Synthesis and pharmacological evaluation of 6-arylpyridazinones as potent vasorelaxants

Article abstract of DOI:10.1002/ddr.21079

Preclinical Research As part of a research program to identify compounds with potent antihypertensive properties, a new series of dihydropyridazin-3(2H)- one analogs was synthesized and evaluated for vasodilator activity in rat thoracic aortic rings. Most

Full text of DOI:10.1002/ddr.21079

DRUG DEVELOPMENT RESEARCH 74 : 296–305 (2013)
Research Article
Synthesis and Pharmacological Evaluation of
6-Arylpyridazinones as Potent Vasorelaxants
Ranju Bansal,1* Dinesh Kumar,1 Deepika Sharma,1 Carmen Calle,2 and Rosalia Carron2
1University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India
2Department of Physiology and Pharmacology, Faculty of Pharmacy, Campus “Miguel de
Unamuno”, University of Salamanca, E-37007 Salamanca, Spain
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT As part of a research program to identify compounds with potent antihypertensive pro-
perties, a new series of dihydropyridazin-3(2H)-one analogs was synthesized and evaluated for vasodi-
lator activity in rat thoracic aortic rings. Most of the newly synthesized compounds displayed good
vasorelaxant activity as compared with SK&F-93741 and hydralazine, with the N2-unsubstituted 4-
isobutyramidophenylpyridazinone derivative 5 being the most potent vasorelaxant, producing vasorelax-
ation greater than that of hydralazine and equipotent to SK&F-93741. A significant effect of the 6-phenyl
substituents and substitution at N-2 position of pyridazinone nucleus on the vasodilatory activity was
observed. Drug Dev Res 74 : 296–305, 2013.
© 2013 Wiley Periodicals, Inc.
Key words: vasodilators; dihydropyridazin-3(2H)-one; hypertension
INTRODUCTION
development of novel pyridazinone-based molecules as
potent vasorelaxants.
Significant efforts have been made to obtain novel
antihypertensive agents acting by diverse mechanisms
to control blood pressure [Demirayak et al., 2004a],
with a wide variety of heterocyclic systems having been
explored [Khalil et al., 1980]. The pyridazinones have
been investigated in recent decades for biological,
medicinal, and agricultural reasons. Pyridazinone
derivatives exhibit diverse pharmacological properties
[Bansal and Thota, 2012]—for example, antidepressant,
antihypertensive, antithrombotic, anticonvulsant, car-
diotonic, antibacterial, and anticancer actions [Rubat
et al., 1990; Matyus, 1998; Coelho et al., 2003; Siddiqui
and Wani, 2004; Thota and Bansal, 2010]. Based on
earlier studies and recent work, it has been observed
that various pyridazinone derivatives possess antihyper-
tensive activity due to their vasorelaxant activity
[Demirayak et al., 2004b; Kumar et al., 2008; Bansal
et al., 2009; Costas et al., 2010; Asif et al., 2012]. In this
context, our research has focused on the discovery and
Because the 6-arylpyridazinone structure is essen-
tial for activity in the cardiovascular system, a number
of studies with regard to substitutions on both pyridazi-
none and aryl residues have been performed [Curran
and Ross, 1974; Lee et al., 2004] that indicate that
amino, acylamino, cyano, and halogen substituents on
the phenyl ring produce interesting antihypertensive
activity. In an effort to identify novel compounds with
Funding/support information: University Grant Commis-
sion and Council for Scientific and Industrial Research, India.
The authors declare no conflict of interest.
*Correspondence to: Ranju Bansal, University Institute of
Pharmaceutical Sciences, Panjab University, Chandigarh-160014,
India.
E-mail: ranju29in@yahoo.co.in
Received 17 January 2013; Accepted 20 February 2013
Published online in Wiley Online Library (wileyonlinelibrary.
com). DOI: 10.1002/ddr.21079
© 2013 Wiley Periodicals, Inc.

VASORELAXANT ACTIVITY OF NEWER 6-ARYLPYRIDAZINONES
297
carried out on a Perkin–Elmer 2400 CHN analyzer.
Thin-layer chromatography (TLC) plates were pre-
pared with silica gel G (E. Merck, India) according to
Stahl’s method using EtOAc as solvent (activated at
110°C for 30 min) and visualized by exposure to iodine
vapors. Anhydrous sodium sulfate was used as drying
agent. All solvents were dried and freshly distilled prior
to use according to standard procedures.
Synthesis
Procedure for synthesis of N-phenyl
isobutyramide (1)
Isobutyryl chloride (2.5 mL, 23.86 mmol) was
added to a stirred solution of aniline (2.0 mL,
21.95 mmol) in dichloromethane (30 mL). The reaction
mixture was stirred at room temperature for 1 h under
anhydrous conditions. TLC was used to monitor the
completion of the reaction. The solvent was removed
under reduced pressure to obtain a solid residue. Ice-
cold water was added to it, and the obtained precipitate
was filtered off, washed with ice-cold water, dried, and
recrystallized from a mixture of acetone and petroleum
ether (60–80°C).
Yield: 44.66%. m.p.: 108–109°C. IR (KBr), u,
per cm: 3299, 2970, 1662, 1600, 1546, 1441, 1099, 758.
1H NMR (400 MHz, CDCl3 + DMSO-d6, TMS): d
9.70 ppm (s, 1H, -NHCOCH(CH3)2, disappeared on
deuterium exchange), 7.53 (d, 2H, aromatic protons
ortho to isobutyramide, Jo = 7.83 Hz), 7.31 (m, 2H,
aromatic protons meta to isobutyramide), 7.09 (t, 1H,
aromatic proton para to isobutyramide, Jo = 7.41 Hz),
2.51 (m, 1H, -CH(CH3)2), 1.25 (d, 6H, -CH(CH3)2,
J = 6.87 Hz).
Fig. 1. Structures of pyridazinone-based vasorelaxants.
vasodilatory and antihypertensive activities, our group
initially focused on the modestly selective structure
of SK&F-93741 (Fig. 1), a dihydropyridazinone deriva-
tive possessing vasodilatory properties [Curran and
Ross, 1974]. We have also reported the vasorelaxant
effect of 6-(4-acetamidophenyl)-2-substituted-4,5-
dihydropyridazin-3(2H)-ones [Kumar et al., 2008]. The
present study extends the exploration of the vasorelax-
ant activity of dihydropyridazinones by synthesizing a
series of derivatives with increased chain length at the
acetamido group of phenyl ring.
Literature reports have also revealed that cyano
group-containing pyridazinones exhibit appreciable
cardiotonic activity with dual inotropic and vasodilatory
properties, as exemplified by levosimendan (Fig. 1), a
drug used for the treatment of congestive heart failure
[Montes et al., 2006]. In view of this and in continuation
of our efforts toward synthesis of pyridazinone-based
vasorelaxants, several newer derivatives possessing a
cyano moiety were also synthesized. As position 2 had
remained relatively unexplored in the context of vasore-
laxant effects, several 2-substituted pyridazinone
derivatives were also prepared.
General procedure for synthesis of N-phenyl
isobutyramide-substituted g-keto acids (2–4)
A mixture of 1 (2.0 g, 12.25 mmol) and the requi-
site anhydride, succinic anhydride (1.4 g, 14.00 mmol)/
methylsuccinic anhydride (1.4 g, 12.27 mmol), was
added to a stirred solution of aluminum chloride (8 g) in
purified carbon disulfide (20 ml). The reaction mixture
was stirred manually under anhydrous conditions for
20 min and allowed to stand at room temperature for
MATERIALS AND METHODS
The m.p. reported is uncorrected; 1H NMR 48 h. The mixture was decomposed with crushed ice.
spectra were recorded on a 300 MHz Brucker AC-300F The solid product obtained was filtered and washed
instrument (Bruker AG, Fallanden, Switzerland) using thoroughly with distilled water. The residual solid was
Me4Si (TMS) as the internal standard (chemical shifts dissolved in 5% aqueous sodium bicarbonate solution
in d, ppm). IR spectra were recorded on a Perkin– and the insoluble part was filtered off. Acidification
Elmer 882 and Perkin–Elmer Spectrum RX 1 FT-IR with concentrated HCl resulted in a precipitate, which
spectrophotometers (Perkin-Elmer Ltd., Beaconsfield, was filtered, washed with ice-cold water, dried, and
Buckinghamshire, UK) using potassium bromide recrystallized from methanol to afford the correspond-
pellets (umax in per cm). Elemental analyses were ing g-keto acids, 2 and 3. Compound 2 (0.5 g, 1.9 mmol)
Drug Dev. Res.

298
BANSAL ET AL.
was added in small portions to the stirred ice-cold 6-(4-Isobutyramidophenyl)-4,5-dihydropyridazin-
mixture of concentrated HNO3–H2SO4 (3:1, 8 mL). 3(2H)-one (5)
The reaction mixture was further stirred in ice for
Recrystallization: methanol. Yield: 57.88%. m.p.:
30–45 min and then at room temperature for 15 min to 260–262°C. IR (KBr), u, per cm: 3309, 2971, 1672, 1615,
1
obtain a yellow, sticky product, 4-(4-isobutyramido-3- 1590, 1515, 1346, 843. H NMR (400 MHz, CDCl3 +
nitrophenyl)-4-oxobutyric acid (4), which was used in DMSO-d6, TMS): d 9.80 ppm (s, 1H, -NH, pyridazi-
further reactions.
none, disappeared on deuterium exchange), 9.18 (s,
1H, -NHCOCH(CH3)2, disappeared on deuterium
exchange), 7.68 (m, 4H, aromatic), 2.96 (t, 2H, 5-CH2,
J = 8.57 Hz), 2.60 (m, 3H, 4-CH2 and -NHCOCH
(CH3)2), 1.22 (d, 6H, -NHCOCH(CH3)2, J = 7.06 Hz).
Analysis calculated for C14H17N3O2: C 64.84, H 6.60, N
16.20; found: C 64.92, H 6.55, N 16.42.
4-(4-Isobutyramidophenyl)-4-oxobutyric acid (2)
Yield: 40.29%. m.p.: 192–193°C. IR (KBr), u, per
cm: 3350, 3290, 2900, 1725, 1678, 1600, 1410, 1210,
840. 1H NMR (400 MHz, CDCl3 + DMSO-d6, TMS): d
9.49 ppm (s, 1H, -NH, disappeared on deuterium
exchange), 7.91 (d, 2H, aromatic protons meta to
isobutyramide, Jo = 8.75 Hz), 7.75 (d, 2H, aromatic
protons ortho to isobutyramide, Jo = 8.73 Hz), 3.25
(t, 2H, -COCH2CH2COOH, J = 6.61 Hz), 2.70 (t, 2H,
-COCH2CH2COOH, J = 6.57 Hz), 2.66 (m, 1H,
-CH(CH3)2), 1.21 (d, 6H, -CH(CH3)2, J = 6.93 Hz).
6-(4-Isobutyramidophenyl)-2-phenyl-4,5-
dihydropyridazin-3(2H)-one (6)
Recrystallization: chloroform and methanol. Yield:
59.66%. m.p.: 170–171°C. IR (KBr), u, per cm: 3330,
1
1660, 1600, 1530, 1325, 680. H NMR (400 MHz,
CDCl3 + DMSO-d6, TMS): d 7.77 ppm (d, 2H, 3-CH
and 5-CH, aromatic protons meta to isobutyramide,
Jo = 8.60 Hz), 7.59 (m, 4H, 2-CH and 6-CH, aromatic,
N-phenyl; aromatic protons ortho to isobutyramide),
4-(4-Isobutyramidophenyl)-3-methyl-4-oxobutyric
acid (3)
Yield: 47.08%. m.p.: 188–190°C. IR (KBr), u, 7.41 (t, 2H, 3-CH and 5-CH, aromatic, N-phenyl,
per cm: 3308, 2976, 1728, 1670, 1535, 1409, 1172, Jo = 7.84 Hz), 7.27 (m, 1H, 4-CH, aromatic, N-phenyl),
832. 1H NMR (400 MHz, CDCl3 + CF3COOD, 3.07 (t, 2H, 5-CH2, J = 8.01 Hz), 2.78 (t, 2H, 4-CH2,
TMS): d 8.01 ppm (d, 2H, aromatic protons meta J = 8.01 Hz), 2.52 (sept, 1H, -NHCOCH(CH3)2, J =
to isobutyramide, Jo = 8.36 Hz), 7.87 (s, 1H, -NH, 6.83 Hz), 1.26 (d, 6H, -NHCOCH(CH3)2, J = 6.89 Hz).
disappeared on deuterium exchange), 7.67 (d, 2H, Analysis calculated for C20H21N3O2: C, 71.62; H, 6.31; N,
aromatic protons ortho to isobutyramide, Jo = 12.53; found: C 71.96, H 6.22, N 12.91.
8.58 Hz), 3.57 (m, 1H, -COCH(CH3)-CH2COOH),
3.20 (m, 2H, -COCH(CH3)CH2COOH), 2.71 (sept,
6-(4-Isobutyramidophenyl)-2-(4-fluorophenyl)-4,
5-dihydropyridazin-3(2H)-one (7)
1H, -NHCOCH(CH3)2, J = 6.11 Hz), 1.39 (d, 3H,
-COCH(CH3)CH2COOH, J = 6.92 Hz), 1.30 (d, 6H,
Recrystallization: methanol. Yield: 46.36%. m.p.
-NHCOCH(CH3)2, J = 6.80 Hz).
213–214°C. IR (KBr), u, per cm: 3295, 2968, 1670, 1604,
1509, 1407, 1330, 1213, 837, 504. 1H NMR (400 MHz,
CDCl3 + DMSO-d6, TMS): d 7.77 ppm (d, 2H, aromatic
General procedure for synthesis of 6-(4-
isobutyramidophenyl)-substituted 4,5-
dihydropyridazin-3(2H)-ones (5–11)
The requisite hydrazine derivative (3.5 mmol) was
added to a stirred and refluxing solution of substituted
protons meta to isobutyramide, Jo = 8.71 Hz), 7.61 (d,
2H, aromatic protons ortho to isobutyramide,
Jo = 8.76 Hz), 7.55 (m, 2H, 2-CH and 6-CH, aromatic,
N-p-fluorophenyl), 7.10 (t, 2H, 3-CH and 5-CH, aro-
matic, N-p-fluorophenyl, Jo = 8.70 Hz), 3.07 (t, 2H,
g-keto acids 2–4 (3.8 mmol) in aldehyde-free ethanol
5-CH2, J = 7.51 Hz), 2.77 (t, 2H, 4-CH2, J = 8.03 Hz),
(25 ml). The reaction mixture was further refluxed with
2.53 (sept, 1H, -NHCOCH(CH3)2, J = 6.13 Hz),1.27 (d,
stirring for 7 h (12 h for compounds 6 and 7). The
6H, -NHCOCH(CH3)2, J = 6.75 Hz). Analysis calcu-
completion of the reaction was monitored by TLC.
lated for C20H20N3O2F: C 67.97, H 5.70, N 11.89; found:
The resulting solution was concentrated to half of the
C 67.63, H 5.38, N 11.89.
volume and left overnight in the refrigerator to yield
crystals of the corresponding dihydropyridazinone
derivatives, 5–11. The crystals obtained were washed 2-(4,5-Dihydro-1H-imidazol-2-yl)-6-(4-
with cold ethanol, dried, and recrystallized from suit- isobutyramidophenyl)-4,5-dihydropyridazin-3(2H)-
able solvent. The yields of compounds 10 and 11 were one (8)
calculated from compound 2, as the nitro derivative 4
was obtained as an oily residue.
Recrystallization: methanol. Yield: 48.25%. m.p.:
170–171°C. IR (KBr), u, per cm: 3394, 1658, 1542,
Drug Dev. Res.

VASORELAXANT ACTIVITY OF NEWER 6-ARYLPYRIDAZINONES
299
1398, 1324, 845. 1H NMR (400 MHz, CDCl3+ -NHCOCH(CH3)2, disappeared on deuterium
CF3COOD, TMS): d 9.50 ppm (s, 1H, -NHCOCH exchange), 8.92 (d, 1H, aromatic proton meta to nitro,
(CH3)2, disappeared on deuterium exchange), 7.78 Jo = 9.18 Hz), 8.59 (d, 1H, aromatic proton ortho to
(d, 2H, aromatic protons meta to isobutyramide, nitro, Jm = 2.16 Hz), 8.11 (dd, 1H, aromatic proton para
Jo = 8.77 Hz), 7.66 (d, 2H, aromatic protons ortho to to nitro, Jo = 9.09 Hz, Jm = 2.18 Hz), 7.53 (m, 2H, 2-CH
isobutyramide, Jo = 8.75 Hz), 3.75 (s, 4H, 2¥ -CH2-, and 6-CH, aromatic, N-p-fluorophenyl), 7.12 (m, 2H,
imidazoline), 3.18 (t, 2H, 5-CH2, J = 8.04 Hz), 2.61 (m, 3-CH and 5-CH, aromatic, N-p-fluorophenyl), 3.10 (t,
1H, -CH(CH3)2), 2.40 (t, 2H, 4-CH2, J = 8.01 Hz), 1.20 2H, 5-CH2, J = 7.51 Hz), 2.81 (t, 2H, 4-CH2,
(d, 6H, -CH(CH3)2, J = 6.74 Hz). Analysis calculated for J = 7.81 Hz), 2.68 (m, 1H, -NHCOCH(CH3)2), 1.32 (d,
C17H21N5O2·H2O: C 59.81, H 6.71, N 20.27; found: C 6H, -NHCOCH(CH3)2, J = 6.27 Hz). Analysis calcu-
59.56, H 7.0, N 20.42.
lated for C20H19N4O4F: C 60.29, H 4.81, N 14.06;
found: C 59.99, H 4.70, N 14.40.
6-(4-Isobutyramidophenyl)-5-methyl-4,5-
dihydropyridazin-3(2H)-one (9)
Procedure for synthesis of phenoxyacetonitrile (12)
Chloroacetonitrile (1.0 mL, 15.80 mmol) was
Recrystallization: methanol. Yield: 63.14%. m.p.: added to a stirred and refluxing suspension of phenol
241–242°C. IR (KBr), u, per cm: 3314, 3207, 3055, (1.0 g, 9.08 mmol) and anhydrous potassium carbonate
1
2970, 2930, 1671, 1527, 1345, 840, 568. H NMR (2.0 g, 14.47 mmol) in ethyl methyl ketone (60 ml). The
(400 MHz, CDCl3 + DMSO-d6, TMS): d 10.07 ppm (s, reaction mixture was further refluxed for 12 h with con-
1H, -NH, pyridazinone, disappeared on deuterium tinuous stirring. The completion of the reaction was
exchange), 9.37 (s, 1H, -NHCOCH(CH3)2, disappeared monitored by TLC. The reaction mixture was cooled
on deuterium exchange), 7.67 (m, 4H, aromatic), 3.05 and filtered, and the excess of solvent was removed
(m, 1H, 5-CH(CH3)), 2.59 (m, 3H, -NHCOCH(CH3)2 under reduced pressure to obtain an oily residue of 12,
and 4-CH2), 1.27 (d, 3H, 5-CH(CH3), J = 3.15 Hz), 1.21 which was used as such for further reactions.
(d, 6H, -NHCOCH(CH3)2, J = 6.72 Hz). Analysis calcu-
1H NMR (400 MHz, CDCl3 + DMSO-d6, TMS):
lated for C15H18N3O2: C 66.15, H 6.66, N 15.42; found: d 7.35 ppm (t, 2H, aromatic protons meta to alkoxy,
C 65.93, H 7.01, N 15.63.
J = 7.11 Hz), 7.08 (m, 1H, aromatic proton para to
alkoxy), 6.98 (d, 2H, aromatic protons ortho to alkoxy,
Jo = 8.61 Hz), 4.74 (s, 2H, -OCH2-).
6-(4-Isobutyramido-3-nitrophenyl)-4,5-
dihydropyridazin-3(2H)-one (10)
General procedure for synthesis of
Recrystallization: methanol. Yield: 45.59%. m.p.: 4-(4-cyanomethoxyphenyl)-substituted g-keto acids
240–242°C. IR (KBr), u, per cm: 3360, 3186, 3077, 2926, (13, 14)
1
1686, 1620, 1517, 1332, 1260, 1143, 826. H NMR
A mixture of 12 (1.3 g, 9.76 mmol) and succinic
(400 MHz, CDCl3 + DMSO-d6, TMS): d 10.46 ppm (s, anhydride (1.3 g, 12.99 mmol)/methyl succinic anhy-
1H, -NHCOCH(CH3)2, disappeared on deuterium dride (1 g, 8.76 mmol) was added to a stirred solution of
exchange), 10.38 (s, 1H, -NH, pyridazinone, disap- aluminum chloride (5 g) in nitrobenzene (10 mL). The
peared on deuterium exchange), 8.76 (d, 1H, aromatic reaction mixture was stirred manually under anhydrous
proton meta to nitro, Jo = 9.06 Hz), 8.54 (d, 1H, aromatic conditions for 20 min and allowed to stand for 48 h at
proton ortho to nitro, Jm = 2.13 Hz, J = 6.87 Hz), 8.05 room temperature. Reaction contents were decom-
(dd, 1H, aromatic proton para to nitro, Jo = 9.05 Hz, posed with crushed ice and steam distilled to remove
Jm = 2.12 Hz), 3.0 (t, 2H, 5-CH2, J = 8.40 Hz), 2.69 nitrobenzene. The hot solution was filtered, and the
(m, 1H, -NHCOCH(CH3)2), 2.60 (t, 2H, 4-CH2, filtrate was cooled in ice for complete precipitation. The
J = 8.64 Hz),1.31 (d, 6H, -NHCOCH(CH3)2). Analysis solid product obtained was filtered and washed thor-
calculated for C14H16N4O4: C 55.25, H 5.30, N 18.41; oughly with distilled water. The resulting residue was
found: C 55.38, H 4.96, N 18.52.
dissolved in 10% aqueous sodium bicarbonate solution
and the insoluble part was filtered off. Acidification with
concentrated HCl gave a precipitate, which was fil-
tered, washed with water, dried, and recrystallized from
methanol to afford 13 and 14. The yields of both com-
6-(4-Isobutyramido-3-nitrophenyl)-2-(4-
fluorophenyl)-4,5-dihydropyridazin-3(2H)-one (11)
Recrystallization: ethanol. Yield: 23.22%. m.p.: pounds 13 and 14, obtained from two-step reactions,
238–239°C. IR (KBr), u, per cm: 3354, 2979, 1685, were calculated from the starting material phenol and
1503, 1332, 1269, 1218, 1151, 839. 1H NMR (400 MHz, not from compound 12, which was obtained as an oily
CDCl3 + DMSO-d6, TMS): d 10.56 ppm (s, 1H, residue.
Drug Dev. Res.

300
BANSAL ET AL.
4-(4-Cyanomethoxyphenyl)-4-oxobutyric acid (13)
6-(4-Cyanomethoxyphenyl)-5-methyl-4,5-
Yield: 37.32%. m.p.: 112–114°C. IR (KBr), dihydropyridazin-3(2H)-one (16)
u, per cm: 3020, 1700, 1680, 1600, 1440, 1240, 850.
Recrystallization: acetone. Yield: 54.20%. m.p.:
1H NMR (400 MHz, CDCl3 + DMSO-d6, TMS): d 185–186°C. IR (KBr), u, per cm: 3240, 2928, 1670,
1
8.01 ppm (dd, 2H, aromatic protons meta to alkoxy, 1510, 1346, 1246, 1044, 836. H NMR (400 MHz,
Jo = 6.72 Hz, Jm = 2.09 Hz), 7.05 (dd, 2H, aromatic CDCl3 + DMSO-d6, TMS): d 8.64 ppm (s, 1H, NH,
protons ortho to alkoxy, Jo = 6.75 Hz, Jm = 2.05 Hz), disappeared on deuterium exchange), 7.72 (dd, 2H,
4.87 (s, 2H, -OCH2-), 3.27 (t, 2H, -COCH2CH2COOH, aromatic protons meta to alkoxy, Jo = 8.91 Hz, Jm =
J = 6.64 Hz), 2.75 (t, 2H, -COCH2CH2COOH, 1.82 Hz), 7.01 (dd, 2H, aromatic protons ortho to
J = 6.75 Hz).
alkoxy, Jo = 8.99 Hz, Jm = 2.03 Hz), 4.81 (s, 2H,
-OCH2-), 3.05 (m, 1H, 5-CH(CH3)), 2.62 (m, 2H,
4-CH2), 1.32 (d, 3H, 5-CH(CH3), J = 6.76 Hz). Analysis
calculated for C13H13N3O2: C 64.18, H 5.38, N 17.27;
found: C 64.01, H 5.41, N 16.82.
4-(4-Cyanomethoxyphenyl)-3-methyl-4-oxobutyric
acid (14)
Yield: 30.16%. m.p.: 110–111°C. IR (KBr), u, per
cm: 3020, 1700, 1600, 1440, 1240, 1180, 850. 1H NMR
(400 MHz, CDCl3 + DMSO-d6, TMS): d 8.08 ppm (d,
2H, aromatic protons meta to alkoxy, Jo = 8.78 Hz), 7.03
(d, 2H, aromatic protons ortho to alkoxy, Jo = 8.69 Hz),
4.95 (s, 2H, -OCH2-), 3.44 (m, 1H, -COCH(CH3)
CH2COOH), 2.97 (m, 2H, -COCH(CH3)CH2COOH),
1.28 (d, 3H, -COCH(CH3)CH2COOH, J = 7.12 Hz).
6-(4-Cyanomethoxyphenyl)-2-phenyl-4,5-
dihydropyridazin-3(2H)-one (17)
Recrystallization: methanol. yield: 55.12%. m.p.:
103–104°C. IR (KBr), u, per cm: 3062, 1672, 1599,
1507, 1325, 1246, 1184, 1043, 836, 605. H NMR
(400 MHz, CDCl3 + DMSO-d6, TMS): d 7.81 ppm (d,
2H, aromatic protons meta to alkoxy, Jo = 8.81 Hz),
7.58 (d, 2H, 2-CH and 6-CH, aromatic, N-phenyl,
Jo = 7.74 Hz), 7.42 (t, 2H, 3-CH and 5-CH, aromatic,
N-phenyl, Jo = 7.77 Hz), 7.28 (t, 1H, 4-CH, aromatic,
N-phenyl, Jo = 7.31 Hz), 7.01 (d, 2H, aromatic protons
ortho to alkoxy, Jo = 8.84 Hz), 4.79 (s, 2H, -OCH2-),
3.06 (t, 2H, 5-CH2, J = 8.11 Hz), 2.78 (t, 2H, 4-CH2,
J = 8.01 Hz). Analysis calculated for C18H15N3O2: C
70.80, H 4.95, N 13.76; found: C 70.51, H 4.56, N
13.65.
1
General procedure for synthesis of
6-(4-cyanomethoxyphenyl)-substituted
4,5-dihydropyridazin-3(2H)-one (15–18)
The requisite hydrazine derivative (3.5 mmol) was
added to a stirred and refluxing solution of substituted
g-keto acids 13 and 14 (3.8 mmol) in aldehyde-free
ethanol (25 mL). The reaction mixture was further
refluxed with stirring. The completion of the reaction
was monitored by TLC. The resulting solution was con-
centrated to half of the volume and left overnight in the
refrigerator to afford crystals of corresponding dihydro-
pyridazinone derivatives 15–18. The crystals obtained
were washed with cold ethanol, dried, and recrystal-
lized from suitable solvent.
6-(4-Cyanomethoxyphenyl)-2-(4,5-dihydro-1H-
imidazol-2-yl)-2-phenyl-4,5-dihydropyridazin-
3(2H)-one (18)
Recrystallization: water. Yield: 54.28%. m.p.: 204–
205°C. IR (KBr), u, per cm: 3369, 3063, 2922, 1658,
1
1557, 1399, 1244, 1040, 840. H NMR (400 MHz,
CDCl3+ CF3COOD, TMS): d 7.26 ppm (s, 2H, aro-
matic protons meta to alkoxy), 7.10 (d, 2H, aromatic
protons ortho to alkoxy, Jo = 8.22 Hz), 4.97 (s, 2H,
-OCH2-), 3.90 (brs, 4H, 2 x -CH2-, imidazoline), 3.07
(brs, 2H, 5-CH2), 2.83 (brs, 2H, 4-CH2). Analysis calcu-
lated for C15H15N5O2:.H2O C 60.59, H 5.43, N 22.21;
found: C 60.23, H 5.22, N 22.01.
6-(4-Cyanomethoxyphenyl)-4,5-dihydropyridazin-
3(2H)-one (15)
Recrystallization: ethanol. Yield: 58.14%. m.p.:
162–163°C. IR (KBr), u, per cm: 3256, 2922, 1666,
1622, 1603, 1518, 1435, 1345, 1318, 1113, 1050, 990,
1
771. H NMR (400 MHz, CDCl3 + DMSO-d6, TMS):
d 7.75 (dd, 2H, aromatic protons meta to alkoxy,
Jo = 8.84 Hz, Jm = 2.97 Hz), 7.02 (dd, 2H, aromatic
protons ortho to alkoxy, Jo = 8.87 Hz, Jm = 2.91 Hz),
4.88 (s, 1H, -OCH2-), 2.96 (t, 2H, 5-CH2, J = 8.18 Hz),
2.58 (t, 2H, 4-CH2, J = 7.7 Hz). Analysis calculated for
Vasodilatory Activity
The vasodilatory activity of the newly synthe-
C12H11N3O2: C 62.87, H 4.84, N 18.33; found: C 62.54, sized compounds was studied using descending tho-
H 4.83, N 18.32.
racic aortic rings of Wistar rats precontracted with
Drug Dev. Res.

VASORELAXANT ACTIVITY OF NEWER 6-ARYLPYRIDAZINONES
301
phenylephrine (10-6 M), as previously reported (Olmo methylene protons appeared at d 2.70 and 3.25 ppm,
et al., 2006; Bansal et al., 2009). The pharmacological respectively, in the 1H NMR spectrum of compound 2.
protocol was reviewed by the University of Salamanca, A downfield and separated methine signal at d
and animal usage was approved by the institutional 3.57 ppm in the case of 3 confirmed the formation of a
ethical committee.
b-methyl derivative. As part of the research program,
Wistar rats of either sex weighing 300–400 g were the effect of introducing a nitro group ortho to the
killed by a blow on the head. The descending thoracic 4-acylamino substituent of the 6-phenyl ring on the
aorta was rapidly dissected and placed in a physiological activity profile of pyridazinones was also investigated.
saline solution (PSS) of the following composition: For this, nitration of compound 2 was carried out in an
NaCl (118 mM), KCI (4.75 mM), NaHCO3 (25 mM), ice-cold mixture of concentrated HNO3–H2SO4 (3:1),
MgSO4 (1.2 mM), CaCl2 (1.8 mM), KH2PO4 (1.2 mM), which yielded a yellow, sticky product, 4. Infrared
and glucose (11 mM). After excess fat and connective spectra of compound 4 showed asymmetrical and sym-
tissue were removed, the aortas were cut into rings metrical stretching bands characteristic of a nitro group
(4–5 mm in length), mounted under a basal tension of in conjugation with aromatic ring system near 1510 and
2 g in 5-mL organ baths containing PSS and attached to 1345 per cm, respectively. Further treatment of the
force-displacement transducers to measure isometric g-keto acids 2–4 with the requisite hydrazine hydrate
contractile force. The tissue bath was maintained at resulted in the formation of the corresponding desired
37°C and bubbled with an O2/CO2 (95:5) gas mixture. pyridazinones 5–11, which exhibited clear N-H stretch-
Each preparation was allowed to equilibrate for at least ing absorption frequencies, characteristic of secondary
90 min prior to initiation of experimental procedures, amides near 3300, and amidic C = O stretching bands of
and during this period the incubation media were pyridazinone residue at 1660–1685 per cm.
changed every 20 min. After equilibration, aortic rings
The methylene protons of the parent pyridazi-
were contracted by a single concentration of phenyle- none ring in compound 5 appeared as triplets at d
phrine (10-6 M). When the contractions became stable, 2.60 (4-CH2) and 2.96 (5-CH2), while those of the
compounds were added in progressively increasing 2-substituted pyridazinones 6 and 7 appeared down-
cumulative concentrations (10-8–10-5 M) at 30-min field at 2.77 (4-CH2) and 3.07 ppm (5-CH2). A doublet,
intervals. Only one compound was tested in each ring. integrating for six protons, for two methyl of the isobu-
All compounds were initially dissolved in DMSO to tyramido moiety was recorded at d ~1.2 ppm for all
prepare a 10-2 M stock solution. Further solutions were these 6-(4-isobutyramidophenyl) pyridazinones. For
made in PSS. Results are expressed as means Ϯ SEM. the N-imidazolinyl-substituted compound 8, the meth-
The compound response of the aortic rings was ylene protons of C4 and C5 of the pyridazinone nucleus
expressed as a percentage of the initial contraction to appeared as triplets at d 2.40 and 3.18 ppm, respec-
10-6 M phenylephrine. Dose–response curves were tively, while those of the imidazoline ring resonated
analyzed by a sigmoidal curve-fitting analysis to give the downfield at d 3.75 ppm.
pD2 value (the drug concentration exhibiting 50% of
Compound 9, 6-(4-isobutyramidophenyl)-5-
the Emax expressed as negative log molar) and Emax value methyl-4,5-dihydropyridazin-3(2H)-one, exhibited cha-
(the maximal effect).
racteristic PMR signals at d 1.27 (d, 3H, 5-CH(CH3)),
2.59 (m, 3H, -NHCOCH(CH3)2 and 4-CH2), and
3.05 ppm (m, 1H, 5-CH(CH3)). Aromatic protons of
the 6-phenyl ring displayed distinct signals at d ~8.1
(dd, 1H, aromatic proton para to nitro), 8.55 (d, 1H,
RESULTS AND DISCUSSION
Chemistry
The synthesis of new pyridazinone derivatives aromatic proton ortho to nitro), and 8.80 ppm (d, 1H,
has been carried out according to Figures 2 and 3. aromatic proton meta to nitro) for both of the nitropy-
N-Phenylisobutyramide (1), which was used as starting ridazinones 10 and 11.
compound for the synthesis of pyridazinone derivatives,
The cyano group-containing pyridazinones were
was obtained by reacting aniline with isobutyryl chlo- prepared by Friedel–Crafts acylation of phenoxyaceto-
ride in dichloromethane at room temperature. The nitrile 12 with appropriate anhydride to form g-keto
Friedel–Crafts acylation reaction of compound 1 with acids 13 and 14, which on cyclocondensation with
succinic anhydride or methylsuccinic anhydride in the various hydrazine derivatives gave the target com-
presence of anhydrous aluminum chloride afforded pounds 15–18 (Fig 3). The pyridazinone C4 protons
g-ketoacids, 2 and 3. The 4-(4-isobutyramidophenyl)-4- for 6-(4-cyanomethoxy phenyl)-4,5-dihydropyridazin-
oxobutyric acid (2) and its 3-methyl analog 3 recorded 3(2H)-one (15) were recorded at d ~2.60 ppm. In case
C = O absorption bands for saturated aliphatic acid near of compound 17, protons for the N- substituted aro-
1720 and for amide at 1680 per cm. Triplets of a and b matic ring appeared in the range of d 7.28–7.58 ppm.
Drug Dev. Res.

302
BANSAL ET AL.
Fig. 2. Reagents and conditions: (a) isobutyryl chloride, dichloromethane, stirred at room temperature; (b) aluminum chloride, succinic
anhydride/methyl succinic anhydride, ice-cold mixture of concentrated HNO3–H2SO4 (3:1); (c) hydrazine hydrate/phenylhydrazine
hydrochloride/p-fluorophenylhydrazine hydrochloride/2-hydrazine-2-imidazoline hydrobromide, absolute ethanol.
Similarly, for compound 18, the imidazolinyl protons were expressed as percentage of the maximal control
resonated as a four-proton broad singlet at d 3.90 ppm. phenylephrine-induced responses. Maximal relaxation
(% of control) and pD2 (-log IC50) and IC50 values of
Vasodilatory Activity
various compounds to inhibit the contractions induced
The compounds 5–11 and 15–18 were evaluated by phenylephrine (10-6 M) are shown in Table 1. Imi-
for vasodilatory activity using rat thoracic aortic rings, dazolinyl derivatives 8 and 18 could not be screened for
precontracted with phenylephrine (10-6 M). The results vasodilatory activity because of solubility problems.
Drug Dev. Res.

VASORELAXANT ACTIVITY OF NEWER 6-ARYLPYRIDAZINONES
303
Fig. 3. Reagents and conditions: (a) Chloroacetonitrile and anhydrous potassium carbonate in ethyl methyl ketone; (b) aluminum chloride,
succinic anhydride/methyl succinic anhydride; (c) hydrazine hydrate/phenylhydrazine hydrochloride/2-hydrazine-2-imidazoline hydrobromide,
absolute ethanol.
Concentration–response curves for vasorelaxant (Table 1). However, an unsubstituted nitrogen on the
activity of compounds on aortic rings, obtained pyridazinone nucleus does not seem to be a necessary
from Wistar rats are depicted in Figure 4. In both condition for a compound to be vasorelaxant, as N2-
series of newly synthesized 6-(para-substituted phenyl) p-fluorophenyl derivatives 7 (IC50 = 1.0 Ϯ 0.1 mM)
pyridazinones, most of the compounds produced a and 11 (IC50 = 0.63 Ϯ 0.06 mM) also produced good
concentration-dependent inhibition of the contractile vasorelaxation.
response to phenylephrine and produced vasorelaxation
Methyl substitution at the 5-position of the
comparable with that of hydralazine and SK&F-93741 pyridazinone skeleton in compounds 9 and 16 resulted
(Fig. 4) except the N2-phenyl-substituted derivatives 6 in slightly increased vasorelaxant action than 5-
and 17. The N2-unsubstituted pyridazinone derivatives unsubstituted analogs 5 and 15, respectively. This is in
5 (IC50 = 0.199 Ϯ 0.1 mM) and 9 (IC50 = 0.158 Ϯ 0.2 accord with earlier reports, indicating that methyl sub-
mM) in the isobutyramide series and 15 (IC50 = stitution at the 5-position of the pyridazinone results in
7.94 Ϯ 0.17 mM) and 16 (IC50 = 3.98 Ϯ 0.2 mM) in the increased antihypertensive activity [Curran and Ross,
acetonitrile series exhibited potent vasorelaxant activity 1974].
Drug Dev. Res.

304
BANSAL ET AL.
Fig. 4. Concentration–response curves for vasorelaxant activity of compounds shown on aortic rings obtained from Wistar rats. Each data point
represents the mean Ϯ SEM (n = 4–6).
midophenylpyridazinone derivative 5 emerged as the
TABLE 1. Maximal Response (Emax), pD2, and IC50 (mM) Values
most potent vasorelaxant among the synthesized deriva-
tives, producing vasorelaxation that was greater than
hydralazine and equipotent to SK&F-93741, thus pro-
viding a possible lead structure for development of new
vasorelaxants.
Needed to Inhibit the Contractions Induced by Phenylephrine
(10-6 M)
Compound (code)
Emax (%)
pD2
IC50 (mM)
5 (DPJ-943)
89.8 Ϯ 2.9*
32.58 Ϯ 5.65
53.3 Ϯ 7.5*
85.1 Ϯ 5.6*
93.1 Ϯ 13.1*
70.0 Ϯ 2.2*
61.06 Ϯ 5.74*
80.0 Ϯ 11.31*
6.7 Ϯ 0.1
—
6.0 Ϯ 0.1
6.8 Ϯ 0.2
0.199 Ϯ 0.1
—
1.0 Ϯ 0.1
0.158 Ϯ 0.2
6** (DPJ-RG-1224)
7 (DPJ-RG-1225)
9 (DPJ-965)
10 (DPJ-RG-1351)
11 (DPJ-RG-300)
15 (DPJ-RG-1242)
16 (DPJ-RG-1352)
6.1 Ϯ 0.01 0.794 Ϯ 0.01
ACKNOWLEDGMENTS
6.2 Ϯ 0.06
5.10 Ϯ 0.17
5.4 Ϯ 0.2
—
0.63 Ϯ 0.06
7.94 Ϯ 0.17
3.98 Ϯ 0.2
The authors are thankful to the University Grants
Commission and Council of Scientific and Industrial
Research, India, for providing financial assistance.
17** (DPJ-RG-1279) 45.20 Ϯ 9.07*
DMSO
Hydralazine
SK&F-93741
1.80 Ϯ 7.01
55.0 Ϯ 5.8*
62.0 Ϯ 3.5*
—
—
6.5 Ϯ 0.1
6.7 Ϯ 0.2
0.316 Ϯ 0.1
0.199 Ϯ 0.2
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