Synthesis and biological activities of flavonoid derivatives as A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm950923i

A broad screening of phytochemicals has demonstrated that certain flavone and flavonol derivatives have a relatively high affinity at A₃ adenosine receptors, with K(i) values of ≥1 μM (Ji et al. J. Med. Chem. 1996, 39, 781-788). We have further modified the flavone structure to achieve a degree of selectivity for cloned human brain A₃ receptors, determined in competitive binding assays versus [¹²⁵I]AB-MECA [N⁶-(4-amino-3- iodebenzyl)adenosine-5'-(N-methyluronamide)]. Affinity was determined in radioligand binding assays at rat brain A₁ and A(2A) receptors using [³H]- N⁶-PIA ([³H]-(R)-N⁶-phenylisopropyladenosine) and [³H]CGS21680 [[³H]-2- [[4-(2-carboxyethyl)phenyl]ethylamino]-5'-(N-ethylcarbamoyl)adenosine], respectively. The triethyl and tripropyl ether derivatives of the flavonol galangin, 4, had K(i) values of 0.3-0.4 μM at human A₃ receptors. The presence of a 5-hydroxyl group increased selectivity of flavonols for human A₃ receptors. The 2',3,4',7-tetraethyl ether derivative of the flavonol morin, 7, displayed a K(i) value of 4.8 μM at human A₃ receptors and was inactive at rat A₁/A(2A) receptors. 3,6-Dichloro-2'-(isopropyloxy)-4'- methylflavone, 11e, was both potent and highly selective (~200-fold) for human A₃ receptors (K(i) = 0.56 μM). Among dihydroflavonol analogues, the 2-styryl instead of the 2-aryl substituent, in 15, afforded selectivity for human A₃ vs rat A₁ or A(2A) receptors. The 2-styryl-6-propoxy derivative, 20, of the furanochromone visnagin was 30-fold selective for human A₃ receptors vs either rat A₁ or A(2A) receptors. Several of the more potent derivatives effectively antagonized the effects of an agonist in a functional A₃ receptor assay, i.e. inhibition of adenylyl cyclase in CHO cells expressing cloned rat A₃ receptors. In conclusion, these series of flavonoids provide leads for the development of novel potent and subtype selective A₃ antagonists.

Full text of DOI:10.1021/jm950923i

J . Med. Chem. 1996, 39, 2293-2301
2293
Syn th esis a n d Biologica l Activities of F la von oid Der iva tives a s A3 Ad en osin e
Recep tor An ta gon ists
Yishai Karton,† J i-long J iang,† Xiao-duo J i,† Neli Melman,† Mark E. Olah,‡ Gary L. Stiles,‡ 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, and Departments of Medicine and Pharmacology,
Duke University Medical Center, Durham, North Carolina 27710
Received December 18, 1995X
A broad screening of phytochemicals has demonstrated that certain flavone and flavonol
derivatives have a relatively high affinity at A3 adenosine receptors, with Ki values of g1 µM
(J i et al. J . Med. Chem. 1996, 39, 781-788). We have further modified the flavone structure
to achieve a degree of selectivity for cloned human brain A3 receptors, determined in competitive
binding assays versus [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluron-
amide)]. 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. The triethyl
and tripropyl ether derivatives of the flavonol galangin, 4, had Ki values of 0.3-0.4 µM at
human A3 receptors. The presence of a 5-hydroxyl group increased selectivity of flavonols for
human A3 receptors. The 2′,3,4′,7-tetraethyl ether derivative of the flavonol morin, 7, displayed
a Ki value of 4.8 µM at human A3 receptors and was inactive at rat A1/A2A receptors. 3,6-
Dichloro-2′-(isopropyloxy)-4′-methylflavone, 11e, was both potent and highly selective (∼200-
fold) for human A3 receptors (Ki ) 0.56 µM). Among dihydroflavonol analogues, the 2-styryl
instead of the 2-aryl substituent, in 15, afforded selectivity for human A3 vs rat A1 or A2A
receptors. The 2-styryl-6-propoxy derivative, 20, of the furanochromone visnagin was 30-fold
selective for human A3 receptors vs either rat A1 or A2A receptors. Several of the more potent
derivatives effectively antagonized the effects of an agonist in a functional A3 receptor assay,
i.e. inhibition of adenylyl cyclase in CHO cells expressing cloned rat A3 receptors. In conclusion,
these series of flavonoids provide leads for the development of novel potent and subtype selective
A3 antagonists.
In tr od u ction
and promote the release of inflammatory mediators from
mast cells.8 We have introduced the first selective
agonist ligands for the A3 subtype, including the agonist
IB-MECA [N6-(3-iodobenzyl)adenosine-5′-(N-methylu-
ronamide)].9,12 IB-MECA has been shown to elicit
locomotor depression,9 and following chronic adminis-
tration it acts as a cerebroprotectant in stroke and
seizure models.13
The principal mechanism by which caffeine and other
alkylxanthines act as physiological stimulants is by
blocking the effects of the ubiquitous neuromodulator
adenosine,1 which acts as a “local hormone” within a
given organ. Adenosine is produced locally in response
to increased activity or stress to the system. This
feedback mechanism allows the organ to compensate for
the stress by decreasing energy demand (depressant
activity) and increasing oxygen supply (e.g. by vasodi-
lation).2
Extracellular adenosine activates receptors of the A1
and A2A subtypes, which depresses the action of the
heart, brain, kidneys, and the immune system, as well
as other systems.3 Recently, the novel A3 subtype of
adenosine receptors has been cloned,4,5 and its phar-
macological6-9 and regulatory characteristics10 have
been studied. Activation of A3 receptors requires rela-
tively high, i.e. pathological, concentrations of adeno-
sine; the Ki value of adenosine has been estimated as
∼1 µM at A3 receptors versus 10 and 30 nM at A1 and
A2A receptors, respectively.11 Thus the physiological role
of A3 receptors may be very different from that of the
A1 and A2A subtypes. A3 agonists induce hypotension
In principle, a selective A3 adenosine receptor antago-
nist should serve as a cerebroprotective, antiasthmatic,
or antiinflammatory agent.6,7,11,13 Previously, we have
successfully designed specific antagonists for A1 and A2A
receptors by chemically modifying the structures of
caffeine and other xanthines.3 However, at A3 receptors
xanthines are of particularly low affinity14 and have not
yet provided satisfactory chemical leads for antago-
nists.15 Consequently, we have searched diverse chemi-
cal libraries for lead structures for developing A3
antagonists.16-19 A number of classes of non-xanthine
adenosine antagonists have already been reported,
including various nitrogen heterocycles and several
classes of nonnitrogen heterocycles.16 We recently
reported that tetrahydrobenzothiophenones, e.g. ethyl
3-(benzylthio)-4,5,6,7-tetrahydro-4-oxobenzo[c]thiophene-
1-carboxylate (BTH4), bind to A1 and A2A adenosine
receptors in the micromolar range.17 One member of
this series, 4,5-dihydro-1-(methylthio)benzo[c]thiophene-
3-carboxylic acid hydrazide, I (Figure 1), bound with
slight selectivity for rat A3 receptors. Siddiqi et al.16
have shown that folic acid (II), a pyridopyrimidinone
(III), cytochalasin H (IV), 11-hydroxytetracarbazolenine
* Address correspondence to: Dr. K. A. J acobson, Chief, Molecular
Recognition Section, NIDDK, NIH, Bldg 8A, Rm B1A-19, Bethesda,
MD 20892-0810. Tel: (301) 496-9024. Fax: (301) 480-8422. E-mail:
kajacobs@helix.nih.gov.
† National Institutes of Health.
‡ Duke University Medical Center.
X Abstract published in Advance ACS Abstracts, April 15, 1996.
S0022-2623(95)00923-X
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

2294 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Karton et al.
F igu r e 2. Synthesis of alkylated derivatives of galangin and
morin. Reagents: 2, 3a , 4, and 7, R-I; 3b, Et-Br; 6 and 8, R2-
SO4.
The alkylation of galangin was carried out using the
appropriate alkyl iodide and potassium carbonate in
refluxing acetone (Figure 2), providing the completely
alkylated product, 3a or 4. Partial alkylation leaving
the 5-position hydroxyl group unreacted, to provide 3b,
was accomplished under less forceful conditions, e.g.
alkyl bromide at room temperature. The O-alkylation
of morin (2′,3,4′,5,7-pentahydroxyflavone) was compli-
cated by an even lower reactivity of the 5-hydroxy group
than in galangin, leading to tetra- rather than penta-
substitution (Figure 2). Thus, when ethyl sulfate was
used the fully alkylated morin derivative, 8, was ob-
tained, while reaction with ethyl iodide provided exclu-
sively the tetraethyl derivative, 7. The position of the
nonalkylated hydroxyl group of 3b and 7 was deter-
mined from the NMR chemical shift. The resonance for
the 5-OH proton in flavone derivatives has been re-
ported to appear at roughly 12 ppm.12 The downfield
shift is apparently caused by the adjacent carbonyl to
which it may form a hydrogen bond and thus a nonco-
valent six-membered ring. This proton signal is present
in the spectra of 3b, 5a , and 7 but not 8.
The synthetic routes to flavone derivatives have been
investigated extensively and optimized.22-25 As shown
in Figure 3, a synthetic route used to form the flavone
ring system was via a condensation of a 2-hydroxyac-
etophenone derivative, 25, with the appropriate alde-
hyde, 26, which gave rise to the 2-substituent. The
condensation resulted first in a trans-olefin, 27, which
could be cyclized under basic oxidizing conditions to give
either the 2,3-saturated dihydroflavonol (e.g. 3-hydroxy-
flavanone) analogues, 15 and 16, or at higher temper-
ature the corresponding dehydro derivative, 10, a
flavonol. Cinnamaldehyde (26, R ) PhCHdCH-) used
as the second component, led to styryl substitution at
the 2-position (15). Similarly, use of phenylpropargyl
aldehyde (26, R ) PhCtC-) provided 2-phenylethynyl
substitution of the flavonol (10) or dihydroflavonol (16).
Evidence that the dihydroflavonols obtained were of the
trans configuration were deduced from NMR coupling
constants for the 2- and 3-position protons in compounds
14-16 of ∼12 Hz, characteristic of trans vicinal cou-
pling.26 The 3-chloro substituent was introduced in
flavone derivatives (as in the preparation of 11b and
11d ) using sulfuryl chloride in carbon tetrachloride.37
F igu r e 1. Substances that have selectivity in binding to rat
A3 vs A1 and A2A adenosine receptors. Ki values shown are in
competition for [125I]AB-MECA binding in membranes from
CHO cells expressing the cloned rat A3 receptors.16,17
(V), the adenosine uptake inhibitor dipyridamole (VI),
certain sulfonylpiperazines (e.g. the protein kinase C
inhibitor HA-100, VII), and a number of other hetero-
cyclic substances displace radioligand from adenosine
receptors with low affinity but with apparent selectivity
for rat A3 receptors. Recently, a broad screening of
phytochemicals has demonstrated that certain flavones
and flavonols, members of the larger class of phenolic
natural products known as flavonoids, have a relatively
high affinity at A3 adenosine receptors, with Ki values
in the micromolar range, close to that of the natural
ligand adenosine.19 The affinity of some flavones at A1
and A2A receptors was also noted by Ares et al.20 In
the present study we have investigated the structure-
activity relationships of flavonoids at adenosine recep-
tors in an effort to develop novel A3 adenosine antago-
nists. Selected analogues have been tested in a
functional A3 receptor assay, i.e. inhibition of adenylyl
cyclase in Chinese hamster ovary (CHO) cells express-
ing cloned rat A3 receptors.4
Resu lts
Syn th esis. The structures of the flavonoid deriva-
tives (including flavonols, 1-10; flavones, 11; fla-
vanones, 12 and 13; dihydroflavonols, 14-16; and
furylchromones, 17-24) examined in the present study
are shown in Table 1, and the chemical characterization
is summarized in Table 2. In our previous study,19 the
flavonol galangin (3,5,7-trihydroxyflavone), 1, was shown
to bind to adenosine receptors nonselectively, while its
trimethyl ether derivative, 2, was shown to have
enhanced affinity at A3 receptors. Thus, we synthesized
and compared in receptor binding assays the series of
alkyl ethers of galangin, 2-4, and other 3-substituted
flavone derivatives.

Synthesis and Biological Activities of Flavonoid Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2295
Ta ble 1. Affinities of Flavonoid Derivatives in Radioligand Binding Assays at A1, A2a, and A3 Receptorsa-c
O
O
O
O
R1
5
O
5
8
5
8
R1
R2
R1
R2
3
3
6
7
6
7
3
R3
R3
O
2
2
2
O
R2
(trans)
2–16
17–24
1–11
Ki (µM) or % inhibitionc
rA1/
hA3
compound
R1
R2
R3
rA1
rA2A
hA3
1f (galangin)
2f (MRS928)
3a (MRS1041)
3b (MRS1093)
4 (MRS1042)
5a f (morin)
5b
OH
Ph
Ph
Ph
Ph
Ph
5,7-(OH)2
5,7-(OMe)2
5,7-(OEt)2
5-OH-7-OEt
5,7-(OPr)2
5,7-(OH)2
5-OH-6-CHdCHCH- 9.09 ( 0.91
(CH3)2-7-OMe
5,7-(OMe)2
5-OH-7-OEt
5,7-(OEt)2
H
6-OCH3
H
H
6-Cl
6-Cl
6-Cl
6-Cl
H
H
H
5-OH-7-OMe
H
6-OMe
6-OMe
0.863 ( 0.092
0.509 ( 0.049
0.603 ( 0.022
1.89 ( 0.56
1.10 ( 0.20
13.8 ( 3.1
0.966 ( 0.164 3.15 ( 0.85
0.27
0.42
OMe
OEt
OEt
OPr
OH
CH2CHd
C(CH3)2
OMe
OEt
OEt
OH
OH
H
Cl
H
Cl
Cl
Cl
H
H
H
H
OH
OH
OH
OCH3
OCH3
OCH3
OC2H5
6.45 ( 1.48
3.31 ( 1.24
63.8 ( 18.8
3.22 ( 1.31
17.3 ( 4.1
1.21 ( 0.30
0.364 ( 0.067 1.7
0.748 ( 0.402 2.5
0.317 ( 0.089 3.5
2′,4′-(OH)2Ph
2′,4′-(OH)2Ph
34 ( 4% (10-4
4.59 ( 1.69
)
∼0.1
d (10-4
)
2.0
6f (MRS923)
7 (MRS1063)
8 (MRS1086)
9
10 (MRS1072)
11a f (flavone)
11b (MRS1132)
11c (MRS1131)
11d (MRS1088)
11e (MRS1067)
11f (MRS1089)
12 (flavanone)
13a
2′,4′-(OMe)2Ph
2′,4′-(OEt)2Ph
2′,4′-(OEt)2Ph
2′,4′,6′-(OMe)3Ph
CtCPh
Ph
Ph
Ph
Ph
2′-i-PrO-4′-MePh
2′,4′,6′-Me3Ph
Ph
2′-OHPh
4′-OHPh
4′-OHPh
2′-OHPh
CHdCHPh
CtCPh
CH3
27.6 ( 7.5
d (10-4
46.7 ( 2.7
2.65 ( 0.72
4.83 ( 1.40
7.27 ( 1.88
50.1 ( 7.8
24.0 ( 9.7
16.9 ( 3.8
11.5 ( 3.7
32 ( 5% (10-4
10
>40
4.4
)
d (10-4
26.8
)
32.3 ( 9.1
7.32 ( 1.36
34 ( 9% (10-4
3.28 ( 0.92
2.48 ( 0.72
28 ( 6% (10-5
d (10-4
d (10-4
)
)
0.15
∼8
)
)
3.45 ( 1.16
0.19
0.22
25.1 ( 5.4
d (10-4
)
)
15% (10-4
)
54.5 ( 26.3
0.741 ( 0.325 >100
36 ( 12% (10-4
)
19 ( 9% (10-4
)
0.561 ( 0.129 ∼200
37 ( 5% (10-4
32.0 ( 4.8
)
d (10-4
)
5.24 ( 0.52
50.1 ( 27.1
6.07 ( 1.43
42.8 ( 10.5
3.40 ( 0.18
27 ( 3% (10-4
21.1 ( 9.9
∼20
0.64
0.43
0.27
2.4
nd
2.64 ( 0.56
11.5 ( 3.7
17.6 ( 2.1
13b
35% (10-4
)
13cf (sakuranetin)
14e
8.18 ( 2.53
91.9 ( 0.3
35.6 ( 13.0
d (10-4
d (10-4
d (10-4
)
)
)
)
<1
15e (MRS1061)
16e (MRS1062)
17 (visnagin)
18
d (10-4
)
>8
50.3 ( 17.0
8.17 ( 0.43
60.0 ( 17.8
88.9 ( 27.1
8.28 ( 2.69
1.16 ( 0.45
3.95 ( 1.98
6.2
36 ( 3% (10-4
)
42.4 ( 11.9
25 ( 8% (10-4
11.5 ( 1.3
33.8 ( 14.8
49.0
∼2
CHO
CHdCHPh
CHdCHPh
d (10-4
)
)
>2
19 (MRS1065)
20 (MRS1066)
21 (MRS1084)
22 (MRS1070)
23 (MRS1071)
24 (MRS1078)
32.6 ( 10.5
35.6 ( 12.7
40.0 ( 3.5
d (10-4)
0.37
31
O(CH2)2CH3 CHdCHPh
10
OCH3
OC2H5
OCH3
CHdCHCHdCHPh
CHdCHCHdCHPh
CHdNPh
d (10-4
167 ( 92
d (10-4
)
d (10-4
)
d (10-4
d (10-4
)
)
45.5 ( 10.3
9.18 ( 2.56
>4
>20
)
a
Displacement of specific [3H]PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5), or as a percentage of
specific binding displaced at 100 µM. Displacement of specific [3H]CGS 21680 binding in rat striatal membranes, expressed as Ki (
b
SEM in µM (n ) 3-6), or as a percentage of specific binding displaced at 100 µM. c Displacement of specific [125I]AB-MECA binding in the
presence of 10 mM Mg2+ at human A3 receptors expressed in HEK293 cells, in membranes, expressed as Ki ( SEM in µM (n ) 2-5), or
d
as a percentage of specific binding displaced at 100 µM. Displacement of e10% of specific binding at the indicated concentrated (M).
e 2,3-trans. f Values from ref 19. The systematic name for compounds 17-24 is different from the numbering scheme used in this table
g
and in Results section. nd ) not determined.
An alternate approach to providing olefinic substitu-
tion at the 2-position (Figure 4) was to condense a
2-methylchromone (such as the natural product visna-
gin, 17)27 with an aldehyde, 28, under basic conditions.
This method was used to prepare the 2-styryl derivative,
19. Evidence for a trans-styryl group of 19 was derived
from the NMR spectra with an olefinic coupling constant
of 15 Hz.26 In the presence of sodium ethoxide the
5-methoxy group readily exchanged, resulting in com-
pound 20. The corresponding 5-propoxy derivative, 21,
was obtained using an alternate approach (Figure 4),
in which visnagin was demethylated to give 29, followed
by condensation with benzaldehyde. The 2-methyl
group of visnagin could also be oxidized to an aldehyde
group, as in 18.28
Affin ity a t Ad en osin e Recep tor s. Ki values at A1
and A2A receptors were determined in radioligand
binding assays in rat brain membranes vs [3H]PIA or
[3H]CGS 21680, respectively.29,30 Affinity at human
brain A3 receptors expressed in HEK-293 cells31 was
determined using [125I]AB-MECA [N6-(4-amino-3-iodo-
benzyl)adenosine-5′-(N-methyluronamide)].32 The com-
plete set of compounds was tested at human A3 recep-
tors, and only selected compounds at rat A3 receptors
(stably expressed in CHO cells4,14-16), which would be
preferred for comparison with the A1 and A2A data. We
chose the human A3 receptor for our screen for the
following reasons: (1) The affinity of most known
adenosine receptor antagonists is minimal at rat A3
receptors.14,16 (2) The human A3, being more sensitive,
allowed for a better comparison between compounds.31
(3) The human receptor is more relevant to therapeutic
need. (4) The preliminary indications are that the A3
selectivity of flavones may be at least as great as
indicated in this study when comparison is made
between human receptor homologues. For example, the
Ki value of galangin at human A2A receptors is 4.1 (
0.7 µM (unpublished), compared to 0.97 µM at rat A2A
receptors.19 At cloned human A1 receptors, the IC50
value for galangin in displacement 2 nM [125I]AB-MECA
is 13.6 ( 1.5 µM (unpublished), i.e. weaker than at rat
A1 receptors.
As we have previously reported,19 the flavonol galan-
gin (2-phenyl-3,5,7-trihydroxy-4H-1-benzopyran-4-one),

2296 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Karton et al.
Ta ble 2. Physical Characterization of Flavone, Flavanol, and
Flavanone Derivatives
compd
no.
mp (°C)
201
MS
formula
anal.
2
313 (CI)
C18H16O5‚0.75H2O
21H22O5
19H18O5
24H28O5
20H20O7
23H26O7
25H30O7
18H12O4
15H9O2Cl‚0.25EtOH
15H9O2Cl‚0.1H2O
15H8O2Cl2
18H16O4‚0.25H2O
18H14O4
20H14O4‚0.25H2O
21H16O4
22H18O4
C,H
C,H
C,H
C,H
C,H
C,H
C,H
C,H
C,H
C,H
a
3a
3b
4
6
7
111-114 355 (FAB)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
100-101 337 (CI)
90-93
397 (CI)
153-156 373 (CI)
122-123 415 (FAB)
115-116 443 (CI)
8
10
11b
11c
11d
15
16
19
20
21
22
23
24
oil
121
293 (CI)
256 (EI)
170-172 257 (CI)
184-186 290 (EI)
160-163 297 (CI)
135-138 294 (EI)
175-178 319 (CI)
148-151 333 (CI)
C,H
a,b
C,H
C,H
C,H
C,H
C,H
C,H
132
347 (CI)
162-165 344 (EI)
162-165 358 (EI)
150-154 320 (CI)
22H16O4‚0.5H2O
23H18O4
19H14NO4
a
High-resolution mass in FAB+ mode m/z determined to be
within acceptable limits. 11d : calcd, 289.9901; found, 289.9893.
b
16: calcd, 294.0892; found, 294.0889. C: calcd, 73.46; found,
74.30. H: calcd, 4.80; found, 5.27.
F igu r e 4. Chemical derivatization of visnagin and alkoxy
substitution at the 5-position.
tively), with a Ki value of 2.65 µM at human A3
receptors. The affinities of tetra- and pentaethyl ether
derivatives of morin, 7 and 8, were compared. The
presence of a free 5-hydroxyl group, in 2′,3,4′,7-tetra-
ethylmorin, 7, greatly diminished affinity at A1 and A2A
receptors and slightly enhanced affinity at A3 receptors,
relative to the pentaethyl ether derivative, 8. Thus,
compound 7 appeared to be selective for human A3
receptors vs rat A1 and A2A receptors. A derivative of
2′,4′,5,7-tetrahydroxyflavone, 5b, that was prenylated33
at the 3- and 6-positions and methylated at the 7-hy-
droxyl group had Ki values of 9.1 and 4.6 µM at rat A1
and human A3 receptors, respectively.
A trimethoxyphenyl derivative of flavonol, 9, only
weakly displaced the binding of [125I]AB-MECA binding
at human A3 receptors. Compound 9 also bound weakly
at rat A2A receptors, but with moderate affinity at rat
A1 receptors (Ki ) 7.3 µM). The combination of an
acetylenic substituent at the 2-position and a 6-methyl
ether group (as in compound 10) failed to significantly
enhance potency or selectivity.
The binding affinities of a number of flavones (non-
hydroxylated at the 3-position) were compared. Flavone
itself, 11a , was 5-fold selective for either rat A1 or A2A
receptors vs human A3 receptors. Compound 11e, a 3,6-
dichloro derivative, was particularly potent and selective
for human A3 receptors. The affinities of compound
11a at cloned rat A3 receptors (Ki ) 5.50 ( 2.221 µM)
and at human A1 receptors (at 100 µM, only 35.0 ( 3.8%
displacement of the binding of 2 nM [125I]AB-MECA)
were also measured. Thus, this flavone derivative is
∼20-fold selective for rat A3 vs rat A1 receptors, and
∼200-fold selective for human A3 vs human A1 receptors.
This compound, obtained from a chemical library of the
National Cancer Institute (NCI), was found by Edwards
et al.34 to lack potential as an anticancer agent. It was
selected in this study for its combination of substituent
groups at 3- and 2′-positions.19 The source of the
selectivity apparently resided with 3,6-dichloro substi-
F igu r e 3. Synthesis of 2-phenylethynyl and 2-styryl deriva-
tives of 4-methoxyfuranochromone.
1, inhibited binding at three adenosine receptor sub-
types with Ki values of 1-3 µM. The trialkylation of
galangin (compounds 2, 3a , and 4) caused an increase
in affinity at human A3 receptors, in the order OPr )
OEt > OMe > OH. At rat A1 and A2A receptors the
affinity either was unchanged (A1) or varied in the order
OH > OEt > OPr > OMe (A2A). The most potent and
“A3 receptor selective” of the galangin ethers was the
tripropyl derivative, 4, which had a Ki value of 0.32 µM
at human A3 receptors and was 3-fold selective vs rat
A1 receptors. The presence of a free 5-hydroxyl group,
in 3b, greatly decreased A2A receptor affinity, while A3
receptor affinity was only 2-fold diminished. Morin, 5a ,
was considerably less potent than galangin at all three
adenosine receptor subtypes.19 However, O-alkylation
caused a gain in affinity, primarily at the human A3
receptors. As previously reported,19 pentamethylmorin,
6, appeared to bind with some selectivity (10- and 18-
fold for human A3 vs rat A1 and A2A receptors, respec-

Synthesis and Biological Activities of Flavonoid Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2297
Ta ble 3. Inhibition of Binding of [125I]AB-MECA at Cloned Rat
A3 Receptors and Effects of Flavone Derivatives (50 µM) on the
A3 Agonist Elicited Inhibition of Adenylyl Cyclase
tution, as indicated by the selectivity of compound 11d ,
which had a Ki value of 0.741 µM at human A3 receptors.
This compound was >100-fold selective for human A3
receptors vs rat A1 receptors and 73-fold selective vs rat
A2A receptors. The presence of trimethyl substitution
of the phenyl ring in 11f did not eliminate selectivity,
although the affinity at A3 receptors was somewhat
diminished. Curiously, either 3-chloro or 6-chloro sub-
stitution alone (11b or 11c) did not result in increased
potency or selectivity for human A3 receptors. 3-Chlo-
roflavone, 11b, was more potent than the 6-chloro
analogue, 11c; however, it did not exceed the affinity
of unsubstituted flavone, 11a .
% inhibition of adenylyl cyclasea
Ki (µM) or %
displacement
compdb
IB-MECA (10-7
)
IB-MECA (10-6
)
control
3a
15
23.7 ( 5.1
2.2 ( 2.2
25.9 ( 2.4
21.2 ( 3.9
35.3 ( 7.2
19.1 ( 7.4
39.2 ( 3.0
40.9 ( 3.7
18.2 ( 5.6
43 ( 3% (100 µM)
12.3 ( 6.6
20
a
Assayed in membranes from CHO cells stably expressing the
rat A3 receptor using a previously reported method4,12 in the
b
presence of 1 µM forskolin (n ) 3). For plot of results for
compound 11e, refer to Figure 5.
A number of derivatives of flavanone, 12, showed
moderate affinity at adenosine receptors. Within a
series of 2,3-saturated derivatives,35 a 2′-hydroxyl group
enhanced the adenosine receptor affinity (e.g. the fla-
vanone derivative 13a ). Compound 13b, 4′-hydroxyfla-
vanone, was less potent in binding to all three subtypes
of adenosine receptors than the corresponding 2′-hy-
droxy analogue. Additional substitution of the A ring
of 4′-hydroxyflavanone, as in sakuranetin, 13c, en-
hanced affinity at human A3 receptors. Previously we
reported that reduction of the 2,3-olefinic bond of the
flavonols, as in trans-(()-dihydroquercetin,19 greatly
diminished affinity at adenosine receptors. Consis-
tently, the 3-hydroxyl group of 14 greatly diminished
affinity relative to 13a .
At rat A3 receptors the affinity of flavones and
flavonols, like that of many xanthines, has been shown
to be weaker than at human A3 receptors.19 The Ki
values of 1 and 2 have been shown to be 43.1 and 17.4
µM, respectively, at rat A3 receptors. This represents
a human A3 vs rat A3 affinity ratio of 14-fold in both
cases. The Ki values of several novel derivatives in this
study (Table 3) have been measured in binding at cloned
rat A3 receptors in membranes of transfected CHO
cells.18,32 The Ki value of 11e at rat A3 receptors was
5.50 ( 2.21 µM; thus it was roughly 20-fold selective vs
rat A1 receptors. In the rat compound 20 was only 3-fold
selective for A3 receptors. The ratios between human
and rat affinities were 50-, 9.8-, and 16-fold for 3a , 11e,
and 20, respectively.
F u n ction a l Assa y a t Clon ed Ra t A3 Recep tor s.
We examined the antagonist properties of several fla-
vonoid derivatives in a functional assay at A3 receptors,
i.e. inhibition of adenylyl cyclase in CHO cells express-
ing cloned rat A3 receptors. Among the derivatives
chosen, compounds 11e, 15, and 20 were subtype
selective in binding, and compound 3a was very potent
at human A3 receptors although nonselective. Adenylyl
cyclase was inhibited by IB-MECA over the concentra-
tion range of 10-9-10-4 M in transfected CHO cells. The
maximal extent of inhibition was 50-60% with an IC50
of approximately 0.1 µM, as reported.12 In Table 3 the
concentration of IB-MECA used was either 0.1 or 1.0
µM, i.e. 1- or 10-fold the IC50 for the inhibition of rat A3
receptor mediated inhibition of adenylyl cyclase, respec-
tively. Compound 3a at a concentration of 50 µM nearly
completely abolished the inhibitory effect of 0.1 µM IB-
MECA on adenylyl cyclase (Table 3). Compound 11e
(Figure 5) also reversed the inhibition of adenylyl
cyclase induced by IB-MECA at several concentrations.
Compounds 15 or 16 at concentrations as high as 50
µM showed no significant effect on potency or maximal
effect of agonist-induced inhibition of adenylyl cyclase.
Among dihydroflavonol analogues, the 2-styryl in-
stead of the 2-aryl substituent, 15, afforded >8-fold
selectivity for human A3 vs rat A1 and A2A receptors.
The corresponding dihydroflavonol 2-phenylethynyl de-
rivative, 16, was nearly as selective as 15 (6.2-fold for
human A3 vs rat A1 receptors).
Visnagin, 17, a vasoactive furanochromone deriva-
tive27,36 isolated from Ammi visnaga, a fruit that has
been used in folk medicine for its antispasmodic proper-
ties and in the treatment of angina pectoris,36 bound
weakly to adenosine receptors. The corresponding
2-aldehyde, 18, was a similarly weak ligand at adeno-
sine receptors. The presence of a trans-styryl group at
the 2-styryl position, in 19, caused an 11-fold enhance-
ment of A3 affinity, with less pronounced enhancements
of affinity at A1 and A2A receptors. Elongation of the
alkyl group at the 6-methyl ether of 19 caused a further
affinity enhancement at A3 receptors, thus the corre-
sponding 6-ethyl ether, 20, was 31-fold selective for
human A3 receptors vs rat A1 receptors, with a Ki value
of 1.16 µM. The selectivity of 20 for human A3 vs rat
A2A receptors was 29-fold. Maximal selectivity was
obtained with 20, since the corresponding 6-propyloxy
derivative, 21, was less selective. The 6-ethoxy and
6-propyloxy derivatives, 20 and 21, were 7.1- and 2.1-
fold more potent, respectively, than the 6-methoxy ether,
19. The 1-phenyl-1,3-butadiene derivative, 22, corre-
sponding to an olefinic homologation of compound 19,
was inactive in binding at all three adenosine receptor
subtypes. The predicted enhancement of A3 receptor
affinity upon ethyl substitution (23) of the 6-methyl
ether, by analogy to compound 20, was insignificant.
Discu ssion
The affinity of xanthines at A3 receptors, although
species dependent, is generally extremely weak relative
to their affinity at other subtypes.14 Thus, the naturally
occurring xanthines do not act as antagonists at A3
receptors, as they do at the other subtypes. It is possible
that certain members of another large family of natural
products, the flavonoids, which are even more wide-
spread in the human diet than the xanthines,38 may
serve in this capacity as A3 receptor antagonists.19 More
than 4000 flavonoids have already been isolated and
identified from vascular plants,38 and they display a
Compound 24, the Schiff’s base equivalent of com-
pound 19, i.e. in which the R-methyne group was
formalistically replaced with nitrogen, bound selectively
to human A3 (Ki 9.2 µM) vs rat A1 receptors.

2298 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Karton et al.
to have affinity for many other proteins, it will be
necessary to screen these derivatives in many other
biochemical assays and at other receptors in order to
establish true selectivity.
Conformational insights may be obtained from the
present results. Compound 11e was highly selective for
the human A3 receptor. In this derivative both the 3-
and 2′-positions are highly substituted; thus there is
likely steric hindrance to free rotation of the phenyl ring
and assumption of a planar geometry. The potency and
selectivity were similar to compound 11d , which was
not as highly hindered sterically. Thus, we may con-
clude that a coplanar geometry between the two ring
systems in the flavonols is not required.
Functional antagonism was indicated for derivatives,
compounds 3a and 11e. It will be necessary to study
the pharmacological effects in greater detail, in order
to determine that other sites of interaction, for example
at the level of G-proteins and second messengers, are
not involved in the observed biological effect on adenylyl
cyclase. It is likely that only flavonoids having Ki values
of e1 µM at human A3 receptors will be useful as
pharmacological probes.
In conclusion, these series of flavonoids provide leads
for the development of novel potent and selective A3
adenosine receptor antagonists,14 although more de-
tailed pharmacological characterization is required. The
present findings indicate that the chemical modification
of flavones at the 2- (with styryl groups), 3- (with
hydroxyl or chloro), 6- (with chloro), and 2′-positions
(with sterically bulky groups) may prove fruitful in this
regard. The 2,3-position double bond appears to be
required for recognition by human A3 receptors when
both a 2-phenyl substituent and a 3-hydroxyl group are
present. However the double bond is not an absolute
requirement, since flavanone derivatives 13a -c, which
lack the 3-hydroxyl group, displayed moderate affinity
but not selectivity. Another means of restoring affinity
in the reduced flavonol derivatives was to introduce a
styryl or phenylethynyl group at the 2-position (com-
pounds 15 and 16). Additional stucture-activity stud-
ies will be needed in order to design flavonoid analogues
that are highly A3 receptor selective across species.
F igu r e 5. Inhibition of adenylate cyclase in membranes from
CHO cells stably transfected with rat A3 receptors elicited by
the agonist N6-(3-iodobenzyl)adenosine-5′-(N-methylurona-
mide) (IB-MECA), alone in the presence of 50 µM compound
11e (Ki ) 5.50 ( 2.21 µM at rat A3 receptors). The assay was
carried out as described in Experimental Section in the
presence of 1 µM forskolin. Each data point is shown as mean
( S.E.M. for three determinations. *p < 0.05, **p < 0.001 (t
test).
very wide range of biological activities. For example,
flavonoids are used therapeutically to improve circula-
tion and integrity of blood vessels40 and to suppress
inflammation.41 They have been proposed as a potential
treatment for diabetes, allergic reactions, infections, and
other disorders.42 Even antineoplastic activities have
been claimed for flavonoids.34 Many of these reported
biological effects have not yet been fully explained
mechanistically, although many mammalian enzymes
and other proteins regulating intracellular biochemical
processes, such as protein tyrosine kinases,23,24 are
known to be affected by flavonoids.43,44 Flavones also
inhibit phosphodiesterases and HIV integrase.48,49 Fla-
vones have been studied in relation to the biological
activity of adenosine45 and adenosine deaminase, which
is inhibited at high concentrations of certain flavones.46
In several recent reports, flavones were found to bind
to adenosine receptors,19,20 and certain derivatives have
substantial affinity at the A3 receptor subtype.19 We
identified flavones and flavonols as moderately potent
adenosine receptor ligands as a result of screening a
natural products library. Ares et al.20 independently
discovered the affinity of flavones at A1 and A2A recep-
tors in an investigation of the known gastroprotective
effects of the substances,47 which yet lack a mechanistic
explanation.
Exp er im en ta l Section
Ma ter ia ls. Compounds 1, 11a , 12, and 17 were obtained
from Fluka, Ronkonoma, NY, or from Aldrich, St. Louis, MO.
Compounds 13a , 13c, and 14 were obtained from Apin
Chemicals, Ltd., Oxon, U.K. Compounds 5a and 13b were
obtained from K+K Laboratories, J amaica, NY. Compounds
5b (NSC #241010-z), 9 (NSC #78634-f), 11e (NSC #74899-t),
and 11f (NSC #74931-f) were obtained from NCI (Bethesda,
MD).
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
Microlabs, Inc. (Norcross, GA).
3,5,7-Tr im eth oxyfla von e (2). Galangin (27 mg, 0.1 mmol)
was dissolved in dry acetone (stored over K2CO3, 20 mL), solid
potassium carbonate (0.5 g) and dimethyl sulfate (1 mL) were
added, and the mixture was refluxed for 4 h and then cooled
to room temperature. The solution was filtered and evapo-
rated, water (20 mL) and concentrated ammonium hydroxide
(2 mL) were added, and the solution was extracted with ethyl
acetate (15 mL × 2). The solvent was evaporated, and the
residue was recrystallized from methanol/water to give 21 mg
of product (67%): 1H-NMR (DMSO-d6) δ 3.73 (s, 3H, 3-OCH3),
The most selective compounds for human A3 receptors
identified in this study were compounds 7 and 11d -f,
which were all essentially inactive at A1 and A2A
receptors. The presence of a 5-hydroxyl group, as in 3b
and 7, increased selectivity of flavonols for human A3
receptors. The most potent compounds in binding to A3
receptor (in order of increasing Ki value) were com-
pounds 4, 3a > 11e, 11d g 3b g 20, 2 > 6. The degree
of selectivity achieved was as high as 200-fold for
compound 11e (Table 1). Since flavonoids are known

Synthesis and Biological Activities of Flavonoid Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2299
3.86 (s, 3H, 7-OCH3), 3.86 (s, 3H, 5-OCH3), 6.48 (s, 1H, 8-H),
6.79 (s, 1H, 6-H), 7.99 (m, 3H, Ar), 8.02 (m, 2H, Ar); MS (CI/
NH3) m/z 313 (MH+, base).
2-(P h en ylet h yn yl)-3-h yd r oxy-6-m et h oxyfla von e (10).
1-(2-Hydroxy-5-methylphenyl)-5-phenylpenta-2-en-4-yn-1-
one (compound 27b, 60 mg, 0.2 mmol) was dissolved in ethanol
(1.5 mL). Sodium hydroxide (1 N, 0.4 mL) and hydrogen
peroxide (0.5 g) were added, and the mixture was heated at
90 °C for 35 min. The cooled mixture was diluted with water
(5 mL) and acidified with hydrochloric acid (1 N). A precipitate
was collected and recrystallized from methanol: mass (CI/NH3)
m/z 293 (M+ + 1, base); 1H-NMR (CDCl3) δ 3.95 (s, 3H, CH3),
6.92 (s, 1H), 7.25-8.0 (m, 7H).
3-Ch lor ofla von e (11b). Sulfuryl chloride (85 mg, 0.55 mL)
in 2 mL of carbon tetrachloride was added dropwise to a
solution of flavone (111 mg, 0.5 mmol) in 5 mL of carbon
tetrachloride. The mixture was stirred overnight. The solu-
tion was diluted with CH2Cl2 (5 mL), washed with water (10
mL × 2), saturated sodium bicarbonate solution (10 mL × 2),
and brine (10 mL × 2), and dried over sodium sulfate. The
solvent was evaporated, and the residue was purified on a
preparative TLC plate (silica, ethyl acetate/petroleum ether,
2:8) to give 80 mg of product (63%): 1H-NMR (CDCl3) δ 7.46-
7.58 (m, 5H, 2-C6H5), 7.74 (m, 1H, 7-H), 7.94 (m, 2H, 5,6-H),
8.33 (dd, 1H, J ) 7.8, 2.0 Hz, 8-H); MS (EI) m/z 256 (M+, base),
228 (M - CO)+, 221 (M - Cl)+; mp 121 °C.
6-Ch lor ofla von e (11c). Bromine (185 mg, 1.15 mmol) was
added a solution of 2-hydroxy-5-chlorochalcone (258 mg, 1.0
mmol) in acetic acid (10 mL). After the reaction was stirred
at ambient temperature for 3 h, 1% aqueous sodium bisulfite
solution (20 mL) was added slowly. The resulting precipitate
was filtered off, washed with water, and suspended in ethanol
(10 mL); potassium hydroxide (200 mg, 3.5 mmol) dissolved
in water (3.5 mL) was added; and stirring was continued
overnight. The reaction mixture was acidified with 2 N HCl.
The precipitate formed was filtered off and washed with water
to give 105 mg of residue, which was recrystallized from EtOH/
H2O to give 85 mg of product (33%): 1H-NMR (CDCl3) δ 6.85
(s, 1H, 3-H), 7.54-7.69 (m, 5H, 2-C6H5), 7.93 (m, 2H, 5, 7-H),
8.22 (d, 1H, J ) 2.7 Hz, 8-H); MS (CI/NH3) m/z 257 (MH+,
base) and 259 (M + 2H+) in 3:1 ratio; mp 170-172 °C.
3,6-Dich lor ofla von e (11d ). Sulfuryl chloride (28 µL, 0.28
mmol) in 2 mL of carbon tetrachloride was added dropwise to
a solution of 6-chloroflavone (65 mg, 0.25 mmol) in 5 mL of
carbon tetrachloride. The mixture was stirred overnight. The
solution was diluted with CH2Cl2 (5 mL), washed with water
(5 mL × 2), saturated sodium bicarbonate solution (5 mL ×
2), and brine (5 mL × 2), and dried over sodium sulfate. The
solvent was evaporated, and the residue was purified on a
preparative TLC plate (silica, ethyl acetate/petroleum ether,
2:8) to give 8 mg of product (10%): 1H-NMR (CDCl3) δ 7.49-
7.70 (m, 5H, 2-C6H5), 7.91 (m, 2H, 5,7-H), 8.28 (d, 1H, J ) 2.6
Hz, 8-H); MS (EI) m/z 290, 292, 294 (M+:(M + 2+):(M + 4+) )
9:6:1); mp 184-186 °C.
3,5,7-Tr ieth oxyflavon e (3a). Galangin (30 mg, 0.11 mmol)
was dissolved in dry acetone (45 mL), solid potassium carbon-
ate (0.5 g) and iodoethane were added, and the mixture was
refluxed overnight. The solution was filtered and evaporated,
water (20 mL) and concentrated ammonia (1 mL) were added,
and the solution was extracted with ethyl acetate. The ethyl
acetate was evaporated, and the crude mass was purified on
a preparative silica TLC plate to give 12 mg of 3,5,7-triethoxy-
flavone: mp 111-114 °C; mass (FAB) m/z 355 (M+ + 1, base);
1H-NMR (CDCl3) δ 1.33 (t, 3H, CH3), 1.55 (t, 3H, J ) 7 Hz,
CH3), 1.62 (t, 3H, J ) 7 Hz, CH3), 4.1-4.25 (m, 6H, CH2), 6.4
(s, 1H), 6.55 (s, 1H), 7.5-7.6 (m, 3H), 8.1-8.2.
3,7-Dieth oxy-5-h yd r oxyfla von e (3b). Galangin (27 mg,
0.1 mmol) was dissolved in dry acetone (20 mL), solid potas-
sium carbonate (0.5 g) and bromoethane (1 mL) were added,
and the mixture was stirred overnight, at room temperature.
The solution was filtered and evaporated, water (20 mL) and
concentrated ammonium hydroxide (2 mL) were added, and
the solution was extracted with ethyl acetate (20 mL × 2). The
solution was dried, and the solvent was evaporated. The
residue was purified by preparative TLC plate (silica, ethyl
acetate/petroleum ether (2:8) to give 21 mg of product (64%):
1H-NMR (CDCl3) δ 1.34 (t, 3H, J ) 7.3 Hz, 3-CH3), 1.48 (t,
3H, J ) 7.1 Hz, 7-CH3), 4.14 (m, 4H, 2 × OCH2), 6.38 (s, 1H,
6-H), 6.47 (s, 1H, 8-H), 12.64 (s, 1H, 5-OH); MS (CI/NH3) m/z
327 (MH+, base).
3,5,7-Tr ip r op yloxyfla von e (4). Galangin (30 mg, 0.11
mmol) was dissolved in dry acetone (45 mL), solid potassium
carbonate (0.5 g) and 1-iodopropane were added, and the
mixture was refluxed overnight. The solution was filtered and
evaporated, water (20 mL) and concentrated ammonia (1 mL)
were added, and the solution was extracted with ethyl acetate.
The ethyl acetate was evaporated, and the crude mass was
purified on a preparative TLC plate (silica) to give 25 mg (63%)
of 3,5,7-tripropyloxyflavone: mp 90-93 °C; mass spectra (CI/
NH3) m/z 397 (M+ + 1, base); 1H-NMR (CDCl3) δ 0.85-1.2 (m,
9H), 1.6-2.1 (m, 6H), 3.9-4.1 (m, 6H), 6.32 (s, 1H), 6.5 (s, 1H),
7.4-7.5 (m, 3H), 8.04-8.12 (m, 2H).
2′,3,4′,5,7-P en ta k is(m eth yloxy)fla von e (6). Morin (120
mg, 0.4 mmol) was dissolved in dry acetone (80 mL). Solid
potassium carbonate (2 g) and iodomethane (2 mL) were added,
and the mixture was refluxed overnight and then cooled to
room temperature. The solution was filtered and evaporated,
and the residue was separated by preparative TLC plates
(silica, ethyl acetate) to give 80 mg of product (55%); 1H-NMR
(CDCl3) δ 3.86, 3.88, 3.91, 3.93, 4.02 (5s, 15H, 5 × OCH3), 6.79
(3m, 3H, phenyl), 6.65 (m, 1H, 8-H), 7.42 (m, 1H, 6-H); MS
(EI) m/z 372 (M+), 371 (M - H)+, 341 (M - OCH3)+.
tr a n s-2-Styr yl-3-h yd r oxy-6-m eth oxyfla va n on e (15). 2′-
Hydroxy-5′-methoxyacetophenone (25, 4.1 g, 22 mmol) and
cinnamaldehyde (3.35 g, 25 mmol) were dissolved in minimal
methanol (2.5 mL). Concentrated sodium hydroxide (12.5 mL,
50%) was added, and the mixture was kept on ice for 8 h. The
resulted solid was suspended in water and acidified using HCl
(4 N). The oil that separated was dissolved in ethanol and
crystallized from ethanol/water to give 1-(2-hydroxy-5-meth-
ylphenyl)-5-phenylpenta-2,4-dien-1-one (1.2 g) as a red-brown
powder: mass (CI/NH3) m/z 281 (M+ + 1, base); 1H-NMR
(CDCl3) δ 3.9 (s, 3H, CH3), 6.95-7.8 (m, 12H). The above
compound (170 mg, 0.6 mmol) was dissolved in an mixture of
ethanol (3.5 mL) and acetone (4 mL). Sodium hydroxide (1
N, 1 mL) and hydrogen peroxide (1 mL, 35%) were added, and
the solution was stirred for 6 h at room temperature. The
mixture was precipitated by adding water and HCl and
purified on prep TLC (petroleum ether/ethyl acetate) to give
2′,4′,3,7-Tetr a eth oxy-5-h yd r oxym or in (7). Compound 7
was synthesized according to the above procedure for 6, except
using iodoethane. The product, 7, was separated on a pre-
parative TLC plate with petroleum ether/ethyl acetate and
displayed a mass (FAB) m/z 415 (M+ + 1, base): 1H-NMR
(CDCl3) δ 1.15 (t, 3H, CH3), 1.35 (t, 3H, CH3), 1.45 (dt, 6H,
CH3), 3.9-4.2 (m, 8H, CH2), 6.33 (d, 1H), 6.6 (m, 3H), 7.4 (d,
1H).
2′,3,4′,5,7-P en ta eth oxyfla von e (8). Morin (302 mg, 1
mmol) was dissolved in dry acetone (80 mL), solid potassium
carbonate (5 g) and diethyl sulfate (5 mL) were added, and
the mixture was refluxed overnight and then cooled to room
temperature. The solution was filtered and evaporated, water
(50 mL) and concentrated ammonium hydroxide (10 mL) were
added, and the solution was extracted with ethyl acetate (40
mL × 2). The solution was dried, and the solvent was
evaporated. The residue was crystallized from ethyl acetate
and petroleum ether (1:9) to give 335 mg of product (76%):
1H-NMR (CDCl3) δ 1.12 (t, 3H, J ) 7.1 Hz, CH3), 1.32 (t, 3H,
J ) 7.1 Hz, CH3), 1.45 (2t, overlap, 6H, 2 × CH3), 1.56 (t, 3H,
J ) 7.1 Hz, CH3), 4.10 (m, 10 H, 5 × OCH2),6.32 (d, 1H, J )
1.2 Hz, 2-phenyl), 6.39 (s, 1H, 2-phenyl), 6.53 (s, 1H, 2-phenyl),
6.58 (s, 1H, 6-H), 7.37 (d, 1H, 8-H); MS (CI/NH3) m/z 443 (MH+,
base).
compound 15: mp 160-163 °C; mass (CI/NH3) m/z 297 (M+
+
1
1,base); H-NMR (CDCl3) δ 3.9 (s, 3H, CH3), 4.45 (d, J ) 12
Hz, 1H), 4.8 (m, 1H), 6.55 (dd, 1H), 6.9-7.6 (m, 9H).
tr a n s-2-(P h en ylet h yn yl)-3-h yd r oxy-6-m et h oxyfla va -
n on e (16). 2′-Hydroxy-5′-methoxyacetophenone and phenyl-
propargyl aldehyde were condensed according to the procedure
for the preparation of compound 15 to give 1-(2-hydroxy-
5-methylphenyl)-5-phenylpenta-2-en-4-yn-1-one: mass (CI/

2300 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Karton et al.
1
NH3) m/z 279 (M+ + 1, base); H-NMR (CDCl3) δ 3.9 (s, 3H,
prepared according to ref 28) and aniline (0.5 mL) were
dissolved in toluene and stirred overnight at 25 °C. The course
of the reaction was followed using analytical TLC (silica,
CHCl3/MeOH, 20:1). The solvent was evaporated, and the
excess aniline was removed under high vacuum. The product
(Rf ∼ 0.7) was purified using preparative TLC (silica, ethyl
acetate): mass (CI/NH3) m/z 320 (M+ + 1, base).
CH3), 7.0 (d, 1H), 7.1-7.6 (m, 9H). The above compound was
allowed to react with hydrogen peroxide to give compound 16
as a white powder: mp 135-138 °C; mass (EI) m/z 295 (M+1),
150 (base); 1H-NMR (CDCl3) δ 3.85 (s, 3H, OCH3), 4.6 (d, J )
12 Hz, 1H), 5.09 (d, J ) 12 Hz, 1H), 7.0-7.6 (m, 8H).
4-Met h oxy-7-for m yl-5H -fu r o[3,2-g][1]b en zop yr a n -5-
on e (18) was synthesized from visnagin (4-methoxy-7-methyl-
5H-furo[3,2-g][1]benzopyran-5-one) according to ref 28.
4-(Meth yloxy)-7-tr a n s-styr ylvisn a gin (19) a n d 4-(Eth -
yloxy)-7-tr a n s-styr ylvisn a gin (20). Visnagin (160 mg, 0.7
mmol) and benzaldehyde (120 mg, 1.1 mmol) were dissolved
in ethanol (4 mL). Sodium ethoxide (20% in ethanol, 0.5 mL)
was added, and the mixture was stirred for 10 min at 80 °C.
Disappearance of the starting material was accompanied by
formation of two products, Rf 0.7 and 0.8 in ethyl acetate. Both
were separated on preparative TLC plates (ethyl acetate) to
give compound 19: mp 175-178 °C; mass (CI/NH3) m/z 319
(M+ + 1, base); 1H-NMR (CDCl3) δ 4.2 (s, 3H, OCH3), 6.23 (s,
1H), 6.75 (d, J ) 15 Hz, 1H), 7.05 (s, 1H), 7.3-7.8 (m, 8H).
Compound 20: mp 148-151 °C; mass (CI/NH3) m/z 333 (M+
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.17,18 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% fetal bovine serum and
penicillin/streptomycin (100 units/mL and 100 µg/mL, respec-
tively) at 37 °C in a 5% CO2 atmosphere, and membrane
homogenates were prepared as reported.18 Binding of [125I]-
N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)
([125I]AB-MECA) to rat A3 receptors in stably transfected CHO
cell membranes was performed as described.32 Assays at
human A3 receptors were performed in a buffer containing 50
mM Tris, 10 mM Mg2+, 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
1
+ 1, base); H-NMR (CDCl3) δ 1.55 (t, 3H, CH3), 4.4 (q, 2H,
OCH2), 6.23 (s, 1H), 6.78 (d, J ) 15 Hz, 1H), 7.0 (s, 1H), 7.35-
7.7 (m, 8H).
[
125I]AB-MECA (final concentration 0.3 nM), and 50 µL of a
4-(P r op yloxy)-7-tr a n s-styr ylvisn a gin (21). A mixture of
visnagin (2 g, 8.7 mmol), potassium iodide (10 g), and acetic
acid (50 mL) was refluxed for 7 h. After cooling, the precipitate
was removed by filtration, and the filtrate was evaporated
under reduced pressure, and coevaporated with toluene (10
mL × 2). The residue was crystallized from ethanol to give
1.62 g (81%) of demethylation product of visnagin: 1H-NMR
(CDCl3) δ 2.40 (s, 3H, 7-CH3), 6.12 (s, 1H, 6-H), 7.00 and 7.62
(2d, 2H, J ) 2.9 Hz, 3-H and 2H), 7.05 (s, 1H, 9-H), 13.6 (s,
1H, 4-OH). The mixture of demethylated visnagin (430 mg, 2
mmol), iodopropane (3 mL), and potassium carbonate (5 g) in
dry acetone (80 mL) was refluxed overnight. The solid was
removed by filtration, the solvent was evaporated, water (50
mL) and concentrated ammonium hydroxide (15 mL) were
added, and the solution was extracted with ethyl acetate (40
mL × 2). The organic layer was dried over sodium sulfate,
and the solvent was evaporated. The residue was purified by
preparative TLC plates (silica, ethyl acetate/petroleum ether,
4:6) to give 465 mg (90%) of 4-(propyloxy)visnagin (30): 1H-
NMR (CDCl3) δ 1.10 (t, 3H, J ) 7.5 Hz, CH3), 1.94 (m, 2H),
2.33 (s, 3H, 7-CH3), 4.23 (t, 2H, J ) 6.7 Hz, 4-OCH2), 6.03 (s,
1H, 6-H), 6.97 and 7.60 (2d, 2H, J ) 2.7 Hz, 3-H and 2-H),
7.27 (s, 1H, 9-H). 4-(Propyloxy)visnagin (86 mg, 0.3 mmol)
and benzaldehyde (50 mg, 0.5 mmol) were dissolved in ethanol
(2 mL). While stirring, sodium ethoxide (20% in ethanol, 0.3
mL) was added, and the mixture was stirred for 15 min at 80
°C. The solvent was evaporated, the residue was partitioned
between ice water (20 mL) and ethyl acetate (20 mL), the
aqueous layer was extracted with ethyl acetate (20 mL), and
the combined organic layer was dried over sodium sulfate. The
solvent was evaporated, and the residue was separated by
preparative TLC plate (silica, ethyl acetate/petroleum ether,
4:6) to give 26 mg of the desired product (25%): 1H-NMR
(CDCl3) δ 1.07 (t, 3H, J ) 7.5 Hz, CH3), 1.97 (m, 2H), 4.25 (t,
2H, J ) 6.7 Hz, 4-OCH2), 6.21 (s, 1H, 6-H), 6.78 (d, 2H, J )
14 Hz, 3-H), 7.00 (s, 1H, 9-H), 7.33 (m, 8H); MS (CI/NH3) m/z
347 (MH+, base).
solution of the proposed antagonist. All nonradioactive com-
pounds 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 25 °C19 and were terminated by rapid filtration over
Whatman GF/B filters, using a Brandell cell harvester (Bran-
dell, 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.29,30
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.39 A Kd value of 0.59
nM for [125I]AB-MECA binding at human A3 receptors was
assumed.19
Adenylyl cyclase activity was measured in membranes from
CHO cells stably expressing the rat A3 receptor, prepared as
above, using a previously reported method.4,12 The method
involved addition of [R-32P]ATP to membranes in the presence
of forskolin to stimulate adenylyl cyclase and papaverine as a
phosphodiesterase inhibitor. The reaction was terminated by
addition of a stop solution containing 20 000 cpm/mL [3H]cyclic
AMP. The total radiolabeled cyclic AMP was isolated on
columns of Dowex 50 ion exchange resin and alumina.
Maximal inhibition of adenylyl cyclase activity corresponded
to 40-50% of total activity under conditions of stimulation
(typically 6-8-fold) in the presence of 1 µM forskolin. IC50
values were calculated using InPlot (GraphPad, San Diego,
CA).
4-Meth oxy-7-(4-ph en yl-1,3-p en ta d ien yl)-5H-fu r o[3,2-g]-
[1]ben zop yr a n -5-on e (22)a n d 4-E t h oxy-7-(4-p h en yl-1,3-
pen tadien yl)-5H-fu r o[3,2-g][1]ben zopyr an -5-on e (23). Com-
pound 22 was prepared by dissolving visnagin and cinnam-
aldehyde in ethanol in the presence of sodium ethoxide
according to the above procedure for 19 and 20: mp 162-165
1
°C; mass (EI) m/z 344 (M+, base); H-NMR (CDCl3) δ 4.2 (s,
Ack n ow led gm en t. We thank Marc Glashofer for
technical assistance and helpful discussions and Dr.
George W. Milne of the National Cancer Institute
(Bethesda, MD) for providing samples of several of the
compounds used in this study. We thank Gilead Sci-
ences (Foster City, CA) and the Cystic Fibrosis Founda-
tion for financial support. We thank Dr. Pat Towers of
Amersham (Cardif, U.K.) and Dr. Garth Brown of
3H, OCH3), 6.15 (s, 1H), 6.33 (d, J ) 15 Hz, 1H), 6.95 (s, 1H),
7.0-7.6 (m, 10H).
Compound 23 was also isolated from the above reaction:
mass (EI) m/z 358 (M+), 343 (M - 15, base); 1H-NMR (CDCl3)
δ 1.55 (t, 3H, CH3), 4.4 (q, 2H, OCH2), 6.15 (s, 1H), 6.35 (d, J
) 15 Hz, 1H), 7.4 (d, J ) 15 Hz, 1H), 6.9-7.6 (m, 10H).
4-Met h oxy-7-for m yl-5H -fu r o[3,2-g][1]b en zop yr a n -5-
on e An ilin e Sch iff ba se (24). Compound 18 (400 mg,

Synthesis and Biological Activities of Flavonoid Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2301
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