Efficient synthesis, anticonvulsant and muscle relaxant activities of new 2-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-6-phenyl-4,5-dihydropyridazin-3(2H) -one derivatives

Article abstract of DOI:10.1007/s00044-013-0618-0

A series of 2-(2-(3-(4-chlorophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)yl) acetyl)hydrazine carbothioamide and 2-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-6- (4-chlorophenyl)-4,5-dihydropyridazin-3(2H)-one derivatives were synthesized, characterized, and evaluated for anticonvulsant activity and muscle relaxant activity. The synthesized compounds 5d (82.75 %) and 5e (85.44 %) showed promising anticonvulsant activity by protection against tonic hind limb extensor phase in maximal electroshock model (MES) at (50 mg/kg) compared to standard drug phenytoin and also compounds 5d (82.75 %), and 5e (85.44 %) showed significant anticonvulsant activity by protection against pentylenetetrazole- induced generalized convulsions in pentylenetetrazole model (PTZ) at (100 mg/kg) compared to standard drug diazepam. On the other hand, compound 5e showed significant muscle relaxant activity (84.57 %) by rotarod and traction test model comparing with diazepam as a standard drug.

Full text of DOI:10.1007/s00044-013-0618-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0618-0
ORIGINAL RESEARCH
Efficient synthesis, anticonvulsant and muscle relaxant activities
of new 2-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-6-phenyl-4,5-
dihydropyridazin-3(2H)-one derivatives
•
Bhawna Sharma Amita Verma
•
•
Upendra Kumar Sharma Sunil Prajapati
Received: 10 January 2013 / Accepted: 4 May 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 2-(2-(3-(4-chlorophenyl)-6-oxo-5,6-
dihydropyridazin-1(4H)yl)acetyl)hydrazine
activity (84.57 %) by rotarod and traction test model
comparing with diazepam as a standard drug.
carbothioa-
mide and 2-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-6-(4-
chlorophenyl)-4,5-dihydropyridazin-3(2H)-one derivatives
were synthesized, characterized, and evaluated for anti-
convulsant activity and muscle relaxant activity. The syn-
thesized compounds 5d (82.75 %) and 5e (85.44 %)
showed promising anticonvulsant activity by protection
against tonic hind limb extensor phase in maximal elec-
troshock model (MES) at (50 mg/kg) compared to standard
drug phenytoin and also compounds 5d (82.75 %), and 5e
(85.44 %) showed significant anticonvulsant activity by
protection against pentylenetetrazole-induced generalized
convulsions in pentylenetetrazole model (PTZ) at (100 mg/
kg) compared to standard drug diazepam. On the other
hand, compound 5e showed significant muscle relaxant
Keywords Anticonvulsant ꢀ Convulsion ꢀ
Aryl pyridazinone ꢀ MES model ꢀ PTZ model ꢀ
Muscle relaxant activity
Introduction
Epilepsy is a major neurological disorder and up to 5 % of the
world population develops epilepsy in their lifetime (Sander
and Shorvon, 1996; Smith and Bleck, 1991; Samrjn et al.,
1997), there is a continuing demand for new anticonvulsant
agents as it has not been possible to control every kind of
seizure with the currently available antiepileptic drugs. About
one third of patients do not respond well to current multiple
¨
drugs therapy (Schmidt and Loscher, 2005; Kwan and Brodie,
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0618-0) contains supplementary
material, which is available to authorized users.
2000), studies reveal that 63 % of patients diagnosed and
treated were seizure-free and more than 50 % of epilepsy
patients have experienced unwanted side effects (Dichter and
Brodie, 1996; Brodie and Dichter, 1996; Kwan and Brodie,
2004); phenobarbital (Hardman et al., 1996) and mephobar-
bital are well-known barbituric acid derivatives which are used
for the treatment of epilepsy. These antiepileptic drugs are very
effective in controlling the seizure; however, they are associ-
ated with side effects, dose-related and chronic toxicity, as well
as teratogenic effects, substituted heterocyclic/substituted aryl
systematic pyridazinone nucleus remarkably increases the
antiepileptic activity. Pyridazinone derivatives 3-oxo-5-
substituted benzylidene-6-methyl-(4H)-2-pyridazinylaceta-
mides and 2-pyridazinylacetyl hydrazides and a considerable
number of pyridazin-3(2H)-ones are reported for their pro-
nounced anticonvulsant activity (Rubut et al., 1990;Asif,2010;
Pooja et al., 2009; Siddiqui et al., 2006; Oya et al., 2004;
B. Sharma (&)
Department of Pharmaceutical Chemistry, Institute of Pharmacy,
Bundelkhand University, Jhansi, India
e-mail: bms1928@yahoo.com
A. Verma
Department of Pharmaceutical Sciences, Faculty of Health
Sciences, Sam Higginbottom Institute of Agriculture,
Technology and Sciences, Allahabad 211007, India
U. K. Sharma
Department of Pharmaceutics, Bundelkhand University,
Jhansi, India
e-mail: ups1928@yahoo.com
S. Prajapati
Department of Pharmaceutics, Institute of Pharmacy,
Bundelkhand University, Jhansi, India
123

Med Chem Res
Sivakumar et al., 2003; Edith et al., 2002; Xu et al., 1991a, b;
Hallot et al., 1986; Perio et al., 1986), in hope of getting syn-
ergistic response of pyridazino methyl 1,3,4-thiadiazole
nucleus itself, the present paper reports on the synthesis, anti-
convulsant, and muscle relaxant activity of 2-[(5-amino-1,3,4-
thiadiazole-2yl)methyl]-6-(4-methoxyphenyl)-4,5-dihydropy-
ridazin-3(2H)-one derivatives. The compounds designed so
were found to possess much significant anticonvulsant activity
with significant reduction in toxicity.
different distance from its starting compound. The struc-
tures of newly synthesized compounds were identified
using infrared (IR), proton nuclear magnetic resonance
(1HNMR), and mass spectral data. Elemental analyses of
all synthesized compounds for C, H, N, and S were within
the theoretical values.
The IR Spectra of all synthesized pyridazinomethyl-1,3,
4-thiadiazole derivatives were recorded in terms of wave
numbers (cm-1). In the IR spectra broad OH peak is observed
in the O–H stretching (1,390) for 1a–5a. As a consequence of
reaction with hydrazine hydrate these bands were not
observed for 1b–5b. Only N–H stretching in the range
(3,303.83–3,118), aromatic C–N stretching in the range
(1,540–1,680), and absorption band of C=O characteristic of
aromatic ketone were observed in the range of (1,600–1,775).
The absorption bands of N–H stretching were not observed in
compounds 1c–5c, ester C=O stretching in the range
(1750–1740 cm-1) and C–O stretching at (1240) were
observed, respectively, in compounds 1c–5c. As expected on
reaction with thiosemicarbazide NH2 peak in the range
(3,400–3,150), C=S absorption band in the range (600–700)
and amide absorption band in the range (1,700–1,640) were
observed in 1d–5d derivatives. In the 1e–5e C–N stretching in
the range (1,540–1,680) and C–S–C absorption band were
observed in the range of (700–600).
Results and discussion
Chemistry
Synthesis of the target compounds was carried out
according to the sequence of reaction outlined in Scheme 1.
The key intermediate b-aroyl propionic acid 1a–5a was
synthesized in excellent yield by the friedal craft acylation
in the presence of succinic anhydride and aluminum
chloride (Khan and Siddiqui, 2000), which on cyclization
with hydrazine hydrate form pyridazinone 1b–5b (Siddiqui
and Dogra, 2001; Siddiqui et al., 2004), this pyridazinone
on treatment with ethyl chloro acetate form 6-oxo-3p-
(anisoyl)-5,6-dihydro-4H-pyridazine-1yl)-acetic acid ethyl
ester 1c. Then these acetic acid ethyl ester products upon
stirring with thiosemicarbazide 1d–5d were obtained. Then
these hydrazinecarbothioamide on dehydrative annulations
with conc. sulfuric acid produced 2-[(5-amino-1,3,4-thia-
diazole-2yl)methyl]-6-(substituted aroyl)-4,5-dihydropyri-
dazin 3(2H)-one derivatives 1e–5e. The purities of the
synthesized compounds were checked using thin layer
chromatography (TLC) in three mobile phase system. Each
of the synthesized compounds gave isolated spot at
1
The H NMR Spectra of 1a–5a displayed the OH at
11.00–11.92 ppm; in the 1b–5b the signal of these protons
was not observed in this; N–H peak wase observed at
7.00–8.00; the signal of N–H was not observed in 1c–5c;
methyl and ethyl proton of the ester group showed
3.57–4.16 and 10.10–1.29 in the 1d–5d; methyl and ethyl
proton peak are not observed. NH2 signal and amide proton
peak are observed at 8.00 and 8.56, respectively. In the
1e–5e NH2 signal is observed at 6.00–6.99. The other
(CH2CO)2O
NH2NH2
R
R
OH
R
O
AlCl3
O
1a
O
N
N
H
Aromatic
Hydrocarbon
1b
ClCH2COOC2H5
C2H5OH
R
O
N
H2NNHCSNH
2 R
H2SO4
O
N
R
O
N
N
C2H5OH
N
N
N
N
CH2CONHNHCSNH2
CH2
NH2
S
CH2COOC2H5
1d
1c
1e
Scheme
R
1
2
3
4
5
OCH3
C2H5
CH3
H
Cl
Scheme 1 Synthesis of 2-(5-amino-1,3,4 thiadiazole-2yl)methyl)-6phenyl-4,5-dihydropyridazine-3 (2H)-one derivatives
123

Med Chem Res
Fig. 1 Comparison of
anticonvulsant activity of test
compound by MES model
Fig. 2 Comparison of
anticonvulsant activity of test
compound by PTZ model
Fig. 3 Comparison of muscle
relaxant activity of test
compounds using rotarod
test model
Fig. 4 Comparison of muscle
relaxant activity of test
compounds using traction
test model
protons of the compounds were as expected. The mass
spectrum of compounds 5d and 5e at m/z 339.06 (M?), m/z
321.05 (M?), respectively, and mass spectrum of all other
derivatives were given in the experimental section as
expected. Standard drug used for MES model was phe-
nytoin (Fig. 1), pentylenetetrazole (PTZ) model was diaz-
epam (Fig. 2), muscle relaxant activity by rotarod test was
diazepam (Fig. 3), and traction test (Fig. 4).
123

Med Chem Res
Onset of seizure was assessed by the chi-square test and
expressed as mean SEM. The results are summarized in
Table 2. In the PTZ model again 5d and 5e showed max-
imum protection (81.99 and 84.91, respectively) and rest of
the compounds were protective in delaying of the onset of
the first myoclonic twitches. So it may be considered that
compounds 5d and 5e may be considered promising for the
development of new anticonvulsant agents.
Table 1 The effect of compounds on seizure in tonic hind limb
extensor phase by MES Model
S. no. Compound
(25 mg/kg)
R
Duration of
HLTE SEM
% Inhibition
1.
Control
–
14.906 0.67998*
00 00
–
2.
Phenytoin
–
100
3.
1d
1e
2d
2e
3d
3e
4d
4e
5d
5e
OCH3
OCH3
C2H5
C2H5
CH3
CH3
–
4.438 0.12487* 70.24
3.844 0.04163* 74.2
4.
5.
8.66 0.1144*
43.24
Muscle relaxant activity
6.
7.396 0.00263* 50.38
5.788 0.25932* 61.43
5.024 0.19198* 66.29
10.496 0.06083* 29.585
7.
The effecton motor co-ordination was assessed usingrotarod
apparatus and traction test results are shown in Table 3. The
rotarod test has been performed to detect the motor deficit in
mice. The time each animal was able to maintain its balance
walking on top of the rod was measured. Compounds 1d, 1e,
2e, and 3e showed good muscle relaxant activity (57.43,
65.79, 50.92, and 42.0 %, respectively) in comparison to
standard diazepam (4 mg/kg), whereas compound 5d and 5e
showed maximum muscle relaxant activity (73.23, and
84.57 %, respectively). Rest of the compounds showed the
least muscle relaxant activity compared with the standard
drug diazepam (100 %).
8.
9.
10.
11.
12.
–
9.42 0.1034*
36.80
Cl
2.57 0.17745* 82.75
2.17 0.03245* 85.44
Cl
Values are expressed as mean SEM; statistical significant test for
comparison was done by ANOVA; followed by chi-square test
(n = 5)
* P \ 0.05 when compared to control group
Pharmacology
Anticonvulsant activity (MES model)
Experimental section
Maximal electroshock and pentylenetetrazole induced model
was used for the evaluation of anticonvulsant study of the test
compounds. For the MES model the results are summarized in
Table 1. Compound 2-(2-(3-(4-chlorophenyl)-6-oxo-5,6-di-
hydropyridazin-1(4H)yl)acetyl)hydrazine carbothioamide 5d
and 2-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-6-(4-chloro-
phenyl)-4,5-dihydropyridazin-3(2H)-one 5e showed excel-
lent protection against HLTE phase (82.75 and 85.44 %
protection, respectively), whereas compounds 4d, 4e, 2d, and
2e were found to have the least protection against Hind limb
tonic extensor phase. Compounds 1d, 1e, 3d, and 3e also
showed good anticonvulsant activity by protection against
HLTE phase with a percentage of 70.24, 74.2, 61.43, and
66.29 %, respectively, in comparison to phenytoin.
All the solvents and reagents used were of laboratory grade
(LR). Melting points of all synthesized compounds were
determined using open capillary tube and were uncor-
rected. The purity of the compounds and completion of the
reaction was monitored from time to time by TLC using
E-Merk 0.25 mm silica gel plates. Solvent system used for
running TLC Plates was toluene, ethyl formate, and formic
acid in the ratio of 5:4:1. Visualization was accomplished
with UV light (256 nm) and iodine chamber. Synthesized
compounds were purified by column chromatography. All
the solvents were dried by appropriate drying agents before
use. The IR spectra (KBr, in cm-1) were recorded by using
potassium bromide (KBr) pellet technique on Schimadzu
1
spectrometer. The H NMR spectra (chemical shift in d
Anticonvulsant activity (PTZ model)
ppm) were recorded in deuterated chloroform using tetra-
methylsilane (TMS) as an internal reference standard on
Bruker Avance II 400 NMR spectrometer.
Pentylenetetrazole model for evaluation of anticonvulsant
activity, according to this seizure latency was defined as
the time elapsed from the injection of pentylenetetrazole to
the first two myoclonic jerk of the forelimbs. This has been
concluded to be the first sign of the beginning of the seizure
activity. Animals devoid of generalized convulsion were
considered to be protected and results were represented as
protection (%). These results of the test compound
(100 mg/kg) were compared with that of diazepam (4 mg/
Kg) as a standard for pentylenetetrazole-induced seizures.
General procedure for preparing the compounds in
Scheme 1 from (1a–1e). All the remaining compounds
were synthesized by analogous method.
b-Anisoyl-propionic acid (1a)
A mixture of anisole (30 ml) and anhydrous aluminum
chloride (0.15 mol) was suspended in a two-necked flask
and this mixture was refluxed under anhydrous condition
123

Med Chem Res
Table 2 The effect of
compounds on onset and
duration of seizure and
protection against
pentylenetetrazole-induced
generalized convulsion
S. no.
R
Onset of seizure (s)
Duration of
clonic seizure (s)
Seizure
protection (%)
1d
OCH3
OCH3
C2H5
C2H5
CH3
CH3
-
103.8 2.24*
124.4 1.50*
93.4 1.32*
91.6 1.74*
101 1.51*
105.8 2.28*
85.8 3.02*
73 0.70*
61 1.702*
51.2 0.8*
112 3.30*
99.8 0.8*
95.4 3.86*
70.2 0.66*
156 4.0*
69.65
74.2
1e
2d
44.27
50.34
62.48
65.07
22.2
2e
3d
3e
4d
Values are expressed as
mean SEM; statistical
significant test for comparison
was done by ANOVA; followed
by chi-square test (n = 5)
4e
–
125.4 1.6*
38.2 1.06*
30.2 0.66*
201 1.06*
00 00
38.3
5d
Cl
159 3.67*
161.4 1.77*
76.8 1.06*
00 00
81.99
84.97
00
5e
Cl
Control
Diazepam
–
* P \ 0.05 when compared to
control group
–
100
Table 3 The effect of
compounds on muscle relaxant
activity by rotarod test and
traction test
S. Test
no. compounds
R
Rotarod test fall of Decrease in
time in (s)
Traction test time of Failure to put hind
holding (s)
time (%)
limb (%)
1. 1d
OCH3 105.404 4.54*
57.05
64.96
44.88
50.92
31.59
34.00
11.11
21.67
70.80
83.31
0.00
2.296 0.03*
17.70 16.05*
2.954 0.02*
2.682 0.05*
3.686 0.10*
3.126 0.03*
4.806 0.12*
4.292 0.07*
1.442 0.04*
0.83 0.18*
5.38 0.16*
0.00 00
57.43
65.79
48.88
50.92
31.59
42.00
11.52
20.26
73.23
84.57
0.00
2. 1e
OCH3
C2H5
C2H5
CH3
CH3
–
85.99 2.54*
135.34 1.78*
120.64 3.25*
168.9 4.10*
160.53 3.21*
218.16 2.53*
194.24 1.59*
71.65 0.78*
40.96 1.21*
245.458 2.093*
00.00 00
3. 2d
4. 2e
5. 3d
6. 3e
7. 4d
Values are expressed as
mean SEM; statistical
significant test for comparison
was done by ANOVA; followed
by chi-square test (n = 5)
8. 4e
–
9. 5d
Cl
10 5e
Cl
11 Control
12 Diazepam
–
* P \ 0.05 when compared
to control group
–
100
100
(anhydrous condition was maintained by guard tube) fol-
lowed by addition of succinic anhydride (0.10 mol) in
small quantities with continuous stirring & heating at
80 °C for 4 h. Contents after leaving overnight at room
temperature were poured into ice-cold hydrochloric acid
(2.5 % v/v) followed by steam distillation. After comple-
tion of the distillation the aqueous solution was concen-
trated to a small volume by evaporating on a water bath to
obtain the crude compound. It was purified by dissolving in
5 % w/v sodium bicarbonate solution followed by extrac-
tion with ether. The aqueous layer on acidification with
dilute hydrochloric acid gave anisoyl propionic acid, and
this was then crystallized from aqueous ethanol (Khan and
Siddiqui, 2000).
55.8 (1C, O–CH3), 29.1 (1C, CH2–(=O)–O), 35.0 (1C,
CH2–(=O)–C), ms: m/z 208.07 (M?), ms: m/z 208.07 (M?),
Anal. Calcd. For C11H12O4: C, 63.45; H, 5.81; o, 30.74.
Found: C, 63.52; H, 5.79; O, 30.71.
6-(Anisoyl)2,3,4,5-tetra-hydro-pyridazine-3-ones (1b)
The above acid anisoyl propionic acid 1a (0.1 mol) was
refluxed for 6 h with hydrazine hydrate (1 ml) in methanol
(25 ml) containing sodium acetate (0.5 g). After comple-
tion of the reaction the contents were concentrated and then
poured into ice-cold water. A solid was separated, which
was filtered and crystallized from ethanol to get compound
1b (Siddiqui and Dogra, 2001; Siddiqui et al., 2004).
White powder (65 %); mp 175 °C; Rf value; 0.71, IR:
White crystals (55 %); mp 155 °C; Rf value; 0.66, IR:
750, 1,275, 1,670, 1,027, 1,666–1,698, 1,390 cm-1.1H
NMR, d ppm: 3.26 (2H, t, CH2), 2.7 (2H, t, CH2), 7.96 (2H,
d, H-20,6), 6.97 (2H, d, H-30,5), 11.92 (OH), 3.86 (3H,s);
13C NMR (75 MHz, CDCl3) d: 114.4–168.0 (6C, Aryl),
1
1,275, 3,303.83, 1,540, 1,665, 1,490 cm-1. H NMR, 2.6
(2H, t, CH2), 2.94 (2H, t, CH2), 7.66 (2H, d, H-20,6), 6.93
(2H, d, H-30,5), 3.85 (3H, s), 7.27 (NH); 13C NMR
(75 MHz, CDCl3) d: 114.4–168.0 (6C, Aryl), 55.8 (1C, O–
123

Med Chem Res
CH3), 29.1 (1C, CH2–(=O)–O), 35.0 (1C, CH2–(=O)–C),
167.7 (1C, Pyridazinone and amide), 146.5 (1C, Imine).
ms: m/z 204.09 (M?), Anal. Calcd. For C11H12N2O2: C,
64.06; H, 6.84; o, 15.52; N, 13.58. Found: C, 64.02; H,
6.78; O, 15.54; N, 13.51.
O, 14.31; N, 20.88; Found: C, 50.38; H, 5.10; O, 14.29; N,
20.85.
2-[(5-Amino-1,3,4-thiadiazole-2yl)methyl]-6-(4-
methoxyphenyl)-4,5-dihydropyridazin-3(2H)-one
derivatives (1e)
6-Oxo-3p-(anisoyl)-5,6-dihydro-4H-pyridazine-1yl)-
acetic acid ethyl ester (1c)
(0.08 mol) of compound 1d was added in conc. H2SO4
(10 ml) in a Petri dish and kept overnight at room tem-
perature, neutralized with ammonia and extracted with
ether. The ether was distilled off and the product so
obtained was crystallized from 80 % ethanol to get yel-
lowish leaflets of compound 1e (Namdeo et al., 2009).
Brown powder (85 %); mp 70 °C, Rf value; 0.82, IR:
The appropriate pyridazinone 1b (0.02 mol) was added to
an ethanolic solution (50 ml) of sodium (0.46 g, 0.02 g
atom). This mixture was first refluxed for 30 min, then
ethyl bromoacetate (0.02 mol) was added by drops to the
cooled solution, which was then refluxed for 24 h and then
the mixture was concentrated to a small volume. The res-
idue was triturated with diisopropyl ether and the solid
which was formed was collected by filtration and the dried
compound was recrystallized from a mixture of ethanol and
water (50:50) to get the compound 1c (Rubut et al., 1990).
Yellow powder (75 %); mp 180 °C; Rf value; 0.58, IR:
1
2,918, 3,028, 1,008, 1,698, 1,575, 3,319, 613 cm-1. H
NMR, d ppm: 6.99 (d, 1H, Ar–H, J = 7.5 Hz), 7.95 (d, 1H,
Ar–H, J = 1.5 Hz), 2.59 (2.61 (t, 2H, CH2, J = 7.1 Hz),
3.19 (t, 2H, CH2, J = 7.1 Hz), 4.60 (s, 2H, N–CH2). 13C
NMR (75 MHz, CDCl3) d: 114.4–168 (6C, Ar), 162.4 (1C,
Pyridazinone and amide), 146.5 (1C, Imine), 32.8 (2C,
–(C=O)–N–N), 24.4 (2C, –C=N), 55.8 (1C, O–CH3), 51.0
(2C, CH2–N), 161.4 (1C, Thiadiazole C–NH2), 168.0 (1C,
Thiadiazole –C–S). ms: m/z 335.7 (M?), Anal. Calcd. For
C15H14N5O2S: C, 52.98; H, 4.76; O, 10.08; N, 22.07; S,
10.10 Found: C, 52.90; H, 4.8; O, 10.05.; N, 22.83; S,
10.18.
1
760, 1,280, 1,664, 1,490, 1,740 cm-1. H NMR (d ppm)
2.59 (2H, t, CH2), 2.95 (2H, t, CH2), 7.69 (2H, d, H-20,6),
6.94 (2H, d, H-30,5), 3.94 (2H,s), 3.84 (2H,m), 1.28 (3H,t);
13C NMR (75 MHz, CDCl3) d: 114.4–168.0 (6C, Aryl),
55.8 (1C,O–CH3), 29.1 (1C, CH2–(=O)–O), 35.0 (1C,
CH2–(=O)–C), 167.7 (1C, Pyridazinone and amide), 146.5
(1C, Imine), 52.3 (1C, CH2–C(=O)–O), 61.0 (1C, CH2–O–
C=O), 14.5 (aliphatic-C), 169.5 (1C, Carboxyl). ms: m/z
295.2 (M?), Anal. Calcd. For C15H18N2O4: C, 62.06; H,
6.25; O, 22.04; N, 9.65. Found: C, 62.02; H, 6.22; O, 22.00;
N, 9.60.
b-Ethyl-phenyl-propionic acid (2a)
White crystals (55 %); mp 110 °C; Rf value; 0.69, IR:
1
709.76, 2,781,1,670, 1,309 cm-1. H NMR, d ppm: 2.83
(2H, t, CH2), 3.30 (2H, t, CH2), 7.91 (2H, d, H-20,6), 7.29
(2H, d, H-30,5), 2.73 (2H, m, CH2), 1.25 (3H, t, CH3), 11.0
(1H, O–H). 13C NMR (75 MHz, CDCl3) d: 127.6–148.7
(6C, Aryl), 28.2 (1C, CH2–CH3), 14.5 (1C, CH2–CH3),
29.1 (1C, CH2–(=O)–O), 32.9 (1C, CH2–(=O)–C). ms: m/z
205.1 (M?), Anal. Calcd. For C12H14O3: C, 69.88; H, 6.84;
O, 23.27; Found: C, 69.85; H, 6.7; O, 23.25.
2-(2-(3-(4-Methoxyphenyl)-6-oxo-5,6-
dihydropyridazine-1(4H)-yl)acetyl)hydrazine-1(4H)-
yl)acetyl) hydrazinecarbothioamide (1d)
Take 1c (0.08 mol) 22 and 7 g (0.08 mol) of thiosemi-
carbazide in ethanol 50 ml and stir the mixture for 6 h and
then reflux on steam bath for 3 h. The excess of solvent
was removed under reduced pressure and recrystallized
from chloroform/hexane (3;1) v/v to yield yellow crystals
of compound 1d (Namdeo et al., 2009).
6-(Ethyl phenyl)2,3,4,5 tetra-hydro-pyridazine-3-one
(2b)
White powder (80 %); mp 125 °C; Rf value; 0.73, IR:
White powder (60 %); mp 150 °C, Rf value; 0.72, IR:
1
1
cm-1 2,918, 3,099, 1,022, 1,664, 1,612, 3,318, 1,100, H
700.11, 2,987, 3,303, 1,614, 1,666, 1,483 cm-1. H NMR,
NMR, (d ppm) 7.6 (d, 1H, Ar–H, J = 7.5 Hz), 6.85 (d, 1H,
Ar–H, J = 1.5 Hz), 3.7 (s, 3H, O–CH3,), 2.88 (t, 2H, CH2,
J = 7.1 Hz), 2.44 (t, 2H, CH2, J = 7.1 Hz), 3.2 (s, 2H,
CH2), 7.86 (s, 1H, NH); 13C NMR (75 MHz, CDCl3) d:
114.4–168 (6C, Ar),162.4 (1C, Pyridazinone and amide),
146.5 (1C, Imine), 32.5 (2C, –(C=O)–N–N), 24.4 (2C,
–C=N), 182.5 (1C, Thioamide), 170.3 (1C, Amide), 55.8
(1C, O–CH3), 57.6 (1C, –(C=O)–N–N). ms: m/z 335.7
(M?), Anal. Calcd. For C14H17N5O3S: C, 50.41; H, 5.11;
d ppm: 2.29 (2H, t, CH2), 2.59 (2H, t, CH2) 7.66 (2H, d,
H-20,6), 7.25 (2H, d, H-30,5), 1.24 (3H, t, CH3), 2.68 (2H,
m, CH2), 7.85 (NH,s). 13C NMR (75 MHz, CDCl3) d:
127.6–148.7 (6C, Aryl), 28.2 (1C, CH2–CH3), 14.5 (1C,
CH2–CH3), 35.0 (1C, CH2–(=O)–N–N), 24.1 (1C, CH2–
C=N), 146.6 (1C, C=N), 167.7 (1C, Pyridazinone and
amide), 146.5 (1C, Imine). ms: m/z 202.11 (M?), Anal.
Calcd. For C12H14 N2 O: C, 70.56; H, 7.90; O, 7.83; N,
13.17 Found: C, 70.52; H, 7.8; O, 7.83.; N, 13.15.
123

Med Chem Res
6-Oxo-3-(ethyl phenyl)-5,6-dihydro-4H-pyridazine-1yl)
-acetic-acid-ethyl-ester (2c)
10.17; Found: C, 57.09; H, 5.41; O, 5.02; N, 22.20; S,
10.26.
Yellow powder (75 %); mp 116–120 °C, Rf value; 0.68,
IR: 731, 2,926.2 C–H, 3,212 N–H, 1,280 C–N, 1,505,
1,723.8 cm-1. 1H NMR, d ppm: 2.98 (2H, t, CH2), 2.6 (2H,
t, CH2), 7.64 (2H, d, H-20,6), 7.26 (2H, d, H-30,5), 2.69 (2H,
m, CH2), 1.25 (3H, t, CH3), 3.94 (2H, s, CH2), 4.17 (2H, m,
CH2), 1.4 (3H, t, CH3). 13C NMR (75 MHz, CDCl3) d:
127.6–146.7 (6C, Aryl), 28.2 (1C, CH2–CH3), 14.5 (1C,
CH2–CH3), 32.5 (1C, CH2–(=O)–N–N), 24.4 (1C,
CH2–C=N), 146.5 (1C, C=N), 162.4 (1C, Pyridazinone and
amide), 146.5 (1C, Imine), 52.3 (1C, CH2–C(=O)–O), 61.0
(1C, CH2–O–C=O), 14.5 (aliphatic-C), 169.5 (1C, Car-
boxyl). ms: m/z 288.15 (M?), Anal. Calcd. For C16H20 N2
O3: C, 66.65; H, 6.99; O, 16.65; N, 9.72 Found: C, 66.62;
H, 6.90; O, 16.61; N, 9.71.
b-Toloyl-propionic acid (3a)
White crystals (59 %); mp 125–130 °C, Rf value; 0.79, IR:
2,750, 1,600, 1,250, 1,460 cm-1, 1H NMR, d ppm: 2.4 (2H,
t, CH2, J = 10 (7.1), 2.8 (2H, t, CH2, J = 9 (7.1), 7.8 (CH2,
d, H-20,6), 7.2 (CH2, d, H-30,5), 2.4 (3H, s), 11 (1H, O–H).
13C NMR (75 MHz, CDCl3) d: 128.7–142.8 (6C, Aryl),
21.3 (1C, CH2–CH3), 29.1 (1C, CH2–(=O)–O), 32.9 (1C,
CH2–(=O)–C), 198.3 (1C, Carbonyl). ms: m/z 192.08
(M?), Anal. Calcd. For C11H12O3: C, 68.74; H, 6.29; O,
24.97; Found: C, 68.71; H, 6.25; O, 24.94.
6-(Toloyl)2,3,4,5-tetra-hydro-pyridazine-3-ones (3b)
White powder (66 %); mp 140–144 °C, Rf value -0.65,
1
IR: 2,384.64, 3,400, 1,623, 1,490, 720 cm-1. H NMR, d
2-(3-(4-Ethylphenyl)-6-oxo-5,6-dihydropyridazin-
1(4H)-l)acetyl)hydrazinecarbothioamide (2d)
ppm: 2.9(2H, t, CH2 J = 7.1), 2.6(2H, t, CH2 J = 7.1), 7.6
(2H, d, H-20,6 J = 7.5), 7.21 (2H, d, H-30,5 J = 7.5), 2.34
(3H, s), 7.269 (NH, s). 13C NMR (75 MHz, CDCl3) d:
127.0–140.7 (6C, Aryl), 21.3 (1C, CH2–CH3), 35.0 (1C,
CH2–(=O)–N–N), 24.1 (1C, CH2–C=N), 167.7 (1C, Py-
ridazinone and amide), 146.5 (1C, Imine). ms: m/z 188.09
(M?), Anal. Calcd. For C11H12 N2 O: C, 69.45; H, 7.42; O,
8.41; N, 14.73 Found: C, 69.42; H, 7.40; O, 8.39; N, 14.71.
White powder (85 %); mp 136–140 °C, Rf value 0.81, IR:
2,928, 3,106, 1,022, 1,666, 1,614, 3,212, 622, 3,323 cm-1
.
1H NMR, d ppm: 6.9 (d, 1H, Ar–H, J = 5 (1.5 Hz), 2
(7.5 Hz), 7.23 (d, 1H, Ar–H, J = 3 (7.5 Hz), 6 (1.5 Hz),
2.65 (t, 2H, CH2, J = (11) 7.1 Hz), 2.51 (q, 2H J = 15
(8.0 Hz), 1.25 (t, 3H, J = 14 (8.0 Hz), 3.82 (s, 2H, CH2
J = (11) 7.1 Hz), 7.65 (d, 1H,), 2.56 (s, NH), 7.95 (s, 2H,
NH2). 13C NMR (75 MHz, CDCl3) d: 127.6–146.7 (6C,
Aryl), 28.2 (1C, CH2–CH3), 14.5 (1C, CH2–CH3), 32.5
(1C, CH2–(=O)–N–N), 24.4 (1C, CH2–C=N),146.5 (1C,
C=N), 162.4 (1C, Pyridazinone and amide), 146.5 (1C,
Imines), 57.6 (1C, CH2–C(=O)–N–N), 170.3 (1C, Amide),
182.5 (1C, Thioamide). ms: m/z 333.13 (M?), Anal. Calcd.
For C15H19 N5O2S: C, 54.15; H, 5.36; O, 9.18; N, 21.10
Found: C, 54.11; H, 5.32; O, 9.16; N, 21.08.
6-Oxo-3p-(toloyl)-5,6-dihydro-4H-pyridazine-1yl)-
acetic acid-ethyl ester (3c)
Yellow powder (72 %); mp 148–152 °C, Rf value; 0.77, IR:
1
720, 2,389.64, 1,250, 1,490 cm-1. H NMR, d ppm: 2.57
(2H, t, CH2), 2.95 (2H, t, CH2), 7.6 (2H, d, H-20,6, J = 2
(7.5), 7.2 (2H, d, H-30,5, J = 3 (7.5)), 3.9 (2H, s), 3.7 (2H,
m), 1.23 (3H, t). 13C NMR (75 MHz, CDCl3) d:
127.0–140.7 (6C, Aryl), 21.3 (1C, CH2–CH3), 35.0 (1C,
CH2–(=O)–N–N), 24.1 (1C, CH2–C=N), 162.4 (1C, Pyrid-
azinone and amide), 146.5 (1C, Imine), 52.3 (1C, CH2–
C(=O)–O), 61.0 (1C, CH2–O–C=O), 14.1 (aliphatic-C),
169.5 (1C, Carboxyl). ms: m/z 273.2 (M?), Anal. Calcd. For
C15H18 N2 O3: C, 65.68; H, 6.61; O, 17.50; N, 10.21 Found:
C, 65.66; H, 6.59; O, 17.48; N, 10.20.
2-((5-Amino-1,3,4-thiadiazol-2-yl)methyl)-6-(4-
ethylphenyl)-4,5-dihydropyridazin-3(2H)-one (2e)
Brown powder (80 %); mp 85–88 °C, Rf value; 0.92, IR
cm-1 2,915, 3,063, 1,678, 1,573, 613, 3,351, H NMR, d
1
ppm: 7.3 (d,1H, Ar–H, J = 6 (1.5 Hz), 3 (7.5 Hz), 7.9 (d,
1H, Ar–H, J = 2 (7.5 Hz), 5 (1.5 Hz), 3.2 (t, 2H, J = (11)
7.1 Hz), 2.6 (t, 2H, J = (12) 7.1 Hz), 2.7 (q, 2H, J = (22)
8.0 Hz), 3.8 (s, 2H), 1.2 (t, 3H J = (21), 8.0 Hz), 6.9 (s,
2H, NH2). 13C NMR (75 MHz, CDCl3) d: 127.6–146.7
(6C, Aryl), 28.2 (1C, CH2–CH3), 14.5 (1C, CH2–CH3),
32.5 (1C, CH2–(=O)–N–N), 24.4 (1C, CH2–C=N), 146.5
(1C, C=N), 162.4 (1C, Pyridazinone and amide), 51.0 (1C,
CH2–N), 168.0 (1C, Thiadiazole, C–S), 161.6 (1C, Thia-
diazole, C–NH2). ms: m/z 315.12 (M?), Anal. Calcd. For
C15H17 N5OS: C, 57.12; H, 5.43; O, 5.07; N, 22.21; S,
2-(2-(6-Oxo-3-p-tolyl-5,6-dihydropyridazin-
1(4H)yl)acetyl)hydrazinecarbothioamide (3d)
White powder (80 %); mp 135–140 °C, Rf value; 0.82, IR:
1
730, 2,385, 1,185, 1,490, 1,545, 3,257 cm-1. H NMR, d
ppm: 2.58 (2H, t, CH2, J = 11 (7.1), 2.91 (2H, t, CH2,
J = 12 (7.1), 7.6 (2H, d, H-20,6, J = 2 (7.5), 7.2 (2H, d,
H-30,5, J = 3 (7.5), 2.4 (3H, s), 3.95 (2H, s), 8.09 (NH), 2.2
(NH2). 13C NMR (75 MHz, CDCl3) d: 127.0–140.7 (6C,
123

Med Chem Res
6-Oxo-3p-(aroyl)-5,6-dihydro-4H-pyridazine-1yl)-
acetic acid ethyl ester (4c)
Aryl), 21.3 (1C, CH2–CH3), 35.0 (1C, CH2–(=O)–N–N),
24.1 (1C, CH2–C=N), 162.4 (1C, Pyridazinone and amide),
146.5 (1C, Imine), 57.6 (1C, CH2–C(=O)–N–N), 170.3
(1C, Amide), 182.5 (1C, Thioamide). ms: m/z 319.11 (M?),
Anal. Calcd. For C14H17 N5O2S: C, 52.65; H, 5.38; O,
10.02; N, 21.93 Found: C, 52.58; H, 5.32; O, 10.00; N,
21.91.
Yellow powder (70 %); mp 155 °C, Rf value; 0.65, IR:
1
2,929, 3,066, 1,595, 1,665.1,756,1,205 cm-1. H NMR, d
ppm: 2.44 (2H, t, CH2, J = 12 (7.1), 2.92 (2H, t, CH2,
J = 11 (7.1), 7.75 (2H, d, H-20,6, J = 2 (7.5), 7.40 (2H, d,
H-30,5, J = 3 (7.5), 3.5 (2H, m), 1.9 (3H, t). 13C NMR
(75 MHz, CDCl3) d: 128.2–136.4 (6C, Aryl), 35.0 (1C,
CH2–(=O)–N–N), 24.1 (1C, CH2–C=N), 167.7 (1C, Py-
ridazinone and amide), 146.5 (1C, Imine), 52.3 (1C, CH2–
C(=O)–O), 61.0 (1C, CH2–O–C=O), 14.1 (aliphatic-C),
169.5 (1C, Carboxyl). ms: m/z 263.1 (M?), Anal. Calcd.
For C14H16 N2O3: C, 64.60; H, 6.20; O, 18.44; N, 10.76
Found: C, 64.60; H, 6.20;O, 18.44; N, 10.76.
2-((5-Amino-1,3,4-thiadiazol-2-yl)methyl)-6-p-tolyl-
4,5-dihydropyridazin-3(2H)-one (3e)
Brown powder (75 %); mp 90 °C, Rf value; 0.94, IR:
1
2,925, 3,029,1,682, 1,607, 3,352, 613 cm-1. H NMR, d
ppm: 7.85 (d, 1H, Ar–H, J = 5 (1.5 Hz), 2 (7.5 Hz), 7.30
(d, 1H, Ar–H, J = 3 (7.5 Hz), 6 (1.5 Hz), 2.41 (s, 3H,
CH3), 2.64 (t, 2H, CH2 J = 12 (7.1 Hz), 3.21 (t, 2H, CH2
J = 11 (7.1 Hz), 2.57 (s, 2H, CH2), 7.98 (s, 2H, NH2). 13
C
2-(2-(6-Oxo-3-phenyl-5,6-dihydropyridazine-
NMR (75 MHz, CDCl3) d: 127.0–140.7 (6C, Aryl), 21.3
(1C, CH2–CH3), 35.0 (1C, CH2–(=O)–N–N), 24.1 (1C,
CH2–C=N), 162.4 (1C, Pyridazinone and amide), 146.5
(1C, Imine), 51.0 (1C, CH2–N), 168.0 (1C, Thiadiazole, C–
S), 161.6 (1C, Thiadiazole, C–NH2). ms: m/z 301.10 (M?),
Anal. Calcd. For C14H17 N5OS: C, 55.80; H, 5.02; O, 5.31;
N, 23.24; S, 10.64 Found: C, 55.75; H, 5.00; O, 5.28; N,
23.20; S, 10.70.
1(4H)yl)acetyl) hydrazinecarbothioamide (4d)
White powder (80 %); mp 115–122 °C, Rf value; 0.74, IR
(KBr)- 2,944, 3,098 1,676, 1,676 1,618 (C=N), 3,206, 685,
3,456 cm-1. 1H NMR, d ppm: 7.36 (t, 1H, Ar–H-206 J = 5
(7.5 Hz), 1 (1.5 Hz, 2 (1.5 Hz), 7.70 (d, 1H, Ar–H-305, J = 2
(7.5 Hz), 5 (7.5 Hz, 1 (1.5 Hz), 2.43 (t, 2H, CH2 J = 12
(7.1 Hz), 2.92 (t, 2H, CH2 J = 11 (7.1 Hz), 3.5 (s, 2H, CH2),
8.15 (s, 2H, NH2), 3.38 (s, 2H, CH2), 2.50 (s, NH). 13C NMR
(75 MHz, CDCl3) d: 128.2–136.4 (6C, Aryl), 35.0 (1C, CH2–
(=O)–N–N), 24.1 (1C, CH2–C=N), 167.7 (1C, Pyridazinone
and amide), 146.5 (1C, Imine), 57.6 (1C, CH2–C(=O)–N–N),
170.3 (1C, Amide), 182.5 (1C, Thioamide). ms: m/z 305.1
(M?), Anal. Calcd. For C13H15 N5O2S: C, 51.13; H, 4.95; O,
10.50; N, 12.94 Found: C, 51.10; H, 4.92; O, 10.49; N, 12.93.
b-Aroyl-propionic acid (4a)
White crystals (72 %); mp 129 °C, Rf value; 0.80, IR:
1
2,925, 1,684, 1,250, 1,460 cm-1. H NMR, d ppm: 3.25
(2H, t, CH2, J = 9 (7.1), 2.65 (2H, t, CH2, J = 10 (7.1), 7.96
(CH2, d, H-20,6, J = 6 (7.6), 7.53 (CH2, d, H-30,5, J = 5
(7.5), 7.62 (H, d), 11 (1H, O–H). 13C NMR (75 MHz,
CDCl3) d: 128.6–136.4 (6C, Aryl), 32.9 (1C, CH2–(=O)–C),
198.3 (1C, Carbonyl), 29.1 (1C, CH2–(=O)–O), 177.3 (1C,
Carboxyl). ms: m/z 179.0 (M?), Anal. Calcd. For C10H10O3:
C, 67.41; H, 5.66; O, 26.94; Found: C, 67.38; H, 5.62; O,
26.91.
2-((5-Amino-1,3,4-thiadiazol-2-yl)methyl)-6-phenyl-
4,5-dihydropyridazin-3(2H)-one (4e)
Brown powder (78 %); mp 98–100 °C, Rf value; 0.89, IR:
2,924, 3,028,1,683, 1,594, 614, 3,350 cm-1. 1H NMR, d
ppm: 7.98 (t,1H, Ar–H-305 J = 6 (7.5 Hz), 3 (1.5 Hz, 1
(1.5 Hz), 7.52 (t,1H, Ar–H-4 J = 2 (7.5 Hz), 5 (1.5 Hz, 1
(1.5 Hz), 7.65 (d, 1H, Ar–H-206) 3.24 (t, 2H, CH2 J = 12
(7.1 Hz), 2.63 (t, 2H, CH2 J = 11 (7.1 Hz), 3.63 (2H, s,
CH2), 2.5 (2H, s, NH2). 13C NMR (75 MHz, CDCl3) d:
128.2–136.4 (6C, Aryl), 35.0 (1C, CH2–(=O)–N–N), 24.1
(1C, CH2–C=N), 167.7 (1C, Pyridazinone and amide),
146.5 (1C, Imine), 51.0 (1C, CH2–N), 168.0 (1C, Thiadi-
azole, C–S), 161.8 (1C, Thiadiazole, C–NH2). ms: m/z
289.0 (M?), Anal. Calcd. For C13H15 N5OS: C, 54.34; H,
4.56; O, 5.57; N, 24.37; S, 11.16 Found: C, 54.31; H, 4.54;
O, 5.54; N, 24.35; S, 11.20.
6-(Aroyl)2,3,4,5-tetra-hydro-pyridazine-3-ones (4b)
White powder (68 %); mp 140 °C, Rf value; 0.62, IR:
2,384.64, 3,400, 1,665, 1,490, 720 cm-1. 1H NMR (d ppm)
2.92 (2H, t, CH2 J = 11 (7.1), 2.43 (2H, t, CH2 J = 12
(7.1), 7.70 (2H, d, H-20,6, J = 6 (7.5), 7.35 (2H, d, H-30,5
J = 6 (7.5), 8.1 (NH,s). 13C NMR (75 MHz, CDCl3) d:
128.2–136.4 (6C, Aryl), 35.0 (1C, CH2–(=O)–N–N), 24.1
(1C, CH2–C=N), 167.7 (1C, Pyridazinone and amide),
146.5 (1C, Imine) ms: m/z 175.1 (M?), Anal. Calcd. For
C10H10N2O3: C, 68.16; H, 6.86; O, 9.08; N, 15.90 Found:
C, 68.14; H, 6.83;, O9.00; N, 15.88.
123

Med Chem Res
b-Chloro propionic acid (5a)
C13H14Cl N5O2S: C, 45.94; H, 4.15; O, 9.42; N, 20.61
Found: C, 45.90; H, 4.09; O, 9.30; N, 20.59.
White crystals (70 %); mp 120–125 °C, Rf value; 0.81, IR:
1
2,586, 1,680, 1,250, 3,060, 1,460 cm-1, H NMR, d ppm:
2-((5-Amino-1,3,4-thiadiazol-2-yl)methyl)-6-(4-
3.25 (2H, t, CH2, J = 9 (7.1 Hz), 2.65 (2H, t, CH2, J = 10
(7.1 Hz), 7.9 (CH2, d, H-20,6, J = 2 (7.5 Hz)), 7.4 (CH2, d,
H-30,5, J = 3 (7.5 Hz)), d ppm. 13C NMR (75 MHz,
CDCl3) d: 128.7–138.7 (6C, Cl-aryl), 32.9 (1C, CH2–(=O)–
C), 29.1 (1C, CH2–(=O)–O), 198.3 (1C, Carbonyl), 29.1
(1C, CH2–(=O)–O), 177.3 (1C, Carboxyl). ms: m/z 212.02
(M?), Anal. Calcd. For C10H9ClO3: C, 56.49; H, 4.27; O,
22.57; Found: C, 56.28; H, 4.20; O, 22.5.
chlorophenyl)-4,5-dihydropyridazin-3(2H)-one (5e)
Brown powder (80 %); mp 102–106 °C, Rf value; 0.90, Yield
–80 %., IR: 2,927, 3,060, 1,680, 1,590, 658, 3,348 cm-1. 1H
NMR (d ppm), 7.51 (d, 1H, Ar–H, J = 3 (7.5 Hz), 6 (1.5 Hz),
7.98 (d, 1H, Ar–H, J = 2 (7.5 Hz), 5 (1.5 Hz), 3.25 (t, 2H,
CH2 J = 12 (7.1 Hz), 2.6 (t, 2H, CH2 J = 13 (7.1 Hz), 2.5 (s,
2H, CH2). 13C NMR (75 MHz, CDCl3) d: 128.7–138.7 (6C,
Cl-aryl), 35.0 (1C, CH2–(=O)–N–N), 24.1 (1C, CH2–C=N),
167.7 (1C, Pyridazinone and amide), 146.5 (1C, Imine), 51.0
(1C, CH2–N), 168.0 (1C, Thiadiazole, C–S), 161.8 (1C,
Thiadiazole, C–NH2). ms: m/z 321.05 (M?), Anal. Calcd. For
C13H14 ClN5OS: C, 48.52; H, 3.76; O, 4.97; N, 21.76; S, 9.96
Found: C, 48.50; H, 3.72; O, 4.95; N, 21.72; S, 9.89.
6-(Chloro)2,3,4,5-tetra-hydro-pyridazine-3-ones (5b)
White powder (70 %); mp 178–182 °C, Rf value; 0.79, IR;
2,384.64, 3,400, 1,674 1,250, 1,490, 720 cm-1. 1H NMR, d
ppm: 2.92 (2H, t, CH2 J = 12 (7.1 Hz), 2.45 (2H, t, CH2
J = 13 (7.1 Hz), 7.6 (2H, d, H-20,6 J = (37.5 Hz), 7.21 (2H,
d, H-30,5 J = 2 (7.5 Hz), 2.34 7.269 (NH, s). 13C NMR
(75 MHz, CDCl3) d: 128.7–138.7 (6C, Cl-aryl), 35.0 (1C,
CH2–(=O)–N–N), 24.1 (1C, CH2–C=N), 167.7 (1C, Pyrid-
azinone and amide), 146.5 (1C, Imine). ms: m/z 209.1 (M?),
Anal. Calcd. For C10H9ClN2O3: C, 57.57; H, 4.35; O, 7.67;
N, 13.43 Found: C, 57.51; H, 4.30; O, 7.62; N, 13.44.
Pharmacology
Experimental animals
Anticonvulsant activity of the synthesized compounds was
determined by their ability to provide protection from
convulsions in albino mice. Swiss Male albino mice of
weight (25–30 g) (n = 5) were used in the experiments of
MES-induced seizures. Female animals were excluded
because of the fact that estrus cycle influence the seizure
threshold. Animals were housed in polypropylene cage
with dust-free rice husk as bedding material under labo-
ratory condition with controlled environment of tempera-
ture 25 2 °C and 12 h light/dark schedule, humidity
(60 10 %) and before subjecting them to experimenta-
tion, the animals were given a week of time to get accli-
matized with laboratory condition. They had free access to
standard mouse diet and tap water except during the
experiment. On the day of the experiment, animals were
transferred to individual cases randomly and allowed to
acclimatize for 30 min before injection of drug or vehicle.
All the experimental procedure and protocols used in this
study were reviewed by the Institutional Animal Ethical
Committee (IAEC) of the institute with reference no.BU/
Pharm/IAEC/11/031 (Approved by CPCSEA Regd No.
716/02/a/CPCSEA).
6-Oxo-3p-(chloro)-5, 6-dihydro-4H-pyridazine-1yl)-
acetic acid ethyl ester (5c)
Yellow powder (74 %); mp 170 °C, Rf value; 0.71, IR:
1
2,935, 3,105, 1,677, 1,611, 1,224 cm-1; H NMR, d ppm:
2.43 (2H, t, CH2), 2.92 (2H, t, CH2), 7.98 (2H, d, H-20,6),
7.52 (2H, d, H-30,5), 3.9 (2H, s), 3.7 (2H, m), 1.23 (3H, t).
13C NMR (75 MHz, CDCl3) d: 128.7–138.7 (6C, Cl-aryl),
35.0 (1C, CH2–(=O)–N–N), 24.1 (1C, CH2–C=N), 167.7
(1C, Pyridazinone and amide), 146.5 (1C, Imine), 52.3 (1C,
CH2–C(=O)–O), 61.0 (1C, CH2–O–C=O), 14.1 (aliphatic-
C),169.5 (1C, Carboxyl). ms: m/z 293.3 (M?), Anal. Calcd.
For C14H15Cl N2O3: C, 57.05; H, 5.13; O, 16.29; N, 9.50
Found: C, 57.00; H, 5.11; O, 16.20; N, 9.49.
2-(2-(3-(4-Chlorophenyl)-6-oxo-5,6-dihydropyridazine-
1(4H)yl)acetyl)hydrazinecarbothioamide (5d)
White powder (74 %); mp 170 °C, Rf value; 0.71, Yield
–74 %., IR: 2,935, 3,109, 1,679, 1,611, 1,612, 3,332, 624,
1
3,322 cm-1. H NMR, d ppm: 7.40 (d, 1H, Ar–H, J = 3
(7.5 Hz), 6 (1.5 Hz), 7.74 (d, 1H, Ar–H, J = 2 (7.5 Hz), 5
(1.5 Hz). 13C NMR (75 MHz, CDCl3) d: 128.7–138.7 (6C,
Cl-aryl), 35.0 (1C, CH2–(=O)–N–N), 24.1 (1C, CH2–C=N),
167.7 (1C, Pyridazinone and amide), 146.5 (1C, Imine),
57.6 (1C, CH2–C(=O)–N–N), 170.3 (1C, Amide), 182.5
(1C, Thioamide). ms: m/z 339.06 (M?), Anal. Calcd. For
Acute toxicity study of the test compounds
Acute toxicity studies were performed on Swiss albino
mice according to Organization for Economic Co-opera-
tion and Development (OECD)—425 guidelines (OECD,
2001; Ghosh, 1984; Hardman et al., 1996), the animals
123

Med Chem Res
were kept fasted for 3 h with free access to water. The test
compounds were administered orally at a dose of 50 mg/
kg. The dose at which mortality was observed in two out of
three mice, was considered as toxic dose (OECD, 2001).
However, if no mortality was observed, the procedure was
repeated with higher dose such as 100, 300, 500, 2,000 mg/
kg body weight. Toxic manifestations like abnormal motor
activity, alteration in water or food intake, respiration,
sedation, and moribund stages were observed for 6 h and
mortality for 24 h. There was no mortality among the
graded dose groups of mice up to a dose of 2,000 mg/kg for
duration of 72 h. This finding probably suggests that the
test compounds are relatively safe or non-toxic in mice at
the doses used for this study.
anticonvulsant response the results of test compound were
compared with control and standard drug diazepam (Jatav
et al., 2008, 2010; Tandon and Gupta, 2005; Upmanyu
et al., 2009). The results are reported in Table 2.
Muscle relaxant activity
Rotarod test
The effect on motor co-ordination was assessed using
Rotarod apparatus (Biocraft Scientific System Pvt. Ltd.,
Agra, India). The Rotarod test has been performed to detect
the motor deficit in mice. Mice were placed on a horizontal
metal-coated rod with rubber (3 cm diameter) rotating at an
initial speed of 10 rpm/min. Terminal speed of the rod was
20 rpm in accelerated studies and rotational velocity of the
rod was linearly increased from 10 to 20 rpm within 20 s.
The time at which each animal was able to maintain its
balance walking on top of the rod was measured. Mice
were given two trials with a maximum time of 300 s and a
30–60 min intertribal rest interval (McIlwain et al., 2001),
before the beginning of all experiments, the riding ability
of the animals in the rotarod was checked. Thus, the mice
were initially put on a rotating rod, and mice that imme-
diately dropped off (within 30 s) were removed from the
experiment. Diazepam (4 mg/kg), test compounds, and
normal saline (10 ml/kg) were injected 30, 60, and 60 min,
respectively, before the test. The results are reported in
Table 3.
Anticonvulsant activity (MES model)
The anticonvulsant activities of the compounds were
determined against MES models (Kulkarni, 1999; Tandon
and Gupta, 2005), (Model: Techno Electro Convulsome-
ter). Phenytoin was considered as positive control drug
with anticonvulsant effect in MES models. Test com-
pounds and phenytoin were given intraperitoneally (IP) as
a freshly prepared solution in 5 % gum acacia to give a
concentration of 1 % (w/v), and 50 % sterile normal saline.
Control animals were injected with vehicle only. The
vehicle had no effect on the test system. Test compounds
injected IP at dose level 50 mg/kg body weight to different
groups of five mice each (Asif et al., 2011; Samanta et al.,
2011). Phenytoin was given at the dose of (25 mg/kg).
Thirty minutes after administration of the test compound,
mice were administered the convulsion stimulus (MES,
60 Hz, 37.2 mA for 0.25 s); the abolition of the hind limb
tonic extensor spasm was recorded as a measure of anti-
convulsant activity (Krall et al., 1978; Shaharyar and
Akhter, 2009); mice were considered protected according
to occurrence of HLTE (hind limb tonic extension) in MES
model. The results are reported in Table 1.
Traction test
Forepaws of a mouse were placed on a 15-cm-long twisted
wire rigidly supported and 20 cm above the table top.
Normal mice grasped the wire with forepaws and when
allowed to hang free, placed at least one hind foot on the
wire within 5 s. Inability to put up at least one hind foot
was considered failure to the traction (Villar et al., 1992).
The test was conducted on five groups of previously
screened mice, 1 h after given saline (10 ml/kg) or test
compound (mg/kg) and 30 min after the injection of
diazepam (4 mg/kg). The results are reported in Table 3.
Anticonvulsant activity (PTZ model)
Animals were divided into three groups each comprising 5
animals. One group was used for studying the effect of PTZ
alone (control) (dose 80 mg/kg, IP; a stock solution con-
taining 8 mg/ml of the drug was prepared and 1 ml/100 g
of body weight of mouse was injected) and second group
for studying the protective effect of diazepam. The test
compound (dose 100 mg/kg) and diazepam (dose 4 mg/Kg,
IP; prepare a suspension of diazepam in 1 % (w/v) gum
acacia containing 0.4 mg/ml of the drug and inject 1 ml/
100 g of body weight of mouse). After 30 min of PTZ
administration the onset and severity of convulsion were
noted. The delay onset of convulsion was referred to be
Statistical analysis of the data
The mean value of the readings (the time duration of the
tonic extensor phase of the seizures) was taken and stan-
dard error of mean was calculated. The percent protection
of the standard drug and the test samples were calculated
with respect to the control. Differences between means
were considered to be significant at P \ 0.05 compared to
control. Chi-square test was applied for the data expressed
in percentage using graph Pd prism. In this case the ‘‘P’’
123

Med Chem Res
Ghosh MN (1984) Fundamentals of experimental pharmacology.
Hilton and Company, Kolkata, pp 187–190
Hallot A, Brodin R, Merlier J, Brochard J, Chambon JP, Biziere K
(1986) Synthesis and activity of 6-aryl-3-(hydroxypolymethyle-
neamino) pyridazines in animal models of epilepsy. J Med Chem
29:369–375
Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Goodman AG
(eds) (1996) Goodman and Gilman’s the pharmacological basis
of therapeutics, 9th edn. McGraw-Hill, New York, p 471
Jatav V, Mishra P, Kashaw S (2008) Synthesis and CNS depressant
activity of some novel 3-[5-substituted 1,3,4-thiadiazole-2-yl]-2-
styryl quinazoline-4(3H)-ones. Eur J Med Chem 43:135–141
Javad KMM, Mahmood RM, Narjes O (2010) Anticonvulsant activity
of Teucrium polium against seizure induced by PTZ and MES in
mice. Iran J Pharm Res 9(4):395–401
Khan MSY, Siddiqui AA (2000) Synthesis and anti-inflammatory
activity of some 6-aryl-2,3,4,5-tetrahydro-3-pyridazinones.
Indian J Chem 39(B):614–619
Krall RJ, Penry JK, White BS, Kupferberg HJ, Swinyard EA (1978)
Antiepileptic drug development: II. Anticonvulsant drug screen-
ing. Epilepsia 19:409–428
Kulkarni SK (1999) Handbook of experimental pharmacology, 3rd
edn. Vallabh Prakashan, New Delhi, pp 131–134
Kwan P, Brodie MJ (2000) Early identification of refractory epilepsy.
Engl J Med 342(5):314–319
Kwan P, Brodie MJ (2004) Drug treatment of epilepsy: when does it
fail and how to optimize its use. CNS Spectr 9:110–119
McIlwain KL, Merriweather MY, Yuva-Paylor LA, Paylor R (2001)
The use of behavioral test batteries: effect of training history.
Physiol Behav 73:705–717
Namdeo KP, Singh VK, Prajapati SK (2009) Synthesis of some
2-(substituted)-5-[(N-benzotriazolomethyl)-1,3,4-thiadiazolyl]-
4-thiazolidinones for their anti-fungal activity. Indian J Pharm
Edu Res 43(3):266–271
OECD (2001) Guidelines for testing of chemicals. Acute oral
toxicity—up and down procedure, vol 425, pp 1–26
Oya U, Ayla B, Cenk A, Berna TM, Zafer G (2004) Synthesis and
evaluation of the anticonvulsant activities of some 5-(4-substi-
tutedbenzylidene)-6-methyl-4,5-dihydro pyridazine-3(2H)-ones.
FABAD J Pharm Sci 29:185–194
value less than 0.05 was considered significant when
compared to control.
Percentageinhibition ¼
½ðmeanof controlꢁmeanof treatmentÞ=ðmeancontrolÞꢂ
ꢃ 100
Conclusions
These 6(chloro)2,3,4,5 tetra hydro-pyridazine-3-ones
derivatives of pyridazinone have shown significant anti-
convulsant activity against MES and PTZ induced seizure
in albino mice after IP administration of 50 and 100 mg/kg
body weight dose, respectively. The potency order of the
test compound on the extensor phase and pentylenete-
trazole-induced seizures 5e [ 5d [ 1e [ 1d [ 3e [ 3d.
The compound 5d and 5e have shown maximum recovery
in practice so, the compound 5d and 5e may be regarded as
true anticonvulsant because a substance is known to pos-
sess anticonvulsant property if it reduces or abolishes the
extensor phase in MES convulsions and elapsed from the
injection of pentylenetetrazole to the first two myoclonic
jerk of the forelimbs and devoid of generalized convulsion
were considered to be protected in PTZ Model.
The title compounds thus may have immense potential
for contribution to human benefit. Scientific exploration of
further studies of more derivatives as well as on more
parameters is needed to elucidate the defined role of py-
ridazinones at the anticonvulsive levels.
Acknowledgments The authors are thankful to the Director, SAIF,
Punjab University, Chandigarh for IR, 1H NMR, 13C NMR, and Mass
spectroscopy, Authors are also thankful to the Director and Head of
the department, Institute of pharmacy, Bundelkhand University,
Jhansi for providing facilities to conduct the research work and Mrs.
Shalini pathak, department of pharmacology, for her support during
the pharmacological screening.
Perio A, Chambon JP, Calassi R, Heaulme M, Biziere K (1986)
Evaluation of two anticonvulsant amino-pyridazine derivatives
in the conflict test in rats. J Pharmacol Exp Ther 239:542–547
Pooja SB, Sharma PK, Rajesh KN (2009) Synthesis and anticonvul-
sant activity of pyridazinone derivatives. Int J Chem Tech Res
1:522–525
Rubut C, Coudert P, Rfouvelet B, Tronche P, Bastide P, Bastide
(1990) Anticonvulsant activity of 3-oxo-5-substituted bezylid-
ene-6-methyl-(4H)-2-pyridazinylacetamides and 2-pyridazinyla-
cetylhydrazides. J Chem Pharm Bull 38:3009–3013
References
Samanta KC, Asif M, Pooja Garg V, Sharma P, Singh R (2011)
Synthesis of different substituted pyridazinone derivatives and
their anticonvulsant activity E. J Chem 8(1):245–251
SamrJn EB, vanDuijn CM, Koch S, Hiidesmaa VK, Klepel H, Bardy
AH, Mannagetta GB, Deichl AW, Gaily E, Granstron ML,
Meinardi AH, Grobbee DE, Hofman A, Janz D, Lindhout D (1997)
Maternal use of antiepileptic drugs and the risk of major congenital
malfunction: a joint European prospective study of human
teratogenesis associated with material epilepsy. Epilepsia 38:981
Sander JWAS, Shorvon SD (1996) Epidermiology of epilepsies.
J Neurol Neurosurg Psychiatry 61:433–443
Asif M (2010) Anticonvulsant and comparative structure activity
relationship of pyridazine derivatives with currently clinically
used anticonvulsants. J Adv Sci Res 1:35–45
Asif M, Singh A, Lakshmayya (2011) Anticonvulsant activity of
4-(substituted benzylidene)-6-(3-nitrophenyl)-4,5-dihydro pyri-
dazin-3(2H)-ones against maximal electro shock induced sei-
zure. Middle-East J Sci Res 9(4):481–485
Brodie MJ, Dichter MA (1996) Antiepileptic drugs. New Engl J Med
334:168–175
Dichter MA, Brodie MJ (1996) New antiepileptic drugs. New Engl J
Med 334:1583–1590
¨
Schmidt D, Loscher W (2005) Drug resistance in epilepsy: putative
neurobiologic and clinical mechanisms. Epilepsia 46(6):858–877
Shaharyar M, Akhter MW (2009) Synthesis and anticonvulsant
activity of 2-(4-chlorophenyl) amino-5-(4-pyridyl)-1,3,4-oxadi-
azole. Acta Pol Pharm Drug Res 66(4):393–397
Edith GK, Ales W, Winfried K, Marija (2002) Synthesis and structure
investigations of potential sedative and anticonvulsant hydroxy-
and acetoxy-N-(3-oxobutyl)-pyrido [2,3-d]pyridazinones. Mon-
atshefte fur Chemie 133:1177–1185
123

Med Chem Res
Siddiqui AA, Dogra A (2001) Synthesis and anti-inflammatory
activity of some 2,6-diphenyl 2,3,4,5-tetrahydro-3-pyridazi-
nones. Indian J Heterocycl Chem 10:215–218
Upmanyu N, Gupta SP, Grover J, Mishra P (2009) Synthesis of
valproic acid derivatives and their evaluation for anticonvulsant
activity. Internet J Altern Med 7(1):1
Siddiqui AA, Kushnoor A, Wani SM (2004) Synthesis and anti-
inflammatory activity of 6-(substituted aryl)-2,3,4,5-tetrahydro-
3-thiopyridazinones. Indian J Heterocycl Chem 13:257–260
Siddiqui AA, Abdullah MM, Arora M, Islam M, Ahmad SR (2006)
Synthesis of novel pyridazinones possessing anticonvulsant
activity. Indian Drugs 43:790–794
Sivakumar RN, Anbalagan V, Gunasekaran JT, Leonard (2003)
Synthesis and anticonvulsant activity of novel 1-substituted-1,2-
dihydro-pyridazine-3,6-diones. Biol Pharm Bull 26:1407–1411
Smith MC, Bleck TP (1991) Convulsive disorder: toxicity of
anticonvulsants. Clin Neuropharmacol 14:97–115
Villar R, Laguna MR, Calleja JM, Cadavid I (1992) Effect of
Skeletonema statum extracts on the central nervous system.
Planta Med 38:398–403
Xu P, Wang SY, Chen Y, Liu WQ, Tao C (1991a) Studies on synthesis,
anticonvulsant activity and the structure-activity relationships of
6-(substituted phenyl)-3(2H)pyridazinones. Yao Xue Xue Bao
26:656–660
Xu P, Wang SY, Liu WQ (1991b) Studies on synthesis and
anticonvulsant activity of 3-GABAderivatives of 6-(substi-
tuted-phenyl) pyridazines. Yao Xue Xue Bao 26(9):650–655
Tandon VR, Gupta RK (2005) An experimental evaluation of
anticonvulsant activity of Vitex negundo. Indian J Physiol
Pharmacol 49A(2):199–205
123