2-(2-phenylcyclopropyl)imidazolines: Reversed enantioselective interaction at I1 and I2 imidazoline receptors [1]

Full text of DOI:10.1021/jm991049m

J . Med. Chem. 1999, 42, 2737-2740
2737
Communications to the Editor
2
-(2-P h en ylcyclop r op yl)im id a zolin es:
Ch a r t 1
Rever sed En a n tioselective In ter a ction a t
I1 a n d I2 Im id a zolin e Recep tor s
†
‡
†
Wilma Quaglia, Pascal Bousquet, Maria Pigini,
§
§
Angelo Carotti, Antonio Carrieri,
‡
†
Monique Dontenwill, Francesco Gentili,
Mario Giannella, Francoise Maranca,
Alessandro Piergentili, and Livio Brasili*
†
‡
†
,∇
Dipartimento di Scienze Chimiche, Universit a` di Camerino,
Via S. Agostino 1, 62032 Camerino, Italy, Laboratoire de
Neurobiologie et Pharmacologie Cardiovasculaire,
Universit e´ Louis Pasteur, CNRS, 11 rue Humann,
6
7000 Strasbourg, France, Dipartimento Farmaco-Chimico,
Universit a` di Bari, Via E. Orabona 4, 70125 Bari, Italy,
and Dipartimento di Scienze Farmaceutiche, Universit a` di
Another way of blocking the free rotation around the
central C-C bond of compound 2 is the insertion of a
rigid cyclopropyl ring, which incorporates the ethylenic
unit between the phenyl and imidazoline moieties. This
way of restricting the conformational freedom, which
implies a minimal additional molecular bulk, generates
the cis (4a ) and trans (4b) isomers, each of which may
be resolved in the two enantiomers, thus offering the
opportunity of studying the interaction of this type of
molecules with I receptors also from a stereospecific
point of view. This idea was further supported by our
Modena, Via G. Campi 183, 41100 Modena, Italy
Received March 30, 1999
In tr od u ction . During the past decade, the concept
of imidazoline (I) receptors has been developed and
gained consensus. Findings from different laboratories
have shown that they are widely distributed in different
tissues and species and may participate in the regula-
tion of various physiological functions. Moreover, recent
evidence suggests their involvement in diverse patholo-
gies, and therefore a more definite knowledge of the
structure and function of this receptor system could help
in the search for therapeutic agents useful for treating
efficaciously a variety of disorders such as hypertension,
diabetes mellitus, gastric ulcer, endogenous depression,
and stroke. Different rank order of the affinity of ligands
indicates the existence of at least two major classes: I1
and I2; however, this seems an oversimplification since
COMFA model, recently developed for I2 receptors using
9,10
a large number of compounds,
which predicted the
trans isomer to be more active than the cis isomer, with
the two enantiomers of the former showing a significant
eudismic ratio for I2 receptors. Furthermore, considering
that eudismic analysis is one of the means to discrimi-
nate between receptor subtypes, the pharmacological
testing was performed on rabbit kidney and PC12 cell
membranes for evaluating the affinities at I2 and I1
imidazoline receptors, respectively.
1
-5
additional categories appear to exist (non-I1, non-I2).
Despite the intense efforts in the field, a conclusive
sub)classification and some physiological roles of the I
(
Ch em istr y. The synthesis of geometric isomers of
compound 4, whose stereochemistry was not defined in
its cis/trans ratio, was reported in 1962 within a study
receptors have yet to be assessed, mainly because the
ligands used for their characterization often suffered
from lack of selectivity with respect to R-adrenoceptors.
In this context, a few years ago we began a systematic
study aimed at the discovery of selective I receptor
ligands. Using as a starting lead Cirazoline (1) (Chart
1
1
of new MAO inhibitors. In our work pure cis and trans
isomers 4a and 4b were prepared.
Commercially available trans-2-phenylcyclopropane-
carboxylic acid (5b) was converted into the methyl ester
1
), a potent R1-adrenergic agonist that also binds to I
6
b by a conventional method (CH3OH and H2SO4) and
receptors, we showed that by removing the o-cyclopropyl
6
then condensed with ethylenediamine in the presence
substituent and substituting the oxygen atom with an
1
2
isosteric methylene unit,7,8 high affinity for I2 receptors
of Al(CH3)3 to give the trans-imidazoline 4b. The cis-
1
2
isomer 4a was similarly prepared starting from the
was maintained, while the R1-adrenergic agonist activity
was abolished (compound 2). Conformational restriction
of 2, carried out by inserting a double bond in the bridge,
led to the discovery of Tracizoline (3), a ligand with high
1
3
corresponding acid 5a (Scheme 1).
The two enantiomers of the most active trans isomer,
1
4
14
(
1R,2R)-(-)-4b and (1S,2S)-(+)-4b, were obtained as
7
,8
depicted in Scheme 2, from the corresponding trans
forms of 2-phenylcyclopropanecarboxylic acids (5b),
affinity and unprecedented selectivity for I2 receptors.
*
Author for correspondence: e-mail, brasili@unimo.it.
Universit a` di Camerino.
Universit e´ Louis Pasteur.
Universit a` di Bari.
Universit a` di Modena.
resolved by dehydroabietylamine and quinine as previ-
†
15,16
ously described in the literature (Scheme 2).
The
‡
§
absolute configuration of the two enantiomeric acids (-)-
5a and (+)-5b has been reported in ref 16.
∇
1
0.1021/jm991049m CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/14/1999

2
738 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Communications to the Editor
Sch em e 1a
determined by nonlinear regression analysis of binding
data with the aid of the Graphpad program. Ki values
were calculated by the equation of Cheng and Prusoff20
and reported as pKi ( SEM.
Resu lts a n d Discu ssion . From the binding data
reported in Table 1, it can be seen that Tracizoline, first
described as an I2/R2-selective ligand, was found to be
I2-selective, although to a lesser extent, also when
compared to the I1 subtype. In addition, it can be
observed that the compounds studied bind to the two I
subtypes in a different manner. In fact, while at the I2
subtype a single binding site was revealed, at the I1
subtype high- and low-affinity binding sites were de-
tected (Table 1 and Figure 1). The resolution of I1
binding in two components has been previously observed
and related to the presence of two interconvertible forms
a
(
a) CH3OH/H2SO4; (b) CH2NH2)2/Al(CH3)3.
Sch em e 2a
2
1,22
of the same receptor.
Furthermore, the pharmacological results clearly
show that conformational restriction of compound 2,
resulting from the insertion of a cyclopropyl ring, was
detrimental, with respect to Tracizoline (3), for both I2
receptor affinity and I2/R2 selectivity. However, the
decreased I2 receptor affinity of the cis isomer 4a was
consistently larger than that of the trans isomer 4b
(about 468- vs 12-fold) resulting in a trans/ cis affinity
ratio close to 40. This difference indicates that the
distance between the phenyl and imidazoline rings plays
a crucial role for high affinity. Although compound 4b
retained a relatively good affinity for I2 receptors (pKi
)
7.64), overall our findings parallel those obtained with
a
(
c) Dehydroabietilamine, NaOH, HCl; (d) quinine, NaOH, HCl;
another class of imidazolines, namely, 2-(phenoxymeth-
yl)imidazolines, in which the introduction of a methyl
group on the carbon atom of the oxymethylene bridge
caused an even larger decrease of affinity for I2
(
a) CH3OH/H2SO4; (b) (CH2NH2)2/Al(CH3)3.
Enantiomeric purity of compounds (+)-4b and (-)-
b was indirectly measured by H NMR spectroscopy
of the corresponding diastereomeric carbamates 7a and
1
4
6
-8
receptors.
However, it is worth noting that the lower
affinity of cyclopropyl derivatives of formula 4, compared
to 3, might be due not only to the additional steric
hindrance arising from the cyclopropyl ring but also to
the mutual spatial arrangement of the two terminal
rings. Indeed, the structure of 3 is nearly planar,
whereas in compounds 4 the two rings lie in two distinct
planes. Conformational analyses have shown that in the
minimum-energy conformer the angle between the
planes containing the phenyl and imidazoline rings is
equal to 12.5° for compound 3 and equal to 107° and
7
b, obtained by reacting (+)-4b and (-)-4b with (-)-
menthyl chloroformate, and found to be >98% (detection
limit) for both enantiomers. The difference of chemical
shifts17 of the cyclopropyl protons at C-1 and C-2 was
quite evident and of diagnostic value: compound 7a
showed two multiplets at δ 2.39 and 2.79 for protons at
C-2 and C-1, respectively, whereas for compound 7b the
corresponding signals were at δ 2.49 and 2.94.
1
4
05°, respectively, for (1S,2S)-(+)-4b and (1R,2R)-(-)-
2
3
b.
It is interesting to note that the rank order of binding
affinities of the enantiomers of the trans ligand 4b can
be correctly predicted by our recently refined three-field
(
steric, electrostatic, and lipophilic) CoMFA model.10
P h a r m a cology. The new compounds, in the form of
hydrogen oxalate salts, were evaluated for affinity at
R2-adrenergic receptors and I2 imidazoline receptors
using membranes of rat cortex and rabbit kidney,
Taking into account that this model was derived from
relatively flexible or planar ligands, the predicted pKi
for very rigid, nonplanar compounds, such as the title
imidazolines, may be considered quite satisfactory. The
different binding modes of two low-energy conformers
of the trans-enantiomers 4b can be observed in the
CoMFA coefficient contour maps drawn in Figure 2.
With its phenyl group, the most active ligand (1R,2R)-
(-)-4a is able to establish favorable contacts with
electrostatic (magenta), lipophilic (yellow), and steric
(green) regions, unlike its (1S,2S)-(+)-4b enantiomer
whose phenyl ring may reach sterically hindered re-
gions.
8
respectively, following already described procedures.
The I1-imidazoline receptor affinity was determined on
rat pheochromocytoma cells (PC12) according to the
methods of Separovic et al.18 with slight modifications.
19
3
The radioligands used were [ H]clonidine (2 nM, R2),
[3
H]idazoxan (5 nM, I2), and [125I]p-iodoclonidine (0.5
nM, I1), and nonspecific binding was defined by inclu-
sion of 10 µM phentolamine (R2, 25%), 10 µM cirazoline
(I2, 10%), and 10 µM BDF 6143 (I1, 35%). IC50 were

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2739
Ta ble 1. I1-, I2-, and R2-Receptor Binding Affinities of Compounds 3 and 4
compound
pKi I1a
Eub
pKi I2 predc
pKi I2a
Eub
pKi R2a
Eub
I2/I1d
10
I2/R2d
Tracizoline (3)
7.72 ( 0.14 (35%)
8.72 ( 0.13
4.85 ( 0.15
7413
5
.42 ( 0.04 (65%)
(
(
(
(
()-4a
5.24 ( 0.07
6.05 ( 0.11
4.53 ( 0.04
7
33
1S,2R)-4a
1R,2S)-4a
()-4b
6.48
6.14
7.76 ( 0.10 67%)
7.64 ( 0.05
8.22 ( 0.02
6.91 ( 0.10
6.40 ( 0.03
6.92 ( 0.12
6.62 ( 0.07
0.8
58
17
20
2
5
.26 ( 0.37 (33%)
(
1R,2R)-(-)-4b
1S,2S)-(+)-4b
6.46 ( 0.05
7.64
6.60
2
9
20
2
(
7.93 ( 0.14 (66%)
.51 ( 0.10 (34%)
0.1
4
a
The affinity values (pKi) for I1 and I2 imidazoline and R2-adrenergic receptors were assessed by measuring the ability of the test
1
25
3
3
compounds to displace [ I]p-iodoclonidine (PC12 membranes), [ H]idazoxan (rabbit kidney membranes), and [ H]clonidine (rat cortex
b
membranes), respectively. Eudismic ratio is the antilog of the difference between the pKi values for the eutomer and the distomer.
c
Predicted values by a three-field CoMFA model.10 Antilog of the difference between pKi I2 and pKi I1 (or pKi R2) values.
d
F igu r e 2. Isocontour maps from a previously published steric,
electrostatic, and lipophilic CoMFA model.10 Color code is as
follows: green and red, favorable and unfavorable steric zones,
respectively; magenta and white, favorable and unfavorable
electrostatic zones for electron-rich groups, respectively; yellow
F igu r e 1. Competition binding curves of (1R,2R)-(-)-4b (A)
(filled squares) and I (open
squares) binding sites on PC12 cell membranes or rabbit
kidney membranes, respectively.
and cyan, favorable and unfavorable zones for lipophilic
and (1S,2S)-(+)-4b (B) on I
1
2
moieties, respectively. The two trans-enantiomers (-)-4b
(
white) and (+)-4b (orange) show a different binding mode.
Tracizoline (3) (green) has been added to the color maps for
comparison.
At I1 sites, the same diastereoselectivity seen for I2
sites is also observed, with an even larger (330-fold)
trans/cis ratio. However, in this case the trans isomer
levorotatory enantiomer (1R,2R)-(-)-4b, with a pK of
i
8
.22. There was clearly a reversal of enantioselectivity
4
b displays the same affinity (pKi ) 7.76) as Tracizoline
which is very peculiar and unprecedented. Owing to this
differential interaction at the two I subtypes, the
enantiomer (1S,2S)-(+)-4b proved to be 10-fold selective
for I1 receptors while the enantiomer (1R,2R)-(-)-4b was
about 60-fold selective for I2 receptors. Therefore, de-
pending on the stereochemistry, replacement of the
double bond in the Tracizoline structure, which is 10-
fold selective for the I2 subtype, by a cyclopropyl ring
provides ligands selective for one or the other subtype,
with a 6-fold increase of I2 selectivity and a 100-fold
increase (reversed) of I1 selectivity.
(pKi ) 7.72), indicating that the cyclopropyl ring pro-
vides a good replacement for the double bond of 3.
Therefore, the trans geometry appears to be an essential
requirement for optimum affinity, and steric hindrance,
and deviation from coplanarity of the two ring systems
seems not to have any effects on it, unlike what seems
to be the case at the I2 subtype.
The most important results have been obtained by
resolving the racemate of the trans isomer 4b, the most
active of the two diastereoisomers, to give the enanti-
omers (1R,2R)-(-)-4b and (1S,2S)-(+)-4b which were
tested in order to study further the role of the stereo-
chemistry on binding to both I1 and I2 receptors. At I1
sites, the dextrorotatory enantiomer (1S,2S)-(+)-4b,
with a pKi of 7.93, was more potent than the corre-
sponding optical antipode (1R,2R)-(-)-4b (pKi ) 6.46),
and the resulting eudismic ratio (Eu) of 29 was indica-
tive of a reasonable enantiospecific interaction. At I2
sites, enantioselective binding was also observed, the
Eu being 20, but in this case the eutomer was the
In conclusion, we have shown that the imidazoline I1
and I2 receptor binding sites have stereospecific require-
ments, the two subtypes showing a reversal of enatio-
selectivity, which may offer leads to the development
of selective imidazoline receptor ligands. The couple of
imidazoline enantiomers (1R,2R)-(-)-4b and (1S,2S)-
(+)-4b reported herein, together with other properly
designed chiral ligands, may be considered as valid tools
for a more extensive eudismic analysis aimed at a

2
740 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Communications to the Editor
necessary subtyping of the I receptors. Work along this
line is in progress.
m, CH-Ar); 3.29 (4H, m, NCH
exchangeable with D O); 7.11-7.33 (5H, m, Ar-H). It was
characterized as the hydrogen oxalate salt: mp 185-186 °C
from EtOH). Anal. (C12 ) C, H, N.
2 2
CH N); 3.73 (1H, br s, NH,
2
(
14 2 2 2 4
H N ‚H C O
Ack n ow led gm en t. This work was supported by a
grant from MURST (Rome, Italy) and the National
Research Council (Rome, Italy).
(13) Kaiser, C.; Weinstock, J .; Olmstead, M. P. cis-2-Phenylcyclopro-
panecarboxylic acid (cyclopropanecarboxylic acid, 2-phenyl-, cis).
Org. Synth. 1988, VI, 913-915 and references therein.
22
(
D
14) Compound (1R,2R)-(-)-4b: mp 114-116 °C; [R] ) -320.72
(
c ) 1, CHCl
3
). It was characterized as the hydrogen oxalate
Refer en ces
salt: mp 178-179 °C (from 2-PrOH/EtOH). Anal. (C12H14N ‚
2
H
2
C
2
O
D
4
) C, H, N. Compound (1S,2S)-(+)-4b: mp 114-116 °C;
) +318.80 (c ) 1, CHCl ). It was characterized as
hydrogen oxalate salt: mp 177-179 °C (from 2-PrOH/EtOH).
Anal. (C12 ) C, H, N.
(
(
(
1) Reis, D. J ., Bousquet, P., Parini, A., Eds. The Imidazoline
Receptor: Pharmacology, Functions, Ligands, and Relevance to
Biology and Medicine. Ann. N. Y. Acad. Sci. 1995, 763.
2) Regunathan, S.; Reis, D. J . Imidazoline Receptors and their
Endogenous Ligands. Annu. Rev. Pharmacol. Toxicol. 1996, 36,
2
2
[R]
3
14 2 2 2 4
H N ‚H C O
(
15) Riley, T. N.; Brier, C. G. Absolute Configuration of (+)- and (-)-
trans-2-Phenylcyclopropylamine Hydrochloride. J . Med. Chem.
5
11-544.
1
972, 15, 1187-1188.
3) Molderings, G. J . Imidazoline Receptors: Basic Knowledge,
Recent Advances and Future Prospects for Therapy and Diag-
nosis. Drugs Future 1997, 22, 757-772.
(
16) Inouye, Y.; Sugita, T.; Walborsky, H. M. Cyclopropanes. XVII.
The Absolute Configurations of trans-1,2-Cyclopropanedicar-
boxylic Acid and trans-2-Phenylcyclopropanecarboxylic Acid.
(
(
(
4) Milligan, C. M.; MacKinnon, A. C. Imidazoline Receptor Ligands.
Tetrahedron 1964, 20, 1695-1699.
Drugs New Perspect. 1997, 10, 74-84.
5) Bousquet, P. Imidazoline Receptors. Neurochem. Intern. 1997,
1
(
17) Compound 7a : H NMR (CDCl
3
) δ 0.52-2.01 (20H, m, 3-CH
2
and menthyl H); 2.39 (1H, m, CH-Ar); 2.79 (1H, m, CHCdN);
.77 (4H, m, NCH CH N); 4.57 (1H, m, COOCH); 7.08-7.29 (5H,
m, Ar-H). Compound 7b: H NMR (CDCl
m, 3-CH and H-menthyl); 2.49 (1H, m, CH-Ar); 2.94 (1H, m,
CHCdN); 3.78 (4H, m, NCH CH N); 4.65 (1H, m, COOCH);
.09-7.31 (5H, m, Ar-H).
1
18) Separovic, D.; Kester, M.; Ernsberger, P. Coupling of I -Imid-
3
0, 3-7.
3
2
2
6) Brasili, L.; Pigini, M.; Marucci, G.; Quaglia, W.; Malmusi, L.;
Lanier, M. L.; Lanier, B. Separation of R-Adrenergic and
Imidazoline/Guanidinium Receptive Sites (IGRS) Activity in a
Series of Imidazoline Analogues of Cirazoline. Bioorg. Med.
Chem. 1995, 3, 1503-1509.
1
3
) δ 0.72-2.10 (20H,
2
2
2
7
(
(
(
(
(
7) Brasili, L.; Pigini, M.; Bousquet, P.; Carotti, A.; Dontenwill, M.;
Giannella, M.; Moriconi, R.; Piergentili, A.; Quaglia, W.; Taye-
bati, S. K. Discovery of Highly Selective Imidazoline Receptor
Ligands. In Perspective in Receptor Research; Giardin a` , D.,
Piergentili, A., Pigini, M., Eds.; Elsevier: Amsterdam, 1996; pp
azoline receptors to diacylglyceride accumulation in PC12 rat
pheochromocytoma cells. Mol. Pharmacol. 1996, 49, 668-675.
1
19) Binding experiments on I receptors were performed as described
in ref 18, although crude membrane preparations were used.
Briefly, PC12 cells were homogenized with a Potter homogenizer
in ice-cold Tris-Hepes buffer (5 mM, pH 7.7) containing 0.5 mM
3
61-373.
8) Pigini, M.; Bousquet, P.; Carotti, A.; Dontenwill, M.; Giannella,
M.; Moriconi, R.; Piergentili, A.; Quaglia, W.; Tayebati, S. K.;
Brasili, L. Imidazoline Receptors: Qualitative Structure-Activ-
ity Relationships and Discovery of Tracizoline and Benazoline.
Two Ligands with High Affinity and Unprecedented Selectivity.
Bioorg. Med. Chem. 1997, 5, 833-841.
2
EDTA, 0.5 mM EGTA, and 0.5 mM MgCl and centrifuged twice
during 20 min at 75000g. P2 pellets were resuspended in the
same buffer at 2-4 mg of protein/mL and frozen at -80 °C until
used in binding experiments. Competition experiments were
1
25
carried out with 0.5 nM [ I]p-iodoclonidine (NEN), 50 µg of
-
10
-4
9) Carrieri, A.; Brasili, L.; Leonetti, F.; Pigini, M.; Giannella, M.;
Bousquet, P.; Carotti, A. 2-D and 3-D Modeling of Imidazoline
Receptor Ligands: Insights into Pharmacophore. Bioorg. Med.
Chem. 1997, 5, 843-856.
protein, and incresing concentrations of drugs (10 -10 M).
Incubations were performed for 30 min at 25 °C. Nonspecific
binding was determined with 10 µM BDF 6143.
(20) Cheng, Y. C.; Prusoff, W. H. Relationship between the Inhibition
Constant (K ) and the Concentration of Inhibitor which Causes
(
10) Pigini, M.; Bousquet, P.; Brasili, L.; Carrieri, A.; Cavagna, R.;
i
Dontenwill, M.; Gentili, F.; Giannella, M.; Leonetti, F.; Piergen-
tili, A.; Quaglia, W.; Carotti, A. Ligand Binding to I Imidazoline
2
Receptor: The Role of Lipophilicity in Quantitative Structure-
Activity Relationship Models. Bioorg. Med. Chem. 1998, 6,
50% Inhibition (IC50) of an Enzimatic Reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(21) Molderings, G. J .; Moura, D.; Fink, K.; Bonisch, H.; Gothert, M.
3
Binding of [ H]-Clonidine to I
1
-Imidazoline Sites in Bovine
2
245-2260.
Adrenal Medullary Membranes. Naunyn-Scmiedeberg’s Arch.
Pharmacol. 1993, 348, 70-76.
(
11) Kaiser, C.; Lester, B. M.; Zirkle, C. L.; Burger, A.; Davis, C. S.;
Delia, T. J .; Zirngibl, L. 2-Substituted Cyclopropylamines. I.
Derivatives and Analogues of 2-Phenylcyclopropylamine. J . Med.
Chem. 1962, 5, 1243-1265.
12) Compound 4a : H NMR (CDCl
(22) Piletz, J . E.; Zhu, H.; Chikkala, D. N. Comparison of Ligand
1
Binding Affinities at Human I -Imidazoline Binding Sites and
the High Affinity State of Alpha-2 Adrenoceptor Subtypes. J .
Pharmacol. Exp. Ther. 1996, 279, 694-702.
1
(
3
) δ 1.29 (1H, m, 3-CH
2
); 1.60 (1H,
m, 3-CH
2
); 1.70 (1H, m, CHCdN); 2.45 (1H, m, CH-Ar); 3.60
4H, s, NCH CH N); 3.95 (1H, br s, NH, exchangeable with D O);
.0-7.32 (5H, m, Ar-H). It was characterized as the hydrogen
(23) In the conformations selected for CoMFA studies, however, the
angle values were 29.5°, 114°, and 132° for 3, (1S,2S)-(+)-4b,
and (1R,2R)-(-)-4b, which imply energy differences of 0.50, 1.09,
and 0.60 kcal/mol, respectively.
(
7
2
2
2
oxalate salts: fusion started at 90 °C and was complete at 119-
20 °C (from 2-PrOH). Anal. (C12 ‚H ) C, H, N.
Compound 4b: mp 133-135 °C; H NMR (CDCl ) δ 1.41 (1H,
m, 3-CH ); 1.61 (1H, m, 3-CH ); 2.13 (1H, m, CHCdN); 2.50 (1H,
1
H
14
N
2
2 2 4
C O
1
3
2
2
J M991049M