Water-soluble pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines as human A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm300323t

A relevant problem of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus, an attractive scaffold for the preparation of adenosine receptor antagonists, is the low water solubility. We originally functionalized the C⁵ position with a salifiable 4-pyridylcarbamoyl moiety that conferred good water solubility at low pH (<4.0) but poor solubility at physiologic pH, indicative of the dissociation of the pyridinium species. Here we replaced the pyridin-4-yl moiety with a 1-(substituted)piperidin-4-yl ring to exploit the higher basicity of this nucleus and for the the possibility to generate stable, water-soluble salts. The hydrochloride salt of the 1-(cyclohexylmethyl)piperidin-4-yl derivative (10, K_i(hA₃) = 9.7 nM, IC₅₀(hA₃) = 30 nM, K_i(hA ₁/hA₃) = 351, K_i(hA_2A/hA ₃) > 515, IC₅₀(hA_2B) > 5 μM) showed a solubility of 8 mg/mL at physiological pH and gave a stable aqueous system suitable for intravenous infusion. Molecular modeling studies were helpful in rationalizing the available structure-activity relationships and the selectivity profile of the new ligands.

Full text of DOI:10.1021/jm300323t

Article
pubs.acs.org/jmc
Water-Soluble Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines as
Human A3 Adenosine Receptor Antagonists
Pier Giovanni Baraldi,*,† Giulia Saponaro,† Romeo Romagnoli,† Mojgan Aghazadeh Tabrizi,†
Stefania Baraldi,† Allan R. Moorman,§,# Sandro Cosconati,∥ Salvatore Di Maro,⊥ Luciana Marinelli,⊥
Stefania Gessi,‡ Stefania Merighi,‡ Katia Varani,‡ Pier Andrea Borea,‡ and Delia Preti†
†Dipartimento di Scienze Farmaceutiche and ‡Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universita
di Ferrara, 44100 Ferrara, Italy
̀
§King Pharmaceuticals, Inc., Research and Development, 4000 CentreGreen Way, Suite 300, Cary, North Carolina 27513, United
States
∥Dipartimento di Scienze Ambientali, Seconda Universita
́
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
⊥Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
̀
“Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
S
* Supporting Information
ABSTRACT: A relevant problem of the pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidine nucleus, an attractive scaffold
for the preparation of adenosine receptor antagonists, is the
low water solubility. We originally functionalized the C5
position with a salifiable 4-pyridylcarbamoyl moiety that
conferred good water solubility at low pH (<4.0) but poor
solubility at physiologic pH, indicative of the dissociation of
the pyridinium species. Here we replaced the pyridin-4-yl
moiety with a 1-(substituted)piperidin-4-yl ring to exploit the
higher basicity of this nucleus and for the the possibility to
generate stable, water-soluble salts. The hydrochloride salt of
the 1-(cyclohexylmethyl)piperidin-4-yl derivative (10, Ki(hA3) = 9.7 nM, IC50(hA3) = 30 nM, Ki(hA1/hA3) = 351, Ki(hA2A/hA3)
> 515, IC50(hA2B) > 5 μM) showed a solubility of 8 mg/mL at physiological pH and gave a stable aqueous system suitable for
intravenous infusion. Molecular modeling studies were helpful in rationalizing the available structure−activity relationships and
the selectivity profile of the new ligands.
degenerative diseases, allergy, asthma, and COPD (Thomson
Reuters Integrity source). Our group provided the first
evidence that the A3AR plays a role in colon tumorigenesis
and, more importantly, can potentially be used as a diagnostic
marker or a therapeutic target for colon cancer.11
INTRODUCTION
■
Adenosine, a ubiquitous nucleoside essential for the proper
functioning of every cell in mammalian species is directly linked
to energy metabolism through ATP, ADP, and AMP, while at
the extracellular level it regulates a wide range of biological
functions through activation of specific receptors (adenosine
receptor, AR).1−4 These adenosine receptors belong to the
superfamily of the G-protein-coupled receptors (GPCRs) and
are classified as A1, A2A, A2B, and A3.
The A3AR subtype is the most recently characterized
member of the family.4 Activation of the subtype has been
shown to inhibit adenylate cyclase, to increase phosphatidyli-
nositol-specific phospholipase C and D activity, to elevate
intracellular Ca2+ and IP3 (inositol 1,4,5-trisphosphate) levels,
and to enhance the release of inflammatory and allergic
mediators from mast cells.5,6 The potential therapeutic
applications derived from the modulation of this receptor
subtype have been recently reviewed.7,8 It is becoming
increasingly apparent that an antagonist of A3AR might be
therapeutically useful for the acute treatment of glaucoma.9,10
Specific antagonists are currently undergoing biological testing
for their potential use in the treatment of stroke, neuro-
The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP)
nucleus has been shown repeatedly to serve as an attractive
scaffold for the preparation of adenosine receptor antagonists.
Systematic substitution of the C2-, C5-, N7-, N8-, or C9-positions
of PTPs7,12−16 allowed a structure−activity relationship (SAR)
profile to be delineated for this class of molecules, primarily
defining receptor subtype selectivity. A 2-furyl or a 2-
(substituted)phenyl17,18 ring appeared important for affinity
toward all four AR subtypes. The presence of a free amine at
the C5-position combined with an arylalkyl moiety at the N7-
position was found to significantly promote both affinity and
selectivity at the A2AAR subtype.12 In contrast, introduction of
an arylurea moiety at the C5-position and small alkyl chains
(e.g., methyl or propyl) at the N8-position led to the
Received: March 6, 2012
Published: May 9, 2012
© 2012 American Chemical Society
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Article
identification of highly potent and selective human A3AR
antagonists.19 Notable in this class is the PTP analogue bearing
a 4-(methoxy)phenylcarbamoyl moiety at C5 and an n-propyl
group at N8 (MRE-3008-F20, 1, Figure 1), which displayed a Ki
of 0.29 nM at the human A3 receptors expressed in CHO cells
with high selectivity over hA1 and hA2A ARs (Ki of 1100 and
141 nM, respectively).20
piperidin-4-yl-ring, a suitable compound could be identified
with significant potency at the target adenosine A3 receptor,
adequate selectivity toward other adenosine receptors, and
appropriate aqueous solubility at physiologic pH due to the
increased basicity of the ring nitrogen. For that reason we
prepared a first series of N-[2-(2-furyl)-8-methyl-8H-pyrazolo-
[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]-N′-(1-(substituted)-
piperidin-4-yl)urea derivatives (series A, compounds 6−20,
Figure 1). Representative compounds were then converted into
the corresponding hydrochloride salts, and the solubility at a
physiologic pH of 7.4 (phosphate-buffered saline, PBS) was
evaluated. The SAR study was then extended to a second series
of N-substituted 4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carboxamide derivatives (series B, compounds
21−34, Figure 1), with which we intended to probe the steric
tolerance in the region around the piperidine nitrogen.
CHEMISTRY
■
Scheme 1 depicts the preparation of final compounds 5−34
starting from 2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-amine 3, which was prepared as
a
Scheme 1
Figure 1. Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine: from pyr-
idineurea to piperidineurea derivatives.
This class of molecules was found to synergistically enhance
cytotoxic treatment of melanoma cells countering P-glyco-
protein efflux in multidrug resistance.21 Nevertheless, a relevant
problem of the PTPs was the typically low water solubility,
which also limited their use as pharmacological and diagnostic
tools. To enhance the water solubility, we originally replaced
the phenylcarbamoyl moiety of 1 with a salifiable pyridin-4-
ylcarbamoyl group. This led to the identification of the most
potent hA3 antagonist known to date (MRE-3005-F20, 2,
Figure 1).15 On the basis of our knowledge that the adenosine
A3 receptor was expressed at elevated levels in certain cancers,21
we wished to evaluate this class of adenosine A3 antagonists for
use in conjunction with various cytotoxic agents. We envisioned
that these compounds would be administered by intravenous
infusion. Although the aqueous solubility of 2 (approximately
11 mg/mL) was significantly enhanced, relative to that of 1,
aqueous formulations required a pH below 3.5 to obtain a
homogeneous solution capable of being filter-sterilized. At a
more physiologically acceptable pH (7.0−7.5), the solubility of
2 dropped to less than 0.1 mg/mL, affording a thick,
unfilterable suspension (unpublished data). The present work
is an extension of our SAR studies on 2, in which we focused
our attention on the pyridine ring. Specifically, we hypothesized
that by converting the pyridin-4-yl-moiety to a 1-(substituted)-
a
Reagents and conditions: (i) (a) triphosgene, THF, CH2Cl2, 0 °C, 15
min; (b) 140 °C, 50 min; (ii) TFA, CH3CN/H2O, 80 °C, 2 h; (iii)
RX, K2CO3, DMF, rt, 2−4 h; (iv) primary and secondary amines,
TEA, 1,4-dioxane, 0 °C, 1 h, then rt, 3 h.
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Table 1. Biological Data of the Synthesized Compounds
a
b
c
d
compd
R
R1
A1 Ki (nM)
>5000
A2A Ki (nM)
>5000
>5000
A2B IC50 (nM)
A3 Ki (nM)
5
H
2428 (2037−2894)
>5000
65 (56−75)
6
Me
>5000
253 (211−303)
71 (51−98)
7
propyl
allyl
2794 (2351−3322)
3357 (3045−3701)
802 (707−910)
3408 (3043−3817)
703 (641−770)
1049 (868−1269)
724 (626−838)
1478 (1254−1740)
1156 (1028−1299)
166 (124−224)
371 (290−474)
1329 (1071−1649)
1188 (949−1488)
348 (267−453)
1500 (1320−1690)
1324 (1164−1508)
744 (598−927)
761 (611−949)
805 (665−973)
525 (441−625)
182 (128−259)
112 (80−158)
1193 (992−1434)
1423 (1117−1811)
789 (725−859)
>5000
>5000
8
>5000
51 (39−67)
25 (22−29)
9
propargyl
cyclohexyl-CH2
Bn
>5000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
>5000
9.7 (7.6−12.4)
30 (25−38)
526 (473−585)
>5000
2061 (1589−2672)
>5000
4-F-Bn
24 (19−31)
26 (20−32)
4-Cl-Bn
>5000
>5000
4-CH3-Bn
4-OCH3-Bn
4-CN-Bn
>5000
>5000
13.8 (9.2−20.6)
13.3 (11.9−14.9)
11.8 (8.2−17.0)
12.4 (9.7−16.0)
44 (37−52)
>5000
>5000
2223 (2016−2452)
>5000
>5000
4-NO2-Bn
3-pyridin-CH2
4-pyridin-CH2
EtO2CCH2
Et
>5000
>5000
>5000
>5000
>5000
39 (33−46)
1084 (915−1283)
>5000
>5000
12.2 (8.4−17.7)
30 (22−41)
H
H
H
H
H
H
H
H
H
H
H
H
H
>5000
isopropyl
>5000
>5000
9.8 (7.9−12.2)
9.3 (6.2−13.9)
13.7 (9.8−19.1)
7.7 (5.5−10.8)
8.1 (6.0−10.9)
6.8 (4.8−9.5)
14.5 (11.4−18.5)
4.9 (2.9−8.1)
25 (16−39)
cyclopentyl
cyclohexyl
cycloheptyl
Bn
>5000
>5000
>5000
>5000
>5000
>5000
>5000
1107 (934−1311)
1755 (1567−1967)
1542 (1405−1695)
1042 (897−1210)
505 (398−638)
2122 (1929−2335)
3244 (3028−3476)
3235 (2744−3813)
2-phenylethyl
3-phenylpropyl
4-F-Bn
1842 (1695−2001)
1641 (1467−1835)
3255 (3098−3420)
1137 (1034−1249)
2813 (2584−3061)
>5000
408 (335−499)
614 (490−768)
396 (335−442)
635 (524−771)
1299 (936−1803)
1123 (929−1356)
4-Cl-Bn
4-CH3-Bn
4-OCH3-Bn
4-pyridyl
3.9 (2.2−6.9)
10.4 (6.2−17.4)
21 (14−30)
e
33
e
1856 (1498−2300)
1282 (1050−1565)
34
4-methylpiperazinyl
2144 (1674−2745)
31 (26−38)
a
b
Displacement of specific [3H]DPCPX binding to human A1ARs expressed in CHO cells (Ki, nM). Displacement of specific [3H]ZM241385
c
d
binding to human A2AARs expressed in CHO cells (Ki, nM). cAMP assay in CHO cells expressing hA2BARs (IC50, nM). Displacement of specific
[3H]MRE-3008-F20 binding to human A3ARs expressed in CHO cells (Ki, nM). Data are expressed as geometric mean with 95% confidence limits
e
in parentheses of three or four independent experiments performed in duplicate. Trifluoroacetate salt.
previously described by us.22 In the first step, the commercially
RESULTS AND DISCUSSION
■
available 1-benzyl-4-aminopiperidine was treated with triphos-
All of the synthesized compounds (5−34) were evaluated in
gene at 0 °C for 15 min, leading to the formation of the
radioligand binding assays to determine their affinities for
corresponding 4-isocyanatopiperidine derivative as established
human A1, A2A, and A3 adenosine receptors using [3H]DPCPX
by IR spectroscopy. This was then reacted with the tricyclic
(1,3-[3H]dipropyl-8-cyclopentylxanthine), [3H]ZM241385 (4-
amine (3) at 140 °C for 50 min. Under these conditions we
(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-
observed the formation of the carbamoyl chloride derivative (4)
ylamino]ethyl)phenol), [3H]MRE-3008-F20 (5-N-(4-
as a result of the concurrent cleavage of the N-benzyl moiety
methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-
and conversion into the corresponding carbamoyl chloride
[4,3-e]-1, 2,4-triazolo[1,5-c]pyrimidine), respectively, as radio-
function. This confirmed the known capability of triphosgene
to cleave N-benzyl-protected secondary amines.23,24
ligands. Efficacy of the compounds versus hA2BAR was
investigated by evaluating their capability to inhibit NECA
(5′-(N-ethylcarboxamido)adenosine)-stimulated (100 nM)
cAMP production. Antagonism of selected ligands versus
hA3AR was also assessed through cAMP experiments evaluating
their capability to block the inhibitory effect mediated by Cl-IB-
MECA (2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcar-
boxamide). Affinity data for A1, A2A, and A3 receptors
(expressed as Ki values) and IC50 values for hA2B and hA3
The carbamoyl chloride derivative 4 was then hydrolyzed in
the presence of TFA and a mixture of CH3CN/H2O as solvent
to give the unsubstituted piperidine derivative (5), which was
subjected to N-alkylation under standard conditions with alkyl/
cycloalkyl/(hetero)arylalkyl halides to afford the final com-
pounds 6−20. The bis-urea derivatives 21−34 were obtained in
good yields via the treatment of the carbamoyl chloride 4 with
the appropriate primary or secondary amines.
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subtypes, derived from the cAMP assays, are listed in Tables 1
and 2, respectively.
not seen by replacing the N-cyclohexylmethyl derivative (10)
with the N-benzyl derivative (11).
While the N-benzyl derivative (11) had lower affinity than
the corresponding N-cyclohexylmethyl derivative, this sub-
stitution led to a slight improvement of affinity over the
unsubstituted derivative (see compound 11 vs 5). This effect
was enhanced occasionally by p-substitution of the benzyl
group (derivatives 12−17). In this regard, both electron
withdrawing and electron donating functions have been
examined, but we did not observe a clear correlation between
binding affinity and the electronic properties of the introduced
moieties, as both strong electron-withdrawing (i.e., compound
16, 17) or donating (compound 15) functions seem to
promote comparable outcomes in terms of A3AR affinity.
Compounds 18 and 19, in which a heteroarylalkyl moiety has
been introduced at the piperidine nitrogen, display similar
binding potencies as the corresponding arylalkyl bioisoster 11.
A slight increase of A3AR affinity was observed with compound
20, deriving from the alkylation of 5 (see Scheme 1) with 2-
bromoethylacetate.
The bis-urea derivatives 21−34 (series B, Figure 1) afforded
A3 affinities in the low nanomolar range. Among the N-alkyl-
substituted derivatives 21−25, a slight preference for bulky and
lipophilic cycloalkyl substitution can be noted. For example, the
N-cycloheptyl derivative (25) displayed an almost 4-fold
increase of affinity when compared to the N-ethyl derivative
21. Nevertheless, in light of the affinity data for the N-
(substituted)arylalkyl derivatives 26−31 (Ki values ranging
from 3.9 to 25 nM), it appeared that A3 binding potency seems
poorly related to structural modifications of the terminal
portion of the chain at the C5-position of the pyrazolotriazo-
lopyrimidine nucleus. The maintenance of affinity in the same
range of concentration for N-4-pyridyl and 4-methylpiperazinyl
derivatives, 33 and 34, respectively, support such an
observation.
Table 2. Potency of Selected Novel A3AR Antagonists in
a
hA3CHO Cells on cAMP Production
compd
A3 IC50 (nM)
compd
A3 IC50 (nM)
5
262 (203−338)
910 (846−978)
301 (271−335)
212 (171−263)
105 (83−131)
30 (22−41)
121 (100−146)
82 (66−101)
91 (80−104)
50 (43−58)
45 (37−56)
40 (33−48)
42 (33−54)
180 (162−199)
166 (140−197)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
41 (35−47)
116 (91−148)
34 (24−47)
30 (24−39)
44 (33−59)
26 (17−38)
29 (18−46)
23 (14−37)
46 (30−72)
15 (10−25)
90 (81−99)
12 (8−19)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
34 (28−41)
82 (71−96)
120 (103−140)
a
Data are expressed as geometric mean with 95% confidence limits in
parentheses of three or four independent experiments performed in
duplicate.
Structure−Activity Relationship Analysis: hA3AR Af-
finity. All of the synthesized molecules exhibited good affinities
at the hA3AR subtype with Ki values ranging from 3.9
(compound 31, Table 1) to 253 nM (compound 6).
Nevertheless, compound 5, bearing an unsubstituted piperidine
ring at the C5-position, showed a remarkable (6500-fold)
decrease of hA3AR affinity compared to its unsaturated
congener 2. Considering that piperidine and pyridine have a
comparable steric bulk (with a calculated van der Waals surface
area of 175.57 and 128.26 Å3, respectively),25 the higher
basicity (pKb of 2.7 for piperidine vs 8.7 for pyridine) and lower
lipophilicity (cLogD calculated at pH 7.4 of −2.15 for
piperidine and 0.75 for pyridine) of piperidine could be
responsible for this marked decrease in affinity. With the
perspective of exploiting the higher basicity of piperidine and its
possibility to generate stable and water-soluble salts, we tried to
enhance the affinity/selectivity of compound 5 by substitution
of the piperidine nitrogen.
Where possible, pairwise comparison between the Ki(hA3)
values of compounds of series A and series B resulted in a
substantial maintenance or a slight increase of affinity in most
cases for the bis-urea derivatives (i.e., compounds 11−15 vs 26,
29−32, respectively). Among these, the 4-CH3-benzyl sub-
stituted 31 is identified as the most potent hA3AR ligand
reported herein, with a Ki of 3.9 nM.
Structure−Activity Relationship Analysis: hA3AR vs
hA2AAR and hA1AR Selectivity. All of the examined
molecules exhibited a clear selectivity toward the hA3AR versus
both hA1 and hA2A AR subtypes. The Ki values at the A2A
subtype were, in most cases, higher than 1 μM (except for
compounds 9 and 11, which displayed high nanomolar affinity
for the target). The hA1/hA3 selectivity ratios appeared highly
variable within the two series. While different substitutions at
the piperidine nitrogen did not seem to remarkably affect hA3
affinity, the hA1AR subtype appeared, indeed, quite responsive
to steric/electronic modulation of this part of the molecule.
Among compounds of series A, the affinity for the hA1AR
appeared particularly relevant when the piperidine nitrogen was
substituted with benzyl moieties bearing strong electron
withdrawing groups in the p-position. The 4-CN (16) and
the 4-NO2 (17) derivatives exhibited the highest hA1 affinities
(Ki(hA1) values of 166 and 371 nM, respectively), and
compound 16 stands out as the least selective A3 ligand of
this subset of molecules (Ki(hA1/hA3) = 14). Interestingly, p-
substitution with electron donating groups reduced hA1
affinities, improving hA3AR vs hA1 selectivity (see compounds
The initial strategy was alkylation of the N1-position of the
piperidine ring to obtain the compounds of series A (Figure 1).
Among the N′-(1-alkyl/arylalkylpiperidin-4-yl)urea derivatives
6−20, the introduction of a methyl group at the piperidine
nitrogen, as in compound 6, led to a 4-fold decrease of A3AR
affinity compared to the corresponding N-unsubstituted
derivative 5. Introduction of a propyl moiety resulted in a
substantial maintenance of A3 affinity (see compound 7 vs 5).
The effect of unsaturated chains was evaluated with the N-allyl
derivative 8 and N-propargyl derivative 9. The latter compound
showed a 2.5-fold increase of affinity when compared with 5,
while the N-allyl substitution was almost equipotent. A more
marked effect on binding properties was observed when a bulky
cycloalkylmethyl substituent was introduced. The affinity of the
N-cyclohexylmethyl derivative 10 was almost 7-fold greater
than that of the N-unsubstituted derivative 5. Although
unsaturation in straight-chain aliphatic substituents led to
improved affinity (see compound 7 vs 8 vs 9), this pattern was
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14 and 15 with Ki(hA1/hA3) ratios of 107 and 87, respectively).
N-(Cyclo)alkyl substitution seemed to be less well tolerated
than N-arylalkyl substitution by the hA1 subtype, promoting
hA3 vs hA1 selectivity. The N-cyclohexylmethyl derivative (10)
was the most selective compound of the entire series (Ki(hA1/
hA3) = 351).
To varying degrees, the N-arylalkyl derivatives of series B
were found to be preferred over the corresponding N-
(cyclo)alkyl derivatives for enhancing hA1AR affinity. It was
observed that hA1AR affinity seemed to be related to the
distance between the aromatic nucleus and the ureidic nitrogen.
In particular, the comparison of the binding profiles for the 3-
phenylpropyl-substituted 28 (Ki(hA1) = 112 nM) and the
inferior homologues 2-phenylethyl 27 (Ki(hA1) = 182 nM) and
benzyl 26 (Ki(hA1) = 525 nM) reveals that binding affinity
proportionately increases with the elongation of the alkyl
spacer, which makes compound 28 the least selective A3 ligand
of both of the analyzed series (Ki(hA1/hA3) = 7.7).
Because of the lack of an adequate number of closely related
analogues, it is difficult to identify clear differences in the
selectivity profiles between the two series. However, a
comparison between the binding pattern of compounds 11−
15 (series A) and the analogously substituted 26, 29−32 (series
B) would suggest that the additional urea moiety of series B is
slightly preferred for promoting A3 versus A1 selectivity.
Functional Assays: hA2B and hA3 ARs. As reported in
Table 1, the examined compounds showed very low or no
activity at the hA2BAR subtype, with IC50 > 1 μM (except for
compound 30 with IC50 = 505 nM). For 20 out of 31
compounds tested, IC50 > 5 μM was found.
The compounds of both series were shown to behave as
antagonists of the hA3AR subtype in the cAMP functional assay
(Figure 2B and Table 2). A good correspondence between
binding and functional experiments was observed. The
molecules showing the best affinities for hA3AR also displayed
very high potency in receptor inhibition (IC50 values in the low
nanomolar range). Derivative 31, showing the highest hA3
affinity (Ki = 3.9 nM), emerged as the most potent compound
of the series, with an IC50 of 12 nM.
Figure 2. Competition binding and cAMP assays of selected novel
A3AR compounds: affinity and potency values expressed as Ki (A) and
IC50 (B), respectively.
in the hA3AR, as well as being highly conserved in all of the
ARs (Figure 3).29
In this position the methyl group at N8 points downward
toward the inner part of the receptor, making contacts with
W243 and being surrounded by hydrophobic residues (L91,
M177, I186, L246, and S247). It is noteworthy that the
interaction with W243 should be considered to be responsible
for the antagonist properties of this ligand, as already
demonstrated for other AR antagonists.18 Hydrophobic
contacts are also established by the furyl ring with the receptor
pocket formed by TM helices 2, 3, and 7, made up by L68, A69,
V72, Y265, and I268. However, in this theoretical binding pose,
the R substituent of 10 points toward the external part of the
receptor at the crevice of TM5 and TM6 where the terminal
cyclohexyl ring makes contact with Q261 and Q167.
Interestingly, as observed with other hA3AR antagonists,26 the
favorable interaction with the outer portion of TM5, TM6,
EL2, and EL3 seems to be the reason for the observed
selectivity of these compounds. In particular, in the hA1, hA2A,
and hA2B ARs, salt bridges are established at the interface of
EL2 and EL3, thus making this area narrower and less prone to
be occupied by the ligand substituents (Figure 4a). On the
other hand in the hA3AR this region appears to be wider to host
the R substituent. Interestingly, the influence on A3AR ligand
selectivity of the receptor area at the crevice between EL2and
EL3 was already postulated by us26 and other authors28 for
different selective antagonists. Indeed, the ligand piperidine
ring, while providing further interaction points with the
Molecular Modeling Studies. To rationalize the SAR data
presented, docking simulations were performed using the
hA3AR model previously published by our research group.26 In
this work the recently published crystal structure of the hA2A
receptor (PDB code 3EML)27 in complex with compound
ZM241385 was used to obtain a homology model of the
hA3ARs. The sequence alignment between the three receptors
and the positions of the disulfide bridges were attained
consistently with what was already suggested by Moro and
co-workers.28 The structure of the modeled receptor was used
with Glide to perform docking calculations of compound 10,
which displayed good affinity for the hA3 receptor (9.7 nM)
and the best selectivity profile (Ki(hA1/hA3) = 351, Ki(hA2A/
hA3) > 515) among compounds from both series. The docking
poses obtained were evaluated for their consistency with the
available SAR data, as well as for the Glide scores obtained from
these calculations. Results obtained for 10 revealed that for the
best Glide score (−7.435), the ligand binds to the outer portion
of the A3AR being surrounded by TM helices 3, 5, 6, and 7 and
EL2. This predicted binding pose strongly resembles one
calculated for some analogues reported by other authors.18 In
this position, the core ligand scaffold establishes a π-stacking
interaction with F168 and two H-bonds with the N250 side
chain, which was demonstrated to be critical for ligand binding
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(see Table S1 of the Supporting Information). Most of the
evaluated parameters were shown to be within the range of
values representative of 95% of known drugs. Moreover, in view
of the known metabolic lability of the 2-furan ring of PTPs18 we
performed preliminary ligand-based calculations based on the
SMARTCyp30 approach to predict the sites of cytochrome
P450 mediated metabolism on compound 10. For each ligand
atom this software calculated the propensity to be the site of
metabolism (SOM). Figure 5 depicts compound 10 in which all
Figure 3. Close view of the calculated binding pose for 10 in the
hA3AR model. The ligand and receptor are depicted as green and
sticks, respectively. H-Bonds are depicted as dashed blue lines.
Figure 5. 2D representation of compound 10. Atoms are colored
according to the calculated SMARTCyp score (color coding is also
reported). Arrows indicate the predicted SOMs for 10.
heavy atoms were colored according to the relative
SMARTCyp score. Interestingly, the software calculated for
the piperidine nitrogen and the adjacent carbon atoms the
highest propensity to be SOMs (lowest scores). As mentioned
in the molecular modeling section, this region of the ligand
should point toward the outer part of the receptor, so it can be
postulated that the metabolites of 10 can also positively interact
with the A3AR. Also, one carbon atom of the furyl substituent
appeared to be a putative SOM; nevertheless, from these
calculations this atom does not appear to be as prone to react
with cytochrome P450.
CONCLUSION
■
Figure 4. Superimposition of the structures of the A1 (a),26 A2A (b),27
In the present work we synthesized two series of compounds in
which the 4-pyridinyl moiety of our reference hA3AR
antagonist 215 (Figure 1) was replaced by a 1-(substituted)-
piperidin-4-yl ring. Derivatives of series A (Table 1), in which
the piperidine nitrogen was alkylated with different (cyclo)-
alkyl/arylalkyl moieties, were designed with the perspective of
exploiting the higher basicity of piperidine and its possibility to
form stable, water-soluble salts. This approach was evaluated in
order to overcome solubility problems encountered with the
pyridine derivative 2 while trying to prepare aqueous
formulations at a physiological pH suitable for intravenous
infusion.
All of the synthesized molecules exhibited good affinity and
selectivity at the hA3AR subtype, with Ki in the low nanomolar
range. The 1-(cyclohexylmethyl)piperidin-4-yl derivative 10
showed the best binding profile in terms of hA3 affinity (Ki =
9.7 nM), potency (IC50 = 30 nM), and selectivity (Ki(hA1/hA3)
= 351, Ki(hA2A/hA3) > 515, IC50(hA2B) > 5 μM). Molecular
modeling studies were helpful in rationalizing the observed
structure−activity relationships, along with the selectivity
profiles of the new series of ligands. Docking of compound
A2B (c),42 and hA3 (d) ARs. Receptors are represented as ribbons
26
and orange sticks. 10 is represented as green surface and sticks.
receptor, projects its nitrogen atom toward the external
receptor region. This should explain why it can be N-alkylated
with different substituents without significantly affecting the
A3AR affinity.
Water Solubility and Druglikeness Prediction. A few
representative compounds were converted into the correspond-
ing hydrochloride salts, and the water solubility at a
physiological pH (7.4, PBS) was evaluated. While under these
conditions the solubility of 2 dropped to less than 0.1 mg/mL;
the piperidine derivatives 9, 10, and 12 were found to have a
solubility of 8−10 mg/mL and gave stable aqueous systems
suitable for evaluation as an intravenous infusion. As expected,
the less lipophilic N-methyl derivative 6 was shown to be even
more soluble (15 mg/mL). Nevertheless, considering the better
binding profile, we selected compound 10 as a reference
compound for molecular modeling studies (see above) and
computational calculation of several druglikeness parameters
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mmol). The mixture was stirred at room temperature for 2 h. The
solvent was removed under reduced pressure, and the product was
purified by column chromatography, eluting with EtOAc (ethyl
acetate)/MeOH (9:1).
10 in complex with hA3AR furnished a general model of the
hypothetical binding mode for the newly described derivatives.
Moreover, this compound, as the hydrochloride salt, proved to
have a solubility of 8 mg/mL at physiological pH and gave a
stable aqueous system suitable for intravenous infusion.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-(1-methylpiperidin-4-yl)urea (6).
1
White solid; 27% yield; mp 228 °C (dec); H NMR (DMSO-d6) δ
9.19 (bs, 1H), 8.77 (s, 1H), 8.73 (bd, J = 7.6 Hz, 1H), 7.99−7.97 (m,
1H), 7.30−7.28 (m, 1H), 6.77−6.74 (m, 1H), 4.12 (s, 3H), 3.89 (m,
1H), 3.39 (s, 3H), 3.08−2.90 (m, 2H), 2.44 (m, 2H), 1.99 (m, 2H),
1.89−1.56 (m, 2H). MS (ESI): [MH]+ = 396.3. Anal. (C18H21N9O2)
C, H, N.
EXPERIMENTAL SECTION
■
Chemistry. Materials and Methods. Reaction progress and
product mixtures were monitored by thin-layer chromatography
(TLC) on silica gel (precoated F254 Macherey-Nagel plates) and
visualized by UV lamp (254 nm light source) or with aqueous
potassium permanganate. Chromatography was performed on Merck
230−400 mesh silica gel. After extraction from aqueous phases, the
organic solutions were dried over anhydrous sodium sulfate. Light
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-(1-propylpiperidin-4-yl)urea (7).
1
White solid; 31% yield; mp 171 °C (dec); H NMR (DMSO-d6) δ
9.11 (bs, 1H), 8.81 (bd, J = 7.6 Hz, 1H), 8.69 (s, 1H), 7.96−7.95 (m,
1H), 7.27−7.25 (m, 1H), 6.75−6.73 (m, 1H), 4.09 (s, 3H), 3.71 (m,
1H), 3.37 (m, 2H), 2.81−2.75 (m, 2H), 2.25−2.21(t, J = 7.6 Hz, 2H),
2.18−2.05 (m, 2H), 1.98−1.1.83 (m, 2H), 1.47−1.43 (m, 2H), 0.86 (t,
J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 424.3. Anal. (C20H25N9O2) C, H,
N.
1
petroleum refers to the fractions boiling at 40−60 °C. H NMR data
were determined in CDCl3 or DMSO-d6 solutions with a Varian VXR
200 spectrometer or a Varian Mercury Plus 400 spectrometer. Peak
positions are given in parts per million (δ) downfield from
tetramethylsilane as internal standard, and J values are given in
hertz. Splitting patterns are designated as follows: s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; b, broad. All of the detected signals
were in accordance with the proposed structures. Melting points for
purified products were determined in a glass capillary on a Stuart
Scientific electrothermal apparatus SMP3 and are uncorrected.
Electrospray ionization mass spectrometry (ESI/MS) was performed
with an Agilent 1100 series LC/MSD in positive scan mode using
direct injection of the purified compound solution (MH+). Elemental
analyses were performed by the microanalytical laboratory of
Dipartimento di Chimica, University of Ferrara, Italy, and were within
0.4% of the theoretical values for C, H, and N. All final compounds
revealed a purity of not less than 95%.
N-(1-Allylpiperidin-4-yl)-N′-[2-(2-furyl)-8-methyl-8H-
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]urea (8).
1
White solid; 45% yield; mp 179 °C (dec); H NMR (DMSO-d6) δ
9.11 (bs, 1H), 8.77−8.72 (m, 2H), 7.98−7.97 (m, 1H), 7.30−7.28 (m,
1H), 6.76−6.74 (m, 1H), 5.95−5.76 (m, 1H), 5.23−5.16 (m, 2H),
4.12 (s, 3H), 3.79−3.71 (m, 1H), 2.99 (d, J = 5.8 Hz, 2H), 2.73 (m,
2H), 2.14−2.08 (m, 2H), 1.97−1.91 (m, 2H), 1.62−1.43 (m, 2H). MS
(ESI): [MH]+ = 422.2. Anal. (C20H23N9O2) C, H, N.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-(1-prop-2-yn-1-ylpiperidin-4-yl)urea
(9). White solid; 38% yield; mp 212 °C (dec); 1H NMR (DMSO-d6) δ
9.11 (bs, 1H), 8.75 (s, 1 H), 8.67 (bd, J = 7.6 Hz, 1H), 7.98−7.97 (m,
1H), 7.30−7.28 (m, 1H), 6.76−6.74 (m, 1H), 4.11 (s, 3H), 3.74−3.55
(m, 1H), 3.28 (s, 2H), 3.18 (s, 1H), 2.78−2.72 (m, 2H), 2.37−2.32
(m, 2H), 2.04−1.94 (m, 2H), 1.55−1.51 (m, 2H). MS (ESI): [MH]+
= 420.2. Anal. (C20H21N9O2) C, H, N.
N-[1-(Cyclohexylmethyl)piperidin-4-yl]-N′-[2-(2-furyl)-8-
methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]-
urea (10). White solid; 34% yield; mp 218 °C; 1H NMR (DMSO-d6)
8.97 (bs, 1H), 8.74 (m, 2H), 7.98 (m, 1H), 7.29 (m, 1H), 6.77 (m,
1H), 4.12 (s, 3H), 3.81 (m, 1H), 2.64 (m, 3H), 2.14−2.06 (m, 4H),
1.85 (m, 2H), 1.75−1.38 (m, 6H), 1.21−1.07 (m, 4H), 0.80 (m, 2H).
MS (ESI): [MH]+ = 477.9. Anal. (C24H31N9O2) C, H, N.
Preparation of 4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carbonyl Chloride (4). A solution of 4-amino-1-
benzylpiperidine (1.14 g, 6 mmol) in anhydrous THF (40 mL) was
added in one portion to a solution of triphosgene (3 g, 10 mmol) in
anhydrous CH2Cl2 (13 mL), previously cooled to 0 °C. TEA
(triethylamine, 5 g, 50 mmol) was then added dropwise, and the
resulting mixture was stirred at the room temperature for 15 min. The
precipitate was filtered off and washed with THF. The filtrate was
concentrated to give a brown oil. IR spectroscopy of the crude material
confirmed the formation of the isocyanate species. 2-(2-Furyl)-8-
methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine 3
(0.5 g, 2 mmol) was added to the crude isocyanate, and the mixture
was heated at 140 °C for 50 min without solvents. The mixture was
slowly cooled to 0 °C, followed by the addition of methanol. The
compound that precipitated was collected by filtration and repeatedly
N-(1-Benzylpiperidin-4-yl)-N′-[2-(2-furyl)-8-methyl-8H-
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]urea (11).
1
White solid; 40% yield; mp 167 °C; H NMR (DMSO-d6) δ 8.99
(bs, 1H), 8.79 (m, 2H), 7.98 (m, 1H), 7.53−7.23 (m, 6H), 6.75 (m,
1H), 4.12 (s, 3H), 3.81 (m,1 H), 3.32 (s, 2H), 2.74 (m, 2H), 1.98−
1.24 (m, 6H). MS (ESI): [MH]+ = 472.21. Anal. (C24H25N9O2) C, H,
N.
N-[1-(4-Fluorobenzyl)piperidin-4-yl]-N′-[2-(2-furyl)-8-meth-
yl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]urea
(12). White solid; 42% yield; mp 188 °C; 1H NMR (DMSO-d6) δ 9.12
(bs, 1H), 8.76 (bm, 2H), 7.97 (m, 1H), 7.37−7.14 (m, 5H), 6.76−
6.74 (m, 1H), 4.12 (s, 3H), 3.71 (m, 1H), 3.48 (s, 2H), 2.73 (m, 2H),
2.12−1.83 (m, 4H), 1.59 (m, 2H). MS (ESI): [MH]+ = 490.2. Anal.
(C24H24FN9O2) C, H, N.
N-[1-(4-Chlorobenzyl)piperidin-4-yl]-N′-[2-(2-furyl)-8-meth-
yl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]urea
(13). White solid; 35% yield; mp 183 °C; 1H NMR (DMSO-d6) δ 9.07
(bs, 1H), 8.79−8.75 (bm, 2H), 7.97 (m, 1H), 7.37−7.28 (m, 5H),
6.76−6.74 (m, 1H), 4.12 (s, 3H), 3.78 (m, 1H), 3.49 (s, 2H), 2.72 (m,
2H), 2.20 (m, 2H), 1.95 (m, 2H), 1.56 (m, 2H). MS (ESI): [MH]+ =
506.0. Anal. (C24H24ClN9O2) C, H, N.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-[1-(4-methylbenzyl)piperidin-4-yl]-
urea (14). White solid; 30% yield; mp 213 °C; 1H NMR (DMSO-d6)
δ 9.08 (bs, 1H), 8.76−8.73 (bm, 2H), 7.97 (m, 1H), 7.29−7.11 (m,
5H), 6.76−6.74 (m, 1H), 4.12 (s, 3H), 3.76 (m, 1H), 3.44 (s, 2H),
1
washed with methanol: white solid; 75% yield; mp 247 °C; H NMR
(DMSO-d6) δ 9.25 (bd, J = 10.8 Hz, 1H), 8.77−8.71 (m, 2H), 7.98
(m, 1H), 7.29 (m, 1H), 6.77−6.74 (m, 1H), 4.12 (s, 3H), 3.98 (m,
1H), 3.35 (m, 2H), 3.16 (m, 2H), 2.08 (m, 2H), 1.61 (m, 2H). Anal.
(C18H18ClN9O3) C, H, N. MS (ESI): [MH]+ = 444.85 (0%); 382.0
(100%).
Preparation of N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]-N′-piperidin-4-ylurea (5).
Trifluoroacetic acid (200 μL) was added to a solution of 4 (0.5 g,
1.1 mmol) in a mixture of H2O/CH3CN (1:1, 60 mL), and the
mixture was stirred at 80 °C for 2 h. The solvents were removed under
reduced pressure, and the product was purified by crystallization from
dichloromethane/diethyl ether: white solid; 91% yield; mp 208 °C; 1H
NMR (DMSO-d6) δ 9.25 (bs, 1H), 8.86 (bm, 1H), 8.77−8.73 (m,
2H), 7.98 (m, 1H), 7.29 (m, 1H), 6.77−6.74 (m, 1H), 4.12 (m, 3H),
3.98 (m, 1H), 3.27 (m, 2H), 3.08 (m, 2H), 2.09 (m, 2H), 1.82 (m,
2H). MS (ESI): [MH]+ = 382.0. Anal. (C17H19N9O2) C, H, N.
General Procedure for the Preparation of N-[2-(2-Furyl)-8-
methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]-
N′-(1-substituted-piperidin-4-yl)urea Derivatives 6−20. To a
solution of 5 (115 mg, 0.3 mmol) in DMF (10 mL) were added
K2CO3 (45 mg, 0.33 mmol) and the appropriate alkyl halide (0.33
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2.68 (m, 2H), 2.28−2.11 (m, 5H), 1.98−1.89 (m,2 H), 1.54−1.49 (m,
2H). MS (ESI): [MH]+ = 486.2. Anal. (C25H27N9O2) C, H, N.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-[1-(4-methoxybenzyl)piperidin-4-yl]-
urea (15). White solid; 33% yield; mp 217 °C; 1H NMR (DMSO-d6)
δ 9.08 (bs, 1H), 8.75 (m, 2H), 7.97 (m, 1H), 7.28 (m, 1H), 7.23 (dd, J
= 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.75 (m, 1H), 4.12 (s, 3H),
3.73 (m, 4H), 3.42 (s, 2H), 2.71 (m, 2H), 2.21−2.12 (m, 2H), 1.95−
1.90 (m, 2H), 1.58−1.49 (m, 2H). MS (ESI): [MH]+ = 502.1. Anal.
(C25H27N9O3) C, H, N.
N-[1-(4-Cyanobenzyl)piperidin-4-yl]-N′-[2-(2-furyl)-8-meth-
yl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]urea
(16). White solid; 30% yield; mp 239 °C; 1H NMR (DMSO-d6) δ 8.74
(m, 3H), 7.97 (m, 1H), 7.80 (dd, J = 7.8 Hz, 2H), 7.55 (dd, J = 8 Hz,
2H), 7.28 (m, 1H), 6.77 (m, 1H), 4.12 (s, 3H), 3.79−3.60 (m, 3H),
2.69 (m, 2H), 2.18 (m, 2H), 1.95 (m, 2H), 1.59 (m, 2H). MS (ESI):
[MH]+ = 496.9. Anal. (C25H24N10O2) C, H, N.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-[1-(4-nitrobenzyl)piperidin-4-yl]urea
(17). White solid; 48% yield; mp 228 °C; 1H NMR (DMSO-d6) δ 9.18
(bs, 1H), 8.82−8.80 (bd, J = 7.2 Hz, 1H), 8.75 (s, 1H), 8.21 (dd, J =
8.8 Hz, 2H), 7.97 (m, 1H), 7.64 (dd, J = 8.8 Hz, 2H), 7.28 (m, 1H),
6.75 (m, 1H), 4.12 (s, 3H), 3.66 (m, 3H), 2.77−2.71 (m, 2H), 2.26
(m, 2H), 1.93 (m, 2H), 1.59 (m, 2H). MS (ESI): [MH]+ = 516.9.
Anal. (C24H24N10O4) C, H, N.
3H), 3.78−3.75 (m, 4H), 3.07 (m, 2H), 1.84 (m, 2H), 1.40 (m, 2H),
1.06 (d, J = 6.6 Hz, 6H). MS (ESI): [MH]+ = 467.0. Anal.
(C21H26N10O3) C, H, N.
N-Cyclopentyl-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carboxamide (23). White solid; 50% yield; mp 205
°C; 1H NMR (DMSO-d6) δ 9.20 (bs, 1H), 8.79 (bd, J = 7.6 Hz, 1H),
8.73 (s, 1H), 7.97 (m, 1H), 7.28 (m, 1H), 6.76−6.73 (m, 1H), 6.27
(bd, J = 7 Hz, 1H), 4.10 (s, 3H), 3.93−3.74 (m, 4H), 3.03 (m, 2H),
1.90−1.35 (m, 12H). MS (ESI): [MH]+ = 493.3. Anal. (C23H28N10O3)
C, H, N.
N-Cyclohexyl-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carboxamide (24). White solid; 41% yield; mp 210
1
°C; H NMR (DMSO-d6) δ 9.13 (bs, 1H), 8.75 (bm, 2H), 7.97 (m,
1H), 7.28 (m, 1H), 6.76 (m, 1H), 6.20 (bd, J = 7.6 Hz, 1H), 4.11 (s,
3H), 3.80−3.74 (m, 3H), 3.40 (m, 1H), 3.00 (m, 2H), 1.95−1.12 (m,
14 H). MS (ESI): [MH]+ = 507.2. Anal. (C24H30N10O3) C, H, N.
N-Cycloheptyl-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carboxamide (25). White solid; 38% yield; mp 165
1
°C; H NMR (DMSO-d6) δ 9.21 (bs, 1H), 8.76 (bm, 2H), 7.97 (m,
1H), 7.28 (m, 1H), 6.76−6.74 (m, 1H), 6.23 (d, J = 7.8 Hz, 1H), 4.11
(s, 3H), 3.77 (m, 4H), 3.05−2.94 (m, 2H), 1.90−1.75 (m, 16H). MS
(ESI): [MH]+ = 521.1. Anal. (C25H32N10O3) C, H, N.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-
urea (18). White solid; 29% yield; mp 226 °C; 1H NMR (DMSO-d6)
δ 9.13 (bs, 1H), 8.76−8.74 (bm, 2H), 8.52−8.45 (m, 2H), 7.98−7.97
(m, 1H), 7.77−7.71 (m, 1H), 7.39−7.33 (m, 1H), 7.30−7.28 (m, 1H),
6.76−6.74 (m, 1H), 4.12 (s, 3H), 3.68 (m, 1H), 3.54 (s, 2H), 2.76−
2.70 (m, 2H), 2.27−2.17 (m, 2H), 1.99−1.90 (m, 2H), 1.61−1.51 (m,
2H). MS (ESI): [MH]+ = 473.4. Anal. (C23H24N10O2) C, H, N.
N-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]-N′-[1-(pyridin-4-ylmethyl)piperidin-4-yl]-
urea (19). White solid; 43% yield; mp 247 °C (dec); 1H NMR
(DMSO-d6) δ 9.08 (bs, 1H), 8.80−8.76 (bm, 2H), 8.51 (dd, J = 6.2
Hz, 2H), 7.98 (m, 1H),7.35 (dd, J = 5.8 Hz, 2H), 7.29 (m, 1H), 6.75
(m, 1H),4.12 (s, 3H), 3.69 (m, 1H), 3.55 (s, 2H), 2.76−2.70 (m, 2H),
2.29−2.19 (m, 2H), 1.99−1.93 (m, 2H), 1.64−1.54 (m, 2H). MS
(ESI): [MH]+ = 473.2. Anal. (C23H24N10O2) C, H, N.
N-Benzyl-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carboxamide (26). White solid; 39% yield; mp 227
°C; 1H NMR (DMSO-d6) δ 9.13 (bs, 1H), 8.79 (bd, J = 7.6 Hz, 1H),
8.75 (s, 1H), 7.98−7.97 (m, 1H), 7.34−7.21 (m, 6H), 7.15 (m, 1H),
6.76−6.74 (m, 1H), 4.26 (d, J = 5.6 Hz, 2H), 4.12 (s, 3H), 3.89−3.81
(m, 3H), 3.10−3.04 (m, 2H), 1.93−1.89 (m, 2H), 1.45−1.38 (m, 2H).
MS (ESI): [MH]+ = 515.3. Anal. (C25H26N10O3) C, H, N.
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-(2-
phenylethyl)piperidine-1-carboxamide (27). White solid; 63%
1
yield; mp 216 °C; H NMR DMSO-d6) δ 9.17 (bs, 1H), 8.75 (bm,
2H), 7.97 (m, 1H), 7.31−7.18 (m, 6H), 6.75 (m, 1H), 6.72 (bt, J = 7
Hz, 1H), 4.11 (s, 3H), 3.80 (m, 3H), 3.24 (m, 2H), 3.05 (m, 2H), 2.73
(m, 2H), 1.82 (m, 2H), 1.40 (m, 2H). MS (ESI): [MH]+ = 529.3.
Anal. (C26H28N10O3) C, H, N.
Ethyl {4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-(3-
phenylpropyl)piperidine-1-carboxamide (28). White solid; 39%
triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]piperidin-
1
1-yl}acetate (20). White solid; 50% yield; mp 207 °C; H NMR
1
(DMSO-d6) δ 9.11 (bs, 1H), 8.76 (bm, 2H), 7.98−7.97 (m, 1H),
7.30−7.28 (m, 1H), 6.76−6.74 (m, 1H), 4.15−4.04 (m, 5H), 3.69 (m,
1H), 3.24 (s, 2H), 2.75 (m, 2H), 2.40 (m, 2H), 1.98−1.82 (m, 2H),
1.61−1.42 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ =
468.2. Anal. (C21H25N9O2) C, H, N.
yield; mp 218 °C; H NMR (DMSO-d6) δ 9.13 (bs, 1H), 8.75 (bm,
2H), 7.97 (m, 1H), 7.29−7.16 (m, 6H), 6.76−6.74 (m, 1H), 6.55 (bt,
J = 7 Hz, 1H), 4.10 (s, 3H), 3.81−3.74 (m, 3H), 3.07−3.02 (m, 4H),
2.61−2.53 (m, 2H), 1.95−1.72 (m, 4H), 1.41 (m, 2H). MS (ESI):
[MH]+ = 543.3. Anal. (C27H30N10O3) C, H, N.
General Procedure for the Preparation of N-Substituted 4-
[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-yl]amino}carbonyl)amino]piperidine-1-carboxa-
mide Derivatives 21−34. The chlorocarbonyl derivative 4 (90 mg,
0.2 mmol) was suspended in 1,4-dioxane (3 mL) and cooled to 0 °C.
TEA (0.018 g, 0.18 mmol) and the appropriate amine (0.3 mmol)
were then added, and the mixture was stirred for 1 h at 0 °C and for an
additional 3 h at room temperature. The solvent was removed under
reduce pressure, and the residue was purified by column
chromatography, eluting with EtOAc (21−31), or via HPLC (33, 34).
N-Ethyl-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-
piperidine-1-carboxamide (21). White solid; 30% yield; mp 192
°C; 1H NMR (DMSO-d6) δ 9.14 (bs, 1H), 8.76 (bm, 2H), 7.98−7.97
(m, 1H), 7.29−7.28 (m, 1H), 6.76−6.75 (m, 1H), 6.52 (bm, 1H), 4.11
(s, 3H), 3.92−3.83 (m, 3H), 3.07−3.01 (m, 4H), 1.91 (m, 2H), 1.43
(m, 2H), 1.02 (t, J = 7.2 Hz, 3H). MS (ESI): [MH]+ = 453.3. Anal.
(C20H24N10O3) C, H, N.
N-(4-Fluorobenzyl)-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo-
[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)-
amino]piperidine-1-carboxamide (29). White solid; 30% yield;
1
mp 217 °C; H NMR (DMSO-d6) δ 9.19 (bs, 1H), 8.78 (bd, J = 7.6
Hz, 1H), 8.75 (s, 1H), 7.97 (m, 1H), 7.34−7.26 (m, 3H), 7.19−7.14
(m, 3H), 6.75 (m, 1H), 4.23 (d, J = 5.6 Hz, 2H), 4.12 (s, 3H), 3.86−
3.79 (m, 3H), 3.06 (m, 2H), 1.89 (m, 2H), 1.40 (m, 2H). MS (ESI):
[MH]+ = 533.2. Anal. (C25H25FN10O3) C, H, N.
N-(4-Chlorobenzyl)-4-[({[2-(2-furyl)-8-methyl-8H-pyrazolo-
[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]amino}carbonyl)-
amino]piperidine-1-carboxamide (30). White solid; 36% yield;
mp 186 °C (dec); 1H NMR (DMSO-d6) δ 8.80−8.77 (bm, 3H), 7.98
(m, 1H), 7.50−7.49 (m, 1H), 7.38 (dd, J = 8.4 Hz, 2H), 7.28 (dd, J =
8.4 Hz, 2H), 7.20 (bm, 1H), 6.76−6.75 (m, 1H), 4.23 (d, J = 5.6 Hz,
2H), 4.12 (s, 3H), 3.84−3.80 (m, 3H), 3.06 (m, 2H), 1.92−1.89 (m,
2H), 1.42−1.40 (m, 2H). MS (ESI): [MH]+ = 549.5. Anal.
(C25H25ClN10O3) C, H, N.
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-(4-
methylbenzyl)piperidine-1-carboxamide (31). White solid; 45%
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-isopropylpi-
peridine-1-carboxamide (22). White solid; 30% yield; mp 224 °C;
1H NMR (DMSO-d6) δ 9.19 (bs, 1H), 8.74 (bm, 2H), 7.97 (m, 1H),
1
yield; mp 232 °C; H NMR (DMSO-d6) δ 9.12 (bs, 1H), 8.80−8.75
(bm, 2H), 7.97 (m, 1H), 7.28 (m, 1H), 7.18−7.07 (m, 5H), 6.75 (m,
1H), 4.21 (d, J = 5.6 Hz, 2H), 4.12 (s, 3H), 3.86−3.79 (m, 3H), 3.11−
7.28 (m, 1H), 6.77−6.74 (m, 1H), 6.20 (bd, J = 7.8 Hz, 1H), 4.11 (s,
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3.00 (m, 2H), 2.27 (s, 3H), 1.93−1.88 (m, 2H), 1.43−1.36 (m, 2H).
MS (ESI): [MH]+ = 529.0. Anal. (C26H28N10O3) C, H, N.
Measurement of Cyclic AMP Levels in CHO Cells Trans-
fected with Human A2B and A3 Adenosine Receptors. CHO
cells transfected with human A2BAR or A3AR were washed with
phosphate buffered saline, diluted trypsin and centrifuged for 10 min
at 200g. The pellet containing CHO cells (1 × 106 cells/assay) was
suspended in 0.5 mL of incubation mixture (mM): NaCl 15, KCl 0.27,
NaH2PO4 0.037, MgSO4 0.1, CaCl2 0.1, Hepes 0.01, MgCl2 1, glucose
0.5, pH 7.4 at 37 °C, 2 IU/mL adenosine deaminase, and 4-(3-butoxy-
4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as phosphodies-
terase inhibitor. The mixture was preincubated for 10 min in a shaking
bath at 37 °C. The potency of the antagonists versus hA2BARs was
determined by antagonism of NECA (100 nM) induced stimulation of
cyclic AMP levels.35 In addition, the potency of the antagonists to
hA3ARs was determined in the presence of 1 μM forskolin by
antagonism of Cl-IB-MECA (100 nM) induced inhibition of cyclic
AMP levels.34 The reaction was terminated by the addition of cold 6%
trichloroacetic acid (TCA). The TCA suspension was centrifuged at
2000g for 10 min at 4 °C, and the supernatant was extracted four times
with water saturated diethyl ether. The final aqueous solution was
tested for cyclic AMP levels by a competition protein binding assay.
Samples of cyclic AMP standard (0−10 pmol) were added to each test
tube containing the incubation buffer (0.1 M Trizma base, 8.0 mM
aminophylline, 6.0 mM 2 mercaptoethanol, pH 7.4) and [3H] cyclic
AMP in a total volume of 0.5 mL. The binding protein was added to
the samples and incubated at 4 °C for 150 min. The samples, after the
addition of charcoal, were centrifuged at 2000g for 10 min. The clear
supernatant was counted by using a Packard Tri Carb 2810 TR
scintillation counter (Perkin-Elmer).
Data Analysis. The protein concentration was determined
according to a Bio-Rad method36 with bovine albumin as a standard
reference. Inhibitory binding constants, Ki, were calculated from the
IC50 values according to the Cheng and Prusoff equation37 Ki = IC50/
(1 + [C*]/KD*), where [C*] is the concentration of the radioligand
and KD* its dissociation constant. A weighted nonlinear least-squares
curve fitting program LIGAND38 was also used for computer analysis
of inhibition experiments. Potency values (IC50) obtained in cyclic
AMP assays were calculated by nonlinear regression analysis using the
equation for a sigmoid concentration−response curve (GraphPad
Prism, San Diego, CA, U.S.). All experimental data are expressed as
geometric mean with 95% confidence limits in parentheses of three or
four independent experiments performed in duplicate.
Molecular Modeling. Receptor Docking. Prior to docking
calculations, the Epik software was used to calculate the most relevant
ionization and tautomeric state of compounds 10.39 Then the Glide
program of the Schrodinger package40 was used to dock 10 to the
hA3AR structure. The receptor grid generation was performed for the
box with a center in the putative binding site. The size of the box was
determined automatically. The extra precision mode (XP) of Glide
was used for docking. The ligand scaling factor was set to 1.0. The
geometry of the ligand binding site of the complex between 10 and the
receptor was then optimized. The binding site was defined as 10 and
all amino acid residues located within 8 Å from the ligand. All the
receptor residues located within 2 Å from the binding site were used as
a shell. The following parameters of energy minimization were used:
OPLS2005 force field was used. Water was used as an implicit solvent,
and a maximum of 5000 iterations of the Polak−Ribier conjugate
gradient minimization method was used with a convergence threshold
of 0.01 kJ mol−1 Å−1. All complex pictures were rendered employing
the UCSF Chimera software.41
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-(4-
methoxybenzyl)piperidine-1-carboxamide (32). White solid;
1
45% yield; mp 224 °C; H NMR DMSO-d6) δ 9.18 (bs, 1H), 8.81
(bd, J = 7.8 Hz, 1H), 8.73 (s, 1H), 7.96 (m, 1H), 7.27 (m, 1H), 7.19
(dd, J = 8.8 Hz, 2H), 7.04 (bt, J = 5.6 Hz, 1H), 6.87 (dd, J = 8.8 Hz,
2H), 6.76−6.73 (m, 1H), 4.18 (d, J = 5.6 Hz, 2H), 4.10 (s, 3H), 3.84
(m, 3H), 3.72 (s, 3H), 3.02 (m, 2H), 1.83 (m, 2H), 1.42 (m, 2H). MS
(ESI): [MH]+ = 545.2. Anal. (C26H28N10O4) C, H, N.
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-pyridin-4-ylpi-
peridine-1-carboxamide Trifluoroacetate (33). White solid; 41%
1
yield; mp 157 °C; H NMR (DMSO-d6) δ 10.2 (bs, 1H), 9.22 (bs,
1H), 8.77 (m, 2H), 8.56 (dd, J = 7.2 Hz, 2H), 7.98 (m, 1H), 7.91 (dd,
J = 7.4 Hz, 2H), 7.30 (m, 1H), 6.77−6.75 (m, 1H), 4.11 (s, 3H), 4.02
(m, 3H), 3.39 (m, 2H), 2.17−2.03 (m, 2H), 1.66−1.43 (m, 2H). MS
(ESI): [MH]+ = 502.4. Anal. (C25H23F3N11O5) C, H, N.
4-[({[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl]amino}carbonyl)amino]-N-(4-methylpi-
perazin-1-yl)piperidine-1-carboxamide Trifluoroacetate (34).
White solid; 44% yield; mp 101 °C; 1H NMR (DMSO-d6) δ 9.79 (bs,
1H), 9.16 (bs, 1H), 8.77 (m, 2H), 7.98 (m, 1H), 7.30 (m, 1H), 6.77−
6.74 (m, 1H), 4.11 (s, 3H), 3.81 (m, 3H), 3.71−3.43 (m, 6H), 3.08−
3.02 (m, 4H), 2.82 (s, 3H), 2.01−1.91 (m, 2H), 1.61−1.41 (m, 2H).
MS (ESI): [MH]+ = 507.8. Anal. (C25H29F3N11O5) C, H, N.
Biology. Evaluation of Affinity (Ki) and Potency (IC50) of the
Novel Adenosine Compounds. CHO (Chinese Hamster Ovary)
Membranes Preparation. The human A1, A2A, A2B, and A3
adenosine receptors have been transfected in CHO cells according
to the method previously described.31 The cells were grown adherently
and maintained in Dulbecco’s modified Eagle's medium with nutrient
mixture F12 (DMEM/F12) without nucleosides, containing 10% fetal
calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), L-
glutamine (2 mM), and Geneticin (G418, 0.2 mg/mL) at 37 °C in 5%
CO2, 95% air. For membrane preparation the culture medium was
removed and the cells were washed with PBS and scraped off T75
flasks in ice-cold hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA, pH
7.4). The cell suspension was homogenized with a Polytron, and the
homogenate was centrifuged for 10 min at 1000g. The supernatant was
then centrifuged for 30 min at 100000g. The membrane pellet was
suspended in (a) 50 mM Tris-HCl buffer, pH 7.4, for A1ARs; (b) 50
mM Tris-HCl, 10 mM MgCl2 buffer, pH 7.4, for A2AARs; (c) 50 mM
Tris-HCl, 10 mM MgCl2, 1 mM EDTA buffer, pH 7.4, for A3ARs. The
cell suspension was incubated with 2 IU/mL of adenosine deaminase
for 30 min at 37 °C. The membrane preparation was used to perform
binding experiments.
Human Cloned A1, A2A, and A3 Adenosine Receptor Binding
Assay. All synthesized compounds have been tested for their affinity
to human A1, A2A, and A3 ARs. Displacement binding experiments of
[3H]DPCPX (1 nM) to hA1 CHO membranes (50 μg of protein/
assay) and at least six to eight different concentrations of examined
antagonists were performed for 120 min at 25 °C. Nonspecific binding
was determined in the presence of 1 μM DPCPX, and this was always
≤10% of the total binding.32 Inhibition binding experiments of
[3H]ZM241385 (2 nM) to hA2A CHO membranes (50 μg of protein/
assay) and at least six to eight different concentrations of the studied
compounds were performed for 60 min at 4 °C. Nonspecific binding
was determined in the presence of 1 μM ZM241385 and was about
20% of total binding.33 Competition binding experiments of
[3H]MRE-3008-F20 (1 nM) to hA3CHO membranes (50 μg of
protein/assay) and at least six to eight different concentrations of
examined ligands were performed for 120 min at 4 °C. Nonspecific
binding was defined as binding in the presence of 1 μM MRE-3008-
F20 and was about 25% of total binding.34 Bound and free
radioactivity was separated by filtering the assay mixture through
Whatman GF/B glass fiber filters which were washed three times with
ice-cold buffer. The filter bound radioactivity was counted by using a
Packard Tri Carb 2810 TR scintillation counter (Perkin-Elmer).
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional table reporting the predicted PK parameters. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Journal of Medicinal Chemistry
Article
adenosine receptors in human colorectal cancer is reflected in
peripheral blood cells. Clin. Cancer Res. 2004, 10, 5895−5901.
(12) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.;
Monopoli, A.; Ongini, E.; Varani, K.; Borea, P. A. 7-Substituted 5-
amino-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as
A2A adenosine receptor antagonists: a study on the importance of
modifications at the side chain on the activity and solubility. J. Med.
Chem. 2002, 45, 115−126.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-0532-455921. Fax: +39-0532-455921. E-mail:
baraldi@unife.it.
■
Present Address
#Alta Vetta Pharmaceutical Consulting, LLC, 3 Skipwith Court,
Durham, North Carolina 27707.
(13) Baraldi, P. G.; Tabrizi, M. A.; Romagnoli, R.; El-Kashef, H.;
Preti, D.; Bovero, A.; Fruttarolo, F.; Gordaliza, M.; Borea, P. A.
Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine template: organic and
medicinal chemistry approach. Curr. Org. Chem. 2006, 10, 259−275.
(14) Baraldi, P. G.; Fruttarolo, F.; Tabrizi, M. A.; Preti, D.;
Romagnoli, R.; El-Kashef, H.; Moorman, A.; Varani, K.; Gessi, S.;
Merighi, S.; Borea, P. A. Design, synthesis, and biological evaluation of
C9- and C2-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines
as new A2A and A3 adenosine receptors antagonists. J. Med. Chem.
2003, 46, 1229−1241.
(15) Maconi, A.; Pastorin, G.; Da Ros, T.; Spalluto, G.; Gao, Z. G.;
Jacobson, K. A.; Baraldi, P. G.; Cacciari, B.; Varani, K.; Moro, S.;
Borea, P. A. Synthesis, biological properties, and molecular modeling
investigation of the first potent, selective, and water-soluble human A3
adenosine receptor antagonist. J. Med. Chem. 2002, 45, 3579−3582.
(16) Baraldi, P. G.; Saponaro, G.; Tabrizi, M. A.; Baraldi, S.;
Romagnoli, R.; Moorman, A.; Varani, K.; Borea, P. A.; Preti, D.
Pyrrolo- and pyrazolo-[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as ad-
enosine receptor antagonists. Bioorg. Med. Chem. 2012, 20, 1046−
1059.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank King Pharmaceuticals, Inc., Research and Develop-
ment, NC, for financial support. We also thank Prof. Karl-
Norbert Klotz for cDNA encoding the human adenosine
receptors. The authors thank Dr. Erika Marzola for technical
assistance.
ABBREVIATIONS USED
■
AR, adenosine receptor; CHO, Chinese hamster ovary; Cl-IB-
MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcar-
boxamide; DMEM, Dulbecco’s modified Eagle's medium;
EtOAc, ethyl acetate; IP3, inositol 1,4,5-trisphosphate; NECA,
5′-(N-ethylcarboxamido)adenosine; PTP, pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidine; TEA, triethylamine
(17) Cheong, S. L.; Dolzhenko, A. V.; Paoletta, S.; Lee, E. P. R.;
Kachler, S.; Federico, S.; Klotz, K. N.; Dolzhenko, A. V.; Spalluto, G.;
Moro, S.; Pastorin, G. 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? Bioorg. Med. Chem. 2011, 19, 6120−6134.
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