Synthesis and antihypertensive activity of some novel pyridazinones

Article abstract of DOI:10.13005/ojc/320129

Four pyridazinones (2-5) were synthesized by the reaction of 3-benzoylpropionic acid with the corresponding hydrazines. These pyridazinones were further derivatized with appropriate aromatic aldehydes in the presence of piperidine to obtain the desired compounds (6-13). The chemical structures of the synthesized pyridazinones were elucidated on the basis of their spectral analysis (IR, ¹H-NMR, and ¹³C-NMR). Molecular docking studies were carried out to identify the most active antihypertensive pyridazinone compound by using the crystal structure of human Angiotensin Converting Enzyme (ACE), the enzyme implicated in the pathogenesis of hypertension. The compound (6) was identified as the most active inhibitor of the Angiotensin Converting Enzyme (ACE). This compound was further assessed for its ACE inhibitory activity using Dojindo ACE Kit-WST test kit. The enzymatic ACE inhibitory activity revealed that the compound (6) had IC₅₀ value of 5.78 μg/mL, wherein the standard drug Lisinopril had IC₅₀ value of 0.85 μg/mL. It has been concluded that incorporation of free amino groups and free carboxylic acid groups in the structure of the synthesized pyridazinone (6-13) may provide more active and potent ACE inhibitors.

Full text of DOI:10.13005/ojc/320129

ISSN: 0970-020 X
CODEN: OJCHEG
2016, Vol. 32, No. (1):
Pg. 267-274
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Synthesis and Antihypertensive Activity
of Some Novel Pyridazinones
MOHD. IMRAN* and NAIRA NAYEEM
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Northern Border University,
Rafha – 91911, P.O. BOX 840, Kingdom of Saudi Arabia.
*Corresponding author E-mail: imran_inderlok@yahoo.co.in
http://dx.doi.org/10.13005/ojc/320129
Received: February 23, 2016; Accepted: March 26, 2016
ABSTRACT
Four pyridazinones (2-5) were synthesized by the reaction of 3-benzoylpropionic acid with the
corresponding hydrazines. These pyridazinones were further derivatized with appropriate aromatic
aldehydes in the presence of piperidine to obtain the desired compounds (6-13). The chemical
structures of the synthesized pyridazinones were elucidated on the basis of their spectral analysis
(IR, 1H-NMR, and 13C-NMR). Molecular docking studies were carried out to identify the most active
antihypertensive pyridazinone compound by using the crystal structure of human Angiotensin
Converting Enzyme (ACE), the enzyme implicated in the pathogenesis of hypertension. The
compound (6) was identified as the most active inhibitor of the Angiotensin Converting Enzyme (ACE).
This compound was further assessed for its ACE inhibitory activity using Dojindo ACE Kit-WST test
kit.The enzymatic ACE inhibitory activity revealed that the compound (6) had IC50 value of 5.78 µg/
mL, wherein the standard drug Lisinopril had IC50 value of 0.85 µg/mL. It has been concluded that
incorporation of free amino groups and free carboxylic acid groups in the structure of the synthesized
pyridazinone (6-13) may provide more active and potent ACE inhibitors.
Key words: Benzoylpropionic acid, Pyridazinones, Molecular modelling,
ACE inhibitor, Antihypertyensive agents.
INTRODUCTION
very high in KSA from 1990 to 2010; the number of
deaths resulting from ischemic heart disease and
hypertensive heart disease in the Middle East and
North Africa region (including the GCC countries)
is estimated to be 294/100,000 and 115/100,000
respectively; and the number of disability-adjusted
life years (DALYs) resulting from ischemic and
hypertensive heart disease is 3702/100,000 and
Cardiovascular disease is a major public
health problem worldwide and also accounts
for approximately 30% of all deaths1. It is also
considered as the major cause of death in the
Kingdom of Saudi Arabia2-4.According to the
literature2-4, the burden of hypertension remained

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IMRAN & NAYEEM, Orient. J. Chem., Vol. 32(1), 267-274 (2016)
1389/100,000, respectively, in the same region. Synthesis of 3-Benzoylpropionic acid (1)
These studies further state that KSA has a young To a solution of succinic anhydride (0.1
population with 81% of the population under the mole) in benzene (50 mL), anhydrous aluminium
age of 40 and life expectancy has increased from chloride(0.125 mole) was added in small portions
72.5 and 76.3 years in 1990 to 75.0 and 79.9 years with stirring over a period of 2 hours. The reaction
in 2010 for men and women, respectively. As life mixture was refluxed for 2 hours. After completion
expectancy increases and the size of population is of the reaction, excess of benzene was removed
on the rise, the burden of hypertension and other by steam distillation. The reaction mixture was
chronic diseases, if uncontrolled, will pose major dissolved in sodium hydroxide solution, filtered, and
challenges to the health system. Studies have also hydrochloric acid was added to it. The solid mass
revealed that there is a need for more research in obtained was filtered, washed with cold water, dried
the field of cardiovascular disease in developing and recrystallized from methanol to give a colorless
countries because in these countries there would productwhich gave effervescence with sodium
be an increase in the prevalence of cardiovascular bicarbonate solution23-25
disease in all age group of patients5,6.
.
Synthesis of 6-phenyl-4,5-dihydropyridazin-
There are many cardioactive pyridazinone 3(2H)-one (2)
derivatives, which are either in clinical use or A mixture of 3-benzoylpropionic acid (0.01
have been tested in clinical trials, for example, mole) and hydrazine hydrate (0.015 mole) in ethanol
imazodan7,8, CI-9308,9, pimobendan8,10, indolidan8,11
levosimendan8,12, SK&F-937418,13, Y-5908,14
Meribendan15,NSP-80416,NSP-80516,bemoradan17,18
,
,
,
(25 mL) was refluxed for about 6 hours.The resulting
mixture was cooled at room temperature. The solid
obtained was filtered, washed with dilute solution
senazodan19, amipizone8,18, prinoxodan20, SKF of sodium bicarbonate and then recrystallized from
9565421, siguazodan22 and KF 1523222. Some of ethanol.
these pyridazinone derivatives were found not
suitable for further development because of their Synthesis of 2-benzoyl-6-phenyl-4,5-
side effects and low potency. Keeping this in mind, it dihydropyridazin-3(2H)-one (3)
was thought to synthesize some novel pyridazinone
6-Phenyl-4,5-dihydropyridazin-3(2H)-
derivatives as antihypertensive agents that may be one (2) (0.01 mole) was taken in a 100 mL round
suitable for further development.
MATERIALS AND METHODS
Chemistry
bottom flask. Potassium carbonate (0.015 mole)
and acetone (25 mL) were added to the flask. The
mixture was stirred at room temperature and benzoyl
chloride (0.01 mole) was added dropwise in 20 to 30
minutes. The resulting mixture was stirred at room
Melting points were measured in open temperature for about 15 hours.The resulting mixture
capillary tubes and are uncorrected. IR (KBr) was reduced to half of its volume and then poured
spectra were recorded on a JASCO, FTIR-4100 to crushed ice. The solid separated was washed
1
spectrophotometer. The H-NMR and 13C-NMR with water, dilute sodium bicarbonate solution
spectra were recorded on Bruker Ultrashield 500 and recrystallized from ethanol. Alternatively, this
Plus MHz spectrophotometer. All reagents used compound may also be prepared by the reaction
in the present work were of analytical grade. The of benzoic acid hydrazide and 3-benzoylpropionic
standard drug Lisinopril used for the assessment acid.
of in vitro ACE inhibitory activity was procured from
Sigma Aldrich, USA. Purity of the compounds was Synthesis of 2-(2,4-dinitrophenyl)-6-phenyl-4,5-
checked on silica gel G plates using iodine vapours dihydropyridazin-3(2H)-one (4)
as visualizing agent. Rf value of the compounds
A mixture of 3-benzoylpropionic acid (0.01
was determined by using a mixture of benzene mole) and 2,4-dinitrophenyl hydrazine (0.01 mole)
and acetone (9:1). The synthetic pathway for the in ethanol (30 mL) was refluxed for about 10 hours.
preparation of the title pyridazinones(2-13) is The solid obtained was hot filtered, washed with
provided in Figure 1.

IMRAN & NAYEEM, Orient. J. Chem., Vol. 32(1), 267-274 (2016)
269
dilute solution of sodium bicarbonate and then
recrystallized from ethanol.
All computations were performed on an Intel(R)
Core(TM) i5-2400 CPU @ 3.10 GHz capacity
processor with memory of 8 GB RAM running with the
Window 7 operating system. Glide 5.9 implemented
in the Maestro 9.4 software package (Schrodinger,
LLC, New York, NY, 2013) was used for docking
studies. Angiotensin Converting Enzyme (ACE)
is an enzyme that is involved in the pathogenesis
of hypertension26. The crystal structure of human
Angiotensin Converting Enzyme (ACE) protein (PDB
code: 1O86, Resolution – 2.00 Å) was retrieved
from Protein Data Bank (PDB) and was utilized for
molecular modelling studies27.Protein was prepared
with the Protein Preparation Wizard in Maestro
using options:bond orders were assigned, hydrogen
atoms were added, formal charges were treated and
water molecules were deleted. Hydrogen bonding
network was then optimized using the exhaustive
sampling option and the protein was minimized
to an RMSD limit from the starting structure of
0.3 Å using the Impref module of Impact with the
OPLS_2005 force field. Prepared protein structure
was used to generate Glide scoring grids for the
subsequent docking calculations. Docking grids
were generated with the default settings in Glide
using the co-crystalized ligand to define the center
of the grid box (20×20×20 Å). Default parameters
Synthesis of 2,6-diphenyl-4,5-dihydropyridazin-
3(2H)-one (5)
The compound (5) was also prepared by
same method used for the compound (4), wherein
2,4-dinitrophenyl hydrazine was replaced by phenyl
hydrazine.
Synthesis of 2-benzoyl-4-(4-bromobenzylidene)-
6-phenyl-4,5-dihydropyridazin-3(2H)-one (6)
A mixture of 2-benzoyl-6-phenyl-4,5-
dihydropyridazin-3(2H)-one (4) (0.01 mole) and
p-bromo benzaldehyde (0.01 mole) was taken in
a round bottom flask containing 25 mL of absolute
ethanol. Piperidine (0.5 mL) was added to the
mixture and it was refluxed for about 8 hours. The
mixture was reduced to half of its volume and then
poured to crushed ice. The solid separated was
washed with water, and recrystallized from ethanol.
The compounds (7-13) were also prepared by similar
methods
Molecular Modelling Studies
The compounds (6-13) were subjected
for their molecular modelling docking studies.
H
N
N
NH 2NH 2.H 2O
EtOH
O
(2)
R-Cl
Benzene
HO
O
Anhydrous AlCl3
O
K2CO
+
3
CH 3COCH3
O
(1)
R
O
N
N
(3) R = -COPh
R-NHNH2
EtOH
O
(4) R = 2,4-(NO2)2Ph-
(5) R = -Ph
O
(3-5)
Succinic Anhydride
(6) R = -COPh, Ar = p-BrPh-
(7) R = -COPh, Ar = p-ClPh-
Ar-CHO
Piperidine
(8) R = -COPh, Ar = p-NO2Ph-
(9) R = -COPh, Ar = -Ph
R
(10) R = 2,4-(NO2)2Ph-, Ar = p-BrPh-
(11) R = 2,4-(NO2)2Ph-, Ar = p-ClPh-
(12) R = 2,4-(NO2)2Ph-, Ar = p-NO2Ph-
(13) R = 2,4-(NO2)2Ph-, Ar = -Ph
N
N
O
Ar
(6-13)
Fig.1 :

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IMRAN & NAYEEM, Orient. J. Chem., Vol. 32(1), 267-274 (2016)
were used and no constraints were included during run according to the manufacturer’s instructions
grid generation.The three dimensional coordinates of using a 96-well plate without modification, and the
most active synthesized compound (6) and lisinopril inhibition rate was calculated based on a comparison
was generated using Maestro module of Schrodinger. of the optical absorbance of samples-treated wells
Ligands were prepared using LigPrep 2.6 with Epik (545‘), control wells (545P), and blank wells (545O).
2.4 to expand protonation and tautomeric states at Absorbance was measured at 450 nm using the
7.0 2.0 pH units and energy was minimized using microplate reader Biotek-ELX800 (BioTek, Vermont,
the OPLS 2005 force field. The energy minimized USA). Inhibition rates werecalculated using the
ligands were docked to the protein structure using following equation.
Glide XP docking since it incorporates structural and
energetic information using the scoring function28.
Inhibition rate (%) = [545P– 545‘ / 545P” 545O] ×
100
Angiotensin Converting Enzyme Inhibitory
Assay
Samples were suspected to inhibit the
ACE activity, and therefore inhibit the formation of
The compound (6) that showed highest formazan. The more strongly inhibitory the activity
ACE inhibitory activity according to the molecular of the samples, the less color appeared in the final
modelling studies was subjected to ACE inhibitory solution.
assay using Dojindo ACE Kit-WST test kit, Dojindo
Laboratories, Kumamoto, Japan29,30. Briefly, The Statistical Analysis
enzymatic reaction was initiated by the ACE and
AllACEinhibitoryactivitydataarepresented
aminoacylase in the mixture containing 3HB-GGG as mean standard deviation (SD, n = 3). The data
(3-hydroxybutyrate glycylglycylglycine) and the were analyzed by one-way analysis of variance
ACE-inhibitor. The mixture was then incubated at (ANOVA) with Dunnett’s Multiple Comparison Test
37oC for 60 minutes. During this incubation, the with respect to control group and standard groups
substrate, 3HB-GGG, was enzymatically cut into using GraphPad Prism version 5.00 for Windows
3HB-G and G-G and then 3HB and G. The yield (GraphPad Software, San Diego California USA).
of 3HB was monitored indirectly through formazan The results were considered significantly different at
concentration, which was measured at 450 nm after p < 0.05. The IC50 values were determine by linear
10 minute reaction at 25oC.Testing procedures were regression calculator of GraphPad software.
Table 1:The physical constants of the synthesized compounds
Compd
No.
R
Ar
Molecular
Formula
M.P.
( 2oC)
Yield
(%)
Rf
Value
1
2
3
4
5
6
7
8
-
-
-
-
-
-
-
C10H10O3
C10H10N2O
C17H14N2O2
C16H12N4O5
C16H14N2O
C24H17BrN2O2
C24H17ClN2O2
C24H17N3O4
C24H18N2O2
C23H15BrN4O5
C23H15ClN4O5
C23H15N5O7
C23H16N4O5
115
152
166
188
144
175
155
175
164
210
204
222
196
75
80
55
80
60
60
65
50
60
55
60
60
70
0.82
0.77
0.76
0.63
0.77
0.74
0.76
0.78
0.71
0.61
0.63
0.64
0.63
-COPh
2,4-(NO2)2Ph-
-Ph
-COPh
-COPh
-COPh
-COPh
p-BrPh-
p-ClPh-
p-NO2Ph-
-Ph
p-BrPh-
p-ClPh-
p-NO2Ph-
-Ph
9
10
11
12
13
2,4-(NO2)2Ph-
2,4-(NO2)2Ph-
2,4-(NO2)2Ph-
2,4-(NO2)2Ph-

IMRAN & NAYEEM, Orient. J. Chem., Vol. 32(1), 267-274 (2016)
271
RESULTS AND DISCUSSION
Chemistry
The detailed spectral data of the compounds (2-6)
is provided below.
The compounds (1-13) were prepared
according to the method outlined in Figure 1. The
melting point of the compound (1) was in accordance
with the literature23-25. The physical constants of the
synthesized compounds (1-13) are summarized in
Table 1.
6-Phenyl-4,5-dihydropyridazin-3(2H)-one (2)
IR (KBr) cm-1: 3220 (N-H), 3100 (C-H),
1
1680 (C=O), 1610 (C=N), 1510 (C=C); H-NMR
(DMSO-d6) d ppm: 2.44 (t, J = 8 Hz, 2H, -CO-CH2-),
2.89 (t, J = 8 Hz, 2H, -C(=N)-CH2-), 7.40 (d, J=
11Hz, 3H, Ar-H), 7.74 (d, J= 11Hz, 2H, Ar-H), 10.99
(s, 1H, -NH-); 13C-NMR (DMSO-d6) d ppm: 167.02
(C=O), 149.34, 135.90, 129.19, 128.42 (2C), 125.56,
125.31, 25.94, 21.85.
The structures of the compounds (2-6)
1
were confirmed on the basis of their IR, H-NMR,
and 13C-NMR.The appearance of the IR absorption
peaks from 1685 cm-1 to 1625 cm-1confirmed the
stretching vibration of C=O group present at C-3
of the compounds (2-5). The appearance of the
2-Benzoyl-6-phenyl-4,5-dihydropyridazin-3(2H)-
one (3)
IR (KBr) cm-1: 1680 and 1640 (C=O), 1610
1
1
signals in the H-NMR spectra of the compounds
(C=N), 1505 (C=C); H-NMR (DMSO-d6) d ppm:
(2-5) at d (ppm) values from 2.36 to 2.44 confirmed
two protons of -CO-CH2- and from 2.72 to 3.11
confirmed two protons of -C(=N)-CH2- portion of the
pyridazinone ring. The compound (2) also showed
characteristic signal at d (ppm) value of 10.99
due to the ®“l¾“NH- proton. The structure of the
compound (6) was supported by the appearnce of
two stretching vibrations at 1685 cm-1 and 1645 due
to two C=O groups.The absence of the peak for two
protons of -CO-CH2- portion of the pyridazinone ring
and appearnce of signals at ä (ppm) value of 2.88
due to the two protons of -C(=N)-CH2- portion of
the pyridazinone ring confirmed the structure of the
compound (6).The 13C-NMR of the compounds (2-6)
were also consistent with their assigned structures.
2.44 (t, J = 8 Hz, 2H, -CO-CH2-), 2.90 (t, J = 8 Hz,
2H, -C(=N)-CH2-), 7.30-7.99 (m, 10H, Ar-H); 13C-
NMR (DMSO-d6) d ppm: 167.37 and 167.00 (C=O),
149.33, 135.89, 132.72 (2C), 129.24 (2C), 129.14,
128.43 (2C), 128.37, 125.53 (3C), 25.93, 21.85.
2 - ( 2 , 4 - D i n i t ro p h e ny l ) - 6 - p h e ny l - 4 , 5 -
dihydropyridazin-3(2H)-one (4)
IR (KBr) cm-1: 1625 (C=O), 1600 (C=N),
1510 (C=C), 1310 and 1340 (NO2); 1H-NMR (DMSO-
d6) d ppm: 2.36 (t, J = 8 Hz, 2H, -CO-CH2-), 3.02
(t, J = 8 Hz, 2H, -C(=N)-CH2-), 7.42-7.66 (m, 4H,
Ar-H), 7.84-7.93 (m, 2H, Ar-H), 8.21-8.26 (m, 1H,
Ar-H), 8.78 (s, 1H, Ar-H); 13C-NMR (DMSO-d6) d
ppm:168.02 (C=O), 146.50, 144.40, 143.30, 136.09,
Fig. 2: 2D/3D interaction of Lisinopril with the active sites of the ACE showing hydrogen bond
interaction and non-covalent lipophilic interactions

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IMRAN & NAYEEM, Orient. J. Chem., Vol. 32(1), 267-274 (2016)
129.60, 128.51 (3C), 126.52 (2C), 123.43, 123.08, Molecular Modelling Studies
116.45, 33.53, 23.63. The molecular modelling studies revealed
that almost all the functional groups of Lisinopril
2,6-Diphenyl-4,5-dihydropyridazin-3(2H)-one (5) (docking score: -11.36; Figure 2) were occupied
IR (KBr) cm-1: 1685 (C=O), 1600 (C=N), with either hydrogen bonding or other non-covalent
1510 (C=C); 1H-NMR (DMSO-d6) d ppm: 2.72 (t, J = lipophilic interactions and were important in exerting
8 Hz, 2H, -CO-CH2-), 3.11 (t, J = 8 Hz, 2H, -C(=N)- greater affinity towards ACE enzyme and ultimately
CH2-), 7.28 (t, J = 8Hz, 1H, Ar-H), 7.42-7.46 (m, 5H, ACE inhibition activity. On the other hand, the
Ar-H,), 7.57 (d, J = 11 Hz, 2H, Ar-H), 7.84 (t, J = 8 compound (6) (docking score: -6.14; Figure 3) was
Hz, 2H, Ar-H); 13C-NMR (DMSO-d6) d ppm: 165.25 identified as the most active ACE inhibitor among
(C=O), 151.84, 141.40, 135.40, 129.77, 128.54 (2C), the synthesized compounds. The compound(7)
128.28 (2C), 126.11, 126.05 (2C), 124.84 (2C), (docking score: -4.70), compound (8) (docking
27.32, 22.23.
score: -4.78), compound (9) (docking score: -4.60),
compound (10) (docking score: -5.02), compound
2-Benzoyl-4-(4-bromobenzylidene)-6-phenyl-4,5- (11) (docking score:-4.57), compound (12) (docking
dihydropyridazin-3(2H)-one (6) score:-4.13), and the compound (13) (docking score:
IR (KBr) cm-1: 1685 and 1645 (C=O), 1605 -4.65) also showed some ACE inhibitory activity.The
(C=N), 1500 (C=C);1H-NMR (DMSO-d6) d ppm:2.88 ACE inhibitory activity of the compounds (7-13) by
(s, 2H, -C(=N)-CH2-), 6.86-7.98 (m, 15H, Ar-H); 13C- molecular docking score was not found significant
NMR (DMSO-d6) d ppm: 167.89 and 167.53 (C=O), when compared to the standard drug Lisinopril.In
161.70, 149.87, 136.34, 136.01, 133.19 (2C), 131.21, general terms, larger is the docking score in negative
130.71, 129.70 (4C), 128.87 (4C), 126.01 (2C), value, more is the active binding with the target
119.53, 117.49, 113.31, 26.39.
receptor .e.g. active site of ACE.
The compounds (7-13) were identified Angiotensin Converting Enzyme Inhibitory
on the basis of the difference in the melting points Assay
and Rf values with respect to their reactants. These
Based on the molecular modelling results,
compounds were not subjected for their spectral the compound (6) was selected for further in vitro
analysis as their ACE inhibitory activity results .i.e. ACE inhibitory assay using Dojindo ACE Kit-WST
docking score obtained from the molecular modelling test kit, Dojindo Laboratories, Kumamoto, Japan.The
studies were not significant.
docking score and the ACE inhibitory activity data of
the compound (6)with respect to the standard drug
Lisinopril is provided in Table 2.
Fig. 3: 2D/3D interaction of the compound (6) with the active sites of the ACE showing hydrogen
bond interaction and non-covalent lipophilic interactions

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IMRAN & NAYEEM, Orient. J. Chem., Vol. 32(1), 267-274 (2016)
Table2: In vitroACE inhibitory activity of Compound
(6) and standard drug Lisinopril
Compound Concentration
(μg/mL)
%ACE
Inhibition
(Mean SD)
IC50
(μg/mL)
Docking
Score
1
2
43.20 0.30*
60.30 0.10*
75.63 0.06*
86.66 0.15*
12.50 0.10*
28.80 0.10*
49.70 0.10*
0.85
5.78
-11.36
Lisinopril
4
8
1
2
4
Compound (6)
-6.14
8
58.76 0.15*
*p < 0.0001; n = 3.
The in vitroACE inhibitory activity revealed data, it is evident that the synthesized compounds
that the compound (6) had IC50 value of 5.78 ìg/mL, are not potent inhibitors of ACE enzyme.Therefore,
wherein the standard drug Lisinopril had IC50 value these compounds require further modifications in
of 0.85 ìg/mL.These results were also supported by their chemical structure. It is suggested that the
the results of molecular modelling, wherein it was incorporation of free amino group and free carboxylic
evident that the compound (6) (Figure 3) did not bind acid groups in the structure of the synthesized
effectively with the active site of the ACE enzyme compounds may provide potent and more active
as compared to standard drug Lisinopril (Figure 2). ACE inhibitors. Accordingly, further studies are in
This may be due to the fact that the compound (6) progress in our laboratory.
does not have free functional groups that aid in the
binding with the active site of the ACE enzyme. It is
possible that the incorporation of free amino groups
and free carboxylic acid groups in the structure of
ACKNOWLEDGEMENT
The authors would like to express their
the compounds(6-13) may provide better and more deep gratitude to the Northern Border University,
potent ACE inhibitors.
Kingdom of Saudi Arabia for providing financial
support (Project No. 3/3/1436/5).
CONCLUSION
Based on the molecular modelling results
and the in vitro enzymatic ACE inhibitory activity
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