Novel dihydropyrimidines and its pyrazole derivatives: Synthesis and pharmacological screening

Article abstract of DOI:10.1016/j.ejmech.2011.02.052

In the present study, we have synthesized novel dihydropyrimidines (1a-j), their dimethylated adducts (2a-j), and hydrazine derivatives (3a-j) of 2a-j and subsequently their pyrazole derivatives (4a-j). Elemental analysis, IR, ¹H NMR and mass s

Full text of DOI:10.1016/j.ejmech.2011.02.052

European Journal of Medicinal Chemistry 46 (2011) 1882e1891
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel dihydropyrimidines and its pyrazole derivatives: Synthesis and
pharmacological screening
*
B. Ramesh, Chetan M. Bhalgat
Department of Pharmaceutical Chemistry, S.A.C. College of Pharmacy, B.G. Nagara 571448, Nagamangala (tq), Mandya (dist), Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we have synthesized novel dihydropyrimidines (1aej), their dimethylated adducts
(2aej), and hydrazine derivatives (3aej) of 2aej and subsequently their pyrazole derivatives (4aej).
Elemental analysis, IR, 1H NMR and mass spectral data elucidated structure of newly synthesized
compounds. Some of these novel derivatives showed moderate to potent in vitro antioxidant, anti-
inflammatory, antibacterial, antifungal and anthelmintic activity.
Received 13 January 2011
Received in revised form
19 February 2011
Accepted 21 February 2011
Available online 26 February 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Dihydropyrimidine
Pyrazole fused pyrimidine
Antioxidant activity
Anti-inflammatory activity
Antimicrobial activity
Anthelmintic activity
1. Introduction
analgesic, anti-inflammatory, ulcerogenic [17], antibacterial, anti-
fungal [18,19], antitubercular [20], antimalarial [21], antitumor
Pyrimidine, being an integral part of DNA and RNA, have imparts
diverse pharmacological properties as effective bactericide and
fungicide [1e3]. Certain pyrimidine derivatives were also known to
exhibit antimalarial [4], antifilarial [5], antioxidant [6,7], anthel-
mintic [8] and anti-HIV activities [9]. Some of the dihydropyr-
imidines (DHPM) have emerged as integral backbones of several
calcium channel blockers, antihypertensive agents, adrenergic and
neuropeptide antagonist [10]. Several alkaloids containing dihy-
dropyrimidine have been isolated from marine sources and among
them the batzelladine alkaloids were found to be potent HIV-gp-
120-CD4 inhibitors [11e13].
In addition to the diverse biological activities of pyrimidine,
other heterocycles in association with pyrimidines play an essential
role in several biological processes and have a considerable
chemical and pharmacological importance. Pyrimidines in associ-
ation with pyrazole have occupied prominent place in medicinal
chemistry because of their significant properties as therapeutics in
clinical application [14e16]. A survey of literature revealed that
pyrazole derivatives of pyrimidine have received much attention
during recent years on account of their prominent utilization as
[18,22,23], antioxidant [23], antiproliferative [24], antihypertensive
[25], hypnotic [26] and vasodilator [27].
In the view of the facts mentioned above and as part of our
initial efforts to discover potentially active new agents, we have
synthesized some new dihydropyrimidines 2e3 and its pyrazole
derivatives 4. The novel derivatives were characterized by spectral
data and elemental analysis and these compounds were tested for
their antioxidant, anti-inflammatory, antimicrobial and anthel-
mintic screening.
The required 6-oxo-4-substituted aryl-2-sulfanyl-1,6-dihy-
dropyrimidine-5-carbonitrile (1aej) were prepared in moderate
to good yield by using the method of Ramesh et al. [28]. Ethyl 5-
amino-1-(5-cyano-1-methyl-6-oxo-4-substituted-1,6-dihydropyr-
imidin-2-yl)-1H-pyrazole-4-carboxylates (4aej) were afforded
from 1aej through their dimethylated adducts (2aej) and subse-
quently their hydrazine derivatives (3aej) following a three step
reaction sequence (Scheme 1).
2. Results and discussion
2.1. Chemistry
In the present work the title compounds were synthesized by
the cyclization of three-components like arylaldehydes, thiourea
* Corresponding author. Tel.: þ91 9241752830; fax: þ91 8234287242.
E-mail address: chetanbhalgat2004@yahoo.co.in (C.M. Bhalgat).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.052

B. Ramesh, C.M. Bhalgat / European Journal of Medicinal Chemistry 46 (2011) 1882e1891
1883
O
NC-CH2-COOCH2CH3
K2CO3
Et-OH
NC
H
NH
Ar
+
S
C
O
, 12 h
Δ
Ar
N
SH
H2N
NH2
1a-j
K2CO3
DMF
3 h RT
O
O
NH2-NH2.H2O (80%)
Et-OH
CH3
NC
CH3
NC
N
N
, 6 h
Δ
Ar
N
S
Ar
N
NH
CH3
NH2
3a-j
2a-j
Ar
H5C2O
H
COOEt
CN
Et-OH
, 5-6h
4-OCH3-C6H4-
2-F-C6H4-
(a)
(b)
Δ
2-Furyl-
(c)
(d)
2-Thienyl-
O
4-N-(CH3)2-C6H4-
4-Cl-C6H4-
(e)
(f)
CH3
NC
N
N
N
Ar
N
2-Cl-C6H4-
(g)
(h)
(i)
2,3-(OCH3)-C6H3-
2-OH-C6H4-
H2N
4a-j
COOEt
2,3-(OH)-C6H3-
(j)
Et-OH = ethanol, DMF = N,N- dimethyl formamide, RT = room temperature
Scheme 1. Schematic representation of synthesis of compounds (2e4).
and ethyl cyanoacetate in ethanol using potassium carbonate to
form 6-oxo-4-substituted aryl-2-sulfanyl-1,6-dihydropyrimidine-
5-carbonitriles (1aej). While, room temperature stirring of
1aej with methyl iodide in DMF in presence of potassium
d
2.28e3.31 for SeCH3 in the 1H NMR and peak for CeS in the range
of 774e758 cmꢀ1 in the IR spectra. The hydrazine derivatives (3aej)
showed peak at 3403e3298 cmꢀ1 for the NHeNH2 in the IR and
d
2.06e2.48 for NH and d
2.62e2.95 for NH2 in the 1H NMR spectra.
carbonate
yielded
1-methyl-2-(methylthio)-5-cyano-6-oxo-
IR spectrum of the compounds 4aej showed the appearance of
absorption bands at 3467e3310 cmꢀ1 for the NH2 and absorption
bands at 1772e1672 cmꢀ1 for the C¼O of ester group, their 1H NMR
spectrum showed the presence of characteristic peaks at
4-substituted-1,6-dihydropyrimidines (2aej). The 2-hydrazino
derivatives, 2-hydrazino-1-methyl-5-cyano-6-oxo-4-substituted-
1,6-dihydropyrimidines (3aej) was obtained by heating 2aej with
hydrazine hydrate (80%) in ethanol. Condensation of latter with
ethyl-2-cyano-3-ethyl acrylate in alcohol and in the presence of
acetic acid afforded the corresponding ethyl 5-amino-1-(5-cyano-
1-methyl-6-oxo-4-substituted-1,6-dihydropyrimidin-2-yl)-1H-
pyrazole-4-carboxylates (4aej) (Scheme 1). The physical and
analytical data have been given in Table 1.
The structures assigned to the compounds were substantiated
by their analytical and other spectral data. The IR spectra of
all the synthesized compounds showed characteristic signals
at 2252e2200 cmꢀ1 for C¼N, 1740e1633 cmꢀ1 for C¼O,
1658e1557 cmꢀ1 for C¼N and 1589e1464 cmꢀ1 for C¼C. Similarly
the 1H NMR spectra showed peaks due to NeCH3 proton in the
d
5.83e6.92 for NH2,
d
7.46e8.13 for CH of pyrazole,
d 3.26e4.65 for
CH2 of ester and at
d
1.11e1.56 for CH3 of ester. The mass spectrum
of all the compounds showed molecular ion peak at Mþ1 corre-
sponding to its molecular formula, which confirmed its chemical
structure. The IR, 1H NMR, mass spectra and elemental analysis
supported the structure of various synthesized pyrimidines and its
pyrazole derivatives (Table 2).
2.2. Pharmacological screening
2.2.1. Antioxidant activity
All the synthesized compounds 2e4 were screened for their in
vitro antioxidant activity by various methods such as scavenging of
range of
d 2.96e4.46. The 2aej has showed peak in the range of

Table 1
Physical and analytical characterization of compounds (2e4).
Compounds
Chemical name
Ar
Yield
(%)
M.P. (ꢁC)
Mol. Formula
C14 H13 N3 O2
Rf value
2a
2b
2c
2d
2e
2f
2g
2h
2i
4-(4-Methoxyphenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Fluorophenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Furyl)-1-methyl-2-(methylthio)-5 cyno-6-oxo-1,6-dihydropyrimidine
1-Methyl-2-(methylthio)-5 cyno-6-oxo-4-thien-2-yl-1,6-dihydropyrimidine
4-[4-(Dimethylamino)phenyl]-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(4-Chlorophenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Chlorophenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(3,4-Dimethoxyphenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Hydroxyphenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2,4-Dihydroxyphenyl)-1-methyl-2-(methylthio)-5-cyno-6-oxo-1,6-dihydropyrimidines
4-(4-Methoxyphenyl) 2-hydrazino–1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Fluorophenyl)-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Furyl)-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
2-Hydrazino-1-methyl-5-cyno-6-oxo-4-thien-2-yl-1,6-dihydropyrimidine
4-[4-(Dimethylamino)phenyl]-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(4-Chlorophenyl)-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2-Chlorophenyl)-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(3,4-Dimethoxyphenyl)-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
2-Hydrazino-4-(2-hydroxyphenyl)-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
4-(2,4-Dihydroxyphenyl)-2-hydrazino-1-methyl-5-cyno-6-oxo-1,6-dihydropyrimidine
5-Amino-4-etheylcorboxylaty -1-[5-cyano-4-(4-methoxyphenyl)-1-methyl-6-oxo-1,
6-dihydropyrimidin-2-yl]-1H-pyrazole
4-OCH3$C6H4
2-FeC6H4
2-Furyl
2-Thienyl
4-Ne(CH3)2eC6H4
4-CleC6H4
46
51
58
61
40
61
56
44
48
68
55
59
61
68
69
51
63
69
51
54
55
197
158
95
S
0.56
0.64
0.72
0.46
0.68
0.39
0.43
0.52
0.61
0.49
0.58
0.45
0.60
0.68
0.56
0.69
0.48
0.53
0.63
0.70
0.59
C13 H10 F N3 O S
11 H9 N3 O2
C
S
256
219
209
190
115
190
205
269
218
C11 H9 N3 O S2
C15 H16 N4 O S
C13 H10 Cl N3 O S
C13 H10 Cl N3 O S
C15 H15 N3 O3
C13 H11 N3 O2
C13 H11 N3 O3
C13 H13 N5 O2
C12 H10 F N5O
C10 H9 N5 O2
C10 H9 N5 O S
2-CleC6H4
2,3e(OeCH3)2eC6H3
2-OHeC6H4
2,3-(OH)2eC6H3
4-OCH3$C6H4
2-FeC6H4
2-Furyl
2-Thienyl
4-Ne(CH3)2eC6H4
4-CleC6H4
2-CleC6H4
S
S
S
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
J
S.S.
277
222
260
118
S.S.J
155
248
210
C14 H16 N6 O
C12 H10 Cl N5
C12 H10 Cl N5
C14 H15 N5 O3
C12 H11 N5 O2
C12 H11 N5 O3
C19 H18 N6 O4
O
O
2,3-(OeCH3)2eC6H3
2-OHeC6H4
2,3-(OH)2eC6H3
4-OCH3$C6H4
3j
4a
4b
4c
4d
4e
4f
5-Amino-4-etheylcorboxylaty -1-[5-cyano-4-(-methoxyphenyl)-1-methyl-6-oxo-1,
6-dihydropyrimidin-2-yl]-1H-pyrazole
Ethyl 5-amino-1-[5-cyano-4-(2-furyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl]-1
H-pyrazole-4-carboxylate
Ethyl 5-amino-1-(5-cyano-1-methyl-6-oxo-4-thien-2-yl-1,6-dihydropyrimidin-2-yl)-1
H-pyrazole-4-carboxylate
5-Amino-1-[5-cyano-4-(4-dimethylamino-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-2-yl]-1
H-pyrazole-4-carboxylic acid ethyl ester
Ethyl 5-amino-1-[4-(4-chlorophenyl)-5-cyano-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl]-1
H-pyrazole-4-carboxylate
Ethyl 5-amino-1-[4-(2-chlorophenyl)-5-cyano-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl]-1
H-pyrazole-4-carboxylate
5-Amino-1-[5-cyano-4-(3,4-dimethoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-2-yl]-1
H-pyrazole-4-carboxylic acid ethyl ester
Ethyl 5-amino-1-[5-cyano-4-(2-hydroxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl]-1
H-pyrazole-4-carboxylate
2-FeC6H4
44
49
56
49
60
44
48
59
42
90
C18 H15 F N6 O3
0.48
0.64
0.44
0.55
0.69
0.64
0.43
0.58
0.41
2-Furyl
192
210
295
240
90
C
C
16 H14 N6 O4
16 H14 N6 O3 S
2-Thienyl
4-Ne(CH3)2eC6H4
4-CleC6H4
C20 H21 N7 O3
C18 H15 Cl N6 O3
C18 H15 Cl N6 O3
C20 H20 N6 O5
C18 H16 N6 O4
C18 H16 N6 O5
4g
4h
4i
2-CleC6H4
2,3e(OeCH3)2eC6H3
2eOHeC6H4
2,3-(OH)2eC6H3
80
162
183
4j
Ethyl 5-amino-1-[5-cyano-4-(2,4-dihydroxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl]-1
H-pyrazole-4-carboxylate
J
S.S. ¼ semisolid.

Table 2
Spectral characterization and elemental analysis of compounds (2e4).
Compounds IR
1H NMR
DMSO-
Mass
m/z
% Calculated (Found)
(KBr)
n
(cmꢀ1
)
d
(ppm)
C
H
N
2a
2b
2c
2d
2227 (C^N), 1723 (C]O), 1625 (C]N), 1589 (C]C), 7.18e8.07 (m, 4H, AreH), 2.72 (s, 3H, SeCH3),
1250 (CeN), 769 (CeS), 1263 (CeOeC) 3.46 (s, 3H, NeCH3), 3.87 (s, 3H, OeCH3)
2252 (C^N), 1689 (C]O), 1613 (C]N), 1508 (C]C), 6.98e7.68 (m, 4H, AreH), 2.58 (s, 3H, SeCH3),
1358 (CeN), 766 (CeS), 1018 (CeF) 3.32 (s, 3H, NeCH3)
2216 (C^N), 1740 (C]O), 1609 (C]N), 1546 (C]C), 6.96e8.18 (m, 3H, Furan), 3.31(s, 3H, SeCH3),
1317 (CeN), 765 (CeS), 1255 (CeOeC) 4.24 (s, 3H, NeCH3)
2232 (C^N), 1714 (C]O), 1617 (C]N), 1544 (C]C), 7.20e8.13 (m, 3H, Thiophene), 2.77
288.3
58.52
4.56
14.62
M þ 1 (58.53) (4.51) (14.61)
276.3 56.72 3.66 15.26
M þ 1 (56.71) (3.62) (15.21)
248.3 53.43 3.67 16.99
M þ 1 (53.41) (3.63) (16.91)
264.4 50.17 3.44 15.96
M þ 1 (50.13) (3.41) (15.93)
1340 (CeN), 762 (CeS of SeCH3),
782 (CeS of Thiophene)
(s, 3H, SeCH3), 4.46 (s, 3H, NeCH3)
2e
2f
2223 (C^N), 1716 (C]O), 1606 (C]N), 1568 (C]C), 7.62e8.04 (m, 4H, AreH), 2.96 (s, 3H, SeCH3),
1354 (CeN), 759 (CeS), 1338 (dimethyl amino) 4.22 [s, 6H, Ne(CH3)2], 3.38 (s, 3H, NeCH3),
2212 (C^N), 1694 (C]O), 1610 (C]N), 1538 (C]C), 7.18e7.42 (m, 4H, AreH), 3.06 (s, 3H, SeCH3),
1352 (CeN), 760 (CeS), 770 (CeCl) 3.96 (s, 3H, NeCH3)
2222 (C^N), 1715 (C]O), 1628 (C]N), 1552 (C]C), 6.96e7.57 (m, 4H, AreH), 2.87 (s, 3H, SeCH3),
1326 (CeN), 758 (CeS),776 (CeCl) 3.76 (s, 3H, NeCH3)
2225 (C^N), 1704 (C]O), 1618 (C]N), 1528 (C]C), 6.78e7.35 (m, 3H, AreH), 2.28 (s, 1H, SeCH3),
1354 (CeN), 760 (CeS), 1214 (CeOeC) 2.92 (s, 1H, NeCH3), 3.68 (s, 6H, 2.OeCH3)
2219 (C^N), 1714 (C]O), 1616 (C]N), 1538 (C]C), 7.04e7.72 (m, 4H, AreH), 2.96 (s, 3H, SeCH3),
1314 (CeN), 774 (CeS), 3412 (OH) 4.22 (s, 3H, NeCH3), 5.72 (s, 1H, OH)
2236 (C^N), 1722 (C]O), 1632 (C]N), 1555 (C]C), 6.28e7.27 (m, 3H, AreH), 3.11 (s, 3H, SeCH3),
301.3 59.98 5.37 18.65
M þ 1 (59.93) (5.33) (18.61)
292.6 53.52 3.45 14.40
M þ 1 (53.51) (3.42) (14.42)
292.5 53.52 3.45 14.40
M þ 1 (53.51) (3.42) (14.41)
318.4 56.77 4.76 13.24
M þ 1 (56.72) (4.73) (13.21)
274.3 57.13 4.06 15.37
M þ 1 (57.12) (4.10) (15.31)
290.3 53.97 3.83 14.52
M þ 1 (53.93) (3.79) (14.51)
272.3 57.56 4.83 25.82
M þ 1 (57.51) (4.81) (25.80)
260.3 55.60 3.89 27.02
M þ 1 (55.51) (3.91) (27.03)
232.3 51.95 3.92 30.29
M þ 1 (51.91) (3.91) (30.21)
248.2 48.57 3.67 28.32
M þ 1 (48.51) (3.71) (28.31)
288.3 59.14 5.67 29.56
2g
2h
2i
2j
1316 (CeN), 765 (CeS), 3466 (2.OH)
3.88 (s, 3H, NeCH3), 5.21 (s, 2H, 2.OH)
3a
3b
3c
3d
3e
3f
3325 (NHeNH2), 2225 (C^N), 1738 (C]O),
1618 (C]N), 1532 (C]C), 1221 (CeOeC)
3300 (NHeNH2), 2200 (C^N), 1690 (C]O),
1600 (C]N), 1500 (C]C), 1005 (CeF)
3348 (NHeNH2), 2218 (C^N), 1728 (C]O),
1658 (C]N), 1554 (C]C), 1217 (CeOeC)
3298 (NHeNH2), 2212 (C^N), 1658 (C]O),
1606 (C]N), 1553 (C]C), 716 (CeS)
7.05e7.74 (m, 4H, AreH), 3.24 (s, 3H, NeCH3),
4.17 (s, 3H, OeCH3), 2.21 (s, 1H, NH), 2.62(s, 2H, NH2)
7.24e7.74 (m, 4H, AreH), 3.35 (s, 3H, NeCH3),
2.42 (s, 1H, NH), 2.84(s, 2H, NH2)
6.68e7.56 (m, 3H, Furan), 3.22 (s, 3H, NeCH3),
2.23 (s, 1H, NH), 2.65 (s, 2H, NH2)
7.34e8.08 (m, 3H, Thiophene), 3.24 (s, 3H, NeCH3),
2.48 (s, 1H, NH), 2.78(s, 2H, NH2)
3327 (NHeNH2), 2222 (C^N), 1657 (C]O),
1608 (C]N), 1510 (C]C), 1350 (dimethyl amino)
3313 (NHeNH2), 2218 (C^N), 1678 (C]O),
1610 (C]N), 1537 (C]C), 710 (CeCl)
3337 (NHeNH2), 2226 (C^N), 1712 (C]O),
1654 (C]N), 1574 (C]C), 718 (CeCl)
3403 (NHeNH2), 2237 (C^N), 1724 (C]O),
1646 (C]N), 1578 (C]C), 1232 (CeOeC)
3342 (NHeNH2), 2214 (C^N), 1633 (C]O),
1557 (C]N), 1508 (C]C), 3405 (OH)
3368 (NHeNH2), 2220 (C^N), 1635 (C]O),
1578 (C]N), 1512 (C]C), 3450 (2.OH)
3452 (NH), 2228 (C^N), 1722 (C]O, ester),
1648 (C]O, pyrimidine), 1578 (C]N),
1520 (C]C), 1218 (CeOeC)
6.84e7.52 (m, 4H, AreH), 3.32 (s, 3H, NeCH3),
3.84 [s, 6H, Ne(CH3)2],2.28 (s, 1H, NH), 2.62(s, 2H, NH2) M þ 1 (59.13) (5.62) (29.51)
7.21e7.46 (m, 4H, AreH), 4.06 (s, 3H, NeCH3),
2.34 (s, 1H, NH), 2.88 (s, 2H, NH2)
276.4
M þ 1 (52.10) (3.62) (25.32)
276.4 52.28 3.66 25.40
M þ 1 (52.21) (3.63) (25.41)
302.4 55.81 5.02 23.24
M þ 1 (55.72) (5.07) (23.21)
290.3 56.03 4.31 27.22
M þ 1 (56.08) (4.32) (27.21)
274.3 52.75 4.06 25.63
M þ 1 (52.77) (4.07) (25.61)
395.3 57.86 4.60 21.31
M þ 1 (57.91) (4.58) (21.33)
52.28
3.66
25.40
3g
3h
3i
7.11e7.53 (m, 4H, AreH), 4.42 (s, 3H, NeCH3),
2.32 (s, 1H, NH), 2.95 (s, 2H, NH2)
6.92e7.26 (m, 3H, AreH), 2.96 (s, 1H, NeCH3),
4.16 (s, 6H, 2.OeCH3), 2.18 (s, 1H, NH), 2.68 (s, 2H, NH2)
6.75e7.68 (m, 4H, AreH), 3.38 (s, 3H, NeCH3),
5.42 (s, 1H, OH), 2.33 (s, 1H, NH), 2.78 (s, 2H, NH2)
6.65e7.34 (m, 3H, AreH), 2.96 (s, 3H, NeCH3),
5.22 (s, 2H, 2.OH), 2.06 (s, 1H, NH), 2.84 (s, 2H, NH2)
7.28e7.77 (m, 4H, AreH), 3.46 (s, 3H, NeCH3),
3.82 (s, 3H, OeCH3), 6.92 (s, 2H, NH2),
3j
4a
7.96 (s, 1H, CH of pyrazole), 4.38 (q, 2H, CH2 of ester),
1.28 (t, 3H, CH3 of ester)
4b
4c
4d
4e
3425 (NH), 2208 (C^N), 1710 (C]O, ester),
1650 (C]O, pyrimidine), 1606 (C]N),
1548 (C]C), 1010 (CeF)
3344 (NH), 2214 (C^N), 1687 (C]O, ester),
1619 (C]O, pyrimidine), 1600 (C]N),
1464 (C]C), 1290 (CeOeC)
3467 (NH), 2218 (C^N), 1702 (C]O, ester),
1682 (C]O, pyrimidine), 1637 (C]N),
1545 (C]C), 720 (CeS)
3450 (NH), 2219 (C^N), 1772 (C]O, ester),
1679 (C]O, pyrimidine), 1623 (C]N),
1528 (C]C), 1340 (dimethyl amino)
7.32e7.65 (m, 4H, AreH), 3.18 (s, 3H, NeCH3),
5.83 (s, 2H, NH2), 7.89 (s, 1H, CH of pyrazole),
3.96 (q, 2H, CH2 of ester), 1.11 (t, 3H, CH3 of ester)
6.73e7.45 (m, 3H, Furan), 2.99 (s, 3H, NeCH3),
6.38 (s, 2H, NH2), 7.72 (s, 1H, CH of pyrazole),
4.16 (q, 2H, CH2 of ester), 1.31 (t, 3H, CH3 of ester)
7.11e7.38 (m, 3H, Thiophene), 3.22 (s, 3H, NeCH3),
6.13 (s, 2H, NH2), 8.07 (s, 1H, CH of pyrazole),
4.65 (q, 2H, CH2 of ester), 1.24 (t, 3H, CH3 of ester)
6.68e7.27 (m, 4H, AreH), 3.07 (s, 3H, NeCH3),
3.72 [s, 6H, Ne(CH3)2], 5.95 (s, 2H, NH2),
7.68 (s, 1H, CH of pyrazole), 3.97 (q, 2H, CH2 of ester),
1.18 (t, 3H, CH3 of ester)
383.3
56.54
3.95
21.98
M þ 1 (56.52) (3.92) (21.91)
355.4 54.24 3.98 23.72
M þ 1 (54.19) (4.01) (23.61)
371.4 51.88 3.81 22.69
M þ 1 (51.82) (3.84) (22.61)
408.3 58.96 5.20 24.07
M þ 1 (58.99) (5.25) (24.08)
4f
3460 (NH), 2225 (C^N), 1672 (C]O, ester),
1609 (C]O, pyrimidine), 1594 (C]N),
1561 (C]C), 710 (CeCl)
3378 (NH), 2212 (C^N), 1732 (C]O, ester),
1648 (C]O, pyrimidine), 1575 (C]N),
1510 (C]C), 714 (CeCl)
3408 (NH), 2232 (C^N), 1726 (C]O, ester),
1669 (C]O, pyrimidine), 1576 (C]N),
1520 (C]C), 1224 (CeOeC)
7.18e7.32 (m, 4H, AreH), 3.33 (s, 3H, NeCH3),
6.38 (s, 2H, NH2), 7.59 (s, 1H, CH of pyrazole),
3.86 (q, 2H, CH2 of ester), 1.45 (t, 3H, CH3 of ester)
7.26e7.63 (m, 4H, AreH), 3.54 (s, 3H, NeCH3),
6.23 (s, 2H, NH2), 8.13 (s, 1H, CH of pyrazole),
3.72 (q, 2H, CH2 of ester), 1.39 (t, 3H, CH3 of ester)
6.74e7.17 (m, 3H, AreH), 2.89 (s, 1H, NeCH3),
3.73 (s, 6H, 2.OeCH3), 5.87 (s, 2H, NH2),
399.7
54.21
3.79
21.07
M þ 1 (54.26) (3.82) (21.01)
4g
4h
308.7 54.21 3.79 21.07
M þ 1 (54.26) (3.81) (21.10)
425.3 56.60 4.75 19.80
M þ 1 (56.56) (4.76) (19.81)
7.52 (s, 1H, CH of pyrazole), 4.21 (q, 2H, CH2 of ester),
1.38 (t, 3H, CH3 of ester)
4i
4j
3310 (NH), 2220 (C^N), 1700 (C]O, ester),
1650 (C]O, pyrimidine), 1600 (C]N),
1520 (C]C), 3300 (OH)
6.56e7.08 (m, 4H, AreH), 3.07 (s, 3H, NeCH3),
5.18 (s, 1H, OH), 6.12 (s, 2H, NH2),
7.46 (s, 1H, CH of pyrazole),
3.85 (q, 2H, CH2 of ester), 1.56 (t, 3H, CH3 of ester)
6.43e7.48 (m, 3H, AreH), 2.82 (s, 3H, NeCH3),
5.01 (s, 2H, 2.OH), 6.28 (s, 2H, NH2),
381.3
56.84
4.24
22.10
M þ 1 (56.80) (4.23) (22.11)
3334 (NH), 2196 (C^N), 1677 (C]O, ester),
1624 (C]O, pyrimidine), 1561 (C]N),
1543 (C]C), 3350 (2.OH)
397.3
54.54
4.07
21.20
M þ 1 (54.50) (4.11) (21.19)
7.74 (s, 1H, CH of pyrazole),
3.26 (q, 2H, CH2 of ester), 1.38 (t, 3H, CH3 of ester)

1886
B. Ramesh, C.M. Bhalgat / European Journal of Medicinal Chemistry 46 (2011) 1882e1891
Table 4
hydrogen peroxide, scavenging of nitric oxide radical, lipid perox-
idation inhibitory activity and reducing power determination. In
vitro antioxidant activity of synthesized compound is summarized
in Table 3.
Anti-inflammatory activity (in vitro) of compounds (2e4).
Compounds
Mean Absorbance ꢂ S.D.
% Inhibition of denaturation
Control
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4a
4b
4c
0.1980 ꢂ 0.024
0.2401 ꢂ 0.001
0.3353 ꢂ 0.008
0.3112 ꢂ 0.020
0.3210 ꢂ 0.010
0.2363 ꢂ 0.025
0.3547 ꢂ 0.004
0.3409 ꢂ 0.026
0.2736 ꢂ 0.046
0.3008 ꢂ 0.007
0.2853 ꢂ 0.016
0.2387 ꢂ 0.014
0.2814 ꢂ 0.019
0.2626 ꢂ 0.046
0.2700 ꢂ 0.005
0.2229 ꢂ 0.006
0.3191 ꢂ 0.009
0.3040 ꢂ 0.020
0.2467 ꢂ 0.018
0.2661 ꢂ 0.024
0.2550 ꢂ 0.008
0.2250 ꢂ 0.026
0.2949 ꢂ 0.016
0.2816 ꢂ 0.020
0.2864 ꢂ 0.009
0.2384 ꢂ 0.014
0.3260 ꢂ 0.024
0.3450 ꢂ 0.003
0.2651 ꢂ 0.025
0.3058 ꢂ 0.026
0.3121 ꢂ 0.016
0.3630 ꢂ 0.003
21.26
69.34
57.17
62.12
19.34
79.14
72.17
38.18
51.92
44.09
20.56
42.12
36.63
36.36
12.58
61.16
53.54
24.60
34.39
28.79
13.64
48.94
42.22
44.65
20.40
64.64
74.24
33.89
54.44
57.63
83.33
The investigation of antioxidant screening revealed that some of
the tested compounds showed moderate to good antioxidant
activity. Particularly, hydrazine derivatives (3aej) showed more
promising antioxidant activity as compared to that of standard,
ascorbic acid. This could be due the availability of free hydrazine
group. In scavenging of nitric oxide radical techniques compounds
3j and 3i shown low IC50 value than the standard. While, 3j, 3i and
3a has shown more potent activity by scavenging of hydrogen
peroxide. This may be due to additional OH group present on
benzene ring in the structure. All the compounds showed higher
IC50 value than the standard by lipid peroxidation inhibitory
activity and reducing power determination. Derivatives with OH
group on benzene ring having good antioxidant activity compared
with the other compounds in their series. 2j, 3h, 3a, 2i, 4j, 3f, 3b, 3g
and 3d having moderate to good antioxidant activity.
2.2.2. Anti-inflammatory activity
All of the newly obtained compounds 2e4 were tested for in
vitro anti-inflammatory activity. Compared to the standard, diclo-
fenac sodium, they have shown acceptable anti-inflammatory
activity. In vitro anti-inflammatory activity of compounds is
summarized in Table 4. The results revealed that the compounds,
2f, 4g, 2g and 2b exhibited moderate anti-inflammatory activities.
Amongst all the tested compounds 2f found to be more potent. The
4d
4e
4f
4g
4h
4i
4j
Standard
S.D. ¼ standard deviation (Average of three determination), Standard ¼ Diclofenac
Table 3
sodium.
Antioxidant activity (IC50 values) of compounds (2e4).
Compounds IC50 (Mean ꢂ S.D.)
m
g/mL
compounds 4f, 2d, 3f, 4j, 2c, 4i, 3g and 2i have showed good
activity. While other having weak to moderate activities.
Scavenging of Scavenging Lipid peroxidation Reducing power
nitric oxide
radical
of hydrogen inhibitory activity determination
peroxide
2.2.3. Antimicrobial activity
2a
2b
2c
2d
2e
2f
2g
2h
2i
73 ꢂ 0.087
78 ꢂ 0.121
108 ꢂ 0.318
87 ꢂ 0.058
93 ꢂ 0.780
71 ꢂ 0.082
81 ꢂ 0.162
67 ꢂ 0.082
58 ꢂ 0.162
53 ꢂ 0.082
56 ꢂ 0.052
60 ꢂ 0.066
65 ꢂ 0.453
63 ꢂ 0.183
69 ꢂ 0.318
59 ꢂ 0.453
62 ꢂ 0.045
54 ꢂ 0.024
46 ꢂ 0.333
44 ꢂ 0.279
79 ꢂ 0.318
87 ꢂ 0.183
48 ꢂ 0.121 53 ꢂ 0.333
63 ꢂ 0.024 58 ꢂ 0.183
79 ꢂ 0.318 90 ꢂ 0.453
72 ꢂ 0.087 83 ꢂ 0.121
84 ꢂ 0.279 98 ꢂ 0.066
56 ꢂ 0.333 54 ꢂ 0.024
67 ꢂ 0.453 72 ꢂ 0.121
43 ꢂ 0.066 48 ꢂ 0.162
39 ꢂ 0.279 44 ꢂ 0.333
37 ꢂ 0.087 41 ꢂ 0.183
32 ꢂ 0.318 39 ꢂ 0.066
49 ꢂ 0.121 43 ꢂ 0.318
58 ꢂ 0.318 78 ꢂ 0.045
53 ꢂ 0.066 68 ꢂ 0.087
66 ꢂ 0.162 63 ꢂ 0.162
41 ꢂ 0.087 46 ꢂ 0.453
46 ꢂ 0.024 57 ꢂ 0.279
36 ꢂ 0.121 37 ꢂ 0.318
30 ꢂ 0.183 34 ꢂ 0.087
29 ꢂ 0.087 31 ꢂ 0.121
74 ꢂ 0.333 68 ꢂ 0.121
86 ꢂ 0.082 94 ꢂ 0.024
58 ꢂ 0.333
61 ꢂ 0.183
68 ꢂ 0.453
71 ꢂ 0.121
78 ꢂ 0.066
64 ꢂ 0.024
76 ꢂ 0.121
54 ꢂ 0.162
52 ꢂ 0.333
48 ꢂ 0.183
41 ꢂ 0.066
50 ꢂ 0.318
64 ꢂ 0.045
61 ꢂ 0.087
81 ꢂ 0.162
44 ꢂ 0.453
58 ꢂ 0.279
44 ꢂ 0.318
38 ꢂ 0.087
34 ꢂ 0.024
59 ꢂ 0.087
69 ꢂ 0.453
91 ꢂ 0.333
79 ꢂ 0.024
98 ꢂ 0.279
64 ꢂ 0.121
78 ꢂ 0.318
54 ꢂ 0.318
48 ꢂ 0.183
41 ꢂ 0.087
31 ꢂ 0.183
The antimicrobial activities of the compounds 2e4 were tested
against Escheria coli, Pseudonmonas aeruginosa (gram-negative
bacteria), Bacillus subtillis and Staphylococcus aureus (gram-positive
bacteria) and two fungi Candida albicans and Aspergillus niger and
the results were reported as zone of inhibition. The results of
preliminary antibacterial testing of compounds 2e4 are shown in
Table 5. The results revealed that, all the pyrazole derivatives of
pyrimidines (4aej) were showing good to potent antibacterial
activity against all the tested strains of bacteria. While the entire
derivatives showed moderate to potent activity against Bacillus
subtilis. Amongst all the derivatives in series i.e. 2, 3 and 4, the
halogenated derivatives exhibited potent antibacterial activity. As
compare to standard streptomycin, compounds 4b, 4f and 4g
exhibited good to potent activity against all the tested strains.
While compounds 4i, 4j, 4c, 4d, 4e and 4h were moderately active.
Compound 2b is the most potent against B. subtilis. While
compounds 2g, 2f, 2j, 2i and 3b exhibited moderate to good activity
against B. subtilis. All the above data may reveal that the pyrazole
ring is responsible for the good antibacterial activity. While the
pyrimidine ring may responsible for the good activity against B.
subtilis. The halogenated substituent on benzene ring in the
structure of the synthesized compounds revealed in the increased
antibacterial activity. Moreover, the other compounds were weakly
active against the tested organism.
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4a
4b
4c
4d
4e
4f
4g
4h
4i
108 ꢂ 0.318 104 ꢂ 0.333 116 ꢂ 0.183
98 ꢂ 0.058 109 ꢂ 0.024 97 ꢂ 0.318
115 ꢂ 0.780 119 ꢂ 0.162 111 ꢂ 0.045
87 ꢂ 0.279
93 ꢂ 0.066
69 ꢂ 0.453
64 ꢂ 0.066
58 ꢂ 0.087
47 ꢂ 0.087
94 ꢂ 0.453 88 ꢂ 0.453
98 ꢂ 0.121 102 ꢂ 0.066
67 ꢂ 0.453 57 ꢂ 0.087
58 ꢂ 0.162 57 ꢂ 0.045
53 ꢂ 0.279 49 ꢂ 0.162
33 ꢂ 0.121 26 ꢂ 0.333
4j
Standard
The results of preliminary antifungal testing of the compounds
2e4 are shown in Table 6. All the compounds exhibited moderate to
good antifungal activity. Compounds 3g and 2c exhibited potent
S.D. ¼ standard deviation (Average of three determination), Standard ¼ Ascorbic
acid.

Table 5
Antibacterial activity of the compoundsa(2e4).
Compounds
Escheria coli
25
g mLꢀ1
Pseudomonas aeruginosa
25 50
g mLꢀ1 g mLꢀ1
Bacillus subtilis
25
g mLꢀ1
Staphylococcus aureus
25 50
g mLꢀ1 g mLꢀ1
m
50
m
g mLꢀ1
100
m
g mLꢀ1
m
m
100
m
g mLꢀ1
m
50
m
g mLꢀ1
100
m
g mLꢀ1
m
m
100
m
g mLꢀ1
ꢂS.D.
ꢂS.D.
ꢂS.D.
ꢂS.D.
ꢂS.D.
ꢂS.D.
ꢂS.D.
ꢂ S.D.
ꢂS.D.
ꢂS.D.
ꢂS.D.
ꢂS.D.
2a
2b
2c
2d
2e
2f
2g
2h
2i
5.08 ꢂ 0.26
10.86 ꢂ 1.16
5.74 ꢂ 1.16
5.86 ꢂ 1.73
e
7.19 ꢂ 0.58
13.78 ꢂ 0.58
6.12 ꢂ 1.53
6.29 ꢂ 2.52
e
8.79 ꢂ 0.45
14.23 ꢂ 0.58
7.82 ꢂ 2.09
8.78 ꢂ 2.65
e
6.19 ꢂ 0.26
9.17 ꢂ 1.16
e
8.84 ꢂ 1.16
10.39 ꢂ 0.26
e
9.17 ꢂ 2.09
11.84 ꢂ 0.58
e
11.67 ꢂ 0.61
16.90 ꢂ 0.45
11.12 ꢂ 0.26
10.36 ꢂ 0.47
10.26 ꢂ 1.71
13.76 ꢂ 2.09
14.16 ꢂ 1.16
10.38 ꢂ 1.16
12.85 ꢂ 2.09
13.04 ꢂ 1.53
9.26 ꢂ 1.53
13.12 ꢂ 1.16
10.84 ꢂ 1.53
9.27 ꢂ 1.16
8.10 ꢂ 1.16
10.15 ꢂ 1.53
11.06 ꢂ 1.16
9.90 ꢂ 1.16
9.81 ꢂ 1.53
11.48 ꢂ 1.16
10.19 ꢂ 1.16
14.78 ꢂ 1.53
12.92 ꢂ 1.16
12.88 ꢂ 1.16
11.21 ꢂ 1.53
14.18 ꢂ 2.09
13.42 ꢂ 1.16
11.62 ꢂ 1.53
13.92 ꢂ 1.16
14.42 ꢂ 1.16
15.14 ꢂ 0.78
13.1 ꢂ 0.58
20.16 ꢂ 0.61
12.23 ꢂ 1.53
12.90 ꢂ 1.16
13.95 ꢂ 0.61
18.13 ꢂ 0.58
19.33 ꢂ 0.58
14.36 ꢂ 1.73
16.88 ꢂ 0.45
15.82 ꢂ 0.45
10.33 ꢂ 1.16
15.66 ꢂ 0.58
13.33 ꢂ 0.58
10.10 ꢂ 0.17
10.48 ꢂ 0.58
15.98 ꢂ 0.17
15.10 ꢂ 1.53
12.24 ꢂ 0.17
13.78 ꢂ 0.58
14.49 ꢂ 2.09
14.04 ꢂ 0.58
19.26 ꢂ 0.58
14.11 ꢂ 1.16
15.05 ꢂ 0.58
14.82 ꢂ 0.47
16.71 ꢂ 1.16
17.16 ꢂ 0.58
15.10 ꢂ 0.45
14.23 ꢂ 1.16
15.10 ꢂ 2.09
20.23 ꢂ 0.47
19.11 ꢂ 1.53
29.39 ꢂ 1.16
15.59 ꢂ 1.53
18.52 ꢂ 1.53
20.26 ꢂ 0.45
27.29 ꢂ 2.89
28.80 ꢂ 1.73
20.27 ꢂ 0.45
25.49 ꢂ 1.53
24.37 ꢂ 1.71
13.62 ꢂ 1.53
21.62 ꢂ 1.16
14.19 ꢂ 1.16
15.37 ꢂ 1.53
14.48 ꢂ 2.62
24.15 ꢂ 0.63
22.68 ꢂ 1.53
16.67 ꢂ 0.47
22.21 ꢂ 0.63
22.13 ꢂ 1.53
18.14 ꢂ 1.16
28.00 ꢂ 1.16
18.30 ꢂ 1.53
18.67 ꢂ 0.61
20.90 ꢂ 0.47
25.39 ꢂ 1.53
26.18 ꢂ 2.00
19.25 ꢂ 1.16
23.06 ꢂ 1.53
21.80 ꢂ 0.61
31.82 ꢂ 1.53
8.76 ꢂ 0.58
12.16 ꢂ 0.47
7.06 ꢂ 1.16
6.29 ꢂ 0.45
6.38 ꢂ 0.58
9.68 ꢂ 0.26
9.40 ꢂ 0.58
e
12.80 ꢂ 0.17
15.33 ꢂ 0.58
9.12 ꢂ 2.09
8.92 ꢂ 0.61
8.25 ꢂ 0.17
11.68 ꢂ 0.58
12.32 ꢂ 0.61
e
14.84 ꢂ 0.58
20.92 ꢂ 0.61
12.55 ꢂ 1.53
11.10 ꢂ 0.37
9.38 ꢂ 1.16
15.76 ꢂ 2.09
16.79 ꢂ 0.26
e
e
e
e
6.06 ꢂ 0.58
8.85 ꢂ 1.16
8.52 ꢂ 1.16
5.86 ꢂ 0.26
6.36 ꢂ 0.45
7.67 ꢂ 0.58
5.00 ꢂ 1.00
9.00 ꢂ 2.09
e
8.52 ꢂ 0.47
9.62 ꢂ 0.61
10.37 ꢂ 1.53
8.25 ꢂ 0.58
8.16 ꢂ 0.58
8.12 ꢂ 1.16
6.96 ꢂ 1.53
13.85 ꢂ 0.17
e
10.63 ꢂ 0.61
11.95 ꢂ 0.58
12.02 ꢂ 0.58
9.93 ꢂ 1.16
10.79 ꢂ 1.16
12.68 ꢂ 2.09
7.21 ꢂ 0.54
15.39 ꢂ 0.58
e
7.08 ꢂ 1.16
9.64 ꢂ 0.58
e
9.82 ꢂ 0.58
10.21 ꢂ 0.58
e
11.65 ꢂ 1.53
12.39 ꢂ 1.53
e
6.25 ꢂ 1.16
7.28 ꢂ 0.58
6.28 ꢂ 0.58
9.85 ꢂ 0.26
e
9.16 ꢂ 0.47
8.78 ꢂ 1.16
7.71 ꢂ 1.16
13.48 ꢂ 0.58
e
11.27 ꢂ 0.00
10.39 ꢂ 2.00
7.49 ꢂ 0.58
15.35 ꢂ 1.16
e
8.14 ꢂ 0.17
8.70 ꢂ 0.61
e
9.33 ꢂ 1.16
10.60 ꢂ 0.58
e
11.18 ꢂ 0.26
13.61 ꢂ 0.26
e
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
13.58 ꢂ 0.58
8.02 ꢂ 0.58
9.12 ꢂ 0.26
e
17.30 ꢂ 1.16
10.52 ꢂ 1.16
10.61 ꢂ 0.58
e
22.18 ꢂ 0.61
12.21 ꢂ 0.58
13.38 ꢂ 0.26
e
e
e
e
e
e
e
5.74 ꢂ 0.17
8.18 ꢂ 2.09
9.26 ꢂ 0.45
7.38 ꢂ 0.47
8.39 ꢂ 0.61
8.72 ꢂ 0.26
10.48 ꢂ 0.47
15.10 ꢂ 2.09
12.96 ꢂ 0.17
12.54 ꢂ 1.71
11.10 ꢂ 0.58
14.72 ꢂ 0.45
13.16 ꢂ 0.61
10.26 ꢂ 0.26
13.48 ꢂ 0.58
13.12 ꢂ 0.47
15.78 ꢂ 0.17
7.82 ꢂ 0.58
10.95 ꢂ 2.00
10.11 ꢂ 0.58
7.16 ꢂ 0.58
9.58 ꢂ 1.16
8.16 ꢂ 0.58
14.72 ꢂ 0.58
22.28 ꢂ 1.16
14.18 ꢂ 0.45
13.12 ꢂ 0.58
10.18 ꢂ 0.47
19.48 ꢂ 1.71
20.48 ꢂ 0.58
12.36 ꢂ 0.47
17.27 ꢂ 0.58
16.00 ꢂ 0.58
21.10 ꢂ 1.16
8.28 ꢂ 1.16
12.42 ꢂ 0.58
13.10 ꢂ 1.16
8.12 ꢂ 1.16
11.22 ꢂ 2.09
10.18 ꢂ 1.16
17.39 ꢂ 1.16
27.49 ꢂ 0.47
18.04 ꢂ 1.16
16.11 ꢂ 1.16
14.03 ꢂ 0.17
25.02 ꢂ 1.16
26.90 ꢂ 1.16
16.18 ꢂ 0.61
24.42 ꢂ 1.16
22.90 ꢂ 1.16
28.71 ꢂ 0.58
5.78 ꢂ 1.16
7.64 ꢂ 1.16
8.41 ꢂ 0.26
6.15 ꢂ 1.16
7.27 ꢂ 1.16
8.10 ꢂ 0.45
8.21 ꢂ 2.09
12.67 ꢂ 0.26
9.19 ꢂ 1.16
9.67 ꢂ 1.53
8.29 ꢂ 1.16
11.79 ꢂ 0.61
12.15 ꢂ 1.16
9.90 ꢂ 1.16
10.16 ꢂ 0.17
10.28 ꢂ 1.16
13.36 ꢂ 1.53
6.27 ꢂ 0.58
10.18 ꢂ 2.09
12.65 ꢂ 1.16
8.12 ꢂ 0.17
9.17 ꢂ 1.53
10.10 ꢂ 0.58
10.23 ꢂ 0.58
16.06 ꢂ 0.45
12.12 ꢂ 2.09
12.72 ꢂ 0.58
11.92 ꢂ 0.26
17.12 ꢂ 2.09
16.31 ꢂ 0.58
12.26 ꢂ 0.58
15.00 ꢂ 2.09
12.82 ꢂ 0.58
18.70 ꢂ 0.45
8.83 ꢂ 1.73
14.90 ꢂ 0.58
15.36 ꢂ 0.58
9.15 ꢂ 1.73
13.04 ꢂ 0.58
12.92 ꢂ 0.61
16.12 ꢂ 1.73
26.25 ꢂ 0.61
19.85 ꢂ 0.26
18.82 ꢂ 1.73
16.73 ꢂ 0.58
25.67 ꢂ 0.26
23.88 ꢂ 1.73
17.42 ꢂ 0.58
21.12 ꢂ 0.58
20.33 ꢂ 1.73
30.33 ꢂ 0.61
11.19 ꢂ 0.58
12.82 ꢂ 0.58
8.72 ꢂ 0.58
10.64 ꢂ 0.26
e
15.53 ꢂ 1.16
15.94 ꢂ 0.63
11.15 ꢂ 1.76
12.24 ꢂ 1.16
e
20.60 ꢂ 1.16
19.32 ꢂ 0.26
15.75 ꢂ 0.26
17.68 ꢂ 0.45
e
3j
4a
4b
4c
4d
4e
4f
4g
4h
4i
10.12 ꢂ 0.37
15.76 ꢂ 2.09
10.92 ꢂ 0.45
11.82 ꢂ 0.17
10.12 ꢂ 0.58
13.07 ꢂ 0.63
14.60 ꢂ 2.09
10.33 ꢂ 0.58
12.33 ꢂ 0.61
12.67 ꢂ 0.71
15.39 ꢂ 0.58
15.05 ꢂ 0.26
20.84 ꢂ 0.45
14.10 ꢂ 1.16
12.18 ꢂ 1.71
13.48 ꢂ 0.58
17.06 ꢂ 1.16
18.26 ꢂ 0.61
14.10 ꢂ 0.58
17.38 ꢂ 1.16
16.12 ꢂ 0.45
22.41 ꢂ 2.09
18.65 ꢂ 1.53
26.67 ꢂ 1.16
17.71 ꢂ 1.53
16.49 ꢂ 0.61
16.52 ꢂ 0.58
22.00 ꢂ 0.47
24.32 ꢂ 0.58
18.67 ꢂ 0.17
22.82 ꢂ 0.47
20.30 ꢂ 0.17
29.48 ꢂ 0.17
4j
Standard
S.D. ¼ standard deviation; Standard ¼ Streptomycin.
a
Zone of inhibition.

1888
B. Ramesh, C.M. Bhalgat / European Journal of Medicinal Chemistry 46 (2011) 1882e1891
Table 6
Antifungal activities of compoundsa(2e4).
Compounds
Candida albicans
25
g mLꢀ1
Aspergillus niger
m
50
m
g mLꢀ1
100
m
g mLꢀ1
25
m
g mLꢀ1
50
m
g mLꢀ1
100 m
g mLꢀ1
ꢂ S.D.
ꢂ S.D.
ꢂ S.D.
ꢂ S.D.
ꢂ S.D.
ꢂ S.D.
2a
2b
2c
2d
2e
2f
2g
2h
2i
9.33 ꢂ 0.58
10.76 ꢂ 0.58
13.00 ꢂ 0.00
10.64 ꢂ 1.16
11.82 ꢂ 0.58
9.27 ꢂ 0.58
9.18 ꢂ 1.16
10.48 ꢂ 0.58
8.84 ꢂ 0.53
9.48 ꢂ 0.58
8.14 ꢂ 0.58
11.82 ꢂ 0.58
8.26 ꢂ 0.58
10.68 ꢂ 0.47
9.14 ꢂ 0.58
12.82 ꢂ 0.58
13.44 ꢂ 0.58
10.12 ꢂ 0.47
9.62 ꢂ 0.58
9.35 ꢂ 0.58
9.62 ꢂ 0.58
12.04 ꢂ 0.58
10.86 ꢂ 0.58
9.10 ꢂ 0.58
10.38 ꢂ 0.58
12.19 ꢂ 0.58
11.48 ꢂ 0.58
09.41 ꢂ 0.58
10.58 ꢂ 0.61
10.43 ꢂ 0.58
12.89 ꢂ 0.58
11.18 ꢂ 1.53
12.14 ꢂ 1.16
16.82 ꢂ 1.73
13.84 ꢂ 1.73
14.37 ꢂ 0.58
13.84 ꢂ 1.16
13.82 ꢂ 0.58
13.00 ꢂ 1.00
12.62 ꢂ 0.58
13.92 ꢂ 1.73
10.66 ꢂ 0.47
17.00 ꢂ 1.00
10.15 ꢂ 0.58
13.82 ꢂ 0.61
9.00 ꢂ 2.00
19.79 ꢂ 0.47
16.00 ꢂ 1.00
18.44 ꢂ 1.53
21.78 ꢂ 1.53
17.95 ꢂ 1.73
18.36 ꢂ 1.53
20.75 ꢂ 0.61
19.48 ꢂ 1.53
20.00 ꢂ 1.00
21.00 ꢂ 1.00
15.82 ꢂ 0.27
19.24 ꢂ 1.53
16.00 ꢂ 1.00
15.88 ꢂ 1.53
15.28 ꢂ 1.53
17.28 ꢂ 0.47
21.36 ꢂ 0.61
17.86 ꢂ 1.53
17.68 ꢂ 1.16
18.72 ꢂ 1.53
19.76 ꢂ 1.53
21.16 ꢂ 1.16
18.00 ꢂ 2.00
21.46 ꢂ 1.53
17.72 ꢂ 1.16
21.76 ꢂ 1.16
20.16 ꢂ 1.53
17.00 ꢂ 1.53
15.46 ꢂ 1.53
17.72 ꢂ 0.63
23.10 ꢂ 1.53
11.59 ꢂ 0.58
12.67 ꢂ 0.53
12.27 ꢂ 0.58
13.04 ꢂ 0.58
11.14 ꢂ 0.58
9.92 ꢂ 0.58
10.62 ꢂ 0.47
9.25 ꢂ 0.58
8.00 ꢂ 0.00
8.40 ꢂ 0.61
8.32 ꢂ 0.61
11.14 ꢂ 0.58
9.82 ꢂ 0.58
10.16 ꢂ 1.16
7.14 ꢂ 0.58
14.64 ꢂ 0.58
11.23 ꢂ 0.58
8.38 ꢂ 0.27
8.24 ꢂ 0.58
7.06 ꢂ 0.58
11.56 ꢂ 0.58
11.90 ꢂ 1.73
10.14 ꢂ 0.47
12.10 ꢂ 0.58
10.24 ꢂ 2.08
11.60 ꢂ 0.58
12.16 ꢂ 0.58
10.80 ꢂ 0.58
9.21 ꢂ 0.58
9.48 ꢂ 0.58
14.89 ꢂ 0.47
12.02 ꢂ 0.53
14.92 ꢂ 0.58
15.94 ꢂ 0.61
15.63 ꢂ 0.58
14.54 ꢂ 1.16
11.00 ꢂ 2.00
10.62 ꢂ 0.58
12.00 ꢂ 1.00
9.46 ꢂ 0.58
10.85 ꢂ 0.58
10.00 ꢂ 2.00
14.00 ꢂ 1.00
10.06 ꢂ 0.47
12.84 ꢂ 1.16
9.48 ꢂ 0.58
16.84 ꢂ 1.73
14.00 ꢂ 0.00
10.26 ꢂ 0.58
10.57 ꢂ 0.58
8.44 ꢂ 0.58
13.00 ꢂ 0.00
14.12 ꢂ 0.61
13.10 ꢂ 0.58
15.80 ꢂ 0.61
12.24 ꢂ 1.16
15.58 ꢂ 1.16
15.06 ꢂ 0.63
12.12 ꢂ 0.27
12.62 ꢂ 0.27
11.74 ꢂ 0.61
17.13 ꢂ 0.58
18.04 ꢂ 0.61
22.67 ꢂ 1.53
23.48 ꢂ 0.58
22.58 ꢂ 1.16
19.72 ꢂ 1.53
18.67 ꢂ 0.58
17.54 ꢂ 0.27
19.72 ꢂ 0.27
15.42 ꢂ 1.16
17.10 ꢂ 1.53
17.00 ꢂ 0.00
19.62 ꢂ 0.58
17.62 ꢂ 1.16
15.88 ꢂ 1.16
15.84 ꢂ 0.58
21.42 ꢂ 1.16
19.38 ꢂ 0.27
16.66 ꢂ 0.58
16.48 ꢂ 1.73
14.96 ꢂ 0.58
20.66 ꢂ 0.58
21.16 ꢂ 0.61
20.36 ꢂ 1.53
22.10 ꢂ 0.58
18.26 ꢂ 2.08
23.78 ꢂ 1.53
22.40 ꢂ 0.58
18.94 ꢂ 0.47
18.12 ꢂ 0.58
17.16 ꢂ 1.16
26.14 ꢂ 0.61
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
15.72 ꢂ 0.58
17.88 ꢂ 0.61
11.00 ꢂ 2.00
12.46 ꢂ 1.16
13.75 ꢂ 0.58
12.58 ꢂ 0.58
15.20 ꢂ 0.58
12.30 ꢂ 0.47
13.83 ꢂ 0.61
13.72 ꢂ 1.16
16.02 ꢂ 2.08
14.86 ꢂ 1.16
13.48 ꢂ 1.63
14.46 ꢂ 0.47
13.30 ꢂ 0.27
15.26 ꢂ 1.73
3j
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
Standard
S.D. ¼ standard deviation; Standard ¼ Amphotericin-B.
a
Zone of inhibition.
activity against C. albicans and compounds 3f, 4f, 2e, 3b and 4g
exhibited good activity than the standard. Compound 3f showed
potent activity against A. niger. While the compounds 2d, 4f, 4g, 4d,
2c, 2b, 2a, 2e, 3b, 3g, 4a, and 4b exhibited moderate to good
activity.
[29] in methanol were added to 2 mL of hydrogen peroxide solution
in phosphate buffer saline. The absorbance was measured at
230 nm after 10 min [30].
3.1.2. Nitric oxide scavenging activity
The reaction mixture (6 mL) containing sodium nitroprusside
(10 mM, 4 mL), phosphate buffer saline (pH 7.4, 1 mL) and test
samples or standard, ascorbic acid solution in dimethyl sulphoxide
2.2.4. Anthelmintic
Synthesized compounds 2e4 were also tested for in vitro
anthelmintic activity. All the compounds showed moderate to
potent anthelmintic activity. In vitro anthelmintic activity of
synthesized compounds is summarized in Table 7. Comparison of
anthelmintic activity data revealed that hydrazine derivatives of the
pyrimidines are slightly more active than the others. Compounds
3e, 3d, 3i, 2e, 2d, 2i and 4e showed more potent paralyzing effect
than the standard, albendazole. While in addition to these
compounds 4i, 4c, 3f, 2f and 2c exhibits less time for death than the
standard. Moreover, compounds 3a, 3c, 4f, 2j, 3j, 3g and 3b showed
anthelmintic activity comparable to that of reference compound.
While other compounds showed moderate activity. This whole data
revealed that dihydropyrimidine ring is responsible for the
anthelmintic activity and the compounds with additional hetero
atom in the structure may responsible for increase in the activity.
(1 mL) at various concentrations (12.5, 25, 50, 100
mg/mL) was
incubated at 25 ꢁC for 150 min. After incubation, 0.5 mL of reaction
mixture containing nitrite ion was removed, 1 mL of sulphanillic
acid reagent was added to this, mixed well and allowed to stand for
5 min for completion of diazotization. Then, 1 mL of naphthyl
ethylene diamine dihydrochloride was added, mixed and allowed
to stand for 30 min in diffused light. A pink colored chromophore
was formed. The absorbance was measured at 640 nm [31].
3.1.3. Lipid peroxidation inhibitory activity
Egg lecithin (3 mg/mL phosphate buffer, pH 7.4) was sonicated
in an ultrasonic sonicator for 10 min to ensure proper liposome
formation. Test samples or standard, ascorbic acid (100
different concentrations (12.5, 25, 50, 100 g/mL) were added to
liposome mixture (1 mL); the control was without test sample.
Lipid peroxidation was induced by adding ferric chloride (10 L,
400 mM) and -ascorbic acid (10 L, 200 mM). After incubation for
mL) of
m
3. Pharmacological screening
m
L
m
3.1. Antioxidant screening (in vitro)
1 h at 37 ꢁC the reaction was stopped by adding hydrochloric acid
(2 mL, 0.25 N) containing trichloroacetic acid (150 mg/mL), thio-
barbituric acid (3.75 mg/mL) and butylated hydroxy anisole
(0.50 mg/mL). The reaction mixture was subsequently boiled for
15 min, cooled, centrifuged at 1000 rpm for 15 min and the
absorbance of the supernatant was measured at 532 nm [32].
3.1.1. Hydrogen peroxide scavenging activity
A solution of hydrogen peroxide (20 mM) was prepared in
phosphate buffer saline (pH 7.4). Various concentrations (12.5, 25,
50,100 mg/mL) of 1 mL of the test samples or standard, ascorbic acid

B. Ramesh, C.M. Bhalgat / European Journal of Medicinal Chemistry 46 (2011) 1882e1891
1889
Table 7
Anthelmentic activity of compounds (2e4).
Compounds
Time (in minutes) ꢂ S.D.
For paralysis
For death
% Concentration (w/v)
% Concentration (w/v)
0.1
0.2
0.5
0.1
0.2
0.5
Control
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4a
4b
4c
e
e
e
e
e
e
54.56 ꢂ 0.98
72.34 ꢂ 0.59
42.68 ꢂ 1.12
39.78 ꢂ 0.21
35.25 ꢂ 0.44
48.15 ꢂ 0.52
55.29 ꢂ 0.89
75.54 ꢂ 0.83
40.28 ꢂ 0.67
58.04 ꢂ 0.93
47.23 ꢂ 1.56
54.93 ꢂ 0.80
43.73 ꢂ 1.90
32.35 ꢂ 0.43
28.25 ꢂ 0.14
42.10 ꢂ 0.23
50.65 ꢂ 1.56
55.23 ꢂ 0.24
35.42 ꢂ 0.45
52.22 ꢂ 0.37
52.09 ꢂ 1.44
70.67 ꢂ 0.17
47.26 ꢂ 1.46
42.48 ꢂ 0.82
41.45 ꢂ 2.20
49.28 ꢂ 0.62
58.08 ꢂ 0.45
75.14 ꢂ 1.12
43.09 ꢂ 0.49
60.22 ꢂ 0.67
50.38 ꢂ 0.52
46.56 ꢂ 0.45
71.40 ꢂ 0.84
36.59 ꢂ 0.72
38.48 ꢂ 0.91
44.52 ꢂ 1.43
39.29 ꢂ 0.76
40.27 ꢂ 0.92
57.18 ꢂ 1.28
39.46 ꢂ 0.28
62.28 ꢂ 0.71
45.23 ꢂ 0.88
51.34 ꢂ 0.77
40.73 ꢂ 0.49
29.13 ꢂ 0.43
26.35 ꢂ 0.24
40.13 ꢂ 0.92
52.38 ꢂ 2.03
55.57 ꢂ 0.34
33.85 ꢂ 0.67
48.56 ꢂ 0.72
49.46 ꢂ 0.69
68.48 ꢂ 1.95
42.22 ꢂ 0.94
40.12 ꢂ 1.63
46.58 ꢂ 1.08
41.03 ꢂ 1.34
42.05 ꢂ 0.65
55.10 ꢂ 0.51
40.03 ꢂ 1.23
65.54 ꢂ 0.90
35.58 ꢂ 0.59
42.65 ꢂ 1.44
55.17 ꢂ 1.88
32.55 ꢂ 0.23
33.35 ꢂ 1.65
40.22 ꢂ 1.24
40.51 ꢂ 1.12
40.61 ꢂ 1.91
41.47 ꢂ 1.47
35.14 ꢂ 1.23
50.10 ꢂ 1.08
42.60 ꢂ 1.33
48.77 ꢂ 1.56
37.52 ꢂ 2.23
26.13 ꢂ 1.31
25.64 ꢂ 0.46
37.67 ꢂ 1.90
45.38 ꢂ 0.33
51.42 ꢂ 1.31
30.13 ꢂ 1.06
45.65 ꢂ 1.25
45.02 ꢂ 0.53
48.38 ꢂ 0.74
45.98 ꢂ 1.17
43.59 ꢂ 1.01
42.87 ꢂ 0.49
34.76 ꢂ 0.51
46.18 ꢂ 0.44
43.90 ꢂ 1.02
38.52 ꢂ 1.55
53.34 ꢂ 1.12
30.57 ꢂ 1.26
78.22 ꢂ 1.87
90.40 ꢂ 1.43
60.18 ꢂ 2.17
45.24 ꢂ 1.04
47.57 ꢂ 1.02
60.96 ꢂ 1.04
72.39 ꢂ 0.20
83.02 ꢂ 1.18
50.82 ꢂ 0.78
65.29 ꢂ 0.39
62.32 ꢂ 0.87
68.45 ꢂ 1.86
61.58 ꢂ 1.63
58.24 ꢂ 0.31
54.22 ꢂ 1.22
58.30 ꢂ 0.49
67.33 ꢂ 0.28
71.45 ꢂ 1.76
50.13 ꢂ 1.44
66.22 ꢂ 1.29
67.41 ꢂ 1.28
88.45 ꢂ 1.55
58.76 ꢂ 0.41
47.38 ꢂ 1.03
49.79 ꢂ 0.97
64.72 ꢂ 0.45
72.26 ꢂ 1.12
87.55 ꢂ 0.56
55.20 ꢂ 1.69
70.56 ꢂ 1.40
60.36 ꢂ 1.02
72.67 ꢂ 0.82
85.73 ꢂ 0.49
55.49 ꢂ 1.32
47.82 ꢂ 0.58
55.59 ꢂ 0.41
57.38 ꢂ 0.69
55.80 ꢂ 0.85
60.44 ꢂ 0.89
48.34 ꢂ 0.56
65.50 ꢂ 0.81
60.22 ꢂ 1.75
65.43 ꢂ 0.67
57.48 ꢂ 0.59
44.42 ꢂ 0.35
46.44 ꢂ 0.56
46.45 ꢂ 0.88
66.37 ꢂ 1.44
70.25 ꢂ 0.35
37.10 ꢂ 0.83
63.98 ꢂ 0.86
63.37 ꢂ 0.40
80.74 ꢂ 0.65
55.23 ꢂ 0.79
49.67 ꢂ 0.37
57.55 ꢂ 1.02
58.23 ꢂ 1.26
57.50 ꢂ 0.38
62.12 ꢂ 0.61
49.54 ꢂ 0.20
75.78 ꢂ 0.87
55.25 ꢂ 0.93
62.21 ꢂ 0.82
69.54 ꢂ 0.93
53.98 ꢂ 0.44
42.34 ꢂ 0.62
54.10 ꢂ 0.60
55.12 ꢂ 0.71
50.24 ꢂ 0.55
56.08 ꢂ 0.38
45.30 ꢂ 0.49
61.02 ꢂ 0.96
57.45 ꢂ 0.78
61.02 ꢂ 0.59
50.98 ꢂ 0.97
40.30 ꢂ 1.13
43.34 ꢂ 0.35
42.54 ꢂ 0.76
60.86 ꢂ 1.21
67.22 ꢂ 1.10
44.10 ꢂ 1.02
60.27 ꢂ 0.81
57.93 ꢂ 1.44
60.77 ꢂ 0.87
54.82 ꢂ 0.48
47.44 ꢂ 1.42
58.72 ꢂ 0.88
56.33 ꢂ 0.53
51.70 ꢂ 0.70
52.69 ꢂ 0.31
47.23 ꢂ 1.69
60.74 ꢂ 0.56
53.34 ꢂ 1.62
4d
4e
4f
4g
4h
4i
4j
Standard
S.D. ¼ standard deviation (Average of three determination), Standard ¼ Albendazole.
For all the above antioxidant methods, experiments were done
in triplicate and average is taken, the % inhibition at different
concentration was calculated by the following formula
3.2. Anti-inflammatory screening (in vitro)
The synthesized compounds are screened for anti-inflammatory
activity by using inhibition of albumin denaturation technique
which was studied according to Muzushima and Kabayashi [34]
with slight modification. The standard drug and test compounds
were dissolved in minimum amount of DMF and diluted with
phosphate buffer saline (pH 7.4) in such a way that concentration of
% Inhibition ¼ ½1 ꢀ ðVt=VcÞꢃ ꢄ 100
Where, Vt ¼ mean absorption of test compound, Vc ¼ mean
absorption of control.
The IC50 value was derived from the % inhibition at different
concentration.
DMF in all solutions was less than 2.5%. Test solution (1 mL, 100 mg/
mL) was mixed with 1 mL of 1% albumin solution in phosphate
buffer saline and incubated at 27 ꢂ 1 ꢁC in an incubator for 15 min.
Denaturation was induced by keeping the reaction mixture at
60 ꢂ 1 ꢁC in a water bath for 10 min. After cooling, the turbidity was
measured at 660 nm with UV-vis spectrophotometer. Percentage of
inhibition of denaturation was calculated from control where no
drug was added. Each experiment was done in triplicate and
average is taken. The diclofenac was used as standard drug [35,36].
The percentage of inhibition was calculated using the formula
3.1.4. Reducing power determination
The reducing power of test samples was determined according
to the method of Oyaizu [33]. The test compounds and standard
drug, ascorbic acid were dissolved in N,N-dimethyl formamide
(DMF) to get different concentrations (12.5, 25, 50, 100 mg/mL). This
was mixed with 2.5 mL of (pH 6.6) 0.2 M phosphate buffer and
2.5 mL of 1% potassium ferricyanide. The mixture was incubated at
50 ꢁC for 20 min. 2.5 mL of 10% Trichloroacetic acid was added to
the mixture, which was then centrifuged for 10 min at 1000 rpm.
2.5 mL upper layer of solution was mixed with 2.5 mL of distilled
water and 0.5 mL of 0.1% ferric chloride. The absorbance was
measured at 700 nm. The blank was also carried out in similar
manner.
% Inhibition of denaturation ¼ ½ðVt=VcÞ ꢀ 1ꢃ ꢄ 100
Where, Vt ¼ mean absorption of test compound, Vc ¼ mean
absorption of control.
3.3. Antimicrobial activity
% Inhibition ¼ ½ðVt=VcÞ ꢀ 1ꢃ ꢄ 100
Where, Vt ¼ mean absorption of test compound, Vc ¼ mean
Applying the agar plate diffusion technique [37] all of the newly
synthesized compounds were screened in vitro for antibacterial
activity against E. coli, P. aeruginosa (Gram-negative), S. aureus, B.
absorption of control.
The IC50 value was derived from the % inhibition at different
concentration.
subtilis (Gram-positive) at 25, 50 and 100 mg/mL concentrations,

1890
B. Ramesh, C.M. Bhalgat / European Journal of Medicinal Chemistry 46 (2011) 1882e1891
respectively. Streptomycin (binds to the 16SrRNA of the bacterial
ribosome, interfering with the binding of formylmethionyl-tRNA to
the 30S subunit therefore prevents initiation of protein synthesis
and leads to death of microbial cell) was chosen as a standard drug
[38]. Streptomycin is an antibiotic that inhibits both gram-positive
and gram-negative bacteria, and is therefore a useful broad spec-
trum antibiotic. Similarly, the antifungal screening of the
compounds was carried out in vitro by paper disc method against
two fungi A. niger and C. albicans by using Amphotericin-B (binds to
plasma membrane sterols like ergosterol. The fungi cells have large
amount of ergosterol in plasma membrane. The ergosterol facili-
tates the attachment of amphotericin B, which act as ionophores
and cause leakage of cations like Kþ) as a standard [35,39].
Infra red (IR) spectra was recorded by using KBr disk on a Thermo
Nicolate IR-400 FTIR spectrophotometer, 1H NMR spectra was
recorded on Bruker Avance-300F spectrometer (300 MHz) using
tetramethylsilane as internal standard (chemical shift in
d ppm).
Mass spectra were recorded on a Triple Quadrupole LC-MSMS
(Sciex with ESI source) spectrometer. The elemental analysis was
carried out by using Heraus CHN rapid analyzer. All the compounds
gave C, H and N analysis within ꢂ1.1% of the theoretical values.
6. Material and methods
6.1. General procedure for the synthesis of 6-oxo-4-substituted
aryl-2-sulfanyl-1,6-dihydropyrimidine-5-carbonitrile (1aej)
3.4. Anthelmintic activity
A mixture equimolar quantities of ethyl cyanoacetate (5.7 g,
50 mmol), thiourea (3.8 g, 50 mmol), appropriate aromatic alde-
hyde (50 mmol) and potassium carbonate (6.9 g, 50 mmol) in
absolute ethanol (50 mL) was gently refluxed for 12 h. The reaction
mixture was neutralized with glacial acetic acid to precipitate out
the product. The product was isolated and recrystallized from
ethanol as yellow crystals.
The synthesized compounds were screened for anthelmintic
activity by using earthworms, Perituma posthuma [40]. Six earth-
worms of nearly equal size were placed in standard drug solution
and test compound solutions at room temperature. Normal saline
used as control. The standard drug and test compounds were dis-
solved in minimum quantity of dimethyl formamide (DMF) and
adjusted the volume up to 15 mL with normal saline solution to get
the concentration of 0.1, 0.2 and 0.5% w/v. Albendazole was used as
a standard drug [41]. The compounds were evaluated by the time
taken for complete paralysis and death of earthworms. The mean
lethal time for each test compound was recorded and compared
with standard drug. The time taken by worms to become motion-
less was noted as paralysis time. To ascertain the death of the
motionless worms were frequently applied with external stimuli,
which stimulate and induce movement in the worms, if alive. The
anthelmintic activity data was revealed in the form of mean lethal
time and paralysis time of the earthworms for different test
compounds and the standard drug.
6.2. General procedure for the synthesis 1-methyl-2-(methylthio)-
5-cyano-6-oxo-4-substituted-1,6-dihydropyrimidine (2aej)
To a solution of 6-oxo-4-substituted aryl-2-sulfanyl-1,6-dihy-
dropyrimidine-5-carbonitrile (1aec, 20 mmol) in N,N,dimethyl
formamide (DMF, 30 mL), potassium carbonate (5.52 g, 40 mmol)
and methyl iodide (5.68 g, 40 mmol) were added and stirred for 3 h
at room temperature. Then the reaction mixture was diluted with
cold water and neutralized by glacial acetic acid. The product was
filtered off and recrystallized from ethanol as creamish crystals.
6.3. General procedure for the synthesis of 2-hydrazino-1-methyl-
5-cyano-6-oxo-4-substituted-1,6-dihydropyrimidine (3aej)
4. Conclusion
In conclusion, we have described simple and efficient protocol
for the synthesis of novel dihydropyrimidines (1aej), its dimethy-
lated adducts (2aej), and hydrazine derivatives (3aej) of 2aej and
its pyrazole derivatives (4aej) with moderate to good yields. All the
synthesized compounds 2e4 have been investigated for their in
vitro antioxidant, anti-inflammatory, antibacterial, antifungal and
anthelmintic activity. In our newly synthesized compounds, it is
cleared that the highest antioxidant activity for compounds 3j and
3i, anti-inflammatory activity for compound 2f, antibacterial
activity for compound 4b, antifungal activity for compounds 3g and
2c against C. albicans and 3f against A. niger and anthelmintic
activity for compounds 4e and 2d were observed. Accordingly,
these novel classes of dihydropyrimidines and its pyrazole deriva-
tives presented in our laboratory emerged as a valuable lead series
that might be useful as antioxidant, anti-inflammatory, antibacte-
rial, antifungal and anthelmintic agents and hence promising
candidates for further efficacy evaluation.
A mixture of compound 1-methyl-2-(methylthio)-5-cyano-6-
oxo-4-substituted-1,6-dihydropyrimidine (2aec, 10 mmol) and
hydrazine hydrate (80%, 0.96 g, 30 mmol) in absolute alcohol was
refluxed for 6 h. The reaction mixture was poured into crushed ice.
Then the product was isolated and recrystallized from ethanol/DMF
mixture as yellow crystals.
6.4. General procedure for the synthesis of ethyl 5-amino-1-(5-
cyano-1-methyl-6-oxo-4-substituted-1,6-dihydropyrimidin-2-yl)-
1H-pyrazole-4-carboxylate (4aej)
A mixture of compound 2-hydrazino-1-methyl-5-cyano-6-oxo-
4-substituted-1,6-dihydropyrimidine (3aec, 10 mmol) and ethyl-2-
cyano-3-ethyl acrylate (1.69 g, 10 mmol) in absolute alcohol and
few drops of glacial acetic acid was refluxed for 5e6 h. Excess of
solvent is removed and mixture was added to crushed ice to get
above said products.
5. Experimental section
Unless otherwise noted, materials were obtained from
commercial suppliers and used without further purification.
Melting points were determined by Micro control based melting
point instrument and are uncorrected. All reactions were moni-
tored by thin-layer chromatography on 0.25 mm silica gel (60GF-
254) plates, using ethyl acetate:butanol:chloroform in the ratio of
[1:2:1] as mobile phase and visualized with UV light. Column
chromatography was performed on silica gel (200e300 mesh).
Acknowledgment
The authors are thankful to Principal, Sri Adichunchanagiri
College of Pharmacy, B. G. Nagara for providing laboratory facilities.
Authors are also grateful to Department of University scientific
instrument center, Karnatak University, Dharwad, India and Suven
Life Sciences, Hyderabad for providing spectral and elemental
analysis data.

B. Ramesh, C.M. Bhalgat / European Journal of Medicinal Chemistry 46 (2011) 1882e1891
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