Synthesis, antimicrobial and antioxidant activities of substituted pyrazoles, isoxazoles, pyrimidine and thioxopyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.06.024

A new class of heterocycles, substituted pyrazoles, isoxazoles, pyrimidines, thioxopyrimidines were prepared from Michael adducts, 2-(1,2-diaroylethyl)malononitrile and 2-(1,2-diarylsulfonylethyl)malononitrile by cyclocondensation with appropriate nucleop

Full text of DOI:10.1016/j.ejmech.2009.06.024

European Journal of Medicinal Chemistry 44 (2009) 4557–4566
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial and antioxidant activities of substituted pyrazoles,
isoxazoles, pyrimidine and thioxopyrimidine derivatives
*
A. Padmaja , T. Payani, G. Dinneswara Reddy, V. Padmavathi
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A newclassofheterocycles, substitutedpyrazoles, isoxazoles, pyrimidines, thioxopyrimidineswereprepared
from Michael adducts, 2-(1,2-diaroylethyl)malononitrile and 2-(1,2-diarylsulfonylethyl)malononitrile by
cyclocondensation with appropriate nucleophiles. The lead compounds were screened for the antimicrobial
and antioxidant activities.
Received 8 April 2009
Received in revised form
20 June 2009
Accepted 22 June 2009
Available online 28 June 2009
Ó 2009 Published by Elsevier Masson SAS.
Keywords:
Malononitrile
Diaminopyrazoles
Aminoiminoisoxazoles
Diaminopyrimidines
Diaminothioxopyrimidines
N,N0-Dimethyldiiminopyrimidines
Michael addition
Antimicrobial activity
Antioxidant activity
1. Introduction
synthesis of substituted pyrazoles, isoxazoles and pyrimidines.
However, the development of simple, facile and efficient method-
Over the years pyrazoles, isoxazoles and pyrimidines have
emerged as an interesting class of five and six membered hetero-
cycles with an astonishingly wide range of applications in phar-
maceutical chemistry. Barbituric acid and its derivatives are known
as sedatives, hypnotics since a long time. Substituted barbituric and
thiobarbituric acids show analgesic, antipyretic and anti-inflam-
matory activities [1–3]. Apart from these, pyrazolidine derivatives
are effective in the treatment of rheumatoid arthritis and allied
conditions [4,5]. Isoxazoline derivatives possess potent antith-
rombic effect and improved pharmaco-kinetic properties [6]. In
fact, a variety of pyrazole and isoxazole derivatives exhibit COX-I/
COX-II inhibiting activity. It is found that Celecoxib, a pyrazole
derivative and Valdecoxib, an isoxazole derivative have no effect on
platelet aggregation and does not reduce increased PG levels in
cerebrospinal fluid [7]. In addition, pyrazole and isoxazole deriva-
tives exhibit various biological properties viz., bacteriostatic, anti-
diabetic, analgesic, antiarrhythmic, anti-inflammatory, antifungal
and antiviral [4,8–12]. Many synthetic procedures exist for the
ologies to get five and six membered heterocycles is one of the
major aspects in organic synthesis. In fact, the Michael acceptors
are valuable intermediates in a variety of synthetic transformations
and useful as building blocks in the synthesis of biologically active
heterocycles. The present communication deals with the synthesis
of diaminopyrazole, isoxazole, pyrimidine and thioxopyrimidine
derivatives from Michael adducts by cyclocondensation with
different nucleophiles.
2. Chemistry
The synthetic scheme involves the Michael addition of malono-
nitrile to 1 and 8 in the presence of K2CO3 in methyl ethyl ketone to
get 2 and 9, respectively (Scheme 1). The gem-dicyano functionality
in 2 and 9 was exploited to get the desired heterocycles. The cyclo-
condensation of 2 and 9 with hydrazine hydrate in the presence of
NaOMe in methanol produced 10. Likewise, 4 and 11 were prepared
by the reaction of 2 and 9 with hydroxylamine hydrochloride
(Schemes 1 and2). Similarly, six membered heterocycles, 5, 7,12, and
14 were produced by cyclocondensation reaction of 2 and 9 with
urea and N,N0-dimethyl urea. In addition, 6 and 13 were prepared by
refluxing compounds 2 and 9 with thiourea (Schemes 1 and 2). The
* Corresponding author. Tel.: þ91 877 2289303; fax: þ91 877 2249532.
E-mail address: adivireddyp@yahoo.co.in (A. Padmaja).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.06.024

4558
A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
O
Ar
Ar
O
1
N
5
N
2
N2
3
O
1
5
1
3
NH2
Ar
4
NH
Ar
H2N
O
4
H2N
O
i
1'
1'
Ar
Ar
2'
2'
O
O
3
4
NC
CN
iii
ii
O
Ar
Ar
O
2
O
O
Me
Me
N
N
O
vi
iv
2
v
N1 3N
4
HN
O
NH
Ar
6
5
H2N
O
NH2
Ar
S
Ar
1'
2'
Ar
N
N
O
5
7
H2N
O
NH2
Ar
Ar
O
6
a: Ar = Ph
b: Ar = 4-Me.Ph
c: Ar = 4-Cl.Ph
i. CH2(CN)2 / K2CO3
ii. NH2NH2.H2
iii. NH2
iv. NH2CONH2
v. NH2CSNH2
Scheme 1.
spectral data IR, 1H NMR, 13C NMR and microanalyses were used to
acertain the structures of all the compounds.
4. Results and discussion
We have synthesized a series of heterocycles, 3a–c to 7a–c and
10a–c to 14a–c by the reaction of 2a–c and 9a–c with appropriate
nucleophiles in the presence of sodium methoxide in methanol as
presented in Schemes 1 and 2.
3. Biology
3.1. Antimicrobial activity
Compounds 3–7 and 10–14 were tested for in vitro antimicrobial
activity against the Gram-positive bacteria Staphylococcus aureus
(NCIM No. 5021), Bacillus subtilis (NCIM No. 2063), the Gram-
negative bacteria Klebsiella pneumoniae (NCIM No. 2957), Proteus
vulgaris (NCIM No. 2027) and fungi Fusarium solani (NCIM No.
1330), Curvularia lunata (NCIM No. 716) and Aspergillus niger (NCIM
No. 596). The primary screen was carried out by agar disc-diffusion
method [13] using nutrient agar medium. The minimum inhibitory
concentration for the most active compounds 3c, 4c, 10c and 11c
against the same microorganisms used in the preliminary
screening was carried out using microdilution susceptibility
method [14]. Chloramphenicol and Ketoconazole were used as
control drugs. The observed data on the antimicrobial activity of the
compounds and control drugs are given in Tables 1–5.
4.1. Biological results
The results of preliminary antibacterial testing of compounds 3–
7 are shown in Table 1. The results revealed that, compounds 3c and
4c displayed excellent activity against Gram-positive bacteria
(inhibitory zone >28 mm) and good activity against Gram-negative
bacteria (inhibitory zone >24 mm). Compound
6 displayed
moderate to high activity towards gram (þve) bacteria (16–25 mm)
and moderate activity (16–22 mm) towards gram (ꢀve) bacteria.
Compounds 5 and 7 exhibited least activity against both bacteria.
All the test compounds except 5 and 7 showed moderate to high
inhibitory effect towards tested fungi (Table 2).
On the other hand, the results of preliminary antibacterial
testing of compounds 10–14 as shown in Table 3 revealed that,
compounds 10c and 11c exhibited maximum activity against Gram-
positive bacteria (inhibitory zone >30 mm) and good activity
against Gram-negative bacteria (inhibitory zone >25 mm).
Compound 13 showed moderate to high activity towards gram
(þve) bacteria (18–25 mm) and moderate activity (17–22 mm)
towards gram (ꢀve) bacteria. Compounds 12 and 14 displayed low
3.2. Antioxidant testing
Compounds 3–7 and 10–14 were tested for antioxidant property
by nitric oxide [15,16] and DPPH [17] methods. The observed data
on the antioxidant activity is given in Table 6.

A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
4559
O
O
Ar
O
S
S
Ar
O
8
N
5
N
2
N
O
1
3
NH2
4
NH
H2N
H2N
i
O
O
O
O
Ar
O
S
Ar
O
S
1'
S
Ar
S
Ar
2'
O
O
10
11
NC
O
CN
iii
ii
O
Ar'
S
S
Ar
O
O
O
9
O
O
vi
iv
2
Me
Me
N1 3 N
v
N
O
N
S
6
4
5
H2N
NH2
HN
S
NH
S
O
O
O
Ar
O
S
Ar
O
1'
O
S
Ar
N
O
N
S
2'
Ar
H2N
NH2
12
14
O
Ar
O
S
Ar
O
13
a: Ar = Ph
i. CH2(CN)2 / K2CO3 / MEK (61-66%)
b: Ar = 4-Me.Ph
c: Ar = 4-Cl.Ph
ii NH2NH2.H2O / NaOMe / MeOH (62-68%)
iii. NH2OH.HCl / NaOMe / MeOH (67-74%)
iv. NH2CONH2 / NaOMe / MeOH (64-73%)
v. NH2CSNH2 / NaOMe / MeOH (69-72%)
vi. MeNHCONHMe / NaOMe / MeOH (61-65%)
Scheme 2.
activity against both bacteria. All the test compounds inhibited the
spore germination of tested fungi. All the test compounds exhibited
relatively high inhibitory activity on F. solani, C. lunata, than on
A. niger (Table 4).
Further, the compounds having diaminopyrazole, amino-
iminoisoxazole units displayed pronounced activity. Besides,
compounds having diarylsulfonyl units (10–14) showed compara-
tively high antimicrobial activity than the compounds with diaroyl
units (3–7).
The MIC values were determined as the lowest concentration
that completely inhibited visible growth of the microorganisms
(Table 5). The structure–antimicrobial activity relationship of the
synthesized compounds revealed that diamino substituted pyr-
azoles and amino-imino substituted isoxazoles displayed high
activity. The compounds having N,N0-dimethyl-imino substituted
pyrimidine derivatives exhibited low activity. The maximum
activity was attained with the compounds having chloro substit-
uent in the aryl moiety viz., 3c, 4c, 10c and 11c.
5. Conclusion
new class of heterocycles diaminopyrazoles, amino-
A
iminoisoxazoles, diaminopyrimidines, diaminothioxopyrimidines
and N,N0-dimethyldiiminopyrimidines were prepared from simple
substrates 2-(10,20-diaroylethyl)malononitrile (2) and 2-(1,2-diary-
lsulfonylethyl)malononitrile (9) by cyclocondensation with appro-
priate nucleophiles. The antimicrobial testing showed that
compounds having diaminopyrazoles and aminoiminoisoxazole
units possess excellent activity. Further, the compounds with
sulfone moieties displayed pronounced antimicrobial and antioxi-
dant activities than with diaroyl units. The maximum activity was
observed with compounds having chloro substituent in the aryl
moiety.
6. Experimental
6.1. Chemistry
4.2. Antioxidant testing
Melting points were determined in open capillaries on a Mel-
Temp apparatus and are uncorrected. The purity of the
compounds was checked by TLC (silica gel H, BDH, ethyl acetate/
hexane, 1:2). The IR spectra were recorded on a Thermo Nicolet IR
200 FT-IR spectrometer as KBr pellets and the wave numbers were
given in cmꢀ1. The 1H NMR spectra were recorded in CDCl3/
Compounds 3–7 and 10–14 were tested for antioxidant property
by nitric oxide and DPPH methods. Compounds 3c, 4c, 10a and 11c
exhibited high antioxidant property in both nitric oxide and DPPH
methods at 100 mM concentration (Table 6).

4560
A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
Table 1
Table 2
Antibacterial activity of 3–7.
Antifungal activity of 3–7.
Compound
Concentration Zone of inhibition (mm)
Compound
Concentration (
m
g/disc)
Zone of inhibition (mm)
(mg/disc)
Gram-positive bacteria Gram-negative
bacteria
Fusarium
solani
Curvularia
lunata
Aspergillus
niger
3a
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
31
33
32
35
33
37
30
31
32
34
33
35
13
16
14
18
17
19
16
20
15
19
19
22
11
13
12
15
14
16
38
42
–
30
32
31
33
34
36
30
32
31
33
32
34
16
20
15
17
14
18
19
22
15
18
17
21
13
15
14
16
13
15
41
44
–
29
30
30
31
32
33
27
29
24
27
26
28
14
17
13
15
16
20
14
19
14
16
15
20
12
16
10
12
11
13
36
39
–
Staphylococcus Bacillus Klebsiella
Proteus
aureus
subtilis pneumoniae vulgaris
3b
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
27
30
25
28
31
33
23
26
22
25
29
32
11
14
12
15
13
15
22
24
20
21
25
26
10
13
10
12
12
14
35
41
–
29
31
27
29
28
32
24
27
26
28
28
30
16
15
11
12
12
14
23
24
21
24
23
25
09
11
09
10
11
13
38
44
–
27
29
25
27
28
30
23
25
22
24
25
27
11
12
10
12
11
13
19
21
18
20
19
22
07
09
07
08
09
10
37
42
–
23
26
24
26
25
28
21
24
21
23
25
26
12
14
13
14
10
12
17
19
16
18
20
22
06
08
07
09
09
11
42
45
–
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
Ketoconazole
Control (DMSO)
Chloramphenicol 100
200
Control (DMSO)
Calcd. For C19H14N2O2: C, 75.48; H, 4.67; N, 9.27; Found: C, 75.60; H,
4.65; N, 9.41%.
DMSO-d6 on a Jeol JNM
recorded in CDCl3/DMSO-d6 on a Jeol JNM spectrometer operating
at 75.5 MHz. All chemical shifts are reported in (ppm) using TMS
l
-300 MHz. The 13C NMR spectra were
6.1.1.2. 2-(1,2-Di(4-methylbenzoyl)ethyl)malononitrile (2b). White
solid (2.048 g, 62%); m.p. 135–137 ꢁC; IR (KBr): 1708 (C]O), 2239
d
(C^N) cmꢀ1; 1H NMR (CDCl3)
d 2.21, 2.28 (s, 6H, Ar-CH3), 3.27 (dd,
as an internal standard. The microanalyses were performed on
a Perkin–Elmer 240C elemental analyzer. The starting compounds
(E)-1,4-diarylbut-2-ene-1,4-dione (1) and 1,2-diarylsulfonylethene
(8) were prepared as per the literature procedure [18,19].
1H, C4-H, J ¼ 8.9, 14.1 Hz), 3.39 (dd, 1H, C4-H, J ¼ 4.6, 14.5 Hz), 3.92–
3.95 (m, 1H, C3-H), 4.43 (d, 1H, C2-H, J ¼ 8.7 Hz), 7.15–7.49 (m, 8H,
Ar-H) ppm; 13C NMR (CDCl3)
d 22.4, 22.7 (Ar-CH3), 35.2 (C-3), 42.6
(C-2), 52.6 (C-4), 114.6 and 115.2 (CN), 204.7, 205.8 (Ar-CO), 126.2,
128.4, 129.3, 132.0, 134.1, 134.8, 135.1 (aromatic carbons). Anal.
Calcd. For C21H18N2O2: C, 76.34; H, 5.49; N, 8.48; Found: C, 76.40; H,
5.89; N, 8.60%.
6.1.1. General procedure for the preparation of 2-(1,2-
diaroylethyl)malononitrile 2a–c/2-(1,2-
diarylsulfonylethyl)malononitrile (9a–c)
A mixture of malononitrile (15 mmol), methyl ethyl ketone
(5 ml) and potassium carbonate (10 mmol) was cooled to 5–10 ꢁC.
To this, compound 1/8 (10 mmol) was added and stirred for 3–5 h
maintaining the same temperature. The contents of the flask were
diluted with water and extracted with dichloromethane. The
organic layer was washed with water, brine and dried over anhy-
drous Na2SO4. The solvent was removed in vacuo. The resultant
solid was recrystallized from 2-propanol.
6.1.1.3. 2-(1,2-Di(4-chlorobenzoyl)ethyl)malononitrile (2c). White
solid (2.143 g, 65%); m.p. 150–152 ꢁC; IR (KBr): 1694 (C]O), 2231
(C^N) cmꢀ1; 1H NMR (CDCl3)
d
3.24 (dd, 1H, C4-H, J ¼ 8.5, 14.0 Hz),
3.32 (dd, 1H, C4-H, J ¼ 4.3, 14.2 Hz), 4.01–4.06 (m, 1H, C3-H), 4.47 (d,
1H, C2-H, J ¼ 8.6 Hz), 7.21–7.64 (m, 8H, Ar-H) ppm; 13C NMR (CDCl3)
d
36.2 (C-3), 41.8 (C-2), 51.8 (C-4), 113.2 and 114.8 (CN), 203.9, 204.9
(Ar-CO), 126.1, 127.6, 129.0, 132.2, 133.4, 134.9, 136.4 (aromatic
carbons). Anal. Calcd. For C19H12Cl2N2O2: C, 61.47; H, 3.26; N, 7.55;
Found: C, 61.41; H, 3.28; N, 7.50%.
6.1.1.1. 2-(1,2-Dibenzoylethyl)malononitrile
(1.813 g, 60%); m.p. 122–124 ꢁC; IR (KBr): 1703 (C]O), 2241 (C^N)
cmꢀ1;1HNMR(CDCl3)
3.21 (dd,1H, C4-H, J ¼ 8.2,14.1 Hz), 3.29 (dd,1H,
(2a). White
solid
6.1.1.4. 2-(1,2-Diphenylsulfonylethyl)malononitrile (9a). White solid
d
(2.284 g, 61%); m.p. 136–138 ꢁC; IR (KBr): 1136, 1336 (SO2), 2245
C4-H, J ¼ 4.0, 14.3 Hz), 4.07–4.09 (m, 1H, C3-H), 4.32 (d, 1H, C2-H,
(C^N) cmꢀ1; 1H NMR (CDCl3)
d
3.04 (dd, 1H, C4-H, J ¼ 8.0, 14.0 Hz),
J ¼ 8.3 Hz), 7.11–7.52 (m,10H, Ar-H) ppm; 13C NMR (CDCl3)
d
36.8 (C-3),
3.27 (dd, 1H, C4-H, J ¼ 4.0, 14.1 Hz), 4.05–4.11 (m, 1H, C3-H), 4.49 (d,
41.1 (C-2), 52.2 (C-4), 113.5 and 116.8 (CN), 204.6, 207.9 (Ar-CO), 129.6,
130.2, 131.8, 132.9, 133.6, 134.9, 135.4, 136.2 (aromatic carbons). Anal.
1H, C2-H, J ¼ 8.1 Hz), 7.13–7.64 (m, 10H, Ar-H) ppm; 13C NMR
(CDCl3)
d 37.1 (C-3), 40.7 (C-2), 52.9 (C-4), 115.3 and 116.2 (CN),

A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
4561
Table 3
Table 4
Antibacterial activity of 10–14.
Antifungal activity of 10–14.
Compound
Concentration Zone of inhibition (mm)
Compound
Concentration
g/ml)
Zone of inhibition (mm)
(
mg/disc)
(m
Gram-positive
bacteria
Gram-positive
bacteria
Fusarium
solani
Curvularia
lunata
Aspergillus
niger
10a
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
34
37
30
35
35
39
31
34
28
31
33
35
14
19
13
16
17
20
18
20
19
21
22
25
13
16
11
14
16
19
38
42
–
33
36
31
33
34
38
29
32
28
30
31
33
18
21
16
18
19
23
19
22
21
23
25
28
16
20
13
16
17
21
41
44
–
31
34
28
31
32
35
28
31
26
29
29
32
15
18
11
15
16
19
16
19
17
20
20
23
14
16
10
13
15
17
36
39
–
Staphylococcus Bacillus Klebsiella
Proteus
aureus
subtilis pneumoniae vulgaris
10b
10a
10b
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
14a
14b
14c
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
30
32
27
29
34
36
29
31
26
27
32
33
18
20
21
23
22
24
22
25
21
24
25
27
13
15
11
13
16
18
35
41
–
31
33
26
28
35
37
30
32
26
29
31
34
16
19
22
24
23
25
23
26
20
22
23
25
14
16
12
14
15
17
38
44
–
28
30
23
26
30
32
26
29
25
27
26
29
16
18
17
19
18
20
21
23
19
21
20
22
09
12
09
11
12
14
37
42
–
24
25
22
25
31
33
26
28
24
25
28
30
14
16
16
18
17
19
19
21
20
20
21
23
10
12
08
10
11
13
42
45
–
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
14a
14b
14c
Ketoconazole
Control (DMSO)
Chloramphenicol 100
200
Control (DMSO)
6.1.1.6. 2-(1,2-Di(4-chlorophenylsulfonylethyl)malononitrile (9c).
White solid (2.926 g, 66%); m.p. 159–161 ꢁC; IR (KBr): 1125, 1337
(SO2), 2238 (C^N) cmꢀ1 1H NMR (CDCl3)
; d 3.11 (dd, 1H, C4-H,
129.2, 130.6, 131.3, 132.3, 133.9, 134.2, 135.1, 135.9 (aromatic
carbons). Anal. Calcd. For C17H14N2O4S2: C, 54.53; H, 3.77; N, 7.48;
Found: C, 54.56; H, 3.76; N, 7.57%.
J ¼ 8.3, 14.1 Hz), 3.28 (dd, 1H, C4-H, J ¼ 4.0, 14.2 Hz), 4.11–4.17
(m, 1H, C3-H), 4.36 (d, 1H, C2-H, J ¼ 8.1 Hz), 7.25–7.81 (m, 8H, Ar-H)
ppm; 13C NMR (CDCl3)
d 36.9 (C-3), 42.7 (C-2), 52.9 (C-4), 113.4 and
115.2 (CN), 128.0, 129.4, 131.9, 132.2, 134.9, 135.3, 135.9, 137.8
(aromatic carbons). Anal. Calcd. for C17H12Cl2N2O4S2: C, 46.06; H,
2.73; N, 6.32; Found: C, 46.13; H, 2.71; N, 6.37%.
6.1.1.5. 2-(1,2-Di(p-methylphenylsulfonylethyl)malononitrile (9b).
White solid (2.576 g, 64%); m.p. 136–138 ꢁC; IR (KBr): 1130, 1331
(SO2), 2240 (C^N) cmꢀ1 1H NMR (CDCl3)
; d 2.24, 2.28 (s, 6H, Ar-
CH3), 3.09 (dd, 1H, C4-H, J ¼ 8.2, 14.2 Hz), 3.25 (dd, 1H, C4-H, J ¼ 4.1,
6.1.2. General procedure for the preparation of 3,5-diamino-4-
(10,20-diaroylethyl)-4H-pyrazole (3a–c)/3,5-diamino-4-(10,20-
diarylsulfonylethyl)-4H-pyrazole (10a–c)
14.0 Hz), 4.09–4.15 (m,1H, C3-H), 4.37 (d,1H, C2-H, J ¼ 8.3 Hz), 7.21–
7.72 (m, 8H, Ar-H) ppm; 13C NMR (CDCl3)
d 21.7, 22.3 (Ar-CH3), 37.6
(C-3), 41.2 (C-2), 52.2 (C-4), 114.9 and 115.8 (CN), 128.4, 129.7, 131.4,
132.7, 133.4, 134.8, 135.0, 136.4 (aromatic carbons). Anal. Calcd. For
C19H18N2O4S2: C, 56.70; H, 4.51; N, 6.96; Found: C, 56.77; H, 4.55; N,
6.92%.
To a solution of 2/9 (10 mmol) in methanol (20 ml), hydrazine
hydrate (15 mmol) and 10% NaOMe (5 ml) were added and refluxed for
4–6 h. The contents were cooled and poured onto crushed ice con-
taining HCl. The product obtained was recrystallized from methanol.
Table 5
Minimum inhibitory concentration (MIC), mg/ml of 3c, 4c, 10c and 11c.
Compound
Minimum inhibitory
concentration, MIC (
m
g/ml)
S. aureus
B. subtilis
K. pneumoniae
P. vulgaris
F. solani
C. lunata
A. niger
3c
4c
10c
11c
100
50
25
50
6.25
–
100
50
25
50
6.25
–
100
100
50
100
12.5
–
100
200
50
100
12.5
–
200
100
200
100
–
200
100
100
50
200
100
100
50
Chloramphenicol
Ketoconazole
–
–
12.5
6.25
6.25

4562
A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
6.1.2.4. 3,5-Diamino-4-(10,20-diphenylsulfonylethyl)-4H-pyrazole (10a).
White solid (2.520 g, 62%); m.p. 163–165 ꢁC; IR (KBr): 1134, 1333 (SO2),
1614 (C]N), 3233, 3300 (NH2) cmꢀ1; 1H NMR (DMSO-d6)
3.15 (dd,
Table 6
Antioxidant property of 3–7 and 10–14.
d
Compound
% Inhibition at 100
mM
0
0
1H, C2 -H, J ¼ 4.3, 14.3 Hz), 3.68 (dd, 1H, C2 -H, J ¼ 8.3, 14.2 Hz), 4.15–
Nitric oxide method
DPPH method
0
4.21 (m, 1H, C1 -H), 4.31 (d, 1H, C4-H, J ¼ 5.6 Hz), 5.74 (bs, 4H, NH2),
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
10a
10b
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
14a
14b
14c
71.65
68.15
90.74
43.52
54.33
90.26
26.78
27.19
39.39
35.66
29.39
43.44
22.64
20.26
28.19
59.96
60.48
92.42
84.92
81.49
95.38
29.68
27.94
35.93
45.76
39.86
48.45
26.92
24.59
30.21
73.72
66.52
91.68
42.27
58.10
89.61
24.71
28.64
40.98
37.80
32.28
41.58
23.82
21.74
29.93
58.49
59.37
92.37
85.81
80.84
94.46
28.76
28.15
35.84
46.87
38.74
47.92
27.74
24.82
31.64
7.14–7.69 (m, 10H, Ar-H), ppm; 13C NMR (DMSO-d6)
d
51.4 (C-20), 52.5
(C-10), 62.7 (C-4), 173.8, 174.7 (C-3 and C-5), 128.4, 129.6, 130.4, 131.6,
132.9, 133.5, 134.3, 135.1 (aromatic carbons). Anal. Calcd. For
C17H18N4O4S2: C, 50.23; H, 4.46; N, 13.78; Found: C, 50.28; H, 4.42; N,
13.86%.
6.1.2.5. 3,5-Diamino-4-(10,20-di(p-methylphenyl)sulfonylethyl)-4H-
pyrazole (10b). White solid (2.824 g, 65%); m.p. 171–173 ꢁC; IR
(KBr): 1131, 1339 (SO2), 1611 (C]N), 3245, 3311 (NH2) cmꢀ1 1H
;
0
NMR (DMSO-d6)
d
2.21, 2.26 (s,0 6H, Ar-CH3), 3.13 (dd, 1H, C2 -H,
J ¼ 4.0, 14.1 Hz), 3.59 (dd, 1H, C2 -H, J ¼ 8.1, 14.0 Hz), 4.10–4.17 (m,
0
1H, C1 -H), 4.32 (d, 1H, C4-H, J ¼ 5.7 Hz), 5.76 (bs, 4H, NH2), 7.12–
7.71 (m, 8H, Ar-H), ppm; 13C NMR (DMSO-d6)
d 21.8, 22.4 (Ar-CH3),
52.7 (C-20), 53.4 (C-10), 61.9 (C-4), 172.4, 173.8 (C-3 and C-5), 127.8,
129.1,130.9,131.2,132.4,133.9 134.6,135.9 (aromatic carbons). Anal.
Calcd. For C19H22N4O4S2: C, 52.52; H, 5.10; N, 12.89; Found: C,
52.60; H, 5.13; N, 12.00%.
6.1.2.6. 3,5-Diamino-4-(10,20-di(p-chlorophenyl)sulfonylethyl)-4H-
pyrazole (10c). White solid (3.232 g, 68%); m.p. 167–168 ꢁC; IR
(KBr): 1128, 1336 (SO2), 1617 (C]N), 3232, 3285 (NH2) cmꢀ1
;
1H
0
NMR (DMSO-d6)
d
3.19 (dd, 1H, C2 -H, J ¼ 4.2, 14.3 Hz), 3.61 (dd, 1H,
0
0
C2 -H, J ¼ 8.0, 14.1 Hz), 4.16–4.23 (m, 1H, C1 -H), 4.38 (d, 1H, C4-H,
J ¼ 5.9 Hz), 7.21–7.85 (m, 8H, Ar-H), 5.84 (bs, 4H, NH2) ppm; 13C
NMR (DMSO-d6) d
53.1 (C-20), 54.6 (C-10), 62.3 (C-4), 173.6, 174.2 (C-
3 and C-5), 128.4, 129.7, 131.4, 132.6, 133.8, 134.5 135.7, 137.3
(aromatic carbons). Anal. Calcd. For C17H16Cl2N4O4S2: C, 42.95; H,
3.39; N, 11.79; Found: C, 43.00; H, 3.44; N, 11.85%.
6.1.2.1. 3,5-Diamino-4-(10,20-dibenzoylethyl)-4H-pyrazole (3a).
White solid (2.039 g, 61%); m.p. 162–164 ꢁC; IR (KBr): 1611 (C]N),
1699 (C]O), 3240, 3292 (NH2) cmꢀ1
;
1H NMR (DMSO-d6)
d
3.06
6.1.3. General procedure for the preparation of 3-amino-5-imino-4-
(10,20-diaroylethyl)-4H-isoxazole (4a–c)/3-amino-5-imino-4-(10,20-
diarylsulfonylethyl)-4H-isoxazole (11a–c)
A mixture of compound 2/9 (10 mmol), hydroxylamine hydro-
chloride (15 mmol), methanol (20 ml) and 10% NaOMe (5 ml) was
refluxed for 4–6 h. The solution was cooled and poured onto
crushed ice containing HCl. The solid obtained was recrystallized
from methanol.
0
0
(dd, 1H, C2 -H, J ¼ 4.1, 14.3 Hz), 3.62 (dd, 1H, C2 -H, J ¼ 8.9, 14.1 Hz),
0
4.12–4.18 (m, 1H, C1 -H), 4.29 (d, 1H, C4-H, J ¼ 5.5 Hz), 5.78 (bs, 4H,
NH2), 7.11–7.78 (m, 10H, Ar-H), ppm; 13C NMR (CDCl3)
d
52.8 (C-20),
53.7 (C-10), 61.4 (C-4), 172.6, 173.2 (C-3 and C-5), 204.6, 205.4 (Ar-
CO), 129.6, 130.2, 131.8, 132.9, 133.6, 134.9, 135.4, 136.2 (aromatic
carbons). Anal. Calcd. for C19H18N4O2: C, 68.25; H, 5.43; N, 16.76;
Found: C, 68.32; H, 5.47; N, 16.90%.
6.1.2.2. 3,5-Diamino-4-(10,20-di(p-methylbenzoyl)ethyl)-4H-pyrazole
(3b). White solid (2.319 g, 64%); m.p. 173–175 ꢁC; IR (KBr): 1623
6.1.3.1. 3-Amino-5-imino-4-(10,20-dibenzoylethyl)-4H-isoxazole (4a).
White solid (2.347 g, 70%); m.p. 161–163 ꢁC; IR (KBr): 1602 (C]N),
(C]N), 1701 (C]O), 3245, 3396 (NH2) cmꢀ0 1; 1H NMR (CDCl3)
d
2.21
1708 (C]O), 3242, 3280 (NH2), 3319 (NH) cmꢀ1
;
1H NMR (CDCl3)
0
0
and 2.26 (s, 6H, Ar-CH3), 3.09 (dd, 1H, C2 -H, J ¼ 4.0, 14.1 Hz), 3.64
d
3.08 (dd, 1H, C2 -H, J ¼ 4.2, 14.3 Hz), 3.64 (dd, 1H, C2 -H, J ¼ 8.8,
0
0
0
(dd, 1H, C2 -H, J ¼ 8.5, 14.0 Hz), 4.11–4.16 (m, 1H, C1 -H), 4.36 (d, 1H,
C4-H, J ¼ 5.3 Hz), 5.72 (bs, 4H, NH2), 7.21–7.72 (m, 8H, Ar-H), ppm;
14.1 Hz), 4.10–4.26 (m, 1H, C1 -H), 4.34 (d, 1H, C4-H, J ¼ 5.2 Hz), 5.83
(bs, 2H, NH2), 7.22–7.74 (m, 10H, Ar-H), 9.91 (bs, 1H, NH) ppm; 13C
13C NMR (DMSO-d6)
d
22.6, 22.8 (Ar-CH3), 51.6 (C-20), 52.9 (C-10),
NMR (CDCl3) d
53.4 (C-20), 55.7 (C-10), 62.9 (C-4),176.7,178.4 (C-3 and
60.8 (C-4), 171.4, 172.9 (C-3 and C-5), 205.2, 206.1 (Ar-CO), 128.5,
129.1, 130.6, 131.5, 132.2, 133.5, 134.1, 135.4 (aromatic carbons).
Anal. Calcd. For C21H22N4O2: C, 69.59; H, 6.12; N, 15.46; Found: C,
69.64; H, 6.13; N, 15.57%.
C-5), 205.6, 206.2 (Ar-CO),128.1,129.5,130.1,131.2,132.6,133.2,134.0,
135.2 (aromatic carbons). Anal. Calcd. For C19H17N3O3: C, 68.05; H,
5.11; N, 12.53; Found: C, 68.16; H, 5.08; N, 12.48%.
6.1.3.2. 3-Amino-5-imino-4-(10,20-di-(p-methylbenzoyl)ethyl)-4H-
isoxazole (4b). White solid (2.471 g, 68%); m.p. 176–178 ꢁC; IR
6.1.2.3. 3,5-Diamino-4-(10,20-di(p-chlorobenzoyl)ethyl)-4H-pyrazole
(3c). White solid (2.661 g, 66%); m.p. 178–180 ꢁC; IR (KBr): 1616
(KBr): 1605 (C]N),1710 (C]O), 3237, 3285 (NH2), 3324 (NH) cmꢀ1
;
(C]N), 1705 (C]O), 3250, 3300 (NH2) cmꢀ1; 1H NMR (CDCl3)
d
3.13
1H NMR (CDCl3)
d
2.27, 2.29 (s,0 6H, Ar-CH3), 3.11 (dd, 1H, C2 -H,
0
0
0
(dd, 1H, C2 -H, J ¼ 4.3, 14.2 Hz), 3.58 (dd, 1H, C2 -H, J ¼ 8.3, 14.1 Hz),
J ¼ 4.30, 14.4 Hz), 3.67 (dd, 1H, C2 -H, J ¼ 8.6, 14.2 Hz), 4.14–4.19 (m,
0
4.13–4.18 (m, 1H, C1 -H), 4.32 (d, 1H, C4-H, J ¼ 5.2 Hz), 5.81 (bs, 4H,
1H, C1 -H), 4.31 (d, 1H, C4-H, J ¼ 5.1 Hz), 5.76 (bs, 2H, NH2), 7.18–7.70
NH2), 7.26–7.84 (m, 8H, Ar-H), ppm; 13C NMR (DMSO-d6)
d
52.7 (C-
(m, 8H, Ar-H), 9.89 (bs, 1H, NH) ppm; 13C NMR (CDCl3)
d 21.7, 22.4
20), 53.4 (C-10), 61.3 (C-4), 172.7, 173.4 (C-3 and C-5), 203.6, 205.5
(Ar-CO), 128.9, 129.7, 130.2, 131.2, 132.9, 133.9, 134.9, 136.4
(aromatic carbons). Anal. Calcd. For C19H16Cl2N4O2: C, 56.59; H,
4.00; N, 13.89; Found: C, 56.54; H, 4.05; N, 13.98%.
(Ar-CH3), 52.9 (C-20), 54.9 (C-10), 63.2 (C-4), 177.3, 178.9 (C-3 and C-
5), 206.2, 207.3 (Ar-CO), 128.7, 129.3, 130.9, 131.9, 132.2, 133.9, 134.8,
135.1 (aromatic carbons). Anal. Calcd. For C21H21N3O3: C, 69.41; H,
5.82; N, 11.56; Found: C, 69.47; H, 5.86; N, 11.65%.

A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
4563
6.1.3.3. 3-Amino-5-imino-4-(10,20-di-(p-chlorobenzoyl)ethyl)-4H-iso-
xazole (4c). White solid (2.870 g, 71%); m.p. 181–183 ꢁC; IR (KBr):
135.7 (aromatic carbons). Anal. Calcd. For C20H18N4O3: C, 66.29; H,
5.01; N, 15.46; Found: C, 66.36; H, 5.06; N, 15.59%.
1610 (C]N), 1698 (C]O), 3245, 3282 (NH2), 3311 (NH) cmꢀ1
;
1H
0
0
NMR (CDCl3)
d
3.07 (dd, 1H, C2 -H, J ¼ 4.2, 14.2 Hz), 3.65 (dd, 1H, C2 -
6.1.4.2. 4,6-Diamino-5-(10,20-di-(p-methylbenzoyl)ethyl)-pyrimidine-
2(5H)-one (5b). White solid (2.733 g, 70%); m.p. 199–201 ꢁC; IR
(KBr): 1611 (C]N), 1658 (N–C]O), 1707 (C]O), 3209, 3288 (NH2)
0
H, J ¼ 8.4, 14.1 Hz), 4.11–4.16 (m, 1H, C1 -H), 4.27 (d, 1H, C4-H,
J ¼ 5.0 Hz), 5.79 (bs, 2H, NH2), 7.21–7.82 (m, 8H, Ar-H), 9.82 (bs, 1H,
NH) ppm; 13C NMR (CDCl3)
d
51.5 (C-20), 53.4 (C-10), 62.8 (C-4),176.8,
cmꢀ1; 1H NMR (CDCl3)
d
2.22, 2.27 (s, 6H, Ar-CH3), 3.10 (dd, 1H, C2 -
0
0
177.7 (C-3 and C-5), 205.9, 206.4 (Ar-CO), 128.1, 129.8, 131.4, 132.3,
133.7, 134.8, 135.2, 136.9 (aromatic carbons). Anal. Calcd. For
C19H15Cl2N3O3: C, 56.45; H, 3.74; N, 10.39; Found: C, 56.53; H, 3.71;
N, 10.50%.
H, J ¼ 4.0,014.2 Hz), 3.64 (dd, 1H, C2 -H, J ¼ 8.8, 14.0 Hz), 4.10–4.16
(m,1H, C1 -H), 4.36 (d, 1H, C5-H, J ¼ 5.3 Hz), 5.67 (bs, 4H, NH2), 7.12–
7.69 (m, 8H, Ar-H) ppm; 13C NMR (CDCl3)
d 22.6, 23.4 (Ar-CH3), 51.4
(C-20), 56.3 (C-10), 65.9 (C-5), 158.6 (C-2), 167.6, 168.5 (C-4 and C-6),
205.2, 206.4 (Ar-CO), 126.9, 128.4, 128.9, 130.4, 131.4, 132.7, 133.8,
134.1 (aromatic carbons). Anal. Calcd. For C22H22N4O3: C, 67.68; H,
5.68; N, 14.35; Found: C, 67.62; H, 5.65; N, 14.43%.
6.1.3.4. 3-Amino-5-imino-4-(10,20-diphenylsulfonylethyl)-4H-isoxazole
(11a). White solid (2.934 g, 72%); m.p. 198–200 ꢁC; IR (KBr): 1130,
1338 (SO2), 1612 (C]N), 3240, 3296 (NH2), 3320 (NH) cmꢀ1; 1H NMR
0
0
(CDCl3)
d
3.17 (dd, 1H, C2 -H, J ¼ 4.0, 14.1 Hz), 3.72 (dd, 1H, C2 -H,
6.1.4.3. 4,6-Diamino-5-(10,20-di-(p-chlorobenzoyl)ethyl)-pyrimidine-
2(5H)-one (5c). White solid (3.105 g, 72%); m.p. 220–222 ꢁC; IR
0
J ¼ 8.4, 14.0 Hz), 4.11–4.19 (m, 1H, C1 -H), 4.35 (d, 1H, C4-H, J ¼ 5.0 Hz),
5.77 (bs, 2H, NH2), 7.12–7.68 (m, 10H, Ar-H), 9.86 (bs, 1H, NH) ppm; 13
C
(KBr): 1609 (C]N), 1650 (N–C]O), 1696 (C]O), 3220, 3284 (NH2)
cmꢀ1;01H NMR (CDCl3)
d
3.16 (dd,1H, C2 -H, J ¼ 4.3,14.6 Hz), 3.61 (dd,
0
NMR (CDCl3)
d
52.6 (C-20), 55.1 (C-10), 61.7 (C-4), 175.3, 177.2 (C-3 and
0
C-5), 127.6, 128.4, 129.9, 130.7, 131.5, 132.8, 133.6, 134.8 (aromatic
carbons). Anal. Calcd. For C17H17N3O5S2: C, 50.11; H, 4.21; N, 10.31;
Found: C, 50.16; H, 4.25; N, 10.35%.
1H, C2 -H, J ¼ 8.2,14.3 Hz), 4.14–4.21 (m,1H, C1 -H), 4.32 (d,1H, C5-H,
J ¼ 5.2 Hz), 5.61 (bs, 4H, NH2), 7.23–7.81 (m, 8H, Ar-H) ppm; 13C NMR
(CDCl3)d
52.7(C-20),57.1(C-10), 64.4(C-5),159.5(C-2),168.2,169.3(C-
4 and C-6), 206.4, 207.3 (Ar-CO),128.9,129.4,130.9,131.7,132.3,133.6,
135.8, 139.4 (aromatic carbons). Anal. Calcd. For C20H16Cl2N4O3: C,
55.70; H, 3.74; N, 12.99; Found: C, 55.64; H, 3.75; N, 13.06%.
6.1.3.5. 3-Amino-5-imino-4-(10,20-di-(p-methylphenyl)sulfonylethyl)-
4H-isoxazole (11b). White solid (2.918 g, 67%); m.p. 205–207 ꢁC; IR
(KBr): 1126, 1341 (SO2), 1608 (C]N), 3238, 3298 (NH2), 3315 (NH)
cmꢀ1; 1H NMR (CDCl3)
d
2.27, 2.29 (s, 6H, Ar-CH3), 3.19 (dd,1H, C2 -H,
6.1.4.4. 4,6-Diamino-5-(10,20-diphenylsulfonylethyl)-pyrimidine-2(5H)-
one (12a). White solid (3.172 g, 73%); m.p. 192–194 ꢁC; IR (KBr): 1119,
0
0
J ¼0 4.2,14.3 Hz), 3.47 (dd,1H, C2 -H, J ¼ 8.0,14.1 Hz), 4.18–4.25 (m,1H,
C1 -H), 4.37 (d, 1H, C4-H, J ¼ 5.2 Hz), 5.85 (bs, 2H, NH2), 7.10–7.64 (m,
1340 (SO2), 1618 (C]N), 1675 (C]O), 3242, 3295 (NH2) cmꢀ1 1H
;
8H, Ar-H), 9.84 (bs, 1H, NH) ppm; 13C NMR (CDCl3)
d
21.4, 22.5 (Ar-
NMR (CDCl3)
d
3.10 (dd, 1H, C2 -H, J ¼ 4.0, 14.0 Hz), 3.70 (dd, 1H, C2 -H,
0
0
0
CH3), 51.9 (C-20), 54.8 (C-10), 62.5 (C-4), 174.8, 176.9 (C-3 and C-5),
128.9, 129.1, 129.7,130.3, 132.4,133.1, 134.4,135.6 (aromatic carbons).
Anal. Calcd. For C19H21N3O5S2: C, 52.40;H, 4.86;N, 9.65; Found: C,
52.46; H, 4.87; N, 9.70%.
J ¼ 9.0, 14.1 Hz), 4.17–4.24 (m, 1H, C1 -H), 4.37 (d, 1H, C5-H, J ¼ 5.3 Hz),
5.59 (bs, 4H, NH2), 7.19–7.76 (m, 10H, Ar-H) ppm; 13C NMR (CDCl3)
d
51.5 (C-20), 56.5 (C-10), 63.2 (C-5), 157.8 (C-2), 167.4, 168.2 (C-4 and C-
6), 127.8, 128.7, 129.9, 130.6, 131.9, 132.8, 133.4, 134.6 (aromatic
carbons). Anal. Calcd. For C18H18N4O5S2: C, 49.76; H, 4.18; N, 12.89;
Found: C, 49.80; H, 4.20; N, 12.98%.
6.1.3.6. 3-Amino-5-imino-4-(10,20-di-(p-methylphenyl)sulfonylethyl)-
4H-isoxazole (11c). White solid (3.525 g, 74%); m.p. 221–223 ꢁC; IR
(KBr): 1121, 1339 (SO2), 1603 (C]N), 30242, 3294 (NH2), 3325 (NH)
6.1.4.5. 4,6-Diamino-5-(10,20-di(p-methylphenyl)sulfonylethyl)-pyrim-
idine-2(5H)-one (12b). White solid (3.238 g, 70%); m.p. 205–207 ꢁC;
IR (KBr): 1126, 1332 (SO2), 1613 (C]N), 1664 (C]O), 3235, 3290
cmꢀ1;01H NMR (CDCl3)
d
3.21 (dd,1H, C2 -H, J ¼ 4.3,14.1 Hz), 3.59 (dd,
0
1H, C2 -H, J ¼ 8.2,14.3 Hz), 4.22–4.28 (m,1H, C1 -H), 4.41 (d,1H, C4-H,
J ¼ 5.4 Hz), 5.81 (bs, 2H, NH2), 7.24–7.84 (m, 8H, Ar-H), 9.79 (bs, 1H,
(NH2) cmꢀ1; 1H NMR (CDCl3)
d
2.22, 2.27 (s, 6H, Ar-CH3), 3.12 (dd,1H,
NH) ppm; 13C NMR (CDCl3)
d
52.4 (C-20), 55.7 (C-10), 61.9 (C-4), 173.9,
C2 -H, J ¼ 4.1, 14.2 Hz), 3.68 (dd, 1H, C2 -H, J ¼ 9.2, 14.0 Hz), 4.14–4.22
0
0
0
175.2 (C-3 and C-5),129.1, 130.6, 131.7,132.4, 133.7,134.3,135.6,136.8
(aromatic carbons). Anal. Calcd. For C17H15Cl2N3O5S2: C, 42.86; H,
3.17; N, 8.82; Found: C, 42.83; H, 3.15; N, 8.80%.
(m, 1H, C1 -H), 4.31 (d, 1H, C5-H, J ¼ 5.1 Hz), 5.63 (bs, 4H, NH2), 7.21–
7.72 (m, 8H, Ar-H) ppm; 13C NMR (CDCl3)
d
22.6, 22.9 (Ar-CH3), 52.8
(C-20), 57.2 (C-10), 61.7 (C-5), 158.4 (C-2), 166.9, 167.6 (C-4 and C-6),
128.1,129.4,130.2,131.5,132.3,133.6,134.8,135.3 (aromatic carbons).
Anal. Calcd. For C20H22N4O5S2: C, 51.93; H, 4.79; N, 12.11; Found: C,
52.00; H, 4.77; N, 12.19%.
6.1.4. General procedure for the preparation of 4,6-diamino-5-
(10,20-diaroylethyl)-pyrimidine-2(5H)-one (5a–c)/4,6-diamino-5-
(10,20-diarylsulfonylethyl)-pyrimidine-2(5H)-one (12a–c)
To a solution of 2/9 (10 mmol) in methanol (20 ml), urea
(10 mmol) and 10% NaOMe (5 ml) were added and refluxed for 6–
10 h. The contents were cooled, poured onto crushed ice containing
conc. HCl and extracted with ethyl acetate. The organic layer was
washed with brine and dried over anhydrous Na2SO4. Removal of
the solvent under vacuum gave crude product which was recrys-
tallized from methanol.
6.1.4.6. 4,6-Diamino-5-(10,20-di(p-chlorophenyl)sulfonylethyl)-pyrimi-
dine-2(5H)-one (12c). White solid (3.221 g, 64%); m.p. 230–232 ꢁC;
IR(KBr):1122,1330(SO2),1610(C]N),1668(C]O),3230, 3285(NH2)
cmꢀ1;01H NMR (CDCl3)
d
3.09 (dd,1H, C2 -H, J ¼ 4.0,14.0 Hz), 3.62 (dd,
0
0
1H, C2 -H, J ¼ 9.0,14.1 Hz), 4.16–4.25 (m,1H, C1 -H), 4.36 (d,1H, C5-H,
J ¼ 5.4 Hz), 5.67 (bs, 4H, NH2), 7.25–7.86 (m, 8H, Ar-H) ppm; 13C NMR
(CDCl3)d
51.4(C-20), 56.7 (C-10), 62.2(C-5),159.5 (C-2),167.3,168.3 (C-
4 and C-6), 127.9, 128.4, 131.7, 132.9, 133.8, 134.2, 135.2, 136.8
(aromatic carbons). Anal. Calcd. For C18H16Cl2N4O5S2: C, 42.95; H,
3.20; N, 11.13; Found: C, 42.93; H, 3.24; N, 11.02%.
6.1.4.1. 4,6-Diamino-5-(10,20-dibenzoylethyl)-pyrimidine-2(5H)-one
(5a). White solid (2.682 g, 74%); m.p. 194–196 ꢁC; IR (KBr): 1613
(C]N),1650 (N–C]O),17000 (C]O), 3233, 3297 (NH2) cmꢀ1; 1H NMR
0
(CDCl3)
d
3.09 (dd, 1H, C2 -H, J ¼ 4.1, 14.1 Hz), 3.69 (dd, 1H, C2 -H,
6.1.5. General procedure for the preparation of 4,6-diamino-5-
(10,20-diaroylethyl)-pyrimidine-2-(5H)-thione (6a–c)/4,6-diamino-
5-(10,20-diarylsulfonylethyl)-pyrimidine-2-(5H)-thione (13a–c)
To an equimolar mixture (10 mmol) of 2/9 and thiourea, meth-
anol (20 ml) and 10% NaOMe (5 ml) were added and refluxed for
0
J ¼ 9.1,14.2 Hz), 4.16–4.25 (m,1H, C1 -H), 4.30 (d,1H, C5-H, J ¼ 5.1 Hz),
5.64 (bs, 4H, NH2), 7.20–7.71 (m, 10H, Ar-H) ppm; 13C NMR (CDCl3)
d
52.9 (C-20), 57.7 (C-10), 64.3 (C-5), 159.8 (C-2), 168.8, 169.7 (C-4 and
C-6), 204.6, 205.7 (Ar-CO),126.4,127.2,128.2,130.1,131.5,132.0,134.5,

4564
A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
10–15 h. The reaction mixture was cooled, poured onto crushed ice
containing conc. HCl and extracted with ethyl acetate. The organic
layer was washed with brine and dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure. The resultant solid
was recrystallized from methanol.
6.1.5.6. 4,6-Diamino-5-(10,20-di-(p-chlorophenyl)sulfonylethyl)-
pyrimidine-2-(5H)-thione (13c). White solid (3.015 g, 61%); m.p.
233–235 ꢁC; IR (KBr): 1127, 1335 (SO2), 1495 (C]S), 1617 (C]N),
1
3254, 3294 (NH2) cmꢀ1
;
H NMR (DMSO-d6) 3.17 (dd, 1H, C2 -H,
0
0
J ¼ 4.2, 14.3 Hz), 3.64 (dd, 1H, C2 -H, J ¼ 8.3, 14.4 Hz), 4.16–4.25 (m,
0
1H, C1 -H), 4.45 (d, 1H, C5-H, J ¼ 5.5 Hz), 5.65 (bs, 4H, NH2), 7.27–
6.1.5.1. 4,6-Diamino-5-(10,20-dibenzoylethyl)-pyrimidine-2-(5H)-thi-
one (6a). White solid (2.785 g, 72%); m.p. 200–202 ꢁC; IR (KBr):
7.82 (m, 8H, Ar-H), ppm; 13C NMR (DMSO-d6)
d
51.8 (C-20), 56.9 (C-
10), 62.3 (C-5), 163.4, 164.8 (C-4 and C-6), 172.6 (C-2), 128.2, 129.9,
130.8, 131.3, 133.8, 133.7, 135.4, 136.8 (aromatic carbons). Anal.
Calcd. For C18H16Cl2N4O4S3: C, 41.62; H, 3.10; N, 10.79; Found: C,
41.57; H, 3.11; N, 10.85%.
1485 (C]S), 1611 (C]N), 1711 (C]O), 3207, 3285 (NH2) cmꢀ1
;
1H
0
NMR (DMSO-d6)
d
3.13 (dd, 1H, C2 -H, J ¼ 4.0, 14.0 Hz), 3.71 (dd, 1H,
0
0
C2 -H, J ¼ 8.9, 14.1 Hz), 4.15–4.22 (m, 1H, C1 -H), 4.36 (d, 1H, C5-H,
J ¼ 5.0 Hz), 5.67 (bs, 4H, NH2), 7.24–7.78 (m, 10H, Ar-H) ppm; 13C
NMR (DMSO-d6)
d
52.6 (C-20), 56.4 (C-10), 64.9 (C-5),162.8,163.7 (C-4
6.1.6. General procedure for the preparation of 4,6-diimino-5-(10,20-
diaroylethyl)-1,3-dimethylpyrimidine-2-(5H)-one (7a–c)/4,6-
diimino-5-(10,20-diarylsulfonylethyl)-1,3-dimethylpyrimidine-2-
(5H)-one (14a–c)
A mixture of 2/9 (10 mmol), N,N0-dimethyl urea (10 mmol),
methanol (10 ml) and 10% NaOMe (5 ml) was refluxed for 8–12 h.
The contents were diluted with ice-cold water, acidified with conc.
HCl and extracted with ethyl acetate. The organic layer was washed
with brine and dried over anhydrous Na2SO4. Removal of the
solvent with rotary evaporator afforded crude product which was
purified by recrystallization from methanol.
and C-6), 172.8 (C-2), 205.9, 206.4 (Ar-CO), 127.6, 129.2, 130.6, 131.4,
132.6, 133.2, 134.2, 135.4 (aromatic carbons). Anal. Calcd. For
C20H18N4O2S: C, 63.47; H, 4.79; N,14.80; Found: C, 63.51; H, 4.80; N,
14.85%.
6.1.5.2. 4,6-Diamino-5-(10,20-di-(p-methylbenzoyl)ethyl)-pyrimidine-
2-(5H)-thione (6b). White solid (2.642 g, 65%); m.p. 218–220 ꢁC; IR
(KBr): 1490 (C]S), 1618 (C]N), 1702 (C]O), 3210, 3275 (NH2)
cmꢀ1; 1H NMR (DMSO-d6)
d
2.26, 2.38 (s, 6H, Ar-CH3), 3.11 (dd, 1H,
0
0
C2 -H, J ¼ 4.2, 14.3 Hz), 3.59 (dd, 1H, C2 -H, J ¼ 8.6, 14.0 Hz), 4.17–
0
4.25 (m, 1H, C1 -H), 4.42 (d, 1H, C5-H, J ¼ 5.0 Hz), 5.71 (bs, 4H, NH2),
7.27–7.82 (m, 8H, Ar-H) ppm; 13C NMR (DMSO-d6)
d
21.9, 22.3 (Ar-
6.1.6.1. 4,6-Diimino-5-(10,20-dibenzoylethyl)-1,3-dimethylpyrimidine-
CH3), 51.9 (C-20), 55.8 (C-10), 64.2 (C-5), 163.6, 165.4 (C-4 and C-6),
171.3 (C-2), 204.6, 205.8 (Ar-CO), 127.1, 129.8, 130.9, 131.1, 132.3,
133.5, 134.8, 136.2 (aromatic carbons). Anal. Calcd. For
C22H22N4O2S: C, 65.00; H, 5.46; N,13.78; Found: C, 65.07; H, 5.51; N,
13.72%.
2-(5H)-one (7a). White solid (2.577 g, 66%); m.p. 191–193 ꢁC; IR
(KBr): 1651 (C]O), 1695 (Ar-CO), 3314 (NH) cmꢀ1; 1H NMR (DMSO-
0
d6)
d
2.68 and 2.71 (s,0 6H, N-CH3), 3.15 (dd, 1H, C2 -H, J ¼ 4.1,
0
14.1 Hz), 3.74 (dd, 1H, C2 -H, J ¼ 8.4, 14.3 Hz), 4.13–4.21 (m, 1H, C1 -
H), 4.32 (d, 1H, C5-H, J ¼ 5.1 Hz), 7.09–7.49 (m, 10H, Ar-H), 9.94 (bs,
2H, NH) ppm; 13C NMR (DMSO-d6) 26.2, 27.0 (N-CH3), 51.9 (C-20),
d
6.1.5.3. 4,6-Diamino-5-(10,20-di-(p-chlorobenzoyl)ethyl)-pyrimidine-
2-(5H)-thione (6c). White solid (2.818 g, 63%); m.p. 228–230 ꢁC; IR
(KBr): 1505 (C]S), 1606 (C]N), 1700 (C]O0), 3208, 3280 (NH2)
54.0 (C-10), 63.8 (C-5), 159.9 (C-2), 163.7, 167.2 (C-4 and C-6), 205.8,
206.9 (Ar-CO), 127.2, 129.4, 130.2, 131.6, 132.4, 133.1, 134.9, 135.6
(aromatic carbons). Anal. Calcd. For C22H22N4O3: C, 67.68; H, 5.68;
N, 14.35; Found: C, 67.78; H, 5.70; N, 14.48%.
cmꢀ1
;
1H NMR 0(DMSO-d6)
d
3.15 (dd, 1H, C2 -H, J ¼ 4.4,0 14.5 Hz),
3.65 (dd, 1H, C2 -H, J ¼ 8.7, 14.3 Hz), 4.19–4.27 (m, 1H, C1 -H), 4.39
(d, 1H, C5-H, J ¼ 5.1 Hz), 5.64 (bs, 4H, NH2), 7.25–7.91 (m, 8H, Ar-H)
6.1.6.2. 4,6-Diimino-5-(10,20-di-(p-methylbenzoyl)ethyl)-1,3-dime-
thylpyrimidine-2-(5H)-one (7b). White solid (2.511 g, 60%); m.p.
ppm; 13C NMR (DMSO-d6) 52.4 (C-20), 56.1 (C-10), 65.2 (C-5),162.8,
d
164.8 (C-4 and C-6), 173.1 (C-2), 205.6, 206.9 (Ar-CO), 127.8, 129.2,
130.1, 131.8, 132.4, 133.9, 134.1, 136.8 (aromatic carbons). Anal.
Calcd. For C20H16Cl2N4O2S: C, 53.70; H, 3.61; N, 12.52; Found: C,
53.75; H, 3.59; N, 12.58%.
208–210 ꢁC; IR (KBr): 1658 (C]O), 1704 (Ar-CO), 3320 (NH) cmꢀ1
;
1H NMR (DMSO-d6)
d
2.25, 2.31 (s, 6H, Ar-CH3), 2.60 and 2.69 (s,06H,
0
N–CH3), 3.13 (dd, 1H, C2 -H, J ¼ 4.3, 14.3 Hz), 3.68 (dd, 1H, C2 -H,
0
J ¼ 8.1, 14.1 Hz), 4.19–4.26 (m, 1H, C1 -H), 4.31 (d, 1H, C5-H,
J ¼ 5.0 Hz), 7.14–7.51 (m, 8H, Ar-H), 9.98 (bs, 2H, NH) ppm; 13C NMR
6.1.5.4. 4,6-Diamino-5-(10,20-diphenylsulfonylethyl)-pyrimidine-2-
(5H)-thione (13a). White solid (2.818 g, 65%); m.p. 206–208 ꢁC; IR
(DMSO-d6) d
21.4, 22.6 (Ar-CH3), 25.6, 26.7 (N-CH3), 52.4 (C-20), 54.6
(C-10), 64.5 (C-5),158.4 (C-2),161.7,165.9 (C-4 and C-6), 204.2, 205.6
(Ar-CO), 127.2, 129.9, 130.1, 131.8, 132.2, 133.9, 135.9, 136.3 (aromatic
carbons). Anal. Calcd. For C24H26N4O3: C, 68.88; H, 6.26; N, 13.39;
Found: C, 68.97; H, 6.31; N, 13.50%.
(KBr): 1134, 1336 (SO2), 1487 (C]S), 1614 (C]N), 3250, 3300 (NH2)
cmꢀ1; 1H NMR (DMSO-d6)
d
3.12 (dd,1H, C2 -H, J ¼ 4.3,14.2 Hz), 3.73
0
0
0
(dd, 1H, C2 -H, J ¼ 8.8, 14.0 Hz), 4.19–4.27 (m, 1H, C1 -H), 4.41 (d, 1H,
C5-H, J ¼ 5.4 Hz), 5.72 (bs, 4H, NH2), 7.17–7.71 (m, 10H, Ar-H), ppm;
13C NMR (DMSO-d6)
d
51.8 (C-20), 56.9 (C-10), 63.4 (C-5), 161.4, 164.3
6.1.6.3. 4,6-Diimino-5-(10,20-di-(p-chlorobenzoyl)ethyl)-1,3-dime-
thylpyrimidine-2-(5H)-one (7c). White solid (2.848 g, 62%); m.p.
(C-4 and C-6),171.4 (C-2),128.1,129.8,130.2,131.2,132.4,133.0,134.9,
135.8 (aromatic carbons). Anal. Calcd. For C18H18N4O4S3: C, 47.98; H,
4.03; N, 12.44; Found: C, 47.96; H, 4.01; N, 12.51%.
225–227 ꢁC; IR (KBr): 1654 (C]O), 1712 (Ar-CO), 3317 (NH) cmꢀ1
;
1H NMR (DMSO-d6)
d
2.57 and 2.61 (s, 6H, N-CH3), 3.10 (dd, 1H, C2 -
0
0
H, J ¼ 4.1,14.2 Hz), 3.62 (dd,1H, C2 -H, J ¼ 8.0,14.0 Hz), 4.11–4.24 (m,
6.1.5.5. 4,6-Diamino-5-(10,20-di-(p-methylphenyl)sulfonylethyl)-pyrim-
idine-2-(5H)-thione (13b). White solid (3.015 g, 63%); m.p. 215–
217 ꢁC; IR (KBr): 1129, 1332 (SO2), 1490 (C]S), 1621 (C]N), 3242,
3296 (NH2) cmꢀ1; 1H NMR (DMSO-d6) 2.21, 2.250(s, 6H, Ar-CH3), 3.10
1H, C1 -H), 4.34 (d, 1H, C5-H, J ¼ 5.2 Hz), 7.21–7.84 (m, 8H, Ar-H),
0
9.89 (bs, 2H, NH) ppm; 13C NMR (DMSO-d6)
d 25.9, 26.1 (N-CH3),
53.6 (C-20), 55.2 (C-10), 63.9 (C-5), 157.6 (C-2), 162.4, 164.6 (C-4 and
C-6), 205.5, 206.9 (Ar-CO), 128.6, 129.2, 130.8, 132.7, 133.5, 134.2,
135.3, 137.2 (aromatic carbons). Anal. Calcd. For C22H20Cl2N4O3: C,
57.53; H, 4.39; N, 12.20; Found: C, 57.60; H, 4.42; N, 12.27%.
0
(dd, 1H, C2 -H, J ¼ 4.0, 14.0 Hz), 3.59 (dd, 1H, C2 -H, J ¼ 8.2, 14.1 Hz),
0
4.20–4.29 (m, 1H, C1 -H), 4.39 (d, 1H, C5-H, J ¼ 5.3 Hz), 5.69 (bs, 4H,
NH2), 7.21–7.79 (m, 8H, Ar-H), ppm; 13C NMR (DMSO-d6)
d
22.8, 22.9
(Ar-CH3), 52.6 (C-20), 57.1 (C-10), 61.9 (C-5), 162.9, 165.6 (C-4 and C-6),
173.6 (C-2), 127.9, 128.2, 129.8, 130.6, 131.5, 132.4, 133.3, 134.9
(aromatic carbons). Anal. Calcd. For C20H22N4O4S3: C, 50.19;H, 4.63;N,
11.71; Found: C, 50.26; H, 4.65; N, 11.66%.
6.1.6.4. 4,6-Diimino-5-(10,20-diphenylsulfonylethyl)-1,3-dimethylpyr-
imidine-2-(5H)-one (14a). White solid (3.284 g, 71%); m.p. 189–
190 ꢁC; IR (KBr): 1125, 1331 (SO2), 1665 (C]O), 3314 (NH) cmꢀ1; 1H
0
NMR (DMSO-d6)
d
2.63 and 2.74 (s, 6H, N-CH3), 3.14 (dd, 1H, C2 -H,

A. Padmaja et al. / European Journal of Medicinal Chemistry 44 (2009) 4557–4566
4565
0
J ¼ 4.3, 14.2 Hz), 3.75 (dd, 1H, C2 -H, J ¼ 8.8, 14.1 Hz), 4.20–4.26 (m,
and 48 h for bacteria and fungi, respectively. The minimum inhib-
itory concentrations of the compounds were recorded as the lowest
concentration of each chemical compounds in the tubes with no
turbidity (i.e. no growth) of inoculated bacteria/fungi.
0
1H, C1 -H), 4.45 (d, 1H, C5-H, J ¼ 5.5 Hz), 7.11–7.58 (m, 10H, Ar-H),
9.78 (bs, 2H, NH) ppm; 13C NMR (DMSO-d6)
d 26.9, 27.2 (N-CH3),
52.3 (C-20), 55.6 (C-10), 61.5 (C-5), 158.4 (C-2), 164.6, 166.8 (C-4 and
C-6), 128.6, 129.1, 130.6, 131.2, 132.9, 133.4, 134.0, 135.3 (aromatic
carbons). Anal. Calcd. For C20H22N4O5S2: C, 51.93; H, 4.79; N, 12.11;
Found: C, 51.96; H, 4.84; N, 12.17%.
6.2.4. Antioxidant testing
Compounds 3–7 and 10–14 were tested for antioxidant property
by nitric oxide and DPPH methods.
6.1.6.5. 4,6-Diimino-5-(10,20-di-(p-methylphenyl)sulfonylethyl)-1,3-
dimethylpyrimidine-2-(5H)-one (14b). White solid (3.532 g, 72%);
m.p. 212–214 ꢁC; IR (KBr): 1131, 1339 (SO2), 1662 (C]O), 3317 (NH)
6.2.5. Assay for nitric oxide (NO) scavenging activity
Sodium nitroprusside (5
mM) in phosphate buffer pH 7.4 was
cmꢀ1; 1H NMR (DMSO-d6)
d
2.24, 2.29 (s, 6H, Ar-CH3), 2.59 and 2.75
incubated with 100 M concentration of test compounds dissolved
m
0
0
(s, 6H, N-CH3), 3.08 (dd, 1H, C2 -H, J ¼ 4.1, 14.0 Hz), 3.64 (dd, 1H, C2 -
in a suitable solvent (dioxane/methanol) and tubes were incubated
at 25 ꢁC for 120 min. Control experiment was conducted with equal
amount of solvent in an identical manner. At intervals, 0.5 ml of
incubation solution was taken and diluted with 0.5 ml of Griess
reagent (1% sulfanilamide, 0.1% N-naphthylethylenediamine dihy-
drochloride and 2% o-phosphoric acid dissolved in distilled water).
The absorbance of the chromophore formed during diazotization of
nitrite with sulfanilamide and subsequent N-naphthylethylenedi-
0
H, J ¼ 8.9, 14.2 Hz), 4.21–4.26 (m, 1H, C1 -H), 4.39 (d, 1H, C5-H,
J ¼ 5.6 Hz), 7.14–7.63 (m, 8H, Ar-H), 9.16 (bs, 2H, NH) ppm; 13C NMR
(DMSO-d6) d
21.9, 22.8 (Ar-CH3), 26.3, 28.4 (N-CH3), 52.9 (C-20), 54.4
(C-10), 62.8 (C-5), 157.6 (C-2), 165.8, 167.4 (C-4 and C-6), 127.4, 128.5,
129.6, 130.5, 133.8, 134.8, 135.2, 135.9 (aromatic carbons). Anal.
Calcd. For C22H26N4O5S2: C, 53.86; H, 5.34; N,11.42; Found: C, 53.84;
H, 5.37; N, 11.48%.
amine dihydrochloride was read at
repeated in triplicate.
l 546 nm. The experiment was
6.1.6.6. 4,6-Diimino-5-(10,20-di-(p-chlorophenyl)sulfonylethyl)-1,3-
dimethylpyrimidine-2-(5H)-one (14c). White solid (3.667 g, 69%);
m.p. 228–230 ꢁC; IR (KBr): 1126, 1341 (SO2), 1667 (C]O), 3322 (NH)
6.2.6. Reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free
radical (DPPH method)
The nitrogen centered stable free radical 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH) has often been used to characterize antioxidants.
It is reversibly reduced and the odd electron in the DPPH free radical
cmꢀ1;01H NMR (DMSO-d6)
d
2.51 and 2.68 (s, 6H, N–CH3), 3.10 (dd,
0
1H, C2 -H, J ¼ 4.02,14.1 Hz), 3.68 (dd,1H, C2 -H, J ¼ 8.8,14.3 Hz), 4.24–
4.28 (m, 1H, C1 -H), 4.31 (d, 1H, C5-H, J ¼ 5.7 Hz), 7.25–7.83 (m, 8H,
Ar-H), 9.77 (bs, 2H, NH) ppm; 13C NMR (DMSO-d6)
d 25.7, 27.3 (N-
CH3), 53.4 (C-20), 55.7 (C-10), 64.2 (C-5), 159.2 (C-2), 167.1, 168.6 (C-4
and C-6), 128.3, 129.2, 130.5, 131.8, 132.7, 133.5, 135.6, 137.2
(aromatic carbons). Anal. Calcd. For C20H20Cl2N4O5S2: C, 45.20; H,
3.79; N, 10.54; Found: C, 42.25; H, 3.76; N, 10.58%.
gives a strong absorption maximum at l 517 nm, which is purple in
color. This property makes it suitable for spectrophotometric studies.
A radical scavenging antioxidant reacts with DPPH stable free radical
and converts into 1,1-diphenyl-2-picrylhydrazine. The resulting
decolorization is stoichiometric with respect to the number of elec-
trons captured. The change in the absorbance produced in this
reaction has been used to measure antioxidant properties.
6.2. Biological assays
6.2.1. Compounds
Compounds 3–7 and 10–14 were dissolved in DMSO at different
concentrations of 100, 200 and 800
The solutions of test compounds (100
(100 M) in dioxane/ethanol. The tubes were kept at an ambient
temperature for 20 min and the absorbance was measured at
mM) were added to DPPH
m
mg/ml.
l
517 nm. The difference between the test and the control experi-
ments was taken and expressed as the per cent scavenging of the
DPPH radical.
6.2.2. Cells
Bacterial strains S. aureus, B. subtilis, E. coli, K. pneumoniae and
fungi F. solani, C. lunata and A. niger were obtained from National
Collection of Industrial Microorganisms (NCIM), National Chemical
Laboratory, Pune, India.
Acknowledgements
The authors are thankful to UGC New Delhi, India for the
financial assistance under minor research project.
6.2.3. Antibacterial and antifungal assays
Preliminary antimicrobial activities of compounds 3–7 and 10–
14 were tested by Agar disc-diffusion method. Sterile filter paper
discs (6 mm diameter) moistened with the test compound solution
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