Indenopyrazole oxime ethers: Synthesis and β1-adrenergic blocking activity

Article abstract of DOI:10.1016/j.ejmech.2015.01.037

This paper reports the synthesis and cardiac activity of new β-blockers derived from (Z/E)-indeno[1,2-c]pyrazol-4(1H)-one oximes (5a,b). The latter compounds were allowed to react with epichlorohydrin, followed by reacting the oxiranyl derivatives formed

Full text of DOI:10.1016/j.ejmech.2015.01.037

Accepted Manuscript
Indenopyrazole Oxime Ethers: Synthesis and β -Adrenergic Βlocking Activity
1
Tommaso Angelone, Anna Caruso, Christophe Rochais, Angela Maria Caputo, Maria
Carmela Cerra, Patrick Dallemagne, Elisabetta Filice, David Genest, Teresa Pasqua,
Francesco Puoci, Carmela Saturnino, Maria Stefania Sinicropi, Hussein El-Kashef
PII:
S0223-5234(15)00057-4
10.1016/j.ejmech.2015.01.037
EJMECH 7653
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2014
Revised Date: 16 January 2015
Accepted Date: 19 January 2015
Please cite this article as: T. Angelone, A. Caruso, C. Rochais, A.M. Caputo, M.C. Cerra, P.
Dallemagne, E. Filice, D. Genest, T. Pasqua, F. Puoci, C. Saturnino, M.S. Sinicropi, H. El-Kashef,
Indenopyrazole Oxime Ethers: Synthesis and β -Adrenergic Βlocking Activity, European Journal of
1
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.01.037.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Highlights
► A new heterocyclic system indeno[1,2-c]pyrazole oxime ethers was reported. ►
Some derivatives of this heterocyclic system have been prepared. ► A selected
derivative was screened for its 1-adrenergic blocking activity. ► The oxime ether
(7b) reduces cardiac contractility and selectively antagonizes β1-AR.

ACCEPTED MANUSCRIPT
Indenopyrazole Oxime Ethers: Synthesis and β1-Adrenergic Βlocking Activity
Tommaso Angelonea,§, Anna Carusob,c,§, Christophe Rochaisd,§, Angela Maria
Caputob, Maria Carmela Cerraa,*, Patrick Dallemagned,*, Elisabetta Filicea, David
Genestd, Teresa Pasquaa, Francesco Puocib, Carmela Saturninoe, Maria Stefania
Sinicropib,* and Hussein El-Kasheff
aDepartment of Biology, Ecology and Earth Sciences, University of Calabria, 87036
Arcavacata di Rende, (CS), Italy;
bDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria,
87036 Arcavacata di Rende, (CS), Italy;
cDepartment of Computer Engineering, Modeling, Electronics and Systems,
University of Calabria, 87036 Rende (CS), Italy;
dUniversité de Caen Basse-Normandie, Centre d’Etudes et de Recherche sur le
Médicament de Normandie UPRES EA 4258 – FR CNRS 3038 INC3M, Bd Becquerel,
14032 Caen cedex, France;
eDepartment of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, 84084
Fisciano (SA), Italy;
fChemistry Department, Faculty of Science, Assiut University, 71516, Assiut, Egypt.
§Tommaso Angelone, Anna Caruso and Christophe Rochais have contributed equally
to the manuscript.
*Corresponding authors:
Tel.: +39 0984 493200; fax: +39 0984 493298.
E-mail address: s.sinicropi@unical.it (M.S. Sinicropi).
Tel.: +39 0984 492907; fax: +39 0984 492906.
E-mail address: maria_carmela.cerra@unical.it (M.C. Cerra).
Tel.: +33 (0)2 31566813; fax: +33 (0)2 31566803
E-mail address: patrick.dallemagne@unicaen.fr (P. Dallemagne).
Abstract
This paper reports the synthesis and cardiac activity of new β-blockers derived from
(Z/E)-indeno[1,2-c]pyrazol-4(1H)-one oximes (5a,b). The latter compounds were
allowed to react with epichlorhydrin, followed by reacting the oxiranyl derivatives
formed (6a,b) with some aliphatic amines to give the target compounds (Z/E)-1-
phenyl-1H-indeno[1,2-c]pyrazol-4-one
O-((2-hydroxy-3-(substituted
1

ACCEPTED MANUSCRIPT
amino)propyl)oxime (7a-c) and (Z/E)-1-methyl-1H-indeno[1,2-c]pyrazol-4-one O-
((2-hydroxy-3-(substituted amino)propyl)oxime (8a-c). These final products 7a-c and
8a-c were evaluated for their ability to modulate the cardiac performance of a
prototype mammalian heart. The results showed that, out of these molecules tested,
7b elicits a more potent depressant effect on contractility and relaxation, and
competitively antagonizes β1-adrenergic receptors.
Keywords. β-Adrenergic blocking agents, β-adrenoceptors, indenopyrazoles,
indenopyrazole oximes, hydroxypropanolamines.
1.
Introduction
The β-blockers are an important class of drugs used in the treatment of several
cardiovascular diseases such as: hypertension, angina pectoris, heart attack and certain
arrhythmias [1]. Indeed, their action is characterized by a mechanism of competitive
antagonism on β1-receptor, localized on the heart, on the arteriolar smooth muscle and
on the β-cells of the juxtaglomerular apparatus, thus chronotropic, inotropic and
dromotropic effect is obtained [2]. Drugs, generally, used in therapy and/or
commercially available as β-blockers are compounds with a well-defined structure I.
They are all chiral molecules (optically active or racemic) in which three functions
can be identified: an aryl group linked through -OCH2- (or another group) to a moiety
containing both an alcohol and an amino function bearing an i-propyl, tert-butyl or a
more bulky residue [3] (Figure 1).
Thus, β-adrenoceptor-blocking agents generally belong to the arylethanolamine II and
aryloxypropanolamine classes. Many of the most potent β-blockers used for the
treatment of hypertension, belong to the latter class such as propranolol III [4,5].
Figure 1: Structures of β-blockers.
2

ACCEPTED MANUSCRIPT
Several β-blockers are now available; however some of them show certain side
effects. These side effects are mainly due to the bronchoconstriction action due to the
β2-receptor localized mainly on the bronchial smooth muscle. Moreover, the
stimulation of the β3-receptors, mainly present on the adipose tissue lead to a
consequent block and alteration of the activity of the enzyme lipase interfering with
the triglyceride synthesis. Therefore the synthesis of new specific β1-antagonists is
still needed.
Previous studies [6,7,10-22] demonstrated that the imino group of β-blockers side
chain bearing the required hydroxyalkyamino side chain attached to an oximino
group, did not abolish the β-adrenoceptor activity of these β-blocking agents. In the
same time some of these β-blockers showed β1- and others showed β2-selective
antagonism [6-10].
Several publications have described the discovery and characterization of the
biological, microbiological and pharmacological activities of novel oxime ethers as β-
adrenoceptor ligands [17, 23]. In the light of the potent β-adrenoceptor blocking
activity of fluorenone oxime ether (IPS 339) IV [22], we wish herein to designe a
synthesis in order to rapidly pharmacomodulate this structure using the commercially
available indanedione (Figure 2) as a starting material to obtain the target heterocycles
(7a-c and 8a-c). The biological activities of these novel heterocycles, analogs of IPS
339 IV, were evaluated for their β1-adrenergic blocking activity, in ex vivo
Langendorf rat heart preparation.
Figure 2: Structure of IPS 339 (IV).
2.
Results and discussion
2.1
Chemistry
The synthesis of some unknown oxime ethers 7a-c and 8a-c derived from indeno[1,2-
c]pyrazole could be envisaged by substituting one of the benzene rings of the fluorene
moiety of IV by a pyrazole ring. Thus, we have adopted the procedure reported by
3

ACCEPTED MANUSCRIPT
Schenone et al. [24] to prepare the indeno[1,2-c]pyrazoles 3a-b using indane-1,3-
dione as a starting material (Scheme 1). When this dione was reacted with N,N-
dimethylformamide
dimethyl
acetal
(DMF/DMA)
the
2-(N,N-
dimethylaminomethylene)indane-1,3-dione (2) was obtained. This was reacted with
phenylhydrazine or methylhydrazine in butanol in the presence of acetic acid to give
2-(N'-phenylhydrazinomethylene)indane-1,3-dione
(3a)
and
2-(N'-
methylhydrazinomethylene)indane-1,3-dione (3b) respectively which were cyclised,
upon heating under reflux in anhydrous toluene and in the presence of p-
toluenesulfonic acid, giving 1-phenyl-1H-indeno[1,2-c]pyrazol-4-one (4a), with 42%
yield, and 1-methyl-1H-indeno[1,2-c]pyrazol-4-one (4b), with 35% yield.
The reaction of the latter compound with hydroxylamine hydrochloride in pyridine
gave the corresponding oximes 5a-b (isomeric mixture E/Z). These oximes were then
converted into the corresponding ethers 7a-c and 8a-c in a two-step reaction following
the classical route as indicated in Scheme 1. Thus the oximes 5a-b were reacted with
epichlorohydrin to give the oxiranyl derivatives 6a-b which were allowed to react
with the appropriate amines; i-propylamine, n-butylamine and tert-butylamine to
obtain 7a-c and 8a-c.
Reagents and conditions: (i) N,N-dimethylformamide dimethyl acetal (DMF/DMA), toluene, reflux,
24h; (ii) Phenyl hydrazine or methyl hydrazine, butan-1-ol, AcOH, overnight, 0 °C; (iii) TsOH,
toluene, reflux, 24h; (iv) NH2OH.HCl, pyridine, rt, 3h; (v) Epichlorhydrin, acetone/water, K2CO3,
reflux, 6 days. (vi) RNH2, anhydrous toluene.
Scheme 1. Synthetic pathway of compounds 7a-c and 8a-c.
4

ACCEPTED MANUSCRIPT
2.2 Biology
The present study shows that six newly synthesized molecules, namely 7a-c and 8a-c,
structurally similar to the known beta-blocker fluorenone oxime ether IV (IPS 339),
are able to modulate the mammalian cardiac performance with different order of
potency. On the isolated and Langendorff perfused rat heart, 7b determined a dose-
dependent reduction of basal myocardial contractility (inotropism) and relaxation
(lusitropism). This effect, which is more potent than that elicited by 7a and 7c, is
obtained without changing heart rate (HR) and coronary pressure (CP). In addition, 7b
elicited competitive antagonism against β1-adrenergic receptors. Because of its higher
influence on the cardiac performance, only 7b was included in the study on the
mechanism of action. 8a and 8b did not induce significant effects on cardiac
performance except for a dose-dependent positive inotropism induced by 8c.
2.2.1 Effect of 7a-c and 8a-c on basal cardiac performance
β-Blockers are known by their basal inotropic action on the mammalian heart. An
example is the classic negative inotropism and lusitropism induced by propranolol, a
typical non-selective β-blocker [25]. In our study we found that 7b reduces
myocardial contraction and relaxation, suggesting this molecule to be functionally
similar to β-blockers. Analysis of the IC50 values on left ventricular pressure (LVP)
revealed that the inhibitory concentration of 7b is 5x10-10 M. This is of relevance
since many common β-blockers, such as propranolol, nadolol, metapropol show an
IC50 of 12x10-9 M [26]. Accordingly, we suggest that 7b is able to elicit inhibitory
effects at a concentration lower than that of other β-blockers. Contrarily, 7a, 7c, 8a
and 8b did not affect basal inotropism and lusitropism except for the positive
inotropism induced by 8c. This could be attributed to the different structural
characteristics of the six molecules at the level of the substituent on the nitrogen atom
of the amine portion, important structural region for the β-blocker activity, and also of
the N1 of the pyrazole ring. In particular, compounds 7a, 8a and 7b, 8b contain an i-
propyl- and n-butyl group respectively on the aminic portion, while 7c, 8c have a tert-
butyl chain.
2.2.1.1 Basal conditions
5

ACCEPTED MANUSCRIPT
Langendorff perfused heart- Cardiac parameters, obtained after 20 min equilibration,
are indicated in Materials and Methods. Endurance and stability of the preparations,
analyzed by measuring the performance variables every 10 min, showed that each
heart was stable up to 180 min.
2.2.1.2 7a-c and 8a-c stimulated preparations
Preliminary experiments (data not shown) obtained by repetitive exposure of each
heart to one concentration of 7a, 7b, 7c, 8a, 8b or 8c (1 nM) revealed the absence of
desensitization.
The biological potency of these putative novel beta-blockers was evaluated by
analyzing the hemodynamic performance of rat hearts ex vivo perfused according to
Langendorff. We found that application of 7a from 1 pM to 10 nM resulted in a
negative inotropic effect revealed by the reduction of LVP and +(LVdP/dT)max
which reached a maximum (20%) at the highest doses tested. 7a did not modify HR
and CP. 7b, which is ineffective on HR and CP, induced a strong reduction of
inotropic parameters (LVP and +(LVdP/dT)max) at all concentrations tested (1 pM-
10 nM), reaching a maximun of reduction of 40% at 10 nM. The IC50 of 7b-dependent
negative inotropism was 5 x 10-10 M. In contrast to the other molecules, 7c did not
significantly affect inotropism, while it induced an insignificant increment of HR and
CP (Figure 3-5). Of note, 8a and 8b elicited limited and insignificant effects on
cardiac performance. 8a increased inotropism (LVP and (LVdP/dt)max) only at
concentration of 10-9 M; it enhanced HR at 10-10 M and 10-9 M with a non significant
vasoconstriction. 8b was able to decrease only LVP at 10-9M and increase HR at 10-10
M and 10-9 M. 8c induced dose-dependent positive inotropism at all concentration
tested without changes in HR and CP (Figure 6-8).
6

ACCEPTED MANUSCRIPT
0
-10
-20
-30
-40
*
*
*
0
-10
-20
-30
-40
*
*
*
30
20
10
0
-10
40
30
20
10
0
1pM 10pM 100pM 1nM 10nM
[7a]
Figure 3. Dose-dependent response curves of 7a (1 pM-10 nM) on LVP,
+(LVdP/dT)max, CP and HR, on Langendorff perfused rat heart preparations.
Percentage changes were evaluated as means ± SEM of 7 experiments. Significance
of difference from control values was evaluated by one-way ANOVA; P= 0<0.05.
7

ACCEPTED MANUSCRIPT
IC50= 5 x 10-10
R²= 0,9543
M
IC50
-20
-25
n
o
i
t
i
-30
b
i
h
n
i
-35
P
V
L
-40
%
-45
-13
-12
-11
-10
-9
-8
-7
[6c], log M
[7b], log M
30
20
10
0
-10
-20
1pM 10pM 100pM 1nM 10nM
[7b]
Figure 4. Dose-dependent response curves of 7b (1 pM-10 nM) on LVP,
+(LVdP/dT)max, CP and HR, on Langendorff perfused rat heart preparations.
Percentage changes were evaluated as means ± SEM of 7 experiments. Significance
of difference from control values was evaluated by one-way ANOVA; P= 0<0.05.
8

ACCEPTED MANUSCRIPT
10
0
-10
-20
-30
-40
-50
20
10
0
-10
-20
-30
40
30
20
10
0
-10
40
30
20
10
0
-10
1pM 10pM 100pM 1nM 10nM
[7c]
Figure 5. Dose-dependent response curves of 7c (1 pM-10 nM) on LVP,
+(LVdP/dT)max, CP and HR, on Langendorff perfused rat heart preparations.
Percentage changes were evaluated as means ± SEM of 7 experiments. Significance
of difference from control values was evaluated by one-way ANOVA; P= 0<0.05.
9

ACCEPTED MANUSCRIPT
60
50
40
30
20
10
0
*
-10
-20
60
50
40
30
20
10
0
*
-10
-20
40
30
20
10
0
*
*
-10
110
100
90
80
70
60
50
40
30
20
10
0
-10
10-12 10-11 10-10 10-9 10-8 M
[8a]
Figure 6. Dose-dependent response curves of 8a (10-12 M – 10-8 M) on LVP,
+(LVdP/dT)max, HR and CP, on Langendorff perfused rat heart preparations.
Percentage changes were evaluated as means ± SEM of 7 experiments. Significance
of difference from control values was evaluated by one-way ANOVA; P= 0<0.05.
10

ACCEPTED MANUSCRIPT
60
50
40
30
20
10
0
-10
-20
*
60
50
40
30
20
10
0
-10
-20
40
30
20
10
0
*
*
-10
110
90
70
50
30
10
-10
10-12 10-11 10-10 10-9 10-8 M
[8b]
Figure 7. Dose-dependent response curves of 8b (10-12 M – 10-8 M) on LVP,
+(LVdP/dT)max, HR and CP, on Langendorff perfused rat heart preparations.
Percentage changes were evaluated as means ± SEM of 7 experiments. Significance
of difference from control values was evaluated by one-way ANOVA; P= 0<0.05.
11

ACCEPTED MANUSCRIPT
60
50
40
30
20
10
0
*
*
*
-10
-20
60
50
40
30
20
10
0
*
*
*
*
*
-10
-20
40
30
20
10
0
-10
110
90
70
50
30
10
-10
10-12 10-11 10-10 10-9 10-8 M
[8c]
Figure 8. Dose-dependent response curves of 8c (10-12 M – 10-8 M) on LVP,
+(LVdP/dT)max, HR and CP, on Langendorff perfused rat heart preparations.
Percentage changes were evaluated as means ± SEM of 7 experiments. Significance
of difference from control values was evaluated by one-way ANOVA; P= 0<0.05.
12

ACCEPTED MANUSCRIPT
2.2.2 Anti-adrenergic action of 7b
It is reported that propranolol elicits competitive antagonism against adrenergic
stimulation and this occurs with an EC50 of 30 nM [27]. Our experiments on the rat
heart revealed that, like propranolol, 7b exerted competitive antagonism in the
presence of an isoproterenol-dependent adrenergic stimulation, being able to
counteract both positive inotropism and lusitropism. Notably, this 7b-induced
competitive antagonism is obtained at a concentration of 0.5 nM, lower than that
reported for propranolol (30 nM; [27]). This confirms the β-blocker-like property of
this new synthesized molecule which appears to behave as a classic anti-adrenergic
drug, but at higher potency. This may be of notable pharmacological interest since it
is recognized that the lower is the active dose of a molecule, the lower can be the
possibility of side effects [28].
Of paramount importance in relation to both basic research and clinical application, is
the catecholamine-dependent regulation of cardiac function which occurs through
activation of β1 and β2 types of β-adrenergic receptors [29]. β1-Receptors activates Gs
proteins, with consequent stimulation of adenylate cyclase, increase of intracellular
cAMP and protein kinase-A (PKA) activation. This induces phosphorylation of
phospholamban, troponin I and sarcoplasmic reticulum Ca2+/ATPasi (SERCA), all
these effects contributing to the positive inotropic and the lusitropic actions which
characterize β1-receptors activity [29]. On the other hand, cardiac β2 receptors,
alternatively coupled to Gs and Gi proteins, are associated to positive and negative
inotropic effects, respectively [30]. In recent years the heart was found to express
another class of adrenergic receptors, namely β3 [31]. Stimulation of these receptors
leads to negative inotropism and lusitropism [32,33], and counteracts the effects
elicited by β1 and β2 activation [34]. This cardiodepression depends on Gi, Gi/o
protein, nitric oxide (NO) generation via the endothelial isoform of Nitric Oxide
Synthase (eNOS), the subsequent increase of intracellular cGMP [31,34] and
activation of protein kinase G (PKG) [33]. In this study we observed that, β3-receptors
are inhibited by a specific antagonist (SR59230), 7b efficacy in counteracting the
effects of isoproterenol-induced adrenergic stimulation is still detectable. This
observation may contribute to characterize 7b as a β1-receptor antagonist, excluding
this molecule to function as a partial β3-receptor agonist. β1-receptor selectivity was
13

ACCEPTED MANUSCRIPT
further confirmed in the presence of non-selective α-adrenergic antagonism by
phentolamine.
2.2.2.1 Anti-adrenergic action of 7b-Langendorff perfused heart
To verify the possible anti-adrenergic action of 7b, heart preparations were perfused
with KHs containing increasing concentrations of Isoproterenol (Iso: 0.1 nM to 1 µM)
either alone or in combination with 7b. Iso stimulation induced a significant increase
of RPP from 5 nM to 1 µM (Figure 9). The subsequent analysis of the percentage of
variations of RPP provided the EC50 values in the presence of either increasing
concentrations of Iso alone or of Iso plus 7b (5 x 10-10 M). Results showed that 7b
exerts a competitive antagonism on adrenergic stimulation inducing a dose-dependent
reduction of Iso intrinsic activity. EC50 values (in logM) and the intrinsic activity of
Iso alone and of Iso in the presence of 7b are shown in the legend of Figure 9.
7b
§
Figure 9. The sigmoid concentration-response curves of ISO-mediated stimulation on
RPP of ISO (from 10-10 to 10-6 M) alone and ISO (from 10-10 to 10-6 M) plus a single
concentration of 7b (5x10-10 M) on the isolated and perfused Langendorff rat heart
preparation. Contraction is expressed as a percentage of RPP [baseline= 0%, peak
constriction by Iso and Iso plus 7b=100 %]. The EC50 values (in logM) of Iso alone
was -8.64 ± 0.23 (r2= 0.93) and of Iso plus 7b (5x10-10 M) was -7.6 ± 0.26 (r2= 0.90).
Comparison between groups (n=6 for each group) (ANOVA, Duncan's test); § = p <
0.05.
14

ACCEPTED MANUSCRIPT
2.2.2.2. Beta 2-AR, Beta3-AR and Alpha-AR receptors involvement in the anti-
adrenergic antagonism of 7b-Langendorff perfused heart
To verify the selectivity of β-adrenergic receptor antagonism of 7b, hearts were
perfused with a single concentration of ISO (5 nM) plus 7b (5 nM) plus β2-adrenergic
antagonist (ICI118,551: 100 nM) or Alpha and β3-adrenergic antagonists
(phentolamine: 100 nM; SR59230: 100 nM, respectively). As expected, ISO alone
induced positive inotropism and lusitropism. These effects were abolished by co-
administration of ISO plus 7b. Contrarily, co-administration of ISO plus 7b plus
phentolamine and SR59230 or plus ICI118,551 did not affect the anti-adrenergic
effect of 7b (Figure 10a,b).
15

ACCEPTED MANUSCRIPT
a)
§
§
§
50
§
50
40
30
20
10
0
*
40
*
*
30
20
10
0
§
§
-10
-10
40
40
§
§
*
30
*
30
20
10
0
*
20
10
0
-10
-10
§
b)
50
40
30
20
10
0
50
§
40
*
*
*
*
30
20
10
0
-10
40
-10
40
§
§
*
30
20
10
0
*
30
20
10
0
*
*
-10
-10
Figure 10. Effects of ISO (5 nM) before and after treatment with 7b (5x10-10 M), or
phentolamine (100 nM) plus SR59230 (100 nM), or phentolamine plus SR59230 plus
7b or ICI118,551 (100 nM), or ICI118,551 plus 7b on LVP (left ventricular pressure),
RPP (rate pressure product), LVdP/dtmax and -LVdP/dtmax on the rat isolated and
Langendorff perfused heart. Percentage changes were evaluated as means ± SEM of 5
experiments for each group. Significant difference from control values; * = p < 0.05.
Comparison between groups; § = p < 0,05.
16

ACCEPTED MANUSCRIPT
3.
Conclusion
New β-adrenergic blockers 7a-c and 8a-c derived from the key intermediates 1-
phenylindeno[1,2-c]pyrazol-4(1H)-one oxime (5a) and 1-methylindeno[1,2-c]pyrazol-
4(1H)-one oxime (5b) respectively have been synthesized and evaluated for their
ability to modulate the cardiac performance of a prototype mammalian heart, showing
different degrees of potency. The results obtained showed that 7b is the most potent
derivative in eliciting a dose-dependent reduction of basal myocardial contractility
and relaxation without affecting heart rate and coronary pressure. This is of
physiological relevance since excessive and uncontrolled changes in coronary
pressure and frequency may be detrimental for cardiac homeostasis. Based on our
results, 7b proved to be the best candidate to elicit β-blocker function for both its
potency and effectiveness in counteracting β1-adrenergic stimulation in a competitive
manner. In contrast to classical effects induced by β-adrenergic blockers, 8c showed
positive inotropism suggesting this molecule as a partial β-adrenergic agonist rather
than antagonist. However, this aspect requires further insight.
4.
Experimental section
4.1
Synthesis and characterization
Commercial reagents were purchased from Aldrich, Acros Organics and Alfa Aesar
and used without additional purification. Melting points were determined on a Kofler
melting point apparatus. IR spectra were taken with a Perkin Elmer BX FT-IR. Mass
spectra were taken on a JEOL JMS GCMate spectrometer at ionising potential of 70
eV (EI) or were performed using a spectrometer LC-MS Waters alliance 2695 (ESI+).
13
1H-NMR and C-NMR spectra were recorded on a JEOL Lambda 400 spectrometer
1
400 MHz (400 MHz for H, 100 MHz for the 13C) or on a Bruker 300 MHz
spectrometer (300 MHz for 1H, 75 MHz for the 13C). 1H and 13C NMR chemical shifts
(δ) were reported in parts per million (ppm) and were referenced to the solvent peak;
1
13
1
CDCl3 (7.26 ppm for H and 76.90 ppm for C) and (CD3)2CO-d6 (2.05 ppm for H
and 29.48 ppm for 13C). Multiplicities are represented by s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet). Coupling constants (J) are reported in Hertz
(Hz). Thin layer chromatography (TLC) was performed on silica gel 60F-264
(Merck).
17

ACCEPTED MANUSCRIPT
2-(N,N-Dimethylaminomethylene)indane-1,3-dione (2) was prepared as described in
the literature [24,35].
4.1.1
General
procedure
for
the
preparation
of
2-(N'–
phenyl(methyl)hydrazinomethylene) indane-1,3-diones (3a,b)
Phenylhydrazine or methylhydrazine (7.14 mmol) in 1-butanol (4 mL) was slowly
added with stirring to a solution of 2-(N,N-dimethylaminomethylene)indane-1,3-dione
2 (1.37 g, 6.80 mmol) in 1-butanol (10 mL) and acetic acid (0.5 ml). The resulting
solution was stirred at 0-5 °C for overnight. The resulting mixture was extracted with
dichloromethane (2 x 20 mL). The organic layer was dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The resulting residue was purified
on a silica gel column chromatography in petroleum ether/ethyl acetate (1/1) to give
3a,b.
4.1.1.1 2-(N'–phenylhydrazinomethylene)indane-1,3-dione (3a).
Brown solid, mp 110 °C, (yield 44 %). IR (KBr, cm-1): ν = 3452; 2936; 2358,03;
1
1646; 1621; 1560; 1462; 1340; 1288; 1232; 1166; 1023; 864; 751; 670. H-NMR
(CDCl3, 400 MHz): δ ppm = 4.02-4.08 (m, 1H, NH-NH-Ar); 6.26 (br, 1H, NH-NH-
C6H5), 6.87-6.91 (m, 2H, Ar); 7.03-7.07 (m, 1H, Ar); 7.31-7.35 (m, 2H, Ar); 7.67-7.70
(m, 2H, Ar); 7.79-7.82 (m, 1H, Ar); 7.83-7.85 (m, 1H, Ar); 7.97 (s, 1H, C=CH-NH).
13C-NMR (CDCl3, 100 MHz): δ 104.45; 113.82; 121.71; 122.27; 122.78; 129.67;
133.64; 133.77; 139.83; 140.37; 146.16; 153.67; 189.66; 193.71. MS (ESI+): 265
(M++1). HRMS (ESI+) m/z calcd for C16H12N2O2: 265.0977; found 265.0983.
4.1.1.2 2-(N'–methylhydrazinomethylene)indane-1,3-dione (3b).
Orange solid, mp 210 °C, (yield 30 %). IR (KBr, cm-1): ν = 3442; 2925; 2369; 1631;
1
1570; 1503; 1437; 1370; 1160; 1104; 1038; 859; 711; 593; 511. H-NMR (CDCl3,
400 MHz): δ ppm = 3.52 (s, 3H, CH3); 5.29 (s, 2H, 2NH); 7.32 (s, 1H, CH-NH); 7.57-
7.64 (m, 2H, Ar); 7.70-7.73 (m, 2H, Ar). 13C-NMR (DMSO-d6, 75 MHz): δ 38.65;
99.60; 128.50; 130.40; 132.75; 158.00; 191.98. MS (ESI+): 203 (M++1). HRMS
(ESI+) m/z calcd for C11H10N2O2: 202.2145; found 202.2150.
18

ACCEPTED MANUSCRIPT
4.1.2 General procedure for the preparation of 1-phenyl-1H-indeno[1,2-c]pyrazol-4-
ones (4a,b).
A mixture of hydrazinomethylene-indane-1,3-dione (3a,b) (3.0 mmol) and p-
toluensulfonic acid (0.015 g, 8.7 x 10-2 mmol) in anhydrous toluene (10 mL) was
refluxed in a Dean-Stark apparatus for 24 h. The resulting reaction mixture was
cooled, and extracted with dichloromethane (2 x 50 mL). The combined organic layer
was washed with an aqueous solution of NaOH (1 N, 100 mL) then with brine and
finally dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The resulting residue was purified on silica gel using dichloromethane/ethyl
acetate/petroleum ether (1/8/1) as an eluent to give 4a,b.
4.1.2.1 1-Phenyl-1H-indeno[1,2-c]pyrazol-4-one (4a).
Brown solid, mp 230 °C, (yield 42 %). IR (KBr, cm-1): ν = 3442; 2977; 2930; 2859;
2363; 1657; 1631; 1503; 1472; 1380; 1309; 1176; 1135; 1048; 976; 956; 762; 711;
1
674. H-NMR (CDCl3, 400 MHz): δ ppm = 7.17-7.19 (m, 1H, Ar), 7.29-7.33 (m, 2H,
Ar), 7.49-7.53 (m, 1H, Ar), 7.56-7.60 (m, 3H, Ar), 7.68-7.72 (m, 3H, Ar). 13C-NMR
(CDCl3, 100 MHz): δ 119.62; 123.44; 124.78; 129.05; 129.62; 130.11; 133.00;
136.26; 138.78; 140.79; 158.00; 184.00. MS (ESI+): 247 (M++1). HRMS (ESI+) m/z
calcd for C16H10N2O: 247.0871; found: 247.0883.
4.1.2.2 1-Methyl-1H-indeno[1,2-c]pyrazol-4-one (4b).
Orange solid, mp 140 °C, (yield 35 %). IR (KBr, cm-1): ν = 3442; 2936; 1713; 1611;
1
1554; 1468; 1268; 1196; 1064; 966; 879; 639; 639. H-NMR (CDCl3, 400 MHz): δ
13
ppm = 3.75 (s, 3H, CH3); 6.96-7.45 (m, 4H, Ar); 7.59-7.61 (m, 1H, Ar). C-NMR
(CDCl3, 100 MHz): δ 37.84; 118.70; 121.72; 123.03; 124.79; 129.78; 132.75; 135.04;
140.97; 183.20; 157.50. MS (ESI+): 185 (M++1). HRMS (ESI+) m/z calcd for
C11H8N2O : 185.0715; found: 185.0714.
4.1.3 General procedure for the preparation of (Z/E)-1-phenyl(methyl)-1H-
indeno[1,2-c] pyrazol-4-one oximes (5a,b).
19

ACCEPTED MANUSCRIPT
To a solution of indenopyrazole derivatives 4a,b (1.26 mmol) in pyridine (5 mL)
hydroxylamine hydrochloride (0.096 g, 1.39 mmol) was added and the resulting
mixture was stirred at rt for 3 h. The reaction mixture was then poured onto an ice-
water mixture 0-5 °C and stirred for 2 h. followed by extraction twice with
dichloromethane (2 x 50 mL) and the combined organic layer was washed with a
solution of HCl (1N, 150 mL) and then with brine. The organic layer was dried over
MgSO4, filtered and concentrated under reduced pressure to give 5a,b.
4.1.3.1 (Z/E)-1-Phenyl-1H-indeno[1,2-c]pyrazol-4-one oxime (5a).
Orange crystals, mp 212 °C, (yield 97 %). IR (KBr, cm-1): ν = 3442; 2977; 2930;
1
2859; 2363; 1703; 1636; 1457; 1370; 1207; 1156; 1115; 1040; 961; 723; 685. H-
NMR ((CD3)2CO), 400 MHz): δ ppm = 7.31-7.34 (m, 3H, Ar); 7.44-7.46 (m, 2H, Ar);
7.53-7.57 (m, 2H, Ar); 7.69-7.82 (m, 3H, Ar); 11.25 (s, 1H, C=N-OH). 13C-NMR
((CD3)2CO), 100 MHz): δ 120.65; 123.17; 124.06; 128.83; 129.13; 130.11; 130.44;
130.88; 137.75; 140.55; 141.33; 146.98. MS (ESI+): 262 (M++1). HRMS (ESI+) m/z
calcd for C16H11N3O: 262.0980; found: 262.0974.
4.1.3.2 (Z/E)-1-Methyl-1H-indeno[1,2-c]pyrazol-4-one oxime (5b).
Yellow solid, mp 210 °C, (yield 60 %). IR (KBr, cm-1): ν = 3422; 3217; 2854; 2363;
1
1646; 1564; 1469; 1263; 1166; 1068; 752; 711. H-NMR ((CD3)2CO, 300 MHz): δ
ppm = 3.98 (s, 3H, CH3), 7.10-7.34 (m, 2H, Ar), 7.43-7.67 (m, 3H, Ar), 8.50 (br, 1H,
OH). 13C-NMR (DMSO-d6, 75 MHz): δ 37.56; 119.71; 122.01; 127.61; 129.21;
134.59; 148.00. MS (ESI+): 200 (M++1). HRMS (ESI+) m/z calcd for C11H9N3O:
199.2139; found: 199.2133.
4.1.4 General procedure for the preparation of (Z/E)-1-phenyl(methyl)-1H-
indeno[1,2-c] pyrazol-4-one O-(oxiranylmethyl)oximes (6a,b).
To a solution of 5a or 5b (1.26 mmol) in acetone (10 mL), K2CO3 (0.35 g, 2.52
mmol), epichlorhydrin (0.13 g, 1.38 mmol) and H2O (1 mL) were added. The reaction
mixture was heated under reflux for 6 days [22,36]. The solvent was evaporated under
reduced pressure and the residue was extracted with dichloromethane (3 x 50 mL).
20

ACCEPTED MANUSCRIPT
The organic layer was dried over Na2SO4, filtered and concentrated under reduced
pressure. The crude products 6a,b were used as such for the next reaction.
4.1.4.1 (Z/E)-1-Phenyl-1H-indeno[1,2-c]pyrazol-4-one O-(oxiranylmethyl)oxime (6a).
Orange solid, mp 210 °C, (yield 98 %). 1H-NMR (CDCl3, 400 MHz): δ ppm = 2.73-
2.75 (m, 1H, CH-H-O), 2.89-2.91 (m, 1H, CH-H-O); 3.38-3.42 (m, 1H, CH2-CH-O);
4.31-4.35 (m, 1H, CH-H-O-N=), 4.53-4.57 (m, 1H, CH-H-O-N); 7.32-7.33 (m, 1H,
Ar); 7.42-7.46 (m, 1H, Ar); 7.52-7.56 (m, 3H, Ar); 7.74-7.84 (m, 4H, Ar); 7.92 (s, 1H,
Ar). MS (ESI+): 318 (M++1).
4.1.4.2 (Z/E)-1-Methyl-1H-indeno[1,2-c]pyrazol-4-one O-(oxiranylmethyl)oxime (6b).
Brown oil, (yield 97 %). 1H-NMR ((CD3)2CO, 300 MHz): δ ppm = 3.50-4.15 (5H, -
CH3, -CH2-); 4.30-4.50 (m, 2H, -CH2); 5.20-5.32 (m, 1H, -CH-O-); 7.10-7.38 (m, 3H,
Ar); 7.48-7.70 (m, 2H, Ar).
4.1.5 General procedure for the preparation of (Z/E)-1-phenyl(methyl)-1H-
indeno[1,2-c] pyrazol-4-one O-((2-hydroxy-3-(substituted amino)propyl)oximes (7a-
c) and (8a-c).
To a solution of 6a or 6b (0.1 mmol) in anhydrous toluene (10 mL) was added i-
propylamine, n-butylamine, or tert-butylamine (5 mL). The reaction mixture was
heated at 80 °C in a sealed tube for 7 days [36,37]. The reaction mixture was cooled,
evaporated under reduced pressure, and the solid obtained was purified on silica gel
using dichloromethane/methanol (9:1) as an eluent to give 7a-c (for 6a) and 8a-c (for
6b).
4.1.5.1
(Z/E)-1-Phenyl-1H-indeno[1,2-c]pyrazol-4-one
O-((2-hydroxy-3-
(isopropylamino)propyl)oxime (7a).
Orange solid, mp 145 °C, (yield 10 %). IR (KBr, cm-1): ν = 3466; 3433; 2082; 1635;
1
1531; 1461; 1384; 1261; 1101; 1055; 972; 947; 867; 762; 643; 694; 725. H-NMR
(CDCl3, 400 MHz): δ ppm = 1.22-1.52 (m, 6H, 2CH3); 3.04-3.30 (m, 3H, CH2-NH;
CH); 4.17-4.18 (m, 1H, CH-OH); 4.31-4.50 (m, 2H, CH2-OH); 5.27 (s, 1H, NH); 5.42
(s, 1H, OH); 7.19-7.24 (m, 1H, Ar); 7.27-7.29 (m, 1H, Ar); 7.43-7.45 (m, 1H, Ar);
21

ACCEPTED MANUSCRIPT
7.50-7.54 (m, 2H, Ar); 7.65-7.70 (m, 4H, Ar); 7.87 (s, 1H, Ar). 13C-NMR (CDCl3, 100
MHz): δ 14.38; 19.62; 32.15; 37.04; 50.77; 68.51; 119.37; 119.82; 123.07; 123.25;
128.20; 128.37; 128.77; 129.47; 129.53; 130.25; 130.87; 137.60; 139.29; 140.15;
146.63; 149.08. MS (ESI+): 377 (M++1). HRMS (ESI+) m/z calcd for C22H24N4O2:
377.1978; found: 377.1968.
4.1.5.2
(Z/E)-1-Phenyl-1H-indeno[1,2-c]pyrazol-4-one
O-((3-n-butylamino)-2-
hydroxypropyl)oxime (7b).
Brown solid, mp 215 °C, (yield 25 %). IR (KBr, cm-1): ν = 3943; 3861; 3802; 3585;
3521; 3497; 3400; 3230; 3178; 2934; 2859; 1637; 606. 1H-NMR (CDCl3, 300 MHz):
δ ppm = 0.70-0.90 (m, 3H, CH3); 1.10-1.40 (m, 4H, 2CH2); 1.80-2.10 (m, 2H, CH2-
NH); 1.50 (br, 1H, OH) 2.68-3.10 (m, 2H, CH2-NH-); 3.40-3.50 (m, 3H, CH-OH,
CH2-O-N); 7.35-7.55 (m, 5H, Ar); 7.60-7.70 (m, 4H, Ar); 8.12 (s, 1H, Ar); 9.70-9.75
(m, 1H, NH). 13C-NMR (CDCl3, 100 MHz): δ 14.45; 22.34; 32.46; 37.16; 50.89;
69.78; 120.43; 120.67; 123.11; 123.35; 128.34; 128.45; 128.87; 129.54; 129.67;
130.34; 130.98; 137.87; 139.76; 140.54; 146.32; 150.08. MS (ESI+): 391 (M++1).
HRMS (EI) m/z calcd for C23H26N4O2: 390.2148; found: 390.2157.
4.1.5.3 (Z/E)-1-Phenyl-1H-indeno[1,2-c]pyrazol-4-one O-(3-tert-butylamino)-2-
hydroxypropyl)oxime (7c).
Brown solid, mp 112 °C, (yield 20 %). IR (KBr, cm-1): ν = 3465; 3434; 2926; 2069;
1
1634; 1529; 1495; 1445; 1383; 1260; 1094; 1030; 969; 759; 697; 580. H-NMR
(CDCl3, 300 MHz): δ ppm = 1.15 (s, 9H, (CH3)3); 2.20 (br, 1H, OH); 3.10-3.80 (m,
3H, CH2-NH, CH-OH); 4.30-4.70 (m, 2H, CH2-O-N); 7.00-7.70 (m, 10H, Ar); 7.95-
8.00 (m, 1H, NH).13C-NMR (CDCl3, 100 MHz): δ 15.56; 19.78; 32.45; 37.12; 60.72;
70.59; 119.97; 120.32; 123.12; 123.65; 128.43; 128.56; 129.01; 129.47; 129.65;
130.65; 130.98; 138.60; 139.65; 141.15; 146.78; 149.08. MS (ESI+): 391 (M++1).
HRMS (EI) m/z calcd for C23H26N4O2: 390.2148; found: 390.2158.
4.1.5.4
(Z/E)-1-Methyl-1H-indeno[1,2-c]pyrazol-4-one
O-((2-hydroxy-3-
(isopropylamino)propyl)oxime (8a).
22

ACCEPTED MANUSCRIPT
White solid, mp > 210 °C, (yield 35 %). IR (KBr, cm-1): ν = 3457; 2930; 2839;
2337; 1754; 1642; 1550; 1396; 1237; 1038; 603; 547. 1H-NMR (CDCl3, 300 MHz): δ
ppm = 1.20-1.25 (m, 6H, 2CH3); 1.80-2.10 (m, 3H, CH3); 2.50-2.90 (m, 2H, 2CH);
3.00 (br, 1H, NH); 5.20 (s, 1H, OH); 3.50-5.10 (m, 4H, 2CH2); 7.20-7.35 (m, 2H, Ar);
7.50-7.70 (m, 2H, Ar); 8.00 (s, 1H, Ar). 13C-NMR (CDCl3, 100 MHz): δ 14.43; 19.78;
32.32; 37.14; 37.80; 50.87; 69.23; 118.34; 122.83; 123.10; 129.80; 132.98; 136.65;
139.45; 150.03. MS (ESI+): 315 (M++1). HRMS (EI) m/z calcd for C17H22N4O2:
314.3905; found: 314.3914.
.
4.1.5.5
(Z/E)-1-Methyl-1H-indeno[1,2-c]pyrazol-4-one
O-((3-n-butylamino)-2-
hydroxypropyl)oxime (8b).
Orange solid, mp 208 °C, (yield 25 %). IR (KBr, cm-1): ν = 3463; 2945; 1737; 1215;
1
1158; 1034; 748; 666; 601. H-NMR (CDCl3, 300 MHz): δ 0.70-0.90 (m, 3H, CH3);
1.00-1.20 (m, 4H, 2CH2); 1.90-2.00 (m, 3H, CH3); 2.10-2.30 (m, 2H, CH2-NH); 2.90-
3.90 (m, 6H, OH; CH2-NH, CH-OH, CH2-O-N); 7.10-7.35 (m, 5H, Ar); 9.60 (s, 1H,
NH). 13C-NMR (CDCl3, 100 MHz): δ 14.54; 20.54; 32.34; 37.14; 38.83; 51.21; 67.86;
119.35; 122.76; 123.21; 129.67; 133.11; 136.76; 140.12; 151.12. HRMS (EI) m/z
calcd for C18H24N4O2: 328.4176; found: 328.4182.
4.1.5.6 (Z/E)-1-Methyl-1H-indeno[1,2-c]pyrazol-4-one O-(3-tert-butylamino)-2-
hydroxypropyl)oxime (8c).
Brown solid, mp 210 °C, (yield 28 %). IR (KBr, cm-1): ν = 3460; 2927; 1735; 1230;
1032; 797; 724; 601. 1H-NMR (CDCl3, 300 MHz): δ 1.50 (s, 9H, 3CH3); 2.10 (s, 3H,
CH3); 3.40-3.60 (m, 1H, CH); 3.80-4.10 (m, 4H, 2CH2); 4.60 (br, 1H, NH); 5.00 (s,
1H, OH); 6.90 (s, 1H, Ar); 7.50-7.90 (m, 3H, Ar); 8.00 (s, 1H, Ar). 13C-NMR (CDCl3,
100 MHz): δ 15.21; 21.53; 32.67; 38.11; 39.65; 51.22; 67.98; 120.11; 122.32; 123.87;
130.12; 133.32; 136.87; 140.23; 150.21. HRMS (EI) m/z calcd for C18H24N4O2:
328.4176; found: 328.4183.
4.2 Biological assay
4.2.1 Animals
23

ACCEPTED MANUSCRIPT
Male Wistar rats (HARLAN, Italy), weighing 180-250 g were used. Animal care,
sacrifice and experiments were done in accordance with the U.S. National Institutes of
Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-
23, revised 1996). Isolated and Langendorff perfused heart preparation Rats were
anesthetized by intraperitoneal injection of ethyl carbamate (2 g/kg rat, ip) and the
rapidly excised hearts were immediately transferred in ice-cold buffered Krebs-
Henseleit solution (KHs) for immediate cannulation through the aorta with the use of
a glass cannula. Then, perfusion started at a constant flow-rate (12 ml/min). To avoid
fluid accumulation, the apex of the left ventricle (LV) was pierced. A water-filled
latex balloon, connected to a pressure transducer (BLPR; WRI, Inc., Sarasota, FL),
was inserted through the mitral valve into the LV, which allowed the recording of
isovolumic contractions and continuous mechanical parameters. Another pressure
transducer located just above the aorta was used to record coronary pressure (CP).
The perfusion solution consisted of a modified non-recirculating KHs containing (in
millimoles) NaCl 113, KCl 4.7, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.2,
glucose 11, mannitol 1.1, Na-pyruvate 5 (pH 7.4; 37 °C; 95% O2; 5% CO2).
Hemodynamic parameters were assessed using a PowerLab data acquisition system
and analyzed using Chart software (both purchased by ADInstruments, Oxford,
United Kingdom).
4.2.2 Basal conditions
Cardiac performance was evaluated for inotropism by analyzing the left ventricular
developed pressure (LVP; in mm Hg: an index of contractile activity) and the
maximal value of the first LVP derivative (mm Hg per second: an index of the
maximal rate of LV contraction). Lusitropism was determined by calculating the
maximal rate of LVP decline [-(LVdP/dT)max; mmHg/sec] and T/−t ratio between
the maximal rate of LV contraction (+(LVdP/dT)max) and the maximal rate of LV
relaxation [−(LVdP/dT)max]. After 20 minutes of stabilization, the following basal
recordings were measured: LVP=89±3 mmHg, heart rate=280±7 beats/min,
RPP=2.5±0.1 104 mmHg beats/min, CP=63±3mmHg, +(LVdP/dT)max=2492±129
(mmHg/sec), T/-t=0.08±0.01 (sec), -(LVdP/dT)max = 1663±70 (mmHg/sec),
HTR=0.05±0.01 (sec) and T/-t or +(LVdP/dT)max/ LVdP/dT)max=1.49±1.84
24

ACCEPTED MANUSCRIPT
(mmHg/sec). Endurance and stability of the preparation, analyzed by measuring
performance variables every 10 min, showed that the heart preparation is stable for up
to 180 min on the perfusion apparatus.
4.2.3 Protocols
4.2.3.1 7a-c and 8a-c stimulated preparations
Repetitive exposure of each heart to a single concentration (1 nM) of each of the six
derivatives 7a-c or 8a-c revealed absence of desensitization (data not shown). Thus,
concentration-response curves were generated by perfusing cardiac preparations with
KHs supplemented with increasing concentrations of 7a-c or 8a-c (from 1 pM to 10
nM) for 10 min.
4.2.3.2 Isoproterenol stimulated preparations
To obtain preliminary information on the antagonistic action of 7b (5 x 10-10 M)
toward the Iso-dependent stimulation, dose-response curves were generated by
perfusing heart preparations with KHs enriched with increasing concentrations of Iso
(0.1 nM to 1 µM) alone. These curves were then compared to those obtained by
exposing other cardiac preparations to the same perfusion medium containing
increasing concentrations of Iso (0.1 nM to 1 µM) plus a single concentration of either
7b (5 x 10-10 M).
4.2.3.3 Beta2-AR, Beta3-AR and Alpha-AR receptors involvement
To evaluate the involvement of Beta2-AR, Beta3-AR and Alpha-AR receptors in the
mechanism of anti-adrenergic action of 7b, the hearts were perfused with ISO alone
(5 nM) for 5 min and then washed-out with KHs. After returning to control
conditions, each heart was perfused with ISO (5 nM) containing 7b (5 x 10-10 M) and
then washed-out with KHs. After returning to control conditions, each heart was
perfused with ISO (5 nM) containing 7b (5 x 10-10 M), plus phentolamine (100 nM), a
selective alpha adrenergic antagonist, plus SR59230 (100 nM), a selective beta3
25

ACCEPTED MANUSCRIPT
adrenergic antagonist or ICI118,551 (100 nM), a selective beta2-adrenergic
antagonist.
References
[1] D.H. Barer, J.M. Cruickshank, S.B. Ebraim, J.R.A. Mitchell, B. M. J. 296
(1988) 737-741.
[2] K.B. Walsh, T.B. Beganisich, R.S. Kass, J. Gen. Physiol. 93 (1989) 841-854.
[3] L. Di Nunno, C. Franchini, A. Scilimati, M.S. Sinicropi, P. Tortorella,
Tetrahedron: Asymmetry 11 (2000) 1571-1583.
[4] A. Charaf, M. Bouzoubaa, A. Bouzoubaa, M. Blanc, G. Leclerc, Eur. J. Med.
Chem. 29 (1994) 69-74.
[5] A.M. Soriano-Ursúa, J.G. Trujillo-Ferrara, J. Correa-Basurto, S. Vilar, J.
Med. Chem. 56 (2013) 8207-8223.
[6] G. Leclerc, A. Mann, C.G. Wermuth, J. Med. Chem. 20 (1977) 1657-1662.
[7] B. Jamart-Gregoire, P. Caubere, M. Blanc, J.P. Gnassounou, C. Advenier, J.
Med. Chem. 32 (1989) 315-320.
[8] M. Bouzoubaa, G. Leclerc, N. Decker, J. Schwartz, G. Andermann, J. Med.
Chem. 27 (1984) 1291-1294.
[9] B. Macchia, A. Balsamo, A. Lapucci, F. Macchia, M.C. Breschi, B. Fantoni,
E. Martinotti, J. Med. Chem. 28 (1985) 153-160.
[10]
M. Blanc, A. Tamir, S. Aubriot, M.C. Michel, M. Bouzoubaa, G.
Leclerc, P. Demenge, J. Med. Chem. 41 (1998) 1613-1618.
[11]
A. Fravolini, F. Schiaffella, G. Orzalesi, R. Selleri, I. Volpato, Eur. J.
Med. Chem. 13 (1978) 347-350.
[12]
A. Martani, M. Magli, G. Orzalesi, R. Selleri, Farmaco Ed. Sci. 30
(1975) 370-379.
[13]
J.J. Baldwin, D.E. McClure, D.M. Gross, M. Williams, J. Med. Chem.
25, (1982) 931-936.
[14]
[15]
N. Amlaiky, G. Leclerc, N. Decker, J. Schwartz, Eur. J. Med. Chem.
19 (1984) 341-346.
P.L. Ferrarini, C. Mori, G. Primofiore, A. Da Settimo, M.C. Breschi,
E. Martinotti, P. Nieri, M.A. Ciucci, Eur. J. Med. Chem. 25 (1990) 489-496.
26

ACCEPTED MANUSCRIPT
[16]
P.L. Ferrarini, C. Mori, M. Badawneh, C. Manera, G. Saccomanni, V.
Calderone, R. Scatizzi, P.L. Barili, Eur. J. Med. Chem. 32 (1997) 955-963.
[17]
P.L. Ferrarini, C. Mori, M. Badawneh, V. Calderone, R. Greco, C.
Manera, A. Martinelli, P. Nieri, G. Saccomanni, Eur. J. Med. Chem. 35 (2000)
815-826.
[18]
A. Balsamo, M.C. Breschi, M. Chini, P. Domiano, G. Giannaccini, A.
Lucacchini, B. Macchia, M. Macchia, C. Manera, A. Martinelli, C. Martini, E.
Martinotti, P. Nieri, A. Rossello, Eur. J. Med. Chem., 27 (1992) 751-764.
[19]
B. Macchia, A. Balsamo, M.C. Breschi, G. Chiellini, M. Macchia, A.
Martinelli, C. Martini, C. Nardini, S. Nencetti, A. Rossello, R. Scatizzi, J.
Med. Chem. 37 (1994) 1518-1525.
[20]
A. Balsamo, D. Gentili, A. Lapucci, M. Macchia, A. Martinelli, E.
Orlandini, Farmaco 49 (1994) 759-766.
[21]
A. Balsamo, A. Lapucci, B. Macchia, M. Macchia, E. Orlandini, A.
Rossello, Farmaco 50 (1995) 239-243.
[22]
D. Gentili, A. Lapucci, B. Macchia, M. Macchia, A. Martinelli, S.
Nencetti, E. Orlandini, G. Ferni, M. Pinza, Farmaco 50 (1995) 519-526.
[23]
S. Rakhit, M. Bouzoubaa, G. Leclerc, J.M. Leger, A. Carpy, Eur. J.
Med. Chem. 21 (1986) 411-416.
[24]
1355-1361.
[25]
P. Schenone, L. Mosti, G. Menozzi, J. Heterocycl. Chem. 19 (1982)
J. Coltart, E.L.Alderman, S.C. Robison, D.C. Harrison, Br Heart J. 37
M. Briley, I. Cavero, S.Z. Langer, A.G. Roach, Br J Pharmacol. 69
(1975) 357-364.
[26]
[27]
(1980) 669-673.
B. Tota, T. Angelone, R. Mazza, M.C. Cerra. Curr. Med.Chem. 15
(2008) 1444-1451.
[28]
[29]
[30]
[31]
Q. Ma, A.Y. H. Lu, Pharmacological Reviews 63 (2011) 2437-2459.
S.B. Wachter, E.M. Gilbert, Cardiology 122 (2012) 104-112.
V. Barrese, M. Taglialatela Front Physiol. 14 (2013) 323.
C. Gauthier, G. Tavernier, F. Charpentier, D. Langin , H. Le Marec, J
Clin Invest. 98 (1996) 556-562.
[32]
G. Tavernier, G. Toumaniantz, M. Erfanian, M.F. Heymann, K.
Laurent, D. Langin, C. Gauthier, Cardiovasc. Res. 59 (2003) 288-296.
27

ACCEPTED MANUSCRIPT
[33]
T. Angelone, E. Filice, A.M. Quintieri, S. Imbrogno, A. Recchia, E.
Pulerà, C. Mannarino, D. Pellegrino,M.C. Cerra. Acta Physiol. (Oxf). 193
(2008) 229-239.
[34]
C. Gauthier, V. Leblais, L. Kobzik, J.N. Trochu, N. Khandoudi, A.
Bril, J.L. Balligand, H. Le Marec, J. Clin. Invest. 102 (1998) 1377-1384.
[35]
(2004) 849-856.
[36] P.L. Ferrarini, C. Mori, M. Badawneh, V. Calderone, R. Greco, C.
F. Campagna, F. Palluotto, A. Carotti, E. Maciocco, Il Farmaco 59
Manera, A. Martinelli, A. Nieri, G. Saccomanni, Eur. J. Chem. 35 (1999) 315-
826.
[37]
V.K. Tandon, A. Chandra, P.R. Dua, R.C. Srimal, Arckiv der
Pharmazie. 326 (1992) 221-225.
28