Structure-activity relationships of new 1H-imidazo[4,5-c]quinolin-4-amine derivatives as allosteric enhancers of the A3 adenosine receptor

Article abstract of DOI:10.1021/jm060086s

1H-Imidazo[4,5-c]quinolin-4-amine derivatives have been synthesized as allosteric modulators of the human A₃ adenosine receptor (AR). Structural modifications were made at the 4-amino and 2 positions. The compounds were tested in both binding and functional assays, and many were found to be allosteric enhancers of the action of A₃AR agonists by several different criteria. First, a potentiation of the maximum efficacy of the agonist C1-IB-MECA was observed for numerous derivatives. Also, a number of these compounds decreased the rate of dissociation of the agonist [ ¹²⁵I]I-AB-MECA from the A3AR. Most prominently, compound 43 (LUF6000) was found to enhance agonist efficacy in a functional assay by 45% and decrease dissociation rate similarly without influencing agonist potency. The structural requirements for allosteric enhancement at the A₃AR were distinct from the requirements to inhibit equilibrium binding. Thus, we have prepared allosteric enhancers of the human A₃AR that have an improved allosteric effect in comparison to the inhibition of equilibrium binding at the orthosteric site.

Full text of DOI:10.1021/jm060086s

3354
J. Med. Chem. 2006, 49, 3354-3361
Structure-Activity Relationships of New 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as
Allosteric Enhancers of the A3 Adenosine Receptor
Aniko´ Go¨blyo¨s,‡ Zhan-Guo Gao,† Johannes Brussee,‡ Roberto Connestari,‡ Sabrina Neves Santiago,‡ Kai Ye,‡
Adriaan P. IJzerman,*,‡ and Kenneth A. Jacobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892, and DiVision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research,
Leiden UniVersity, P.O. Box 9502, 2300 RA, Leiden, The Netherlands
ReceiVed January 25, 2006
1H-Imidazo[4,5-c]quinolin-4-amine derivatives have been synthesized as allosteric modulators of the human
A3 adenosine receptor (AR). Structural modifications were made at the 4-amino and 2 positions. The
compounds were tested in both binding and functional assays, and many were found to be allosteric enhancers
of the action of A3AR agonists by several different criteria. First, a potentiation of the maximum efficacy
of the agonist Cl-IB-MECA was observed for numerous derivatives. Also, a number of these compounds
decreased the rate of dissociation of the agonist [125I]I-AB-MECA from the A3AR. Most prominently,
compound 43 (LUF6000) was found to enhance agonist efficacy in a functional assay by 45% and decrease
dissociation rate similarly without influencing agonist potency. The structural requirements for allosteric
enhancement at the A3AR were distinct from the requirements to inhibit equilibrium binding. Thus, we
have prepared allosteric enhancers of the human A3AR that have an improved allosteric effect in comparison
to the inhibition of equilibrium binding at the orthosteric site.
Introduction
The adenosine receptors (ARs), a family of GPCRs consisting
of the A1, A2A, A2B, and A3 subtypes, are activated by the
ubiquitous modulator adenosine in our body. Selective agonists
have been synthesized for three out of the four subtypes of
adenosine receptors (ARs).1-3 Each of these groups of subtype-
selective agonists has representative examples that have already
entered clinical trials.1 A1AR selective agonists are under
development for treating cardiac arrhythmia, pain, and pulmo-
nary disorders.4-6 A2AAR agonists are in clinical trials for
cardiac stress imaging and are also of interest for inflammation.7
A3AR agonists, such as IB-MECA (N6-(3-iodobenzyl)-5′-N-
methylcarboxamidoadenosine, see Figure 1), are in clinical trials
for colon carcinoma and rheumatoid arthritis and have potential
for the treatment of myocardial infarction, stroke, and other
disorders.1
Although newer and more selective AR agonists are being
developed,1,2 there are inherent problems, in general, associated
with the therapeutic use of GPCR agonists.8 The widespread
occurrence of receptors such as the ARs makes selectivity for
some organs or tissues nearly unachievable using directly acting
Figure 1. Structures of reference agonists (IB-MECA and 2-Cl-IB-
(orthosteric) agonists, and thus a number of agonists were
discontinued after initial phases of clinical trials.1,2 In contrast
to directly acting agonists, allosteric modulators act at a separate
site on the receptor protein to modulate the effect of a native
agonist.9 An advantage of a positive allosteric modulator of a
membrane receptor over its native, orthosteric activator is that
greater selectivity, in principle, may be achieved. The positive
allosteric modulator would enhance the action of the native
MECA) and allosteric modulators (DU 124183, amiloride, and VUF5455)
of the A3AR.
agonist, but may have no effect of its own on the unoccupied
receptor. Thus, the effect of endogenous agonist, which may
be insufficient in a particular disease state, may be magnified
in a temporally specific manner through allosteric modulation.
The higher subtype-selectivity commonly exerted by allosteric
modulators and the fact that the allosteric action is ideally
coupled to simultaneous presence of the endogenous ligand both
help to prevent over-dosage compared to the administration of
a conventional, and possibly nonselective, orthosteric agonist.
Allosteric modulation is considered an important mechanism
of controlling receptor function.8,10-12 In the case of the ligand-
gated ion channels, a series of benzodiazepines, including
diazepam, enhance the CNS inhibitory function of the endog-
* To whom correspondence should be addressed. Dr. K. A. Jacobson,
Chief, Molecular Recognition Section, Bldg. 8A, Rm. B1A-19, NIH,
NIDDK, LBC, Bethesda, MD 20892-0810. Tel: 301-496-9024. Fax: 301-
480-8422; e-mail: kajacobs@helix.nih.gov. Ad P. IJzerman, Ph.D., Leiden/
Amsterdam Center for Drug Research, P.O. Box 9502, 2300RA, Leiden,
The Netherlands, Tel: 31-71-5274651, Fax: 31-71-5274565, e-mail:
ijzerman@lacdr.leidenuniv.nl.
† National Institutes of Health.
‡ Leiden/Amsterdam Center for Drug Research.
10.1021/jm060086s CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/15/2006

Allosteric Enhancers of the A3 Adenosine Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3355
Scheme 1a
a Reagents: (i) HCl, HONdCHCH2NO2; (ii) (CH3CO)2O, CH3COOK; (iii) POCl3; (iv) NH3; (v) H2/Pd; (vi) (a) polyphosphoric acid, R′COOH, (b)
trimethyl orthoformate, HCOOH, (c) 1. R′COCl, 2. NaOH; (vii) 3-chloroperoxybenzoic acid; (viii) POCl3; (ix) RNH2.
enous γ-aminobutyric acid, and have become the most widely
prescribed sleep medications. In contrast, the directly acting
agonists have not found clinical applications due to the inherent
side effects. In the GPCR field, cinacalcet, a positive allosteric
modulator of the calcium-sensing receptor (CaR), has recently
been approved for the treatment of secondary hyperparathy-
roidism in dialysis patients suffering from chronic kidney
disease.13,14 In the case of ARs, the A1AR has been the most
studied, and one of its allosteric enhancers, T62 (2-amino-
4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl-(4-chlorophenyl)metha-
none), is now in Phase I clinical trials for the potential treatment
of neuropathic pain.1,15
Several classes of compounds (see also Figure 1) have
previously been reported to be allosteric modulators for the A3-
AR, including the 3-(2-pyridinyl)isoquinoline (VUF5455),16 the
1H-imidazo[4,5-c]quinolin-4-amine (DU124183, 28 in the cur-
rent study),17 and analogues of amiloride such as N,N-hexa-
methyleneamiloride.18 One of the most interesting findings was
that 28 and some of its analogues decreased agonist potency
but enhanced maximum agonist efficacy.17,19
In the present study we have extended our earlier finding
that 1H-imidazo[4,5-c]quinolin-4-amine derivatives act as al-
losteric modulators of the A3AR.17 We have synthesized and
characterized the SAR (structure-activity relationship) of a new
set of analogues based on compound 28. A number of
derivatives were found to be allosteric enhancers of the A3AR.
One of the analogues of 28, compound 43, was found to enhance
agonist efficacy in a functional assay and decrease dissociation
rate in binding but without influencing agonist potency.
3-nitro-4-aminoquinoline (5) with ammonia, which was subse-
quently reduced by catalytic hydrogenation to 3,4-diamino-
quinoline (6) with 10% palladium on charcoal as catalyst. The
next step involved ring-closure, which was carried out on three
different ways. Compounds 8-11 were prepared by ring closure
of the appropriate carboxylic acids and 3,4-diaminoquinoline
(6) in polyphosphoric acid.21 Compounds 12 and 13 were
prepared by ring-closure of 2-furoyl chloride and hexanoyl
chloride, respectively, with 3,4-diaminoquinoline (6).22 Com-
pound 7 was prepared by ring-closure of 3,4-diaminoquinoline
(6) with formic acid in trimethylorthoformate. Oxidation with
3-chloroperoxybenzoic acid afforded 5-oxides 14-20, which
subsequently could be converted with phosphorus oxychloride
into the respective 4-chloro compounds 21-27. Finally, treat-
ment of compounds 21-27 with the appropriate amines afforded
the desired compounds 28-45, respectively.23
Biological Activity. Previously reported compound 28 (ref
17) and newly synthesized imidazoquinolines 29-45 affect AR
binding to or function at ARs as shown in Table 1. We first
tested the effect of these compounds on the equilibrium binding
at A1, A2A, and A3ARs using standard agonist radioligands16,17
[3H]R-N6-[phenylisopropyl]adenosine, [3H]2-[p-(carboxyethyl)-
phenylethylamino]-5′-N-ethylcarboxamidoadenosine, and 125I-
N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide, re-
spectively. We then measured the effect of these compounds
(at 10 µM) on the A2BAR with a cyclic AMP functional assay,24
as an agonist radioligand has not been readily available for this
subtype. Most compounds only induced a rather weak inhibition
of equilibrium binding at the concentration used. For compounds
28-32 we also determined Ki values, if possible. It is not known
whether the observed inhibition is of allosteric or nonallosteric
character.
Results
Chemistry. The novel derivatives 28-45 were synthesized
as shown in Scheme 1. Condensation of anthranilic acid
hydrochloride (1) with 2-nitroacetaldehyde oxime (HONd
CHCH2NO2), prepared in situ from CH3NO2 and NaOH,20
resulted in 2-(2-nitroethylideneamino)benzoic acid (2), which
was dehydrated in acetic anhydride in the presence of potassium
acetate to give 3-nitro-4-hydroxyquinoline (3).20 3-Nitro-4-
hydroxyquinoline (3) was treated with phosphorus oxychloride
to afford 3-nitro-4-chloroquinoline (4). This was converted to
We further measured the capability of these compounds to
influence the dissociation of an agonist radioligand, e.g. [125I]I-
AB-MECA at the A3AR (Figure 2), which is a commonly used
approach in the study of allosteric modulators8,10 and perturba-
tion of the dissociation rate reflects the potency of the allosteric
modulators. It has been reported previously that compound 28
decreased the dissociation rate of [125I]I-AB-MECA from the
human A3ARs.17 Here it is shown that the dissociation rates of

3356 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Go¨blyo¨s et al.
Table 1. Potency of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives in Binding Assays at Human A1, A2A, A2B, and A3ARs Expressed in CHO Cells
and Allosteric Effects at the Human A3ARa
a All experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human ARs. Binding at human A1, A2A and
A3ARs in this study was carried out as described in Experimental Procedures using [3H]R-PIA, [3H]CGS 21680, or [125I]I-AB-MECA as a radioligand.
Values from the present study are expressed as mean ( SEM, n ) 3-5. Percentage inhibition at A1, A2A, or A3 receptors is expressed as the mean value
b
from two to four separate experiments with similar results performed in duplicate. A2BAR: effect of compounds at 10 µM on NECA (150 nM)-induced
cyclic AMP accumulation from one experiment performed in triplicate, CGS15943 (10 µM) ) 100%. c Dissociation: % decrease of [125I]I-AB-MECA
dissociation at 30 min (control ) 100%). d Increase of efficacy: compared to maximal effect by 2-Cl-IB-MECA alone (control).

Allosteric Enhancers of the A3 Adenosine Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3357
important target in drug development. Thus, allosteric modula-
tion of GPCRs is of particular importance.
The present study is actually an important extension of an
earlier pharmacological study.17 Previously it was found that
functional enhancement of agonist action at the A3AR by 28
(see Figure 1 and Table 1) and its analogues has potential for
further development in the direction of therapeutic applications.
A3AR mutagenesis studies18 have implicated amino acids
F1825.43 and N2747.45 in the action of 28. The mutagenesis data
were interpreted using a rhodopsin-based A3AR molecular
model, suggesting multiple binding modes of the enhancers
either at the orthosteric site or at a distinct putative allosteric
site. Here we synthesized and systematically studied a new series
of allosteric modulators for the A3AR. Allosteric enhancers of
A3AR activation may be predicted to be useful against a number
of disorders, including ischemic conditions and cancer.
Figure 2. Radioligand binding studies on the human A3AR. Study of
the dissociation kinetics of the agonist radioligand [125I]I-AB-MECA
under control conditions and in the presence of 10 µM of compound
42, 43, or 44.
It is interesting that compounds within this series showed a
remarkable variety of allosteric modulatory effects, with positive,
neutral, and maybe slightly negative effects on both agonist
efficacy and ligand dissociation. It was shown that these
compounds influenced agonist binding, potency, and efficacy
in a separate manner. The identification of the important
correlation between the ability of this series of compounds to
affect ligand dissociation and their capability to enhance agonist
efficacy suggested the possibility of further structural refinement
to design more potent and efficacious allosteric enhancers.
Figure 3. Functional assay of the human A3AR. The % inhibition of
forskolin-stimulated cAMP production by increasing concentrations of
2-Cl-IB-MECA under control conditions or in the presence of 10 µM
of compound 28 or 43.
Allosteric modulators that separately influence agonist affinity
and efficacy at several GPCRs have been reported. For example,
T62, which is now under clinical trails for potential treatment
of neuropathic pain, has recently been suggested to enhance
agonist efficacy rather than agonist potency as demonstrated in
a functional assay of GTPγS binding to rat brain membranes.15
CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carbox-
ylate ethyl ester), a noncompetitive metabotropic glutamate
receptor 1 antagonist, allosterically inhibits receptor signaling
without affecting glutamate binding.28 PIT (2,2′-pyridylisatogen
tosylate) antagonizes P2Y1 receptor signaling without inhibiting
nucleotide binding.29 Compound 28 enhanced agonist efficacy
but decreased agonist potency (ref 17 and this study). In previous
studies of modulation of various GPCRs by several series of
compounds, allosteric potencies determined from dissociation
kinetics have not been found to correlate with binding potency
from equilibrium experiments.19,26,27 However, to our knowl-
edge, the findings from the current study represent the first
evaluation of the correlation among the effects on agonist
efficacy, dissociation kinetics, agonist potency, and affinity.
[
125I]I-AB-MECA from the A3AR in the presence of 10 µM 42
and 43 were 0.038 ( 0.004 and 0.036 ( 0.005 min-1
,
respectively, which were significantly reduced and different from
control (no further compound present) and that in the presence
of 10 µM 44 (0.061 ( 0.006 min-1). By comparing the ligand
dissociation at 30 min in the absence or presence of 10 µM
imidazoquinoline, a number of these newly synthesized com-
pounds (compounds 28-33, 37-40, 43) were found to decrease
the dissociation significantly (Table 1). The ability of these
compounds to affect the dissociation was found not to correlate
with their ability to displace the equilibrium ligand binding at
the A3AR (Table 1, r ) 0.34). For example, although 28 was
more potent than 43 in displacing [125I]I-AB-MECA binding,
their ability to affect the dissociation rate was similar (Table
1).
We further examined the ability of these compounds to
influence agonist function by using a cyclic AMP functional
assay in intact CHO cells stably expressing the human A3AR.
The selective A3 agonist Cl-IB-MECA concentration-depen-
dently inhibited forskolin-stimulated cyclic AMP accumulation
(Figure 3), corresponding to an EC50 of 3.8 ( 1.2 nM.
Compound 28 concentration-dependently increased the maxi-
mum agonist efficacy but somewhat decreased agonist potency.
Interestingly, one of the newly synthesized compounds (43) was
found to increase agonist efficacy but did not influence agonist
potency. The ability of compounds to influence agonist efficacy
did not correlate with their ability to inhibit equilibrium ligand
binding at the human A3AR (Table 1, r ) 0.25), further evidence
of a complex mechanism of interaction. However, the ability
of each compound to decrease the dissociation rate was
positively correlated with its efficacy-enhancing effect (Table
1, r ) 0.88).
Agonist binding site(s) on a GPCR used to be conceived of
as an orthosteric site in contrast to the site(s) at which allosteric
modulators bind. In that framework, allosteric modulators are
without effect in the absence of a ligand binding to the
orthosteric site. However, recently, a number of compounds
were found to be allosteric agonists. AC-42 (4-n-butyl-1-[4-(2-
methylphenyl)-4-oxo-1-butyl]piperidine) is an allosteric agonist
for the M1 muscarinic receptor.30 Alcuronium has been shown
to induce muscarinic receptor activation, which is not prevented
by the classical muscarinic antagonist quinuclidinyl benzilate.31
Thus, the activation mechanisms and structural and pharmaco-
logical modeling of GPCRs should also be reconstructed to
accommodate the complex nature of GPCR activation. Allosteric
modulation of GPCRs has recently been modeled in pharma-
cological terms,32,33 to provide a theoretical basis for an allosteric
modulator separately affecting affinity, potency, and efficacy.
Hall32 suggested that CPCCOEt is positively cooperative with
the binding of glutamate but negatively cooperative with its
Discussion
Allosteric modulation is considered of growing importance
in controlling biological systems.25 GPCRs are the single most

3358 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Go¨blyo¨s et al.
3,4-Diaminoquinoline (6). Prepared as described in the litera-
ture.23 Yield: 2.66 g (98%): mp 183-185 °C.
functional activation of the receptor. There is also evidence that
some allosteric enhancers of agonist binding to the A1AR have
intrinsic activity themselves at that receptor.9,34 However, the
allosteric enhancers in the present study were not demonstrated
to be allosteric agonists, as they did not show any effects by
themselves.
1H-Imidazo[4,5-c]quinoline (7). Prepared as described in the
literature.23 Yield: 0.44 g (81%): mp 263-265 °C.
General Procedure for 2-Substituted 1H-Imidazo[4,5-c]-
quinolines (8-11). Polyphosphoric acid (1.3 mL/mmol) was added
to 3,4-diaminoquinoline (6) and the appropriate carboxylic acid (1.2
equiv). The mixture was stirred at 100 °C for 5 h. Then it was
cooled to 0 °C, and to it was added slowly NH4OH till pH ) 8-9.
The mixture was extracted with ethyl acetate (3 × 15 mL), washed
with water, brine, and again water, and dried over MgSO4. The
solution was filtered, the solvent was evaporated, and the residue
was dried.
Structure-activity relationships for the allosteric enhancing
effects, based on slower dissociation of an A3AR agonist
radioligand, for the present series were evident. At the 4-amino
position, a phenyl ring or substituted monocyclic phenyl ring
was more enhancing than bicyclic aryl rings. Also, by the same
criterion a phenyl group was more highly enhancing than the
corresponding benzyl and phenylethyl groups. As noted previ-
ously17 and consistent with compound 44, substitution at the
2-position was necessary for allosteric enhancement. At this
position, medium-sized cycloalkyl substituents (cyclopentyl and
cyclohexyl) were most favorable for enhancement. Analogues
bearing smaller or larger rings or an acyclic alkyl group were
considerably less enhancing, and similarly an aromatic five-
membered ring was not conducive to enhancement. With the
criterion of increased maximal efficacy of a potent A3AR
agonist, benzyl and 3,4-dichlorophenyl groups at the 4-amino
position were most enhancing.
In summary, by chemical structural modification we have
surpassed the allosteric enhancement observed in the previous
series of 1H-imidazo[4,5-c]quinolin-4-amine derivatives. Two
compounds, 2-cyclopentyl-4-benzylamino (38) and 2-cyclo-
hexyl-4-(3,4-dichlorophenyl)amino (43) analogues, potentiated
the maximum efficacy of the agonist Cl-IB-MECA by 45-50%.
Moreover, 43 enhanced agonist efficacy in a functional assay
and decreased agonist dissociation rate without influencing
agonist potency, probably because of its experimentally observed
decreased interaction with the orthosteric binding site on the
A3AR (compare DU124128 (28) and 43). In fact, most allosteric
enhancers in the present study were generally weak in inhibition
of ligand binding, an otherwise complicating factor in pharma-
cological evaluation. Thus, members of this series are useful
as pharmacological probes for further studies of the A3AR. Since
the structural requirements for allosteric enhancement at the A3-
AR are distinct from the requirements to inhibit equilibrium
binding, there is hope of achieving even greater selectivity upon
future structural manipulation.
2-Cyclobutyl-1H-imidazo[4,5-c]quinoline (8). Scale: 6.2 mmol.
Eluent for column chromatography was ethyl acetate:petroleum
ether ) 1:1. Yield: 0.88 g (63%): mp 191-192 °C.
2-Cyclopentyl-1H-imidazo[4,5-c]quinoline (9). Scale: 4.0 mmol.
Eluent for column chromatography was 5% methanol in dichloro-
methane. Yield: 0.77 g(81%): mp 191-192 °C.
2-Cyclohexyl-1H-imidazo[4,5-c]quinoline (10). Scale: 6.3 mmol.
Eluent for column chromatography was 1-5% methanol in di-
chloromethane. Yield: 0.60 g (38%): mp 205-206 °C.
2-Cycloheptyl-1H-imidazo[4,5-c]quinoline (11). Scale: 6.3
mmol. Eluent for column chromatography was 1-5% methanol in
dichloromethane. Yield: 0.45 g (27%): mp 225-226 °C.
2-(2-Furyl)-1H-imidazo[4,5-c]quinoline (12). 2-Furoyl chloride
(1.1 g, 0.8 mL, 8.1 mmol) in dry dichloromethane (15 mL) was
added dropwise to a solution of to 3,4-diaminoquinoline (1.0 g,
6.0 mmol) in dry pyridine (6.2 mL) under an atmosphere of
nitrogen. The solution was stirred for 2 h at room temperature.
Water (15 mL) was added to quench the reaction, and the solvent
was evaporated under reduced pressure to afford an orange solid.
This crude solid was refluxed for 2 h in 2 M NaOH (15 mL). After
cooling on ice, the pH was adjusted to 7 using concentrated HCl.
The solid was filtered off, washed with water and ether, extracted
with ethyl acetate (3 × 15 mL), and washed with water (3 × 15
mL) again, and dried over MgSO4. After evaporation, the residue
was dried. Eluent for column chromatography was 1-5% methanol
in dichloromethane. Yield: 0.62 g (44%): mp 236-238 °C.
2-Pentyl-1H-imidazo[4,5-c]quinoline (13). Prepared as de-
scribed for the furyl compound (12) using hexanoyl chloride (1.75
g, 13 mmol). Eluent for column chromatography was ethyl acetate:
petroleum ether 1:4 to 4:1. Yield: 0.85 g (41%): mp 142-143
°C.
General Procedure for 2-Substituted 1H-Imidazo[4,5-c]-
quinolin-5-oxide (14-20). Starting material was almost completely
dissolved (with heating) in chloroform (2.5 mL/mmol), dichloro-
methane (2.5 mL/mmol), and methanol (0.25 mL/mmol). 3-Chlo-
roperoxybenzoic acid (2.5 equiv) was added, and the solution was
refluxed. After 30 min Na2CO3 (0.04 g/mmol) was added, and the
mixture was refluxed for an additional 1 h. The reaction mixture
was cooled, and the solvent was evaporated. Column chromatog-
raphy was needed for purification and removal of 3-chloroperoxy-
benzoic acid.
Experimental Procedures
Instrumentation and Analysis. Microwave reactions were
performed in an Emrys Optimizer (Biotage AB). Wattage was
automatically adjusted so as to maintain the desired temperature.
1H NMR spectra were measured at 200 MHz with a Bruker AC
200 or Bruker DMX 600 spectrometer. Chemical shifts for 1H are
given in ppm (δ) relative to tetramethylsilane (TMS) as internal
standard, 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 Laborato-
ries, Leiden University (The Netherlands) and are within 0.4% of
theoretical values unless otherwise specified.
1H-Imidazo[4,5-c]quinolin-5-oxide (14). Scale: 8.3 mmol.
Eluent for column chromatography was 2% methanol in dichloro-
methane. Yield: 0.55 g (36%): mp 290-295 °C.
2-Cyclobutyl-1H-imidazo[4,5-c]quinolin-5-oxide (15). Scale:
1.8 mmol. Eluent for column chromatography was 2-6% methanol
in dichloromethane. Yield: 0.18 g (42%): mp 123-130 °C.
2-Cyclopentyl-1H-imidazo[4,5-c]quinolin-5-oxide
(16).
Scale: 0.8 mmol. Eluent for column chromatography was 5%
methanol in dichloromethane. Yield: 0.19 g (95%): mp 155-157
°C.
Synthesis. 2-(2-Nitroethylideneamino)benzoic Acid (2). Pre-
pared as described in the literature.20 Yield: 18.40 g (89%): mp
196-197 °C.
2-Cyclohexyl-1H-imidazo[4,5-c]quinolin-5-oxide (17). Scale:
2.4 mmol. Eluent for column chromatography was 5-10% metha-
nol in dichloromethane. Yield: 0.59 g (92%): mp 160-165 °C.
3-Nitro-4-hydroxyquinoline (3). Prepared as described in the
literature.20 Yield: 6.93 g (49%): mp >300 °C.
3-Nitro-4-chloroquinoline (4). Prepared as described in the
literature.23 Yield: 5.05 g (81%): mp 118-119 °C.
2-Cycloheptyl-1H-imidazo[4,5-c]quinolin-5-oxide
(18).
Scale: 2.0 mmol. Eluent for column chromatography was 3-8%
methanol in dichloromethane. Yield: 0.22 g (39%): mp 115-120
°C.
3-Nitro-4-aminoquinoline (5). Prepared as described in the
literature.23 Yield: 6.1 g (95%): mp 255-257 °C.

Allosteric Enhancers of the A3 Adenosine Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3359
2-(2-Furyl)-1H-imidazo[4,5-c]quinolin-5-oxide (19). Scale: 2.2
mmol. Eluent for column chromatography was 1-10% methanol
in dichloromethane. Yield: 0.36 g (66%): mp >280 °C.
2-Pentyl-1H-imidazo[4,5-c]quinolin-5-oxide (20). Scale: 3.34
mmol. Eluent for column chromatography was 1-5% methanol in
dichloromethane. Yield: 0.12 g (14%).
General Procedure for Substituted 4-Chloro-1H-imidazo[4,5-
c]quinolines (21-27). A mixture of dry toluene (0.45 mL/mmol)
and dry dimethylformamide (0.90 mL/mmol) was cooled in an ice
bath, and phosphorus oxychloride (2.6 equiv) was added. After 10
min, the appropriate1H-imidazo[4,5-c]quinolin-5-oxide was added,
and the solution was stirred at room temperature for 10 min.
Subsequently the solution was heated to 100 °C for 30 min. Upon
cooling, the solvent was evaporated, and the resulting syrup was
poured on chipped ice while stirring. The mixture was then warmed
to room temperature and carefully adjusted to pH 6-7 with solid
NaHCO3. After 2 h, the formed solid was filtered off, washed with
water and diisopropyl ether, and subsequently dried.
tography was 1% methanol in dichloromethane. Yield: 120 mg
(81%): mp 142-145 °C.
N-(1H-Indazol-6-yl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-
4-amine (35). Scale: 0.4 mmol. Eluent for column chromatography
was 4% methanol in dichloromethane. Yield: 110 mg (74%): mp
235-236 °C.
N-(4-Methoxy-benzyl)-2-cyclopentyl-1H-imidazo[4,5-c]quino-
lin-4-amine (36). Scale: 0.4 mmol. Eluent for column chroma-
tography was diisopropyl ether. Yield: 33 mg (22%): mp 245-
247 °C.
N-(1H-Indol-6-yl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-
4-amine (37). Scale: 0.4 mmol. Eluent for column chromatography
was ethyl acetate:petroleum ether ) 1:4, increased to 3:7. Yield:
30 mg (20%): mp 260-261 °C.
N-Benzyl-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine (38).
Scale: 0.8 mmol. Eluent for column chromatography was 2%
methanol in dichloromethane. Yield: 70 mg (25%), product was
oily.
N-(Phenethyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-
amine (39). Scale: 0.8 mmol. Eluent for column chromatography
was 1-4% methanol in dichloromethane. Yield: 160 mg (56%):
mp 95-97 °C.
N-(3,4-Dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quin-
olin-4-amine (40). Scale: 0.8 mmol. Eluent for column chroma-
tography was ethyl acetate:petroleum ether ) 1:9, increased to
100% ethyl acetate. Yield: 164 mg (50%): mp 236-238 °C.
N-(3,4-Dichloro-phenyl)-2-(2-furyl)-1H-imidazo[4,5-c]quino-
lin-4-amine (41). Scale: 0.4 mmol. Eluent for column chroma-
tography was ethyl acetate:petroleum ether ) 1:4. Yield: 96 mg
(64%): mp 135-138 °C.
N-(3,4-Dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quin-
olin-4-amine (42). Scale: 0.7 mmol. Eluent for column chroma-
tography was ethyl acetate:petroleum ether ) 1:4. Yield: 121 mg
(48%): mp 131-134 °C.
N-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quin-
olin-4-amine (43). Scale: 0.9 mmol. Eluent for column chroma-
tography was ethyl acetate:petroleum ether ) 15:85, increased to
30:70. Yield: 160 mg (44%): mp 237-240 °C.
4-Chloro-1H-imidazo[4,5-c]quinoline (21). Scale: 3.5 mmol.
Eluent for column chromatography was 2% methanol in dichlo-
romethane. Yield: 0.31 g (44%): mp 257-258 °C.
4-Chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinoline
Scale: 0.6 mmol. Yield: 0.16 g (99%): mp 142-145 °C.
4-Chloro-2-cyclopentyl-1H-imidazo[4,5-c]quinoline
Scale: 12.7 mmol. Yield: 2.65 g (75%): mp > 265 °C.
4-Chloro-2-cyclohexyl-1H-imidazo[4,5-c]quinoline
Scale: 2.2 mmol. Yield: 0.65 g (97%): mp 245-250 °C.
4-Chloro-2-cycloheptyl-1H-imidazo[4,5-c]quinoline
Scale: 0.8 mmol. Yield: 0.38 g (86%): mp 195-200 °C.
(22).
(23).
(24).
(25).
4-Chloro-2-(2-furyl)-1H-imidazo[4,5-c]quinoline (26). Scale:
1.4 mmol. Eluent for column chromatography was ethyl acetate:
petroleum ether ) 25:75. Yield: 0.1 g (26%): mp 235-238 °C.
4-Chloro-2-pentyl-1H-imidazo[4,5-c]quinoline (27). Scale: 0.45
mmol. Yield: 0.052 g (41%): mp 236-237 °C.
General Procedure for N-Substituted 1H-Imidazo[4,5-c]-
quinolin-4-amines (28-45). These compounds were prepared by
means of microwave-assisted chemistry. Absolute ethanol (2.5-
3.0 mL) was added to the appropriate 4-chloro-1H-imidazo[4,5-c]-
quinoline and the appropriate aniline (2-3 equiv) under nitrogen.
Conditions: prestirring 60 s, temperature 120 °C, time 2400 s,
normal sample absorption, fixed hold time. After the reaction was
completed, the solvent was evaporated and the remaining product
was purified by column chromatography and recrystallized (MeOH/
H2O).
N-Phenyl-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine (28).
Scale: 0.8 mmol. Eluent for column chromatography was 2.5-
10% methanol in dichloromethane. Yield: 40 mg (15%): mp 155-
157 °C.
N-(4-Methyl-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quino-
lin-4-amine (29). Scale: 0.7 mmol. Eluent for column chroma-
tography was 1% methanol in dichloromethane. Yield: 80 mg
(35%): mp 125-126 °C.
N-(3,4-Dichloro-phenyl)-1H-imidazo[4,5-c]quinolin-4-amine
(44). Scale: 1.0 mmol. Eluent for column chromatography was ethyl
acetate:petroleum ether ) 1:4. Yield: 160 mg (50%): mp 167-
172 °C.
N-(3,4-Dichloro-phenyl)-2-pentyl-1H-imidazo[4,5-c]quinolin-
4-amine (45). Scale: 0.2 mmol. Eluent for column chromatography
was dichloromethane. Yield: 44 mg (73%): mp 195-200 °C.
Pharmacological Methods. [125I]N6-(4-Amino-3-iodobenzyl)-
adenosine-5′-N-methyluronamide (I-AB-MECA; 2000 Ci/mmol),
[3H]R-PIA (R-N6-[phenylisopropyl]adenosine, 34 Ci/mmol), [3H]-
CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcar-
boxamidoadenosine, 47 Ci/mmol) and [3H]cyclic AMP (40 Ci/
mmol) were from Amersham Pharmacia Biotech (Buckinghamshire,
UK).
Cell Culture and Membrane Preparation. CHO (Chinese
hamster ovary) cells expressing the recombinant human ARs (HEK-
293 cells were used for the human A2AAR) were cultured in DMEM
and F12 (1:1) supplemented with 10% fetal bovine serum, 100 units/
mL penicillin, 100 µg/mL streptomycin, and 2 µmol/mL glutamine.
Cells were harvested by trypsinization. After homogenization and
suspension, cells were centrifuged at 500g for 10 min, and the pellet
was resuspended in 50 mM Tris‚HCl buffer (pH 7.4) containing
10 mM MgCl2. The suspension was homogenized with an electric
homogenizer for 10 s, and was then recentrifuged at 20 000g for
20 min at 4 °C. The resultant pellets were resuspended in buffer in
the presence of 3 Units/mL adenosine deaminase, and the suspen-
sion was stored at -80 °C until the binding experiments. The
protein concentration was measured using the Bradford assay.35
Binding Assays to the Human A1 and A2A ARs. For binding
to the human A1 AR, [3H]R-PIA (2 nM) was incubated with
membranes (40 µg/tube) from CHO cells stably expressing the
human A1 AR at 25 °C for 60 min in 50 mM Tris‚HCl buffer (pH
7.4; MgCl2, 10 mM) in a total assay volume of 200 µL. Nonspecific
N-(4-Methoxy-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quino-
lin-4-amine (30). Scale: 0.7 mmol. Eluent for column chroma-
tography was 1% methanol in dichloromethane. Yield: 90 mg
(38%): mp 106-107 °C.
N-(3,4-Dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quin-
olin-4-amine (31). Scale: 0.7 mmol. Eluent for column chroma-
tography was 0-2% methanol in dichloromethane. Yield: 150 mg
(54%): mp 114-115 °C.
N-(4-Chloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quino-
lin-4-amine (32). Scale: 1.0 mmol. Eluent for column chroma-
tography was 0.5-2% methanol in dichloromethane. Yield: 120
mg (33%): mp 189-190 °C.
N-(3-Hydroxymethyl-phenyl)-2-cyclopentyl-1H-imidazo[4,5-
c]quinolin-4-amine (33). Scale: 0.4 mmol. Eluent for column
chromatography was ethyl acetate:petroleum ether ) 1:1, later
increased to 7:3. Yield: 33 mg (25%): mp 228-230 °C.
N-([3,4-c]Indan-5-yl)-2-cyclopentyl-1H-imidazo[4,5-c]quino-
lin-4-amine (34). Scale: 0.4 mmol. Eluent for column chroma-

3360 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Go¨blyo¨s et al.
1
binding was determined using 10 µM of N6-cyclopentyladenosine.
For human A2A AR binding, membranes (20 µg/tube) from HEK-
293 cells stably expressing the human A2A AR were incubated with
15 nM [3H]CGS21680 at 25 °C for 60 min in 200 µL of 50 mM
Tris.HCl, pH 7.4, containing 10 mM MgCl2. N-5′-Ethyluronami-
doadenosine (10 µM) was used to define nonspecific binding.
Reaction was terminated by filtration with GF/B filters.
Supporting Information Available: Tables with H and 13C
NMR spectroscopic data and elemental analyses are available for
selected compounds including all target compounds (28-45). This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
Binding Assay to the Human A3 AR. Each tube in the
competitive equilibrium binding assay contained 100 µL of
membrane suspension (20 µg protein), 50 µL of [125I]I-AB-MECA
(0.5 nM), and 50 µL of increasing concentrations of the test ligands
in Tris‚HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl2, 1
mM EDTA. Nonspecific binding was determined using 10 µM of
5′-N-ethylcarboxamidoadenosine in the buffer. The mixtures were
incubated at 25 °C for 60 min. Binding reactions were terminated
by filtration through Whatman GF/B filters under reduced pressure
using a MT-24 cell harvester (Brandell, Gaithersburg, MD). Filters
were washed three times with 9 mL of ice-cold buffer. Radioactivity
was determined in a Beckman 5500B γ-counter.
Dissociation Kinetics of [125I]I-AB-MECA from Human
A3ARs. The dissociation of [125I]I-AB-MECA was measured as
follows. Membranes (20 µg) were preincubated at 25 °C with 0.5
nM [125I]I-AB-MECA, in a total volume of 100 µL of Tris‚HCl
buffer (50 mM, pH 8.0) containing 10 mM MgCl2, and 1 mM
EDTA for 60 min. The dissociation was then initiated by the
addition of 3 µM Cl-IB-MECA with or without allosteric modula-
tors. The time course of dissociation of total binding was measured
by rapid filtration at appropriate time intervals. Nonspecific binding
was measured after 60-min incubation in the presence of 3 µM
Cl-IB-MECA. Binding reactions were terminated as described
above.
Cyclic AMP Accumulation Assay. Intracellular cyclic AMP
levels were measured with a competitive protein binding method.24
CHO cells that expressed recombinant human A3ARs were
harvested by trypsinization. After centrifugation and resuspension
in medium, cells were plated in 24-well plates in 0.5 mL of medium.
After 24 h, the medium was removed and cells were washed three
times with 1 mL of DMEM, containing 50 mM HEPES, pH 7.4.
Cells were then treated with agonists and/or test compounds in the
presence of rolipram (10 µM) and adenosine deaminase (3 units/
mL). After 45 min, forskolin (10 µM) was added to the medium,
and incubation was continued an additional 15 min. The reaction
was terminated by removing the supernatant, and cells were lysed
upon the addition of 200 µL of 0.1 M ice-cold HCl. The cell lysate
was resuspended and stored at -20 °C. For determination of cyclic
AMP production, protein kinase A (PKA) was incubated with
[3H]cyclic AMP (2 nM) in K2HPO4/EDTA buffer (K2HPO4, 150
mM; EDTA, 10 mM), 20 µL of the cell lysate, and 30 µL of 0.1
M HCl or 50 µL of cyclic AMP solution (0-16 pmol/200 µL for
standard curve). Bound radioactivity was separated by rapid
filtration through Whatman GF/C filters and washed once with cold
buffer. Bound radioactivity was measured by liquid scintillation
spectrometry.
Statistical Analysis. Binding and functional parameters were
calculated using Prism 5.0 software (GraphPAD, San Diego, CA).
IC50 values obtained from competition curves were converted to
Ki values using the Cheng-Prusoff equation.36 Data were expressed
as mean ( standard error. The Pearson correlation coefficients (r)
between efficacy, binding, and dissociation were calculated using
SYSTAT 11 (SYSTAT Software Inc.). The Pearson correlation
coefficient is between -1 and 1, with 1 meaning that two series
are positively correlated, 0 meaning that they are completely
uncorrelated, and -1 meaning they are perfectly negatively
correlated.
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