Imidazoline Binding Sites and Their Ligands an Overview of the Different Chemical Structures - Med...

Imidazoline Binding Sites andTheirLigands: An Overview of theDi¡erent Chemical Structures

Christophe Dardonville, Isabel Rozas

1Instituto de Quımica Medica (CSIC). Juan de la Cierva 3, 28006-Madrid, Spain2Centre for Synthesis and Chemical Biology, Department of Chemistry, Trinity College

Dublin, Dublin 2, Ireland

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20007

!

Abstract: Since Bousquet et al. discovered the imidazoline binding sites (IBS) two decades ago,

when they realized that the antihypertensive drug clonidine interacts not only with the a2-adrenenoceptors (a2-AR) but also with a distinct imidazoline preferring binding site, these

receptors have been paid a great deal of attention. At least two subtypes, I1 and I2, have been

characterised based on their binding affinity for different radioligands, but their structures still

remain unknown. The pharmacological profile of these IBSs has been the objective of several and

very thorough reviews. However, a medicinal chemistry overview of the different IBS ligands

prepared to date has never been attempted. In this study, we attempt to compile all the different

chemical structures reported to date as IBS ligands and classify them in function of their chemical

structure and binding affinity for the different IBS subtypes. Thus, we comment on the different

endogenous IBS ligands known as well as the drugs described to interact with the I1-IBS which

have found application as antihypertensive drugs. Then, we review those compounds described in

the literature to interact with the I2-IBS, classifying them by their chemical families (imidazolines,

guanidines, 2-aminoimidazolines, b-carbolines). Finally, some conclusions are drawn.

� 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 5, 639–661, 2004

Key words: imidazoline binding site; ligands; structure activity relationship; guanidine; imidazo-

line; carboline

1 . I N T R O D U C T I O N

The concept of imidazoline receptors (IR), also known as imidazoline binding sites (IBS) or

imidazoline/guanidinium receptive sites (IGRS), has gained a great deal of interest in the last two

Contract grant sponsor: Spanish Ministerio de Educacion, Cultura y Deporte; Contract Grant number: SB2001-

0174. Contract grant sponsor: University of Dublin, Trinity College for funds (Start-Up Project No. 504/R01056).

Correspondence to:ChristopheDardonville, Instituto deQui¤micaMe¤ dica (CSIC). Juande la Cierva 3, 28006-Madrid,Spain.

E-mail: [email protected]

Medicinal Research Reviews, Vol. 24, No. 5, 639^661, 2004

� 2004 Wiley Periodicals, Inc.

decades since Bousquet et al.1 discovered that the well known antihypertensive drug clonidine and

its analogues produced their pharmacological effect in the central nervous system (CNS) by

interaction not only with the a2-adrenenoceptors (a2-AR) but also with an imidazoline preferring

binding site. Such IBS were pharmacologically distinct from the a-AR because they were not

activated by catecholamines. To date, pharmacological studies have allowed the characterisation of

two subtypes of IBS, I1 and I2, based on their binding affinity for different radioligands.2 I1-IBS

are characterised by their high affinity for 2-aminoimidazolines (or 2-iminoimidazolidines) such as

[3H]-clonidine, medium affinity for imidazolines such as [3H]-idazoxan, and low affinity for

guanidines such as amiloride. I2-IBS are preferentially bound by imidazolines ([3H]-idazoxan)

and guanidines and show lower affinity for 2-aminoimidazolines. Further pharmacological

evidences demonstrated the heterogeneous nature of the I2-IBS and allowed the subdivision into

two subtypes, I2A and I2B, depending on their affinity, high and low, respectively, for the guanidine

drug amiloride.3

In addition, some pharmacologically atypical receptors present in pancreatic b-cells and

involved in insulin secretion were identified and putatively designated as I3-IBS.4–6 The imidazoline

efaroxan is a selective agonist at the I3 receptor and its imidazole analogue (KU14R) is an antagonist.

Various proteins from rat and human brain were identified as IBS by immunological studies

which confirmed the heterogeneous nature of IBS.7 However, the IBS protein structures have not yet

been solved to date. It is known that I1-IBS are G-protein coupled receptors (GPCR) whereas I2-IBS

are not. The best candidate identified for the human brain I1-IBS is a protein of approximately 43 kD.8

I1-sites are present in synaptic plasmamembranes9 whereas I2-IBS are located principally in the

outer membrane of mitochondria of peripheral and central tissues.10,11 In humans, I2-IBS are

localized in high density in the CNS (especially in glia12), blood platelets, liver, adipocytes, and in

minor quantity in the kidney. Based on biochemical and pharmacological studies, it has been

suggested that I2-IBS could be an allosteric binding site of monoamine oxidase B (MAO-B),13,14

although, these findings are still a matter of controversy.15,16

Various substances such as CDS (‘‘clonidine displacing substance’’),17,18 the guanidine

agmatine,19 or the b-carboline harmane20 have been proposed as putative endogenous substrates of

IBS. This will be developed further in the next section.

Although there is still little knowledge on the pharmacological functions of IBS, a few

applications for which these receptors are being studied can be mentioned. I1-IBS are involved in the

control of blood pressure,21 and selective ligands such as moxonidine and rilmenidine are used

clinically to reduce blood pressure with minimum sedative effects which are mediated by a2-AR. Todate, no clear pharmacological functions have been discovered for I2-IBS. Nevertheless, alteration of

I2-IBS have been associatedwith eating behavior in rats22 and variousmental pathological conditions

such as major depression,7,23,24 neurodegenerative disorders,9,25,26 and tolerance and dependence

to opioids.27–29 Spinal antinociception was also studied in relation with different I2-IBS ligands

(i.e., agmatine,30 BU22431), showing a possible implication of these receptors.

From a chemical point of view, the nature of known IBS ligands seems relatively restricted and

the pharmacophoric moiety is generally related to guanidine- and imidazoline-like structures.

However, many different families of IBS ligands (i.e., heterocyclic, hetero-aromatic, aliphatic)

bearing such guanidine or imidazoline moieties have been constantly reported in the literature for the

last decade. In addition, new IBS ligands differing in their structures from the classical ‘‘guanidine/

imidazoline’’ paradigm such as endogenous amines or carbolines have been discovered. We thought

that it would be useful to compile all the different chemical structures reported to date as IBS ligands

and classify them in function of their chemical structure and binding affinity for the different IBS

subtypes.

The aim of this study is to give an overview of the chemical diversity and progress made in the

SAR studies of IBS ligands. For that reason, it is not intended to be an exhaustive literature review on

the IBS topic. Formore information on IBS pharmacology, the reader is refereed to thevast amount of

640 * DARDONVILLE AND ROZAS

studies published on the subject in the last years.25,32–44 Of special interest is the Issue 881 of the

Annals of New York Academy of Science (‘‘Imidazoline receptors and their endogenous ligands.

Current concepts and therapeutic potential’’), published in 1999.

2 . E N D O G E N O U S L I G A N D S

Several endogenous substances have been identified as imidazoline binding site ligands. Initially, a

CDS was detected but not identified.17,18 Later this substance was characterized as agmatine event

though other CDSs have been found. More recently, a b-carboline harmane (1) has been detected to

bind to the IBSs (Fig. 1).20

Agmatine (1-amino-4-guanidinobutane), an intermediate in polyamine biosynthesis, is bio-

synthesized by decarboxylation of L-arginine by arginine decarboxylase andmetabolized by diamine

oxidase. This metabolite was identified in 1994 in mammalian brains as an endogenous ligand of

IBSs.19 Agmatine (2) is widely distributed in the body and binds, like clonidine, to a2-AR (pKi< 5)

and IBS of all subclasses (pKi I1¼ 7.48, pKi I2< 5). The fact that agmatine and its biosynthetic

enzyme are present in different neuronal pathways and that its concentration was similar to that of

classic neurotransmitters suggested that agmatine could be a neurotransmitter itself.45 During the

90s, several studies on the possibilities of agmatine were carried out. Thus, it was demonstrated that

agmatine displayed more preferential affinity for I1 receptors than for different a2. Further, itcompetitively inhibited the activity of all isoenzymes of NO synthase.46 More recently it has been

shown that the CDSmediated cardiovascular activity appears not to be due to agmatine47 and that this

endogenous ligand may play an important role in regulating morphine tolerance/dependence and

withdrawal symptoms in chronic morphine treatments.48

Other CDSs than agmatine have been detected but not characterized. There are several endo-

genous ligands that bind to IBSs. Among them, the classic CDS, a partially purified entity that

displaces ligands binding both a2-AR and IBS and the immunoreactive CDS, a moiety that binds to

antibodies against clonidine, p-aminoclonidine, or idazoxan.49 In 1998 a CDS was isolated and

partially identified from bovine lung and brain indicating that this substance was related to

guanosine.50However, in 2000 another CDSwas isolated fromNG108-15 cells supporting the notion

of the existence of several CDSs.51

The third group of endogenous IBS ligands includes the b-carboline harmane (Fig. 1).

b-Carboline alkaloids result from the condensation between indoleamines, such as tryptamine,

and short-chain carboxylic acids (e.g., pyruvic acid) or aldehydes (e.g., acetaldehyde). These

compounds have been endogenously found in human tissues and could be formed as by-products of a

secondarymetabolism. It has been shown that harmane acts as an endogenous ligand at certain I1-IBS

and has central effects on blood pressure.20 Other studies have demonstrated that b-carbolinessuch as harmane stimulate locus coeruleus neuron activity by a mechanism independent of I1 and I2IBS.52

Figure 1.

IMIDAZOLINE BINDING SITES AND THEIR LIGANDS * 641

3 . I 1 - I B S L I G A N D S

I1-IBS can be distinguished from I2-IBS because they can be labeled by [3H]-clonidine and

[3H]-idazoxan whereas I2 sites can only be labeled by [3H]-idazoxan. A 43-kd protein has been

purified from the human brainstem and further considered to be the I1-binding protein. Binding

studies have shown that these types of non-adrenergic I1 receptors are involved in the

hypotensive action of imidazoline drugs. Therefore, I1-IRs have become therapeutic targets for

antihypertensives.53

A. 2-Aminoimidazoline Derivatives

Clonidine (3) was the first compound identified that exhibited affinity towards IBS showing

antihypertensive activity (Fig. 2). Hypotension has been associated to action at the I1-IBS; thus, it is

consistent that when considering the different subsites, clonidine seems to have higher affinity

towards the I1-IBS type, with a pKi of 7.25, than towards the I2-IBS (pKi¼ 6.02).26 The introduction

of a iodine atom in para-position of the dichlorophenyl moiety of clonidine yielded the compound

known as p-iodoclonidine (4). This structural change increased the affinity towards I1-IBS and the

selectivity versus I2-IBS (pKi I1¼ 8.31, pKi I2< 5).Unfortunately, such amodification also increased

the potency of binding to a2-ARs (pKi a2A¼ 8.65, pKi a2B¼ 8.13, and pKi a2C¼ 9.10).26,54 Further

and deeper changes in the structure of clonidine yielded moxonidine (5, Fig. 2). In this derivative the

phenyl ring of clonidine has been substituted by a pyrimidine ring, one of the chloro substituent has

been replaced by methoxy and the para-position of the aromatic ring is occupied by a methyl group.

With all these modifications it was possible to obtain a compound more potent versus I1-IBS

(pKi¼ 8.37) and much more selective versus I2-IBS (pKi< 5) and the three a2-AR subtypes (pKi

a2A< 5, pKi a2B< 5, and pKi a2C< 5).26,54

Figure 2.


B. Rilmenidine and its Analogues

It was with the intention of looking for new treatments for hypertension that rilmenidine (Fig. 2)

became the first I1-IBS selective antihypertensive agent to enter the therapeutic arena. Rilmenidine

(6) is an antihypertensive agent with selectivity for I1-IBS (pKi I1¼ 7.22; I1/I2¼ 18; I1/a2¼ 2)a that

acts both centrally by reducing sympathetic over activity and in the kidney by inhibiting theNaþ /Hþ

antiport.55 Experience from clinical trials and especially its efficacy/tolerance ratio not only

highlights the clinical and metabolic acceptability for the treatment of hypertension of rilmenidine,

but also supports its role as a first-line antihypertensive.

However, although rilmenidine has a low selectivity for I1-IBS this drug still binds to I2-IBS

(pKi¼ 5.96) and a2-AR (pKi¼ 6.90). This fact and the simple structure of this dicyclopropylamino-

oxazoline derivative has opened the possibilities for new compounds more I1-selective.

A family of aminopyrrolines analogues of rilmenidine (7) was prepared and evaluated versus I1-

IBS, I2-IBS, and a2-AR (Fig. 2).36 In this family, the oxazoline ring was substituted by a pyrroline

ring with different substituents (5-methyl, 3-methyl, 4-methyl, cis/trans-4,5-dimethyl, 5-ethyl and

cis-4,5-(CH2)4). Itwas not possible to improve the affinity towards I1-IBS: the best pKi (6.77)was that

of the 5-ethyl derivative.However, these compounds had amuch lower affinity towards a2-AR and I2-

IBS (all of them<5) implying an improvement in selectivity. This study revealed the importance of

the O atom of rilmenidine to distinguish between the pharmacophores of I1 and a2 receptors: theisosteric substitution of rilmenidine with a pyrrolinic ring abolished the binding affinity to a2-ARswhereas I1-IBS affinity was hardly affected. One of the compounds prepared, the cis/trans-dimethyl

derivative, was selective for I1-IBS (pKi I1¼ 6.27; pKi I2< 5; pKi a2< 5) and showed cardiovascular

activity.

C. 2-Arylimidazoline Derivatives

Even though the naphtylimidazoline benazoline (8) was described originally to have affinity towards

I2-IBS and, therefore, it will be thoroughly discussed in the following section, it has been demons-

trated recently that this compound is able to activate the I1-IBS (as an agonist) in a highly selective

way.56

Following this kind of structure, Anastassiadou et al.57 replaced the naphtyl moiety by other

aryl groups (naphtyl differently substituted and/or connected, heterocycles or phenyl rings

differently substituted) and then, these compounds were tested in vitro at I1- and I2-IBS, and a1-and a2-AR. Several of these derivatives were potent ligands of I1-IBS and especially the 2 0-methoxyphenyl-2-imidazoline derivative 9 (see Fig. 2) showed not only a very high I1 affinity

(pKi¼ 8.53) but also an excellent selectivity over both I2 sites and a-ARs. In general, it seems that I1-

IBS bind preferablywith phenyl-imidazoline analogues substitutedwith amethyl or amethoxy group

in the 2 0 or 3 0 positions.The heterocyclic 3-methyl-2-thiophene imidazoline derivative (10, Fig. 2) displayed high I1-IBS

affinity but low selectivity with respect to I2-IBS (pKi I1¼ 8.37; I1/I2¼ 8.5). No cardiovascular

effects were mentioned.

D. 2-Imidazolines

After the rational design of idazoxan (a benzodioxan-2-imidazoline derivative), several structural

modifications were made in order to enhance both its selectivity and affinity. As a result of these

studies and by substitution of the dioxane ring by a dihydrofuran onewith an ethyl substitution, (� )-

efaroxan (11, Fig. 2) was obtained.58 This compound is a mixed a2-adrenoceptor antagonist/I1-IBS

aThe selectivity is expressed as the antilog of the difference between the pKi values.


ligand (pKi I1¼ 7.28 in human platelets).26,54 According to some (but not all) studies, the affinity of

efaroxan for I1-receptors is much higher than its affinity for a2-ARs.59 The unsubstituted

hydrobenzofuran-2-imidazoline analogue, RX 801080 (12), was prepared as well showing a good

affinity towards I2-IBS (pKi¼ 7.32) and a2-AR (pKi¼ 7.31).60

Based on the structure of idazoxan (17) and efaroxan (11), new phenyl ethylene imidazoline

derivativeswere prepared byGentili et al. A significant improvement in selectivity between I1 and I2-

IBS was obtained by replacing the ethylene bridge by a cyclopropane ring. This substitution yielded

two optical isomers, and whereas the (1S,2S)-(þ ) form of 13 was more potent for the I1-IBS (pKi

I1¼ 7.93; pKi I2¼ 6.91) with an eudismic ratio2 of 29,61 the (1R,2R)-(�) onewasmore potent for the

I2-IBS (pKi I1¼ 6.46; pKi I2¼ 8.22) with an eudismic ratiob of 20. These results highlighted some

stereospecific requirements of the I1-IBS and I2-IBS subtypes and were the origin for the preparation

of other chiral agents.62 With this intention, the a-methyl derivative of the phenylethylen-2-

imidazoline (14) was prepared and the two enantiomers separated. Thus, the (S)-(�) isomer of

14 showed a high selectivity towards I1-IBS (pKi I1¼ 8.97; pKi I2¼ 6.84)with a selectivity ratio I1/a2of 4,667. The (R)-(þ ) enantiomer bound only to the IBSs andwith very lowaffinity (pKi I1¼ 5.2; pKi

I2¼ 4.9). These results show the importance of the chiral center with respect to I1 affinity. In vivo

studies showed that the (S)-(�) isomer had no significant cardiovascular effects. However, since (S)-

(�)-14 was selective for I1-IBS and able to prevent the hypotensive effects of clonidine, the authors

suggested it could be considered a useful new I1-IBS antagonist.62

Another compound showing high affinity for the I1-IBS (pKi¼ 7.66) and a high selectivity

towards adrenoceptors (145 over a2-AR and 250 over a1-AR) is the imidazoline analogue of

hydroxyindane, PMS 952 (15), described by Godfroid et al.63 This compound also exhibits a

moderate selectivity over I2-IBS (I1/I2¼ 5.4). In order to understand the affinity of this compound

towards IBS, its molecular flexibility and different conformations were analyzed using ab initio

calculations. It was concluded that PMS 952 presents an important conformational flexibility which

could have a great repercussion on its affinity towards IBS. However,many other factors are probably

involved in the discrimination between IBSs and a-ARs. No mention is made to the potential

antihypertensive properties of this compound, however.

E. Others

Compound AGN 192403 (16, Fig. 2) was the first agent equipotent to moxonidine in IBS binding

assays and nonpotent in adrenergic binding assays to be prepared. This compound showed an affinity

of 42 nM (pKi¼ 7.38) for the I1-IBS which is comparable to that of moxonidine and only 5-fold less

potent than clonidine. This amine derivative showed no binding activity at either a-AR or I2-IBS

evaluated.AlthoughAGN1924023 can be considered as the first ligand reported to bind selectively at

the I1-IBS,64 it is devoid of any antihypertensive activity.

4 . I 2 - I B S L I G A N D S

Since no structural data are available (i.e., these receptors have not been cloned), the better

understanding of the IBS functions and the pharmacological characterization of these receptors relies

on the discovery of selective I2-IBS ligands devoid of I1-IBS and a-AR affinity. Various research

teams took the challenge and made possible the discovery of very selective I2-IBS ligandsc over

2Eudismic ratio¼ antilog of the difference between the pKi values for the eutomer and the distomer.cThe most commonly used radioligands for identification of I2-IBS are [3H]-idazoxan or [3H]-2-BFI in the presence of

rauwolscine, yohimbine, or epinephrine to block a2-AR. [125I]-AMIPI and [125I]-AZIPI are high affinity photosensitive

derivatives of the imidazoline cirazoline which proved useful for autoradiographic studies.


I1-IBS and a2-ARs. The I2-IBS ligands described to date belong to four chemical families:

imidazolines, which are the most selective ligands discovered until now, guanidines, 2-

aminoimidazolines, and carbolines.

In this section, unless mentioned, the binding affinity reported for IBS relates to rabbit kidney

preparations in the case of I2-IBS, bovine adrenal gland for I1IBS, and rat calf cerebral cortex for

a-AR.3,65,66

A. Imidazolines and Amidines

1. Benzo Fused Heterocyclic (Benzofuran, Benzoxazine, Benzodioxane)

Idazoxan (17), the I2-IBS prototype, is an imidazoline antagonist at a2-adrenergic receptors whosebinding properties to I2-sites were first mentioned in 1987.43 This 2,3-dihydro-benzo[1,4]dioxane

imidazoline is a good ligand for I2-IBS (pKi I2¼ 8.37) and a2-AR (pKi a2¼ 7.72) with only a slight

selectivity for the I2-sites (I2/a2¼ 4.5).67 This prototype of I2-IBS ligand opened the route to the

discovery of various families of fused aromatic heterocyclic compounds such as benzodioxane,60

benzofuran (e.g., 2-BFI),60,68 or benzoxazine69 derivatives with more potent and selective binding

affinities (Fig. 3).

Figure 3.


During the development of idazoxan (17) and related a2-AR antagonists in the early 80’s,

Chapleo et al. prepared many imidazoline drugs58,70 which would be later assayed as I2-IBS ligands.

Benzodioxane, benzodioxolane, and benzofuran analogues of idazoxan were tested on whole rabbit

brain membranes which allowed the discovery of the potent and selective I2-IBS ligand 2-BFI (25).60

The study of the substitution of the benzene ring of idazoxan revealed that halogen substituents (Cl,

Br) at the 6- or 7-position tended to slightly increase the selectivity over a2-AR and were clearly

preferred to 6- or 7- methyl, methoxy, or 6,7-benzo derivatives (18). However, these results reflected

mainly a decrease in a2-AR affinity more than an increase in I2-IBS activity, except for 6,7-

dichloroidazoxan (18, R¼ 6-Cl; R 0 ¼ 7-Cl) that was more active (pKi I2¼ 8.06) and selective (I2/

a2¼ 41.3) than idazoxan (pKi I2¼ 7.97; I2/a2¼ 5.2). Modifications in the benzodioxane ring were

also studied affording the 1,3-benzodioxane isomer of idazoxan RX821029which ismore potent and

selective (19: pKi I2¼ 8.57; I2/a2¼ 161.5) than the parent compound (Fig. 3). On the contrary, the

benzodioxolyl analogues RX791042 (20) and RX801024 (21) showed low affinity and a moderate

selectivity towards a2-AR. The results of the assay of chromanyl analogues showed that removal of

the oxygen atom at position 4 (22) did not change neither the affinities nor the selectivity, whereas

removal of the 2-oxygen (23: pKi I2¼ 7.4; I2/a2¼ 10) decreased affinity for I2-IBS (4-fold) and a2-AR (10-fold) compared to idazoxan. Another interesting feature was the clear influence for affinity

and selectivity of the introduction of a double bond (i.e., rigidity and planarity) into the chromane

cycle. This was demonstrated the same year by Pigini et al. who showed the increased affinity and

selectivity of the chromene analogue 24 (pKi I2¼ 8.43, pKi a2¼ 6.15; I2/a2¼ 191).71

The highly active and selective ligand 2-BFI (25: pKi I2¼ 8.89; pKi a2¼ 4.57; I2/a2¼ 2,874)

was discovered by Hudson et al. in their effort to find new selective ligands for central I2-IBS relative

to a2-AR.60 Since this molecule proved also to be selective in different species, the authors pointed

out at least certain pharmacological homogeneity of I2-IBS between species. As it appears for the

chromene derivative (24) of idazoxan (vide infra), the loss of planarity in the dihydrobenzofuran

analogue RX801080 (12, Fig. 2) led to a total loss in selectivity towards I2-sites (pKi I2¼ 7.32; pKi

a2¼ 7.31; I2/a2¼ 1). Regarding the aromatic ring of 2-BFI, electron withdrawing substituents such

as halogens were well tolerated and 7-chloro-2-BFI (26) retained almost the same affinity (pKi

I2¼ 8.55) and selectivity (I2/a2¼ 2,192) as the parent compound. Finally, 4,6-dibromo-2-BFI (28),

although slightly less potent and selective than 2-BFI, was of interest because it was used as a

precursor for the synthesis of the radioligand [3H]-2-BFI by catalytic tritium gas exchange.

5-Isothiocyanato-2-BFI (27, BU99006) is an irreversible I2-IBS ligandwhich proved very active

and selective for I2-sites (greater than three orders of magnitude compared to I1-IBS and a2-AR) inwhole rat brain membranes in vitro (pKi I2¼ 8.6)72 and in vivo.73 This compound was an

improvement compared to another irreversible ligand, 2-(4-isothiocyanatobenzyl)-imidazoline

(IBI), which had only moderate affinities for I2-IBS.74,75

More recently, Touzeau et al. chose the dihydro[1,4]benzoxazine skeleton as a bioisostere of the

dihydro[1,4]benzodioxanemoiety.69 They prepared three series of benzoxazine derivatives aswell as

tricyclic analogues and evaluated the binding affinities for IBS, a1- and a2-adrenoceptors of 29

compounds (Fig. 4). The cardiovascular effect of these molecules was also evaluated in vivo. A few

important conclusions regarding the SAR of this series of compounds could be summarized as

follows:

(a) the bioisosteric replacement of the 4-oxygen of idazoxan with a nitrogen atom (compound 29,R1¼R2¼H) did not affect significantly the binding affinity for I2-IBS (pKi I2¼ 7.29) but did

increase considerably the selectivity over a2-AR (I2/a2 > 2,000) compared to the non-selective

idazoxan;

(b) 2,2-disubstituted benzoxazinic derivatives (29, R1¼Me, R2¼Me, n-Pr) were hardly active:

introduction of alkyl groups on the carbon-2 bearing the imidazoline moiety considerably

decreased IBS activity and almost totally suppressed a-AR affinity. This behavior should be


compared with the enhanced a2-AR activity and reduced I2-IBS affinity produced by the

introduction of a methoxy substituent at the same position in idazoxan.76 In the benzofuran

series, introduction of an ethyl substituent in a to the imidazoline ring in efaroxan reduced the I2-

IBS affinity compared to RX801080 (12, Fig. 2);

(c) substitution of the benzoxazine nitrogen atom by a methyl group (29, R1¼Me, R2¼H)

increased the affinity for both IBS and a-AR but did also reduce the selectivity (I2/I1¼ 7.1, I2/

a2¼ 10). Larger substituents (29, R1¼Bn, n-Pr; R2¼H) clearly reduced the affinity towards IBS

and a2-AR by one or two orders of magnitude;

(d) mono substitution on the aromatic ring seemed to benefit IBS affinity, giving a 6-methyl

substituent (30, R¼Me) the best activities on all the receptors (pKi I1¼ 7.66, pKi I2¼ 8.7, and

pKi a2¼ 7.15) and a moderate selectivity towards I2-IBS (I2/I1¼ 10.96, I2/a2¼ 35.3). Other

substituents in 6-position (30, R¼OMe, CF3, CH2OH) were detrimental to the affinity for both

IBS and a-AR. Substitution at the position 6 by halogens such as F or Cl atoms did not havemuch

influence on the activity. However, the most remarkable result of the 6-substitution was the

increased selectivity towards I2-IBS (I2/a2> 126) foundwith the 6-methoxycarbonyl substituent

(30, R¼CO2Me) with no detrimental effect on the I2-IBS activity compared to the non

substituted analogue (30, R¼H);

(e) effect of the disubstitution of the benzene ring with two methyl groups was dependent on the

relative position of the substituents. Thus, 6,7-Me analogue 31 was a good ligand, whereas the

6,8-Me counterpart was not active. In addition, fused additional cyclohexyl or benzene ring (32)

reduced the affinity for both IBS;

(f) oxazinoindole and oxazinoquinoline tricyclic analogue (33) binding affinity depends largely onthe structure of the ring: only the five-member ring analogue (33, n¼ 3) retained high IBS and a2-AR activity whereas introduction of a double bond (i.e., indole-like compound 34) led to themost

active (pKi I2¼ 8.45, pKi I1¼ 7.88, and pKi a2¼ 6.44) and selective molecule towards I2receptors (I2/I1¼ 3.7 and I2/a2¼ 102). In these tricyclic analogues, increasing the size of the ring

or decreasing the basic character of the oxazino nitrogen atom (i.e., introducing a keto moiety)

was detrimental to the activity.

Figure 4.


2. 2-Phenoxymethyl and Phenethyl Imidazolines (Cirazoline Derivatives)

Cirazoline (35), a potent a1-AR agonist and a2-AR antagonist belonging to the 2-phenoxymethyl

imidazoline family,77 is a good I2-IBS ligand (pKi I2¼ 7.90) which is also selective over a1-AR (I2/

a1¼ 25).78 Since the first report of the possible separation of a-AR and IBS activity by simple

structural modifications of cirazoline,78 the group of Pigini put a large synthetic effort and carried out

qualitative67,71 and quantitative79 SAR studies on these series of molecules (Fig. 5). They first found

out that the cyclopropyl moiety was not essential for either a-AR or I2-IBS affinity. Hence, the

cyclopropyl ring could be replaced by a linear alkyl group (36: R¼Me, Et, n-Pr, allyl; R 0 ¼H) hardly

modifying I2-IBS affinity, whereas a branch alkyl or alkoxy group (36: R¼ i-Pr, t-Bu, OMe, Oi-Pr,

R 0 ¼H) was detrimental for I2-IBS activity.78 They carried out structural modification on the

aromatic ring and on the oxymethylene-bridge linking the imidazoline to the phenyl moiety. From

these studies it appeared that the insertion of a second phenyl ring in the structure of cirazoline (37:

R1¼ Ph, Bn;R2¼H;X¼NHorOH)was detrimental to the affinity and selectivity at I2-IBS.80 It also

appeared that the bridge linking the imidazoline to the phenyl ring played a key role in themodulation

of the affinity and selectivity between IBS and a-AR. Hence, methylation at the b-position (e.g., 39:pKi I2¼ 5.57; I2/a2¼ 0.036) dramatically decreased the affinity and selectivity of the cirazoline

derivative 38 (pKi I2¼ 9.05; I2/a2¼ 58.8).80 Isosteric replacement of the oxygen atom by a

methylene group (compound 40) reduced (a2-AR) or eliminated (a1-AR) a-adrenergic activity whileconserving I2-IBS affinity (pKi I2¼ 8.6; I2/a2¼ 794).71 Compound 40 was a new I2-IBS lead which

was further modified by the same team affording a new family of highly potent and selective ligands

for the I2-IBS with respect to the a2-AR (e.g., tracizoline (44), benazoline (8)). These 2-trans-styrylimidazolines and congeners will be discussed in the next section.

The reverse affinity and selectivity observed for the a-methylated (14: pKi I1¼ 8.3; I2/

I1¼ 0.005; I2/a2¼ 4) with respect to the b-methylated (41: pKi I2¼ 6.91; I2/I1¼ 49; I2/a2¼ 10)

analogue of 40 emphasize the key role played by the ethylene bridge in modulating I1- and I2-IBS

selectivity in these series of molecules.62 Another remarkable feature was the distinct stereospecific

requirements of the I1- and I2-IBS sites already discussed in section 3. The resolved enantiomer (S)-

(�)-14 proved to be the eutomerd with respect to I1-IBS (pKi I1¼ 8.97; pKi I2¼ 6.84; I2/I1¼ 0.007;

Figure 5.

dEutomer: the most efficacious of the enantiomeric pair.


I1/a2¼ 4,667) whereas the distomer (R)-(þ )-14 was a weak IBS ligand (pKi I1¼ 5.20; pKi

I2¼ 4.90). These results were in agreement with the findings for the 2-(2-phenylcyclopropyl)imi-

dazoline 13 (Fig. 2), which also showed a reversal enantioselectivity at the two IBS subtypes (see

section 3).

Noteworthy is the effect ofmethyl-substitution on the imidazoline ring (Fig. 5)which provoked a

reduction (42) or even eliminated (43) I2-IBS activity while reducing a1-adrenergic agonist

activity.71 Such unfavorable behavior for the a1-AR activity had been previously described for a

different series of imidazoline derivatives.81

3. 2-Trans-Styryl-Imidazolines and Heteroaromatic Congeners

The discovery of 2-trans-styryl-imidazolines as highly selective I2-IBS ligands with respect to a-ARwas the result of the intense SAR studies carried out on the a1-AR agonist cirazoline by Pigini and co-

workers (vide infra). They successively removed the a1-adrenergic component of cirazoline by

isosteric replacement of the oxygen atom by amethylene group to obtain 40 and then discovered high

affinity and selective I2-IBS ligands by conformational restriction of 40. Reduction of the

conformational flexibility of 40 with a trans-styryl bridge afforded the very potent and selective

tracizoline (pKi I2¼ 8.74; I2/a2¼ 7,762; I2/a1¼ 2,344) whereas the even more rigid ‘‘naphthalene-

like’’ molecule named benazoline (8) displayed high affinity and unprecedented I2-IBS selectivity

(pKi I2¼ 9.07; I2/a2¼ 18,621; I2/a1¼ 2,691) with respect to a-AR (Fig. 6).71 The study of various 2-

trans-styryl analogues revealed that the relative position of the aromatic and imidazoline rings played

a crucial role in the I2 binding (e.g., 2-naphtalen-1-yl-imidazoline was less active than benazoline)

and the region accommodating the aromaticmoiety of the ligandswas of limited size as demonstrated

by the lower activity of some polyaromatic derivatives (e.g., 2-phenanthren-9-yl-, 2-(2,2,-diphenyl-

ethyl)-, 2-biphenyl-4-yl-imidazoline congeners).

However, benazoline was unable to discriminate between IBS subtypes.82 This was resolved

with the isosteric substitution of the naphtyl ring by a quinoline (45, BU224) or isoquinoline (46,

BU226) nucleus, affording potent I2-IBS ligands (pKi I2¼ 8.7 and 8.85, respectively) selective with

respect to I1-IBS (I2/I1¼ 832 and 380, respectively).83

The authors of these SAR studies attempted to rationalize their findings and developed 3-D

models (CoMFA analysis) in order to understand the key structural features (steric and electrostatic)

governing the binding of a large set of I2-IBS ligands and the structural requirements for I2-IBS/a2-AR selectivity. The first modeling studies,67 involving a set of 40 compounds (i.e., cirazoline,

Figure 6.


tracizoline, idazoxan, and benazoline derivatives), revealed that planar ligand conformations seemed

to characterize the binding to I2-IBS whereas more puckered conformations could be involved in the

binding to a2-AR. A refinement of this model, taking into account the molecular liphophilicity

potential (MLP) as an additional field, was carried out using a new series of 2-trans-styryl

imidazolines related to tracizoline (47, 48, and 49).79 Regarding the SARof these series of analogues,

all the para-substituted derivatives (R¼ 4-Cl, -I, -Me, -OMe, -OH, -NH2, -NO2) showed high I2-IBS

affinity (pKi I2¼ 7.61–8.54), although lower than the parent compound tracizoline (44: pKi

I2¼ 8.74). On the contrary, ortho-substituents such as 2-N3 and especially 2-Me groups afforded

ligands with higher I2-IBS affinity and selectivity (pKi I2¼ 8.96 and 9.43, respectively; I2/

a2¼ 2,455) than tracizoline. The only meta-substituted analogue (48, R¼ 3-Me) showed also high

affinity for I2-IBS (pKi I2¼ 9.05). The substitution of the phenyl ring of tracizoline by azo-

heterocycles (49, R¼ 2-pyridyl, 2-pyrrolyl) or alkyl groups (49, R¼ i-Pr, c-hexyl) reduced I2-IBS

affinity whereas oxo- (49, R¼ 2-furyl) and sulpho- (49, R¼ 2-thienyl) heterocycles retained high I2affinity. All these tracizoline derivatives were poor a2-AR ligands (pKi< 6).

The results of activity of these tracizoline congeners taken together with a set of cirazoline

analogues were used to develop a tri-dimensional model that revealed the most relevant steric,

electrostatic, and lipophilic interactions accounting for high I2-IBS affinity.79 The main interactions

important for I2-binding affinity could be summarized as follows:

(a) favorable lipophilic (and steric) interaction close to the 2-ortho and 3-meta positions of the

phenyl ring;

(b) hydrophilic/electronic interaction on the 5-meta region;

(c) unfavorable steric hindrance in two regions: one near and above the imidazoline ring and the

other in the distal part of planar polycyclic ligands;

(d) unfavorable high energy density in an area above the ortho-region.

More recently, in an effort to develop an I2 tri-dimensional pharmacophore, Baurin et al. carried

out a 3D-QSAR CoMFA analysis on 119 imidazolinergic I2 ligands. The compounds studied

represented the main families of I2 imidazoline ligands described at that time (i.e., molecules

described in this section A), although guanidines (aliphatic or aromatic), 2-aminoimidazolines, or

carbolines (i.e., molecules presented in section B, C, and D) were not taken into account. The first

elements of the I2 pharmacophore presented in that study were a nonsubstituted imidazoline cycle

(plane p1) and a 2.5 A wide aromatic zone (plane p2) with a dihedral angle of 142� such as the

imidazoline C2 atom belongs to both planes p1 and p2. A set of electronic and steric favorable or

unfavorable tri-dimensional regions were also defined.84

4. 2-Aryl and 2-Heterocyclic Imidazolines and Amidines

An insight into the IBS affinity of 2-aryl and 2-heterocyclic imidazolines was possible thanks to the

work of Anastassiadou et al. who tested 35 substituted phenyl compounds and 11 heterocyclic

imidazolines as IBS and a-AR ligands (Fig. 7).57 Overall, it appeared that the linkage of the

imidazoline nucleus at the 2-position with an aromatic substituent eliminated the a-AR activity.

In addition, I2-IBS seemed more accessible by the 2-phenyl imidazolines with a substituent in

para-position (compound 50) whereas I1-IBS were more accessible by phenyl rings substituted in

ortho- or meta-position by a methyl or a methoxy group (e.g., compound 9, Fig. 2, pKi I1¼ 8.53; I1/

I2¼ 3,388). The best example of this was the high I2-IBS affinity and selectivity of the p-methyl

substituted compound 51 (pKi I2¼ 8.53; I2/I1> 3,388) compared to the o-methyl substituted

analogue 52 which was selective for I1-sites (pKi I1¼ 7.64; I2/I1¼ 0.0046).

Among the branched-heterocyclic imidazolines tested, pyridine, pyridazine, 2-furan, 9 0-xanthene, and 2-thiophene derivatives had low affinity for both I-sites (pKi< 7) and no a-AR activity.


An interesting result was the similar affinity and selectivity found for the 5 0-indolyl imidazoline

derivative 53 (pKi I2¼ 8.57; I2/I1¼ 20) compared to the benzofuran 2-BFI. Regarding the branching

of the imidazoline nucleus on the heterocycle, the 2-position (a to the heteroatom) seemed most

favored and loss of affinity for both IBS was observed when moving the imidazoline moiety to the 3-

or 4-position in the case of chromanyl (22 and 23; Fig. 3),60 quinoline (BU224 and 3-regional isomer;

Fig. 6), and pyridine derivatives.57

It should also be pointed out that in this study, benzo-fused analogues (e.g., quinoline and

benzofuran) were more potent and selective compared to their simple heterocyclic parents (i.e.,

pyridine and furan derivatives). Although this observation is limited to three pairs of compounds, it

might be relevant for the design of new selective I2-IBS ligands.

The I2-IBS binding affinity of amidines was reported in only few cases. The amidine analogues

of RX821029 and idazoxan (55 and 56, Fig. 7) showed about 145- and 20-times less affinity towards

I2 sites than the parent compounds, respectively.84 In another study, Wood et al. showed that the

amidine pentamidine (54, Fig. 7) displayed potent inhibition of [3H]-idazoxan binding to I2-IBS in a

rat livermembrane preparation comparable to that of its imidazoline analogue.85 The full structure of

this 1,5-bis(4-amidinophenoxy)pentane was necessary to get high I2-IBS affinity since removal of

one benzamidine moitey led to a 228-fold decrease in affinity. In addition, the effect of meta-

substitution by a methoxy group led to significant loss of I2-IBS affinity, which is in agreement with

the observationmade for the 2-arylimidazoline series (vide infra). The authors suggested that affinity

for I2-IBS, although not related to antimicrobial activity, may be related to mechanisms of toxicity of

this drug which is used for the treatment of African trypanosomiasis and Leishmaniasis.86

B. Guanidines

1. Aliphatic Guanidines: ‘‘Agmatine-Like’’ Ligands

As commented in section 2 of this review, agmatine (2) was first reported as putative endogenous IBS

ligand by Li et al. in 1994.19 Agmatine is an example of aliphatic guanidine that binds to I1-IBS

Figure 7.


(pKi¼ 7.48) and I2-IBS although with low affinity (pKi< 5).54 However, in regard to the importance

of this molecule in the CNS,45 it results quite surprising that so few aliphatic guanidines (i.e.,

‘‘agmatine-like’’ ligands) have been reported or tested as IBS ligands until now.87,88

A set of bis-guanidines and their mono-guanidine counterparts bearing different chain lengths

(57), structurally related to agmatine, were tested88 in membranes from postmortem human frontal

cortex, a brain area that shows an important density of I2-IBS and a2-ARs (Fig. 8).89–91 A few

compounds displayed good I2-IBS affinity (57: n¼ 5, 8) and in general, the longer the aliphatic chain

used, the better both affinity and selectivity. The bis-guanidine derivative with the longest alkylic

chain (57, n¼ 8) showed the highest affinity (pKi I2¼ 7.48), similar to idazoxan, and a better

selectivity (I2/a2¼ 15.5 compared to 1.35 for idazoxan). The presence of two guanidines was also

important since themono-guanidine analogues (58) were inactive at both receptors. Thus, it appeared

that the lipophilicity played a significant role in this series of alkylic bis-guanidines. Nevertheless,

since I1-IBS are in very low density in human postmortem brain, it was not possible to evaluate in that

study the I2/I1 selectivity of these compounds.

Recent findings by our group92 confirmed the potential of aliphatic methylenic compounds as

potent I2-IBS ligands; thus, fentanyl derivatives bearing a guanidine at the end of a long alkylic chain

(Fig. 8) showed high affinity for I2-IBS, increasing with the chain length (59, n¼ 10, pKi I2¼ 8.24 in

membranes of postmortem human frontal cortex).

2. ‘‘Aromatic’’ Guanidines

Historically, guanidines and aminoguanidines N-substituted with an aromatic or heteroaromatic

nucleus (i.e., ‘‘aromatic’’ guanidines) such as amiloride and guanabenz (60 and 65, Fig. 9) have been

important ligands used for the characterization of IBS. In particular, amiloride and its analogueswere

able to discriminate between I2-IBS subtypes whereas imidazoline drugs (e.g., idazoxan, 2-BFI) had

similar affinities for both subtypes.74 Alkylation of the 5-amino-pyrazine group tended to slightly

reduce affinity at the I2A-IBS (rabbit cerebral cortex) and increase affinity at I2B-IBS (rat cerebral

cortex) compared to amiloride in the following order: amiloride> 61> 62> 63> 64 (Fig. 9).

A series of 12 Schiff bases of 1-(benzylidenamino)-3,3-dimethylguanidine (66), structurally

related to guanabenz and substituted on the aromatic nucleus, were tested as I2-IBS and a2-ARligands in guinea pig cerebral cortex.93 To the difference with guanabenz (which shows 10-fold

selectivity for a2-AR), all of the dimethylguanidines were selective for I2-IBS with respect to a2-AR.

Figure 8.


The authors came to the conclusion that the two N-methyl susbtituents were crucial for I2 selectivity.

The highest I2-IBS affinity and selectivity (pKi I2¼ 8.3; I2/a2¼ 110) was observed for the 2-bromo

derivative WAP8 (66, R1¼Br, R2¼R3¼R4¼H). In general, best affinity and selectivity were

observed for 2-substituted and 2,6-disubstituted benzene ring with the following order: 2-Br> 2-

Cl> 2-Me> 2-F> 2-NH2. In the case of 2,6-disubstituted aromatic nucleus, a2-AR activity slightly

increased leading to less selective compounds. The effect of para-substitution with a methyl group

seemed to be unfavorable to I2-IBS activity. Interestingly, this result is opposite to the effect of para-

methyl substitution in the 2-arylimidazoline series (see compound 50, R¼Me; Fig. 7) reported by

Anastassiadou et al.

The aminoguanidine aganodine (67, Fig. 9), with a 1,3-dihydroisoindoline scaffold, could be

considered as a guanabenz-likemolecule. This compound proved to be a very good ligand for both I2-

IBS subtypes (pKi¼ 8.22 and 8.70 for I2A and I2B).74

C. 2-Aminoimidazolines (2-Iminoimidazolidines)

Classical 2-aminoimidazolines drugs such as clonidine (3), p-aminoclonidine or brimonidine (70)

have been traditionally used for their agonistic action at central a2-adrenergic receptors. Only laterwas their affinity for IBS discovered. Clonidine-like drugsmainly bind to I1-IBSwhich could explain

why we found only very few data on the study of 2-aminoimidazoline compounds binding with high

affinity to I2-IBS. The 5-bromoquinoxaline brimonidine (70, Fig. 10) is an example of 2-

aminoimidazoline showing a good binding affinity to I2-IBS (pKi¼ 7.47) and different selectivity

with respect to a2-AR subtypes (I2/a2A¼ 6.3; I2/a2B¼ 28; I2/a2C¼ 57).26,94

The 2,3-dihydro-1H-isoindolyl imidazoline RS45041 (68, Fig. 10), a 2-aminoimidazoline

compoundwhose 2-amino group is included into the isoindoline nucleus, is a ligand very selective for

I2-IBS over a2-AR (pKi I2¼ 9.37; I2/a2 > 23,000).95,96 A possible explanation for this atypical I2-

affinity and selectivity (for a 2-aminoimidazoline derivative) could be the conformational restriction

induced by the isoindoline ring, or the methylene group separating the 2-aminoimidazoline moiety

from the phenyl ring. Nevertheless, more data on analogous series would be needed in order to

understand the SAR of RS45041.

A series of 2-(4,5-dihydro-1H-imidazol-2-yl)indazole derivatives of indazim (69, Fig. 10), were

prepared by Saczewski et al. and tested as I2-IBS ligands. The parent compound indazim exhibited a

moderate affinity to I2-IBS whereas its 4-chloro derivative (69, R¼Cl) showed good affinity (pKi

Figure 9.


I2¼ 7.5) and excellent selectivity with respect to a2-adrenergic receptor (I2/a2> 3,076).97 In these

series, the methyl or chloro substitution in position 4- or 7- of the indazole heterocyle appeared to be

favorable for I2-IBS affinity. Moreover, the 4,5,6,7-tetrahydro-2H-indazole analogue of indazim

showed lower affinity than the parent compound, possibly due to additional unfavorable steric

interaction of the cyclohexyl compared to a planar phenyl ring.

Finally, interesting results were obtained with a series of symmetric bis-2-aminoimidazoline

compounds structurally related to clonidine.88 Their affinity for I2-IBS, tested in membranes from

postmortem human frontal cortex, was moderate (e.g., 71; X¼NH; pKi¼ 6.29) and varied

depending on the bridge linking both phenyl rings (NH>CH2>CO>> SO2). These molecules,

however, weremore selective towards a2-AR, with themethylene bridge giving a highly selectivea2-AR ligand (71; X¼CH2; pKi a2¼ 8.80; I2/a2¼ 0.002). The guanidine analogues of these molecules

were also tested but proved less active at I2-IBS than the 2-aminoimidazoline parents.

D. b-Carbolines

As mentioned in section 2, the b-carboline harmane (1) was recently identified as a ligand for IBS.20

This has opened an avenue in the search for new compounds interacting with this IBSs. Husbands

et al. examined the binding of an extended series of b-carbolines to imidazoline sites with the

objective to understand the structural motifs important for this interaction. Thus, they havemeasured

not only the I1-, I2-IBS and a2-AR affinities of harmane and some of its derivatives (norharmane,

harmine, harmalan, harmalol, and harmaline), but also of a number of differently substituted dihydro-

and tetrahydro-b-carbolines prepared by them.98 They found that, from the harmane family (Fig. 11),

harmine (72, a b-carbolinewith R2¼OMe and R5¼Me) has a high affinity towards I2-IBS (pKi¼ 8)

and good selectivity towards I1-IBS and a2-AR (pKi I1¼ 6.20; pKi a2< 5).

Figure 10.

Figure 11.


One of the dihydro-b-carbolines (73, R2¼OMe; R3¼H; R5¼H) showed an interesting affinity

towards I2-IBS (pKi¼ 7.74) and some selectivity versus I1-IBS (pKi¼ 5.52) and a2-AR (pKi< 5).

However, the best affinity towards I2-IBS was that exhibited by a tetrahydro-b-carboline (74,

R1¼R2¼R3¼R5¼R6¼R7¼H; R4¼Me) with a pKi¼ 8.27 and a low I1-IBS affinity

(pKi¼ 6.70). From all their studies they concluded a number of structure–activity relationships:

(a) substitution on the aryl ring is well tolerated or even beneficial for I2-IBS;

(b) substitution at C8 (R3) results in poor affinity;

(c) replacing R4¼H by R4¼Me results in an increase in I1-IBS affinity with no change in I2-IBS

one;

(d) I2-IBS affinity is dramatically lowered if R5 is out of the plane as in the case of tetrahydro

derivatives; therefore, a planar structure is preferred for I2 binding;

(e) replacing H by Me at R6 position lowers I2-IBS affinity with no change in I1;

(f) when R7¼COOH affinity is abolished in the tetrahydro-b-carboline series. Whether this is

relevant to the pharmacological profile of b-carbolines has yet to be determined.

In order to get an insight into themode of binding of b-carbolines relative to the 2-imidazoline 2-

BFI, Glennon et al. prepared a fused imidazopyridoindolemolecule (75) that can be seen as an hybrid

of 1,2,3,4-tetrahydro-b-carboline and 2-BFI. Compound 75 displayed an I2-IBS affinity similar (pKi

I2¼ 8.14) to that of the tetrahydro-b-carboline 74 (R1–R7¼H, pKi I2¼ 8.02), however, its

selectivity towards I1-IBS and a2-adrenergic receptors was somewhat lower. The authors attributed

these results to the embedded imidazoline structure.99

E. Miscellaneous

The relative prominence of the sections dedicated to imidazolines and guanidines as I2-IBS ligands

reflect the larger amount of data regarding the IBS activity of such structures. However, other

structures such as imidazoles, amines, or 2-alkylimidazolines were also found to bind the I2-IBS.

Since there were only a few examples of such compounds, we decided to classify them as mis-

cellaneous I2-IBS ligands (Fig. 12).

As we mentioned in the ‘‘Introduction,’’ two subtypes of I2-IBS were proposed, I2A and I2B (in

rabbit and rat cerebral cortex, respectively), on the basis of their differential susceptibility to binding

the guanidine drug amiloride. Olmos and co-workers tested the affinity of various drugs such as the

imidazole medetomidine and various MAO inhibitors (e.g., chlordimeform, clorgyline).74 The

imidazole (þ )-Medetomidine (76: pKi I2A¼ 5.95; pKi I2B¼ 7.26; I2B/I2A¼ 20.4) and the ir-

reversible MAO inhibitor Clorgyline (81: pKi I2A¼ 5.45; pKi I2B¼ 7.27; I2B/I2A¼ 66.7)

discriminated between both receptors subtypes and were more potent on the high affinity I2B-sites.

On the contrary, the formamidine pesticide chlordimeform (reversible MAO inhibitor) showed high

affinity for both I2-IBS subtypes (pKi I2A/B� 8.22). However, other MAO inhibitors amino

derivatives such as 2-phenethylhydrazine (Tranylcypromine) or trans-(� )-2-phenylcyclopropana-

mine (Phenelzine) displayed low affinity for both I2-IBS subtypes.

Several endogenous indolamines were also tested as I2-IBS ligands in rat and rabbit brain

membranes.100 Isatin (85) and histamine (86) displayed low to moderate binding affinity for I2-sites

in both preparations, whereas tryptamine (84) was of higher affinity (pKi¼ 4.57 and 5.52 in rat and

rabbit brain, respectively). The presence of a hydroxyl group in 5-hydroxytryptamine (83) reduced I2-

IBS affinity by about 7-fold compared to the parent compound. Remarkably, in this study, the

endogenous tryptamine was a better ligand than agmatine in rabbit central I2-IBS (280-fold).

A few imidazoles were described as selective I2-IBS. The imidazole analogue of 2-BFI,

LSL60101 (77), was moderately active and selective for I2-IBS (pKi I2¼ 6.45; I2/a2> 28)101

and so was the 1-methyl-5-n-heptylimidazole S15674 (78).13 Another atypical molecule is the


2-heptylimidazoline S15430 which represents the only example of linear aliphatic 2-imidazoline

described as selective ligand for I2-IBS (pKi I2� 6.9; I2/a2� 110) in the literature.13,93 Its cyclic

counterpart (80) displays a better affinity (pKi I2¼ 7.4) although its selectivity was not reported.84

5 . C O N C L U S I O N A N D O U T L O O K

The area of IBS is still a very novel and open field of research for biologists, pharmacologists, and

chemists. Advancement in the IBS research field is limited by the lack of knowledge of the structure

of IBSs. The discovery of the structures of I1- and I2-IBS is a great challenge for biologists who will

require more selective I1 or I2 ligands as tools for such studies. Up to now, insight into the IBS

structures relies on SAR studies of various families of ligands. In this sense, it should be mentioned

that the important efforts made to computationally develop an I2 pharmacophore have been mainly

restricted to the 2-phenoxymethyl and 2-trans-styryl families of ligands.67,84 Thus, these studies

should be extended to other families of compounds (e.g., carbolines, alkylguanidines, 2-amino-

imidazolines, etc.) in order to obtain more generally applicable results.

The pharmacological profile of I1-IBS has been more or less determined to be involved in

hypertension and this has allowed focusing the research on new compounds that could be tested as

antihypertensive drugs. A better knowledge of the pharmacology of I2-IBS would help to target it

more specifically, opening the door for new families of compounds.

From the chemical point of view, many possibilities remain to be explored. For example, all the

imidazoline derivatives have always been connected by the position 2 of the imidazoline ring. This

Figure 12.


leaves open the question of whether the 3-, 4-, or 5-connected derivatives would be good ligands. As

shown in this study, new chemical structures differing from the guanidine/imidazoline paradigmhave

been reported as IBS ligands (e.g., carbolines). Hence, applying updated medicinal chemistry

techniques such as the high-throughput screening and, therefore, testing more diverse chemical

structures would probably allow the discovery of new IBS ligands with more different structures and

shapes than those known to date.

A C K N O W L E D G M E N T S

The authors thank the SpanishMinisterio deEducacion,Cultura yDeporte for a grant (SB2001-0174)

and the University of Dublin, Trinity College for funds (Start-Up Project No. 504/R01056).

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Christophe Dardonville graduated at the Universite Pierre et Marie Curie (Paris) and obtained his Master

degree in chemistry (D.E.A.) at the University of Montpellier (France). He moved to Spain with a Marie Curie

Fellowship and obtained his Ph.D. degree from the Universidad Complutense (Madrid) in 2000. From 2000 to

2003, he was a postdoctoral fellow with Dr. Ian Gilbert (Welsh School of Pharmacy, Cardiff University, UK). He

presently works at the Instituto de Quımica Medica of the CSIC as a postdoctoral fellow. His current research

interests include the design and study of new ligands for imidazoline binding sites and opioid receptors.

Isabel Rozas obtained her Ph.D. degree from the Universidad Complutense (Spain) and was a postdoctoral

fellow with Prof. Paul G. Mezey (University of Saskatchewan, Canada, 1989–1991) and Prof. Donald F. Weaver

(Queen’s University, Canada, 1993–1994). She worked as a researcher at the Instituto de Quımica Medica,

CSIC (Spain) from 1987 until 2000 and presently is a Lecturer of Medicinal Chemistry at the Department of

Chemistry of the University of Dublin, Trinity College (Ireland). Her current research interests include the

design and study of new ligands for imidazoline binding sites as well as for different alpha-adrenoceptors and

their pharmacological applications.


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Imidazoline Binding Sites and Their Ligands an Overview of the Different Chemical Structures - Med...

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