1,3-Dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives as highly potent and selective human A2B adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2007.11.058

A new series of 1,3-dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives has been identified as potent A_2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A_2B, A₁, A_2A, and A₃ adenosine receptors. N-(4-chloro-phenyl)-2-[3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl] (11c) showed a high affinity for the human A_2B adenosine receptor K_i = 7 nM and good selectivity (A₁, A_2A, A₃/A_2B > 140). Synthesis and SAR of this novel class of compounds is presented herein.

Full text of DOI:10.1016/j.bmc.2007.11.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2419–2430
1,3-Dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine
derivatives as highly potent and selective human A2B
adenosine receptor antagonists
Mojgan Aghazadeh Tabrizi,a Pier Giovanni Baraldi,a,* Delia Preti,a
Romeo Romagnoli,a Giulia Saponaro,a Stefania Baraldi,a Allan R. Moorman,c
Abdel Naser Zaid,d Katia Varanib and Pier Andrea Boreab
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
b
`
Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universita di Ferrara, 44100 Ferrara, Italy
cKing Pharmaceuticals, Inc., Research and Development, 4000 CentreGreen Way, Suite 300, Cary, North Carolina 27513, Italy
dCollege of Pharmacy, An-Najah National University, Nablus, Palestine
Received 20 September 2007; revised 14 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—A new series of 1,3-dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives has been identified as potent
A2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A2B, A1, A2A
,
and A3 adenosine receptors. N-(4-chloro-phenyl)-2-[3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-5-methyl-pyrazol-
1-yl] (11c) showed a high affinity for the human A2B adenosine receptor Ki = 7 nM and good selectivity (A1, A2A, A3/A2B > 140).
Synthesis and SAR of this novel class of compounds is presented herein.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
receptor in all brain regions tested,5 whereas, high levels
can be found in the cecum, large intestine, and urinary
bladder. Lower levels of the A2B adenosine receptor
can be detected in spinal cord, vas deferens, pituitary,
in a variety of skin cells, and fibroblasts.6 It has been re-
ported the presence of A2B receptors in HMC-1 cells
(human mast cells). Several compounds have been stud-
ied in this cell line and evaluated for their potency to de-
crease NECA-stimulated cAMP production showing a
high potency.7 It has been also investigated the expres-
sion and functional coupling of A2B receptors in periph-
eral blood cells by using a novel selective antagonist
radioligand [3H]MRE-2029F20.8,9
The biological activity of adenosine occurs through the
activation of specific receptors located on cell mem-
branes and belonging to the extensive family of G-pro-
tein coupled receptors.1,2 Adenosine activates four
subtypes of receptors (ARs) named as A1, A2A, A2B
,
and A3.1,3 Adenosine receptors are transmembrane G-
protein coupled with approximately 21–28 amino acids
in each transmembrane region. The N-terminus of the
receptor is extracellular and all receptors, with the
exception of the A2A adenosine receptor, have a palmi-
toylation site near the C-terminus.4 While A1 and A3
receptors are coupled to Gi-protein, A2A and A2B aden-
osine receptors are linked to adenylate cyclase-stimula-
tory Gs-proteins.1 Reverse transcription coupled to the
polymerase chain reaction (RT-PCR) and in situ hybrid-
ization of rat tissue demonstrates low levels of A2B
The A2B adenosine receptor has been also implicated in
several biological events. This receptor plays a role in
mediating vasodilatation in some vessels, such as guinea
pig and mouse aorta.10,11 Inhibition of growth of rat
aortic smooth muscle cells has been achieved through
selective A2B receptor activation and appears to be sec-
ondary to blockade of the mitogen-activated protein ki-
nase (MAPK) pathway.12,13 Recently it has been
revealed the presence of A2B adenosine receptors in hu-
man lung peripheral parenchyma to investigate the
Keywords: Xanthine; Phenylacetamide; Adenosine receptors; A2B
antagonists; Biological activity; Binding; Functional; Human.
*
Corresponding author. Tel.: +39 0532 455921; fax: +39 0532
291296; e-mail: baraldi@dns.unife.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.058

2420
M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
H
O
O
H
N
H
N
N
R1
O
OCH3
OCH3
N
N
N
O
N
N
N
O
N
N
R2
3, MRE2028F20
Ki (hA2B) = 38 nM, Ki (hA1, hA2A, hA3) > 1000 nM
1, Theophylline: R1 = R2 = Methyl
2, Enprophylline: R1 = H, R2 = propyl
O
O
O
O
N
H
N
O
N
H
N
N
N
O
N
4, MRE2029F20
Ki (hA2B) = 5.5 nM, Ki (hA1) = 200 nM, (hA2A, hA3) > 1000nM
Figure 1. Representative structures of xanthines as A2B adenosine receptor antagonists.
expression, affinity, and density of adenosine receptors
in peripheral lung parenchyma from age-matched smok-
ers with chronic obstructive pulmonary disease (COPD)
and smokers with normal lung function.14 In addition a
role of adenosine in asthma has been demonstrated on
the basis of key experimental evidence such as the in-
crease of adenosine concentration in hypoxia and cellu-
lar inflammation in bronchoalveolar fluids of asthmatics
and in plasma (upon contact with allergens).15,16 More-
over, adenosine (in the form of AMP) induces broncho-
constriction in asthmatics, but not in healthy
individuals.17,18 Finally, theophylline, a dimethylxan-
thine with a well-established role in the therapy of asth-
ma, was shown to block selectively the AMP-induced
bronchoconstriction.16 The bronchodilating activity of
theophylline 1 and its structural analog enprofylline 2
(Fig. 1) recently has been attributed to a selective, albeit
small, antagonism at the A2BAR.15 These findings
prompted several groups to design and test a large num-
ber of xanthine derivatives in the search for new, more
potent, and A2B-selective ligands.
The affinity of the synthesized products was evaluated in
human CHO cells by using specific radioligands of A2B
A1, A2A, and A3 receptors; all compounds were tested
for the inhibition of NECA-induced cAMP accumula-
tion mediated by the adenosine A2B receptor.
,
2. Chemistry
The synthetic routes to obtain the target compounds
11a–q are outlined in Scheme 1. Appropriately substi-
tuted anilines are acylated with bromoacetyl bromide
to provide the corresponding bromoacetanilides. N-
alkylation of pyrazoles 5–7 in anhydrous ethanol with
bromoacetanilides in the presence of sodium ethoxide
gave an approximately 1:4 mixture of the N1-3/N1-5-
(un)substituted pyrazoles (compounds 8a–q and 9a–q).
The two isomers were separated by flash chromatogra-
phy. Hydrolysis of pyrazole ethyl esters 9a–q in 2N
KOH yielded the corresponding carboxylic acids 10a–
q. The coupling reaction of derivatives 10a–q with
1,3-dipropyl-5,6-diaminouracil was achieved by a two-
step sequence involving reaction in methanol solution
using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide
hydrochloride (EDAC) as condensing agent, followed
by ring closure in the presence of sodium hydroxide at
70 ꢁC yielding the target xanthines 11a–q. The low yields
of this step are due to the partial hydrolysis of the amide
group, which occurs during the cyclization in the
presence of NaOH. The target compounds 16a–c were
synthesized by the same procedures except using the
4-halo-pyrazoles 12a–c, which are prepared by haloge-
nation of pyrazole ester 5 in the presence of the appro-
priate N-halo-succinamide (Scheme 2).
We have recently discovered a number of 8-heterocycle-
substituted xanthine derivatives as potent and selective
A2BAR antagonists.19 These MRE compounds (Fig. 1)
shared high affinity for the A2B adenosine receptor.
The dimethoxy compound MRE2028F20 (3) had a very
high selectivity and affinity of 38 nM. The highest affin-
ity was obtained with MRE2029F20 (4), Ki = 5.5 nM
combined with strong selectivity (>36-fold) with respect
to the other three adenosine receptor subtypes.
Herein we report some of the SAR we developed and the
characterization of a new series of compounds (11a–q)
obtained by replacement of the side chain from the 3-po-
sition of the pyrazole ring to the 1-position. Next to
these derivatives, we have also prepared analogs 16a–c
to investigate the presence of a halogen atom at the 4-
position of pyrazole ring.
In Table 1 are reported the 13C NMR signals of several
alkyl pyrazoles examined. The comparison of the carbon
chemical shifts of the two N-alkyl-pyrazole series (8acd,
14a, 9acd, 13a) showed that the C-3 and C-5 signals of

M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
2421
O
O
O
O
O
R
R
O
R
i
N
N
N
N
R1
HN
N
R1
8a-q
R = CH3, 5
R = H, 6
9a-q
R = C2H5, 7
ii
O
N
H
N
R
O
N
O
iii
OH
R
N
N
R1
N
N
N
R1
11a-q
10a-q
O
R'
Comp
R1 =
N
H
R
11
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
R’
phenyl
4-F-phenyl
4-Cl-phenyl
4-Br-phenyl
4-I-phenyl
4-tolyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
4-methoxy-phenyl
4- (CH3)2-phenyl
4-sec-butyl-phenyl
3-methoxy-phenyl
3-Cl-phenyl
N
3,4-dichloro-phenyl
3,4-dimethoxy-phenyl
3,4-dimethyl-phenyl
1-naphtyl
CH3CH2
H
4-Cl-phenyl
4-Cl-phenyl
Scheme 1. Reagents and conditions: (i) appropriate bromoacetanilides, C2H5 ONa, C2 H5 OH, rt; (ii) KOH, dioxan temperature, (iii) a—1,3-
dipropyl-5,6-diamino-uracil, EDAC, CH3OH, room temperature; b—NaOH, 80 ꢁC.
O
R
N
O
R
N
O
O
O
R
O
N
O
O
N
i
ii
N
HN
N
O
HN
NH
O
12a-c
a: R = Cl, b: R = Br, c: R = I
HN
Cl
Cl
13a-c
14a-c
iii
O
R
H
N
Cl
H
N
R
O
O
N
iv
N
N
N
HO
NH
N
O
N
N
O
Cl
15a-c
16a-c
Scheme 2. Reagents and conditions: (i) N–Cl/Br/I-succinimide, DMF; (ii) 2-bromo-N-(4-chloro-phenyl)-acetamide, C2H5ONa,C2H5OH, room
temperature; (iii) KOH, dioxane, rt; (iv) a—1,3-dipropyl-5,6-diamino-uracil, EDAC,CH3OH, room temperature; b—NaOH, 80 ꢁC.

2422
M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
Table 1. 13C NMR parameters of several pyrazole isomers
electron-withdrawing groups (EWG). The substitution
at the para-position of the phenyl ring of 11a with elec-
tron-withdrawing (EWG) groups, such as halogen
atoms (11b–e), resulted in an increase of selectivity while
retaining affinity at the A2B receptor. The 4-chloro-
phenyl derivative 11c was found to be the most potent
(KiA2B = 7 nM) and selective compound within the ser-
ies (A1, A2A, A3/A2B > 140). The substitution at the
para-position of the phenyl ring of 11a with electron-
donating groups (EDG), such as CH3 (11f, Ki = 56)
and OCH3 (11g, Ki = 12), also showed good affinity at
the A2B, retaining selectivity at AR subtypes (Table 2).
Bulky lipophilic groups, such as the 4-sec-butyl (11i)
and dimethylamino (11h) groups, led to a loss of affinity.
Shifting of substituents from the para to the meta posi-
tion of the phenyl ring resulted in compounds with low-
er binding affinity at the A2B receptor compared to the
corresponding para-chloro (11c) and para-methoxy
(11g) analogs, (11j: m-methoxy Ki = 65) and (11k: m-
chloro, Ki = 28). The m-methoxy derivative also showed
a slight loss of selectivity versus the A1 adenosine (hA1
Ki = 480 nM). Disubstituted phenyl analogs which con-
tain a 3,4-substituent (3,4-dichloro 11l, dimethoxy 11m,
and dimethyl 11n derivatives) were found to have lower
A2B AR affinity than mono-substituted analogs. In par-
ticular the dichloro and dimethoxy derivatives showed a
5- to 6-fold decrease of affinity compared to that of com-
pounds 11c and 11g (Table 2). The replacement of the
phenyl group on the N-position of acetamido-chain with
a naphthyl ring resulted in compound 11o which dis-
played a loss of affinity at ARs. Hence the analogs 11p
and 11q were investigated to determine the effect of sub-
stitution at the 5-position of pyrazole ring in compari-
son to the reference compound 11c. Introduction of an
Compound
C3
C5
C4
N-CH2
8a
9a
146.55
141.56
149.60
143.99
149.40
143.97
144.86
139.58
133.13
141.61
135.80
142.18
135.18
142.19
137.44
137.20
110.91
107.60
112.30
108.94
111.70
108.93
112.80
109.22
54.95
52.63
54.86
53.32
54.96
53.29
55.70
53.63
8d
9d
8c
9c
13a
14a
the 1-alkyl-3-methyl derivatives (8acd, 13a), in accor-
dance with Pugmire and Grant’s principle,20 resonate
upfield and downfield, respectively, with respect to the
corresponding signals in the spectrum of the 1-alkyl-
5-methyl derivatives (9acd, 14a). For the pyrazole
derivatives 9acd, 14a the 13C NMR resonance of C-5 is
downfield of the corresponding signals in the spectrum
of the N1-3-methyl-pyrazole isomer. These data coincide
with our previous studies.21
3. Results and conclusions
As depicted in Table 2 a number of selected analogs
were prepared, showing good binding affinities for the
A2B receptor in the radioligand assay, as well as good
selectivity versus A1, A2A, and A3 ARs. Unsubstituted
phenylacetamide chain on the N-position of pyrazole
ring (compound 11a) was found to show high potency
and selectivity. The derivatives 11b–o were prepared to
study the effect of substituting the 3- and/or 4-position
of the phenyl ring with electron-donating (EDG) and
Table 2. Adenosine receptor affinities and functional data of A2B adenosine derivatives 11a–q, 16a–c
Compound
Ki (nM)
IC50 (nM)
a
b
c
d
e
hA1
hA2A
hA3
hA2B
hA2B
11a
11b
11c
11d
11e
11f
11g
11h
11i
350 (275–482)
500 (420–595)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
15 (10–21)
14 (10–20)
7.0 (5.0–9.0)
35 (27–45)
28 (18–43)
56 (45–70)
12 (8–18)
>1000
58 (45–74)
68 (51–91)
43 (31–60)
156 (123–198)
115 (84–159)
212 (162–277)
61 (44–84)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
11j
480 (444–518)
675 (569–800)
>1000
65 (56–75)
28 (19–41)
40 (25–66)
60 (44–82)
48 (35–65)
>1000
266 (216–327)
106 (81–139)
188 (166–213)
222 (203–242)
200 (182–219)
>1000
11k
11l
11m
11n
11o
11p
11q
16a
16b
16c
>1000
>1000
>1000
>1000
>1000
>1000
140 (123–159)
>1000
>1000
20 (15–26)
222 (181–273)
250 (205–306)
>1000
80 (63–100)
>1000
>1000
>1000
>1000
a Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells (n = 3–6).
b Displacement of specific [3H]ZM241385 binding at human A2A receptors expressed in CHO cells (n = 3–6).
c Displacement of specific [3H]MRE3008-F20 binding at human A3 receptors expressed in CHO cells (n = 3–6).
d Displacement of specific [3H] MRE2029F20 binding at human A2B receptors expressed in HEK293 cells (n = 3–6).
e cAMP assay in CHO cells expressing human A2B adenosine receptors IC50 (nM). Data are expressed as geometric means with 95% confidence limits.

M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
2423
5-methyl substituted compounds have the highest affinity,
5-ethyl leads to a marked reduction in affinity,
5-unsubstituted leads to loss of affinity
Replacement of hydrogen by
halogen atoms decrases affinity
EWG groups increas affinity; small EDG
groups confer good affinity; bulky lipophilic
substituents lead to loss of affinity
O
R3
R4
R1
R2
5
H
N
4
O
Disubstitution produces lower affinity
N
N
H
N
1
3
N
2
N
O
N
Substitution at the
m-position leads to lower binding
affinity with respect to the o-position
Figure 2. Illustration of structure–activity relationship of synthesized compounds.
ethyl moiety at the 5-position (11p) led to a loss of affin-
ity and the derivative with a hydrogen atom at the same
5. Experimental
5.1. General information
position (11q) displayed a 3-fold decrease of affinity and
selectivity (hA2B Ki = 20, hA1 Ki = 140 nM) compared
to that of compound 11c. We also explored the effect
of 4-substituted pyrazoles on A2B AR affinity with the
4-halo-derivatives 16a–c. This resulted in a loss of affin-
ity for compound 16c (4-I-derivative) and a decrease of
affinity (30-fold) for analogs 16a and 16b relative to that
of the unsubstituted derivative 11c. The structure–activ-
ity relationship (SAR) of synthesized compounds is
illustrated in Figure 2.
Reagent grade solvents were dried according to standard
techniques. Sodium sulfate was used as a drying agent
for water containing organic phases. Reactions were rou-
tinely monitored by thin-layer chromatography (TLC)
on silica gel (precoated F245 Merck plates). Chromato-
graphic spots were visualized by UV light. Purification
of crude compounds and separation of reaction mixtures
were carried out by column chromatography on silica gel
60 (230–400 mesh from Merck) using appropriate elu-
ents. Melting points (uncorrected) were determined in a
Buchi-Tottoli melting point apparatus. Chemical shifts
(d) are reported in parts per million (ppm) relative to
the solvent central peak. 1H NMR spectra were recorded
at 200 MHz on a Bruker AC 200 spectrometer. Electro-
spray Ionization Mass Spectrometry (ESI/MS) was per-
formed with an Agilent 1100 Series LC/MSD model in
positive scan mode. The molecular weights from the
MS spectra (not reported) were in full agreement with
the proposed chemical structures of target compounds.
Elemental analyses were performed by the microanalyti-
cal laboratory of Dipartimento di Chimica University of
Ferrara. All final products had satisfactory C, H, N anal-
yses (within 0.40 of theoretical values). Unambiguous
assignment for all proton and carbon resonances of the
two isomeric N1-3/N1-5-substituted or unsubstituted pyr-
azoles (compounds 8a–q and 9a–q) was achieved via 13C
NMR chemical shifts and via a strong NOE between N-
CH2 of the side chain and CH3 on the 5 position of the
pyrazole ring.
Figure 3A reports the competition curves of specific
[3H]-MRE 2029F20 binding to human A2B receptors
of new, potent, and selective A2B adenosine antagonists
showing affinity values in the range 7–40 nM. The log
dose-response curves of cAMP accumulation for se-
lected antagonists to human A2B receptors representing
the capability of the new compounds to block the stim-
ulatory effect of 100 nM NECA on cAMP levels are
shown in Figure 3B. All adenosine antagonists are able
to inhibit cAMP accumulation, displaying an order of
potency strictly similar to that observed in binding affin-
ities. The new A2B adenosine antagonists 11c and 11g
are potent in binding (Ki = 7 and 12 nM, respectively)
and in functional assays (IC50 = 43 and 61 nM, respec-
tively). The comparison of Ki and IC50 values
(Fig. 3C) indicates that an high correlation exists be-
tween data obtained from binding and functional assays
(r = 0.99, P < 0.01).
4. Conclusion
1H-Pyrazole-3-carboxylic acid ethyl ester 6 was commer-
cially available. The 5-methyl-1H-pyrazole-3-carboxylic
acid ethyl ester 5 and 5-ethyl-1H-pyrazole-3-carboxylic
acid ethyl ester 7 were prepared by cyclization of 2,4-
dioxo-pentanoic- or -hexanoic acid ethyl esters with
hydrazine salts in ethanol at reflux.
Synthesis of a new class of 8-(pyrazol-3-yl) xanthine
derivatives led to the identification of several A2B AR
antagonists with high affinity and very good selectivity
for the adenosine receptor subtypes. 8-[5-Methyl-(4-
chlorophenylacetamido-1H-pyrazol)-3-yl]-1,3-dipropyl-
xanthine (11c) had the highest affinity and selectivity in
this series that will be useful in understanding the phys-
iological role of the A2B AR, allowing for the targeted
synthesis of potent and selective antagonist of the A2B
AR.
5.2. General synthesis of 2-bromo-N-phenylacetamide
A solution of aniline (0.016 mol) in 40 mL of dichloro-
methane was cooled on ice; 1.0 equiv of bromoacetyl

2424
M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
5.3. General procedure for preparation of 5-(alkyl)-1-
(substituted)phenylcarbamoylmethyl-1H-pyrazole-3-car-
boxylic acid ethyl esters 8a–q and 9a–q
A
100
80
60
40
20
0
11c Ki = 7.0 nM
11g Ki = 12 nM
11b Ki = 14 nM
11q Ki = 20 nM
11l Ki = 40 nM
To a magnetically stirred mixture of pyrazole 5–7
(0.02 mol) and sodium ethoxide (0.22 mol) in anhydrous
ethanol (20 mL) was added the appropriate 2-bromo-N-
phenyl-acetamide (0.02 mol). The resulting suspension
was stirred at room temperature for 3 h. After concen-
tration in vacuo, EtOAc was added, and the resulting
solution was washed twice with water and dried over
Na2SO4. After filtration and concentration, the residue
that contained the alkylated pyrazoles 8 and 9a–q as a
separable mixture of N1-3- and N1-5-substituted isomers
(ratio approximately 1:4) was further purified by flash
chromatography (ethyl acetate/hexane).
-11
-10
-9
-8
-7
-6
-5
-4
log drug concentration (M)
B
100
80
60
40
20
0
11c IC50 = 43 nM
11g IC50 = 61 nM
11b IC50 = 68 nM
11q IC50 = 80 nM
11l IC50 = 188 nM
5.3.1. 5-Methyl-1-phenylcarbamoylmethyl-1H-pyrazole-
3-carboxylic acid ethyl ester (9a). Yield: 65%; mp: 139–
1
140 ꢁC; H NMR DMSO-d6 d 1.26–1.29 (t, 3H); 2.28
(s, 3H); 4.23–4.25 (q, 2H); 5.06 (s, 2H); 6.56 (s, 1H);
7.07 (m, 1H); 7.32 (m, 2H); 7.57 (m, 2H); 10.41 (s, 1H).
5.3.2. 1-[(4-Fluoro-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9b). Yield:
-9
-8
-7
-6
-5
1
log drug concentration (M)
62%; mp: 149–153 ꢁC; H NMR DMSO-d6 d 1.40–1.42
(t, 3H); 2.57 (s, 3H); 4.41–4.43 (q, 2H); 4.95 (s, 2H);
6.67 (s, 1H); 6.98 (m, 2H); 7.39 (m, 2H); 8.09 (s, 1H).
8.0
7.5
7.0
6.5
6.0
C
P<0.01, r=0.99
n= 15
5.3.3. 1-[(4-Chloro-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9c). Yield:
1
62%; mp: 216–218 ꢁC; H NMR DMSO-d6 d 1.37–1.39
(t, 3H); 2.38 (s, 3H); 4.38 (q, 2H); 5.07 (s, 2H); 6.65 (s,
1H); 7.18 (d, 2H); 7.33 (d, 2H); 8.78 (s, 1H).
5.3.4. 1-[(4-Bromo-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9d). Mp:
6.0
6.5
7.0
7.5
8.0
8.5
9.0
1
Yield: 62%; 195–198 ꢁC; H NMR DMSO-d6 d1.37 (t,
[3H]-MRE 2029F20 binding
(-logKi, nM)
3H); 2.38 (s, 3H); 4.39 (q, 2H); 5.06 (s, 2H); 6.65 (s,
1H); 7.27 (m, 2H); 7.32 (m, 2H); 8.77 (s, 1H).
Figure 3. Affinity and potency of selected new A2B adenosine
antagonists: (A) Competition curves of specific [3H]-MRE 2029F20
binding to human A2B receptors; (B) Inhibition curves of cAMP
accumulation to human A2B receptors representing the capability of
the antagonists to block the stimulatory effect of 100 nM NECA on
cAMP levels. Curves are the mean of three independent experiments;
(C) Comparison between affinity values (Ki) of [3H]-MRE 2029F20
binding to human A2B receptors and potency values (IC50) obtained in
cAMP assays to human A2B receptors.
5.3.5. 1-[(4-Iodo-phenylcarbamoyl)-methyl]-5-methyl-1H-
pyrazole-3-carboxylic acid ethyl ester (9e). Yield: 60%;
mp: 209–210 ꢁC; 1H NMR DMSO-d6 d1.26–1.28 (m,
3H); 2.27 (s, 3H); 4.23 (q, 2H); 5.06 (s, 2H); 6.56 (s,
1H); 7.41 (m, 2H); 7.66 (m, 2H); 10.52 (s, 1H).
5.3.6. 5-Methyl-1-(p-tolylcarbamoyl-methyl)-1H-pyra-
zole-3-carboxylic acid ethyl ester (9f). Yield: 65%; mp:
1
244–246 ꢁC; H NMR DMSO-d6 d 1.24–1.28 (m, 3H);
2.25 (s, 3H); 2.28 (s, 3H); 4.20–4.24 (q, 2H); 5.04 (s,
2H); 6.55 (s, 1H); 7.12 (d, 2H); 7.46 (d, 2H); 10.32
(s, 1H).
bromide in 3 mL of dichloromethane was added
dropwise, followed by 0.019 mol of triethylamine. The
solution turned dark and a precipitate formed, as the
reaction mixture was warmed to ambient temperature
over 3 h. The mixture was concentrated and taken up
in ethyl acetate and extracted with water; the organic
phase was dried over Na2SO4. After filtration and con-
centration, the solid was purified by crystallization
(ethyl acetate), yield: 80–95%. All anilines are commer-
cially available. Analytical data of bromo-phenyl-aceta-
mides are reported in Table 3.
5.3.7. 1-[(4-Methoxy-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9g). Yield: 65%;
mp: 239–241 ꢁC; 1H NMR DMSO-d6 d 1.44–1.47 (t, 3H);
2.47 (s, 3H); 3.69 (s, 3H); 4.45 (q, 2H); 4.91 (s, 2H); 6.49 (s,
1H); 6.77 (m, 2H); 7.52 (m, 2H); 9.83 (s, 1H).
5.3.8. 1-[(4-Dimethylamino-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (9h).
Yield: 55%; mp: 168–171 ꢁC; 1H NMR DMSO-d6
d

M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
Table 3. Analytical data of bromophenylacetamides
2425
Compound
Mp (ꢁC)
1H NMR: CDCl3 (d)
2-Bromo-N-phenyl-acetamide
131–135
137–141
153–155
163–165
183–185
155–159
125–127
212–214
90–93
4.01 (s, 2H); 7.18 (m, 1H); 7.36 (m, 2H); 7.54 (2H); 8.09 (s, 1H)
4.01 (s, 2H); 7.05 (m, 2H); 7.49 (m, 2H); 8.14 (s, 1H)
4.02 (s, 2H); 7.31 (m, 2H); 7.49 (m, 2H); 8.09 (s, 1H)
4.05 (s, 2H); 7.36 (m, 2H); 7.54 (2H); 8.09 (s, 1H)
2-Bromo-N-(4-fluoro-phenyl)-acetamide
2-Bromo-N-(4-chloro-phenyl)-acetamide
2-Bromo-N-(4-bromo-phenyl)-acetamide
2-Bromo-N-(4-iodo-phenyl)-acetamide
2-Bromo-N-p-tolyl-acetamide
4.01 (s, 2H); 7.32 (m, 2H); 7.67 (m, 2H); 8.09 (s, 1H)
2.24 (s, 3H); 4.06 (s, 2H); 7.25 (d, 2H); 7.29 (d, 2H); 8.09 (s, 1H)
3.75 (s, 3H); 4.04 (s, 2H); 6.85 (d, 2H); 7.58 (d, 2H); 8.05 (s, 1H)
2.85 (s, 6H); 4.03 (s, 2H); 6.71 (d, 2H); 7.47 (d, 2H); 8.04 (s, 1H)
0.81 (t, 3H); 1.19 (d, 3H); 1.54 (m, 2H); 2.54 (m, 1H); 4.95 (s, 2H);
7.11 (d, 2H); 7.35 (d, 2H); 8.01 (s, 1H)
2-Bromo-N-(4-methoxy-phenyl)-acetamide
2-Bromo-N-(4-dimethylamino-phenyl)-acetamide
2-Bromo-N-(4-sec-butyl-phenyl)-acetamide
2-Bromo-N-(3-methoxy-phenyl)-acetamide
2-Bromo-N-(3-chloro-phenyl)-acetamide
2-Bromo-N-(3,4-dichloro-phenyl)-acetamide
2-Bromo-N-(3,4-dimethoxy-phenyl)-acetamide
2-Bromo-N-(3,4-dimethyl-phenyl)-acetamide
78–81
66–68
3.69 (s, 3H) 4.02 (s, 2H); 7.14–7.66 (m, 4H); 8.09 (s, 1H)
4.22 (s, 2H); 7.20–7.61 (m, 4H); 8.02 (s, 1H)
4.24 (s, 2H); 7.210 (d, 1H); 7.26 (d, 1H); 7.32 (s, 1H); 8.01 (s, 1H)
98–99
153–155
118–120
3.38 (d, 6H); 4.01 (s, 2H); 6.82 (d, 1H); 7.92 (d, 1H); 7.26 (1H); 8.09 (s, 1H)
2.23 (s, 3H); 2.25 (s, 3H); 4.01 (s, 2H); 7.10 (d, 1H); 7.24 (d, 1H);
7.29 (s, 1H); 8.09 (s, 1H)
2-Bromo-N-naphthalen-1-yl-acetamide
153–154
4.22 (s, 2H); 7.31–8.10 (m, 7H); 8.09 (s, 1H)
1.22–1.24 (t, 3H); 2.80 (s, 6H); 3.29 (s, 3H); 4.19 (q, 2H);
4.96 (s, 2H); 6.51 (s, 1H); 6.65–6.67 (d, 2H); 7.31–7.34
(d, 2H); 10.06 (s, 1H).
1.40–1.42 (t, 3H); 2.19 (s, 6H); 2.37 (s, 3H); 4.40 (q,
2H); 4.94 (s, 2H); 6.66 (s, 1H); 7.04–7.18 (m, 3H); 7.90
(s, 1H).
5.3.9. 1-[(4-sec-Butyl-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9i). Yield: 58%;
mp: 116–118 ꢁC; 1H NMR DMSO-d6 d 0.78 (t, 3H);
1.18 (d, 3H); 1.39–1.42–1.45 (t, 3H); 1.54 (m, 2H); 2.36
(s, 3H); 2.54 (m, 1H); 4.42 (q, 2H); 4.95 (s, 2H); 6.66
(s, 1H); 7.10–7.14 (d, 2H); 7.31–7.35 (d, 2H); 10.55
(s, 1H).
5.3.15. 5-Methyl-1-(naphthalen-1-ylcarbamoylmethyl)-
1H-pyrazole-3-carboxylic acid ethyl ester (9o). Yield:
65%; mp: 170–174 ꢁC; H NMR DMSO-d6 d 1.24–1.31
(t, 3H); 2.32 (s, 3H); 4.20–4.30 (q, 2H); 5.28 (s, 2H);
6.58 (s, 1H); 7.46–8.18 (m, 7H); 10.56 (s, 1H).
1
5.3.16. 1-[(4-Chloro-phenylcarbamoyl)-methyl]-5-ethyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9p). Yield:
1
5.3.10. 1-[(3-Methoxy-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9j). Yield: 65%;
mp: 133–135 ꢁC; 1H NMR DMSO-d6 d 1.26–1.29 (t, 3H);
2.28 (s, 3H); 3.71 (s, 3H); 4.23–4.31 (q, 2H); 5.06
(s, 2H); 6.56 (s, 1H); 6.65 (m, 1H); 7.07–7.30 (m, 3H); 0.43
(s, 1H).
60%; mp: 203–208 ꢁC; H NMR DMSO-d6 d 1.16–1.25
(m, 6H); 2.54–2.58 (q, 2H); 4.20–4.25 (q, 2H); 5.18 (s,
2H); 6.75 (s, 1H); 7.35–7.38 (d, 2H); 7.60 (m, 2H);
10.56 (s, 1H).
5.3.17. 1-[(4-Chloro-phenylcarbamoyl)-methyl]-1H-pyra-
zole-3-carboxylic acid ethyl ester (9q). Yield: 65%; mp:
1
5.3.11. 1-[(3-Chloro-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid ethyl ester (9k). Yield:
209–211 ꢁC; H NMR DMSO-d6 d 1.24–1.31 (t, 3H);
4.24–4.27 (q, 2H); 5.14 (s, 2H); 6.76 (d, 1H); 7.36–7.41
(d, 2H); 7.59–7.63 (d, 2H); 7.89 (d, 1H); 10.56 (s, 1H).
1
60%; mp: 184–186 ꢁC; H NMR DMSO-d6 d 1.24–1.28
(m, 3H); 2.28 (s, 3H); 4.21–4.26 (q, 2H); 5.08 (s, 2H);
6.56 (s, 1H); 7.13–7.16 (m, 1H); 7.34–7.45 (m, 2H);
7.78 (d, 1H), 10.65 (s, 1H).
5.4. General procedure for preparation of 5-(alkyl)-1-
(substituted)phenylcarbamoylmethyl-1H-pyrazole-3-
carboxylic acids 10a–q
5.3.12.
1-[(3,4-Dichloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (9l).
To a mixture of pyrazole esters 9a–q (3.0 mmol) in diox-
Yield: 58%; mp: 207–209 ꢁC; 1H NMR DMSO-d6
1.24–1.28 (m, 3H); 2.27 (s, 3H); 4.20–4.26 (q, 2H);
5.09 (s, 2H); 6.56 (s, 1H); 7.46 (d, 1H); 7.58 (d, 1H);
7.96 (d, 1H); 10.7 5 (s, 1H).
d
ane (50 mL) was added a 2 N KOH solution (5 mL) and
the resulting mixture was stirred at room temperature
for 3 h. After concentration in vacuo, the residue was
cooled and acidified with a 10% HCl and the resulting pre-
cipitate was collected by filtration (quantitative yield).
5.3.13. 1-[(3,4-Dimethoxy-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (9m).
5.4.1. 5-Methyl-1-phenylcarbamoylmethyl-1H-pyrazole-
3-carboxylic acid (10a). Mp: 273–275 ꢁC; 1H NMR
DMSO-d6 d 2.28 (s, 3H); 5.06 (s, 2H); 6.56 (s, 1H);
7.07 (m, 1H); 7.32 (m, 2H); 7.57 (m, 2H); 10.41 (s,
1H); 12.50 (s,1H).
Yield: 65%; mp: 133–135 ꢁC; 1H NMR DMSO-d6
d
1.39–1.42 (t, 3H); 2.38 (s, 3H); 3.83 (s, 6H), 4.41 (q,
2H); 4.96 (s, 2H); 6.67 (s, 1H); 6.78 (m, 2H); 7.14 (s,
1H); 7.98 (s, 1H).
5.3.14.
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (9n).
Yield: 65%; mp: 104–109 ꢁC; 1H NMR DMSO-d6
1-[(3,4-Dimethyl-phenylcarbamoyl)-methyl]-5-
5.4.2. 1-[(4-Fluoro-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid (10b). Mp: 236–241 ꢁC;
1H NMR DMSO-d6 d 2.58 (s, 3H); 4.90 (s, 2H); 6.63
d

2426
M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
(s, 1H); 6.95 (m, 2H); 7.43 (m, 2H); 8.11 (s, 1H); 12.04 (s,
1H).
5.4.13. 1-[(3,4-Dimethoxy-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid (10m). Mp: 204–
207 ꢁC; 1H NMR DMSO-d6 d 2.39 (s, 3H); 3.85 (s,
6H), 4.99 (s, 2H); 6.76 (s, 1H); 6.88 (m, 2H); 7.13 (s,
1H); 7.99 (s, 1H); 12.02 (s, 1H).
5.4.3. 1-[(4-Chloro-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid (10c). Mp: 254–257 ꢁC;
1H NMR DMSO-d6 d 2.40 (s, 3H); 5.12 (s, 2H); 6.71
(s, 1H); 7.38 (d, 2H); 7.63 (d, 2H); 10.61 (1H); 12.98
(s, 1H).
5.4.14. 1-[(3,4-Dimethyl-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3 carboxylic acid ethyl ester (10n).
1
Mp: 232–236 ꢁC; H NMR DMSO-d6 d 2.21 (s, 6H);
5.4.4. 1-[(4-Bromo-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid (10d). Mp: 288–291 ꢁC;
1H NMR DMSO-d6 d 2.36 (s, 3H); 5.03 (s, 2H); 6.50
(s, 1H); 7.41 (d, 2H); 7.66 (d, 2H); 10.51 (1H); 12.55
(s, 1H).
2.40 (s, 3H); 4.99 (s, 2H); 6.68 (s, 1H); 7.05–7.25 (m,
3H); 7.94 (s, 1H); 12.20 (s, 1H).
5.4.15. 5-Methyl-1-(naphthalen-1-ylcarbamoylmethyl)-
1H-pyrazole-3-carboxylic acid (10o). Mp: 227–229 ꢁC;
1H NMR DMSO-d6 d 2.32 (s, 3H); 5.28 (s, 2H); 6.58
(s, 1H); 7.46–8.18 (m, 7H); 10.56 (s, 1H); 12.09 (s, 1H).
5.4.5. 1-[(4-Iodo-phenylcarbamoyl)-methyl]-5-methyl-1H-
pyrazole-3-carboxylic acid (10e). Mp: 238–244 ꢁC; 1H
NMR DMSO-d6 d 2.25 (s, 3H); 5.04 (s, 2H); 6.58 (s,
1H); 7.45 (d, 2H); 7.69 (d, 2H); 10.50 (s, 1H); 12.35 (s,
1H).
5.4.16. 1-[(4-Chloro-phenylcarbamoyl)-methyl]-5-ethyl-
1H-pyrazole-3-carboxylic acid (10p). Mp: 189–195 ꢁC;
1H NMR DMSO-d6 d 1.22 (t, 3H); 2.60 (q, 2H); 4.98
(s, 2H); 6.46 (s, 1H), 7.40 (d, 2H); 7.56 (d, 2H); 10.43
(s, 1H); 12.45 (1H).
5.4.6. 5-Methyl-1-(p-tolylcarbamoyl-methyl)-1H-pyrazole-
3-carboxylic acid (10f). Mp: 253–255 ꢁC; 1H NMR
DMSO-d6 d 2.24 (s, 3H); 2.27 (s, 3H); 5.021 (s, 2H);
6.49 (s, 1H); 7.12 (d, 2H); 7.16 (d, 2H); 10.32 (s, 1H);
12.54 (s, 1H).
5.4.17. 1-[(4-Chloro-phenylcarbamoyl)-methyl]-1H-pyra-
1
zole-3-carboxylic acid (10q). Mp: 239–242 ꢁC; H NMR
DMSO-d6 d 5.12 (s, 2H); 6.79 (s, 1H); 7.40 (d, 2H); 7.63
(d, 2H); 7.88 (m, 1H); 10.54 (s, 1H); 12.31 (s, 1H).
5.4.7. 1-[(4-Methoxy-phenylcarbamoyl)-methyl]-5-methyl-
1H-pyrazole-3-carboxylic acid (10g). Mp: 230–234 ꢁC;
1H NMR DMSO-d6 d 2.49 (s, 3H); 3.68 (s, 3H); 4.89
(s, 2H); 6.46 (s, 1H); 6.75 (d, 2H); 7.49 (d, 2H); 9.80
(s, 1H); 12.03 (s, 1H).
5.5. General procedure for preparation of 2-[3-(2,6-dioxo-
1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-5-(substi-
tuted)-pyrazol-1-yl]-N-(substituted)phenyl-acetamides
(11a–q)
5.4.8. 1-[(4-Dimethylamino-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid (10h). Mp: 189–
201 ꢁC; 1H NMR DMSO-d6 d 2.82 (s, 6H);3.30 (s,
3H); 4.98 (s, 2H); 6.51 (s, 1H); 6.67 (d, 2H); 7.36 (d,
2H); 10.10 (s, 1H); 12.01 (s, 1H).
To
a
solution of 1,3-dipropyl-5,6-diaminouracil
(2.20 mmol), in methanol (10 mL), was added an equi-
molar amount of the appropriate carboxylic acid (10a–
q) and EDAC (2.21 mmol). The reaction mixture was
stirred at room temperature for 4–5 h with being moni-
tored by TLC. Methanol was concentrated and the
amide intermediate derivative was precipitated by the
addition of water. After filtration, the solid was dis-
solved in methanol (10 mL) and NaOH (2.5 N, 15 mL)
and warmed at 50–60 ꢁC for 1 h. The methanol was dis-
tilled off, and the residue was taken up in H2O and acid-
ified with HCl to pH 4–5. The precipitate was filtered
off, washed with water, and purified by flash chromatog-
raphy by elution with different mixtures of ethyl acetate-
petroleum ether. Compounds 11a–q were also purified
by crystallization from methanol or dioxane.
5.4.9. 1-[(4-sec-Butyl-phenylcarbamoyl)-methyl]-5-methyl-
1
1H-pyrazole-3-carboxylic acid (10i). Mp: 160–162 ꢁC; H
NMR DMSO-d6 d 0.76 (t, 3H); 1.16 (d, 3H); 1.51
(m, 2H); 2.52 (m, 4H); 5.09 (s, 2H); 6.76 (s, 1H);
7.09 (d, 2H); 7.38 (d, 2H); 10.09 (s, 1H); 12.06 (s,
1H).
5.4.10. 1-[(3-Methoxy-phenylcarbamoyl)-methyl]-5-methyl-
1
1H-pyrazole-3-carboxylic acid (10j). Mp: 226–230 ꢁC; H
NMR DMSO-d6 d 2.29 (s, 3H); 3.74 (s, 3H); 5.08 (s, 2H);
6.53 (s, 1H); 6.66 (m, 1H); 7.06–7.32 (m, 3H); 10.46 (s,
1H); 12.04 (s, 1H).
5.5.1.
2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-phenyl-acetam-
ide (11a). Yield: 45%; mp: 255–257 ꢁC; 1H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.70 (q, 2H);
2.32 (s, 3H); 3.84 (m, 2H); 3.98 (m, 2H); 5.08 (s, 2H);
6.74 (s, 1H); 7.08 (m, 1H); 7.33 (m, 2H); 7.59 (m, 2H);
10.41 (s, 1H); 13.85 (s, 1H).
5.4.11. 1-[(3-Chloro-phenylcarbamoyl)-methyl]-5-methyl-
1
1H-pyrazole-3-carboxylic acid (10k). Mp: >300 ꢁC; H
NMR DMSO-d6 d 2.37 (s, 3H); 5.07 (s, 2H); 6.51 (s,
1H); 7.12–7.48 (m, 3H); 7.79 (s, 1H); 10.76 (s, 1H);
12.06 (s, 1H).
5.4.12.
1-[(3,4-Dichloro-phenylcarbamoyl)-methyl]-5-
5.5.2. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-fluoro-phenyl)-
acetamide (11b). Yield: 38%; mp: 205–207 ꢁC; H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.70 (q, 2H);
2.31 (s, 3H); 3.84 (t, 2H); 3.97 (t, 2H); 5.07 (s, 2H);
methyl-1H-pyrazole-3-carboxylic acid (10l). Mp: 219–
220 ꢁC; 1H NMR DMSO-d6 d 2.27 (s, 3H); 5.07 (s,
2H); 6.51 (s, 1H); 7.50–7.97 (m, 3H); 10.77 (s, 1H);
12.04 (s, 1H).
1

M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
2427
6.74 (s, 1H); 7.17 (d, 2H); 7.61 (d, 2H); 10.48 (s, 1H);
13.80 (s, 1H).
(q, 2H); 2.31 (s, 3H); 3.71 (s, 3H); 3.84 (t, 3H); 3.97 (t,
2H); 5.06 (s, 2H); 6.63–6.68 (m, 1H); 6.72 (s, 1H);
7.08–7.31 (m, 3H); 10.48 (s, 1H); 13.80 (s, 1H).
5.5.3. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-chloro-phenyl)-
acetamide (11c). Yield: 40%; mp: 261–262 ꢁC; H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H ); 1.70 (q, 2H);
2.31 (s, 3H); 3.82 (m, 2H); 3.95 (m, 2H); 5.07 (s, 2H);
6.73 (s, 1H); 7.37 (d, 2H); 7.60 (d, 2H); 10.55 (s, 1H);
13.68 (s, 1H).
5.5.11. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(3-chloro-phenyl)-
acetamide (11k). Yield: 28%; mp: 304–305 ꢁC; 1H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.70 (q, 2H);
2.31 (s, 3H); 3.84 (t, 2H); 3.95 (t, 2H); 5.09 (s, 2H);
6.73(s, 1H);7.15–7.80(m, 4H);10.48(s, 1H);13.80(s, 1H).
1
5.5.4. 2-[3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-
purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-bromo-phenyl)-
acetamide (11d). Yield: 42%; mp: 270–271 ꢁC; H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.70 (q, 2H);
2.31 (s, 3H); 3.85 (m, 2H); 3.98 (m, 2H); 5.08 (s, 2H);
6.75 (s, 1H); 7.51 (d, 2H); 7.57 (d, 2H); 10.56 (s, 1H);
13.69 (s, 1H).
5.5.12. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(3,4-dichloro-
phenyl)-acetamide (11l). Yield: 30%; mp: 298 ꢁC; 1H
NMR DMSO-d6 d 0.87 (m, 6H); 1.57 (q, 2H); 1.73 (q,
2H); 2.31 (s, 3H); 3.86 (t, 2H); 3.95 (t, 2H); 5.01 (s,
2H); 6.75 (s, 1H); 7.47–7.59 (m, 2H); 7.97 (d, 1H);
10.73 (s, 1H); 13.75 (s, 1H).
1
5.5.5.
2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
5.5.13. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(3,4-dimethoxy-
phenyl)-acetamide (11m). Yield: 35%; mp: 289–290 ꢁC;
1H NMR DMSO-d6 d 0.88 (m, 6H); 1.57 (q, 2H); 1.70
(q, 2H); 2.32 (s, 3H); 3.71 (s, 6H); 3.84 (t, 2H); 3.88 (t,
2H); 5.03 (s, 2H); 6.73 (s, 1H); 6.90 (d, 1H); 7.05 (d,
1H); 7.32 (s, 1H); 10.29 (s, 1H); 13.88 (s, 1H).
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-iodo-phenyl)-
acetamide (11e). Yield: 42%; mp: 287 ꢁC; 1H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.71 (q, 2H);
2.30 (s, 3H); 3.84 (t, 2H); 3.97 (t, 2H); 5.07 (s, 2H);
6.74 (s, 1H); 7.42 (d, 2H); 7.66 (d, 2H); 10.52 (s, 1H);
13.69 (s, 1H).
5.5.6. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-
purin-8-yl)-5-methyl-pyrazol-1-yl]-N-p-tolyl-acetamide (11f).
Yield: 32%; mp: >300 ꢁC; H NMR DMSO-d6 d 0.88
(m, 6H); 1.57 (q, 2H); 1.72 (q, 2H); 2.25 (s, 3H); 2.31
(s, 3H); 3.84 (t, 2H); 3.99 (t, 2H); 5.05 (s, 2H); 6.73
(s, 1H); 7.20 (d, 2H); 7.46 (d, 2H); 10.32 (s, 1H); 13.85
(br s, 1H).
5.5.14. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(3,4-dimethyl-
phenyl)-acetamide (11n). Yield: 43%; mp: 260–263 ꢁC;
1H NMR DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.70
(q, 2H); 2.17 (m, 6H); 2.31 (s, 3H); 3.84 (t, 2H); 3.97
(t, 2H); 5.03 (s, 2H); 6.70 (s, 1H); 7.06 (d, 1H); 7.29
(d, 1H); 7.37 (s, 1H) 10.24 (s, 1H); 13.80 (s, 1H).
1
5.5.7. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-
purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-methoxy-phenyl)-
acetamide (11g). Yield: 39%; mp: 295–296 ꢁC; H NMR
DMSO-d6 d 0.87 (m, 6H); 1.56 (q, 2H); 1.70 (q, 2H);
2.31 (s, 3H); 3.71 (s, 3H); 3.84 (t, 2H); 3.97 (t, 3H)
5.03 (s, 2H); 6.72 (s, 1H); 7.90 (d, 2H); 7.49 (d, 2H);
10.28 (s, 1H); 13.80 (s, 1H).
5.5.15. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-naphthalen-1-yl-
acetamide (11o). Yield: 35%; mp >300 ꢁC; 1H NMR
DMSO-d6 d 0.89 (m, 6H); 1.59 (q, 2H); 1.72 (q, 2H);
2.36 (s, 3H); 3.85 (t, 2H); 3.97 (t, 2H); 5.30 (s, 2H);
6.76 (s, 1H); 7.46–7.81 (m, 5H); 7.93–78.19 (m, 2H);
10.36 (s, 1H); 13.72 (s, 1H).
1
5.5.8.
2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
5.5.16. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-ethyl-pyrazol-1-yl]-N-(4-chlorophenyl)
acetamide (11p). Yield: 37%; mp: 256–257 ꢁC; H NMR
DMSO-d6 d 0.88 (m, 6H); 1.25 (m, 3H); 1.55 (q, 2H);
1.70 (q, 2H); 2.66 (q, 2H); 3.84 (t, 2H); 3.99 (t, 2H);
5.08 (s, 2H); 6.77 (s, 1H); 7.40 (d, 2H); 7.63 (d, 2H);
10.56 (s, 1H); 13.67 (s, 1H).
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-dimethyl-
amino-phenyl)-acetamide (11h). Yield: 28%; mp >
300 ꢁC; H NMR DMSO-d6 d 0.88 (m, 6H); 1.56 (q,
2H); 1.70 (q, 2H); 2.31 (s, 3H); 2.84 (s, 6H); 3.85 (t,
2H); 3.94 (t, 2H); 5.01 (s, 2H); 6.67 (s, 1H); 6.72 (d,
2H); 7.42 (d, 2H); 10.11 (s, 1H); 13.70 (s, 1H).
1
1
5.5.9. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-
purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-sec-butyl-phenyl)-
acetamide (11i). Yield: 25%; mp: 269 ꢁC; 1H NMR
DMSO-d6 d 0.74 (t, 3H);0.88 (m, 6H); 1.15 (q, 3H);
1.48–1.55 (m, 7H); 2.31 (s, 3H); 3.85 (t, 2H); 3.97 (t,
2H); 5.06 (s, 2H); 6.74 (s, 1H); 7.14 (d, 2H); 7.49 (d,
2H);10.33 (s, 1H); 13.75 (s, 1H).
5.5.17. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-pyrazol-1-yl]-N-(4-chlorophenyl)acetamide
(11q). Yield: 33%; mp: 283–285 ꢁC; 1H NMR DMSO-d6 d
0.87 (m, 6H); 1.52 (q, 2H); 1.66 (q, 2H); 3.85 (t, 2H); 3.98
(t, 2H); 5.14 (s, 2H); 6.94 (d, 1H); 7.37 (d, 2H); 7.60 (d,
2H); 7.89 (d, 1H); 10.57 (s, 1H); 13.69 (s, 1H).
5.6. Synthesis of 4-halogeno-5-methyl-1H-pyrazole-3-
carboxylic acid ethyl esters 12a–c
5.5.10. 2-[3-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-
1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(3-methoxyl-
phenyl)-acetamide (11j). Yield: 29%; mp: 276–277 ꢁC; 1H
NMR DMSO-d6 d 0.87 (m, 6H); 1.58 (q, 2H); 1.68
To a magnetically stirred solution of pyrazole ester 5
(3 mmol) in 10 mL anhydrous DMF at 0 ꢁC was added

2428
M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
N-halogensuccinimide (4 mmol). The solution was stir-
red at room temperature for 5 h. The solvent was evap-
orated to a half of the original volume, water was added
and the reaction mixture was allowed to cool over ice.
The precipitate was collected by filtration and purified
by crystallization to afford the desired compounds
12a–c as a white solid.
2H); 7.41 (d, 2H); 7.61 (d, 2H); 10.60 (s, 1H); 12.96 (s,
1H).
5.8.3. 4-Iodo-1-[(4-chloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid (15c). Mp:
248 ꢁC; 1H NMR DMSO-d6 d 2.30 (s, 3H); 5.17 (s,
2H); 7.37 (d, 2H); 7.61 (d, 2H ); 10.58 (s, 1H); 12.96
(s, 1H).
5.6.1. 4-Chloro-5-methyl-1H-pyrazole-3-carboxylic acid
ethyl ester (12a). Yield: 85%, mp: 106–107 ꢁC; 1H NMR
DMSO-d6 d 1.25–1.32 (t, 3H); 2.21 (s, 3H); 4.22–4.32 (q,
2H); 13.59 (s, 1H).
5.9. General procedure for preparation of 2-[4-halo-3-
(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)- 5-methyl-pyrazol-1-yl]-N-(4-chlorophenyl)-aceta-
mides 16a–c
5.6.2. 4-Bromo-5-methyl-1H-pyrazole-3-carboxylic acid
ethyl ester (12b). Yield: 88%, mp: 150–152 ꢁC; 1H NMR
DMSO-d6 d 1.25–1.33 (t, 3H); 2.24 (s, 3H); 4.22–4.31 (q,
2H); 13.61 (s, 1H).
Same procedure as 11a–q, except used 15a–c.
5.9.1. 2-[4-Chloro-3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra-
hydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-chloro-
phenyl)-acetamide (16a). Yield: 58%; mp: 297–298 ꢁC;
1H NMR DMSO-d6 d 0.88 (m, 6H); 1.62 (q, 2H); 1.70
(q, 2H); 2.31 (s, 3H); 3.85 (t, 2H); 4.00 (t, 2H); 5.15 (s,
2H); 7.39 (d, 2H), 7.61 (d, 2H); 10.59 (s, 1H); 13.89 (s,
1H).
5.6.3. 4-Iodo-5-methyl-1H-pyrazole-3-carboxylic acid
ethyl ester (12c). Yield: 80%, mp: 113–114 ꢁC; 1H
NMR DMSO-d6 d 1.25–1.35 (t, 3H); 2.24 (s, 3H);
4.20–4.32 (q, 2H); 13.62 (s, 1H).
5.7. General procedure for preparation of 4-halogeno-1-
[(4-chloro-phenylcarbamoyl)-methyl]-5-methyl-1H-pyra-
zole-3-carboxylic acid ethyl esters 14a–c
5.9.2. 2-[4-Bromo-3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra-
hydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-chloro-
phenyl)-acetamide (16b). Yield: 58%; mp: 298–299 ꢁC;
1H NMR DMSO-d6 d 0.88 (m, 6H); 1.57 (q, 2H); 1.72
(q, 2H); 2.321 (s, 3H); 3.91 (t, 2H); 4.02 (t, 2H); 5.19
(s, 2H); 7.39 (d, 2H), 7.61 (d, 2H); 10.68 (s, 1H); 13.79
(s, 1H).
Same procedure as 9a–q, except used 12a–c.
5.7.1. 4-Chloro-1-[(4-chloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (14a).
1
Mp: 208–210 ꢁC; H NMR DMSO-d6 d 1.26–1.29 (t,
3H); 2.25 (s, 3H); 4.26–4.28 (q, 2H); 5.14 (s, 2H); 7.39
(d, 2H); 7.60 (d, 2H); 10.60 (s, 1H).
5.9.3. 2-[4-Iodo-3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra-
hydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl]-N-(4-chloro-
phenyl)-acetamide (16c). Yield: 55%; mp: 296–297 ꢁC;
1H NMR DMSO-d6 d 0.90 (m, 6H); 1.59 (q, 2H); 1.74
(q, 2H); 2.35 (s, 3H); 3.87 (t, 2H); 4.03 (t, 2H); 5.19 (s,
2H); 7.40 (d, 2H), 7.59 (d, 2H); 10.58 (s, 1H); 13.87 (s,
1H).
5.7.2. 4-Bromo-1-[(4-chloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (14b).
1
Mp: 221–223 ꢁC; H NMR DMSO-d6 d 1.25–1.28 (t,
3H); 2.27 (s, 3H); 4.26–4.28 (q, 2H); 5.16 (s, 2H); 7.39
(d, 2H); 7.60 (d, 2H); 10.60 (s, 1H).
5.10. Biology experiments
5.7.3. 4-Iodo-1-[(4-chloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid ethyl ester (14c).
Mp: 234–235 ꢁC; H NMR DMSO-d6 d 1.26–1.30 (t,
3H); 2.30 (s, 3H); 4.26–4.28 (q, 2H); 5.19 (s, 2H); 7.39
(d, 2H); 7.60 (d, 2H); 10.59 (s, 1H).
5.10.1. Membrane preparation. The human A1, A2A,
1
A2B, and A3 receptors were transfected in CHO or
HEK cells, were grown adherently and maintained in
Dulbecco’s modified Eagle’s medium with nutrient mix-
ture F12 (DMEM/F12) without nucleosides, containing
10% fetal calf serum, penicillin (100 U/ml), streptomycin
(100 lg/ml), L-glutamine (2 mM), and Geneticin (G418,
0.2 mg/ml) at 37 ꢁC in 5% CO2/95% air.9,22 For mem-
brane 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 cen-
trifuged for 30 min at 100,000g. The membrane pellet
was suspended in 50 mM Tris–HCl buffer, pH 7.4, for
A1 adenosine receptors; in 50 mM Tris–HCl, 10 mM
MgCl2, pH 7.4, for A2A adenosine receptors; in
50 mM Tris–HCl, 10 mM MgCl2, 1 mM EDTA, pH
7.4, for A2B and A3 adenosine receptors.
5.8. General procedure for preparation of 4-halo-1-[(4-
chloro-phenylcarbamoyl)-methyl]-5-methyl-1H-pyrazole-
3-carboxylic acids 15a–c
Same procedure as 10a–q, except used 14a–c.
5.8.1. 4-Chloro-1-[(4-chloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid (15a). Mp: 244–
245 ꢁC; 1H NMR DMSO-d6 d 2.24 (s, 3H); 5.11 (s,
2H); 7.39 (d, 2H); 7.60 (d, 2H); 10.60 (s, 1H); 12.98 (s,
1H).
5.8.2. 4-Bromo-1-[(4-chloro-phenylcarbamoyl)-methyl]-5-
methyl-1H-pyrazole-3-carboxylic acid (15b). Mp: 247–
248 ꢁC; 1H NMR DMSO-d6 d 2.26 (s, 3H); 5.14 (s,

M. A. Tabrizi et al. / Bioorg. Med. Chem. 16 (2008) 2419–2430
2429
5.10.2. Human cloned A1, A2A, A2B, and A3 adenosine
receptor binding assay. The synthesized compounds have
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 incu-
bated at 4 ꢁC for 150 min, and after the addition of char-
coal centrifuged at 2000g for 10 min. The clear
supernatant was counted in a Packard scintillation
counter TR2500 with an efficiency of 57%.
been tested to evaluate their affinity to human A1, A2A
,
A2B, and A3 adenosine receptors. Displacement experi-
ments of [3H]-DPCPX to CHO cells transfected with
the human recombinant A1 adenosine receptors were
performed for 120 min at 25 ꢁC incubating diluted mem-
branes (50 lg of protein/assay) and at least 6–8 different
concentrations of examined antagonists.23 Non specific
binding was determined in the presence of 10 lM of
DPCPX and this was always 6 10% of the total binding.
Binding of [3H]-ZM 241385 to CHO cells transfected
with the human recombinant A2A adenosine receptors
was performed using a suspension of membranes
(50 lg of protein/assay) and at least 6–8 different con-
centrations of studied antagonists for an incubation
time of 60 min at 4 ꢁC. Non specific binding was deter-
mined in the presence of 1 lM ZM 241385 and was
about 20% of total binding.24 Competition binding
experiments of [3H]-MRE 2029F20 to CHO cells trans-
fected with the human recombinant A2B adenosine
receptors were carried out incubating for 120 min at
4 ꢁC diluted membranes (50 lg of protein/assay) and
at least 6–8 different concentrations of examined com-
pounds.9 Non-specific binding was defined as binding
in the presence of 1 lM MRE 2029F20 and was about
25% of total binding. Competition binding experiments
of [3H]-MRE 3008F20 to CHO cells transfected with the
human recombinant A3 adenosine receptors were car-
ried out incubating for 120 min at 4 ꢁC diluted mem-
branes (50 lg of protein/assay) and at least 6–8
different concentrations of examined ligands.22 Non-spe-
cific binding was defined as binding in the presence of
1 lM MRE 3008F20 and was about 25% of total bind-
ing. Bound and free radioactivity were separated by fil-
tering the assay mixture through Whatman GF/B glass
fiber filters using a Micro-Mate 196 cell harvester (Pack-
ard Instrument Co). The filter bound radioactivity was
counted on a Top Count (efficiency 58%) with Micro-
Scint 20.
5.10.4. Data analysis. The protein concentration was
determined according to a Bio-Rad method with
bovine albumin as a standard reference.25 Inhibitory
binding constant, Ki, values were calculated from
those of IC50 accꢀording to Cheng & Prusoff equation
Ki ¼ IC50=ð1 þ ½C ꢁ=KꢀDÞ, where [C*] is the concentra-
tion of the radioligand and KꢀD its dissociation
constant.26 A weighted non linear least-squares curve
fitting program LIGAND was also used for computer
analysis of inhibition experiments.27 IC50 values ob-
tained in cyclic AMP assay were calculated by non
linear regression analysis using the equation for a
sigmoid concentration-response curve (GraphPad
Prism, San Diego, CA, USA). Data are expressed as
geometric means with 95% confidence limits in
parentheses.
Acknowledgments
We thank King Pharmaceuticals, Inc., Research and
Development, 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.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.11.058.
5.10.3. Measurement of cyclic AMP levels in CHO cells
transfected with human A2B adenosine receptors. CHO
cells transfected with human A2B adenosine receptors
were washed with phosphate-buffered saline and centri-
fuged for 10 min at 200g. The pellet containing the CHO
cells (1 · 106 cells/assay) was suspended in 0.5 ml of
incubation mixture (mM): NaCl 15, KCl 0.27, NaH2-
PO40.037, 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-methoxybenzyl)-2-imi-
dazolidinone (Ro 20-1724) as phosphodiesterase inhibi-
tor, and preincubated for 10 min in a shaking bath at
37 ꢁC. The potency of antagonists studied was deter-
mined by antagonism of NECA (100 nM)-induced stim-
ulation of cyclic AMP levels. 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 ex-
tracted 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 (0–10 pmole) were added to
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