Synthesis of new 4,5-3(2H)pyridazinone derivatives and their cardiotonic, hypotensive, and platelet aggregation inhibition activities

Article abstract of DOI:10.1007/s12272-010-2222-x

4,5-dihydro-3(2H)pyridazinones such as CI-914, CI-930 and pimobendan along with tetrahydropyridopyridazine (endralazine) and perhydropyridazinodiazepine (cilazopril) have been used as potent positive inotropes, antihypertensives as well as platelet aggreg

Full text of DOI:10.1007/s12272-010-2222-x

Arch Pharm Res Vol 33, No 1, 25-46, 2010
DOI 10.1007/s12272-010-2222-x
Synthesis of New 4,5-3(2H)pyridazinone Derivatives and Their Car-
diotonic, Hypotensive, and Platelet Aggregation Inhibition Activities
Enas Nashaat Amin1, Abdel-Alim M. Abdel-Alim1, Samia G. Abdel-Moty1, Abdel-Naser A. El-Shorbagi1, and
Mahran Sh. Abdel-Rahman2
1Department of Pharmaceutical Organic Chemistry, Faculty of Medicine, Assiut University, Assiut 71526, Egypt and
2Faculty of Pharmacy and Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut 71526, Egypt
(Received August 20, 2009/Revised October 19, 2009/Accepted October 20, 2009)
4,5-dihydro-3(2H)pyridazinones such as CI-914, CI-930 and pimobendan along with tetrahy-
dropyridopyridazine (endralazine) and perhydropyridazinodiazepine (cilazopril) have been
used as potent positive inotropes, antihypertensives as well as platelet aggregation inhibitors.
Accordingly, the present work involves the synthesis of 24 target compounds; 4,5-dihydro-
3(2H)pyridazinones in addition to seven reported intermediates. The chemical structures of
1
the new compounds were assigned by microanalysis, IR, H-NMR spectral analysis and some
representatives by mass spectrometry. The positive inotropic effect of the final compounds and
the intermediates 12a-12d as well as the reported intermediate compound 10 was determined
in-vitro on isolated rabbit heart in comparison to digoxin. Data obtained revealed that twelve
of the test compounds exhibited higher effective response than digoxin, nine compounds elic-
ited comparable effects to digoxin and eight compounds were less active than digoxin. In addi-
tion, four compounds approved marked significant hypotensive effect better than that of the
previously reported compound 10. Moreover, two compounds induced complete platelet aggre-
gation inhibition. The last two compounds were also subjected to determination of their LD50
and they showed no signs of toxicity up to the dose level 300 mg/kg (i.p.), while the reported
oral LD50 of digoxin is 17.78 mg/kg. Correlation of cardiotonic and hypotensive activities with
structures of compounds was tried and pharmacophore models were computed to get useful
insight onto the essential structural features required for inhibiting phosphodiesterase-III in
the heart muscles and blood vessels.
Key words: Positive inotropic (Cardiotonic), 4,5-dihydropyridazinones, Hypotensive, Platelet
aggregation inhibition, Acute toxicity, Pharmacophore models
use due to arrhythmogenic and chronotropic liabilities
respectively. In addition, sympathomimetic agents
Selected by Editors
lack oral efficacy (Erhardt, 1987; Hansen, 1984; Forest
et al., 1992)
INTRODUCTION
Also, a series of pyridazine and pyridazinone deri-
Cardiac glycosides (digoxin, digitoxin) and sym- vatives were introduced into clinical practice as posi-
pathomimetic agents such as dopamine and dobuta- tive inotropes and vasodilators such as imazodan
1
mine are well known positive inotropes and have been (Erhardt, 1987; Forest et al., 1992; Bristol et al., 1984;
used for the treatment of congestive heart failure. Mertens et al., 1990; Sircar et al., 1985; Weishaar et
However, these agents have got a limited therapeutic al., 1985; Moss et al., 1987). CI-930
2 (Erhardt, 1987;
Sircar et al., 1985; Moss et al., 1987), pimobendan
3
(Sweetman, 2002; O' Neil et al., 2006; Rüegg et al.,
1984; Rüegg, 1986; Fujino et al., 1988; Von Meel,
Correspondence to: Abdel-Alim M. Abdel Alim, Departement of
Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
Tel: 0127370730, Fax: 002 088 2332776
E-mail: alimorg43@yahoo.com
1985), endralazine
2006) and cilazopril
2006; Calam, 2001) (Chart 1). In addition, (Thyes et
4
(Sweetman, 2002; O' Neil et al.,
5
(Sweetman, 2002; O' Neil et al.,
25

26
E. N. Amin et al.
Chart 1. Structures of different pyridazine and pyridazinone positive inotropic agents
al., 1983) reported some 6-aryl-4,5-dihydro-3(2H) pyri- characterize their physicochemical and spectroscopic
dazinones with platelet aggregation inhibition and properties since no data are available about them in
6
hypotensive activities. Moreover, (Okushima et al., this aspect. These compounds were, also, tested for
1987) reported the synthesis of a class of 6-[4-(sub- their cardiotonic effect on isolated rabbit heart, hypo-
stituted-amino)phenyl]-4,5-dihydro-3(2H)-pyridazinones tensive activity on anesthetized normotensive rabbits
7
with extremely potent positive inotropic activity and platelet aggregation inhibition activity in-vitro on
along with vasodilating effect. In addition, compounds human blood. Activity of these compounds was also
12a-d were reported in an EP-patent (Haikala et al., taken as references for our new target compounds.
1990) as positive inotropes in dogs and guinea pigs
heart muscle.
Accordingly, the present work aims at the synthesis
of 6-[4-(2-substituted hydrazino) phenyl]-4,5-dihydro-
Although compounds 12a-d were reported as car- 3(2H)-pyridazinones to be assessed as positive inotro-
diotonics on dogs, we undertook their synthesis to pes, vasodilators, and platelet aggregation inhibitors.

New Cardiotonic Pyridazinones
27
Preparation of 6-{4-[2-(Disubstitutedmethyli-
dene)hydrazino]-phenyl}-4,5-dihydro-3(2H)-pyri-
dazinones (12a-d)
MATERIALS AND METHODS
The starting materials and solvents required in this
paper are commercially available
.
Anhydrous alumi-
Sodium nitrite (0.393 g, 0.0057 mole) was added,
num chloride was purchased from Sigma-Aldrich. little at a time in the course of 30 min to the ice cooled
Melting points were determined on an electrothermal solution of compound 10 (1 g, 0.0053 mole) in conc.
Stuart Scientific SMPI and were uncorrected. Pre- hydrochloric acid/water mixture (2:1). (Solution A).
coated silica gel plates Kieselgel 0.25 mm 60G F254 Appropriate amount from the active methylene deri-
(Merck) were used for monitoring the reactions using vatives 11a-d (0.0053 mole) was added to an ice-
(ethyl acetate: hexane) (9:1) as a mobile phase unless cooled solution of sodium acetate (1.195 g, 0.015 mole)
otherwise stated. Visualization was effected by ultra- in 2.7 mL of water and 2.7 mL of absolute ethanol.
violet lamp Spectroline ENF-240C/F (model CM-10) at (Solution B). Solution A was added, portionwise, with
short wavelength (
λ = 254 nm) and/or iodine stain. All stirring to solution B during 1 h with cooling. The
chemical yields are unoptimized and generally repre- precipitate formed was left under stirring in the ice
sent a single experiment. The IR spectra were re- bath for 15 min and then, 2.7 mL of ethanol and 1.3
corded on a Shimadzu 200-91527 spectrophotometer mL of water were added. The precipitate obtained was
1
as potassium bromide discs. H-NMR spectra were filtered, washed with water and dried. Compound 12a
scanned on a Varian EM-360 L NMR spectrophoto- was purified by column chromatography using chloro-
meter (60 MHz). Chemical shifts are expressed in
δ-
form as mobile phase. Compounds 12b and 12c were
values (ppm) relative to tetramethylsilane (TMS) as crystallized from aqueous ethanol and compound 12d
an internal standard, using CDCl3 as a solvent unless was crystallized from ethyl acetate. Physical, micro-
otherwise specified, and deuterium oxide was used for analytical and spectral data are listed in Table I.
the detection of the exchangeable protons. Electron-
Impact mass were done on a JEOL JMS600 mass
spectrometer. The microanalysis for C, H and N were
performed on a Perkin Elmer 240 elemental analyzer.
Compounds 10 (Thyes et al., 1983) and 12a-d (Haikala
Preparation of 6-{4-[2-(3-Methyl-5-oxo-1(un)sub-
stituted-4-pyrazolinylidene)-hydrazino]phenyl}-
4,5-dihydro-3(2H)-pyridazinones (13a-c)
A mixture of compound 12a (1 g, 0.003 mole) and
et al., 1990) were prepared according to reported pro- the appropriate hydrazine derivative (0.03 or 0.003
cedures. The physical and spectral data for compounds mole) was heated under reflux in ethanol for 26 h. The
12a-d are not found in the available literatures, while reactions were monitored by TLC using the system of
that of compound 10 is compatible with the reported (CHCl3:CH3OH) (9:1). The reaction mixtures were
(Thyes et al., 1983). Positive inotropic, hypotensive cooled and the solids obtained were filtered and
activities and toxicity determinations were done at the washed with water. Compound 13a was purified by
department of Pharmacology, Faculty of Medicine, column chromatography using CHCl3:CH3OH (9.25:
Assiut University. The platelet aggregation inhibition 0.75), 13b was crystallized from aqueous ethanol, and
activity was performed at the department of Clinical 13c was crystallized from glacial acetic acid. Physical,
Pathology, Assiut University Hospital.
micro-analytical and spectral data are listed in Table
All the animals used in this work were obtained II.
from the house of laboratory animals, Faculty of
Medicine, Assiut University.
Preparation of 6-{4-[3, 5-Dimethyl-1(un)substi-
tuted-4-pyrazolylazo]phenyl}-4,5-dihydro-3(2H)-
Preparation of 6-(4-Aminophenyl)-4,5-dihydro- pyridazinones (14a, b)
Compound 12c (1 g, 0.003 mole) was stirred with
3(2H)–pyridazinone (10)
A mixture of 4-(4-aminophenyl)-4-oxobutanoic acid the appropriate hydrazine derivative (0.03 or 0.003
compound
9 (2 g, 0.01 mole) and hydrazine hydrate mole) in absolute ethanol for 24 or 10h respectively to
98% (0.75 g, 0.015 mole) in absolute ethanol was re- give 14a or 14b. Reactions were monitored by TLC
fluxed for 24 h. After cooling, the faint brown crystals using (CHCl3:CH3OH) (9:1). The reaction mixtures
produced were filtered off, washed with ethanol and were cooled and the solids obtained were filtered,
dried. Physical and spectral data are compatible with washed with water and dried. Compound 14a was
the reported, yield (92%), m.p. 236-238ºC with decom- purified by column chromatography and compound
position as reported (Thyes et al., 1983).
14b was crystallized from ethyl acetate. Physical,
micro-analytical and spectral data are listed in Table
II.

28
E. N. Amin et al.
Table I. Physicochemical and spectral data of compounds 12a-d
Microanalysis
Calc. Found
Molecular
formula
(M. wt.)
Comp. Yield M.p.
1H-NMR
IR Spectra
No.
%
oC
(DMSO-d6,
δ
ppm)*
(KBr, cm-1)**
%
%
1.37 (3H, t,
J
= 7.5 Hz, CH2CH3); 2.73 (7H, m, COCH3,
1720
C=O ester)
and 1697
178
-
180
C
H
N
58.17 58.35 COCH2CH2C=N); 4.50 (3H, br q,
25.49 25.49 C6H5NH, CH2CH3); 7.67 (2H, d, = 8.7 Hz, C'2H,
16.95 16.65 C'6H); 8.10 (2H, d, = 8.7 Hz, C'3H, C'5H) and
11.37 (1H, s, CONHN).
J = 7.4 Hz,
***
12a
C16H18N4O4
(330.30)
(
υ
95
J
J
(
υ
C=O ketone).
1.33 (6H, t, = 7.5 Hz, 2CH2CH3); 2.77 (4H, m,
55.28 54.99 COCH2CH2C=N); 3.70 (1H, s, NHN=C); 4.45 (4H, q,
25.73 25.26 = 7.5 Hz, 2CH2CH3); 7.60 (2H, d, = 8.5 Hz, C'2H,
15.16 14.96 C'6H); 8.10 (2H, d, = 8.5 Hz, C'3H, C'5H) and
11.18 (1H, s, CONHN).
2.60 (6H, s, 2COCH3); 2.68 (2H, t,
59.99 59.85 COCH2CH2C=N); 3.10 (3H, m, COCH2 CH2 C=N
25.37 25.13 and NHN=C); 7.78 (2H, d, = 8.4 Hz, C'2H, C'6H);
J
180 C17H20N4O5
C
H
N
1689
C=O ester).
12b
12c
12d
80
92
94
-
1/2 H2O
J
J
(
υ
182
(369.34)
J
J
= 8.0 Hz,
228
-
230
C
H
N
C15H16N4O3
(300.28)
1680
C=O ketone).
J
(
υ
18.64 18.58 8.03 (2H, d,
CONHN).
J = 8.4 Hz, C'3H, C'5H); 11.28 (1H, s,
1.38 (3H, t,
J = 7.5 Hz, CH2CH3); 2.53 (2H, t, J = 7.8
2185
(υ C≡N)
and 1700
242
-
243
Hz, COCH2CH2 C=N); 3.10 (3H, m, COCH2CH2 C=N
C15H15N5O3
(313.27)
C
H
57.50 57.35
24.83 14.68
and NHN=C)); 4.43 (2H, q,
7.70 (2H, d, = 8.6 Hz, C'2H, C'6H); 8.08 (2H, d,
= 8.6 Hz, C'3H, C'5H); and 11.20 (1H, s, CONHN).
J = 7.5 Hz, COOCH2CH3);
J
J
(υ
C=O ester).
*
Protons of NH groups are exchangeable by D2O.
**IR spectra (KBr, cm-1) showed the following common bands: 3445 (
1223-1188 ( C-N), 1129, 1101 ( C-O) and 800-840 ( para disubstituted aromatic).
υ
NH), 1665 (υ C=O amide), 1614, 1556 (υ C=N),
υ
υ
δ
***EI-Mass Spectrum of Compound 12a m/z (%): 54.58 (14.7), 91.07 (24.6), 117.05 (29.0), 145.11(14.0), 187.07(80.0),
188.07 (60.2), 214.08(100.0) {base peak}, 215.09 (23.2), 330.07 (87.0) {Molecular ion peak M•+} and 331.82 (20.5)
{M•++1}.
Preparation of 6-{4-[2-(3-Amino-5 oxo-pyrazolin- Preparation of amides or amide/imine deriva-
4-ylidene)hydrazino]phenyl}-4,5-dihydro-3(2H)- tives (16c, 16e, 16f and 16h)
Compound 12a (0.5 g, 0.002 mole) was refluxed with
pyridazinone (15a)
A mixture of compound 12d (1 g, 0.003 mole) and either propylamine (0.05 mole) or cyclohexylamine
hydrazine hydrate (98%) (0.25 g, 0.005 mole) was (0.5 g, 0.005 mole) or isopropylamine (1.18 g, 0.02
refluxed in absolute ethanol for 24 h and the reaction mole) or 2-phenethylamine (0.005 mole) for 15 min
was monitored by TLC. The reaction mixture was then absolute ethanol (10-20 mL) was added. The
cooled and the precipitate obtained was filtered, reactions were refluxed for five days as monitored by
washed with water and dried; yield 73%. The product TLC. The reaction mixtures were cooled and the
was crystallized from glacial acetic acid to give dark precipitates formed were filtered, washed with water
reddish brown crystals. Physical, micro-analytical and and dried. Compounds 16c and 16h were purified by
spectral data are listed in Table II.
column chromatography. Compounds 16e and 16f
were crystallized from aqueous ethanol. Physical,
micro-analytical and spectral data are listed in Table
III.
Preparation of 6-{4-[2-(1-N-methylcarbamoyl-1-
{1-(N-methylimino)ethyl}-methylidene)-hydrazi-
no]phenyl}-4,5-dihydro-3(2H)-pyridazinone (16a)
Compound 12a (0.5 g, 0.002 mole) was refluxed with
methylamine 30% (1.7 mL, 0.05 mole) for two days as
monitored by TLC. The reaction mixture was cooled
and the precipitate formed was filtered, washed with
Preparation of 6-{4-[2-(1-Acetyl-1-N-n-butylcar-
bamoylmethylidene)hydrazino]-phenyl}-4,5-di-
hydro-3(2H)-pyridazinone (16d)
Compound 12a (0.5 g, 0.002 mole) was dissolved in
water and dried. The product obtained was crystalliz- 3 mL hot dimethylformamide and n-butylamine (0.37
ed from chloroform. Physical, micro-analytical and g, 0.005 mole) was added. The reaction mixture was
spectral data are listed in Table III.
refluxed with stirring for a week where the reaction

New Cardiotonic Pyridazinones
29
Table II. Physicochemical and spectral data of compounds 13a-c; 14a-b; 15a
Microanalysis
Calc. Found
Molecular
formula
(M. wt.)
Comp. Yield M.p.
1H-NMR
IR Spectra
(KBr, cm-1)
No.
%
oC
(DMSO-d6,
δ
ppm)*
%
%
2.23 (3H, s, CH3C=N); 2.53 (3H, t,
J = 7.0 Hz,
COCH2CH2C=N and NHN=C); 3.00 (2H, t,
54.72 54.21 = 7.0 Hz, COCH2CH2C=N); 7.67 (2H, d,
14.92 15.00 8.5 Hz, C'2H, C'6H); 8.03 (2H, d, = 8.5 840 (δ
J
3500-3450 (
= ( C=O amide) and 800-
para-disubstitut-
υ NH); 1665
290 C14H14N6O2
C
H
J
υ
13a
66
-
1/2 H2O
J
292
(307.26)
Hz,C'3H, C'5H); 10.90 (1H, s, CONHN) and ed aromatic)
11.50 (1H, br. s, CONHN).
2.23 (3H, s, CH3C=N); 2.53 (3H, br. t,
J
= 7.0 The same data as 13a in
Hz, COCH2CH2C=N and NHN=C); 3.00 (2H, addition to: 697, 770 (
t, = 7.0 Hz, COCH2CH2C=N); 7.8 (9H, m, mono-substituted aro-
C6H4 and C6H5) and 11.20 (1H, s, CONHN). matic).
293 C20H18N6O2
C
H
N
62.65 62.41
14.99 14.94
21.90 21.63
δ
13b
13c
88
80
-
1/2 H2O
J
295
(383.33)
The same data as 13a in
addition to: 1570, 1380
300
-
302
1H-NMR (CF3COOD,
δ ppm)
C20H16N8O6
(464.33)
C
H
51.71 51.70
13.47 14.15
3.10 (7H, m, CH3C=N, COCH2CH2C=N) and (
υ
NO2) and 900 & 800
1, 2, 4-tri-substituted
7.50 – 8.70 (8H, m, C6H4, C6H3 and NHN=C). (
δ
aromatic).
3500-3450 (
C=O amide); 1614,
3.45 (10H, m, 2CH3C=N and COCH2 CH2C= 1556 ( C=N); 1223-1188
N) and 8.62 (6H, m, C6H4; CONHN and ( C-N); 1129, 1101 ( C-
O) and 800-840 ( para-
disubstituted aromatic).
υ NH); 1665
1H-NMR (CF3COOD,
δ
ppm)
(υ
162 C15H16N6O
57.32 57.54
15.77 15.68
26.73 26.43
C
HN
υ
14a
14b
15a
81
80
73
-
1moleH2O
(314.28)
υ
υ
164
NHN=C).
δ
3500-3450 ( NH); 2460
N+); 1665 (C=O amide);
para-disub-
N); 7.60 (5H, s, C6H5); 8.00 (4H, s, C6H4) and stituted aromatic) and
9.23 (1H, s, CONHN). 691, 760 ( mono-substi-
tuted aromatic).
υ
1H-NMR (CDCL3,
2.95 (10H, m, 2COCH3 and COCH2 CH2C= 800-840 (
δ
ppm)
(υ
238 C21H20N6O
C
H
N
56.69 56.83
15.66 15.66
18.88 19.02
δ
-
HCl.2H2O
(444.87)
240
δ
2.68 (2H, t,
3.30 (2H, t,
J
J
= 7.5 Hz, COCH2CH2C=N);
= 7.5 Hz, COCH2CH2C=N); 3500-3450 (
C=O amide), and 800-
= 8.0 Hz,C'2H, C'6H); 8.50 840 ( para-disubstitut-
= 8.0 Hz, C'3H, C'5H); 11.40 (1H, ed aromatic).
br s, CONHN) and 11.76 (1H, s, CONHN).
υ
NH), 1665
308 C13H13N7O2
C
H
50.65 50.33 3.82 (1H, br s, NHNC); 6.28 (2H, br s, NH2); (
14.58 14.22 8.16 (2H, d,
(2H, d,
υ
-
1/2 H2O
(308.23)
J
δ
310
J
*Protons of NH groups are exchangeable with D2O.
was monitored by TLC. The warm mixture was pour- gm crushed ice. The precipitates formed were filtered,
ed onto 50 gm crushed ice. The formed precipitate was washed thoroughly with water and dried. Compound
filtered, washed thoroughly with water and dried. The 17a was crystallized from chloroform and compound
product was purified by column chromatography. 17b was purified by column chromatography. Physi-
Physical, micro-analytical and spectral data are listed cal, micro-analytical and spectral data are listed in
in Table III.
Table IV.
Preparation of 6-{4-[2-(1, 1-Bis-N-methyl (ethyl) Preparation of diamides or amide/ester; com-
carbamoylmethylidene)-hydrazino]phenyl}-4,5-
dihydro-3(2H) – pyridazinones (17a, b)
pounds (17c and 17e-h)
Compound 12b (0.5 g, 0.002 mole) was fused with
Compound 12b (0.5 g, 0.002 mole) was dissolved in the appropriate amine (0.04 mole of n-propyl and iso-
3mL hot dimethylformamide and methylamine 30% propyl amines and 0.005 mole of other amines) for 15
(1.8 mL, 0.05 mole) or ethylamine 70% (1.3 mL, 0.02 min. Absolute ethanol (10-20 mL) was added and the
mole) was added respectively. The reaction mixture mixtures were refluxed for the appropriate time ranges
was refluxed with stirring for a week as been moni- from 4 to ten days. The reactions were monitored by
tored by TLC. The warm mixture was poured onto 50 TLC using the usual system in all compounds except

30
E. N. Amin et al.
Table III. Physicochemical and spectral data of compounds 16a, 16c-f, 16h
Microanalysis
Calc. Found
Molecular
formula
(M. wt.)
Comp. Yield M.p.
1H-NMR
IR Spectra
No.
%
oC
(DMSO-d6,
δ
ppm)*
(KBr, cm-1)**
%
%
3.14 (14H, m, CH3N=C, CH3NH, COCH2, CH2C=N,
244
-
246
C16H20N6O2
(328.32)
C
H
58.52 58.32 CH3C=N and NHN=C); 8.13 (2H, d,
16.14 16.30 C'6H); 8.43 (2H, d, = 8.5 Hz, C'3H, C'5H); 9.48 (1H,
s, CONHN) and 12.13 (1H, br s, CONHCH3).
J = 8.5 Hz,C'2H,
16a
73
J
1H-NMR (DMSO-d6,
ppm)
δ
1.33 (14H, m, NHCH2CH2CH3 and NCH2CH2 CH3);
61.76 61.72 3.18 (7H, m, CH3C=N and COCH2CH2 C=N); 7.68
17.38 16.97 (2H, d, J = 8.4 Hz, C'2H, C'6H); 8.00 (2H, d, J = 8.4
202 C20H28N6O2
C
H
16c
16d
72
-
1/4 H2O
204
(388.92)
Hz, C'3H, C'5H); 8.5 (2H, m, CONHN and NHN=C)
and 11.1 (1H, br s, CONHCH2CH2).
1.03 (3H, t,
60.49 60.90 CH2CH2CH3); 3.32 (8H, m, CH3CO, COCH2CH2C=N
16.48 16.48 and NHN=C); 8.00 (2H, d, = 8.6 Hz,C'2H, C'6H);
19.58 19.07 8.48 (2H, d, = 8.6 Hz, C'3H, C'5H); 9.63 (1H, s,
J = 7.5 Hz, CH2CH3); 1.77 (6H, m, CH2-
152
-
154
C
H
N
C18H23N5O3
( 357.36)
1697
C=O ketone).
92
95
J
(
υ
J
CONHN) and 10.18 (1H, br s, NHCH2CH2).
1.68 (10H, m, 5CH2 cyclohexyl); 3.10 (7H, m, CH3CO,
COCH2CH2C=N); 4.18 (2H, m, NHN=C and NHCH
216
-
218
***
16e
C20H25N5O3
(383.39)
C
H
62.65 62.95
16.57 16.82
1697
C=O ketone).
cyclohexyl); 7.68 (2H, d,
(2H, d, = 8.0 Hz, C'3H, C'5H); 10.00 (2H, m,
CONHCH cyclohexyl and CONHN).
J = 8.0 Hz, C'2H, C'6H); 8.58
υ
J
1H-NMR (DMSO-d6,
ppm)
55.81 55.90 1.20 (6H, d, = 8.0 Hz, CH (CH3)2); 2.82 (9H, m,
16.47 15.61 CH3CO, COCH2CH2C=N, CH (CH3)2 and NHN=C);
δ
200 C17H21N5O3
-
202
C
H
N
J
1697
C=O ketone).
16f
77
84
11/4 H2O
(365.84)
(
υ
19.13 18.78 7.84 (5H, m, C6H4 and CONHN) and 11.38 (1H, d,
= 6.0 Hz, CONHCH (CH3)2).
J
2.95 (12H, m, 2CH2C6H5 CH3C=N, COCH2 CH2C=N
and NHN=C); 3.9 (4H, m, 2CH2 C6H5); 8.10 (15H, m,
C6H4, 2C6H5 and CONHN) and 9.4 (1H, br s,
CONHCH2).
194 C30H32N6O2
C
H
N
68.43 68.85
16.51 16.11
15.95 16.37
743 and 694
monosubsti-
tuted aromatic).
16h
-
1 mole H2O
(526.55)
(δ
196
*Protons of NH groups are exchangeable by D2O.
**IR spectra (KBr, cm-1) showed the following common bands: 3500-3450 (
C=N), 1223-1188 ( C-N), 1129, 1101 ( C-O) and 800-840 (
υ
NH), 1665 (
υ C=O amide), 1614, 1556 (υ
υ
υ
δ para-disubstituted aromatic)
***EI-Mass Spectrum of Compound 16e m/z (%): 54.57 (31.9), 98.06 (14.6), 116.99 (9.2), 187.01 (15.6), 195.07 (9.1), 214.01
(29.6), 383.00 (100.0) {M•+ and base peak} and 383.99 (26.1) {M•+ + 1}.
in case of compound 17h where CH3Cl:CH3OH (9:1) isopropylamine or 0.004 mole of other amines) was
was used. Reaction mixtures were cooled and the pre- added and the reaction mixtures were refluxed with
cipitates formed were filtered, washed thoroughly stirring for the appropriate time ranging from four to
with water and dried. Compounds 17c and 17e were ten days as monitored by TLC. Reaction mixtures
crystallized from ethyl acetate and compounds 17g were cooled and the precipitates formed were filtered,
and 17h were purified by column chromatography. washed thoroughly with water and dried. Compounds
Physical, micro-analytical and spectral data are listed 18d
,
18f and 18g were crystallized from ethyl acetate,
in Table IV.
chloroform and aqueous ethanol respectively. Com-
pounds 18e and 18h were purified by column chroma-
tography. Physical, micro-analytical and spectral data
are listed in Table V.
Preparation of 6-{4-[2-(1-Acetyl-1-[1-N-alkyl(aryl)
iminoethyl]methylidene)-hydrazino]-phenyl}-4,5-
dihydro-3(2H)-pyridazinones (18d-h)
Compound 12c (0.5 g, 0.002 mole) was dissolved in
absolute ethanol. Two drops of glacial acetic acid were
Positive inotropic activity
Experiments were done on the isolated hearts of
added and the reaction mixtures were refluxed for 15 rabbits. The isolated hearts were prepared for per-
min then the appropriate amine (1.18 g, 0.02 mole fusion according to Langendorff (Abdel-Rahman, 1989).

New Cardiotonic Pyridazinones
31
Table IV. Physicochemical and spectral data of compounds 17a-c, 17e-h
Microanalysis
Calc. Found
Molecular
formula
(M. wt.)
Comp. Yield M.p.
1H-NMR
IR Spectra
(KBr, cm-1)**
No.
%
oC
(DMSO-d6,
δ
ppm)*
%
%
1H-NMR (DMSO-d6,
54.54 54.28 2.75 (4H, m, COCH2CH2C=N); 3.40 (7H, m, 2NHCH3
15.49 15.37 and NHN=C); 8.30 (5H, m, C6H4 and CONHN); 10.40
(1H, m, CONHCH3) and 11.00 (1H, m, CONHCH3).
δ
ppm)
294
-
296
C15H18N6O3
(330.2922)
C
H
17a
65
1.30 (6H, t,
COCH2CH2C=N and NHN); 3.64 (4H, q,
J
= 7.5 Hz, 2 CH2CH3); 3.03 (5H, m,
= 7.5 Hz,
2CH2CH3); 7.70 (1H, s, CONHN); 7.68 (2H, d, = 8.0
Hz, C'2H, C'6H); 8.35 (2H, d, = 8.0 Hz,C'3H, C'5H);
J
272
-
274
C
H
N
56.98 56.34
16.18 16.00
23.44 23.30
C17H22N6O3
(358.34)
J
17b
17c
17e
70
95
62
J
9.43 (1H, br s, CONHCH2) and 10.76 (1H, br s,
NHCH2).
1.00 (6H, t,
J = 7.5 Hz, CH2CH2CH3); 1.80 (4H, m,
239
-
241
C
H
N
59.05 58.93 CH2CH2CH3); 3.09 (8H, m, COCH2 CH2C=N and 2
16.78 16.73 NCH2CH2CH3); 8.05 (6H, m, C6H4, NHN=C and
21.73 21.82 CONHN); 9.90 (1H, br s, CONH pr.) and 10.90 (1H,
br s, NH pr.).
1H-NMR (DMSO-d6,
1.58 (20H, m, 2(5CH2) cyclohexyl); 2.80 (2H, t,
64.35 64.33 8.0 Hz, COCH2CH2C=N); 3.23 (2H, t, = 8.0 Hz,
C19H26N6O3
(386.39)
δ ppm)
J
=
198
-
200
C25H34N6O3
(466.52)
C
H
J
17.34 17.67 COCH2CH2C=N); 4.96 (3H, m, 2CH of cyclohexyl
and NHN=C); 8.15 (5H, m, C6H4 and CONHN) and
11.52 (2H, d,
J = 6.5 Hz, 2 CONHCH cyclohexyl).
1.40 (9H, m, CH (CH3)2 and CH2CH3); 2.80 (5H, m,
COCH2CH2C=N and CH (CH3)2); 4.63 (3H, m,
170 C18H23N5O4
C
H
N
57.21 57.02
16.27 15.86
18.53 18.08
COOCH2CH3 and NHN=C); 8.00 (2H, d,
C'2H, C'6H); 8.40 (2H, d, = 8.4 Hz, C'3H, C'5H);
9.58 (1H, d, = 6.0 Hz, CONHCH (CH3)2 and 9.88
J
= 8.4 Hz,
1710
(υ C=O ester).
17f
41
-
1/4 H2O
J
172
(377.86)
J
(1H, s, CONHN).
3.00 (4H, m, COCH2CH2C=N); 4.98 (4H, d,
Hz, 2CH2C6H5); 8.29 (16H, m, C6H4; 2C6H5; NHN=C
J = 8.0
214
-
216
C
H
N
67.21 66.82
15.43 15.15
17.41 17.31
795 and 693 (δ
monosubstitute
d aromatic).
C27H26N6O3
(482.48)
17g
17h
75
67
and CONHN); 9.70 (1H, t,
J = 6.5 Hz, NHCH2) and
11.13 (1H, t, = 6.5 Hz, NHCH2).
J
C29H30N6O3
1/2 H2O
(519.53)
C
H
N
67.04 66.93 3.30 (13H, m, COCH2CH2C=N); 2CH2CH2 C6H5 and 795 and 693 (
16.02 16.32 NHN=C); 7.93 (14H, m, C6H4 and 2C6H5); 9.60 (2H, monosubstitute
δ
206 -
208
16.17 15.65 br s, 2NHCH2) and 10.10 (1H, br s, CONHN).
d aromatic).
*Protons of NH groups are exchangeable with D2O.
**IR spectra (KBr, cm-1) showed the following common bands: 3500-3450 (
υ
NH), 1665 (
υ
C=O amide), 1614, 1556 (υ
C=N), 1223-1188 (υ C-N), 1129, 1101 (υ C-O) and 800-840 (δ para-disubstituted aromatic).
Rabbits of either sex weighing (1-1.2 Kg) were sacrifi- adjusted at 38oC. The rate of flow of the perfusion
ed. The heart with at least 1cm of the aorta attached fluid was kept constant at 3 mL/minute by adjusting
was removed as quickly as possible and placed in a the pressure exerted on the fluid by means of a
dish containing Lock-Ringer solution at 37oC. The mercury manometer connected to the perfusion
heart was gently squeezed several times in the system. The aorta was then tied onto the rubber tip of
solution so as to remove as much blood as possible. the glass cannula. Care must be taken to see that air
The aorta afterwards was located and dissected free bubbles do not enter the aorta and any bubbles which
and all other vessels connected to the heart were have been formed in the cannula should be removed.
trimmed away. The aorta was cut just below the point A thread was attached to the ventricle by a hook and
where it divides and the heart was transferred to the the other end of the thread was passed over pulley
perfusion apparatus that contains a bottle filled with wheels and was attached to the displacement transdu-
perfusion fluid that is an oxygenated Lock-ringer cer connected to oscillograph (400 MD 2C Bioscience).
solution warmed to 37oC by means of water jacket The contractions of the perfused heart were then

32
E. N. Amin et al.
Table V. Physicochemical and spectral data of compounds 18d-h
Microanalysis
Calc. Found
Molecular
formula
(M. wt.)
Comp. Yield M.p.
1H-NMR
IR Spectra
No.
%
oC
(DMSO-d6,
δ
ppm)*
(KBr, cm-1)**
%
%
1.57 (7H, m, CH2CH2CH3); 3.03 (10H, m, CH3C=N,
176 C19H25N5O2
C
H
N
63.40 63.42 CH3C=O, COCH2CH2C=N); 3.97 (3H, m, NHN=C
17.14 17.25 and C=NCH2); 8.10 (2H, d, = 8.0 Hz, C'2H, C'6H);
= 8.0 Hz, C'3H, C'5H) and 9.63 (1H, s,
18d
81
-
1/4 H2O
J
178
(359.89)
19.45 18.94 8.43 (2H, d,
CONHN).
J
2.12 (20H, m, 5CH2 cyclohexyl, CH3C=N, CH3C=O
64.59 64.74 and COCH2CH2C=N); 3.91 (2H, m, NHN=C and
210 C21H27N5O2
C
H
N
18e
18f
79
70
-
1/2 H2O
17.23 16.70 =NCH cyclohexyl); 7.73 (2H, d,
J
= 8.4 Hz, C'2H,
212
(390.42)
17.93 17.78 C'6H); 8.10 (2H, d,
(1H, s, CONHN).
J = 8.4 Hz, C'3H, C'5H) and 9.18
1.28 (6H, d,
62.50 62.45 COCH2CH2C=N, NHN=C, CH3C=N and CH3C=O);
16.85 16.57 4.17 (1H, m, CH3CHCH3); 7.88 (2H, d, = 8.5 Hz,
= 8.5 Hz, C'3H, C'5H) and
J = 8.0 Hz, CH3CHCH3; 2.76 (11H, m,
186 C18H23N5O2
C
H
N
-
1/4 H2O
J
188
(345.86)
20.24 19.77 C'2H, C'6H); 8.50 (2H, d,
9.50 (1H, s, CONHN).
J
200
-
202
C
H
N
67.85 67.31 2.96 (11H, m, CH3C=N, CH3C=O, COCH2CH2C=N 731 and 691 (
15.95 15.76 and NHN=C); 5.03 (2H, s, C=NCH2C6H5); 8.14 (9H, monosubstitute
δ
C22H23N5O2
(389.40)
18g
18h
46
52
17.98 17.62 m, C6H4 and C6H5) and 9.60 (1H, s, CONHN).
3.03 (12H, m, CH3C=N, CH3C=O, COCH2CH2C=N
d aromatic).
204 C23H25N5O2
-
206
731 and 691 (δ
monosubstituted
aromatic).
and C=NCH2CH2C6H5); 4.2 (2H, t,
J = 8.0 Hz,
1/2 H2O
(412.43)
N
16.97 16.86
CH2CH2C6H5); 8.15 (9H, m, C6H4 and C6H5); 9.2 (1H,
s, NHN=C) and 9.8 (1H, s, CONHN).
*
Protons of NH groups are exchangeable by D2O.
**IR spectra (KBr, cm-1) showed the following common bands: 3500-3450 (
amide), 1614, 1556 ( C=N), 1223-1188 ( C-N), 1129, 1101 ( C-O) and 800-840 (δ
υ
NH), 1680 (
υ C=O ketone), 1665 (υ C=O
υ
υ
υ
para-disubstituted aromatic).
recorded at a constant chart speed (Abdel-Rahman, blood pressure transducer. Heparin was placed in the
1989). tip of the cannula to prevent clotting. Blood pressure
Normal contraction was recorded during a period of was recorded using an oscillograph (400 MD 2C Bio-
5 min until it was stabilized and become con- science). The transducer was calibrated and the test
sistent. Thereafter, the definite concentration (0.5, compounds were injected i.v. in the ear vein. Blood
1.5, 2.5, 5 and 10 µM) of the test compounds was then pressure was recorded before and after administration
injected in the rubber tip of the cannula and myocar- of the test compounds over a period of 30 min and the
dial contractility was recorded for a definite time in- results were given as a mean change in percent.
terval. The percentage changes in the amplitude re- Screening of the effects of the test com- pounds on
presenting the force of contraction and the percentage blood pressure was done on groups of rabbits; each of
changes in frequency of myocardial contractility repre- five animals. Test compounds were injected i.v. in the
senting the heart rate from the normal contractility rabbit’s ear veins as solutions in saline/NaCMC (0.5%
were calculated. Each experiment was done 5 times aqueous solution) mixture at doses of 0.1 and 0.3 mg/
and the results were expressed as a mean change in kg.
percent. The test compounds and digoxin were used as
solutions or suspensions in saline or saline/NaCMC
(0.5% in water) mixture.
Acute toxicity studies
Groups of adult albino mice (either sex), each of five
animals weighing (20-30 g) were used. Graded doses
starting from 10 mg/kg of each of the test compounds
Hypotensive activity
Adult healthy rabbits of either sex weighing (1.2-1.5 were injected i.p. and the toxic effects were noticed for
Kg) were anaesthetized with an intraperitoneal (i.p.) 72 h latter to injection. Both compounds 12a and 16d
injection of urethane in a dose of 1.6 g/kg (6 mL) (Hui were injected (i.p.) as solutions in a mixture of saline/
et al., 2001). Arterial blood pressure was recorded via NaCMC (0.5% in water).
the carotid artery; the latter was cannulated to Burden

New Cardiotonic Pyridazinones
33
Fig. 1. Cardiotonic activity of all test compounds in descending order according to their mean percent change in amplitude
of contractility in comparison to digoxin
Fig. 2. Platelet aggregation after ADP (PAF) addition
incubated for 5 min with different concentrations from
Screening of the effects of the test compounds on the test compounds. The reported derivatives (Sircar
platelet aggregation was done on human platelet-rich et al., 1989) were used as 300-0.001 M. So, at first,
plasma that were prepared from whole blood, freshly compound 12a was used in 0.6 M/20 L and 1.5 M/
drawn from healthy volunteers who had not ingested 50 L respectively, then compound 16d was tested
aspirin or other non steroidal anti-inflammatory using smaller concentrations 0.1 M/50 L and 0.04
drugs within the preceding two weeks and who were M/20 L respectively. After the addition of 2.5
Platelet aggregation inhibition activity
µ
µ
µ
µ
µ
µ
µ
µ
µ
µM
fasting for 9 h before blood draw (Sircar et al., 1989) adenosine diphosphate (ADP) used as platelet activat-
Aggregation was measured in a 4-channel PAP4 ing factor (PAF), aggregation was determined, before
Biodata aggregometer. Aliquots, at a final of 450
and 480 L platelets were equilibrated to 37ºC before change in absorbance monitored for 4 min (Murray et
being placed in the sample chamber. They were then al., 1992). Experiments were repeated on three samples
µ
L
and after addition of the test compounds, by the
µ

34
E. N. Amin et al.
Fig. 3. Inhibition of platelet aggregation after addition of 0.6 µM/20 µL from compound 12a
Fig. 4. Addition of 0.04 µM/20 µL from compound 16d showing 5 & 10% platelet aggregation for two volunteer’s samples
from three volunteers for each compound.
Sherbooke St. West, Suite 910, Montreal, Quebec,
H3A 2R7, Canada. The program operated under
“Microsoft Windows XP” operating system installed on
an Intel Pentium IV PC with a 2.8 GHz processor and
Pharmacophore building and pharmacophore
identifications
All the computational works were carried out at the 512 Mb of RAM. Two Pharmacophore models were
Department of Medicinal Chemistry, Faculty of Phar- constructed, one for the cardiotonic activity and the
macy, Assiut University, Assiut, Egypt. Receptor other for the hypotensive one. Construction of the
building and pharmacophore identification were per- models was based on the reported active compounds
formed on Molecular Operating Enviornment (MOE) (Thyes et al., 1983; Okushiima et al., 1987). The aim
version 2007.09, Chemical Computing Group Inc. 1010 of this approach is to gain useful insights into ligand

New Cardiotonic Pyridazinones
35
receptor interactions, to identify pharmacophoric build up the hypothetical model using molecular
structural features of the active ligands, and to use builder of MOE program. Each structure was sub-
this model for searching molecular databases in order jected to energy minimization up to gradient of 0.01
to find new structural categories, a process known as kcal/mol Ao using MMffyn force field. The training set
virtual screening.
Eight reported active ligands compounds 10 and ment. Alignment had the lowest strain energy, U, and
19a-g (Fig. 5) in addition to pimobendan were the highest S value was selected to build the phar-
molecules were aligned using MOE’s flexible align-
3
selected as the training set for cardiotonic activity, to macophore query.
Fig. 5. Training set used in building the cardiotonic hypothetical model, compounds 10 and 19a-g

36
E. N. Amin et al.
A pharmacophore query was created with the them in Ao are shown in Fig. 6. Under this scheme,
pharmacophore query editor of MOE, using PPCH-All ML denotes metal ligator. Hyd S denotes non planar
(Planar-Polarity-Charge-Hydrophobicity) scheme. The hydrophobic region (sp3). Hyd P denotes planar
pharmacophore query was composed of five common hydrophobic region (sp2). Acc P denotes planar H-
chemical features (F1-F5) that would be considered in bond acceptor (sp2). Don P denotes planar H-bond
the generation of the hypothetical model within the donor (sp2).
training set.
The performance of the obtained hypothetical model
The pharmacophoric features and distances between was evaluated by fitting of all the target compounds
Fig. 6. Cardiotonic pharmacophore features and distances
Fig. 7. Mapping of compound 13b onto the cardiotonic pharmacophore

New Cardiotonic Pyridazinones
37
12a-d; 13a-c; 14a, b; 15a; 16a, c-f, h; 17a-c, e-h and the hypotensive activity using ten reported com-
18d-h. Fitting operations to the hypothetical model pounds (Thyes et al., 1983) as the training set 20a-i
were accomplished through pharmacophore output Fig. 8. The same procedures used for building the car-
contains RMSD, i.e., the root mean square distance diotonic activity pharmacophore model was followed.
between the query features and their corresponding The pharmacophore query composed of five common
ligand target points. The smaller the RMSD, the chemical features and the pharmacophore search was
better fitting the query compound has.
done for the synthesized target compounds.
Another pharmacophoric model was constructed for
Fig. 8. Training set used in building the hypotensive hypothetical model, compounds 10 and 20a-i

38
E. N. Amin et al.
ed product 14a, and reduction of the azo bond took
place to give the starting compound 10 as has been
identified by 1H-NMR.
RESULTS AND DISCUSSION
Chemistry
The key intermediates 12a-d were prepared by
Also, aminopyrazolinonopyridazinone 15a was pre-
coupling the diazonium chloride of compound 10 with pared by cyclization of compound 12d with hydrazine
the different active methylene containing compounds hydrate in absolute ethanol by reflux for 24 h (Chart
11a-d in presence of sodium acetate (Chart 2). The 3a).
formed intermediates 12a-d are reported compounds
(Haikala et al., 1990) but their physical and spectral aralkyl amines (Chart 3b) afforded the target amide/
data are not available so their structures have been imine derivatives 16a, c and only on using methyl-,
established by IR, 1H-NMR, and mass spectrometry in n-propyl-, and phenethyl-amines, respectively. How-
addition to elemental analysis. ever, on using other bulky or branched amines such as
Moreover, reaction of compound 12a with alkyl or
h
The target compounds 13a-c were prepared by n-butyl-, cyclohexyl- or isopropyl- amines, aminolysis
cyclization of compound 12a with hydrazine hydrate, of the ester group of 12a only occurred leaving the keto
phenylhydrazine hydrochloride or 2,4-dinitrophenyl- group of the acetyl moiety intact to afford compounds
hydrazine by reflux in absolute ethanol for about 26 h 16d
(Chart 3a). attributed to the steric hindrance induced by such
On the other hand, pyrazolopyridazinones 14a and bulky or branched amines at the site of the reaction.
were prepared by cyclization of compound 12c with Also, reaction of the di-ester intermediate 12b with
, 16e and 16f respectively. This finding may be
b
either hydrazine hydrate (24%) or phenylhydrazine alkyl- or aralkyl amines (Chart 3b) in DMF or ethanol
hydrochloride in absolute ethanol by refluxing for 24 h afforded the target di-amides 17a-c, e, g, h except
or 10 h, respectively (Chart 3a). Trials to cyclise 12c compound 17f where only one molecule of isopro-
with 98% hydrazine hydrate, didn’t afford the requir- pylamine was involved in the aminolysis of only one
Chart 2. Synthetic pathway for the preparation of compounds 10 and 12a-d

New Cardiotonic Pyridazinones
39
Chart 3a. Synthetic pathway for the preparation of compounds 13a-c; 14a, b; 15a
ester group of 12b due to branching. Decoupling of the and only one molecule of the used amines attack only
benzyl methylene of compound 17g was done in order one ketonic group of 12c as confirmed by analytical
to notice the change in the nearby NH- splitting and it techniques.
showed change in the splitting from triplet to singlet
Several attempts were carried out to form imines
at (9.70 ppm) and to hump at (11.13) ppm. The two from 12c with methyl-, ethyl-, and n-propyl- amines
methylene groups of the benzyl moieties are not but they were unsuccessful. This might be attributed
equivalent thus the nearby NH groups differ in their to the aqueous nature of the first two amines and the
appearance. This is confirmed from the pharma- volatilization of the n-propyl one (B.P. 48oC). All the
cophoric studies where one of the two amidic chains is obtained compounds are crystalline solids and their
1
nearby the (C6H5NHN), thus forms an intramolecular structures were confirmed by IR, H-NMR and mass
hydrogen bond and consequently appears more down- spectrometry along with microanalysis.
field than the other.
Reaction of the diketopyridazinone derivative 12c
Pharmacological Screening
with amines in absolute ethanol under reflux (Chart
Positive Inotropic Effect: Twenty four new com-
3b) yielded the corresponding iminoketones 18d-h pounds 13a-c; 14a, b; 15a; 16a, c-f & h; 17a-c, e-h

40
E. N. Amin et al.
Chart 3b. Synthetic pathway for the preparation of compounds 16a, c-f, h; 17a-c, e-h; 18d-h
and 18d-h in addition to the five reported inter-
mediates compounds 10 and 12a-d were evaluated for of compound 12a was the best of the tested inter-
their positive inotropic effect (Abdel-Rahman, 1989) in mediates, since it exceeds that of both digoxin and 10
comparison to digoxin1). It was found that the first This finding indicates that presence of an acetyl and
effective dose was 5 M, thus was used thereafter. ester moieties at the methylidene carbon atom linked
Results are listed in Fig. 1 and Tables VI-IX. to the hydrazine substituent at the para position of
It is noteworthy to mention that the inotropic effect
.
µ
Study of results listed in Tables VI-IX reveals that the 6-phenyl seemed to be crucial for the inotropic
the inotropic activity of the tested intermediates 12a- effect of this class of pyridazinones.
d
surpassed that of digoxin and more or less similar to
It is also of interest to mention that cyclization of
the intermediate 12a into pyrazolinone derivatives
13a-c (Table VIII) resulted in either elevation 13b or
reduction 13a and 13c of the activity. Superiority of
the inotropic activity of 13b than its congeners 13a
and 13c could be explained by the presence of a
that of the reported compound 10
.
1) Pimobendan, Imazodan or any phosphodiesterase acting
drugs are not available in the middle-east area to be used as
positive inotropic reference thus digoxin was used instead in
comparison.

New Cardiotonic Pyridazinones
41
Table VII. Cardiotonic activity of compounds 16a, c-f, h;
17a-c, e-h; 18d-h and digoxin using 5 µM concentration
Table VI. Cardiotonic activity of compounds 10; 12a-d;
13a-c; 14a, b; 15a and digoxin at 5
µM concentration
Mean
change in
frequency/
(min)
% mean
change in change in
amplitude frequency/
Mean
Mean change
% mean
% mean
change in
frequency
Mean change
% mean
change in
frequency
Comp.
No.
in amplitude change in
No.
in amplitude
(cm) a
amplitude
(cm) a
(cm)
(min)
16a 3.14 ± 0.095**
16c 3.10 ± 0.025**
16d 3.12 ± 0.025**
16e 3.34 ± 0.020**
31.93
30.25
31.09
40.34
27.73
26.05
16.80
26.05
30.25
35.29
15.12
55.46
26.89
27.73
36.97
30.25
31.09
47.05
31.93
34.8 ± 0.96 -14.47
27.4 ± 0.75 1-9.87b
33.4 ± 0.75 -09.87
39.8 ± 0.96 -30.92
31.6 ± 0.59 -03.95
34.8 ± 0.96 14.47
30.4 ± 0.75 -00
Normal 2.38 ± 0
10 3.32 ± 0.057**
12a 3.62 ± 0.100*** 52.10
···
39.49
30.4
···
25.0 ± 0.541 -17.76b
35.4 ± 0.751 -16.45
33.4 ± 0.751 -09.87
36.2 ± 0.921 -19.07
25.0 ± 0.541 -17.76
35.8 ± 0.961 -17.76
36.8 ± 0.961 -21.05
37.2 ± 0.921 -22.37
35.8 ± 0.961 -17.76
33.4 ± 0.751 -09.87
16.2 ± 0.375 -46.71b
12b 3.24 ± 0.057**
12c 3.28 ± 0.112**
12d 3.30 ± 0.185**
13a 3.26 ± 0.150**
13c 3.14 ± 0.003**
14a 3.14 ± 0.043**
14b 3.12 ± 0.049**
15a 2.80 ± 0.144*
Digoxin 3.14 ± 0.011**
36.13
37.81
38.65
36.97
31.93
31.93
31.09
17.64
31.93
16f
3.04 ± 0.134*
16h 3.00 ± 0.0190**
17a 2.78 ± 0.057*
17b 3.00 ± 0.055**
17c 3.10 ± 0.191*
17e 3.22 ± 0.104**
26.0 ± 0.54 -14.47b
30.4 ± 0.75 -00
31.8 ± 0.96 -04.61
26.0 ± 0.54 -14.47b
32.4 ± 0.75 -06.57
35.8 ± 0.96 -17.76
36.2 ± 2.05 -19.07
38.2 ± 2.05 -25.65
32.4 ± 0.75 -06.57
39.8 ± 0.96 -30.92
35.8 ± 0.96 -17.76
16.2 ± 0.38 b-46.71b
17f
2.74 ± 0.023*
17g 3.70 ± 0.095***
17h 3.02 ± 0.120*
18d 3.04 ± 0.091**
18e 3.26 ± 0.07**
aEach result represents the mean of five observations ±
S.E.M.
b(-) Negative chronotropic effect (i.e. decrease in heart rate).
*
Significant at
compared to control.
**Significant at 0.01 (degree of 99% confidence limits)
compared to control.
p ≤ 0.05 (degree of 95% confidence limits)
18f
3.10 ± 0.028**
18g 3.12 ± 0.085**
18h 3.50 ± 0.185**
Digoxin 3.14 ± 0.011**
p
≤
***Significant at
p ≤ 0.005 (degree of 99.5% confidence
limits) compared to control.
aEach result represents the mean of five observations ±
S.E.
b(-) Negative chronotropic effect (i.e. decrease in heart rate).
phenyl substituent at N1 of the pyrazolinone ring
rather than hydrogen 13a which might be attributed
to the best fitting of 13b with the appropriate recep-
tors. Further increase in bulkiness led to steric hin-
drance of the resulted N1-(2,4-dinitrophenyl)pyrazo-
linone derivative 13c at the receptors and hence bad
fitting and decrease of the observed inotropic effect.
*
Significant at
compared to control.
**Significant at 0.01 (degree of 99% confidence limits)
compared to control.
***Significant at 0.005 (degree of 99.5% confidence
limits) compared to control.
p ≤ 0.05 (degree of 95% confidence limits)
p
≤
p
≤
On the other hand, either amide formation 16d, 16e of bulkiness of the alkyl moieties on the amide
and 16f (Table III) or amide and imine formation 16a, nitrogen atoms with a noticeable drop of activity of
16c and 16h (Table VIII) of the intermediate 12a the isopropyl containing amide 17f than its analog
resulted in marked decrease of activity in comparison 17c, although the former is a monosubstituted amide.
to the parent 12a
.
This observation may be attributed to the steric
Also, results of Table VIII show that the activity of hindrance which might be produced at the receptor
the intermediate 12b is 1.13 times as active as digoxin due to branching of the propyl moiety 17f and in turn
which is almost comparable to that of the reported bad fitting with the receptor.
compound 10. Compound 12b contains di-ester moieties
It is worth noting to discuss the activity of com-
at the methylidene carbon atom linked to the pounds 17g and 17h in comparison to 12b. It is clear
hydrazino group at the p-position of the phenyl ring at that substitution of the methyl group of the ethyl
position 6 of the pyridazinone ring system. Substi- moiety at the amide nitrogen by a phenyl one 17g
tution of the di-ester moieties by di-amide ones 17a, augmented the activity to more than the double, while
17b, 17c, 17e, 17g and 17h (Table VIII) resulted in substitution of such methyl by a benzyl one 17h
diminishing of activity compared to the parent retains the same activity of 12b. Also, it is of interest
compound 12b except compound 17g which exhibited to mention that presence of benzyl substituents at the
1.72 fold as active as digoxin.
amide nitrogens 17g rather than phenethyl ones 17h
Study of the observed activities of the amide series might lead to better fitting on the receptor of the
17a–h reveals that activity increases by the increase former than the latter, which indicates the importance

42
E. N. Amin et al.
Table IX. Rmsd values of the synthesized compounds and
their positive inotropic activity
Table VIII. % mean changes in amplitude of contractility
of isolated cardiac muscles of all test compounds in
comparison to digoxin and 10 at 5
µ
M concentration
Comp.
No.
% increase in contractility
of cardiac muscle
RMSD
value *
% mean change in Activity in folds in
Comp.
No.
amplitude of
contractility
comparison to
digoxin
12a
12b
12d
13a
13b
16e
17g
18h
52
36
39
37
59
40
56
47
0.1992
0.2818
0.2730
0.2349
0.2144
0.2855
0.2643
0.2589
Digoxin
10
31.93
39.39
52.00
36.00
38.00
39.00
37.00
31.93
31.09
17.64
31.93
30.25
31.09
40.00
27.73
26.05
16.80
26.05
30.25
35.00
15.12
55.00
26.89
27.73
37.00
30.25
31.09
47.00
···
1.23
1.63
1.13
1.20
1.22
1.16
1.00
0.97
0.55
1.00
0.95
0.97
1.25
0.87
0.82
0.53
0.82
0.95
1.10
0.47
1.72
0.84
0.87
1.16
0.95
0.97
1.47
12a
12b
12c
12d
13a
14a
14b
15a
16a
16c
16d
16e
16f
16h
17a
17b
17c
17e
17f
*
Rmsd values of the most aligned eight compounds are listed.
In addition, cyclization of the diketo intermediate
12c into pyrazoles 14a and 14b didn’t show a marked
decrease of activity than the parent 12c.
Also, inspection of results listed in Table VIII indi-
cates that intermediate 12d which embody a cyano
group and an ester moiety at the methylidene carbon,
exhibited a comparable inotropic effect as 10 and
slightly exceeds that of digoxin. Cyclization of 12d
into pyrazolinone 15a resulted in 50% decrease of
activity.
Hypotensive activity
Thirteen selected new pyridazinones 13a-c; 16a, c-f,
h; 17f, g, h; and 18d in addition to the intermediates
10 and 12a-d were tested for their effect on blood
pressure of normotensive rabbits.
It was noticed that there are fluctuations of the
effect of the test compounds on blood pressure as
there are two competing forces, the cardiotonic and
the vasodilator effects, and the net result was due to
the stronger effect. Results are listed in Tables X-XII.
Study of results listed in Tables X and XI indicates
17g
17h
18d
18e
18f
18g
18h
of the presence of a phenyl group at just a distance of
one methylene group rather than two from the NH of that the maximum decrease in blood pressure induced
the amide moiety. This finding was verified by the by this series of compounds appears at 15 min after
obtained data of the corresponding rmsd values of i.v. injection. It was, also, noticed that the decrease of
these compounds (Table IX).
BP of compounds 16d and 17g remains up to 30 min,
Results cited in Table VIII show that the intermedi- while the effect of most of the other compounds
ate 12c is 1.2 times as active as digoxin and also showed a significant drop at 30 min. On the other
comparable to 10. Transformation of 12c into keto hand, compounds 12b, 16a, 16e, 16f and 16h showed
imines resulted in final compounds 18d–h which a noticeable increase in BP at 30 min interval from
revealed inotropic effect ranging from 0.95 to 1.47 as injection, the latter observation may be explained by
active as digoxin. These results indicate that increase the dominance of their inotropic effect.
of bulkiness of the substituent at the imine nitrogen
Also, it is especially worth noting that the hypoten-
from alkyl to cyclohexyl or phenethyl exerted a sive effect of compounds 16c, 16d, 16e and 17g at 15
prompt effect on the activity of these compounds (c.f. min in comparison to the reported compound 10
18e and 18h). Superiority of cardiotonic activity of ranges from 1.07 to 2.38 as active as 10
compound 18h over 18g, also, was in a good agree- These data also show that an acetyl and an ester
ment with the determined rmsd values of these substituents at the methylidene carbon linked to the
compounds (Table IX). hydrazino moiety at the para position of the phenyl
.

New Cardiotonic Pyridazinones
43
Table X. % mean changes in BP of test compounds in comparison to 10
% mean change in BP at 0.1 mg/kg dosea
Comp. No.
Time (min)
D'
5'
15'
30'
10
0-8.00 ± 0.080**
0-5.57 ± 0.051*
-01.66 ± 0.090
-03.00 ± 0.094
0-2.00 ± 0.100*
0-3.50 ± 0.060**
0-2.00 ± 0.100*
0-9.00 ± 0.090***
0-3.60 ± 0.036**
-02.55 ± 0.069*
-03.66 ± 0.042**
-08.91 ± 0.058*
-02.30 ± 0.050
0-4.00 ± 0.037***
0-3.00 ± 0.088
-04.04 ± 0.051*
0-5.00 ± 0.090*
0-1.50 ± 0.040
0-9.00 ± 0.070***
0-1.52 ± 0.084*
-04.03 ± 0.050*
-09.00 ± 0.080**
0-4.20 ± 0.040
0-4.00 ± 0.040**
0-3.00 ± 0.060
-11.00 ± 0.090***
-01.00 ± 0.050
-13.77 ± 0.020***
-16.09 ± 0.077**
-06.05 ± 0.038*
0-3.80 ± 0.100
-01.20 ± 0.050
0-4.00 ± 0.084
-04.04 ± 0.059*
-02.00 ± 0.070*
-03.40 ± 0.140
-13.00 ± 0.070
0-9.96 ± 0.040*
-02.00 ± 0.04**
no change
0-4.00 ± 0.070
no change
-28.00 ± 0.070
0-4.79 ± 0.038*
-05.00 ± 0.040
no change
-00
no change
12a
12b
12c
12d
13a
13b
13c
16a
16c
16d
16e
16f
16h
17f
17g
17h
18d
coagulation
coagulation
coagulation
coagulation
-04.00 ± 0.037***
-14.54 ± 0***
-31.46 ± 0.055***
-14.85 ± 0.052**
-02.00 ± 0.096
-02.00 ± 0.050
-03.00 ± 0.084
-13.93 ± 0.034*
-03.00 ± 0.060*
0-5.00 ± 0.150
-04.00 ± 0.037***
0-2.55 ± 0.029*
-22.19 ± 0.02***
-07.88 ± 0.064*
-02.00 ± 0.096
-02.00 ± 0.048
0-7.00 ± 0.074**
-20.09 ± 0.06**
0-7.00 ± 0.06***
0-8.00 ± 0.11**
aEach result represents the mean of five observations ± S.E.
D: Direct after injection.
*
Significant at
p
p
p
≤
≤
≤
0.05(degree of 95% confidence limits).
0.01(degree of 99% confidence limits).
0.005(degree of 99.5% confidence limits).
**Significant at
***Significant at
N.B.: (-) negative sign means decrease in blood pressure.
group induced the highest hypotensive effect (c.f. 12a,
Table XI. % mean changes in BP after injection of all test
compounds at 15 min and 30 min in comparison to 10 at
0.1 mg/kg
12b, 12c and 12d)
.
Moreover, comparison of the activity of the inter-
mediates 12a and 12b with their corresponding amides
(final compounds) 16c–16e and 17g respectively show
that amide formation improves the hypotensive
activity of this series of pyridazinones.
% mean change in BP
Comp. No.
15 min
30 min
10
-13
-10
-12
-28
1-5
-15
12a
12b
12c
12d
13a
13b
13c
16a
16c
16d
16e
16f
The results obtained comply with the pharma-
cophoric studies rmsd values (Table XII).
no change
1-4
no change
coagulation
coagulation
-14
no change
-10
no change
coagulation
coagulation
-14
Platelet aggregation inhibition
Since compounds 12a and 16d are the most signifi-
cant cardiotonic and hypotensive, respectively. There-
fore, these two compounds were, also, tested as platelet
aggregation inhibitors at different concentrations. The
results observed for compound 12a were complete
inhibition to the previously aggregated platelets in
both cases nevertheless how much the percent of
aggregation was (i.e. whether the percent was 68, 74
or 80% aggregation) for different volunteers. The
normal percent of aggregation after addition of PAF
should be more than 50%.
-15
-31
-15
-12
1-3
-22
-18
-12
16h
17f
-12
-13
-12
1-7
17g
17h
18d
-14
-14
1-5
-20
1-3
1-8
On the other hand, results for compound 16d re-
vealed complete inhibition with the higher concen-
tration and possessed only 5 and 10% aggregation
N.B.: the negative sign means decrease in blood pressure.

44
E. N. Amin et al.
Table XII. Rmsd values of the most aligned synthesized
compounds onto the hypotensive pharmacophore
Pharmacophore building and pharmacophore
identifications
Study of the results (Table IX) revealed that 12a
was the most active compound according to its rmsd
and this also was confirmed from the percent increase
in contractility that decreased by increasing the rmsd
value.
Comparing the cyclized derivatives 13a and 13b, it
is clear that compound 13b was more active than 13a
and this also was in accordance with their rmsd
values. Also, comparing the amide derivative 16e with
Comp. no.
RMSD value
12a
12d
16c
16e
17g
18g
0.4651
0.4477
0.4686
0.3879
0.4262
0.4114
with the smaller one, which is also considered as a its parent 12a, the parent was more active due to the
neglected percent. Test was done as described in the smaller rmsd value that makes better fitting to the
experimental protocol using ADP as platelet acti- hypothetical model. Similarly, compounds 17g and
vating factor PAF on platelet rich plasma (Murray et 18h showed better fitting than their parent 12b and
al., 1992) of human volunteers. It is worth noting that 12c respectively.
increasing concentration of PAF from 2.5
showed no effect on platelet aggregation inhibition model is shown in Fig. 7. It can be seen that all the
percent observed with the test compounds. chemical features of the hypothesis are matched by
Results are shown in Fig. 2-4 which indicate com- the chemical groups of the molecule: F1: -phenyl
plete inhibition of platelet aggregation at all concen- substituted, CH2-CH2 of the pyridazinone ring and
µM to 5
µM
Mapping of compound 13b onto the pharmacophore
p
trations.
C6H5-NHN=C; F2: NH of the pyridazinone ring; F3:
C=NNH of the pyridazinone ring; F4: oxygen of the
carbonyl group of the pyridazinone ring; and F5:
Toxicity studies
No signs of toxicity were noticed up to the dose level carbonyl in the pyrazolinone ring.
300 mg/kg concentration for both test compounds,
As for the hypotensive pharmacophore, it was inter-
while the reported oral LD50 of digoxin is 17.78 mg/kg esting to find that almost all compounds showed well
(Roche, through Internet).
fitting onto the pharmacophore hypothesis with RMSD
values range between (0.3879-0.4686). Mapping of
compound 17g onto the pharmacophore model is
Fig. 9. Mapping of compound 17g onto the hypotensive pharmacophore

New Cardiotonic Pyridazinones
45
failure. Eur. Pat. Appl. 383449, (1990), Through Chem.
Abst. 114, 228967, 1991 and through internet.
shown in Fig. 9. Also, here it was noticed that all the
chemical functionalities of the hypothesis are matched
by the chemical groups of the synthesized compounds
Hansen, O., Interaction of cardiac glycosides with (Na+ + K+)
Activated ATPase, A Biochemical link to Digitalis-In-
duced Inotropy. Pharmacol. Rev., 36, 143-160 (1984).
Hui, X., Fink, G. D., Chen, A., Watts, S., and Galligan, J. J.,
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and NHN of the pyridazinone ring; F2: oxygen of the
carbonyl group of the pyridazinone ring; F3: COCH2 of
the pyridazinone ring; F4: carbonyl group of the ester
moiety; and F5: N=CCH2 of the pyridazinone ring.
ACKNOWLEDGEMENTS
Mertens, A., Friebe, W., Müller-Beckmann, B., Kampe, W.,
Kling, L., and Von der saal, W., Nonsteroidal Cardioto-
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Stutchbury, C. A., Patel, B., Knowles, A., Worby, A., Lynham,
J. A., and Coates, W. J., The effect of SK and F 95654, a
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The authors are greatly indebted to Prof. Dr. Nabila
M. Thabet and Dr. Ola A. Halim Afifi, Department of
Clinical Pathology, Assiut University Hospital, for
their true and sincere help in the determination and
supervision of the platelet aggregation inhibition
studies. Our cordial thanks and gratitude to the De-
partment of Medicinal Chemistry, Faculty of Phar-
macy, Assiut University for permitting to carry out
the pharmacophore studies that assess a lot in the
success of this work.
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