Does the combination of optimal substitutions at the C2-, N 5- and N8-positions of the pyrazolo-triazolo-pyrimidine scaffold guarantee selective modulation of the human A3 adenosine receptors?

Article abstract of DOI:10.1016/j.bmc.2011.08.026

In an attempt to study the optimal combination of a phenyl ring at the C²-position and different substituents at the N⁵- and N⁸-positions towards the selective modulation of human A₃ adenosine receptors (hA₃AR), we synthesized a new series of 2-para-(un)substituted-phenyl-pyrazolo-triazolo-pyrimidines bearing either a methyl or phenylethyl at N⁸ and chains of variable length at N ⁵. Through biological evaluation, it was found that the majority of the compounds had good affinities towards the hA₃AR in the low nanomolar range. Compound 16 possessed the best hA₃AR affinity and selectivity profile (K_ihA₃ = 1.33 nM; hA ₁/hA₃ = 4880; hA_2A/hA₃ = 1100) in the present series of 2-(substituted)phenyl-pyrazolo-triazolo-pyrimidine derivatives. In addition to pharmacological characterization, a molecular modeling investigation on these compounds further elucidated the effect of different substituents at the pyrazolo-triazolo-pyrimidine scaffold on affinity and selectivity to hA₃AR.

Full text of DOI:10.1016/j.bmc.2011.08.026

Bioorganic & Medicinal Chemistry 19 (2011) 6120–6134
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Does the combination of optimal substitutions at the C2-, N5- and N8-positions
of the pyrazolo-triazolo-pyrimidine scaffold guarantee selective modulation
of the human A3 adenosine receptors?
Siew Lee Cheong a, , Anton V. Dolzhenko a, , Silvia Paoletta b, Evelyn Pei Rong Lee a, Sonja Kachler c,
Stephanie Federico d, Karl-Norbert Klotz c, Anna V. Dolzhenko a, Giampiero Spalluto d,
,
⇑
a,
Stefano Moro b, , Giorgia Pastorin
⇑
⇑
a Department of Pharmacy, National University of Singapore, 3 Science Drive 2, Block S15, #05-PI-03, 117543 Singapore, Singapore
b Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche, Università di Padova, via Marzolo 5, I-35131 Padova, Italy
c Institut für Pharmakologie, Universität of Würzburg, Versbacher Straße 9, D-97078 Würzburg, Germany
d Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
In an attempt to study the optimal combination of a phenyl ring at the C2-position and different substit-
uents at the N5- and N8-positions towards the selective modulation of human A3 adenosine receptors
(hA3AR), we synthesized a new series of 2-para-(un)substituted-phenyl-pyrazolo-triazolo-pyrimidines
bearing either a methyl or phenylethyl at N8 and chains of variable length at N5. Through biological
evaluation, it was found that the majority of the compounds had good affinities towards the hA3AR in
the low nanomolar range. Compound 16 possessed the best hA3AR affinity and selectivity profile
(KihA3 = 1.33 nM; hA1/hA3 = 4880; hA2A/hA3 = 1100) in the present series of 2-(substituted)phenyl-
pyrazolo-triazolo-pyrimidine derivatives. In addition to pharmacological characterization, a molecular
modeling investigation on these compounds further elucidated the effect of different substituents at
the pyrazolo-triazolo-pyrimidine scaffold on affinity and selectivity to hA3AR.
Article history:
Received 12 June 2011
Revised 10 August 2011
Accepted 11 August 2011
Available online 23 August 2011
Keywords:
2-Phenyl-pyrazolo-triazolo-pyrimidines
Human A3 adenosine receptor antagonists
Structure–affinity relationship
Affinity
Ó 2011 Elsevier Ltd. All rights reserved.
Selectivity
1. Introduction
senger, adenylyl cyclase, with a subsequent decrease in cyclic
adenosine monophosphate (cAMP) levels; other signaling trans-
Among the four human adenosine receptor subtypes (A1, A2A
,
duction pathways are also reported for this receptor subtype.2
Many studies have been performed to identify its pharmacological
roles in our body, showing that the modulation of hA3AR is associ-
ated with a number of pathophysiological conditions. Explicitly,
the inhibition of the hA3AR is implicated in the treatment of sev-
eral diseases, such as glaucoma, asthma and cancer.3,4 In light of
the biological importance of such receptor, diverse classes of poly-
heterocyclic scaffolds have been designed and discovered as
potential hA3AR antagonists to-date.5,6
Among these derivatives, the pyrazolo-triazolo-pyrimidines
(PTPs) have been identified as highly potent and moderately selec-
tive hA3AR antagonists.7–9 From our previous studies, we showed
that, among the N5-unsubstituted PTPs, the presence of a small
group at the N8-position was crucial for selective binding to
hA3AR.8 Concurrently, the introduction of an amide group, particu-
larly an arylcarbamoyl chain at the N5-position further improved
hA3AR affinity.10 In a recent study, we also reported that the new
series of PTPs, bearing a phenyl group in place of the C2-furyl
ring, not only conferred a good pharmacological profile, but also
A
2B, A3), human A3 adenosine receptor (hA3AR) is the least charac-
terized.1 Biochemically, it is coupled to Gi-protein in the cellular
membrane, which leads to inhibition of activity of a second mes-
Abbreviations: APCI, atmospheric pressure chemical ionization; ATP, adenosine
triphosphate; cAMP, cyclic adenosine monophosphate; CHO cells, Chinese hamster
ovary cells; DIPEA, diisopropylethylamine; EL, extracellular loop; EGTA, ethylene
glycol tetraacetic acid; Gi, inhibitory G protein; hA1/A2A/A2B/A3AR, human A1/A2A
/
A
2B/A3 adenosine receptor; [3H]-CCPA, [3H]-2-chloro-6-cyclopentyl adenosine;
[3H]-NECA, [3 H]-N-ethylcarboxamido adenosine; HPLC, high performance liquid
chromatography; MOE, molecular operating environment software; PTP, pyrazolo-
triazolo-pyrimidine; rmsd, root mean square deviation; R-PIA, R-N6-phenylisopro-
pyladenosine; SAR, structure–affinity relationship; TM, transmembrane; TLC,
thin-layer chromatography.
⇑
Corresponding authors. Tel.: +39 040 558 3726; fax: +39 040 52572 (G.S.); tel.:
+39 049 8275704; fax: +39 049 8275366 (S.M.); tel.: +65 6516 1876; fax: +65 6779
1554 (G.P.).
E-mail addresses: spalluto@univ.trieste.it (G. Spalluto), stefano.moro@unipd.it
(S. Moro), phapg@nus.edu.sg (G. Pastorin).
These authors contributed equally to the manuscript.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.026

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6121
X
X = H, F, OCH3
O
N
N
H
N
HN
5
N
R1
2
N
N
7
N
II
8
H3C
R1 = Cl, Br, CF3, Ph
R
N
N
N
N
R1
N
O
O
N
I
R2
R =
OR
HN
C
HN
H2
R
5
N
N
N
N
R1
2
N
7
N
8
III
R1 = H, F, Cl, Br
Chart 1. Rational design of new 2-(substituted)phenyl PTPs.
overcame the metabolic transformation of the furan ring into toxic
intermediates11 (I in Chart 1 and compounds 1–3, 6, 7, 20–25 and
28 in Table 1).
of 4-cyano-1-methyl-pyrazol-3-ylamine 38 via a more practical
and chromatographic-free process was preferred. The condensa-
tion reaction between hydrazine and benzaldehyde gave the inter-
mediate benzenyl hydrazone 35 with the benzenyl group serving
as a protecting group. Subsequent reaction with ethoxymethylene
malononitrile, selective methylation with methyl iodide and
deprotection with ring closure provided only 4-cyano-1-methyl-
pyrazol-3-ylamine 38 in good yield.
Imidate 39 was prepared from 4-cyano-1-methyl-pyrazolo-3-
ylamine 38 and triethyl orthoformate using a known procedure.12
A novel method that has been recently reported by our group14
was employed for the synthesis of 40, in which imidate 39 was re-
acted with hydrazine in ethanol to isolate pure 40. In the previous
study, intermediates 41 and 42 (with R1 = Cl or Br) were prepared
by cyclocondensation of hydrazides with imidate 39.12 This meth-
od required harsh conditions (260 °C) and chromatographic purifi-
cation of the products. The poor solubility of 41–44, particularly
when C2-moiety is a substituted phenyl group, made chromato-
Based on these observations, in this study we thus hypothesized
that the combination of a (substituted)phenyl group at the C2-po-
sition, together with substituents at the N5- and the N8-positions
deemed optimal for interaction with the hA3AR, would effectively
give rise to new potent and selective A3AR antagonists. In order
to test our hypothesis, a new series of 2-(substituted)phenyl-
pyrazolo-triazolo-pyrimidines bearing either a methyl (II) or a
phenylethyl group (III) at the N8-position, in conjunction with
arylcarbamide, phenylacetamide or benzamide chains at the
N5-position (Chart 1) was designed and synthesized in the present
study. This new series of compounds (compounds 4, 5, 8–19, 26, 27
and 29–33) was aimed at further exploring the effect of these sub-
stituents at each position of the PTP tricyclic scaffold towards
hA3AR affinity and selectivity. Moreover, to better clarify the effect
of these substituents on the binding modes of the new compounds
at the hA3AR,
performed.
a
molecular modeling investigation was also
graphic purification a time- and solvent-consuming process.
Hence, we adopted a more practical process14 with mild reaction
conditions, as shown in steps vi and vii in Scheme 1, to obtain rea-
sonable yields and good purity of intermediates 41–44.
2. Chemistry
Subsequent steps were performed using established proce-
dures.9,15,16 Treatment of 41–44 with hydrochloric acid at reflux
temperature induced pyrimidine ring opening to furnish the
1-methyl-4-(3(5)-aryl-1,2,4-triazol-5(3)-yl)-pyrazol-3-ylamines
45–48 in good yield. These derivatives 45–48 were converted
to 2-(substituted)phenyl-8-methyl-pyrazolo[4,3-e][1,2,4]triazol-
o[1,5-c]pyrimidin-5-yl amines 2–5 by cyclocondensation with
an excess of cyanamide in 1-methyl-2-pyrrolidone at 160 °C.12
Synthesis of new N8-methyl derivatives 4, 5 and 8–19 was
performed using a combination of new steps as well as known
procedures (Scheme 1). In order to obtain a methyl group at the
N8-position of the PTP scaffold in the substituted ureas, the prepa-
ration of intermediate 38 is a critical step. Several methods have
been reported thus far, but all with their limitations.12,13 Therefore,
a regioselective synthetic procedure that allowed the preparation

Table 1
Binding affinity (Ki) at hA1AR, hA2AAR, hA2BAR and hA3AR and selectivity against hA1AR and hA2AAR
R
5
N
N
N
N
R1
2
N
7
N
8
R2
Compd
R
R1
R2
Ki, nM (95% CI)
Selectivity
a
b
c
d
hA1
hA2A
hA2B
hA3
hA1/hA3
hA2A/hA3
1e
2e
3e
4
NH2
NH2
NH2
NH2
H
Cl
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
N8–CH3
339 (319–359)
121 (100–147)
2020 (1060–3840)
1500 (1370–1640)
6930 (4340–11,100)
>30,000
8320 (6180–11,200)
>100,000
987 (615–1580)
2360 (2160–2570)
36,000 (33,900–38,300)
>30,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
75.0 (63.1–90.4)
72.4 (71.3–73.6)
38.6 (35.9–41.5)
304 (237–391)
4.52
67.1
74.9
>328.9
>680
19,600
70,700
3250
>4000
3750
>5090
1.61
27.9
38.9
22.8
>204
33,500
>290,000
577
944
10,500
>5090
4860 (3360–7010)
2890 (2230–3730)
>100,000
>100,000
4850 (3680–6400)
24,400 (13,900–42,900)
5550 (4050–7600)
>10,000
12,900 (9180–18,200)
>30,000
Br
CF3
Ph
Cl
Br
Cl
Br
CF3
Ph
5
NH2
147 (114–188)
6e
7e
8
NH–COCH2Ph
NH–COCH2Ph
NH–CO–NH–Ph
NH–CO–NH–Ph
NH–CO–NH–Ph
NH–CO–NH–Ph
0.248 (0.211–0.292)
0.345 (0.313–0.381)
1.71 (1.20–2.44)
2.50 (1.89–3.32)
3.44 (2.34–5.05)
5.90 (3.33–10.5)
9
10
11
12
13
14
15
16
17
18
19
NH–CO–NH–(4-F)Ph
NH–CO–NH–(4-F)Ph
NH–CO–NH–(4-F)Ph
NH–CO–NH–(4-F)Ph
NH–CO–NH–(4-OCH3)Ph
NH–CO–NH–(4-OCH3)Ph
NH–CO–NH–(4-OCH3)Ph
NH–CO–NH–(4-OCH3)Ph
NH2
NH2
NH2
NH2
NH2
NH–COPh
NH–COPh
NH–COPh
NH–COPh
NH–COPh
NH–COCH2Ph
NH–COCH2Ph
Cl
N8–CH3
>30,000
>30,000
4420 (1700–11,500)
>100,000
6490 (3360–12,500)
>10,000
1060 (864–1310)
5930 (3270–10,800)
>30,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>30,000
>30,000
>10,000
>10,000
>30,000
>30,000
5.30 (4.35–6.45)
1.72 (0.91–3.26)
1.91 (1.01–3.59)
4.06 (3.17–5.19)
1.33 (0.91–1.95)
1.95 (1.64–2.31)
2.36 (1.46–3.80)
2.10 (1.41–3.14)
76.7 (59.5–99.0)
50.6 (32.9–78.0)
79.7 (67.9–93.6)
221 (152–320)
25 (17.5–35.6)
23.9 (20.3–28.1)
17.1 (14.9–19.8)
11.4 (9.94–13.0)
153 (120–195)
28.9 (22.0–37.9)
3.02 (1.49–6.11)
3.34 (3.16–3.53)
>5660
>17,400
2310
>24,600
4880
>5130
>42,400
>47,600
0.975
0.773
2.56
2.25
11.6
13.1
23.1
19.4
1.76
28.8
63.6
200
Br
CF3
Ph
Cl
Br
CF3
Ph
H
F
Cl
Br
OCH3
H
N8–CH3
3450
>15,700
>24,600
1100
4310
>42,400
>47,600
2.56
2.51
27.4
>136
56.0
40.3
N8–CH3
N8–CH3
>100,000
N8–CH3
1460 (1050–2040)
8400 (5220–13,500)
>100,000
N8–CH3
N8–CH3
>100,000
>100,000
N8–CH3
>100,000
20e
21e
22e
23e
24e
25e
26
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
74.8 (51.9–108)
39.1 (31.9–47.9)
204 (159–262)
498 (327–758)
289 (232–359)
313 (209–468)
395 (318–492)
221 (196–251)
270 (191–382)
831 (530–1300)
192 (167–220)
232 (209–259)
196 (121–317)
127 (106–151)
2180 (1400–3410)
>30,000
1400 (895–2210)
963 (749–1240)
4040 (3500–4620)
19,400 (11,900–31,400)
>100,000
F
Cl
Br
OCH3
H
236
27
1700
>654
872
203
171
28e
29
25,200 (22,300–28,400)
614 (574–656)
571 (507–642)
30
31
F
69.5
32
33
NH–COCH2Ph
NH–COCH2Ph
Cl
Br
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
932 (688–1260)
>10,000
3090 (2680–3560)
>10,000
>30,000
>10,000
8.48 (4.81–14.9)
>10,000
110
NA
364
NA
a
b
c
Displacement of specific [3H]-CCPA binding at hA1AR expressed in CHO cells, (n = 3–6).
Displacement of specific [3H]-NECA binding at hA2AAR expressed in CHO cells, (n = 3–6).
Ki values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells, (n = 3–6).
d
e
Displacement of specific [3H]-NECA binding at hA3AR expressed in CHO cells, (n = 3–6). Data are expressed as geometric means, with 95% confidence limits.
Data taken from Ref. 11.

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6123
NC
CN
NC
CN
CN
NH2
Me
i
iii
iv
ii
v
NH2
NH2
NH2
N
HN
N
N
N
N
Me
N
Ph
35
Ph
36
Ph
37
R1
34
38
NH
N
CN
vii
viii
R1
vi
N
NH2
OEt
N
N
N
N
Me
N
N
Me
Me
Me
N
N
N
N
N
R1
R1
39
40
41-44
2
N
N
N
ix
x
N
N
N
8
N
H
N
N
N
N
Me
N
Me
5
N
N
N
NH2
N
N
NH2
R
7
45-48
2-5
8-19
R1
R
R
R
R1
Cl
Br
8
9
NH-CO-NH-Ph
NH-CO-NH-Ph
NH-CO-NH-Ph
NH-CO-NH-Ph
12
13
14
15
NH-CO-NH-(4-F)Ph
NH-CO-NH-(4-F)Ph
NH-CO-NH-(4-F)Ph
NH-CO-NH-(4-F)Ph
16 NH-CO-NH-(4-OCH3)Ph
17 NH-CO-NH-(4-OCH3)Ph
18 NH-CO-NH-(4-OCH3)Ph
19 NH-CO-NH-(4-OCH3)Ph
2
3
4
5
Cl
Br
CF3
Ph
CF3 10
Ph 11
Scheme 1. Synthetic schemes for compounds 2–5 and 8–19. Reagents: (i) PhCHO; (ii) (NC)2C@CH(OEt); (iii) MeI, NaOH; (iv) conc. HCl, reflux; (v) HC(OEt)3; (vi) H2NNH2; (vii)
(1) R1CHO, Et3N, (2) PhI(OAc)2; (viii) HCl, reflux; (ix) H2NCN, TsOH; (x) R2N@C@O.
Substituted ureas 8–19 were obtained by reaction of the substi-
tuted amines 2–5 with the appropriate isocyanate in toluene under
reflux.
and the residue was subjected to chromatographic purification,
to afford the desired compounds (26, 27 and 29–33).
The synthetic pathways for the new series of 5-amide-8-phen-
ylethyl-2-(para-(un)substituted)phenyl-pyrazolo-[4,3-e]1,2,4-tri-
azolo[1,5-c]pyrimidine derivatives 26, 27 and 29–33 were based
on known procedures as described in Refs. 11,17,18 (Scheme
2).11,17,18 The inseparable mixtures of N1 and N2 regioisomers for
alkylated 3(5)-amino-4-cyano-pyrazoles (50 and 51) were synthe-
sized through alkylation reaction between (2-iodoethyl)benzene
and 3(5)-amino-4-cyano-pyrazole (49). These intermediates were
directly refluxed in triethyl orthoformate to give the corresponding
imidates (52 and 53), and they were subsequently reacted with the
appropriate (para-substituted)benzoic hydrazides in refluxing
2-methoxy-ethanol to afford the intermediates 54–58. Next, these
compounds were subjected to a thermally induced cyclization in
diphenyl ether at about 260 °C. After the chromatographic separa-
tion of N7 (minor product) and N8 (major product) regioisomers,
they provided the tricyclic compounds (59–63) in rather good yield
(about 60% for N8 derivatives and 30% for N7 derivatives). Hydroly-
sis in 20% HCl gave rise to the corresponding aminotriazoles
(64–68), which were subsequently converted into the 5-amino-
8-phenylethyl-2-[(para-substituted)phenyl]pyrazolo-[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidine derivatives (20–24).
3. Biology
3.1. Binding assay at hA1, hA2A, and hA3 ARs
The affinity of an antagonist towards its receptor (expressed in
Chinese Hamster Ovary cells (CHO) for hA1, hA2A, hA3 AR) was eval-
uated through the measurement of displacement of selective
radioligands,11,19–22 which were previously bound to the receptor
expressed at the cell surface. In this assay, the displacement was
evaluated of: (i) specific [3H]CCPA binding at the hA1AR; and (ii)
specific [3H]NECA binding at both the hA2AAR and hA3AR. The data
were expressed as Ki (dissociation constant), which was calculated
from the Cheng and Prusoff equation,22 with geometric means of at
least three experiments including 95% confidence intervals.
3.2. Adenylyl cyclase activity assay at hA2BAR
Due to the lack of a suitable radioligand for hA2BAR in binding
assay, the potency of antagonists on hA2BAR (expressed on CHO
cells) was determined in adenylyl cyclase experiments instead.11
The procedure was carried out as described previously in Klotz
et al. with minor modifications.19,20 In this assay, an adenosine
agonist (NECA) at known concentration was subjected to inhibitory
effect of tested antagonist. As a consequence, the activation of
adenylyl cyclase was inhibited by such tested antagonist with a
following decrease of the cAMP level.
Some of these 2-(substituted)phenyl-pyrazolo-[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidin-5-amines were dispersed in toluene and
treated with either benzoic anhydride or phenylacetyl chloride
(Scheme 3). The mixture was heated under reflux and stirred for
12–24 h. The solvent was later removed under reduced pressure

6124
S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
CN
CN
CN
2
2
N
N
ii
i
2
N
N
1
N
1
N
OEt
NH2
PhEt
PhEt
N
H
NH2
1
49
50, 51
52, 53
R1
H
N
N
N
iii
iv
v
N
N
N
N
N
R1
O
7
N
NH
N
N
8
PhEt
PhEt
54-58
59-63
NH2
N
N
N
HN
N
NH2
N
R1
R1
vi
N
7
N
N
N
N
8
PhEt
PhEt
R1
20-24
64-68
20
H
F
21
22
23
Cl
Br
24 OCH3
Scheme 2. Synthetic schemes for compounds 20–24. Reagents: (i) NaH, PhCH2CH2I, dimethylformamide; (ii) HC(OEt)3, reflux; (iii) 2-(4-substituted)benzoic hydrazide,
MeO(CH2)2OH; (iv) Ph2O, 260 °C, flash chromatography; (v) HCl, reflux; (vi) NH2CN, 1-methyl-2-pyrrolidone, pTsOH, reflux.
4. Molecular modeling
5. Results and discussion
A receptor-driven molecular modeling investigation has been
performed in order to rationalize the results obtained from the
pharmacological evaluation. To that purpose, we conducted molec-
ular docking simulations of the new PTP derivatives on the recently
published hA3AR model.23,24 As previously described, this hA3AR
model was built based on the template of the hA2AAR crystallo-
graphic structure in complex with the high affinity antagonist
ZM-241385 (PDB code: 3EML)25 (methodological details were
summarized in the Section 7).23,24
In the process of selecting a reliable docking protocol to be em-
ployed in the following docking studies of these new derivatives,
we have evaluated the ability of different docking softwares in
reproducing the crystallographic pose of ZM-241385 inside the
binding cavity of hA2AAR. Among the four different types of pro-
grams used to calibrate our docking protocol, the Gold programs
was finally chosen since it showed the best performance in regards
to the calculated root mean square deviation (RMSD) values rela-
tive to the crystallographic pose of ZM-241385.23 Consequently,
based on the selected docking protocol, all the newly synthesized
5.1. Structure–affinity relationship studies
A
new series of 2-(para-(un)substituted)phenyl-pyrazolo[4,
3-e]1,2,4-triazolo-[1,5-c]-pyrimidines was successfully synthe-
sized and characterized. We introduced only a few modifications
to the original PTP scaffold, in order to focus our investigation on
the combination of optimal substituents at the C2-, N5- and N8-
positions. To that end, we selected a (substituted)phenyl group at
the C2-position and either a methyl (compounds 4, 5 and 8–19)
or a phenylethyl (compounds 26, 27 and 29–33) group at N8, with
concurrent introduction of different amide moieties at position N5.
Table 1 summarizes the receptor binding affinities of these
compounds determined to hA1AR, hA2AAR and hA3AR and the
corresponding adenylyl cyclase activity in CHO cells that express
hA2BAR. The results showed that the new series of 2-(substi-
tuted)phenyl-pyrazolo-triazolo-pyrimidines presented good affin-
ity to hA3AR, as indicated by the nanomolar range of Ki values
and, most importantly, improved selectivities over other AR
subtypes.
phenyl-pyrazolo[4,3-e]1,2,4-triazolo-[1,5-c]-pyrimidines
docked into the orthosteric transmembranes (TMs) binding cavity
of hA3AR.
Besides that, in order to analyze the possible ligand–receptor
recognition mechanism in a more quantitative manner, we also cal-
culated the individual electrostatic and hydrophobic contributions
to the interaction energy of each receptor residues involved in
the binding interaction with ligands.
were
5.1.1. N8-Methyl derivatives
From our recent study, it was observed that the bioisosteric
replacement of the furan ring at the C2-position with a phenyl ring
resulted in a 3- to 8-fold increase in affinity towards hA3AR and a
significant improvement in selectivity (of 2–3 orders of magnitude)
over other AR subtypes, namely hA1AR, hA2AAR and hA2BAR. In
other words, compounds with a phenyl ring at the C2-position

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6125
O
HN
N
N
N
R1
N
N
N
i
26, 27, 29
PhEt
R1
NH2
26
27
F
N
N
N
R1
Cl
N
29 OCH3
N
N
PhEt
20-24
ii
O
HN
N
N
N
R1
N
N
30-33
R1
N
PhEt
30
31
32
33
H
F
Cl
Br
Scheme 3. Synthetic schemes for compounds 26, 27, 29–33. Reagents: (i) benzoic anhydride, toluene, reflux; (ii) phenylacetyl chloride, DIPEA, toluene, reflux.
demonstrated better affinity and selectivity profiles towards
hA3AR as compared with the 2-furyl counterparts. Nonetheless,
the binding results, the substituents (e.g. Cl, Br, CF3, Ph) at the
para-position of the C2-phenyl ring were found to modulate hA3AR
affinity to a certain extent. The introduction of functional groups
with relatively high molecular volume, such as a 4-trifluoromethyl
(e.g. compound 4, with KihA3 = 304 nM) and a 4-phenyl ring (e.g.
compound 5, with KihA3 = 147 nM) on the 2-phenyl ring of some
N5-unsubstituted derivatives seemed unfavourable for hA3AR
affinity, as shown by their high nanomolar Ki values. From these
findings we could infer that the presence of bulky groups at the
para-position of the C2-phenyl ring was undesirable for the hA3AR
affinity. In contrast, the situation improved remarkably when the
same derivatives were further substituted at the N5-position. The
additional chains at N5 (as shown in compounds 10 and 11) en-
abled an increase of affinity to hA3AR with a concomitant decrease
of binding to hA1AR and hA2AAR. This observation implied that the
hA3AR binding cavity around the N5-position was spacious enough
11
in the present investigation we noticed a different trend of hA3AR
affinities in the new series of 2-(para-(un)substituted)phenyl-8-
methyl-PTPs bearing arylcarbamoyl moieties at the N5-position
(4, 5 and 8–19), despite the fact that these compounds still main-
tained good hA3AR affinity at low nanomolar range (1.3–5.9 nM). It
was found that the bioisosteric replacement of the existing furan
ring with a phenyl ring in this group of compounds caused 7- to
37-fold decrease in affinity to hA3AR. Notably, the selectivity over
other AR subtypes of these 2-phenyl-PTP derivatives (e.g. com-
pound 10, with KihA3 = 3.44 nM; hA1/hA3 = 3750; hA2A/hA3 =
10,500 and compound 18, with KihA3 = 2.36 nM; hA1/hA3
>42,400; hA2A/hA3 >42,400) was still greatly improved in compar-
ison with that of 2-furyl analogues (e.g. compound 70, with KihA3 =
0.16 nM; hA1/hA3 = 3713; hA2A/hA3 = 2381 and compound 71, with
KihA3 = 0.20 nM; hA1/hA3 = 5495; hA2A/hA3 = 6950) (Table 2).
These findings suggested that for this group of N8-methyl-PTP-
N5-arylcarbamides, the 2-furyl ring was more favourable than
the 2-phenyl ring for hA3AR affinity. Even so, the introduction of
a phenyl ring at the C2-position was deemed crucial to confer high-
er selectivity towards hA3AR in comparison with the 2-furyl ring,
while maintaining good affinity at the hA3AR.
to accommodate extended chains including the phenylacetyl
11
(compounds 6 and 7)
and arylcarbamoyl groups (compounds
8–19). Among these substituents, the relatively flexible phenylace-
tyl group (e.g. compound 7, with KihA3 = 0.345 nM, hA1/hA3 =
70,700; hA2A/hA3 >290,000)11 showed a better binding profile than
the arylcarbamoyl chain (e.g. compound 9, with KihA3 = 2.50 nM,
hA1/hA3 >4000; hA2A/hA3 = 944).
In addition, we also evaluated the effect of para-substituents on
the 2-phenyl ring towards the affinity profile of hA3AR. Based on
Further incorporation of para-substituents such as 4-fluoro
(compounds 12–15) and 4-methoxy (compounds 16–19) groups

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S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
Table 2
Binding affinity (Ki) at hA3AR and selectivity against hA1AR and hA2AAR for compounds 10, 18, 20, 30 (2-para-(un)substituted-phenyl-PTPs) and 69–72 (2-furyl-PTPs)
R
N
N
N
R1'
N
N
N
R
2
0
Compd
R
R1
R2
hA3 (Ki, nM)
hA1/hA3
hA2A/hA3
10
69
18
70
20
71
30
72
NH–CO–NH–Ph
NH–CO–NH–Ph
NH–CO–NH–(4-OCH3)Ph
NH–CO–NH–(4-OCH3)Ph
NH2
NH2
NH–CO–CH2–Ph
NH–CO–CH2–Ph
Ph-p-CF3
Furyl
Ph-p-CF3
Furyl
Ph
Furyl
Ph
Furyl
N8–CH3
N8–CH3
N8–CH3
N8–CH3
3.44
0.16a
2.36
0.20a
76.7
300b
3.02
45.0c
3750
10,500
2381a
>42,400
6950a
2.56
3713a
>42,400
5485a
0.975
0.33b
63.6
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
N8–CH2–CH2–Ph
0.009b
203
2.67c
1.33c
a
b
c
Data taken from Ref. 10.
Data taken from Ref. 17.
Data taken from Ref. 26.
on the phenyl ring of arylcarbamoyl moiety seemed tolerable for
hA3AR affinity giving values in the range of 1.33–5.30 nM. Except
for derivative 12, compounds with 4-fluoro and 4-methoxy groups
on the phenyl ring of arylcarbamoyl moiety generally possessed
better hA3AR affinities in comparison with the N5-phenyl-
carbamoyl-substituted analogues (e.g. compound 13, with KihA3 =
1.72 nM, hA1/hA3 >17,400; hA2A/hA3 = 3450 vs compound 9, with
KihA3 = 2.50 nM, hA1/hA3 >4000; hA2A/hA3 = 944). Nonetheless,
they were still less potent than the N5-phenylacetamidic deriva-
tives (e.g. compound 7, with KihA3 = 0.345 nM, hA1/hA3 =
70,700; hA2A/hA3 >290,000). Furthermore, the 4-methoxy-phe-
nylcarbamoyl chain at the N5-position was shown to confer good
selectivity against the hA1AR, hA2AAR and hA2B AR as well. In
general, there were no substantial differences between the elec-
tron-withdrawing (e.g. 4-fluoro in compounds 12–15) and the
electron-donating (e.g. 4-methoxy in compounds 16–19) effects
of such para-substituents on hA3AR affinity, thus implying that
the steric effects might be more prominent on the hA3AR binding
profiles of these new PTPs.
hA2A/hA3 = 203). However, the hA3AR affinities and selectivities
of such N8-phenylethyl derivatives were found to be remarkably
lower than those of the N8-methyl analogues: compound 32,
bearing a N5-phenylacetamide, a 4-chlorophenyl at C2 and an
N8-phenylethyl groups showed a 30-fold drop in hA3AR affinity
(KihA3 = 8.48 nM; hA1/hA3 = 110; hA2A/hA3 = 364) in comparison
with the N8-methyl analogue (compound 6, with KihA3 =
0.248 nM; hA1/hA3 = 19,600; hA2A/hA3 = 33,500). This might be
due to the reason that in the N5-unsubstituted derivatives, there
was still enough space for the bulky phenylethyl group to orient it-
self inside the hA3AR binding cavity. Once an additional group was
introduced at the N5-position, there was limited space remaining
to anchor the phenylethyl, thus causing a considerable decrease
in hA3AR affinity. Between the two N5-phenylacetyl and N5-ben-
zoyl moieties, the relatively flexible phenylacetyl group (e.g. com-
pounds 31, with KihA3 = 3.34 nM; hA1/hA3 = 69.5; hA2A/hA3 = 171
and 32, with KihA3 = 8.48 nM; hA1/hA3 = 110; hA2A/hA3 = 364)
showed relatively better binding profiles than those with the
shorter benzoyl chain (e.g. compounds 26, with KihA3 = 17.1 nM;
hA1/hA3 = 23.1; hA2A/hA3 = 236 and 27, with KihA3 = 11.4 nM;
hA1/hA3 = 19.4; hA2A/hA3 = 1700). Such observations were consis-
tent with the findings obtained for the N8-methyl series of 2-phe-
nyl-PTP derivatives reported previously.14
We also investigated the impact of 2-furyl ring substitution
with a 2-phenyl ring in the current N8-phenylethyl series of
2-phenyl-PTPs towards hA3AR affinities and selectivities. The bind-
ing assays results were compared between the new 2-(substi-
tuted)phenyl derivatives (e.g. compounds 20 and 30) and the
2-furyl counterparts (e.g. compounds 72 and 73)17,26 (Table 2). It
was observed that the bioisosteric replacement of existing furan
ring with a phenyl ring resulted in about 4- to 15-fold increase
in affinity towards hA3AR and considerable improvements in selec-
tivity (of 2- to 3 orders of magnitude) over the other AR subtypes.
Such compounds with a phenyl ring at C2 and a phenylethyl group
at N8 showed better affinity and selectivity profiles towards the
hA3AR in comparison with their 2-furyl counterparts, and this fur-
ther strengthened our previous finding that the substituted-phenyl
at the C2-position indeed played a crucial role on the hA3AR affinity
and selectivity against other ARs.
5.1.2. N8-Phenylethyl derivatives
As reported in our recent study, it was found that when a small
alkyl group, like a methyl, was present at the N8-position, com-
pounds (1–19) showed a preference for hA3AR, regardless of sub-
stitutions at the C2- and N5-positions (e.g. compounds 3 [(with a
4-bromophenyl at C2 and a free amino at N5), KihA3 = 38.6 nM;
hA1/hA3 = 74.9; hA2A/hA3 = 38.9] 7 [(with a 4-bromophenyl at C2
and
a
phenylacetamide at N5), KihA3 = 0.345 nM; hA1/
hA3 = 70,700; hA2A/hA3 >290,000] and 9 [(with a 4-bromophenyl
at C2 and a phenylcarbamide at N5), KihA3 = 2.50 nM; hA1/hA3
>4000; hA2A/hA3 = 944]). Conversely, when a longer chain (e.g.
phenylethyl group) was introduced at N8, we observed a different
trend for derivatives bearing a free or substituted amino group at
the N5-position. When a free amino group was present at the N5-
position, the presence of an N8-phenylethyl group seemed to be
responsible for the lower affinities and selectivities of compounds
20–24 for hA3AR as compared with the N8-methyl analogues (e.g.
compound 22, with KihA3 = 79.7 nM; hA1/hA3 = 2.56; hA2A
/
hA3 = 27.4 vs compound 2, with KihA3 = 72.4 nM; hA1/hA3 = 67.1;
hA2A/hA3 = 27.9). On the other hand, when the same derivatives
were further substituted at the N5-position, the presence of such
additional groups at N5 seemed to be always favourable for hA3AR
affinity (e.g. compound 30, with KihA3 = 3.02 nM; hA1/hA3 = 63.6;
In particular, among the substituents introduced at the para-
position of the C2-phenyl ring, both the fluoro and chloro groups
exerted more favourable effects on hA3AR affinity in the N5-
benzamide-substituted (e.g. compound 26, with KihA3 = 17.1 nM;

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6127
hA1/hA3 = 23.1; hA2A/hA3 = 236) and N5-phenylacetamide-substi-
tuted (e.g. compound 31, with KihA3 = 3.34 nM; hA1/hA3 = 69.5;
hA2A/hA3 = 171) derivatives. Moreover, the presence of two para-
substituents on the 2-phenyl ring was shown to confer good
selectivity against hA1AR, hA2AAR and hA2BAR. Although all the
compounds with a 4-bromo group in the N8-methyl series showed
good affinities at hA3AR (e.g. compound 3, with KihA3 = 38.6 nM;
hA1/hA3 = 74.9; hA2A/hA3 = 38.9 nM; and compound 7, with
KihA3 = 0.345 nM; hA1/hA3 = 70,700; hA2A/hA3>290,000), its pres-
ence in the N8-phenylethyl-substituted derivatives (e.g. compound
23, with KihA3 = 221 nM; hA1/hA3 = 2.25; hA2A/hA3 >136; and com-
pound 33, with KihA3 >10,000 nM; KihA1, KihA2A >10,000) had the
opposite effect on the affinity to hA3AR. This could be possibly
due to the additional steric hindrance caused by the phenylethyl
group, which compromised the accommodation of relatively bulky
bromo groups within the binding site and resulted in detrimental
effects on hA3AR affinities.
5.2. Molecular modeling studies
In order to rationalize the hA3AR affinity profile observed for
this new series of compounds, a receptor-driven molecular model-
ing investigation was carried out. All of the 2-(substituted)phenyl-
pyrazolo[4,3-e]1,2,4-triazolo-[1,5-c]-pyrimidines were docked into
the transmembrane (TM) binding cavity of the recently published
hA3AR model. Additionally, for the selected binding poses, individ-
ual electrostatic and hydrophobic contributions to the whole inter-
action energy of each receptor residue were calculated.
5.2.1. N8-Methyl derivatives
All of the N8-methyl-2-(substituted)phenyl-PTPs reported in
this study (compounds 1–19) showed affinities to hA3AR in the
nanomolar range. Compound 16 showed the highest affinity
(KihA3 = 1.33 nM). The hypothetical binding mode of this com-
pound at hA3AR is shown in Figure 1: ligand-recognition occurred
in the upper region of the TM bundle, and the pyrazolo-triazolo-
pyrimidine scaffold was surrounded by TMs 3, 5, 6 and 7 with
the phenyl ring at the C2-position oriented towards TM2. Com-
pound 16 was anchored by two stabilizing hydrogen-bonding
interactions with the side chain of Asn250 (6.55) and an aromatic
Figure 1. Hypothetical binding mode of the N8-methyl derivative 16 at the hA3AR
binding site obtained after docking simulation. Pose is viewed from the membrane
side facing TM6, TM7 and TM1. The view of TM7 is voluntarily omitted. Side chains
of some amino acids important for ligand recognition and H-bonding interactions
are highlighted. Hydrogen atoms are not displayed.
Figure 2. (A) Electrostatic interaction energy values (in kcal/mol) and (B) hydrophobic interaction scores (in arbitrary hydrophobic units) between the ligand and each single
amino acid involved in ligand recognition calculated from the hypothetical binding mode of compound 16 inside the hA3AR binding sites.

6128
S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
p
–
p
stacking interaction with Phe168 (EL2). The asparagine resi-
and presented comparable interactions with the residues of the
due 6.55, conserved among all AR subtypes, was already found to
be important for ligand binding to hA3AR.27 Moreover, compound
16 formed hydrophobic interactions with many residues of the
hA3AR binding site including Leu91 (3.33), Phe168 (EL2), Leu246
(6.51), Ile268 (7.39) and the highly conserved Trp243 (6.48), an
important residue in receptor activation and antagonist recogni-
tion27; while the phenyl ring at the C2-position interacted with
Val65 (2.57), Leu68 (2.60), Ala69 (2.61), Val72 (2.64), Thr87
(3.29), Leu90 (3.32); and the phenyl ring of the phenylcarbamoyl
chain at N5 made hydrophobic contacts with Val169 (EL2),
Leu264 (7.35) and Tyr265 (7.36). In addition, another H-bond
was observed between the 4-methoxy group of the phenylcarba-
moyl chain at N5 and the amide group of the side chain of
Gln167 (EL2).
binding site.11
Considering the N5-unsubstituted N8-methyl PTP derivatives,
even if they showed binding modes to hA3AR similar to the one ob-
served for compound 16, they lost some stabilizing hydrophobic
interactions with Val169 (EL2), Leu264 (7.35) and Tyr265 (7.36)
and this could explain the lower affinities to hA3AR of the N5-
unsubstituted derivatives as compared with the N5-substituted
analogues. In addition, better hA3AR selectivity profile against
the other AR subtypes was also found in such N5-substituted deriv-
atives. This could be attributed to the lack of space to accommo-
date bulky substituents at that position for the other AR
subtypes. In particular, the weak binding of these compounds at
hA2AAR could be due to the different orientations that they ac-
quired inside the hA2A binding cleft as compared with the one ob-
The observed interactions were confirmed by the analysis of the
calculated electrostatic and hydrophobic contributions per residue
to the whole interaction energy for the complex between hA3AR
and compound 16. As shown in Figure 2, the main stabilizing con-
tributions were associated with Asn250 (6.55) and Gln167 (EL2)
(strong negative electrostatic interaction energy); while the stron-
gest hydrophobic contributions were related to Phe168 (EL2),
Leu264 (6.51), Leu246 (7.35), Leu90 (3.32) and Ile268 (7.39) (high
hydrophobic interaction score). Overall, the hereby proposed bind-
ing mode of compound 16 to hA3AR was very similar to the binding
poses already described for previously reported PTP derivatives
served inside hA3AR. As already proposed for the previously
11
reported PTP derivatives,
the presence of Glu169 (EL2) in
hA2AAR, which is mutated to Val169 (EL2) in hA3AR, seemed to
influence the binding poses of these compounds and led to the loss
of good interactions with key residue Asn253 (6.55). Among the
substituents at the N5-position, the phenylacetamide chain was
more preferable than the phenylcarbamoyl chain for hA3AR affin-
ity, probably because of its higher flexibility that allowed better
accommodation and stronger interaction in the binding cleft.
Regarding the effect of different para-substituents on the 2-phe-
nyl ring towards affinity to hA3AR, it appeared that the lateral cleft,
• R = NH2
moderate affinity at hA3AR; low selectivity against hA1AR and
hA2AAR, high selectivity against hA2BAR.
Figure 1
• R = NH-COPh
• R = NH-COCH2Ph
high hA3AR affinity and selectivity against hA1AR, hA2AAR and
hA2BAR.
confers better hA3AR affinity in comparison to R = NH-COPh
and high selectivity against hA1AR, hA2AAR and hA2BAR.
• R = NH-CONH-(4-F/OMe)Ph
high hA3AR affinity and selectivity against hA1AR,
hA2AAR and hA2BAR; 4-F/OMe on N5-Ph is
tolerable for hA3AR affinity.
R
5
•
Small alkyl chain (CH3) gives
good hA3 affinity and selectivity
against hA1AR, hA2AAR and
hA2BAR; longer and bulky alkyl
chain (PhCH2CH2) is disfavoured
for hA3AR affinity.
N
N
N
N
N
7
N
2
R1'
8
R2
* Replacement of 2-furyl ring with 2-aryl ring in derivatives bearing N5-NH2, -NH-
COPh and –NH-COCH2Ph improvement of hA3AR affinity and significant
increase of selectivity against hA1AR, hA2AAR and hA2BAR.
* Replacement of 2-furyl ring with 2-aryl ring in derivatives with N5-NH-CONHPh
decrease of hA3AR affinity but maintained in low nanomolar range; selectivity
against hA1AR, hA2AAR and hA2BAR is greatly improved.
• R1’ = (4-F/Cl/Br/OMe/CF3/Ph)-Ph
4-F/Cl/OMe are tolerable in all derivatives; 4-F/OMe gives higher hA3AR.
affinity; 4-OMe gives high selectivity against hA1AR, hA2AAR and hA2BAR.
4-Br is tolerable in N8-CH3 series, intolerable in N8-CH2CH2Ph series.
4-CF3/Ph is tolerable in derivatives with N5-arylcarbamide, intolerable in
derivatives with N5-amino.
Figure 3. Structure–affinity relationship profile for the series of 2-(substituted)phenyl PTP derivatives 1–33.

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6129
which accommodated the 2-phenyl ring, was not sufficiently spa-
cious to host bulky substituents; this could account for the general
decrease in hA3AR affinity observed with the increase in molecular
volume of the para-substituent on the 2-phenyl ring.
with an (un)substituted)phenyl group could also potentially
overcome the metabolic instability associated with the C2-furan
ring. The present work has provided a complete structure–affin-
ity relationship for 2-phenyl PTPs as potent and selective hA3AR
antagonists. Furthermore, the binding affinity profiles have also
been successfully rationalized through molecular modeling
investigations.
5.2.2. N8-Phenylethyl derivatives
The N8-phenylethyl derivatives hereby reported, in general,
presented lower affinities to hA3AR as compared with the N8-
methyl analogues. Among them, the most affine derivative to this
receptor was compound 30 (KihA3 = 3.02 nM). The docking simula-
tions on hA3AR performed for this series of compounds showed a
binding mode similar to the one observed for the N8-methyl deriv-
atives, but with the PTP nucleus shifted up towards the entrance of
the binding cavity. In fact, due to the presence of the bulkier N8-
phenylethyl chain, these compounds bound less deeply into the
binding site. As a consequence, these ligands were still able to form
some interactions with the key residue Asn250 (6.55), but they
were weaker than those observed for the N8-methyl derivatives.
This finding seemed to explain the lower affinities of the N8-phen-
ylethyl derivatives to hA3AR as compared with the N8-methyl
analogues.
7. Experimental section
7.1. Chemistry
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) on silica gel plate (precoated 60 F254 Merck plate). Col-
umn chromatographies were performed using Silica Gel 60
(Merck, 70–230 mesh). Melting points were determined on a Gal-
lenkamp instrument and were uncorrected. Compounds were dis-
solved in HPLC (high performance liquid chromatography)-grade
methanol for determination of mass to charge ratio (m/z) via the
LCQ Finnigan MAT mass spectrometer (source of ionization: atmo-
spheric pressure chemical ionization (APCI) probe). Purity of com-
pounds was detected by elemental analyses performed at the
laboratory of microanalysis of the Department of Chemistry, Uni-
versity of Ferrara (Italy), and all the compounds were confirmed
to achieve P95% purity. 1H spectra were determined in deuterated
chloroform (CDCl3) or deuterated dimethylsulfoxide (DMSO-d6)
through a Bruker DPX 300 spectrometer, with chemical shifts given
in parts per million (d) downfield relative to the central peak of the
solvents, and J values (coupling constants) given in hertz. The fol-
lowing abbreviations were used: s, singlet; br s, broad singlet; d,
doublet; dd, double doublet, br d, broad doublet; t, triplet; m,
multiplet.
For the substituents at both the N5-position of the PTP nucleus
and the para-position of C2-phenyl ring, hypotheses similar to the
ones reported above for the N8-methyl derivatives could be made.
In fact, the introduction of a benzoyl or phenylacetyl group at the
N5-position (compounds 25–33) caused an increase in affinities to
hA3AR as compared with the N5-unsubstituted analogues (com-
pounds 20–24), mainly due to the increase in hydrophobic interac-
tions with residues at the entrance of the hA3AR binding site, such
as Val169 (EL2), Ile253 (6.58), Val259 (EL3), Leu264 (7.35) and
Tyr265 (7.36).
On the other hand, the presence of para-substituents on the 2-
phenyl ring in these N8-phenylethyl derivatives was shown to be
tolerable for binding to hA3AR, provided they were not too bulky.
As an illustration, the presence of a relatively bulky bromo group
at the para-position in compound 33 was detrimental to hA3AR
affinity; this could probably be attributed to the additional steric
hindrance caused by the phenylethyl group, which reduced the
space to accommodate bulky substituents at the para-position of
the 2-phenyl ring.
On the whole, the above-mentioned results have further de-
fined the structure–affinity profiles of hA3AR for the new 2-(substi-
tuted)phenyl-PTP scaffold (Fig. 3) and emphasized the importance
of (i) a longer chain such as a benzamide or phenylacetamide group
at the N5-position to confer higher affinity and better selectivity
especially towards hA1AR, hA2AAR and hA2BAR; (ii) a small methyl
group at the N8-position in order to enhance both affinity and
selectivity to the hA3AR; (iii) a 2-(para-(un)substituted) phenyl
ring at the C2-position to improve affinity and selectivity profiles
to hA3AR relative to their 2-furyl counterparts. Among the newly
synthesized 2-(substituted)phenyl-PTP derivatives, compound 16,
with a 4-chlorophenyl at C2, a small methyl group at N8 and a 4-
methoxyphenylcarbamoyl chain at N5, showed the best hA3AR
affinity profile (KihA3 = 1.33 nM) and good selectivities against
the other ARs (hA1/hA3 = 4880; hA2A/hA3 = 1100).
7.1.1. {[(2Z)-2-Benzylidenehydrazino]methylene}malononitrile
(A) and {[(2E)-2 benzylidenehydrazino]methylene}malono-
nitrile (B) (4:1) (36)
To solution of hydrazine hydrate (9.7 ml, 200 mmol) in absolute
EtOH (350 ml) at 0 °C, benzaldehyde (21.2 ml, 200 mmol) was
added. After 1.5 h, ethoxymethylenemalononitrile (24.4 g,
200 mmol) was added and the resulting solution was stirred for
1 h at room temperature. After cooling overnight, the yellowish
precipitate was collected, washed with cold EtOH and recrystal-
lized from CHCl3.
Yield 82% 1H NMR (300 MHz, DMSO-d6): d 7.43–7.52 (3H, m,
aromatic HA+B), 7.64–7.73 (2H, m, aromatic HB), 7.76–7.84 (2H,
m, aromatic HA), 7.86 (1H, s, CHA), 8.20 (1H, s, CHA), 8.43 (1H, s,
CHB), 8.45 (1H, s, CHB), 12.45 (1H, br s, NHA+B).
7.1.2. {[(2Z)-2-Benzylidene-1-methylhydrazino]methylene}-
malononitrile (37)
To solution of (19.6 g, 100 mmol) (2-benzylidenehydrazi-
no)methylenemalononitrile in absolute EtOH (200 ml) and 1 M
aqueous NaOH (110 mmol), methyl iodide (12.5 ml, 200 mmol)
was added and the resulting solution was stirred at rt for 24 h.
After cooling overnight, the yellowish product was filtered, washed
with cold EtOH and recrystallized from EtOH.
6. Conclusions
The NMR characterization of intermediate 37 has been previ-
ously described in Ref. 18.
Consistent with our previous findings, the bioisosteric
replacement of the furan ring with a phenyl ring at the C2-posi-
7.1.3. 4-Cyano-1-methylpyrazol-3-amine (38), 4-cyano-3-[(etho-
xymethylene)amino]-1-methylpyrazole (39) and 5-amino-4-
imino-2-methylpyrazolo[3,4-d]pyrimidine (40)
Both the preparation procedures and the NMR characterizations
of intermediates 38, 39 and 40 have been previously described in
Refs. 12,18 and 14, respectively.
tion led to the identification of
a new series of 2-(para-
(un)substituted)phenyl-5-amide-8-substituted-pyrazolo-triazolo-
pyrimidine derivatives with hA3AR affinities and (or) selectivity
profiles against the other AR subtypes better than those of the
2-furyl PTP derivatives. In addition, such substitution of 2-furyl

6130
S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
7.1.4. General procedure for the preparation of 2-(substituted)-
phenyl-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimi-
dines (41–44)
The procedures for the preparation of intermediates 41–44 have
been previously described in Ref. 14.
7.50 (2H, t, J = 7 Hz, aromatic HA+B), 7.73 (2H, d, J = 8 Hz, aromatic
HA), 7.78 (2H, d, J = 8 Hz, aromatic HA+B), 7.87 (2H, d, J = 8 Hz, aro-
matic HB-30 and HB-50), 7.90 (1H, s, pyrazolo H-5A+B), 8.13 (2H, d,
J = 8 Hz, aromatic HB), 8.16 (2H, d, J = 8 Hz, aromatic HA), 13.86
(1H, s, NHA), 14.29 (1H, s, NHB).
7.1.4.1. 2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triaz-
olo[1,5-c]pyrimidine (41). Yield 45% 1H NMR (300 MHz, DMSO-
d6): d 4.21 (3H, s, Me), 7.64 (2H, d, J = 9 Hz, aromatic H), 8.23
(2H, d, J = 9 Hz, aromatic H), 8.92 (1H, s, pyrazolo-H), 9.46 (1H, s,
pyrimidine-H).
7.1.6. General procedure for the preparation of 2-(substituted)-
phenyl-8-methyl-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
5-amines (2–5)
To a solution of pyrazole derivatives 45–48 (10 mmol) in
N-methylpyrrolidinone (40 ml), cyanamide (0.42 g, 60 mmol) and
p-toluenesulfonic acid (2.85 g, 15 mmol) were added, and the
mixture was heated at 160 °C for 4 h. Then cyanamide (0.42 g,
60 mmol) was added again and the solution was heated overnight.
Then, the solution was diluted with hot water (100 ml) and
triturated for 30 min and the precipitate (excess of cyanamide)
was filtered off and washed with hot water (100 ml) again.
Compounds 2 and 3 have already been reported elsewhere.11
7.1.4.2. 2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triaz-
olo[1,5-c]pyrimidine (42). Yield 64% 1H NMR (300 MHz, DMSO-
d6): d 4.21 (3H, s, Me), 7.78 (2H, d, J = 8 Hz, aromatic H), 8.16
(2H, d, J = 8 Hz, aromatic H), 8.92 (1H, s, pyrazolo-H), 9.46 (1H, s,
pyrimidine-H).
7.1.4.3. 8-Methyl-2-(4-trifluoromethylphenyl)-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidine (43). Yield 50% 1H NMR
(300 MHz, DMSO-d6): d 4.21 (3H, s, Me), 7.95 (2H, d, J = 8 Hz, aro-
matic H), 8.43 (2H, d, J = 8 Hz, aromatic H), 8.94 (1H, s, pyrazolo-H),
9.50 (1H, s, pyrimidine-H).
7.1.6.1. 8-Methyl-2-(4-trifluoromethylphenyl)-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-amine (4). Yield 63%, pale yel-
low solid, mp 299–300 °C (MeOH). 1H NMR (300 MHz, DMSO-d6): d
4.04 (3H, s, Me), 7.70 (2H, s, NH2), 7.95 (2H, d, aromatic H, J = 8 Hz),
8.43 (2H, d, aromatic H, J = 8 Hz), 8.61 (1H, s). MS-APCI (methanol)
m/z: 334.2 (M+1)+ Anal. (C14H10N7F3) C, H, N.
7.1.4.4. 2-(4-Biphenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,
5-c]pyrimidine (44). Yield 50% 1H NMR (300 MHz, DMSO-d6): d
4.21 (3H, s, Me), 7.42 (1H, t, J = 7 Hz, aromatic H), 7.52 (2H, t,
J = 8 Hz, aromatic H), 7.78 (2H, d, J = 8 Hz, aromatic H), 7.89 (2H,
d, J = 8 Hz, aromatic H), 8.32 (2H, d, J = 8 Hz, aromatic H), 8.93
(1H, s, pyrazolo-H), 9.48 (1H, s, pyrimidine-H).
7.1.6.2. 2-(4-Biphenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,
5-c]pyrimidin-5-amine (5). Yield 85%, pale yellow solid, mp
>300 °C (EtOH) 1H NMR (300 MHz, DMSO-d6): d 4.04 (3H, s, Me),
7.42 (1H, t, J = 7 Hz), 7.52 (2H, t, J = 7 Hz), 7.65 (2H, s, NH2), 7.78
(2H, d, J = 8 Hz), 7.88 (2H, d, J = 8 Hz), 8.32 (2H, d, J = 8 Hz), 8.61
(1H, s). MS-APCI (methanol) m/z: 342.4 (M+1)+. Anal. (C19H15N7)
C, H, N.
7.1.5. General procedure for the preparation of 5(3)-[3-amino-
2-methylpyrazol-4-yl]-3(5)-(phenyl)-1,2,4-triazoles (45–48)
A solution of the mixture of 41–44 (10 mmol) in 10% HCl
(50 ml) was refluxed for 4–8 h depending on the substituent. Then
the solution was cooled and basified with concentrated ammo-
nium hydroxide. The resulting precipitate was filtered and washed
with H2O to afford the desired compounds 45–48 as solids.
7.1.7. General procedure for the preparation of N-aryl-N0-(2-
aryl-8-methyl-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-
yl)-ureas (8–19)
A mixture of amino compounds 2–5 (1 mmol) and the appropri-
ate phenylisocyanate (4.5 mmol) in toluene (10 ml) was heated
under reflux overnight. After cooling, the resulting mixture was fil-
tered and washed with EtOH to afford the final compounds 8–19.
7.1.5.1.
5(3)-[3-Amino-2-methylpyrazol-4-yl]-3(5)-(4-chloro-
phenyl)-[1,2,4]-1H-triazole (A:B 4.5:1) (45). Yield 87% 1H NMR
(300 MHz, DMSO-d6): d 3.69 (3H, s, MeB), 3.71 (3H, s, MeA), 5.27
(2H, s, NH2/B), 5.53 (2H, s, NH2/A), 7.52 (2H, d, J = 8 Hz, aromatic
HA), 7.63 (2H, d, J = 8 Hz, aromatic HB), 7.87 (1H, s, pyrazolo-
7.1.7.1. N-(2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e]-1,2,4-tri-
azolo[1,5-c]pyrimidin-5-yl)-N0-phenylurea (8). Yield 72%, white
solid, mp >300 °C (EtOH) 1H NMR (300 MHz, DMSO-d6): d 4.15 (3H,
s, Me), 7.13 (1H, t, aromatic H, J = 7 Hz), 7.41 (2H, t, aromatic H,
J = 8 Hz), 7.60 (2H, d, aromatic H, J = 8 Hz), 7.67 (2H, d, aromatic
H, J = 9 Hz), 8.32 (2H, d, aromatic H, J = 9 Hz), 8.82 (1H, s, pyrazolo
H), 9.94 (1H, s, N0H), 10.94 (1H, s, NH). MS-APCI (methanol) m/z:
419.5 (M+1)+. Anal. (C20H15N8OCl) C, H, N.
HA+B), 8.08 (2H, d, J = 9 Hz, aromatic HA+B), 13.88 (1H, s, NHA),
14.30 (1H, s, NHB).
7.1.5.2.
5(3)-[3-Amino-2-methylpyrazol-4-yl]-3(5)-(4-bromo-
phenyl)-[1,2,4]-1H-triazole (A:B 4.1:1) (46). Yield 84% 1H NMR
(300 MHz, DMSO-d6): d 3.71 (3H, s, MeA+B), 5.51 (2H, s, NH2/
A+B), 7.66 (2H, d, J = 8 Hz, aromatic HA), 7.76 (2H, m, aromatic
HA), 7.88 (1H, s, pyrazolo-HA+B), 8.02 (2H, d, J = 8 Hzaromatic
7.1.7.2. N-(2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e]-1,2,4-
HA+B), 13.90 (1H, s, NHA) and 14.31 (1H, s, NHB).
triazolo[1,5-c]pyrimidin-5-yl)-N0-phenylurea (9)
. Yield 87%,
white solid, mp >300 °C (EtOH) 1H NMR (300 MHz, DMSO-d6): d
4.15 (3H, s, Me), 7.13 (1H, t, aromatic H, J = 8 Hz), 7.41 (2H, t, aro-
matic H, J = 8 Hz), 7.60 (2H, d, aromatic H, J = 8), 7.81 (2H, d, aro-
matic H, J = 8 Hz), 8.25 (2H, d, aromatic H, J = 8 Hz), 8.82 (1H, s,
pyrazolo H), 9.95 (1H, s, N0H), 10.97 (1H, s, NH). MS-APCI (metha-
nol) m/z: 464.3 (M+1)+. Anal. (C20H15N8OBr) C, H, N.
7.1.5.3. 5(3)-[3-Amino-2-methylpyrazol-4-yl]-3(5)-(4-trifluoro-
methylphenyl)-1,2,4-triazole (A:B 6.1:1) (47). Yield 88% 1H
NMR (300 MHz, DMSO-d6): d 3.67 (3H, s, MeB), 3.71 (3H, s, MeA),
5.26 (2H, s, NH2/B), 5.54 (2H, s, NH2/A), 7.83 (2H, d, J = 8 Hz, aro-
matic HA), 7.89 (1H, s, pyrazolo-HA+B), 7.94 (2H, d, J = 8 Hz, aro-
matic HB), 8.24 (2H, d, J = 8 Hz, aromatic HB), 8.27 (2H, d, J = 8 Hz,
aromatic HA), 14.00 (1H, s, NHA), 14.50 (1H, s, NHB).
7.1.7.3. N-(8-Methyl-2-(4-trifluoromethylphenyl)-pyrazolo[4,3-
e]-1,2,4-triazolo[1,5-c]pyrimidin-5-yl)-N0-phenylurea
(10).
7.1.5.4. 5(3)-[3-Amino-2-methylpyrazol-4-yl]-3(5)-(4-biphenyl)-
1,2,4-triazole (A:B 2.5:1) (48). Yield 87% 1H NMR (300 MHz,
DMSO-d6): d 3.69 (3H, s, MeB), 3.72 (3H, s, MeA), 5.30 (2H, s,
NH2/B), 5.57 (2H, s, NH2/A), 7.39 (1H, t, J = 7 Hz, aromatic HA+B),
Yield 74%, white solid, mp 213–214 °C (EtOH) 1H NMR (300 MHz,
DMSO-d6): d 4.15 (3H, s, Me), 7.14 (1H, t, aromatic H, J = 7 Hz),
7.41 (2H, t, aromatic H, J = 8 Hz), 7.60 (2H, d, aromatic H,
J = 8 Hz), 7.97 (2H, d, aromatic H, J = 8 Hz), 8.52 (2H, d, aromatic

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6131
H, J = 8 Hz), 8.84 (1H, s, pyrazolo H), 10.04 (1H, s, N0H), 10.97 (1H, s,
NH). MS-APCI (methanol) m/z: 453.3 (M+1)+. Anal. (C21H15N8OF3)
C, H, N.
7.1.7.10. N-(2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-5-yl)-N0-(4-methoxyphenyl)-urea
(17). Yield 84%, white solid, mp >300 °C (EtOH) 1H NMR
(300 MHz, DMSO-d6): d 3.77 (3H,s, OMe), 4.14 (3H, s, Me), 6.99
(2H, d, aromatic H, J = 9 Hz), 7.50 (2H, d, aromatic H, J = 9 Hz),
7.81 (2H, d, aromatic H, J = 8 Hz), 8.25 (2H, d, aromatic H,
J = 8 Hz), 8.82 (1H, s, pyrazolo H), 9.85 (1H, s, N0H), 10.83 (1H, s,
NH). MS-APCI (methanol) m/z: 494.2 (M+1)+. Anal. (C21H17N8O2Br)
C, H, N.
7.1.7.4.
N-(2-(4-Biphenyl)-8-methylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-5-yl)-N0-phenylurea (11). Yield 59%,
pale brown solid, mp, >300 °C (EtOH) 1H NMR (300 MHz, DMSO-
d6): d 4.16 (3H, s, Me), 7.14 (1H, t, aromatic H, J = 7 Hz), 7.37–
7.47 (3H, m, aromatic H), 7.53 (2H, t, aromatic H, J = 8 Hz), 7.61
(2H, d, aromatic H, J = 8 Hz), 7.80 (2H, d, aromatic H, J = 8 Hz),
7.91 (2H, d, aromatic H, J = 8 Hz), 8.41 (2H, d, aromatic H,
J = 8 Hz), 8.84 (1H, s, pyrazolo H), 9.93 (1H, s, N0H), 11.00 (1H, s,
NH). MS-APCI (methanol) m/z: 460.4 (M+)+. Anal. (C26H20N8O) C,
H, N.
7.1.7.11. N-(4-Methoxyphenyl)-N0-8-methyl-2-(4-trifluorometh-
ylphenyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidin-5-yl)-
urea (18). Yield 59%, white solid, mp 211–213 °C (EtOH) 1H NMR
(300 MHz, DMSO-d6): d 3.77 (3H, s, OMe), 4.15 (3H, s, Me), 6.98
(2H, d, aromatic H, J = 9 Hz), 7.50 (2H, d, aromatic H, J = 9 Hz),
7.96 (2H, d, aromatic H, J = 8 Hz), 8.51 (2H, d, aromatic H,
J = 8 Hz), 8.82 (1H, s, pyrazolo H), 9.91 (1H, s, N0H), 10.82 (1H, s,
NH). MS-APCI (methanol) m/z: 483.2 (M+1)+. Anal. (C22H17N8O2F3)
C, H, N.
7.1.7.5. N-(2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-5-yl)-N0-(4-fluorophenyl)-urea
(12). Yield 73%, white solid, mp >300 °C (EtOH) 1H NMR
(300 MHz, DMSO-d6): d 4.14 (3H, s, Me), 7.25 (2H, dd, aromatic
H, J = 9 Hz, J = 9 Hz), 7.61 (2H, dd, aromatic H, J = 9 Hz, J = 5 Hz),
7.66 (2H, d, aromatic H, J = 9 Hz), 8.31 (2H, d, aromatic H,
J = 9 Hz), 8.82 (1H, s, pyrazolo H), 9.97 (1H, s, N0H), 10.95 (1H, s,
NH). MS-APCI (methanol) m/z: 437.6 (M+1)+. Anal. (C20H14N8OFCl)
C, H, N.
7.1.7.12. N-(2-(4-Biphenyl)-8-methylpyrazolo[4,3-e]-1,2,4-triaz-
olo[1,5-c]pyrimidin-5-yl)-N0-(4-methoxyphenyl)-urea
(19).
Yield 55%, white solid, mp >300 °C (EtOH) 1H NMR (300 MHz,
DMSO-d6): d 3.77 (3H,s, OMe), 4.15 (3H, s, Me), 6.99 (2H, d, aro-
matic H, J = 9 Hz), 7.43 (1H, t, aromatic H, J = 7 Hz), 7.51 (2H, d, aro-
matic H, J = 9 Hz), 7.52 (2H, t, aromatic H, J = 7 Hz), 7.79 (2H, d,
aromatic H, J = 8 Hz), 7.90 (2H, d, aromatic H, J = 8 Hz), 8.40 (2H,
d, aromatic H, J = 8 Hz), 8.83 (1H, s, pyrazolo H), 9.81 (1H, s, N0H),
10.85 (1H, s, NH). MS-APCI (methanol) m/z: 490.2 (M+)+. Anal.
(C27H22N8O2) C, H, N.
7.1.7.6. N-(2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-5-yl)-N0-(4-fluorophenyl)-urea
(13). Yield 72%, white solid, mp >300 °C (EtOH) 1H NMR
(300 MHz, DMSO-d6): d 4.14 (3H, s, Me), 7.25 (2H, dd, aromatic
H, J = 9 Hz, J = 9 Hz), 7.61 (2H, dd, aromatic H, J = 9 Hz, J = 5 Hz),
7.80 (2H, d, aromatic H, J = 8 Hz), 8.24 (2H, d, aromatic H,
J = 8 Hz), 8.82 (1H, s, pyrazolo H), 9.98 (1H, s, N0H), 10.94 (1H, s,
NH). MS-APCI (methanol) m/z: 481.5 (M+)+. Anal. (C20H14N8OBrF)
C, H, N.
7.1.8. General procedure for thesynthesis of N-(2-(substituted)-
phenyl-8-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-
pyrimidin-5-yl)benzamides (26, 27, 29)
To a suspension of 1 mmol of 2-(substituted)phenyl-8-phen-
ylethylpyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidin-5-amine (21,
22, 24) in 10 mL of toluene, 1.81 g (8 mmol) of benzoic anhydride
was added. The mixture was refluxed and stirred for 12 h at
120 °C. Then, the solvent was removed under reduced pressure
and the residue was purified through column chromatography
(AcOEt/petroleum ether in different ratios), to obtain the pure
N5-substituted pyrazolo-triazolo-pyrimidine derivatives (26, 27
and 29).
7.1.7.7. N-(4-Fluorophenyl)-N0-8-methyl-2-(4-trifluoromethyl-
phenyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidin-5-yl)-
urea (14). Yield 74%, pale yellow solid, mp 298–299 °C (EtOH) 1H
NMR (300 MHz, DMSO-d6): d 4.14 (3H, s, Me), 7.24 (2H, dd, aro-
matic H, J = 9 Hz, J = 9 Hz), 7.60 (2H, dd, aromatic H, J = 9 Hz,
J = 5 Hz), 7.95 (2H, d, aromatic H, J = 8 Hz), 8.49 (2H, d, aromatic
H, J = 8), 8.81 (1H, s, pyrazolo H), 10.02 (1H, s, N0H), 10.94 (1H, s,
NH). MS-APCI (methanol) m/z: 470.5 (M+)+. Anal. (C21H14N8OF4)
C, H, N.
7.1.8.1. N-[2-(4-Fluorophenyl)-8-phenylethyl-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidin-5-yl]benzamide (26). Yield 38%,
pale yellow solid, mp 109–111 °C (AcOEt–petroleum ether) 1H
NMR (300 MHz, CDCl3) d: 3.34 (2H, t, Ph-CH2, J = 7 Hz), 4.61 (2H,
t, N8–CH2, J = 7 Hz), 7.07 (2H, d, aromatic H, J = 6 Hz), 7.12–7.24
(5H, m, aromatic H), 7.54–7.71 (3H, m, aromatic H), 7.95 (1H, s,
pyrazolo-H), 8.07 (2H, d, aromatic H, J = 7 Hz), 8.27 (2H, dd, aro-
matic H, J = 6 Hz, J = 6 Hz), 9.81 (1H, s, NH). MS-APCI (methanol)
m/z: 478.8 (M+1)+ Anal. (C27H20N7OF) C, H, N.
7.1.7.8. N-(2-(4-Biphenyl)-8-methylpyrazolo[4,3-e]-1,2,4-triazolo
[1,5-c]pyrimidin-5-yl)-N0-(4-fluorophenyl)-urea (15). Yield 84%,
white solid, mp >300 °C (EtOH) 1H NMR (300 MHz, DMSO-d6): d
4.15 (3H, s, Me), 7.25 (2H, dd, aromatic H, J = 9 Hz, J = 9 Hz), 7.43
(1H, t, aromatic H, J = 7 Hz), 7.52 (2H, t, aromatic H, J = 7 Hz), 7.62
(2H, dd, aromatic H, J = 9 Hz, J = 5 Hz), 7.79 (2H, d, aromatic H,
J = 8 Hz), 7.90 (2H, d, aromatic H, J = 8 Hz), 8.39 (2H, d, aromatic
H, J = 8 Hz), 8.83 (1H, s, pyrazolo H), 9.94 (1H, s, N0H), 10.97 (1H,
s, NH). MS-APCI (methanol) m/z: 479.5 (M+1)+. Anal. (C26H19N8OF)
C, H, N.
7.1.8.2. N-[2-(4-Chlorophenyl)-8-phenylethyl-pyrazolo[4,3-e]1,
2,4-triazolo[1,5-c]pyrimidin-5-yl]benzamide (27). Yield 50%,
pale yellow solid, mp 112–114 °C (AcOEt–petroleum ether) 1H
NMR (300 MHz, CDCl3) d: 3.33 (2H, t, Ph-CH2, J = 7 Hz), 4.61 (2H,
t, N8–CH2, J = 7 Hz), 7.07 (2H, d, aromatic H, J = 6 Hz), 7.21–7.24
(3H, m, aromatic H), 7.49 (2H, d, aromatic H, J = 8 Hz), 7.58–7.68
(3H, m, aromatic H), 7.95 (1H, s, pyrazolo-H), 8.07 (2H, d,
aromatic H, J = 7 Hz), 8.27 (2H, d, aromatic H, J = 8 Hz), 9.80 (1H,
s, NH). MS-APCI (methanol) m/z: 494.9 (M+)+ Anal. (C27H20N7OCl)
C, H, N.
7.1.7.9. N-(2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-5-yl)-N0-(4-methoxyphenyl)-urea (16).
Yield 94%, white solid, mp 252–253 °C (EtOH) 1H NMR
(300 MHz, DMSO-d6): d 3.77 (3H, s, OMe), 4.14 (3H, s, Me), 6.99
(2H, d, aromatic H, J = 9 Hz), 7.50 (2H, d, aromatic H, J = 9 Hz),
7.67 (2H, d, aromatic H, J = 8 Hz), 8.32 (2H, d, aromatic H,
J = 8 Hz), 8.82 (1H, s, pyrazolo H), 9.85 (1H, s, N0H), 10.83 (1H, s,
NH). MS-APCI (methanol) m/z: 448.4 (M+)+. Anal. (C21H17N8O2Cl)
C, H, N.

6132
S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
7.1.8.3. N-[2-(4-Methoxyphenyl)-8-phenylethyl-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidin-5-yl]benzamide (29). Yield 22%,
pale yellow solid, mp 113–115 °C (AcOEt–petroleum ether) 1H
NMR (300 MHz, CDCl3) d: 3.34 (2H, t, Ph-CH2, J = 7 Hz), 3.89 (3H,
s, OCH3), 4.61 (2H, t, N8–CH2, J = 7 Hz), 7.03 (2H, d aromatic H,
J = 9 Hz), 7.07 (2H, d, aromatic H, J = 7 Hz), 7.22–7.24 (3H, m, aro-
matic H), 7.58–7.68 (3H, m, aromatic H), 7.95 (1H, s, pyrazolo-H),
8.07 (2H, d, aromatic H, J = 8 Hz), 8.21 (2H, d, aromatic H,
J = 8 Hz), 9.84 (1H, s, NH) MS-APCI (methanol) m/z: 491.2 (M+2)+
Anal. (C28H23N7O2) C, H, N.
7.2. Biology
7.2.1. CHO membrane preparation
All the pharmacological methods involved in membrane prepa-
ration for radioligand binding experiments and adenylyl cyclase
activity assays followed the procedures as described earlier.19
Membranes for radioligand binding were prepared from cells
stably transfected with the human adenosine receptor subtypes
(hA1AR, hA2AAR, and hA3AR expressed on CHO cells) in a two-step
procedure. In the first low-speed step (1000g for 4 min), the cell
fragments and nuclei were removed. After that, the crude mem-
brane fraction was sedimented from the supernatant at 100,000g
for 30 min. The membrane pellet was then resuspended in the spe-
cific buffer used for the respective binding experiments, frozen in
liquid nitrogen and stored at ꢀ80 °C. For the measurement of the
adenylyl cyclase activity in hA2BAR expressed on CHO cells, only
one step of centrifugation was used, in which the homogenate
was sedimented for 30 min at 54,000g. The resulting crude mem-
brane pellet was resuspended in 50 mM Tris/HCl, pH 7.4 and
immediately used for the adenylyl cyclase assay.
7.1.9. General procedure for the synthesis of N-(2-(substituted)-
phenyl-8-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyri-
midin-5-yl)phenylacetamides (30–33)
To
a suspension of 1 mmol of 2-(substituted)phenyl-8-
phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidin-5-amine
(20–23) in 10 mL of toluene, 1.1 mL (8 mmol) of phenylacetyl
chloride was added, followed by 1.4 mL (8 mmol) of DIPEA. The
mixture was refluxed and stirred for 24 h at 120 °C. Then, the sol-
vent was removed under reduced pressure and the residue was
purified via column chromatography (AcOEt: Petroleum Ether in
different ratios), to obtain the pure N5-substituted pyrazolo-
triazolo-pyrimidine derivatives (30–33).
7.2.2. Human cloned A1, A2A, A3 AR binding assay
Binding of [3H]-CCPA to CHO cells transfected with the human
recombinant A1AR was performed as previously described.19,20
Displacement experiments were performed for 3 h at 25 °C in
7.1.9.1. N-[2-(Phenyl)-8-phenylethyl-pyrazolo[4,3-e]-1,2,4-triaz-
olo[1,5-c]pyrimidin-5-yl]phenylacetamide (30). Yield 19%, yel-
low solid, mp 92–94 °C (AcOEt–petroleum ether) 1H NMR
(300 MHz, CDCl3) d: 3.34 (2H, t, Ph-CH2, J = 7 Hz), 4.51 (2H, s,
CH2–CO), 4.61 (2H, t, N8–CH2, J = 7 Hz), 7.07 (2H, d, aromatic H,
J = 6 Hz), 7.18–7.23 (3H, m, aromatic H), 7.32–7.43 (5H, m, aro-
matic H), 7.49–7.51 (3H, m, aromatic H), 7.95 (1H, s, pyrazolo-H),
8.16–8.20 (2H, br d, aromatic H), 9.18 (1H, s, NH). MS-APCI (meth-
anol) m/z: 475.2 (M+2)+. Anal. (C28H23N7O) C, H, N.
200
l
L of buffer containing 1 nM [3H] CCPA, 0.2 U/mL adenosine
deaminase, 20
l
g of membrane protein in 50 mM Tris/HCl, pH
7.4 and tested compound in different concentrations. Nonspecific
binding was determined in the presence of 1 mM theophylline.21
Binding of 30 nM [3H]-NECA to CHO cells transfected with the
human recombinant A2AARs was performed following the condi-
tions as that described for the hA1AR binding assay.19,20 In the dis-
placement experiments, sample with 50 lg of membrane protein
in 50 mM Tris/HCl, 10 mM MgCl, pH 7.4 and tested compound in
different concentrations was incubated for 3 h at 25 °C. Nonspecific
7.1.9.2. N-[2-(4-Fluorophenyl)-8-phenylethyl-pyrazolo[4,3-e]-1,
2,4-triazolo[1,5-c]pyrimidin-5-yl]phenylacetamide (31). Yield
26%, yellow solid, mp 107–109 °C (AcOEt–petroleum ether) 1H
NMR (300 MHz, CDCl3) d: 3.33 (2H, t, Ph-CH2, J = 7 Hz), 4.50 (2H,
s, CH2–CO), 4.60 (2H, t, N8–CH2, J = 7 Hz), 7.07 (2H, d, aromatic H,
J = 6 Hz), 7.15–7.23 (5H, m, aromatic H), 7.32–7.43 (5H, m, aro-
matic H), 7.94 (1H, s, pyrazolo-H), 8.17 (2H, dd, aromatic H,
J = 6 Hz, J = 6 Hz), 9.14 (1H, s, NH). MS-APCI (methanol) m/z:
492.6 (M+1)+. Anal. (C28H22N7OF) C, H, N.
binding was determined in the presence of 100
l
M R-PIA (R-N6-
phenylisopropyladenosine).21
Binding of [3H]-NECA to CHO cells transfected with the human
recombinant A3ARs was carried out as previously described.19,20
The displacement experiments were performed for 3 h at 25 °C in
buffer solution containing 10 nM [3H]-NECA, 20
lg of membrane
protein in 50 mM Tris/HCl, 1 mM EDTA (ethylenediaminotetraace-
tate), 10 mM MgCl2, pH 8.25 and tested compound in different con-
centrations. Nonspecific binding was determined in the presence of
100 l
M R-PIA.21
7.1.9.3. N-[2-(4-Chlorophenyl)-8-phenylethyl-pyrazolo[4,3-e]-1,
2,4-triazolo[1,5-c]pyrimidin-5-yl]phenylacetamide (32). Yield
20%, yellow solid, mp 108–110 °C (AcOEt–petroleum ether) 1H
NMR (300 MHz, CDCl3) d: 3.33 (2H, t, Ph-CH2, J = 7 Hz), 4.49 (2H,
s, CH2–CO); 4.61 (2H, t, N8–CH2, J = 7 Hz), 7.07 (2H, d, aromatic
H, J = 7 Hz), 7.17–7.23 (3H, m, aromatic H), 7.33–7.43 (5H, m, aro-
matic H), 7.47 (2H, d, aromatic H, J = 8 Hz), 7.93 (1H, s, pyrazolo-H),
8.11 (2H, d, aromatic H, J = 8 Hz), 9.14 (1H, s, NH). MS-APCI (meth-
anol) m/z: 508.6 (M+)+. Anal. (C28H22N7OCl) C, H, N.
Bound and free radioactivity was separated by filtering the as-
say mixture through Whatman GF/B glass-fiber filters using a Mi-
cro-Mate 196-cell harvester (Packard Instrument Company). The
filter bound radioactivity was counted on Top Count (efficiency
of 57%) with Micro-Scint 20. The protein concentration was deter-
mined according to the Bio-Rad method28 with bovine albumin
used as a reference standard.
7.2.3. Adenylyl cyclase activity
Due to the lack of a suitable radioligand for hA2BAR in binding
assay, the potency of antagonists at hA2BAR (expressed on CHO
cells) was determined in adenylyl cyclase experiments instead.
The procedure was carried out as described previously with minor
modifications.19,20 Membranes were incubated with about
7.1.9.4. N-[2-(4-Bromophenyl)-8-phenylethyl-pyrazolo[4,3-e]-1,
2,4-triazolo[1,5-c]pyrimidin-5-yl]phenylacetamide (33). Yield
24%, yellow solid, mp 279–281 °C (AcOEt–petroleum ether) 1H
NMR (300 MHz, CDCl3) d: 3.34 (2H, t, Ph-CH2, J = 7 Hz), 4.50 (2H,
s, CH2–CO), 4.61 (2H, t, N8–CH2, J = 7 Hz), 7.07 (2H, d, aromatic
H, J = 8 Hz), 7.19–7.23 (3H, m, aromatic H), 7.33–7.45 (5H, m,
aromatic H), 7.64 (2H, d, aromatic H, J = 8 Hz), 7.93 (1H, s,
pyrazolo-H), 8.05 (2H, d, aromatic H, J = 8 Hz), 9.13 (1H, s, NH).
MS-APCI (methanol) m/z: 552.5 (M+)+. Anal. (C28H22N7OBr) C,
H, N.
150,000 cpm of [a-
32P]ATP for 20 min in the incubation mixture
as described19,20 without EGTA and NaCl. The IC50 values of tested
antagonists for concentration-dependent inhibition of NECA-stim-
ulated adenylyl cyclase were calculated accordingly. Dissociation
constants (Ki) of the antagonists were then calculated from the
Cheng and Prusoff equation.22

S. L. Cheong et al. / Bioorg. Med. Chem. 19 (2011) 6120–6134
6133
7.3. Molecular modeling
cal group that minimized the total free energy of the system (tak-
ing titration into account).33 Protein stereochemistry evaluation
was then performed by several tools (Ramachandran plot; back-
bone bond lengths, angles and dihedral plots; clash contacts re-
port; rotamers strain energy report) implemented in MOE suite.29
7.3.1. General
All the modeling studies were carried out on a 20 CPU (Intel
Core2 Quad CPU 2.40 GHz) Linux cluster. Homology modeling, en-
ergy calculation, and analyses of docking poses were performed
using the Molecular Operating Environment (MOE, version
2010.10) suite.29 The software package MOPAC,30 implemented
in MOE suite, was utilized for all quantum mechanical calculations.
Docking simulations were performed using GOLD suite 5.0.1.31
7.3.3. Molecular docking of hA3AR antagonists
Ligand structures were built using MOE-builder tool, part of the
MOE suite,29 and were subjected to MMFF94x energy minimiza-
tion until the rms of conjugate gradient was <0.05 kcal molꢀ1
Å
ꢀ1. Partial charges for the ligands were calculated using PM3/ESP
methodology. Four different programs have been used to calibrate
our docking protocol: MOE-Dock,29 GOLD,31 Glide,34 and PLANTS.35
In particular, ZM-241385 was re-docked into the crystal structure
of the hA2AAR (PDB code: 3EML) with different docking algorithms
and scoring functions, as already described.23,24 Then, RMSD values
between predicted and crystallographic poses of ZM-241385 were
calculated for each of the docking algorithms. The results showed
that docking simulations performed with Gold gave the lowest
RMSD value, the lowest mean RMSD value and the highest number
of poses with RMSD value <2.5 Å.
On the basis of the best docking performance, all antagonist
structures were docked into the hypothetical TM binding site of
the hA3AR model using the docking tool of the GOLD suite.32
Searching was conducted within a user-specified docking sphere
using the Genetic Algorithm protocol and the GoldScore scoring
function. GOLD performs a user-specified number of independent
docking runs (25 in our specific case) and writes the resulting con-
formations and their energies in a molecular database file. The
resulting docked complexes were subjected to MMFF94x energy
minimization until the rms of conjugate gradient was <0.1 kcal -
molꢀ1 Åꢀ1. Charges for the ligands were imported from the MOPAC
output files using PM3/ESP methodology.
Prediction of antagonist-receptor complex stability (in terms of
corresponding pKi value) and the quantitative analysis of non-
bonded intermolecular interactions (H-bonds, transition metal,
water bridges, hydrophobic, electrostatic) were calculated and
visualized using several tools implemented in MOE suite.29 Electro-
static and hydrophobic contributions to the binding energy of indi-
vidual amino acids have been calculated as implemented in MOE
suite.29 In order to estimate the electrostatic contributions, atomic
charges for the ligands were calculated using PM3/ESP methodol-
ogy. Partial charges for protein amino acids were calculated on
the basis of the AMBER99 force field.
7.3.2. Homology model of hA3AR
Based on the assumption that GPCRs share similar TM bound-
aries and overall topology, a homology model of the hA3AR was
constructed as previously reported,23,24 based on a template of
the crystallographic structure of hA2AAR in complex with the
antagonist ZM-241385 (PDB code: 3EML).25 The numbering of
the amino acids follows the arbitrary scheme by Ballesteros and
Weinstein. According to this scheme, each amino acid identifier
starts with the helix number, followed by the position relative to
a reference residue among the most conserved amino acid in that
helix. The number 50 is arbitrarily assigned to the reference
residue.32
Firstly, the amino acid sequences of TM helices of the hA3AR
were aligned with those of the template, guided by the highly con-
served amino acid residues, including the DRY motif (Asp3.49,
Arg3.50, and Tyr3.51) and three proline residues (Pro4.60,
Pro6.50, and Pro7.50) in the TM segments of GPCRs. The same
boundaries were applied for the TM helices of hA3AR as they were
identified from the 3D structure of the template, the coordinates of
which were used to construct the seven TM helices for hA3AR.
Then, the loop domains were constructed by the loop search meth-
od implemented in MOE on the basis of the structure of compatible
fragments found in Protein Data Bank. In particular, loops were
modeled first in random order. For each loop, a contact energy
function analyzed the list of candidates collected in the segment
searching stage, taking into account all atoms already modeled
and any atoms specified by the user as belonging to the model
environment. These energies were then used to make
a
Boltzmann-weighted choice from the candidates, the coordinates
of which were then copied to the model. Subsequently, the side
chains were modeled using a library of rotamers generated by sys-
tematic clustering of the Protein Data Bank data via the same pro-
cedure. Side chains of residues whose backbone coordinates were
copied from a template were modeled first, followed by side chains
of modeled loops. Gaps and their side chains were modeled last.
Special caution has to be given to the second extracellular loop
(EL2) because amino acids of this loop could be involved in direct
interactions with the ligands. A driving force to the peculiar fold of
EL2 might be the presence of a disulfide bridge between cysteines
in TM3 and EL2. Since this covalent link is conserved in both
hA2AAR and hA3AR, the EL2 loop was modeled using a constrained
geometry around the EL2–TM3 disulfide bridge. The constraints
were applied before the construction of the homology model, espe-
cially during the sequences alignment, by selecting the cysteine
residues involved in the disulfide bridge of hA2AAR to be con-
strained with the corresponding cysteine residues in hA3AR se-
quence. In particular, Cys166 (EL2) and Cys77 (3.25) of the
hA2AAR were constrained with Cys166 (EL2) and Cys83 (3.25) of
the hA3AR, respectively. During the alignment, MOE-Align at-
tempted to minimize the number of constraint violations. At the
end of the homology modeling, the presence of the conserved
disulfide bridge in the model was manually checked.
Acknowledgments
This work has been supported by the National University of Sin-
gapore (FRC grant, R-148-000-129-112), Ministry of Education
(MOE2009-T2-2-011 project, R-398-000-068-112) and A-STAR
(TSRP project, R-148-001-435-305). The molecular modeling work
coordinated by S.M. has been carried out with financial support
from the University of Padova, Italy, and the Italian Ministry for
University and Research, Rome, Italy (MIUR, PRIN2008: protocol
number 200834TC4L_002). S.M. is also very grateful to Chemical
Computing Group for the scientific and technical partnership.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.08.026.
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