Synthesis, antimicrobial and cytotoxic activities of sulfone linked bis heterocycles

Article abstract of DOI:10.1016/j.ejmech.2012.06.014

A new class of sulfone linked bis heterocycles viz., pyrrolyl/pyrazolyl arylaminosulfonylmethyl 1,3,4-oxadiazoles, 1,3,4-thiadiazoles, and 1,2,4-triazoles were prepared and tested for antimicrobial activity and cytotoxicity. The chloro-substituted compounds 5c, 8c and 14c showed comparable antibacterial activity to chloramphenicol against Pseudomonasaeruginosa and compound 5c exhibited comparable antifungal activity to ketoconazole against Penicilliumchrysogenum. One of the compounds, vinylsulfonyl oxadiazole showed appreciably cytotoxic activity on A549 lung carcinoma cells with an IC ₅₀ at a concentration of 31.7 μM.

Full text of DOI:10.1016/j.ejmech.2012.06.014

European Journal of Medicinal Chemistry 54 (2012) 605e614
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial and cytotoxic activities of sulfone linked bis heterocycles
,
b
b
A. Muralikrishna a, B.C. Venkatesh a, V. Padmavathi a, A. Padmaja a , P. Kondaiah , N. Siva Krishna
*
a Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India
b Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore 560 012, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
new class of sulfone linked bis heterocycles viz., pyrrolyl/pyrazolyl arylaminosulfonylmethyl
Received 12 December 2011
Received in revised form
6 June 2012
Accepted 7 June 2012
Available online 17 June 2012
1,3,4-oxadiazoles, 1,3,4-thiadiazoles, and 1,2,4-triazoles were prepared and tested for antimicrobial
activity and cytotoxicity. The chloro-substituted compounds 5c, 8c and 14c showed comparable anti-
bacterial activity to chloramphenicol against Pseudomonasaeruginosa and compound 5c exhibited
comparable antifungal activity to ketoconazole against Penicilliumchrysogenum. One of the compounds,
vinylsulfonyl oxadiazole showed appreciably cytotoxic activity on A549 lung carcinoma cells with an IC50
at a concentration of 31.7 mM.
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Pyrroles
Pyrazoles
Oxadiazoles
Thiadiazoles
Triazoles
1,3-Dipolar cycloaddition
Antimicrobial activity
Cytotoxicity
1. Introduction
investigate the antimicrobial and cytotoxic activity of sulfone
linked pyrrolyl-oxadiazoles/thiadiazoles/triazoles and pyrazolyl-
oxadiazoles/thiadiazoles/triazoles.
In the past years considerable evidence has been accumulated to
demonstrate the efficacy of five membered heterocycles viz.,
pyrrole, pyrazole, oxadiazole, thiadiazole, triazole and their deriv-
atives. In fact tolmetin, ketorolac, the pyrrole derivatives were of
pharmacological relevance due to their anti-inflammatory and
analgesic properties [1]. Celecoxib, a pyrazole derivative is widely
used in the market as an anti-inflammatory drug [1]. As a scaffold in
medicinal chemistry, 1,3,4-oxadiazoles were established as
a member of the privileged structures class [2]. In particular a few
differently substituted 1,3,4-oxadiazoles have been found to exhibit
anticancer activity [3e5]. 1,3,4-Thiadiazole nucleus constitutes the
active part of several biologically active compounds including
antibacterial [6e8], antimycotic [9,10] and anti-inflammatory
agents [11e13]. The most frequently used triazoles are
fluconazole and itraconazole that display a broad spectrum of
antifungal activity and reduced toxicity when compared with
imidazole antifungals [14e19]. Based on the above facts and our
continued interest in the synthesis of biologically potent
heterocyclic systems, the present work has been taken up to
2. Chemistry
The general synthetic pathway discussed hereafter is depicted in
Scheme. The synthetic Scheme involves the cyclocondensation
of arylaminosulfonylacetic acid hydrazide (1) and E-arylsulfonyle-
thenesulfonylacetic acid (2) in the presence of POCl3 to get 2-(aryla-
minosulfonylmethyl)-5-[E-(2-arylsulfonylvinylsulfonylmethyl)]-1,3,
4-oxadiazole (3). The oxadiazole moiety was interconverted
to thiadiazole by treating with thiourea in THF. Thus 2-(arylamino-
sulfonylmethyl)-5-[E-(2-arylsulfonylvinylsulfonylmethyl)]-1,3,4-thia
diazole (4) was prepared. However, compound 4-amino-3-(arylam
inosulfonylmethyl)-5-[E-(2-arylsulfonylvinylsulfonylmethyl)]- 1,2,4-
triazole (5) was obtained by the reaction of 3 with hydrazine hydrate
in the presence of KOH in n-butanol (Scheme 1). The 1H NMR spectra
of 3a and 4a displayed two singlets at d 5.50, 5.41 and 4.88, 5.12 due to
methylene protons attached to C-2 and C-5 while 5a at 5.30 and
5.15 ppm due to methylene protons attached to C-3 and C-5. The
downfield signal was assigned to the one adjacent to sulfonamide
moiety. In addition to this, one doublet was observed at 7.16, 6.83 and
6.77 ppm in these compounds due to olefin proton HB. The olefin
proton, HA displayed a signal at downfield region, merged with
* Corresponding author. Tel.: þ91 877 2289303; fax: þ91 877 2249532.
E-mail address: adivireddyp@yahoo.co.in (A. Padmaja).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.014

606
A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
O
O
H
H
N
OH
S
N
S
S
NH2
O
O
O
+
O
O
O
R
O
R
R
2
1
i
R
HB
O
N
N
O
O
O
O
S
S
N
H
S
O
3
HA
iii
ii
R
R
R
R
N
N
N N
O
O
O
O
O
O
O
O
S
S
S
S
N
H
S
N
H
S
S
N
O
O
O
O
NH2
4
5
R a = H
b = Me
c = Cl
i. POCl3
ii. NH2CSNH2 / THF
iii. NH2NH2.H2O / KOH / n-Butanol
Scheme 1. Synthesis of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles.
aromatic protons. The coupling constant value J z 14.3 Hz indicated
thattheypossesstrans geometry. Thebroad signals appeared at10.24,
10.32 and 10.28 ppm in 3a, 4a and 5a was assigned to NH which
disappeared on deuteration. Compound 5a also exhibited another
broad signal at 5.68 ppm due to NH2 which disappeared on
deuteration.
The olefin moiety present in 3, 4 and 5 was exploited to develop
five membered heterocycles, pyrroleand pyrazole. Thereaction of 3, 4
and 5 with tosylmethyl isocyanide (TosMIC) in the presence of
sodium hydride in a solvent mixture of DMSO and ether yielded 2-
(arylamino-sulfonylmethyl)-5-(40-arylsulfonyl- 10H-pyrrol-30-ylsul-
fonylmethyl)-1,3,4-oxadiazole (6), 2-(aryl-aminosulfonylmethyl)-
5-(40-arylsulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,3,4-thiadiazole
(7) and 4-amino-3-(arylaminosulfonylmethyl)-5-(40-arylsulfonyl-
10H-pyrrol-30-ylsulfonylmethyl)-1,2,4-triazole (8) (Scheme 2). The 1H
Oxidation of compound 9, 10 and 11 was effected with chloranil in
xylene to afford the aromatized compounds, 2-(arylaminos
ulfonylmethyl)-5-(30-arylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-
1,3,4-oxadiazole (12), 2-(arylaminosulfonylmethyl)-5-(30-arylsul-
fonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thiadiazole (13) and 4-
amino-3-(arylaminosulfonylmethyl)-5-(30-arylsulfonyl-10H-pyrazol-
40-ylsulfonylmethyl)-1,2,4-triazole (14) (Scheme 2). The absence of
AMX splitting pattern due to pyrazoline ring protons in 12, 13 and 14
indicated that aromatization took place. The structures of these
compounds were further established by IR and 13C NMR spectra.
3. Biology
3.1. Antimicrobial activity
NMR spectra of 6a, 7a and 8a displayed two singlets at
d
5.38, 5.29,
Compounds 3e14 were tested for antimicrobial activity at two
5.37 and 5.08, 5.18, 5.11 ppm due to two methylene protons attached
to C-2 and C-5 of oxadiazole/thiadiazole and C-3 and C-5 of triazole
units. Apart from these, one singlet was observed at 6.59, 6.65 and
different concentrations 50 and 100 mg/ml.
3.2. Cytotoxicity
0
0
6.73 due to C5 eH of pyrrole ring whereas C2 eH appeared at down-
field region and merged with aromatic protons. Compounds 6a, 7a
and 8a also showed two broad singlets at 8.35, 8.39, 8.37 and at 10.40,
10.34, 10.35 ppm due to NH of pyrrole ring and NHeSO2 which dis-
appeared on deuteration. On the other hand, the 1,3-dipolar cyclo-
addition of diazomethane to 3, 4 and 5 in the presenceof Et3N in ether
at ꢀ20 ꢁC for 40e48 h resulted in 2-(arylaminosulfonylmethyl)-5-(30-
arylsulfonyl-40,50-dihydro-10 H-pyrazol-40-ylsulfonylmethyl)-1,3,4-
oxadiazole (9), 2-(arylaminosulfonylmethyl)-5-(30-arylsulfonyl-40,50-
dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thiadiazole (10) and
4-amino-3-(arylaminosulfonylmethyl)-5-(30-arylsulfonyl-40,50-dihy-
dro-10H-pyrazol-40-ylsulfonylmethyl)-1,2,4-triazole (11). The pyr-
azoline ring protons displayed an AMX splitting pattern in the 1H
NMR spectra of 9a,10a and 11a. Thus three double doublets observed
Compounds 3e14 were subjected to MTT assay to determine
cytotoxic capability.
4. Result and discussion
4.1. Antimicrobial activity
The results of antibacterial activity shown in Table 1 indicated
that Gram-negative bacteria were more susceptible towards the
tested compounds than Gram-positive ones. When compared to
the standard drug Chloramphenicol it was seen that the chloro-
substituted compounds 5c, 8c and 14c were effective particularly
against Pseudomonas aeruginosa at 100
mg/ml. Amongst bis
at
d
3.57, 4.24, 4.60 in 9a, at 3.58, 4.29, 4.64 in 10a and at 3.52, 4.21,
heterocyclic compounds, the aromatized bis heterocycles 12,13 and
14 were effective than the non-aromatized compounds 9, 10 and 11.
Amongst pyrrole and pyrazole containing bis heterocycles, the
latter compounds 12, 13 and 14 displayed greater activity. It was
4.64 ppm in 11a were assigned to Hx, HM and HA, respectively. The
coupling constant values JAx z 6.1, JMx z 10.2 and JAM z 12.1 Hz
indicatedthatHA, HM are cis, HA, Hx are trans while HM, Hx are geminal.

A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
607
R
N
N
O
O
O
O
O
O
ii
HA
S
S
S
3 / 4 / 5
N
H
X
MH
N
9 / 10 / 11
R
N
HX
iii
H
i
R
N
N
R
R
O
O
O
O
O
O
N
N
S
S
S
O
O
S
O
O
O
O
N
H
X
3'
4'
5'
S
S
N
2'
X
1'
4' 3'
2'
H
N
5'
N
1'
H
R
N
6 / 7 / 8
12 / 13 / 14
H
i. TosMIC / NaH / DMSO / Et2O
ii. CH2N2 / Et3N / Et2O
iii. Chloranil / Xylene
R a = H
b = Me
c = Cl
3, 6, 9, 12 : X = O
4, 7, 10, 13 : X = S
5, 8, 11, 14 : X = N-NH2
Scheme 2. Synthesis of sulfonamido pyrrolyl/pyrazolyl 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles.
also observed that the compounds having thiadiazole 4, 7, 13 and
triazole rings 5, 8, 14 were effective when compared with those
having oxadiazole unit 3, 6, 12.
All the tested compounds inhibited the spore germination
against tested fungi except compound 9. In general, most of the
compounds showed slightly higher antifungal activity towards
Table 1
The in vitro antibacterial activity of compounds 3e14.
Compound
Diameter of zone of inhibition (mm)
Gram-positive bacteria
S. aureus
Gram-negative bacteria
B. subtilis
P. aeruginosa
K. pneumoniae
50
100
50
100
50
100
50
100
mg/well
mg/well
mg/well
mg/well
mg/well
mg/well
m
g/well
m
g/well
3a
3b
10
e
13
e
12
e
14
e
e
e
e
e
e
e
e
e
3c
4a
4b
4c
5a
5b
5c
6a
14
19
18
29
21
20
32
12
e
17
21
20
32
23
22
35
14
e
15
22
21
27
24
23
36
13
e
17
24
23
29
26
25
38
15
e
16
16
14
25
17
15
31
e
19
17
16
27
18
17
33
e
15
24
23
33
26
25
39
e
17
26
25
35
28
27
40
e
6b
e
e
e
e
6c
7a
7b
7c
8a
8b
8c
9a
18
15
14
26
17
16
30
e
20
17
16
27
19
18
33
e
17
18
17
27
20
19
29
e
21
20
19
29
22
21
33
e
18
14
13
23
15
13
25
e
20
15
14
25
16
15
28
e
23
20
19
29
22
21
34
e
25
22
21
30
24
23
36
e
9b
e
e
e
e
e
e
e
e
9c
10a
10b
10c
11a
11b
11c
12a
17
12
11
24
13
13
25
10
e
18
14
13
27
15
15
27
12
e
17
14
14
25
15
16
24
13
e
20
16
16
26
17
18
27
15
e
17
e
19
e
22
e
24
e
11
22
12
12
21
10
e
13
23
14
15
24
12
e
16
25
17
18
28
15
e
18
28
19
20
30
17
e
12b
12c
13a
13b
13c
14a
14b
14c
20
16
12
27
19
16
31
33
e
23
17
14
29
21
17
34
35
e
21
15
10
28
19
18
33
34
e
22
17
13
31
23
20
35
38
e
19
10
8
24
15
12
30
27
e
21
12
11
26
17
14
32
30
e
23
14
13
30
24
20
37
40
e
24
16
15
33
26
22
39
42
e
Chloramphenicol
Control (DMSO)
(e) No activity.

608
A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
Table 2
The in vitro antifungal activity of compounds 3e14.
Compound
Diameter of zone of inhibition (mm)
A. niger
P. chrysogenum
50
100
50
100
mg/well
m
g/well
m
g/well
mg/well
3a
e
e
e
e
3b
e
e
e
e
3c
4a
4b
4c
5a
5b
5c
6a
19
19
18
31
21
19
32
e
22
22
21
33
24
23
34
e
21
21
20
32
23
22
35
e
25
24
23
35
26
25
38
e
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
10a
10b
10c
11a
11b
e
e
e
e
Fig. 1. The cytotoxic activity 3c as measured by MTT assay on A549 lung carcinoma
cells. X-axis shows the concentration of the compound and Y-axis, cell viability.
21
16
14
29
18
18
32
e
24
18
17
31
20
19
34
e
22
17
16
30
19
18
33
e
26
20
19
33
22
21
36
e
P. chrysogenum. However, the other compounds showed the
bactericidal and fungicidal effects greater than 2 ꢂ MIC. The
structureeantimicrobial activity relationship of the synthesized
compounds revealed that the compounds having extended conju-
gation 3, 4 and 5 exhibited greater activity than bis heterocycles
6e14. Amongst bis heterocyclic compounds, the compounds having
oxadiazole moiety exhibited least activity when compared with
those having thiadiazole and triazole units. The compounds having
chloro substituent on the aromatic ring showed greater activity.
e
e
e
e
20
e
23
e
22
e
24
e
11
28
13
13
28
10
e
14
29
15
16
30
13
e
13
28
14
15
29
12
e
16
31
17
18
32
15
e
11c
12a
12b
4.2. Cytotoxicity
12c
13a
13b
13c
14a
14b
14c
20
15
10
30
19
16
30
33
e
23
17
13
32
23
19
34
36
e
21
16
11
31
20
14
33
36
e
24
19
13
34
23
18
37
38
e
Compounds 3e14 were subjected to MTT assay to determine
cytotoxic capability. Only compound 3c showed appreciable cyto-
toxic activity on A549 cells (IC50 ¼ 31.7
mM). However, all the other
compounds did not show any cytotoxicity when used upto 0.2 mM
concentration. Fig. 1 shows the results of cytotoxicity of 3c using
MTT assay. The cytotoxic activity observed with compound 3c is
concentration dependent. Compound 3c at concentrations
Ketoconazole
Control (DMSO)
100e200
75% was observed when this compound was used at concentrations
below 12.50 M. This suggests compound 3c is a potential lead
mM showed lowest viability, while viability more than
(e) No activity.
m
molecule for cytotoxic activity against tumour cells. Further
modifications to 3c may provide a more potent molecule that could
be developed as therapeutic drug. Future efforts are in progress
towards this goal.
Penicillium chrysogenum than Aspergillus niger. Compounds 5c and
14c displayed comparable activity particularly against P. chrys-
ogenum almost equivalent to the standard drug Ketoconazole
(Table 2). This may be due to the presence of more electronegative
atom viz., chlorine in the aromatic ring which may enhance the
biological potency, bioavailability, metabolic stability and lip-
ophilicity. Enhanced lipophilicity may lead to easier absorption and
transportation of molecules within the biological systems.
The MIC, MBC and MFC values of the compounds tested are
listed in Table 3. Compound 5c exhibited low MIC values when
compared with 8c and 14c. In addition MBC value is 2 ꢂ MIC in case
of Bacillussubtilis and MFC value is 2 ꢂ MIC in case of
5. Conclusion
In conclusion we have prepared a new class of sulfonamido
mono and bis heterocycles and studied their antimicrobial and
cytotoxic activities. The chloro-substituted compounds 5c, 8c and
14c showed moderate and comparable antibacterial activity to
chloramphenicol against P. aeruginosa and compound 5c exhibited
Table 3
MIC, MBC and MFC of compounds 5c, 8c and 14c.
Compound
Minimum inhibitory concentration
MIC (MBC/MFC)
m
g/ml
B. subtilis
S. aureus
P. aeruginosa
K. pneumoniae
A. niger
P. chrysogenum
5c
8c
14c
12.5(50)
50(200)
50(200)
6.25
12.5(25)
25(100)
12.5(50)
6.25
12.5(50)
50(>200)
25(100)
6.25
25(100)
100(>200)
50(200)
12.5
12.5(100)
25(100)
12.5(50)
e
25(50)
50(>200)
25(100)
e
Chloramphenicol
Ketoconazole
e
e
e
e
6.25
12.5

A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
609
comparable antifungal activity to ketoconazole against
C18H15Cl2N3O7S3: C, 39.17; H, 2.74; N, 7.61; Found: C, 39.17; H, 2.77;
P. chrysogenum. It was observed that compound 3c displayed
N, 7.58%.
appreciable cytotoxic activity that could be used as
compound in the future studies.
a lead
6.1.2. General procedure for the synthesis of 2-(arylaminosulfonyl
methyl)-5-[E-(2-arylsulfonylvinylsulfonylmethyl)]-1,3,4-thiadiazole
4aec
6. Experimental
In a sealed test tube, compound 3 (5 mmol), thiourea (20 mmol)
and tetrahydrofuran (5 ml) were taken and heated at 120e150 ꢁC in
an oil bath for 18e21 h. After the reaction was completed, it was
extracted with dichloromethane. The organic layer was washed
with water, brine solution and dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure and the resultant
solid was recrystallized from methanol.
6.1. Chemistry
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, hexane/ethyl
acetate, 3:1). The IR spectra were run 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 DMSO-d6 on
6.1.2.1. 2-(Phenylaminosulfonylmethyl)-5-[E-(2-phenylsulfonylvinyl
sulfonylmethyl)]-1,3,4-thiadiazole (4a). White solid (1.63 g, 68%); m.p.
165e167 ꢁC; IR (KBr): 1138, 1321 (SO2), 1568 (C]N), 1632 (C]C), 3248
a Jeol JNM
DMSO-d6 on a Jeol JNM spectrometer operating at 100 MHz. All
chemical shifts are reported in (ppm) using TMS as an internal
l
-400 MHz. The 13C NMR spectra were recorded in
d
(NH) cmꢀ1; 1H NMR (DMSO-d6):
d
5.12 (s, 2H, SO2eCH2), 5.41 (s, 2H,
CH2eSO2eNH), 6.83 (d, 1H, HB, J ¼ 14.2 Hz), 7.20e7.74 (m, 11H, HA
AreH),10.32 (bs,1H, NH) ppm; 13C NMR (DMSO-d6):
44.3 (SO2eCH2),
standard. The microanalyses were performed on a PerkineElmer
240C elemental analyser.
&
d
52.2 (CH2eSO2eNH), 123.8 (CeHB), 143.0 (CeHA), 157.9 (C-5), 158.2 (C-
2), 126.8, 127.3, 128.1, 128.8, 129.5, 131.2, 132.6, 134.6 (aromatic carbons)
ppm; Anal. Calcd. for C18H17N3O6S4: C, 43.27; H, 3.43; N, 8.41; Found: C,
43.23; H, 3.45; N, 8.51%.
6.1.1. General procedure for the synthesis of 2-(arylaminosulfonyl
methyl)-5-[E-(2-arylsulfonylvinylsulfonylmethyl)]-1,3,4-oxadiazole
3aec
A
mixture of arylaminosulfonylacetic acid hydrazide (1)
(10 mmol), E-arylsulfonylethenesulfonylacetic acid (2) (10 mmol)
and POCl3 (7 ml) was heated under reflux for 3e5 h. The excess
POCl3 was removed under reduced pressure and the residue was
poured onto crushed ice. The resulting precipitate was filtered,
washed with saturated sodium bicarbonate solution and then with
water. It was dried and recrystallized from ethanol.
6.1.2.2. 2-(p-Methylphenylaminosulfonylmethyl)-5-[E-(2-p-methyl-
phenylsulfonylvinyl-sulfonylmethyl)]-1,3,4-thiadiazole (4b). White
solid (1.71 g, 65%); m.p. 180e182 ꢁC; IR (KBr): 1131, 1319 (SO2), 1561
(C]N), 1625 (C]C), 3237 (NH) cmꢀ1; 1H NMR (DMSO-d6):
d
2.26 &
2.31 (s, 6H, AreCH3), 5.10 (s, 2H, SO2eCH2), 5.40 (s, 2H,
CH2eSO2eNH), 6.79 (d, 1H, HB, J ¼ 14.6 Hz), 7.16e7.71 (m, 9H, HA
AreH), 10.30 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6):
21.1 & 22.4
&
d
6.1.1.1. 2-(Phenylaminosulfonylmethyl)-5-[E-(2-phenylsulfonylvinyl
sulfonylmethyl)]-1,3,4-oxadiazole (3a). White solid (3.48 g, 72%);
m.p. 132e134 ꢁC; IR (KBr): 1152, 1312 (SO2), 1574 (C]N), 1635 (C]
(AreCH3), 43.6 (SO2eCH2), 50.8 (CH2eSO2eNH), 122.6 (CeHB), 142.8
(CeHA), 158.2 (C-5), 159.8 (C-2), 126.4, 127.1, 127.7, 128.4, 129.7, 130.2,
130.9, 132.8 (aromatic carbons) ppm; Anal. Calcd. for C20H21N3O6S4:
C, 45.52; H, 4.01; N, 7.96; Found: C, 45.58; H, 3.99; N, 8.02%.
C), 3231 (NH) cmꢀ1 1H NMR (DMSO-d6):
; d 4.88 (s, 2H, SO2eCH2),
5.50 (s, 2H, CH2eSO2eNH), 7.16 (d, 1H, HB, J ¼ 14.3 Hz), 7.23e8.00
(m, 11H, HA & AreH), 10.24 (bs, 1H, NH) ppm; 13C NMR (DMSO-
6.1.2.3. 2-(p-Chlorophenylaminosulfonylmethyl)-5-[E-(2-p-chloroph
d6):
d
47.1 (SO2eCH2), 49.5 (CH2eSO2eNH), 120.7 (CeHB), 142.9
enylsulfonylvinyl-sulfonylmethyl)]-1,3,4-thiadiazole
solid (1.90 g, 67%); m.p. 192e194 ꢁC; IR (KBr): 1128, 1327 (SO2), 1572
(C]N), 1628 (C]C), 3255 (NH) cmꢀ1; 1H NMR (DMSO-d6):
5.19 (s,
(4c). White
(CeHA), 157.8 (C-5), 159.4 (C-2), 125.1, 125.8, 126.3, 126.9, 128.0,
129.4, 130.1, 131.5 (aromatic carbons) ppm; Anal. Calcd. for
C18H17N3O7S3: C, 44.71; H, 3.54; N, 8.69; Found: C, 44.79; H, 3.52; N,
8.80%.
d
2H, SO2eCH2), 5.46 (s, 2H, CH2eSO2eNH), 6.89 (d, 1H, HB,
J ¼ 14.8 Hz), 7.19e7.65 (m, 9H, HA & AreH), 10.35 (bs, 1H, NH) ppm;
13C NMR (DMSO-d6):
d 43.5 (SO2eCH2), 51.8 (CH2eSO2eNH), 124.2
6.1.1.2. 2-(p-Methylphenylaminosulfonylmethyl)-5-[E-(2-p-methylph
enylsulfonylvinyl-sulfonylmethyl)]-1,3,4-oxadiazole (3b). White solid
(3.58 g, 70%); m.p. 114e116 ꢁC; IR (KBr): 1148, 1308 (SO2), 1562 (C]
(CeHB), 143.6 (CeHA), 157.5 (C-5), 158.4 (C-2), 126.5, 127.4, 128.1,
128.8, 129.3, 130.4, 132.1, 134.6 (aromatic carbons) ppm; Anal.
Calcd. for C18H15Cl2N3O6S4: C, 38.03; H, 2.66; N, 7.39; Found: C,
38.07; H, 2.70; N, 7.32%.
N), 1624 (C]C), 3217 (NH) cmꢀ1; 1H NMR (DMSO-d6):
d 2.24 & 2.28
(s, 6H, AreCH3), 4.84 (s, 2H, SO2eCH2), 5.39 (s, 2H, CH2eSO2eNH),
6.46 (d, 1H, HB, J ¼ 14.3 Hz), 7.14e7.92 (m, 9H, HA & AreH), 10.25
6.1.3. General procedure for the synthesis of 4-amino-3-(arylamino
sulfonylmethyl)-5-[E-(2-arylsulfonylvinylsulfonylmethyl)]-1,2,4-tria
zole 5aec
To a solution of 3 (5 mmol) in n-butanol (25 ml), hydrazine
hydrate (15 mmol) was added and refluxed for 7e9 h. Then KOH
(0.01 mol) was added to the reaction media and the precipitate
formed was filtered. The solid obtained was acidified with conc. HCl
to pH ¼ 3 and washed with water. It was dried and recrystallized
from ethanol.
(bs, 1H, NH) ppm; 13C NMR (DMSO-d6):
d 20.6 & 21.5 (AreCH3), 46.8
(SO2eCH2), 45.5 (CH2eSO2eNH), 124.8 (CeHB), 143.2 (CeHA), 157.3
(C-5), 158.8 (C-2), 123.5, 124.1, 124.8, 125.2, 126.4, 127.3, 129.1, 132.4
(aromatic carbons) ppm; Anal. Calcd. for C20H21N3O7S3: C, 46.95; H,
4.14; N, 8.21; Found: C, 46.90; H, 4.15; N, 8.28%.
6.1.1.3. 2-(p-Chlorophenylaminosulfonylmethyl)-5-[E-(2-p-chloroph
enylsulfonylvinyl-sulfonylmethyl)]-1,3,4-oxadiazole (3c). White solid
(4.14 g, 75%); m.p. 154e156 ꢁC; IR (KBr): 1157, 1316 (SO2), 1575 (C]
N), 1631 (C]C), 3238 (NH) cmꢀ1; 1H NMR (DMSO-d6):
d
4.92 (s, 2H,
6.1.3.1. 4-Amino-3-(phenylaminosulfonylmethyl)-5-[E-(2-phenylsulf
onylvinylsulfonyl-methyl)]-1,2,4-triazole (5a). White solid (1.71 g,
69%); m.p. 170e172 ꢁC; IR (KBr): 1132, 1324 (SO2), 1559 (C]N),1624
SO2eCH2), 5.45 (s, 2H, CH2eSO2eNH), 6.99 (d, 1H, HB, J ¼ 14.6 Hz),
7.24e8.00 (m, 9H, HA & AreH), 10.39 (bs, 1H, NH) ppm; 13C NMR
(DMSO-d6):
d
44.8 (SO2eCH2), 49.3 (CH2eSO2eNH), 126.1 (CeHB),
(C]C), 3251 (NH), 3358, 3474 (NH2) cmꢀ1 1H NMR (DMSO-d6):
;
145.5 (CeHA), 158.5 (C-5), 159.2 (C-2), 125.6, 126.1, 128.4, 129.0,
d
5.15 (s, 2H, SO2eCH2), 5.30 (s, 2H, CH2eSO2eNH), 5.68 (bs, 2H,
129.8, 130.8, 132.4, 135.7 (aromatic carbons) ppm; Anal. Calcd. for
NH2), 6.77 (d, 1H, HB, J ¼ 14.4 Hz), 7.11e7.78 (m, 11H, HA & AreH),

610
A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
10.28 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6):
d
45.8 (SO2eCH2),
(DMSO-d6): d 21.5 & 22.9 (AreCH3), 44.8 (SO2eCH2), 51.2
51.3 (CH2eSO2eNH), 121.8 (CeHB), 143.2 (CeHA), 158.1 (C-5),
159.3 (C-3), 125.4, 126.1, 126.7, 128.4, 129.0, 130.2, 132.1, 133.4
(aromatic carbons) ppm; Anal. Calcd. for C18H19N5O6S3: C, 43.45; H,
3.85; N, 14.08; Found: C, 43.41; H, 3.82; N, 14.17%.
(CH2eSO2eNH), 105.6 (C-40), 110.2 (C-30), 116.5 (C-50), 121.2 (C-20),
155.8 (C-5), 158.1 (C-2), 126.3, 127.4, 128.0, 128.7, 129.3, 130.4, 132.6,
134.5 (aromatic carbons) ppm; Anal. Calcd. for C22H22N4O7S3: C,
47.99; H, 4.03; N, 10.18; Found: C, 47.93; H, 4.00; N, 10.28%.
6.1.3.2. 4-Amino-3-(p-methylphenylaminosulfonylmethyl)-5-[E-(2-
6.1.4.32-(p-Chlorophenylaminosulfonylmethyl)-5-(40-p-chlorophenyl
sulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,3,4-oxadiazole
p-methylphenyl-sulfonylvinylsulfonylmethyl)]-1,2,4-triazole
. White solid (1.73 g, 66%); m.p. 195e197 ꢁC; IR (KBr): 1126, 1321
(SO2), 1561 (C]N), 1622 (C]C), 3254 (NH), 3356, 3470 (NH2) cmꢀ1
1H NMR (DMSO-d6):
2.22 & 2.26 (s, 6H, AreCH3), 5.14 (s, 2H,
(5b)
(6c). White solid (0.41 g, 71%); m.p.162e164 ꢁC; IR (KBr): 1144,1336
;
(SO2), 1585 (C]N), 1634 (C]C), 3265 (NH) cmꢀ1; 1H NMR (DMSO-
d
d6):
d
5.13 (s, 2H, SO2eCH2), 5.35 (s, 2H, CH2eSO2eNH), 6.62 (s, 1H,
0
0
SO2eCH2), 5.35 (s, 2H, CH2eSO2eNH), 5.59 (bs, 2H, NH2), 6.74 (d,
1H, HB, J ¼ 14.2 Hz), 7.18e7.90 (m, 9H, HA & AreH), 10.26 (bs, 1H,
C5 eH), 7.25e7.94 (m, 9H, C2 eH & AreH), 8.40 (bs,1H, NH),10.45 (bs,
1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d 46.4 (SO2eCH2), 51.7
NH) ppm; 13C NMR (DMSO-d6):
d
20.8 & 21.4 (AreCH3), 45.1
(CH2eSO2eNH), 106.9 (C-40), 112.3 (C-30), 117.8 (C-50), 123.5 (C-20),
156.3 (C-5), 159.4 (C-2), 127.3, 128.1, 129.5, 130.8, 132.7, 133.2, 134.8,
136.7 (aromatic carbons) ppm; Anal. Calcd. for C20H16Cl2N4O7S3: C,
40.61; H, 2.73; N, 9.47; Found: C, 40.65; H, 2.76; N, 9.52%.
(SO2eCH2), 51.0 (CH2eSO2eNH), 122.7 (CeHB), 140.6 (CeHA), 158.5
(C-5), 159.7 (C-3), 126.3, 127.3, 127.9, 128.6, 130.1, 130.8, 131.4, 133.2
(aromatic carbons) ppm; Anal. Calcd. for C20H23N5O6S3: C, 45.70; H,
4.41; N, 13.32; Found: C, 45.73; H, 4.45; N, 13.38%.
6.1.4.42-(Phenylaminosulfonylmethyl)-5-(40-phenylsulfonyl-10H-pyrr
ol-30-ylsulfonyl-methyl)-1,3,4-thiadiazole (7a). White solid (0.38 g,
72%); m.p. 167e169 ꢁC; IR (KBr): 1141, 1324 (SO2), 1591 (C]N), 1627
6.1.3.3. 4-Amino-3-(p-chlorophenylaminosulfonylmethyl)-5-[E-(2-p-
chlorophenylsulfonyl-vinylsulfonylmethyl)]-1,2,4-triazole (5c). White
solid (1.98 g, 70%); m.p. 206e208 ꢁC; IR (KBr): 1137,1330 (SO2),1567
(C]C), 3241 (NH) cmꢀ1 1H NMR (DMSO-d6):
; d 5.18 (s, 2H,
(C]N), 1630 (C]C), 3267 (NH), 3369, 3479 (NH2) cmꢀ1
;
1H NMR
SO2eCH2), 5.29 (s, 2H, CH2eSO2eNH), 6.65 (s, 1H, C5 eH), 7.10e7.81
0
0
(DMSO-d6):
(bs, 2H, NH2), 6.82 (d, 1H, HB, J ¼ 14.7 Hz), 7.14e7.67 (m, 9H, HA
AreH), 10.31 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6):
46.1
(SO2eCH2), 51.5 (CH2eSO2eNH), 123.0 (CeHB), 142.3 (CeHA), 158.2
(C-5), 158.8 (C-3), 127.3, 127.9, 128.4, 129.0, 129.6, 130.2, 131.4, 133.7
(aromatic carbons) ppm; Anal. Calcd. for C18H17Cl2N5O6S3: C, 38.17;
H, 3.02; N, 12.36; Found: C, 38.22; H, 3.00; N, 12.44%.
d
5.14 (s, 2H, SO2eCH2), 5.32 (s, 2H, CH2eSO2eNH), 5.72
(m, 11H, C2 eH & AreH), 8.39 (bs, 1H, NH), 10.34 (bs, 1H, NHeSO2)
&
ppm; 13C NMR (DMSO-d6):
d 45.9 (SO2eCH2), 51.3 (CH2eSO2eNH),
d
104.3 (C-40), 112.2 (C-30), 115.3 (C-50), 121.8 (C-20), 154.8 (C-5), 158.4
(C-2), 126.8, 127.5, 128.1, 129.4, 130.3, 130.9, 132.5, 136.4 (aromatic
carbons) ppm; Anal. Calcd. for C20H18N4O6S4: C, 44.60; H, 3.37; N,
10.40; Found: C, 44.57; H, 3.38; N, 10.47%.
6.1.4.52-(p-Methylphenylaminosulfonylmethyl)-5-(40-p-methylpheny
lsulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,3,4-thiadiazole
6.1.4. General procedure for the synthesis of 2-(arylaminosulfonyl
methyl)-5-(40-arylsulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,3,4-ox
adiazole 6aec/2-(arylaminosulfonylmethyl)-5-(40-arylsulfonyl-10H-
pyrrol-30-ylsulfonylmethyl)-1,3,4-thiadiazole 7aec/4-amino-3-(aryl
aminosulfonylmethyl)-5-(40-arylsulfonyl-10H-pyrrol-30-ylsulfonyl
methyl)-1,2,4-triazole 8aec
(7b). White solid (0.36 g, 65%); m.p. 175e177 ꢁC; IR (KBr): 1133,
1322 (SO2), 1583 (C]N), 1622 (C]C), 3237 (NH) cmꢀ1 1H NMR
;
(DMSO-d6):
d 2.28 & 2.32 (s, 6H, AreCH3), 5.12 (s, 2H, SO2eCH2),
0
5.26 (s, 2H, CH2eSO2eNH), 6.68 (s, 1H, C5 eH), 7.08e7.86 (m, 9H,
C2 eH & AreH), 8.32 (bs, 1H, NH), 10.29 (bs, 1H, NHeSO2) ppm; 13C
0
Compound 3/4/5 (1 mmol) and TosMIC (1 mmol) in Et2OeDMSO
(2:1) were added dropwise under stirring to a suspension of NaH
(50 mg) in Et2O (20 ml) at room temperature and stirring was
continued for 6e8 h. Then, water was added and the reaction mass
was extracted with Et2O. The ethereal layer was dried over anhy-
drous Na2SO4 and filtered. Evaporation of the solvent in vacuo gave
a crude product which was purified by column chromatography
using silica gel (hexaneeethyl acetate; 4:1).
NMR (DMSO-d6): d 20.6 & 21.8 (AreCH3), 44.6 (SO2eCH2), 50.9
(CH2eSO2eNH), 103.8 (C-40), 111.5 (C-30), 112.4 (C-50), 120.5 (C-20),
154.6 (C-5), 158.3 (C-2), 126.5, 127.4, 128.1, 128.9, 131.2, 132.4, 133.5,
136.7 (aromatic carbons) ppm; Anal. Calcd. for C22H22N4O6S4: C,
46.63; H, 3.91; N, 9.89; Found: C, 46.68; H, 3.90; N, 9.84%.
6.1.4.62-(p-Chlorophenylaminosulfonylmethyl)-5-(4-p-chlorophenyl
sulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,3,4-thiadiazole
(7c). White solid (0.44 g, 73%); m.p. 192e194 ꢁC; IR (KBr): 1143,
6.1.4.12-(Phenylaminosulfonylmethyl)-5-(40-phenylsulfonyl-10H-pyrr
ol-30-ylsulfonyl-methyl)-1,3,4-oxadiazole (6a). White solid (0.35 g,
67%); m.p. 140e142 ꢁC; IR (KBr): 1141, 1335 (SO2), 1581 (C]N), 1631
1331 (SO2), 1594 (C]N), 1631 (C]C), 3245 (NH) cmꢀ1 1H NMR
;
(DMSO-d6): d 5.16 (s, 2H, SO2eCH2), 5.33 (s, 2H, CH2eSO2eNH), 6.71
0
0
(s, 1H, C5 eH), 7.13e7.78 (m, 9H, C2 eH & AreH), 8.36 (bs, 1H, NH),
(C]C), 3250 (NH) cmꢀ1
;
1H NMR (DMSO-d6):
d
5.08 (s, 2H,
10.33 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d 46.8
0
SO2eCH2), 5.38 (s, 2H, CH2eSO2eNH), 6.59 (s,1H, C5 eH), 7.22e7.81
(SO2eCH2), 52.1 (CH2eSO2eNH), 105.2 (C-40), 112.9 (C-30), 116.7 (C-
50), 122.7 (C-20), 155.0 (C-5), 159.8 (C-2), 127.1, 128.4, 129.6, 130.2,
130.8, 132.2, 133.8, 136.2 (aromatic carbons) ppm; Anal. Calcd. for
C20H16Cl2N4O6S4: C, 39.54; H, 2.65; N, 9.22; Found: C, 39.50; H,
2.62; N, 9.26%.
0
(m, 11H, C2 eH & AreH), 8.35 (bs, 1H, NH), 10.40 (bs, 1H, NHeSO2)
ppm; 13C NMR (DMSO-d6):
d 45.6 (SO2eCH2), 50.8 (CH2eSO2eNH),
106.2 (C-40), 111.8 (C-30), 117.2 (C-50), 122.6 (C-20), 155.2 (C-5), 158.7
(C-2), 126.4, 127.1, 127.6, 128.2, 129.2, 130.4, 132.8, 135.2 (aromatic
carbons) ppm; Anal. Calcd. for C20H18N4O7S3: C, 45.97; H, 3.47; N,
10.72; Found: C, 46.00; H, 3.50; N, 10.67%.
6.1.4.74-Amino-3-(phenylaminosulfonylmethyl)-5-(40-phenylsulfon
yl-10H-pyrrol-30-ylsulfonylmethyl)-1,2,4-triazole (8a). White solid
(0.39 g, 74%); m.p. 179e181 ꢁC; IR (KBr): 1135, 1334 (SO2), 1596 (C]
6.1.4.22-(p-Methylphenylaminosulfonylmethyl)-5-(40-p-methylphen
ylsulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,3,4-oxadiazole
(6b)
N), 1630 (C]C), 3266 (NH), 3318, 3445 (NH2) cmꢀ1 1H NMR
;
. White solid (0.35 g, 65%); m.p. 153e155 ꢁC; IR (KBr): 1136, 1327
(DMSO-d6): d 5.11 (s, 2H, SO2eCH2), 5.37 (s, 2H, CH2eSO2eNH), 5.50
(SO2), 1577 (C]N), 1628 (C]C), 3252 (NH) cmꢀ1; 1H NMR (DMSO-
(bs, 2H, NH2), 6.73 (s, 1H, C5 eH), 7.15e7.80 (m, 11H, C2 eH & AreH),
0
0
d6): d 2.23 & 2.28 (s, 6H, AreCH3), 5.16 (s, 2H, SO2eCH2), 5.37 (s, 2H,
8.37 (bs, 1H, NH), 10.35 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-
d
47.0 (SO2eCH2), 52.8 (CH2eSO2eNH), 105.7 (C-40), 108.3 (C-
0
0
CH2eSO2eNH), 6.53 (s, 1H, C5 eH), 7.19e7.74 (m, 9H, C2 eH &
d6):
30), 114.3 (C-50), 123.4 (C-20), 155.6 (C-5), 158.7 (C-3), 127.1, 128.4,
AreH), 8.38 (bs, 1H, NH), 10.38 (bs, 1H, NHeSO2) ppm; 13C NMR

A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
611
128.9, 129.5, 131.7, 132.4, 133.3, 137.5 (aromatic carbons) ppm; Anal.
Calcd. for C20H20N6O6S3: C, 44.77; H, 3.76; N,15.66; Found: C, 44.72;
H, 3.77; N, 15.75%.
4.92 (s, 2H, SO2eCH2), 5.26 (s, 2H, CH2eSO2eNH), 7.10e7.72 (m,
8H, AreH), 9.98 (bs, 1H, NH), 10.33 (bs, 1H, NHeSO2) ppm; 13C
NMR (DMSO-d6):
d 20.6 & 21.7 (AreCH3), 46.5 (SO2eCH2), 52.4
(CH2eSO2eNH), 52.8 (C-50), 68.5 (C-40), 152.7 (C-30), 156.5 (C-5),
158.7 (C-2), 126.7, 127.4, 128.1, 128.7, 129.3, 130.4, 132.6, 134.8
(aromatic carbons) ppm; Anal. Calcd. for C21H23N5O7S3: C, 45.56;
H, 4.19; N, 12.65; Found: C, 45.52; H, 4.18; N, 12.72%.
6.1.4.84-Amino-3-(p-methylphenylaminosulfonylmethyl)-5-(40-p-met
hylphenylsulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,2,4-triazole
(8b). White solid (0.42 g, 76%); m.p. 187e189 ꢁC; IR (KBr): 1132,
1326 (SO2), 1583 (C]N), 1627 (C]C), 3262 (NH), 3312, 3437 (NH2)
cmꢀ1; 1H NMR (DMSO-d6):
d
2.25 & 2.30 (s, 6H, AreCH3), 5.16 (s, 2H,
6.1.5.32-(p-Chlorophenylaminosulfonylmethyl)-5-(30-p-chlorophenyl
sulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-
oxadiazole (9c). White solid (2.11 g, 71%); m.p.166e168 ꢁC; IR (KBr):
1148,1342 (SO2),1597 (C]N), 3264 (NH) cmꢀ1; 1H NMR (DMSO-d6):
SO2eCH2), 5.33 (s, 2H, CH2eSO2eNH), 5.52 (bs, 2H, NH2), 6.68 (s,
0
0
1H, C5 eH), 7.11e7.76 (m, 9H, C2 eH & AreH), 8.29 (bs, 1H, NH),
10.38 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d
21.4 & 22.7
(AreCH3), 46.6 (SO2eCH2), 52.2 (CH2eSO2eNH), 104.3 (C-40), 107.5
(C-30), 113.8 (C-50), 121.1 (C-20), 154.2 (C-5), 158.5 (C-3), 127.4, 128.1,
128.6,129.2, 130.0,131.2,133.4,135.8 (aromatic carbons) ppm; Anal.
Calcd. for C22H24N6O6S3: C, 46.80; H, 4.28; N, 14.88; Found: C,
46.86; H, 4.30; N, 14.94%.
d
3.69 (dd, 1H, Hx, JAx ¼ 6.5 Hz, JMx ¼ 10.7 Hz), 4.27 (dd, 1H, HM,
JAM ¼ 12.5 Hz), 4.62 (dd, 1H, HA), 5.00 (s, 2H, SO2eCH2), 5.27 (s, 2H,
CH2eSO2eNH), 7.18e7.81 (m, 8H, AreH), 10.08 (bs, 1H, NH), 10.45
(bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d 47.7 (SO2eCH2), 52.1
(CH2eSO2eNH), 53.5 (C-50), 67.4 (C-40), 153.6 (C-30), 158.2 (C-5),
159.5 (C-2), 127.1, 127.7, 128.3, 129.4, 130.8, 131.6, 132.4, 135.6
(aromatic carbons) ppm; Anal. Calcd. for C19H17Cl2N5O7S3: C, 38.39;
H, 2.88; N, 11.78; Found: C, 38.43; H, 3.00; N, 11.75%.
6.1.4.9. 4-Amino-3-(p-chlorophenylaminosulfonylmethyl)-5-(40-p-ch
lorophenylsulfonyl-10H-pyrrol-30-ylsulfonylmethyl)-1,2,4-triazole
(8c). White solid (0.43 g, 72%); m.p. 206e208 ꢁC; IR (KBr): 1139,
1338 (SO2), 1598 (C]N), 1634 (C]C), 3272 (NH), 3327, 3448 (NH2)
6.1.5.42-(Phenylaminosulfonylmethyl)-5-(30-phenylsulfonyl-40,50-dihy
dro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thiadiazole (10a). White
solid (1.86 g, 69%); m.p.182e184 ꢁC; IR (KBr): 1128, 1327 (SO2), 1602
cmꢀ1
;
1H NMR (DMSO-d6):
d
5.14 (s, 2H, SO2eCH2), 5.39 (s, 2H,
0
CH2eSO2eNH), 5.54 (bs, 2H, NH2), 6.79 (s, 1H, C5 eH), 7.17e7.84 (m,
9H, C2 eH & AreH), 8.33 (bs, 1H, NH), 10.42 (bs, 1H, NHeSO2) ppm;
(C]N), 3268 (NH) cmꢀ1 1H NMR (DMSO-d6):
; d 3.58 (dd, 1H, Hx,
0
13C NMR (DMSO-d6):
d
48.2 (SO2eCH2), 53.0 (CH2eSO2eNH), 106.2
JAx ¼ 5.8 Hz, JMx ¼ 9.9 Hz), 4.29 (dd, 1H, HM, JAM ¼ 11.7 Hz), 4.64 (dd,
1H, HA), 4.95 (s, 2H, SO2eCH2), 5.24 (s, 2H, CH2eSO2eNH),
7.24e7.71 (m, 10H, AreH), 9.95 (bs, 1H, NH), 10.42 (bs, 1H,
(C-40), 109.2 (C-30), 116.2 (C-50), 124.0 (C-20), 155.9 (C-5), 159.1 (C-3),
127.3, 127.9, 128.5, 129.4, 130.7, 131.8, 132.5, 136.6 (aromatic
carbons) ppm; Anal. Calcd. for C20H18Cl2N6O6S3: C, 39.67; H, 3.00;
N, 13.88; Found: C, 39.72; H, 3.01; N, 13.84%.
NHeSO2) ppm; 13C NMR (DMSO-d6):
d 48.3 (SO2eCH2), 51.6
(CH2eSO2eNH), 52.7 (C-50), 65.2 (C-40), 152.8 (C-30), 156.4 (C-5),
158.4 (C-2), 126.1, 127.2, 127.8, 128.7, 129.9, 132.1, 134.7, 137.3
(aromatic carbons) ppm; Anal. Calcd. for C19H19N5O6S4: C, 42.13; H,
3.54; N, 12.93; Found: C, 42.18; H, 3.58; N, 13.00%.
6.1.5. General procedure for the synthesis of 2-(arylaminosulfonylm
ethyl)-5-(30-arylsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylme
thyl)-1,3,4-oxadiazole 9aec/2-(arylaminosulfonyl-methyl)-5-(30-ar
ylsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thi
adiazole 10aec/4-amino-3-(arylaminosulfonylmethyl)-5-(30-aryl
sulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,2,4-
triazole 11aec
6.1.5.52-(p-Methylphenylaminosulfonylmethyl)-5-(30-p-methylpheny
lsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-
thiadiazole (10b). White solid (1.90 g, 67%); m.p. 187e189 ꢁC; IR
(KBr): 1124, 1322 (SO2), 1589 (C]N), 3262 (NH) cmꢀ1 1H NMR
;
To a cooled solution of compound 3/4/5 (5 mmol) in dichloro-
methane (20 ml), an ethereal solution of diazomethane (40 ml,
0.4 M) and triethylamine (0.12 g) were added. The reaction mixture
was kept at ꢀ20 to ꢀ15 ꢁC for 48 h. The solvent was removed
under reduced pressure and the resultant residue was passed
through a column of silica gel using hexane and ethyl acetate (3:1)
as eluent.
(DMSO-d6): d 2.28 & 2.33 (s, 6H, AreCH3), 3.50 (dd, 1H, Hx,
JAx ¼ 5.9 Hz, JMx ¼ 9.8 Hz), 4.19 (dd, 1H, HM, JAM ¼ 11.5 Hz), 4.58 (dd,
1H, HA), 4.93 (s, 2H, SO2eCH2), 5.36 (s, 2H, CH2eSO2eNH),
7.18e7.68 (m, 8H, AreH), 9.90 (bs, 1H, NH), 10.39 (bs, 1H,
NHeSO2) ppm; 13C NMR (DMSO-d6):
d 21.1 & 21.8 (AreCH3), 47.6
(SO2eCH2), 50.5 (CH2eSO2eNH), 52.3 (C-50), 64.3 (C-40), 152.4 (C-
30), 156.2 (C-5), 157.9 (C-2), 126.3, 128.1, 128.9, 129.5, 131.4, 133.2,
134.3,136.4 (aromatic carbons) ppm; Anal. Calcd. for C21H23N5O6S4:
C, 44.27; H, 4.07; N, 12.29; Found: C, 44.34; H, 4.04; N, 12.35%.
6.1.5.1. 2-(Phenylaminosulfonylmethyl)-5-(30-phenylsulfonyl-40,50-di
hydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-oxadiazole
(9a). White solid (1.83 g, 70%); m.p. 152e154 ꢁC; IR (KBr): 1141,
6.1.5.62-(p-Chlorophenylaminosulfonylmethyl)-5-(30-p-chloropheny
lsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-
thiadiazole (10c). White solid (2.22 g, 73%); m.p. 198e200 ꢁC; IR
1335 (SO2), 1586 (C]N), 3253 (NH) cmꢀ1 1H NMR (DMSO-d6):
;
d
3.57 (dd, 1H, Hx, JAx ¼ 6.3 Hz, JMx ¼ 10.8 Hz), 4.24 (dd, 1H, HM,
JAM ¼ 12.1 Hz), 4.60 (dd, 1H, HA), 4.99 (s, 2H, SO2eCH2), 5.31 (s, 2H,
(KBr): 1131, 1334 (SO2), 1606 (C]N), 3276 (NH) cmꢀ1 1H NMR
;
CH2eSO2eNH), 7.17e7.79 (m, 10H, AreH), 10.03 (bs, 1H, NH), 10.35
(DMSO-d6):
d
3.55 (dd, 1H, Hx, JAx ¼ 6.0 Hz, JMx ¼ 10.2 Hz), 4.24 (dd,
(bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d
47.1 (SO2eCH2), 51.6
1H, HM, JAM ¼ 11.8 Hz), 4.67 (dd, 1H, HA), 4.98 (s, 2H, SO2eCH2), 5.28
(s, 2H, CH2eSO2eNH), 7.25e7.80 (m, 8H, AreH), 9.98 (bs, 1H, NH),
(CH2eSO2eNH), 53.2 (C-50), 67.1 (C-40), 153.2 (C-30), 157.6 (C-5),
159.2 (C-2), 127.4, 128.8, 129.6, 130.9, 131.7, 132.5, 133.4, 134.9
(aromatic carbons) ppm; Anal. Calcd. for C19H19N5O7S3: C, 43.42; H,
3.64; N, 13.33; Found: C, 43.48; H, 3.66; N, 13.39%.
10.44 (bs,1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d 49.6 (SO2eCH2),
52.8 (CH2eSO2eNH), 53.9 (C-50), 66.7 (C-40),154.1 (C-30),157.9 (C-5),
160.5 (C-2), 127.8, 128.3, 130.2, 131.6, 132.4, 133.4, 136.2, 137.7
(aromatic carbons) ppm; Anal. Calcd. for C19H17Cl2N5O6S4: C, 37.38;
H, 2.81; N, 11.47; Found: C, 37.42; H, 2.82; N, 11.42%.
6.1.5.22-(p-Methylphenylaminosulfonylmethyl)-5-(30-p-methylphen
ylsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-oxadi
azole (9b). White solid (1.82 g, 66%); m.p. 159e161 ꢁC; IR (KBr):
6.1.5.7. 4-Amino-3-(phenylaminosulfonylmethyl)-5-(30-phenylsulfon
yl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-1,2,4-triazole
(11a). White solid (1.94 g, 72%); m.p. 184e186 ꢁC; IR (KBr): 1135,
1314 (SO2),1610 (C]N), 3251 (NH), 3372, 3485 (NH2) cmꢀ1; 1H NMR
1136, 1331 (SO2), 1588 (C]N), 3256 (NH) cmꢀ1 1H NMR (DMSO-
;
d6):
d
2.27 & 2.32 (s, 6H, AreCH3), 3.55 (dd, 1H, Hx, JAx ¼ 6.2 Hz,
JMx ¼ 10.3 Hz), 4.15 (dd, 1H, HM, JAM ¼ 12.0 Hz), 4.53 (dd, 1H, HA),

612
A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
(DMSO-d6):
d
3.52 (dd, 1H, Hx, JAx ¼ 6.2 Hz, JMx ¼ 10.4 Hz), 4.21 (dd,
(DMSO-d6): d 2.23 & 2.28 (s, 6H, AreCH3), 5.07 (s, 2H, SO2eCH2),
1H, HM, JAM ¼ 12.3 Hz), 4.64 (dd,1H, HA), 5.07 (s, 2H, SO2eCH2), 5.29
(s, 2H, CH2eSO2eNH), 5.72 (bs, 2H, NH2), 7.20e7.83 (m, 10H, AreH),
9.87 (bs,1H, NH),10.26 (bs,1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
5.32 (s, 2H, CH2eSO2eNH), 6.42 (bs, 1H, NH), 6.84e7.51 (m, 9H,
C5 eH & AreH), 10.32 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
0
d
21.6 & 22.4 (AreCH3), 49.8 (SO2eCH2), 53.9 (CH2eSO2eNH), 133.8
d
46.9 (SO2eCH2), 51.7 (CH2eSO2eNH), 53.1 (C-50), 66.8 (C-40),153.3
(C-40), 139.6 (C-50), 148.6 (C-30), 158.7 (C-5), 161.4 (C-2), 127.8, 128.4,
129.8, 131.6, 132.7, 133.5, 135.8, 136.9 (aromatic carbons) ppm; Anal.
Calcd. for C21H21N5O7S3: C, 45.72; H, 3.84; N,12.70; Found: C, 45.70;
H, 3.85; N, 12.64%.
(C-30), 158.6 (C-5), 159.5 (C-3), 127.1, 128.2, 129.3, 130.7, 131.9, 133.5,
135.3, 137.2 (aromatic carbons) ppm; Anal. Calcd. for C19H21N7O6S3:
C, 42.29; H, 3.92; N, 18.17; Found: C, 42.25; H, 3.90; N, 18.37%.
6.1.5.84-Amino-3-(p-methylphenylaminosulfonylmethyl)-5-(30-p-met
hylphenylsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-
1,2,4-triazole (11b). White solid (1.90 g, 67%); m.p. 197e199 ꢁC; IR
(KBr): 1131, 1308 (SO2), 1603 (C]N), 3252 (NH), 3364, 3481 (NH2)
6.1.6.32-(p-Chlorophenylaminosulfonylmethyl)-5-(30-p-chlorophen
ylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-oxadiazole
(12c). White solid (0.43 g, 73%); m.p. 219e221 ꢁC; IR (KBr): 1135,
1334 (SO2), 1593 (C]N), 1642 (C]C), 3278 (NH) cmꢀ1 1H NMR
;
cmꢀ1; 1H NMR (DMSO-d6):
d
2.21 & 2.26 (s, 6H, AreCH3), 3.57 (dd,
(DMSO-d6):
6.46 (bs, 1H, NH), 7.01e7.66 (m, 9H, C5 eH & AreH), 10.37 (bs, 1H,
NHeSO2) ppm; 13C NMR (DMSO-d6):
50.7 (SO2eCH2), 54.8
d 5.10 (s, 2H, SO2eCH2), 5.38 (s, 2H, CH2eSO2eNH),
0
1H, Hx, JAx ¼ 6.3 Hz, JMx ¼ 10.3 Hz), 4.20 (dd, 1H, HM, JAM ¼ 12.1 Hz),
4.62 (dd, 1H, HA), 4.99 (s, 2H, SO2eCH2), 5.24 (s, 2H, CH2eSO2eNH),
5.64 (bs, 2H, NH2), 7.16e7.79 (m, 8H, AreH), 9.89 (bs, 1H, NH), 10.24
d
(CH2eSO2eNH), 135.1 (C-40), 141.2 (C-50), 150.4 (C-30), 160.3 (C-5),
162.9 (C-2), 127.8, 128.2, 129.3, 130.6, 131.8, 132.4, 134.6, 137.8
(aromatic carbons) ppm; Anal. Calcd. for C19H15Cl2N5O7S3: C, 38.52;
H, 2.55; N, 11.82; Found: C, 38.58; H, 2.53; N, 11.88%.
(bs,1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
d 21.1 & 22.5 (AreCH3),
46.1 (SO2eCH2), 51.5 (CH2eSO2eNH), 52.8 (C-50), 66.4 (C-40), 152.9
(C-30), 158.1 (C-5), 159.7 (C-3), 128.1, 128.9, 129.8, 131.2, 132.7, 133.4,
135.2, 137.9 (aromatic carbons) ppm; Anal. Calcd. for C21H25N7O6S3:
C, 44.43; H, 4.44; N, 17.27; Found: C, 44.49; H, 4.48; N, 17.34%.
6.1.6.42-(Phenylaminosulfonylmethyl)-5-(30-phenylsulfonyl-10H-pyr-
azol-40-ylsulfonyl-methyl)-1,3,4-thiadiazole
(13a). White
(0.37 g, 69%); m.p. 232e234 ꢁC; IR (KBr): 1129, 1342 (SO2), 1584
(C]N), 1641 (C]C), 3254 (NH) cmꢀ1; 1H NMR (DMSO-d6):
5.11 (s,
solid
6.1.5.94-Amino-3-(p-chlorophenylaminosulfonylmethyl)-5-(30-p-chlo
rophenylsulfonyl-40,50-dihydro-10H-pyrazol-40-ylsulfonylmethyl)-
1,2,4-triazole (11c). White solid (2.28 g, 75%); m.p. 218e220 ꢁC; IR
(KBr): 1136, 1321 (SO2), 1608 (C]N), 3272 (NH), 3376, 3492 (NH2)
d
2H, SO2eCH2), 5.33 (s, 2H, CH2eSO2eNH), 6.53 (bs, 1H, NH),
6.99e7.62 (m,11H, C5 eH & AreH),10.42 (bs,1H, NHeSO2) ppm; 13C
0
cmꢀ1
;
1H NMR (DMSO-d6):
d
3.53 (dd, 1H, Hx, JAx ¼ 6.1 Hz,
NMR (DMSO-d6): d 49.6 (SO2eCH2), 53.5 (CH2eSO2eNH), 133.5 (C-
JMx ¼ 10.5 Hz), 4.29 (dd, 1H, HM, JAM ¼ 12.3 Hz), 4.67 (dd, 1H, HA),
5.18 (s, 2H, SO2eCH2), 5.32 (s, 2H, CH2eSO2eNH), 5.70 (bs, 2H,
NH2), 7.22e7.69 (m, 8H, AreH), 9.84 (bs, 1H, NH), 10.28 (bs, 1H,
40), 138.7 (C-50), 149.4 (C-30), 158.5 (C-5), 161.3 (C-2), 126.4, 127.5,
128.8, 129.5, 131.6, 132.8, 135.1, 138.2 (aromatic carbons) ppm; Anal.
Calcd. for C19H17N5O6S4: C, 42.29; H, 3.18; N, 12.98; Found: C, 42.36;
H, 3.20; N, 12.94%.
NHeSO2) ppm; 13C NMR (DMSO-d6):
d 47.0 (SO2eCH2), 51.9
(CH2eSO2eNH), 53.6 (C-50), 67.2 (C-40), 154.1 (C-30), 158.5 (C-5),
159.3 (C-3), 127.8, 129.1, 129.9, 130.7, 131.5, 133.4, 135.2, 138.1
(aromatic carbons) ppm; Anal. Calcd. for C19H19Cl2N7O6S3: C, 37.50;
H, 3.15; N, 16.11; Found: C, 37.55; H, 3.18; N, 16.20%.
6.1.6.52-(p-Methylphenylaminosulfonylmethyl)-5-(30-p-methylphen
ylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thiadiazole
(13b). White solid (0.40 g, 71%); m.p. 240e242 ꢁC; IR (KBr): 1126,
1335 (SO2), 1589 (C]N), 1636 (C]C), 3251 (NH) cmꢀ1 1H NMR
;
6.1.6. General procedure for the synthesis of 2-(arylaminosulfonylm
ethyl)-5-(30-arylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-ox
adiazole 12aec/2-(arylaminosulfonylmethyl)-5-(30-arylsulfonyl-
10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thiadiazole 13aec/4-amino-
3-(arylaminosulfonylmethyl)-5-(30-arylsulfonyl-10H-pyrazol-40-ylsu
lfonylmethyl)-1,2,4-triazole 14aec
A mixture of 9/10/11 (1 mmol) and chloranil (1.4 mmol) in
xylene (10 ml) was refluxed for 20e23 h. Then, the reaction mixture
was treated with a 5% NaOH solution. The organic layer was sepa-
rated and repeatedly washed with water. It was dried over anhy-
drous Na2SO4 and the solvent was removed on a rotary evaporator.
The resultant solid was recrystallized from methanol.
(DMSO-d6): d 2.23 & 2.27 (s, 6H, AreCH3), 5.13 (s, 2H, SO2eCH2),
5.36 (s, 2H, CH2eSO2eNH), 6.59 (bs, 1H, NH), 6.97e7.59 (m, 9H,
C5 eH & AreH), 10.38 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
0
d
21.0 & 21.9 (AreCH3), 48.5 (SO2eCH2), 52.8 (CH2eSO2eNH), 132.8
(C-40), 137.9 (C-50), 148.6 (C-30), 157.6 (C-5), 159.8 (C-2), 126.6, 127.2,
128.1, 128.7, 130.1, 131.5, 133.2, 137.4 (aromatic carbons) ppm; Anal.
Calcd. for C21H21N5O6S4: C, 44.43; H, 3.73; N,12.34; Found: C, 44.48;
H, 3.76; N, 12.40%.
6.1.6.62-(p-Chlorophenylaminosulfonylmethyl)-5-(30-p-chlorophen
ylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-thiadiazole
(13c). White solid (0.44 g, 73%); m.p. 247e249 ꢁC; IR (KBr): 1134,
1344 (SO2), 1596 (C]N), 1644 (C]C), 3267 (NH) cmꢀ1 1H NMR
;
6.1.6.12-(Phenylaminosulfonylmethyl)-5-(30-phenylsulfonyl-10H-pyr-
azol-40-ylsulfonyl-methyl)-1,3,4-oxadiazole (12a). White solid (0.34 g,
66%); m.p. 200e202 ꢁC; IR (KBr): 1141, 1327 (SO2), 1596 (C]N), 1638
(DMSO-d6):
6.55 (bs, 1H, NH), 7.04e7.62 (m, 9H, C5 eH & AreH), 10.45 (bs, 1H,
NHeSO2) ppm; 13C NMR (DMSO-d6):
49.7 (SO2eCH2), 54.1
d 5.17 (s, 2H, SO2eCH2), 5.40 (s, 2H, CH2eSO2eNH),
0
d
(C]C), 3281 (NH) cmꢀ1; 1H NMR (DMSO-d6):
d
5.09 (s, 2H, SO2eCH2),
(CH2eSO2eNH), 133.9 (C-40), 139.2 (C-50), 149.2 (C-30), 158.8 (C-5),
161.7 (C-2), 128.1, 128.9, 131.5, 132.6, 133.3, 134.1, 137.3, 137.9
(aromatic carbons) ppm; Anal. Calcd. for C19H15Cl2N5O6S4: C, 37.50;
H, 2.48; N, 11.51; Found: C, 37.44; H, 2.46; N, 11.48%.
5.26 (s, 2H, CH2eSO2eNH), 6.47 (bs, 1H, NH), 6.92e7.57 (m, 11H,
C5 eH & AreH), 10.35 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-d6):
0
d
50.2 (SO2eCH2), 54.5 (CH2eSO2eNH), 134.6 (C-40), 140.2 (C-50), 149.4
(C-30), 159.3 (C-5), 162.3 (C-2), 126.5, 127.4, 128.3, 129.7, 131.6, 132.8,
135.9, 138.5 (aromatic carbons) ppm; Anal. Calcd. for C19H17N5O7S3: C,
43.59; H, 3.27; N, 13.38; Found: C, 43.63; H, 3.25; N, 13.44%.
6.1.6.74-Amino-3-(phenylaminosulfonylmethyl)-5-(30-phenylsulfo
nyl-10H-pyrazol-40-ylsulfonylmethyl)-1,2,4-triazole
(14a). White
solid (0.39 g, 74%); m.p. 234e236 ꢁC; IR (KBr): 1140, 1330 (SO2),
1611 (C]N), 1638 (C]C), 3265 (NH), 3335, 3458 (NH2) cmꢀ1 1H
NMR (DMSO-d6): 5.08 (s, 2H, SO2eCH2), 5.34 (s, 2H,
6.1.6.22-(p-Methylphenylaminosulfonylmethyl)-5-(30-p-methylpheny
lsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,3,4-oxadiazole
;
d
(12b). White solid (0.37 g, 68%); m.p. 211e213 ꢁC; IR (KBr): 1134,
CH2eSO2eNH), 5.68 (bs, 2H, NH2), 6.53 (bs, 1H, NH), 7.09e7.76 (m,
1319 (SO2), 1587 (C]N), 1636 (C]C), 3275 (NH) cmꢀ1
;
1H NMR
11H, C5 eH & AreH), 10.31 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-
0

A. Muralikrishna et al. / European Journal of Medicinal Chemistry 54 (2012) 605e614
613
d6):
d
48.8 (SO2eCH2), 51.6 (CH2eSO2eNH), 133.6 (C-40), 139.5 (C-
diluted 100 folds in nutrient broth (100
m
l bacterial cultures in
50), 148.5 (C-30), 158.7 (C-5), 160.1 (C-3), 127.1, 128.3, 129.0, 129.8,
131.4, 132.5, 133.6, 136.4 (aromatic carbons) ppm; Anal. Calcd. for
C19H19N7O6S3: C, 42.45; H, 3.56; N, 18.24; Found: C, 42.50; H, 3.59;
N, 18.32%.
10 ml NB). Increasing concentrations of the test samples (6.25,
.200 g) were added to the test tubes containing the bacterial and
m
fungal cultures. All the tubes were incubated at 37 ꢁC for 24 h for
bacteria and at 28 ꢁC 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.
6.1.6.84-Amino-3-(p-methylphenylaminosulfonylmethyl)-5-(30-p-m
ethylphenylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,2,4-triazole
(14b). White solid (0.39 g, 69%); m.p. 254e256 ꢁC; IR (KBr): 1133,
1326 (SO2), 1603 (C]N), 1629 (C]C), 3257 (NH), 3329, 3463 (NH2)
To determine the Minimum Bactericidal Concentration (MBC)
[24] and Minimum Fungicidal Concentration (MFC) [25] 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 ꢁC for 24 h and at 28 ꢁC for 48 h, respectively. After
incubation, that concentration was noted as MBC (for bacteria) or
MFC (for fungi) at which no visible growth was observed.
cmꢀ1; 1H NMR (DMSO-d6):
d 2.25 & 2.30 (s, 6H, AreCH3), 5.10 (s, 2H,
SO2eCH2), 5.32 (s, 2H, CH2eSO2eNH), 5.65 (bs, 2H, NH2), 6.52 (bs,
0
1H, NH), 7.11e7.81 (m, 9H, C5 eH & AreH), 10.34 (bs, 1H, NHeSO2)
ppm; 13C NMR (DMSO-d6):
d 20.4 & 21.6 (AreCH3), 47.9 (SO2eCH2),
51.8 (CH2eSO2eNH), 133.0 (C-40), 139.4 (C-50), 147.6 (C-30), 157.9 (C-
5), 159.7 (C-3), 128.4, 129.1, 129.9, 131.2, 132.6, 133.2, 135.3, 136.7
(aromatic carbons) ppm; Anal. Calcd. for C21H23N7O6S3: C, 44.59; H,
4.10; N, 17.33; Found: C, 44.55; H, 4.09; N, 17.39%.
6.1.6.94-Amino-3-(p-chlorophenylaminosulfonylmethyl)-5-(30-p-chlo
rophenylsulfonyl-10H-pyrazol-40-ylsulfonylmethyl)-1,2,4-triazole
(14c). White solid (0.47 g, 78%); m.p. 260e262 ꢁC; IR (KBr): 1144,
1337 (SO2), 1614 (C]N), 1636 (C]C), 3262 (NH), 3341, 3476 (NH2)
6.2.4. MTT assay for cell viability
The cytotoxicity of the compounds was tested using A549 lung
carcinoma cells. 5 ꢂ104 cells were plated in each well of a 96-well
tissue culture cluster (Nunc Inc Germany) and incubated at 37 ꢁC in
a medium containing DMEM, 10% foetal bovine serum and antibi-
otics (Invitrogen, USA), in 5% CO2 atmosphere [26,27]. After
attachment of the cells (usually 3e4 h), different concentrations of
the compound were added and incubated for 72 h. MTT solution
cmꢀ1 1H NMR (DMSO-d6):
; d 5.15 (s, 2H, SO2eCH2), 5.39 (s, 2H,
CH2eSO2eNH), 5.72 (bs, 2H, NH2), 6.57 (bs, 1H, NH), 7.19e7.87 (m,
9H, C5 eH & AreH), 10.43 (bs, 1H, NHeSO2) ppm; 13C NMR (DMSO-
0
d6):
d
48.7 (SO2eCH2), 52.5 (Ch2eSO2eNH), 134.3 (C-40), 140.8(C-
50), 149.1 (C-30), 159.2 (C-5), 160.5 (C-3), 127.1, 128.4, 129.5, 131.4,
132.6, 133.8, 135.2, 137.8 (aromatic carbons) ppm; Anal. Calcd. for
C19H17Cl2N7O6S3: C, 37.63; H, 2.83; N, 16.17; Found: C, 37.70; H,
2.86; N, 16.14%.
(20 ml of 5 mg/ml) was added to each well and the incubation
continued for additional 3 h. The dark blue formazan crystals
formed within the healthy cells were solubilized with DMSO and
the absorbance was estimated in ELISA plate reader (7520 Micro-
plate reader, Cambridge technologies, Inc) at 550 nm and the
absorbance was correlated with the cell number. Experiments were
performed in triplicates and the values are the average of three
(n ¼ 3) independent experiments. The inhibitory concentration
(IC50) of the compound was assessed by Graph Pad Prism software.
6.2. Biological assays
6.2.1. Compounds
Compounds 3e14 were dissolved in DMSO at different
concentrations of 50 & 100 mg/ml.
Acknowledgements
6.2.2. Cells
Bacterial strains Staphylococcus aureus, B. subtilis, P. aeruginosa,
Klebsiella pneumoniae and fungi A. niger & P. chrysogenum were
obtained from Department of Microbiology, S.V University, Tirupati,
India.
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 A. Muralikrishna is thankful to
Council of Scientific and Industrial Research (CSIR), New Delhi,
India for the sanction of Senior Research Fellowship.
6.2.3. Antibacterial and antifungal assays
The in vitro antimicrobial studies were carried out by agar well
diffusion method against test organisms [20,21]. Nutrient broth
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