Interactions of flavonoids and other phytochemicals with adenosine receptors.

Article abstract of DOI:10.1021/jm950661k

Flavone derivatives and other phytochemicals were found to bind to three subtypes of adenosine receptors in the micromolar range. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-N6-PIA ([3H]-(R)-N6-phenyl

Full text of DOI:10.1021/jm950661k

J . Med. Chem. 1996, 39, 781-788
781
In ter a ction s of F la von oid s a n d Oth er P h ytoch em ica ls w ith Ad en osin e
Recep tor s
Xiao-duo J i, Neli Melman, and Kenneth A. J acobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes,
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810
Received September 6, 1995X
Flavone derivatives and other phytochemicals were found to bind to three subtypes of adenosine
receptors in the micromolar range. Affinity was determined in radioligand binding assays at
rat brain A1 and A2A receptors using [3H]-N6-PIA ([3H]-(R)-N6-phenylisopropyladenosine) and
[3H]CGS21680 ([3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)adenosine),
respectively. Affinity was determined at cloned human and rat brain A3 receptors using [125I]-
AB-MECA [N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)]. A structure-activity
analysis indicated that the hydroxyl groups of naturally occurring flavones are not essential
for affinity at adenosine receptors. Galangin, 14, displayed Ki values of 1 µM at both rat A1
and A2A receptors and 3 µM at human A3 receptors. Methylation but not acetylation of the
hydroxyl groups of galangin enhanced A3 affinity. Pentamethylmorin, 20, appeared to bind
with 14-17-fold selectivity for human A3 receptors vs rat A1 and A2A receptors, with a Ki value
of 2.65 µM. Two flavone derivatives (14 and 15) showed 14-fold greater affinity at human vs
rat A3 receptors. Reduction of the 2,3-olefinic bond, as in (()-dihydroquercetin, or glycosidation,
as in robinin, greatly diminished affinity. An isoflavone, genistein, also bound only very weakly
at A3 receptors. R-Naphthoflavone had greater receptor affinity (0.79 µM at A1 receptors) than
the â-isomer. Other natural products of plant origin, including oxogalanthine lactam,
hematoxylin, and arborinine were found to bind to A1 adenosine receptors with Ki values of
3-13 µM. These findings indicate that the flavones, flavonols, flavanones, and other
phytochemicals may provide leads for the development of novel adenosine antagonists. The
unexpected finding of considerable affinity of flavones at both rat and human A3 receptors
may explain some of the previously observed biological effects of these compounds.
In tr od u ction
search for non-xanthine antagonists is that most xan-
thines bind only very weakly to A3 receptors,14 for which
no selective antagonists have yet been reported.
Adenosine receptors mediate many of the physiologi-
cal actions of adenosine in the periphery1,2 and in the
central nervous system.3 There are four subtypes of
adenosine receptors: A1, A2A, A2B, and A3.4 Activation
of A1 receptors results in the bradycardiac,5 cerebro-
protective,3 and antilipolytic effects of adenosine.7 Ac-
tivation of A2A receptors results in the hypotensive and
antiplatelet aggregatory effects of adenosine.8 There are
not yet any selective ligands with which to characterize
physiological effects of selective activation of A2B recep-
tors. Several years ago a new subtype, A3 receptors,
was discovered9 while screening cDNA libraries for
protein sequences resembling the G-protein-coupled
receptors through the use of PCR (polymerase chain
reaction). Activation of this receptor results in hypoten-
sion and promotion of release of inflammatory mediators
from mast cells.10 We have introduced the first selective
agonist ligands for the A3 subtype, including the agonist
IB-MECA (N6-(3-iodobenzyl)adenosine-5′-(N-methyl-
uronamide).11,12 In general adenosine antagonists are
sought as renal protective,6 cognition enhancing,3 cere-
broprotective,3 antiasthmatic,13 and antiinflammatory
agents.13
A
number of classes of non-xanthine adenosine antago-
nists (Figure 1) have already been reported. These
include the nitrogen heterocycles11 triazoloquinazo-
lines,16 9-methyladenines,17 pyrazolotriazolepyrimidines,18
and triazolotriazines.19 Many of the non-xanthine an-
tagonists are relatively nonselective,11,20 although se-
lectivity for A1 receptors17 or A2 receptors18,19 has been
achieved. Several non-nitrogen heterocycles have also
been reported to bind to adenosine receptors (Figure 1),
including the protein tyrosine kinase inhibitor genistein
(5,7-dihydroxy-4′-hydroxyisoflavone), 1,21 a natural prod-
uct benzofurancarbaldehyde derivative 5-(3-hydroxy-
propyl)-7-methoxy-2-(3′-methoxy-4′-hydroxyphenyl)-3-
benzo[b]furancarbaldehyde, 2,22 and the recently re-
ported tetrahydrobenzothiophenes, e.g. BTH4, 3.23
A broad screening effort of phytochemicals in our
laboratory, similar to that recently reported,20 turned
the focus to the flavones, members of the larger class
of flavonoids, as a result of their relatively high affinity
at adenosine receptors. Several of these derivatives
even displayed significant affinity for A3 receptors.
Flavonoids are phenolic natural products derived largely
from fruits, vegetables, bark, and flowers and are
consumed in large doses in the human diet.24 They have
biological properties ranging from vascular protective
and antioxidative properties25 to antiinflammatory26
and antineoplastic.27 Since a related compound,
genistein, 1, an isoflavone derivative, was shown to be
Xanthines are the classical adenosine antagonists;2
however, it is highly desirable to identify additional
classes of antagonists. One reason for the current
* Address correspondence to: Dr. K. A. J acobson, Chief, Molecular
Recognition Section, NIDDK, NIH, Bldg. 8A, Rm. B1A-19, Bethesda,
MD 20892-0810. Telephone: (301) 496-9024. FAX: (301) 402-4182.
E-mail: kajacobs@helix.nih.gov.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

782 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
J i et al.
F igu r e 1. Structures of non-xanthine and non-nitrogen-containing derivatives studied previously as adenosine receptor
antagonists.
a competitive adenosine antagonist at A1 receptors in
FRTL (thyroid) cells,21 we investigated the interactions
of flavonoids in general as leads for developing novel
adenosine antagonists.
Resu lts
Affinity at Adenosine Receptors. Ki values at A1 and
A2A receptors were determined in radioligand binding
assays in brain membranes vs [3H]PIA or [3H]CGS
21680, respectively.31,32 Affinity at human brain A3
receptors expressed in HEK-293 cells43 and at rat brain
A3 receptors stably expressed in Chinese hamster ovary
(CHO) cells30 was determined using [125I]AB-MECA [N6-
(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluron-
amide)].30 A typical saturation curve for this radio-
ligand at human A3 receptors is shown in Figure 2. A
Kd value of 0.59 nM was obtained, and the Bmax was
0.159 pmol/mg of protein. Comparable curves at rat A3
receptors are shown in ref 30, in which the Kd value
was determined to be 1.48 nM. Thus, [125I]AB-MECA
is of slightly greater affinity at human vs rat A3
receptors. In contrast to the rat receptor, binding at
human A3 receptors is optimal at 25 °C.
Flavone and its hydroxylated derivatives (compounds
4-21) bound to rat A1 and A2A adenosine receptors in
the low micromolar range (Table 1). A structure-
activity analysis indicated that the hydroxyl groups of
naturally occurring flavones are not essential for affinity
at adenosine receptors, since unsubstituted flavone, 4,
displayed Ki values of 3.3 and 3.8 µM at A1 and A2A
receptors, respectively. In general, there was a ten-
dency toward lack of selectivity among the flavones
between A1 and A2A receptors, except for compound 9,
which had A1 selectivity of 8-fold. The flavonol galangin
F igu r e 2. (A) Representative saturation curve for binding of
(2-phenyl-3,5,7-trihydroxy-4H-1-benzopyran-4-one), 14,
[
125I]AB-MECA [N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-
displayed the highest affinity at A1 and A2A receptors
(e1 µM). Tetramethylscutallarein, 11, circimaritin,28b
13, galangin, 14, its trimethyl derivative, 15, and
tetramethylkaempferol, 17, also had relatively high
affinity among the analogues at the A1 subtype (0.8-
1.3 µM).
methyluronamide)]30 at human brain A3 receptors expressed
in HEK-293 cells43 at 37 °C, showing nonspecific (squares) and
specific (circles) binding. (B) Corresponding Scatchard analysis.
A Kd value ( SEM of 0.59 ( 0.02 nM was obtained (Bmax 0.157
( 0.08 pmol/mg of protein).
Typical inhibition curves for displacement of [125I]AB-
MECA binding at human A3 receptors are shown in
Figure 3A. The Ki values of many of the flavone
derivatives at human A3 adenosine receptors was in the
range of 1-10 µM, with the most potent being the
trimethyl derivative of galangin, 15, rhamnetin, 18, and
circimaritin, 13. Thus, methylation of the hydroxyl
groups of galangin enhanced affinity at A3 receptors,
while acetylation of the hydroxyl groups of galangin (e.g.
16) reduced affinity at all receptor subtypes. Unlike at
A1 and A2A receptors, at A3 receptors affinity of flavone
derivatives was often (e.g. 13-15, 17-19) enhanced by
the presence of multiple hydroxyl and/or methoxy
substitution. Pentamethylmorin, 20, appeared to bind
with some A3 receptor selectivity (10- and 18-fold for
human A3 vs rat A1 and A2A receptors, respectively),
with a Ki value of 2.65 µM. Substitution at the 3-posi-
tion was not required (e.g. 13) to achieve micromolar
affinity at adenosine receptors. The more selective A3
ligands tended to contain multiple substitutions of the
flavone, especially methoxy, of the phenyl ring, as in 7
and 20. In these compounds, affinity was diminished
only at A1 and A2A receptors, thus enhancing selectivity
for A3 receptors.
Quercetin, 19, had an unexpected effect on the bind-
ing of [125I]AB-MECA binding at human A3 receptors.

Interactions of Flavonoids with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 783
Ta ble 1. Affinities of Flavone Analogues Determined in Radioligand Binding Assays at A1, A2A, and A3 Receptorsa-e
O
5
R3
6
R1
2′
7
3′
O
8
R2
4′
5′
4-21
Ki (µM) or % inhibition at the indicated concn (M)
R1 R2 R3
b
compound
4 (flavone)
5
6
(rA1)a
(rA2∆
)
(hA3)c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.28 ( 0.92
2.17 ( 0.06
3.03 ( 0.49
18.8 ( 3.6
3.45 ( 1.16
6.20 ( 1.24
2.68 ( 0.68
35.4 ( 7.3
7.58 ( 1.23
28.0 ( 7.35
16.9 ( 3.8
5-OH
7-OH
7-OH
5,7-(OH)2
5-OH-7-Me
5-OH-7-MeO
47 ( 12% (10-4
)
41 ( 7% (10-4
32 ( 4% (10-4
63 ( 1% (10-5
6.70 ( 1.78
)
)
)
7
3′,4′-(MeO)2
4′-OH
4′-OMe
4′-OMe
4′-Me
4′-OH
4′-OH
H
8 (apigenin)
9
10
3.00 ( 0.29
3.40 ( 0.35
d (10-4
)
d (10-4
)
69 ( 7% (10-5
4.48 ( 0.14
)
)
11 (tetramethylscutallarein) 5,6,7-(Me)3
1.29 ( 0.08
1.61 ( 0.29
1.20 ( 0.36
nd
12 (hispidulin)
13 (cirsimaritin)
14 (galangin)
15
16
17 (tetramethylkaempferol)
18 (rhamnetin)
19 (quercetin)
20 (pentamethylmorin)
21 (hexamethylmyricetin)
5,7-(OH)2-6-MeO
5-OH-6,7-(MeO)2
5,7-(OH)2
6.48 ( 0.65
3.00 ( 0.70
63 ( 2% (10-5
1.72 ( 0.19
OH
0.863 ( 0.092 0.966 ( 0.164 3.15 ( 0.85
5,7-(Me)2O
5,7-(AcO)2
5,7-(MeO)2
7-OMe
5,7-(OH)2
5,7-(MeO)2
5,7-(MeO)2
H
H
OMe
OAc
OMe
OH
OH
OMe
OMe
0.509 ( 0.049 6.45 ( 1.48
1.21 ( 0.30
17.5 ( 2.0
3.37 ( 1.83
1.38 ( 0.18
see text
2.65 ( 0.72
16.2 ( 2.2
11.6 ( 4.4
1.07 ( 0.56
nd
2.47 ( 0.64
27.6 ( 7.5
d (10-6)
56.5 ( 9.5
nd
4′-OMe
3′,4′-(OH)2
3′,4′-(OH)2
2′,4′-(OMe)2
3′,4′,5′-(OMe)3
nd
6.99 ( 0.89
46.7 ( 2.7
nd
a
b
Displacement of specific [3H]PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5). Displacement of specific
[3H]CGS 21680 binding in rat striatal membranes, expressed as Ki ( SEM in µM (n ) 3-6). c Displacement of specific [125I]AB-MECA
binding at human A3 receptors expressed in HEK-293 cells, in membranes, expressed as Ki ( SEM in µM (n ) 2-3), or as a percentage
d
of specific binding displaced at the specified concentration (M). Displacement of <10% of specific binding at the specified concentration
(M). nd ) not determined.
Rather than inhibiting the specific binding as in Figure
3A, it raised the level of binding of the radioligand. As
shown in Figure 3B, at 30 µM quercetin the binding of
the A3 receptor radioligand was dramatically enhanced,
with >10× the level of control binding in membranes
of A3-transfected HEK-293 cells. Curiously, cyanidin
chloride, 25, although a very weak displacer in A1
receptor binding (Table 2), also enhanced [125I]AB-
MECA binding (Figure 3C). Since the additional bind-
ing occurred also in the presence of 200 µM NECA (540
( 110% of control binding at 30 µM 25), it probably does
not represent selective binding enhancement at A3
receptors. Instead, cyanidin likely causes enhanced
binding of [125I]AB-MECA to a nonreceptor site on the
membranes. It is not clear which structural features
are necessary for this [125I]AB-MECA effect. Rhamne-
tin, 18, a flavone also bearing the 3′,4′-dihydroxy groups,
did not behave similarly (Table 1).
At rat A3 receptors the affinity of flavone derivatives,
like that of many xanthines,14 was weaker than at
human A3 receptors. The Ki values of 14 and 15 were
43.1 and 17.4 µM, respectively, at rat A3 receptors
(Figure 4). This represents a human A3 vs rat A3 ratio
of 14-fold in both cases. Thus, within the same species
(rat) compound 20 is likely nonselective; however, it may
indeed prove to be selective when comparison is made
between human A3 and A1/A2A subtypes.
coside rutin, 26 (derived from quercetin, 19), and the
3,7-diglycoside robinin, 27 (derived from kaempferol),
greatly diminished affinity at A1 receptors. Similarly,
catechin (a flavan-3-ol), 24, and cyanidin (a flavylium
salt), 25, did not appreciably displace binding from A3
receptors. The effects of fused aromatic substitution of
the flavone ring system on receptor binding affinity were
studied. R-Naphthoflavone, 28, had greater affinity at
A1 (0.8 µM), A2A, and A3 receptors than the correspond-
ing â-isomer, 29. The isoflavone, genistein, 1, had very
low affinity at human A3 receptors.
A library of natural products of plant origin and
related intermediates was screened for affinity at ad-
enosine receptors. The compounds found to have con-
siderable affinity for at least one subtype of adenosine
receptors (Table 2) are shown in Figure 6. Included
among the hits are alkaloids of the amaryllidaceae, 30
and 31,34 oxogalanthine lactam, 32,35 acetylhaemanth-
amine methiodide, 33,36 hematoxylin, 35,37 and arbori-
nine, 36.38 Hematoxylin, 35, a pigment isolated from
logwood, was relatively potent at A1 receptors, with a
Ki value of 3.1 µM, and thus was highly selective for A1
vs A2A receptors. A synthetic intermediate (2,3-meth-
ylenedioxyfluoren-9-one), 34, with a ring system slightly
similar to hematoxylin, bound to A1 receptors, with a
Ki value of 8.9 µM. Compound 33, was the weakest in
binding among these derivatives. Affinities of the non-
flavones at A3 adenosine receptors (Table 2) were
generally weak. There were several compounds that
showed some degree of A1 selectivity. In addition to 35,
mentioned above, 32 was specific, and 34 was 10-fold
selective for A1 receptors. Only compound 36 was
slightly selective for A2A receptors (2-fold).
Reduction of the 2,3-olefinic bond of a flavonol, as in
the trans-dihydroflavonol (()-dihydroquercetin (Figure
5, Table 2), 22, greatly diminished affinity at adenosine
receptors. However a combination of reduction and
removal of the 3-hydroxy group, as in the flavanone
sakuranetin, 23, restored affinity at A1 and A3 receptors
(Table 2). Other stereoisomers were not studied for
comparison to 22. Glycosidation, as in the 3-monogly-

784 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
J i et al.
F igu r e 4. Representative competition curves comparing
inhibition of binding of [125I]AB-MECA at human (solid
symbols) and at rat (open symbols) A3 receptors. Inhibition
by compounds 14 (squares) and 15 (triangles) is shown.
Human brain A3 receptors were expressed in HEK-293 cells,
and rat brain A3 receptors were expressed in CHO cells.
Binding to rat brain A3 receptors was carried out as by the
published method.30
creasing the permeability and fragility of peripheral
blood vessels.
The biochemical pathways with which flavonoids
interact are numerous. Active flavonoids tend to inhibit
cell activation at various levels. Many flavonoids, such
as quercetin, 19,40 inhibit protein kinase C in the
micromolar range. Inhibition by flavonoids of many
other mammalian enzymes, including phosphodiester-
ases46 and transport ATPases, has been studied. At
very high concentrations, certain flavonoids, such as
quercetin, 19, but not apigenin, 8, have been found to
inhibit adenosine deaminase.47 Some flavonoids are
carcinogenic and turn on genes or activate cells via fatty
acid mobilization. It is also clear that some of the
biological effects of flavonoids, such as inhibition of
platelet function,41 are not fully explicable by any
previously identified mechanism. Quercetin, 19, has
been found to act synergisitically with â-adrenergic
agonists to promote cAMP accumulation and lipolysis
in isolated rat adipocytes.46
We have shown that many flavones have considerable
affinity for adenosine receptors. It is an historical
coincidence that Albert Szent-Gyorgi,1 the first inves-
tigator to detect the biological effects of adenosine, was
also one of the first to investigate the biological effects
of flavonoids.39 The fact that many of the flavones have
micromolar Ki values at adenosine receptors suggests
that perhaps these receptors may be important in the
spectrum of biological activities reported for flavones.
By comparison, caffeine has a Ki value of 29 µM at A1
receptors.2 Middleton40 has commented: “It is esti-
mated that the flavonoids have been persistent in
nature for somewhat over one billion years and, there-
fore, it seems entirely likely that they have been
interacting with evolving animal forms/species over the
eons”. Purinoceptors are also thought to be among the
earliest G-protein-coupled receptors to appear in evolu-
tion.45 Thus, just as the biological stimulant properties
of the naturally occurring methylxanthines, such as
caffeine and theophylline, were known long before their
interaction with adenosine was suspected, we raise the
possibility that the flavone derivatives are another class
of natural products with considerable activity as a result
of interaction with adenosine receptors. Moreover, the
F igu r e 3. (A) Representative competition curves for inhibition
of binding of
125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-
[
adenosine-5′-(N-methyluronamide)] by 15 (triangles), 17 (circles),
and 21 (diamonds) at human brain A3 receptors expressed in
HEK-293 cells at 25 °C. (B) Effects of quercetin, 19, on the
binding of [125I]AB-MECA to membranes from human A3-
transfected HEK-293 cells at 25 °C, expressed as percent of
control specific binding. Each point represents the average (
SEM of three determinations done in duplicate. (C) Effects of
cyanidin, 25, in the absence (b) or presence (O) of 200 µM
NECA, on the binding of [125I]AB-MECA to membranes from
human A3-transfected HEK-293 cells at 25 °C, expressed as
percent of control specific binding. Each point represents the
average ( SEM of 3 determinations done in duplicate.
Discu ssion
The flavonoids are a ubiquitous family of phytochemi-
cals that serve to protect plants from environmental
stress. The biological effects of flavonoids in mammals
have been studied for over 60 years,39 and currently
more than 100 preparations containing flavonoids are
marketed in Europe,42 mainly for their action of de-

Interactions of Flavonoids with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 785
Ta ble 2. Affinities of Various Phytochemicals and Related Derivatives Determined in Radioligand Binding Assays at A1, A2A, and A3
Receptorsa-e
b
compound
(rA1)a
(rA2∆
)
(hA3)c
1 (genistein)
5.021
3621
nd
35.6 ( 13.0
1.32 ( 0.43
d (10-4)
20 ( 2% (10-4
34.1 ( 10.1
)
22 ((()-dihydroquercetin)
23 (sakuranetin)
28 (R-naphthoflavone)
29 (â-naphthoflavone)
30
d (10-5)
8.18 ( 2.53
0.786 ( 0.018
8.8 ( 3.6
7.10 ( 0.10
7.59 ( 2.14
5.55 ( 0.83
52.5 ( 3.7
8.89 ( 2.15
3.10 ( 0.60
12.7 ( 1.2
3.40 ( 0.18
71 ( 2% (10-5
12 ( 1% (10-5
)
)
29.9 ( 10.1
d (10-4
)
31
6.98 ( 2.21
19 ( 3% (10-4
)
32 (oxogalan thine lactam)
33 (acetylhaem anthamine methiodide)
34
35 (hematoxylin)
36 (arborinine)
d (10-4
d (10-4
)
)
d (10-4
d (10-4
)
)
84.0 ( 6.9
52 ( 6% (10-4
)
)
28 ( 2% (10-4
6.47 ( 1.90
)
d (10-4
63 ( 7% (10-4
)
a
Displacement of specific [3H]PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5). The following compounds
at a concentration of 10-5 M displaced less than 10% of the specific binding at A1 receptors: 24 d-(+)-catechin (3,3′,4′,5,7-flavanpentol),
b
25 cyanidin (3,3′,4′,5,7-pentahydroxy-2-phenylbenzopyrylium chloride), and 27 robinin (kaempferol 3-robinoside 7-rhamnoside). Dis-
placement of specific [3H]CGS 21680 binding in rat striatal membranes, expressed as Ki ( SEM in µM (n ) 3-6). 26 rutin (quercetin
3-rutinoside) at a concentration of 10-4 M displaced less than 10% of the specific binding at A2A receptors. c Displacement of specific
[
125I]AB-MECA binding at human A3 receptors expressed in HEK-293 cells, in membranes, expressed as Ki ( SEM in µM (n ) 2-3), or
as a percentage of specific binding displaced at the specified concentration (M). 26 (rutin) at a concentration of 10-4 M displaced 25 ( 3%
d
of the specific binding at A3 receptors. Displacement of <10% of specific binding at the specified concentration (M). nd ) not determined.
F igu r e 5. Structures of other flavonoid derivatives studied for affinity at adenosine receptors.
degree of activation of the receptors by endogenous
adenosine may be more dependent on dietary factors
than previously realized.
effects of flavones are related to A3 or other adenosine
receptor action.
The structure-activity analysis of the flavonoids
indicates that a carbonyl group at the 4-position pro-
motes affinity at adenosine receptors. A comparison of
R- and â-naphthoflavone also suggests that to achieve
optimal receptor interaction the side of the molecule
bearing the carbonyl group not be sterically crowded,
as in the less active â-isomer. The various phytochemi-
cals in Figure 5 were selected for their moderate
adenosine receptor affinity by screening a library of
several hundred phytochemicals. A feature common to
all of the more potent adenosine receptors ligands is a
phenyl ring that is fused to a five- or six-membered ring,
which contains an R-carbonyl group. Compound 33,
which is lacking the R-carbonyl group, was the weakest
in this series in binding to A1 receptors. This structural
pattern, shared also with the flavones, suggests that
there is a common pharmacophore among all of these
phytochemicals. More detailed structure-activity stud-
ies are in progress in our laboratory.
Most curious is the relatively high affinity among
many of the flavones and flavonols at A3 receptors.
Unlike xanthines and nearly all other classes of non-
xanthine adenosine antagonists,14 the affinity of flavone
derivatives at the newly cloned A3 receptor is generally
in the same range as the affinity at A1 and A2A
receptors. The A3 receptors have already been impli-
cated in vascular effects, inflammation, and cancer,44
three areas in which flavonoids are also biologically
active.25-27 For example, flavone derivatives such as 8
and 19 have been shown to diminish the release of
histamines from mast cells.26 Adenosine agonists,
presumably acting via A3 receptors, have been shown
to promote such a release.13 Thus, the unexpected
finding of considerable affinity of flavone derivatives at
both rat and human A3 receptors may explain some of
the previously observed biological effects of these com-
pounds. Further pharmacological experiments will be
required to determine to which degree these known
The presence of multiple hydroxyl groups on the

786 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
J i et al.
7.9 (m, 2H, Ar), 7.65 and 7.17 (each, d, 1H, J ) 2 Hz, Ar, ortho
to AcO), 2.34 (s, 6H, Ac), 2.30 (s, 3H, Ac). CHN analysis.
(B) Meth yla tion of th e F la von es. Methylation of hy-
droxyflavones (galangin, kaempferol, morin, and myricetin)
and mass spectral analysis using ammonia gas ionization were
carried out by the general methods reported28 and as below.
2′,3,4′,5,7-P en ta bis(m eth yloxy)fla von e, 20. Morin hy-
drate (25 mg, 82 µmol, Aldrich) was dissolved in dry acetone
(20 mL), in which potassium carbonate (1.0 g) was suspended.
Dimethyl sulfate (1.0 mL, 11 mmol) was added, and the
mixture was refluxed for 4 h under nitrogen. After cooling in
an ice bath, 2 mL of concentrated ammonium hydroxide were
added in aliquots followed by 20 mL of water. The solution
was extracted with ethyl acetate. The organic layer was dried
and evaporated, and the white solid residue was recrystallized
from methanol/water to yield 11 mg (36%) of 20, which was
homogeneous by thin layer chromatography (Rf 0.32, silica,
chloroform/methanol/acetic acid, 95:4:1). MS: 373 (M + 1),
359 (M + 1 - CH2), 343. Mp: 153-156 °C. NMR: δ 7.35 (d,
1H, J ) 8 Hz, Ar, 6-Ph), 6.66 (dd, 1H, J ) 2, 8 Hz, Ar, 5-Ph),
6.70, 6.62 and 6.48 (each, d, 1H, J ) 2 Hz, Ar), 3.84 (s, 9H,
Me), 3.80 (s, 3H, Me), 3.61 (s, 3H, Me). CHN analysis.
1-Keto-8,9-dim eth yloxy-1,2,3,4-tetr ah ydr odiben zo[b,d]6-
p yr on e, 30. Compound 30 was synthesized by Dr. Henry
Fales by a modification of a literature procedure.34 6-Bromo-
3,4-dimethoxybenzoic acid (6-bromoveratric acid, 2.0 g, 7.7
mmol, Spectrum Chem. Corp., New Brunswick, NJ ) was
dissolved in 50% EtOH/H2O (100 mL), and treated with
resorcinol (0.85 g, 7.7 mmol), 50 mg of copper powder, 50 mg
of cupric acetate, and sodium hydroxide (0.31 g, 7.7 mmol).
The mixture was heated to reflux overnight. The reaction
mixture was extracted once with ether, and insolubles solids
were removed by filtration. The aqueous layer was acidified
with 1 N HCl to form the lactone. The precipitate was
removed by filtration and dried. In order to remove unreacted
6-bromoveratric acid from this solid containing the product,
it was suspended and partially dissolved in cold saturated
aqueous NaHCO3. The remaining insoluble solids were col-
lected by filtration and the washings discarded. The dried
residue was recrystallized from EtOH to give 0.74 g of the pure
product (35% yield). NMR (CDCl3) δ 8.68 and 7.65 (each s,
1H, Ar), 4.06 and 3.99 (each s, 3H, Me), 2.95 and 2.67 (each t,
2H, CH2), 2.18 (m, 2H, CH2). Mp: 236-237 °C. CHN
analysis.
F igu r e 6. Structures of various phytochemicals and related
derivatives studied for affinity at adenosine receptors. These
compounds were identified through the screening of a natural
products library. All were found to have considerable affinity
for at least on subtype of adenosine receptors, except for
compound 33, which was weaker in binding.
flavones tended to increase A3 affinity and either had
no effect on or decreased A1 and A2A affinity. The best
A3 selectivity was achieved with pentamethylmorin, 20,
which contained a 2′-methoxy group on the phenyl ring
that may impede free rotation of the ring.
In conclusion, this series of flavone derivatives may
provide important leads for the development of potent
and selective A3 antagonists, which are as yet un-
known.14 The present findings indicate that the chemi-
cal modification of flavonoids and other phytochemicals,
in general, may lead to the development of novel
adenosine antagonists.
2,3-(Meth ylen ed ioxy)flu or en -9-on e, 34, was synthesized
by Dr. H. Fales (NIH) by the following method: Gaseous HF
was condensed by collecting in a Teflon vial placed in a dry
ice/acetone bath. 6-Phenylpiperonylic acid48 (100 mg, 0.41
mmol) was added to HF (2 mL) and the solution let stand
overnight. The HF was evaporated leaving the pure fluo-
renone derivative in quantitative yield (92 mg). Mp: 156-
157 °C. CHN analysis.
Exp er im en ta l Section
Ma ter ia ls. Compounds 4, 7-14, 28, and 29 were obtained
from Fluka, Ronkonoma, NY, or from Aldrich, St. Louis, MO.
Compounds 18 and 22 were obtained from Apin Chemicals,
Ltd., Oxon, U.K. Compounds 5, 6, 19, and 23-27 were
obtained from K+K Laboratories, J amaica, NY. Compound
1 was obtained from Sigma, Milwaukee, WI. Compound 32
was from Fisher, New York, NY. Compounds 31-33 and 36
(1-hydroxy-2,3-dimethoxy-10-methyl-9(10H)-acridinone) were
obtained from Dr. Henry Fales (NIH) and were synthesized
as described.34-36,38
Syn th esis. Proton nuclear magnetic resonance spectros-
copy was performed on a Varian GEMINI-300 spectrometer
and spectra were taken in DMSO-d6. Electron-impact mass
spectrometry was performed with a VG7070F mass spectrom-
eter at 6 kV. Elemental analysis was performed by Atlantic
Microlab Inc. (Norcross, GA).
3,5,7-Tr ia cetoxyfla von e, 16. (A) Acetyla tion of Ga la n -
gin , 14. Galangin (9.0 mg, 33 µmol) was dissolved in 1 mL of
DMF and treated with acetic anhydride (0.2 mL) and 4-(di-
methylamino)pyridine (3 mg). After stirring for 10 min, 2 mL
of 1 N NaH2PO4 was added. A white precipitate was removed
by filtration and recrystallized from methanol/water to yield
11.4 mg of a solid (87%), which was homogeneous by thin layer
chromatography (Rf 0.56, silica, chloroform/methanol/acetic
acid, 95:4:1). MS: 414 (M + 1 + NH3), 397 (m+1), 355 (M +
1 + NH3 - OAc). Mp: 128-129 °C. NMR: δ 7.6 (3H, Ar),
P h a r m a cology. Ra d ioliga n d Bin d in g Stu d ies. The
radioligand binding data for the novel compounds were
determined as described previously.12,23
Membranes prepared from HEK-293 cells stably expressing
the human A3 receptor were obtained from Receptor Biology,
Inc. (Baltimore MD). CHO cells stably expressing the rat A3
adenosine receptor were grown in F-12 medium containing
10% FBS and penicillin/streptomycin (100 units/mL and 100
µg/mL, respectively) at 37 °C in a 5% CO2 atmosphere, and
membrane homogenates were prepared as reported.29 Binding
of
[
125I]-N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyl-
uronamide) ([125I]AB-MECA) to rat A3 receptors in stably
transfected CHO cell membranes (generous gift of Prof. Gary
L. Stiles, Duke University, Durham, NC) was performed as
described.30 Assays at human A3 receptors were performed
in a Mg2+-free buffer containing 50 mM Tris and 1 mM EDTA,
at pH 8.0. The glass tubes contained 100 µL of the membrane
suspension (0.3 mg of protein/mL, stored at -80 °C in the same
buffer), 50 µL of [125I]AB-MECA (final concentration 0.3 nM),
and 50 µL of a solution of the proposed antagonist. All
nonradioactive compounds were initially dissolved in DMSO
and diluted with buffer to the final concentration, where the
amount of DMSO never exceeded 1%. Duplicate incubations
were carried out for 1 h at 37 or 25 °C (the latter temperature

Interactions of Flavonoids with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 787
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provided roughly twice the level of specific binding for the
human, but not rat, A3 receptor) and were terminated by rapid
filtration over Whatman GF/B filters, using a Brandell cell
harvester (Brandell, Gaithersburg, MD). The tubes were
rinsed three times with 3 mL of buffer each. The radioactivity
on the filters was determined with a Beckman 5500B γ-counter.
Nonspecific binding was determined in the presence of 100 µM
N6-phenylisopropyladenosine ((R)-PIA).
Binding of [3H]-(R)-PIA to A1 receptors from rat cortical
membranes and of [3H]CGS 21680 to A2A receptors from rat
striatal membranes was performed as described previously.31,32
Adenosine deaminase (3 units/mL) was present during the
preparation of the brain membranes, in which an incubation
of 30 min at 30 °C is carried out, and during the incubation
with the radioligands. At least five different concentrations
spanning 3 orders of magnitude, adjusted appropriately for
the IC50 of each compound, were used. IC50 values, computer-
generated using the nonlinear regression method implemented
in the InPlot program (Graph-PAD, San Diego, CA), were
converted to apparent Ki values using Kd values of 1.0 and 14
nM for [3H]PIA and [3H]CGS 21680 binding, respectively,
applying the Cheng-Prusoff equation.33
Ack n ow led gm en t. We thank Dr. Henry Fales of the
Laboratory of Chemistry, NHLBI (Bethesda, MD), and
Dr. David Kingston of Virginia Polytech (Blacksburg,
VA) for providing samples of many of the compounds
used in this study and for helpful discussions. We thank
Gilead Sciences (Foster City, CA) and the Cystic Fibro-
sis Foundation for financial support. We thank Dr. Pat
Towers of Amersham and Dr. Garth Brown of DuPont
NEN for the synthesis of [125I]I-AB-MECA.
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Glossa r y
[
125I]AB-MECA
[
125I]-N6-(4-amino-3-iodobenzyl)adenosine-
5′-(N-methyluronamide)
ADA
adenosine deaminase
BTH4
ethyl 3-(benzylthio)-4-oxo-4,5,6,7-tetrahy-
dro-benzo[c]thiophen-1-carboxylate
CGS21680
2-[[4-(2-carboxyethyl)phenyl]ethylamino]-
5′-(N-ethylcarbamoyl)adenosine
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CHO
DMF
DMSO
FBS
Ki
(R)-PIA
Tris
Chinese hamster ovary
N,N-dimethylformamide
dimethyl sulfoxide
fetal bovine serum
equilibrium inhibition constant
(R)-N6-phenylisopropyladenosine
tris(hydroxymethyl)aminomethane
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