfate (25.5 g, 0.2 mol),
and the mixture was heated at 60 °C for 16 h under nitrogen.
The mixture was then cooled and added to a saturated aqueous
solution of K2CO3 (about 150 mL). This solution was extracted
with ether, which was dried and evaporated to give, after
distillation under reduced pressure, the corresponding 2-meth-
oxypyrroline 3a -d .
2-Meth oxy-4,5-d ih yd r o-3H-p yr r ole (3a ):39 63%; bp 59-
60 °C/80 mmHg; MS m/z 99(84), 84(15), 71(21), 69(34), 56-
(80), 42(53), 41(100).
2-Meth oxy-5-m eth yl-4,5-d ih yd r o-3H-p yr r ole (3b): 63%;
bp 66-68 °C/85 mmHg; MS m/z 113(32), 98(100), 70(47), 56-
(37), 41(23).

1592 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Schann et al.
CH2), 0.96 (t, 3H, CH3, J ) 7.4); MS m/z 130(14), 129(67), 100-
(100), 71(10), 67(30), 42(13), 41(21).
clonidine binding in the presence of 1 µM of (5-(2-methyl-
phenoxy-methyl)-1,3-oxazoline-2-yl)amine (S22687)45 (high af-
finity competition drug, Ki ) 4.98 nM) and represented 55%
of the total binding.
Octa h yd r o-in d ole-2-th ion e (4g): 52%; mp 94-95 °C
1
(EtOAc-cyclohexane); H NMR (CDCl3) δ 8.12 (br, 1H, NH),
I2BS Bin d in g Assa ys. Tissue used was prepared as fol-
lows: renal cortex from male New Zealand rabbits were
homogenized in ice-cold preparation buffer (20 mM NaHCO3).46
The homogenate was centrifuged at 40000g for 30 min at 4
°C. The pellet was resuspended in buffer (50 mM Tris HCl,
0.5 mM EDTA, pH 7.4), centrifuged again, and resuspended
in the same buffer. The pellet was stored at -80 °C until use.
Affinities of drugs for I2 binding sites were determined as
reported by Pigini et al.23 with [3H]idazoxan as radioligand;
2-BFI (10-5 M) was used to define the nonspecific binding.
R2-Ad r en ocep tor s Bin d in g Assa y. Tissue used was pre-
pared from calf frontal cortex membranes according to Liefde
et al.47 The membranes (0.5 mg protein/mL) were incubated
for 60 min at 25 °C with 0.8 nM [3H]RX821002 in the presence
of 0.3 µM 5HT to mask 5HT1A receptors in 50 mM sodium
phosphate buffer, pH 7.4, with increasing concentrations of
drugs (10-9 to 10-4 M). Nonspecific binding was defined with
10 µM phentolamine.
In these three assays, incubations were terminated by rapid
filtration under vacuum through Whatman GF/C glass fiber
filters followed by rapid washing of the tubes and filters three
times with ice-cold binding buffer. Radioactivity remaining on
the filters was counted in a Packard Tricarb counter. Results
are given as the mean ( standard error mean (sem) of three
experiments, each performed in triplicate. Competition experi-
ments were analyzed using the iterative nonlinear least-
squares curve fitting program GraphPad. Ki were determined
using the method of Cheng and Prussof.48
Ca r d iova scu la r Exp er im en ts. Cardiovascular evalua-
tions of compound cis-/trans-1e were performed according to
published procedures:33 normotensive male rabbits (Zika
strain) weighing 2.5 to 3.5 kg were anaesthetized with pen-
tobarbital sodium (40 mg/kg), paralyzed with pancuronium
bromide (1 mg/kg), and artificially ventilated. Mean arterial
pressure (MAP) was calculated as diastolic pressure plus one-
third of the differential pressure. In experiments during which
drugs were injected intracisternally, 0.2 mL of drug solutions
were injected after removal of the same volume of cerebrospi-
nal fluid. In control experiments, vehicle alone was injected
the same way in the same volume and never modified MAP
significantly, as shown in Figure 2 (n ) 6).
3.87-3.96 (m, 1H, H-7a), 2.39-2.93 (m, 3H, H-3a+H-3), 1.15-
1.87 (m, 8H, 4 × CH2); MS m/z 156(52), 155(100), 112(54), 99-
(27), 81(16), 67(19), 41(14).
Am in op yr r olin es 1e-g. An excess of iodomethane (14 g,
98 mmol) was added to a solution of thiolactam 4e-g (14
mmol) and 2-PrOH (100 mL). The mixture was stirred under
nitrogen for 12 h, and then 2-PrOH was removed in vacuo.
The residue was treated with water and saturated K2CO3 and
extracted with ether (3 × 50 mL). After this solution has been
dried and evaporated, the residue was treated with C,C-
dicyclopropylmethylamine hydrochloride (1.98 g, 14 mmol) in
10 mL ethanol under nitrogen and at reflux for 24 h. Solvent
was removed in vacuo, and then water and saturated K2CO3
were added. This basic aqueous solution was extracted with
ether (3 × 50 mL), and the organic layer was dried and
evaporated to give, after crystallization, the products 1e-g.
Mixtu r e of cis- a n d tr a n s-d icyclop r op ylm eth yl-(4,5-
d im eth yl-4,5-d ih yd r o-3H-p yr r ol-2-yl)-a m in e h yd r och lo-
r id e (1e‚HCl): 56%; mp base 65-66 °C (hexane), mp hydro-
1
chloride 143-145 °C (2-PrOH-ether); H NMR (D2O) δ 4.09
(m, 0.5H, H-5), 3.64 (m, 0.5H, H-5), 2.50-3.10 (m, 3.5H, H-4
+ H-3 + CH), 2.18 (m, 0.5H, H-4), 1.00-1.20 (m, 8H, 2 × CH3
+ 2 × CH), 0.30-0.70 (m, 8H, 4 × CH2); 13C NMR (D2O) δ
167.1 (C2), 63.4 (C5), 61.8 (CH-NH), 59.1 (C5), 38.5 (C3), 38.3
(C4), 37.9 (C3), 33.3 (C4), 19.0 (CH3), 16.8 (CH3), 15.0 (CH3),
13.7 (CH3), 14.3 + 3.0 + 1.9 (C3H5); MS m/z 206(41), 191(100),
177(34), 165(32), 151(16), 137(16), 126(15), 113(27), 97(22), 95-
(45), 79(20), 67(31), 55(21), 41(31). Anal. (C13H22N2‚HCl) C, H,
N.
Dicyclop r op ylm eth yl-(5-eth yl-4,5-d ih yd r o-3H-p yr r ol-
2-yl)-a m in e h yd r och lor id e (1f‚HCl): 39%; mp base 72-73
°C (cyclohexane), mp hydrochloride 108-110 °C (2-PrOH-
ether); 1H NMR (D2O) δ 3.99 (m, 1H, H-5), 2.81-2.95 (m, 3H,
H-3 + CH), 2.22-2.41 (m, 1H, H-4), 1.53-1.92 (m, 3H, H-4 +
CH2), 1.01-1.18 (m, 2H, 2 × CH), 0.93 (t, 3H, CH3, J ) 7.5
Hz), 0.30-0.70 (m, 8H, 4 × CH2); 13C NMR (D2O) δ 167.8 (C2),
62.4 (CH-NH), 61.8 (C5), 30.6 (C3), 27.8 (C4), 25.9 (CH2), 9.17
(CH3), 14.3 + 3.0 + 1.9 (C3H5); MS m/z 206(22), 191(54), 177-
(87), 165(32), 151(11), 137(14), 126(13), 113(21), 95(100), 83-
(43), 79(21), 67(45), 55(25), 41(33). Anal. (C13H22N2‚HCl) C, H,
N.
Dicyclop r op ylm eth yl-(3a ,4,5,6,7,7a -h exa h yd r o-3H-in -
d ol-2-yl)-a m in e h yd r och lor id e (1g‚HCl): 49%; mp base
93-95 °C (cyclohexane-isopentane), mp hydrochloride 204-
206 °C (ethanol-EtOAc); 1H NMR (D2O) δ 3.97-4.04 (m, 1H,
H-5), 2.80-3.01 (m, 2H, H-3 + CH), 2.39-2.58 (m, 2H, H-3 +
H-4), 1.04-1.80 (m, 10H, 4 × CH2 + 2 × CH), 0.30-0.75 (m,
8H, 4 × CH2); 13C NMR (D2O) δ 168.3 (C2), 62.0 (CH-NH), 59.2
(C5), 37.9 (C3), 34.7 (C4), 27.2 (CH2), 26.9 (CH2), 22.6 (CH2),
20.5 (CH2), 14.3 + 3.0 + 1.9 (C3H5); MS m/z 233(36), 232(63),
217(88), 203(46), 191(60), 189(57), 152(25), 139(33), 137(25),
95(100), 67(45), 41(37). Anal. (C15H24N2‚HCl) C, H, N.
Octa h yd r o-in d ol-2-on e (2g). Oxindole (3 g, 22.6 mmol)
was reduced in t-BuOH (60 mL) with Rh/Al2O3 under 15 atm
of H2 during 12 h at the temperature of 50 °C. The mixture
was then filtered through Celite and the Celite bed rinsed with
EtOH (50 mL). After evaporation, 2g41-43 (2.14 g, 68%) was
Ack n ow led gm en t. S.S. was supported by a CIFRE
grant from the Association Nationale de la Recherche
Technique (ANRT) and Institut de Recherches Inter-
nationales Servier (IRIS). We thank IRIS for financial
support. The compounds presented in this paper are
part of French patent 99.14434. The authors also thank
Dr. Monique Dontenwill and Dr. Hugues Greney for
their critical reading of the manuscript. We are grateful
to Tatjana Silec for her proofreading of the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
of compounds 1a -g. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
Refer en ces
obtained as a colorless oil, which crystallized in the cold. H
NMR (CDCl3) δ 6.17 (br, 1H, NH), 3.55-3.80 (m, 1H, H-7a),
1.81-2.55 (m, 3H, H-3a + H-3), 1.05-1.82 (m, 8H, 4 × CH2);
MS m/z 139(30), 96(100), 83(9), 68(9), 55(12), 41(10).
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