Allosteric modulation of A1-adenosine receptor: a review

TECHNOLOGIES

DRUG DISCOVERY

TODAY

Allosteric modulation of A1-adenosinereceptor: a reviewMarıa Kimatrai-Salvador, Pier Giovanni Baraldi*, Romeo RomagnoliDipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17/19, 44121 Ferrara, Italy

Drug Discovery Today: Technologies Vol. 10, No. 2 2013

Editors-in-Chief

Kelvin Lam – Simplex Pharma Advisors, Inc., Arlington, MA, USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Allosteric modulation

Allosteric modulators of adenosine receptors repre-

sent an alternative to direct-acting adenosine agonists

and nucleoside uptake blockers, preferably those can

selectively modulate the response to adenosine in only

those organs or localized areas of a given organ in

which production of adenosine is increased. Allosteric

enhancers at the adenosine A1 receptor have received

attention as anti-arrhythmic cardiac agents, and, more

recently, as anti-lipolytic agents. In addition, this class

of compounds has therapeutic potential as analgesics

and neuroprotective agents.

Introduction

The term ‘allostery’ meaning that an allosteric site of a

regulatory protein is physically distinct from the binding site

for the endogenous ligand which is referred to as the orthos-

teric site. In this setting, an allosteric modulator is a small

molecule that binds at a topographically distinct allosteric

site and either potentiates or inhibits the binding and/or

signaling of an orthosteric ligand [1–4]. Clinical success of

the well known allosteric modulator drugs in the ionotropic

receptor field such as benzodiazepines, which potentiate the

effect of the neurotransmitter g-aminobutyric acid (GABA) at

the ionotropic GABAA [5] receptor, has fuelled the develop-

ment of other ion channels, kinases, phospholipases and

seven transmembrane spanning receptors (7TMRs, also

known as G-protein coupled receptors (GCPRs)) allosteric

modulators.

*Corresponding author: P.G. Baraldi ([email protected])

1740-6749/$ � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddtec.2012

Section editors:Ad P. IJzerman – Leiden/Amsterdam Center for DrugResearch, Leiden, The Netherlands. Rob Lane – MonashInstitute of Pharmaceutical Sciences, Victoria, Australia

In the past few years there have been substantial and

increasing scientific efforts directed toward the detection

and characterization of allosteric sites on several GPCRs as

well as the accompanying discovery and development of

molecules that begin to define the pharmacology of those

sites. In this review we want to summarize the medicinal

chemistry of such compounds, the latest developments and

the structure activity relationship of one of the most active

ones.

Adenosine receptors

GCPRs constitute the most interesting class of receptors for

drug discovery and are direct targets of about 30% of all

medicines currently on the market [6]. Historically, such

drugs have produced their effects by interaction with the

orthosteric site, defined as the primary binding site for the

endogenous ligand, and regulating the receptor function by

classical agonism (directly stimulating a receptor response),

inverse agonism (blocking constitutive receptor activity) or

competitive antagonism (blocking the binding of the native

agonist). The character of these interactions may lead to

receptor desensitization, internalization, and down regula-

tion due to being ‘turned on’ for prolonged period, or may be

even toxic [2–4]. The extraordinary progress made in chemi-

cal optimization, pharmacology understanding and develop-

ment of functional assays has allowed discover ligands that

do not bind at the orthosteric site. These new allosteric

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http://dx.doi.org/10.1016/j.ddtec.2012.08.005


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Drug Discovery Today: Technologies | Allosteric modulation Vol. 10, No. 2 2013

ligands include PAMs (positive allosteric modulators, which

potentiate the endogenous ligand receptor response), NAMs

(negative allosteric modulators, which decrease the endogen-

ous ligand receptor response), SAMs (silent or neutral allos-

teric ligands, which bind at allosteric sites and can block

PAMs and NAMs but have no effect on orthosteric ligand

responses), and bitopic ligands, that is, hybrid orthosteric/

allosteric ligands that bind to both the orthosteric and allos-

teric site [2–4,7–9]. The idea of allosteric modulation of

agonistic activity is an approach with many potential bene-

fits; allosteric enhancers (AE) would enhance the activation of

the receptor from the orthosteric site, enhancing the potency

of the endogenous agonist avoiding the adverse affects

observed by the use of exogenous agonist [10].

Allosteric ligands offer numerous advantages; they will

always exert their effects when the endogenous agonist is

present (referred as state dependence) or temporal and spatial

activity of the endogenous ligand resulted as a consequence

of the presence of the allosteric ligand. The effects of an

allosteric modulator are saturable, once allosteric sites have

been occupied, no additional effects will be observed (ceiling

effect), and those binding allosteric sites may be under less

evolutionary pressure for their conservation, enabling high

subtype selectivity to be achieved [11–15]. Nevertheless these

advantages can be in turn the main drawbacks, for instance,

the lack of that evolutionary pressure on allosteric sites

can complicate preclinical pharmacodynamics or toxicity

studies in mice or rats because of significant species differ-

ences. Even the state dependence produced by allosteric

modulators could be a handicap some degenerative pathol-

ogies could present progressive loss of endogenous orthos-

teric tone [2–8,11–19].

7TMRs or GPCRs commonly are divided into three main

families or classes named A, B and C on the basis of sequence

homology and functional roles. Class A, also termed rhodop-

sin-like after the prototypic visual pigment, constitutes the

largest group of GPCRs which adenosine receptors belong to.

Four subtypes of adenosine receptors (AR) have been identi-

fied: A1, A2A, A2B and A3. Adenosine A1 and A3 receptors are

coupled to a Gi protein, thereby inhibiting the production of

cAMP via adenylate cyclase. By contrast, adenosine A2A and

A2B receptors are coupled to a Gs protein, thereby stimulating

production of cAMP. It is important to study the distribution

of receptors because this will tell us where agonists and

antagonists given to the intact organism can act. [20].

Adenosine is the main agonist at this receptor class. The

endogenous levels of adenosine are sufficient to tonically

activate inhibitory A1-AR and caffeine, perhaps the most

commonly used drug in the world [21], mediates its excita-

tory effects through the antagonism of this inhibition. The

therapeutic areas for which there is growing interest in these

receptors include immune function and inflammation, cen-

tral nervous system (CNS) disorders, and pulmonary and

e286 www.drugdiscoverytoday.com

cardiovascular diseases. This nucleoside has been shown to

play a role in the regulation of coronary and systemic vascular

tone, platelet function and lipolysis in adipocytes [22]. Ade-

nosine itself is being marketed for purposes such as restoring

normal rhythm in patients with paroxysmal supraventricular

tachycardia (Adenocard) or being adjunct to thallium cardiac

imagining in the evaluation of coronary artery disease in

patients (Adenoscan) [20]. Adenosine has been implicated

in the regulation of asthma and chronic obstructive pulmon-

ary disease (COPD) [23]. Levels of adenosine are increased in

the lungs of asthmatics, in which elevations correlate with

the degree of inflammatory insult [24]. Theophylline is

widely used as an antiasthmatic drug, and a related xanthine,

enprofylline (3-propylxanthine), is also therapeutically effi-

cacious in the treatment of asthma. Enprofylline only blocks

A2B receptors with a Ki (7 mM) similar to that of theophylline

(13 mM) and well within its therapeutic plasma levels

(5–25 mM) [25]. The literature indicates that the release of

allergic mediators from mast cells is mediated by A3 and/or

A2B receptors [26,27].

Activation of A2A receptors protects tissues from injury by

reducing inflammation during reperfusion following ische-

mia. The selective A2A agonist, CGS 21689 inhibits neutrophil

accumulation and protects the heart from reperfusion injury

[28]. Similar cardiac protection has also attributed to A3

receptor activation [29]. The localization of A2A receptors

to the dopamine rich areas of the brain and the effect of

A2A receptor antagonist in a synergic manner with dopamine

receptor agonist have led the development of antagonists,

and some of which (KW6002) are undergoing clinical trials as

anti-Parkinson agents [20].

The A1-adenosine receptor (A1-AR) is responsible for seda-

tive, anticonvulsant, anxiolytic and locomotor depressant

effects induced by adenosine. Importantly it has been

reported to play a role in adenosine-mediated analgesia

[30]. At cardiovascular level A1-ARs mediate negative chron-

otropic, dromotropic and inotropic effects. A1-AR subtypes

located on sinoatrial and atrioventricular nodes cause brady-

cardia and heart block, respectively, while the negative ino-

tropic effects include a decrease in atrial contractility and

action potential duration. Stimulation of A1-ARs in the heart

exerts cardioprotective effects by inhibiting norepinephrine

release from sympathetic nerve endings [31]. Activation of

A1-ARs, protein kinase C and mitochondrial KATP channels is

responsible of the protection of myocardium against infarc-

tion from a subsequent prolonged ischemic insult [32–34]. In

the kidney, A1-ARs mediate vasoconstriction, decrease glo-

merular filtration, inhibit renin secretion and inhibit neuro-

transmitter release. A1-AR antagonists represent a novel class

of agents for potential use in the treatment of hypertension

and edema [35,36]. Efforts to selectively target these receptors

with modified adenosine analogues have resulted in thera-

peutics that are limited by side effects due to their ubiquitous

Vol. 10, No. 2 2013 Drug Discovery Today: Technologies | Allosteric modulation

nature and poor receptor subtype selectivity. Compounds

that are able to enhance the activity of ARs by the endogen-

ous ligand within specific tissues may have potential

therapeutic advantages over non-endogenous agonists inter-

acting with the classical agonist/orthosteric site; the idea is

that the AE binds to a site that shows greater sequence

divergence between adenosine receptor subtypes and also

preferentially acts in the presence of elevated levels of endo-

genous adenosine. Thus, this approach can lead to greater

selectivity in action as a consequence of both site- and

receptor-specific modulation. Such an opportunity for inter-

vention is provided by the concept of allosteric modulation of

GPCRs [37].

Pharmacological characteristic of GPCRs allosterism

GPCR allosterism is characterized by several properties that

reflect the underlying reciprocity of effect between two dif-

ferent ligands. These key properties are saturability, probe

dependence, stimulus-bias (functional selectivity), and dif-

ferential effects on orthosteric ligand affinity versus efficacy.

Saturability of effect

Two classic examples of cooperative binding proteins, the

enzyme hemoglobin and the GABAA ion channel-receptor

complex illustrate the phenomenon of cooperativity. The

binding of oxygen to hemoglobin or GABA to the GABAA

receptor is characterized distinctly by sigmoid curves when

plotted on a linear scale, reflecting that the binding of one

equivalent of ligand alters the affinity of the subsequent

binding of the next equivalent(s) of ligand to the same

protein complex. Studies such as these conducted on a variety

of ion channel-linked receptors, thus, led to the conclusion

that certain receptors can possess more than one binding site

for ligands, leading to the idea of allosteric binding sites.

The allosteric mechanism possesses an obvious limit of

pharmacological effect due to the saturation of the allosteric

site that is governed by the cooperativity between the orthos-

teric and allosteric ligands. Allosteric interactions character-

ized by limited degrees of either positive or negative

cooperativity best illustrate the propensity of allosteric

ligands to fine-tune physiological signaling. This phenom-

enon will provide an additional advantage, the minimization

of risk associated with overdose at the level of the receptor

target because no additional pharmacological effect would be

observed over and above that defined by cooperativity [38].

Probe dependence

The phenomenon of probe dependence is directly related to

the cooperativity between orthosteric and allosteric ligands,

which refers to the fact that the extent and direction of an

allosteric interaction can vary with the nature of the orthos-

teric ligand used as a probe of receptor function For example,

the allosteric modulator, LY2033298, positively modulates

the binding affinity of the orthosteric agonist, ACh, at the M4

mAChR, but is essentially neutral when tested against two

structurally distinct orthosteric antagonists, [3H]N-methyls-

copolamine ([3H]NMS) and [3H]quinuclidinyl benzylate

([3H]QNB), at the same receptor and in the same assay. This

highlights the requirement for careful consideration in the

choice of orthosteric ligands to assess the effects of an allos-

teric modulator, as seen in the previous example, the use of

the endogenous agonist in a compound screen would reveal

the allosteric activity of a ligand such as LY2033298, whereas

a radioligand-based screen using [3H]QNB as the probe would

fail to identify LY2033298. Some GPCRs have more than one

endogenous orthosteric agonist but that may not at all

respond the same way to allosteric ligands, as seen recently

at the glucagon-like peptide 1 (GLP1) receptor, where the

Novo Nordisk’s small molecule allosteric agonist had no

effect on the signaling on the endogenous orthosteric peptide

agonist GLP1, but significantly potentiated the signaling

of the oxyntomodulin, another endogenous GLP1 receptor

peptide [38–41].

Functional selectivity or stimulus-bias

When an allosteric ligand binds to a 7TMR, the receptor

adopts a unique, novel conformation, enabling it to activate

any number of downstream signaling cascade to the exclu-

sion of other possible receptor states. Here, probe dependence

manifested at the level of cytosolic proteins would result in

signal pathway-dependent allosteric modulation, termed as

‘stimulus bias’, ‘stimulus trafficking’, ‘differential receptor

trafficking’ or ‘functional selectivity’. For instance,

VU0029767, an allosteric modulator of the M1 muscarinic

receptor potentiates ACh-mediated intracellular calcium

mobilization but not phospholipase D activation; therefore,

depending on the pathway/assay assessed, VU0029767 would

alternatively be classed as a PAM or a SAM, respectively

[38,41].

Differential effects on affinity versus efficacy

The ability of an allosteric ligand to modulate the affinity of

an orthosteric ligand need not track with the effects of the

modulator on orthosteric ligand efficacy, and vice versa. This

has indeed been noted with the allosteric modulator

Org27569 and related compounds when tested against

orthosteric agonist binding and function at the cannabinoid

CB1 receptor. Despite acting as positive modulators of agonist

affinity, the Org compounds all act as negative modulators of

agonist signaling efficacy in both recombinant and native

tissue bioassays [38].

All these properties must be taken into account to

understand the mode of action of allosteric modulators.

Operationally, 7TMR allosteric modulators exhibit affinity

modulation, efficacy modulation, or varying degrees of

both modes of modulation. With affinity modulation, the

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conformational change in the 7TMR upon allosteric ligand

binding can affect either the association or dissociation rate

(or both) of the orthosteric ligand. For efficacy modulation,

the conformational change in the 7TMR upon allosteric

ligand binding leads to a change in signaling capacity (also

termed intrinsic efficacy) and thereby either facilitates or

inhibits receptor coupling to downstream effectors.

There are two models proposed to account for the inter-

actions between the allosteric and orthosteric ligand, the

‘allosteric hot wire’ model and ‘global allosteric modulation’

model. In the first one, the allosteric modulation site is

directly linked to the orthosteric site through specific path-

ways. In the ‘global allosteric modulation’ model, allosteric

communication to the orthosteric site relies on long range

interaction through order/disorder transitions from multiple

receptor conformations (i.e., population dynamics). Several

mass-action schemes, based on variants of the ternary com-

plex model, have been presented to describe the molecular

effects of allosteric ligands on orthosteric pharmacology in

terms of one or more ‘cooperativity factors’, which indicate

the magnitude and direction of an allosteric modulator

mediated stabilization of different 7TMR states. From these

models, it can be appreciated that the functional potency of

an allosteric modulator will depend not only on its affinity for

the allosteric site but also on the degree of cooperativity with

the orthosteric ligand. Most mass-action-based molecular

models of allosteric modulation contain too many para-

meters to be fitted to real experimental data and thus cannot

be used to rationalize structure–activity studies in a manner

that can inform drug candidate selection matrices. A useful

means for placing these issues on a more practical quantita-

tive level is through the use of an ‘operational’ model of

allosterism and agonism, which has been developed to

describe allosteric effects in terms of a minimum number

of experimentally accessible parameters. The equation

describing the signaling of an orthosteric agonist in the

presence of an allosteric modulator according to the model

is as follows:

E ¼ fEmðtA½A�ðKB þ ab½B�Þ þ tB½B�KAÞn

fð½A�KB þ KAKB þ ½B�KA þ a½A�½B�Þn þ ðtA½A�ðKB þ ab½B�Þ þ tB½B�KAÞng

where E is the effect, [A] and [B] are the concentrations, KA

and KB are the equilibrium dissociation constants of the

orthosteric and allosteric ligand, respectively, a is the coop-

erativity factor describing the allosteric effect of each ligand

on the other’s binding affinity, b is a scaling factor (from zero

to infinity) that quantifies the magnitude by which the

allosteric modulator modifies the efficacy of the orthosteric

agonist at a given signal pathway, and the parameters tA and

tB relate to the ability of the orthosteric and allosteric ligands,

respectively, to promote receptor activation (direct agonism).

These latter parameters incorporate the intrinsic efficacy of

each ligand, the total density of receptors, and the efficiency


of stimulus–response coupling. The parameters Em and n

denote the maximal possible system response and the slope

factor of the transducer function that links occupancy to

response, respectively. Three key parameters can be routinely

derived from application of this model to most functional

screening data, as long as full concentration response and

curve–shift relationships are determined. These three para-

meters are the allosteric modulator KB, which provides infor-

mation on the interaction of the allosteric ligand with the

allosteric binding pocket on the free receptor, the composite

cooperativity parameter ab, which provides information on

the overall allosteric effect on the orthosteric agonist in the

chosen functional assay, and the modulator efficacy para-

meter tB, which provides information on the ability of the

allosteric ligand to promote agonism in its own right in the

absence of orthosteric ligand. May these parameters ask

questions such as: How much cooperativity (ab) or allosteric

agonism (tB) is required to achieve in vivo efficacy? How do

structural modifications affect compound affinity (KB) versus

cooperativity (ab)? The latter is important because these

properties are not correlated, and thus, different structural

manipulations can change them in different directions. Probe

dependence will manifest as different ab values depending on

the orthosteric agonist used and/or the signal pathway being

assessed as a read out of receptor activation. In terms of drug

discovery programs, these insights can be used to more

rationally inform the design of candidate selection matrices

for drugs acting allosterically [38,41].

In recent years, compounds that behave as molecules that

engaged both, orthosteric and allosteric sites on a GPCR to

achieve functionally selective signaling have been discovered

or designed. These compounds have been named ‘bitopic’,

‘dualsteric’ or ‘multivalent’. The compound McN-A-343

represents one of the earlier examples of bitopic mode of

action at the M2 muscarinic acetylcholine receptor

(M2mAChR), and has been shown to display stimulus-bias

at the M2 mAChR. This molecule is composed by two frag-

ments, tetramethylammonium (TMA), which behaves as a

robust orthosteric agonist of the M2 mAChR, and 3-chloro-

phenylcarbamate, which is a negative allosteric modulator of

TMA signaling efficacy, thus accounting for the partial agon-

ism of the parent compound and, potentially, for its functional

selectivity. It is easy to say that McN-A-343 concomitantly

engages both the orthosteric and an allosteric site on the M2

mAChR, a postulate supported by the fact that a single point

mutation in an allosteric pocket of the M2 mAChR known to

reduce the potency of prototypical M2 mAChR modulators also

blunts the negative signaling effects of the negative allosteric

modulator DDBL4 fragment of McN-A-343, leading to an

increase in efficacy consistent with an orthosteric action of

TMA only. Bitopic ligand pharmacology gives us a novel tool to

rationally design new compounds using appropriate orthos-

teric and allosteric building blocks [38].


Medicinal chemistry of allosteric enhancers for A1

adenosine receptors

A comprehensive review of allosteric modulators for the

A1-AR has been published not long ago [37]; nevertheless

we will provide an overview of them since the first allosteric

modulators published in the early 1990s to present.

Bruns and colleagues introduced the first allosteric mod-

ulators of A1-AR in 1990s [42,43]. These initial reports

described benzoylthiophene derivatives such as PD 117,975

(1), PD 71,605 (2) and PD 81,723 (3) (Fig. 1). Synthesized

originally as intermediates of benzodiazepine-like com-

pounds, the discovery of their allosteric enhancer (AE) char-

acter was serendipitous. The authors observed that these

compounds increased rather than decreased the binding of

the agonist radioligand [3H]N6-cyclohexyladenosine

([3H]CHA) to A1 adenosine receptors in rat brain membranes.

These so-called enhancers were next shown to slow the

dissociation of the agonist [3H]CHA from the A1 receptor,

indicating an allosteric mechanism of action. They also

reported that these compounds were capable of acting as

S

NNH2

O

Cl

S

PD 117,975 (1) PD 7

SR5

R4

NH2

O

Ro R0=halo R4=H, CR5=CH3 R4, R5= 4a, R0=4b or T-

SR5

R4

NH2

O

S

5

SR5

R4

NH2

O

S

6

R4, R5=CH 3, -(CH2)n- with n=3 or 45a, R4, R5=-(CH 2)4-6a, R4, R5=-(CH 2)4-

Figure 1. Representative adenosine A1 receptor allosteric modulators with struc

competitive antagonism at the same receptor subtype at

higher concentrations.

Among the compounds tested by Bruns, PD 81,723 (3)

represents the first specific and selective allosteric enhancer

of agonist binding to the A1 receptor, with the most favor-

able ratio of allosteric enhancement to antagonism at this

receptor [44]. By the synthesis of the compounds, it became

clear that the tetrahydropyridine ring was not essential,

because it can be replaced with a tetrahydrobenzene

nucleus or even eliminated entirely without loss of activity.

PD 81,723 was shown to enhance agonist binding and the

functional activation of the A1 receptor in brain [45] and

cardiovascular tissues [46]. PD 81,723 is selective for A1-AR,

having no effect on other adenosine receptors subtypes or

on other classes of receptors. The compounds discovered

by Bruns and coworkers have been shown to enhance

adenosine binding and the functional activation of the

A1-AR in heart and cardiovascular tissues, thus they could

be useful both as antiarrythmic [47] and cardioprotective

[48] agents.

NH2

O

Cl

S NH2

O

CF3

1,605 (2) PD 81,723 (3)

gen, alkyl, alkoxy, CF3, CO2H, NO 2H3, C2H5

, n-C3H7, cyclopentyl, cyclohexyl-(CH2)4-3,4-diCl; R4, R5=-(CH 2)4-62, R0=4-Cl; R4, R5=-(CH2)4-;

SR5

R4

NH2

O

O

7

SR5

R4

NH2

O

8

N

Drug Discovery Today: Technologies

tures 1–4 and general structures of 2-amino-3-heteroaryl thiophenes 5–8.



To study the role of various substitutions on the 3-aroyl

moiety and the importance of the 4,5-dimethyl group on the

thiophene ring, Bruns [43], Baraldi [49], Van der Klein and

Kourounakis [50,51], Tranberg [52] and Lutjens [53] have

directed significant effort to refining the SAR of the 3-, 4-

and 5-positions of the 2-aminothiophene moiety (Fig. 1).

Bruns et al. had previously reported that the lack of the 2-

amino group resulted in a complete loss of activity, implying

that this group was essential for activity [54]. The molecules

were evaluated for both as allosteric enhancers and as possible

antagonists on the adenosine A1 receptor. In the structure

activity relationship (SAR) studies of 4,5-dialkyl derivatives

with general structure 4, chloro substitution on either the

meta or the para position of the benzoyl ring led to activities

comparable to that of the meta-trifluoromethyl counterpart

PD 81,723.

The extension of this series was the ‘cyclized’ PD 81,723

analogues or tetrahydrobenzothiophene derivatives. Com-

paring compounds with the same substituent on 3-aroyl

moiety, bridging the 4- and 5-positions with a methylene

chain [–(CH2)n–], with n = 4, caused an increase of activity

which was comparable to that reported for the corresponding

4,5-dimethyl analogues. In particular, compound 4a, (LUF

5484), in which a meta/para substitution are combined hav-

ing an additive effect, was found to be 2.4-times more potent

than PD 81,723 as AEs, showing comparable antagonistic

activities. King Pharmaceuticals has initiated a phase II clin-

ical trial program evaluating the efficacy and safety of deri-

vative 4b or T-62 [(2-amino-4,5,6,7-tetrahydrobenzo[b]

thiophen-3-yl)(4-chlorophenyl) methanone] for the treat-

ment of neuropathic pain [55]. Lipophilic meta and/or para

substituents such as halogen are preferred for enhancing

activity, whereas more hydrophilic groups such as nitro

and carboxylate are not favorable.

The synthesis and biological evaluation of 5-alkyl-2-

amino-3-benzoyl thiophenes derivatives by van der Klein

[50] and Kourounakis [51] have allowed to establish that

bulky 5-alkyl substituents (such as cyclopentyl, cyclohexyl

or phenyl) increased 10-fold antagonistic properties. By con-

trast, substitution on the 4-position of the thiophene ring

increased AE activity (evaluated as the decrease of [3H]CCPA

dissociation), causing a decrease in antagonist binding.

A series of 2-amino-3-heteroarylthiophenes with general

formula 5–8 (Fig. 1) were also synthesized by Baraldi et al.

[56]. Only compounds 5a and 6a, with a 3- or 2-thienyl

moiety, respectively, maintained a comparable activity to

that of PD 81,723. Unfortunately, derivatives with general

structures 7 and 8, these last one with a pyridine moiety

which may improve the hydrophilic properties of the 2-

amino-3-benzoylthiophenes compounds, were significantly

less potent than the reference compound PD 81,723.

Nikolakopoulos et al. studied 2-aminothiophene-3-carbox-

ylates and carboxamides [57] with general formula 9 (Fig. 2)


as AE at the A1-AR. Compound 9a was more potent and

efficacious than PD 81,723. Allosteric enhancement was

evaluated using an in vitro assay which measures their ability

to stabilize the agonist–A1AR–G–protein ternary complex in

three phases: (1) binding to equilibrium of the agonist125I-ABA to the A1-AR; (2) addition of the putative AE, and

(3) dissociation of the complex by adding a combination of

an A1 adenosine receptor and GTPgS to accelerate agonist

radioligand dissociation. An enhancing activity (AE) score of

100% means no dissociation and a score of zero complete

dissociation. PD 81,723 had an AE score of 28%, while for

compound 9a it was 79.5%. Interestingly, ethyl-2-amino-

4,5,6,7-tetrahydro-1-benzoselenophene-3-carboxylate (Fig. 2,

compound 10) also proved to be an allosteric enhancer of

A1-AR. Compound 10 had an AE score of 64% and it was

significantly more potent than its thiophene analogue as AE

of A1-AR. However it is noteworthy, that this compound is not

stable under mildly acidic conditions [58].

The same authors have also reported the synthesis and

biological evaluation of analogues of PD 117,975 (1), where

substituents in 3- and 6-positions of 2-amino-4,5,6,7-tetra-

hydrothieno[2,3-c]pyridines were modified (Fig. 2). The

greatest degree of allosteric stabilization of agonist-receptor

complexes was achieved by derivatives that contained an

ethyl ester in the 3-position together with N-Boc amino acid

substitution in the 6-position (compounds 11b and 11c), or a

methyl substitution of the 6-position together with an amide

or hydrazide substitution in the 3-position (compounds 11g

and 11i). The potency of these compounds was similar or

slightly better than that displayed by PD 81,723. Although

the compounds posses the ability to recognize an allosteric

site on the agonist-occupied A1-AR at high concentrations, a

functional assay indicate that the compounds are more

potent as orthosteric antagonists than allosteric modulators

of agonist dissociation. The potential of the compounds to

interact allosterically with the A1-AR was initially screened by

evaluating their ability to stabilize an orthosteric agonist–

A1AR–G protein ternary complex in an in vitro dissociation

kinetic binding assay, using 125IABA as the orthosteric radi-

oligand and promoting dissociation by addition of GTPgS in

the presence of an orthosteric antagonist. The resulting K-

score (‘kinetic score’) reported in Fig. 2 denotes the percen-

tage of the ternary complex remaining after 10 min of radi-

oligand dissociation, and is a measure of the strength of the

allosteric interaction between a given concentration of test

ligand and the pre-equilibrated [125I]-ABA-A1 receptor com-

plex; the higher this K-score percentage, the greater the

ability of the test ligand to allosterically stabilize the binding

of the orthosteric radioligand [59].

However, recently, it has been reported that the agonist

dissociation assay may be interpreted in an alternative man-

ner that would shed doubt on the location of the putative

allosteric site; agonist dissociation would slow down if the


S NH2

OX

R3

Se NH2

O

EtO

R4

R5

9

X=O or NHR3=H, 3-CF3C6H4, CH2C6H5, 3-CF3C6H4CH 2R4, R5=-(CH 2)n- with n=4 or 59a, X=O, R3=H, R4, R5=-(CH 2)5- 10

S

NNH2

11a-i

R6

R3

Compound R3 R6 K-score (%) EC50 ( M)PD117,915 (1) p-ClC6H4CO Bn 32.6±3.8 2011a CO2Et Bn 44.9±3.4 10.3±0.111b CO2Et Boc-L-Phe 82.5±12.4 7.2±3.911c CO2Et Boc-L-Cys(Trt) 93.2±0.4 7.9±5.511d CO2H Bn 68.3±4.7 8.0±2.911e CONHNH2 Bn 82.9±13.0 8.9±3.911f CONHNHPh Bn 55.4±11.1 7.8±3.411g CONHBn Me 87.2±7.7 12.0±6.311h CONHNH2 Me 85.7±9.2 4.4±3.411i CONHNHPh Me 83.1±11.9 11.5±2.8

S NH2

O

F3C

12a

S NH2

O

F3C

12b

S NH2

F3C

12cCl

13 14

X, R=Halogen, alkyl, alkoxy

S

O

X

NH2S

R

NH2

O

X

CO2Et


Figure 2. 2-Aminothiophene-3-carboxylate and carboxamide derivatives 9, ethyl-2-amino-4,5,6,7-tetrahydro-1-benzoselenophene-3-carboxylate

10, 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate derivatives (11), 5-aryl-2-aminothiophene derivatives 12a–c,

(2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl) methanone (13) and 1-aminoindeno[1,2-c]thiophen-8-ones (14).

complex were stabilized through the inhibition of GTP

uptake into the G protein a-subunit. Indeed, benzoyl sub-

stituted 2-aminothiophenes have been reported to function

as direct G protein inhibitors [60]. Thus some of the com-

pounds may not act as allosteric enhancers but as G-protein

antagonists.

Aurelio et al. synthesized two more series of 5-substituted 2-

aminothiophenes (Fig. 2). In the 3-benzoyl series, a 5-phenyl

group was found to give the greatest potency (compound

12b, ED50 = 2.1 mM, AE score = 18%). However the analogue

with no 5-substituent (12a) ED50 = 15.8 mM, AE score = 77%)

proved to be the most efficacious. In the 3-ethoxycarbonyl

series, the 5-(4-chlorophenyl) analogue was the most potent

and efficacious (12c: ED50 = 6.6 mM, AE score = 57%). The

activity of the target compounds was evaluated in a kinetic

assay measuring agonist dissociation, initiated by the addition

of A1-AR antagonist, CPX and GTPgS. A score of 0 corresponds

to near complete dissociation of agonist radioligand binding

when no AE is added. A score of 100 would be assigned to an AE

that completely blocks radioligand dissociation [61].

Moreover, a series of 4-substituted 2-amino-3-benzoylthio-

phenes and 2-amino-3-(4-chlorobenzoyl)thiophenes were

also studied using a functional assay of A1-AR-mediated

phosphorylation of ERK 1/2 in intact CHO cells, testing



two concentrations (3 and 10 mM) in the presence of an EC50

concentration of the orthosteric agonist R-PIA. It was shown

that 3-trifluoromethylphenyl (12a) and 3,5-di-trifluoro-

methylphenyl substitution at the 4-position of thiophene

supported robust activity [62].

Thus conformationally restricted analogues of 12a, in

which the 4-phenyl group is effectively locked in a coplanar

conformation relative to the thiophene ring, can be achieved

tethering the 4-phenyl group to the 5-position of thiophene

ring via a methylene unit (general structure 13). Another

series of compounds with general structure 14 can be synthe-

sized tethering the 3-benzoyl group of 2-amino-3-ben-

zoylthiophes to the 4-position of thiophene ring. However,

the major finding of that study was that all the 1-aminoin-

deno[1,2-c]thiophen-8-ones with general structure 14 proved

to be inactive or behaved as antagonists. By contrast, some

members of the (2-aminoindeno[2,1-b]thiophen-3-yl)(phe-

nyl) methanone series with general formula 13 indicated a

positive AE effect [63].

Tranberg [52] and Baraldi [64] also reported the AE activity

of a series of 2-amino-3-naphthoylthiophenes with general

structure 15 and 16 (Fig. 3), characterized by the replacement

of the 3-benzoyl with a naphtoyl moiety. Because previous

studies are suggesting that the receptor environment neigh-

boring the benzoyl binding site is lipophilic, the substituents

at the 40-position on the naphtalene were varied from H to

groups with a different degree of lipophilicity (CH3, CH3O,

and halogens). Comparing the activities of derivatives bear-

ing the same substituents on the 4- and 5-position of the

thiophene ring, it may be concluded that the 1-naphthoyl

derivatives with general structure 15 are generally more

potent than the corresponding 2-naphtoyl analogues with

general formula 16, and in particular, cycloalkylthiophenes

tended to be more potent than 4,5-dimethyl analogues. A

lipophilic halogen or a methyl group, rather than an hydro-

philic group such as the methoxy, were preferred for AE

activity at the 4-position of naphtoyl moiety.

It has been shown that a hydrophobic group at the 4-

position of thiophene ring increased AE activity [43,64].

For this reason Romagnoli and colleagues identified a series

of 2-amino-3-(4-chlorobenzoyl)-4-[4-(arylpiperazin-yl]thio-

phene derivatives with general structure 17 (Fig. 3), charac-

terized by a common 2-amino-3-(4-chlorobenzoyl)thiophene

core, with various arylpiperazine moieties attached to the 4-

position of the thiophene ring by a methylene unit. The

results obtained by the synthesis of a wide series of com-

pounds modifying both the number and position of electron-

releasing or electron-withdrawing substituents in all five

positions the phenyl portion of arylpiperazine moiety, iden-

tified the 4-chloro (17a), 4-trifluoro (17b), 3,4-difluoro (17c),

3-chloro, 4-fluoro (17d) and 4-trifluoromethoxy (17e) deri-

vatives as the most active compounds in binding (saturation

displacement experiments) and functional cAMP studies


[65,66]. It may be possible that the phenyl ring of the phe-

nylpiperazine moiety seemed to exert an important influence

on AE potency, contributing to activity with a p–p interac-

tion (such as charge transfer), interacting with a lipophilic

pocket at the A1-AR. This was confirmed by replacement of

the phenyl moiety with electron-deficient heteroaromatic

rings, such as pyridine or pyrimidine, which incorporate

one or two basic nitrogens in the crucial region of binding,

causing a reduction of the AE activity. The results obtained

presumably reflect the importance of the relative position in

space between the piperazine and phenyl rings, and suggest a

detrimental ortho-effect that can be overcome by the number

and position of other substituents on the aryl moiety.

Chordia et al. [67,68] identified another class of AE for A1-

AR, characterized by a 2-aminothiazole core (2-aminothiazo-

lium salts), whose structure shares with the 2-aminothio-

phenes the five-membered aromatic ring containing

sulphur and an exocyclic amine (compounds with general

formula 18 and 19, Fig. 3), such as the catechol derivative

(18d), and the acetate ester (18c), which decreased the

agonist dissociation rates from A1AR with EC50 values of

1.2 and 3.8 mM respectively. Derivative 18c was the most

potent and efficacious compound of this series, and its intrin-

sic activity was twice that of PD 81,723. Whereas the AE

activity of the 2-aminothiophenes depended on both, the 2-

amino and the 3-aroyl groups, these aminothiazoles lack a

substituent that corresponds to the aroyl group. However

superimposing the two heterocycles suggests that the thia-

zole nitrogen could act as a surrogate for the benzoyl, acting

as a hydrogen-bond acceptor. The aryl group of the 2-amino-

3-aroylthiophenes can occupy a position near, but not over-

lapping, the benzene moiety of the indenothiazole. Never-

theless, 2-aminothiazoles often show a strong antagonistic

activity. Some of the 2-aminothiazoles prepared and tested by

Goblyos et al. proved to antagonists rather than allosteric

modulators [69]. Moreover, amides derived from the 2-ami-

nothiazoles 18 tested in radioligand binding studies, proved

to have affinity for adenosine A1 and/or A2 receptors, and

they were also classified as antagonists based on the results

obtained in a functional assay based on cAMP production.

We can therefore conclude that, up to now, 2-amino-3-

aroylthiophene derivatives proved to be the only class of

compounds that have been reported to be AEs at the A1-ARs.

Fawzi et al. reported an allosteric modulator of both agonist

and antagonist binding to GPCRs termed SCH-202676 (N-

(2,3-diphenyl-[1,2,4]thiadiazole-5(2H)-ylidene) methana-

mine) (Fig. 3, compound 20). It appeared to modulate the

activity of many GPCRs, including opioid, muscarinic, adre-

nergic and dopaminergic receptors [70]. The research group

of (Goblyos) Ijzerman synthesized several 2,3,5-substituted

[1,2,4]thiadiazole analogues of SCH-202676 (a non selective

GPCR allosteric modulator) and tested these ones as potential

allosteric modulators of adenosine receptors. On A1-AR,


S NH2R5

R4 O

S NH2R5

R4 O

R0

R1 R4, R5 =Me and -(CH2)n with n=3 or 4R0=H, CH 3, halogen, OCH 3R1=H, Cl

15 16

S NH2

O

Cl

NNR

R=H, halogen, CH3, CF 3, OCH3, OCF 317a, R=4-Cl17b, R=4-CF317c, R=3,4-diF17d, R=3-Cl, 4-F17e, R=4-OCF3

17

S

N NH2

18

1

2

34

5

67

8

18a, R=6-OMe18b, R=6-OCOCH318c, R=7-OCOCH318d, R=6,7-diOCH318e, R=7-Me

S

N NH2

R19

19a, R=4-OMe19b, R=4-OPh19c, R=4-N(C H3)219d, R=3,4-diOCH319e, R=3,4-OC H2O-

R

N

SN N

20, SCH202676

N

SN N

MeO21


Figure 3. General structure of 2-amino-3-naphthoylthiophene analogues 15 and 16, 2-amino-3-(4-chlorobenzoyl)-4-[4-arylpiperazin-yl]thiophene

derivatives 17a–e, 2-aminothiazole derivatives 18a–e and 19 a–e and [1,2,4]thiadizole analogues 20–21.

compound 21 was shown to be an AE of antagonist

([3H]DPCPX) binding [71]. However, one year later it turned

out that these type of compounds are sulfhydryl modifying

agents rather than allosteric modulators as they appear to

reversibly modify the sulfhydryl groups of cysteine residues

in cell membrane preparations [72].

In a previous study presented by Tranberg et al. [32], it was

shown that increasing the ring size of 2-amino-4,5-cycloalk-

yl[b]thiophenes from five to seven membered rings improved

the ability of these 2-aminothiophenes to act as AEs to

stabilize the agonist-receptor-G protein ternary complex in

an in vitro dissociation kinetic binding assay of the A1-AR. By

extrapolation, Aurelio and colleagues synthesized and eval-

uated a novel series of 2-amino-4,5,6,7,8,9-hexahydrocy-

cloocta[b]thiophenes with general structure 22 as AEs at

the A1-AR to check if further increases in ring size may

improve allosteric ligand activity; an assumption that unfor-

tunately did not hold [73] (Fig. 4).

An alternative approach to achieving selectivity of signal-

ing at GPCRs is associated with ligand directed signaling

outcomes manifested as changes in rank orders of potency

and or maximal effects relative to a reference (e.g., the



S NH2

R

O

22

R=NHNH2,NHNHPh, NHBn,NH2, 4-CH3Ph, 4-ClPh, Ph

S NH2

O

Cl

F3C

23, VCP520

S NH2

ON

Cl

O

O

24, VCP333

S NH2

O

Cl

Cl

25, LUF5484

S NH2

O

R

26a, R=H26b, R=Cl

F3C

26


Figure 4. General structure of 2-amino-4,5,6,7,8,9-hexahydrocyclooctal[b]thiophenes (22), 2-amino-3-aroyl-4-aryl thiophenes 23, 26a and 26b, VCP333

(24) and LUF5484 (25).

endogenous) agonist (stimulus bias or functional selectivity).

Valant et al. demonstrated this to be the case with the

compound 12a, which differentially modulated the effects

of the orthosteric agonist, R-PIA, in mediating ERK1/2 phos-

phorylation (pERK1/2) relative to [35S]GTPgS binding to acti-

vated G proteins. They also found that the well-characterized

compound T62 (4b) and two novel modulators/agonists,

VCP520 (23) and VCP333 (24) exhibited stimulus bias toward

cAMP inhibition compared to pERK1/2, whereas the orthos-

teric agonist, R-PIA, was strongly biased toward the latter

pathway. In both cases, the compounds under comparison

possessed a 2-amino group and the same substituent in the 3-

position [3-benzoyl group or 3-(4-chlorobenzoyl)], with the

point of divergence being in the 4/5 positions of the thio-

phene core. However, in numerous earlier studies, the nature

of the 3-benzoyl group has been shown to have a profound

effect on allosteric activity. Examples of substituents that

impart favorable activity include the 3-(3-trifluoromethyl)-

benzoyl present in PD 81,723 (3), the 3-(3,4-dichlorobenzoyl)

in LUF 5484 (25) as well as the 3-(4-chlorobenzoyl) in T62

(4b). Valant and coworkers studied the influence of further

substitution on the 3-benzoyl group of allosteric modulation

and biased signaling, by targeting analogues of compound

12a that possesses a 4-phenyl with electronegative substitu-

ents, namely (3-trifluoromethyl) or 4-(3,5-ditrifluoromethyl).

They found that only compounds 26a and 26b, differing

only with regards to the absence (26a) or presence (26b) of a

halogen atom in the 4-position of the benzoylthiophene ring,

promote functionally biased states of the A1-AR. In compar-

ison to the orthosteric agonist, R-PIA, 26a (alone) was biased


as an allosteric agonist toward cAMP accumulation over

ERK1/2 phosphorylation, whereas 26b showed minimal bias.

By contrast, when combined with R-PIA, 26a allosterically

modulated the activity of the orthosteric agonist at the two

pathways in a nonbiased manner, whereas the combination

of 26b and R-PIA resulted in the generation of pathway-

biased allosteric modulation. Collectively, these results high-

light how a GPCR bound with both an allosteric modulator

and an orthosteric agonist should be viewed as a unique

protein state that differs from those promoted by either

orthosteric or allosteric agonist alone [74].

Conclusions

Allosteric modulation is now being employed as a new way to

fine-tune the response of a receptor. Although an allosteric

modulator may not possess efficacy by itself (at least not at

low-medium concentrations), it can selectively influence the

tissue responses providing a powerful therapeutic advantage

over orthosteric ligands. Agonist-based compounds may not

be selective enough for the receptor subtype that produces a

desired therapeutic effect. In addition, high doses of an

agonist-based drug might result in a level of receptor activa-

tion that causes dangerous side effects. With respect to ade-

nosine receptors, allosteric enhancers for A1-AR have been

most studied.

The research on AE of A1-AR was focused on the prepara-

tion of compounds characterized by the lack of antagonist

activity at higher concentrations, which has most commonly

been interpreted as reflecting a recognition of the orthosteric

site on the receptor in addition to an allosteric site. The


compounds are claimed for uses including protection against

hypoxia, ischemia-induced injury and treatment of adeno-

sine-sensitive cardiac arrhythmias. The derivative (2-amino-

4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-(4-chloro-phe-

nyl)-methanone (4b, also known by its abbreviation T62)

normalized hyperexcited sensory nerve functions in a model

of neuropathic pain [43]. King Pharmaceuticals has initiated a

clinical trial program for the evaluation of derivative 4b for

the treatment of neuropathic pain, which progressed into

phase IIB before failing due to the lack of efficacy.

The mutagenesis and modeling studies might help the

rational design of more effective allosteric modulators to

improve classical pharmacological responses.

Conflict of interest

The authors declare no conflict of interest.

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Allosteric modulation of A1-adenosine receptor: a review

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