Antihypertensive profile of 2-thienyl-3,4-methylenedioxybenzoylhydrazone is mediated by activation of the A2A adenosine receptor

Article abstract of DOI:10.1016/j.ejmech.2012.06.056

Several N-acylhydrazone derivatives synthesized from safrole have been found to promote intense vasodilation and antihypertensive activity. The present work describes the synthesis and antihypertensive profile of 2-thienyl-3,4-methylenedioxybenzoylhydrazone (LASSBio-1027), a new analogue of the lead compound 3,4-methylenedioxybenzoyl-2-thienylhydrazone. Thoracic aortas from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. Noninvasive blood pressure measurements were made during 14 days of intraperitoneal (10 mg/kg) or oral (20 mg/kg) administration of LASSBio-1027. LASSBio-1027 exhibited partially endothelium-dependent vasorelaxant activity, which was attenuated in the presence of l-NAME, glibenclamide, or ZM 241385. LASSBio-1027 exhibited an antihypertensive effect in SHR during 14 days of intraperitoneal or oral administration, but did not induce a hypotensive effect in normotensive WKY rats. LASSBio-1027-induced vascular relaxation of aortas from WKY rats was mediated by the activation of A_2A adenosine receptors. Docking studies and binding assays suggested that LASSBio-1027 has affinity for A _2A and A₃ adenosine receptors. This new N-acylhydrazone derivative represents a potential strategy for the treatment of arterial hypertension.

Full text of DOI:10.1016/j.ejmech.2012.06.056

European Journal of Medicinal Chemistry 55 (2012) 49e57
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antihypertensive profile of 2-thienyl-3,4-methylenedioxybenzoylhydrazone
is mediated by activation of the A2A adenosine receptor
,
,
,
,1
Carla Moreira Leal a 1, Sharlene Lopes Pereira a 1, Arthur Eugen Kümmerle b 2, Daniella Moreira Leal a
,
Roberta Tesch c 3, Carlos M.R. de Sant’Anna b 2, Carlos Alberto M. Fraga a 3, Eliezer Jesus Barreiro a
,
,
,
,
c
,
1,
,c,1,3
Roberto Takashi Sudo a 3, Gisele Zapata-Sudo a
,
c,
1,
,c, ,1,3
*
a Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590,
RJ, Brazil
b Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, Seropédica 23890-000, RJ, Brazil
c Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de
Janeiro 21941-590, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Several N-acylhydrazone derivatives synthesized from safrole have been found to promote intense
vasodilation and antihypertensive activity. The present work describes the synthesis and antihyper-
tensive profile of 2-thienyl-3,4-methylenedioxybenzoylhydrazone (LASSBio-1027), a new analogue of the
lead compound 3,4-methylenedioxybenzoyl-2-thienylhydrazone. Thoracic aortas from Wistar-Kyoto
(WKY) rats and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording.
Noninvasive blood pressure measurements were made during 14 days of intraperitoneal (10 mg/kg) or
oral (20 mg/kg) administration of LASSBio-1027. LASSBio-1027 exhibited partially endothelium-
Received 29 February 2012
Received in revised form
26 June 2012
Accepted 28 June 2012
Available online 7 July 2012
Keywords:
dependent vasorelaxant activity, which was attenuated in the presence of L-NAME, glibenclamide, or
N-acylhydrazone
LASSBio-1027
Vasodilator
Antihypertensive
Adenosine agonist
ZM 241385. LASSBio-1027 exhibited an antihypertensive effect in SHR during 14 days of intraperitoneal
or oral administration, but did not induce a hypotensive effect in normotensive WKY rats. LASSBio-1027-
induced vascular relaxation of aortas from WKY rats was mediated by the activation of A2A adenosine
receptors. Docking studies and binding assays suggested that LASSBio-1027 has affinity for A2A and A3
adenosine receptors. This new N-acylhydrazone derivative represents a potential strategy for the
treatment of arterial hypertension.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Arterial hypertension is the most common risk factor for
Abbreviations: DMSO, dimethylsulfoxide; WKY, Wistar-Kyoto; SHR, spontane-
ously hypertensive rats; -NAME, N-nitro- -arginine methyl ester; KATP, ATP-sensi-
cardiovascular disease, which is the leading cause of death in
industrialized societies [1,2]. More than 25% of the adult population
worldwide had hypertension in 2000, and almost 30% are projected
to have this condition by 2025 [3]. The prevalence of arterial
hypertension is increasing worldwide, both in developed and
developing countries [4]. Thus, the prevention, detection, and
treatment of this disease should be a high priority. Moreover, even
with apparently adequate blood pressure (BP) control with
conventional antihypertensive drugs, cardiovascular disease risks
in hypertensive populations remain increased above those in
normotensive populations [5]. This latter finding highlights the
importance of the discovery of new drugs for the treatment of
arterial hypertension.
L
L
tive potassium channels; NO, nitric oxide; i.p., intraperitoneal; BP, blood pressure;
SP, systolic pressure; DP, diastolic pressure; HR, heart rate; IC50, concentration
necessary to reduce the phenylephrine-induced contraction by 50%; SEM, standard
error of the mean; SR, sarcoplasmic reticulum; VSM, vascular smooth muscle; NAH,
N-acylhydrazone.
* Corresponding author. Universidade Federal do Rio de Janeiro, Centro de
Ciencias da Saude, Instituto de Ciencias Biomedicas, Bloco J, Sala 14, Rio de Janeiro
21941-590, Brazil. Tel./fax: þ55 21 25626505.
E-mail addresses: ludaca@uol.com.br (C.M. Leal), sharlene.pereira@
yahoo.com.br (S.L. Pereira), akummerle@yahoo.com.br (A.E. Kümmerle), daniella-
leal@hotmail.com (D.M. Leal), robertatesch@yahoo.com.br (R. Tesch), santanna@
ufrrj.br (C.M.R. de Sant’Anna), cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga),
ejbarreiro@ccsdecania.ufrj.br (E.J. Barreiro), rtsudo@icb.ufrj.br (R.T. Sudo), gsudo@
icb.ufrj.br, gsudo@farmaco.ufrj.br (G. Zapata-Sudo).
1
Tel./fax: þ55 21 25626505.
2
Recently, our group studied a series of new N-acylhydrazones
(NAH) with vasodilator activity synthesized from a Brazilian natural
Tel./fax: þ55 21 26821872.
3
Tel./fax: þ55 21 25626503.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.056

50
C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
product obtained from sassafras oil, known as safrole. The lead
compound for this series was LASSBio-294 [6], an NAH with potent
positive cardiac inotropic action [7]. The activity of this compound
was related to its ability to increase Ca2þ accumulation in the
sarcoplasmic reticulum, which promoted vasodilation in aortic
rings mediated by the guanylate cyclase/cyclic guanylate mono-
phosphate pathway [8]. Considering the bioprofile of LASSBio-294,
we obtained synthetic analogues by changing the electronic
density of the thienyl subunit and modifying the stereoelectronic
behavior of the acylhydrazone group through its N-alkylation.
These analogues were evaluated in vascular smooth muscle (VSM),
in an effort to identify new drug candidates with vasodilator
properties and fewer side effects. The results showed an important
pharmacophoric profile for the thienyl ring [9].
N-acylhydrazone compounds [11e13]. The analytical results for C, H,
and N for LASSBio-1027 were within ꢁ0.4% of the theoretical values.
3. Pharmacology
3.1. Effects of LASSBio-1027 on VSM
The vasodilator activity of LASSBio-1027 was investigated in
aortic rings from WKY rats. The maximal contractile response
induced by phenylephrine (10 mM) in aortic rings with and without
endothelium was recorded after exposure to increasing concen-
trations of LASSBio-1027, which induced relaxation of the precon-
tracted aorta in a concentration-dependent manner (Fig. 2). The
concentration necessary to induce 50% relaxation (IC50) of aortic
Through knowledge of retroisosterism and inversion of the
functional groups present in the lead compound structure (Fig. 1),
in this work we describe the discovery of a new analogue of the
lead compound, 2-thienyl-3,4-methylenedioxybenzoylhydrazone
(LASSBio-1027). We evaluated the in vitro effects of LASSBio-1027
on VSM, as well as the possible mechanisms involved in its
effects. We also investigated its effects on BP during the treatment
of normotensive Wistar-Kyoto (WKY) rats and spontaneously
hypertensive rats (SHR). Docking experiments and binding assays
were performed to confirm the possible molecular mechanisms
involved in the vascular effects of the new N-acylhydrazone
derivative.
rings with endothelium was 6.9 ꢁ 1.4 M (n ¼ 6). Removal of the
m
endothelium reduced the vasodilator response induced by the
compound, which indicates that its vasorelaxant effect was
partially dependent on the integrity of vascular endothelium.
Considering the endothelial involvement in the relaxation of
aortic rings induced by LASSBio-1027, we investigated which
signaling pathways were involved in its mechanism of vasodilation.
Endothelium-intact aortic rings were pretreated with L-NAME,
a nitric oxide synthase inhibitor, which significantly reduced the
maximal relaxation to 9.1 ꢁ1.8% (Fig. 3A). Pretreatment of the aorta
with intact endothelium with ZM 241385, a selective antagonist of
the A2A adenosine receptor, induced a rightward shift of the
concentrationeresponse curve and reduced the maximal relaxa-
tion to 3.6 ꢁ 2.0% (Fig. 3B). This finding indicates that LASSBio-1027
promotes vasorelaxation through NO production mediated by
activation of the A2A adenosine receptors.
We also investigated the mechanism of LASSBio-1027-induced
vasodilation when the endothelium-denuded aorta was pre-
treated with glibenclamide, a KATP channel blocker. Glibenclamide
completely abolished vascular relaxation of the aorta (Fig. 4). This
result suggests that these channels have an important role in the
vascular effect of LASSBio-1027, because the activation of the A2A
adenosine receptors in VSM promotes the opening of KATP chan-
nels, inducing hyperpolarization and vasodilation.
2. Chemistry
The synthetic route used to generate the NAH target compound
LASSBio-1027 is shown in Scheme 1. The derivative was prepared in
ca. 65% overall yield from the thiophene-2-carboxaldehyde 1. By
employing the oxidative Yamada’s procedure [10], 1 was ‘one-pot’
converted, in 89% yield, to the corresponding methyl ester 2 by
treatment with 2.6 eq. of KOH and 1.3 eq. of iodine in methanol at
0
ꢀC. Next, the key acylhydrazine intermediate 3 was obtained at
91% yield by treatment of an ethanolic solution of the ester 2 with
hydrazine hydrate at 70 ꢀC for 3.5 h [11].
Maximal relaxation values for LASSBio-1027 in phenylephrine-
contracted aortic rings from WKY rats are summarized in Table 1.
LASSBio-1027 was obtained, in good yield (90%), by condensing
compound 3 with piperonal in ethanol, using hydrochloric acid as
a catalyst [11] (Scheme 1).
Presence of the (E)-diastereomer was detected by analyzing the
1H NMR spectra of LASSBio-1027, on the basis of several previous
reports from our group describing the configuration of bioactive
3.2. Effects of LASSBio-1027 during prolonged treatment of WKY
rats and SHR
LASSBio-1027 was administrated intraperitoneally (i.p.) (10 mg/
kg/day) to WKY rats and SHR for 14 days. LASSBio-1027 had no
significant effect on BP in WKY rats (Fig. 5, Table 2). However, i.p.
treatment of SHR for 14 days significantly reduced both systolic and
diastolic BP (SP and DP) (Fig. 5, Table 2). Prolonged treatments with
LASSBio-1027 did not alter significantly the heart rate (HR) of WKY
rats or SHR. Oral administration of LASSBio-1027 (20 mg/kg/day) to
SHR for 14 days resulted in significant reduction of SP and DP, but
did not change the HR of the rats (Fig. 6, Table 3).
3.3. Docking of LASSBio-1027 in A2A and A3 adenosine receptors
To confirm the interaction of LASSBio-1027 with the adenosine
receptors, we performed a docking study using GOLD 5.1 (CCDC).
The crystal structure of A2A receptor was obtained from the RCSB
Protein Data Bank (PDB code: 3EML) and until now no crystal
structure of A3 receptor is available on this Data Bank. Thus, an A3
receptor model was construct by homology modeling using the
Swiss Model Server [14,15] using the A2A crystal structure as
template (PDB code: 3EML). To validate the GoldScore fitness
function, a redocking of the cocrystallized antagonist ZM 241385
Fig. 1. Retroisosterism applied to the design of LASSBio-1027.

C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
51
Scheme 1. General pathway for the synthesis of LASSBio-1027: (a) I2, KOH, 0 ꢀC, 2 h; (b) NH2NH2$H2O, EtOH, 70 ꢀC, 3.5 h; (c) piperonal, HCl cat, EtOH, 1 h.
The average of the three docking scores was calculated for
LASSBio-1027 in A2A and A3 receptors and for ZM 241385 in A2A
receptor. The obtained values were 51.6, 49.9 and 62.7, respectively.
This result indicates that ZM 241385 has more favorable interac-
tions than LASSBio-1027, which will be the subject of further
studies.
To compare the binding modes of ZM 241385 and LASSBio-1027,
the crystal structure was superimposed with one of the confor-
mations (Fig. 9). Based on the superpositions, LASSBio-1027 seems
to bind in the same active site as ZM 241385, which corroborates
the in vitro pharmacological findings in the VSM of rat aorta.
Score values of the GoldScore fitness function did not identify
differences in the affinity of LASSBio-1027 for A2A and A3 receptors.
One possible explanation is that the hydrogen-bond energy terms
of GoldScore fitness function could have a higher representative
value than the van der Waals energy terms. Indeed the A3 receptor
has more hydrophobic amino acid residues in the active site, which
may explain why the score value of LASSBio-1027 was lower than in
A2A receptor (Fig. 10).
With endothelium
Without endothelium
100
80
60
40
20
0
*
*
*
*
*
-7
-6
-5
-4
-3
LASSBio-1027 (Log M)
Fig. 2. Concentration-response curves for LASSBio-1027 in rat aorta with and without
endothelium, precontracted with phenylephrine. Data are mean
ꢁ
SEM (n
¼
6).
*P < 0.05 vs. with endothelium.
LASSBio-1027 had greater affinity for A3 adenosine receptor and
it could be explained by the hydrophobic nature of the A3 receptor
active site in comparison to the A2A receptor active site, which sum
of hydrophobic interactions may be more energetic favorable than
isolated hydrogen-bond interactions.
was done. Redocking results show the ability of the function to
predict an experimental binding mode. Redocked ZM 241385 and
crystal ZM 241385 of the complex were located very close to each
ꢀ
other in the active site, with an RMSD of 0.52 A for all atoms (Fig. 7).
To perform the docking of LASSBio-1027, three consecutive runs
were conducted and the highest-scoring conformations from each
run were analyzed. Water molecules were removed from the A2A
pdb file and the A3 model was constructed without water mole-
cules. The docking of ZM 241385 was performed with the same
protocol.
3.4. Binding assay
Binding of the agonist [3H]CCPA (1 nM) to the A1 receptor (n ¼ 2)
was inhibited by an average of 29.1% by LASSBio-1027 (10
mM).
Binding of agonist [3H]CGS21680 (6 nM) to the A2A receptor (n ¼ 2)
and binding of agonist [125I]AB-MECA (0.15 nM) to the A3 receptor
All of the docking runs showed the same pattern, except for the
orientation of the thiophene ring at the acyl unit of LASSBio-1027.
(n ¼ 2) were inhibited by 47.5% and 67.5% by LASSBio-1027 (10
mM).
According to the results, the 1,3-benzodioxole ring interacts by
ꢀ
a hydrogen bond with ASN253 (distance of 2.5 A) and through
p
ep
4. Discussion
stacking contacts with PHE168. Moreover, the N-acylhydrazone
hydrogen interacts with GLU169 through hydrogen bond
(distance of 1.9 A), as shown in Fig. 8A. In the A3 receptor the
GLU169 is substituted by a hydrophobic amino acid residue VAL169
which might be responsible for the change in the orientation of
LASSBio-1027 in the active site showed in Fig. 8B.
a
Adenosine modulates various physiological processes in
mammals. Many of the responses mediated by adenosine are
caused by its interaction with specific membrane-bound receptors.
From pharmacological and molecular biology studies, four adeno-
ꢀ
sine receptor subtypes have been characterized, named A1, A2A, A2B
,
A
B
100
100
80
60
80
60
With endothelium
40
40
20
0
With endothelium
+ ZM 241385 (100 nM)
+ L-NAME (100µM)
20
*
*
*
*
*
*
*
* *
0
*
-7
-6
-5
-4
-3
-7
-6
-5
-4
-3
LASSBio-1027 (Log M)
LASSBio-1027 (Log M)
Fig. 3. Concentration-response curves for LASSBio-1027 in aorta with endothelium precontracted with phenylephrine in the presence of (A) L-NAME and (B) ZM 241385. Data are
mean ꢁ SEM (n ¼ 6). *P < 0.05 vs. with endothelium.

52
C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
Table 1
100
80
60
40
20
0
Without endothelium
+ Glibenclamide (5µM)
Maximal relaxation induced by LASSBio-1027 in phenylephrine-contracted aortic
rings from WKY rats.
With endothelium
Without endothelium
Control
100.0 ꢁ 0.0
9.1 ꢁ 1.8*
8.3 ꢁ 6.0*
ND
28.4 ꢁ 2.5#
þ
-NAME (100 mM)
ND
ND
þLZM 241385 (100 nM)
þGlibenclamide (5
m
M)
3.6 ꢁ 2.0*
*P < 0.05 vs. control; #P < 0.05 vs. with endothelium.
All values are mean ꢁ SEM, n ¼ 6.
ND: not determined.
*
*
-7
-6
-5
-4
-3
which include cytokine production, degranulation, chemotaxis,
cytotoxicity, apoptosis and proliferation [23].
LASSBio-1027 (Log M)
LASSBio-1027-induced concentration-dependent relaxation of
the aorta mainly by activating A2A adenosine receptors and
releasing NO. These actions were observed in pharmacological
experiments and confirmed in the molecular docking study, which
demonstrated an interaction between LASSBio-1027 and the A2A
receptor. The compound induced hypotension and an antihyper-
tensive effect in SHR during prolonged treatment, but it had no
effect on the BP of normotensive WKY rats. Moreover, LASSBio-
1027 had no effect on the HR of either WKY rats or SHR, which
suggests the selectivity of the compound for the A2A receptor
subtype relative to the A1 subtype, which was shown by the low
affinity of LASSBio-1027 for the A1 receptor in the binding assay.
Both docking studies and binding assays suggest that LASSBio-1027
could be an agonist of A2A and A3 subtypes but the vasodilator
activity of LASSBio-1027 is probably mediated by the activation of
the A2A receptor.
Fig. 4. Concentration-response curves for LASSBio-1027 in endothelium-denuded
aorta precontracted with phenylephrine in the presence of glibenclamide. Data are
mean ꢁ SEM (n ¼ 6). *P < 0.05 vs. without endothelium.
and A3 [16]. These receptors belong to the large family of G protein
coupled receptors. Activation of A1 and A3 receptors leads to inhi-
bition of adenylate cyclase by an inhibitory G protein, whereas A2A
and A2B receptors stimulate the enzyme through a stimulatory G
protein [17]. In the cardiovascular system, activation of the A1
receptor subtype produces an inhibitory action on the heart,
promoting bradycardia, negative dromotropy, and inotropy, and
consequently reduces the cardiac output [18e20]. Stimulation of
the A2A receptor subtype elicits various effects, including vasodi-
lation, inhibition of both platelet aggregation and neutrophil
adhesion, and reduction in the generation of oxygen free radicals
[20e22]. A3 adenosine receptors are expressed on the surface of
most immune cell types, including neutrophils, macrophages,
dendritic cells, lymphocytes and mast cells [23]. A3 activation on
immune cells governs a broad array of immune cell functions,
The activation of A3 receptor improves myocardial function after
ischemia-reperfusion protocol in guinea-pig, reduces myocardial
injury in rat and also helps protecting isolated cardiomyocytes from
cell death [24,25]. Additionally, A3 agonist exerts anti-apoptotic and
A
B
300
300
250
250
WKY
SHR
WKY
SHR
200
150
100
50
200
150
100
50
*
*
*
*
*
*
*
*
*
*
0
3
6
9
12 15
0
3
6
9
12 15
Time (day)
Time (day)
C
500
400
300
200
WKY
SHR
0
3
6
9
12 15
Time (day)
Fig. 5. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were treated with i.p. LASSBio-1027 (10 mg/kg/day) for 14 days. Effects on (A) systolic blood pressure,
(B) diastolic blood pressure, and (C) heart rate are shown. Data are mean ꢁ SEM (n ¼ 6). *P < 0.01 vs. day zero.

C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
53
Table 2
Intraperitoneal treatment of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) with LASSBio-1027 (10 mg/kg/day) for 14 days.
WKY
SHR
Day 0
Day 7
Day 14
Day 0
Day 7
Day 14
SP (mmHg)
DP (mmHg)
HR (bpm)
121.2 ꢁ 4.5
72.6 ꢁ 4.7
109.0 ꢁ 3.3
79.4 ꢁ 7.8
109.0 ꢁ 2.8
88.0 ꢁ 7.1
226.0 ꢁ 11.0
175.9 ꢁ 7.3
419.0 ꢁ 11.0
126.2 ꢁ 3.2*
106.2 ꢁ 4.1*
376.0 ꢁ 18.3
118.0 ꢁ 1.2*
95.5 ꢁ 4.3*
383.0 ꢁ 21.0
379.0 ꢁ 17.9
398.0 ꢁ 11.8
344.8 ꢁ 14.7
*P < 0.01 vs. day 0.
All values are mean ꢁ SEM, n ¼ 6.
SP, systolic pressure; DP, diastolic pressure; HR, heart rate.
anti-necrotic effects after ischemia/reoxygenation protocol in iso-
lated adult rat myocytes [24]. Thus, A3 agonists, such as LASSBio-
1027, are of great interest in cardioprotection. On the other hand,
activation of A3 receptors could promote hyper responsiveness of
mast cells [26] which would be contraindicated in patients with
asthma or chronic obstructive pulmonary disease.
pathway [28,29]. In addition, activation of A2A receptors in the VSM
increases activation of cAMP and PKA, which leads to phosphory-
lation and opening of KATP channels. This effect, in turn, causes
hyperpolarization and vasodilation [30]. Therefore, blocking of
these channels by glibenclamide prevented the vasodilator action
of LASSBio-1027.
According to the results, LASSBio-1027 caused pronounced
vascular relaxation of the endothelium-intact aortic rings, which
was significantly reduced in endothelium-denuded aorta. The
relaxation was also reduced when the aortas were incubated with
Dubey and collaborators [31] reported lower levels of extracel-
lular adenosine in the smooth muscle cells of conduit arteries
(aorta) and resistance microvessels (renal arterioles) from SHR
versus normotensive WKY rats, due to the increased activity of
adenosine deaminase, which is responsible for the degradation of
adenosine. Dysregulation of extracellular adenosine in the VSM
cells of SHR contributes to the enhanced proliferative response that
is involved in the pathophysiology of vascular remodeling induced
by arterial hypertension [32], because vascular-derived adenosine
inhibits cell growth [33]. Therefore, the development of new
adenosine receptor agonists, especially A2 agonists, is of great
importance, given their vasodilator and antiproliferative effects.
Several reports have described the synthesis and antihyper-
tensive effects of adenosine A1 and A2A agonists in conscious SHR
[34e39]. In these studies, the adenosine analogues were synthe-
sized from structural modifications of adenosine. Hypotension with
a NO synthase inhibitor (L-NAME) or with a selective antagonist of
the A2A adenosine receptor (ZM 241385). LASSBio-1027 induced
a minor vasodilator effect on endothelium-denuded aorta, and this
effect was completely abolished in the presence of glibenclamide,
a KATP channel blocker. These findings indicate that LASSBio-1027
activates the A2A receptor located in the vascular endothelium
and VSM of the rat aorta, thereby promoting vasorelaxant activity.
The A2A adenosine receptor is located not only in the vascular
endothelium but also in the VSM of the rat aorta [27], and its
activation is involved in vasodilation [21,22]. Activation of endo-
thelial A2A receptors, which are coupled with a G stimulatory
protein, induces NO release by activating the adenylate cyclase-PKA
A
B
300
300
250
250
200
200
*
#
150
*
*
*
150
*
*
*
*
*
100
50
*
100
50
*
0
3
6
9
12 15
0
3
6
9
12 15
Time (day)
Time (day)
C
500
400
300
200
0
3
6
9
12 15
Time (day)
Fig. 6. Spontaneously hypertensive rats (SHR) were treated with oral LASSBio-1027 (20 mg/kg/day) for 14 days. Effects on (A) SP, (B) DP, and (C) HR are shown. Data are mean ꢁ SEM
(n ¼ 6). #P < 0.05 vs. day zero; *P < 0.01 vs. day zero.

54
C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
Table 3
Oral treatment of spontaneously hypertensive rats (SHR) with LASSBio-1027 (20 mg/
kg/day) for 14 days.
SHR
Day 0
Day 7
Day 14
SP (mmHg)
DP (mmHg)
HR (bpm)
121.2 ꢁ 4.5
72.6 ꢁ 4.7
109.0 ꢁ 3.3*
79.4 ꢁ 7.8*
379.0 ꢁ 17.9
109.0 ꢁ 2.8*
88.0 ꢁ 7.1*
398.0 ꢁ 11.8
383.0 ꢁ 21.0
*P < 0.01 vs. day 0.
All values are mean ꢁ SEM, n ¼ 6.
SP, systolic pressure; DP, diastolic pressure; HR, heart rate.
bradycardia or tachycardia was induced after administration of A1
or A2A agonists, respectively. In addition, Webb and collaborators
[40] reported the development of tolerance to the antihypertensive
effect of adenosine A2A agonists after two weeks of administration.
The present study describes an innovative compound, LASSBio-
1027, which is classified as an NAH derivative and does not have
structural similarity to adenosine. The activation of the A2A receptor
is responsible for the vasodilator and antihypertensive actions of
the new compound. Chronic administration of the substance in SHR
did not induce tolerance to the antihypertensive effect, suggesting
a new candidate for the prolonged oral treatment of hypertensive
subjects.
Fig. 7. Superposition of ZM 241385 in the crystal structure of the A2A receptor (orange)
and superposition obtained after redocking (blue) with the program GOLD.
ꢀ
RMSD ¼ 0.52 A. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
a Bruker Avance-500 at 500 MHz 13C NMR spectra were obtained in
the same spectrometer at 125 MHz.
Microanalyses were performed with
a Perkin Elmer 240
analyzer and Perkin Elmer AD-4 balance. The progress of all reac-
tions was monitored by thin-layer chromatography (TLC), which
was performed on 2.0 cm ꢂ 6.0 cm aluminum sheets precoated
with silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm. The
developed chromatograms were viewed under ultraviolet light
(254e365 nm) and treated with iodine vapor. Reagents and
solvents were purchased from commercial suppliers and used as
received.
5. Conclusions
We describe the successful synthesis of a new NAH derivative,
LASSBio-1027, which exhibited vasodilator and antihypertensive
actions that were mediated by activation of the A2A adenosine
receptor. Molecular docking studies and binding assays showed
interaction of the compound with both A2A and A3 receptors. This
work highlights the importance of this new class of compounds,
which could be explored as an original structural pattern for the
activation of adenosine receptors.
6.1.1. Synthesis of methyl thiophene-2-carboxylate (2)
Methanolic solutions (each 20 mL) of iodine (1.00 g, 3.9 mmol)
and KOH (0.51 g, 9.0 mmol) at 0 ꢀC were added successively to
a solution of thiophene-2-carboxaldehyde 1 (0.34 g, 3.0 mmol) in
absolute methanol (15 mL) that was cooled to 0 ꢀC. After stirring for
2 h at 0 ꢀC, small amounts of saturated NaHSO3 solution were added
until the brown color disappeared [10]. Next, the methanol was
almost totally evaporated under reduced pressure. Water (30 mL)
was added to the residue, followed by a partition with CH2Cl2
(3 ꢂ 30 mL). The desired methyl thiophene-2-carboxylate 2 was
obtained after the organic phase was dried with Na2SO4 and
evaporated under vacuum, to 89% yield, as brown oil. 1H NMR
6. Experimental section
6.1. Chemistry
Melting points were determined with a Quimis 340 apparatus.
Uncorrected melting points are reported. 1H NMR spectra were
determined in deuterated chloroform and dimethylsulfoxide con-
taining ca. 1% tetramethylsilane as an internal standard, with
(300 MHz CDCl3-d6)
d
3.87 (s, 3H), 7.08 (dd, 1H, Jab ¼ 4.5 Hz,
Fig. 8. Possible conformations of the compound LASSBio-1027 (yellow, blue, pink) in (A) A2A adenosine receptor (PDB ID: 3EML) and (B) A3 adenosine receptor model. Amino acid
residues in green are part of the active site of these receptors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
55
precipitation was visualized. Next, the solvent was partially
concentrated at reduced pressure, and the resulting mixture was
poured into cold water. After neutralization with 10% aqueous
sodium bicarbonate solution, the precipitate that formed was
filtered out and dried under vacuum to give the desired acylhy-
drazone derivative, LASSBio-1027 (90%) as a cream-colored solid;
m.p. 228e231 ꢀC. 1H NMR (300 MHz, DMSO-d6)
OCH2O), 6.98 (d, H3,4-metilenodioxy 7.9 Hz), 7.15e7.27
d 6.08 (s, 2H,
,
J
¼
(m, 1H3,4-metilenodioxy, 1Htiophene), 7.51 (d, 1H, HeAr, J ¼ 8.1 Hz),
7.27 (s, H3,4-metilenodioxy), 7.37 (s, H3,4-metilenodioxy), 7.84e8.00
(m, 2Htiophene), 8.02 (s, 1H, NCH), 8.32 (s, 1H, NCH), 11.70 (s, 1H,
CONH), 11.75 (s, 1H, CONH); 13C NMR (75 MHz, DMSO-d6)
d 102.1,
105.7, 105.8,109.0,123.8,124.0,127.1, 128.6,129.0,129.1, 129.3,132.2,
133.4,135.2,135.4,138.9,144.3,147.9,148.5,149.5,149.6,158.1,161.6;
MS (ESI) m/z 275 ([M þ H]þ). Anal. Calcd for: C13H10N2O3S: C 56.92;
H 3.67; N 10.21; Found: C 56.87; H 3.69; N 10.15.
Fig. 9. Superposition of the A2A adenosine receptor antagonist ZM 241385 (orange)
with the agonist LASSBio-1027 (blue). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.).
6.2. Pharmacology
Jac ¼ 3.9 Hz), 7.53 (d, 1H, Jab ¼ 3.9 Hz), 7.79 (d, 1H, J ¼ 4.5 Hz); 13
C
The protocols used in the present study were approved by the
Animal Care and Use Committee at Universidade Federal do Rio de
Janeiro.
NMR (75 MHz CDCl3-d6)
d 52.1, 127.7, 132.3, 133.5, 133.6, 162.7.
6.1.2. Synthesis of thiophene-2-carbohydrazide (3)
A 0.6-mL aliquot of 100% hydrazine monohydrate was added to
a solution of 0.30 g (1.24 mmol) of 2 in 6 mL of ethanol [11]. The
reaction mixture was maintained at 70 ꢀC for 3.5 h, when TLC
indicated the end of the reaction. The media was poured on ice. The
resulting precipitate was partitioned with AcOEt (5 ꢂ 30 mL) to
generate the title compound with 81% yield, as a brown solid, m.p.
6.2.1. In vitro experiments
Thoracic aortas were removed from 13-week-old male WKY
rats. The aortas were cleaned of connective tissue and prepared for
isometric tension recording, as previously described [41]. After
a 2-h equilibrium period of 1 g resting tension, the aortic rings were
136e138 ꢀC. 1H NMR (300 MHz, DMSO-d6)
d 4.46 (br, 2H), 7.11 (dd,
contracted with phenylephrine (10
mM), followed by exposure to
1H, Jab ¼ 4.8 Hz, Jac ¼ 3.9 Hz), 7.69e7.72 (m, 2H), 9.74 (s, 1H); 13C
acetylcholine (10 M) to test the integrity of the endothelium.
m
Acetylcholine-induced relaxation of >80% denoted the presence of
intact endothelium. In some experiments, LASSBio-1027 was tested
in aortas in which the endothelium had been mechanically
removed [41].
To investigate the ability of LASSBio-1027 to induce relaxation of
the aortic rings, intact or endothelial-denuded rings were precon-
tracted with a single concentration of phenylephrine (10
exposed to increasing concentrations of LASSBio-1027 (1e300
Control experiments were performed in the presence of dime-
thylsulfoxide (DMSO) alone.
NMR (75 MHz, DMSO-d6)
d 127.2, 127.6, 130.0, 138.0, 160.9.
6.1.3. Synthesis of (E)-N0-(benzo[d][1,3]dioxol-5-ylmethylene)
thiophene-2-carbohydrazide (LASSBio-1027)
Piperonal (0.174 g, 1.16 mmol) was added to a solution of thio-
phene-2-carbohydrazide 3 (0.150 g, 1.01 mmol) in absolute EtOH
(7 mL) containing 2 drops of 37% hydrochloric acid [11]. The mixture
mM) and
mM).
was stirred at room temperature for
1 h, until extensive
To evaluate the possible mechanisms involved in the effects of
the derivative, aortic rings were pretreated with: N-nitro-
methyl ester ( -NAME), a nitric oxide synthase inhibitor (100
[42,43]; glibenclamide, a KATP channel blocker (5 M) [44]; and ZM
L-arginine
L
mM)
m
241385, a selective antagonist of the A2A adenosine receptor
(100 nM) [29,42]. The antagonists were added to the solution
20 min before the contraction with phenylephrine.
6.2.2. In vivo experiments
Male WKY rats and SHR (13-week old) were treated i.p. daily for
14 days with a single dose of LASSBio-1027 (10 mg/kg). In addition,
SHR were treated orally daily for 14 days with LASSBio-1027
(20 mg/kg). Noninvasive blood pressure measurements were per-
formed through tail-cuff plethysmography (Letica model LE 5001)
to acquire SP, DP, and HR. The BP of rats was measured before and at
1, 3, 5, 7, 11, and 14 days of treatment. The BP measurements were
performed at 4 h after administration.
6.2.3. Drugs
Phenylephrine, acetylcholine, glibenclamide, and L-NAME were
purchased from Sigma Chemical (St Louis, MO, USA). ZM 241385
was purchased from Tocris Bioscience (Ellisville, MO, USA).
Fig. 10. Superposition of LASSBio-1027 and some amino acid residues in A2A adenosine
receptor active site (blue); LASSBio-1027 and some amino acid residues in A3 adeno-
sine receptor model (green). The amino acid in black is conserved in both receptors.
(For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
LASSBio-1027, glibenclamide, and
L-NAME were dissolved in
dimetylsulphoxide (DMSO) (Merck Darmstadt, Germany). Phenyl-
ephrine and acetylcholine were dissolved in distilled water.

56
C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
6.2.4. Statistics
References
All data are expressed as the mean ꢁ standard error of the mean
(SEM). The LASSBio-1027 concentration necessary to reduce the
phenylephrine-induced contraction by 50% (IC50) was determined
for each experiment. The concentrationeresponse curve was fitted
to the following equation: ymax ¼ ymin þ a/(1 þ eꢃxꢃx0/b), where y is
the percentage of isometric tension; a ¼ ymax ꢃ ymin; b ¼ slope; and
x0 ¼ IC50. Differences between 2 groups were determined with the
unpaired Student’s t-test and were considered significant when P
was <0.05.
[1] World Health Organization, The World Health Report 2002: Reducing
Risks, Promoting Healthy Life, World Health Organization, Geneva, Swit-
zerland, 2002, Available at: http://www.who.int/whr/2002/en/ (accessed
19.02.10).
[2] A. Mosterd, R.B. D’ Agostino, H. Silbershatz, P.A. Sytkowski, W.B. Kannel,
D.E. Grobbee, Trends in the prevalence of hypertension, antihypertensive
therapy, and left ventricular hypertrophy from 1950 to 1989, N. Engl. J. Med.
340 (1999) 1221e1227.
[3] P.M. Kearney, M. Whelton, K. Reynolds, P. Muntner, P.K. Whelton, J. He, Global
burden of hypertension: analysis of worldwide data, Lancet 365 (2005)
217e223.
[4] M. Pereira, N. Lunet, A. Azevedo, Differences in prevalence, awareness,
treatment and control of hypertension between developing and developed
countries, J. Hypertens. 27 (2009) 963e975.
6.3. In silico experiments
[5] American Heart Association, Cardiovascular diseases in the United States:
2009. Available at: www.yourethecure.org (accessed 21.01.11).
[6] E.J. Barreiro, Estratégia de simplificação molecular no planejamento racional
de fármacos: A descoberta de novo agente cardioativo, Quim. Nova 25 (2002)
1172e1180.
[7] R.T. Sudo, G. Zapata-Sudo, E.J. Barreiro, The new compound, LASSBio 294,
increases the contractility of intact and saponin-skinned cardiac muscle from
Wistar rats, Br. J. Pharmacol. 134 (2001) 603e613.
[8] C.L.M. Silva, F. Noel, E.J. Barreiro, Cyclic GMP-dependent vasodilatory prop-
erties of LASSBio 294 in rat aorta, Br. J. Pharmacol. 135 (2002) 293e298.
[9] A.G. Silva, G. Zapata-Sudo, A.E. Kummerle, C.A. Fraga, E.J. Barreiro, R.T. Sudo,
Synthesis and vasodilatory activity of new N-acylhydrazone derivatives,
designed as LASSBio-294 analogues, Bioorg. Med. Chem. 13 (2005)
3431e3437.
[10] S. Yamada, D. Morizono, K. Yamamoto, Mild oxidation of aldehydes to the
corresponding carboxylic acids and esters: alkaline iodine oxidation revisited,
Tetrahedron Lett. 33 (1992) 4329e4332.
[11] P.C. Lima, L.M. Lima, K.C.M. da Silva, P.H.O. Léda, A.L.P. de Miranda,
C.A.M. Fraga, E.J. Barreiro, Synthesis and analgesic activity of novel N-acylar-
ylhydrazones and isosters, derived from natural safrole, Eur. J. Med. Chem. 35
(2000) 187e203.
[12] C.A.M. Fraga, E.J. Barreiro, Medicinal chemistry of N-acylhydrazones: new
lead-compounds of analgesic, antiinflammatory and antithrombotic drugs,
Curr. Med. Chem. 13 (2006) 167e198.
[13] A.E. Kümmerle, J.M. Raimundo, C.M. Leal, G.S. da Silva, T.L. Balliano,
M.A. Pereira, C.A. de Simone, R.T. Sudo, G. Zapata-Sudo, C.A.M. Fraga,
E.J. Barreiro, Studies towards the identification of putative bioactive confor-
mation of potent vasodilator arylidene N-acylhydrazone derivatives, Eur. J.
Med. Chem. 44 (2009) 4004e4009.
The docking experiment was performed with the crystal struc-
ture of the A2A adenosine receptor (PDB ID: 3EML) [45] and the A3
adenosine receptor homology model made by our group using the
same crystal structure as the template. The docking program used
was GOLD 5.1 (CCDC) and its fitness function GoldScore [46]. The
GoldScore fitness function is calculated from the sum of the
following energy terms: hydrogen bond of the proteineligand
complex, van der Waals energy of the proteineligand complex,
van der Waals energy of the ligand, and ligand internal torsional
energy. Empirical parameters were used for the calculation. The
resulting nondimensional score represents the relative affinity
when comparing ꢄ2 ligands. A higher score represents more
favorable interactions in the ligandereceptor complex [46]. Before
the experiment, energy minimization was performed on LASSBio-
1027 with the semi-empirical AM1 method [47].
The set of amino acid residues selected as the binding site to
ꢀ
perform docking studies was determined by a distance of 10 A from
the conserved amino acid residue ASN253 located in the A2A and A3
receptors. Three consecutive runs were conducted, each of which
generated 10 conformations. The highest-scoring conformation of
each run was chosen and analyzed.
[14] M.C. Peitsch, Protein modeling by E-mail, Nat. Biotechnol. 13 (1995) 658e660.
[15] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace:
a web-based environment for protein structure homology modeling, Bio-
informatics 22 (2006) 195e201.
6.4. Binding assay
A binding assay was performed between LASSBio-1027 (10 mM)
[16] B.B. Fredholm, A.P. Ijzerman, K.A. Jacobson, K. Klotz, J. Linden, International
Union of Pharmacology XXV. Nomenclature and classification of adenosine
receptors, Pharmacol. Rev. 53 (2001) 527e552.
[17] M.E. Olah, G.L. Stiles, Adenosine receptor subtypes: characterization and
therapeutic regulation, Annu. Rev. Pharmacol. Toxicol. 35 (1995) 581e606.
[18] R.A. Olsson, J.D. Pearson, Cardiovascular purinoceptors, Physiol. Rev. 70 (1990)
761e845.
[19] R.L. Webb, R.B. Mcneal Jr., B.W. Barclay, G.D. Yasay, Hemodynamic effects of
adenosine agonists in the conscious spontaneously hypertensive rat,
J. Pharmacol. Exp. Ther. 254 (1990) 1090e1099.
[20] J.C. Shryock, L. Belardinelli, Adenosine and adenosine receptors in the
cardiovascular system: biochemistry, physiology, and pharmacology, Am. J.
Cardiol. 79 (1997) 2e10.
and the A1, A2A or A3 receptors of human recombinant CHO cells
(A1) or human recombinant HEK-293 cells (A2A and A3). [3H]CCPA
(1 nM), [3H]CGS21680 (6 nM) and [125I]AB-MECA (0.15 nM) were
used as the A1, A2A and A3 agonists radioligands, respectively. The
data were expressed as a percent inhibition of control specific
binding obtained in the presence of LASSBio-1027 using the
equation: % inhibition ¼ 100 ꢃ [(measured specific binding/control
specific binding) ꢂ 100].
Disclosure statement
[21] C.D. Lewis, S.M. Hourani, C.J. Long, M.G. Collis, Characterization of adeno-
sine receptors in the rat isolated aorta, Gen. Pharmacol. 25 (1994)
1381e1387.
[22] D.J. Prentice, S.M. Hourani, Activation of multiple sites by adenosine analogues
in the rat isolated aorta, Br. J. Pharmacol. 118 (1996) 1509e1517.
[23] B. Koscsó, B. Csóka, P. Pacher, G. Haskó, Investigational A3 adenosine receptor
targeting agents, Expert Opin. Investig. Drugs 20 (2011) 757e768.
[24] H.L. Maddock, M. Mocanu, D. Yellow, Adenosine A3 receptor activation
protects the myocardium from reperfusion/reoxygenation injury, Am. J.
Physiol. Heart Circ. Physiol. 283 (2002) H1307eH1313.
[25] H.L. Maddock, N.M. Gardner, N. Khandoudi, A. Bril, K.J. Broadley, Protection
from myocardial stunning by ischaemia and hypoxia with the adenosine A3
receptor agonist, IB-MECA, Eur. J. Pharmacol. 477 (2003) 235e245.
[26] N. Rudich, K. Ravid, R. Sagi-Eisenberg, Mast cell adenosine receptors function:
a focus on the A3 adenosine receptor and inflammation, Front. Immunol. 3
(2012) 1e12.
The authors declared no conflict of interest. All co-authors have
agreed with the submission of the final manuscript and all authors
participated in the research and article preparation.
Acknowledgments
This work was supported by Conselho Nacional de Desenvolvi-
mento Cientifico e Tecnológico (CNPq), Coordenação de Aperfei-
çoamento de Pessoal de Nível Superior (CAPES), PRONEX, Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ), Instituto Nacional de Ciência e Tecnologia (INCT).
[27] S. Leal, C. Sá, J. Gonçalves, P. Fresco, C. Diniz, Immunohistochemical charac-
terization of adenosine receptors in rat aorta and tail arteries, Microsc. Res.
Tech. 71 (2008) 703e709.
Appendix A. Supplementary data
[28] U. Ikeda, K. Kurosaki, K. Ohya, K. Shimada, Adenosine stimulates nitric oxide
synthesis in vascular smooth muscle cells, Cardiovasc. Res. 35 (1997)
168e174.
Supplementary data associated with this article can be found,
online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.056.

C.M. Leal et al. / European Journal of Medicinal Chemistry 55 (2012) 49e57
57
[29] C.J. Ray, J.M. Marshall, The cellular mechanisms by which adenosine evokes
release of nitric oxide from rat aortic endothelium, J. Physiol. 570 (2006)
85e96.
[39] C. Sala, A. Monopoli, C. Alberti, C. Casati, E. Ongini, A. Zanchetti, A. Morganti,
Humoral effects of selective adenosine agonists in spontaneously hyperten-
sive rats, J. Hypertens. 14 (1996) 75e79.
[30] E.A. Ko, J. Han, I.D. Junq, W.S. Park, Physiological roles of Kþ channels in
vascular smooth muscle cells, J. Smooth Muscle Res. 44 (2008) 65e81.
[31] R.K. Dubey, Z. Mi, D.G. Gillespie, E.K. Jackson, Dysregulation of extracellular
adenosine levels by vascular smooth muscle cells from spontaneously
hypertensive rats, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 249e254.
[32] R.K. Dubey, E.K. Jackson, H. Rupperecht, R.B. Sterzel, Factors controlling
growth and matrix production in vascular smooth muscle and glomerular
mesangial cells, Curr. Opin. Nephrol. Hypertens. 6 (1997) 88e105.
[33] R.K. Dubey, D.G. Gillespie, Z. Mi, F. Suzuki, E.K. Jackson, Smooth muscle cell
derived adenosine inhibits cell growth, Hypertension 27 (1996) 766e773.
[34] T. Abiru, T. Yamaguchi, Y. Watanabe, K. Kogi, K. Aihara, A. Matsuda, The
antihypertensive effect of 2-alkynyladenosines and their selective affinity for
adenosine A2 receptors, Eur. J. Pharmacol. 196 (1991) 69e76.
[40] R.L. Webb, M.A. Sills, J.P. Chovan, J.V. Peppard, J.E. Francis, Development of
tolerance to the antihypertensive effects of highly selective adenosine A2A
agonists upon chronic administration, J. Pharmacol. Exp. Ther. 267 (1993)
287e295.
[41] G. Zapata-Sudo, S.L. Pereira, H.J.V. Beiral, A.E. Kümmerle, J.M. Raimundo,
F. Antunes, Pharmacological characterization of (3-thienylidene)-3,4-meth-
ylenedioxybenzoylhydrazide: a novel muscarinic agonist with antihyperten-
sive profile, Am. J. Hypertens. 23 (2010) 135e141.
[42] C.J. Ray, M.R. Abbas, A.M. Coney, J.M. Marshall, Interactions of adenosine,
prostaglandins and nitric oxide in hypoxia-induced vasodilatation: in vivo and
in vitro studies, J. Physiol. 544 (2002) 195e209.
[43] X.M. Zhu, L.H. Fang, Y.J. Li, G.H. Du, Endothelium-dependent and independent
relaxation induced by pinocembrin in rat aortic rings, Vascul. Pharmacol. 46
(2007) 160e165.
[35] C. Casati, A. Monopoli, A. Forlani, E. Bonizzoni, E. Ongini, Telemetry moni-
toring of hemodynamic effects induced over time by adenosine agonists in
spontaneously hypertensive rats, J. Pharmacol. Exp. Ther. 275 (1995)
914e919.
[44] J. Duarte, F. Perez-Vizcaino, A.I. Torres, A. Zarzuelo, J. Jimenez, J. Tamargo,
Vasodilator effects of visnagin in isolated vascular smooth muscle, Eur. J.
Pharmacol. 286 (1995) 115e122.
[36] E. Bonizzoni, S. Milani, E. Ongini, C. Casati, A. Monopoli, Modeling hemody-
namic profiles by telemetry in the rat. A study with A1 and A2A adenosine
agonists, Hypertension 25 (1995) 564e569.
[45] V.P. Jaakola, M.T. Griffith, M.A. Hanson, V. Cherezov, E.Y. Chien, J.R. Lane,
A.P. Ijerman, R.C. Stevens, The 2.6 angstrom crystal structure of a human
A2A adenosine receptor bound to an antagonist, Science 322 (2008)
1211e1217.
[37] D.R. Borcherding, N.L. Lentz, P.M. Weintraub, M.W. Dudley, R. Secrest,
P.R. Kastner, N.P. Peet, The synthesis and biological activity of
selective adenosine A2A receptor agonist, Nucleosides Nucleotides 18 (1998)
2175e2191.
a
highly
[46] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997)
727e748.
[38] E. Camaioni, E. Di Francesco, S. Vittori, R. Volpini, G. Cristalli, Adenosine
receptor agonists: synthesis and biological evaluation of the diastereoisomers
of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA, Bioorg. Med. Chem. 5 (1997)
2267e2275.
[47] M.J.S. Dewar, E.G. Zoebisch, E.F. Healy, J.J.P. Stewart, Development and use of
quantum mechanical molecular models 76. AM1: a new general purpose
quantum mechanical molecular model, J. Am. Chem. Soc. 107 (1985)
3902e3909.