(E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide protecting rat heart tissues from isoproterenol toxicity: Evidence from in vitro and in vivo tests

Article abstract of DOI:10.1016/j.ejphar.2020.173137

The current study was aimed to assess the protective effect of a new molecule (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide, denoted 1c, against cardiac remodeling process in isoproterenol (Isop) induced myocardial infarction (MI) in

Full text of DOI:10.1016/j.ejphar.2020.173137

Journal Pre-proof
(E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide protecting rat
heart tissues from isoproterenol toxicity: Evidence from in vitro and in vivo tests
Khdhiri Emna, Kais Mnafgui, Lakhdar Ghazouani, Anouar Feriani, Raouf Hajji, Walid
Bouzanna, Noureddine Allouche, Jean-Pierre Bazureau, Houcine Ammar, Souhir
Abid
PII:
S0014-2999(20)30229-6
DOI:
https://doi.org/10.1016/j.ejphar.2020.173137
EJP 173137
Reference:
To appear in: European Journal of Pharmacology
Received Date: 18 October 2019
Revised Date: 10 April 2020
Accepted Date: 20 April 2020
Please cite this article as: Emna, K., Mnafgui, K., Ghazouani, L., Feriani, A., Hajji, R., Bouzanna,
W., Allouche, N., Bazureau, J.-P., Ammar, H., Abid, S., (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-
yl)ethylidene)benzohydrazide protecting rat heart tissues from isoproterenol toxicity: Evidence from
in vitro and in vivo tests, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/
j.ejphar.2020.173137.
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Graphical Abstract
(E)- N'-[1-(3-Oxo-3H-benzo[f]chromen-2-yl)ethylidene benzohydrazide protecting rat heart tissues
from isoproterenol toxicity: Evidence from in vitro and in vivo tests
Khdhiri Emna 1, Lakhdar Ghazouani 2, Kais Mnafgui 3, Anouar Feriani 2, Raouf Hajji 4, Walid Bouzanna 5,
Noureddine Allouche 6, Jean-Pierre Bazureau 7,*, Houcine Ammar 1 and Souhir Abid 1,8
1Laboratoire de Chimie Appliquée "Hétérocycles Corps Gras & Polymères", Faculté des Sciences, Université de Sfax, 3038 Sfax,
Tunisie.
2 Unité de Recherche en Biochimie Macromoléculaire et de Génétique, Faculté des Sciences de Gafsa, 2112 Gafsa, Tunisie
3 Laboratoire de Physiologie Animale, Faculté des Sciences de Sfax, Université de Sfax, P.O. Box 95, Sfax 3052, Tunisie
4 Département de Médecine Interne, Hôpital de Sidi Bouzid, Faculté de Médecine d'Ibn Eljazzar, Université de Sousse, Sousse,
Tunisie
5 École d'Enseignement Hospitalier d'Habib Thamer, 8 Ali Ben Ayed St., Montefleury, 1089, Tunis, Tunisie
6 Laboratoire de Chimie des Substances Naturelles (UR11/ES74), Faculté des Sciences de Sfax, Université de Sfax, Route Soukra,
BP1171, 3000 Sfax, Tunisie
7 Institut des Sciences Chimiques de Rennes, ISCR UMR CNRS 6226, Université de Rennes 1, Bât. 10A, Room 207, Campus de
Beaulieu, CS 74205, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France
8 Département de Chimie, Collège des Sciences et des Arts, Université de Jouf, Al Qurayyat, Al Jawf, KSA
H
N
N
O
O
O
Protection of rat heart tissues by 1c from
1c: (E)- N'-[1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene
benzohydrazide
isoproterenol toxicity
1

Title page
(E)-N’-(1-(3-Oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide
protecting rat heart tissues from isoproterenol toxicity:
Evidence from in vitro and in vivo tests
Khdhiri Emna 1, Kais Mnafgui 2, Lakhdar Ghazouani 3, Anouar Feriani 3, Raouf Hajji 4, Walid
Bouzanna 5, Noureddine Allouche 6, Jean-Pierre Bazureau 7,*, Houcine Ammar 1 and Souhir Abid 8,1
1
Laboratoire de Chimie Appliquée "Hétérocycles Corps Gras & Polymères", Faculté des Sciences,
Université de Sfax, 3038 Sfax, Tunisie.
2
Laboratoire de Physiologie Animale, Faculté des Sciences de Sfax, Université de Sfax, P.O. Box 95, Sfax
3052, Tunisie.
3
Unité de Recherche en Biochimie Macromoléculaire et de Génétique, Faculté des Sciences de Gafsa, 2112
Gafsa, Tunisie.
4
Département de Médecine Interne, Hôpital de Sidi Bouzid, Faculté de Médecine de Ibn Eljazzar,
Université de Sousse, Sousse, Tunisie
5
École d'Enseignement Hospitalier d'Habib Thamer, 8 Ali Ben Ayed St., Montefleury, 1089, Tunis, Tunisie
6
Laboratoire de Chimie des Substances Naturelles (LR17/ES08), Faculté des Sciences de Sfax, Université
de Sfax, Route Soukra, BP1171, 3000 Sfax, Tunisie
7
Institut des Sciences Chimiques de Rennes, ISCR UMR CNRS 6226, Université de Rennes 1, Bât. 10A,
Room 207, Campus de Beaulieu, CS 74205, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France
8
Département de Chimie, Collège des Sciences et des Arts, Université de Jouf, Al Qurayyat, Al Jawf, KSA
Corresponding author: Jean-Pierre Bazureau*
Mailing address: Institut des Sciences Chimiques de Rennes, ISCR UMR CNRS 6226, Université de Rennes
1, Bât. 10A, Room 207, Campus de Beaulieu, CS 74205, 263 Avenue du Général Leclerc, 35042 Rennes
Cedex, France
Phone: +33 223 236 603
E-mail: jean-pierre.bazureau@univ-rennes1.fr
1

Abstract
The current study was aimed to assess the protective effect of a new molecule (E)-N’-(1-(3-oxo-3H-
benzo[f]chromen-2-yl)ethylidene)benzohydrazide, denoted 1c, against cardiac remodeling process
in isoproterenol (Isop) induced myocardial infarction (MI) in rats. Male Wistar rats were randomly
divided into four groups, control, Isop (85 mg/kg body weight was injected subcutaneously into rats
at an interval of 24 h for 2 days (6th and 7th day) to induce MI and pretreated animals with
acenocoumarol (Ace) (150 µg/kg bw) and 1c (150 µg/kg bw) by oral administration during 7 days
and injected with isoproterenol (Isop + Ace) and (Isop + 1c) groups. Results in vitro showed that 1c
is endowed with potent inhibition of angiotensin-converting enzyme (ACE) with an IC50 39.12
µg/ml. The in vivo exploration evidenced alteration in the ECG pattern, notable cardiac hypertrophy
and increase in plasma level of fibrinogen, troponin-T, CK-MB and LDH, AST and ALT by 171%,
300%, 50%, 64% and 75% respectively with histological myocardium necrosis and cells
inflammatory infiltration. However, pre-treatment with 1c improved the ECG pattern reduced
significantly the cardiac dysfunction markers and ameliorated the thrombolytic process by
decreasing fibrinogen level as compared to untreated infracted rats. Overall, (E)-N’-(1-(3-oxo-3H-
benzo[f]chromen-2-yl)ethylidene)benzohydrazide 1c could be used as anticoagulant agent to
prevent thrombosis in acute myocardial infarction.
Keywords: (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide; isoproterenol;
myocardial infarction; ACE; thrombolytic.
2

1. Introduction
Myocardial infarction (MI) is a frequent cardiovascular disease and the main cause of
cardiovascular deaths, which is estimated to reach 23.3 million by 2030 (World Health
Organization, 2019). Myocardial infarction is the result of prolonged imbalance between the
myocardial oxygen supply and demand of the myocardium leading to myocardial necrosis (Ahsan
et al., 2014). Abnormalities in the electrocardiogram (ECG) pattern are detected during infarctions
and reflect the severity of cardiac injuries (Nabel et al., 2012). Moreover, MI seriously induces
ventricular remodeling process as an adaptive response in which damaged tissue is replaced by
collagen. The left ventricle remodeling process is endowed with loss of cardiac contractility and
hemodynamic properties and relates to mural hypertrophy (Sutton et al, 2000). Experimentally, the
induction of MI was performed through surgical procedures or the administration of sub-acute dose
of isoproterenol (Isop) in animal, which offered rapid myocardial damage similar to that seen in
acute MI in humans (Mnafgui et al., 2015). Hence, isoproterenol (Fig. 1) is a synthetic
catecholamine, and is a non-selective β-agonist (Sahu et al., 2014). Supra maximal administration
induces myocardial damage typically in the sub-endocardial layer of the left ventricle and evidences
severe myofibrillar degeneration (Shukla et al. 2015). Several previous studies reported that
subcutaneous treatment with Isop induced remarkable increase in reactive oxygen species (ROS)
such as superoxide anion, hydrogen peroxide, and hydroxyl radicals and lipid peroxidation (LPO),
which further exacerbate the myocardial damage (Ramya et al., 2014).
The design and construction of novel poly-heterocycles and/or poly-functional containing different
privileged substructures within a same molecule represent an important strategy in medicinal
chemistry (Abdallah et al., 2017; Chekir et al., 2018; Dong et al., 2014). In fact, it is generally
proven that the presence of two or more pharmacophores linked and/or fused within a same
framework could contribute to provide a significant positive effect on the overall biological
efficiency in the resulting poly-functions (Filali et al., 2016). Coumarins are a class of outstanding
organic molecules with a wide array of biological activities and therapeutic functions (Jameel et al.,
2016), due to their significant applications such as antimicrobial (Kovvuri et al., 2018), antifungal
(Elias et al., 2019), antiviral (Conti et al., 2019), anti-tyrosinase (Pintus et al., 2017), anticancer
(Chen et al., 2013; Kostova, 2005), anti-thrombotic (Kirsch et al., 2016), anti-inflammatory
(Fylaktakidou et al., 2004; Gagliotti Vigil de Mello and Frode, 2018), anticoagulant (Razavi et al.,
2013) and anti-oxidant properties (Pérez-Cruz et al., 2018). They are predicted to play a crucial role
of the MI management. In particularly, various researches suggest that the presence of functional
groups at the 3-position of coumarin skeleton include specific activities (Keri et al., 2015). A
similar trend is seen for hydrazones have been shown to be active in several pharmacological tests
both in vitro and/or in vivo such as antiplatelet and antithrombotic activities (Chelucci et al., 2014).
3

Several hydrazone derivatives synthesized have been shown to promote intense vasodilatation and
antihypertensive activity, for instance, 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-
294) (Braga et al., 2012) and its analogue 2-thienyl-3,4-methylenedioxybenzoylhydrazone
(LASSBio-1027) (Leal et al., 2012).
This encouraged us to prepare a new class of hybrid conjugate associating these two
pharmacophores within a same molecular target, namely the hydrazone coumarin and study their
biological activity. In this context, the present study aims to examine the anti-thrombotic, anti-
inflammatory effect and the potential anti-ischemic properties of a new synthetic hydrazone
coumarin (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide 1c on infracted
rats.
4

2. Material and methods
2.1 Chemistry section
General: Thin-layer chromatography (TLC) was accomplished on 0.2-mm pre-coated plates of
silica gel 60 F-254 (Merck) with appropriate eluent. Visualization was made with ultraviolet light
(254 and 365 nm) or with a fluorescence indicator. Melting points were determined on a Kofler
melting point apparatus and were uncorrected. HPLC analysis was performed on a Perkin Elmer
chromatographic system equipped with a 200 series pump, a 20 µl automatic loop injector and UV-
Visible detector. Analysis was realized on a Eurospher 100-10 C18 column (250 x 4 mm) using
MeOH/water as mobile phase with appropriate composition and flow rate. The analytical process is
managed by TotalChrom Navigator workstation. Infrared (IR) spectra were registered on a Perkin
Elmer spectrum 100 FTIR Spectrometer apparatus using KBr pellets. 1H NMR spectra were
recorded on BRUKER AC 400 P (400 MHz) spectrometer and 13C NMR spectra on BRUKER AC
400 P (100 MHz) spectrometer. Chemical shifts are expressed in parts per million downfield from
tetramethylsilane TMS as an internal standard. Data are given in the following order: δ value,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint, quintuplet; m, multiplet; br, broad),
number of protons, coupling constants J is given in Hertz. The high-resolution mass spectra
(HRMS) were recorded in positive mode using direct Electrospray infusion on a Thermo Fisher
Scientific Q-Exactive spectrometer at the "Centre Régional de Mesures Physiques de l’Ouest"
ScanMAT platform (CRMPO platform, UMS 2001 CNRS ScanMAT, Rennes, France).
Benzoylhydrazine b, 2-hydroxy-1-naphthaldehyde 1 purchased from Sigma-Aldrich (Tunisia),
piperidine and methyl 3-oxobutanoate a purchased from Fluka (Tunisia), ethanol (purchased from
LOBA Chemie, Tunisia) were analytical grade and used as received.
2.1.1 2-Acetyl-3H-benzo[f]chromen-3-one (1a).
In a round bottomed flask (25 ml) equipped with a condenser and magnetic stirring, piperidine (14
mg, 0.17 mmol, 10% mol) was added directly in a stirred (37 g) solution of substituted 2-hydroxy-
1-naphthaldehyde 1 (0.336 g, 1.7 mmol) and ethyl 3-oxobutanoate a (0.2 g, 1.7 mmol) in 10 ml of
ethanol pa. The resulting mixture was stirred at 80°C during 30 min. (monitored by thin layer
chromatography on 0.2 mm plates of silica gel 60 F254 Merck using appropriate eluent). After
cooling down to room temperature, the insoluble material was collected by filtration in a Buchner
funnel (porosity N°4) and the precipitate was washed with cooled (4°C) ethanol (3 x 5 ml). The
desired solid was further dried under high vacuum (10-3 Torr) and afforded the desired 2-acetyl-3H-
benzo[f]chromen-3-one 1a in 87% yield as yellowish powder, which was used later without further
purification. Mp = 194-196°C. IR (KBr, cm-1): 1737 (C=O, ArOCO), 1676 (C=O, MeCO), 1624
(C=C). 1H NMR (CDCl3): δ = 9.26 (s, 1H, H4), 8.32 (d, J = 7.8 Hz, 1H, H5’), 8.07 (d, J = 8.9 Hz,
5

1H, H7), 7.90 (d, J = 7.8 Hz, 1H, H6’), 7.73 (t, J = 7.8 Hz, 1H, H5’'), 7.60 (t, J = 7.8 Hz, 1H, H6’’),
7.44 (d, J = 8.9 Hz, 1H, H8), 2.77 (s, 3H, CH3). 13C NMR (CDCl3): δ = 195.6, 159.4, 156.1 , 143.3,
136.3, 130.2, 129.8, 129.2, 126.6, 122.3, 121.8, 116.5, 112.7, 30.7.
2.1.2 (E)-N’-(1-(3-Oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide (1c).
In a round bottomed flask (25 ml) equipped with a condenser and magnetic stirring, a mixture of 2-
acetyl-3H-benzo[f]chromen-3-one 1a (0.3 g, 1.25 mmol), benzoylhydrazine (b) (0.17 g, 1.25 mmol)
in ethanol pa (10 ml) and a catalytic amount of glacial acetic acid (0.1% mol.) was refluxed during
12 h under vigorous magnetic stirring (37 g). Progress of the reaction mixture was monitored by
thin layer chromatography on 0.2 mm plates of silica gel 60 F254 Merck using appropriate eluent.
After cooling down to room temperature, the insoluble material was collected by filtration in a
Buchner funnel (porosity N°4) and the precipitate was washed with cooled (4°C) ethanol (3 x 5 ml).
Purification of the desired compound 1c was conducted by recrystallization from dry ethanol, then
(E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide 1c was further dried under
high vacuum (10-3 Torr) during 30 min. at room temperature which gave 1c as pale white powder in
82% yield. Mp: 240-242°C. IR (KBr, cm-1): 1694 (C=O, ArOCO), 1676 (C=O, NHCOPh), 1633
(C=N), 3302 (NH). 1H NMR (DMSO-d6): δ = 10.93 (br s, 1H, NH), 8.89 (s, 1H, H4), 8.51 (d, J =
7.8 Hz, 1H, H5’), 8.20 (d, J = 8.8 Hz, 1H, H7), 7.91 (d, J = 7.2 Hz, 2H, H11), 8.04 (d, J = 7.8 Hz, 1H,
H6’), 7.73 (t, J = 7.8 Hz, 1H, H5’’), 7.63-7.52 (m, 5H, H12, H13, H8, H6’‘), 2.41 (s, 3H, CH3) ppm. 13C
NMR (DMSO-d6): δ = C
2
(159.6), C
3
(126.3), C3’ (153.8), C
4
(137.8), C4a (129.4), C
5
(129.4), C5’
(122.6), C5’’ (130.3), C (113.2), C6’ (129.3), C6’’ (126.6), C
6
7
(134.2), C (116.8), C8a (153.8), C
8
9
(159.6), C10 (129.0), C11 (128.5), C12 (128.5), C12’ (132.1), C13 (128.8) ppm. HRMS (ES+,
MeOH/CH2Cl2 9:1), m/z: 379.1054 found (calculated for C22H16N2O3Na [M+Na]+ requires
379.1053); 395.0788 found (calculated for C22H16N2O3K [M+K]+ requires 395.0793).
2.2 Biology section
2.2.1 In vitro ACE inhibitory activity assays
The ACE inhibitory activity was assayed by the method reported in literature (Wu et al., 2002) with
captoril as positive control. The total reaction volume of 100 µl was made up of 50 µl of 2.17 mM
Hippuryl-L-histidyl-L-leucine (HHL), 25 µl of 2 mU of ACE, 25 µl tested samples in different
concentrations (all prepared with 100mM borate buffer, containing 300 mM NaCl, pH 8.3) or 25 µl
borate buffer for control. The mixture of HHL and sample of 1c or ACE alone was incubated at
37°C in shaking water bath type (HETO, SBD-50, Denmark) with continuous agitation at 30 g for
10 min to achieve thermal equilibrium. The reaction was started by adding ACE to tube containing
HHL and samples or borate buffer. The reaction was terminated after 30 min incubation by addition
6

of 300 µl of HCl (1 M) and the solution filtered through a 0.45 nm nylon syringe filter for reversed-
phase (RP)-HPLC analysis. HPLC analysis was performed on a LC-10ATvp system equipped with
a diode array detector (Shimadzu, Japan). ZORBAX SB-C18 column (4.6 mm id x150 mm, 5 µm
particle size) was used and HA (hippuric acid) and HHL were detected at 228 nm. External standard
of HA sample was prepared freshly on a daily basis and used for the qualification of HA. The peak
area of HA was recorded and the percentage inhibition of ACE activity was calculated using
equation:
2.2.2 Animals
In the current study, a total of 24 adult male Wistar rats with 12 weeks of age weighing 260 ± 23 g
were purchased from SIPHAT (Tunisia). Their standard pellet diet was purchased from the
Industrial Society of Rodents’ Diet (SNA, Sfax, Tunisia). The experiments were carried out
according to the local Institute Ethical Committee Guidelines for the Care and Use of laboratory
animals issued by the University of Gafsa, Tunisia. After one weeks period of adaptation in the
laboratory, animals were maintained under constant conditions in polypropylene cages on a 12 h
light/dark cycle and at a temperature of 22 ± 2°C; humidity: 60 ± 5 % with free access to food and
water. Approval for this study was received from the Ethics Committee of the Faculty of Sciences
of Gafsa, Tunisia, according to the Medical Ethical Committee for the Care and Use of Laboratory
Animals of Pasteur Institute of Tunis (Approval No: LNFP/Pro 152012).
2.2.3 Experimental animals
After acclimatization, animals were assigned into four groups consisting of six rats per group.
Treatment was then carried out as follows:
•
Group I: served as negative control (Control) rats received standard laboratory diet and drinking
saline water ad libtum.
•
Group II: (Isop) Animals received saline water for 7 days, were subcutaneously injected at the
sixth day with isoproterenol (85 mg/kg/day) once per day at an interval of 24 h for 2 continued
days for induction of myocardial infarction and served as isoproterenol group (Ghazouani et al.,
2019).
•
Group III: (Isop + Ace) Animals were pre-co treated with Acenocoumarol (150 µg/kg body
weight), by gastric gavage for 7 days and injected with isoproterenol (85 mg/kg body weight) on
the sixth and seventh day.
7

•
Group IV: (Isop + 1c) Animals received 1c pre-co treatment (150 µg/kg body weight) by gastric
gavage for 7 days and were injected with isoproterenol (85 mg/kg body weight) at the sixth day
once per day, for 2 consecutive days.
2.2.4 Experimental induction of MI
Isoproterenol (Isop) was dissolved in normal saline and injected to rats (85 mg/kg BW)
subcutaneously at an interval of 24 h for 2 consecutive days to induce experimental myocardial
infarction (Mnafgui et al., 2015). Animals were killed 48 h after the first dose of isoproterenol.
2.2.5 Electrocardiography
At the end of the experimental period, after 24h of the second dose of isoproterenol, all rats from
the four groups included in the present study were anesthetized intraperitoneally with ketamine
hydrochloride (100 mg/kg bw). Needle electrodes were then inserted under the skin of the animals
in lead II position. ECG recordings were taken using veterinary electrocardiograph (ECG VET 110,
Biocare, China).
2.2.6 Sample collection and biochemical analysis in plasma
After recording the electrocardiogram, all the rats were killed by cervical decapitation. Blood was
collected and plasma was removed from each sample, stored at - 20°C and used thereafter for
various biochemical and enzymatic estimations such CK-MB (Ref. 97217 from Biolabo, France)
and (LDH (Ref. 20011), ALT (Ref. 20046), AST (Ref. 20042) are purchased from Biomaghreb,
Tunisia) or determination of the lipid profile (total cholesterol and triglycerides) according to the
manufacturer’s instructions of the commercial reagent kits purchased from Biomaghreb (Tunisia).
The levels of plasma cardiac troponin-T were measured using a standard kit by
electrochemiluminescence immunoassay (Roche Diagnostics, Switzerland, Ref, 07007302 119).
The amount of fibrinogen was assessed using chronometry-based methods and according to the
manufacturer’s instructions in the commercial reagent kits (Biolabo, France, Ref. 13450). The
technique is based on the work of Clauss et al. (1957), validated by Destaing et al. (1960). In the
presence of an excess of thrombin, the coagulation time of previously diluted plasma is inversely
proportional to the fibrinogen concentration. Immediately after kill, the heart was excised out,
washed with saline and fixed in a Bouin solution for 24 h, and then embedded in paraffin. Sections
of 5 µm thickness were stained with hematoxylin-eosin. The slides were photographed with an
Olympus U-TU1X-2 camera connected to an Olympus CX41 microscope (Tokyo, Japan).
8

2.2.7 Statistical analysis
All data were expressed as mean ± standard deviation (S.D) of six rats in each group. Statistical
differences between the means of the different groups were examined using a one-way analysis of
variance (ANOVA) followed by the Fisher test using Statistical Package for Social Sciences (SPSS)
Software Package Version 17.0. A P-value < 0.05 was considered as statistically significant.
3. Results
3.1. Chemistry: Synthesis and structure of (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-
yl)ethyliden)benzohydrazide (1c)
The synthetic route used for the preparation of the title compound 1c was achieved only in two
steps, as presented in Scheme 1. In the first step, reaction of commercial 2-hydroxy-1-
naphthaldehyde 1 with methyl 3-oxobutanoate a produced mainly the corresponding 2-acetyl-3H-
benzo[f]chromen-3-one 1a in a 87% yield as yellowish powder after purification by simple washing
with ethanol pa. This reaction involving a Knoevenagel condensation and a ring closure
(Knoevenagel, 1898) was catalyzed by amount of piperidine. For the second step, the 2-acetyl-3H-
benzo[f]chromen-3-one 1a derivative was converted into its hydrazone coumarin 1c in the presence
of glacial acetic acid (0.1% mol.) as catalyst. The desired compound 1c was purified by classical
recrystallization from ethanol in 82% yield (71% overall yield) as pale white powder. Purity of
compound 1c was controlled by HPLC analysis using Eurospher 100-10 C18 column (250 x 4 mm,
5 µm spherical particle size) with UV detector at 254 nm using MeOH/water 8:2 v/v as mobile
phase (flow rate = 0.5 ml/min., injection = 10 µl). For 1c, we obtained only one peak, the retention
time was measured at 2.86 min. (the corresponding chromatogram was reported in Supplementary
Materials).
IR spectra comparison between the starting 2-acetyl-3H-benzo[f]chromen-3-one 1a and the product
of condensation 1c showed the following points: - firstly, the disappearance of the absorption band
of 1a initially located at 1673 cm-1 which indicated the participation of the exocyclic ketone group
in the nucleophilic addition process; - secondly, the appearance of the characteristic band for the
C=N bond at 1678 cm-1 and the absorption band at 3300 cm-1 can be attributed to the NH vibrator in
the desired compound 1c.
In 1H NMR spectrum of compound 1c recorded in DMSO-d6 solution, showed the presence of a
broad singlet at d 10.93 ppm for NH proton (I = 1H), a doublet centered at δ 7.91 ppm with a
coupling constant J = 7.2 Hz (I = 2H) for H11 and a multiplet at 7.63 < δ < 7.52 ppm attributable to
the respective protons H-12, H-13, H-8 and H-6”. These attributions were based on the study of the
2D-1H-13C (H-H COSY, HSQC and HMBC) spectroscopy and the NOESY spectra. In 13C NMR
9

spectrum, conversion of 2-acetyl-3H-benzo[f]chromen-3-one 1a into its phenylhydrazone coumarin
derivative 1c was confirmed by disappearance of signal for MeCO moiety in compound 1a and
appearance of a signal at δ 153.86 ppm can be attributed to the imino C=N group in 1c. The Z- or E-
stereochemistry of final compound 1c was determined by NOESY experiments. In the NOESY-
map, it showed correlation between NH/HMe and it was in agreement for a geometric (E)-
configuration in this sole isomer for this C=N bond. The steric hindrance of the phenylamino
moiety on this C=N bond is certainly the major factor that induces this unique E-stereochemistry
and was in agreement with results of the literature (Syakaev et al., 2006; Patorski et al., 2013;
Ershov et al., 2009; Gonzaga et al., 2016; Glidewell et al., 2004). HRMS of 1c completed the
analytical data and was in accordance with this structure.
3.2 Biological assays
3.2.1 Angiotensin II- Converting Enzyme (ACE) inhibition, in vitro assay
Table 1 data showed that the compound 1c caused a dose dependent inhibition of angiotensin
converting enzyme activity with an IC50 39.12 µg/ml.
3.2.2 ECG patterns of experimental groups
As shown in Fig. 2, control rats evidenced normal ECG pattern with regular sinus rhythm.
However, irregular sinus rhythm with significant elevation of ST-segment and disappearance of
Pardee wave were observed in ECG of untreated infracted rats as compared to normal animals.
While, pretreatment with acenocoumarol (Ace, Fig. 1) did not ameliorate the ST-segment elevation.
Moreover, pre-cotreatment with 1c mitigated the ECG pattern abnormalities of infracted rats and
restored the regular sinus rhythm.
3.2.3 Effect of 1c on heart weight to body weight ratio of experimental rats
The effects of 1c and acenocoumarol (Ace) pre-co treatment on heart weight to body weight ratio at
the end of experiment are presented in Table 2. The heart weight to body weight ratio markedly
increased (P < 0.05) in isoproterenol-induced rats when compared to control group by 28%.
Whereas, a significant decrease (P < 0.05) in cardiac hypertrophy by 13 % was observed in
infracted rats pre-co treated with 1c as compared to myocardial infarction of untreated rats.
3.2.4 Effect of 1c on the levels of cardiac injury marker enzymes in rats
As shown in Fig. 3, remarkable increase (P < 0.05) in plasma levels of CK-MB, LDH, troponin-T,
AST and ALT by 50%, 26 %, 300%, 64% and 75% respectively in infracted rats as compared to
control group. Though, prior administration of 1c followed by isoproterenol-induced MI in rats has
significantly prevented the depletion of these myocardial markers as evidenced by a significant
10

decrease (P<0.05) in the levels of all the above mentioned biomarkers by 31 %, 6%, 39%, 14 % and
40 % respectively when compared to isoproterenol (Isop) induced infracted rats. Nearly, the same
effect was shown by pre-co treatment with acenocoumarol (Ace).
3.2.5 Plasma fibrinogen level in the experimental groups
As shown in Fig. 4, our results showed that Isop-induced rats exhibited a significant rise (P < 0.05)
in plasma fibrinogen by 171 % when compared to control group. However, pre-co treatment with 1c
or Ace significantly reduced the plasma fibrinogen level (P < 0.05) by 42 % and 40 %, respectively,
in comparison to Isop alone-administered rats.
3.2.6 Effect of 1c on lipid profile
As shown in Table 3, animals treated with Isop showed remarkable increases (P < 0.05) in total
cholesterol and triglycerides levels by 111% and 21 % respectively as compared to normal control
group. However, pre-co treatment either with 1c or Ace induced significant decrease in these two
parameters activity by 35% and 15 % respectively.
3.2.7. Histopathological observations of heart tissues
Normal myocardium architecture was observed in the control group. However, the non-treated
infarcted group showed myocardial cells necrosis, separation of cardiac myofibrillar and large
inflammatory cells infiltration. While pre-co treatment with 1c improved the normal myocardial
arrangement and transverse striations (Fig. 5).
4. Discussion
Myocardial infarction (MI) is a leading life-menacing manifestation of ischemic heart disease
(IHD) causing mortality worldwide (Benjamin et al., 2017). Clinical searches have shown that
statins therapy can prevent or ameliorate cardiac injury. Rather, evidence has shown that statins is
able to cause various adverse effect (Attalla et al., 2019). Nowadays, the coumarins and its
derivatives have being used for the counteracting effect and treatment of cardiac dysfunction
(Mnafgui et al., 2019). These bioantioxidants may play key role in help in cardiac remodeling and
preventing disease by its anti-inflammatory, anticoagulant, antithrombotic, antioxidant, and
hypolipidemic effects (Kontogiorgis et al. 2015; Paracatu et al. 2016; Srikrishna et al. 2018).
Hence, based on the aforementioned literature, the present study was designed to investigate, for the
first time, the potential preventive capacity of a novel synthetic hydrazone coumarin, (E)-N’-(1-(3-
oxo-3H-benzo[f]chromen-2-yl) ethylidene) benzohydrazide “1c”, against MI induced in
experimental male Wistar rats.
In vitro, results showed that the compound 1c caused a dose dependent inhibition of angiotensin
converting enzyme activity with an IC50 39.12 µg/ml. Previous studies have reported that ACE
11

inhibitors were widely used in treatment of myocardial infarction to prevent left ventricular
remodeling process (Mnafgui et al., 2016; Daoud et al., 2017). In fact, the inhibition of ACE
activity inactivated expression of cytokine transforming growth factor (TGF-β1), a major mediator
of the remodeling process and fibrosis tissues (Border et al., 1994; Borghi et al., 2006).
An electrocardiogram (ECG) is a medical test that detects infarction by measuring the electrical
activity generated by the heart as it contracts (Daoud et al., 2017). ECG recordings in Isop treated
groups revealed ischemic and conduction abnormalities as demonstrated by significant elevation of
ST segments and unidentifiable P wave. These conduction abnormalities indicate the presence
myocardial necrosis of consequent loss of cell membrane in an injured myocardium as reported by
Mnafgui et al., 2016. Pre-treatment with the synthesized hydrazone coumarin 1c restored normal
ECG pattern, suggestive of its cell membrane protective effects.
In this work, all treated groups showed insignificant change in the body weight of the rats, and
increase in heart weight to body weight ratio indicating hypertrophy which was significantly
improved only by 1c pre-treatment. The observed effect in relative heart weight could be related to
invasion of inflammatory cells and increased fibrosis (Mnafgui et al., 2015) as confirmed by
biochemical and histological examinations in the present work.
Elevated cardiac enzymes are well-known markers of myocardial infarction. Isop induced MI
promotes rupture of cardiac membrane and subsequent leakage of these enzymes into the plasma
(Khalil et al., 2015; Paula et al., 2017). The current findings revealed deterioration in the cardiac
function as shown by increased plasma levels of LDH, ALT, AST and CK-MB activities, in
accordance with recent studies of Paula et al. 2017 and Khan et al. 2018. Isop treated rats showed
also elevated troponin levels, which predict the risk of both cardiac death and subsequent infarction
(Saravanan, et al., 2013). Interestingly, 1c pre-treatment revealed a significant decrease in plasma
amounts of all these cardiac markers. The decreased amounts observed in these enzymes were close
to these pretreated in with acenocoumarol.
This data indicating that the synthesized antioxidant compound might could prevent rats against
isoproterenol toxicity in the heart, by maintaining myocardial membrane integrity and restricting
the leakage of cardiac enzymes into the circulation, as signaled by Ghazouani et al., 2019.
Hyperlipidaemia and hypercholesterolaemia has been reported to be the major pathogenesis in the
progression and development of the pathogenesis of heart and myocardial infarction (Abbas 2016).
In the current study, results showed also that the levels of TC, TGs increased in plasma of treated
rats compared to the control group. The increased level of cholesterol in Isop treated rats is due to
increased level of LDL-C taken from the blood circulation (Anandan et al., 2007). For TGs, may be
due to increased hydrolysis of stored triglyceride via hormone-sensitive lipase activity (Khalil et al.,
2015). However, a strong inhibition of the alteration of lipids profile by 1c was observed in plasma
12

of treated rats, which may be due to the hypocholesterolaemic and hypolipidemic activities of 1c
(Pari et al., 2014).
Several prospective epidemiological studies and clinical observations have reported a wide range of
data regarding the potential role of fibrinogen as a biomarker of cardiovascular disease (Danesh et
al., 2005). Indeed, fibrinogen is one of the most common hemostatic factors that contributes to
thrombosis and development of atherosclerosis (Machlus et al., 2011). Further, several factors
involved in coagulation or thrombosis are associated with acute myocardial infarction and
inflammation, including high fibrinogen concentrations by promoting thrombus formation and
altering coagulation kinetics (Mohseni et al., 2014).
Results of the current study showed that isoproterenol intoxication induced increase in the amount
of this glycoprotein, which could explain in part the observed cardiac damage. However,
pretreatment with 1c lessened the increased level of fibrinogen produced by isoproterenol
metabolism thereby inhibited the thrombosis phenomenon, which prevented cardiac damage, and
myocardial injury. Similar results were noted by Daoud et al., 2017 and Ghazouani et al., 2019. In
fact, 1c could inhibit the thrombotic process by acting as a vitamin K antagonist (VKA) through
preventing carboxylation of glutamic acid residues of vitamin K-dependent coagulation factors II,
VII, IX, and X, a crucial step in the coagulation process (Ufer, 2005; Ghazouani et al., 2019;
Mnafgui et al., 2019).
Histopathology study of the MI group evidenced focal lesions in the cardiac tissue, separation of
cardiac myofibrillar and large inflammatory cells infiltration, which are in accordance with previous
searchs (Mnafgui et al., 2016). The preventive effects of 1c on the abolition of myocardium cells
necrosis could be attributed to their antioxidant potential (Attalla et al., 2018). In fact, the
composition of this new synthesized hydrazone coumarin containing different privileged
substructures within a same molecule (coumarin) and function (hydrazone), represent a good
strategy to fight against oxidative stress (Mnafgui et al., 2019).
5. Conclusion
In conclusion, the present study revealed that isoproterenol induced in rats an increased level of
plasma cardiac troponin-T, plasmatic cardiac markers and elevated ST-segments, as well as,
increased levels of plasma cholesterol and triglyceride. Pre- and co-treatment with 1c ameliorated
cardiac injury, dyslipidemia and elevated ST-segments in isoproterenol induced myocardial
infarcted rats. Histopathology of myocardium correlated with these biochemical findings. Thus, the
compound named (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene) benzohydrazide 1c
could be used as an anticoagulant agent to prevent thrombosis in acute myocardial infarction.
13

Declaration of interest
The authors of the present work declare no conflict of interest The authors alone are responsible for
the content and writing of this article.
Acknowledgments
The Ministry of Higher Education and Scientific Research of Tunisia and the Ministry of Public
Health of Tunisia supported this research program. The authors are grateful to the assistance of the
staff (P. Jéhan, F. Lambert and N. Le Yondre) of the CRMPO analytical chemistry core facility for
HRMS analysis (CRMPO platform, ScanMAT UMS 2001 CNRS, Université de Rennes 1, Bât.
11A, Campus de Beaulieu, Rennes, France).
Supplementary Material
The following contents as IR, 1H and 13C NMR, H-H COSY, HSQC, HMBC, NOESY for
compounds 1a and 1c are available online at https://dx.doi.org/10.1016/j.ejphar.2019.xx.xxx.
14

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19

Scheme, Figures and Table legends
Scheme 1: Preparation of (E)-N’-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)benzohydrazide 1c
Fig 1: Structures of isoproterenol (Isop) and acenocoumarol (Ace)
Fig 2: Effect of 1c on electrocardiographic (ECG) pattern in normal and experimental rats. Control:
Recording of the control rats. Isop: Recordings of the rats injected with isoproterenol (85 mg/kg). Isop +
Ace: Recording of rats treated with acenocoumarol (Ace) and isoproterenol (Isop). Isop + 1c: Recording of
rats treated with 1c and isoproterenol.
Fig 3: Variation of plasma markers of the myocardial infarction in control and treated rats. Values are given
as mean ± standard deviation / S.D. for groups of 6 animals each. The values are statistically introduced as
follows: * P < 0.05 significant difference compared to the control rats; # P < 0.05 significant difference
compared to isoproterenol (Isop) groups; @ P < 0.05 significant difference compared to treated rats with
Acenocoumarol (Ace). Fig. 3 (A): The creatine kinase (CK-MB), Fig. 3 (B): the lactate deshydrogenase
(LDH) and Fig.3 (C): the troponin (cTn-1), Fig.3 (D): the aspartate aminotransferase (AST), and Fig.3 (E):
the alanine aminotransferase (ALT).
.
Fig 4: Plasma fibrinogen level variation in control and treated rats. Values are given as mean ± standard
deviation/ S.D. for groups of 6 animals each. The values are statistically presented as follows: * P < 0.05
significant difference compared to the control rats; # P < 0.05 significant differences compared to
isoproterenol groups.
Fig 5: Impact of 1c pre-co-treatment on histology of myocardial tissue (H&E X 400). Control group
revealed normal myocardial structure with clear transverse striations. Isop treated group showing myocardial
cells necrosis with separation of cardiac myofibrillar and excessive leukocyte infiltration.. Isop+Ace treated
group showing moderate injury with few inflammatory cell infiltration. Isop+1c showing normal myocardial
arrangement and limited focal neutrophil infiltration in a small area.
leucocyte infiltration;
separation of cardiac myofibrillar;
cells necrosis
Table 1: Angiotension converting enzyme inhibition activity of compound 1c
Table 2: Effect of 1c pre-treatment on heart weight index (HWI) in isoproterenol (Isop) induced myocardial
infraction in rats. (HWI) was computed as HWI = heart weight (HW)/body weight (BW) × 100. Values are
given as mean ± standard deviation/S.D. for groups of 6 animals each.
Table 3: Effect of (1c) on biochemical parameters in plasma of experimental rats. (TC) total cholesterol,
(TG) triglyceride.
20

Schemes
Scheme 1.
O
NH2
O
O
N
H
H
N
Piperidine
OMe
b
N
O
O
EtOH, reflux
30 min.
AcOH cat.
O
O
O
OH
O
O
EtOH, 80°C,
12 h
a
1c
1
1a
21

Fig.1
O
OH
OH
O
Me
H
HO
HO
N
NO2
O
Isoproterenol (Isop)
Acenocoumarol (Ace)
22

Fig. 2
23

Fig. 3.
250
200
150
100
50
(B)
*
400
350
300
250
200
150
100
50
*
(A)
#,@
*
#
#
0
Control
Isop + Ace
Isop + 1c
Isop Groups
0
Control
Isop
Isop + Ace
Isop + 1c
Groups
250
200
150
100
50
(D)
2.0
(C)
*
*
*,#
*,#,@
1.5
1.0
0.5
0.0
*,#
*,#
0
Control
Isop + Ace
Isop + 1c
Isop Groups
IsopGroupsIsop + Ace
Control
Isop + 1c
180
(E)
*
160
140
120
100
80
*,#
*,#,@
60
40
20
0
Control
Isop + 1c
IsopGroupsIsop + Ace
24

Fig. 4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
*
*,#
*,#
Control
Isop
Isop + Ace
Isop + 1c
Groups
25

Fig. 5
Isop
Control
Isop+Ace
Isop+1c
26

Tables
Table 1: Angiotension converting enzyme inhibition activity of compound 1c
% Inhibition
31.33 ± 1.44
63.90 ± 1.89
70.53 ± 1.22
80.55 ± 1.11
81.70 ± 1.08
Concentration of 1c (µg/ml)
IC50 (µg/ml)
25
50
75
39.12
100
200
The data are expressed in mean ± S.E.M. n = 3 in each tested concentration.
27

Table 2: Effect of 1c pre-treatment on heart weight index (HWI) in isoproterenol (Isop) induced myocardial
infraction in rats. (HWI) was computed as HWI = heart weight (HW)/body weight (BW) × 100. Values are
given as mean ± standard deviation/S.D. for groups of 6 animals each.
Parameters
Control
Isop
Isop + Ace
Isop + 1c
Initial BW (g)
Final BW (g)
261.63 ± 20.11
272.70 ± 20.71
261.33 ± 19.71
273.70 ± 21.00
260.83 ± 20.14
283.60 ± 20.00
261.83 ± 19.90
280.20 ± 20.90
0.95 ± 0.02a
0.35 ± 0.02a
0.86 ± 0.02a, b
0.30 ± 0.01b
0.80 ± 0.01a, b, c
0.29 ± 0.01b
Heart weight (g)
HWI
0.73 ± 0.03
0.28 ± 0.04
BW: Body Weight (g)
Values are statistically introduced as follows: a P < 0.05 significant difference compared to control rats, b P < 0.05
significant difference compared to treated rats with isoprotenol (Isop), c P < 0.05 significant difference compared to
treated rats with acenocoumarol (Ace).
28