Novel 2-and 4-Substituted 1H-Imidazo[4,5-c]quinolin-4-amine derivatives as allosteric modulators of the A3 adenosine receptor

Article abstract of DOI:10.1021/jm801659w

4-Arylamino and 2- cycloalkyl (including amino substitution) modifications were made in a series of 1Himidazo[ 4,5-c]quinolin-4-amine derivatives as allosteric modulators of the human A₃ adenosine receptor (AR). In addition to allosteric modula

Full text of DOI:10.1021/jm801659w

2098
J. Med. Chem. 2009, 52, 2098–2108
Novel 2- and 4-Substituted 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as Allosteric
Modulators of the A3 Adenosine Receptor
Yoonkyung Kim,†,‡,§ Sonia de Castro,†,‡ Zhan-Guo Gao,† 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 December 31, 2008
4-Arylamino and 2- cycloalkyl (including amino substitution) modifications were made in a series of 1H-
imidazo[4,5-c]quinolin-4-amine derivatives as allosteric modulators of the human A3 adenosine receptor
(AR). In addition to allosteric modulation of the maximum functional efficacy (in [35S]GTPγS G protein
binding assay) of the A3AR agonist Cl-IB-MECA (15), some analogues also weakly inhibited equilibrium
radioligand binding at ARs. 4-(3,5-Dichlorophenylamino) (6) or 2-(1-adamantyl) (20) substitution produced
allosteric enhancement (twice the maximal agonist efficacy), with minimal inhibition of orthosteric AR
binding. 2-(4-Tetrahydropyranyl) substitution abolished allosteric enhancement but preserved inhibition of
orthosteric binding. Introduction of nitrogen in the six-membered ring at the 2 position, to improve aqueous
solubility and provide a derivatization site, greatly reduced the allosteric enhancement. 2-(4-(Benzoylami-
no)cyclohexyl) analogues 23 and 24 were weak negative A3AR modulators. Thus, consistent with previous
findings, the allosteric and orthosteric inhibitory A3AR effects in imidazoquinolines are structurally separable,
suggesting the possible design of additional derivatives with enhanced positive or negative allosteric A3AR
activity and improved selectivity in comparison to inhibition of orthosteric binding.
Chart 1. Structures of a Series of 2-Cycloalkylimidazoquinoline
Derivatives Previously Found To Be Positive Allosteric
Modulators of the Human A3AR
Introduction
The adenosine receptors (ARsa), of which A1, A2A, A2B, and
A3 subtypes have been defined, represent a physiologically
important family of G-protein-coupled receptors (GPCRs).1,2
ARs are important pharmacological targets in the treatment of
a variety of diseases because of their key roles in controlling
numerous cellular processes. For example, A3AR agonists are
of interest for the treatment of cardiac ischemia, bowel inflam-
mation, protection of skeletal muscle, cancer, and rheumatoid
arthritis.3-7 However, therapeutic intervention using a selective
AR agonist is subject to side effects related, in part, to the
widespread occurrence of the corresponding receptor throughout
the body.
Native agonists of a given GPCR bind at a principal
(orthosteric) site on the receptor protein to effect its activation.
However, an allosteric modulator would bind to a distinct site
on the receptor to either enhance (positive modulator) or impede
(negative modulator) the action of a native agonist.8-11 The
therapeutic application of allosteric modulation has advantages
over directly acting orthosteric GPCR agonists. In a particular
disease state, the effect of an endogenous agonist, which may
be insufficient to fully compensate for an imbalance, may be
magnified in a temporally and/or spatially specific manner by a
positive allosteric modulator for therapeutic benefit.12 The
allosteric modulator theoretically would have no effect of its
own on the unoccupied receptor. An additional advantage of
allosteric modulators is that they typically are found to display
higher subtype-selectivity than orthosteric ligands of the same
receptor. Thus, allosteric action that is dependent on the
simultaneous presence of an endogenous ligand ideally can
produce a more selective drug action and prevent side effects
and possible overdosage associated with the administration of
a conventional orthosteric agonist.
Allosteric modulators of several subtypes of ARs have been
reported and their structure-activity relationships (SARs)
explored.8,13,14 N-(3,4-Dichlorophenyl)-2-cyclohexyl-1H-imi-
dazo[4,5-c]quinolin-4-amine (LUF6000, 3, Chart 1)15-17 is an
allosteric modulator of the human A3AR that increases the
maximum efficacy of the agonist 2-chloro-N6-(3-iodobenzyl)-
adenosine-5′-N-methylcarboxamide (Cl-IB-MECA, 15). A six-
membered ring provided the optimal A3AR enhancement in the
homologous series of 2-cycloalkyl derivatives 1-4. Compound
3 enhanced the A3AR agonist efficacy in a functional assay and
decreased the agonist dissociation rate without influencing
* To whom correspondence should be addressed. Phone: 301-496-9024.
Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov.
†
National Institute of Diabetes and Digestive and Kidney Diseases.
Contributed equally to this work.
Current address: Biomedical Research Center, Korea Institute of Science
‡
§
and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul, 136-791,
Korea.
|
Leiden/Amsterdam Center for Drug Research.
a Abbreviations: AR, adenosine receptor; Cl-IB-MECA, 2-chloro-N6-(3-
iodobenzyl)adenosine-5′-N-methylcarboxamide; CCPA, 2-chloro-N6-cyclo-
pentyladenosine; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-
propanesulfonate; DMEM, Dulbecco’s modified Eagle’s medium; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; ESI, electrospray
ionization; GPCR, G-protein-coupled receptor; GTPγS, guanosine 5′-(γ-
thiotriphosphate); I-AB-MECA, N6-(4-amino-3-iodobenzyl)adenosine-5′-
N-methylcarboxamide; m-CPBA, 3-chloroperoxybenzoic acid; NECA, 5′-
N-ethylcarboxamidoadenosine; SAR, structure-activity relationship; TLC,
thin layer chromatography.
10.1021/jm801659w CCC: $40.75
2009 American Chemical Society
Published on Web 03/13/2009

Quinolin-4-amine DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2099
Scheme 1. Synthesis of Novel 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives with Structural Variation at the 4 Positiona
a Reagents: (i) m-CPBA, CHCl3/CH2Cl2/MeOH; (ii) POCl3, toluene/DMF; (iii) R-PhNH2, DMF.
agonist potency, because of decreased interaction with the
orthosteric binding site on the A3AR. Since the structural
requirements for allosteric enhancement at the A3AR are distinct
from the requirements to inhibit equilibrium binding, structural
manipulation of this family of imidazoquinolines might achieve
even greater selectivity.
In the present study we have extended our search for highly
effective allosteric enhancer ligands for the A3AR, by modifying
the substitutions around the 4-arylamino and 2-cycloalkyl
moieties of the imidazoquinoline scaffold. We have also
identified weak negative allosteric modulators in the same
structural series.
To obtain a general procedure for the aniline substitution
reaction, we first attempted the synthesis following the method
recently described by Go¨blyo¨s et al.,15 using a microwave
reactor (in ethanol under nitrogen, prestirring for 60 s, at 120
°C for 40 min, normal sample absorption, fixed hold time).
Although the reaction was performed on a very small scale, we
were able to determine preferred conditions to be N,N-
dimethylformamide (DMF) as a solvent and using 2-3 equiv
of corresponding aniline with heating at 140 °C overnight under
a N2 atmosphere in a tightly sealed Biotage reaction vial. In an
earlier attempted reaction at 105 °C under the same conditions,
no desired product was obtained.18
The structures of the imidazoquinoline derivatives were
analyzed by NMR in dimethyl sulfoxide (DMSO-d6). In all final
compounds except the 2-exo-norbornyl-4-(3,4-dichlorophenyl)-
amino analogue 18 that was attached in an axial position, the
central imidazoquinoline system was clearly attached to the six-
membered ring in an equatorial position. This was demonstrated
Results and Discussion
The novel 4-substituted imidazoquinoline derivatives 5-14
(Scheme 1) were prepared from a key 4-chloro-2-cyclohexyl
intermediate 29. Oxidation of 2715 with 3-chloroperoxybenzoic
acid (m-CPBA) afforded the 5-oxide derivative 28, which was
subsequently converted with phosphorus oxychloride into a
4-chloro species 29.15,18 Reaction of 29 with the appropriately
substituted aniline provided the desired 4-amino derivatives
5-14 in varying yields (4-100%), where the 3-aminopyridyl
derivative 14 has the lowest yield. Attempts to prepare an
o-pyridyl derivative (i.e., using 2-aminopyridine) under the same
reaction condition with 29 failed.
The novel 2-substituted imidazoquinoline derivatives 16-26
(Scheme 2) were prepared similarly. However, here different
2-cycloalkyl groups were appended, each as its carboxylic acid
form, to a common intermediate 30 in an earlier synthetic step
to give the corresponding imidazoquinoline derivatives 31a-f.
In fact, compound 30 also served as a precursor for 27.15 Next,
compounds 31a-f were treated with m-CPBA to afford 5-oxide
derivatives 32a-f, followed by phosphorus oxychloride to give
the respective 4-chloro compounds 33a-f.15,18 Reaction of the
4-chloro-2-cycloalkyl derivatives 33a-f with 3,4-dichloroani-
line afforded the desired compounds (16-21, 23). Aniline was
used instead in the synthesis of compound 24.
based on the J coupling of the proton at the junction (Jax-ax
≈
12-11 and Jax-eq ≈ 5-3). We also noted the fact that potentially
there are two possible annular tautomers (1H or 3H at the
imidazole ring), which may affect the GPCR binding equilibrium
depending on the stability of each form in the aqueous media.
2D NOESY experiments in DMSO-d6 (see Supporting Informa-
tion) suggested the 3H-tautomer as an exclusive form for
compounds 10 and 11. Interestingly, for compound 14, two
tautomers were observed as correlated by NOE cross-peaks
(1H-/3H- ≈ 88:12 by NMR integration in DMSO-d6). Unlike
other compounds, detection of two competing tautomers on the
NMR time scale could be due to the formation of a hydrogen
bond between the pyridyl nitrogen (acceptor) and the imidazole
NH (donor) as a less preferred 3H-tautomer.
All of the imidazoquinoline derivatives were evaluated for
interaction with the human A3 receptors and two other ARs as
listed in Tables 1 and 2. We first tested the effect of these
compounds on the equilibrium binding at A1, A2A, and A3ARs
using standard agonist radioligands19-21 [3H]2-Chloro-N6-cyclo-
pentyladenosine ([3H]CCPA, 34), [3H]2-[4-(2-carboxylethyl)phe-
nylethylamino]-5′-N-ethylcarboxamidoadenosine ([3H]CGS21680,
35), and [125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-meth-
ylcarboxamide ([125I]I-AB-MECA, 36), respectively. For com-
pounds 5-14 and 16-26, only the percent inhibition of
orthosteric radioligand binding was reported rather than Ki
values because the affinity at all three subtypes is weak and
close to the solubility limits of the compounds. It is unknown
Several alkylamino derivatives, 22 and 26, were included.
In the case of a 2-(4-cis-aminocyclohexyl) analogue 22, it was
necessary to remove the amine-protecting group of 21 in order
to obtain the final compound. The deprotection was ac-
complished using methylamine in ethanol. In the case of the
piperidine derivative 26, the reduction of the carbonyl group
of 23 with lithium aluminum hydride afforded the N-benzyl
derivative 25, which was subsequently converted into the desired
compound 26 by hydrogenation.

2100 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kim et al.
Scheme 2. Synthesis of Novel 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives with Structural Variation at the 2 Positiona
a Reagents: (i) polyphosphoric acid, R1CO2H; (ii) m-CPBA, CHCl3/CH2Cl2/MeOH; (iii) POCl3, toluene/DMF; (iv) R2-PhNH2, DMF; (v) LiAlH4, THF;
(vi) MeNH2, EtOH; (vii) H2/Pd, MeOH.
Table 1. Potency of 4-Anilino Substituted 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives in Binding Assays at the Human A1 and A3ARs Expressed in
CHO Cells and at the Human A2A in HEK-293 Cells and the Allosteric Effects at the Human A3ARa
A1AR, %
displ at 10 µM
A2AAR, %
displ at 10 µM
A3AR, %
displ at 10 µM
A3AR ag dissociation,b
[35S]GTPγS binding in A3AR cells,c
% at 10 µM
compd
R
% at 10 µM
3
5
6
7
8
9
10
11
12
13
14
3,4-Cl2-PhNH
3-Cl-PhNH
3,5-Cl2-PhNH
2,4-Cl2-PhNH
3,4-F2-PhNH
37 ( 9
-11.2 ( 2.8
42.8 ( 3.7
-11.4 ( 10.9
67.2 ( 2.4
26.5 ( 7.3
17.2 ( 2.1
15.6 ( 0.3
37.2 ( 1.5
93.8 ( 1.6
49.0 ( 0.7
78.1 ( 1.8
40.5 ( 13.4
79.2 ( 1.3
32.7 ( 4.8
43.9 ( 0.1
68.8 ( 1.2
70.1 ( 7.9
21.8 ( 1.4
42.2 ( 2.9
98.4 ( 0.1
83.9 ( 1.1
64.8 ( 6.9
192 ( 7
194 ( 3
184 ( 5
174 ( 5
187 ( 1
188 ( 3
137 ( 5
129 ( 7
173 ( 1
185 ( 6
114 ( 5
208 ( 14
200 ( 13
209 ( 22
187 ( 20
197 ( 17
171 ( 7
143 ( 3
138 ( 7
182 ( 14
166 ( 11
119 ( 1
53.3 ( 3.8
16.7 ( 2.9
48.0 ( 1.1
14.6 ( 4.8
36.6 ( 5.3
7.2 ( 2.3
3,5-F2-PhNH
3,5-(CF3)2-PhNH
3,4-(CN)2-PhNH
3,4-(OMe)2-PhNH
3,4-O2CH2-PhNH
3-pyridyl-NH
33.1 ( 4.1
75.6 ( 2.1
65.4 ( 3.3
73.1 ( 2.5
a All experiments were performed using adherent mammalian 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 the Experimental Procedures using [3H]34, [3H]35, or [125I]36 as a radioligand.19-21 Values from
the present study are expressed as the mean ( SEM, n ) 3-5. Percentage of inhibition at A1, A2A, or A3 receptors is expressed as the mean value from two
b
to four separate experiments with similar results performed in duplicate. Dissociation: % decrease of [125I]36 dissociation at 60 min (control ) 100%).
c Increase of efficacy in the stimulation of the binding of [35S]GTPγS compared to maximal effect induced by 10 µM 15 alone (set to 100%). It is noted that
the Emax of 15 in this functional assay was recently demonstrated to be about 50% of that of 37.17 In the adenylate cyclase assay, 15 and 37 were both full
agonists.15
whether the observed inhibition is of an allosteric or nonallos-
teric character.
Ability to allosterically modulate the A3AR was determined
using two methods: effects of the imidazoquinoline on the
dissociation rate of 36 ([125I]I-AB-MECA) and on the binding
to the G protein of the stable GTP analogue [35S]guanosine-
5′-(γ-thiotriphosphate) ([35S]GTPγS).22 Depending on the
functional assay used, 15 (Cl-IB-MECA) may appear to be
either a full15 or partial agonist17,23-25 at the A3AR. The
earlier series of imidazoquinoline derivatives displayed dual

Quinolin-4-amine DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2101
Table 2. Potency of 2-Cycloalkyl Substituted 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives in Binding Assays at the Human A1 and A3ARs
Expressed in CHO Cells and at the Human A2A in HEK-293 Cells and the Allosteric Effects at the Human A3ARa
a All experiments were performed using adherent mammalian 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 the Experimental Procedures using [3H]34, [3H]35, or [125I]36 as a radioligand.19-21 Values from
the present study are expressed as the mean ( SEM, n ) 3-5. Percentage inhibition at A1, A2A, or A3 receptors is expressed as the mean value from two
to four separate experiments with similar results performed in duplicate. b Values from Go¨blyo¨s et al.,15 the functional enhancement of which was measured
with the cyclic AMP assay.26 c Dissociation: % decrease of [125I]36 dissociation at 60 min (control ) 100%). d Increase of efficacy in the stimulation of the
binding of [35S]GTPγS compared to maximal effect induced by 10 µM 15 alone (set to 100%). It should be noted that the Emax of 15 in this functional assay
was recently demonstrated to be about 50% of that of 37.17 In the adenylate cyclase assay, 15 and 37 were both full agonists.15 e Ki value in a binding
assay:15 4690 ( 970 nM.

2102 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kim et al.
Figure 2. Allosteric modulation of the human A3AR by compound
20 or 24. (a) Functional assay of the human A3AR: % stimulation of
binding of [35S]GTPγS by increasing concentrations of 15 under control
conditions or in the presence of 10 µM compound 3 or 20. (b)
Radioligand binding studies on the human A3AR: study of the
dissociation kinetics of the agonist radioligand [125I]36 under control
conditions and in the presence of 10 µM compound 20 or 24.
Figure 1. Allosteric modulation of the human A3AR by compound 6.
(a) Functional assay of the human A3AR: % stimulation of binding of
[35S]GTPγS by increasing concentrations of 15 under control conditions
or in the presence of 10 µM compound 3 or 6. (b) Radioligand binding
studies on the human A3AR: study of the dissociation kinetics of the
agonist radioligand [125I]36 under control conditions and in the presence
of 10 µM compound 3 or 6.
and apparently opposite actions as positive allosteric modula-
tors of agonist action and as inhibitors of radioligand
binding.15 In the previous study, compound 3 induced a
substantial functional enhancement of the effects of the A3AR
agonist 15 as determined using an agonist radioligand
dissociation kinetic assay and a cyclic AMP assay,15,26 while
there was only a weak inhibition of equilibrium radioligand
binding at ARs at the concentration used.
The potentiation of the maximum efficacy of the agonist 15
by arylhalo derivatives 5-9 (Table 1) was high and similar to
that observed with the lead compound 3. The percent of maximal
functional effect ranged from 171% for 9 to 209% for 6. Among
these halo analogues, 6 had the least inhibition of orthosteric
binding to ARs. The degree of enhancement of [35S]GTPγS
binding by 6 over a range of concentrations of 15 up to 10 µM
was indistinguishable from the effect of the lead compound 3
(Figure 1a). Similarly, the marked decrease in dissociation rate
of the A3AR agonist radioligand [125I]36 produced by 6 was
indistinguishable from the effect of compound 3 (Figure 1b).
A 3,5-di(trifluoromethyl) analogue 10 and another aniline
derivative 11 that was disubstituted with electron withdrawing
groups displayed an intermediate degree of allosteric enhance-
ment. Other 3,4-disubstituted anilines 12 and 13 that contained
electron-donating groups displayed a high degree of allosteric
enhancement but also substantial inhibition of orthosteric
binding to the A3AR and other ARs. The 3-pyridinylamine 14
had a low degree of allosteric enhancement.
allosteric potentiation. Multiply bridged cycloalkyl substitution
in the 2-(1-adamantyl)-4-(3,4-dichlorophenyl)amino analogue
20 resulted in high allosteric potentiation with minimal effects
on the binding of orthosteric ligands at A1, A2A, and A3ARs.
The 2-exo-noradamantyl-4-(3,4-dichlorophenyl)amino analogue
19 displayed only moderate allosteric potentiation. The (de-
creasing) order of allosteric enhancement by these bridged
cycloalkyl derivatives was 20, 17 > 18 > 19. The adamantyl
derivative 20 increased [35S]GTPγS binding over a range of
concentrations of 15 up to 10 µM in a manner indistinguishable
from the effect of 3 (Figure 2a).
Introduction of a nitrogen atom in (or as substituted at) the
six-membered ring in 21-26 in an attempt to improve aqueous
solubility and to provide a site for further derivatization greatly
reduced the allosteric enhancement of this series. Variable
degrees of inhibition of orthosteric binding at the AR were
observed in this group with the most pronounced inhibition
found for 21 and 24 at the A2AAR and for 25 and 26 at the
A3AR. The slightly decreased agonist efficacy (91-93% of
control in both functional assays, Table 2) of 2-(N-benzoyl-4-
piperidinyl) analogues 23 and 24 was suggestive of possible
negative allosteric modulation of the A3AR. The effect of
compound 24 on the dissociation kinetics of the agonist
radioligand was only slightly different from that of the control
(Figure 2b).
Conclusions
Highly variable biological activities were observed for the 2
position derivatives 16-26 (Table 2). Replacement of the distal
methylene group of the 2-cyclohexyl ring of 3 with an ether
oxygen in the tetrahydropyranyl derivative 16 abolished allos-
teric modulation of the A3AR but retained a similar degree of
orthosteric inhibition. Inclusion of a methylene bridge across
the 2-cyclohexyl ring of 3 in 17 and 18 resulted in considerable
In this study, structural modification of the 1H-imidazo[4,5-
c]quinolin-4-amine 3 has demonstrated that a limited set of
substituents at the 2 and 4 positions are tolerated to preserve
the allosteric enhancement of agonist action at the A3AR.
Notably, the haloanilino derivatives 5, 6, and 8 and the bridged
2-cycloalkyl analogues 17 and 20 approximately doubled the

Quinolin-4-amine DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2103
maximum efficacy of the agonist 15 in the [35S]GTPγS assay.
The highest potentiation of the maximum efficacy of the agonist
15, without increased inhibition of orthosteric binding, was
observed for the 2-(1-adamantyl)-4-(3,4-dichlorophenyl)amino
analogue 20, with 210% activity of control in the [35S]GTPγS
binding assay. Compounds 6 and 20 were preferred as selective
allosteric enhancers of the A3AR because of the minimal effect
on binding at the orthosteric sites of the three ARs examined.
Substitution of a 4-tetrahydropyran moiety at the 2 position
completely abolished allosteric enhancement but preserved
inhibition of othosteric binding. Thus, as extension of previous
findings, the allosteric and orthosteric inhibitory effects at the
A3AR in this series of imidazoquinolines are structurally
separable. These biological results suggest that it will be possible
to design additional derivatives that would display enhanced
positive or negative allosteric activity at this receptor and
improved selectivity in comparison to inhibition of orthosteric
binding.
washed with water, brine, and again with water, and then dried
over MgSO4. The solution was filtered, the solvent was evaporated,
and the residue was dried in vacuo. The residue obtained was
subjected to preparative silica gel column chromatography (100:1
to 8:1 CHCl3/MeOH).
2-(Tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinoline (31a).
1
Yield: 107.6 mg (67%). H NMR (300 MHz, CD3OD/CDCl3) δ
13.40 (s, 1H), 9.13 (s, 1H), 8.35 (s, 1H), 8.08 (m, 1H), 7.65 (m,
2H), 3.98 (m, 2H), 3.35 (dd, 2H, J ) 11.4, 10.2 Hz), 3.28 (t, 1H,
J ) 11.3 Hz), 2.05 (m, 2H), 1.95 (dd, 1H, J ) 11.6, 4.2 Hz), 1.93
(dd, 1H, J ) 11.6, 4.2 Hz); 13C NMR (75 MHz, CD3OD/CDCl3) δ
143.4, 129.5, 126.3, 121.6, 66.7, 31.07; HRMS (ESI) calcd for
C15H16N3O+ (M + H+) 254.1288, found 254.1293.
2-(2-Norbornanyl)-1H-imidazo[4,5-c]quinoline (31b). Yield:
1
86.43 mg (52%). H NMR (300 MHz, CD3OD/CDCl3) δ 13.33
(bs, 1H), 9.11 (s, 1H), 8.32 (s, 1H), 8.08 (m, 1H), 7.64 (m, 2H),
3.45 (m, 1H), 3.36 (s, 2H), 3.17 (s, 2H), 3.06 (s, 1H), 2.72 (m,
1H), 2.58 (m, 1H), 2.50 (s, 1H), 2.38 (m, 1H), 1.44 (m, 7H); 13C
NMR (75 MHz, CD3OD/CDCl3) δ 143.85, 129.57, 125.98, 121.43,
48.59, 42.59, 36.61, 35.78, 29.10, 28.59, 23.60; HRMS (ESI) calcd
for C17H18N3 (M + H+) 264.1495, found 264.0561.
+
Experimental Procedures
2-(4-(1,3-Dioxoisoindolin-2-yl)cyclohexyl)-1H-imidazo[4,5-
c]quinoline (31c). Yield: 88.0 mg (35%). H NMR (300 MHz,
General. Glassware was oven-dried and cooled in a desiccator
before use. All reactions were carried out under a dry nitrogen
atmosphere. Solvents were purchased as anhydrous grade and used
without further purification. Suppliers of some commercial com-
pounds are listed as follows: 3,4-diaminoquinoline (30) was
purchased from Tyger Scientific, Inc.; 3,5-bis(trifluoromethyl)aniline
and 4-aminophthalonitrile were purchased from Acros Organics;
polyphosphoric acid, m-CPBA, phosphorus oxychloride (POCl3),
chloroform (CHCl3), methylene chloride (CH2Cl2), DMF, toluene,
diisopropyl ether, methanol (MeOH), tetrahydrofuran (THF), and
most of other reagents and solvents were purchased from Sigma-
Aldrich; dimethyl sulfoxide (DMSO-d6), chloroform-d (CDCl3), and
CD3OD were purchased from Cambridge Isotope Laboratories. All
reagents were of commercial grade and were used without further
purification unless otherwise noted.
1
CD3OD/CDCl3) δ 13.35 (bs, 1H), 9.13 (s, 1H), 8.35 (s, 1H), 8.07
(m, 1H), 7.69 (m, 6H), 4.16 (m, 1H), 3.06 (t, 1H, J ) 12.2 Hz),
2.36 (m, 4H), 1.88 (m, 4H); 13C NMR (75 MHz, CD3OD/CDCl3)
δ 167.9, 167.7, 143.4, 134.3, 129.5, 126.1, 122.9, 121.7, 49.5, 36.9,
30.7, 28.8; HRMS (ESI) calcd for C24H21N4O2 (M + H+)
+
397.1659, found 397.1234.
2-(1-Benzoylpiperidin-4-yl)-1H-imidazo[4,5-c]quinoline (31d).
Yield: 142 mg (67%). 1H NMR (300 MHz, CD3OD/CDCl3) δ 13.38
(s, 1H), 9.13 (s, 1H), 8.35 (s, 1H), 8.09 (s, 1H), 7.66 (s, 1H), 7.46
(s, 1H), 4.56 (s, 1H), 3.71 (s, 1H), 3.13 (s, 1H), 2.17 (s, 1H), 1.91
(s, 1H); 13C NMR (75 MHz, CD3OD/CDCl3) δ 169.1, 143.4, 136.3,
129.4, 128.5, 126.6, 121.4, 59.8, 46.8, 41.2, 35.8, 30.4; HRMS (ESI)
calcd for C22H21N4O+ (M + H+) 357.1710, found 357.1732.
NMR spectra were recorded on either a Varian Inova/Gemini
300 or a Bruker DRX-600 spectrometer at 25.0 °C under an
optimized parameter setting for each sample, unless otherwise
2-(3-Noradamantanyl)-1H-imidazo[4,5-c]quinoline (31e).
1
Yield: 118.7 mg (68%). H NMR (300 MHz, CD3OD/CDCl3) δ
8.98 (s, 1H), 8.36 (s, 1H), 8.03 (d, 1H, J ) 7.8 Hz), 7.52 (m, 2H),
2.77 (t, 1H, J ) 6.9 Hz), 2.33 (s, 2H), 2.20 (m, 2H), 2.06 (m, 3H),
1.99 (m, 2H), 1.68 (m, 4H); 13C NMR (75 MHz, CD3OD/CDCl3)
δ 162.7, 143.2, 128.3, 121.5, 45.6, 43.6, 38.1, 37.8, 37.5, 37.3,
34.3, 30.4; HRMS (ESI) calcd for C19H20N3+ (M + H+) 290.1652,
found 290.1670.
1
mentioned. For compounds 5-14, 28, and 29, H NMR chemical
shifts were measured relative to the residual solvent peak at 2.50
ppm in DMSO-d6 and at 3.31 ppm in CD3OD or in a mixture of
CD3OD/CDCl3. For compounds 16-26, 31a-f, 32a-f, and 33a-f,
1H NMR chemical shifts were measured relative to tetramethylsilane
at 0.00 ppm in CDCl3 and the residual water peak at 3.30 ppm in
CD3OD. 13C NMR chemical shifts were measured relative to the
residual solvent peak at 49.15 ppm in CD3OD or in a mixture of
CD3OD/CDCl3. Suggested NMR peak assignments of some target
compounds are shown in Supporting Information and are based on
2D COSY and NOESY experiments.
2-(1-Adamantanyl)-1H-imidazo[4,5-c]quinoline (31f). Yield:
1
75.8 mg (39%). H NMR (300 MHz, CD3OD/CDCl3) δ 9.11 (s,
1H), 8.30 (s, 1H), 8.15 (d, 1H, J ) 8.4 Hz), 7.53 (t, 1H, J ) 7.2
Hz), 7.43 (d, 1H, J ) 7.2 Hz), 2.21 (s, 6H), 2.05 (s, 4H), 1.72 (m,
6H); 13C NMR (75 MHz, CD3OD/CDCl3) δ 143.6, 129.1, 127.4,
126.6, 121.9, 41.6, 36.5, 36.0, 228.3, 1.2; HRMS (ESI) calcd for
Analytical or preparative thin layer chromatography (TLC) was
performed on either 0.2 mm silica coated sheets with F254 indicator
(Sigma-Aldrich) or 0.2 mm reversed-phase C18 silica coated sheets
(pore size of 60 Å, Whatman Inc.). Visualization of the products
on the TLC plate was aided by the use of UV light, ninhydrin, or
potassium permanganate. Column chromatography was performed
on 230-400 mesh silica gel (pore size of 60 Å, Sigma-Aldrich).
The tested imidazoquinoline derivatives were confirmed by HPLC
to possess a g96% purity.
The electrospray ionization (ESI) MS experiments were per-
formed on a Micromass/Waters LCT Premier electrospray time-
of-flight (TOF) mass spectrometer coupled with a Waters HPLC
system at the Mass Spectrometry Facility, NIDDK, NIH.
General Procedure for 2-Substituted 1H-Imidazo[4,5-c]quin-
oline (31a-f). Polyphosphoric acid (1.3 mL/mmol) was added to
3,4-diaminoquinoline (30) (100 mg, 0.63 mmol) and the appropriate
carboxylic acid (1.2 equiv). The mixture was stirred at 100 °C for
5 h. Then the mixture was cooled to 0 °C and to it was slowly
added NH4OH till pH 8-9. The mixture was extracted with ethyl
acetate (3 × 15 mL), and the combined organic extracts were
C20H22N3 (M + H+) 304.1808, found 304.1823.
+
General Procedure for 2-Substituted 1H-Imidazo[4,5-c]quin-
oline 5-Oxide (28, 32a-f). The appropriate 2-substituted 1H-
imidazo[4,5-c]quinoline derivatives (27, 31a-f) in a mixture of
CHCl3 (2.5 mL/mmol), CH2Cl2 (2.5 mL/mmol), and MeOH (0.25
mL/mmol) was heated for dissolution. To this mixture was added
m-CPBA (2.5 equiv), which was then refluxed for 30 min. The
mixture was cooled to room temperature, Na2CO3 (0.04 g/mmol)
was added in one portion as a solid, and then the mixture was
refluxed for 1 h. The solvent was removed in vacuo, and the crude
product was chromatographed on silica gel (20:1 to 7:1 CH2Cl2/
MeOH for 28, 100:1 to 8:1 CHCl3/MeOH for 32a-f) to give the
desired compound.
2-Cyclohexyl-1H-imidazo[4,5-c]quinoline 5-Oxide (28).15
Scale: 0.612 mmol. Yield: 150 mg (91%). Rf ) 0.43 [silica gel,
10:1 CH2Cl2/MeOH]. 1H NMR (300 MHz, CD3OD) δ 9.03 (s, 1H),
8.78-8.72 (m, 1H), 8.47 (m, 1H), 7.89-7.83 (m, 2H), 3.06 (tt,
1H, J ) 11.8, 3.5 Hz), 2.20-2.14 (m, 2H), 1.98-1.91 (m, 2H),
1.86-1.69 (m, 3H), 1.60-1.32 (m, 3H); 13C NMR (75 MHz,

2104 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kim et al.
CD3OD) δ 130.9, 130.6, 130.4, 129.0, 123.5 121.2, 40.2, 32.9, 27.2,
27.0; HRMS (ESI) calcd for C16H18N3O (M + H+) 268.1450, found
268.1451.
4-Chloro-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quin-
oline (33a). Scale: 0.27 mmol. Yield: 41 mg (53%). 1H NMR (300
MHz, CD3OD/CDCl3) δ 13.66 (br s, 1H), 8.35 (m, 1H), 8.02 (m,
1H), 7.70 (m, 2H), 4.04 (m, 2H), 3.52 (dd, 2H, J ) 11.1, 10.8 Hz),
3.25 (m, 1H), 1.95 (m, 4H); 13C NMR (75 MHz, CD3OD/CDCl3)
δ 143.2, 128.1, 126.8, 121.2, 67.6, 36.1, 31.1; HRMS (ESI) calcd
for C15H15ClN3O+ (M + H+) 288.0898, found 288.0713.
2-(Tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinoline
1
5-Oxide (32a). Scale: 0.42 mmol. Yield: 65 mg (57%). H NMR
(300 MHz, CD3OD/CDCl3) δ 9.18 (s, 1H), 8.80 (m, 1H), 8.53 (m,
1H), 7.85 (m, 2H), 4.17 (m, 2H), 3.68 (m, 2H), 3.36 (m, 1H), 2.15
(m, 4H); HRMS (ESI) calcd for C15H16N3O2+ (M + H+) 270.1237,
found 270.1032.
4-Chloro-2-(2-norbornanyl)-1H-imidazo[4,5-c]quinoline (33b).
1
Scale: 0.25 mmol. Yield: 44.4 mg (60%). H NMR (300 MHz,
2-(2-Norbornanyl)-1H-imidazo[4,5-c]quinoline 5-Oxide (32b).
CD3OD/CDCl3) δ 8.29 (m, 1H), 7.96 (m, 2H), 7.56 (m, 2H), 3.31
(m, 1H), 3.05 (s, 2H), 2.91 (s, 2H), 2.83 (m, 2H), 2.41 (m, 1H),
2.06 (m, 1H), 1.62 (m, 7H); 13C NMR (75 MHz, CD3OD/CDCl3)
δ 143.0, 127.8, 126.5, 121.4, 42.8, 42.0, 36.3, 36.1, 29.6, 28.6;
1
Scale: 0.25 mmol. Yield: 44.4 mg (60%). H NMR (300 MHz,
CD3OD/CDCl3) δ 8.77 (s, 1H), 8.05 (s, 1H), 7.95 (s, 1H), 7.81 (m,
2H), 3.13 (mm, 1H), 2.81 (m, 1H), 2.68 (s, 1H), 2.52 (s, 1H), 2.14
(m, 1H), 1.93 (m, 1H), 1.58 (m, 7H); HRMS (ESI) calcd for
C17H18N3O+ (M + H+) 280.1444, found 280.1443.
HRMS (ESI) calcd for C17H17ClN3 (M + H+) 298.1106, found
+
298.1104.
2-(4-(1,3-Dioxoisoindolin-2-yl)cyclohexyl)-1H-imidazo[4,5-
c]quinoline 5-Oxide (32c). Scale: 0.40 mmol. Yield: 65 mg (44%).
1H NMR (300 MHz, CD3OD/CDCl3) δ 8.96 (s, 1H), 8.63 (m, 1H),
8.32 (m, 1H), 7.75 (m, 6H), 4.18 (m, 1H), 3.04 (t, 1H, J ) 11.6
Hz), 2.24 (m, 4H), 1.84 (m, 4H); HRMS (ESI) calculated for
4-Chloro-2-(4-(1,3-dioxoisoindolin-2-yl)-1H-imidazo[4,5-c]quin-
oline (33c). Scale: 0.27 mmol. Yield: 87 mg (74%). 1H NMR (300
MHz, CD3OD/CDCl3) δ 8.56 (m, 1H), 8.22 (d, 1H, J ) 7.9 Hz),
8.09 (d, 1H, J ) 8.5 Hz), 7.70 (m, 6H), 4.25 (m, 1H), 3.35 (m,
1H), 2.42 (m, 4H), 1.85 (m, 41H); 13C NMR (75 MHz, CD3OD/
CDCl3) δ 168.5, 158.7, 144.2, 143.6, 134.1, 132.5, 132.3, 132.2,
129.0, 128.8, 123.3, 50.0, 37.8, 31.3, 29.3; HRMS (ESI) calcd for
C24H21N4O2 (M + H+) 413.1608, found 413.1463.
+
2-(1-Benzoylpiperidin-4-yl)-1H-imidazo[4,5-c]quinoline 5-Ox-
1
C24H20ClN4O2 (M + H+) 431.1269, found 431.1260.
+
ide (32d). Scale: 0.30 mmol. Yield: 74 mg (67%). H NMR (300
MHz, CD3OD/CDCl3) δ 8.79 (s, 1H), 8.57 (m, 1H), 8.18 (bs, 1H),
7.76 (s, 1H), 7.68 (m, 1H), 7.54 (m, 5H), 4.59 (m, 1H), 3.76 (m,
1H), 2.93 (m, 2H), 2.02 (m, 2H), 1.85 (m, 4H); HRMS (ESI) calcd
4-Chloro-2-(1-benzoylpiperidin-4-yl)-1H-imidazo[4,5-c]quino-
line (33d). Scale: 0.20 mmol. Yield: 42.4 mg (54%). 1H NMR (300
MHz, CD3OD/CDCl3) δ 7.99 (s, 1H), 7.86 (d, 1H, J ) 7.5 Hz),
7.48 (m, 3H), 7.26 (m, 5H), 4.63 (m, 1H), 3.76 (m, 1H), 3.18 (m,
1H), 2.87 (m, 1H), 2.05 (m, 2H), 1.92 (m, 4H); 13C NMR (75 MHz,
CD3OD/CDCl3) δ 135.9, 129.9, 128.6, 128.3, 128.0, 127.0, 126.7,
121.4, 53.6, 36.8, 30.6; HRMS (ESI) calcd for C22H20ClN4O+ (M
+ H+) 391.1320, found 391.1317.
for C22H20N4O2 (M + H+) 372.1586, found 373.1665.
+
2-(3-Noradamantanyl)-1H-imidazo[4,5-c]quinoline 5-Oxide
(32e). Scale: 0.41 mmol. Yield: 89 mg (71%). 1H NMR (300 MHz,
CD3OD/CDCl3) δ 8.77 (s, 1H), 8.52 (m, 1H), 8.29 (s, 1H), 7.71
(m, 1H), 7.53 (m, 2H), 2.67 (t, 1H, J ) 6.7 Hz), 2.27 (s, 2H), 2.09
(d, 2H, J ) 11.4 Hz), 1.95 (d, 1H, J ) 10.8 Hz), 1.87 (m, 3H),
1.58 (m, 4H); HRMS (ESI) calcd for C19H20N3O+ (M + H+)
306.1601, found 306.1606.
4-Chloro-2-(3-noradamantanyl)-1H-imidazo[4,5-c]quino-
1
line (33e). Scale: 0.23 mmol. Yield: 60 mg (79%). H NMR (300
MHz, CD3OD/CDCl3) δ 8.37 (s, 1H), 8.10 (d, 1H, J ) 7.8 Hz),
7.78 (m, 1H), 7.38 (m, 2H), 2.67 (m, 1H), 2.21 (s, 2H), 2.08 (m,
2H), 1.90 (m, 5H), 1.54 (s, 4H); 13C NMR (75 MHz, CD3OD/
CDCl3) δ 162.9, 142.9, 128.2, 127.7, 121.4, 45.5, 43.5, 38.1, 37.7,
2-(1-Adamantanyl)-1H-imidazo[4,5-c]quinoline 5-Oxide (32f).
Scale: 0.25 mmol. Yield: 63 mg (78%). 1H NMR (300 MHz,
CD3OD/CDCl3) δ 8.91 (m, 1H), 8.60 (m, 1H), 8.31 (m, 1H), 7.90
(s, 1H), 7.80 (d, 1H, J ) 7.5 Hz), 7.54 (m, 1H), 2.08 (s, 10H),
1.76 (s, 6H); HRMS (ESI) calcd for C20H22N3O+ (M + H+)
320.1757, found 320.1753.
+
37.4, 37.2, 36.4, 34.3, 31.2; HRMS (ESI) calcd for C19H19ClN3
(M + H+) 324.1262, found 324.1268.
4-Chloro-2-cyclohexyl-1H-imidazo[4,5-c]quinoline (29).15 Com-
pound 28 (150 mg, 560 µmol) was suspended in a mixture of
toluene (0.30 mL) and DMF (0.60 mL) and then was treated with
140 µL (1.50 mmol) of POCl3 at 0 °C with stirring. The ice bath
was removed, and the mixture was heated at 100 °C for 1.5 h. The
mixture was cooled to room temperature, and a few small pieces
of ice were added with stirring. Subsequently, the pH was adjusted
to 6-7 with solid NaHCO3. The mixture was sonicated and filtered,
rinsing with water and diisopropyl ether, and the collected solid
was dried in vacuo to give 97.1 mg (340 µmol, 61%) of 29 as a
4-Chloro-2-(1-adamantanyl)-1H-imidazo[4,5-c]quinoline (33f).
Scale: 0.20 mmol. Yield: 39 mg (60%). 1H NMR (300 MHz,
CD3OD/CDCl3) δ 8.34 (s, 1H), 7.95 (m, 2H), 7.45 (m, 2H), 2.15
(m, 10H), 1.78 (m, 6H); 13C NMR (75 MHz, CD3OD/CDCl3) δ
163.1, 143.0, 132.6, 127.8, 126.5, 121.5, 40.8, 36.4, 36.2, 35.8,
32.2, 28.1; HRMS (ESI) calcd for C20H21ClN3 (M + H+)
+
338.1419, found 338.1414.
General Procedure for N-Substituted 2-Cyclohexyl-1H-imi-
dazo[4,5-c]quinolin-4-amine (5-14). A solution of compound 29
and an appropriate aniline in DMF was heated at 140 °C overnight
under a dry nitrogen atmosphere in a tightly sealed Biotage
microwave vial (size, 0.2-0.5 mL or 0.5-2.0 mL) equipped with
a magnetic stir bar. The reaction mixture was cooled, the solvent
was removed in vacuo, and the crude product was purified by a
preparative TLC.
N-(3-Chlorophenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-
4-amine (5). Compound 29 (6.7 mg, 23 µmol) was reacted with
3-chloroaniline (10 µL, 94 µmol) in DMF (50 µL). The crude
product was chromatographed on silica gel (3:1 petroleum ether/
EtOAc) to give 5.5 mg (15 µmol, 62%) of 5. Rf ) 0.43 [silica gel,
2:1 petroleum ether/EtOAc]; 1H NMR (600 MHz, 4:1 CDCl3/
CD3OD) δ 8.30 (s, 1H), 7.94 (d, 1H, J ) 7.4 Hz), 7.89 (d, 1H, J
) 8.5 Hz), 7.75 (d, 1H, J ) 8.0 Hz), 7.48 (t, 1H, J ) 7.5 Hz), 7.38
(t, 1H, J ) 7.3 Hz), 7.24 (t, 1H, J ) 7.9 Hz), 6.94 (d, 1H, J ) 8.3
Hz), 2.92 (t, 1H, J ) 11.9 Hz), 2.13 (d, 2H, J ) 12.6 Hz), 1.90 (d,
2H, J ) 13.3 Hz), 1.78 (d, 1H, J ) 12.6 Hz), 1.67 (q, 2H, J )
11.7 Hz), 1.44 (q, 2H, J ) 12.7 Hz), 1.32 (q, 1H, J ) 12.9 Hz);
13C NMR (150 MHz, 4:1 CDCl3/CD3OD) δ 147.1, 143.9, 134.7,
130.1, 127.7, 127.6, 123.3, 121.8, 120.7, 118.8, 117.0, 115.7, 38.8,
32.2, 26.4, 26.1; HRMS (ESI) calcd for C22H22ClN4 (M + H+)
377.1533, found 377.1528.
1
beige solid. Rf ) 0.56 [silica gel, 10:1 CH2Cl2/MeOH]; H NMR
(600 MHz, DMSO-d6) δ 8.38-8.35 (m, 1H), 8.02-7.99 (m, 1H),
7.71-7.62 (m, 2H), 3.07 (tt, 1H, J ) 12.0, 3.5 Hz), 2.16-2.10 (m,
2H), 1.97-1.91 (m, 2H), 1.88-1.75 (m, 3H), 1.59-1.35 (m, 3H);
13C NMR (75 MHz, CD3OD) δ 129.5, 129.3, 128.2, 122.8, 40.4,
33.0, 27.4, 27.0; HRMS (ESI) calcd for C16H17ClN3 (M + H+)
286.1111, found 286.1106.
General Procedure for 2-Substituted 4-Chloro-1H-imidazo[4,5-
c]quinolines (33a-f). A mixture of toluene (0.45 mL/mmol) and
DMF (0.90 mL/mmol) was cooled in an ice bath, and phosphorus
oxychloride (2.6 equiv) was added. After 10 min, the appropriate
1H-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. When the mixture was cooled,
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. The residue obtained was
subjected to preparative silica gel column chromatography (100:1
to 8:1 CHCl3/MeOH).

Quinolin-4-amine DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2105
2-Cyclohexyl-N-(3,5-dichlorophenyl)-1H-imidazo[4,5-c]quino-
lin-4-amine (6). Compound 29 (7.4 mg, 26 µmol) was reacted with
3,5-dichloroaniline (13 mg, 80 µmol) in DMF (50 µL). The crude
product was chromatographed on silica gel (3:1 petroleum ether/
EtOAc) to give 6.0 mg (15 µmol, 56%) of 6. Rf ) 0.50 [silica gel,
2:1 petroleum ether/EtOAc]; 1H NMR (600 MHz, 4:1 CDCl3/
CD3OD) δ 8.05 (s, 2H), 7.95 (d, 1H, J ) 7.6 Hz), 7.91 (d, 1H, J
) 8.3 Hz), 7.49 (t, 1H, J ) 7.6 Hz), 7.32 (t, 1H, J ) 7.4 Hz), 6.94
(s, 1H), 2.91 (t, 1H, J ) 12.0 Hz), 2.13 (d, 2H, J ) 11.9 Hz), 1.89
(d, 2H, J ) 13.9 Hz), 1.78 (d, 1H, J ) 12.2 Hz), 1.66 (q, 2H, J )
12.5 Hz), 1.44 (q, 2H, J ) 13.0 Hz), 1.32 (q, 1H, J ) 13.1 Hz);
13C NMR (150 MHz, 4:1 CDCl3/CD3OD) δ 158.1, 146.7, 143.7,
143.2, 135.1, 134.8, 127.8, 127.7, 126.7, 123.6, 121.4, 120.8, 116.9,
115.8, 38.8, 32.2, 26.4, 26.1; HRMS (ESI) calcd for C22H21Cl2N4
(M + H+) 411.1143, found 411.1140.
2-Cyclohexyl-N-(2,4-dichlorophenyl)-1H-imidazo[4,5-c]quino-
lin-4-amine (7). Compound 29 (8.2 mg, 29 µmol) was reacted with
2,4-dichloroaniline (14 mg, 87 µmol) in DMF (50 µL). The crude
product was chromatographed on silica gel (3:1 hexane/EtOAc) to
give 5.0 mg (12 µmol, 42%) of 7. Rf ) 0.63 [silica gel, 2:1
petroleum ether/EtOAc]; 1H NMR (600 MHz, 4:1 CDCl3/CD3OD)
δ 8.89 (d, 1H, J ) 9.4 Hz), 7.98 (d, 1H, J ) 7.7 Hz), 7.84 (d, 1H,
J ) 8.1 Hz), 7.47 (t, 1H, J ) 7.6 Hz), 7.39 (d, 1H, J ) 2.2 Hz),
7.33 (t, 1H, J ) 7.4 Hz), 7.27 (dd, 1H, J ) 8.6, 2.3 Hz), 2.96 (tt,
1H, J ) 11.9, 3.5 Hz), 2.11 (d, 2H, J ) 12.1 Hz), 1.89 (d, 2H, J
) 13.7 Hz), 1.77 (d, 1H, J ) 12.3 Hz), 1.70 (q, 2H, J ) 12.3 Hz),
1.44 (q, 2H, J ) 12.8 Hz), 1.32 (q, 1H, J ) 12.7 Hz); 13C NMR
(150 MHz, 4:1 CDCl3/CD3OD) δ 146.7, 136.1, 135.0, 129.0, 127.7,
127.6 (127.615), 127.6 (127.570), 123.6, 122.1, 120.9, 116.1, 39.1,
32.2, 26.4, 26.1; HRMS (ESI) calcd for C22H21Cl2N4 (M + H+)
411.1143, found 411.1132.
2-Cyclohexyl-N-(3,4-difluorophenyl)-1H-imidazo[4,5-c]quino-
lin-4-amine (8). Compound 29 (7.0 mg, 24 µmol) was reacted with
3,4-difluoroaniline (20 µL, 200 µmol) in DMF (30 µL). The crude
product was chromatographed on silica gel (7:4 petroleum ether/
EtOAc) to give 7.1 mg (19 µmol, 77%) of 8. Rf ) 0.36 [silica gel,
2:1 petroleum ether/EtOAc]; 1H NMR (600 MHz, 4:1 CDCl3/
CD3OD) δ 8.31 (m, 1H), 7.93 (d, 1H, J ) 7.9 Hz), 7.87 (d, 1H, J
) 8.4 Hz), 7.47 (t, 1H, J ) 7.7 Hz), 7.44 (d, 1H, J ) 8.8 Hz), 7.30
(t, 1H, J ) 7.4 Hz), 7.09 (q, 1H, J ) 9.3 Hz), 2.91 (t, 1H, J ) 11.8
Hz), 2.13 (d, 2H, J ) 11.9 Hz), 1.89 (d, 2H, J ) 13.2 Hz), 1.78 (d,
1H, J ) 12.7 Hz), 1.66 (q, 2H, J ) 11.4 Hz), 1.44 (q, 2H, J )
12.8 Hz), 1.32 (q, 1H, J ) 13.0 Hz); 13C NMR (150 MHz, 4:1
CDCl3/CD3OD) δ 175.5, 163.8, 158.0, 147.1, 143.9, 134.7, 127.6,
123.2, 120.8, 117.2, 117.1, 115.7, 114.4, 108.3, 108.1, 38.8, 32.2,
26.4, 26.1; HRMS (ESI) calcd for C22H21F2N4 (M + H+) 379.1734,
found 379.1739.
2-Cyclohexyl-N-(3,5-difluorophenyl)-1H-imidazo[4,5-c]quino-
lin-4-amine (9). Compound 29 (5.1 mg, 18 µmol) was reacted with
3,5-difluoroaniline (9.5 mg, 72 µmol) in DMF (50 µL). The crude
product was chromatographed on silica gel (20:1 CH2Cl2/MeOH,
20:1 CHCl3/MeOH, and then 15:1 CH2Cl2/MeOH) to give 7.4 mg
(20 µmol, 100%) of 9. Rf ) 0.54 [silica gel, 20:1 CH2Cl2/MeOH];
1H NMR (600 MHz, DMSO-d6) δ 13.19 (s, 1H), 9.58 (s, 1H), 8.16
(m, 3H), 7.83 (d, 1H, J ) 8.2 Hz), 7.53 (t, 1H, J ) 7.7 Hz), 7.41
(t, 1H, J ) 8.0 Hz), 6.73 (t, 1H, J ) 8.8 Hz), 3.00 (tt, 1H, J )
11.7, 3.3 Hz), 2.08 (d, 2H, J ) 12.2 Hz), 1.87 (d, 2H, J ) 13.1
Hz), 1.77-1.71 (m, 3H), 1.44 (qt, 2H, J ) 12.8, 3.0 Hz), 1.32 (qt,
1H, J ) 12.5, 3.3 Hz); 13C NMR (150 MHz, 4:1 CDCl3/CD3OD)
δ 164.6, 164.4, 162.9, 162.8, 146.8, 143.7, 143.6, 134.8, 127.8,
127.7, 123.5, 120.8, 115.8, 101.6, 101.4, 96.8, 96.6, 96.5, 38.8,
32.2, 26.4, 26.1; HRMS (ESI) calcd for C22H21F2N4 (M + H+)
379.1734, found 379.1740.
DMSO-d6) δ 13.25 (s, 1H), 10.00 (s, 1H), 9.17 (s, 2H), 8.19 (d,
1H, J ) 7.7 Hz), 7.76 (d, 1H, J ) 8.2 Hz), 7.58 (s, 1H), 7.56 (t,
1H, J ) 7.9 Hz), 7.44 (t, 1H, J ) 7.6 Hz), 3.02 (tt, 1H, J ) 11.8,
3.4 Hz), 2.11 (d, 2H, J ) 11.9 Hz), 1.88 (d, 2H, J ) 13.5 Hz),
1.78-1.72 (m, 3H), 1.45 (qt, 2H, J ) 12.8, 3.3 Hz), 1.32 (qt, 1H,
J ) 12.6, 3.3 Hz); 13C NMR (150 MHz, 4:1 CDCl3/CD3OD) δ
143.6, 142.9, 135.0, 128.0, 127.8, 123.8, 120.8, 118.2, 115.9, 114.3,
38.8, 32.2, 26.4, 26.1; HRMS (ESI) calcd for C24H21F6N4 (M +
H+) 479.1670, found 479.1680.
4-(2-Cyclohexyl-1H-imidazo[4,5-c]quinolin-4-ylamino)phtha-
lonitrile (11). Compound 29 (4.50 mg, 15.7 µmol) was reacted
with 4-aminophthalonitrile (11.6 mg, 78.8 µmol) in DMF (50 µL).
The crude product was chromatographed on reversed-phase C18
silica gel (9:1 MeOH/H2O) and normal-phase silica gel (15:1
CHCl3/MeOH) to give 0.45 mg (1.1 µmol, 7.3%) of 11. Rf ) 0.80
[silica gel, 10:1 CHCl3/MeOH]; 1H NMR (600 MHz, DMSO-d6) δ
13.30 (s, 1H), 10.19 (s, 1H), 9.07 (s, 1H), 8.78 (d, 1H, J ) 7.6
Hz), 8.21 (d, 1H, J ) 7.2 Hz), 8.04 (d, 1H, J ) 8.9 Hz), 7.89 (d,
1H, J ) 8.2 Hz), 7.58 (t, 1H, J ) 7.6 Hz), 7.48 (t, 1H, J ) 7.1
Hz), 3.02 (tt, 1H, J ) 12.1, 3.4 Hz), 2.09 (d, 2H, J ) 10.7 Hz),
1.87 (d, 2H, J ) 13.2 Hz), 1.78-1.72 (m, 3H), 1.45 (qt, 2H, J )
12.8, 3.3 Hz), 1.32 (qt, 1H, J ) 12.9, 3.5 Hz); 13C NMR (150 MHz,
4:1 CDCl3/CD3OD) δ 158.5, 146.2, 145.7, 143.3, 134.6, 130.0,
128.2, 128.0, 124.4, 122.6, 121.8, 120.9, 116.9, 116.7, 116.5, 116.1,
38.8, 32.2, 26.4, 26.1; HRMS (ESI) calcd for C24H21N6 (M + H+)
393.1828, found 393.1835.
2-Cyclohexyl-N-(3,4-dimethoxyphenyl)-1H-imidazo[4,5-c]quin-
olin-4-amine (12). Compound 29 (7.8 mg, 27 µmol) was reacted
with 3,4-dimethoxyaniline (13 mg, 83 µmol) in DMF (50 µL). The
crude product was chromatographed on silica gel (2:3 hexane/
EtOAc) to give 2.5 mg (6.2 µmol, 23%) of 12. Rf ) 0.33 [silica
gel, 1:1 petroleum ether/EtOAc]; 1H NMR (600 MHz, 4:1 CDCl3/
CD3OD) δ 7.98 (s, 1H), 7.92 (d, 1H, J ) 6.6 Hz), 7.80 (d, 1H, J
) 8.6 Hz), 7.45 (t, 1H, J ) 7.6 Hz), 7.33 (d, 1H, J ) 9.7 Hz), 7.27
(t, 1H, J ) 7.1 Hz), 6.87 (d, 1H, J ) 8.1 Hz), 3.95 (s, 3H), 3.84
(s, 3H), 2.92 (t, 1H, J ) 11.9 Hz), 2.13 (d, 2H, J ) 11.0 Hz), 1.89
(d, 2H, J ) 13.2 Hz), 1.78 (d, 1H, J ) 11.6 Hz), 1.67 (q, 2H, J )
12.1 Hz), 1.44 (q, 2H, J ) 13.0 Hz), 1.32 (q, 1H, J ) 13.0 Hz);
13C NMR (150 MHz, 4:1 CDCl3/CD3OD) δ 149.3, 144.3, 144.1,
135.1, 127.5, 127.3, 122.7, 120.7, 112.4, 111.0, 104.5, 56.6, 56.0,
38.9, 32.2, 26.4, 26.1; HRMS (ESI) calcd for C24H27N4O2 (M +
H+) 403.2134, found 403.2100.
N-(Benzo[d][1,3]dioxol-5-yl)-2-cyclohexyl-1H-imidazo[4,5-
c]quinolin-4-amine (13). Compound 29 (13.7 mg, 47.9 µmol) was
reacted with 3,4-(methylenedioxy)aniline (16.3 mg, 115 µmol) in
DMF (100 µL). The crude product was chromatographed on silica
gel (2:3 and 1:1 hexane/EtOAc) to give 5.4 mg (14 µmol, 29%) of
13. Rf ) 0.59 [silica gel, 1:1 hexane/EtOAc]; 1H NMR (600 MHz,
4:1 CDCl3/CD3OD) δ 7.91 (br s, 1H), 7.82 (d, 1H, J ) 8.6 Hz),
7.80 (s, 1H), 7.44 (t, 1H, J ) 7.7 Hz), 7.26 (t, 1H, J ) 6.8 Hz),
7.21 (d, 1H, J ) 7.3 Hz), 6.78 (d, 1H, J ) 8.5 Hz), 5.91 (s, 2H),
2.91 (t, 1H, J ) 11.6 Hz), 2.12 (d, 2H, J ) 12.0 Hz), 1.89 (d, 2H,
J ) 13.5 Hz), 1.77 (d, 1H, J ) 13.4 Hz), 1.66 (q, 2H, J ) 11.9
Hz), 1.43 (q, 2H, J ) 12.7 Hz), 1.31 (q, 1H, J ) 12.8 Hz); 13C
NMR (150 MHz, 4:1 CDCl3/CD3OD) δ 147.9, 144.0, 135.6, 127.5,
127.2, 122.8, 120.7, 115.6, 112.1, 108.5, 102.2, 101.2, 38.9, 32.2,
26.4, 26.1; HRMS (ESI) calcd for C23H23N4O2 (M + H+) 387.1821,
found 387.1814.
2-Cyclohexyl-N-(pyridin-3-yl)-1H-imidazo[4,5-c]quinolin-4-
amine (14). Compound 29 (6.67 mg, 23.3 µmol) was reacted with
3-aminopyridine (11.0 mg, 116 µmol) in DMF (50 µL). The crude
product was chromatographed on normal-phase silica gel (10:1 and
3:1 CHCl3/MeOH and 90:3:1 CHCl3/MeOH/triethylamine), on
reversed-phase C18 silica gel (4:1 MeOH/H2O), and again on
normal-phase silica gel (15:1 CHCl3/MeOH) to give 0.35 mg (0.99
µmol, 4.2%) of 14. Rf ) 0.57 [silica gel, 200:10:1 CHCl3/MeOH/
N-(3,5-Bis(trifluoromethyl)phenyl)-2-cyclohexyl-1H-imidazo[4,5-
c]quinolin-4-amine (10). Compound 29 (4.35 mg, 15.2 µmol) was
reacted with 3,5-bis(trifluoromethyl)aniline (10.0 µL, 62.7 µmol)
in DMF (50 µL). The crude product was chromatographed on silica
gel (2:1 hexane/EtOAc, 30:1 CHCl3/MeOH, 80:20:1 hexane/EtOAc/
triethylamine, 5:2 hexane/EtOAc, and 35:1 CHCl3/MeOH with a
trace amount of citric acid) to give 1.40 mg (2.93 µmol, 19%) of
10. Rf ) 0.43 [silica gel, 2:1 hexane/EtOAc]; 1H NMR (600 MHz,
1
triethylamine]; H NMR (600 MHz, DMSO-d6) [major tautomer]
δ 13.16 (s, 1H), 9.36 (d, 1H, J ) 2.6 Hz), 9.31 (s, 1H), 8.73 (ddd,
1H, J ) 8.2, 2.5, 1.3 Hz), 8.17 (dd, 1H, J ) 4.7, 1.5 Hz), 8.14 (dd,
1H, J ) 8.2, 1.0 Hz), 7.78 (d, 1H, J ) 7.7 Hz), 7.50 (td, 1H, J )
7.7, 1.7 Hz), 7.38 (td, 1H, J ) 7.4, 1.1 Hz), 7.35 (dd, 1H, J ) 8.3,

2106 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kim et al.
4.7 Hz), 3.00 (tt, 1H, J ) 12.1, 3.5 Hz), 2.09 (d, 2H, J ) 12.7 Hz),
1.87 (dt, 2H, J ) 13.2, 3.4 Hz), 1.78-1.72 (m, 3H), 1.45 (qt, 2H,
J ) 13.0, 3.2 Hz), 1.32 (qt, 1H, J ) 12.8, 3.3 Hz); 13C NMR (150
MHz, 2:1 CDCl3/CD3OD) δ 158.4, 142.0, 140.3, 139.0, 137.2,
131.5, 130.4, 130.2, 127.9, 127.8, 126.7, 124.5, 123.7, 121.1, 116.1,
39.1, 32.4, 26.6, 26.3; HRMS (ESI) calcd for C21H22N5 (M + H+)
344.1875, found 344.1873.
N-(3,4-Dichlorophenyl)-2-(1-benzoylpiperidin-4-yl)-1H-imi-
dazo[4,5-c]quinolin-4-amine (23). Scale: 0.108 mmol. Yield: 25
mg (44%). H NMR (300 MHz, CD3OD/CDCl3) δ 8.37 (s, 1H),
7.85 (d, 1H, J ) 7.3 Hz), 7.82 (d, 1H, J ) 8.4 Hz), 7.66 (dd, 1H,
J ) 8.7, 2.4 Hz), 7.43 (m, 1H), 7.31 (m, 7H), 4.69 (m, 12H), 3.82
(m, 2H), 3.17 (m, 1H), 3.00 (s, 1H), 1.92 (m, 4H); 13C NMR (75
MHz, CD3OD/CDCl3) δ 171.2, 143.5, 140.4, 135.3, 132.2, 130.1,
128.6, 127.5, 127.3, 126.5, 124.0, 123.2, 120.4, 119.9, 117.9, 42.1,
36.4, 31.0, 30.5; HRMS (ESI) calcd for C28H24Cl2N5O+ (M + H+)
516.1352, found 516.1352.
1
General Procedure for N-Substituted 1H-Imidazo[4,5-c]quin-
olin-4-amine (16-21, 23). A solution of the appropriate 4-chloro-
1H-imidazo[4,5-c]quinoline and 3,4-dichloroaniline in DMF was
heated at 140 °C overnight under a dry nitrogen atmosphere. The
reaction mixture was cooled, and the solvent was evaporated. The
residue obtained was subjected to preparative silica gel column
chromatography (100:1 to 8:1 CHCl3/MeOH).
N-Phenyl-2-(1-benzoylpiperidin-4-yl)-1H-imidazo[4,5-c]quin-
olin-4-amine (24). A solution of 33d (61.4 mg, 0.156 mmol) and
aniline (43 µL, 0.47 mmol) in DMF (0.8 mL) was heated at 140
°C overnight under N2 atmosphere. The reaction mixture was
cooled, and the solvent was evaporated. The residue obtained was
subjected to preparative silica gel column chromatography (100:1
N-(3,4-Dichlorophenyl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imi-
dazo[4,5-c]quinolin-4-amine (16). Scale: 0.14 mmol. Yield: 20
1
to 8:1 CHCl3/MeOH). Yield: 40 mg (57%). H NMR (300 MHz,
1
mg (35%). H NMR (300 MHz, CD3OD/CDCl3) δ 8.44 (s, 1H),
CD3OD/CDCl3) δ 7.92 (d, 1H, J ) 7.5 Hz), 7.81 (s, 2H), 7.75 (d,
1H, J ) 8.1 Hz), 7.33 (dd, 1H, J ) 7.2, 1.5 Hz), 7.21 (t, 1H, J )
8.0 Hz), 7.12 (d, 1H, J ) 7.3 Hz), 6.86 (t, 1H, J ) 7.5 Hz), 4.62
(m, 12H), 3.60 (m, 2H), 3.07 (d, 1H, J) 11.4 Hz), 1.92 (m, 4H);
13C NMR (75 MHz, CD3OD/CDCl3) δ 170.7, 154.8, 143.6, 140.6,
135.6, 129.8, 128.7, 128.5, 127.2, 127.1, 126.6, 122.5, 121.6, 120.5,
118.7, 53.4, 20.8, 13.9; HRMS (ESI) calcd for C28H26N5O+ (M +
H+) 448.2132, found 448.2137.
7.89 (d, 2H, J ) 9.0 Hz), 7.74 (dd, 1H, J ) 9.0, 2.5 Hz), 7.50 (m,
1H), 7.37 (m, 2H), 4.10 (tdt, 2H, J ) 11.4, 3.3 Hz), 3.59 (m, 2H),
3.21 (m, 1H), 2.04 (m, 4H); 13C NMR (75 MHz, CD3OD/CDCl3)
δ 143.4, 140.4, 132.2, 127.4, 127.2, 124.0, 123.2, 120.4, 119.9,
117.9, 115.3, 67.5, 35.5, 31.1; HRMS (ESI) calcd for
C21H19Cl2N3O+ (M + H+) 413.0930, found 413.0952.
N-(3,4-Dichlorophenyl)-2-(exonorbornanyl)-1H-imidazo[4,5-
c]quinolin-4-amine (17). Scale: 0.139 mmol. Yield: 6.4 mg (11%).
1H NMR (300 MHz, CD3OD/CDCl3) δ 9.54 (s, 1H), 8.49 (s, 1H),
8.00 (d, 1H, J ) 8.1 Hz), 7.83 (t, 1H, J ) 9.6 Hz), 7.57 (t, 2H, J
) 8.4 Hz), 7.31 (m, 1H), 7.27 (m, 12H), 3.99 (m, 1H), 3.59 (s,
1H), 3.42 (dd, 1H, J ) 11.4, 6.0 Hz), 2.71 (s, 1H), 2.47 (s, 1H),
2.09 (m, 2H), 1.47 (m, 5H); 13C NMR (75 MHz, CD3OD/CDCl3)
δ 130.4, 127.8, 123.4, 120.6, 120.0, 118.4, 42.3, 41.2, 40.5, 37.3,
2-((1s,4s)-4-Aminocyclohexyl)-N-(3,4-dichlorophenyl)-1H-imi-
dazo[4,5-c]quinolin-4-amine (22). A 33% solution of methylamine
in absolute ethanol (0.5 mL) is added to a stirred solution of 21
(10 mg, 0.017 mmol) in ethanol (0.25 mL). The solution was
refluxed for 2 h. The mixture was then cooled to room temperature,
and the solvent was evaporated under reduced pressure. The residue
was purified by flash column chromatography using a mixture of
10:1 EtOAc/MeOH, by volume. Yield: 5 mg (70%). 1H NMR (300
MHz, CD3OD/CDCl3) δ 8.40 (s, 1H), 8.08 (d, 1H, J ) 8.1 Hz),
7.87 (d, 1H, 8.7 Hz), 7.75 (dd, 1H, J ) 8.7, 2.5 Hz), 7.47 (t, 2H,
J ) 6.9 Hz), 7.29 (m, 4H), 3.40 (m, 1H), 3.12 (m, 1H), 2.27 (m,
2H), 1.95 (m, 2H), 1.89 (m, 4H); 13C NMR (75 MHz, CD3OD/
CDCl3) δ 143.4, 140.4, 132.1, 130.0, 127.4, 124.0, 123.2, 120.8,
119.9, 117.9, 110.7, 105.7, 31.6, 27.4, 25.6; HRMS (ESI) calcd
33.5, 29.9, 24.2; HRMS (ESI) calcd for C23H21Cl2N4 (M + H+)
+
423.1138, found 423.1140.
N-(3,4-Dichlorophenyl)-2-(endonorbornanyl)-1H-imidazo[4,5-
c]quinolin-4-amine (18). Scale: 0.139 mmol. Yield: 12.6 mg (21%).
1H NMR (300 MHz, CD3OD/CDCl3) δ 9.81 (s, 1H), 8.44 (s, 1H),
7.98 (d, 1H, J ) 8.4 Hz), 7.78 (m, 2H), 7.55 (t, 1H, J ) 8.5 Hz),
7.36 (s, 1H), 2.99 (dd, 1H, J ) 8.7, 4.9 Hz), 2.60 (s, 1H), 2.48 (s,
1H), 2.23 (m, 1H), 1.82 (m, 1H), 1.66 (m, 2H), 1.30 (m, 5H); 13C
NMR (75 MHz, CD3OD/CDCl3) δ 130.2, 127.4, 123.1, 120.1,
118.1, 42.7, 41.9, 36.4, 36.3, 36.1, 29.7, 28.9; HRMS (ESI) calcd
for C22H22Cl2N5 (M + H+) 426.1247, found 426.4203.
+
2-(1-Benzylpiperidin-4-yl)-N-(3,4-dichlorophenyl)-1H-imida-
zo[4,5-c]quinolin-4-amine (25). A solution of compound 23 (18
mg, 0.035 mmol) in dry THF (0.18 mL) was added dropwise to a
stirred slurry of lithium aluminum hydride (0.89 mL of 0.2 M in
THF, 0.175 mmol). After the mixture was heated under reflux
overnight, decomposition of excess hydride was effected by cautious
addition of water. The inorganic solids were removed by filtration,
the organic layer was dried over anhydrous sodium sulfate and
filtered, and the solvent was removed under reduced pressure. The
final residue was purified by flash column chromatography using a
for C23H21Cl2N4 (M + H+) 423.1138, found 423.1151.
+
N-(3,4-Dichlorophenyl)-2-(3-noradamantanyl)-1H-imidazo[4,5-
c]quinolin-4-amine (19). Scale: 0.186 mmol. Yield: 58.2 mg (70%).
1H NMR (300 MHz, CD3OD/CDCl3) δ 8.23 (s, 1H), 8.06 (d, 1H,
J ) 8.4 Hz), 7.80 (d, 1H, J ) 8.4 Hz), 7.63 (dd, 1H, J ) 8.7, 2.4
Hz), 7.43 (m, 1H), 7.31 (m, 2H), 2.76 (t, 1H, J ) 6.3 Hz), 2.38 (s,
2H), 2.20 (d, 2H, J ) 10.5 Hz), 2.08 (m, 2H), 2.02 (m, 4H), 1.70
(m, 4H); 13C NMR (75 MHz, CD3OD/CDCl3) δ 160.5, 145.9, 142.0,
139.9, 139.8, 132.4, 132.4, 130.3, 127.6, 125.9, 124.9, 123.5, 120.9,
118.8, 115.5, 45.8, 43.8, 37.6, 34.5; HRMS (ESI) calcd for
1
mixture of 10:1 EtOAc/MeOH, by volume. H NMR (300 MHz,
CD3OD/CDCl3) δ 11.79 (s, 1H), 8.50 (s, 1H), 8.01 (m, 2H), 7.87
(dd, 1H, J ) 9.0, 2.6 Hz), 7.78 (m, 1H), 7.53 (m, 7H), 7.37 (s,
2H), 3.59 (m, 2H), 3.09 (m, 2H), 2.06 (m, 2H), 1.76 (s, 4H); 13C
NMR (75 MHz, CD3OD/CDCl3) δ 140.4, 136.4, 129.4, 128.1,
127.8, 123.0, 120.3, 119.8, 117.8, 100.2, 61.6, 52.9, 30.1, 28.8;
C25H23Cl2N4 (M + H+) 449.1294, found 449.1291.
+
N-(3,4-Dichlorophenyl)-2-(1-adamantanyl)-1H-imidazo[4,5-
c]quinolin-4-amine (20). Scale: 0.115 mmol. Yield: 38 mg (71%).
1H NMR (300 MHz, CD3OD/CDCl3) δ 8.33 (s, 1H), 8.04 (d, 1H,
J ) 7.5 Hz), 7.81 (d, 1H, J ) 8.4 Hz), 7.66 (m, 1H), 7.42 (m, 1H),
7.31 (m, 3H), 2.09 (s, 6H), 1.78 (s, 4H); 13C NMR (75 MHz,
CD3OD/CDCl3) δ 161.3, 146.1, 142.4, 140.1, 132.3, 124.5, 120.6,
HRMS (ESI) calcd for C28H26Cl2N5 (M + H+) 502.1560, found
+
502.1541.
N-(3,4-Dichlorophenyl)-2-(piperidin-4-yl)-1H-imidazo[4,5-
c]quinolin-4-amine (26). A solution of 25 (6 mg, 0.011 mmol) in
dry MeOH (0.5 mL) was hydrogenated at 1 atm in the presence of
palladium black (0.011 mg). After 2 h, the catalyst was removed
by filtration, and the solvent was evaporated under reduced pressure.
The final residue was purified by flash column chromatography
using a mixture of 10:1:0.1 EtOAc/MeOH/NH4OH, by volume. 1H
NMR (300 MHz, CD3OD/CDCl3) δ 8.51 (m, 1H), 8.03 (m, 2H),
7.72 (m, 1H), 7.60 (m, 1H), 7.43 (m, 2H), 3.38 (m, 2H), 2.35 (m,
2H), 1.19 (m, 12H), 0.90 (m, 12H); HRMS (ESI) calcd for
+
41.0, 36.3, 35.5, 28.6, 27.6; HRMS (ESI) calcd for C26H25Cl2N4
(M + H+) 463.1451, found 463.1456.
N-(3,4-Dichlorophenyl)-2-((1s,4s)-4-(1,3-dioxoisoindolin-2-yl)-
cyclohexyl)-1H-imidazo[4,5-c]quinolin-4-amine (21). Scale: 0.044
mmol. Yield: 40 mg (78%). 1H NMR (300 MHz, CD3OD/CDCl3)
δ 8.47 (m, 1H), 8.23 (s, 1H), 7.78 (m, 7H), 7.39 (m, 14H), 4.28
(m, 1H), 3.09 (m, 1H), 2.52 (m, 4H), 1.91 (m, 4H); 13C NMR (75
MHz, CD3OD/CDCl3) δ 168.5, 160.1, 143.3, 140.4, 134.0, 132.0,
131.6, 130.2, 129.9, 127.2, 122.9, 121.3, 120.3, 119.8, 118.9, 117.8,
C21H20Cl2N5 (M + H+) 412.1090, found 412.1086.
+
+
100.2, 37.0, 30.9, 28.9; HRMS (ESI) calcd for C30H24Cl2N5O2
HPLC Analysis of Compounds 5-14 and 16-26. Purity of
(M + H+) 556.1302, found 556.1290.
compounds was checked using a Hewlett-Packard 1100 HPLC

Quinolin-4-amine DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2107
equipped with a Zorbax SB-Aq 5 µm analytical column (50 mm
× 4.6 mm; Agilent Technologies, Palo Alto, CA). System A: linear
gradient solvent system; 5 mM TBAP (tetrabutylammonium
dihydrogenphosphate)-CH3CN from 50:50 to 0:100 in 13 min; the
flow rate was 0.5 mL/min. System B: linear gradient solvent system;
10 mM TEAA (triethylammonium acetate)-CH3CN from 65:35
to 0:100 in 13 min; the flow rate was 0.5 mL/min. Peaks were
detected by UV absorption with a diode array detector. The
compounds eluted at the following retention times: 5, 7.0 min
(system A), 9.4 min (system B); 6, 8.9 min (system A), 10.44 min
(system B); 7, 9.2 min (system A), 10.9 min (system B); 8, 7.5
min (system A), 9.3 min (system B); 9, 7.3 min (system A), 10.1
min (system B); 10, 9.4 min (system A), 11.7 min (system B); 11,
7.1 min (system A), 9.8 min (system B); 12, 3.3 min (system A),
8.0 min (system B); 13, 4.7 min (system A), 8.6 min (system B);
14, 7.2 min (system A), 4.7 min (system B); 16, 5.7 min (system
A), 8.6 min (system B); 17, 8.4 min (system A), 10.9 min (system
B); 18, 8.7 min (system A), 11.2 min (system B); 19, 9.3 min
(system A), 11.3 min (system B); 20, 9.7 min (system A), 12.0
min (system B); 21, 9.2 min (system A), 11.5 min (system B); 22,
2.4 min (system A), 10.3 min (system B); 23, 6.5 min (system A),
9.6 min (system B); 24, 2.9 min (system A), 6.9 min (system B);
25, 7.06 min (system A), 10.8 min (system B); 26, 1.93 min (system
A), 10.6 min (system B).
described.22 [35S]GTPγS binding was measured in 200 µL of buffer
containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM MgCl2,
1 µM GDP, 1 mM dithiothreitol, 100 mM NaCl, 3 U/mL adenosine
deaminase, 0.2 nM [35S]GTPγS, 0.004% 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate (CHAPS), and 0.5% bovine
serum albumin. Incubations were started by addition of the
membrane suspension (10 µg of protein/tube) to the test tubes and
carried out in duplicate for 30 min at 25 °C. The reaction was
stopped by rapid filtration through Whatman GF/B filters, presoaked
in 50 mM Tris-HCl and 5 mM MgCl2 (pH 7.4) containing 0.02%
CHAPS. The filters were washed twice with 3 mL of the buffer
mentioned before, and retained radioactivity was measured using
liquid scintillation counting. Nonspecific binding of [35S]GTPγS
was measured in the presence of 10 µM unlabeled GTPγS.
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.28 Data were expressed
as the mean ( standard error.
Acknowledgment. This research was supported in part by
the Intramural Research Program of the NIH, National Institute
of Diabetes and Digestive and Kidney Diseases, and by the
European Union (Grant QLK3-CT-2001-51963). We thank Dr.
Aniko´ Go¨blyo¨s for preparation of compound 27, Dr. John Lloyd
for collecting MS data, and Dr. Herman Yeh and Wesley White
for the helpful advice on the NMR experiments. S.d.C. thanks
Ministerio de Educacio´n y Ciencia (Spain) for financial support.
Y.K. thanks the Can-Fite Biopharma for financial support.
Pharmacological Methods. [3H]35 (47 Ci/mmol) was from GE
Healthcare Bio-Sciences Corp. (Piscataway, NJ). [3H]34 (CCPA,
42.6 Ci/mmol), [125I]36 (I-AB-MECA, 2000 Ci/mmol), and
[35S]GTPγS (1068 Ci/mmol) were from Perkin-Elmer Life Sciences
(Waltham, MA).
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 1000g 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
20000g for 20 min at 4 °C. The resultant pellets were resuspended
in buffer in the presence of 3 U/mL adenosine deaminase, and the
suspension was stored at -80 °C until the binding experiments.
The protein concentration was measured using the Bradford assay.27
Binding to the Human A1AR and the A2AAR. For binding to
the human A1AR, [3H]34 (2 nM) was incubated with membranes
(40 µg/tube) from CHO cells stably expressing the human A1AR
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.19 Nonspecific binding
was determined using 10 µM of N6-cyclopentyladenosine. For
human A2AAR binding, membranes (20 µg/tube) from HEK-293
cells stably expressing the human A2AAR were incubated with 15
nM [3H]35 at 25 °C for 60 min in 200 µL of 50 mM Tris-HCl, pH
7.4, containing 10 mM MgCl2.20 5′-N-Ethylcarboxamidoadenosine
(NECA, 37, 10 µM) was used to define nonspecific binding.
Reaction was terminated by filtration with GF/B filters.
Supporting Information Available: Selected 1H and 2D COSY
and NOESY spectra with peak assignments. This material is
available free of charge via the Internet at http://pubs.acs.org.
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