Synthesis of hybrid analogues of caffeine and eudistomin D and its affinity for adenosine receptors

Article abstract of DOI:10.1016/j.bmc.2009.05.036

Four bis-N-n-propyl analogues (3-6) in the uracil ring of two hybrid molecules (1 and 2) of caffeine and eudistomin D, a β-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A₁

Full text of DOI:10.1016/j.bmc.2009.05.036

Bioorganic & Medicinal Chemistry 17 (2009) 4280–4284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of hybrid analogues of caffeine and eudistomin D and its affinity
for adenosine receptors
Haruaki Ishiyama a, Hikaru Nakajima a, Hiroyasu Nakata b, Jun’ichi Kobayashi a,
*
a Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
b Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Four bis-N-n-propyl analogues (3–6) in the uracil ring of two hybrid molecules (1 and 2) of caffeine and
eudistomin D, a b-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and
selectivity for adenosine receptors A1, A2A, and A3 were examined. All the compounds (3–6) showed bet-
ter potency as adenosine receptor ligands than caffeine. Bis-N-n-propylation (3 and 4, respectively) of the
uracil ring in 1 and 2 resulted in higher affinity for A1 and A2A adenosine receptors. Furthermore, it was
found that a compound (5) possessing a n-propyloxy group at C-7 in compound 3 with a nitrogen at the
b-position of the pyridine ring (b-N type) enhanced remarkably affinity for adenosine receptor A3 sub-
type, while n-propyloxy substitution (compound 6) at C-5 in compound 4 with a nitrogen at the d-posi-
tion of the pyridine ring (d-N type) reduced affinity for all the adenosine receptor, A1, A2A, and A3. Among
all the compounds (1–6) examined, compound 5 showed the most potent affinity for adenosine receptor
Received 27 April 2009
Revised 13 May 2009
Accepted 14 May 2009
Available online 18 May 2009
Keywords:
Hybrid analogues
Caffeine
Adenosine receptor
SAR
A3 subtype (Ki value, 0.00382 lM).
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
bis-N-alkyl group in the uracil ring on affinity for adenosine recep-
tors (Scheme 3), while n-propyloxy substituted analogues (5 and 6,
respectively) of the pyridine ring in 3 and 4 were synthesized to
study the effects of the substituent in the pyridine ring on the affinity
for adenosine receptors (Scheme 4).
Caffeine shows a various activities (or action) including effects to
the blood pressure, urine volume, respiratory functioning, gastric
and colonic activity, and exercise performance.1 The mechanism of
actions of caffeine is involved in competitive antagonism to A1 and
A2A adenosine receptors,2a inducing of Ca2+-release from sarcoplas-
mic reticulum (SR), inhibition of phosphodiesterase, and so on. Dur-
ing our continuing search for bioactive compounds from marine
organisms, we have found that eudistomin D, a b-carboline alkaloid
isolatedfrom a marinetunicate Eudistomaolivaceum3 and its analogs
are potent inducer of Ca2+-release from SR4 as well as inhibitors of
phosphodiesterase.5 Previously, our group has found that a com-
pound (2) of two types of hybrid molecules (1 and 2) of caffeine
and eudistomin D showed a potent affinity for adenosine receptor
A3 subtype (Scheme 1).6 Selective ligands for adenosine receptor
subtypes, A1, A2A, A2B, and A3, are expected as novel drug candidates,
for example, anticancer agents for A3 agonists and anti-asthma
agents for A3 antagonists.2b In the present study, to obtain more po-
tent ligand to adenosine receptor A3 subtype, we synthesized bis-N-
n-propyl analogues (3–6) in the uracil ring of compounds 1 and 2,
O
N
O
O
N
N
N
N
N
N
H
N
H
O
4
6
3
O
O
N
N
O
N
O
N
N
N
N
O
N
H
H
O
5
2. Results and discussion
2.1. Chemistry
and their affinity and selectivity for adenosine receptors A1, A2A
and A3 were examined. The bis-N-n-propyl derivatives (3 and 4,
respectively) of 1 and 2 were prepared to examine effects of the
,
Retrosynthetic analysis of 3–6 were outlined in Scheme 2. A key
step to prepare both b-N type and d-N type analogues (7 and 8,
respectively) with a nitrogen at the b- or d-position of the pyridine
* Corresponding author. Tel.: +81 011 706 3239; fax: +81 011 706 4989.
E-mail address: jkobay@pharm.hokudai.ac.jp (J. Kobayashi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.036

H. Ishiyama et al. / Bioorg. Med. Chem. 17 (2009) 4280–4284
4281
Br
H
O
N
O
O
N
N
N
O
N
a
b
HO
N
N
HN
N
N
H
O
O
caffeine
12
13
eudistomin D
O
N
O
O
N
N
O
N
O
N
c
+
N
N
N
N
H
N
H
N
N
O
NH2
NO2
O
O
O
1
2
15
14
Scheme 1. Hybrid compounds (1 and 2) of caffeine and eudistomin D.
d
N
O
N
R1
N
R3
+
N
NH2
Cl
O
N
N
16
15
R2
N
H
R1
N
O
O
N
7
P
N
R2
N
H
R3
O
N
O
N
R1
N
O
9
O
N
N
N
H
N
R2
N
R3
17
H
O
XPhos
R1
8
N
O
N
+
N
Cl
R3
N
O
R2
NH2
O
N
N
N
H
11
10
O
e
Scheme 2. Retrosynthetic analysis of compounds 7 and 8.
3 (6%)
+
17
ring in one reaction is photocyclization of 9. Compound 9 could be
derived from 5-aminouracil derivative (10) and 3-chloropyridine
derivative (11) employing Buchwald’s X-Phos catalyzed amination
reaction7 (Scheme 2).
The synthesis of 3 and 4 is summarized in Scheme 3. Treatment
of uracil (12) with 1-bromopropane and NaH in DMF yielded 1,3-
bis-n-propyluracil (13),8 which was nitrated using fuming nitric
acid to give 5-nitro-1,3-bis-n-propyluracil (14).9 Reduction of nitro
group in 14 using Fe provided 5-amino-1,3-bis-n-propyluracil
(15).10 Coupling of 15 and 3-chloropyridine (16) in DMF with
Pd2(dba)3, X-Phos,7 and Cs2CO3 afforded 17 in 67% yield. Photocyc-
lization of 17 in toluene afforded 3 and 4 in 6%, respectively
(Scheme 3).
O
N
O
N
N
N
H
4 (6%)
Scheme 3. Synthesis of compounds 3 and 4. Reagents and conditions: (a) 1-
bromopropane, NaH/DMF, 70 °C, 3 h (92%); (b) HNO3 (fuming)/H2SO4, 0 °C–rt, 6 h
(98%); (c) Fe/AcOH, rt, 4 h (86%); (d) Pd2(dba)3, XPhos, Cs2CO3/DMF, 65 °C, 10 h
(67%); (e) hv/toluene, rt, 9 h.
summarized in Table 1. When the affinity of the test compounds
In Scheme 4 is shown the synthesis of 5 and 6. Alkylation of 3-
chloro-5-hydroxypiridine (18) with 1-bromopropane and NaH pro-
vided 3-chloro-5-n-propyloxypyridine (19).11 Buchwald’s X-Phos
catalyzed amination7 of 19 with 15 yielded aminopyridine (20).
Photocyclization of 20 in toluene gave 5 and 2112 in 14% and 12%
yield, respectively, and treatment of 21 with DDQ furnished 6.
were very low, percentage of inhibition at 100 lM was shown.
Affinities of reference ligands, that is, the non-selective caffeine
and 50-(N-ethylcarboxamido)adenosine (NECA), the A1-selective
xanthine amine congener (XAC) and the A2A-selective CGS21680,
determined under the same conditions, are also shown for compar-
ison. These compounds showed reasonable affinity for each recep-
tor confirming the validity of our assay.
2.2. Biological evaluation
Previously, our group has found that a d-N type compound 2
exhibited potent affinity to all the adenosine receptors, A1, A2A
,
The potency of these newly synthesized compounds as adeno-
sine receptor ligands was investigated by radioligand binding as-
and A3, especially high affinity for A3. The Ki value of 2 to adenosine
A3 receptor was comparable to that of a known A3 agonist, NECA
(Table 1). The d-N type compound (2) showed better affinity to
all the adenosine A1, A2A, and A3 receptors than that of the corre-
sponding b-N type compound (1).
says using human recombinant adenosine A1, A2A
, and A3
receptors expressed in cell membranes of HEK293T cells as de-
scribed under ‘Section 3’. The results expressed as Ki values are

4282
H. Ishiyama et al. / Bioorg. Med. Chem. 17 (2009) 4280–4284
it was found that a compound (5) possessing a n-propyloxy group
N
N
a
at C-7 in compound 3 with a nitrogen at the b-position of the pyr-
idine ring (b-N type) enhanced remarkably affinity for adenosine
receptor A3 subtype, while n-propyloxy substitution (compound
6) at C-5 in compound 4 with a nitrogen at the d-position of the
pyridine ring (d-N type) reduced affinity for all the adenosine
receptor A1, A2A, and A3.
Among all the compounds (1–6) examined, compound 5 exhib-
ited the most potent affinity for the adenosine receptor A3. The Ki
value (0.00382 lM) of 5 to adenosine A3 receptor was more potent
Cl
O
OH
Cl
N
O
18
19
N
b
+
N
O
Cl
O
NH2
O
than that (Ki = 0.020 lM) of a known A3 agonist, NECA.
19
15
3. Experimental
3.1. Chemistry
c
N
N
N
3.1.1. Instruments and analyses
N
H
O
The IR spectrum was taken on a JASCO FT/IR-230 spectrometer.
Proton and carbon NMR spectra were recorded on a JEOL 400 and/
or 500 MHz spectrometer. EI mass spectra were obtained on a JEOL
JMS-FABmate spectrometer.
O
20
O
H
H
3.1.2. 1,3-Bis-n-propyluracil (13)
N
O
N
O
N
O
+
To a suspension of uracil (12; 1.12 g, 10 mmol) in DMF (2 mL)
was added NaH (60% dispersion in mineral oil, 0.96 g, 24 mmol)
and 1-bromopropane (2.2 mL, 24 mmol). After 3 h at 70 °C, the
reaction mixture was purified by column chromatography on silica
gel (n-hexane/EtOAc, 2:1) to give 13 (1.82 g, 92%).
N
N
N
N
N
H
O
H
O
5 (14%)
21 (12%)
Compound 13: 1H NMR (400 MHz, CDCl3) d 0.94 (3H, t, J = 7.2,
7.7 Hz), 0.95 (3H, t, J = 7.2 Hz), 1.65 (2H, ddq, J = 7.2, 7.7 Hz), 1.72
(2H, ddq, J = 7.2, 7.7 Hz), 3.70 (2H, dd, J = 7.2 Hz), 3.90 (2H, dd,
J = 7.7 Hz), 5.71 (1H, d, J = 8.2 Hz), 7.09 (1H, d, J = 8.2 Hz); 13C NMR
(100 MHz, CDCl3) d 10.8, 11.2, 20.7, 22.2, 42.6, 51.22, 101.3, 142.1,
151.3, 163.0.
O
N
N
d
N
21
N
O
H
O
6
3.1.3. 5-Nitro-1,3-bis-n-propyluracil (14)
To a stirred suspension of 1,3-bis-n-propyluracil (13: 1.65 g,
8.41 mmol) in concentrated sulfuric acid (20 mL) at 0 °C was added
fuming nitric acid (1.5 mL) dropwise. Stirring was continued for 1 h
at 0 °C and then at room temperature for 5 h. The mixture was
poured onto ice, neutralized with solid Na2CO3, extracted with
EtOAc, washed with brine, dried over Na2SO4, concentrated under
reduced pressure. Purification by flash column chromatography on
silica gel (n-hexane/EtOAc, 2:1) gave nitrated 14 (1.99 g, 98%) as a
red oil.
Scheme 4. Synthesis of compounds 5 and 6. Reagents and conditions: (a) 1-
bromopropane, K2CO3/DMF, rt, 12 h (77%); (b) Pd2(dba)3, XPhos, Cs2CO3/DMF, 65 °C,
12 h (37%); (c) hv/toluene, rt, 10 h; (d) DDQ, MeOH/CHCl3 (1:3), rt, 30 min (47%).
Table 1
Affinities of caffeine and eudistomin
D hybrid analogues (1–6) and its related
compounds at human adenosine A1, A2A, and A3 receptors
Compound
Kia
A2A
(
l
M) or % inhibitionb
A1
A3
Compound 14: IR (neat) 2921, 2851, 1673, 1623, 1509, 1455,
1321, and 1239 cmꢀ1 1H NMR (400 MHz,CDCl3) d 0.97 (3H, t,
;
1
2
3
4
5
6
11.7 6.2
0.379 0.043
1.41 0.07
0.132 0.011
0.346 0.025
0.921 0.125
49.0 19.6
0.009 0.001
nd
10.1 0.01
0.893 0.086
3.63 0.18
0.570 0.041
0.164 0.023
1.47 0.16
18.1 5.9
nd
0.526 0.156
0.0139 0.0032
0.290 0.040
0.0168 0.0021
0.00382 0.00064
0.209 0.048
9%
J = 7.7 Hz), 1.03 (3H, t, J = 7.2 Hz), 1.68 (2H, ddq, J = 7.2, 7.7 Hz),
1.84 (2H, ddq, J = 7.2, 7.7 Hz), 3.90 (2H, dd, J = 7.2, 7.7 Hz), 3.97
(2H, dd, J = 7.2, 7.7 Hz), 8.67 (1H, s); 13C NMR (100 MHz, CDCl3) d
10.9, 11.3, 20.7, 22.5, 44.1, 53.1, 125.1, 146.9, 149.5, 154.4; HREIMS
m/z 241.1060 (M+), calcd for C10H15N3O4 241.1058.
Caffeine
XAC
CGS21680
NECA
nd
nd
0.0462 0.0084
nd
3.1.4. 5-Amino-1,3-bis-n-propyluracil (15)
nd
0.020 0.009
To a stirred solution of 5-nitro-1,3-bis-n-propyluracil (14:
200 mg, 0.83 mmol) in acetic acid (37 mL) was added iron powder
(4.4 g) and stirred at rt for 4 h. The reaction mixture was neutral-
ized with solid Na2CO3, extracted with ethyl acetate, washed with
brine, dried over Na2SO4, and evaporated in vacuo. The residue was
purified by flash column chromatography on silica gel (n-hexane/
EtOAc, 1:3) to give 15 (150 mg, 86%).
Compound 15: IR (neat) 3329, 2961, 2359, 2342, 1686, 1670, and
1629 cmꢀ1; 1H NMR (400 MHz,CDCl3) d 0.92 (3H, t, J = 7.2 Hz), 0.92
(3H, t, J = 7.7 Hz), 1.66 (4H, m), 3.63 (2H, dd, J = 7.2 Hz), 3.92 (2H,
dd, J = 7.7 Hz), 6.62 (1H, s); 13C NMR (100 MHz, CDCl3) d 10.8, 11.2,
20.7, 22.0, 43.1, 50.7, 120.8, 121.0, 149.6, 160.6.
a
The Ki values are mean SEM of two or three separate assays.
b
Percentage of inhibition (%) of specific [3H]DPCPX (for A1), [3H]CGS21680 (for
A2A), or [3H]NECA (for A3) binding by test compounds at 100
lM concentration. The
binding of each radioactive ligand to membranes prepared from HEK293T cells
expressing human A1, A2A, or A3 adenosine receptors was best-fitted to a one-site
competition model of binding with estimated Kd (dissociation constant) values of 5,
52, and 6.5 nM, respectively, and Bmax values of 8600, 7000, and 310 fmol/mg
protein, respectively. nd; not determined.
In this study, bis-N-n-propylation (compounds 3 and 4, respec-
tively) of the uracil ring in compounds 1 and 2 resulted in higher
affinity for A1 and A2A adenosine receptors (Table 1). Furthermore,

H. Ishiyama et al. / Bioorg. Med. Chem. 17 (2009) 4280–4284
4283
3.1.5. 1,3-Bis-n-propyl-5-(pyridin-3-ylamino)uracil (17)
Compound 20: 1H NMR (400 MHz, CDCl3)
d
0.95 (3H, t,
To an oven-dried Schlenk tube charged with Pd2(dba)3 (130 mg,
142 lmol), X-Phos (69.8 mg, 146 lmol), and Cs2CO3 (521 mg,
1.60 mmol) was added a solution of 5-amino-1,3-bis-n-propylura-
cil (15) (78.0 mg, 0.5 mmol) in DMF (3 mL). After stirring for
J = 7.7 Hz), 0.97 (3H, t, J = 7.2 Hz), 1.03 (3H, t, J = 7.2 Hz), 1.74 (6H,
m), 3.72 (2H, dd, J = 7.2, 7.2 Hz), 3.93 (2H, t, J = 6.3 Hz), 3.96 (2H,
dd, J = 7.2, 7.7 Hz), 5.89 (1H, s), 6.71 (1H, br s), 7.12 (1H, s), 7.85
(1H, s), 7.94 (1H, s); 13C NMR (125 MHz, CDCl3) d 10.3, 10.9, 11.2,
20.8, 22.2, 22.4, 43.5, 51.4, 69.9, 109.1, 117.3, 125.3, 129.6, 131.2,
140.2, 149.4, 155.8, 160.7; HREIMS m/z 346.1994 (M+), calcd for
C18H26N4O3 346.1983.
10 min at rt, 3-chloropyridine (16; 152 lL, 1.6 mmol) was added
and then stirred for 10 h at 65 °C. The reaction mixture was filtered
through Celite and the residue was purified by flash column chro-
matography on silica gel (CHCl3/MeOH, 80:1?50:1) to give 17
(274 mg, 67%) as a brown oil.
3.1.9. 2,4-Bis-n-propyl-4,9-dihydro-5-n-propyloxy-2,4,7,
9-tetraaza-fluorene-1,3-dione (5) and 2,4-bis-n-propyl-1a,4,4a,-
9-tetrahydro-7-n-propyloxy-2,4,5,9-tetraaza-fluorene-1,-
3-dione (21)
Compound 17: IR (neat) 3319, 2964, 1698, 1638, 1584, 1456
cmꢀ1 1H NMR (400 MHz, CDCl3) d 0.94 (3H, t, J = 7.2 Hz), 0.96
;
(3H, t, J = 7.2 Hz), 1.70 (4H, m), 3.71 (2H, dd, J = 7.2, 7.7 Hz), 3.96
(2H, dd, J = 7.7 Hz), 5.95 (1H, s), 7.10 (1H, s), 7.17 (1H, ddd,
J = 0.5, 4.5, 8.2 Hz), 7,21 (1H, ddd, J = 1.4, 2.7, 8.2 Hz), 8.15 (1H, d,
J = 3.2 Hz), 8.33 (1H, s); 13C NMR (100 MHz, CDCl3) d 10.8, 11.2,
20.7, 22.2, 43.4, 51.3, 117.3, 123.0, 123.7, 124.7, 138.9, 142.0,
149.3, 160.7; HREIMS m/z 288.1583 (M+), calcd for C15H20N4O2
288.1580.
A solution of 20 (75.3 mg, 0.22 mmol) in toluene (100 mL) was
irradiated with tungsten lamp for 10 h at rt. The mixture was con-
centrated in vacuo and purified by column chromatographies on
silica gel (CHCl3/MeCN, 10:1?1:1 and MeCN/MeOH 5:1?1:1)
and amino silica gel (n-hexane/acetone, 5:1?1:1) to give 5
(10.8 mg, 14%) and 21 (8.8 mg, 12%) as brown oil, respectively.
Compound 5: 1H NMR (400 MHz, CDCl3) d 0.94 (3H, t, J = 7.7 Hz),
1.01 (3H, t, J = 6.8 Hz), 1.11 (3H, t, J = 7.2 Hz), 1.78 (4H, m), 1.99
(2H, tq, J = 6.8, 7.2 Hz), 4.16 (2H, dd, J = 7.2, 7.7 Hz), 4.27 (2H, t,
J = 7.2 Hz), 4.69 (2H, dd, J = 7.2 Hz), 7.96 (1H, s), 8.72 (1H, s); UV
3.1.6. 2,4-Bis-n-propyl-4,9-dihydro-2,4,5,9-tetraaza-fluorene-1,
3-dione (3) and 2,4-bis-n-propyl-4,9-dihydro-2,4,7,-
9-tetraaza-fluorene-1,3-dione (4)
A solution of 17 (94 mg, 326
l
mol) in toluene (100 mL) was
(MeOH) kmax 203 (e 163,00), 242 (22,100), 284 (5900), 294
irradiated with tungsten lamp for 9 h at rt. The mixture was con-
centrated in vacuo and purified by column chromatographies on
silica gel (n-hexane/acetone, 5:1?1:1) and amino silica gel
(MeCN/MeOH, 1:0?1:3) to give 3 (5.9 mg, 6%) and 4 (5.6 mg, 6%)
as brown oil, respectively.
(5800), 354 (5500), 369 (5000); HREIMS m/z 344.1847 (M+), calcd
for C18H24N4O3 344.1848.
3.1.10. 2,4-Bis-n-propyl-4,9-dihydro-7-n-propyloxy-2,4,5,-
9-tetraaza-fluorene-1,3-dione (6)
Compound 3: 1H NMR (400 MHz, DMSO-d6) d 0.89 (3H, t,
J = 7.2 Hz), 0.98 (3H, t, J = 7.7 Hz), 1.62 (2H, m), 1.75 (2H, m), 3.94
(2H, dd, J = 7.2, 7.7 Hz), 4.24 (2H, dd, J = 7.2, 7.7 Hz), 7.84 (1H, dd,
J = 0.91, 5.9 Hz), 8.26 (1H, d, J = 5.9 Hz), 8.89 (1H, d, J = 0.91 Hz);
To a stirred solution of 21 (3.8 mg, 15
lmol) in CHCl3 and MeOH
(3:1, 1.2 mL) was added DDQ (3.8 mg, 17
lmol) and stirred at rt for
30 min. The reaction mixture was evaporated in vacuo and purified
by flash column chromatography on amino silica gel (n-hexane/
CHCl3, 3:1?1:1) to give 6 (2.4 mg, 47%).
UV (MeOH) kmax 203 (
e 10,800), 227 (9000), 287 (3300), 297
(3600), 350 (2400); HREIMS m/z 286.1418 (M+), calcd for
C15H18N4O2 286.1406.
Compound 6: 1H NMR (500 MHz, CDCl3) d 1.02 (3H, t, J = 6.9 Hz),
1.03 (3H, t, J = 6.9 Hz), 1.09 (3H, t, J = 6.9 Hz), 1.76 (2H, tq, J = 6.9,
7.6 Hz), 1.89 (4H, m), 4.02 (2H, t, J = 6.9 Hz), 4.13 (2H, dd, J = 7.6,
7.6 Hz), 4.65 (2H, dd, J = 7.6, 7.6 Hz), 7.16 (1H, d, J = 3.1 Hz), 8.30
Compound 4: 1H NMR (400 MHz, CDCl3) d 1.03 (6H, t, J = 7.2,
7.7 Hz), 1.80 (2H, m), 1.91 (2H, m), 4.16 (2H, dd, J = 7.2, 7.7 Hz),
4.71 (2H, dd, J = 7.2, 7.7 Hz), 7.36 (1H, dd, J = 4.5, 8.6 Hz), 7.83 (1H,
dd, J = 1.4, 8.6 Hz), 8.57 (1H, dd, J = 1.4, 4.5 Hz), 10.3 (1H, s); UV
(1H, d, J = 2.3 Hz), 9.98 (1H, s); UV (MeOH) kmax 203 (e 15,600),
222 (16,000), 261 (14,600), 268 (13,600), 322 (11,800); HREIMS
(MeOH) kmax 221 (
e
15,600), 253 (11,900), 312 (10,400), 341
m/z 344.1850 (M+), calcd for C18H24N4O3 344.1848.
(4000); HREIMS m/z 286.1417 (M+), calcd for C15H18N4O2 286.1404.
3.2. Biological assays
3.1.7. 3-Chloro-5-n-propyloxypyridine (19)
To
a
solution of 3-chloro-5-hydroxypyridine (18; 300 mg,
3.2.1. Radioligand materials
2.32 mmol) in DMF (3 mL) was added K2CO3 (385 mg, 2.78 mmol)
and 1-bromopropane (0.25 mL, 2.78 mmol). After stirring 12 h, the
reaction was quenched with H2O (10 mL), extracted with Et2O,
washed with brine, dried over Na2SO4, and under reduced pres-
sure. The mixture was purified by flash column chromatography
on silica gel (n-hexane/EtOAc, 6:1) to give 19 (305 mg, 77%).
Compound 19: 1H NMR (500 MHz, CDCl3) d 1.04 (3H, t, J = 7.4 Hz),
1.82 (2H, tq, J = 6.9, 7.4 Hz), 3.95 (2H, t, J = 6.9 Hz), 7.19 (1H, dd,
J = 2.3, 2.3 Hz), 8.16 (1H, d, J = 2.3 Hz), 8.19 (1H, d, J = 2.3 Hz).
[3H]-8-Cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX), [3H]-2-
[4-(2-carboxyethyl)phenethylamino]-50-N-ethylcarboxamidoad-
enosine ([3H]CGS21680), and [3H]-50-N-ethyl-carboxamidoadeno-
sine ([3H]NECA) were purchased from PerkinElmer (Boston, MA,
USA). Unless otherwise stated, all other materials used for ligand
binding assay were purchased from Sigma–Aldrich (St. Louis, MO,
USA).
3.2.2. Membrane preparations
HEK293T cells transiently expressing human adenosine A1, A2A
,
3.1.8. 1,3-Bis-n-propyl-5-(5-n-propyloxypyridin-3-ylamino)
uracil (20)
To an oven-dried Schlenk tube charged with Pd2(dba)3 (65 mg,
71 lmol), X-Phos (34.9 mg, 73 lmol), and Cs2CO3 (280 mg,
0.86 mmol) was added a solution of 5-amino-1,3-bis-n-propylura-
cil (15) (150 mg, 0.71 mmol) in DMF (2 mL). After stirring for
10 min at rt, 3-chloro-5-n-propyloxypyridine (19; 150 mg,
0.86 mmol) was added and then stirred for 12 h at 65 °C. The reac-
tion mixture was filtered through Celite and the residue was puri-
fied by column chromatography on silica gel (CHCl3/MeOH,
1:0?80:1) to give 20 (90.3 mg, 37%) as a brown oil.
and A3 receptors were used as the receptor source in this study.
Plasmids encoding human adenosine A1, A2A, or A3 receptor con-
struct obtained from UMR cDNA Resource Center (Rolla, MO,
USA; www.cdna.org) were transiently transfected into HEK293T
cells using Effectene (Quiagen). Cells were maintained at 37 °C in
humidified air containing 5% CO2 in Dulbecco’s modified Eagle’s
medium supplemented with 10% FBS (Fatal Bovine Serum),
100 lg/mL kanamycin for 48 h. The cells were harvested and
homogenized in lysis buffer containing 50 mM Tris–HCl buffer
(pH 7.4) with a protease-inhibitor mixture (Roche Diagnostics)
and subjected to low-speed centrifugation to remove organelles

4284
H. Ishiyama et al. / Bioorg. Med. Chem. 17 (2009) 4280–4284
and nuclei. The resulting supernatant was subjected to centrifuga-
tion at 30,000 g for 30 min, and precipitated cell membranes were
collected, washed twice, resuspended in the lysis buffer, and used
immediately.
partly supported by a Grant-in-aid from the Akiyama Foundation
and Grant-in-Aid for Scientific Research from the Ministry of Edu-
cation, Science, Sports, and Culture of Japan.
References and notes
3.2.3. Adenosine receptor binding assays
Radioligand binding experiments to adenosine A1, A2A, and A3
receptors were carried out by using [3H]DPCPX, [3H]CGS21680,
and [3H]NECA, respectively, as the radioactive ligands. Cell mem-
branes expressing adenosine A1, A2A, and A3 receptors were incu-
bated with 4 nM [3H]DPCPX, 15 nM [3H]CGS21680, or 32 nM
[3H]NECA, respectively, in the presence of 9–10 different concen-
1. Laura, M. J.; Roland, R. G. Psychopharmacology 2004, 176, 1.
2. (a) Jacobson, K. A.; Gao, Z.-G. Nat. Rev. Drug Disc. 2006, 5, 247; (b) Jeong, L. S.;
Choe, S. A.; Gunaga, P.; Kim, H. O.; Lee, H. W.; Lee, S. K.; Tosh, D. K.; Patel, A.;
Palaniappan, K. K.; Gao, Z.-G.; Jacobson, K. A.; Moon, H. R. J. Med. Chem. 2007,
50, 3159.
3. (a) Kobayashi, J.; Harbour, G. C.; Gilmore, J.; Rinehart, K. L., Jr. J. Am. Chem. Soc.
1984, 106, 1526; (b) Rinehart, K. L., Jr.; Kobayashi, J.; Harbour, G. C.; Gilmore, J.;
Mascal, M.; Holt, T. G.; Shield, L. S.; Lafargue, F. J. Am. Chem. Soc. 1984, 106,
1524; (c) Rinehart, K. L., Jr.; Kobayashi, J.; Harbour, G. C.; Gilmore, J.; Mascal,
M.; Holt, T. G.; Shield, L. S.; Lafargue, F. J. Am. Chem. Soc. 1987, 109, 3378.
4. Nakamura, Y.; Kobayashi, J.; Gilmore, J.; Mascal, M.; Rinehart, K. L., Jr.;
Nakamura, H.; Ohizumi, Y. J. Biol. Chem. 1986, 261, 4139.
5. Kobayashi, J.; Taniguchi, M.; Hino, T.; Ohizumi, Y. J. Pharm. Pharmacol. 1988, 40,
62.
6. (a) Ohshita, K.; Ishiyama, H.; Oyanagi, K.; Nakata, H.; Kobayashi, J. Bioorg. Med.
Chem. 2007, 15, 3235; (b) Ishiyama, H.; Ohshita, K.; Abe, T.; Nakata, H.;
Kobayashi, J. Bioorg. Med. Chem. 2008, 16, 3825.
trations of test compounds in 250 lL of assay buffer containing
50 mM Tris–acetate buffer, pH 7.4, 5 mM MgCl2, 1 mM EDTA, and
1 U/mL adenosine deaminase for 60 min at 25 °C. The incubated
mixture was then harvested on Whatman GF/B filters pre-soaked
in 0.1% polyethyleneimine by a cell harvester. The filters were
quickly washed three times with cold 50 mM Tris–HCl buffer (pH
7.4) and the radioactivity on the filter was measured by a scintilla-
tion counter. All experiments were carried out two or three times.
The nonspecific binding for adenosine A1, A2A, and A3 receptors was
defined as the binding activity in the presence of XAC, CGS21680,
7. (a) Strieter, E. R.; Blackmond, D. G.; Buchwald, S. L. J. Am. Chem. Soc. 2003, 125,
13978; (b) Huang, X.; Anderson, K. W.; Zim, D.; Jiang, L.; Klapars, A.; Buchwald,
S. L. J. Am. Chem. Soc. 2003, 125, 6653; (c) Kim, Y. M.; Yu, S. J. Am. Chem. Soc.
2003, 125, 1696.
and NECA, respectively, at 10 lM each. Ki values in the inhibition
8. Itahara, T. Bull Chem. Soc. Jpn. 1996, 69, 3239.
studies were determined using one-site competition binding mod-
el by non-linear regression analysis (GraphPad Prism 4; GraphPad,
San Diego, CA, USA).
9. Dolman, N. P.; Troop, H. M.; More, J. C. A.; Alt, A.; Knauss, J. L.; Nistico, R.; Jack,
S.; Morley, R. M.; Bortolotto, Z. A.; Roberts, P. J.; Bleakman, D.; Collingridge, G.
L.; Jane, D. E. J. Med. Chem. 2005, 48, 7867.
10. Tapolcsanyi, P.; Krajsovszky, G.; Ando, R.; Lipcsey, P.; Horvath, G.; Matyus, P.;
Riedl, Z.; Hajos, G.; Maes, B. U. W.; Lemiere, G. L. F. Tetrahedron 2002, 58, 10137.
11. Congreve, M.; Aharony, D.; Albert, J.; Callaghan, O.; Campbell, J.; Carr, R. A. E.;
Chessari, G.; Cowann, S.; Edwards, P. D.; Frederickson, M.; McMenamin, R.;
Murray, C. W.; Patel, S.; Wallis, N. J. Med. Chem. 2007, 50, 1124.
Acknowledgments
We thank S. Oka and N. Owada, Center for Instrumental Analy-
sis, Hokkaido University, for EIMS measurements. This work was
12. Photocyclization under Ar provided
a dihydro compound. Benchekroun-
Mounir, N.; Dugat, D.; Gramain, J.-C.; Husson, H.-P. J. Org. Chem. 1993, 58, 6457.