One-Pot Reaction to Obtain N,N′-Disubstituted Guanidines of Pyrazolo[4,3- e ][1,2,4]triazolo[1,5- c ]pyrimidine Scaffold as Human A3 Adenosine Receptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.5b00551

In this paper we describe an extension SAR study of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus as A₃AR antagonist. Our initial aim was to replace the phenylcarbamoyl moiety at the 5 position of PTP nucleus with a thiourea functiona

Full text of DOI:10.1021/acs.jmedchem.5b00551

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Brief Article
One-Pot Reaction to obtain N,N’-disubstituted Guanidines
of Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine
Scaffold as Human A3 Adenosine Receptor Antagonists
Pier Giovanni Baraldi, Stefania Baraldi, Giulia Saponaro, Mojgan Aghazadeh Tabrizi, Romeo
Romagnoli, Emanuela Ruggiero, Fabrizio Vincenzi, Pier Andrea Borea, and Katia Varani
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2015
Downloaded from http://pubs.acs.org on June 7, 2015
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Journal of Medicinal Chemistry
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One-Pot Reaction to obtain N,N’-disubstituted Guanidines of
Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine Scaffold as
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Human A Adenosine Receptor Antagonists
3
*,§
§
§
§
Pier Giovanni Baraldi, Stefania Baraldi, Giulia Saponaro, Mojgan Aghazadeh Tabrizi, Romeo
§
§
#
#
#
Romagnoli, Emanuela Ruggiero, Fabrizio Vincenzi , Pier Andrea Borea , Katia Varani
§
Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Ferrara, Via Fossato di Mortara 17,
4
4121, Italy
#
Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli Studi di Ferrara, Via Fossato di
Mortara 17, 44121, Italy
ABSTRACT: In this paper we describe an extension SAR study of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus as
A AR antagonist. Our initial aim was to replace the phenylcarbamoyl moiety at the 5 position of PTP nucleus with a
3
thiourea functionality, in order to evaluate the contribute of new structural modification against the A AR. The
3
synthesized compounds 12-25 were not characterized by the predicted side chain but with a 1,3 disubstituted guanidine,
which showed to be interesting A AR antagonists.
3
INTRODUCTION
an attractive scaffold for the preparation of adenosine re-
ceptor antagonists.
Adenosine, a ubiquitous nucleoside, regulates many
physiological functions through the activation of four
specific receptor subtypes, classified as A , A , A and A3
Our research group synthesized a large number of
7-9
compounds, characterized by the PTP nucleus, with
1
2A
2B
(
ARs), belonging to the superfamily of G-protein-coupled
very good binding profile as ligands for ARs. To perform a
structure-activity relationship (SAR) investigation, the
effect of substitutions at various positions of this scaffold
was evaluated. From these studies substituents responsi-
receptors (GPCRs). The A AR subtype is the most recently
characterized member of the family. Activation of A AR
subtype has been displayed to inhibit adenylate cyclase,
3
1
3
to increase the activity of phospholipase C and D, to pro-
ble of affinity for the human A AR were identified. In par-
3
++
mote intracellular Ca
and IP3 (inositol 1,4,5-
ticular the best potency and affinity towards the hA AR
3
trisphosphate) levels and to improve the release of in-
flammatory and allergic mediators from mast cells. The
was obtained by substitution at the C-2 position with a 2-
furyl or substituted phenyl moieties, combined with small
alkyl chain at the N-8 and substituted phenylcarbamoyl
2,3
potential therapeutic application, resulting from the acti-
vation of this receptor subtype, have been recently re-
7,10
moiety at the C-5 position. The 2-furyl moiety at posi-
4
viewed.
tion C-2, the methyl group at position N-8 combined with
the phenylcarbamoyl moiety at the C-5 position allowed
us to identified one of the best compound with high affin-
ity and selectivity for A AR (1, Chart 1, hA = 0.16 nM, hA
The A AR antagonists are presently undergoing biologi-
3
cal testing for their potential use in the treatment of
stroke, neurodegenerative diseases, allergy, asthma and
COPD (Chronic Obstructive Pulmonary Disease,Thomson
Reuters Integrity source). It is becoming increasingly evi-
3
3
1
11
=
594 nM, hA2A = 381 nM, hA = 222 nM).
2B
Chart 1. Preliminary rational design for the synthesis
dent that the A AR antagonists could be therapeutically
3
of new A antagonists.
3
5
useful for the acute treatment of glaucoma. It has been
also observed that in central nervous system, the pro-
longed A AR stimulation decrease the currents generated
3
by gamma aminobutyrric acid in epileptic tissues suggest-
ing that A AR antagonists may offer therapeutic opportu-
3
6
nities in various forms of human epilepsy.
The
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine
(
PTP) nucleus has been revealed constantly to provide as
1
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Page 2 of 7
In light of this, our aim was to synthesize new hA AR
antagonists by replacing the urea group with a thiourea
moiety at C-5 position of PTP scaffold to evaluate the ef-
fect of this substitution on biological activity.
Scheme 1
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
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1
1
2
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3
4
4
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5
5
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5
5
5
5
5
6
Chart 2.
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0
a
Reagents and conditions: i) CH NHNH , EtOH, 80 °C,
3
2
3h; ii) ethoxymethylenemalononitrile, benzene, 80 °C, 1h; iii)
EtOH, HCl 37%, 80 °C, 1h; iv) (EtO) CH, reflux, 20h; v) 2-
furoic hydrazide, 2-methoxyethanol, reflux, 24h; vi) Ph O,
2
260 °C, 1h; vii) HCl aq. 10%, reflux, 1h; viii) 1-methyl-2-
pyrrolidone, pTsOH, cyanamide, 160 °C, 6h.
3
Nevertheless, applying the classical synthetic approach
used for the synthesis of the urea derivatives (appropriat-
11
ed isocyanate, THF/ Rfx or dioxane/ Rfx) for the prepara-
tion of thiourea compounds, in presence of the appropri-
ate isothiocyanate, no reaction was observed (Chart 2).
The reaction between benzaldehyde and N-
methylhydrazine, commercially available, gave the corre-
sponding hydrazone 2. Subsequent reaction with ethox-
ymethylenemalononitrile and hydrolysis with concentrat-
ed HCl provided 4 in good yield. This synthetic procedure
starting from benzaldehyde allowed us to obtain only one
isomer of 3-amino-1-methyl-1H-pyrazole-4-carbonitrile
(4), to the contrary of which could be obtain by classical
pyrazole alkylation route. Consequently this synthetic
approach furnished us only the 2-(2-furyl)-8-methyl-8H-
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (9)
and not two isomers of tricycle amine N-7 and N-8 meth-
In the attempt to force the reaction by employing
stronger conditions, high temperature (160 °C) and an ex-
cess of phenyl isothiocyanate, compound 12 was achieved.
The structural investigation of the new compound
showed the presence of a guanidine moiety at the 5 posi-
tion of PTP nucleus rather than the predicted thiourea
functionality. Applying the new stronger conditions to
commercial appropriate isothiocyanate or available car-
bodiimide, choosen in agreement with the best results
obtained in phenylcarbamoyl series, compounds 13-23 and
1
5
1
6
2
3-25 were obtained according to method A or method B
respectively.
ylalkylated respectively.
The free amino group of 4 was transformed into the im-
idate 5 by refluxing in HC(OEt) that was reacted with 2-
furoic acid hydrazide in refluxing 2-methoxyethanol to
provide 6, following the method of Gatta et al. The pyra-
zolo[4,3-e]pyrimidine derivative 6 was converted through
a thermally-induced ciclyzation in diphenylether to the
tricyclic derivative 7 in good yield. Treatment of 7 with
diluted HCl induced pyrimidine ring opening to give 8
which was converted in 9 by treatment with an excess of
cyanamide and 1-methyl-2-pyrrolidone at 160 °C.
The guanidine functionality represents an important
moiety which turns up as a structural part in various drug
and agrochemicals and are actually top sold pharmaceuti-
cals appearing moreover in the top 200 prescription list of
3
1
7
1
2
2
010. Considering the importance of the new structural
motif (N,N’-disubstituted guanidines) we tested these de-
rivatives in binding and cAMP functional assays as hA AR
3
antagonists, using Chinese Hamster Ovary (CHO) cells.
CHEMISTRY
The synthetic route to achieve N’’-[2-(2-furyl)-8-
methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
5-yl]-N,N’-disubstituted guanidine 12-25 is depicted in
Scheme 2 [Method A, via isothiocyanate (12-23) or Meth-
od B, via carbodiimide(23-25)].
All final compounds were synthesized starting from 2-
2-furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-amine (9) prepared by known regioselec-
(
tive procedure reported in Scheme 1 (for experimental
13,14
section
see
supporting
information).
In view of the first attempt in which 9 was reacted with
phenyl isothiocyanate (10a) in mild condition without
achieving any product, the reaction was carried out in
stronger condition. Indeed, performing the reaction neat
11,13
(without solvent) at high temperature (160 °C) and using
an excess of commercial phenyl isothiocyanate yielded
the guanidine derivative 12. Applying the same approach
and different isothiocyanates, compounds 13-23 were syn-
2
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Journal of Medicinal Chemistry
Scheme 3. Proposed reaction mechanism to achieve
thesized, all characterized by 1,3-disubstituted guanidine
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
moiety at the 5 position of PTP nucleus.
compound 12-23
Scheme 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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2
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8
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0
1
2
3
4
5
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7
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9
0
RESULTS AND DISCUSSION
All the final compounds N’’-[2-(2-furyl)-8-methyl-8H-
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)-N,N’-
disubstituted guanidine (12-24, except 25) are character-
ized by a symmetric 1,3-guanidine moiety at the 5 position
of PTP.
The synthesized compounds (12-25) were evaluated in
radioligand binding assays to determine their affinities for
3
hA , A
and A ARs using [ H]DPCPX (1,3-dipropyl-8-
1
2A,
3
a
3
Reagents and conditions: i) neat, 160 °C, 18 h (except
cyclopentylxanthine), [ H]ZM241385 ([4-(2-[7-amino-2-(2-
furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-
ylamino]ethyl)phenol),
methoxyphenylcarbamoyl)
for 23, 36 h); ii) neat, 160 °C, 6h (except for 25, 12h).
3
[ H]MRE-3008-F20
(5-N-(4-
amino-8-propyl-2-(2-
To confirm the chemical structure of the new mole-
cules, we decided to perform the reaction of 9 in presence
of N,N’-dicyclohexylcarbodiimide (DCC, 11a) which per-
mitted us to obtain compound 23, previously achieved us-
ing cyclohexyl isothiocyante 10l. The analytical data of 23,
reached both via isothiocyanate (method A, 45% yield)
and via carbodiimide (method B, 60% yield), matched.
We extended this methodology using other commercially
available carbodiimide as starting material which led to
compounds 24 and 25 (Scheme 2, method B).
furyl)pyrazolo-[4,3-e]-[1,2,4]triazolo[1,5-c]pyrimidine), as
radioligands, respectively. Efficacy of the compounds ver-
sus hA AR was investigated by evaluating their capability
2B
to inhibit NECA (5′-(N-ethylcarboxamido)adenosine)-
stimulated (100 nM) cAMP production. Antagonism of
selected ligands versus hA AR was also assessed through
3
cAMP experiments evaluating their capability to block the
inhibitory effect mediated by 100 nM Cl-IBMECA (2-
6
chloro-N -(3-iodobenzyl)adenosine-5′-N-
All final compounds 12-25 were obtained in moderate
yield and was not observed the complete conversion of
the starting material 9 which was recovered as well.
methylcarboxamide).
Affinity data expressed as K values for A , A , and A
3
i
1
2A
receptors, and K values for hA and hA in the functional
B
2B
3
In Scheme 3 is reported the proposed reaction mecha-
nism to achieve compounds 12-23. During the reaction
between 9 and 10a-l, sulfidric acid was released (whose
characteristic smell was developed during Schlenk open-
ing) and the intermediate A, with PTP structure and ap-
propriate carbodiimidic functionality at position 5, was
observed by TLC and MS monitoring. In addition, the
strong conditions are probably responsible for the release
of the amine from the corresponding isothiocyanate. Sub-
sequent addition of the amine species to the carbodiimid-
ic intermediate provided the final compounds N,N’-
disubstituted guanidine (12-23). This mechanism was fur-
ther supported by formation of corresponding 1,3-
disubstituted symmetric thiourea as side product which
contributes to the low yield of the final compounds.
assays are listed in Table 1.
All
the
N’’-[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)-N,N’-disubstituted
guanidine 12-25 exhibited a good selectivity towards the
other AR subtypes (hA , hA : K >1000, hA : K >1000)
1
2A
i
2B
B
and 12 out of 18 showed a K values for hA AR lower than
i
3
89 nM. Derivative 12 is characterized by an unsubstituted
phenyl ring at 1,3 positions of guanidine moiety and pre-
sented a good binding profile against hA AR (hA : K = 79
3
3
i
nM, hA , hA : K > 1000, hA : K >1000). The introduc-
1
2A
i
2B
B
tion of different substituents both electron withdrawing
and electron donating functions have been examined. The
substitution of position 4 of phenyl ring with electron do-
nating moieties maintained or improved the hA AR affini-
3
ty (13, Me, hA : K = 63 nM and 14, OMe, hA : K = 42 nM)
3
i
3
i
respect to the unsubstituted 12 (hA : K = 79 nM).
3
i
The presence of a chlorine residue at the para position
(
16, 4-Cl-Ph, hA : Ki= 71 nM) did not show any improve-
3
ments in binding affinities respect to the unsubstituted
analogue 12. The introduction of different halogens, such
3
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Page 4 of 7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Biological Data of the Synthesized Compounds
hA ARs
hA ARs
hA3ARs
hA ARs
hA ARs
2B
1
2A
3
compd
R
R1
a
b
c
d
d
K (nM)
K (nM)
i
K (nM)
i
K (nM)
B
K (nM)
B
i
1
2
Ph
Ph
>1000 (10%)
>1000 (6%)
>1000 (7%)
>1000 (1%)
>1000 (8%)
>1000 (1%)
>1000 (1%)
>1000 (1%)
>1000 (1%)
>1000 (17%)
>1000 (3%)
>1000 (25%)
>1000 (4%)
>1000 (2%)
>1000 (1%)
>1000 (1%)
>1000 (16%)
>1000 (11%)
>1000 (10%)
>1000 (1%)
>1000 (1%)
>1000 (18%)
>1000 (1%)
>1000 (1%)
>1000 (3%)
>1000 (1%)
79 ± 8
63 ± 5
42 ± 5
67 ± 6
57 ± 5
>1000 (1%)
>1000 (3%)
>1000 (4%)
>1000 (1%)
>1000 (2%)
>1000 (1%)
>1000 (1%)
>1000 (1%)
>1000 (1%)
>1000 (1%)
>1000 (2%)
>1000 (4%)
>1000 (3%)
13
4-Me-Ph
4-OMe-Ph
4-F-Ph
4-Me-Ph
4-OMe-Ph
4-F-Ph
14
42 ± 4
129 ± 10
63 ± 5
1
5
122 ± 10
71 ± 6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
4-Cl-Ph
4-Cl-Ph
17
4-Br-Ph
4-Br-Ph
345 ± 36
30± 3
362 ± 34
33 ± 3
1
8
9
4-CN-Ph
4-NO2-Ph
3-OMe-Ph
3-Cl-Ph
4-CN-Ph
4-NO2-Ph
3-OMe-Ph
3-Cl-Ph
1
18 ± 2
13 ± 2
20
155 ± 11
89 ± 8
520 ± 57
29 ± 3
141 ± 12
74 ± 6
537 ± 49
32 ± 3
2
1
2
2
3-CF3-Ph
cyclohexyl
isopropyl
3-CF3-Ph
cyclohexyl
isopropyl
23
2
4
33 ± 4
36 ± 3
N,N-dimethyl-
aminopropyl
>1000
(19%)
3
25
ethyl
>1000 (22%)
>1000 (3%)
>1000 (3%)
>1000 (1%)
3
3
Affinity values obtained from displacement of specific [ H]DPCPX (a), [ H]ZM241385 (b) or [ H]MRE3008F20 (c) binding to hA
1
d
ARs, hA2A ARs or h A ARs, respectively (n=3-6). Potency (K ) calculated by using the modified Cheng-Prusoff analysis proposed
3
B
1
8
by Leff and Dougall. In parentheses are reported the % of inhibition to hA , A A2B or A CHO cells. Data are expressed as
1
2A,
3
means ± SEM.
A
B
1
20
00
80
120
1
9
3
2
1
100
80
60
40
20
0
1
12
4
19
2
3
4
60
1
40
12
20
0
-
11 -10
-9
-8
-7
-6
-5
-4
-11 -10
-9
-8
-7
-6
-5
-4
log [Drug] (M)
log [Drug] (M)
3
Figure 1. Competition curves of specific [ H]MRE 3008F20 binding to hA AR by selected compounds (A). Inhibition curves rep-
3
resenting the capability of the antagonists to block the inhibitory effect of 100 nM Cl-IB-MECA on Forskolin-induced cAMP ac-
cumulation (B).
The functionalization of the 3 position of the phenyl
ring was also investigated and from the binding data
emerged that the substitution in 4 is preferred for the
hA AR affinity (20, 3-OMe-Ph, hA : K = 155 nM vs 14, 4-
We evaluated the introduction of an alkyl cyclic sub-
stituent, such as cyclohexyl, that conferred a greater affin-
ity for A than the corresponding unsaturated cycle. In
3
fact, the cyclohexyl derivative 23 showed 2,5 folds high
affinity than that of compound 12 (23, hA : K = 29 nM vs
3
3
i
OMe-Ph, hA : K = 42 nM). Compound 22 (3-CF3-Ph, hA :
3
i
3
3
i
K = 520 nM) functionalized with a sterically hindrance
12, hA : K = 79 nM). Also compound 24 bearing an iso-
3 i
i
group, showed a dropped affinity against hA AR.
propyl alkyl chain, exhibited comparable affinity for A AR
3
3
with a K value of 33 nM.
i
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Journal of Medicinal Chemistry
The asymmetric alkyl guanidine 25, resulted completely
inactive against all four ARs subtypes (hA , hA , hA : K >
General Procedures for the Synthesis of N’’-[2-(2-
furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-yl]-N,N’-disubstituted guanidine (12-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
2A
i
1
000, hA : K >1000).
2B B
2
5)
Method A (via isothiocyanate, 12-23)
mixture of 2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-
Functional experiments showed that all examined de-
rivatives proved no activity at the hA AR with K values
2B
B
major than 1 µM (Table 1). Moreover, a good correlation
A
between affinity and potency values to hA AR was detect-
3
e][1,2,4]-triazolo[1,5-c]pyrimidin-5-amine 9 (0.2 mmol)
and the appropriate substituted aryl/ alkyl isothiocya-
nates (10a-k and 10l respectively, 1.0 mmol except for
compound 23 which required 2.0 mmol) was stirred neat
ed (Figure 1). The majority of the novel compounds dis-
played good affinity for the A AR subtype and high poten-
3
cy as expressed by the KB values. Compound 19 is the
most affine, potent and selective of this series as indicated
by the K value of 18 nM vs hA ARs closely similar to the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(without solvent) at 160 °C in a Schlenk tube for 18 hours
i
3
(except for compound 23 for which the reaction time in-
creased to 36 hours). The final compounds (12-23) were
purified by flash chromatography, eluting with EtOAc
(ethyl acetate) /Petroleum Ether (7:3).
K value of 13 nM. Interestingly this compound is not able
B
to bind the other AR subtypes (Table 1) showing a com-
plete selectivity versus hA ARs.
3
CONCLUSION
N’’-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]-N,N’-
diphenylguanidine (12)
We synthesized by one-pot reaction a series of new
compound characterized by a guanidine function at posi-
tion 5 of the PTP scaffold. From our biological data
emerged that the synthesized molecules showed a good
Pale white solid; C H N O; 40% yield; mp 241 °C. MS
27
19
9
+
1
(ESI): [MH] = 450.5. H NMR (200 MHz, DMSO-d ): δ
6
affinity and potency for the hA AR with a complete selec-
3
4.05 (s, 3H), 6.70-6.73 (m, 1H), 7.03-7.10 (m, 2H), 7.16-7.19
(m, 1H), 7.27-7.35 (m, 4H), 7.54-7.58 (m, 4H), 7.93-7.96
(m, 1H), 8.59 (s, 1H), 10.15 (bs, 2H).
tivity versus the other subtypes (hA , hA : K > 1000, hA :
1
2A
i
2B
K > 1000).
B
EXPERIMENTAL SECTION
Experimental data for compounds 13-23 are reported in
the SI (S5-S6).
Chemistry. Materials and Methods. All commercially
available reagents and anhydrous solvents were furnished
by Sigma-Aldrich or Alfa Aesar and used without further
purification. Reaction progress and product mixtures
were monitored by TLC on silica gel (precoated F254 Ma-
cherey-Negel plates) and visualized by UV lamp (254 nm
light source) or with ninidrine ethanolic solution. Chro-
matoghraphy was performed on Merck 230-400 mesh sili-
ca gel. The organic layer obtained after extraction from
Method B (via carbodiimide, 23-25)
A
mixture of 2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-
e][1,2,4]-triazolo[1,5-c]pyrimidin-5-amine 9 (0.2 mmol)
and the appropriate commercial carbodiimide (11a-b, 0.6
mmol; 11c, 1.0 mmol) was stirred neat at 160 °C in a
Schlenk tube for 6 hours (except for compound 25 for
which the reaction time increased to 12 hours). The final
desired compounds (23-25) were purified by flash chro-
matography, eluting with EtOAc /Petroleum Ether (7:3).
acquous phases were dried over anhydrous sodium sul-
1
fate. H-NMR data were resolute in DMSO-d solution
6
N’’-[2-(2-Furyl)-8-methyl-8H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl]-N,N’-
diisopropylguanidine (24)
with a Varian VXR 200 spectrometer and peack position
are given in ppm (δ) downfield from tetramethylsilane as
internal standard, and J values are given in Hertz. All of
the identified signals were in accordance with the pro-
posed structure and the splitting patterns are designated
as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; b, broad. Electrospray ionization mass spec-
trometry (ESI/MS) were performed with an Agilent 1100
series LC/ MSD in positive scan mode using a direct injec-
White solid; C H N O; 44% yield; mp 135-136 °C. MS
1
8
23
9
1
+
(ESI): [MH] = 382.4. H NMR (200 MHz, DMSO-d ): δ
6
1.20 (d, 12H, J = 6.4 Hz), 3.84- 3.87 (m, 2H), 3.99 (s, 3H),
4.12 (bs, 2H) 6.66-6.69 (m, 1H), 7.13-7.15 (m, 1H), 7.83-7.85
(m, 1H), 8.42 (s, 1H).
Experimental data for compound 25 is reported in the
SI (S6).
+
tion of the purified compound solution (MH ). Elemental
analyses were performed by the microanalytical laborato-
ry of Dipartimento di Scienze Chimiche e Farmaceutiche,
University of Ferrara, and were within ± 0.4% of the theo-
retical values for C, H, and N. All final compounds re-
vealed a purity of not less than 95%. Melting points for
purified derivatives were determined in a glass capillary
on a Stuart Scientific electrothermal apparatus SMP3 and
are uncorrected.
3
3
Biology Experiments. Materials. [ H]DPCPX ([ H]1,3-
dipropyl-8-cyclopentyl-xanthine, specific activity, 120
Ci/mmol) was obtained from Perkin Elmer Research
3
3
Products (Boston, MA). [ H]ZM 241385 ([ H](4-(2-[7-
amino-2-(2-furil)[1,2.4]triazolo[2,3-a][1,3,5]triazin-5-
ylamino]ethyl)phenol), specific activity, 17 Ci/mmol) was
3
obtained from Biotrend (Cologne, Germany). [ H]MRE-
3
3
008-F20 ([ H]5-N-(4-methoxyphenylcarbamoyl)amino-8-
propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo
[1,5-
c]pyrimidine, specific activity, 67 Ci/mmol) was obtained
from Amersham International (Buckinghamshire, UK).
5
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Journal of Medicinal Chemistry
Page 6 of 7
DPCPX (1,3-dipropyl-8-cyclopentyl-xanthine), R-PIA ((R)-
washed with phosphate-buffered saline, diluted trypsine
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N -(L-2-Phenylisopropyl)adenosine) and CCPA (2-chloro-
and centrifuged for 10 min at 200 g. The pellet containing
6
6
N -cyclopentyladenosine) were obtained from Sigma (St.
CHO cells (1x10 cells /assay) was suspended in 0.5 ml of
Louis, MO, USA). All other reagents were of analytical
grade and obtained from commercial sources.
incubation mixture (mM): NaCl 15, KCl 0.27, NaH PO
2
4
0.037, MgSO 0.1, CaCl 0.1, Hepes 0.01, MgCl 1, glucose
4
2
2
0
.5, pH 7.4 at 37°C, 2 IU/ml adenosine deaminase and 4-
CHO Membranes Preparation. The human A , A
1
2A,
(
3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-
A2B and A adenosine receptors (ARs) were transfected in
3
1
1
724) as phosphodiesterase inhibitor and preincubated for
0 min in a shaking bath at 37 °C. The potencies of antag-
CHO cells which were grown adherently and maintained
in Dulbecco's modified Eagles medium with nutrient mix-
ture F12 (DMEM/F12) without nucleosides, containing
onists to hA ARs were determined by antagonism of
2B
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NECA (100 nM)-induced stimulation of cyclic AMP lev-
1
0% fetal calf serum, penicillin (100 U/ml), streptomycin
21
els. The potency of the antagonists to hA ARs was de-
3
(
100 ꢀg/ml), L-glutamine (2 mM) and Geneticin (G418, 0.2
termined in the presence of Forskolin 1µM by antagonism
of Cl-IB-MECA (100 nM)-induced inhibition of cyclic
mg/ml) at 37 °C in 5% CO , 95% air. For membrane prep-
2
aration 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
1
9
AMP levels. The reaction was terminated by the addition
of cold 6% trichloroacetic acid (TCA). The TCA suspen-
sion was centrifuged at 2000 g for 10 min at 4 °C and the
supernatant was extracted four times with water saturat-
ed 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 pmoles) were added
to each test tube containing the incubation buffer (trizma
base 0.1 M, aminophylline 8.0 mM, 2 mercaptoethanol 6.0
7.4). The cell suspension was homogenized with Polytron
and the homogenate was spun for 10 min at 1000 x g. The
supernatant was then centrifuged for 30 min at 100000 x
g. The membrane pellet was suspended in: a) 50 mM Tris
HCl buffer pH 7.4 for A ARs; b) 50 mM Tris HCl, 10 mM
1
MgCl buffer pH 7.4 for A ARs; c) 50 mM Tris HCl, 10
2
2A
19
mM MgCl , 1 mM EDTA buffer pH 7.4 for A ARs. The
2
3
3
mM, pH 7.4) and [ H] cyclic AMP. The binding protein
cell suspension were incubated with 2 UI/ml of adenosine
deaminase for 30 min at 37 °C. The membrane prepara-
tion was used to perform competition binding experi-
ments.
previously prepared from beef adrenals, was added to the
samples previously incubated at 4 °C for 150 min, and af-
ter the addition of charcoal were centrifuged at 2000 g for
10 min. The clear supernatant was counted in a 2810-TR
Packard scintillation counter.
Competition binding experiments to ARs. All syn-
thesized compounds have been tested for their affinity to
hA , A and A ARs. Displacement binding experiments of
Data Analysis. The protein concentration was deter-
1
2A
3
22
3
mined according to a Bio-Rad method with bovine al-
[
H]-DPCPX (1 nM) to hA CHO membranes (50 ꢀg of pro-
1
bumin as a standard reference. Inhibitory binding con-
tein/assay) and at least 6-8 different concentrations of an-
tagonists studied were performed for 120 min at 25 °C.
Non-specific binding was determined in the presence of 1
stants, Ki values, were calculated from those of IC50 ac-
23
cording to Cheng
&
Prusoff equation
Ki
=
IC /(1+[C*]/K *), where [C*] is the concentration of the
5
0
D
ꢀ
ing.
M of DPCPX and this was always ≤ 10% of the total bind-
20
radioligand and K * its dissociation constant. A weighted
D
24
non-linear least-squares curve fitting program LIGAND
3
Inhibition binding experiments of [ H]-ZM 241385 (2
was also used for computer analysis of inhibition experi-
ments. IC50 values obtained in cyclic AMP assays were
calculated by non-linear regression analysis using the
equation for a sigmoid concentration-response curve
nM) to hA CHO membranes (50 ꢀg of protein/assay) and
2A
at least 6-8 different concentrations of antagonists stud-
ied were performed for 60 min at 4 °C. Non-specific bind-
ing was determined in the presence of 1 ꢀM ZM 241385
(
(
Graph PAD Prism, San Diego, CA, USA). Potency values
21
and was about 20% of total binding.
K ) were calculated by using the modified Cheng-Prusoff
B
3
18
Competition binding experiments of [ H]-MRE 3008F20
analysis proposed by Leff and Dougall. Affinity (K ) and
i
(
1 nM) to hA CHO membranes (50 ꢀg of protein/assay)
potency (K ) values are expressed as the arithmetic mean
B
3
and at least 6-8 different concentrations of examined lig-
ands were performed for 120 min at 4 °C. Non-specific
binding was defined as binding in the presence of 1 ꢀM
± SEM.
ASSOCIATED CONTENT
Supporting Information.
Experimental details for chemical synthesis of 2-(2-furyl)-8-
methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-
amine(9); experimental data for compounds 13-23 and 25.
This material is available free of charge via the Internet at
http://pubs.acs.org.
19
MRE 3008F20 and was about 25% of total binding.
Bound and free radioactivity were separated by filtering
the assay mixture through Whatman GF/B glass fiber fil-
ters using a Brandel cell harvester (Brandel Instruments,
Unterföhring, Germany). The filter bound radioactivity
was counted by Packard Tri Carb 2810 TR scintillation
counter (Perkin Elmer).
Effect of the novel compounds in cyclic AMP as-
says. CHO cells transfected with hA2B or A ARs were
3
6
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Page 7 of 7
Journal of Medicinal Chemistry
as human A
3
adenosine receptor antagonists. J. Med. Chem. 2002,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AUTHOR INFORMATION
45, 770-780.
(12) Rauws, T. R. M.; Maes, B. U. W. Transition metal-
catalyzed N-arylations of amidines and guanidines. Chem. Soc.
Rev. 2012, 41, 2463–2497.
(13) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.;
Klotz, K. N.; Leung, E.; Varani, K.; Gessi, S.; Merighi, S.; Borea, P.
A. Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]- pyrimidine derivatives as
highly potent and selective human A adenosine receptor antag-
3
onists J. Med. Chem. 1999, 42, 4473-4478.
(14) Baraldi, P.G.; Bovero, A.; Fruttarolo, F.; Romagnoli, R.;
Aghazadeh Tabrizi, M.; Preti, D.; Varani, K.; Borea, P.A.; Moor-
Corresponding Author
Phone: +39-0532455921. Fax: +39-0532455921 . E-mail:
*
baraldi@unife.it
Notes
The authors declare no competing finantial interest.
ABBREVIATIONS
hA AR, human A adenosine receptor; hA AR, human A
2A
1
1
2A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
adenosine receptor; hA AR, human A adenosine receptor;
2B
2B
man A. R. New strategies of the synthesis of A
tors antagonists. Bioorg. Med. Chem. 2003, 11, 4161-4169.
15) Baraldi, P.G.; Chiarini, A.; Budriesi, R.; Roberti, M.; Caso-
3
adenosine recep-
hA AR, human A adenosine receptor.
3
3
(
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Table of Contents graphic
(11) Baraldi, P. G.; Cacciari, B.; Moro, S.; Spalluto, G.; Pastorin,
O
G.; Da Ros, T.; Klotz, K. N.; Varani, K.; Gessi, S.; Borea, P. A. Syn-
thesis, biological activity and molecular modelling investigation
of new pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives
N
N
N
N
HN
R
N
N
N
HN R1
7
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