1,2,4-Triazolo[5,1-i]purine derivatives as highly potent and selective human adenosine A3 receptor ligands

Article abstract of DOI:10.1021/jm010570p

A series of triazolopurines showed structural similarity to human adenosine A₃ receptor antagonist, 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino] [1,2,4]triazolo[1,5-c]quinazoline (MRS 1220, 1). In this study, we found novel 1,2,4-triazolo[5,1- i]purine derivatives (2) showing human adenosine A₃ receptor affinities. The compounds were obtained in two steps from 5-amino-4-cyanoimidazole (33). The affinity was determined in radioligand binding assays for the cloned human adenosine A₁, A_2A, A_2B, and A₃ receptors. After the structure-activity relationship was analyzed, we determined that there was a mild parabolic relationship between the length of alkyl groups at the 5-position and the affinities at the A₃ receptor and positive correlation between the length of the substituents on phenyl groups at the 8-position and the affinities at the A_2A receptor. These investigations led to potent and selective human adenosine A₃ receptor ligands. The most potent A₃ receptor ligand (5-n-butyl-8-(4-methoxyphenyl)-3H-[1,2,4]triazolo-[5,1-i]purine (27, K_i= 0.18 nM) and the most selective A₃ receptor ligand against A₁, A_2A, and A_2B receptors, (5-n-butyl-8 -(4-n-propoxyphenyl)-3H-[1,2,4]triazolo[5,1-i]purine (29, > 19 600), were discovered.

Full text of DOI:10.1021/jm010570p

J . Med. Chem. 2002, 45, 3703-3708
3703
1,2,4-Tr ia zolo[5,1-i]p u r in e Der iva tives a s High ly P oten t a n d Selective Hu m a n
Ad en osin e A3 Recep tor Liga n d s
Takashi Okamura,*,† Yasuhisa Kurogi,† Hiroshi Nishikawa,† Kinji Hashimoto,† Hiroshi Fujiwara,† and
Yoshimitsu Nagao‡
Nutrition Research Institute, Otsuka Pharmaceutical Factory, Inc. Tateiwa, Muya-cho, Naruto, Tokushima 772-8601 J apan,
and Faculty of Pharmaceutical Sciences, The University of Tokushima, Sho-machi 1, Tokushima 770-8505 J apan
Received December 17, 2001
A series of triazolopurines showed structural similarity to human adenosine A3 receptor
antagonist, 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]triazolo[1,5-c]quinazoline (MRS
1220, 1). In this study, we found novel 1,2,4-triazolo[5,1-i]purine derivatives (2) showing human
adenosine A3 receptor affinities. The compounds were obtained in two steps from 5-amino-4-
cyanoimidazole (33). The affinity was determined in radioligand binding assays for the cloned
human adenosine A1, A2A, A2B, and A3 receptors. After the structure-activity relationship was
analyzed, we determined that there was a mild parabolic relationship between the length of
alkyl groups at the 5-position and the affinities at the A3 receptor and positive correlation
between the length of the substituents on phenyl groups at the 8-position and the affinities at
the A2A receptor. These investigations led to potent and selective human adenosine A3 receptor
ligands. The most potent A3 receptor ligand (5-n-butyl-8-(4-methoxyphenyl)-3H-[1,2,4]triazolo-
[5,1-i]purine (27, Ki ) 0.18 nM) and the most selective A3 receptor ligand against A1, A2A, and
A2B receptors, (5-n-butyl-8-(4-n-propoxyphenyl)-3H-[1,2,4]triazolo[5,1-i]purine (29, >19 600),
were discovered.
In tr od u ction
Adenosine is an important regulator for homeostasis
of the brain, heart, kidney, and other organs.1 Adenosine
interacts with four different G-protein-coupled receptors
classified as A1, A2A, A2B, and A3 receptor subtypes.2 The
A1 and A3 subtypes inhibit adenylate cyclase (AC)
coupling to Gi protein, whereas A2A and A2B subtypes
stimulate AC via Gs protein. Because the adenosine A3
receptors were characterized in 1992,3 the physiological
roles of the adenosine A3 receptors have been investi-
gated.
In 1996, J acobson and co-workers disclosed potent
and selective adenosine A3 receptor antagonists of 1,4-
dihydropyridines,4-6 triazoloquinazolines (e.g., com-
pound 1),7,8 and flavonoids.9 Recently, triazolonaph-
thyridines,10 isoquinolines,11,12 quinazolines,12 pyrazolo-
triazolopyrimidines,13 and thiazoles14 have been re-
ported as new adenosine A3 receptor antagonists. Selec-
tive adenosine A3 receptor antagonists are considered
as potential antiinflammatory,15 antiasthmatic,16 anti-
ischemic,17 and antiglaucoma agents.18,19
A series of 1,2,4-triazolo[5,1-i]purine derivatives (2)
were synthesized as hybrid scaffolds of adenine and 5-n-
butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]-
pyrimidine (OT-7100, 3). Compound 3 was synthesized
in our laboratory. In experiments using rats, 3 has a
unique profile with the effect of normalizing the noci-
ceptive threshold in peripheral neuropathic pain20 and
diabetic neuropathy.21
Although 1,2,4-triazolo[5,1-i]purines (2) did not show
analgesic activity in vivo, the structural similarity
between 1 and 1,2,4-triazolo[5,1-i]purines (2) led us to
evaluate adenosine A3 receptor affinities. In this paper,
selective adenosine A3 receptor affinities of novel 1,2,4-
triazolo[5,1-i]purine derivatives (2) are described includ-
ing structure-activity relationships (SAR).
* To whom correspondence should be addressed. Tel: +81-88-684-
2353. Fax: +81-88-686-8176. E-mail: okamurtk@otsukakj.co.jp.
† Otsuka Pharmaceutical Factory, Inc.
‡ The University of Tokushima.
10.1021/jm010570p CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/16/2002

3704 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Okamura et al.
Ta ble 1. Affinities of 1,2,4-Triazolo[5,1-i]purine Derivatives in
Radioligand Binding Assays at Human A2A and A3
Sch em e 1. Synthesis of 1,2,4-Triazolo[5,1-i]purine
Derivativesa
IC50 (nM) or
% inhibitionc
a
b
compd
R1
CH3
C2H5
n-C3H7 Ph
n-C4H9 Ph
n-C5H11 Ph
n-C6H13 Ph
R2
hA2A
hA3
hA2A/hA3
4
5
6
7
8
Ph
Ph
550
360
120
71
200
7800
23
1.0
550
800
520
280
670
0.45
0.23
0.25
0.30
0.61
0.41
9
13 000
56
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Ph
Ph
n-C4H9 CH3
n-C4H9 PhCH2
n-C4H9 3-pyridyl
n-C4H9 2-furyl
n-C4H9 2-Cl-Ph
n-C4H9 3-Cl-Ph
n-C4H9 4-Cl-Ph
n-C4H9 4-F-Ph
n-C4H9 4-Br-Ph
n-C4H9 3-CH3-Ph
n-C4H9 4-CH3-Ph
n-C4H9 4-t-C4H9-Ph
n-C4H9 4-CF3-Ph
n-C4H9 4-Biphenyl
n-C4H9 4-HO-Ph
n-C4H9 3-CH3O-Ph
n-C4H9 4-CH3O-Ph
n-C4H9 4-C2H5O-Ph
n-C4H9 4-n-C3H7 O-Ph
46 610
47 680
0.075
0.069
750
36
62
900
1.2
210
18
56
5.9
0.29
1.1
51
2600
510
3300
188
180
20%
28%
12%
58
0.41
0.25
1.9
0.27
0.33
1.2
0.61 >16 000
5.0
1.8
6300
2000
1700
700
550
>8300
>2000
32
67
0.22
300
1600 <0.1 >16 000
3800
9%
0.21
18 000
0.30 >33 000
1.1
3.3
n-C4H9 3,4,5-(CH3O)3-Ph 2500
n-C4H9 4-CH3S-Ph
n-C4H9 4-(CH3)2N-Ph
2300
>3000
42%
0%
0.67 >15 000
a
Displacement of specific [3H]CGS 21680 binding at human A2A
receptors expressed in HEK-293 cells, in membranes, expressed
as IC50 in nanomolar (n ) 2); CV ) 4.3%. b Displacement of specific
[
125I]AB-MECA binding at human A3 receptors expressed in HEK-
293 cells, in membranes, expressed as IC50 in nanomolar (n ) 2);
CV ) 7.2%. c Displacement of specific binding at 10 000 nM
concentration.
a
Reagents: (i) R1C(OY)3, DMF, 80 °C. (ii) R2CONHNH2, DMF
or diglyme, reflux. (iii) 10% HCl, reflux. (iv) R1COCl, pyridine. (v)
TMSCl, Et3N, THF, reflux.
was performed by treatment with trimethylsilyl chloride
to provide the desired compounds 8 and 9. Yields and
characterizations are shown in the Experimental Sec-
tion.
Ch em istr y
1,2,4-Triazolo[5,1-i]purines (2) were prepared follow-
ing the synthetic strategy (Scheme 1), which was
modified from that of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidines.22 5-Amino-4-cyanoimidazole (33) was
transformed into imidates 34-38 by treatment with
commercially available ortho esters at 80 °C in dimethyl
formamide (DMF). The desired compounds (4-7 and
10-32) were obtained by refluxing the imidates with
the corresponding acylhydrazines in DMF or diglyme
(method A).
On the other hand, 8 and 9 with a long alkyl chain at
the R1 position were synthesized from 5-methyl-8-
phenyl-3H-[1,2,4]triazolo[5,1-i]purine (4) using com-
mercially available n-hexanoyl and n-heptanoyl chloride
(method B), because of the unavailability of trialkyl
ortho-n-hexanoate or ortho-n-heptanoate. Compound 4
was converted into an aminoimidazole derivative (39)
by hydrolysis with aqueous 10% HCl. The acylation of
39 with the corresponding acyl chlorides afforded the
intermediates 40 and 41. The cyclization of 40 and 41
Resu lts a n d Discu ssion
The binding affinities of 4-32 at human adenosine
A2A and A3 receptors expressed in HEK-293 cells are
shown in Table 1. As initially observed among com-
pounds 4-10 bearing an unsubstituted phenyl group
at the R2 position, there was a mild parabolic relation-
ship between the length of the R1 position and the
affinities to the A3 receptor. Among compounds (7 and
11-14) with a butyl group at the R1 position and
structurally diverse substituents at the R2 position, 7
bearing a phenyl group at the R2 position resulted in
the highest A3 receptor affinity.
Furthermore, compounds 15-32 bearing a substi-
tuted phenyl group at the R2 position were evaluated.
By analyzing SAR of these compounds, the length of
substituents at the para position on phenyl group seems
to correlate positively with the binding affinities (IC50)

1,2,4-Triazolo[5,1-i]purine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3705
Ta ble 2. Binding Affinities at Adenosine A1, A2A, A2B, and A3 Receptors of Compounds 1, 23, 27-29, 32, 42, and 43
Ki (nM)a or % inhibitionb
c
d
e
f
compd
hA1
hA2A
hA2B
hA3
0.95
hA1/hA3
hA2A/hA3
hA2B/hA3
23
4 ( 2%
31 ( 4%
6 ( 11%
>10 500
>10 500
>10 500
(0.72-1.24)
27
28
29
32
398
892
1030
0.18
2210
459
4960
636
5720
931
(256-620)
(811-982)
(772-1390)
(0.17-0.20)
0.90
(0.58-1.40)
413
572
838
(335-509)
32 ( 0%
(438-749)
49 ( 2%
(640-1100)
21 ( 3%
0.51
>19 600
1050
>19 600
>8000
>19 600
5330
(0.35-0.74)
1.25
(0.82-1.91)
0.65 ( 0.25
0.80
1310
49 ( 3%
6660
(983-1730)
305 ( 51g
1200
(3580-12 400)
1h
52.0 ( 8.8g
140
470g
1500
80g
175
42h
2056
2570
(1030-1400)
>10 000
(120-155)
>10 000
(1640-2580)
(0.63-1.00)
18
43h
>556
>556
a
b
Data are expressed as geometric means, with 95% confidence intervals. A percentage of specific binding displaced at 10 000 nM
concentration, mean ( SEM (n ) 2-3). c Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells, in
membranes, expressed as Ki in nanomolar (n ) 3). Displacement of specific [3H]CGS 21680 binding at human A2A receptors expressed
d
in HEK cells, in membranes, expressed as Ki in nanomolar (n ) 3). e Displacement of specific [3H]DPCPX binding at human A2B receptors
expressed in HEK cells, in membranes, expressed as Ki in nanomolar (n ) 3). f Displacement of specific [125I]AB-MECA binding at human
g
h
A3 receptors expressed in HEK cells, in membranes, expressed as Ki in nanomolar (n ) 3). At rat A1 and A2A receptors. Values taken
from refs 7, 10, and 22.
at A2A receptors. In fact, a series of unsubstituted (7),
fluoro (18), chloro (17), bromo (19), and trifluoromethyl
(23) derivatives and a series of hydroxy (25), methoxy
(27), ethoxy (28), and n-propoxy (29) derivatives showed
a positive correlation between the length and the A2A
affinity. On the other hand, no significant difference was
observed regarding the affinity at A3 receptors. Electron-
donating and -withdrawing substituents on the phenyl
group at the R2 position were also unaffected by the
binding affinities at A2A and A3 receptors.
phenyl)amino]carbonyl]amino-8-propyl-2-(2-furyl)pyra-
zolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine (MRE3008F20,
42)22 and 5-amino-3-(4-methoxyphenyl)thiazolo[3,2-a]-
pyrimidin-7-one (L268605, 43)10 indicated hA3 ) 0.80
nM, hA1/hA3 ) 1496, hA2A/hA3 ) 175, hA2B/hA3 ) 2570,
and hA3 ) 18 nM, hA1/hA3 > 556, hA2A/hA3 > 556,
respectively. Very recently, Baraldi et al. reported new
human adenosine A3 receptor antagonists with the good
profile (e.g., compound 44, hA3 ) 0.16 nM, hA1/hA3 )
3713, hA2A/hA3 ) 2381, and hA2B/hA3 ) 1388).23
Con clu sion
Novel 1,2,4-triazolo[5,1-i]purine derivatives (2) were
synthesized by the modified method of pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidines, which showed high and
selective adenosine A3 receptor affinity. As a result of
SAR analysis, the most potent and selective human
adenosine A3 receptor ligands, 27 and 29, were found.
These potent human adenosine A3 receptor ligands
might be useful as pharmacological probes and good
therapeutic agents.
Exp er im en ta l Section
Column chromatography was performed on silica gel 60
(Merck, particle size 63-200 mm). All melting points were
determined on a Yamato micromelting point apparatus (MP-
21). 1H nuclear magnetic resonance (NMR) spectra were
measured on a J EOL GX-270 (270 MHz) and a J EOL J NM-
AL400 (400 MHz; compounds 32, 40, and 41) spectrometer,
and chemical shifts are indicated in δ units from tetramethyl-
silane (TMS) as an internal standard. Elemental analyses were
performed by the analytical department of Wako Pure Chemi-
cal Industries, Ltd. or the University of Tokushima and were
within (0.4% of the calculated values.
Meth od A. Gen er a l P r oced u r e for P r ep a r a tion of
Im id a tes 34-38. A mixture of 5-amino-4-cyano-1H-imidazole
(33, 0.14 mol) and the appropriate ortho ester derivative (0.21
mol) in 30-40 mL of DMF was heated at 90 °C for 0.5-5 h.
After the solvent was evaporated, product was recrystallized
from ethyl acetate (AcOEt) and hexane.
Potent and highly selective compounds (23, 27-29,
and 32) were evaluated for the binding affinity as Ki
values at four human adenosine receptor subtypes
including A1 and A2B, as shown in Table 2.
The potent and selective affinities to human adeno-
sine A3 receptors against A1, A2A, and A2B receptors were
observed. Compound 27 was the most potent A3 ligand
(Ki ) 0.18 nM), and compound 29 was the most selective
A3 ligand against other subtypes (>19 600) in this
series. In comparison to the most selective adenosine
A3 receptor antagonists contained in the literature,
compound 29 had potent and selective affinities to hu-
man adenosine A3 receptors. For example, 5-[[(4-methoxy-
Meth yl N-(4-Cya n o-1H-im id a zol-5-yl)a cetim id a te (34).
Yield 93%; colorless powder. 1H NMR (DMSO-d6): δ 2.04 (3H,
s), 3.78 (3H, s), 7.67 (1H, s), 12.5-13.0 (1H, brs).
Eth yl N-(4-Cyan o-1H-im idazol-5-yl)pr opion im idate (35).
1
Yield 66%; colorless powder. H NMR (CDCl3): δ 1.17 (3H, t,

3706 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Okamura et al.
J ) 7.9 Hz), 1.34 (3H, t, J ) 7.4 Hz), 2.42 (2H, q, J ) 7.9 Hz),
4.26 (2H, q, J ) 7.4 Hz), 7.49 (1H, s), 10.9-11.7 (1H, brs).
Meth yl N-(4-Cyan o-1H-im idazol-5-yl)bu tyr im idate (36).
NMR (DMSO-d6): δ 0.96 (3H, t, J ) 7.4 Hz), 1.4-1.5 (2H, m),
1.9-2.0 (2H, m), 3.36 (2H, t, J ) 7.5 Hz), 7.5-7.7 (3H, m),
8.0-8.2 (1H, m), 8.47 (1H, s). Anal. (C16H15N6Cl‚0.5H2O) C,
H, N.
1
Yield 79%; colorless powder. H NMR (CDCl3): δ 0.89 (3H, t,
J ) 7.2 Hz), 1.5-1.7 (2H, m), 2.40 (2H, t, J ) 7.4 Hz),3.84
5-n -Bu tyl-8-(3-ch lor op h en yl)-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (16). Yield 80%; colorless powder; mp 218-221 °C. 1H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.4-1.6 (2H, m),
1.8-2.0 (2H, m), 3.34 (2H, t, J ) 7.7 Hz), 7.6-7.7 (2H, m),
8.2-8.3 (2H, m), 8.43 (1H, s). Anal. (C16H15N6Cl) C, H, N.
5-n -Bu tyl-8-(4-ch lor op h en yl)-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (17). Yield 88%; colorless powder; mp 273-275 °C. 1H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 6.9 Hz), 1.4-1.6 (2H, m),
1.8-2.0 (2H, m), 3.35 (2H, t, J ) 7.4 Hz), 7.64 (2H, d, J ) 8.4
Hz), 8.27 (2H, d, J ) 8.4 Hz), 8.43 (1H, s), 13.6-14.0 (1H, brs).
Anal. (C16H15N6Cl) C, H, N.
5-n -Bu t yl-8-(4-flu or op h en yl)-3H -[1,2,4]t r ia zolo[5,1-i]-
p u r in e (18). Yield 85%; colorless powder; mp 259-260 °C. 1H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.4 Hz), 1.4-1.6 (2H, m),
1.9-2.0 (2H, m), 3.36 (2H, t, J ) 7.7 Hz), 7.41 (2H, t, J ) 8.9
Hz), 8.31 (2H, dd, J ) 6.4, 8.9 Hz), 8.43 (1H, s). Anal.
(C16H15N6F) C, H, N.
8-(4-Br om op h en yl)-5-n -bu tyl-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (19). Yield 81%; colorless needles; mp 280-282 °C.
1H NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.4 Hz), 1.4-1.6 (2H,
m), 1.9-2.0 (2H, m), 3.35 (2H, t, J ) 7.4 Hz), 7.78 (2H, d, J )
8.4 Hz), 8.21 (2H, d, J ) 8.4 Hz), 8.43 (1H, s). Anal.
(C16H15N6Br) C, H, N.
5-n -Bu tyl-8-(3-tolyl)-3H-[1,2,4]tr iazolo[5,1-i]pu r in e (20).
Yield 70%; colorless powder; mp 201-204 °C. 1H NMR (DMSO-
d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.4-1.6 (2H, m), 1.8-2.0 (2H,
m), 2.43 (3H, s), 3.34 (2H, t, J ) 7.7 Hz), 7.35 (1H, d, J ) 7.7
Hz), 7.45 (1H, t, J ) 7.7 Hz), 8.07 (1H, d, J ) 7.7 Hz), 8.08
(1H, s), 8.42 (1H, s), 13.5-14.0 (1H, brs). Anal. (C17H18N6‚H2O)
C, H, N.
(3H, s), 7.50 (1H, s), 10.7-11.3 (1H, brs).
Met h yl N-(4-Cya n o-1H -im id a zol-5-yl)p en t a n im id a t e
(37). Yield 96%; colorless powder. 1H NMR (CDCl3): δ 0.86
(3H, t, J ) 6.9 Hz), 1.2-1.4 (2H, m), 1.5-1.7 (2H, m), 2.43
(2H, t, J ) 7.4 Hz), 3.83 (3H, s), 7.46 (1H, s), 10.1-10.5 (1H,
brs).
Meth yl N-(4-Cya n o-1H-im id a zol-5-yl)ben zim id a te (38).
Yield 90%; colorless powder. 1H NMR (CDCl3): δ 4.01 (3H, s),
7.2-7.5 (6H, m), 10.2-10.7 (1H, brs).
Gen er a l P r oced u r e for th e Syn th esis of 1,2,4-Tr ia zolo-
[5,1-i]p u r in e Der iva tives 4-7 a n d 10-32. A mixture of the
imidate 34-38 (4.85 mmol) and the corresponding acylhydra-
zine (5.34 mmol) in 10 mL of DMF or 2-methoxyethyl ether
(diglyme) was refluxed for 3-24 h. One hundred milliliters of
aqueous 50% ethanol was added to the reaction mixture at 80
°C. After it was cooled at room temperature, the precipitate
was filtrated and washed with aqueous 50% ethanol. The
filtrate was recrystallized from aqueous ethanol or aqueous
methanol.
5-Met h yl-8-p h en yl-3H-[1,2,4]t r ia zolo[5,1-i]p u r in e (4).
Yield 90%; colorless powder; mp >285 °C. 1H NMR (DMSO-
d6): δ 3.03 (3H, s), 7.5-7.7 (3H, m), 8.3-8.4 (2H, m), 8.48 (1H,
s), 13.6-14.1 (1H, brs). Anal. (C13H10N6) C, H, N.
5-Eth yl-8-ph en yl-3H-[1,2,4]tr iazolo[5,1-i]pu r in e (5). Yield
1
87%; colorless powder; mp 253-255 °C. H NMR (DMSO-d6):
δ 1.46 (3H, t, J ) 7.4 Hz), 3.39 (2H, q, J ) 7.4 Hz), 7.5-7.7
(3H, m), 8.2-8.3 (2H, m), 8.43 (1H, s), 13.6-14.0 (1H, brs).
Anal. (C14H12N6‚0.75H2O) C, H, N.
8-P h en yl-5-n -p r op yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (6).
Yield 88%; colorless powder; mp 230-233 °C. 1H NMR (DMSO-
d6): δ 1.07 (3H, t, J ) 7.4 Hz), 1.9-2.1 (2H, m), 3.33 (2H, t, J
) 7.7 Hz), 7.5-7.7 (3H, m), 8.2-8.3 (2H, m), 8.43 (1H, s), 13.6-
14.1 (1H, brs). Anal. (C15H14N6‚H2O) C, H, N.
5-n -Bu tyl-8-p h en yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (7).
Yield 92%; colorless powder; mp 238-240 °C. 1H NMR (DMSO-
d6): δ 1.05 (3H, t, J ) 7.4 Hz), 1.5-1.6 (2H, m), 1.9-2.1 (2H,
m), 3.43 (2H, t, J ) 7.4 Hz), 7.6-7.7 (3H, m), 8.3-8.4 (2H, m),
8.50 (1H, s), 13.6-14.2 (1H, brs). Anal. (C16H16N6‚1.2H2O) C,
H, N.
5-n -Bu tyl-8-(4-tolyl)-3H-[1,2,4]tr iazolo[5,1-i]pu r in e (21).
Yield 74%; colorless powder; mp 254-255 °C. 1H NMR (DMSO-
d6): δ 0.98 (3H, t, J ) 7.4 Hz), 1.4-1.6 (2H, m), 1.8-2.0 (2H,
m), 2.40 (3H, s), 3.35 (2H, t, J ) 7.4 Hz), 7.38 (2H, d, J ) 7.9
Hz), 8.16 (2H, d, J ) 7.9 Hz), 8.42 (1H, s), 13.5-14.0 (1H, brs).
Anal. (C17H18N6) C, H, N.
5-n -Bu tyl-8-(4-t-bu tylp h en yl)-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (22). Yield 67%; colorless powder; mp 242-244 °C. 1H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.35 (9H, s), 1.4-
1.5 (2H, m), 1.9-2.0 (2H, m), 3.36 (2H, t, J ) 7.7 Hz), 7.60
(2H, d, J ) 8.2 Hz), 8.21 (2H, d, J ) 8.2 Hz), 8.42 (1H, s).
Anal. (C20H24N6) C, H, N.
5,8-Dip h en yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (10). Yield
1
46%; colorless powder; mp 268-269 °C. H NMR (DMSO-d6):
d. 7.5-7.6 (3H, m), 7.6-7.7 (3H, m), 8.2-8.3 (2H, m), 8.3-8.4
(2H, m), 8.54 (1H, s), 13.7-14.2 (1H, brs). Anal. (C18H12N6‚
0.25H2O) C, H, N.
5-n -Bu tyl-8-(4-tr iflu or om eth ylph en yl)-3H-[1,2,4]tr iazolo-
[5,1-i]p u r in e (23). Yield 69%; colorless powder; mp 278-280
1
°C. H NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.4-1.6
(2H, m), 1.8-2.0 (2H, m), 3.33 (2H, t, J ) 7.7 Hz), 7.92 (2H, d,
J ) 7.9 Hz), 8.43 (1H, s), 8.44 (2H, d, J ) 7.9 Hz). Anal.
(C17H15N6F3) C, H, N.
8-(Bip h en yl-4-yl)-5-n -bu tyl-3H-[1,2,4]tr ia zolo[5,1-i]p u -
r in e (24). Yield 79%; colorless powder; mp 244-246 °C. 1H
NMR (DMSO-d6): δ 0.99 (3H, t, J ) 7.2 Hz), 1.4-1.6 (2H, m),
1.8-2.0 (2H, m), 3.38 (2H, t, J ) 7.7 Hz), 7.3-7.6 (3H,m), 7.78
(2H, d, J ) 8.2 Hz), 7.89 (2H, d, J ) 7.7 Hz), 8.36 (2H, d, J )
7.7 Hz), 8.43 (1H, s). Anal. (C22H20N6) C, H, N.
5-n -Bu tyl-8-(4-h yd r oxyp h en yl)-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (25). Yield 87%; colorless powder; mp >285 °C. 1H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.4 Hz), 1.4-1.6 (2H, m),
1.9-2.0 (2H, m), 3.34 (2H, t, J ) 6.9 Hz), 6.94 (2H, d, J ) 8.9
Hz), 8.11 (2H, d, J ) 8.9 Hz), 8.40 (1H, s), 9.7-10.2 (1H, brs),
13.3-14.2 (1H, brs). Anal. (C16H16N6O‚1.8H2O) C, H, N.
5-n -Bu tyl-8-(3-m eth oxyp h en yl)-3H-[1,2,4]tr ia zolo[5,1-
i]p u r in e (26). Yield 74%; colorless prisms; mp 183-185 °C.
1H NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.4-1.6 (2H,
m), 1.9-2.0 (2H, m), 3.36 (2H, t, J ) 7.9 Hz), 3.88 (3H, s),
7.12 (1H, d, J ) 8.4 Hz), 7.49 (1H, dd, J ) 7.7, 8.4 Hz), 7.79
(1H, s), 7.86 (1H, d, J ) 7.7 Hz), 8.43 (1H, s), 13.6-14.0 (1H,
brs). Anal. (C17H18N6O) C, H, N.
5-n -Bu tyl-8-m eth yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (11).
Yield 45%; colorless powder; mp 252-254 °C. 1H NMR (DMSO-
d6): δ 0.95 (3H, t, J ) 7.2 Hz), 1.3-1.5 (2H, m), 1.8-2.0 (2H,
m), 2.54 (3H, s), 3.25 (2H, t, J ) 7.9 Hz), 8.38 (1H, s). Anal.
(C11H14N6) C, H, N.
8-Ben zyl-5-n -bu tyl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (12).
Yield 80%; colorless prisms; mp 197-200 °C. 1H NMR (DMSO-
d6): δ 0.95 (3H, t, J ) 7.2 Hz), 1.3-1.5 (2H, m), 1.8-2.0 (2H,
m), 3.28 (2H, t, J ) 7.9 Hz), 4.25 (2H, s), 7.2-7.5 (5H, m),
8.38 (1H, s). Anal. (C17H18N6) C, H, N.
5-n -Bu t yl-8-(3-p yr id yl)-3H -[1,2,4]t r ia zolo[5,1-i]p u -
1
r in e (13). Yield 38%; colorless powder; mp >220 °C (dec). H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.4 Hz), 1.4-1.6 (2H, m),
1.8-2.0 (2H, m), 3.38 (2H, t, J ) 7.4 Hz), 7.62 (1H, dd, J )
5.0, 7.9 Hz), 8.44 (1H, s), 8.59 (1H, d, J ) 7.9 Hz), 8.75 (1H, d,
J ) 5.0 Hz), 9.42 (1H, s). Anal. (C15H15N7) C, H, N; C: calcd,
61.42; found, 52.29. N: calcd, 33.43; found, 28.30.
5-n -Bu tyl-8-(2-fu r yl)-3H-[1,2,4]tr iazolo[5,1-i]pu r in e (14).
Yield 64%; colorless powder; mp 255-256 °C. 1H NMR (DMSO-
d6): δ 0.97 (3H, t, J ) 7.4 Hz), 1.4-1.5 (2H, m), 1.8-2.0 (2H,
m), 3.33 (2H, t, J ) 7.4 Hz), 6.75 (1H, dd, J ) 2.0, 3.5 Hz),
7.29 (1H, d, J ) 3.5 Hz), 7.97 (1H, d, J ) 2.0 Hz), 8.43 (1H, s),
13.6-14.0 (1H, brs). Anal. (C14H14N6O) C, H, N.
5-n -Bu tyl-8-(4-m eth oxyp h en yl)-3H-[1,2,4]tr ia zolo[5,1-
i]p u r in e (27). Yield 70%; colorless powder; mp 237-240 °C.
1H NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.4 Hz), 1.4-1.6 (2H,
5-n -Bu tyl-8-(2-ch lor op h en yl)-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (15). Yield 80%; colorless powder; mp 177-179 °C. 1H

1,2,4-Triazolo[5,1-i]purine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3707
m), 1.8-2.0 (2H, m), 3.34 (2H, t, J ) 7.4 Hz), 3.86 (3H, s),7.11
(2H, d, J ) 8.9 Hz), 8.20 (2H, d, J ) 8.9 Hz), 8.41 (1H, s).
Anal. (C17H18N6O) C, H, N.
5-n -P en tyl-8-p h en yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (8).
Yield 40%; colorless powder; mp 219-220 °C. 1H NMR (DMSO-
d6): δ 0.91 (3H, t, J ) 6.9 Hz), 1.3-1.5 (4H, m), 1.9-2.0 (2H,
m), 3.36 (2H, t, J ) 7.4 Hz), 7.5-7.6 (3H, m), 8.2-8.3 (2H, m),
8.43 (1H, s). Anal. (C16H16N6‚1.2H2O) C, H, N.
5-n -Hexyl-8-p h en yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e (9).
Yield 65%; colorless powder; mp 205-209 °C. 1H NMR (DMSO-
d6): δ 0.88 (3H, t, J ) 7.2 Hz), 1.2-1.6 (6H, m), 1.9-2.0 (2H,
m), 3.36 (2H, t, J ) 7.7 Hz), 7.4-7.7 (3H, m), 8.2-8.3 (2H, m),
8.43 (1H, s), 13.6-14.1 (1H, brs). Anal. (C17H18N6) C, H, N.
Hu m a n Clon ed Ad en osin e A1, A2A, A2B, a n d A3 Recep -
tor Bin d in g Assa y. Binding of [3H]DPCPX to Chinese
hamster ovary cells transfected with the human recombinant
A1 adenosine receptor was performed as previously described.24
Displacement experiments were performed for 60 min at 22
°C in 0.25 mL of 50 mM Tris-HCl buffer, 5 mM MgCl2, 1 mM
EDTA at pH 7.4, 2 units/mL adenosine deaminase containing
1 nM [3H]DPCPX, diluted membranes (20 µg of protein/assay),
and eight different concentrations of examined compounds.
Nonspecific binding was determined in the presence of 1 µM
DPCPX.
Binding of [3H]CGS 21680 to HEK-293 cells transfected with
the human recombinant A2A adenosine receptor was performed
as previously described.25 Displacement experiments were
performed for 90 min at 22 °C in 0.25 mL of 50 mM Tris-HCl
buffer, 10 mM MgCl2 at pH 7.4, 2 units/mL adenosine
deaminase containing 6 nM [3H]CGS 21680, diluted mem-
branes (50 µg of protein/assay), and at least 3-8 different
concentrations of examined compounds. Nonspecific binding
was determined in the presence of 10 µM NECA.
5-n -Bu tyl-8-(4-eth oxyp h en yl)-3H-[1,2,4]tr ia zolo[5,1-i]-
p u r in e (28). Yield 71%; colorless powder; mp 241-243 °C. 1H
NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.38 (3H, t, J )
6.9 Hz),1.4-1.6 (2H, m), 1.9-2.0 (2H, m), 3.34 (2H, t, J ) 7.4
Hz), 4.12 (2H, q, J ) 6.9 Hz),7.09 (2H, d, J ) 8.7 Hz), 8.19
(2H, d, J ) 8.7 Hz), 8.41 (1H, s). Anal. (C18H20N6O) C, H, N.
5-n -Bu tyl-8-(4-n -p r op oxyp h en yl)-3H-[1,2,4]tr ia zolo[5,1-
i]p u r in e (29). Yield 87%; colorless powder; mp 233-235 °C.
1H NMR (DMSO-d6): δ 0.9-1.1 (6H, m), 1.4-1.6 (2H, m), 1.7-
1.8 (2H, m), 1.9-2.0 (2H, m), 3.34 (2H, t, J ) 7.7 Hz), 4.02
(2H, t, J ) 6.4 Hz), 7.11 (2H, d, J ) 8.4 Hz), 8.19 (2H, d, J )
8.4 Hz), 8.41 (1H, s). Anal. (C19H22N6O) C, H, N.
5-n -Bu tyl-8-(3,4,5-tr im eth oxyph en yl)-3H-[1,2,4]tr iazolo-
[5,1-i]p u r in e (30). Yield 82%; colorless powder; mp 191-193
1
°C. H NMR (DMSO-d6): δ 0.99 (3H, t, J ) 7.2 Hz), 1.4-1.6
(2H, m), 1.8-2.0 (2H, m), 3.33 (2H, t, J ) 7.7 Hz), 3.78 (3H,
s), 3.92 (6H, s), 7.52 (2H, s), 8.41 (1H, s), 13.5-14.1 (1H, brs).
Anal. (C19H22N6O3‚H2O) C, H, N.
5-n -Bu t yl-8-(4-m et h ylt h iop h en yl)-3H -[1,2,4]t r ia zolo-
[5,1-i]p u r in e (31). Yield 56%; colorless powder; mp 264-265
1
°C. H NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.2 Hz), 1.4-1.6
(2H, m), 1.9-2.0 (2H, m), 2.56 (3H, s), 3.35 (2H, t, J ) 7.4
Hz), 7.44 (2H, d, J ) 8.7 Hz), 8.19 (2H, d, J ) 8.7 Hz), 8.41
(1H, s). Anal. (C17H18N6S) C, H, N.
5-n -Bu tyl-8-(4-dim eth ylam in oph en yl)-3H-[1,2,4]tr iazolo-
[5,1-i]p u r in e (32). Yield 79%; colorless powder; mp 236-240
1
°C. H NMR (DMSO-d6): δ 0.98 (3H, t, J ) 7.5 Hz), 1.4-1.5
(2H, m), 1.9-2.0 (2H, m), 3.01 (6H, s), 3.33 (2H, t, J ) 7.9
Hz), 6.84 (2H, d, J ) 8.7 Hz), 8.08 (2H, d, J ) 8.7 Hz), 8.38
(1H, s), 13.4-14.2 (1H, brs). Anal. (C18H21N7) C, H, N.
Meth od B. 5-Am in o-4-(3-p h en yl-1H-1,2,4-tr ia zol-5-yl)-
1H-im id a zole (39). Compound 4 (1.6 g, 6.4 mmol) in 16 mL
of 10% hydrochloric acid was heated at reflux temperature for
1 h. After it was cooled, the mixture was carefully basified
with 25% aqueous ammonia solution. The resulting precipitate
was collected by filtration and then recrystallized with aqueous
ethanol. Yield 1.2 g (83%); colorless powder. 1H NMR (DMSO-
d6): δ 5.4-5.8 (2H, brs), 7.2-7.6 (4H, m), 8.06 (2H, d, J ) 7.9
Hz), 11.4-11.8 (1H, brs), 13.5-14.0 (1H, brs).
Gen er a l P r oced u r e for th e Syn th esis of 5-(N-Alk a n oyl-
a m in o)-4-(3-p h en yl-1H-1,2,4-tr ia zol-5-yl)-1H-im id a zole 40
a n d 41. n-Hexanoyl or n-heptanoyl chloride (30.9 mmol) was
added dropwise to a suspension of 39 (8.8 mmol) in 20 mL of
pyridine at 0 °C. The reaction mixture was stirred for 0.5-1
h at 0 °C and then for 16 h at room temperature. Followed by
the addition of methanol (20-40 mL), the mixture was refluxed
for 1 h. After it was cooled, a precipitate was collected and
washed with methanol.
5-(N-n -Hexa n oyla m in o)-4-(3-p h en yl-1H-1,2,4-tr ia zol-5-
yl)-1H-im id a zole (40). Yield 66%; colorless powder. 1H NMR
(DMSO-d6): δ 0.8-1.0 (3H, brs), 1.2-1.4 (4H, brs), 1.6-1.8
(2H, brs), 2.3-2.6 (2H, brs), 7.4-7.6 (4H, m), 8.0-8.2 (2H, m),
9.8-10.2 (1H, brs), 12.4-13.0 (1H, brs), 14.0-14.4 (1H, brs).
5-(N-n -Hep ta n oyla m in o)-4-(3-p h en yl-1H-1,2,4-tr ia zol-
5-yl)-1H-im id a zole (41). Yield 67%; colorless powder. 1H
NMR (DMSO-d6): δ 0.8-1.0 (3H, brs), 1.2-1.4 (4H, brs), 1.3-
1.5 (2H, brs), 1.6-1.8 (2H, brs), 2.3-2.6 (2H, brs), 7.4-7.6 (4H,
m), 8.0-8.2 (2H, m), 9.8-10.3 (1H, brs), 12.4-13.0 (1H, brs),
13.9-14.5 (1H, brs).
Gen er a l P r oced u r e for th e Syn th esis of 5-Alk yl-8-
p h en yl-3H-[1,2,4]tr ia zolo[5,1-i]p u r in e 8 a n d 9. Chlorotri-
methylsilane (4.9 mmol) was added dropwise to a suspension
of the N-acylated aminoimidazole 40 or 41 (1.2 mmol) in 8 mL
of tetrahydrofurane (THF) and triethylamine (9.9 mmol). The
reaction mixture was refluxed for 24-72 h. After it was cooled,
the reaction was quenched by adding cold water. The crude
product was extracted with AcOEt and dried with sodium
sulfate. The solvent was evaporated, and the residue was
purified by column chromatography using CHCl3/CH3OH (25:
1) as the eluent. Recrystallization from aqueous methanol gave
the corresponding product 8 or 9 as a colorless powder.
Binding of [3H]DPCPX to HEK-293 cells transfected with
the human recombinant A2B adenosine receptor was performed
as previously described.26 Displacement experiments were
performed for 120 min at 22 °C in 0.50 mL of 10 mM Hepes-
Tris buffer, 1 mM MgCl2, 1 mM EDTA at pH 7.4, containing
5 nM [3H]DPCPX, diluted membranes (500 µg of protein/
assay), and eight different concentrations of examined com-
pounds. Nonspecific binding was determined in the presence
of 100 µM NECA.
Binding of [125I]AB-MECA to HEK-293 cells transfected with
the human recombinant A3 adenosine receptor was performed
as previously described.27 Displacement experiments were
performed for 90 min at 22 °C in 0.25 mL of 50 mM Tris-HCl
buffer, 5 mM MgCl2, 1 mM EDTA at pH 7.4, 2 units/mL
adenosine deaminase containing 0.1 nM [125I]AB-MECA, di-
luted membranes (20 µg of protein/assay), and at least 3-8
different concentrations of examined compounds. Nonspecific
binding was determined in the presence of 1 µM IB-MECA.
Ack n ow led gm en t. We thank Dr. S. Sato, Dr. K.
Miyata, Mr. K. Kiryu, and Mr. Eric Hasegawa for their
support of this effort.
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