New pyrrolo[2,1-f]purine-2,4-dione and imidazo[2,1-f]purine-2,4-dione derivatives as potent and selective human A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm058008c

Compounds presenting an additional fused ring on the xanthine nucleus have been reported to exhibit antagonistic activity with various levels of affinity and selectivity toward the four adenosine receptors subtypes A₁, A_2A, A_2B<

Full text of DOI:10.1021/jm058008c

J. Med. Chem. 2005, 48, 4697-4701
4697
Brief Articles
New Pyrrolo[2,1-f]purine-2,4-dione and Imidazo[2,1-f]purine-2,4-dione
Derivatives as Potent and Selective Human A3 Adenosine Receptor Antagonists
Pier Giovanni Baraldi,*,§ Delia Preti,§ Mojgan Aghazadeh Tabrizi,§ Francesca Fruttarolo,§ Romeo Romagnoli,§
Naser Abdel Zaid,† Allan R. Moorman, Stefania Merighi,# Katia Varani,# and Pier Andrea Borea#
Dipartimento di Scienze Farmaceutiche, and Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia,
Universita` di Ferrara, 44100 Ferrara Italy, College of Pharmacy, An-Najah National University, Nablus, Palestine, and
King Pharmaceutical R & D, 4000 CentreGreen Way, Suite 300 Cary, North Carolina 27513
Received February 10, 2005
Compounds presenting an additional fused ring on the xanthine nucleus have been reported
to exhibit antagonistic activity with various levels of affinity and selectivity toward the four
adenosine receptors subtypes A1, A2A, A2B, and A3. This paper reports synthesis and biological
evaluation of new 1-benzyl-3-propyl-1H,6H-pyrrolo[2,1-f]purine-2,4-diones and 1-benzyl-3-
propyl-1H,8H-imidazo[2,1-f]purine-2,4-diones, among which we identified potent and selective
A3 adenosine receptors antagonists. In particular, 1-benzyl-7-methyl-3-propyl-1H,8H-imidazo-
[2,1-f]purine-2,4-dione (11e) shows a Ki (hA3) value from binding assay of 0.8 nM.
Introduction
efflux in multidrug resistance.17 Furthermore, A3 recep-
tor antagonists may be useful in the treatment of
glaucoma.18
Adenosine exerts a number of physiological functions
through the activation of cell membrane G-protein
coupled receptors classified into four different subtypes
named A1, A2A, A2B, and A3.1 The A3 adenosine receptor
is able to cause inhibition of forskolin-induced cAMP
accumulation, to increase phosphatidylinositol-specific
phospholipase C and D activity, and to elevate IP3 levels
and intracellular Ca2+ pools.2 As a therapeutic target,
it is the subject of intensive pharmacological charac-
terization due to its significant involvement in several
pathophysiological processes, such as inflammation,
neurodegeneration,3,4 cardiac and brain ischaemic dam-
age,5,6 asthma,7 and cancer.8
A3 receptor agonists appear to exert dual and opposite
effects, either cytoprotective or cytotoxic, depending on
the cell type and on the level of receptor activation.9,10
A3 receptors and their ability to regulate cell survival
represent a promising therapeutic target in diseases in
which excessive cell death is either undesirable, such
as neurodegeneration, or desirable, such as cancer and
inflammation.11,12 Adenosine acts as a potent regulator
of both normal and tumor cell growth.13,14 Evidence of
high levels of expression of A3 adenosine receptor
subtype has been provided in Jurkat cells,15 a human
leukemia cell line originating from the immune system,
in the human melanoma A375 cell line,16 and in human
pancreatic, breast, prostate, colon, lung, and ovarian
carcinoma cells.17 A3 antagonists seem to synergistically
enhance cytotoxic treatment and counter P-glycoprotein
In the past few years, different classes of compounds
with nonxanthine structures have been reported to be
A3 adenosine receptor antagonists.19-21 In a recent work,
the approach based on the annelation of xanthine
derivatives for the development of adenosine receptors
antagonists has been extensively considered.22 In par-
ticular, 1H,3H-pyrido[2,1-f]purine-2,4-diones23 and imi-
dazo[2,1-i]purin-5-ones24 have been claimed as potent
A3 adenosine receptor antagonists. Recently, we re-
ported a series of 1,3-dipropyl-7-aryl/heteroaryl-1H,6H-
pyrrolo[2,1-f]purine-2,4-dione derivatives which were
conceived as rigid analogues of KF17837, a known A2A
adenosine receptor antagonist belonging to the class of
styryl xanthines.25 Unfortunately, the synthesized com-
pounds did not show significant affinity for the inves-
tigated targets.
The report by Priego et al.23 about the mentioned
1H,3H-pyrido[2,1-f]purine-2,4-diones highlighted the
importance of a benzyl and a propyl moieties at the 1
and 3 positions, respectively. In light of this we thought
that the lack of activity of our reported 1,3-dipropyl-
pyrrolo[2,1-f]purine-2,4-dione derivatives might be par-
tially due to the presence of a propyl chain, instead of
the benzyl moiety at the 1 position. We therefore
evaluated the effect of the introduction of a benzyl and
a propyl at the 1 and 3 position, respectively, in our
previous series and in a new series of fused xanthine
derivatives. In particular, we performed the synthesis
of 1-benzyl-3-propyl-7-aryl/alkyl-1H,6H-pyrrolo[2,1-f]pu-
rine-2,4-dione (7a-d, Table 1) and 1-benzyl-3-propyl-
7-aryl/alkyl-1H,8H-imidazo[2,1-f]purine-2,4-dione (11a-
n, Table 1). We report the synthesis of these new
tricyclic structures and the evaluation of their affinity
and activity for the human adenosine A1, A2A, A2B, and
* To whom correspondence should be addressed. Tel: +39-0532-
291293. Fax: +39-0532-291296. E-mail: baraldi@dns.unife.it.
§ Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
# Dipartimento di Medicina Clinica e Sperimentale-Sezione di
Farmacologia, Universita` di Ferrara.
† An-Najah National University.
King Pharmaceutical R & D.
10.1021/jm058008c CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/21/2005

4698 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Brief Articles
Table 1. Structures and Physiochemical Parameters of the
n) were prepared following the general synthetic strategy
depicted in Scheme 1. The 6-amino-1-benzyl-3-propyl-
uracil 1 was synthesized starting from 1-benzyl-6-
aminouracil according to a known procedure for the
alkylation at the N3 position via protection of the amino
group at the 6-position as N-[(dimethylamino)methyl-
ene] derivative.26 Subsequent nitrosation at the 5-posi-
tion in acetic acid with NaNO2 furnished compound 2,
and then the reduction of the nitroso group with sodium
dithionite27 gave 5,6-diamino-1-benzyl-3-propyl-uracil 3
in good yield.
The synthesis of the final 1-benzyl-3-propyl-1H,6H-
pyrrolo[2,1-f]purine-2,4-dione derivatives 7a-d required
the conversion of intermediate 3 into the 3-benzyl-8-
hydroxymethyl-1-propyl-3,7-dihydro-purine-2,6-dione 4
by a two-step reaction. Refluxing derivative 3 with
glycolic acid, followed by cyclization of the resulting
amide intermediate by heating in a solution of aqueous
NaOH, afforded the desired product 4.25 Alkylation at
the N7-position with the appropriate R-halo-ketone
using K2CO3 in DMF as solvent provided the 3-benzyl-
8-hydroxymethyl-7-(2-oxo-alkyl)-1-propyl-3,7-dihydro-
purine-2,6-dione derivatives 5a-d in good yield. The
obtained 7-(2-oxo-alkyl)-8-hydroxymethyl derivatives
were converted into the corresponding 8-bromomethyl-
purine-2,6-dione intermediates 6a-d via treatment
with PBr3 in anhydrous benzene.
Synthesized Compounds
compd
R
R′
mp (°C)
MW
formula
anal.
7a
H
H
H
Ph
235
180
148
398.46
336.39
350.41
C
C
C
C
C
24H22N4O2
19H20N4O2
20H22N4O2
20H22N4O2
23H21N5O2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
7b
CH3
7c
7d
CH2CH3
CH3 CH3
114-115 350.41
255 399.45
4-OCH3-Ph 257
11a
11b
11c
11d
11e
11f
11g
11h
11i
11l
H
H
H
H
H
H
H
H
H
H
Ph
429.47 C24H23N5O3
4-Ph-Ph
4-F-Ph
CH3
CH2CH3
CH(CH3)2
C(CH3)3
cyclopropyl 244-245 363.41
cyclohexyl 130-132 405.49
272
250
303
285
475.54
417.44
337.38
351.17
C
C
C
C
C
C
C
C
C
C
29H25N5O2
23H20FN5O2 C, H, N
18H19N5O2
19H21N5O2
20H23N5O2
21H25N5O2
20H21N5O2
23H27N5O2
19H21N5O2
20H23N5O2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
128-130 365.43
230 379.46
11m CH3 CH3
11n CH3 CH2CH3
259
239
351.17
365.19
A3 receptors through radioligand binding assays and
cAMP assays.
Results and Discussion
To obtain the cyclization which furnished the pyrrole
ring condensed at the N7-C8 link of the purinone
nucleus, we employed a strategy involving an intramo-
Chemistry. 1-Benzyl-3-propyl-1H,6H-pyrrolo[2,1-f]-
purine-2,4-dione derivatives (7a-d) and 1-benzyl-3-
propyl-imidazo[2,1-f]purine-2,4-dione derivatives (11a-
Scheme 1a
a Reagents: (i) NaNO2, CH3COOH, EtOH, 40 °C, 30 min; (ii) Na2S2O4, H2O, 85 °C, 30 min; (iii) (a) HOCH2CO2H, dioxane, 100 °C, 1
h; (b) NaOH, EtOH/H2O, reflux, 3 h; (iv) R-halo-ketones, K2CO3, DMF, rt, 6-10 h; (v) PBr3, benzene, rt, 4-6 h; (vi) (a) PPh3, benzene,
reflux, 5 h; (b) CH3ONa, CH3OH, 0 °C, 10′; (vii) (a) HCO2H, reflux, 1 h; (b) NaOH, EtOH/H2O, reflux, 1 h; (viii) Br2, CH3CO2H, CH3CO2Na,
45 °C, 1 h; (ix) liquid ammonia, EtOH, 120 °C, ON.

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4699
their affinities for human A1, A2A, and A3 adenosine
receptors. Potency of the compounds versus hA2B ad-
enosine receptors were studied, evaluating their capa-
bility to inhibit (100 nM) NECA-stimulated cAMP
production. Basal and NECA stimulation of cAMP levels
were 15 ( 2 and 80 ( 9 pmoles cAMP/106 cells,
respectively. NECA was able to stimulate cAMP levels
in hA2BCHO cells with an EC50 value of 145 ( 15 nM.
Moreover, the compounds showing high affinity to hA3
receptors were also studied through cAMP experiments
performed in hA3CHO cells evaluating their capability
to block, in the presence of forskolin 10 µM, the
inhibitory effect mediated by (100 nM)-Cl-IB-MECA
(Figure 1). Basal, forskolin stimulation, and Cl-IB-
MECA inhibition of cAMP levels were 14 ( 2, 75 ( 8,
and 40 ( 5 pmoles cAMP/106 cells, respectively. Cl-IB-
MECA was able to inhibit forskolin stimulated cAMP
levels with an IC50 value of 8.7 ( 0.9 nM. Affinity data
for A1, A2A and A3 receptors, expressed as Ki values, and
IC50 values derived from the cAMP assay carried out
for hA2B subtypes, are listed in Table 2.
Figure 1. Inhibitory curves of cAMP accumulation in human
A3 adenosine receptors by adenosine antagonists blocking the
effect of 100 nM Cl-IB-MECA.
lecular Wittig reaction between the carbonyl moiety of
the introduced N7-chain and the bromomethyl function
at the 8-position. Thus, we treated the 8-bromomethyl
derivatives 6a-d with triphenylphosphine in benzene,
heating the mixture at reflux for 5 h to allow the
formation of the intermediate phosphonium salts. The
crude material was easily cyclized into the correspond-
ing pyrrolo[2,1-f]purine-2,4-diones 7a-d by treatment
with sodium methoxide.25
The 3-benzyl-1-propyl-3,7-dihydro-purine-2,6-dione 8
was obtained by reacting the diamino derivative 3 with
formic acid28 according to the same procedure followed
for preparation of compound 4. Bromination at the
8-position with Br2 and sodium acetate in acetic acid
at 60 °C for about 1 h led to formation of the key
8-bromo-intermediate 9 in excellent yield. Alkylation at
the N7-position with different R-halo-ketones under the
same conditions employed for the synthesis of 5a-d
supplied 7-(2-oxo-alkyl)-1-propyl-3,7-dihydro-purine-2,6-
dione derivatives 10a-n. Treatment of these intermedi-
ates with liquid ammonia in a sealed tube at 120 °C
overnight in ethanol effected, at first, the substitution
of the bromine at the 8-position followed by the in situ
cyclization of the amino group with the N7 carbonyl
function to give the desired 1-benzyl-3-propyl-1H,8H-
imidazo[2,1-f]purine-2,4-dione derivatives 11a-n.
Biological Evaluation and Structure-Affinity
Relationships. All the synthesized compounds were
evaluated in radioligand binding assays to determine
In the reported series of compounds we evaluated the
effect of different heterocycles fused on the N7-C8
positions of the xanthine nucleus. The fundamental
feature of these molecules lies in their practically
complete selectivity in binding A3 receptor versus A1,
A2A and A3 subtypes, as reflected by the notable Ki (hA1-
hA2A/hA3) and IC50 (hA2B)/Ki (hA3) ratios (Table 2). The
Ki values related to the interaction with the adenosine
A3 receptor are strictly dependent on the nature of the
substituents at the 7-position of the tricyclic structures
while the ability to discriminate between the different
AR subtypes is not generally affected by such structural
modification. The synthesized compounds include both
7-(4-substituted-aryl)-pyrrolo/imidazo[2,1-f]purine-2,4-
dione and 7-(cyclo)alkyl-pyrrolo/imidazo[2,1-f]purine-
2,4-dione derivatives.
Among the examined tricycles, the imidazo[2,1-f]-
purine-2,4-dione derivatives 11a, 11e, 11f, and 11m
were 2- to 10-fold more active than the corresponding
substituted-pyrrolo[2,1-f]purine-2,4-dione derivatives
7a-d toward the adenosine A3 receptor subtype. Both
series had Ki values in the low nanomolar range (Ki )
0.8-200 nM). This indicates a possible involvement of
Table 2. Binding and Functional Parameters of Synthesized 1H,6H-Pyrrolo[2,1-f]purine-2,4-dione Derivatives (7a-d) and
Imidazo[2,1-f]purine-2,4-dione Derivatives (11a-n) Toward hA1, hA2A, hA2B, and hA3 Adenosine Receptors
a
b
c
d
compd
hA1
>1000
hA2A
hA2B
hA3
hA1/hA3
hA2A/hA3
hA2B/hA3
7a
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
-
200 (134-297)
8.0 (7.1-9.1)
3.5 (2.7-4.4)
80 (63-100)
115 (89-150)
55 (28-104)
>1000
22 (19-26)
0.8 (0.6-0.9)
15 (9-27)
31 (25-38)
99 (77-129)
23 (18-29)
555 (467-660)
36 (31-43)
60 (53-69)
>5
>5
-
50
7b
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
400 (323-496)
>1000
>1000
-
>125
>290
>13
>9
>125
>290
>13
>9
7c
>290
>13
-
7d
11a
11b
11c
11d
11e
11f
11g
11h
11i
11l
11m
11n
-
>18
-
>18
-
-
-
-
-
>45
>1250
>67
>15
>10
15
>45
>1250
>67
>32
>10
>44
>2
-
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1250
>67
>32
>10
>44
>2
460 (424-498)
>1000
350 (299-411)
>1000
>2
>28
>17
>1000
>1000
>28
>17
>28
>17
a Displacement of specific [3H]-DPCPX binding to human A1 receptors expressed in CHO cells (Ki, nM). b Displacement of specific [3H]-
ZM 241385 binding to human A2A receptors expressed in CHO cells (Ki, nM). c cAMP assay in CHO cells expressing hA2B receptors (IC50
nM). d Displacement of specific [3H]-MRE3008F20 binding to human A3 receptors expressed in CHO cells (Ki, nM).
,

4700 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Brief Articles
the N8-position in the interaction of the molecules with
the receptor, suggesting an opportunity to establish a
hydrogen bond.
pound 11e represents a significant improvement over
MRE3008F20, in particular with regard to the signifi-
cant increase of selectivity toward adenosine A2A sub-
type.
Interestingly, a notable concordance between binding
and functional experiments performed with the hA3
receptor has been revealed. Among the examined com-
pounds, the molecules showing the best affinities for the
hA3 adenosine receptor have also proved to have very
high potency in functional assays (Figure 1). In par-
ticular, derivative 11e can be considered the most potent
compound, exhibiting an IC50 value of 5 nM.
Among the 7-aryl-substituted series, it was observed
that substitution at the 4-position of the phenyl ring
with a methoxy function or especially with the small
electron-withdrawing fluorine atom, which is also able
to form hydrogen bonds, produces an increase in affinity,
while the introduction of a p-phenyl group leads to the
total loss of affinity. This indicates that the presence of
a large aromatic and lipophilic moiety, such as the
biphenyl, at the 7-position of the corresponding tricyclic
derivative establishes repulsive interactions with the
receptor.
Experimental Section
We then decided to evaluate the effect of replacing
the phenyl ring at the 7-position of compounds 7a and
11a-d with various (cyclo)alkyl chains. Compounds
7b-d and 11e-l contain at the 7-position alkyl chains
with different length such as -methyl (7b and 11e),
-ethyl (7c and 11f), branched alkyl chains such as
-isobutyl (11g), -tert-butyl (11h), and cycloalkyl chains
such as -cyclopropyl (11i) and -cyclohexyl (11l). The best
results were obtained with the introduction of small
linear alkyl chains, in particular a methyl group (11e,
Ki(hA3) ) 0.8 nM with a surprising selectivity pattern
versus the other AR subtypes). Longer chains or branch-
ing led to a loss of activity (11g Ki(hA3) ) 99 nM and
11l Ki(hA3) ) 555 nM), supporting the observation with
the 7-biphenyl derivative (11c), which indicates that a
sterically demanding, lipophilic moiety at the 7-position
would be detrimental to binding. The synthesis of
compounds 7d and 11m,n permitted us to estimate the
effect of the introduction of an additional methyl group
at the 6-position of the tricyclic derivatives. In all the
examples, this kind of structural modification decreased
the affinity of the molecules for the receptor binding site,
inducing a significant increase of the related Ki(hA3)
values (7d 10-fold less active than 7b, 11m 45-fold less
active than 11e, 11n 4-fold less active than 11f).
However, modification of this side of the molecule did
not seem to affect the selectivity versus A1, A2A, and A2B
receptors.
General Procedure for Preparation of 1-Benzyl-3-
propyl-1H,6H-pyrrolo[2,1-f]purine-2,4-dione Derivatives
(7a-d, Intramolecular Wittig Reaction). A solution of the
corresponding bromide 6a-d (0.42 mmol) and PPh3 (0.46
mmol) in anhydrous benzene (5 mL) was refluxed for 5 h. After
this time, the resulting mixture was concentrated to half-
volume and the precipitates collected by filtration. The inter-
mediate phosphonium salts (0.26 mmol) were then added to
an ice-cooled and stirred solution of sodium methoxide (0.29
mmol) in anhydrous methanol (5 mL). The reaction was stirred
at 0 °C for 10 min, the solvent was evaporated, and the
products were purified by column chromatography on silica
gel eluting with the appropriate mixture of light petroleum-
EtOAc (6:4 for 7a, 1:1 for 7b-d).
General Procedure for Preparation of 1-Benzyl-3-
propyl-1H,8H-imidazo[2,1-f]purine-2,4-dione Derivatives
(11a-n). A solution of the appropriate 7-(2-oxo-alkyl)-3,7-
dihydro-purine-2,6-dione derivatives 10a-n (0.4 mmol) in
EtOH (4 mL) was cooled at -40 °C. Liquid ammonia (3-4 mL)
was then added to the mixture. The mixture was heated in a
sealed tube overnight at 100-120 °C. The reaction was finally
allowed to cool at room temperature, and then the solvent and
the excess of ammonia were evaporated to obtain a residue
that was suspended with water and extracted with EtOAc (3
× 25 mL). The organic phase was dried with anhydrous sodium
sulfate, and the solvent was evaporated to give a residue,
which was purified by column chromatography on silica gel,
eluting with the appropriate mixture of light petroleum-
EtOAc.
Acknowledgment. We thank King Pharmaceutical
R & D 4000 Centre Green Way, Suite 300 Cary, NC
27513, for financial support. We also thank Prof. Karl-
Norbert Klotz for cDNA encoding the human adenosine
receptors.
Conclusions
In conclusion the present study can be considered an
innovative contribution to the previously reported22
approach based on annelation of xanthine derivatives.
Some of the newly reported imidazo[2,1-f]purine-2,4-
dione and pyrrolo[2,1-f]purine-2,4-dione derivatives rep-
resent, to the best of our knowledge, the most potent
and selective hA3 adenosine receptor antagonists con-
taining a xanthine nucleus. In particular 1-benzyl-7-
methyl-3-propyl-1H,8H-imidazo[2,1-f]purine-2,4-dione
(11e) shows a subnanomolar affinity toward the desired
receptor target with a noteworthy selectivity versus the
other adenosine receptors subtypes (Ki(hA3) ) 0.8 nM,
Ki(hA1/hA3) ) 3163, Ki(hA2A/hA3) > 6250, IC50 (hA2B)/
Ki(hA3) ) 2570). These data are even more sur-
prising when compared with the binding profile of
MRE3008F20,29 a potent A3 adenosine receptor antago-
nists belonging to the family of pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidines (Ki (hA3) ) 0.85 nM, Ki(hA1/
hA3) ) 1294, Ki(hA2A/hA3) ) 165, Ki(hA2B/hA3) ) 2471).
From the selectivity pattern, it is apparent that com-
Supporting Information Available: Detailed experi-
mental procedures for the synthesis and the biological assays
1
of the reported compounds, C, H, N analytical data, H NMR
data. This material is available free of charge via Internet at
http://pubs.acs.org.
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