"Reversine" and its 2-substituted adenine derivatives as potent and selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm050221l

The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)- N⁶-cyclohexyladenine 2] was found to be a moderately potent antagonist for the human A₃ adenosine receptor (AR) with a K _i value of 0.66 μM. This result pro

Full text of DOI:10.1021/jm050221l

4910
J. Med. Chem. 2005, 48, 4910-4918
“Reversine” and Its 2-Substituted Adenine Derivatives as Potent and Selective
A3 Adenosine Receptor Antagonists
Melissa Perreira,†,‡ Jian-kang Jiang,†,‡ Athena M. Klutz,§ Zhan-Guo Gao,§ Asher Shainberg,§,| Changrui Lu,§
Craig J. Thomas,† and Kenneth A. Jacobson*,†,§
Chemical Biology Core Facility, and Molecular Recognition Section, Laboratory of Biological Chemistry, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, and Faculty of Life
Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel
Received March 9, 2005
The dedifferentiation agent “reversine” [2-(4-morpholinoanilino)-N6-cyclohexyladenine 2] was
found to be a moderately potent antagonist for the human A3 adenosine receptor (AR) with a
Ki value of 0.66 µM. This result prompted an exploration of the structure-activity relationship
of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with
selected nucleophiles. Optimization of substituents at these two positions identified 2-(phenyl-
amino)-N6-cyclohexyladenine (12), 2-(phenylamino)-N6-cycloheptyladenine (19), and 2-phenyl-
amino-N6-endo-norbornyladenine (21) as potent A3 AR ligands with Ki values of 51, 42, and 37
nM, respectively, with 30-200-fold selectivity in comparison to A1 and A2A ARs. The most
selective A3 AR antagonist (>200-fold) was 2-(phenyloxy)-N6-cyclohexyladenine (22). 9-Methyl-
ation of 12, but not 19, was well-tolerated in A3 AR binding. Extension of the 2-phenylamino
group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-(phenylamino),
2-(phenyloxy), and 2-(phenylthio) substitutions, the order of affinity at the A3 AR was oxy g
amino > thio. Selected derivatives, including reversine (KB value of 466 nM via Schild analysis),
competitively antagonized the functional effects of a selective A3 AR agonist, i.e., inhibition of
forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells.
These results are in agreement with other studies suggesting the presence of a lipophilic pocket
in the AR binding site that is filled by moderately sized cycloalkyl rings at the N6 position of
both adenine and adenosine derivatives. Thus, the compound series reported herein comprise
an important new series of selective A3 AR antagonists. We were unable to reproduce the
dedifferentiation effect of reversine, previously reported, or to demonstrate any connection
between A3 AR antagonist effects and dedifferentiation.
Chart 1. Structures of Selected Adenine Derivatives
Reported as AR Antagonists (1a-c) and Reversine (2)
Introduction
The A1, A2A, A2B, and A3 adenosine receptors (ARs)
are G-protein-coupled receptors that are specifically
distributed throughout various tissues and cell types of
the human body.1 The ubiquitous nature of the natural
ligand, adenosine, has prompted countless studies aimed
to delineate the extent to which each subtype of the
receptor could be selectively modulated and if such a
modulation could be exploited for some therapeutic
advantage. Over the past decade, it has become appar-
ent that control of the four AR subtypes has a wide-
ranging impact on stroke and other ischemic conditions,
as well as inflammation, neurodegenerative diseases,
diabetes, sleep regulation, and other therapeutic fronts.2
The structure-activity relationships (SAR) of adeno-
sine derivatives as selective agonists at three of the four
AR subtypes (except for A2B) is well-developed.4-8
Among AR antagonists, several classes of small non-
nucleoside heterocycles, including xanthines, deaza-
adenines, pyrazolopyrimidines, and adenines, have
varying degrees of potency and receptor subtype selec-
tivity.1,9 Various 9-alkyladenine derivatives have dis-
played high selectivity for the A1 AR and more modest
selectivity for A2A and A3 ARs.10-13 For example, 8-[(N-
methylisopropyl)amino]-N6-(endo-2′-(endo-5′-hydroxy)-
norbornyl)-9-methyladenine (WRC-0571, 1a) (Chart 1)
displayed a Ki value of 1.7 nM at the human A1 AR.14
Compound 1b was 10-fold more potent at antagonizing
* Corresponding author. Tel: 301-496-9024. Fax: 301-480-8422.
E-mail: kajacobs@helix.nih.gov.
† Chemical Biology Core Facility, NIH.
‡ Contributed equally to this work.
§ Molecular Recognition Section, Laboratory of Biological Chemistry,
NIH.
| Bar-Ilan University.
10.1021/jm050221l CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/06/2005

Reversine and Its 2-Substituted Adenine Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4911
Scheme 1. Synthesis of 2,6-Disubstituted Purine
the A2A AR in guinea pig coronary artery than a putative
A
Derivativesa
2B AR in the aorta.15 An attempt to convert known A3
AR agonists, such as Cl-IB-MECA [2-chloro-N6-(3-iodo-
benzyl)-5′-(N-methylcarboxamido)adenosine], into selec-
tive antagonists at the same subtype through truncation
of the ribose moiety in stages greatly reduced receptor
affinity.13 A series of 8-substituted adenine derivatives,
such as 9-ethyl-8-(phenylethynyl)-9H-adenine 1c, dis-
played selectivity as antagonists of the human A3 AR.11
A3 AR antagonists are of interest as antiglaucoma
agents and in inflammatory diseases.32-34
a Reagents and conditions: (i) R-NH2 (0.9 equiv.), Hunig’s base,
n-BuOH, 80 °C, 24 h; (ii) R′-NH2, R′-OH, or R′-SH (2 equiv), EtOH,
110 °C, 48 h; (iii) MeI, K2CO3, DMF 0 °C 1-2 h
Adenine derivatives are also important pharmacologi-
cal probes of other biochemical processes such as kinase
inhibition,16 and the AR affinity of such derivatives has
not been fully explored. An 9-alkyladenine derivative,
SQ22536, that inhibits another signaling enzyme, aden-
ylate cyclase, was also shown to antagonize the A1 AR.17
Recently, Chen et al. reported that “reversine” [2-(4-
morpholinoanilino)-N6-cyclohexyladenine, 2] (Chart 1),
selected from a large chemical library, stimulated the
dedifferentiation of lineage-committed cells (myoblasts)
back to progenitor cells, which were capable of being
redirected via osteogenic-inducing agents or adipogenic-
inducing agents into osteoblasts or adipocytes, respec-
tively.18 This remarkable discovery has vast conse-
quences throughout biological and medical research.
The mechanism by which this extraordinary transfor-
mation takes place is as yet unknown and subject to
speculation and investigation. This project will no doubt
be a formidable undertaking, given the breadth of the
cellular landscape and the complexity of the pheno-
typical response. We noted the similarity of the substi-
tution pattern of 2 in relation to various known AR
antagonists and considered that, while not knowing the
significance of AR modulation on the cell cycle and/or
dedifferentiation, reversine might selectively bind at one
or more of the ARs.
Following the protocols set forth by Ding et al., we
synthesized several 2,6-disubstituted purine analogues
including reversine (2).19 Our initial results showed that
2 bound with moderate affinity to the A3 AR. Our
selection of derivatives was motivated by a desire to
initially explore the SAR for this series of compounds
at the ARs rather than to expand on the theme of
dedifferentiation. Accordingly, we diverged from the
parent structure in several fundamental ways, i.e., the
size of the cycloalkyl ring at N,6 the substitution pattern
of the aromatic ring at C2, heteroatom substitution of
the 2-amino group, and methylation at the 9 position.
Table 1. Binding Affinities of N6-Substituted Derivatives of
2-(4-Morpholinophenylamino)adenine at Human A1, A2A, and A3
ARs Expressed in CHO Cellsa
Ki (hA1 AR) Ki (hA2A AR)
(µM) or
% inhibn
at 10 µM
(µM) or
% inhibn
at 10 µM
Ki (hA3 AR)
(µM)a
compd
R1
4
5
6
ethyl
1.53 ( 0.47
1.87 ( 0.89
1.81 ( 0.50
1.39 ( 0.48
0.66 ( 0.14
23%
22%
29%
22%
16%
25%
18%
20%
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
(reversine)
cycloheptyl
cyclooctyl
7
2b
>10 (36%) >10 (30%)
8
9
10
0.42 ( 0.17
2.77 ( 0.83
6%
0%
38%
17%
39%
2-endo-norbornyl 0.122 ( 0.010 34%
a All A3AR experiments were performed using adherent CHO
cells stably transfected with cDNA encoding one of the human
adenosine receptor. Binding at human A1, A2A, and A3ARs in this
study was carried out as described in the methods using [3H]-
DPCPX, [3H]ZM241,385 or [125I]AB-MECA as radioligand. Values
from the present study are expressed as Ki values (mean ( SEM,
n ) 3) or as percent displacement of radioligand. b Demonstrated
to be antagonist of the human A3 AR.
yields of the purified products ranged typically from 30
to 60% for the two-step procedure. We noted that the
substitution of 2,6-dichloropurine resulted in the same
transformation; however, the use of 6-chloro-2-fluoro-
purine was more facile and provided a convenient
method to ensure that the correct 2,6-substitution
pattern was achieved via mass analysis of the fluori-
nated intermediate. We also noted the success of
microwave-assisted organic synthesis in the facile prepa-
ration of similar 2,6-disubstituted purine analogues
(data not shown). Selected analogues were methylated
at the N9 position in quantitative yield via treatment
with methyl iodide in basic DMF at 0 °C over 1-2 h.
Biological Characterization. Binding affinity at
the human A3 AR and in selected cases the rat A3 AR
was studied using the radioiodinated agonist [125I]-
N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluron-
amide (I-AB-MECA).20 Binding at the human A1 and
A2A receptors utilized the selective antagonists [3H]-8-
cyclopentyl-1,3-dipropylxanthine (DPCPX) and [3H]-4-
2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-
5-yl-amino]ethylphenol(ZM241,385),respectively.Functional
assays of the human A3 receptor consisted of measuring
inhibition of forskolin-stimulated cAMP production in
intact Chinese hamster ovary (CHO) cells expressing
the ARs.21
Results and Discussion
Chemical Synthesis. The preparation of 2 and its
analogues (4-24) was achieved via the established
protocols of Ding et al. (Scheme 1).19 Substitution of
6-chloro-2-fluoropurine (3) with a series of amines,
including ethylamine and several cyclic amines of
varying sizes from cyclopropyl to 2-endo-norbornyl, was
accomplished in n-butanol with Hunig’s base at 80 °C
over 24 h. Following solvent removal, the crude reaction
mixture was treated with an excess of a second series
of amines, including ethylamine and several amines
with various aromatic groups (see Table 2), phenol, or
thiophenol, in ethanol and heated in a sealed tube at
110 °C for 48 h. Following flash chromatography the

4912 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Perreira et al.
Table 2. Binding Affinities of 2-Substituted
N6-Cyclohexyladenine Derivatives at Human A1, A2A, and A3
ARs Expressed in CHO Cellsa
amino (13), and (2-phenylethyl)amino (14) substitutions,
there was a pronounced preference for the shortest
analogue 12. In comparison to substituted phenylamino
derivatives 15-18, the unsubstituted 12 displayed the
highest A3 AR affinity.
The compelling finding that 2-phenylamino analogues
bound strongly at the A3 AR motivated us to further
explore a series of analogues that maintained the
2-phenylamino moiety while optimizing the N6-cyclo-
alkyl ring (Table 3). We also replaced the 2-NH with
either oxygen or sulfur as the heteroatom. The results
of these analogues supported the SAR regarding ring
size at the N6 position; i.e., cyclohexyl 12 and cycloheptyl
19 rings provided analogues with strong binding prop-
erties while the cyclooctyl derivative 20 showed 7-8-
fold weaker affinity (Table 3). The N6-endo-norbornyl
analogue 21 appeared to be the most potent in the series
in binding to the A3 AR. In the series of N6-cyclohexyl
derivatives having phenylamino (12), phenyloxy (22),
and phenylthio (24) substitutions, the order of affinity
at the A3 AR was oxy g amino > thio. This also matched
the order of affinity of 2-substituted adenosine deriva-
tives at the A3 AR, which was oxy > amino > thio.22
Thus, the replacement of the NH with other hetero-
atoms helped to optimize the SAR for the A3 AR only
when NH was replaced with O; however, the phenylthio
analogue maintained a moderate affinity for the A3 AR.
We further altered analogues 12 and 19 by a simple
methylation based upon numerous reports describing
9-alkyl adenine derivatives as AR antagonists.11,23 While
9-methylation of the N6-cyclohexyl analogue 12 to yield
25 (Ki ) 0.070 µM) provided a derivative that main-
tained its affinity for the A1, A2A, and A3 ARs, N9
alkylation on the cycloheptyl analogue to yield 26 (Ki )
0.96 µM) was detrimental, leading to an unanticipated
23-fold loss of A3 AR affinity in comparison to 19 with
no significant change in affinity at the A1 and A2A ARs
(Table 3).
The affinity at human A1 and A2A ARs was low with
most derivatives binding only weakly at 10 µM. Various
2-phenylamino derivatives displayed measurable affin-
ity at the A1 AR with Ki values in the range of 2-3 µM,
i.e., 12 (N6-cyclohexyl), 19 (N6-cycloheptyl), 21 (N6-endo-
norbornyl), 25 (N6-cyclohexyl-9-methyl), and 26 (N6-
cycloheptyl-9-methyl). The following are Ki values of the
same derivatives (in µM) at the A2A AR: 12 (1.70), 19
(4.12), 21 (7.73), 25 (1.20), and 26 (2.95). Thus, the most
selective A3 AR antagonists in this study was 22, with
selectivity ratios of >200 in comparison to the A1 and
A2A AR subtypes. The very potent compounds 12 and
21 were 54- and 60-fold selective, respectively, in
comparison to the A1 AR. A comparison of N6-cyclohexyl
derivatives 12 and 22 indicated that the 2-aryloxy group
was more advantageous for A3 AR selectivity than the
corresponding 2-arylamino group. Thus, within this
series compound 22 displayed the most favorable com-
bination of high affinity (Ki 47 nM) and selectivity for
the human A3 AR.
Ki (hA1 AR) Ki (hA2A AR)
(µM) or
% inhibn
at 10 µM
(µM) or
% inhibn
at 10 µM
Ki (hA3 AR)
µMa
compd
R2
11
12
13
14
15
16
17
18
ethyl
phenyl
benzyl
1.12 ( 0.61
51%
42%
0.051 ( 0.017 2.73 ( 0.58 1.70 ( 0.95
0.920 ( 0.121 42% 34%
3.17 ( 1.02 46%
2-phenylethyl
2-naphthyl
biphenyl-1-yl
1.46 ( 0.11
2.42 ( 1.44
5.40 ( 1.00
39%
14%
16%
52%
22%
50%
4-piperidinophenyl 1.57 ( 0.56
4-(dimethylamino)- 0.070 ( 0.025 3.90 ( 0.96 6.1 ( 2.5
phenyl
a All A3 AR experiments were performed using adherent CHO
cells stably transfected with cDNA encoding one of the human
adenosine receptors. Binding at human A1, A2A, and A3 ARs in
this study was carried out as described in the methods using [3H]-
DPCPX, [3H]ZM241,385, or [125I]AB-MECA as radioligand. Values
from the present study are expressed as Ki values (mean ( SEM,
n ) 3) or as a percent displacement of radioligand.
An initial screen of the dedifferentiation agent re-
versine (2) at the ARs indicated binding at the A3 AR
with a Ki value of 0.66 µM. At the A1 and A2A ARs,
binding was much weaker, with only 36% and 30% of
radioligand binding inhibited, respectively, at 10 µM of
2. Following the identification of 2 as a moderately
potent ligand in binding at the A3 AR, we sought to
explore the SAR of related 2,6-disubstituted purines in
order to enhance potency and A3 AR selectivity. Our
initial notion was to individually consider the two
structurally distinct moieties on the purine ring system,
i.e., the N6-cyclohexyl ring and the 2-(4-morpholino-
anilino) moiety. The binding results for these two sets
of analogues are displayed in Tables 1 and 2.
N6-Alkylation of both adenosine and adenine ana-
logues is well-established as a means to increase the
potency and selectivity at the ARs.4-7,10,12 Clearly, as
seen in Table 1, the size of the N6-cycloalkyl ring had
important consequences for binding at the A3 AR. The
A3 AR affinity increased in stages upon enlarging the
ring size from the cyclopropyl derivative 5 (Ki ) 1.9 µM)
to the cycloheptyl derivative 8 (Ki ) 0.42 µM), strongly
suggesting that a bulky hydrophobic alkyl group was
favored at the N6 position. The trend appeared to be
limited to cycloalkyl rings smaller than eight carbons
in size, as demonstrated by a reduced tolerance for the
cyclooctyl ring in 9. The most potent 2-(4-morpholino-
phenylamino)adenine derivative contained the rigid N6-
(2-endo-norbornyl) substituent 10 (Ki ) 0.122 µM).
Exploration of the moiety at the 2-NH suggested that
smaller aromatic analogues were more apt to bind
strongly at the A3 receptor subtype, as demonstrated
by the high affinity of the 2-phenylamino analogue 12
(Ki ) 0.051 µM) and the 2-(4-dimethylamino)phenyl-
amino analogue 18 (Ki ) 0.070 µM) (Table 2). The
2-ethylamino derivative 11 was less potent than 2 at
the A3 AR. In the series of phenylamino (12), benzyl-
Selected derivatives (2, 12, and 18) were tested in
binding to the rat A3 AR and were shown to be inactive
at 10 µM. Therefore, the previously noted species
dependence of the affinity of most known A3 AR
antagonists (human . rat)1,7,32 also applied to this series
of A3 AR antagonists.

Reversine and Its 2-Substituted Adenine Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4913
Table 3. Binding Affinities of 2,6-Disubstituted Adenine Derivatives at Human A1, A2A, and A3 ARs Expressed in CHO Cellsa
Ki (hA1AR)
(µM) or % inhibn
at 10 µM
Ki (hA2AAR)
(µM) or % inhibn
at 10 µM
Ki (hA3AR)
µMa
compdb
X
Y
R1
12
19
20
21
22
23
24
25
26
NH
NH
NH
NH
O
H
H
H
H
H
H
H
cyclohexyl
cycloheptyl
cyclooctyl
2-endo-norbornyl
cyclohexyl
2-endo-norbornyl
cyclohexyl
cyclohexyl
cycloheptyl
0.051 ( 0.017
0.042 ( 0.010
0.340 ( 0.016
0.037 ( 0.009
0.047 ( 0.012
0.074 ( 0.007
0.134 ( 0.007
0.070 ( 0.021
0.96 ( 0.51
2.73 ( 0.58
1.72 ( 0.02
4.47 ( 0.01
2.22 ( 0.58
26%
1.47 ( 0.01
3%
1.70 ( 0.95
4.12 ( 0.48
46%
7.73 ( 0.13
16%
25%
14%
1.20 ( 0.15
2.95 ( 1.00
O
S
NH
NH
methyl
methyl
1.88 ( 0.01
1.94 ( 0.41
a All A3AR experiments were performed using adherent CHO cells stably transfected with cDNA encoding one of the human adenosine
receptor. Binding at human A1, A2A, and A3 ARs in this study was carried out as described in the methods using [3H]DPCPX, [3H]ZM241,385,
or [125I]AB-MECA as radioligand. Values from the present study are expressed as Ki values (mean ( SEM, n ) 3) or as percent displacement
of radioligand. b 12, MRS3767; 19, MRS3769; 22, MRS3777.
adenylate cyclase assay.6 5′-(N-Ethylcarboxamido)-
adenosine (NECA, 100 nM) was used as agonist, and
each adenine derivative was tested at a concentration
of 10 µM. Results are reported for each of the following
compounds as percent inhibition of the NECA-elicited
production of cAMP: 2 and 12 <10%; 21, 31 ( 4%; 22,
13 ( 2%; and 25, 12 ( 3%. The adenine derivatives in
the absence of agonist had no effect on cAMP levels in
the A2B AR-expressing CHO cells.
We attempted to investigate whether other AR an-
tagonists might also display the dedifferentiation effect
of 2 according to the reported procedure.18 However, the
experiments were inconclusive since there were difficul-
ties obtaining a definitive effect with 2, itself, which
displayed toxic effects. Similar results were obtained
with compound 12 (5 µM). Mouse C2C12 myogenic cells
in culture normally fuse to form myotubes. We found
that exposure of the cells for 4 days to 5 µM 2 and then
for 7 days to a differentiating medium, consisting of 0.1
µM dexamethasone, 50 µg/mL ascorbic acid-2-phos-
phate, and 10 mM â-glycerophosphate under the condi-
tions reported,18 produced extensive cell death. The
remaining live cells did not proliferate, and some cells
still fused to form myotubes (Figure 2B), although to a
lesser degree than control cells (Figure 2A). Only a few
percent of the mononucleated cells stained positive for
alkaline phosphatase (not shown), which is one indicator
of osteogenic differentiation,18 but this staining was also
obtained in the cells not treated with differentiating
medium (Figure 2C). Other AR antagonists of low
selectivity,1 including XAC (8-[4-(carboxymethyloxy)-
phenyl]-1,3-di-n-propylxanthine), CGS15943 (N-[9-chloro-
2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine), and
MRS1523 (5-propyl-2-ethyl-4-propyl-3-[(ethylsulfanyl)-
carbonyl]-6-phenylpyridine-5-carboxylate), each at 5 µM,
were not toxic to the cells and did not show dedifferen-
tiating effects using the same protocol.18 When, im-
mediately after treatment with 2, the cells were trans-
ferred to medium containing 2-5% horse serum, which
is a condition normally used for their differentiation
(myogenesis), the cells differentiated to myotubes to a
similar extent as control cells.
Figure 1. Schild analysis for antagonism of the human A3
AR in stably transfected CHO cells by compound 2. Cl-IB-
MECA is used as a potent A3 AR agonist. The experiment was
performed in the presence of 10 µM rolipram. Forskolin (10
µM) was used to stimulate cyclic AMP levels. The level of
cAMP corresponding to 100% was 220 ( 30 pmol mL-1
.
Functional antagonism of the human A3 AR by this
series of adenine derivatives was demonstrated in CHO
cells stably expressing the receptor. A functional assay
consisting of human A3 AR-mediated inhibition of
forskolin-stimulated adenylate cyclase was used. A
Schild analysis of inhibition by 2 of the effects of an A3
AR agonist Cl-IB-MECA at the human A3 AR confirmed
that this adenine derivative was a competitive antago-
nist with a KB value of 466 nM (Figure 1). This value
was in close agreement with the Ki value determined
in the binding assay.
Selected compounds were examined for antagonism
of the human A2B AR. As with the A3 AR, the A2B AR
was stably expressed in CHO cells for a standard

4914 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Perreira et al.
AR family, in some cases with high selectivity toward
the A3 subtype. Functional analysis of these compounds
has demonstrated that they can produce a potent
antagonistic effect at the human A3 AR. The initial SAR
profile for this class of molecules suggested that among
the variety of reported adenine derivatives, the 2-
(phenylamino)-N6-cyclohexyl/cycloheptyl substitution pat-
tern offered the highest affinity for binding at the A3
AR. While these adenine-based compounds demon-
strated a reduced affinity compared to nucleoside-based
ligands, they constitute an important advance, particu-
larly with respect to A3 AR antagonists. A more exten-
sive SAR study of this series, e.g. probing the effects of
substitution pattern of the 2-phenylamino ring or the
introduction of polar groups to increase aqueous solubil-
ity, is warranted.
More extensive SAR studies have been carried out for
adenosine derivatives as agonists at the ARs than for
adenine derivatives as antagonists. In general, a com-
parison of the SAR within these two classes provides
parallels in some aspects (e.g. N6-cycloalkyl groups at
the A1 AR) but not all characteristics.13 These results
are in agreement with other studies suggesting the
presence of a lipophilic pocket in the A1 and A3 AR
binding sites that is filled by moderately sized cycloalkyl
or other hydrophobic rings at the N6 position of adeno-
sine derivatives.8 Data described herein demonstrated
an important commonality between the SAR of the N6
cycloalkyl ring seen within this study of adenine deriva-
tives and previous work9,12 on A1 AR antagonists. The
dependence of A3 AR affinity on the ring size seemed to
mirror the SAR profile for a class of N6-cycloalkyl-9-
benzyl-2-phenyladenines reported to antagonize the A1
receptor by Lucacchini and co-workers.9 Within that
study it was shown that the N6-cycloalkyl ring size was
optimized at either cyclohexyl or cycloheptyl rings, while
the cyclooctyl analogues showed a marked decrease in
affinity. These findings might represent a common
feature of the ligand-receptor interaction characteristic
of both receptor subtypes. Curiously, the N6-(2-endo-
norbornyl) substituent led to high A3 AR affinity and
selectivity in the present series of 2-substituted ad-
enines, while it led to high affinity and selectivity at
the A1 AR subtype for adenines not substituted at the
2 position14 and for adenosine derivatives.22 Integration
of novel 2 position side-chains provided an initial SAR.
The discovery of the seemingly favorable effect on the
A3 AR of inclusion of either phenyl or 4-(dimethyl-
amino)phenyl moieties at the 2 position represents a
new finding.
On the theme of dedifferentiation by reversine (2),
ARs are known to have effects on growth and prolifera-
tion; thus, we considered a possible connection between
dedifferentiation ascribed to 2 and its activity at one or
more of the ARs. Each of the four ARs is coupled to
mitogen-activated protein kinases (MAPK).17 Specific
activation of one receptor subtype versus another has
been shown to initiate a myriad of biological events,
including alterations to the cell cycle.17 For example,
expression of the A1 receptor within ob17 preadipocytes
stimulated differentiation while slowing proliferation
relative to unaltered cells.24 Stimulation of the A2B
receptors by known agonists 2-chloroadenosine and 5′-
(N-methylcarboxamido)adenosine was shown to down-
Figure 2. Micrograph of the effects of differentiating medium
and AR antagonists on mouse C2C12 myogenic cells. Shown
are (A) control differentiated myotubes and (B) cells after 4-day
treatment with 5 µM of the adenine derivative 2 followed by
maintaining the cells for an additional 7 days in differentiating
medium containing 0.1 µM dexamethasone, 50 µg/mL ascorbic
acid-2-phosphate, and 10 mM â-glycerophosphate (see Experi-
mental Section). (C) Cells treated identically to the description
in B, except lacking differentiating medium. Arrows indicate
cells that stained positive for alkaline phosphatase activity
using a semiquantitative method. Bar in part A indicates 20
µm.
Discussion
It was apparent from Tables 1-3 that 2-(phenyl-
amino)-N6-cycloadenines are capable of binding to the

Reversine and Its 2-Substituted Adenine Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4915
General Procedure for the Preparation of 2 and 4-21.
Diisopropylethylamine (1.2 equiv) and the appropriate primary
amine for N6 substitution (0.9 equiv) were added to a solution
of 6-chloro-2-fluoropurine (3) (1 equiv) in n-butanol (2-4 mL),
and the mixture was heated for 15-24 h at 80 °C. The solution
was allowed to cool, and the solvent was removed under
reduced pressure to yield a yellow solid. An aliquot was
removed for LC-MS analysis. The remaining solid was
transferred to a sealed reaction vessel containing EtOH (2-4
mL) and the appropriate amine (2 equiv) for substitution at
the 2 position, where the mixture was heated for 48 h at 110
°C. The solution was allowed to cool, and the solvent was
removed under reduced pressure. The resulting solid was
purified via column chromatography (EtOAc) to yield com-
pounds 2 and 4-21 as white or off-white solids.
2-(4-Morpholinoanilino)-6-(cyclohexylamino)purine (2):
yield 63 mg (55%); purity analysis was achieved by C8
reversed-phase LC-MS using a linear gradient of H2O con-
taining increasing amounts of CH3CN (0-10 min, linear
gradient from 10% to 30% CH3CN; 10-15 min, linear gradient
from 30% to 90% CH3CN at a flow rate of 1 mL/min; tR 14.6
min); 1H NMR (DMSO-d6) δ 1.11-1.43 (m, 6H), 1.58-1.80 (m,
3H), 1.82-1.99 (m, 2H), 2.90-3.28 (m, 4H), 3.71-3.78 (m, 4H),
4.08 (bs, 1H), 6.81 (d, JHH ) 9.0 Hz, 2H), 7.02 (bs, 1H), 7.63
(d, JHH ) 9.0 Hz, 2H), 7.72 (s, 1H), 8.51 (s, 1H); TOFMS m/z
(M + H+) 394.2355 (calculated for C18H23N6+) 394.2350.
2-(4-Morpholinoanilino)-6-(ethylamino)purine (4): yield
93 mg (95%); purity analysis was achieved by C8 reversed-
phase LC-MS using a linear gradient of H2O containing
increasing amounts of CH3CN (0-15 min, linear gradient from
25% to 90% CH3CN at a flow rate of 1 mL/min; tR 3.1 min); 1H
NMR (DMSO-d6) δ 1.29 (t, JHH ) 7.2 Hz, 3H), 3.08-3.11 (m,
4H), 3.23-2.29 (m, 2H), 3.63 (bs, 1H), 3.81-3.84 (m, 4H), 6.93
(d, JHH ) 8.4 Hz, 2H), 7.76 (d, JHH ) 8.7 Hz, 2H), 7.82 (s, 1H),
7.39 (bs, 1H), 8.60 (s, 1H); TOFMS m/z 340.1886 (M + H+)
(calculated for C17H22N7O+) 340.1880.
2-(4-Morpholinoanilino)-6-(cyclopropylamino)pu-
rine (5): yield 58 mg (57%); purity analysis was achieved by
C8 reversed-phase LC-MS using a linear gradient of H2O
containing increasing amounts of CH3CN (0-5 min, linear
gradient from 25% to 50% CH3CN; 5-10 min, linear gradient
from 50% to 90% CH3CN at a flow rate of 1 mL/min; tR 4.7
min); 1H NMR (DMSO-d6) δ 0.45-0.50 (m, 2H), 0.61-0.68 (m
2H), 0.70-0.78 (m, 1H), 2.97-3.08 (m, 4H), 3.69-3.76 (m, 4H),
7.48 (bs, 1H), 7.75 (d, JHH ) 9.6 Hz, 2H), 7.92 (d, JHH ) 9.0
Hz, 2H), 8.35 (s, 1H), 9.18 (s, 1H); TOFMS m/z 352.1885 (M +
H+) (calculated for C18H22N7O+) 352.1880.
regulate DNA synthesis and slow cellular prolifera-
tion.25 Treatment of rat lymphoma cells with adenosine
and the specific A3 agonist IB-MECA was shown to
down-regulate DNA synthesis, proliferation, and telo-
meric signaling.26 A3 AR agonists appear to have a net
cytostatic effect triggered through activation of the ERK
1/2 pathway.17 Even from this small sampling of studies
it is apparent that the function of the ARs in cell
signaling is complex and still in the process of being
elucidated.
A relationship between the antagonistic effects on the
A3 AR demonstrated by 2 and the observed cellular
dedifferentiation reported by Chen et al.18 could not be
demonstrated in the present study. The analogues of 2
reported in the present study might be of use in further
delineating the role that the ARs play during the
dedifferentiation process; however, we were unsuccess-
ful in our attempts to recreate an appreciable degree of
myoblast dedifferentiation with various AR antagonists,
including 2. Obviously, further study is needed to
advance this series of compounds as modulators of the
function of the ARs and within the delineation of the
individual roles of the ARs on the cell cycle, including
dedifferentiation. However, A3 adenosine antagonists
are not to be considered general dedifferentiation re-
agents. Even the preliminary information about the
SAR of reversine congeners presented by Chen et al.18
did not appear to match the structural requirements for
binding to the A3 receptor.
In conclusion, on the basis of the relatively simple 2,6-
disubstituted purine template, we have designed highly
selective A3 AR antagonists reaching high affinity.
Among the most potent and selective A3 AR antagonists
in comparison to A1 and A2A ARs were compounds 12,
19, and 22. Compound 19 and other analogues were also
demonstrated to be nearly inactive at the A2B AR. It will
now be worthwhile to examine such antagonists as
potential antiglaucoma agents, although the species
selectivity of these antagonists will limit the experi-
mental models that may be used.32 Although reversine
was previously identified as a dedifferentiating agent
and we have presently shown it to be a moderately
potent AR antagonist, we were unable to demonstrate
a link between AR antagonism and dedifferentiation.
2-(4-Morpholinoanilino)-6-(cyclobutylamino)purine (6):
yield 64 mg (60%); purity analysis was achieved by C8
reversed-phase LC-MS using a linear gradient of H2O con-
taining increasing amounts of CH3CN (0-15 min, linear
gradient from 25% to 90% CH3CN at a flow rate of 1 mL/min;
1
tR 2.9 min); H NMR (DMSO-d6) δ 1.57-1.74 (m, 2H), 2.06-
Experimental Section
2.16 (m, 2H), 2.20-2.32 (m, 3H), 2.99-3.02 (m, 4H), 3.71-
3.74 (m, 4H), 4.65 (bs, 1H), 6.85 (d, JHH ) 9.0 Hz, 2H), 7.58
(bs, 1H), 7.66 (d, JHH ) 9.0 Hz, 2H), 7.75 (s, 1H), 8.45 (s, 1H);
TOFMS m/z 366.2042 (M + H+) (calculated for C19H24N7O+)
366.2037.
Reagents and Instrumentation. 6-Chloro-2-fluoropurine
(3) was purchased from Oakwood Products, Inc. [125I]-N6-(4-
Amino-3-iodobenzyl)adenosine-5′-N-methyluronaminde (I-AB-
MECA, 2000 Ci/mmol), [3H]DPCPX (80-130 Ci/mmol), and
[3H]cyclic-AMP (40 Ci/mmol) were purchased from Amersham
Biosciences Corp. (Piscataway, NJ). [3H]ZM241385 (4-2-[7-
amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]-
ethylphenol; 17 Ci/mmol) was purchased from Tocris Cookson
Inc. (Ellisville MO). All other reagents and solvents were
purchased from Sigma-Aldrich. Selected intermediates and
products were analyzed on an Agilent 1100 series LC/MSD
SL using a Zorbax Eclipse XDB-C8 reverse phase column (4.6
× 150 mm, 5 m). High-resolution mass spectroscopy measure-
ments were performed on a Micromass/Waters LCT Premier
Electrospray TOF mass spectrometer. 1H NMR data was
recorded on a Varian Gemini300, and spectra were recorded
in DMSO-d6 and/or CDCl3 and were referenced to the residual
solvent peaks at 2.50 and 7.26, respectively. Elemental
analyses (C, H, N) were performed by Atlantic Microlabs, Inc.
(Norcross, GA).
2-(4-Morpholinoanilino)-6-(cyclopentylamino)pu-
rine (7): yield 77 mg (70%); purity analysis was achieved by
C8 reversed-phase LC-MS using a linear gradient of H2O
containing increasing amounts of CH3CN (0-12 min, linear
gradient from 5% to 25% CH3CN; 12-20 min, linear gradient
from 25% to 90% CH3CN at a flow rate of 1 mL/min; tR 16.5
min); 1H NMR (DMSO-d6) δ 1.42-1.77 (m, 6H), 1.81-2.03 (m,
3H), 2.97-3.06 (m, 4H), 3.70-3.75 (m, 4H), 4.05 (bs, 1H), 6.83
(d, JHH ) 9.3 Hz, 2H), 7.26 (bs, 1H), 7.67 (d, JHH ) 9.0 Hz,
2H), 8.45 (s, 1H), 9.04 (s, 1H); TOFMS m/z 380.2199 (M + H+)-
(calculated for C20H26N7O+) 380.2193.
2-(4-Morpholinoanilino)-6-(cycloheptylamino)pu-
rine (8): yield 33 mg (28%); purity analysis was achieved by
C8 reversed-phase LC-MS using a linear gradient of H2O
containing increasing amounts of CH3CN (0-5 min, linear
gradient from 10% to 50% CH3CN; 5-20 min, linear gradient

4916 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Perreira et al.
from 50% to 70% CH3CN at a flow rate of 1 mL/min; tR 8.6
min); 1H NMR (DMSO-d6) δ 1.46-1.72 (m, 10H), 1.87-1.98
(m, 3H), 2.98-3.01 (m, 4H), 3.72-3.75 (m, 4H), 4.22 (bs, 1H),
6.83 (d, JHH ) 9.0 Hz, 2H), 7.25 (bs, 1H), 7.66 (d, JHH ) 9.0
Hz, 2H), 7.73 (s, 1H), 8.45 (s, 1H); TOFMS m/z 408.2512 (M +
H+) (calculated for C22H30N7O+) 408.2506.
Hz, 2H); TOFMS m/z 392.2576 (M + H+) (calculated for
C22H30N7+) 392.2563.
2-[[4-(Dimethylamino)phenyl]amino]-6-(cyclohexyl-
amino)purine (18): yield 20 mg (25%); 1H NMR (CDCl3) δ
1.10-1.50 (m, 6H), 1.60-1.85 (m, 4H), 2.04-2.14 (m, 2H), 2.92
(s, 6H), 4.10 (bs, 1H), 5.53 (bs, 1H), 6.56 (s, 1H), 6.74 (d, JHH
)
2-(4-Morpholinoanilino)-6-(cyclooctylamino)purine (9):
yield 87 mg (71%); purity analysis was achieved by C8
reversed-phase LC-MS using a linear gradient of H2O con-
taining increasing amounts of CH3CN (0-15 min, linear
gradient from 30% to 90% CH3CN at a flow rate of 1 mL/min;
9.0 Hz, 2H), 7.38 (d, JHH) 9.0 Hz, 2H); TOFMS m/z 352.2247
(M + H+) (calculated for C19H26N7+) 352.2250
2-(Phenylamino)-6-(cycloheptylamino)purine (19): yield
1
36 mg (48%); H NMR (CDCl3) δ 1.40-1.80 (m, 12H), 2.00-
2.20 (m, 2H), 4.30 (bs, 1H), 5.60 (bs, 1H), 6.90 (s, 1H), 7.05 (t,
JHH) 7.5 Hz, 1H), 7.32 (t, JHH) 7.5 Hz, 2H), 7.57 (d, JHH) 7.5
Hz, 2H); TOFMS m/z 323.1982 (M + H+) (calculated for
C18H23N6+) 323.1984.
1
tR 8.5 min); H NMR (DMSO-d6) δ 0.78-0.92 (m, 1H), 1.08-
1.35 (m, 8H), 1.45-1.88 (m, 6H), 2.95-3.06 (m, 4H), 3.67-
3.79 (m, 4H), 4.38 (bs, 1H), 6.83 (d, JHH ) 9.0 Hz, 2H), 7.19
(bs, 1H), 7.65-7.72 (m, 3H), 8.48 (s, 1H); TOFMS m/z 422.2668
(M + H+) (calculated for C23H32N7O+) 422.2662.
2-(Phenylamino)-6-(cyclooctylamino)purine (20): yield
78 mg (80%); purity analysis was achieved by C8 reversed-
phase LC-MS using a linear gradient of H2O containing
increasing amounts of CH3CN (0-15 min, linear gradient from
30% to 90% CH3CN at a flow rate of 1 mL/min; tR 10.1 min);
1H NMR (DMSO-d6) δ 1.49-1.87 (m, 15H), 3.22-3.44 (bs, 1H),
2-(4-Morpholinoanilino)-6-[(2-endo-norbornyl)amino]-
purine (10): yield 32 mg (27%); 1H NMR (CDCl3) δ 0.77-0.95
(m, 2H), 1.18-1.78 (m, 8H), 2.50-2.66 (m, 2H), 3.11 (dd, JHH
)
4.8, 4.5 Hz, 4H), 3.86 (dd, JHH) 4.8, 4.5 Hz, 4H), 4.38 (bs, 1H),
5.70 (bs, 1H), 6.68 (s, 1H), 6.90 (d, JHH) 8.7 Hz, 2H), 7.45 (d,
JHH) 8.7 Hz, 2H); TOFMS m/z 406.2369 (M + H+) (calculated
for C22H28N7O+) 406.2355.
7.17-7.22 (m, 3H), 7.79-7.84 (m, 3H), 8.02 (s, 1H), 8.75 (s,
1H); TOFMS m/z 337.2141 (M + H+) (calculated for C19H25N6
)
+
337.2135.
2-(Ethylamino)-6-(cyclohexylamino)purine (11): yield
72 mg (95%); purity analysis was achieved by C8 reversed-
phase LC-MS using a linear gradient of H2O containing
increasing amounts of CH3CN (0-12 min, linear gradient from
5% to 30% CH3CN; 12-20 min, linear gradient from 30% to
90% CH3CN at a flow rate of 1 mL/min; tR 16.6 min); 1H NMR
(DMSO-d6) δ 1.09 (t, JHH ) 7.2 Hz, 3H), 1.12-1.36 (m, 6H),
1.58-1.63 (m, 1H), 1.65-1.76 (m, 2H), 1.83-1.91 (m, 2H),
3.18-3.27 (m, 2H), 3.42 (bs, 1H), 4.02 (bs, 1H), 7.65 (s, 1H),
6.81 (bs, 1H); TOFMS m/z 261.1827 (M + H+) (calculated for
C13H21N6+) 261.1822.
2-(Phenylamino)-6-(cyclohexylamino)purine (12): yield
43 mg (60%); 1H NMR (CDCl3) δ 1.20-1.50 (m, 6H), 1.60-
1.85 (m, 4H), 2.05-2.20 (m, 2H), 4.10 (bs, 1H), 5.60 (bs, 1H),
6.95 (s, 1H), 7.06 (t, JHH) 7.8 Hz, 1H), 7.33 (t, JHH) 7.8 Hz,
2H), 7.56 (d, JHH) 7.8 Hz, 2H); TOFMS m/z 309.1823 (M +
H+) (calculated for C17H21N6+) 309.1828.
2-(Phenylamino)-6-(2-endo-norbornylamino)purine (21):
yield 31 mg (42%); 1H NMR (CDCl3) δ 0.77-0.95 (m, 2H),
1.18-1.78 (m, 8H), 2.50-2.66 (m, 2H), 4.38 (bs, 1H), 5.80 (bs,
1H), 6.90 (s, 1H), 7.07 (t, JHH) 7.5 Hz, 1H), 7.21 (dd, JHH
)
7.8, 7.5 Hz, 2H), 7.54 (d, JHH) 7.8 Hz, 2H); TOFMS m/z
321.1828 (M + H+) (calculated for C18H21N6+) 321.1828.
2-Phenoxy-6-(cyclohexylamino)purine (22). Cyclohexyl-
amine (30 µL, 0.26 mmol) and diisopropylethylamine (61 µL,
0.35 mmol) were added to a solution of 6-chloro-2-fluoropurine
(3) (50 mg, 0.29 mmol) in n-butanol (2 mL), and the mixture
heated for 15 h at 80 °C. The solution was allowed to cool,
and the solvent was removed under reduced pressure to yield
a yellow solid. An aliquot was removed for LC-MS analysis.
The remaining solid was transferred to a sealed reaction vessel
with EtOH (5 mL), and phenol (48 mg, 0.5 mmol) and
potassium tert-butoxide (47 mg, 0.42 mmol) were added. The
mixture was heated for 48 h at 110 °C. The solution was
allowed to cool, and the solvent was removed under reduced
pressure. The resulting solid was purified via column chro-
matography (EtOAc) to yield 22 as a white powder: yield 6
mg (8%); purity analysis was achieved by C8 reversed-phase
LC-MS using a linear gradient of H2O containing increasing
amounts of CH3CN (0-15 min, linear gradient from 25% to
2-(Benzylamino)-6-(cyclohexylamino)purine (13): yield
35 mg (47%); 1H NMR (CDCl3) δ 1.10-1.50 (m, 6H), 1.56-
1.82 (m, 4H), 2.00-2.12 (m, 2H), 4.66 (d, JHH) 6.0 Hz, 2H),
4.82 (bs, 1H), 5.58 (bs, 1H), 7.09 (s, 1H), 7.22-7.40 (m, 5H);
TOFMS m/z 323.1975 (M + H+) (calculated for C18H23N6
323.1984.
)
+
1
2-(2-Phenethylamino)-6-(cyclohexylamino)purine (14):
yield 51 mg (65%); 1H NMR (CDCl3) δ 1.10-1.50 (m, 6H),
1.60-1.85 (m, 4H), 2.04-2.15 (m, 2H), 2.97 (t, JHH) 6.9 Hz,
2H), 3.69 (m, JHH) 6.9, 6.0 Hz, 2H), 4.88 (bs, 1H), 5.50 (bs,
1H), 7.22-7.36 (m, 5H), 7.40 (s, 1H); TOFMS m/z 337.2126
(M + H+) (calculated for C19H25N6+) 337.2141
90% CH3CN at a flow rate of 1 mL/min; tR 7.3 min); H NMR
(DMSO-d6) δ 1.14-1.46 (6H), 1.67-1.98 (m, 5H), 4.13 (bs, 1H),
7.22-7.30 (m, 3H), 7.46-7.51 (m, 2H), 7.61 (bs, 1H), 7.99 (s,
1H); TOFMS m/z 310.1668 (M + H+) (calculated for C17H20N5O+)
310.1662.
2-Phenoxy-6-(2-endo-norbornylamino)purine (23). 2-
Aminonorborane hydrochloride (46 mg, 0.31 mmol) and diiso-
propylethylamine (73 µL, 0.42 mmol) were added to a solution
of 6-chloro-2-fluoropurine (3) (60 mg, 0.35 mmol) in n-butanol
(2 mL), and the mixture was heated for 15 h at 80 °C. The
solution was allowed to cool, and the solvent was removed
under reduced pressure to yield a yellow solid. An aliquot was
removed for LC-MS analysis. The remaining solid was
transferred to a sealed reaction vessel with EtOH (5 mL), and
phenol (66 mg, 0.70 mmol) and postassium tert-butoxide (79
mg, 0.70 mmol) were added. The mixture was heated for 48 h
at 110 °C. The solution was allowed to cool, and the solvent
was removed under reduced pressure. The resulting solid was
purified via column chromatography (EtOAc) to yield 23 as a
white powder: yield 4.0 mg (4.3%); purity analysis was
achieved by C8 reversed-phase LC-MS using a linear gradient
of H2O containing amounts of CH3CN (0-15 min, linear
gradient from 25% to 90% CH3CN at a flow rate of 1 mL/min;
tR 10.2 min); 1H NMR (DMSO-d6) δ 1.22-1.62 (m, 10H), 1.80-
1.95 (m, 1H), 4.16 (bs, 1H), 7.13-7.20 (m, 3H), 7.36-7.41 (m,
2H), 7.77 (bs, 1H), 7.92 (s, 1H); TOFMS m/z (M + H+) 322.1668
(calculated for C18H20N5O+ 322.1662).
2-(2-Naphthylamino)-6-(cyclohexylamino)purine (15):
1
yield 37 mg (45%); H NMR (DMSO-d6) δ 1.10-1.50 (m, 6H),
1.60-1.85 (m, 4H), 1.94-2.04 (m, 2H), 4.20 (bs, 1H), 7.28 (t,
JHH) 8.1 Hz, 1H), 7.42 (t, JHH) 8.1 Hz, 1H), 7.65-7.70 (m,
4H), 7.85 (s, 1H), 8.53 (s, 1H), 9.05 (bs, 1H); TOFMS m/z
359.1994 (M + H+) (calculated for C21H23N6+) 359.1984.
2-(Biphen-1-ylylamino)-6-(cyclohexylamino)purine (16):
yield 46 mg (41%); purity analysis was achieved by C8
reversed-phase LC-MS using a linear gradient of H2O con-
taining increasing amounts of CH3CN (0-12 min, linear
gradient from 5% to 25% CH3CN; 12-15 min, linear gradient
from 25% to 60% CH3CN at a flow rate of 1 mL/min; tR 12.4
min); 1H NMR (DMSO-d6) δ 1.15-1.39 (m, 6H), 1.59-1.78 (m,
3H), 1.92-2.01 (m, 2H), 3.71 (bs, 1H), 7.31-7.33 (m, 1H),
7.42-7.47 (m, 3H), 7.56-7.66 (m, 4H), 7.79 (s, 1H), 8.15 (d,
JHH ) 9.0 Hz, 2H), 9.42 (s, 1H); TOFMS m/z 385.2141 (M +
H+) (calculated for C23H25N6+) 385.2135.
2-(4-Piperidinoanilino)-6-(cyclohexylamino)purine (17):
yield 25 mg (27%); 1H NMR (CDCl3) δ 1.10-1.80 (m, 16H),
2.00-2.20 (m, 2H), 3.00-3.25 (m, 4H), 4.13 (bs, 1H), 5.70 (bs,
1H), 6.68 (s, 1H), 6.92 (d, JHH) 9.0 Hz, 2H), 7.41 (d, JHH) 9.0

Reversine and Its 2-Substituted Adenine Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4917
2-(Phenylthio)-6-(cyclohexylamino)purine (24). Cyclo-
hexylamine (30 µL, 0.26 mmol) and diisopropylethylamine (61
µL, 0.35 mmol) were added to a solution of 6-chloro-2-
fluoropurine (3) (50 mg, 0.29 mmol) in n-butanol (2 mL), and
the mixture was heated for 15 h at 80 °C. The solution was
allowed to cool, and the solvent was removed under reduced
pressure to yield a yellow solid. An aliquot was removed for
LC-MS analysis. The remaining solid was transferred to a
sealed reaction vessel, where EtOH (2 mL) and thiophenol (51
µL, 0.5 mmol) were added, and the mixture was heated for 48
h at 110 °C. The solution was allowed to cool, and the solvent
was removed under reduced pressure. The resulting solid was
purified via column chromatography (EtOAc) to yield 24 as a
white powder: yield 43 mg (63%); purity analysis was achieved
by C8 reversed-phase LC-MS using a linear gradient of H2O
containing increasing amounts of CH3CN (0-15 min, linear
gradient from 25% to 90% CH3CN at a flow rate of 1 mL/min;
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 using a
Beckman γ-counter, and the percent inhibition was calculated.
The antagonists [3H]DPCPX (0.5 nM) and [3H]ZM241,385
(2 nM) were used as radioligands for A1 and A2A ARs,
respectively. Each assay tube used to measure competitive
binding for human A1 and A2A ARs contained 100 µL of
membrane suspension (20 µg of protein), 50 µL of radioligand,
and 50 µL of 10 µM test ligand in the Tris-HCl buffer.
Nonspecific binding was measured using 10 µM NECA in the
buffer. Each assay was incubated at 25 °C for 60 min and was
terminated by filtration through Whatman GF/B filters under
reduced pressure using a MT-24 cell harvester. Filters were
washed three times with ice-cold buffer and placed in scintil-
lation vials. Each vial also contained 5 mL of Hydrofluor
scintillation buffer. All compounds that gave more than 60%
inhibition at 10 µM for either A1 or A2A ARs were also tested
using a full concentration curve between 10-9 and 10-5 M in
order to determine a Ki value.
1
tR 9.6 min); H NMR (DMSO-d6) δ 1.12-1.30 (m, 6H), 1.50-
1.89 (m, 5H), 3.66 (bs, 1H), 7.41-7.45 (m, 3H), 7.58-7.62 (m,
3H), 7.93 (s, 1H); TOFMS m/z 326.1439 (M + H+) (calculated
for C17H20N5S+) 326.1434.
9-Methyl-2-(phenylamino)-6-(cyclohexylamino)pu-
rine (25). 12 (17 mg, 0.06 mmol) was added to a slurry of
potassium carbonate (9.7 mg, 0.07 mmol) in DMF (0.5 mL).
The mixture was cooled to 0 °C, and methyl iodide (5.6 µL,
0.09 mmol) was added. The reaction was allowed to stir for 1
h at 0 °C followed by the addition of water (1 mL) and EtOAc
(1 mL). The mixture was extracted with EtOAc (3 × 5 mL).
The organic layers were collected and dried (Na2SO4), and the
solvent was removed under reduced pressure to yield 25 as a
white solid: yield 18 mg (95%); purity analysis was achieved
by C8 reversed-phase LC-MS using a linear gradient of H2O
containing increasing amounts of CH3CN (0-5 min, linear
gradient from 25% to 50% CH3CN; 5-10 min, linear gradient
from 50% to 90% CH3CN at a flow rate of 1 mL/min; tR 9.8
min); 1H NMR (DMSO-d6) δ 1.12-1.81 (m, 6H), 1.24-1.43 (m,
3H), 1.91-1.99 (m, 2H), 3.64 (s, 3H), 4.10 (bs, 1H), 7.19-7.24
(m, 3H), 7.86 (d, JHH ) 8.4 Hz, 2H), 7.96 (s, 1H); TOFMS m/z
323.1984 (M + H+) (calculated for C18H23N6+) 323.1979.
9-Methyl-2-(phenylamino)-6-(cycloheptylamino)pu-
rine (26). 19 (20 mg, 0.062 mmol) was added to a slurry of
potassium carbonate (10.3 mg, 0.075 mmol) in DMF (0.5 mL).
The mixture was cooled to 0 °C, and methyl iodide (5.8 µL,
0.093 mmol) was added. The reaction was allowed to stir for
1 h at 0 °C followed by the addition of water (1 mL) and EtOAc
(1 mL). The mixture was extracted with EtOAc (3 × 5 mL).
The organic layers were collected and dried (Na2SO4), and
solvent was removed under reduced pressure to yield 26 as a
white solid: yield 18 mg (86%); 1H NMR (CDCl3) δ 1.40-1.80
(m, 12H), 2.00-2.20 (m, 2H), 3.73 (s, 3H), 4.30 (bs, 1H), 6.97
Cyclic AMP Accumulation Assay. Intracellular cyclic
AMP levels were measured with a competitive protein binding
method.28,29 CHO cells expressing the recombinant human A2B
AR or A3 AR were harvested by trypsinization. After centrifu-
gation 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 the appropriate agonist, NECA (100 nM) for the
A2B AR or variable concentrations of Cl-IB-MECA for the A3
AR and/or adenine derivatives in the presence of rolipram (10
µM). For the A2B AR, incubation was carried out for 1 h. For
the A3 AR, after an initial incubation of 45 min, forskolin (10
µM) was added to the medium, and incubation was continued
for 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 0.1M
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. A Schild constant (KB) was calcu-
lated as described using the equation.30
(t, JHH) 7.5 Hz, 1H), 7.31 (t, JHH) 7.5 Hz, 2H), 7.72 (d, JHH
)
7.5 Hz, 2H), 8.02 (s, 1H); TOFMS m/z 337.2148 (M + H+)
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.31
Data were expressed as the mean ( standard error.
(calculated for C19H25N6+) 337.2141.
Cell Culture and Membrane Preparation. CHO (Chi-
nese hamster ovary) cells expressing the recombinant human
ARs were cultured in Dulbecco’s modified Eagle medium
(DMEM) and F12 (1:1) supplemented with 10% fetal bovine
serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and
2 µmol/mL glutamine. After harvesting, cells were homog-
enized and suspended. Cells were then 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 and was then recentrifuged at 20 000g for
20 min at 4 °C. The resultant pellets were resuspended in Tris
buffer, and the suspension was stored at -80 °C until the
binding experiments. The protein concentration was measured
using the Bradford assay.27
Radioligand Binding Assays. Each tube in the A3 AR
competitive binding assay20 contained 100 µL of membrane
suspension (20 µg of protein), 50 µL of [125I]-4-amino-3-
iodobenzyl)adenosine-5′-N-methyluronamide (0.5 nM), and 50
µL of increasing concentrations of the test ligands in Tris-HCl
buffer (50 mM, pH 7.4) containing 10 mM MgCl2 and 1 mM
EDTA. Nonspecific binding was determined using 10 mM 5′-
Studies of Cellular Differentiation. Mouse C2C12 myo-
genic cells (ATCC, Manassas, VA) were plated in 6-well plates
in growth medium (DMEM with 10% fetal bovine serum), and
after overnight incubation (during which time cells attach to
the bottom of the plate), 5 µM adenine derivative or other AR
antagonists were added. After 4 days, medium containing the
antagonists was removed and replaced with differentiating
medium containing 50 µg/mL ascorbic acid-2-phosphate, 0.1
µM dexamethasone, and 10 mM â-glycerophosphate in 10%
fetal bovine serum (to direct the cells from myogenic dif-
ferentiation to osteogenic). The culture was maintained for an
additional 4-7 days. Cells were stained for alkaline phos-
phatase activity using a semiquantitative method (“Leukocyte”
Alkaline Phosphatase kit, Procedure No. 85, Sigma), observed
using a Nikon microscope, and compared with control cells
subjected to the same treatment except not exposed to dif-
ferentiating medium or AR antagonist.

4918 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Perreira et al.
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Acknowledgment. The authors thank Dr. John
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Supporting Information Available: Characterization of
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