Synthesis and anticonvulsant activity of some 2-(2-{1-[substituted phenyl]ethylidene}hydrazinyl)-4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine- 5-carbonitrile

Article abstract of DOI:10.3109/14756366.2011.618129

A series of dihydro-pyrimidine-5-carbonitrile derivatives (3-16) were synthesized and evaluated for their anticonvulsant activity against MES and scPTZ models. Motor impairment screening was carried out by rotarod test method and CNS depressant effect was determined by Porsolt's force swim pool method. Compounds 4 and 9 having p-substituted bromo and m-substituted nitro groups, respectively, were found to be most active showing activity both in MES and scPTZ screen at lower doses of 30 mgkg^-1 at 0.5h and 100 mgkg ^-1 at 4h. In the rotarod motor impairment screen, compound 4 did not show any motor impairment even at the maximum dose of 300 mgkg^-1; however, compound 9 showed motor impairment at 300 mgkg^-1 dose after 4.0h. The compounds were also tested for their CNS depression effect. The compounds 4 and 9 showed 41.38 and 43.44% increase in immobility time with respect to control. The pharmacophore hypothesis also fits best for compounds 4 and 9.

Full text of DOI:10.3109/14756366.2011.618129

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(6): 825–831
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.618129
ReseaRch aRtIcle
Synthesis and anticonvulsant activity of some 2-(2-{1-
[substituted phenyl]ethylidene}hydrazinyl)-4-(4-methoxy-
phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-carbonitrile
Mohammad Shaquiquzzaman, Suroor Ahmad Khan, Mohammad Amir, and
Mohammad Mumtaz Alam
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi, India
abstract
A series of dihydro-pyrimidine-5-carbonitrile derivatives (3–16) were synthesized and evaluated for their
anticonvulsant activity against MES and scPTZ models. Motor impairment screening was carried out by rotarod test
method and CNS depressant effect was determined by Porsolt’s force swim pool method. Compounds 4 and 9 having
p-substituted bromo and m-substituted nitro groups, respectively, were found to be most active showing activity
both in MES and scPTZ screen at lower doses of 30 mgkg−1 at 0.5h and 100 mgkg−1 at 4h. In the rotarod motor
impairment screen, compound 4 did not show any motor impairment even at the maximum dose of 300 mgkg−1;
however, compound 9 showed motor impairment at 300 mgkg−1 dose after 4.0h. The compounds were also tested
for their CNS depression effect. The compounds 4 and 9 showed 41.38 and 43.44% increase in immobility time with
respect to control. The pharmacophore hypothesis also fits best for compounds 4 and 9.
Keywords: Cyanopyrimidine derivatives, Schiff’s Base, Anticonvulsant, Neurotoxicity, CNS depression activity
Introduction
important anticonvulsant agents, Schiff’s bases have
played an important role.
Epilepsy is a common neurological disorder character-
ized by excessive temporary neuronal discharges result-
ing in unpredictable recurrence of unprovoked seizures.
At present, phenytoin is still a drug of choice after its dis-
covery over a period of more than half century1. Although
several new anticonvulsants are already in clinical use,
some types of seizure are still not adequately treated with
current therapy. Current drug therapy is accompanied
by numerous side effects including drowsiness, ataxia,
gastrointestinal disturbances, gingival hyperplasia, hir-
sutism and megaloblastic anaemia2,3. ese facts give the
field of anticonvulsant drug discovery high priority.
ese observations place new emphasis on the need as
well as search for alternative new and more effective anti-
convulsant agents with reduced neurotoxicity and CNS
depression effect. Among a wide variety of compounds
that have been explored for developing pharmaceutically
In recent years, Schiff’s bases have gained much atten-
tion due to its wide spectrum of biological activities like
anticonvulsant4,5, antidepressant6, anti-inflammatory7,
antimalarial8, antimycobacterial9 etc. Nifuroxazide
(Figure 1), an intestinal antiseptic10, is a Schiff’s base.
Cakir B. et al.11 have synthesized fifteen Schiff’s
bases and out of them N’-(4-fluoro-benzylidene)-2-(2-
oxobenzo[d]oxazol-3(2H)-yl) aceto-hydrazide (Figure 2)
was found to be a very active anticonvulsant.
According to the literature12, the compounds with at
least one aromatic ring (R), one electron donor (D) and a
hydrogenbondacceptorordonor(HBD)combinationwere
found to possess very good anticonvulsant activity. e gen-
erated pharmacophore using 3.1 module of Schrodinger-913
showed the presence of all crucial structural components
required for a compound to be an anticonvulsant.
Address for Correspondence: Mohammad Shaquiqquzzaman, Department of Pharmaceutical chemistry, Faculty of pharmacy, Jamia
Hamdard (Hamdard University), New Delhi-110 062, India. Tel: +91-11-26059681; 26059688, Ext. 5667; Mobile- +91-9990663405.
Fax: +91-11-26059686. E-mail: shaqiq@gmail.com
(Received 05 March 2011; revised 04 August 2011; accepted 23 August 2011)
825

826 M. Shaquiquzzaman et al.
Owing to the versatility of Schiff’s bases and the
support of pharmacophore modeling, we have syn-
thesized fourteen new 2-(2-{substituted benzylidene}
hydrazinyl)-4-(4-methoxyphenyl)-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrileas outlined in Scheme 1. All
the final compounds were structurally confirmed on the
basis of IR, 1H-NMR and mass spectral data and the final
synthesized compounds were evaluated for their anti-
convulsant effect in both MES14,15 and scPTZ method16.
ey were also tested for their side effects i.e. neurotoxic-
ity effect by rotarod test17 and CNS depression effect by
Porsolt’s swim pool test18.
Methods
Chemistry
Chemicals were purchased from Merck and Sigma-
Aldrich as ‘synthesis grade’ and used without further
purification. Melting points were determined in open
capillary tubes and are uncorrected. Elemental analy-
ses were performed on a Perkin-Elmer model 240 ana-
lyzer and found within 0.4% of theoretical values. e
IR spectra were measured as potassium bromide pel-
lets using a Buck Scientific M-500 Infrared spectropho-
1
tometer. H-NMR spectra were recorded in DMSO as a
solvent (using TMS as an internal standard). 13C-NMR
of compound 3 and 4 were recorded as a prototype
using DMSO as a solvent. e NMR and mass spectra
were recorded on Bruker avance-400 MHz and JEOL BX
102/DA-6000 mass spectrometers, respectively. Purity
of the compounds was checked by TLC on silica gel G
plates using toluene: ethyl acetate: formic acid (5:4:1)
as solvent system and the spots were located either
under ultra violet light or through exposure to iodine
vapors.
e synthesis of title compounds involves three steps
and is exemplified by the synthesis of compound 3.
Figure 1. Nifuroxazide.
Synthesis of 2-Mercapto-4-(4-methoxy-phenyl)-6-oxo-
1,6-dihydro-pyrimidine-5-carbonitrile (1)
Anisaldehyde (1 mmol), ethyl cyanoacetate (1 mmol)
and thiourea (1 mmol) were dissolved in absolute alco-
hol. Potassium carbonate (3 mmol) was added to this
reaction mixture and refluxed for 2 h. e solvent was
concentrated and poured into ice cold water with stir-
ring. e solution was neutralized with glacial acetic
acid, which causes the separation of compound 1 which
Figure 2. N’-(4-fluoro-benzylidene)-2-(2-oxobenzo[d]oxazol-3
(2H)-yl) aceto-hydrazide.
Scheme 1. Reagents and Conditions: (i) K2CO3, Abs. alcohol, reflux, (ii) Hydrazine hydrate, Abs. alcohol, reflux, (iii) Aromatic ketone,
Glacial acetic acid, reflux.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and anticonvulsant activity 827
was filtered, washed with water and recrystallized from
methanol. Yield:90%; mp 139–40°C; Rf 0.6;IR (cm−1): 3245
(-NH of amide), 3215 (-NH), 2225 (C≡N), 1674 (C=O),
1165 (C=S);1H-NMR (δ, ppm): 3.83(s, 3H, OCH3), 6.87 (d,
2H, J = 8.4 Hz, H-3,5, Phenyl), 7.86 (d, 2H, J = 8.4 Hz, H-2,6,
Phenyl), 9.60 (s, 1H, NH), 12.06 (bs, 1H, NH-C=O).
3308 (CONH), 3234 (NH), 2225 (C≡N), 1676 (C=O), 1604
(C=N); H-NMR (δ, ppm):2.42 (s, 3H, CH3), 3.84 (s, 3H,
1
OCH3), 6.99 (d, 2H, J = 8.8 Hz, H3,5), 7.37 (dd, 1H, J = 8.0,
7.6 Hz, H5′), 7.51 (d, 1H, J = 8.0 Hz, H6′), 7.57 (d, 1H, J = 7.6
Hz, H4′), 7.79 (s, 1H, H2′), 7.95 (d, 2H, J = 8.4 Hz, H2,6), 11.26
(bs, 1H, NH), 11.96 (bs, 1H, CONH); Mass (m/z) 438(M+),
440 (M+2). Anal. Calcd. for C20H18BrN5O2:C, 54.56; H,
4.12; N, 15.98. Found: C, 54.72; H, 4.11; N, 15.98.
Synthesis of 2-Hydrazinyl-4-(4-methoxy-phenyl)-6-
oxo-1,6-dihydro-pyrimidine-5-carbonitrile (2)
2-(2-{1-[4-Fluoro-phenyl]ethylidene}hydrazinyl)-4-
-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile (6) mp 271–72°C, Rf−0.64, %Yield-60; IR
(cm−1):3319(CONH), 3284(NH), 2220(C≡N), 1672(C=O),
Compound 1 (1 mmol) was dissolved absolute ethanol
and to it hydrazine hydrate (99%; 4 mmol) was added
and refluxed for 1 h. e reaction mixture was allowed
to cool which causes the separation of solid. e precipi-
tate product was filtered and washed with water. It was
recrystallized with ethanol. Yield:82%; mp 180°C; Rf 0.2;IR
(cm−1): 3285–3218 (2NH+NH2), 2210 (C≡N), 1680 (C=O),
1065 (C-O-C);1H-NMR (δ, ppm): 3.84(s, 3H, OCH3), 3.97
(bs, 2H, NHNH2),6.93 (d, 2H, J = 8.4 Hz, H-3,5, phenyl),
7.77 (d, 2H, J = 8.4 Hz, H-2,6, phenyl), 10.34 (bs, 1H, NH )
11.71 (bs, 1H, NH-C=O).
1
1605 (C=N); H-NMR (δ, ppm):2.43 (s, 3H, CH3), 3.90 (s,
3H, OCH3), 7.03 (d, 2H, J = 8.4 Hz, H3,5), 7.16 (t, 2H, J = 8.4
Hz, H3′,5′), 7.93 (d, 2H, J = 8.4 Hz, H2,6), 8.07 (d, 2H, J = 8.4
Hz, H2′,6′), 10.49 (bs, 1H, NH), 11.78 (bs, 1H, CONH); Mass
(m/z) 377(M+). Anal. Calcd. for C20H16FN5O2:C, 63.66; H,
4.27; N, 18.56. Found: C, 63.75; H, 4.28; N, 18.57.
2-(2-{1-[4-Chloro-phenyl]ethylidene}hydrazinyl)-4-
-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile (7) mp 235–36°C, Rf-0.64, %Yield-75; IR
(cm−1): 3316 (CONH), 3281 (NH), 2219 (C≡N), 1678
Synthesis of 2-(2-{1-phenyl-ethylidene}hydrazinyl)-4-
(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitrile (3)
1
(C=O), 1607 (C=N); H-NMR (δ, ppm):2.42 (s, 3H, CH3),
3.79 (s, 3H, OCH3), 6.95 (d, 2H, J = 8.4 Hz, H3,5), 7.38 (d,
2H, J = 8.0 Hz, H2′,6′), 7.69 (d, 2H, J = 8.0 Hz, H3′,5′), 7.83 (d,
2H, J = 8.4 Hz, H2,6), 10.52 (bs, 1H, NH), 11.46 (bs, 1H,
CONH); Mass (m/z) 394(M+), 396(M+2). Anal. Calcd. for
C20H16ClN5O2:C, 61.00; H, 4.09; N, 17.78. Found: C, 61.13;
H, 4.08; N, 17.77.
Compound 2 (1 mmol) was dissolved in a mixture of gla-
cial acetic acid and alcohol (2:8). To this solution, alco-
holic solution of acetophenone (1.1 mmol) was added
and refluxed for 2–3 h. Solvent was concentrated to half
of its volume, poured into ice water. e precipitate
obtained was filtered, washed with water and recrystal-
lized from methanol. Mp 203–04°C, Rf-0.66, %Yield-68;
IR (cm−1): 3310 (CONH), 3240 (NH), 2218 (C≡N), 1679
2-(2-{1-[4-Nitro-phenyl]ethylidene}hydrazinyl)-4-
(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile (8) mp 241–42°C, Rf-0.65, %Yield-65;
IR (cm−1): 3353 (CONH), 3273 (NH), 2224 (C≡N), 1668
1
(C=O), 1604 (C=N); H-NMR (δ, ppm):2.44 (s, 3H, CH3),
1
3.89 (s, 3H, OCH3), 7.04 (d, 2H, J = 8.8 Hz, H3,5), 7.41–7.43
(m, 3H, H3′,4′,5′), 7.93 (d, 1H, J = 8.4 Hz, H6′), 7.97 (d, 2H,
J = 8.8 Hz, H2,6), 8.05 (d, 2H, J = 8.0 Hz, H2′), 11.43 (bs, 1H,
NH), 11.71 (bs, 1H, CONH);
13C-NMR (δ, ppm): 15.13, 55.61, 113.73, 127.30, 128.34,
129.88, 130.68, 137.50, 162.05; Mass (m/z) 359(M+). Anal.
Calcd. for C20H17N5O2:C, 66.84; H, 4.77; N,19.49. Found: C,
67.02; H, 4.78; N, 19.51.
(C=O), 1613 (C=N); H-NMR (δ, ppm):2.36 (s, 3H, CH3),
3.85 (s, 3H, OCH3), 6.96 (d, 2H, J = 8.4 Hz, H3,5), 7.68
(d, 2H, J = 8.8 Hz, H2′,6′), 7.94 (d, 2H, J = 8.4 Hz, H2,6),
8.26 (d, 2H, J = 8.8 Hz, H3′,5′), 11.39 (bs, 1H, NH), 11.92
(bs, 1H, CONH); Mass (m/z) 404(M+). Anal. Calcd. for
C20H16N6O4:C, 59.40; H, 3.99; N, 20.78. Found: C, 59.27;
H, 3.98; N, 20.77.
2-(2-{1-[3-Nitrophenyl]ethylidene}hydrazinyl)-4-(4-
methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile (9) mp 245–46°C, Rf-0.64, %Yield-75; IR (cm−1):
3326 (CONH), 3276 (NH), 2227 (C≡N), 1683 (C=O), 1607
e remaining compounds were synthesized with
analogous procedure.
2-(2-{1-[4-Bromo-phenyl]ethylidene}hydrazinyl)-
4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimi-
dine-5-carbonitrile(4) mp 255–56°C, Rf-0.65, %Yield-66;
IR (cm−1): 3346 (CONH), 3311 (NH), 2213 (C≡N), 1675
1
(C=N); H-NMR (δ, ppm):2.39 (s, 3H, CH3), 3.86 (s, 3H,
OCH3), 7.04 (d, 2H, J = 8.8 Hz, H3,5), 7.62 (dd, 1H, J = 8.8,
8.4 Hz, H5′), 7.93 (d, 2H, J = 8.8 Hz, H2,6), 8.09 (d, 1H, J = 8.8
Hz, H6′), 8.24 (d, 1H, J= 8.4 Hz, H4′), 8.37 (s, 1H, H2′), 10.36
(bs, 1H, NH), 12.21 (bs, 1H, CONH); Mass (m/z) 404(M+).
Anal. Calcd. for C20H16N6O4:C, 59.40; H, 3.99; N, 20.78.
Found: C, 59.25; H, 3.98; N, 20.79.
1
(C=O), 1608 (C=N); H-NMR (δ, ppm):2.40 (s, 3H, CH3),
3.80 (s, 3H, OCH3), 6.94 (d, 2H, J = 8.8 Hz, H3,5), 7.39 (d, 2H,
J = 8.0 Hz, H3′,5′), 7.62 (d, 2H, J = 8.0 Hz, H2′,6′), 7.91 (d, 2H,
J = 8.8 Hz, H2,6), 11.08 (bs, 1H, NH), 11.68 (bs, 1H, CONH);
13C-NMR (δ, ppm): 14.87, 55.56, 113.66, 123.68, 129.08,
130.66, 131.28, 136.54, 162.04; Mass (m/z) 43 8(M+), 440
(M+2). Anal. Calcd. for C20H16BrN5O2:C, 54.81; H, 3.68; N,
15.98. Found: C, 54.96; H, 3.67; N, 15.97.
2-(2-{1-[3-Bromo-phenyl]ethylidene}hydrazinyl)-4-
(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitrile(5)mp265–66°C,Rf-0.65,%Yield-72;IR(cm−1):
2-(2-{1-[2-Nitro-phenyl]ethylidene}hydrazinyl)-4-(4-
methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile (10) mp 241–42°C, Rf-0.64, %Yield-80; IR (cm−1):
3342 (CONH), 3251 (NH), 2222 (C≡N), 1689 (C=O), 1602
1
(C=N); H-NMR (δ, ppm):2.36 (s, 3H, CH3), 3.90 (s, 3H,
OCH3), 6.97 (d, 2H, J = 8.4 Hz, H3,5), 7.58 (t, 1H, J = 7.6 Hz,
H4′), 7.73 (t, 1H, J = 8.0 Hz, H5′), 7.82 (d, 1H, J = 8.4 Hz, H6′),
© 2012 Informa UK, Ltd.

828 M. Shaquiquzzaman et al.
(bs, 1H, CONH); Mass (m/z) 419(M+). Anal. Calcd. for
C22H21N5O4:C, 63.00; H, 5.05; N, 16.70. Found: C, 63.12; H,
5.04; N, 16.69.
7.89 (d, 2H, J = 8.44 Hz, 44 Hz, H2,6), 8.13 (d, 1H, J = 8.0 Hz,
H3′), 1110.21 (bs, 1H, NH), 11.64 (bs, 1H, CONH); Mass
(m/z) 404(M+). Anal. Calcd. for C20H16N6O4:C, 59.40; H,
3.99; N, 20.78. Found: C, 59.31; H, 4.00; N, 20.77.
2-(2-{1-[4-Methyl-phenyl]ethylidene}hydrazinyl)-4-
-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile(16) mp 215–16°C, Rf-0.64, %Yield-74; IR
(cm−1): 3320 (CONH), 3274 (NH), 2223 (C≡N), 1681 (C=O),
1601 (C=N); 1H-NMR (δ, ppm):2.31 (s, 3H, CH3), 2.39 (s, 3H,
CH3), 3.81 (s, 3H, OCH3), 6.98 (d, 2H, J=8.4 Hz, H3,5), 7.21 (d,
2H, J=7.6 Hz, H3′,5′), 7.47 (d, 2H, J=7.6 Hz, H2′,6′), 7.89 (d, 2H,
J=8.4 Hz, H2,6), 11.27 (bs, 1H, NH), 11.81 (bs, 1H, CONH);
Mass (m/z) 373(M+). Anal. Calcd. for C21H19N5O2:C, 67.55;
H, 5.13; N, 18.75. Found: C, 67.45; H, 5.14; N, 18.76.
2-(2-{1-[4-Hydroxy-phenyl]ethylidene}hydrazinyl)-
4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile (11) mp 251–52°C, Rf-0.66, %Yield-76; IR
(cm−1): 3418 (OH), 3345 (CONH), 3246 (NH), 2215 (C≡N),
1
1674 (C=O), 1606 (C=N); H-NMR (δ, ppm):2.26 (s, 3H,
CH3), 3.78 (s, 3H, OCH3), 6.71 (d, 2H, J = 8.4 Hz, H3′,5′), 7.00
(d, 2H, J = 8.4 Hz, H3,5), 7.80 (d, 2H, J = 8.4 Hz, H2′,6′), 7.85
(d, 2H, J = 8.4 Hz, H2,6), 9.96 (s, 1H, OH), 11.25 (bs, 1H,
NH), 11.73 (bs, 1H, CONH); Mass (m/z) 375(M+). Anal.
Calcd. for C20H17N5O3:C, 63.99; H, 4.56; N, 18.66. Found:
C, 63.79; H, 4.57; N, 18.67.
Pharmacology
Swiss albino mice used in the present study were housed
and kept in accordance with the Hamdard University
Animal Care Unit, which applies the guidelines and
rules laid down by the Committee for the Purpose of
Control and Supervision of Experiments on Animals
(CPCSEA), Ministry of Social Justice and Empowerment,
Government of India. Albino mice of either sex weighing
22–25 g were used. e animals were housed in groups
of six and acclimatized to room conditions for at least 2
days before the experiments. Food and water were freely
available up to the time of experiments except during the
short time they were removed from the cages for testing.
2-(2-{1-[2-Hydroxy-phenyl]ethylidene}hydrazinyl)-
4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile (12) mp 213–14°C, Rf-0.67, %Yield-70; IR
(cm−1): 3426 (OH), 3334 (CONH), 3264 (NH), 2215 (C≡N),
1
1683 (C=O), 1606 (C=N); H-NMR (δ, ppm):2.28 (s, 3H,
CH3), 3.85 (s, 3H, OCH3), 6.95–7.02 (m, 4H, H3,5,3′,5′), 7.42
(t, 1H, J = 7.6 Hz, H4′), 7.73 (d, 1H, J = 8.0 Hz, H6′), 7.87 (d,
2H, J = 8.4 Hz, H2,6), 10.334 (s, 1H, OH), 11.13 (bs, 1H, NH),
12.01 (bs, 1H, CONH); Mass (m/z) 375(M+). Anal. Calcd.
for C20H17N5O3:C, 63.99; H, 4.56; N, 18.66. Found: C, 63.74;
H, 4.55; N, 18.67.
2-(2-{1-[4-Methoxy-phenyl]ethylidene}hydrazinyl)-
4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-
5-carbonitrile (13) mp 223–24°C, Rf-0.67, %Yield-68;
IR (cm−1): 3287 (CONH), 3229 (NH), 2216 (C≡N), 1671
Anticonvulsant activity
e synthesized compounds were evaluated for their
anticonvulsant activity using maximal electroshock
(MES14,15) and scPTZ method16. e convulsion was mea-
sured using Convulsiometer.
1
(C=O), 1603 (C=N); H-NMR (δ, ppm):2.39 (s, 3H, CH3),
3.81 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.90 (d, 2H, J = 7.6
Hz, H3′,5′), 6.97 (d, 2H, J = 8.4 Hz, H3,5), 7.76 (d, 2H, J = 7.6
Hz, H2′,6′), 7.84 (d, 2H, J = 8.4 Hz, H2,6), 11.13 (bs, 1H, NH),
11.84 (bs, 1H, CONH); Mass (m/z) 389(M+). Anal. Calcd.
for C21H19N5O3:C, 64.77; H, 4.92; N, 17.98. Found: C, 64.93;
H, 4.91; N, 17.97.
Maximal electroshock (MES) test
Maximal electroshock seizure14,15 was elicited with a 60
cycle altering current of 50mA intensity delivered for 0.25
s via ear clip electrodes. Animals were previously given
the test drug i.p. Abolition of the hind limb tonic exten-
sion spasm was recorded as the anticonvulsant activity. In
preliminary screening, each compound was administered
through an i.p. injection at three dose levels (30, 100 and
300 mgkg−1 body mass) and the anticonvulsant activity
was assessed after 0.5 and 4h intervals of administration.
2-(2-{1-[3,4-Dimethoxy-phenyl]ethylidene}
hydrazinyl)-4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrile (14) mp 167–68°C, Rf-0.67,
%Yield-78; IR (cm−1): 3318 (CONH), 3265 (NH), 2221
1
(C≡N), 1677 (C=O), 1610 (C=N); H-NMR (δ, ppm):2.41
(s, 3H, CH3), 3.88 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.93
(s, 3H, OCH3), 6.93 (d, 1H, J = 8.4 Hz, H5′), 6.99 (d, 2H,
J = 8.8 Hz, H3,5), 7.47 (s, 1H, H2′), 7.58 (d, 1H, J = 8.4 Hz,
H6′), 7.93 (d, 2H, J = 8.8 Hz, H2,6), 11.29 (bs, 1H, NH), 12.13
(bs, 1H, CONH); Mass (m/z) 419(M+). Anal. Calcd. for
C22H21N5O4:C, 63.00; H, 5.05; N, 16.70. Found: C, 63.17; H,
5.04; N, 16.71.
Subcutaneous pentylenetetrazole (scPTZ) seizure
threshold test
scPTZ16 was conducted by administering PTZ dissolved
in 0.9% sodium chloride solution in the posterior midline
of the animals. A minimal time of 30 min subsequent to
s.c. administration of PTZ was used for seizure detection.
Protection was referred to as the failure to observe an
episode of clonic spasms of at least 5 s during this time
period.
2-(2-{1-[2,4-Dimethoxy-phenyl]ethylidene}
hydrazinyl)-4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrile (15) mp 161–62°C, Rf-0.66,
%Yield-82; IR (cm−1): 3319 (CONH), 3236 (NH), 2218
1
(C≡N), 1674 (C=O), 1609 (C=N); H-NMR (δ, ppm):2.40
(s, 3H, CH3), 3.88 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.92
(s, 3H, OCH3), 6.81 (s, 1H, H3′), 6.91 (d, 1H, J = 7.6 Hz, H5′),
6.99 (d, 2H, J = 8.4 Hz, H3,5), 7.32 (d, 1H, J = 7.6 Hz, H6′),
7.91 (d, 2H, J = 8.4 Hz, H2,6), 11.11 (bs, 1H, NH), 11.72
Neurotoxicity
e synthesized drugs interference with motor coordi-
nation was checked by the rotarod test at a dose level of
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and anticonvulsant activity 829
Table 1. Anticonvulsant, neurotoxicity screening and CNS depression study of compounds 3–16.
Anticonvulsant activity Neurotoxicity screening
MES scPTZ NT
CNS depression study
Duration of immobility % Increase of
Compound
After 0.5 h
After 4 h
300
100
300
300
300
(-)
After 0.5 h
After 4 h
300
100
300
300
300
300
100
-
After 0.5 h
(-)
After 4 h
300
300
300
300
300
300
300
300
(-)
(Mean SEM)c
110 1.41***
82.16 0.94***
X
immobility
89.22
41.38
X
3
100
30
300
100
300
100
100
300
30
4
(-)
5
(-)
(-)
6
100
100
300
30
300
300
(-)
54.92
67.70
X
90 1.06***
97.5 1.38***
X
7
8
9
100
300
300
300
(-)
100
100
(-)
43.44
52.55
116.05
137.30
X
83.34 0.42***
88.67 1.14***
125.5 1.17***
137.84 0.94***
X
10
100
100
100
(-)
-
11
-
-
12
300
-
300
-
(-)
(-)
13
(-)
300
300
X
14
100
300
300
30
300
300
300
30
300
300
300
-
300
300
300
-
(-)
106.53
X
120 1.29***
X
15
X
16
300
(-)
300
(-)
X
X
Phenytoinb
Carbamazepineb
Control
-
-
30
100
100
100
(-)
(-)
45.18
84.34 1.85***
58.17 1.01***
-
aDoses of 30, 100, 300 mgkg−1 were administered i.p. Test compounds were suspended in 0.5% methylcellulose/water mixture or in
polyethylene glycol (PEG). Figures in the table indicate the minimum dose with bioactivity demonstrated in half or more of the mice.
e dash (-) indicates the absence of activity at maximum dose administered (300 mgkg−1). X denotes not tested.
bData from references20,21
.
cDose=300 mgkg−1; n=6; ***p<0.001, Data were analysed by unpaired students “t” test.
Table 2. Comparison of distance between the essential structural
elements of existing drugs and synthesized compounds.
Distance of
Average distances
for existing drugs (12)
synthesized
compounds
Site 1
HA5
HA5
HA5
D6
Site 2
D6
3.59 1.04
2.41 0.31
4.73 0.61
4.15 1.43
5.00 1.68
4.92 1.25
5.51
2.46
6.20
3.50
6.03
6.61
HD7
R12
HD7
R12
R12
D6
HD7
30, 100 and 300 mgkg−1 body mass. In that test, mice were
trained to stay on the knurled plastic rod having diam-
eter of 3.2 cm. Normal mice could maintain equilibrium
on the rotating rod for longer period of time. e neuro-
toxicity17 was indicated by inability of mice to maintain
equilibrium on the rod for at least 1 min in each of the
three trials. e dose which impairs the ability of 50% of
the animals to remain on the revolving rod was consid-
ered the end point.
Figure 3. ADDRRR-24 hypothesis and the distance between
critical pharmacophoric elements.
(100 mgkg−1) of the test compounds 30 min before the
test session. e period of immobility (passive floating
without struggling, making only those movements which
are necessary to keep its head above the surface of water)
during the 5-min test period was measured.
CNS depressant study
e forced swim pool method (Porsolt’s swim pool
test) was followed to study CNS depression18. Male
albino mice were placed in a chamber (diameter 45cm,
height: 20 cm) containing water up to a height of 15 cm
at 25 2°C. Two swim sessions were conducted, an ini-
tial 15 min pre-test, followed by a 5-min test session 24 h
later. e animals were administered an i.p. injection
Pharmacophore modeling
Various 3D pharmacophore13 hypotheses have been
generated using Phase 3.1 module of Schrodinger-9 and
pharmacophore distance was compared with average
distance for existing anticonvulsant drugs12. e data
related to this are presented in Table 2.
© 2012 Informa UK, Ltd.

830 M. Shaquiquzzaman et al.
Figure 4. Fitness of the most active compounds 4 and 9 on the Pharmacophore.
300 mgkg−1; however, compound 9 showed motor
impairment at 300 mgkg−1 dose after 4.0 h.
Result and discussion
A series of 2-(2-{1-phenyl-ethylidene}hydrazinyl)-4-(4-
methoxy-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile derivatives (3–16) were synthesized by refluxing
2-hydrazino-4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrile (2) with different substituted
acetophenones in glacial acetic acid and alcohol mixture
2:8).
e required intermediate compound 2 was synthe-
sizedfrom2-mercapto-4-(4-methoxy-phenyl)-6-oxo-1,6-
dihydro-pyrimidine-5-carbonitrile 1 upon nucleophilic
attack by the hydrazine hydrate. e compound 1 was
synthesizedbymodifiedBiginellecondensation19 method
using anisaldehyde, ethyl cyanoacetate and thiourea in
presence of potassium carbonate.
In general, the IR spectral data of all the compounds
showed characteristic peaks around 3346 cm−1 for -NH
of amide group, 2224 cm−1 for C≡N and 1669 cm−1 for
C=O indicatingthe formation of cyanopyrimidinone.
Similarly, peak around 1617 cm−1 indicates formation of
Schiff’s base. In 1H-NMR spectral data, all the compounds
showed characteristic peak at appropriate δ-values.
e synthesized compounds were screened for their
anticonvulsant activity against two standard models MES
and scPTZ for their ability to reduce seizure spread and
to evaluate seizure threshold, respectively. Motor impair-
ment screening was carried out by rotarod test method
and CNS depressant effect of the compounds was deter-
mined by Porsolt’s force swim pool method. e anti-
convulsant activity was reported after 0.5 and 4.0 h time
intervals at dose levels of 30, 100 and 300 mgkg−1 body
weight. e CNS depressant was studied at a dose level of
100 mgkg−1 body weight. Phenytoin and carbamazepine
were used as standard drugs.
Some selected compounds having significant anticon-
vulsant activity were also tested for their CNS depressant
effect. e compounds 4 and 9 showed 41.38 and 43.44%
increase in immobility time with respect to control where
as standard drug carbamazepine showed 45.18% increase
in the immobility time (Table 1). Rest of the tested com-
pounds also showed a good increase in immobility time.
e generated pharmacophore using Phase 3.1 mod-
ule of Schrodinger-9 also showed the presence of all
crucial structural components i.e. the aryl ring centre
or the lipophilic group (R12), an electron donor atom
(D6), a hydrogen bond acceptor (HA) and a hydrogen
bond donor (HD7) required for a compound to be an
anticonvulsant. On the basis of fitness of the highest
active ligands i.e. compound 4 and 9, ADDRRR-24 phar-
macophore hypothesis was selected (Figure 3) and the
selected pharmacophore hypothesis was found to fit
best for most active ligands which can be visualized by
Figure 4.
e result indicates that compounds 4 and 9 are the
most active compounds of this study as it is effective in
both MES and scPTZ screen showing no motor impair-
ment effect. e compound 4 and 9 also showed reduced
CNS depressant effect in comparison to standard drug
carbamazepine.
conclusion
Fourteen new 2-(2-{1-phenyl-ethylidene}hydrazinyl)-
4-(4-methoxy-phenyl)-6-oxo-1,6-dihydro-pyrim-
idine-5-carbonitrile derivatives were successfully
synthesized. Biological evaluation results showed that
the compounds are promising anti-convulsant agents
with no motor impairment and good decrease in immo-
bility time indicating that the synthesized molecules
have lesser CNS depressant effect.
Among the newer derivatives, two compounds 4 and
9 emerged as lead compounds. e synthesized com-
pounds were also supported by the pharmocophore
model.
In this series, compounds 4 and 9 having p-substi-
tuted bromo and m-substituted nitro groups, respec-
tively, were found to be most active of the series showing
activity both in MES and scPTZ screen at lower doses of
30 mgkg−1 at 0.5 h and 100 mgkg−1 at 4 h. In the rotarod
motor impairment screen, compound 4 did not show
any motor impairment even at the maximum dose of
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and anticonvulsant activity 831
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Recková Z et al. A new modification of anti-tubercular active
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acknowledgements
e authors are thankful to Jamia Hamdard (New Delhi)
for providing research facilities to pursue this work.
Declaration of interest
11. Cakir B, Dag O, Yildirim E, Erol K, Sahin MF. Synthesis and
anticonvulsant activity of some hydrazones of 2-[(3H)-
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
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