New 2-heterocyclyl-imidazo[2,1- i ]purin-5-one derivatives as potent and selective human A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm2004738

A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/ethyl-2-[(substituted) isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-ones has been synthesized and evaluated in radioligand binding assays to determine their affinities at the human A₁, A<

Full text of DOI:10.1021/jm2004738

ARTICLE
pubs.acs.org/jmc
New 2-Heterocyclyl-imidazo[2,1-i]purin-5-one Derivatives as Potent
and Selective Human A3 Adenosine Receptor Antagonists
Pier Giovanni Baraldi,*,† Delia Preti,*,† Abdel Naser Zaid,§ Giulia Saponaro,† Mojgan Aghazadeh Tabrizi,†
Stefania Baraldi,† Romeo Romagnoli,† Allan R. Moorman,|| Katia Varani,‡ Sandro Cosconati,#
Salvatore Di Maro,^ Luciana Marinelli,^ Ettore Novellino,^ and Pier Andrea Borea‡
†Dipartimento di Scienze Farmaceutiche, ‡Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia,
Universitꢀa di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara Italy
§College of Pharmacy, An-Najah National University, Nablus, Palestine
King Pharmaceutical R & D, 4000 CentreGreen Way, Suite 300 Cary, North Carolina 27513, United States
^Dipartimento di Chimica Farmaceutica e Tossicologica, Universitꢀa “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
#Dipartimento di Scienze Ambientali, Seconda Universitꢀa di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
S Supporting Information
b
ABSTRACT: A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/
ethyl-2-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]-
purin-5(4H)-ones has been synthesized and evaluated in radi-
oligand binding assays to determine their affinities at the human
A1, A2A, and A3 adenosine receptors. Efficacy at the hA2B AR and
antagonism of selected ligands at the hA3 AR were also assessed
through cAMP experiments. All of the synthesized molecules
exhibited high affinity at the hA3 AR (Ki values ranging from 1.46
to 44.8 nM), as well as remarkable selectivity versus A1, A2A, and
A2B AR subtypes. Compound (R)-4-allyl-8-ethyl-7,8-dihydro-
2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-imidazo[2,1-i]purin-
5(4H)-one (R-33) was found to be the most potent and selective
ligand of the series (Ki hA3 = 1.46 nM, Ki hA2A/Ki hA3 > 3425;
IC50 hA2B/Ki hA3 > 3425; Ki hA1/Ki hA3 = 1729). Molecular modeling studies were helpful in rationalizing the available
structureꢀactivity relationships along with the selectivity profiles of the new series of ligands.
’ INTRODUCTION
Although many different chemical classes have been identified
as A3 AR antagonists, few have reached the preclinical phase of
investigation, possibly due to the enigmatic physiological role of
A3 AR, whose activation seems related to opposite effects con-
cerning tissue protection in inflammatory and cancer cells. A3 AR
antagonists are currently under biological testing for their promis-
ing therapeutic potential in the treatment of inflammation, neu-
rodegeneration, ischemia, asthma, glaucoma, and cancer.8ꢀ11
A wide number of compounds exerting high potency and
selectivity in antagonizing the hA3 AR has been identified.12,13
Aromatic nitrogen-containing monocyclic (i.e., thiazoles, thiadiazoles,14
1,4-dihydropyridines,15 pyridines,16 2-mercaptopyrimidines17), bicyclic
(i.e., flavonoid,18 isoquinoline, quinozalines,19 (aza)adenines20), tri-
cyclic systems (i.e., pyrazoloquinolines,21,22 triazoloquinoxalines,23
pyrazolotriazolopyrimidines,24 triazolopurines,25 tricyclic xan-
thines26ꢀ28), and nucleoside-derived antagonists29ꢀ31 have been
reported as A3 AR ligands.
Adenosine exerts a number of physiological functions through
activation of four cell membrane G-protein-coupled receptors
classified as A1, A2A, A2B, and A3 (ARs).1 The A3 AR has been the
subject of intensive investigations over the past decade as a
potential therapeutic target due to its contribution to important
pathophysiological processes.2
The clarification of the role of adenosine and its receptors in
cancer development may hold great promise for the chemother-
apeutic treatment of patients affected by malignancies.3 It has
been suggested that adenosine inhibits tumor cell growth while
maintaining bone marrow cell proliferation through the involve-
ment of the A3 receptors.4 Evidence of high levels of expression
of A3 adenosine receptor subtype has been provided in several
tumor cell lines, and A3 specific antagonists seem to synergisti-
cally enhance cytotoxic treatment and counter P-glycoprotein
efflux in multidrug resistance.5ꢀ7 A recent study provides the first
evidence that the A3 AR plays a role in colon tumorigenesis and,
more importantly, can potentially be used as a diagnostic marker
or a therapeutic target for colon cancer.8
Received: April 20, 2011
Published: June 15, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of the Described 2-Heterocyclyl-
imidazo[2,1-i]purin-5-one Derivativesa
a Reagents: (i) (a) XCOOH, EDAC, HOBt, DMF, rt, 24 h; (b) NaOH
10%, reflux, 1.5 h; (ii) P2S5, pyridine, 140 ꢀC, 5 h; (iii) CH3I, NaOH/
EtOH, rt, 3 h; (iv) (R/S/R,S)-2-amino-butan/propan-1-ol, DMSO,
150 ꢀC, 1 h; (v) SOCl2, CH2Cl2, reflux, 18 h.
Figure 1. 2-Substituted-imidazo[2,1-i]purin-5-one derivatives as ARs
antagonists.
in binding studies in CHO cells expressing recombinant hA3 ARs
(IC50 = 4 nM).
We focused our attention on the imidazo[2,1-i]purin-5-one
scaffold, obtained by the fusion of an imidazoline ring on the
xanthine bicycle, as an interesting tricyclic structure useful for the
development of ARs ligands. Particular attention has been paid to
substitution at the 2-position of the imidazo[2,1-i]purinone
nucleus, which theoretically corresponds to the 8-position of
the original xanthine core. It has been established that the 2-sub-
stituent plays a crucial role in the subtype selectivity of the AR
antagonists reported so far.32ꢀ37 Compound 1a (KF20274,
Figure 1),32 substituted at the 2-position with a 3-noradamantyl
moiety, can be structurally associated to the previously reported A1
antagonist 1,3-dipropyl-8-(3-noradamantyl)xanthine.32 Thus, the
3-noradamantyl function was shown to induce A1 selective antago-
nist activity in both the tricycle 1a and the corresponding xanthine
analogue. The main advantage claimed for the annelation of the
xanthine core into the imidazo[2,1-i]purinone scaffold is the
enhancement of water solubility afforded by the imidazoline basic
nitrogen, which has been reported to be subject to protonation
at physiological pH. Compound 1b, (R)-7,8-dihydro-8-ethyl-
2-(4-bicyclo[2.2.2]octan-1-ol)-4-propyl-1H-imidazo[2,1-i]purin-
5(4H)-one,33 is a particularly potent A1 AR antagonist with good
selectivity over the other three AR subtypes and high water
solubility (>100 mg/mL) and showed a good in vivo profile after
oral administration in a rat diuresis model. M€uller and co-worker
explored the imidazopurinone nucleus, introducing at the 2-posi-
tion substituents previously known to promote A2A or A3 AR
activity in the corresponding 8-substituted xanthine analogues.35
Compound 2 (Figure 1) was conceived as a water-soluble tricyclic
congener of 8-styrylxanthines with A2A AR antagonist properties,
while derivative 3 (PSB11,35 (R)-4-methyl-8-ethyl-2-phenyl-
4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one) exhibited a Ki
value of 2.3 nM for A3 receptor. The radiolabeled derivative of this
compound exhibited a KD value of 4.9 nM and a Bmax value
of 3500 fmol/mg of protein.36 The 2-(2,3,5-trichlorophenyl)
substituted analogue, PSB10,37 showed inverse agonist activity
In a recent study, we synthesized a wide series of 8-hetero-
cyclyl-substituted xanthine derivatives, among which we identi-
fied very potent and selective human A2B AR antagonists.38 With
this series, whose design was based on the structure of the
8-phenyl-xanthine derivative MRS1754 (N-(4-cyanophenyl)-
2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-
phenoxy]acetamide),39 we demonstrated that the phenyl and
the pyrazole rings may occasionally behave as bioisosters. Given
these findings, in the present study, we evaluated the effect of the
replacement of the 2-phenyl ring of 3 and congeners with
differently substituted 5-membered heterocycles, in particular
1,3- and 1,5-disubstituted pyrazoles or a 3-substituted isoxazole
(Figure 1). At the 4-position, an allyl or a benzyl group have been
introduced, while the 8-position has been functionalized with a
methyl or an ethyl group, as the efficacy of such substituents was
suggested by previous SAR studies on a different series of
xanthine-related ARs antagonists. To verify a possible enantio-
selective interaction between the newly reported series of
imidazo[2,1-i]purinones and ARs, the pharmacological proper-
ties of the optically pure enantiomers have been compared to
those of the corresponding racemates for a select number of
compounds. Moreover, molecular modeling studies were helpful
in rationalizing the available structureꢀactivity relationships
along with the selectivity profiles of the new series of ligands.
’ CHEMISTRY
The synthesis of the 2-heterocyclyl tricyclic purinone deriva-
tives has been performed, in analogy to described procedures, as
depicted in Scheme 1.32,35,37,40 1-Subsituted-5,6-diaminouracils
4a,b41 and the appropriate pyrazole/isoxazole carboxylic acids
were reacted in DMF solution in the presence of 1-ethyl-3-[3
(dimethylamino)propyl]carbodiimide hydrochloride (EDAC)
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Table 1. Binding (hA1, hA2A, and hA3) and Functional (hA2B, hA3) Parameters of the Synthesized 2-Heterocyclyl-imidazo[2,1-
i]purin-5-one Derivatives Towards Adenosine Receptors
hA1
(Ki nM)a
hA2A
(Ki nM)b
A2B
(IC50 nM)c
hA3
(Ki nM)d
hA3
(IC50 nM)e
compd
X
Y
R
28
Me
allyl
Me
2040
(1710ꢀ2450)
2510
>5000
(7%)
>5000
(30%)
>5000
(10%)
>5000
(12%)
>5000
(20%)
>5000
(8%)
4.56
(3.44ꢀ6.05)
5.04
34.2
(27.4ꢀ42.6)
35.9
R-28
S-28
29
Me
Me
Et
allyl
allyl
allyl
Bn
Me
>5000
(1%)
(2050ꢀ3060)
1960
(5.14ꢀ6.01)
6.03
(29.9ꢀ43.0)
40.9
Me
>5000
(4%)
(1540ꢀ2480)
515
(5.64ꢀ6.38)
3.01
(34.9ꢀ47.9)
30.3
Me
>5000
(8%)
(437ꢀ608)
>5000
(2.29ꢀ3.96)
17.2
(26.0ꢀ35.2)
ndf
30
Me
Et
Me
>5000
(1%)
(20%)
(12.2ꢀ24.0)
10.3
31
Bn
Me
615
>5000
(5%)
>5000
(20%)
>5000
(10%)
>5000
(13%)
>5000
(13%)
>5000
(16%)
>5000
(21%)
>5000
(14%)
>5000
(11%)
>5000
(19%)
>5000
(22%)
>5000
(22%)
>5000
(17%)
>5000
(14%)
>5000
(13%)
>5000
(12%)
>5000
(9%)
nd
nd
nd
(536ꢀ707)
803
(7.9ꢀ13.4)
12.6
R-31
S-31
32
Et
Bn
Me
>5000
(3%)
(654ꢀ986)
545
(9.4ꢀ16.9)
13.2
Et
Bn
Me
>5000
(4%)
(468ꢀ635)
>5000
(9.7ꢀ17.9)
2.36
Me
Et
allyl
allyl
allyl
allyl
Bn
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OBn
OBn
OBn
OBn
>5000
(1%)
14.5
(10.5ꢀ20.1)
12.2
(30%)
(1.60ꢀ3.49)
1.96
33
1530
>5000
(1%)
(1280ꢀ1830)
2525
(1.22ꢀ3.13)
1.46
(8.4ꢀ17.7)
23.4
R-33
S-33
34
Et
>5000
(13%)
>5000
(15%)
>5000
(1%)
(2106ꢀ3027)
1078
(0.88ꢀ2.42)
2.37
(16.7ꢀ32.9)
10.5
Et
(868ꢀ1340)
1900
(1.48ꢀ3.83)
3.11
(7.4ꢀ14.9)
27.6
Me
Me
Me
Et
(1720ꢀ2090)
2550
(2.01ꢀ4.80)
2.38
(23.3ꢀ32.7)
15.9
R-34
S-34
35
Bn
>5000
(11%)
>5000
(1%)
(2060ꢀ3170)
1730
(1.79ꢀ3.17)
2.52
(12.6ꢀ20.1)
17.5
Bn
(1470ꢀ2030)
405
(1.64ꢀ3.88)
3.85
(13.4ꢀ22.9)
20.4
Bn
>5000
(9%)
(338ꢀ486)
>5000
(3.14ꢀ4.72)
24.7
(14.3ꢀ29.1)
nd
36
Me
Et
allyl
allyl
Bn
>5000
(25%)
>5000
(5%)
(47%)
(17.0ꢀ35.8)
21.5
37
1200
nd
nd
nd
nd
nd
(1030ꢀ1400)
2560
(17.0ꢀ27.3)
44.8
38
Me
Et
2010
(2300ꢀ2860)
1680
(1720ꢀ2360)
2130
(36.5ꢀ55.0)
39.8
39
Bn
(1380ꢀ2040)
>5000
(1870ꢀ2430)
>5000
(1%)
(31.6ꢀ50.1)
13.9
40
Me
Et
allyl
allyl
(13%)
(10.7ꢀ18.1)
12.5
41
>5000
>5000
(1%)
>5000
(16%)
(20%)
(8.6ꢀ18.2)
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Table 1. Continued
hA1
(Ki nM)a
hA2A
(Ki nM)b
A2B
(IC50 nM)c
hA3
(Ki nM)d
hA3
(IC50 nM)e
compd
X
Y
R
R-41
Et
allyl
>5000
(12%)
>5000
(3%)
>5000
(14%)
>5000
(8%)
12.2
(8.4ꢀ17.7)
9.20
nd
nd
nd
nd
S-41
42
Et
allyl
Bn
>5000
>5000
(1%)
(29%)
(7.61ꢀ11.12)
12.20
Me
Et
>5000
>5000
(9%)
>5000
(24%)
>5000
(27%)
>5000
(13%)
>5000
(25%)
>5000
(11%)
>5000
(18%)
(22%)
(8.4ꢀ17.7)
15.0
43
Bn
>5000
>5000
(13%)
>5000
(11%)
1520
(28%)
(13.3ꢀ16.9)
3.58
44
Me
Et
allyl
allyl
Bn
>5000
24.2
(19.1ꢀ30.7)
14.6
(26%)
(2.84ꢀ4.51)
1.93
45
3023
(2388ꢀ3827)
>5000
(1126ꢀ2051)
>5000
(5%)
(1.28ꢀ2.93)
2.68
(12.0ꢀ17.8)
20.4
46
Me
Et
(35%)
(2.11ꢀ3.41)
2.71
(14.3ꢀ29.1)
25.3
47
Bn
485
>5000
(407ꢀ578)
(12%)
(2.20ꢀ3.33)
(22.7ꢀ28.2)
a Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells (n = 3ꢀ6). b Displacement of specific [3H]ZM241385
binding at human A2A receptors expressed in CHO cells (n = 3ꢀ6). c Potency (IC50) of examined compounds to inhibit 100 nM NECA stimulation
cAMP levels in hA2B CHO cells. d Displacement of specific [3H]MRE3008F20 binding at human A3 receptors expressed in CHO cells (n = 3ꢀ6). Data
are expressed as geometric means with 95% confidence limits. In parentheses are reported the % of inhibition to hA1, A2A, A2B, and A3 CHO cells.
e Potency (IC50) of selected compounds to inhibit 100 nM Cl-IB-MECA inhibition of cAMP levels in hA3 CHO cells. f Not determined.
as condensing agent, followed by ring closure with sodium hydro-
xide at reflux to afford the desired 3-allyl/benzyl-8-[(substituted)-
isoxazol/pyrazol-3/5-yl]-1H-purine-2,6(3H,7H)-dione derivatives
(5aꢀj).38 The diamino uracils 4a,b were obtained by reduction of
the corresponding nitroso uracils using sodium dithionite.38
The substituted pyrazole/isoxazole carboxylic acids were
prepared according to procedures reported in literature.42 The
3-methoxy-isoxazole-5-carboxylic acid was obtained from the
reaction of dimethyl acetylenedicarboxylate with hydroxylamine,
followed by O-alkylation with methyliodide in presence of
K2CO3, followed by classical saponification.43
The 8-heterocyclyl-xanthine derivatives 5aꢀj were treated
with phosphorus pentasulfide in dry pyridine at reflux to give
the corresponding 6-thioxanthine 6aꢀj. The subsequent reac-
tion with methyliodide in the presence of sodium hydroxide
furnished the S-methyl-derivatives 7aꢀj in good yields. Com-
pounds 7aꢀj were then treated with the appropriate amino
alchol (2-amino-butan/propan-1-ol purchased both as racemic
mixtures and optically active reagents) in anhydrous DMSO at
150 ꢀC for 1.5 h. Final cyclizations were performed in dichlor-
omethane solution by treatment with thionyl chloride heating at
reflux for 18 h. This kind of cyclization is known to yield final
compounds with retained stereochemistry.35
hA2B AR was investigated, evaluating their capability to inhibit
(100 nM) NECA stimulated cAMP production. Antagonism of
selected ligands versus hA3AR was also assessed through cAMP
experiments evaluating their capability to block the inhibitory
effect mediated by Cl-IB-MECA. Affinity data for A1, A2A, and A3
receptors (expressed as Ki values), and IC50 values for hA2B and
hA3 subtypes, derived from the cAMP assays, are listed in Table 1.
StructureꢀActivity Relationships Analysis: hA3AR Affi-
nity. All the synthesized molecules exhibited high affinities at
the hA3AR subtype with Ki values ranging from 1.46 (compound
R-33) to 44.8 nM (compound 38). The different kind of
heterocycle introduced at the 2-position gave various contribu-
tions to the binding profile of the examined imidazo[2,1-i]purin-
5-one derivatives. The affinities at hA3AR of 4-allyl-2-(10,50-
dimethyl-pyrazole) derivatives (28, 29) showed to be about 3-
to 4-fold higher than that of the corresponding 4-allyl-2-(10,30-
dimethyl-pyrazole) isomers (40, 41, respectively), whereas
4-benzyl-2-(10,50-dimethyl-pyrazole) derivatives (30, 31) show
hA3 AR Ki binding values similar to those of the corresponding
4-benzyl-2-(10,50-dimethyl-pyrazole) derivatives 42 and 43. This
suggests that 10,50-disubstitution of the 2-pyrazole ring is equiva-
lent or slightly detrimental, with regard to hA3 binding affinity, if
compared with 10,30-disubstitution. The comparison between
hA3 affinities of 2-(10-methyl-30-methoxy-pyrazole) derivatives
32ꢀ35 and 2-(30-methoxy-isoxazole) derivatives 44ꢀ47 indi-
cated a substantial bioisosterism of these heterocycles as regards
the interaction with hA3 AR of the examined ligands. As indicated
by the affinity values of compounds 28ꢀ39, the type of sub-
stitutions at the 30-position of the 2-pyrazole ring appreciably
affects the affinity for the hA3 AR. The order of efficacy OMe
(compounds 32ꢀ35) > Me (28ꢀ31) > OBn (36ꢀ39) is
maintained in the whole subset of molecules. For example, the
30-OCH3-derivative 32 (Ki hA3 = 2.36 nM) appeared 2- and 10-
fold more potent in binding hA3 subtype than the analogously
’ RESULTS AND DISCUSSION
All the 2-heterocyclyl-imidazo[2,1-i]purin-5-one derivatives
28ꢀ47 (Table 1) were evaluated in radioligand binding assays to
determine their affinities for human A1, A2A, and A3 adenosine
receptors using [3H]-DPCPX (1,3-[3H]-dipropyl-8-cyclopentyl-
xanthine), [3H]-ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-
triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol), [3H]-
MRE3008F20 (5-N-(4-methoxyphenylcarbamoyl)amino-8-pro-
pyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine),
respectively, as radioligands. Efficacy of the compounds versus
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substituted 30-CH3-derivative 28 (Ki hA3 = 4.56 nM) and 30-
OBn-derivative 36 (Ki hA3 = 24.7 nM), respectively. Similarly,
the 30-OCH3-derivative 33 (Ki hA3 = 1.96 nM) was found to be
1.5- and 11-fold more potent then the corresponding 30-CH3
derivative 29 (Ki hA3 = 3.01 nM) and 30-OBn derivative 37
(Ki hA3 = 21.5 nM), respectively, at the hA3AR. This trend led us
to synthesize 30-OCH3-isoxazole derivatives 44ꢀ47, which con-
firmed the efficacy of the introducing a methoxy group at the 30-
position of the 2-heterocycle. From the set of molecules we
analyzed, it emerged that the kind of substitutions of the
5-membered heterocycle at the 2-position of the imidazo[2,1-
i]purin-5-one tricycle is particularly important for hA3 AR
binding affinity. A steric control seems to take place around
the 30-position of the 2-heterocycle, as suggested by the decrease
of affinities of the 30-OBn derivatives 36ꢀ39 (Ki hA3 ranging
from 21.5 to 44.8 nM). Moreover, the angularity component of
the 30-OCH3, in association with the possibility of this function
to establish a hydrogen bond, could contribute to the formation
of particular ligandꢀreceptor interactions.
of the series because of the concomitant small reduction of hA3
affinity. The most selective derivatives have been obtained with
the introduction of the 2-(10-methyl-30-methoxy-pyrazole) or the
2-(30-methoxy-isoxazole) functions (derivatives 32ꢀ35 and
44ꢀ47, respectively). In particular, compounds 32 (4-allyl-7,8-
dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-8-methyl-1H-
imidazo[2,1-i]purin-5(4H)-one) and 46 (4-benzyl-8-methyl-
7,8-dihydro-2-(3-methoxyisoxazol-5-yl)-1H-imidazo[2,1-i]purin-
5(4H)-one) appeared to be the most selective of the series of the
2-heterocyclyl-imidazo[2,1-i]purin-5-ones described, displaying
more than 2100- and 1800-fold selectivity, respectively, for the
hA3 AR over the other AR subtypes. The kind of substituent at
the 30-position of the 2-(10,30-disubstituted-pyrazole) moiety
seems to affect the selectivity of the ligands for the hA3 vs hA1
AR. In fact, the related affinity ratios (Ki hA1/Ki hA3) clearly
suggested that the selectivity follows the sequence 30-OMe > 30-
Me > 30-OBn. For example, compounds 32 (30-OMe), 28 (30-
Me), and 36 (30-OBn) were 2100-, 450-, and 202-fold selective
for hA3 vs hA1, respectively. The same can be noticed in the
comparison of compounds 33 (30-OMe, Ki hA1/Ki hA3 = 780),
29 (30-Me, Ki hA1/Ki hA3 =170),and37 (30-OBn, Ki hA1/Ki hA3 =56)
as well as between compounds 34 (Ki hA1/Ki hA3 = 610), 30
(Ki hA1/Ki hA3 = 291), 38 (Ki hA1/Ki hA3 = 57), and derivatives
35 (Ki hA1/Ki hA3 = 105), 31 (Ki hA1/Ki hA3 = 60), and 39
(Ki hA1/Ki hA3 = 42).
In many of the evaluated examples, the 4-allyl substitution
provides greater selectivity for Ki hA1/Ki hA3 in comparison with
4-benzyl substitution, for example, compounds 32 (Ki hA1/Ki
hA3 = 2119), which is 3.5-fold more selective then the corre-
sponding 4-benzyl substituted 34 (Ki hA1/Ki hA3 = 610).
Similarly, the 4-allyl derivative 28 (Ki hA1/Ki hA3 = 450) is
1.5-fold more selective for hA3 vs hA1 than the corresponding
4-benzyl derivative 30 (Ki hA1/Ki hA3 > 290). The only
exceptions to this leaning are compounds 40 (Ki hA1/Ki hA3 >
359) compared to 42 (Ki hA1/Ki hA3 > 400), in which the
4-substitution with a benzyl or an allyl functions seems practically
equivalent, and compounds 44 (Ki hA1/Ki hA3 > 1397) com-
pared to 46 (Ki hA1/Ki hA3 > 1866), in which the effect on hA3 vs
hA1 selectivity of the two substituents at the 4-position appeared
slightly reversed.
While practically equivalent for their effect on hA3 affinity, the
introduction of a methyl or an ethyl at the 8-position appears to
influence the affinity of the examined molecules toward the hA1
AR subtype. The choice of the substituent at this position was
therefore important for the optimization of hA3 over hA1
selectivity. The general selectivity pattern would quite strongly
suggest a preference by A1 adenosine receptor for the 8-ethyl
substitution. This effect is fairly evident if the 8-methyl derivative
46 (Ki hA1 g 5000, Ki hA1/Ki hA3 > 1866) was compared to the
8-ethyl analogue 47 (Ki hA1 = 485, Ki hA1/Ki hA3 = 179). With
the exception of compound 43, the molecules exerting higher
affinity at the hA1 subtype were concomitantly substituted at the
4- and 8-positions with a benzyl and an ethyl moieties, respec-
tively, indicating a synergistic detrimental effect of the two
substituents on hA3 selectivity. On the contrary, the 4-allyl-8-
methyl-imidazo[2,1-i]purin-5-one derivatives displayed the best
Ki hA1/Ki hA3 ratios of the series.
Introduction of a benzyl or an allyl substituent at the 4-posi-
tion of the imidazo[2,1-i]purin-5-one core was also examined.
The pairwise comparison between the Ki (hA3) values of 4-allyl
and 4-benzyl derivatives resulted in most cases in preferred
affinity for the allyl-substituted compounds (i.e., compounds
30, 31, and 35 were 3.8-, 1.6- and 2-fold less potent then the
correspondent 4-allyl derivatives 28, 29, and 33 as hA3 ligands).
Nevertheless, some exceptions have been observed (see com-
pounds 42, 43, and 46 versus 40, 41, and 44, respectively).
The substitution of the 8-position with a methyl or an ethyl
seems to be essentially equivalent in terms of hA3 affinity, as can
be deduced from the equipotent binding of the 8-methyl and
8-ethyl derivatives. For example compounds 28 exhibited a binding
affinity similar to that of the correspondent 8-ethyl derivative 29.
The same can be observed, for example, for compounds 36, 38, and
40 versus 37, 39, and 41, respectively.
The monosubstitution of C8 generated an asymmetric carbon.
For selected compounds (28, 31, 33, 34, and 41), both racemic
mixtures and optically active derivatives have been prepared in
order to determine if there is a stereoselective preference in the
interaction with hA3 AR. The affinities of racemates at the A3 AR
appeared generally comparable to those of the respective R and S
pure isomers. Only the possibility of a very weak preference for
the R isomer can be noted (R-28 compared to S-28 or R-33
compared to S-33). The data related to the 8-position seem to
suggest that this side of the molecule may not be involved in
forming strong interactions with the hA3 receptor binding site. In
fact, neither the kind of substitution nor the stereoisomeric
configuration of C8 seem to particularly affect the affinity of
the ligands for the hA3 AR.
StructureꢀActivity Relationships Analysis: hA3AR vs hA2A
and hA1 ARs Selectivity. Very high levels of hA3 vs hA2A
selectivity have been obtained with all compounds. Indeed,
with the exception of derivatives 38 (Ki hA2A = 2010 nM), 39
(Ki hA2A = 2130 nM), and 45 (Ki hA2A = 1520 nM), the Ki values
calculated in the binding assay at the hA2A AR were greater than
5 μM. However, the affinity at the hA1 AR subtype was
occasionally considered relevant (lower than 1 μM for com-
pounds 29, 31, 35, and 47). Comparing the type of heterocycle at
the 2-position, the 1,5-disubstituted pyrazole seems to ensure
a low affinity at the hA1 AR. However, derivatives 40ꢀ43
(Ki hA1 > 5 μM), displaying from about 330- to 540-fold selecti-
vity for the hA3 AR over the hA2A AR, are not the most selective
A slight level of enantioselectivity seems to concern the
interaction of the described ligands with the hA1 AR subtype.
The affinities of the S isomers of compounds 28, 31, 33, and 34
were 1.3- to 2.3-fold higher than those of the corresponding R
isomers, thus resulting in a higher hA3 vs hA1 selectivity of the R
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Docking calculations obtained for R-33 in complex with hA3
AR revealed that in the best Glide score (ꢀ6.75), the ligand binds
to the outer portion of the A3 AR being surrounded by TMs 3, 5,
6,7 helices and EL2 (Figure 3). In this position, the imidazo[2,1-
i]purin-5-one scaffold establishes a π-stacking interaction with
F168 and a H-bond between its N3 atom and N250 side chain.
Interestingly, the latter residue was found to be critical for ligand
binding at both the A3 and A2A ARs.47,48
In addition, the 4-allyl substituent points toward the outer part
of the receptor taking favorable hydrophobic contacts with V169,
M174, M177, and I253 (herein referred as L1 pocket). Docking
calculations on compound 35 (see Figure S1 in Supporting
Information) indicated that while the imidazo[2,1-i]purin-5-one
scaffold occupies the same position in the hA3 AR, now the bulky
4-benzyl substituent points outward being less prone to effi-
ciently occupy the L1 cleft. This might explain why compounds
featuring a 4-allyl substituent are generally characterized by a
higher affinity at the hA3 receptor if compared with the corre-
sponding 4-benzyl compounds.
In the same calculated binding pose, the R-33 8-ethyl group
points toward transmembrane helices 2 and 3, although this
substituent is too distant from this region of the receptor to play a
critical role in the receptor binding. This result is in accordance
with SAR data indicating that the substitution of the 8-position
with a methyl or an ethyl and the inversion of the chiral C8 does
not significantly affect the binding at the hA3 AR, these data were
further confirmed by docking results achieved for compound S-
33 (see Figure S2 in Supporting Information).
Figure 2. Inhibition curves of cAMP accumulation in hA3CHO cells by
adenosine antagonists blocking the effect of 100 nM Cl-IB-MECA.
isomers with reference to the corresponding S-configured mol-
ecules. This data resulted in agreement with the findings
previously described by M€uller and co-workers35 and appeared
to give evidence for a direct involvement of the additional
imidazolidine ring in critical interactions between the 2-hetero-
cyclyl-imidazo[2,1-i]purinones and the hA1 AR binding pocket.
This could be the reason for the responsiveness of hA1 AR
binding affinities to the kind of substitution and/or the config-
uration of the C8.
Compound R-33 was found to be the most potent hA3 AR
ligand of the 2-heterocyclyl-imidazo[2,1-i]purin-5-one deriva-
tives herein described. It also confirmed to have very high
selectivity versus A2A, A2B, and also at A1 AR, with one of the
highest value of Ki hA1/Ki hA3 ratio of the series (1729).
Functional Assays: hA2B and hA3 ARs. All the compounds
If compared with the 8-ethyl group, the 10-methyl-30-methoxy-
pyrazole ring in the 2-position is able to make favorable hydro-
phobic contact with the receptor, being embedded in a rather
profound pocket (herein referred as L2) made up by L91, M177,
I186, W243, L246, and S247. Here, the 30-methoxy accepts an
H-bond from the latter residue and the interaction with W243
should be considered to be responsible for the antagonist
properties of this ligand, as already demonstrated for other AR
antagonists.47 Also in this case, the position of the pendant ring in
position 2- in the A3 AR seems to explain the SAR data achieved
for this portion of the molecule. Indeed, as indicated above, the
substitution of the methoxy group with a methyl one
(compounds R-28, S-28, 29, 30, R-31, and S-31) leads to a
decrease of affinity due to the loss of a H-bond with S247 (see
Figure S3 in Supporting Information), while a further decrease of
affinity is recorded for the bulkier benzyloxy derivatives that are
less easily hosted in the L2 pocket (compounds 36ꢀ39).
Docking results were also helpful in rationalizing the lower
affinity at the A3AR of compounds 40ꢀ43. In fact, when docking
R-41 in the A3AR, the resulting binding pose was similar to the
one predicted for R-33 (see Figure S4 in Supporting In-
formation), nevertheless the presence of two methyl groups on
the pendant pyrazole ring results in the loss of the H-bond
interaction with S247. On the other hand, replacement of the
pendant 10-methyl-30-methoxy-pyrazole ring with the 30-meth-
oxy-isoxazole one (compounds 44ꢀ47) still allows the afore-
mentioned interaction with the receptor (see Figure S5 in
Supporting Information) explaining why the two series display
the same binding and selectivity properties at the ARs.
resulted practically inactive at the hA2B AR subtype (IC50
>
5 μM, see Table 1).
The tested compounds confirmed to operate as antagonists of
the hA3 AR subtype in the cAMP functional assay. Interestingly, a
good accordance between binding and functional experiments
performed with hA3 receptor has been revealed. The molecules
showing the best affinities for hA3 adenosine receptor have also
proved to have very high potency (IC50 values in the low
nanomolar range) in the functional assay (Figure 2, Table 1).
Derivative R-33, showing the highest hA3 affinity, also emerged
as the most potent compound of the series with an IC50 value of
10.5 nM.
Molecular Modeling Studies. Molecular modeling studies
were performed with the aim of rationalizing the aforementioned
SAR data both in terms of affinity for the hA3 AR and selectivity
of binding for this receptor versus the hA1 and hA2A ARs. To this
end, the recently published crystal structure of the hA2A receptor
(PDB code 3EML)44 in complex with compound ZM241385
was used to obtain a homology model of the hA1 and hA3 ARs.
The sequence alignment between the three receptors as well as
the positions of the disulfide bridges were attained consistently
with what was already suggested by Moro and co-workers.45 The
structures of the modeled receptors were used to perform
docking calculations of the most potent and selective compound
R-33 employing the Glide program of the Schrodinger package
that was already used to successfully dock a series of ARs
antagonists.46 The docking poses obtained were evaluated for
their consistency with the available SAR data as well as for the
Glide scores obtained from these calculations.
R-33 was predicted to bind at the hA1 and hA2A AR structures
in a different fashion if compared with the calculated R-33/hA3
AR complex (Figure 4). In fact, in the best scoring pose (Glide
scores of ꢀ5.43 and ꢀ5.81 for hA1 and hA2A ARs, respectively),
the 8-ethyl group of the ligand points toward outer part of the
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Figure 3. (a) View of the R-33/hA3 AR complex. (b) Close view of the calculated binding pose for R-33 in the hA3 AR mode.
Figure 4. Close-up view of the calculated binding pose for R-33 in the hA1 AR (a) and hA2A AR structures.
receptors, while the 4-allyl substituent is in the vicinity of the
TM2 and TM3 helices. In this position, the critical N254 (hA1)
and N253 (hA2A) residues are H-bonded by the 20 nitrogen of
the 10-methyl-30-methoxy-pyrazole ring, while F171 (hA1)
and F168 (hA2A) residues are in contact with the imidazo-
[2,1-i]purin-5-one core through a π-stacking interaction.
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Figure 5. Close-up view of some important residues of the L1 pocket in the hA1 (a), hA2A and hA3 ARs.
A comparison of the R-33/hA3 with the R-33/hA1 and R-33/hA2A
complexes reveals that, for the latter complexes, both the L1 and
L2 pockets are not efficiently contacted by the ligand. This
behavior should be ascribed to the different amino acid composi-
tion of the L1 pocket. Indeed, in the hA1 and hA2A AR
respectively, the E172 and E169 residues belonging to the EL2
region are replaced by V169 in the hA3 AR, resulting in different
electrostatic properties of the L1 cleft. Moreover, the presence of
a glutamate residue makes this cleft narrower and less prone to be
occupied be the ligand substituents. In principle, it may be argued
that the flexible EL2 domain could undergo a conformational
rearrangement to allow the opening of the L1 pocket in hA1
and hA2A ARs. Nevertheless, such an event seems unlikely to
happen given that in the hA2A AR, the conformation of E169 is
stabilized by an electrostatic interaction with H264 in EL3. The
same holds true for E172 in hA1, which is involved in the same sort
of interaction with K265 (Figure 5). In this respect, the position of
H264 seems to be relatively constrained, as demonstrated experi-
mentally by Stevens and co-workers.44 Similar results were also
recently achieved by Moro and co-workers, postulating the different
residue composition of the L1 pocket should influence not only the
binding pose but also the access of the ligands to the TM region of
the receptors.45
’ EXPERIMENTAL SECTION
Chemistry. Reaction progress and product mixtures were mon-
itored by thin-layer chromatography (TLC) on silica gel (precoated
F254 Merck plates) and visualized with aqueous potassium permanga-
nate. 1H NMR data were determined in CDCl3 or DMSO-d6 solutions
with a Varian VXR 200 spectrometer or a Varian Mercury Plus 400
spectrometer. Peak positions are given in parts per million (δ) downfield
from tetramethylsilane as internal standard, and J values are given in
hertz. Light petroleum refers to the fractions boiling at 40ꢀ60 ꢀC.
Melting points were determined on a BuchiꢀTottoli instrument and are
uncorrected. Chromatography was performed on Merck 230ꢀ400 mesh
silica gel. Organic solutions were dried over anhydrous sodium sulfate.
Elemental analyses were performed by the microanalytical laboratory
of Dipartimento di Chimica, Universitꢀa di Ferrara, and were within
(0.4% of the theoretical values for C, H, and N. All final compounds
revealed a purity of not less than 95%. The mass spectra were obtained
on a ESI Micromass ZMD 2000 mass spectrometer. Chiral amino
alcohols were purchasedfrom Alfa Aesar orAldrich in the highest available
purity grade.
General Procedure for the Synthesis of 3-Allyl/benzyl-
8-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-purine-2,6(3H,7H)-
dione Derivatives (5aꢀj). To a solution of the appropriately
substituted isoxazole/pyrazole-carboxylic acid (2.5 mmol) in DMF
(7 mL), EDAC HCl (2.5 mmol, N-ethyl-N0-(3-dimethylaminopro-
3
Thus, the different residue composition of the L1 pocket also
prevents the ligand to make contacts with the L2 cleft in which a
tryptophan residue (W243, W247, and W246 in the hA3, hA1, and
hA2A ARs, respectively) is believed to play a crucial role in receptor
activation and in antagonist binding.47 This should explain why the
great majority of the 2-heterocyclyl-imidazo[2,1-i]purin-5-ones
described herein are endowed with high binding selectivity toward
the hA3 AR.
pyl)carbodiimide hydrochloride) and HOBt (2.5 mmol, hydroxybenzo-
triazole) were added. The mixture was stirred at room temperature
for 10 min, and then 5,6-diamino-1-allyl/benzyl-1H-pyrimidine-2,4-dione
(2.5 mmol) was added. The mixture was stirred for further 24 h, and then
the solvent was evaporated and the residue was suspended with H2O
(30 mL) to favor the precipitation of a solid, which was subsequently
filtered and washed with cold H2O. The solid was suspended with a 10%
aqueous solution of NaOH, and the mixture was refluxed for 1.5 h. The
reaction was cooled to 0 ꢀC and acidified with a 10% aqueous solution of
HCl to obtain a white precipitate, which was filtered, washed with cold
water, and finally crystallized from DMFꢀH2O.
’ CONCLUSIONS
3-Allyl-8-(1,3-dimethyl-1H-pyrazol-5-yl)-1H-purine-2,6(3H,7H)-dione
(5a). Pale-white solid; 65% yield; mp >300 ꢀC. H NMR (200 MHz,
In conclusion, a new series of 2-heterocyclyl-imidazo[2,1-i]purin-
5-one derivatives has been synthesized and tested at the four known
ARs. The 2-heterocyclyl substitution proved to induced excellent
affinity and selectivity toward hA3 AR subtype. Molecular modeling
studies were helpful in rationalizing the observed structureꢀactivity
relationships along with the selectivity profiles of the new series of
ligands. Docking of the most potent compound (R-33) in complex
with hA3 AR furnished a general survey of the hypothetical binding
mode of the newly described derivatives. The A3 AR antagonists
herein described, standing out for their remarkable potency and
selectivity, will be further investigated to clarify their potential for
future drug development and clinical application.
1
DMSO-d6) δppm13.83 (bs, 1H), 11.24 (bs, 1H),6.77(s, 1H),5.97ꢀ5.88
(m, 1H), 5.22ꢀ5.14 (m, 2H), 4.56 (m, 2H), 4.11 (s, 3H), 2.18 (s, 3H).
3-Benzyl-8-(1,3-dimethyl-1H-pyrazol-5-yl)-1H-purine-2,6(3H,7H)-
dione (5b). White solid; 57% yield; mp >300 ꢀC. 1H NMR (200 MHz,
DMSO-d6) δ ppm 13.90 (bs, 1H), 11.25 (s, 1H), 7.32 (m, 5H), 6.75 (s,
1H), 5.15 (s, 2H), 4.10 (s, 3H), 2.17 (s, 3H).
3-Allyl-8-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-purine-2,6(3H,7H)-
dione (5c). White solid; 81% yield; mp >300 ꢀC. 1H NMR (200 MHz,
DMSO-d6) δ ppm 13.93 (bs, 1H), 11.27 (s, 1H), 6.45 (s, 1H),
6.11ꢀ5.92 (m, 1H), 5.22ꢀ5.13 (m, 2H), 4.57 (d, J = 5 Hz, 2H), 4.06
(s, 3H), 3.79 (s, 3H).
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3-Benzyl-8-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-purine-2,6-
1
244 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 13.89 (bs, 1H), 12.50
(s, 1H), 7.47ꢀ7.30 (m, 10H), 6.72 (s, 1H), 5.19 (s, 2H), 5.17 (s, 2H),
4.08 (s, 3H).
(3H,7H)-dione (5d). White solid; 60% yield; mp >300 ꢀC. H NMR
(200 MHz, DMSO-d6) δ ppm 13.89 (bs, 1H), 11.32 (s, 1H), 7.43ꢀ7.22
(m, 5H), 6.41 (s, 1H), 5.15 (s, 2H), 4.05 (s, 3H), 3.79 (s, 3H).
3-Allyl-8-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-1H-purine-2,6-
(3H,7H)-dione (5e). White solid; 57% yield; mp 296 ꢀC dec. 1H NMR
(200 MHz, DMSO-d6) δ ppm 13.87 (bs, 1H), 11.27 (s, 1H), 7.43ꢀ7.36
(m, 5H), 6.46 (s, 1H), 6.02ꢀ5.87 (m, 1H), 5.16ꢀ5.13 (m, 4H), 4.57 (d,
J = 5 Hz, 2H), 4.07 (s, 3H).
3-Benzyl-8-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-1H-purine-
2,6(3H,7H)-dione (5f). White solid; 58% yield; mp >300 ꢀC. 1H NMR
(200 MHz, DMSO-d6) δ ppm 13.94 (bs, 1H), 11.32 (s, 1H), 7.43ꢀ7.29
(m, 10H), 6.45 (s, 1H), 5.16 (s, 4H), 4.06 (s, 3H).
3-Allyl-8-(1,5-dimethyl-1H-pyrazol-3-yl)-1H-purine-2,6(3H,7H)-dione
(5g). White solid; 66% yield; mp >300 ꢀC. 1H NMR (200 MHz, DMSO-
d6) δ ppm 13.61 (bs, 1H), 11.08 (s, 1H), 6.65 (s, 1H), 5.87 (m, 1H),
5.15ꢀ5.04 (m, 2H), 4.56 (m, 2H), 3.80 (s, 3H), 2.29 (s, 3H).
3-Benzyl-8-(1,5-dimethyl-1H-pyrazol-3-yl)-1H-purine-2,6(3H,7H)-
dione (5h). White solid; 77% yield; mp >300 ꢀC. 1H NMR (200 MHz,
DMSO-d6) δ ppm 13.45 (bs, 1H), 11.14 (s, 1H), 7.33ꢀ7.10 (m, 5H),
6.65 (s, 1H), 5.15 (s, 2H), 3.79 (s, 3H), 2.29 (s, 3H).
3-Allyl-1,6-dihydro-8-(1,5-dimethyl-1H-pyrazol-3-yl)-6-thioxo-3H-
1
purin-2(7H)-one (6g). Yellow solid; yield 83%; mp 280ꢀ282 ꢀC. H
NMR (200 MHz, DMSO-d6) δ ppm 13.60 (bs, 1H), 12.23 (bs, 1H),
6.86 (s, 1H), 5.97ꢀ5.89 (m, 1H), 5.18ꢀ5.06 (m, 2H), 4.58 (d, J = 4.6
Hz, 2H), 3.82 (s, 3H), 2.31 (s, 3H).
3-Benzyl-1,6-dihydro-8-(1,5-dimethyl-1H-pyrazol-3-yl)-6-thioxo-3H-
1
purin-2(7H)-one (6h). Yellow solid; yield 87%; mp 288 ꢀC. H NMR
(200 MHz, DMSO-d6) δ ppm 13.63 (bs, 1H), 12.26 (bs, 1H), 7.30 (m,
5H), 6.84 (s, 1H), 5.15 (s, 2H), 3.78 (s, 3H), 2.28 (s, 3H).
3-Allyl-1,6-dihydro-8-(3-methoxyisoxazol-5-yl)-6-thioxo-3H-purin-
2(7H)-one (6i). Pale-yellow solid; 75% yield; mp 254 ꢀC dec. 1H NMR
(200 MHz, DMSO-d6) δ ppm 14.51 (bs, 1H), 12.61 (bs, 1H), 8.63 (s,
1H), 6.12ꢀ5.82 (m, 1H), 5.21 (m, 2H), 4.62 (m, 2H), 4.02 (s, 3H).
3-Benzyl-1,6-dihydro-8-(3-methoxyisoxazol-5-yl)-6-thioxo-3H-pur-
in-2(7H)-one (6j). Pale-green solid; 96% yield; mp 205 ꢀC dec. 1H NMR
(200 MHz, DMSO-d6) δ ppm 14.45 (bs, 1H), 12.62 (bs, 1H), 8.59 (s,
1H), 7.43ꢀ7.19 (m, 5H), 5.17 (s, 2H), 3.97 (s, 3H).
General Procedure for the Synthesis of 3-Allyl/benzyl-
8-[(substituted)isoxazol/pyrazol-3/5-yl]-6-(methylthio)-3H-
purin-2(7H)-one Derivatives (7aꢀj). The appropriate 3-allyl/
benzyl-1,6-dihydro-8-[(substituted)isoxazol/pyrazol-3/5-yl]-6-thioxo-
3H-purin-2(7H)-one derivative 6aꢀj (1 mmol) was dissolved in 10 mL
of NaOH 0.5 N and EtOH (5 mL). After cooling at 0 ꢀC, CH3I (1.5
mmol) was added and the reaction was stirred at room temperature for 3
h. The mixture was acidified with HCl 5% to obtain a precipitate, which
was collected by filtration, washed with cold H2O, and dried under
vacuum.
3-Allyl-8-(1,3-dimethyl-1H-pyrazol-5-yl)-6-(methylthio)-3H-purin-
2(7H)-one (7a). Pale-white solid; 68% yield; mp 250ꢀ252 ꢀC. 1H NMR
(200 MHz, DMSO-d6) δ ppm 13.63 (bs, 1H), 6.82 (s 1H), 6.17ꢀ5.85
(m, 1H), 5.19 (m, 2H), 4.68 (d, J = 5.2 Hz, 2H), 4.14 (s, 3H), 2.63 (s,
3H), 2.21 (s, 3H).
3-Benzyl-8-(1,3-dimethyl-1H-pyrazol-5-yl)-6-(methylthio)-3H-pur-
in-2(7H)-one (7b). Yellow solid; 93% yield; mp 163ꢀ165 ꢀC. 1H NMR
(200 MHz, DMSO-d6) δ ppm 13.76 (bs, 1H), 7.30ꢀ7.26 (m, 5H), 6.69
(s, 1H), 5.25 (s, 2H), 3.83 (s, 3H), 2.56 (s, 3H), 2.32 (s, 3H).
3-Allyl-8-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-6-(methylthio)-3H-
purin-2(7H)-one (7c). Pale-yellow solid; 78% yield; mp 243 ꢀC dec. 1H
NMR (200 MHz, DMSO-d6) δ ppm 13.63 (bs, 1H), 6.39 (s, 1H),
6.15ꢀ5.86 (m, 1H), 5.17ꢀ5.11 (m, 2H), 4.66 (d, J = 4.8 Hz, 2H), 4.07
(s, 3H), 3.80 (s, 3H), 2.62 (s, 3H).
3-Benzyl-8-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-6-(methylthio)-
3H-purin-2(7H)-one (7d). White solid; 48% yield; mp 268ꢀ270 ꢀC.
1H NMR (200 MHz, DMSO-d6) δ ppm 13.73 (bs, 1H), 7.39ꢀ7.27 (m,
5H), 6.46 (s, 1H), 5.25 (s, 2H), 4.08 (s, 3H), 3.81 (s, 3H), 2.63 (s, 3H).
3-Allyl-8-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-6-(methylthio)-
3H-purin-2(7H)-one (7e). Pale-yellow solid; 85% yield; mp 224 ꢀC. 1H
NMR (200 MHz, DMSO-d6) δ ppm 13.79 (bs, 1H), 7.4ꢀ7.34 (m, 5H),
6.53 (s, 1H), 6.15ꢀ5.87 (m, 1H), 5.19ꢀ5.11 (m, 4H), 4.68 (d, J = 4.4
Hz, 2H), 4.09 (s, 3H), 2.62 (s, 3H).
3-Allyl-8-(3-methoxyisoxazol-5-yl)-1H-purine-2,6(3H,7H)-dione (5i).
Pale-white solid; 45% yield; mp 297 ꢀC. 1H NMR (200 MHz, DMSO-
d6) δ ppm 14.55 (bs, 1H), 11.37 (s, 1H), 6.84 (s, 1H), 6.01ꢀ5.87 (m,
1H), 5.16ꢀ5.06 (m, 2H), 4.56 (m, 2H), 3.96 (s, 3H).
3-Benzyl-8-(3-methoxyisoxazol-5-yl)-1H-purine-2,6(3H,7H)-dione
(5j). Pale-yellow solid; 40% yield; mp 284 ꢀC dec. 1H NMR (200 MHz,
DMSO-d6) δ ppm 14.45 (bs, 1H),11.41 (bs, 1H), 7.34ꢀ7.22 (m, 5H),
6.84 (s, 1H), 5.15 (s, 2H), 3.96 (s, 3H).
General Procedure for the Synthesis of 3-Allyl/benzyl-1,6-
dihydro-8-[(substituted)isoxazol/pyrazol-3/5-yl]-6-thioxo-
3H-purin-2(7H)-one derivatives (6aꢀj).35 The appropriate 3-al-
lyl/benzyl-8-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-purine-2,6(3H,7H)-
dione derivative 5aꢀj (1.5 mmol) was dissolved with pyridine (10 mL),
and 2.55 mmol of P2S5 were added. The reaction was vigorously stirred
at 140 ꢀC for 5 h and then cooled at 0 ꢀC. After the addition of water
(30 mL), a pale-green solid precipitated, which was filtered, washed with
cold water, and crystallized from DMF/H2O.
3-Allyl-1,6-dihydro-8-(1,3-dimethyl-1H-pyrazol-5-yl)-6-thioxo-3H-
1
purin-2(7H)-one (6a). Pale-yellow solid; 95% yield; mp 272 ꢀC. H
NMR (200 MHz, DMSO-d6) δ ppm 13.80 (bs, 1H), 12.40 (s, 1H), 7.01
(s, 1H), 6.10ꢀ5.80 (m, 1H), 5.25ꢀ5.16 (m, 2H), 4.59 (d, 2H, J = 5.8),
4.12 (s, 3H), 2.19 (s, 3H).
3-Benzyl-1,6-dihydro-8-(1,3-dimethyl-1H-pyrazol-5-yl)-6-thioxo-3H-
1
purin-2(7H)-one (6b). Yellow solid; 87% yield; mp 265 ꢀC. H NMR
(200 MHz, DMSO-d6) δ ppm 13.66 (bs, 1H), 12.28 (bs, 1H), 7.32 (m,
5H), 6.86 (s, 1H), 5.17 (s, 2H), 3.81 (s, 3H), 2.30 (s, 3H).
3-Allyl-1,6-dihydro-8-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-6-thioxo-
3H-purin-2(7H)-one (6c). Pale-yellow; 82% yield; mp 272 ꢀC dec. 1H
NMR (200 MHz, DMSO-d6) δ ppm 13.81 (bs, 1H), 12.43 (bs, 1H),
6.68 (s, 1H), 6.11ꢀ5.97 (m, 1H), 5.25ꢀ5.16 (m, 2H), 4.60 (d, J = 4.6
Hz, 2 H), 4.07 (s, 3H), 3.80 (s, 3H).
3-Benzyl-1,6-dihydro-8-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-
6-thioxo-3H-purin-2(7H)-one (6d). Pale-yellow solid; 84% yield; mp
293ꢀ295 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 13.92 (bs, 1H),
12.48 (bs, 1H), 7.40ꢀ7.30 (m, 5H), 6.68 (s, 1H), 5.18 (s, 2H), 4.06 (s,
3H), 3.80 (s, 3H).
3-Benzyl-8-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-6-(methylthio)-
3H-purin-2(7H)-one (7f). White solid; 87% yield; mp 228 ꢀC. 1H NMR
(200 MHz, DMSO-d6) δ ppm 13.79 (bs, 1H), 7.43ꢀ7.27 (m, 5H), 6.47
(s, 1H), 5.26 (s, 2H), 5.18 (s, 2H), 4.10 (s, 3H), 2.63 (s, 3H).
3-Allyl-8-(1,5-dimethyl-1H-pyrazol-3-yl)-6-(methylthio)-3H-purin-
3-Allyl-8-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-1,6-dihydro-6-
thioxo-3H-purin-2(7H)-one (6e). Pale-yellow solid; 71% yield; mp
1
2(7H)-one (7g). White solid; 95% yield; mp 242ꢀ244 ꢀC. H NMR
1
241 ꢀC dec. H NMR (200 MHz, DMSO-d6) δ ppm 13.82 (bs, 1H),
(200 MHz, DMSO-d6) δ ppm 13.99 (bs, 1H), 6.71 (s, 1H), 6.11ꢀ5.85
(m, 1H), 5.16ꢀ5.03 (m, 2H), 4.67 (d, J = 4.6 Hz, 2H), 3.84 (s, 3H), 2.61
(s, 3H), 2.32 (s, 3H).
12.43 (bs, 1H), 7.44ꢀ7.36 (m, 5H), 6.71 (s, 1H), 6.15ꢀ5.82 (m, 1H),
5.25ꢀ5.16 (m, 2H), 4.60 (d, J = 4 Hz, 2H), 4.08 (s, 3H).
3-Benzyl-8-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-1,6-dihydro-
6-thioxo-3H-purin-2(7H)-one (6f). Pale-yellow solid; 96% yield; mp
3-Allyl-8-(1,5-dimethyl-1H-pyrazol-3-yl)-6-(methylthio)-3H-purin-
1
2(7H)-one (7h). White solid; 82% yield; mp 183 ꢀC. H NMR (200
5213
dx.doi.org/10.1021/jm2004738 |J. Med. Chem. 2011, 54, 5205–5220

Journal of Medicinal Chemistry
ARTICLE
MHz, DMSO-d6) δ ppm 13.80 (bs, 1H), 7.30 (m, 5H), 6.69 (s, 1H),
5.25 (s, 2H), 3.83 (s, 3H), 2.56 (s, 3H), 2.31 (s, 3H).
1.69ꢀ1.67 (m, 1H), 1.52ꢀ1.49 (m, 1H), 0.92 (t, J = 7.2 Hz, 3H). MS
(ESI): [MH]+ = 408.2.
(S)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(1,3-dimethyl-1H-
3-Allyl-8-(3-methoxyisoxazol-5-yl)-6-(methylthio)-3H-purin-2(7H)-
one (7i). Pale-yellow solid; 73% yield; mp 271 ꢀC dec. 1H NMR (200
MHz, DMSO-d6) δ ppm 14.38 (bs, 1H), 6.90 (s, 1H), 6.09ꢀ5.80 (m,
1H), 5.11 (m, 2H), 4.65 (m, 2H), 3.97 (s, 3H), 2.67 (s, 3H).
3-Benzyl-8-(3-methoxyisoxazol-5-yl)-6-(methylthio)-3H-purin-2(7H)-
one (7j). Pale-yellow solid; 75% yield; mp 249ꢀ251 ꢀC dec. 1H NMR
(200 MHz, DMSO-d6) δ ppm 14.62 (bs, 1H), 7.35ꢀ7.28 (m, 5H), 6.91
(s, 1H), 5.25 (s, 2H), 3.97 (s, 3H), 2.68 (s, 3H).
General Procedure for the Synthesis of 6-(1-Hydroxybu-
tan/propan-2-ylamino)-3-allyl/benzyl-8-[(substituted)iso-
xazol/pyrazol-3/5-yl]-3H-purin-2(7H)-one Derivatives (8ꢀ27).
The appropriate 3-allyl/benzyl-8-[(substituted)isoxazol/pyrazol-3/5-yl]-
6-(methylthio)-3H-purin-2(7H)-one derivative 7aꢀj (0.3 mmol) was
suspended in anhydrous DMSO (0.3 mL) and, after cooling at 0 ꢀC, the
opportune (R/S/R,S)-2-amino-butan/propan-1-ol (1.5 mmol) was added.
The reaction was heated at 150 ꢀC for 1 h. The solvent was evaporated
under vacuum and the product was precipitated from CH2Cl2ꢀEt2O,
filtered, washed with Et2O, and finally purified by column chromatography
on silica gel eluting with EtOAc/CH3OH 9.5:0.5.
pyrazol-5-yl)-3H-purin-2(7H)-one (S-11). White solid; 48% yield;
1
mp 174ꢀ174 ꢀC. H NMR (200 MHz, DMSO-d6) δ ppm 12.61
(bs, 1H), 7.28ꢀ7.19 (m, 6H), 6.53 (s, 1H), 5.11 (s, 2H), 4.83
(m, 1H), 4.20ꢀ4.10 (m, 1H), 3.79 (s, 3H), 3.47 (m, 2H), 2.28
(s, 3H), 1.81ꢀ1.42 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H). MS (ESI):
[MH]+ = 408.2.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(3-methoxy-1-meth-
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (12). White solid; 58% yield;
mp 225 ꢀC dec. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.72 (bs, 1H),
7.53 (bs, 1H), 6.12ꢀ5.83 (m, 2H), 5.19ꢀ5.02 (m, 3H), 4.62.4.55 (m,
2H), 4.21 (m, 1H), 4.07 (s, 3H), 3.78 (s, 3H), 3.56ꢀ3.44 (m, 2H),
1.35ꢀ1.19 (m, 3H). MS (ESI): [MH]+ = 360.4
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(3-methoxy-1-meth-
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (13). White solid; 52% yield;
mp 231 ꢀC dec. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.89 (bs, 1H),
7.51 (bs, 1H), 6.11ꢀ5.82 (m, 2H), 5.19ꢀ5.02 (m, 2H), 4.99ꢀ4.80
(m, 1H), 4.59 (m, 2H), 4.07 (m, 4H), 3.77 (s, 3H), 3.62ꢀ3.41
(m, 2H),1.79ꢀ1.52 (m, 2H), 0.98ꢀ0.82 (m, 3H). MS (ESI):
[MH]+ = 374.4.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(1,3-dimethyl-1H-
pyrazol-5-yl)-3H-purin-2(7H)-one (8). White solid; 56% yield; mp
255ꢀ256 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.98 (bs,
1H), 7.43 (bs, 1H), 6.45 (s, 1H), 6.18ꢀ5.83 (m, 1H), 5.18ꢀ5.03 (m,
3H), 4.59 (d, J = 3 Hz, 2H), 4.22 (m, 1H), 4.13 (s, 3H), 3.58 (m, 2H),
2.18 (s, 3H), 1.22 (m, 3H). MS (ESI): [MH]+ = 344.3.
(R)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(3-methoxy-1-meth-
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (R-13). White solid; 55%
yield; mp 230 ꢀC dec. 1H NMR (200 MHz, CDCl3) δ ppm 12.12 (bs,
1H), 7.53 (bs, 1H), 6.14 (s, 1H), 6.07ꢀ5.95 (m, 1H), 5.26ꢀ5.14 (m,
2H), 4.81ꢀ4.79 (m, 2H), 4.15 (s, 3H), 3.87 (s, 3H), 3.87ꢀ3.75 (m, 1H),
3.44 (m, 2H), 1.40ꢀ1.20 (m, 2H), 0.69ꢀ0.65 (m, 3H). MS (ESI):
[MH]+ = 374.4.
(S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(3-methoxy-1-methyl-
1H-pyrazol-5-yl)-3H-purin-2(7H)-one (S-13). White solid; 43% yield;
mp 230 ꢀC dec. 1H NMR (200 MHz, CDCl3) δ ppm 12.13 (bs, 1H),
7.55 (bs, 1H), 6.13 (s, 1H), 6.07ꢀ5.85 (m, 1H), 5.26ꢀ5.14 (m, 2H),
4.81ꢀ4.79 (m, 2H), 4.15 (m, 3H), 3.87 (s, 3H), 3.87ꢀ3.75 (m, 1H),
3.41 (m, 2H), 1.40ꢀ1.20 (m, 2H), 0.69ꢀ0.65 (m, 3H). MS (ESI):
[MH]+ = 374.4
(R)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(1,3-dimethyl-1H-
pyrazol-5-yl)-3H-purin-2(7H)-one (R-8). White solid; 66% yield; mp dec
253ꢀ254 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.38 (bs, 1H),
7.43 (bs, 1H), 6.45 (s, 1H), 6.03ꢀ5.84 (m, 1H), 5.14ꢀ5.08 (m, 3H),
4.57 (d, J = 4.8 Hz, 2H), 4.21 (m, 1H), 4.11 (s, 3H), 3.51 (m, 2H), 2.17
(s, 3H), 1.22ꢀ1.16 (m, 3H). MS (ESI): [MH]+ = 344.3.
(S)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(1,3-dimethyl-1H-
pyrazol-5-yl)-3H-purin-2(7H)-one (S-8). White solid; 52% yield;
mp dec 253ꢀ254 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.80
(bs, 1H), 7.42 (bs, 1H), 6.48 (s, 1H), 6.05ꢀ5.82 (m, 1H), 5.15ꢀ
4.92 (m, 3H), 4.58 (d, J = 5.0 Hz, 2H), 4.22 (m, 1H), 4.12 (s, 3H),
3.51 (m, 2H), 2.19 (s, 3H), 1.22ꢀ1.17 (m, 3H). MS (ESI): [MH]+ =
344.3.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(3-methoxy-1-
methyl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (14). White solid; 53%
yield; mp 258ꢀ260 ꢀC dec. 1H NMR (400 MHz, DMSO-d6) δ
ppm 13.88 (bs, 1H), 7.51 (bs, 1H), 7.39ꢀ7.25 (m, 5H), 6.02 (s, 1H),
5.16 (s, 2H), 5.08 (s, 1H), 4.24 (m, 1H), 4.07 (s, 3H), 3.78 (s, 3H), 3.49
(m, 2H), 1.26ꢀ1.17 (m, 3H). MS (ESI): [MH]+ = 410.3.
(R)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(3-methoxy-1-meth-
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (R-14). White solid; 57% yield;
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(1,3-dimethyl-1H-
pyrazol-5-yl)-3H-purin-2(7H)-one (9). White solid; 62% yield; mp dec
253ꢀ255 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.47 (bs, 1H),
7.52 (bs, 1H), 6.45 (s, 1H), 6.10ꢀ5.85 (m, 1H), 5.20ꢀ5.10 (m, 2H),
5.00ꢀ4.80 (bs, 1H), 4.57 (d, J = 4.6 Hz, 2H), 4.12 (m, 4H), 4.75ꢀ4.67
(m, 2H), 2.16 (m, 3H), 1.80ꢀ1.60 (m, 2H), 1.12ꢀ0.80 (m, 3H). MS
(ESI): [MH]+ = 357.5.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(1,3-dimethyl-
1H-pyrazol-5-yl)-3H-purin-2(7H)-one (10). White solid; 46% yield; mp
230ꢀ233 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.70 (bs, 1H),
7.31ꢀ7.20 (m, 6H), 6.55 (s, 1H), 5.14 (s, 2H), 4.95 (bs, 1H), 4.19 (m,
1H), 3.82 (s, 3H), 3.47 (m, 2H), 2.30 (s, 3H), 1.18 (d, 3H, J = 6.6 Hz).
MS (ESI): [MH]+ = 394.5.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(1,3-dimethyl-1H-
pyrazol-5-yl)-3H-purin-2(7H)-one (11). White solid; 72% yield; mp
171ꢀ172 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.65 (bs, 1H),
7.40ꢀ7.26 (m, 6H), 6.56 (s, 1H), 5.16 (s, 2H), 4.89ꢀ4.81 (m, 1H), 4.12
(m, 4H), 3.60ꢀ3.40 (m, 2H), 2.19ꢀ2.15 (m, 3H), 1.80ꢀ1.67 (m, 2H),
0.87 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 408.2.
(R)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(1,3-dimethyl-1H-
pyrazol-5-yl)-3H-purin-2(7H)-one (R-11). White solid; 45% yield; mp
170ꢀ172 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.70 (bs, 1H),
7.31ꢀ7.26 (m, 6H), 6.56 (s, 1H), 5.16 (s, 2H), 4.88ꢀ4.79 (m, 1H),
4.06ꢀ4.04 (m, 1H), 3.82 (s, 3H), 3.532ꢀ3.43 (m, 1H), 2.31 (s, 3H),
1
mp 257ꢀ259 ꢀC. H NMR (400 MHz, DMSO-d6) δ ppm 12.83 (bs,
1H), 7.56 (bs, 1H), 7.39ꢀ7.25 (m, 5H), 6.02 (s, 1H), 5.16 (s, 2H), 5.06
(bs, 1H), 4.23 (m, 1H), 4.07 (s, 3H), 3.77 (s, 3H), 3.50 (m, 2H),
1.26ꢀ1.19 (m, 3H). MS (ESI): [MH]+ = 410.3.
(S)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(3-methoxy-1-meth-
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (S-14). White solid; 71% yield;
mp 259 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.93 (bs, 1H), 7.56
(bs, 1H), 7.39ꢀ7.25 (m, 5H), 6.02 (s, 1H), 5.16 (s, 2H), 5.09 (bs, 1H),
4.23 (m, 1H), 4.07 (s, 3H), 3.78 (s, 3H), 3.50 (m, 2H), 1.26ꢀ1.19 (m,
3H). MS (ESI): [MH]+ = 410.3.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(3-methoxy-1-meth-
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (15). White solid; 60% yield;
1
mp 259ꢀ260 ꢀC. H NMR (400 MHz, DMSO-d6) δ ppm 12.85 (bs,
1H), 7.67 (bs, 1H), 7.40ꢀ7.30 (m, 5H), 6.02 (s, 1H), 5.17 (s, 2H), 5.02
(bs, 1H), 4.07 (m, 4H), 3.77 (s, 3H), 3.61 (m, 2H), 1.79ꢀ1.45 (m, 2H),
0.93 (m, 3H). MS (ESI): [MH]+ = 424.3.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(3-(benzyloxy)-1-
methyl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (16). White solid; 56%
yield; mp 180ꢀ182 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.89
(bs, 1H), 7.43ꢀ7.35 (m, 6H), 6.02ꢀ5.87 (m, 2H), 5.16ꢀ5.10 (m, 5H),
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Journal of Medicinal Chemistry
ARTICLE
4.58 (d, J = 4.4 Hz, 2H), 4.22 (m, 1H), 4.08 (s, 3H), 3.56ꢀ3.43 (m, 2H),
1.23 (m, 3H). MS (ESI): [MH]+ = 436.5.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(3-(benzyloxy)-1-meth-
4.58 (d, J = 4.8 Hz, 2H), 4.22 (m, 1H), 3.92 (s, 3H), 3.50 (m, 2H),
1.24ꢀ1.19 (m, 3H). MS (ESI): [MH]+ = 347.2.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(3-methoxyisoxazol-
5-yl)-3H-purin-2(7H)-one (25). White solid; 54% yield; mp 270ꢀ
yl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (17). White solid; 54% yield;
1
1
mp 224ꢀ225 ꢀC. H NMR (200 MHz, DMSO-d6) δ ppm 12.91 (bs,
271 ꢀC. H NMR (200 MHz, DMSO-d6) δ ppm 8.58 (bs, 1H), 7.71
1H), 7.43ꢀ7.36 (m, 6H), 6.19ꢀ5.76 (m, 2H), 5.16ꢀ5.00 (m, 4H), 4.92
(bs, 1H), 4.59 (m, 2H), 4.08 (m, 4H), 3.51ꢀ3.42 (m, 2H), 1.79ꢀ1.46
(m, 2H), 1.08ꢀ0.86 (m, 3H). MS (ESI): [MH]+ = 450.5.
(bs, 1H), 6.42 (s, 1H), 6.02ꢀ5.88 (m, 1H), 5.16ꢀ4.91 (m, 3H), 4.58
(d, J = 4.6 Hz, 2H), 4.11 (m, 1H), 3.93 (s, 3H), 3.61ꢀ3.52 (m, 2H),
1.62ꢀ1.59 (m, 2H), 0.97ꢀ0.86 (m, 3H). MS (ESI): [MH]+ = 361.5.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(3-methoxyisox-
azol-5-yl)-3H-purin-2(7H)-one (26). White solid; 40% yield; mp 292ꢀ
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(3-(benzyloxy)-
1-methyl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (18). White solid; 63%
yield; mp 121 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.83 (bs,
1H), 7.61 (bs, 1H), 7.45ꢀ7.25 (m, 10H), 6.10 (s, 1H), 5.16 (s, 4H), 5.01
(bs, 1H), 4.22 (m, 1H), 4.08 (s, 3H), 3.50 (m, 2H), 1.26ꢀ1.19 (m, 3H).
MS (ESI): [MH]+ = 486.3.
1
293 ꢀC. H NMR (200 MHz, DMSO-d6) δ ppm 8.68 (bs, 1H), 7.78
(bs, 1H), 7.31 (m, 5H), 6.43 (s, 1H), 5.17 (s, 2H), 5.16ꢀ5.00 (m, 1H),
4.22 (m, 1H), 3.92 (s, 3H), (m, 2H), 1.35ꢀ1.24 (m, 3H). MS (ESI):
[MH]+ = 397.5.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(3-(benzyloxy)-1-
methyl-1H-pyrazol-5-yl)-3H-purin-2(7H)-one (19). White solid; 61%
yield; mp 248 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.98 (bs,
1H), 7.56 (bs, 1H), 7.44ꢀ7.30 (m, 10H), 6.08 (s, 1H), 5.17 (s, 2H), 5.14
(s, 2H), 5.02 (m, 1H), 4.86 (m, 1H), 4.08 (s, 3H), 3.61ꢀ3.45 (m, 2H),
1.79ꢀ1.47 (m, 2H), 0.95ꢀ9.91 (m, 3H). MS (ESI): [MH]+ = 500.4.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(1,5-dimethyl-1H-
pyrazol-3-yl)-3H-purin-2(7H)-one (20). Pale-yellow solid; 57% yield;
mp 177ꢀ180 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.53
(bs, 1H), 7.26 (d, J = 7.4 Hz, 1H), 6.55 (s, 1H), 5.93ꢀ5.85 (m,
1H), 5.09ꢀ4.97 (m, 3H), 4.54 (d, J = 4.8 Hz, 2H), 4.19 (m, 1H),
3.82 (s, 3H), 3.47 (m, 2H), 2.31 (s, 3H), 1.18 (d, J = 6.8 Hz, 3H). MS
(ESI): [MH]+ = 344.4.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(1,5-dimethyl-1H-
pyrazol-3-yl)-3H-purin-2(7H)-one (21). White solid; 52% yield; mp
169ꢀ170 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.61 (bs, 1H),
7.25 (d, J = 8 Hz, 1H), 6.56 (s, 1H), 6.02ꢀ5.81 (m, 1H), 5.10ꢀ4.98 (m,
3H), 4.54 (d, J = 4.8 Hz, 2H), 4.17ꢀ3.99 (m, 1H), 3.82 (s, 3H), 3.50-
3.48 (m, 2H), 2.31 (s, 3H), 2.79ꢀ2.61 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H).
MS (ESI): [MH]+ = 358.4.
(R)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(1,5-dimethyl-1H-pyr-
azol-3-yl)-3H-purin-2(7H)-one (R-21). White solid; 62% yield; mp
168ꢀ170 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.66 (bs,
1H), 7.21 (bs, 1H), 6.55 (s, 1H), 5.95ꢀ5.88 (m, 1H), 5.08ꢀ5.05 (m,
2H), 4.89 (bs, 1H), 4.54 (d, J = 4.8 Hz, 2H), 4.05ꢀ4.04 (m, 1H), 3.82 (s,
3H), 3.51 (m, 2H), 2.31 (s, 3H), 1.72ꢀ1.66 (m, 1H), 1.54ꢀ1.47 (m,
1H), 0.92 (t, 7.6 Hz, 3H). MS (ESI): [MH]+ = 358.6.
(S)-6-(1-Hydroxybutan-2-ylamino)-3-allyl-8-(1,5-dimethyl-1H-pyr-
azol-3-yl)-3H-purin-2(7H)-one (S-21). White solid; 59% yield; mp
167ꢀ169 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.63 (bs,
1H), 7.21ꢀ7.18 (bd, J = 7.4, Hz 1H), 6.54 (s, 1H), 5.98ꢀ5.84 (m, 1H),
5.14ꢀ4.82 (m, 3H), 4.54 (d, 2H), 4.15ꢀ3.93 (m, 1H), 3.80 (s, 3H), 3.49
(m, 2H), 2.92 (s, 3H), 1.79ꢀ1.43 (m, 2H), 0.9 (t, J = 7 Hz, 3H). MS
(ESI): [MH]+ = 358.6.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(3-methoxyisoxa-
zol-5-yl)-3H-purin-2(7H)-one (27). White solid; 67% yield; mp 284ꢀ
1
286 dec ꢀC. H NMR (200 MHz, DMSO-d6) δ ppm 12.91 (bs, 1H),
7.80 (bs, 1H), 7.31 (m, 5H), 6.44 (s, 1H), 5.15 (s, 2H), 4.90ꢀ5.00
(s, 1H), 4.22 (m, 1H), 3.20 (s, 3H), 3.60ꢀ3.50 (m, 2H), 1.75ꢀ1.55
(m, 2H), 0.91 (t, J = 7.6 Hz, 3H). MS (ESI): [MH]+ = 411.5.
General Procedure for the Preparation of 4-Allyl/benzyl-
7,8-dihydro-8-methyl/ethyl-2-[(substituted)isoxazol/pyra-
zol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-one derivatives
(28ꢀ47) . The opportune 6-(1-hydroxybutan/propan-2-ylamino)-3-
allyl/benzyl-8-[(substituted)isoxazol/pyrazol-3/5-yl]-3H-purin-2(7H)-
one derivative 8ꢀ27 (0.2 mmol) was dissolved in freshly distilled
CH2Cl2 (7 mL) and, after cooling at 0 ꢀC, 0.3 mL of SOCl2 were added
to the mixture. The reaction was stirred for further 10 min at room
temperature, then heated at reflux for 18 h. The solvent and the excess of
SOCl2 were removed under vacuum, and the residue was purified by
column chromatography on silica gel eluting with EtOAc/CH3OH 9:1.
(R,S)-4-Allyl-7,8-dihydro-8-methyl-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (28). White solid; 64% yield; mp
232 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.17 (bs, 1H), 6.40 (s,
1H), 6.05ꢀ5.83 (m, 1H), 5.22ꢀ5.13 (m, 2H), 4.62 (d, J = 5 Hz, 2H),
4.32 (m, 2H), 4.15 (s, 3H), 3.78 (m, 1H), 2.14 (s, 3H), 1.34 (d, J = 5.6
Hz, 3H). MS (ESI): [MH]+ = 326.3. Anal. (C16H19N7O) C, H, N.
(R)-4-Allyl-7,8-dihydro-8-methyl-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (R-28). White solid; 64% yield; mp
233 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.17 (bs, 1H), 6.40 (s,
1H), 5.97ꢀ5.88 (m, 1H), 5.23ꢀ5.13 (m, 2H), 4.62 (d, J = 5.2 Hz, 2H),
4.37ꢀ4.22 (m, 2H), 4.15 (s, 3H), 3.75ꢀ3.67 (m, 1H), 2.14 (s, 3H), 1.34
(d, J = 5.8 Hz, 3H). MS (ESI): [MH]+ = 325.8. Anal. (C16H19N7O)
C, H, N.
(S)-4-Allyl-7,8-dihydro-8-methyl-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (S-28). White solid; 48% yield; mp
233 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.17 (bs, 1H), 6.40 (s,
1H), 6.02ꢀ5.88 (m, 1H), 5.22ꢀ5.13 (m, 2H), 4.62 (d, J = 5.2 Hz, 2H),
4.39ꢀ4.22 (m, 1H), 4.15 (s, 3H), 3.75ꢀ3.67 (m, 1H), 2.14 (s, 3H), 1.34
(d, J = 5.8 Hz, 3H). MS (ESI): [MH]+ = 325.8. Anal. (C16H19N7O)
C, H, N.
(R,S)-4-Allyl-8-ethyl-7,8-dihydro-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (29). White solid; 51% yield; mp
188 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.17 (bs, 1H), 6.40 (s,
1H), 6.02ꢀ5.88 (m, 1H), 5.22ꢀ5.13 (m, 2H), 4.62 (d, J = 5.2 Hz, 2H),
4.25ꢀ4.22 (m, 2H), 4.15 (s, 3H), 3.75ꢀ3.67 (m, 1H), 2.14 (s, 3H), 1.68
(m, 2H), 0.94 (t, J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 340.8. Anal.
(C17H21N7O) C, H, N.
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-benzyl-8-(1,5-dimethyl-1H-
pyrazol-3-yl)-3H-purin-2(7H)-one (22). White solid; 56% yield; mp
212 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.84 (bs, 1H),
7.30ꢀ7.22 (m, 6H), 6.55 (s, 1H), 5.13 (s, 2H), 5.00 (bs, 1H), 4.20 (m,
1H), 3.81 (s, 3H), 3.45 (m, 2H), 2.30 (s, 3H), 1.18 (d, 3H, J = 6.6). MS
(ESI): [MH]+ = 394.7.
(R,S)-6-(1-Hydroxybutan-2-ylamino)-3-benzyl-8-(1,5-dimethyl-1H-
pyrazol-3-yl)-3H-purin-2(7H)-one (23). White solid; 47% yield; mp
199ꢀ201 ꢀC dec. 1H NMR (200 MHz, DMSO-d6) δ ppm 12.78 (bs,
1H), 7.31ꢀ7.24 (m, 6H), 6.56 (s, 1H), 5.14 (s, 2H), 4.90 (bs, 1H), 4.10
(m, 1H), 3.82 (s, 3H), 3.45 (m, 2H), 2.30 (s, 3H), 1.8ꢀ1.6 (m, 2H), 0.92
(t, 3H, J = 7.6). MS (ESI): [MH]+ = 408.7.
(R,S)-4-Benzyl-7,8-dihydro-8-methyl-2-(1,3-dimethyl-1H-pyrazol-5-
yl)-1H-imidazo[2,1-i]purin-5(4H)-one (30). White solid; 57% yield; mp
244 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.07 (bs, 1H),
7.38ꢀ7.24 (m, 5H), 6.41 (s, 1H), 5.19 (s, 2H), 4.38ꢀ4.17 (m, 2H),
3.73 (s, 3H), 3.69ꢀ3.60 (m, 1H), 2.26 (s, 3H), 1.32 (d, J = 6.4 Hz, 3H).
MS (ESI): [MH]+ = 376.6. Anal. (C20H21N7O) C, H, N
(R,S)-6-(1-Hydroxypropan-2-ylamino)-3-allyl-8-(3-methoxyisoxazol-
5-yl)-3H-purin-2(7H)-one (24). White solid; 64% yield; mp 289ꢀ
290 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 8.52 (bs, 1H),
7.78 (bs, 1H), 6.44 (s, 1H), 6.03ꢀ5.84 (m, 1H), 5.15ꢀ5.10 (m, 3H),
5215
dx.doi.org/10.1021/jm2004738 |J. Med. Chem. 2011, 54, 5205–5220

Journal of Medicinal Chemistry
ARTICLE
(R,S)-4-Benzyl-8-ethyl-7,8-dihydro-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (31). White solid; 48% yield; mp
295 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.12 (bs, 1H),
7.42ꢀ7.28 (m, 5H), 6.68 (s, 1H), 5.19 (s, 2H), 4.20ꢀ4.00 (m, 5H), 3.80
(m, 1H), 2.15 (s, 3H), 1.65 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). MS (ESI):
[MH]+ = 390.2. Anal. (C21H23N7O) C, H, N.
(R)-4-Benzyl-8-ethyl-7,8-dihydro-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (R-31). White solid; 48% yield; mp
294 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.12 (bs, 1H),
7.39ꢀ7.27 (m, 5H), 6.42 (s, 1H), 5.20 (s, 2H), 4.20ꢀ4.18 (m, 2H), 3.73
(m, 4H), 2.26 (s, 3H), 1.65 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). MS (ESI):
[MH]+ = 390.2. Anal. (C21H23N7O) C, H, N.
(R,S)-4-Benzyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyr-
azol-5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one (35). White solid; 53%
yield; mp 140 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.38 (bs,
1H), 7.43ꢀ7.25 (m, 5H), 6.08 (s, 1H), 5.21 (s, 2H), 4.27ꢀ4.23 (m, 2H),
4.08 (s, 3H), 3.81ꢀ3.80 (m, 1H), 3.76 (s, 3H), 1.70ꢀ1.66 (m, 2H), 0.93
(t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 406.2. Anal. (C21H23N7O2)
C, H, N.
(R,S)-4-Allyl-2-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-7,8-dihy-
dro-8-methyl-1H-imidazo[2,1-i]purin-5(4H)-one (36). White solid;
59% yield; mp 214 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.18
(bs, 1H), 7.45ꢀ7.38 (m, 5H), 6.03 (s, 1H), 5.98ꢀ5.84 (m, 1H),
5.20ꢀ5.14 (m, 4H), 4.62 (d, J = 5.2 Hz, 2H), 4.37ꢀ4.25 (m, 2H),
4.11 (s, 3H), 3.74ꢀ3.70 (m, 1H), 1.34 (d, J = 6.4 Hz, 3H). MS (ESI):
[MH]+ = 418.2. Anal. (C22H23N7O2) C, H, N.
(R,S)-4-Allyl-2-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-8-ethyl-7,8-
dihydro-1H-imidazo[2,1-i]purin-5(4H)-one (37). White solid; 60%
yield; mp 223 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.25 (bs,
1H), 7.43ꢀ7.38 (m, 5H), 6.03 (s, 1H), 5.98ꢀ5.91 (m, 1H), 5ꢀ20ꢀ5.14
(m, 4H), 4.62 (d, J = 5.6 Hz, 2H), 4.28ꢀ4.21 (m, 2H), 4.11 (s, 3H),
3.81ꢀ3.78 (m, 1H), 1.70ꢀ1.66 (m, 2H), 0.94 (t, J = 7.2 HZ, 3H). MS
(ESI): [MH]+ = 432.2. Anal. (C23H25N7O2) C, H, N.
(R,S)-4-Benzyl-2-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-7,8-di-
hydro-8-methyl-1H-imidazo[2,1-i]purin-5(4H)-one (38). White solid;
42% yield; mp 143 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.22
(bs, 1H), 7.45ꢀ7.31 (m, 10H), 6.05 (s, 1H), 5.20 (s, 2H), 5.14 (s, 2H),
4.37ꢀ4.26 (m, 2H), 4.11 (s, 3H), 3.75ꢀ3.71 (m, 1H), 1.34 (d, J = 6 Hz,
3H). MS (ESI): [MH]+ = 468.2. Anal. (C26H25N7O2) C, H, N.
(R,S)-4-Benzyl-2-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-8-ethyl-
7,8-dihydro-1H-imidazo[2,1-i]purin-5(4H)-one (39). White solid; 49%
yield; mp 120 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.22 (bs,
1H), 7.45ꢀ7.29 (m, 10H), 6.05 (s, 1H), 5.20 (s, 2H), 5.14 (s, 2H),
4.26ꢀ4.20 (m, 2H), 3.82ꢀ3.78 (m, 2H), 4.11 (s, 1H), 3.82ꢀ3.78
(m,1H), 1.70ꢀ1.66 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+
= 482.2. Anal. (C27H27N7O2) C, H, N.
(S)-4-Benzyl-8-ethyl-7,8-dihydro-2-(1,3-dimethyl-1H-pyrazol-5-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (S-31). White solid; 43% yield;
1
mp 295 ꢀC. H NMR (200 MHz, DMSO-d6) δ ppm 10.21 (bs, 1H),
7.35ꢀ7.27 (m, 5H), 6.42 (s, 1H), 5.20 (s, 2H), 4.17 (m, 2H), 3.73 (m,
4H), 2.26 (s, 3H), 1.79ꢀ1.62 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). MS
(ESI): [MH]+ = 390.2. Anal. (C21H23N7O) C, H, N.
(R,S)-4-Allyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-
8-methyl-1H-imidazo[2,1-i]purin-5(4H)-one (32). White solid; 55%
yield; mp 121 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.17 (bs,
1H), 5.99 (s, 1H), 5.95ꢀ5.91 (m, 1H), 5.20ꢀ5.14 (m, 2H), 4.62 (d, J =
5.2 Hz, 2H), 4.4.38ꢀ4.25 (m, 2H), 4.10 (s, 3H), 3.77 (s, 3H), 3.75ꢀ3.70
(m, 1H), 1.34 (d, J = 6 Hz, 3H). MS (ESI): [MH]+ = 342.4. Anal.
(C16H19N7O2) C, H, N.
(R,S)-4-Allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-
5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one (33). White solid; 47% yield;
mp 133 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.23 (bs, 1H), 6.00
(s, 1H), 5.98ꢀ5.89 (m, 1H), 5.20ꢀ5.14 (m, 2H), 4.62 (d, J = 5.2 Hz,
2H), 4.26ꢀ4.24 (m, 2H), 4.10 (s, 3H), 3.83ꢀ3.78 (m, 1H), 3.77 (s, 3H),
1.68 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 356.2. Anal.
(C17H21N7O2) C, H, N.
(R)-4-Allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-
5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one (R-33). White solid; 54% yield;
mp 133 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.23 (bs, 1H), 6.00
(s, 1H), 5.98ꢀ5.89 (m, 1H), 5.22ꢀ5.13 (m, 2H), 4.62 (d, J = 5.2 Hz,
2H), 4.26ꢀ4.24 (m, 2H), 4.10 (s, 3H), 3.83ꢀ3.78 (m, 1H), 3.77 (s, 3H),
1.68 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 356.2. Anal.
(C17H21N7O2) C, H, N.
(S)-4-Allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-
5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one (S-33). White solid; 51% yield;
mp 133 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.23 (bs, 1H), 6.02
(s, 1H), 5.98ꢀ5.89 (m, 1H), 5.18ꢀ5.13 (m, 2H), 4.62 (d, J = 5.2 Hz,
2H), 4.26ꢀ4.24 (m, 2H), 4.10 (s, 3H), 3.83ꢀ3.78 (m, 1H), 3.77 (s, 3H),
1.68 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 356.2. Anal.
(C17H21N7O2) C, H, N.
(R,S)-4-Benzyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-
8-methyl-1H-imidazo[2,1-i]purin-5(4H)-one (34). White solid; 57%
yield; mp 243 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.21 (bs,
1H), 7.44ꢀ7.25 (m, 5H), 5.98 (s, 1H), 5.19 (s, 2H), 4.35ꢀ4.22 (m, 2H),
4.10 (s, 3H), 3.76 (s, 3H), 3.70ꢀ3.66 (m, 1H), 1.32 (d, J = 6.4 Hz, 3H).
MS (ESI): [MH]+ = 392.2. Anal. (C20H21N7O2) C, H, N.
(R)-4-Benzyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-
8-methyl-1H-imidazo[2,1-i]purin-5(4H)-one (R-34). White solid; 67%
yield; mp 242 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.19 (bs,
1H), 7.42ꢀ7.23 (m, 5H), 5.98 (s, 1H), 5.18 (s, 2H), 4.35ꢀ4.24 (m, 2H),
4.08 (s, 3H), 3.75 (s, 3H), 3.73ꢀ3.68 (m, 1H), 1.32 (d, J = 6 Hz, 3H).
MS (ESI): [MH]+ = 392.2. Anal. (C20H21N7O2) C, H, N.
(S)-4-Benzyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-
8-methyl-1H-imidazo[2,1-i]purin-5(4H)-one (S-34). White solid; 60%
yield; mp 244 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.23 (bs,
1H), 7.43ꢀ7.24 (m, 5H), 6.06 (s, 1H), 5.20 (s, 2H), 4.39ꢀ4.26 (m, 2H),
4.08 (s, 3H), 3.76 (s, 3H), 3.73ꢀ3.71 (m, 1H), 1.34 (d, J = 6.4 Hz, 3H).
MS (ESI): [MH]+ = 392.2. Anal. (C20H21N7O2) C, H, N.
(R,S)-4-Allyl-7,8-dihydro-8-methyl-2-(1,5-dimethyl-1H-pyrazol-3-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (40). White solid; 49% yield; mp
1
214 ꢀC dec. H NMR (400 MHz, DMSO-d6) δ ppm 10.43 (bs, 1H),
6.41 (s, 1H), 5.98ꢀ5.91 (m, 1H), 5.14ꢀ5.11 (m, 2H), 4.60 (d, J = 5.2
Hz, 2H), 4.39ꢀ4.18 (m, 2H), 3.74 (s, 3H), 3.67ꢀ3.62 (m, 1H), 2.26 (s,
3H), 1.31 (d, J = 6 Hz, 3H). MS (ESI): [MH]+ = 326.2. Anal.
(C16H19N7O) C, H, N.
(R,S)-4-Allyl-8-ethyl-7,8-dihydro-2-(1,5-dimethyl-1H-pyrazol-3-yl)-1H-
imidazo[2,1-i]purin-5(4H)-one (41). White solid; 46% yield; mp
249 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.43 (bs, 1H), 6.45
(s, 1H), 5.98ꢀ5.91 (m, 1H), 5.15ꢀ5.11 (m, 2H), 4.61 (d, J = 5.2 Hz,
2H), 4.22ꢀ4.20 (m, 2H), 3.76 (m, 4H), 2.27 (s, 3H), 1.68ꢀ1.65 (m,
2H), 0.95 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 340.2. Anal.
(C17H21N7O) C, H, N.
(R)-4-Allyl-8-ethyl-7,8-dihydro-2-(1,5-dimethyl-1H-pyrazol-3-yl)-1H-
imidazo[2,1-i]purin-5(4H)-one (R-41). White solid; 48% yield; mp
249 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.11 (bs, 1H), 6.41
(s, 1H), 5.99ꢀ5.87 (m, 1H), 5.14ꢀ5.09 (m, 2H), 4.60 (d, J = 4.8 Hz,
2H), 4.17ꢀ4.60 (m, 2H), 3.80ꢀ3.70 (m, 4H), 2.26 (s, 3H), 1.65 (m,
2H), 0.94 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 339.9. Anal.
(C17H21N7O) C, H, N.
(S)-4-Allyl-8-ethyl-7,8-dihydro-2-(1,5-dimethyl-1H-pyrazol-3-yl)-1H-
imidazo[2,1-i]purin-5(4H)-one (S-41). White solid; 47% yield; mp
249 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.08 (bs, 1H), 6.41
(s, 1H), 6.00ꢀ5.88 (m, 1H), 5.16ꢀ5.07 (m, 2H), 4.60 (d, J = 4.8 Hz,
2H), 4.20ꢀ4.17 (m, 2H), 3.74 (s, 3H), 3.70 (m, 1H), 2.26 (s, 3H),
1.68ꢀ1.62 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 340.2.
Anal. (C17H21N7O) C, H, N.
(R,S)-4-Benzyl-7,8-dihydro-8-methyl-2-(1,5-dimethyl-1H-pyrazol-3-
yl)-1H-imidazo[2,1-i]purin-5(4H)-one (42). White solid; 65% yield; mp
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245 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.20 (bs, 1H),
7.35ꢀ7.27 (m, 5H), 6.42 (s, 1H), 5.20 (s, 2H), 4.40ꢀ4.20 (m, 2H),
3.80ꢀ3.60 (m, 4H), 2.26 (s, 3H), 1.31 (d, J = 5.8 Hz, 3H). MS (ESI):
[MH]+ = 376.9. Anal. (C20H21N7O) C, H, N.
e10% of the total binding.50 Inhibition binding experiments of [3H]-
ZM 241385 (2nM) to hA2ACHO membranes (50 μg of protein/assay)
and at least 6ꢀ8 different concentrations of antagonists studied were
performed for 60 min at 4 ꢀC. Nonspecific binding was determined in
the presence of 1 μM ZM 241385 and was about 20% of total binding.51
Competition binding experiments of [3H]-MRE 3008F20 (1nM) to
hA3CHO membranes (50 μg of protein/assay) and at least 6ꢀ8
different concentrations of examined ligands were performed for 120
min at 4 ꢀC. Nonspecific binding was defined as binding in the presence
of 1 μM MRE 3008F20 and was about 25% of total binding.52 Bound
and free radioactivity were separated by filtering the assay mixture
through Whatman GF/B glass fiber which were washed three times with
ice-cold buffer. Filter bound radioactivity was measured by scintillation
spectrometry (Tri Carb Packard 2500-TR) after addition of Aquassure
liquid.
(R,S)-4-Benzyl-8-ethyl-7,8-dihydro-2-(1,5-dimethyl-1H-pyrazol-3-yl)-
1H-imidazo[2,1-i]purin-5(4H)-one (43). White solid; 61% yield; mp
275 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.20 (bs, 1H),
7.35ꢀ7.27 (m, 5H), 6.42 (s, 1H), 5.20 (s, 2H), 4.20ꢀ4.10 (m, 2H),
3.80ꢀ3.60 (m, 4H), 2.26 (s, 3H), 1.70ꢀ1.60 (m, 2H), 0.94 (t, J = 7.2 Hz,
3H). MS (ESI): [MH]+ = 390.9. Anal. (C21H23N7O) C, H, N.
(R,S)-4-Allyl-7,8-dihydro-2-(3-methoxyisoxazol-5-yl)-8-methyl-1H-
imidazo[2,1-i]purin-5(4H)-one (44). White solid; 57% yield; mp
169 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.74 (bs, 1H), 6.53
(s, 1H), 5.93ꢀ5.88 (m, 1H), 5.21ꢀ5.13 (m, 2H), 4.63 (d, J = 4.8 Hz,
2H), 4.45ꢀ4.26 (m, 2H), 3.94 (s, 3H), 3.80ꢀ3.72 (m, 1H), 1.36 (d, J = 6
Hz, 3H). MS (ESI): [MH]+ = 329.2. Anal. (C15H16N6O3) C, H, N.
(R,S)-4-Allyl-8-ethyl-7,8-dihydro-2-(3-methoxyisoxazol-5-yl)-1H-
imidazo[2,1-i]purin-5(4H)-one (45). White solid; 51% yield; mp 171 ꢀC
dec. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.79 (bs, 1H), 6.50 (s,
1H), 6.02ꢀ5.88 (m, 1H), 5.20ꢀ5.12 (m, 2H), 4.63 (d, J = 4.8 Hz, 2H),
4.30ꢀ4.27 (m, 2H), 3.94 (s, 3H), 3.84ꢀ3.82 (m, 1H), 1.74ꢀ1.67 (m,
2H), 0.95 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 343.3. Anal.
(C16H18N6O3) C, H, N.
(R,S)-4-Benzyl-8-methyl-7,8-dihydro-2-(3-methoxyisoxazol-5-yl)-1H-
imidazo[2,1-i]purin-5(4H)-one (46). White solid; 38% yield; mp
111 ꢀC. 1H NMR (200 MHz, DMSO-d6) δ ppm 10.64 (bs, 1H),
7.41ꢀ7.21 (m, 5H), 6.47 (s, 1H), 5.22 (s, 2H), 4.43ꢀ4.26 (m, 2H), 3.94
(m, 4H), 1.36 (d, J = 5.6 Hz, 3H). MS (ESI): [MH]+ = 379.1. Anal.
(C19H18N6O3) C, H, N.
(R,S)-4-Benzyl-7,8-dihydro-8-ethyl-2-(3-methoxyisoxazol-5-yl)-1H-
imidazo[2,1-i]purin-5(4H)-one (47). White solid; 39% yield; mp
157 ꢀC. 1H NMR (200 MHz, CDCl3) δ ppm 10.64 (bs, 1H),
7.60ꢀ7.56 (m, 2H), 7.32ꢀ7.28 (m, 3H), 6.46 (s, 1H), 5.35 (s, 2H),
4.63ꢀ4.31 (m, 2H), 4.01 (s, 3H), 3.90ꢀ3.85 (m, 1H), 1.60ꢀ1.40 (m,
2H), 0.74 (t, J = 7.6 Hz, 3H). MS (ESI): [MH]+ = 393.1. Anal.
(C20H20N6O3) C, H, N.
Evaluation of Affinity (Ki) and Potency (IC50) of the Novel
Adenosine Compounds. CHO Membranes Preparation. The
human A1, A2A, A2B, and A3 receptors have been transfected in CHO cells
according with the method previously described.49 The cells were grown
adherently and maintained in Dulbecco’s Modified Eagles Medium with
nutrient mixture F12 (DMEM/F12) without nucleosides, containing
10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/
mL), L-glutamine (2 mM), and Geneticin (G418, 0.2 mg/mL) at 37 ꢀC
in 5% CO2, 95% air. Cells were split 2 or 3 times weekly at a ratio
between 1:5 and 1:20. For membrane preparation, the culture medium
was removed and the cells were washed with PBS and scraped off T75
flasks in ice-cold hypotonic buffer (5 mM Tris HCl, 2 mM EDTA, pH
7.4). The cell suspension was homogenized with Polytron, and the
homogenate was spun for 10 min at 1000g. The supernatant was then
centrifuged for 30 min at 100000g. The membrane pellet was suspended
in: (a) 50 mM Tris HCl buffer pH 7.4 for A1 adenosine receptors, (b)
50 mM Tris HCl, 10 mM MgCl2 buffer pH 7.4 for A2A adenosine
receptors, and (c) 50 mM Tris HCl, 10 mM MgCl2, 1 mM EDTA buffer
pH 7.4 for A3 adenosine receptors. The cell suspension were incubated
with 2 UI/mL of adenosine deaminase for 30 min at 37 ꢀC. The
membrane preparation was used to perform binding experiments.
Human Cloned A1, A2A, and A3 Adenosine Receptor Binding Assay.
All synthesized compounds have been tested for their affinity at human
A1, A2A, and A3 adenosine receptors. Displacement binding experiments
of [3H]-DPCPX (1nM) to hA1CHO membranes (50 μg of protein/
assay) and at least 6ꢀ8 different concentrations of antagonists studied
were performed for 120 min at 25 ꢀC. Nonspecific binding was
determined in the presence of 1 μM of DPCPX, and this was always
Measurement of Cyclic AMP Levels in CHO Cells Transfected with
Human A2B and A3 Adenosine Receptors. CHO cells transfected with
human A2B or A3 adenosine receptors were washed with phosphate-
buffered saline and diluted trypsine and centrifuged for 10 min at 200g.
The pellet containing CHO cells (1 ꢁ 106 cells/assay) was suspended in
0.5 mL of incubation mixture (mM): NaCl 15, KCl 0.27, NaH2PO4
0.037, MgSO4 0.1, CaCl2 0.1, Hepes 0.01, MgCl2 1, glucose 0.5, pH 7.4
at 37 ꢀC, 2 IU/mL adenosine deaminase, and 4-(3-butoxy-4-meth-
oxybenzyl)-2-imidazolidinone (Ro 20-1724) as phosphodiesterase in-
hibitor and preincubated for 10 min in a shaking bath at 37 ꢀC. The
potencies of antagonists to hA2B adenosine receptors were determined
by antagonism of NECA (100 nM)-induced stimulation of cyclic AMP
levels.53 The potencies of antagonists to hA3 adenosine receptors were
determined in the presence of Forskolin 1 μM by antagonism of Cl-IB-
MECA (100 nM)-induced inhibition of cyclic AMP levels.52 The
reaction was terminated by the addition of cold 6% trichloroacetic acid
(TCA). The TCA suspension was centrifuged at 2000g for 10 min at
4 ꢀC, and the supernatant was extracted four times with water saturated
diethyl ether. The final aqueous solution was tested for cyclic AMP levels
by a competition protein binding assay. Samples of cyclic AMP standard
(0ꢀ10 pmoles) were added to each test tube containing the incubation
buffer (trizma base 0.1 M, aminophylline 8.0 mM, 2 mercaptoethanol
6.0 mM, pH 7.4) and [3H] cyclic AMP in a total volume of 0.5 mL. The
binding protein, previously prepared from beef adrenals, was added to
the samples previously incubated at 4 ꢀC for 150 min and after the
addition of charcoal were centrifuged at 2000g for 10 min. The clear
supernatant was counted in a 2500-TR Packard scintillation counter.
Data Analysis. The protein concentration was determined according
to a Bio-Rad method54 with bovine albumin as a standard reference.
Inhibitory binding constants, Ki values, were calculated from those of
IC50 according to Cheng and Prusoff equation55 Ki = IC50/(1 + [C*]/
KD*), where [C*] is the concentration of the radioligand and KD* its
dissociation constant. A weighted nonlinear least-squares curve fitting
program LIGAND56 was also used for computer analysis of inhibition
experiments. Potency values (IC50) obtained in cyclic AMP assays were
calculated by nonlinear regression analysis using the equation for a
sigmoid concentrationꢀresponse curve (Graph PAD Prism, San Diego,
CA, USA). Affinity values are expressed as geometric mean, with 95% or
99% confidence limits in parentheses and IC50 values are expressed as
the arithmetic mean ( SEM.
Molecular Modeling. The homology model of the hA1 and hA3 ARs
were built using Schr€odinger Prime57 software accessible through the
Maestro interface.58 Unless otherwise stated, default parameters were
used throughout. The sequence of the hA1 (P30542) and hA3 (P33765)
ARs were aligned against the sequence of hA2A AR (P29274). Alignment
of anchor residues within each TM domain were attained according to
the sequence alignment suggested by Moro and co-workers (Figure 6).45
During the homology model building, Prime keeps the backbone
rigid for the cases in which the backbone does not need to be
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Figure 6. Sequence alignment of hA1, hA3 and hA2A ARs. Conserved and semiconserved residues are identified by black boxes and bold characters,
respectively. Transmembrane portions are identified by green boxes.
reconstructed due to gaps in the alignment. The loop refinements were
carried out using Prime with default parameter settings, if not mentioned
otherwise. Prior to refinement, the protein structures were subjected to a
protein preparation step to reorientate side chain hydroxyl groups and
alleviate potential steric clashes. The implemented loop modeling
protocol consists of several steps.59 First, large numbers of loops are
created by a conformational search in dihedral angle space. Clustering of
loop conformations and side chain optimization is being used to select
representative solutions. On the basis of the user parameters, a limited
number of structures is then processed using complete energy mini-
mization. The top ranked solution in terms of Prime energy was
considered best. The short loops (ELI and ELIII) were refined using
default sampling rates in the initial step, while the extended, highest
sampling rate was chosen for the larger amino acid loops (ELII). Side
chains were unfrozen within 7.5 Å of the corresponding loop, and the
energy cutoff was set to 10 kcal.
molꢀ1 Åꢀ1. All complexes pictures were rendered employing the UCSF
Chimera software.60
’ ASSOCIATED CONTENT
S
Supporting Information. Additional molecular model-
b
ing pictures for 35, S-33, 29, R-41, and 45. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For P.G.B.: phone, +39-0532-455921; Fax, +39-0532-455921;
E-mail, baraldi@dns.unife.it. For D.P.: phone, +39-0532-455302;
fax, +39-0532-455921; E-mail, prtdle@unife.it.
The Glide program of the Schrodinger package was used to dock R-33
to the hA1, hA2A, and hA3 ARs structures and compounds 29, S-33, 35,
R-41, and 45 to the hA3AR. The receptor grid generation were
performed for the box with a center in the putative binding site. The
size of the box was determined automatically. The extra precision mode
(XP) of Glide was used for the docking. The ligand scaling factor was set
to 1.0. The geometry of the ligand binding site of the complex between
R-33 and the three receptors was then optimized. The binding site was
defined as R-33 and all amino acid residues located within 8 Å from the
ligand All the receptor residues located within 2 Å from the binding site
were used as a shell. The following parameters of energy minimization
were used: OPLS2005 force field, water was used as an implicit solvent, a
maximum of 5000 iterations of the PolakꢀRibier conjugate gradient
minimization method was used with a convergence threshold of 0.01 kJ 3
’ ACKNOWLEDGMENT
We thank King Pharmaceuticals, Inc., Research & Develop-
ment, 4000 CentreGreen Way, Suite 300, Cary, North Carolina
27513, for financial support. We also thank Prof. Karl-Norbert
Klotz for cDNA encoding the human adenosine receptors.
’ ABBREVIATIONS USED
ARs, adenosine receptors; cAMP, 30-50-cyclic adenosine mono-
phosphate; DMF, dimethylformamide; CHO, Chinese hamster ovary;
Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-50-N-methyl-
carboxamide; DMEM, Dulbecco’s Modified Eagles Medium;
DMSO, dimethylsulfoxide; EDAC, 1-ethyl-3-[3 (dimethylamino)-
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propyl]carbodiimide hydrochloride; EDTA, ethylenediaminete-
traacetic acid;HOBt, hydroxybenzotriazole; NECA, 50-(N-ethyl-
carboxamido)adenosine; PBS, phosphate buffered saline; PDB,
Protein Data Bank; TCA, trichloroacetic acid
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