Synthesis and antimicrobial activity of amine linked bis- and tris-heterocycles

Article abstract of DOI:10.1016/j.ejmech.2014.06.001

A series of amine linked bis- and tris-heterocycles were prepared from heteroaryl cinnamamides and tested for antimicrobial activity. The compounds 11c and 12c exhibited excellent antibacterial activity while 12a and 12c displayed excellent antifungal act

Full text of DOI:10.1016/j.ejmech.2014.06.001

European Journal of Medicinal Chemistry 82 (2014) 347e354
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activity of amine linked bis- and
tris-heterocycles
T. Bhanu Prakash, G. Dinneswara Reddy, A. Padmaja, 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 series of amine linked bis- and tris-heterocycles were prepared from heteroaryl cinnamamides and
tested for antimicrobial activity. The compounds 11c and 12c exhibited excellent antibacterial activity
while 12a and 12c displayed excellent antifungal activity.
Received 29 January 2014
Received in revised form
16 May 2014
Accepted 1 June 2014
Available online 3 June 2014
© 2014 Published by Elsevier Masson SAS.
Keywords:
Oxazole
Thiazole
Imidazole
Pyrazole
Antimicrobial activity
1. Introduction
prepared by the reaction of 4-aryloxazol-2-ylamine, 4-arylthiazol-
2-ylamine and 4-aryl-1H-imidazol-2-ylamine with cinnamoyl
Oxazoles are widely distributed in natural products including
phenoxan [1], noricumazoles [2], hennoxazoles [3] and leiodolides
A & B [4] and many of them possess significant biological activities
such as antifungal, cytotoxic, anthelmintic etc., [5e7]. Thiazoles and
their derivatives exhibit antibacterial [8], antifungal [9] and anti-
inflammatory [10] activities. Multi-substituted imidazoles are sig-
nificant core structures used in medicinal chemistry due to their
remarkable activities such as antifungal, antibacterial, antiviral and
anti-inflammatory [11e13]. Besides, cyclooxygenase-2 (COX-2) in-
hibitor Celecoxib [14], herbicide Fluazolate [15] and fungicide
Pyraclostrobin [16] contains pyrazole moiety. The potent biological
activity and the prevalence of azoles in natural products and as
drug candidates stimulated intensive interest in the synthesis of
hitherto unknown bis- and tris-heterocycles and to study their
antimicrobial activity.
chloride in the presence of toluene. The cyclocondensation reaction
of 1, 2 and 3 with thiosemicarbazide in the presence of sodium hy-
droxide in ethanol resulted in 30-(4-aryloxazol-2-ylamino)-50-aryl-
40,50-dihydropyrazole-10-carbothioamide (4), 30-(4-arylthiazol-2-
ylamino)-50-aryl-40,50-dihydropyrazole-10-carbothioamide (5) and
30-(4-aryl-1H-imidazol-2-ylamino)-50-aryl-40,50-dihydropyrazole-
10-carbothioamide (6), respectively. The 1H NMR spectra of 4a, 5a
and 6a displayed an AMX splitting pattern for the methine and
methylene protons of pyrazoline ring. The double doublets observed
at
3.14 ppm in 6a were attributed to HA, HM and Hx, respectively. In
addition two broad singlets were observed at 5.54, 5.68 in 4a, at
5.52, 5.64 in 5a and at 5.62, 5.76 ppm in 6a due to NH and NH2.
Besides, a broad singlet observed at 11.94 ppm in 6a was due to NH
d 5.32, 3.85, 3.18 in 4a, at 5.30, 3.79, 3.16 in 5a and at 5.33, 3.86,
d
d
of imidazole. The signals of highly acidic protons disappeared on
deuteration. Oxidation of compounds 4, 5 and 6 with chloranil
afforded the aromatized products 30-(4-aryloxazol-2-ylamino)-50-
aryl-10H-pyrazole-10-carbothioamide (7), 30-(4-arylthiazol-2-yl-
amino)-50-aryl-10H-pyrazole-10-carbothioamide (8) and 30-(4-aryl-
1H-imidazol-2-ylamino)-50-aryl-10H-pyrazole-10-carbothioamide
2. Chemistry
The synthetic intermediates (E)-N-(4-aryloxazol-2-yl)cinnama-
mide (1) [17], (E)-N-(4-arylthiazol-2-yl)cinnamamide (2) [17] and
(E)-N-(4-aryl-1H-imidazol-2-yl)cinnamamide (3) [17] were
(9). The 1H NMR spectra of 7a, 8a and 9a showed a singlet at
d 7.04,
0
7.02 and 6.88 ppm due to C4 eH in addition to the signals of other
protons. The thioamide group in 7, 8 and 9 was utilized to develop
thiazole ring bycyclocondensationwith phenacyl bromide. Thus the
* Corresponding author.
E-mail address: vkpuram2001@yahoo.com (V. Padmavathi).
compounds
50-aryl-N-(4-aryloxazol-2-yl)-10-(400-arylthiazol-200-
http://dx.doi.org/10.1016/j.ejmech.2014.06.001
0223-5234/© 2014 Published by Elsevier Masson SAS.

348
T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
yl)-10H-pyrazol-30-amine (10), 50-aryl-N-(4-arylthiazol-2-yl)-10-
(400-arylthiazol-200-yl)-10H-pyrazol-30-amine (11) and 50-aryl-N-(4-
aryl-1H-imidazol-2-yl)-10-(400-arylthiazol-200-yl)-10H-pyrazol-30-
amine (12) were prepared. The 1H NMR spectra of 10a, 11a and 12a
0
arylthiazol-200-yl)-10H-pyrazol-30-amine
(10),
50-aryl-N-(4-
arylthiazol-2-yl)-10-(400-arylthiazol-200-yl)-10H-pyrazol-30-amine
(11) and 50-aryl-N-(4-aryl-1H-imidazol-2-yl)-10-(400-arylthiazol-200-
yl)-10H-pyrazol-30-amine (12) displayed greater activity than the
corresponding bisheterocycles, 30-(4-aryloxazol-2-ylamino)-50-
exhibited a singlet at
d
6.95, 6.88 and 6.93 ppm due to C4 eH. How-
ever, the signals of C5eH and C5 eH appeared at downfield region
aryl-10H-pyrazole-10-carbothioamide
(7),
30-(4-arylthiazol-2-
00
and merged with aromatic protons. In addition a broad singlet at
ylamino)-50-aryl-10H-pyrazole-10-carbothioamide (8) and 30-(4-
aryl-1H-imidazol-2-ylamino)-50-aryl-10H-pyrazole-10-carbothioa-
mide (9). In fact, 50-(4-chlorophenyl)-N-(4-(4-chlorophenyl)thia-
zol-2-yl)-10-(400-(4-chlorophenyl)thiazol-200-yl)-10H-pyrazol-30-
amine (11c) and 50-(4-chlorophenyl)-N-(4-(4-chlorophenyl)-1H-
imidazol-2-yl)-10-(400-(4-chlorophenyl)thiazol-200-yl)-10H-pyrazol-
30-amine (12c) exhibited higher antibacterial activity greater than
the standard drug Chloramphenicol particularly against Staphylo-
coccus aureus. The compounds 8a, 8c, 9c, 11a, and 12a exhibited
good antibacterial activity whereas the compounds 7c, 8b, 9a, 9b,
10c, 11b and 12b showed moderate activity. The presence of elec-
tron withdrawing chloro substituent on the aromatic ring increases
the activity when compared with those having methyl and
unsubstituted compounds.
All the tested compounds inhibited spore germination of the
tested fungi except 4, 5 and 6. In general, most of the compounds
showed slightly higher antifungal activity towards Aspergillus
niger than Penicillium chrysogenum. The trisheterocyclic compounds
having imidazole unit 50-phenyl-N-(4-phenyl-1H-imidazol-2-yl)-
10-(400-phenylthiazol-200-yl)-10H-pyrazol-30-amine (12a) and 50-(4-
chlorophenyl)-N-(4-(4-chlorophenyl)-1H-imidazol-2-yl)-10-(400-
(4-chlorophenyl)thiazol-200-yl)-10H-pyrazol-30-amine (12c) exhibi-
ted excellent activity particularly against A. niger greater than the
d
5.47, 5.42 and 5.58 ppm was observed in these compounds due to
NH. The compound 12a displayed another broad singlet at
12.06 ppm due to NH of imidazole. The signals of NH disappeared
d
on deuteration. The structures of all the new compounds were
further ascertained by IR, 13C NMR, mass and elemental analyses
(see Scheme 1).
3. Biology
3.1. In vitro antimicrobial activity
The compounds 4e12 were screened for antimicrobial activity
at three different concentrations 25, 50 and 100
4. Results and discussion
mg/well.
4.1. Antimicrobial activity
The results are presented in Table 1. The data revealed that
Gram-positive bacteria were more susceptible towards the tested
compounds than Gram-negative ones. It was observed that trish-
eterocyclic
compounds,
50-aryl-N-(4-aryloxazol-2-yl)-10-(400-
Scheme 1. Synthesis of bis- and tris-heterocycles.

T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
349
Table 1
The in vitro antibacterial activity of compounds 4e12.
Compound
Zone of inhibition (mm)
Gram-positive bacteria
Staphylococcus aureus
Gram-negative bacteria
Bacillus subtilis
Pseudomonas aeruginosa
Klebsiella pneumoniae
25
m
g/well 50
m
g/well 100
mg/well 25
m
g/well 50
m
g/well 100
mg/well 25
m
g/well 50
m
g/well 100
mg/well 25
m
g/well 50
m
g/well 100 mg/well
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
6
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
7
e
e
e
e
e
e
e
e
6
e
e
e
e
e
e
6
e
e
e
e
e
e
2
e
e
e
2
1
e
e
e
2
2
2
1
1
1
2
1
2
2
1
1
2
2
2
3
1
2
2
2
7
12
8
2
3
2
2
2
2
1
2
1
1
2
2
1
2
1
1
2
1
1
3
6
e
1
8
e
2
10
6
1
3
7
e
1
9
7
1
2
2
2
2
1
2
1
1
2
2
1
3
3
1
2
2
2
3
11
8
2
9
7
1
2
3
3
2
2
2
2
2
2
1
2
1
2
1
2
3
1
e
15
22
11
24
18
10
21
8
2
2
2
1
3
3
1
2
17
25
13
26
20
12
23
10
6
18
31
15
36
28
14
33
33
e
1
1
3
2
2
2
2
2
3
3
2
2
2
1
2
3
1
19
26
15
29
23
14
25
13
8
21
32
18
38
30
17
35
35
e
1
3
3
1
2
2
2
3
2
3
2
1
3
2
2
2
2
15
21
10
22
18
9
19
8
6
16
25
14
29
24
12
27
32
e
2
2
1
1
1
2
1
2
2
2
2
1
2
2
2
1
3
16
22
12
25
20
10
21
10
8
18
26
15
32
26
13
29
34
e
19
24
14
26
22
13
23
12
9
21
28
18
35
27
16
30
38
e
8
12
e
2
2
9
15
6
16
12
e
2
2
2
1
2
12
17
8
18
14
7
1
1
16
23
11
26
19
10
20
8
2
1
1
2
1
2
2
3
3
1
3
2
2
1
1
2
1
17
25
13
28
20
12
22
10
8
19
32
15
37
29
14
34
40
e
20
28
16
31
24
14
26
13
10
22
35
18
41
33
17
38
42
e
2
1
1
1
2
2
14
10
e
1
1
9b
9c
11
e
2
13
e
1
15
6
10a
10b
10c
11a
11b
11c
12a
12b
12c
e
e
e
e
6
17
29
14
34
27
12
32
2
3
3
2
2
2
1
3
9
17
7
20
15
6
18
25
e
1
1
2
2
2
1
2
2
10
18
8
22
17
7
19
27
e
1
1
1
2
2
1
2
3
13
21
11
25
20
10
23
30
e
2
3
2
2
2
1
2
1
17
30
14
35
28
13
32
38
e
Chloramphenicol 30
Control (DMSO)
e
(e) No activity. ( ) Standard deviation.
standard drug Ketoconazole. The compounds 8c, 9c and 11a dis-
played good activity. However, the compounds 7a, 7c, 8a, 8b, 9a, 9b,
10a, 10b, 10c, 11b and 12b, showed moderate activity (Table 2).
The minimum inhibitory concentration (MIC), minimum
bactericidal concentration (MBC) and minimum fungicidal con-
centration (MFC) values of the tested compounds are listed in
Table 3. MIC is the lowest concentration of an antimicrobial that
will inhibit the visible growth of a microorganism (But it is not sure
that the microorganisms are completely killed). The MBC/MFC is
the lowest concentration of antibiotic required to kill a particular
bacterium/fungi. The MBC/MFC involves an additional set of steps
performed once the minimum inhibitory concentration (MIC) is
determined. The antimicrobials are usually regarded as bacteri-
cidal/fungicidal if the MBC/MFC is not greater than four times the
trisheterocycles. All the compounds were tested for antimicrobial
activity. The compounds thiazolyl/imidazolyl thiazolyl pyrazolyl
amines 11c and 12c exhibited excellent antibacterial activity
whereas imidazolyl thiazolyl pyrazolyl amines 12a and 12c dis-
played excellent antifungal activity. The presence of chloro sub-
stituent on the aromatic ring enhanced the activity.
6. Experimental
6.1. Chemistry
Melting points were determined in open capillaries on a Mel-
Temp apparatus and are uncorrected. The purity of the com-
pounds was checked by TLC (silica gel H, BDH, ethyl acetate/hexane,
1:3). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR
spectrometer as KBr pellets and the wave numbers were given in
cmꢁ1. The 1H NMR spectra were recorded in DMSO-d6 on a Bruker-
400 spectrometer (400 MHz). The 13C NMR spectra were recorded
in DMSO-d6 on a Bruker spectrometer operating at 100 MHz. All
MIC
[18].
The
compounds
50-(4-chlorophenyl)-N-(4-(4-
chlorophenyl)thiazol-2-yl)-10-(400-(4-chlorophenyl)-thiazol-200-yl)-
10H-pyrazol-30-amine (11c) and 50-(4-chlorophenyl)-N-(4-(4-
chlorophenyl)-1H-imidazol-2-yl)-10-(400-(4-chlorophenyl)thiazol-
200-yl)-10H-pyrazol-30-amine (12c) exhibited low MIC values when
compared with 50-phenyl-N-(4-phenylthiazol-2-yl)-10-(400-phenyl-
thiazol-200-yl)-10H-pyrazol-30-amine (11a) and 50-phenyl-N-(4-
phenyl-1H-imidazol-2-yl)-10-(400-phenylthiazol-200-yl)-10H-pyr-
azol-30-amine (12a). It was observed that in compounds 11c and
12c the MBC value 2 ꢀ MIC in case of S. aureus whereas in 12c the
MFC value is 2 ꢀ MIC in case of A. niger.
chemical shifts are reported in
standard. The mass spectra were recorded on Jeol JMS-D 300 and
Finnigan Mat 1210 B at 70 eV with an emission current of 100 A.
d (ppm) using TMS as an internal
m
The microanalyses were performed on a PerkineElmer 240C
elemental analyzer.
6.1.1. General procedure for the synthesis of 30-(4-aryloxazol-2-
ylamino)-50-aryl-40,50-dihydropyrazole-10-carbothioamide (4aec)/
30-(4-arylthiazol-2-ylamino)-50-aryl-40,50-dihydropyrazole-10-
carbothioamide (5aec)/30-(4-aryl-1H-imidazol-2-ylamino)-50-aryl-
40,50-dihydropyrazole-10-carbothioamide (6aec)
5. Conclusion
A series of amine linked bisheterocycles were prepared from
heteroaryl cinnamamides by reaction with thiosemicarbazide fol-
lowed by oxidation. The thioamide group was exploited to develop
thiazole ring on treatment with phenacyl bromide to get
To an equimolar (1 mmol) mixture of compound 1/2/3 and
thiosemicarbazide in ethanol (2 ml), sodium hydroxide (1.5 mmol)

350
T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
Table 2
The in vitro antifungal activity of compounds 4e12.
Compound
Zone of inhibition (mm)
Aspergillus niger
Penicillium chrysogenum
25
mg/well
50
m
g/well
100
mg/well
25
m
g/well
50
m
g/well
100 mg/well
4a
e
e
e
e
e
e
4b
e
e
e
e
e
e
4c
e
e
e
e
e
e
5a
e
e
e
e
e
e
5b
e
e
e
e
e
e
5c
e
e
e
e
e
e
6a
e
e
e
e
e
e
6b
e
e
e
e
e
e
6c
e
e
e
e
e
e
7a
7b
7c
8a
8b
8c
9a
9b
9c
10a
10b
10c
11a
11b
11c
12a
8
e
1
9
6
1
2
2
1
2
1
2
2
3
1
1
1
2
2
1
2
3
1
2
11
7
2
6
e
1
8
e
2
9
6
3
3
2
2
1
2
1
1
1
3
1
1
2
3
1
2
2
3
2
3
1
3
1
1
2
3
2
2
2
3
1
2
2
1
1
2
3
10
17
12
20
19
14
22
9
2
1
3
1
1
2
1
1
2
2
3
1
2
1
2
2
3
11
19
13
22
20
15
24
10
8
12
28
16
31
34
17
36
33
e
14
22
17
25
23
18
27
12
10
16
30
19
33
37
21
39
36
e
8
1
2
1
3
2
2
2
2
10
20
13
21
20
15
22
9
2
1
2
2
3
1
1
1
1
1
2
3
1
2
2
1
2
13
22
16
23
22
18
25
11
8
15
26
19
29
31
21
32
38
e
16
12
19
17
13
21
7
6
e
7
11
25
15
29
32
16
33
31
e
10
22
14
25
27
15
29
35
e
2
1
1
2
1
3
2
1
11
24
16
27
29
18
30
36
e
12b
12c
Ketoconazole
Control (DMSO)
(e) No activity. ( ) Standard deviation.
was added and refluxed for 10e12 h. The contents were poured
onto crushed ice. The solid separated was filtered and recrystallized
from 2-propanol.
(C]N), 3243 (NH), 3336, 3444 (NH2) cmꢁ1 1H NMR (DMSO-d6):
;
2.28 & 2.31 (s, 6H, AreCH3), 3.15 (dd, 1H, Hx, JAx ¼ 6.5 Hz,
JMx ¼ 11.6 Hz), 3.78 (dd, 1H, HM, JAM ¼ 14.7 Hz, JMx ¼ 11.6 Hz), 5.36
(dd, 1H, HA, JAM ¼ 14.7 Hz, JAx ¼ 6.5 Hz), 5.51 (bs, 1H, C2eNH), 5.69
(bs, 2H, CeNH2), 7.02e7.75 (m, 9H, AreH & C5eH) ppm; 13C NMR
(DMSO-d6): 24.1 & 24.4 (AreCH3), 43.2 (C-40), 66.0 (C-50), 137.5 (C-
5), 140.8 (C-4), 151.9 (C-2), 158.9 (C-30), 178.1 (C]S), 125.9, 127.2,
128.9, 131.2, 131.9, 133.2, 134.8, 139.6 (aromatic carbons) ppm; MS
6.1.1.1. 30-(4-Phenyloxazol-2-ylamino)-50-phenyl-40,50-dihydropyr-
azole-10-carbothioamide (4a). Light yellow solid (0.18 g, 74%); m.p.
206e208 ꢂC; IR (KBr): 1342 (C]S), 1570 (C]N), 3248 (NH), 3341,
3450 (NH2) cmꢁ1; 1H NMR (DMSO-d6): 3.18 (dd, 1H, Hx, JAx ¼ 6.7 Hz,
JMx ¼ 11.9 Hz), 3.85 (dd, 1H, HM, JAM ¼ 14.8 Hz, JMx ¼ 11.9 Hz), 5.32
(dd, 1H, HA, JAM ¼ 14.8 Hz, JAx ¼ 6.7 Hz), 5.54 (bs, 1H, C2eNH), 5.68
(bs, 2H, CeNH2), 6.92e7.80 (m, 11H, AreH & C5eH) ppm; 13C NMR
(DMSO-d6): 43.6 (C-40), 66.5 (C-50), 138.8 (C-5), 141.2 (C-4), 151.6 (C-
2), 158.3 (C-30), 178.4 (C]S), 126.2, 127.6, 128.4, 130.1, 132.7, 133.5,
ꢃ
(m/z): 391.49 [Mþ ]; Anal. Calcd. for C21H21N5OS: C, 64.43; H, 5.41;
N, 17.89. Found: C, 64.51; H, 5.40; N, 18.01%.
6.1.1.3. 30-(4-(4-Chlorophenyl)oxazol-2-ylamino)-50-(4-chloro-
phenyl)-40,50-dihydro-pyrazole-10-carbothioamide (4c). Light yellow
solid (0.18 g, 76%); m.p. 232e234 ꢂC; IR (KBr): 1345 (C]S), 1566
ꢃ
135.3, 140.2 (aromatic carbons) ppm; MS (m/z): 363.44 [Mþ ]; Anal.
(C]N), 3255 (NH), 3338, 3435 (NH2) cmꢁ1 1H NMR (DMSO-d6):
;
Calcd. for C19H17N5OS: C, 62.79; H, 4.71; N,19.27. Found: C, 62.90; H,
4.73; N, 19.43%.
3.20 (dd, 1H, Hx, JAx ¼ 6.8 Hz, JMx ¼ 12.0 Hz), 3.84 (dd, 1H, HM,
JAM ¼ 15.0 Hz, JMx ¼ 12.0 Hz), 5.35 (dd, 1H, HA, JAM ¼ 15.0 Hz,
JAx ¼ 6.8 Hz), 5.58 (bs, 1H, C2eNH), 5.72 (bs, 2H, CeNH2), 7.05e7.68
(m, 9H, AreH & C5eH) ppm; 13C NMR (DMSO-d6): 42.9 (C-40), 66.7
(C-50), 139.2 (C-5), 141.5 (C-4), 152.0 (C-2), 157.8 (C-30), 178.7 (C]S),
6.1.1.2. 30-(4-(4-Methylphenyl)oxazol-2-ylamino)-50-(4-methyl-
phenyl)-40,50-dihydro-pyrazole-10-carbothioamide (4b). Light yellow
solid (0.16 g, 69%); m.p. 218e220 ꢂC; IR (KBr): 1350 (C]S), 1576
Table 3
MIC, MBC and MFC of compounds 11a, 11c, 12a and 12c.
Compound
Minimum inhibitory concentration
MIC (MBC/MFC)
m
g/well
B. subtilis
S. aureus
P. aeruginosa
K. pneumoniae
A. niger
P. chrysogenum
11a
11c
12a
12c
12.5 (50)
6.25 (12.5)
12.5 (50)
6.25 (12.5)
6.25
50 (200)
25 (100)
50 (200)
50 (200)
6.25
50 (>200)
50 (200)
50 (>200)
50 (200)
6.25
50 (>200)
25 (100)
50 (>200)
50 (200)
12.5
25 (100)
12.5 (100)
12.5 (100)
6.25 (12.5)
e
50 (200)
50 (200)
25 (100)
25 (100)
e
Chloramphenicol
Ketoconazole
e
e
e
e
6.25
12.5
(e) No activity.

T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
351
127.1, 128.3, 129.4, 131.6, 132.2, 134.3, 136.5, 140.8 (aromatic car-
C5eH),11.98 (bs, 1H, C5eNH) ppm; 13C NMR (DMSO-d6): 24.1 & 24.2
(AreCH3), 43.2 (C-40), 66.3 (C-50), 120.5 (C-5), 137.2 (C-2), 141.7 (C-
4), 158.6 (C-30), 177.8 (C]S), 127.3, 128.0, 129.4, 131.7, 132.3, 133.8,
ꢃ
bons) ppm; MS (m/z): 432.33 [Mþ ]; Anal. Calcd. for C19H15Cl2N5OS:
C, 52.78; H, 3.50; N, 16.20. Found: C, 52.91; H, 3.53; N, 16.38%.
ꢃ
135.6, 140.6 (aromatic carbons) ppm; MS (m/z): 390.50 [Mþ ]; Anal.
6.1.1.4. 30-(4-Phenylthiazol-2-ylamino)-50-phenyl-40,50-dihydropyr-
azole-10-carbothioamide (5a). Light yellow solid (0.17 g, 71%); m.p.
214e216 ꢂC; IR (KBr): 1336 (C]S), 1560 (C]N), 3246 (NH), 3345,
3446 (NH2) cmꢁ1; 1H NMR (DMSO-d6): 3.16 (dd,1H, Hx, JAx ¼ 6.6 Hz,
JMx ¼ 11.5 Hz), 3.79 (dd, 1H, HM, JAM ¼ 14.5 Hz, JMx ¼ 11.5 Hz), 5.30
(dd, 1H, HA, JAM ¼ 14.5 Hz, JAx ¼ 6.6 Hz), 5.52 (bs, 1H, C2eNH), 5.64
(bs, 2H, CeNH2), 7.15e7.74 (m, 11H, AreH & C5eH) ppm; 13C NMR
(DMSO-d6): 43.3 (C-40), 66.1 (C-50), 103.2 (C-5), 149.2 (C-4), 158.0
(C-30), 163.8 (C-2), 177.8 (C]S), 126.5, 127.8, 128.8, 129.6, 131.9,
Calcd. for C21H22N6S: C, 64.59; H, 5.68; N, 21.52. Found: C, 64.66; H,
5.67; N, 21.66%.
6.1.1.9. 30-(4-(4-Chlorophenyl)-1H-imidazol-2-ylamino)-50-(4-
chlorophenyl)-40,50-dihydropyrazole-10-carbothioamide
(6c).
Light yellow solid (0.18 g, 75%); m.p. 263e265 ꢂC; IR (KBr):
1351(C]S), 1575 (C]N), 3249 (NH), 3338, 3458 (NH2) cmꢁ1 1H
;
NMR (DMSO-d6): 3.15 (dd, 1H, Hx, JAx ¼ 7.0 Hz, JMx ¼ 11.8 Hz), 3.87
(dd, 1H, HM, JAM ¼ 14.6 Hz, JMx ¼ 11.8 Hz), 5.34 (dd, 1H, HA,
JAM ¼ 14.6 Hz, JAx ¼ 7.0 Hz), 5.58 (bs, 1H, C2eNH), 5.78 (bs, 2H,
CeNH2), 7.14e7.70 (m, 9H, AreH & C5eH), 12.06 (bs, 1H, C5eNH)
ppm; 13C NMR (DMSO-d6): 43.5 (C-40), 65.6 (C-50), 121.3 (C-5), 137.8
(C-2), 142.6 (C-4), 157.9 (C-30), 177.1 (C]S), 127.0, 128.5, 129.4, 130.7,
132.4, 133.5, 135.6, 139.3 (aromatic carbons) ppm; MS (m/z): 431.34
ꢃ
133.2, 135.7, 141.4 (aromatic carbons) ppm; MS (m/z): 379.50 [Mþ ];
Anal. Calcd. for C19H17N5S2: C, 60.13; H, 4.52; N, 18.45. Found: C,
60.43; H, 4.54; N, 18.60%.
6.1.1.5. 30-(4-(4-Methylphenyl)thiazol-2-ylamino)-50-(4-
ꢃ
methylphenyl)-40,50-dihydro-pyrazole-10-carbothioamide
(5b).
Light yellow solid (0.19 g, 78%); m.p. 241e243 ꢂC; IR (KBr): 1324
(C]S), 1552 (C]N), 3238 (NH), 3332, 3440 (NH2) cmꢁ1 1H NMR
[Mþ ]; Anal. Calcd. for C19H16Cl2N6S: C, 52.91; H, 3.74; N, 19.48.
Found: C, 53.00; H, 3.76; N, 19.60%.
;
(DMSO-d6): 2.25 & 2.31 (s, 6H, AreCH3), 3.19 (dd, 1H, Hx, JAx ¼ 6.5
Hz, JMx ¼ 11.3 Hz), 3.75 (dd, 1H, HM, JAM ¼ 14.3 Hz, JMx ¼ 11.3 Hz),
5.32 (dd, 1H, HA, JAM ¼ 14.3 Hz, JAx ¼ 6.5 Hz), 5.48 (bs, 1H, C2eNH),
5.65 (bs, 2H, CeNH2), 7.10e7.68 (m, 9H, AreH & C5eH) ppm; 13C
NMR (DMSO-d6): 24.0 & 24.2 (AreCH3), 43.8 (C-40), 65.7 (C-50),
102.8 (C-5), 148.6 (C-4), 159.5 (C-30), 163.2 (C-2), 176.3 (C]S), 126.1,
127.3, 128.2, 129.3, 130.9, 132.4, 134.5, 138.8 (aromatic carbons)
6.1.2. General procedure for the synthesis of 30-(4-aryloxazol-2-
ylamino)-50-aryl-10H-pyrazole-10-carbothioamide (7aec)/30-(4-
arylthiazol-2-ylamino)-50-aryl-10H-pyrazole-10-carbothioamide
(8aec)/30-(4-aryl-1H-imidazol-2-ylamino)-50-aryl-10H-pyrazole-
10-carbothioamide (9aec)
A
solution of compound 4/5/6 (1 mmol) and chloranil
(1.2 mmol) in xylene (10 ml) was refluxed for 23e25 h. Then it was
treated with 5% NaOH solution. The organic layer was separated,
repeatedly washed with water and dried over an. Na2SO4. The
solvent was removed in vacuo. The solid obtained was purified by
recrystallization from 2-propanol.
ꢃ
ppm; MS (m/z): 407.55 [Mþ ]; Anal. Calcd. for C21H21N5S2: C, 61.89;
H, 5.19; N, 17.18. Found: C, 61.83; H, 5.20; N, 17.31%.
6.1.1.6. 30-(4-(4-Chlorophenyl)thiazol-2-ylamino)-50-(4-
chlorophenyl)-40,50-dihydro-pyrazole-10-carbothioamide
(5c).
Light yellow solid (0.19 g, 81%); m.p. 248e250 ꢂC; IR (KBr): 1328
(C]S), 1571 (C]N), 3250 (NH), 3339, 3455 (NH2) cmꢁ1 1H NMR
6.1.2.1. 30-(4-Phenyloxazol-2-ylamino)-50-phenyl-10H-pyrazole-10-
carbothioamide (7a). Yellow solid (0.13 g, 66%); m.p. 228e230 ꢂC;
IR (KBr): 1337 (C]S), 1568 (C]N), 1637 (C]C), 3257 (NH), 3337,
;
(DMSO-d6): 3.12 (dd, 1H, Hx, JAx ¼ 6.8 Hz, JMx ¼ 11.8 Hz), 3.83 (dd,
1H, HM, JAM ¼ 14.9 Hz, JMx ¼ 11.8 Hz), 5.29 (dd, 1H, HA, JAM ¼ 14.9 Hz,
JAx ¼ 6.8 Hz), 5.55 (bs, 1H, C2eNH), 5.69 (bs, 2H, CeNH2), 7.18e7.70
(m, 9H, AreH & C5eH) ppm; 13C NMR (DMSO-d6): 42.4 (C-40), 66.4
(C-50), 103.5 (C-5), 149.7 (C-4), 158.6 (C-30), 164.1 (C-2), 178.1 (C]S),
127.4, 128.6, 129.1, 131.6, 132.8, 133.9, 135.3, 139.6 (aromatic car-
3447 (NH2) cmꢁ1
;
1H NMR (DMSO-d6): 5.52 (bs, 1H, C2eNH), 5.65
0
(bs, 2H, CeNH2), 7.04 (s, 1H, C4 eH), 7.14e7.68 (m, 11H, AreH &
C5eH) ppm; 13C NMR (DMSO-d6): 93.6 (C-40), 137.8 (C-5), 139.3 (C-
4), 144.2 (C-50), 146.4 (C-30), 147.1 (C-2), 178.2 (C]S), 127.2, 128.5,
129.6, 131.2, 132.5, 133.9, 135.3, 136.2 (aromatic carbons) ppm; MS
ꢃ
ꢃ
bons) ppm; MS (m/z): 448.39 [Mþ ]; Anal. Calcd. for C19H15Cl2N5S2:
(m/z): 361.42 [Mþ ]; Anal. Calcd. for C19H15N5OS: C, 63.14; H, 4.18; N,
C, 50.89; H, 3.37; N, 15.62. Found: C, 50.97; H, 3.35; N, 16.79%.
19.38. Found: C, 63.21; H, 4.22; N, 19.50%.
6.1.1.7. 30-(4-Phenyl-1H-imidazol-2-ylamino)-50-phenyl-40,50-dihy-
dropyrazole-10-carbo-thioamide (6a). Light yellow solid (0.19 g,
77%); m.p. 226e228 ꢂC; IR (KBr): 1346 (C]S), 1579 (C]N), 3240
(NH), 3352, 3451 (NH2) cmꢁ1; 1H NMR (DMSO-d6): 3.14 (dd, 1H, Hx,
JAx ¼ 6.9 Hz, JMx ¼ 12.0 Hz), 3.86 (dd, 1H, HM, JAM ¼ 14.4 Hz,
JMx ¼ 12.0 Hz), 5.33 (dd, 1H, HA, JAM ¼ 14.4 Hz, JAx ¼ 6.9 Hz), 5.62 (bs,
1H, C2eNH), 5.76 (bs, 2H, CeNH2), 7.12e7.62 (m, 11H, AreH &
C5eH), 11.94 (bs,1H, C5eNH) ppm; 13C NMR (DMSO-d6): 43.9 (C-40),
65.8 (C-50), 120.8 (C-5), 137.4 (C-2), 142.0 (C-4), 157.4 (C-30), 177.6
(C]S), 126.7, 127.1, 127.9, 128.5, 131.4, 134.6, 136.3, 139.6 (aromatic
6.1.2.2. 30-(4-(4-Methylphenyl)oxazol-2-ylamino)-50-(4-
methylphenyl)-10H-pyrazole-10-carbothioamide (7b). Yellow solid
(0.13 g, 68%); m.p. 246e248 ꢂC; IR (KBr): 1334 (C]S), 1573 (C]N),
1632 (C]C), 3260 (NH), 3332, 3439 (NH2) cmꢁ1 1H NMR (DMSO-
;
d6): 2.26 & 2.28 (s, 6H, AreCH3), 5.55 (bs, 1H, C2eNH), 5.63 (bs, 2H,
0
CeNH2), 7.03 (s, 1H, C4 eH), 7.12e7.63 (m, 9H, AreH & C5eH) ppm;
13C NMR (DMSO-d6): 24.2 & 24.4 (AreCH3), 94.3 (C-40), 137.5 (C-5),
138.8 (C-4), 143.8 (C-50), 145.2 (C-30), 147.8 (C-2), 177.8 (C]S), 127.0,
128.7, 129.2, 130.7, 131.9, 133.5, 134.6, 135.8 (aromatic carbons)
ꢃ
ppm; MS (m/z): 389.47 [Mþ ]; Anal. Calcd. for C21H19N5OS: C, 64.76;
ꢃ
carbons) ppm; MS (m/z): 362.45 [Mþ ]; Anal. Calcd. for C19H18N6S:
H, 4.92; N, 17.98. Found: C, 64.85; H, 4.90; N, 18.13%.
C, 62.96; H, 5.01; N, 23.19. Found: C, 63.08; H, 5.04; N, 23.39%.
6.1.2.3. 30-(4-(4-Chlorophenyl)oxazol-2-ylamino)-50-(4-
chlorophenyl)-10H-pyrazole-10-carbothioamide (7c). Yellow solid
(0.14 g, 72%); m.p. 258e260 ꢂC; IR (KBr): 1345 (C]S), 1558 (C]N),
6.1.1.8. 30-(4-(4-Methylphenyl)-1H-imidazol-2-ylamino)-50-(4-
methylphenyl)-40,50-dihydropyrazole-10-carbothioamide
(6b).
Light yellow solid (0.18 g, 73%); m.p. 244e246 ꢂC; IR (KBr): 1338
(C]S), 1584 (C]N), 3236 (NH), 3346, 3447 (NH2) cmꢁ1 1H NMR
1625 (C]C), 3252 (NH), 3348, 3456 (NH2) cmꢁ1
;
1H NMR (DMSO-
0
;
d6): 5.48 (bs, 1H, C2eNH), 5.66 (bs, 2H, CeNH2), 7.07 (s, 1H, C4 eH),
7.18e7.60 (m, 9H, AreH & C5eH) ppm; 13C NMR (DMSO-d6): 94.8
(C-40), 138.1 (C-5), 139.7 (C-4), 144.7 (C-50), 146.8 (C-30), 148.1 (C-2),
178.4 (C]S), 127.5, 128.8, 129.4, 130.9, 132.4, 133.5, 134.2, 136.8
(DMSO-d6): 2.27 & 2.30 (s, 6H, AreCH3), 3.17 (dd, 1H, Hx,
JAx ¼ 6.8 Hz, JMx ¼ 11.7 Hz), 3.77 (dd, 1H, HM, JAM ¼ 14.2 Hz,
JMx ¼ 11.7 Hz), 5.27 (dd, 1H, HA, JAM ¼ 14.2 Hz, JAx ¼ 6.8 Hz), 5.60 (bs,
1H, C2eNH), 5.78 (bs, 2H, CeNH2), 7.09e7.68 (m, 9H, AreH &
ꢃ
(aromatic carbons) ppm; MS (m/z): 430.31 [Mþ ]; Anal. Calcd. for

352
T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
C19H13Cl2N5OS: C, 53.03; H, 3.05; N, 16.28. Found: C, 52.97; H, 3.09;
N, 16.19%.
(0.13 g, 68%); m.p. 285e287 ꢂC; IR (KBr): 1347 (C]S), 1556 (C]N),
1630 (C]C), 3258 (NH), 3351, 3466 (NH2) cmꢁ1
;
1H NMR (DMSO-
0
d6): 5.56 (bs, 1H, C2eNH), 5.76 (bs, 2H, CeNH2), 6.94 (s, 1H, C4 eH),
7.12e7.74 (m, 9H, AreH & C5eH), 11.95 (bs, 1H, C5eNH) ppm; 13C
NMR (DMSO-d6): 94.8 (C-40), 121.1 (C-5), 138.2 (C-2), 139.5 (C-4),
146.9 (C-50), 148.7 (C-30), 177.3 (C]S), 126.3, 127.5, 128.7, 129.3,
130.6, 131.1, 133.8, 135.5 (aromatic carbons) ppm; MS (m/z): 429.33
6.1.2.4. 30-(4-Phenylthiazol-2-ylamino)-50-phenyl-10H-pyrazole-10-
carbothioamide (8a). Yellow solid (0.14 g, 70%); m.p. 237e239 ꢂC;
IR (KBr): 1340 (C]S), 1565 (C]N), 1640 (C]C), 3241 (NH), 3336,
3442 (NH2) cmꢁ1
;
1H NMR (DMSO-d6): 5.49 (bs, 1H, C2eNH), 5.60
ꢃ
(bs, 2H, CeNH2), 7.02 (s, 1H, C4 eH), 7.08e7.65 (m, 11H, AreH &
C5eH) ppm; 13C NMR (DMSO-d6): 94.2 (C-40), 102.8 (C-5), 143.8 (C-
50), 146.1 (C-30), 147.3 (C-4), 162.7 (C-2), 177.5 (C]S), 126.4, 127.6,
128.2, 128.9, 130.6, 132.2, 133.9, 135.6 (aromatic carbons) ppm; MS
[Mþ ]; Anal. Calcd. for C19H14Cl2N6S: C, 53.15; H, 3.29; N, 19.57.
0
Found: C, 53.26; H, 3.33; N, 19.74%.
6.1.3. General procedure for the synthesis of 50-aryl-N-(4-
aryloxazol-2-yl)-10-(400-arylthiazol-200-yl)-10H-pyrazol-30-amine
(10aec)/50-aryl-N-(4-arylthiazol-2-yl)-10-(400-arylthiazol-200-yl)-
10H-pyrazol-30-amine (11aec)/50-aryl-N-(4-aryl-1H-imidazol-2-
yl)-10-(400-arylthiazol-200-yl)-10H-pyrazol-30-amine (12aec)
To a solution of compound 7/8/9 (1 mmol) in ethanol (10 ml),
phenacyl bromide (1 mmol) was added and refluxed for 4e6 h. The
solid separated on cooling was filtered and purified by column
chromatography using ethyl acetateehexane (1:1) as eluent.
ꢃ
(m/z): 377.49 [Mþ ]; Anal. Calcd. for C19H15N5S2: C, 60.45; H, 4.01; N,
18.55. Found: C, 60.53; H, 3.99; N, 18.69%.
6.1.2.5. 30-(4-(4-Methylphenyl)thiazol-2-ylamino)-50-(4-
methylphenyl)-10H-pyrazole-10-carbothioamide (8b). Yellow solid
(0.13 g, 67%); m.p. 261e263 ꢂC; IR (KBr): 1326 (C]S), 1561 (C]N),
1628 (C]C), 3247 (NH), 3327, 3436 (NH2) cmꢁ1 1H NMR (DMSO-
;
d6): 2.25 & 2.27 (s, 6H, AreCH3), 5.46 (bs, 1H, C2eNH), 5.58 (bs, 2H,
0
CeNH2), 6.98 (s, 1H, C4 eH), 7.06e7.66 (m, 9H, AreH & C5eH) ppm;
13C NMR (DMSO-d6): 24.0 & 24.3 (AreCH3), 94.9 (C-40), 102.4 (C-5),
142.5 (C-50),144.7 (C-30),146.0 (C-4),162.3 (C-2),176.2 (C]S),126.6,
127.3,128.7,130.3,131.8,132.7,134.3,135.1 (aromatic carbons) ppm;
6.1.3.1. 50-Phenyl-N-(4-phenyloxazol-2-yl)-10-(400-phenylthiazol-200-
yl)-10H-pyrazol-30-amine (10a). Yellow solid (0.16 g, 63%); m.p.
236e238 ꢂC; IR (KBr): 1574 (C]N), 1634 (C]C), 3252 (NH) cmꢁ1
;
ꢃ
MS (m/z): 405.54 [Mþ ]; Anal. Calcd. for C21H19N5S2: C, 62.19; H,
H NMR (DMSO-d6): 5.47 (bs, 1H, C2eNH), 6.95 (s, 1H, C4 eH),
1
0
7.12e7.66 (m, 17H, AreH, C5eH & C5 eH) ppm; 13C NMR (DMSO-
00
4.72; N, 17.27. Found: C, 62.29; H, 4.77; N, 17.40%.
d6): 94.4 (C-40), 112.8 (C-500), 137.5 (C-5), 138.6 (C-4), 146.0 (C-30),
150.2 (C-50), 153.5 (C-400), 160.8 (C-200), 162.8 (C-2), 126.5, 127.2,
127.9, 128.9, 129.6, 130.8, 131.5, 132.4, 133.1, 134.7, 135.5, 136.7 (ar-
6.1.2.6. 30-(4-(4-Chlorophenyl)thiazol-2-ylamino)-50-(4-
chlorophenyl)-10H-pyrazole-10-carbothioamide (8c). Yellow solid
(0.14 g, 73%); m.p. 269e271 ꢂC; IR (KBr): 1348 (C]S), 1573 (C]N),
ꢃ
omatic carbons) ppm; MS (m/z): 461.54 [Mþ ]; Anal. Calcd. for
1632 (C]C), 3244 (NH), 3342, 3452 (NH2) cmꢁ1
;
1H NMR (DMSO-
C27H19N5OS: C, 70.26; H, 4.15; N, 15.17. Found: C, 70.33; H, 4.13; N,
0
d6): 5.50 (bs, 1H, C2eNH), 5.59 (bs, 2H, CeNH2), 7.05 (s, 1H, C4 eH),
7.14e7.60 (m, 9H, AreH & C5eH) ppm; 13C NMR (DMSO-d6): 94.7
(C-40), 103.1 (C-5), 142.2 (C-50), 146.9 (C-30), 147.7 (C-4), 163.0 (C-2),
176.6 (C]S), 126.9, 127.7, 128.4, 130.1, 132.5, 133.3, 134.6, 135.8
15.29%.
6.1.3.2. 50-(4-Methylphenyl)-N-(4-(4-methylphenyl)oxazol-2-yl)-10-
(400-(4-methylphenyl)-thiazol-200-yl)-10H-pyrazol-30-amine
(10b).
ꢃ
(aromatic carbons) ppm; MS (m/z): 446.38 [Mþ ]; Anal. Calcd. for
Yellow solid (0.16 g, 61%); m.p. 251e252 ꢂC; IR (KBr): 1586 (C]N),
C
19H13Cl2N5S2: C, 51.12; H, 2.94; N,15.69. Found: C, 51.19; H, 2.97; N,
1639 (C]C), 3240 (NH) cmꢁ1
;
1H NMR (DMSO-d6): 2.26, 2.28 &
0
15.80%.
2.29 (s, 9H, AreCH3), 5.43 (bs, 1H, C2eNH), 6.92 (s, 1H, C4 eH),
7.06e7.63 (m, 14H, AreH, C5eH & C5 eH) ppm; 13C NMR (DMSO-
00
6.1.2.7. 30-(4-Phenyl-1H-imidazol-2-ylamino)-50-phenyl-10H-pyr-
azole-10-carbothioamide (9a). Yellow solid (0.12 g, 62%); m.p.
250e252 ꢂC; IR (KBr): 1336 (C]S), 1577 (C]N), 1622 (C]C), 3254
d6): 24.1, 24.2 & 24.3 (AreCH3), 93.2 (C-40), 112.5 (C-500), 137.1 (C-5),
138.9 (C-4), 145.7 (C-30), 151.9 (C-50), 153.3 (C-400), 160.5 (C-200),
162.4 (C-2), 126.3, 126.9, 127.6, 128.1, 128.5, 130.4, 131.1, 132.6, 133.2,
ꢃ
(NH), 3340, 3458 (NH2) cmꢁ1
;
1H NMR (DMSO-d6): 5.48 (bs, 1H,
133.8, 134.7, 136.2 (aromatic carbons) ppm; MS (m/z): 503.62 [Mþ ];
0
C2eNH), 5.74 (bs, 2H, CeNH2), 6.88 (s, 1H, C4 eH), 7.10e7.68 (m,
11H, AreH & C5eH), 11.90 (bs, 1H, C5eNH) ppm; 13C NMR (DMSO-
d6): 94.3 (C-40), 121.6 (C-5), 137.4 (C-2), 138.8 (C-4), 146.7 (C-50),
148.2 (C-30), 177.6 (C]S), 125.6, 127.1, 129.7, 131.3, 132.5, 133.6,
Anal. Calcd. for C30H25N5OS: C, 71.55; H, 5.00; N, 13.91. Found: C,
71.61; H, 5.03; N, 14.05%.
6.1.3.3. 50-(4-Chlorophenyl)-N-(4-(4-chlorophenyl)oxazol-2-yl)-10-
ꢃ
135.2, 136.3 (aromatic carbons) ppm; MS (m/z): 360.44 [Mþ ]; Anal.
Calcd. for C19H16N6S: C, 63.31; H, 4.47; N, 23.32. Found: C, 63.36; H,
4.56; N, 23.40%.
(400-(4-chlorophenyl)-thiazol-200-yl)-10H-pyrazol-30-amine
(10c).
Yellow solid (0.18 g, 69%); m.p. 267e269 ꢂC; IR (KBr): 1578 (C]N),
1636 (C]C), 3234 (NH) cmꢁ1 1H NMR (DMSO-d6): 5.49 (bs, 1H,
;
0
C2eNH), 7.00 (s, 1H, C4 eH), 7.18e7.72 (m, 14H, AreH, C5eH &
00
13
6.1.2.8. 30-(4-(4-Methylphenyl)-1H-imidazol-2-ylamino)-50-(4-
methylphenyl)-10H-pyrazole-10-carbothioamide (9b). Yellow solid
(0.13 g, 65%); m.p. 276e278 ꢂC; IR (KBr): 1331 (C]S), 1565 (C]N),
C5 eH) ppm; C NMR (DMSO-d6): 93.7 (C-40), 113.2 (C-500), 137.8
(C-5),138.4 (C-4),146.4 (C-30),151.4 (C-50),153.7 (C-400),161.2 (C-200),
163.1 (C-2), 127.1, 127.8, 128.7, 129.4, 130.6, 131.7, 132.4, 132.9, 133.5,
ꢃ
1636 (C]C), 3243 (NH), 3347, 3452 (NH2) cmꢁ1
;
1H NMR (DMSO-
134.2,134.8,136.2 (aromatic carbons) ppm; MS (m/z): 564.87 [Mþ ];
d6): 2.26 & 2.29 (s, 6H, AreCH3), 5.54 (bs, 1H, C2eNH), 5.72 (bs, 2H,
Anal. Calcd. for C27H16Cl3N5OS: C, 57.41; H, 2.85; N, 12.40. Found: C,
57.51; H, 2.87; N, 12.56%.
0
CeNH2), 6.86 (s, 1H, C4 eH), 7.08e7.62 (m, 9H, AreH & C5eH), 11.93
(bs, 1H, C5eNH) ppm; 13C NMR (DMSO-d6): 24.1 & 24.3 (AreCH3),
94.1 (C-40), 121.3 (C-5), 137.8 (C-2), 138.2 (C-4),146.4 (C-50),147.7 (C-
30), 178.2 (C]S), 125.2, 126.8, 128.4, 129.1, 131.6, 133.2, 134.8, 135.3
6.1.3.4. 50-Phenyl-N-(4-phenylthiazol-2-yl)-10-(400-phenylthiazol-200-
yl)-10H-pyrazol-30-amine (11a). Yellow solid (0.17 g, 70%); m.p.
ꢃ
(aromatic carbons) ppm; MS (m/z): 388.49 [Mþ ]; Anal. Calcd. for
239e241 ꢂC; IR (KBr): 1563 (C]N), 1628 (C]C), 3230 (NH) cmꢁ1
;
1
0
C
21H20N6S: C, 64.92; H, 5.19; N, 21.63. Found: C, 65.01; H, 5.25; N,
H NMR (DMSO-d6): 5.42 (bs, 1H, C2eNH), 6.88 (s, 1H, C4 eH),
7.08e7.62 (m, 17H, AreH, C5eH & C5 eH) ppm; 13C NMR (DMSO-
00
21.79%.
d6): 93.8 (C-40), 102.5 (C-5), 112.4 (C-500), 145.7 (C-30), 147.0 (C-4),
149.8 (C-50), 153.2 (C-400), 159.4 (C-200), 162.4 (C-2), 126.2, 126.9,
127.5, 128.6, 129.2, 130.5, 131.2, 131.8, 132.6, 133.3, 134.0, 135.8
6.1.2.9. 30-(4-(4-Chlorophenyl)-1H-imidazol-2-ylamino)-50-(4-
chlorophenyl)-10H-pyrazole-10-carbothioamide (9c). Yellow solid

T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
353
ꢃ
(aromatic carbons) ppm; MS (m/z): 477.60 [Mþ ]; Anal. Calcd. for
C
14.79%.
6.2. Biological assays
6.2.1. Cells
27H19N5S2: C, 67.90; H, 4.01; N, 14.66. Found: C, 67.99; H, 4.05; N,
The compounds 4e12 were dissolved in DMSO at different
concentrations of 25, 50 and 100 g/well. Bacterial strains S. aureus,
Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and
fungi A. niger, P. chrysogenum were obtained from the Department
of Microbiology, S.V. University, Tirupati.
6.1.3.5. 50-(4-Methylphenyl)-N-(4-(4-methylphenyl)thiazol-2-yl)-10-
m
(400-(4-methylphenyl)-thiazol-200-yl)-10H-pyrazol-30-amine
(11b).
Yellow solid (0.16 g, 65%); m.p. 280e282 ꢂC; IR (KBr): 1570 (C]N),
1620 (C]C), 3226 (NH) cmꢁ1
;
1H NMR (DMSO-d6): 2.24, 2.26 &
0
2.28 (s, 9H, AreCH3), 5.43 (bs, 1H, C2eNH), 6.84 (s, 1H, C4 eH),
7.02e7.60 (m, 14H, AreH, C5eH & C5 eH) ppm; 13C NMR (DMSO-
00
6.2.2. Antibacterial and antifungal assays
The in vitro antimicrobial studies were carried out by agar well
diffusion method against test organisms [19,20]. Nutrient broth
(NB) plates were swabbed with 24 h old broth culture (100 ml) of
test bacteria. Using the sterile cork borer, wells (6 mm) were made
into each petriplate. Various concentrations of DMSO dissolved
d6): 24.0, 24.2 & 24.3 (AreCH3), 92.4 (C-40), 101.3 (C-5), 112.0 (C-500),
144.2 (C-30), 146.6 (C-4), 149.5 (C-50), 152.8 (C-400), 160.1 (C-200),
162.7 (C-2), 126.7, 127.1, 128.2, 128.9, 130.1, 130.7, 131.4, 132.2, 132.9,
ꢃ
133.5, 134.6, 135.2 (aromatic carbons) ppm; MS (m/z): 519.68 [Mþ ];
Anal. Calcd. for C30H25N5S2: C, 69.33; H, 4.85; N, 13.48. Found: C,
69.25; H, 4.83; N, 13.57%.
compounds (25, 50 100 mg/well) were added into the wells by using
sterile pipettes. The standard antibiotics, Chloramphenicol, for
antibacterial activity and Ketoconazole, for antifungal activity (as
positive control) were simultaneously tested against the pathogens.
The samples were dissolved in DMSO which showed no zone of
inhibition acts as a negative control. The plates were incubated at
37 ꢂC for 24 h for bacteria and at 28 ꢂC for 48 h for fungi. After
appropriate incubation, the diameter of zone of inhibition of each
well was measured. Duplicates were maintained and the average
values were calculated for eventual antibacterial activity.
6.1.3.6. 50-(4-Chlorophenyl)-N-(4-(4-chlorophenyl)thiazol-2-yl)-10-
(400-(4-chlorophenyl)-thiazol-200-yl)-10H-pyrazol-30-amine
(11c).
Yellow solid (0.17 g, 67%); m.p. 288e290 ꢂC; IR (KBr): 1565 (C]N),
1632 (C]C), 3237 (NH) cmꢁ1 1H NMR (DMSO-d6): 5.45 (bs, 1H,
;
0
C2eNH), 6.87 (s, 1H, C4 eH), 7.14e7.68 (m, 14H, AreH, C5eH &
13
C5 eH) ppm; C NMR (DMSO-d6): 94.1 (C-40), 101.9 (C-5), 112.7 (C-
00
500), 146.1 (C-30), 147.8 (C-4), 150.3 (C-50), 153.5 (C-400), 160.7 (C-200),
163.7 (C-2), 126.5, 127.3, 128.3, 129.4, 130.9, 131.7, 132.1, 132.3, 132.9,
ꢃ
133.7, 135.2, 136.2 (aromatic carbons) ppm; MS (m/z): 580.94 [Mþ ];
Anal. Calcd. for C27H16Cl3N5S2: C, 55.82; H, 2.78; N, 12.06. Found: C,
55.91; H, 2.82; N, 12.19%.
6.2.3. Minimum inhibitory concentration assay
Broth dilution test was used to determine Minimum Inhibitory
Concentration (MIC) of the above mentioned samples [21,22].
Freshly prepared nutrient broth was used as diluents. The 24 h old
culture of the test bacteria S. aureus, B. subtilis, P. aeruginosa and K.
pneumoniae and the test fungi A. niger and P. chrysogenum were
6.1.3.7. 50-Phenyl-N-(4-phenyl-1H-imidazol-2-yl)-10-(400-phenyl-
thiazol-200-yl)-10H-pyrazol-30-amine (12a). Yellow solid (0.17 g,
68%); m.p. 272e274 ꢂC; IR (KBr): 1580 (C]N), 1637 (C]C), 3240
(NH) cmꢁ1
;
1H NMR (DMSO-d6): 5.58 (bs, 1H, C2eNH), 6.93 (s, 1H,
0
00
C4 eH), 7.12e7.74 (m, 17H, AreH, C5eH & C5 eH), 12.06 (bs, 1H,
diluted 100 fold in nutrient broth (100 ml bacterial cultures in 10 ml
13
C5eNH) ppm; C NMR (DMSO-d6): 94.2 (C-40), 113.0 (C-500), 121.6
NB). The stock solution of the synthesized compounds was pre-
pared in DMSO by dissolving 5 mg of the compound in 1 ml of
DMSO. Increasing concentrations of the test samples (1.25, 2.5, 5,
(C-5), 137.4 (C-2), 138.9 (C-4), 146.5 (C-30), 151.7 (C-50), 154.2 (C-400),
161.6 (C-200), 127.6, 128.6, 129.4, 130.8, 131.3, 132.5, 133.1, 134.6,
135.1, 135.7, 136.3, 137.6 (aromatic carbons) ppm; MS (m/z): 460.55
10, 20, 40 ml of stock solution contains 6.25, 12.5, 25, 50, 100, 200 mg
ꢃ
[Mþ ]; Anal. Calcd. for C27H20N6S: C, 70.41; H, 4.38; N, 18.25. Found:
of the compounds) were added to the test tubes containing the
bacterial and fungal cultures. All the tubes were incubated at 37 ꢂC
for 24 h for bacteria and at 28 ꢂC for 48 h for fungi. The tubes were
examined for visible turbidity and using NB as control. Control
without test samples and with solvent was assayed simultaneously.
The lowest concentration that inhibited visible growth of the tested
organisms was recorded as MIC.
C, 70.48; H, 4.41; N, 18.19%.
6.1.3.8. 50-(4-Methylphenyl)-N-(4-(4-methylphenyl)-1H-imidazol-2-
yl)-10-(400-(4-methyl-phenyl)thiazol-200-yl)-10H-pyrazol-30-amine
(12b). Yellow solid (0.18 g, 71%); m.p.: 263e265 ꢂC; IR (KBr): 1588
(C]N), 1633 (C]C), 3248 (NH) cmꢁ1 1H NMR (DMSO-d6): 2.23,
;
2.25 & 2.26 (s, 9H, AreCH3), 5.63 (bs, 1H, C2eNH), 6.90 (s, 1H,
0
00
C4 eH), 7.08e7.66 (m, 14H, AreH, C5eH & C5 eH), 12.11 (bs, 1H,
C5eNH) ppm; 13C NMR (DMSO-d6): 24.1, 24.3 & 24.4 (AreCH3), 93.8
(C-40), 112.4 (C-500), 121.2 (C-5), 137.2 (C-2), 139.1 (C-4), 146.8 (C-30),
152.3 (C-50), 153.9 (C-400), 160.5 (C-200), 127.3, 128.1, 128.9, 129.7,
130.3, 131.3, 132.5, 132.9, 134.2, 135.7, 136.1, 137.3 (aromatic car-
6.2.4. Minimum bactericidal/fungicidal concentration
To determine the Minimum Bactericidal Concentration (MBC)
[23] and Minimum Fungicidal Concentration (MFC) [24] for each
set of test tubes in the MIC determination, a loopful of broth was
collected from those tubes which did not show any growth and
inoculated on sterile nutrient broth (for bacteria) and PDA (for
fungi) by streaking. Plates inoculated with bacteria and fungi were
incubated at 37 ꢂC for 24 h and at 28 ꢂC for 48 h, respectively. After
incubation, the lowest concentration was noted as MBC (for bac-
teria) or MFC (for fungi) at which no visible growth was observed.
ꢃ
bons) ppm; MS (m/z): 502.63 [Mþ ]; Anal. Calcd. for C30H26N6S: C,
71.69; H, 5.21; N, 16.72. Found: C, 71.64; H, 5.22; N, 16.82%.
6.1.3.9. 50-(4-Chlorophenyl)-N-(4-(4-chlorophenyl)-1H-imidazol-2-
yl)-10-(400-(4-chloro-phenyl)thiazol-200-yl)-10H-pyrazol-30-amine
(12c). Yellow solid (0.17 g, 66%); m.p. 277e279 ꢂC; IR (KBr): 1576
(C]N), 1635 (C]C), 3243 (NH) cmꢁ1; 1H NMR (DMSO-d6): 5.65 (bs,
0
1H, C2eNH), 6.96 (s, 1H, C4 eH), 7.18e7.70 (m, 14H, AreH, C5eH &
00
Acknowledgments
C5 eH), 12.08 (bs, 1H, C5eNH) ppm; 13C NMR (DMSO-d6): 94.7 (C-
40), 113.8 (C-500), 120.8 (C-5), 136.7 (C-2), 139.7 (C-4), 147.2 (C-30),
152.8 (C-50), 155.2 (C-400), 162.3 (C-200), 126.8, 127.4, 128.8, 129.1,
130.6,131.5,132.8,133.1,133.9,134.5,136.1,137.8 (aromatic carbons)
The authors are grateful to Council of Scientific and Industrial
Research (CSIR), New Delhi for financial assistance under major
research project. One of the authors, T. Bhanu Prakash is thankful to
University Grants Commission (UGC), New Delhi for the sanction of
UGC-BSR fellowship.
ꢃ
ppm; MS (m/z): 563.89 [Mþ ]; Anal. Calcd. for C27H17Cl3N6S: C,
57.51; H, 3.04; N, 14.90. Found: C, 57.61; H, 2.98; N, 15.02%.

354
T. Bhanu Prakash et al. / European Journal of Medicinal Chemistry 82 (2014) 347e354
[12] M. Lhassani, O. Chavignon, J.M. Chezal, J.C. Teulade, J.P. Chapat, R. Snoeck,
Appendix A. Supplementary data
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271e274.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.001.
[13] H. Nakano, T. Inoue, N. Kawasaki, H. Miyataka, H. Matsumoto, T. Taguchi,
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