A series of 2,4-disubstituted quinolines as a new class of allosteric enhancers of the adenosine A3 receptor

Article abstract of DOI:10.1021/jm8014052

The adenosine receptor subfamily consists of the adenosine A₁, A_2A, A_2B, and A₃ receptors, which are localized in a variety of tissues throughout the human body. It is, therefore, a challenge to develop receptor specific ligands with improved tissue selectivity. Allosteric modulators could have these therapeutic advantages over orthosteric ligands. In the present study, a series of 2,4-disubstituted quinolines were synthesized on the basis of the structure of LUF6000 (34). Compound 27 (LUF6096) was able to allosterically enhance agonist binding to a similar extent as 34. In addition, this new compound showed low, if any, orthosteric affinity for any of the adenosine receptors. In a functional assay, compound 27 showed improved activity in comparison to 34, as it increased both the intrinsic efficacy and the potency of the reference agonist Cl-IB-MECA at the human adenosine A ₃ receptor.

Full text of DOI:10.1021/jm8014052

926
J. Med. Chem. 2009, 52, 926–931
A Series of 2,4-Disubstituted Quinolines as a New Class of Allosteric Enhancers of the
Adenosine A3 Receptor
Laura H. Heitman,† Aniko´ Go¨blyo¨s,† Annelien M. Zweemer, Rene´e Bakker, Thea Mulder-Krieger, Jacobus P.
D. van Veldhoven, Henk de Vries, Johannes Brussee, and Adriaan P. IJzerman*
DiVision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden UniVersity,
P.O. Box 9502, 2300 RA Leiden, The Netherlands
ReceiVed NoVember 7, 2008
The adenosine receptor subfamily consists of the adenosine A1, A2A, A2B, and A3 receptors, which are localized
in a variety of tissues throughout the human body. It is, therefore, a challenge to develop receptor specific
ligands with improved tissue selectivity. Allosteric modulators could have these therapeutic advantages
over orthosteric ligands. In the present study, a series of 2,4-disubstituted quinolines were synthesized on
the basis of the structure of LUF6000 (34). Compound 27 (LUF6096) was able to allosterically enhance
agonist binding to a similar extent as 34. In addition, this new compound showed low, if any, orthosteric
affinity for any of the adenosine receptors. In a functional assay, compound 27 showed improved activity
in comparison to 34, as it increased both the intrinsic efficacy and the potency of the reference agonist
Cl-IB-MECA at the human adenosine A3 receptor.
Introduction
Adenosine receptors belong to the class A family of membrane-
bound G-protein-coupled receptors (GPCRsa).1 The endogenous
ligand adenosine and its receptors play an important physi-
ological role, and together they mediate a large variety of effects,
for example, on the cardiovascular, immune, and central nervous
systems.2 The adenosine receptor family consists of four
subtypes, namely, the adenosine A1, A2A, A2B, and A3 receptors,
which are localized throughout the human body.
Several compound classes, both agonists and antagonists, have
been described as (orthosteric) ligands for the adenosine
receptors.3-6 Over the years, medicinal chemists succeeded in
Figure 1. Structure of the reference allosteric enhancer (34) and the
general structure of the new allosteric enhancers (2,4-disubtituted
quinolines) of the adenosine A3 receptor. The arrow points to the bond
deletion that led to the new series.
the development of subtype selective ligands, with the possible
exception of agonists for the adenosine A2B receptor.5 However,
many organs and tissues in the body express adenosine receptors,
which has compromised the development of clinical candidates
that have true in vivo selectivity of action. Allosteric modulation
of a receptor target may have the therapeutic advantage of
greater receptor subtype selectivity and tissue specificity.7 In
the past 2 decades, it has been observed that several GPCRs
from different classes can be allosterically modulated, such as
muscarinic receptors (class A), the corticotropin-releasing factor1
(class B), and glutamate (class C) receptors.8,9 Both allosteric
enhancers and inhibitors have been described that can either
enhance or inhibit the affinity and/or potency of the endogenous
ligand.8 A similar situation has emerged for adenosine receptors.
Nonselective modulators such as sodium ions and amiloride
derivatives influence orthosteric ligand binding to adenosine A1,
A2A, and A3 receptors.10,11 More selective allosteric modulators
also exist, such as PD81,723, an allosteric enhancer of agonist
binding at the adenosine A1 receptor.12 On human adenosine
A3 receptors, 3-(2-pyridinyl)isoquinoline (VUF5455)13 and 1H-
imidazo[4,5-c]quinolin-4-amine (DU124183) were shown to be
allosteric enhancers.14 However, these compounds also inhibited
equilibrium binding at the orthosteric site of the receptor.
Recently, in a series of imidazoquinolinamines a novel allosteric
enhancer of this receptor, (2-cyclohexyl-3H-imidazo[4,5-c]-
quinolin-4-yl)-(3,4-dichlorophenyl)amine (34), stood out because
it had improved allosteric over orthosteric properties.15
Our aim for the present study was to find a new chemical
template as the basis for novel allosteric modulators of the
adenosine A3 receptor with negligible, if any, orthosteric effects,
and selectivity over the A1 receptor. Therefore, the imidazo-
quinoline heterocyclic ring system of reference compound 34
was opened at the position indicated with an arrow (Figure 1),
which resulted in a new compound class, namely, the 2,4-
disubstituted quinolines. A series of these compounds with
* To whom correspondence should be addressed. Phone: +31- (0)71
527 4651. Fax: +31- (0)71 527 4565. E-mail: ijzerman@lacdr.leidenuniv.nl.
†
Both authors contributed equally to this study.
a Abbreviations: BSA, bovine serum albumin; cAMP, cyclic adenosine
5′-monophosphate; CGS 21680, 2-p-(2-carboxyethyl)phenethylamino-5′-
N-ethylcarboxamidoadenosine; CHAPS, 3-[(3- cholamidopropyl)dimethy-
lammonio]-1-propanesulfonate; CHO, Chinese hamster ovary; Cl-IB-MECA,
2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine; DCM, dichlo-
romethane; DMSO, dimethyl sulfoxide; DPCPX, 8-cyclopentyl-1,3-dipro-
pylxanthine; EDTA, ethylenediaminetetraacetic acid; GPCR, G-protein-
coupled receptor; I-AB-MECA, N6-(4-amino-3-iodobenzyl)-5′-N-methyl-
carbamoyladenosine; LUF6000, (2-cyclohexyl-3H-imidazo[4,5-c]quinolin-
4-yl)-(3,4-dichlorophenyl)amine; MRS1754, N-(4-cyanophenyl)-2-[4-(2,3,6,7-
tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide; NECA,
adenosine-5′-N-ethylcarboxamide; PBS, phosphate-buffered saline; R-PIA,
R-phenylisopropyl; SAR, structure-activity relationship; SEM, standard
error of the mean; TR-FRET, time-resolved fluorescence resonance energy
transfer; ZM241385, 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5,}-
triazin-5-yl amino]ethyl)phenol.
10.1021/jm8014052 CCC: $40.75
2009 American Chemical Society
Published on Web 01/22/2009

Quinolines as Allosteric Enhancers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 927
Scheme 1a
µM. For example, compounds 22 and 23 showed the highest
inhibition of equilibrium radioligand binding at the adenosine
A1 and A3 receptor, respectively. Notably, compound 30
inhibited radioligand binding at both the adenosine A2A and A2B
receptor. In addition, compound 17 seemed to increase radio-
ligand binding at the adenosine A2B receptor; however, the same
effect was observed on membranes of CHO cells without the
A2B receptor expressed (data not shown). The results obtained
for the reference modulator 34 in this study were comparable
to previously reported data,15 although some inhibition of
binding at the adenosine A1 receptor was observed in the present
study (39% vs -2%, respectively).
a Reagents and conditions: (i) CH3COOH, H2O2, 70 °C; (ii) H2SO4,
HNO3, 65 °C; (iii) POBr3, 0 °C; (iv) CH3COOH, Fe, 65 °C; (v) pyridine,
acid chloride, 115 °C; (vi) amines, EtOH (140 °C, 80 min) or without solvent
(180 °C, 90 min) in microwave.
As compounds 16-33 were synthesized as possible allosteric
modulators, their effect on the dissociation rate of 125I-AB-
MECA was investigated, as a measure for allosterism (Table
1).15 It was previously reported that 34 decreased the dissocia-
tion rate of 125I-AB-MECA from human adenosine A3 receptors.7
This was confirmed in the present study, as the amount of
radioligand bound to the receptor was increased in comparison
to the control condition (Table 1). Compound 34 did not
influence the dissociation rate of 125I-AB-MECA from the
adenosine A1 receptor, while equilibrium binding was inhibited
to some extent on both adenosine A1 and A3 receptors. The
latter strengthens the hypothesis that the allosteric site, and not
the orthosteric site, is not evolutionarily constrained, which may
result in a higher selectivity within a receptor subfamily.15
Several compounds with a cyclopentyl substituent (16-23) at
the R1 position decreased the dissociation rate of 125I-AB-MECA
from the adenosine A3 receptor, while the dissociation rate from
the adenosine A1 receptor was not influenced (Table 1).
Typically, combination with a (substituted) phenyl ring on the
R2 position resulted in the most potent allosteric enhancers.
However, introduction of a benzyl (18), cyclopentyl (22), or
indazole (23) group on the R2 position abolished the effect on
the dissociation rate from the adenosine A3 receptor. The most
potent allosteric enhancer with a cyclopentyl substituent at R1
was obtained in combination with a 3,4-dichlorophenyl group
at R2 (17). Subsequently, some analogues were synthesized with
a smaller cyclobutyl (24 and 25) and a larger cyclohexyl
(26-29) moiety at the R1 position. Introduction of cyclobutyl
instead of cyclopentyl resulted in compounds with lower
modulating potency for a phenyl (24 vs 16) or a 3,4-dichlo-
rophenyl (25 vs 17) substituent at R2. In combination with a
cyclohexyl at R1, however, these substituents resulted in a
strongly increased potency, 26 and 27, respectively. A 4-me-
thylphenyl (28) and an indane (29) substituent also resulted in
highly potent allosteric enhancers. In general, the cyclohexyl
group at R1 yielded compounds with a higher potency than with
a cyclopentyl one. Notably, compound 27 was the most potent
enhancer of these series and showed the highest structural
similarity to 34. Moreover, compound 27 showed a decreased
orthosteric effect on the adenosine A1 and A3 receptors when
compared to 34 (Table 1). In addition, analogues were synthe-
sized with a furan (30 and 31) or a phenyl ring (32 and 33).
Although these rings have a similar size as the cyclopentyl (16
and 17) or cyclohexyl ring (26 and 27), respectively, the potency
of the corresponding compounds was completely abolished.
Apparently, an aromatic instead of an aliphatic substituent at
the R1 position is not tolerated.
Scheme 2a
a Reagents and conditions: (i) POCl3, reflux; (ii) NH4OH, 160 °C, 2.5 h
in microwave; (iii) pyridine, acid chloride, 60 °C; (iv) amines, EtOH (140
°C, 80 min) or without solvent (180 °C, 90 min) in microwave.
similar substituents as described by Go¨blyo¨s et al. were
synthesized, and their structure-activity relationship (SAR) was
examined.15 The results obtained from radioligand displacement
and kinetic dissociation experiments as well as functional
(cAMP-based) assays were compared with the effects of 34.
Results and Discussion
Chemistry. The novel derivatives 16-20 were synthesized
as shown in Scheme 1. Oxidation of quinoline resulted in
quinoline 1-oxide (2),16,17 which was nitrated to give 4-nitro-
quinoline 1-oxide (3).18 4-Nitroquinoline 1-oxide (3) was treated
with phosphorus oxybromide to afford 2-bromo-4-nitroquinoline
(4).19,20 This was converted into 4-amino-2-bromoquinoline (5)
with iron powder in acetic acid.21 Reaction of compound 5 with
carbonyl chlorides22 and then subsequently with the appropriate
amines afforded the desired compounds 16-20.15 Derivatives
21-33 were obtained as depicted in Scheme 2. Ring-closure
reaction of malonic acid with aniline in phosphorus oxychloride
gave 2,4-dichloroquinoline (8),23 which was subsequently treated
with ammonia under microwave conditions to give 2-amino-
4-chloroquinoline (9).24,25 Reaction of compound 9 with car-
bonyl chlorides22 and then subsequently with the appropriate
amines afforded the desired compounds 21-33.15
Structure-Activity Relationships. All newly synthesized
compounds (16-33) and the reference compound (34, Fig-
ure 1) were tested in equilibrium and kinetic radioligand binding
assays (Table 1). At first, equilibrium displacement assays were
performed with 125I-AB-MECA on CHO cells expressing the
human adenosine A1 or A3 receptor. In addition, their effect on
[3H]ZM241385 and [3H]MRS1754 equilibrium binding at the
adenosine A2A and A2B receptors was examined. From Table 1
it follows that compounds 16-33 only moderately inhibited
radioligand binding to the adenosine A1 or A3 receptor at 10
In the present study, compound 27 was the most potent
allosteric enhancer of the adenosine A3 receptor. Therefore, the
effect of 27 on the dissociation rate of 125I-AB-MECA from
the A3 receptor was further investigated in comparison to 34
(Figure 2 and Table 2). Dissociation was induced by the addition

928 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Heitman et al.
Table 1. Affinity and Modulatory Potency of Compounds 16-33 and That of the Reference Allosteric Enhancer 34 at the Human Adenosine A1, A2A
,
A2B, and A3 Receptor Subtypes, Expressed as % Displacement and % Enhancement at 10 µM, Respectivelyd
a Displacement of specific 125I-AB-MECA (A1 and A3), [3H]ZM241385 (A2A), or [3H]MRS1754 (A2B) binding at their respective cell membranes.
b Dissociation of 125I-AB-MECA induced by an excess DPCPX (A1) or Cl-IB-MECA (A3) in the absence (control) and presence of modulator.
Enhancement of radioligand binding was determined after 90 (A1) or 120 min (A3) of dissociation, where the control was set at 100%. Values are the
mean of two separate assays performed in duplicate. c See Figure 1 for the chemical structure of 34. d All experiments were performed using CHO
cells stably transfected with the human adenosine A1, A2B, or A3 receptor or HEK293 cells stably transfected with the human adenosine A2A recep-
tor.

Quinolines as Allosteric Enhancers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 929
Table 3. Inhibitory Effect (Potency and Intrinsic Activity) of
Cl-IB-MECA on cAMP Production in CHO Cells Expressing the
Human Adenosine A3 Receptor in the Absence and Presence of Either
27 or 34 (10 µM)
activity in cAMP Assaya
compd
EC50 (nM)
Emax (%)
100 ( 1
223 ( 10***
286 ( 39**
Cl-IB-MECA
+10 µM 27
+10 µM 34
31 ( 3
9 ( 1***
22 ( 4
a Inhibition of forskolin-induced cAMP production in CHO cells that
stably express the human adenosine A3 receptors by Cl-IB-MECA in the
absence (control) and presence of modulator, with 100% as the amount of
cAMP production in the presence of 10 µM Cl-IB-MECA and 0% as the
amount of forskolin-stimulated cAMP production. Values are the mean
((SEM) of three separate assays performed in duplicate: (**) p < 0.005;
(***) p < 0.001 versus control.
Figure 2. Dissociation kinetics of 125I-AB-MECA binding to human
adenosine A3 receptors stably expressed in CHO cell membranes.
Dissociation was initiated by either the addition of 10 µM Cl-IB-MECA
mixed with either buffer (control) or modulator. Shown are representa-
tive results from one experiment performed in duplicate (see Table 2
for kinetic parameters).
present study. Under those conditions 34 was also capable of
enhancing the intrinsic activity of Cl-IB-MECA but now to
approximately 150%.15 Interestingly, in the present study 27
also enhanced the potency (expressed as EC50 value) of Cl-IB-
MECA, from 31 to 9 nM, whereas 34 did not, the latter in line
with previously reported findings.15 Notably, 27 and 34 do not
display intrinsic efficacy at the human adenosine A3 receptor
when applied alone (see point along y-axis, Figure 3) and can,
therefore, be classified as true allosteric enhancers.26 Similarly,
two classes of allosteric enhancers have been reported for the
GABAB receptor that increase agonist potency and efficacy
without having intrinsic efficacy.27,28 Urwyler and co-workers
postulated that these compounds will have a better side effect
profile because a synergistic effect will only arise in the presence
of the endogenous agonist. As the adenosine receptor is
expressed throughout the body, an allosteric enhancer such as
compound 27 may therefore be more beneficial than an
(allosteric) ligand that has intrinsic efficacy on its own.
Table 2. Influence of 27 and 34 on the Dissociation Kinetics of the
Radiolabeled Agonist 125I-AB-MECA from the Human Adenosine A3
Receptor Expressed on Membranes of Stably Transfected CHO Cells
dissociation assaya
condition
control
koff (min-1)
shift koff
0.089 ( 0.043 (fast)
0.0072 ( 0.0011 (slow)
0.029 ( 0.014
+10 µM 27
+10 µM 34
3.1
2.5
0.035 ( 0.008
a The value of the kinetic dissociation rate constant was obtained by
analysis of the exponential dissociation curve of 125I-AB-MECA bound to
human adenosine A3 receptors in the absence (control) or presence of
modulator. The shift is defined as the ratio of koff values in the presence
and absence (control, fast component) of modulator, respectively. Values
are the mean ((SEM) of three separate assays performed in duplicate.
Conclusions
In summary, it was shown that scission of a chemical bond
in the structure of the reference compound 34 led to the
discovery of a new series of 2,4-disubstituted quinolines as
allosteric enhancers of the adenosine A3 receptor. In particular
compound 27 (LUF6096) was able to decrease the dissociation
rate of 125I-AB-MECA to a similar extent as 34. In addition,
compound 27 had a decreased orthosteric effect at both the
adenosine A3 and A1 receptors. In a functional assay, 27 and
34 increased the intrinsic efficacy of the agonist Cl-IB-MECA.
Moreover, the presence of 27 significantly increased the potency
of Cl-IB-MECA, unlike 34.
Figure 3. Modulatory effects of 27 and 34 on the effect-concentration
curve of Cl-IB-MECA for the inhibition of the forskolin-stimulated
cAMP production in CHO cells expressing the human adenosine A3
receptor. Representative graphs from one experiment performed in
duplicate (see Table 3 for EC50 and Emax values).
Experimental Section
of an excess of Cl-IB-MECA, which resulted in dissociation
rates of 0.089 ( 0.043 and 0.0072 ( 0.0011 min-1 for a fast
and slow phase, respectively. In the presence of 34 the
dissociation rate decreased by 3.1-fold (koff ) 0.029 min-1) when
compared to the fast phase of dissociation in the absence of
modulator, which corresponds to the previously reported effect
of the same compound.15 In a similar fashion, the dissociation
rate was decreased 2.5 times (koff ) 0.035 min-1) in the presence
of 27.
The effects of 27 and 34 were also investigated in a functional
assay by measuring the inhibition of cAMP production by the
reference agonist Cl-IB-MECA (Figure 3 and Table 3). Both
compounds (10 µM) significantly and dramatically enhanced
the intrinsic activity of Cl-IB-MECA to 223% for 27 and 286%
for 34. A similar study for 34 had been performed previously,
in the presence of 10 µM rather than 1 µM forskolin as in the
Chemistry: Material and Methods. Microwave-assisted chem-
istry was performed on an Emrys Optimizer with Emrys Optimizer
software. For the reactions, vials with a volume of 2-5 mL were
1
used. H NMR spectra were measured at 200 MHz with a Bruker
AC 200 or Bruker DMX 600 spectrometer. 13C NMR spectra were
measured at 50 or 150 MHz. Chemical shifts for H and 13C are
1
given in ppm (δ) relative to tetramethylsilane (TMS) as internal
standard, and coupling constants are given in Hz. Melting points
were determined with a Bu¨chi capillary melting point apparatus
and are uncorrected. Combustion analyses of new target compounds
were performed by the analytical department of the Gorlaeus
Laboratories, Leiden University (The Netherlands), and are within
0.4% of theoretical values unless otherwise specified.
General Procedure for the Preparation of 2,4-Substituted
Quinolines (16-33). Method A. Compounds 10-15 were dis-
solved/suspended in absolute ethanol (1.5 mmol/2.5 mL), and the
appropriate amines (3 equiv) were added. The mixture was heated

930 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Heitman et al.
in the microwave at 140 °C for 80 min. After the reaction was
completed, ethanol was evaporated and the residue was dissolved
in DCM (100 mL) and washed with 1 M NaOH (3 × 100 mL).
The organic layer was dried on MgSO4. The products were purified
by column chromatography and recrystallized.
Method B. Compounds 10-15 and the appropriate amines (10
equiv) were heated in the microwave without any solvent at 180
°C for 90 min. After the reaction was completed, the reaction
mixture was dissolved in DCM (100 mL) and washed with water
(2 × 50 mL) and brine (1 × 50 mL). The organic layer was dried
on MgSO4. The products were purified by column chromatography
and recrystallized
medium consisting of Dulbecco’s modified Eagle’s medium
(DMEM) and Ham’s F12 medium (1:1) supplemented with 10%
(v/v) normal adult bovine serum, streptomycin (50 µg/mL),
penicillin (50 IU/mL), and G418 (0.4 mg/mL) at 37 °C in 5% CO2.
The cells were subcultured twice weekly at a ratio of 1:20. All cell
membranes were prepared as described previously.29
Radioligand Displacement Assays. Adenosine A1 and A3
Receptor. Membrane aliquots containing 20 µg (A1) or 10 µg of
protein (A3) were incubated in a total volume of 100 µL assay buffer
(50 mM Tris HCl, pH 8.0, supplemented with 10 mM MgCl2, 1
mM EDTA, and 0.01% (w/v) CHAPS) at 25 °C for 60 min (A1) or
120 min (A3). Displacement experiments were performed using 10
µM competing ligand in the presence of 45 000 cpm (∼0.1 nM)
125I-AB-MECA. Nonspecific binding was determined in the pres-
ence of 10 µM DPCPX (A1) or 100 µM R-PIA (A3). Incubations
were terminated by dilution with ice-cold assay buffer. Separation
of bound from free radioligand was performed by rapid filtration
through Whatman GF/B filters using a Brandel harvester. Filters
were subsequently washed three times with 2 mL of ice-cold buffer.
Filter-bound radioactivity was determined in a γ-counter (Wizard
1470, PerkinElmer Life and Analytical Sciences).
Adenosine A2A Receptor. Membrane aliquots containing 30 µg
of protein were incubated in a total volume of 200 µL of assay
buffer (50 mM Tris-HCl, pH 7.4) at 25 °C for 120 min.
Displacement experiments were performed using 10 µM competing
ligand in the presence of ∼1.7 nM [3H]ZM241385. Nonspecific
binding was determined in the presence of 100 µM CGS 21680.
Incubations were terminated, and samples were obtained as
described under Adenosine A1 and A3 Receptor. Filter-bound
radioactivity was determined in a ꢀ-counter (Tri-Carb 2900 TR,
PerkinElmer Life and Analytical Sciences) after addition of 3.5
mL of PerkinElmer Emulsifier Safe.
Adenosine A2B Receptor. Membrane aliquots containing 20 µg
of protein were incubated in a total volume of 100 µL of assay
buffer (50 mM Tris-HCl, pH 7.4, supplemented 0.1% (w/v)
CHAPS) at 25 °C for 60 min. Displacement experiments were
performed using 10 µM competing ligand in the presence of ∼1.2
nM [3H]MRS1754. Nonspecific binding was determined in the
presence of 1 mM NECA. Incubations were terminated, and samples
were obtained and analyzed as described under Adenosine A2A
Receptor.
Radioligand Kinetic Dissociation Assays. Dissociation experi-
ments were performed by preincubating membrane aliquots con-
taining 20 µg (A1) or 10 µg of protein (A3) in a total volume of
100 µL of assay buffer at 25 °C for 60 min (A1) or 120 min (A3)
with 45 000 cpm (∼0.1 nM) 125I-AB-MECA. After preincubation,
dissociation was initiated by addition of 10 µM DPCPX (A1) or
10 µM Cl-IB-MECA (A3) in the absence (control) or presence of
allosteric modulators in a volume of 5 µL, of which 25% (v/v) is
DMSO. The amount of radioligand still bound to the receptor was
measured after 90 (A1) and 120 min (A3) of dissociation. The
obtained amount of radioligand binding determined at control
conditions was set at 100%. Incubations were terminated and
samples were obtained and analyzed as described under Radioligand
Displacement Assays.
cAMP Determination. Intracellular cAMP levels were measured
using a LANCE cAMP 384 kit (PerkinElmer, The Netherlands) as
described previously.30 In short, to each well different concentra-
tions of Cl-IB-MECA in stimulation buffer (PBS with 5 mM Hepes,
pH 7.4, supplemented with 0.1% BSA, rolipram (50 µM), and
cilostamide (50 µM)) were added in a volume of 5 µL in the absence
(control) or presence of allosteric modulators. Then 4.5 µL of
CHOhA3 cell suspension in stimulation buffer was seeded into a
384-well plate (approximately 5000 cells/well), which was followed
by incubation for 15 min at room temperature. Subsequently, 2.5
µL of forskolin (1 µM) was added and the mixture was incubated
for 30 min at room temperature. Finally, 3 h after addition of
detection mix (6 µL) and cAMP antibody solution (6 µL),
intracellular cAMP levels were measured using a TR-FRET assay
on a Victor spectrometer (PerkinElmer, The Netherlands) according
to instructions of the supplier.
N-(2-Anilinoquinolin-4-yl)cyclopentanecarboxamide (16).
Method A. An amount of 0.36 mmol of N-(2-bromoquinolin-4-
yl)cyclopentanecarboxamide (10) was used, and the eluent was
3-10% MeOH in DCM. The product was recrystallized from
methanol to give yellow crystals. Yield: 0.039 g, 33%. MS (ESI)
1
m/z: 331.2 [M + H]1+. H NMR (CDCl3) δ 1.72-2.04 (m, 8H,
4CH2), 2.80-2.98 (m, 1H, CH), 6.93 (br s, 1H, NH), 7.07 (t, 1H,
J ) 5.74, 8.06 Hz, Ar), 7.25-7.41 (m, 3H, Ar), 7.57-7.67 (m,
5H, m, Ar), 7.78-7.83 (m, 1H, Ar), 8.01 (s, 1H, NH). 13C NMR
(CDCl3) δ 25.63, 30.18, 46.89, 100.90, 116.18, 118.36, 119.73,
122.50, 127.25, 129.46, 139.77, 140.96, 147.54, 154.51, 174.87.
Anal. (C21H21N3O·0.3H2O) C, H, N.
N-{2-[(3,4-Dichlorophenyl)amino]quinolin-4-yl}cyclohexa-
necarboxamide (27). Method B. An amount of 0.17 mmol of
N-(2-chloroquinolin-4-yl)cyclohexanecarboxamide (12) was used,
and the eluent was 1% MeOH in DCM. The product was
recrystallized from ethyl acetate to give white crystals. Yield: 0.16 g,
43%. 1H NMR (CDCl3) δ 1.23-2.17 (m, 10H, 5CH2), 2.35-2.50
(m, 1H, CH), 6.77 (bs, 1H, NH), 7.32-7.41 (m, 2H, Ar), 7.49 (dd,
1H, J ) 6.22, 2.56 Hz, Ar), 7.58-7.67 (m, 2H, Ar), 7.84-7.88
(m, 2H, Ar, NH), 7.95 (s, 1H, Ar), 8.09 (d, 1H, J ) 2.56 Hz, Ar).
13C NMR (DMSO-d6) δ 25.23, 25.48, 29.27, 38.30, 44.46, 103.69,
117.58, 118.24, 119.03, 121.55, 122.00, 122.55, 127.07, 129.67,
130.28, 130.80, 141.89, 141.98, 147.50, 154.26, 175.87. Anal.
(C22H21Cl2N3O·0.5H2O) C, H, N.
Biology: Material and Methods. NECA, Cl-IB-MECA, R-PIA,
and DPCPX were purchased from Sigma Aldrich Chemie B.V.
(Zwijndrecht, The Netherlands). Compound 34 was synthesized in
our laboratory as described previously.15 125I-AB-MECA (specific
activity 2200 Ci/mmol) was purchased from PerkinElmer Life and
Analytical Sciences (Groningen, The Netherlands). [3H]ZM241385
(specific activity 17 Ci/mmol) and [3H]MRS1754 (specific activity
37 Ci/mmol) were obtained from Tocris Cookson, Ltd. (U.K.), and
CGS 21680 was a gift from Dr. R. A. Lovell (Novartis, Summit,
NJ). CHO (Chinese hamster ovary) cells stably expressing the
human adenosine A1 (CHOhA1) or A3 receptor (CHOhA3) were
obtained from Dr. A. Townsend-Nicholson (University College
London, U.K.) and Dr. K.-N. Klotz (University of Wu¨rzburg,
Germany), respectively. HEK293 (human embryonic kidney) cells
stably expressing the human adenosine A2A receptor (HEK293hA2A)
were obtained from Dr. J. Wang (Biogen Idec, Cambridge, MA).
CHO cells stably expressing the human adenosine A2B receptor and
a secreted placental alkaline phosphatase (SPAP) reporter gene
(CHOhA2B_SPAP) were obtained from Dr. S. J. Dowell (GSK,
Stevenage, U.K.). All other chemicals and cell culture materials
were obtained from standard commercial sources.
Cell Culture and Membrane Preparation. CHOhA1 cells were
cultured as described previously.29 HEK293hA2A cells were grown
in culture medium consisting of Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% (v/v) normal adult
bovine serum, streptomycin (50 µg/mL), penicillin (50 IU/mL), and
G418 (0.5 mg/mL) at 37 °C in 7% CO2. The cells were subcultured
twice weekly at a ratio of 1:8. CHOhA2B_SPAP cells were grown
in culture medium consisting of Dulbecco’s modified Eagle’s
medium (DMEM) and Ham’s F12 medium (1:1) supplemented with
10% (v/v) normal adult bovine serum, streptomycin (50 µg/mL),
penicillin (50 IU/mL), G418 (1.0 mg/mL), and hygromycin B (0.4
mg/mL) at 37 °C in 5% CO2. The cells were subcultured twice
weekly at a ratio of 1:10. CHOhA3 cells were grown in culture

Quinolines as Allosteric Enhancers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 931
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Data Analysis. All binding data was analyzed using the nonlinear
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Supporting Information Available: Experimental details of the
synthesis of the compounds described in this paper, their 1H NMR
and 13C NMR spectroscopic data, and their elemental analysis
results. This material is available free of charge via the Internet at
http://pubs.acs.org.
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