Synthesis, in vitro cytotoxicity, and anti-microbial studies of 1,4-bis(4-substituted-5-mercapto-1,2,4-triazol-3-yl)butanes

Article abstract of DOI:10.1007/s00044-010-9517-9

Synthesis and evaluation of cytotoxicity and anti-microbial activity of a series of 1,4-bis(4-substituted- 5-mercapto-1,2,4-triazol-3-yl)butane derivatives comprising thioether functionality and other pharmacophore modifications are described. All the new

Full text of DOI:10.1007/s00044-010-9517-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:174–184
DOI 10.1007/s00044-010-9517-9
ORIGINAL RESEARCH
Synthesis, in vitro cytotoxicity, and anti-microbial studies
of 1,4-bis(4-substituted-5-mercapto-1,2,4-triazol-3-yl)butanes
•
Madhusudan Purohit Yergeri C. Mayur
Received: 26 March 2010 / Accepted: 15 November 2010 / Published online: 8 December 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Synthesis and evaluation of cytotoxicity and
anti-microbial activity of a series of 1,4-bis(4-substituted-
5-mercapto-1,2,4-triazol-3-yl)butane derivatives compris-
ing thioether functionality and other pharmacophore
modifications are described. All the newly synthesized
compounds were characterized by IR, NMR, elemental
analyses, and mass spectral studies. The compounds 4a–f,
5a–f, and 6a–f were evaluated for in vitro cytotoxicity
potential using the standard MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay against a
panel of three human cancer cell lines: Lung carcinoma
A-549, Colon carcinoma HT-29, and Breast Cancer MDA
MB-231. All the compounds were subjected to in vitro anti-
bacterial activity against Bacillus subtilus (ATCC 6633),
Staphylococcus aureus (ATCC-25923), Escherichia coli
(ATCC-25922), and Pseudomonas aeruginosa (ATCC-
27853) and their minimal inhibitory concentrations were
determined.
Introduction
Over the last few years the increasing number of neoplastic
diseases together with the accompanied high mortality
rates has stimulated an unparallel level of research directed
towards the development of new lead molecules that might
be of use in designing novel anti-neoplastic agents.
Dimeric analogues of various heterocyclic compounds are
drawing much attention in the recent past. Many dimeric
compounds designed as bis-DNA intercalators were eval-
uated as anti-cancer agents. Dimers of more lipophilic
compounds have shown potent and broad spectrum activity
against human solid tumor cell lines both in culture and as
xenografts in nude mice. Some of the bis-intercalators were
found to possess high selective toxicity against human
colon carcinoma (Yong et al., 2005; Denny, 2003). In
addition to DNA intercalation, the bis-heterocyclic mole-
cules were also shown to exhibit such diverse pharma-
cological activity as anti-microbial (Holla et al., 1998),
anti-protozoal (Coro et al., 2005), anti-inflammatory
(Sondhi et al., 2007), anti-HIV (Pomarnacka and Kornicka,
2001), and cytotoxicity (Antonini et al., 2008; Brana et al.,
1997; Rewcastle et al., 1987; Gamage et al., 1999; Spicer
et al., 2000; Dabholkar and Ansari, 2008). Many of the bis-
1,2,4-triazoles have also been reported to possess wide
spectrum of biological activity (Holla et al., 2000; Holla
et al., 2002; Ghorab et al., 2000; Al-Soud and Al-Masoudi,
2004). The triazole derivatives vorozole, letrozole, and
anastrozole are non-steroidal drugs used for the treatment
of breast cancer (Clemons et al., 2004) (Fig. 1).
Keywords 1,2,4-Triazoles ꢀ Cytotoxicity ꢀ
Anti-microbial activity ꢀ MTT assay
M. Purohit
Department of Pharmaceutical Chemistry,
JSS College of Pharmacy, JSS University,
Mysore 570015, Karnataka, India
Y. C. Mayur
Medicinal Chemistry Research Division,
V.L. College of Pharmacy, Raichur, India
Based on the above literature, the authors have tried to
design the bis-triazole derivatives by incorporating various
modifications using groups such as diethyl carbamoyl,
thioester, and hydrazide functional groups. The incorpo-
ration of thioester moiety being attached to different
Y. C. Mayur (&)
Center for Drug Design, SVERI’s College of Pharmacy,
Pandharpur, India
e-mail: mayuryc@rediff.com; mayuryc@hotmail.com
123

Med Chem Res (2012) 21:174–184
175
Fig. 1 Clinically useful triazole
derivatives
N
CH3
CN
N
N
N
N
CH3
N
N
N
N
H3C
N
N
N
NC
Cl
CH3
CH3
Cl
CN
Anastrozole
Vorozole
Letrozole
heterocyclic systems such as 1,2,4-triazolo[4,3-a]quinoxa-
line (Gulerman et al., 2001; Badran et al., 2003) and 1,3,4-
oxadiazole has shown to enhance the anti-microbial
activity (Berghot, 2001). The well-known anti-filarial agent
diethylcarbamazine contains the diethylcarbamoyl moiety;
moreover, the fact that hydrazino derivatives are them-
selves capable of exerting anti-cancer activity by alkylation
of DNA through free radical intermediate (Remers, 2004).
These studies prompted the authors to synthesize some
more novel bis-triazole derivatives hooked with thioester,
diethylcarbamoyl, and hydrazine moieties and carry out
their evaluation as cytotoxic agents.
such type of infections continue to be the driving force for
the search and discovery of novel, more potent and selec-
tive non-traditional anti-microbial agents with the less
likeliness of development of cross-resistance.
Keeping these observations in mind and in continuation
of our study on the synthesis of bis-heterocyclic com-
pounds (Purohit et al., 2006); we report herein the syn-
thesis and in vitro cytotoxic activity and anti-bacterial
studies of certain novel bis-1,2,4-triazole derivatives.
Results and discussion
On the other hand, the effectiveness of commercially
available anti-microbials has become unreliable, due to the
emergence of resistant microorganisms like methicillin-
resistant Staphylococcus aureus (MRSA), chloroquine-
resistant Plasmodium falciparam, multi-drug-resistant
Mycobacterium tuberculosis, and vancomycin-resistant
Enterococcus faecium (VRE) (Rostom et al., 2009). Hence
Chemistry
Synthesis of the intermediate and target compounds was
accomplished according to the steps depicted in Scheme 1.
The bis-1,2,4 triazoles 3a–f, which served as the key inter-
mediates were prepared starting from adipoyldihydrazide 1
Scheme 1 Scheme of synthesis
N
N
N N
i)RNCS (2a-f)
(CH2)4
H2NHNOC (CH2)4 CONHNH2
HS
SH
N
R
N
R
ii) 2N NaOH
iii) 2N HCl
1
3a-f
(C2H5)2N-COCl
(C2H5)2N
N
N
N N
O
(CH2)4
O
3a-f
S
S
N
N
R
N(C2H5)2
4a-f
R
BrCH2COOC2H5/K2CO3
N
N
N N
R
(CH2)4
phenyl
a
b
c
d
H5C2OOCH2CS
SCH2COOC2H5
N
R
N
R
p-tolyl
5 a-f
m-tolyl
NH2NH2.H2O/Ethanol
p-ethoxy
phenyl
N
N
N
N
(CH2)4
cyclohexyl
n-butyl
e
f
H2NHNOCH2CS
SCH2CONHNH2
N
R
N
R
6 a-f
123

176
Med Chem Res (2012) 21:174–184
according to the previously reported reaction conditions
(Mahmoud Omar, 1997) using six different substituted
isothiocyanates namely phenyl, p-tolyl, m-tolyl, p-ethoxy-
phenyl, cyclohexyl, and n-butyl. Stirring 3a–f with N,N-
diethyl carbamoyl chloride in the presence of dry acetone
and anhydrous potassium carbonate at reflux temperature
yielded respective diethyl carbamoyl thio derivatives 4a–
f in moderate yields. Similarly, when 3a–f were reacted with
ethyl bromoacetate under similar reaction conditions as
above-yielded corresponding thioesters 5a–f in moderate to
good yield. Refluxing 5a–f with excess of hydrazine hydrate
in absolute ethanol afforded respective acid hydrazides 6a–
f in good yield. The structures of all the compounds were
elucidated on the basis of elemental analysis, IR, 1H-NMR,
13C-NMR, and mass spectral data.
The carbon atoms of –OCH2 and –SCH2 of the side chain
resonated at 61.20 and 33.97 ppm. The chemical shifts at
32.37 and 25.30 ppm were assigned to the methylene
carbons (C2 and C3) and (C1 and C4) of the butyl bridge.
The aliphatic methyl carbon of the ester resonated at
13.72 ppm.
1
The H-NMR spectrum of compound 6a showed a sin-
glet at 9.31 and 4.34 ppm which were assigned to –CONH
and NH2 of the acid hydrazide group. The absence of the
characteristic group signals of the ethoxy group of the ester
(a quartet at 4.1 ppm and a triplet at 0.9 ppm) also con-
firmed the formation of the acid hydrazide. The protons of
the methylene bridge appeared at 2.44 and 1.45 ppm. The
protons of –SCH2 group resonated as singlet at 3.8 ppm.
The multiplet signal between 7.3 and 7.6 ppm was attrib-
uted due to ten aromatic protons of the phenyl ring. The
13C-NMR spectrum of the compound 6a showed a signal at
d 166.11 ppm due to the –CONH functional group. The
signal at 155.18 and 149.41 ppm was attributed to the C3
and C5 of the triazole moiety. The chemical shifts at
132.94, 130.88, 129.94, and 127.18 were assigned to the
aromatic carbon atoms of the phenyl ring. The carbon atom
of –SCH2 of the side chain resonated at 35.06 ppm. The
chemical shifts at 34.24 and 25.33 ppm were assigned to
the methylene carbons (C2 and C3) and (C1 and C4) of the
butyl bridge. Similar explanation for assigning the carbon
holds good for the rest of the compounds. In conclusion,
1H-NMR and 13C-NMR spectral data were consistent with
the proposed structures. The mass spectra of all the triazole
derivatives were analyzed under ESI conditions. Molecular
ions were observed in the form of M ? H. Most of the
compounds yield abundant molecular ions in the form of
M ? H peaks. Similarly, the elemental analyses of all the
compounds have been performed and the data given under
the physical data in Table 1.
The IR spectra of compound 4a clearly showed the C=O
stretching band at 1675 cm-1, which is absent in the IR
spectrum of its starting compound 3a. Further, the
1H-NMR spectrum shows the signal due to –NCH2 protons
at 3.3 ppm as quartet and the terminal methyl protons
resonated at 1.0 ppm as triplet. The absence of tautomeric
form of mercapto proton signal at 12.3 ppm also supports
the formation of 4a. The protons of the methylene bridge
appeared at 2.45 and 1.54 ppm. The aromatic protons of
the phenyl ring were observed as multiplet between 7.2 and
7.5 ppm. In the 13C-NMR spectrum of the same compound,
the chemical shift was observed at 163.30 ppm, this was
assigned to the carbonyl carbon of the side chain. The
signals at 155.22 and 148.56 ppm were due to the C3 and
C5 of the triazole moiety. The signals at 43.72 and
13.09 ppm were assigned to carbon atom of the NCH2
and CH3 groups of the diethyl carbamoyl side chain,
respectively. The chemical shifts at 132.56, 129.89, 128.95,
and 127.15 were assigned to the aromatic carbon atoms of
the phenyl ring. The chemical shifts at 32.33 and
25.10 ppm were assigned to the methylene carbons (C2 and
C3) and (C1 and C4) of the butyl bridge, respectively.
The IR spectrum of compounds 5a–f showed C=O
stretching bands between 1685 and 1715 cm-1. The
1H-NMR spectrum of compound 5a shows a quartet at
4.1 ppm and a triplet at 0.9 ppm assigned to the –OCH2
and –CH3 group of ethyl ester, respectively. The protons of
the methylene bridge appeared at 2.4 and 1.5 ppm. The
–SCH2 protons resonated as singlet at 3.8 ppm. The ten
aromatic protons of the phenyl ring appeared between 7.1
and 7.4 ppm as multiplet. The 13C-NMR spectrum of the
compound 5a showed 12 signals corresponding to the 12
magnetically different carbon atoms. The signal at d
168.09 ppm was assigned to carbonyl carbon of the ester
functional group. The signals at 155.27 and 148.88 ppm
were due to the C3 and C5 of the triazole moiety. The
chemical shifts at 132.86, 129.99, 129.04, and 127.11 were
assigned to the aromatic carbon atoms of the phenyl ring.
Biological activity
Lipophilicity
The efficiency of the cytotoxicity of the drug depends on
the accumulation of the compound into the cell and thus
lipophilic character plays a major role in the cytotoxic
effect of the compounds. The partition coefficient (log10P)
of the compounds which is a measure of lipophilicity was
calculated using the software Bioloom (version 1) from
Biobyte Corp. (201, West 4th St. Suite 204, Claremont, CA
91711). The lipophilicity data of all the compounds vary
between 0.35 and 5.82, which is expressed in log10P, are
given along with other physical data in Table 1. Presence
of a methyl group or ethoxy group on the phenyl ring
markedly increased the log10P values, while the replace-
ment of aryl ring with cyclohexyl moiety slightly decreased
123

Med Chem Res (2012) 21:174–184
177
Table 1 Physical data of synthesized compounds
Compound
R
Mol. wt Mol. formula
Clog Pa Yield MP(°C)b Elemental analysis calculated (found)
C
H
N
4a
4b
4c
4d
4e
4f
Phenyl
p-Tolyl
m-Tolyl
607
635
635
C30H38N8O2S2
4.50
5.50
5.50
5.57
4.39
3.50
4.75
5.75
5.75
5.82
4.64
3.75
57
55
50
52
48
45
65
72
70
65
60
63
78
75
75
65
70
68
165
183
180
197
110
104
197
210
214
235
182
176
215
224
240
257
205
190
59.38 (59.25) 6.31 (6.12) 18.47 (18.28)
60.54 (60.40) 6.67 (6.35) 17.65 (17.74)
60.54 (60.56) 6.67 (6.55) 17.65 (17.55)
58.76 (58.80) 6.67 (6.65) 16.12 (16.10)
58.22 (58.34) 8.14 (8.18) 18.11 (18.20)
55.09 (55.24) 8.18 (8.23) 19.77 (19.82)
57.91 (57.85) 5.55 (5.65) 14.47 (14.55)
59.19 (59.30) 5.96 (6.05) 13.80 (13.75)
59.19 (59.43) 5.96 (5.87) 13.80 (13.73)
57.47 (57.65) 6.03 (6.10) 12.57 (12.45)
56.73 (56.87) 7.48 (7.65) 14.18 (14.24)
56.73 (56.85) 7.48 (7.54) 14.18 (14.32)
51.04 (51.22) 7.14 (7.23) 24.80 (24.65)
53.77 (53.86) 5.55 (5.47) 24.12 (23.95)
53.77 (53.65) 5.55 (5.45) 24.12 (24.32)
52.48 (52.34) 5.66 (5.76) 21.86 (21.65)
51.04 (51.23) 7.14 (6.95) 24.80 (24.26)
56.73 (56.34) 7.48 (7.70) 14.18 (14.35)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
32H42N8O2S2
32H42N8O2S2
34H46N8O4S2
30H50N8O2S2
26H46N8O2S2
28H32N6O4S2
30H36N6O4S2
30H36N6O4S2
32H40N6O6S2
28H44N6O4S2
24H40N6O4S2
p-Ethoxy phenyl 695
Cyclohexyl
n-butyl
619
567
580
608
608
5a
5b
5c
5d
5e
5f
Phenyl
p-Tolyl
m-Tolyl
p-Ethoxy phenyl 668
Cyclohexyl
n-Butyl
Phenyl
592
540
552
580
580
6a
6b
6c
6d
6e
6f
24H28N10O2S2 0.46
26H32N10O2S2 1.46
26H32N10O2S2 1.46
28H36N10O4S2 1.53
24H40N10O2S2 0.35
20H36N10O2S2 0.54
p-Tolyl
m-Tolyl
p-Ethoxy phenyl 640
Cyclohexyl
564
512
n-Butyl
a
Determined using software Bioloom (BioByte Corp, USA)
Average of three trials
b
Cytotoxicity studies
the log10P values. The diethylcarbamoyl and thioesters
derivatives (4a–f and 5a–f, respectively) were found to be
more lipophilic as indicated by higher log10P values. The
conversion of thioester group into the acid hydrazide
drastically decreased the log10P values due to more polar
hydrazide functionality. The triazoles 4b and 4c, 5b and 5c,
and 6b and 6c are the positional isomers which exhibit
identical log10P values. It is clear from log10P data given in
Table 1 that the triazoles have shown lipophilicity in the
following order, 5d [ 5b [ 4d [ 4b [ 5a [ 5e [ 4a [
4e [ 5f [ 4f [ 6d [ 6b [ 6f [ 6a [ 6e. The analysis of
the relationship between log10P values and the efficiency of
the compounds cytotoxicity in cancer cells showed a poor
correlation. The hydrazide derivative 6d with ethoxy
phenyl substitution having a log10P value (1.53) showed
significant cytotoxicity, while the diethyl carbamoyl
derivative 4d having same substitution and a higher
log10P value was not effective against cancer cell lines. It
is also speculated that the bis-triazole nucleus with sub-
stitution may also exhibit higher affinity for membranes or
be more readily taken up into cells than that with hydrogen
atom present. Therefore, we can conclude that the degree
of lipophilicity of each drug would seem to be important,
but it is not the sole determinant of potency for the anti-
cancer activity of the triazoles.
The cytotoxicity of all the compounds was evaluated in
vitro against the following human cancer cell lines: A-549
lung carcinoma, HT-29 colon adenocarcinoma, and MDA-
MB-231 breast carcinoma. The standard MTT assay was
used to determine IC50 values, i.e., the drug concentration
that causes 50% cell growth inhibition after 72 h of
continuing exposure to the test compounds and the mean of
the results obtained from triplicate assays are shown in
Table 2 (Molinari et al., 2009; Manjula et al., 2009). The
IC50 values were compared with that of anti-cancer anti-
biotic doxorubicin. From the evaluation of the data repor-
ted in Table 2, these observations can be made. All the
synthesized compounds were far less potent cytotoxic than
the standard drug doxorubicin (IC50 value 0.07–0.1 lM) as
evident from higher IC50 values. However, among the
synthesized compounds, the triazoles with diethylcarba-
moyl and thioester functionality were found to be more
effective than the acid hydrazides possibly due to their
higher lipophilicity and higher membrane penetrating
potency. The compounds which possess aromatic phenyl or
substituted phenyl group at the 4th position of the triazole
moiety are found to be more effective against the three cell
lines assayed. The methyl and ethoxy substitution at the
123

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Med Chem Res (2012) 21:174–184
Table 2 In vitro anti-cancer activity of bis-1,2,4-triazoles
Table 3 In vitro anti-bacterial potential of bis-1,2,4-triazoles
Compounds Minimal inhibitory concentrations (MIC, in lg/ml)
Compound
IC50 (lM)a
HT-29c
A-549b
MDA-MB-231d
B.substilus
S. aureus
E. coli
P. aeruginosa
4a
19.31 0.98
3.23 2.23
15.25 2.02
51.87 2.07
51.81 3.07
63.06 2.80
34.47 1.46
9.13 3.33
23.27 1.35
9.30 3.16
51.87 1.10
61.50 2.78
63.16 1.87
36.94 1.72
39.15 1.39
9.13 2.53
65.26 2.49
69.33 1.60
0.09 0.01
14.32 1.89
3.45 1.67
10.23 3.53
75.03 1.09
69.85 1.84
64.52 2.34
51.25 2.45
13.52 1.62
25.81 2.53
13.18 5.75
65.31 4.72
69.82 2.15
65.31 1.48
31.18 2.49
38.25 2.66
5.16 1.75
66.19 3.11
63.39 1.65
0.07 0.01
14.91 1.05
8.17 2.53
17.21 1.26
71.63 3.90
66.17 1.74
70.34 2.44
32.17 1.13
24.31 2.03
25.19 3.51
15.13 3.68
70.36 4.09
71.39 1.51
69.90 2.08
32.20 4.50
40.17 1.48
13.21 1.33
70.20 4.16
59.82 3.83
0.10 0.02
4a
25
50
12.5
12.5
12.5
100
100
ND
50
50
4b
4b
12.5
25
25
25
4c
4c
50
25
4d
4d
50
ND
ND
ND
100
100
100
50
ND
ND
ND
100
100
100
100
ND
ND
100
100
ND
12.5
ND
ND
12.5
4e
4e
100
ND
100
100
50
4f
4f
5a
5a
5b
5b
100
50
5c
5c
5d
5d
25
25
5e
5e
100
100
25
ND
ND
25
100
100
25
5f
5f
6a
6a
6b
6b
12.5
25
12.5
25
12.5
25
6c
6c
6d
6d
6.25
100
100
12.5
12.5
ND
ND
6.25
12.5
50
6e
6e
6f
6f
100
6.25
Doxorubicin
Ampicillin
a
The drug concentration that causes 50% cell growth inhibition
Lung carcinoma
ND not determined due to solubility problem
b
c
d
Colon adenocarcinoma
Breast carcinoma
not show good anti-bacterial activity when compared to
Ampicillin, while compounds 4a, 4b, 4c, and 6a exhibited
fairly good anti-bacterial activity. The compounds 6b and
6d were found to be more potent anti-bacterial agents with
MIC value closer to that of the reference standard. The
lipophilic nature of the compounds appears to be an
important factor as higher lipophilicity of 4d, 4e, and 5a–
f failed to exhibit anti-bacterial activity. However, the
lipophilicity is not the sole determinant for the activity
since the compounds 4a, 4b, and 4c having higher lipo-
philicity showed moderate to fairly good anti-bacterial
activity. This clearly indicates that the thioester moiety is
not essential for anti-bacterial activity, while diethyl car-
bamoyl moiety and hydrazide functional group are essen-
tial for better anti-bacterial activity of the triazole
derivatives. The compound 6d showed better anti-bacterial
activity against gram positive B. subtilus with lowest MIC
of (6.25 lg/ml) compared to the other compounds. The
acid hydrazide 6b also showed good anti-bacterial activity
with MIC of (12.5 lg/ml) against B. subtilus, E. coli, and
S. aureus. The investigation of structure activity relation-
ship clearly revealed that the compounds 4b, 6b, and 6d
showed better anti-bacterial activity and this may be
attributed due to the presence of electron donating groups
like methyl and ethoxy substituents on the para position of
SEM: average of three experiments
para position of the phenyl ring appears to increase the
effectiveness of the molecule, while methyl group at meta
position slightly decreased the activity. The compounds
with cyclohexyl and n-butyl substitution exhibited poor
cytotoxicity. Among the six acid hydrazides synthesized
para ethoxy phenyl-substituted triazole 6d only exhibited
significant cytotoxic activity.
Anti-microbial studies
All the compounds were tested for in vitro anti-microbial
activity against the following microorganisms: Bacillus
subtilus (ATCC 6633), Staphylococcus aureus (ATCC-
25923) (Gram positive bacteria), Pseudomonas aeruginosa
(ATCC-27853), Escherichia coli (ATCC-25922) (Gram
negative bacteria). Ampicillin was used as a reference
standard. The minimal inhibitory concentration (MIC)
values for compounds tested, defined as the lowest con-
centration of the compound preventing the visible growth,
were determined using serial dilution method and are given
in Table 3 (Rostom et al., 2009). From the data given in the
Table 3, it is clear that the compounds 4d, 4e, and 5a–f did
123

Med Chem Res (2012) 21:174–184
179
Synthesis of 1,4-bis[5-(N,N-diethylcarbamoyl)-thio-4-
phenyl-1,2,4-triazol-3-yl]-butane 4a
the phenyl group and also due to polar hydrazide functional
groups.
A mixture of 3a (0.5 g, 0.0012 mol), diethylcarbamoyl
chloride (0.34 g, 0.32 ml, 0.0025 mol), and anhydrous
K2CO3 (0.5 g, 0.0036 mol) in dry acetone (15 ml) was
heated under reflux for 8 h with continuous stirring. The
resulting mixture was filtered. The residue was washed
twice with 5 ml each of hot acetone and the washings were
added to the filtrate. The excess solvent was concentrated
under reduced pressure and the residue was poured into the
crushed ice. The precipitate obtained was filtered and
washed with cold methanol, dried, and crystallized using
DMF. The physical data is presented in Table 1.
Conclusion
Eighteen bis-1,2,4-traizole derivatives containing dieth-
ylcarbamoyl, thioester, and hydrazide functional groups
were prepared and characterized. The compounds were
evaluated for their in vitro cytotoxicity against three human
cell lines (A-549 lung carcinoma, HT-29 colon adenocar-
cinoma, and MDA-MB-231) and anti-microbial potential
against B. subtilus, S. aureus, E. coli, and P. aeruginosa.
The triazoles substituted with p-tolyl and p-ethoxy phenyl
groups showed greater effectiveness than other com-
pounds. The triazoles 4b, 5b, 5d, and 6d were found to be
potent cytotoxic molecules. Probably the cytotoxicity is
conferred via DNA intercalation and further studies are
necessary to confirm the possible mechanism of cytotox-
icity and to validate the lead molecule. The anti-microbial
activity evaluations suggest as indicated by higher MIC
values that they lack any significant anti-bacterial activity
probably due to higher lipophilicity. However, anti-bacte-
rial activity of triazoles 6b and 6d is probably attributed to
polar carbohydrazide functional group and electron
donating groups like methyl and ethoxy substituents on the
para position of the phenyl ring.
1
IR: 1675 cm-1 (C=O stretching); H-NMR d: 7.2–7.5
(m, Ar–H, 10H), 3.3 (q, 8H, NCH2), 2.45 (t, 4H, C1, and C4
methylene protons of butyl chain), 1.54 (t, 4H, C2 and C3
methylene protons of butyl chain), 1.0 (t, 12H, CH3);
13C-NMR d: 163.3 (C=O), 155.22 (C3 of triazole), 148. 56
(C5 of triazole), 132.56 (C1 of phenyl ring), 129.89 (C2 and
C6 of phenyl ring), 128.95 (C3 and C5 of phenyl ring),
127.15 (C4 of phenyl ring), 43.72 (NCH2), 32.33 (C2 and
C3 of butyl chain), 25.10 (C1 and C4 of butyl chain), 13.09
(terminal methyl); MS m/z: 608 [M ? H]? (75).
1,4-bis[5-(N,N-diethylcarbamoyl)-thio-4-(p-tolyl)-1,2,
4-triazol-3-yl]-butane 4b
Experimental
The procedure used for preparation of 4a was followed
using 3b (0.52 g, 0.0012 mol), diethylcarbamoyl chloride
(0.34 g, 0.32 ml, 0.0025 mol) and anhydrous K2CO3
(0.5 g, 0.0036 mol). The physical data is presented in
Table 1.
Materials and methods
The melting points were determined in open glass capil-
laries and are uncorrected. IR spectra were recorded on
Shimadzu FT-IR 8400-S spectrophotometer by KBr pellet
technique. Elemental analyses were performed and found
values are within 0.4% of theoretical values unless other-
1
IR: 1672 cm-1 (C=O stretching); H-NMR d: 7.0–7.6
(m, Ar–H, 8H), 3.4 (q, 8H, NCH2), 2.44 (t, 4H, C1, and C4
methylene protons of butyl chain), 2.25 (s, 6H, tolyl
methyl), 1.51 (t, 4H, C2 and C3 methylene protons of butyl
chain), 0.92 (t, 12H, CH3); 13C-NMR d: 165.3 (C=O),
155.34 (C3 of triazole), 147. 86 (C5 of triazole), 132.67 (C1
of phenyl ring), 129.99 (C2 and C6 of phenyl ring), 127.95
(C3 and C5 of phenyl ring), 127.10 (C4 of phenyl ring),
43.62 (NCH2), 32.33 (C2 and C3 of butyl chain), 25.10 (C1
and C4 of butyl chain), 20.27 (tolyl methyl), 13.12 (ter-
minal methyl); MS m/z: 636 [M ? H]? (79).
1
wise noted. H-NMR and 13C-NMR spectra were recorded
on AMX-400 NMR spectrophotometer at 400 MHz using
DMSO-d6 as the solvent and tetramethylsilane (TMS) as
internal standard. The chemical shifts are expressed in d
ppm. The splitting patterns were designated as follows; s:
singlet; d: doublet; q: quartet; m: multiplet. LCMS were
recorded by using Shimadzu LCMS-2010A instrument by
ESI.
1,4-bis[5-(N,N-diethylcarbamoyl)-thio-4-(m-tolyl)-1,2,
4-triazol-3-yl]-butane 4c
Chemistry
The starting compounds 1,4-bis-[5-mercapto-4-substituted-
1,2,4-triazol-3-yl]-butane 3a–f were synthesized from adi-
poyl dihydrazide 1 by reacting with six different substi-
tuted isothiocyantes 2a–f as per the reported method
(Mahmoud Omar 1997).
The experimental steps for the 4a was repeated using 3c
(0.52 g, 0.0012 mol), diethylcarbamoyl chloride (0.34 g,
0.32 ml, 0.0025 mol) and anhydrous K2CO3 (0.5 g,
0.0036 mol).
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Med Chem Res (2012) 21:174–184
IR: 1671 cm-1 (C=O stretching); H-NMR d: 7.1–7.6
(m, Ar–H, 8H), 3.33 (q, 8H, NCH2), 2.42 (t, 4H, C1, and C4
methylene protons of butyl chain), 2.23 (s, 6H, tolyl
methyl),1.53 (t, 4H, C2 and C3 methylene protons of butyl
chain), 0.95 (t, 12H, CH3); 13C-NMR d: 165.43 (C=O),
155.39 (C3 of triazole), 147. 86 (C5 of triazole), 126.30 (C1
of phenyl ring), 124.83 (C2 of phenyl), 138.78 (C3 of
phenyl ring), 129.89 (C4 of phenyl ring), 128.12 (C5 of
phenyl), 126.89 (C6 of phenyl ring), 127.90 (C3 and C5
of phenyl ring), 127.17 (C4 of phenyl ring), 43.76 (NCH2),
32.33 (C2 and C3 of butyl chain), 25.13 (C1 and C4 of butyl
chain), 20.25 (tolyl methyl), 13.15 (terminal methyl); MS
m/z: 636 [M ? H]? (79).
butyl chain), 0.93 (t, 12H, CH3); MS m/z: 620 [M ? H]?
(45).
1
1,4-bis[5-(N,N-diethylcarbamoyl)-thio-4-(n-butyl)-1,2,
4-triazol-3-yl]-butane 4f
The experimental steps for the 4a was repeated using 3f
(0.5 g, 0.0013 mol), diethylcarbamoyl chloride (0.34 g,
0.32 ml, 0.0025 mol), and anhydrous K2CO3 (0.5 g,
0.0036 mol) in dry acetone (15 ml).
1
IR: 1663 cm-1 (C=O stretching); H-NMR d: 3.79 (t,
4H, C1 protons of n-butyl), 3.35 (q, 8H, NCH2), 2.45 (t, 4H,
C1 and C4 methylene protons of butyl chain), 1.65 (m, 4H,
C2 protons of n-butyl), 1.47 (t, 4H, C2 and C3 methylene
protons of butyl chain), 1.30 (m, 4H, C3 protons of
n-butyl), 1.03 (t, 12H, CH3), 0.91(t, 6H, C4 protons of
n-butyl); MS m/z: 568 [M ? H]? (45).
1,4-bis[5-(N,N-diethylcarbamoyl)-thio-4-(p-ethoxyphenyl)-
1,2,4-triazol-3-yl]-butane 4d
A mixture of 3d (0.5 g, 0.001 mol), diethylcarbamoyl
chloride (0.27 g, 0.25 ml, 0.002 mol), and anhydrous
K2CO3 (0.5 g, 0.0036 mol) was treated similar to the
method described in 4a. The residue obtained was washed
three times with 10 ml each of diethyl ether to obtain the
product. Crystallization was done using dichloromethane.
1,4-bis-[5-(carbethoxy-methyl)-thio-4-phenyl-1,2,4-triazol-
3-yl]-butane 5a
A suspension of 3a (0.5 g, 0.0012 mol) in 10 ml dry ace-
tone was mixed with a solution of ethyl bromoacetate
(0.67 g, 0.5 ml, 0.004 mol) in 10 ml of acetone in an
Erlenmeyer flask and freshly fused anhydrous K2CO3
(0.5 g, 0.0036 mol) was added. The resulting reaction
mixture was refluxed for 8 h on boiling water bath with
constant stirring. After the completion of the reaction
(monitored by TLC), the reaction mixture was cooled and
filtered. The solid residue was washed four times with
10 ml of each of hot acetone. The filtrate and the washings
were mixed and the excess solvent was evaporated under
reduced pressure. The mother liquor was kept in a refrig-
erator over night whereby the ester was solidified. The
product was filtered and washed with cold methanol, dried,
and recrystallized with dichloromethane.
1
IR: 1681 cm-1 (C=O stretching); H-NMR d: 7.1–7.6
(m, 8H, Ar–H), 4.3(q, 4H, OCH2), 3.30 (q, 8H, NCH2),
2.46 (t, 4H, C1, and C4 methylene protons of butyl chain),
1.50 (t, 4H, C2 and C3 methylene protons of butyl chain),
1.13 (t, 6H, CH3 of ethoxy group), 0.93 (t, 12H, CH3); 13C-
NMR d: 165.55 (C=O), 155.35 (C3 of triazole), 147. 87 (C5
of triazole), 132.65 (C1 of phenyl ring), 129.88 (C2 and C6
of phenyl ring), 127.93 (C3 and C5 of phenyl ring), 127.18
(C4 of phenyl ring), 62.37 (OCH2), 43.76 (NCH2), 32.33
(C2 and C3 of butyl chain), 25.15 (C1 and C4 of butyl
chain), 15.11 (methyl of ethoxy group), 13.11 (terminal
methyl); MS m/z: 696 [M ? H]? (65).
1
1,4-bis[5-(N,N-diethylcarbamoyl)-thio-4-cyclohexyl-1,2,
4-triazol-3-yl]-butane 4e
IR: 1680 cm-1 (C=O stretching); H-NMR d: 7.0–7.6
(m, Ar–H, 10H), 4.1(q, 4H, OCH2), 3.8 (s, 4H, SCH2), 2.45
(t, 4H, C1 and C4 methylene protons of butyl chain), 1.54
(t, 4H, C2 and C3 methylene protons of butyl chain), 0.90
(t, 6H, CH3); 13C-NMR d: 168.09 (C=O), 155.27 (C3 of
triazole), 148.88 (C5 of triazole), 132.86 (C1 of phenyl
ring), 129.99 (C2 and C6 of phenyl ring), 129.04 (C3 and C5
of phenyl ring), 127.11 (C4 of phenyl ring), 61.20 (OCH2),
33.97 (SCH2), 32.37 (C2 and C3 of butyl chain), 25.30 (C1
and C4 of butyl chain); MS m/z: 581 [M ? H]? (100).
A mixture of 3e (0.5 g, 0.0012 mol), diethylcarbamoyl
chloride (0.27 g, 0.25 ml, 0.002 mol), and anhydrous
K2CO3 (0.5 g, 0.0036 mol) was treated similar to the
method described in the preparation of 4a. The residue
obtained was washed three times with 10 ml each of die-
thyl ether to obtain the product. Crystallization was done
using mixture of DMF and ethanol.
1
IR: 1660 cm-1 (C=O stretching); H-NMR d: 3.69 (m,
2H, C1 protons of cyclohexyl), 3.25 (q, 8H, NCH2), 2.44 (t,
4H, C1 and C4 methylene protons of butyl chain), 2.05 (m,
8H, C2 and C6 protons of cyclohexyl), 1.75 (m, 8H, C3 and
C5 protons of cyclohexyl), 1.64 (m, 4H, C4 protons of
cyclohexyl), 1.45 (t, 4H, C2 and C3 methylene protons of
1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-tolyl)-1,2,
4-triazol-3-yl]-butane 5b
The experimental procedure was followed as per the
synthesis of 5a using 3b (0.52 g, 0.0012 mol), ethyl
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Med Chem Res (2012) 21:174–184
181
bromoacetate (0.67 g, 0.5 ml, 0.004 mol) and anhydrous
K2CO3 (0.5 g, 0.0036 mol).
0.5 ml, 0.004 mol), and anhydrous K2CO3 (0.5 g,
0.0036 mol).
1
1
IR: 1673 cm-1 (C=O stretching); H-NMR d: 7.2–7.4
IR: 1670 cm-1 (C=O stretching); H-NMR d: 4.12 (q,
(m, 8H, ArH), 4.1 (q, 4H, OCH2), 4.0 (s, 4H, SCH2), 2.5 (t,
4H, C1 and C4 methylene protons of butyl chain), 2.4 (s,
6H, tolyl CH3), 1.4 (t, 4H, C2 and C3 methylene protons of
butyl chain), 1.1 (t, 6H, ester CH3); 13C-NMR d: 168.07
(C=O), 155.31 (C3 of triazole), 148.95 (C5 of triazole),
129.46 (C1 of phenyl ring), 130.38 (C2 and C6 of phenyl
ring), 126.81 (C3 and C5 of phenyl ring), 139.77 (C4 of
phenyl ring), 61.16 (OCH2), 33.89 (S-CH2), 32.34 (C2 and
C3 of butyl chain), 25.29 (C1 and C4 of butyl chain), 20.70
(tolyl CH3), 13.9 (CH3); MS m/z: 609 [M ? H]? (100).
4H, OCH2), 3.9 (s, 4H, SCH2), 3.65 (m, 2H, protons of C1
of cyclohexyl), 2.5 (t, 4H, C1 and C4 methylene protons of
butyl chain), 2.10 (m, 8H, C2 and C6 protons of cyclo-
hexyl), 1.75 (m, 8H, C3 and C5 protons of cyclohexyl), 1.64
(m, 4H, C4 protons of cyclohexyl), 1.4 (t, 4H, C2 and C3
methylene protons of butyl chain), 1.1 (t, 6H, CH3 of ester
group); MS m/z: 593 [M ? H]? (68).
1,4-bis[5-(carbethoxy-methyl)-thio-4-butyl-1,2,4-triazol-3-
yl]-butane 5f
1,4-bis[5-(carbethoxy-methyl)-thio-4-(m-tolyl)-1,2,4-
triazol-3-yl]-butane 5c
The method described in the synthesis of 5a was followed
using 3d (0.5 g, 0.001 mol), ethyl bromoacetate (0.67 g,
0.5 mL, 0.004 mol), and anhydrous K2CO3 (0.5 g,
0.0036 mol).
The procedure described in the synthesis of 5a was repe-
ated using 3c (0.52 g, 0.0012 mol), ethyl bromoacetate
(0.67 g, 0.5 ml, 0.004 mol) and anhydrous K2CO3 (0.5 g,
0.0036 mol).
1
IR: 1673 cm-1 (C=O stretching); H-NMR d: 4.1 (q,
4H, OCH2), 3.9 (s, 4H, SCH2), 3.73 (t, 4H, C1 protons of n-
butyl), 2.35 (t, 4H, C1 and C4 methylene protons of butyl
chain), 1.75 (m, 4H, C2 protons of n-butyl), 1.47 (t, 4H, C2
and C3 methylene protons of butyl chain), 1.30 (m, 4H, C3
protons of n-butyl), 1.03 (t, 6H, CH3), 0.91 (t, 6H, C4
protons of n-butyl); 13C-NMR d: 169.07 (C=O), 154.33 (C3
of triazole), 149.19 (C5 of triazole), 62.16 (OCH2), 36.21
(C1 of butyl), 33.88 (S-CH2), 32.90 (C2 of butyl), 32.10 (C2
and C3 of butyl chain), 25.2 (C1 and C4 of butyl chain),
20.12 (C3 of butyl), 14.5 (CH3 of ester), 12.32 (C4 of
butyl); MS m/z: 541 [M ? H]? (55).
IR: 1678 cm-1 (C=O stretching); 1H-NMR d: 7.2–7.5
(m, 8H, ArH), 4.15 (q, 4H, OCH2), 3.85 (s, 4H, SCH2),
2.54 (t, 4H, C1 and C4 methylene protons of butyl chain),
2.42 (s, 6H, tolyl CH3), 1.54 (t, 4H, C2 and C3 methylene
protons of butyl chain), 1.1 (t, 6H, ester CH3); 13C-NMR d:
167.98 (C=O), 155.18 (C3 of triazole), 148.75 (C5 of tria-
zole), 127.29 (C1 of phenyl ring), 123.99 (C2 of phenyl),
139.72 (C3 of phenyl ring), 132.71 (C4 of phenyl ring),
130.54 (C5 of phenyl), 129.61 (C6 of phenyl), 61.09
(OCH2), 33.88 (S-CH2), 32.15 (C2 and C3 of butyl chain),
25.26 (C1 and C4 of butyl chain), 20.59 (tolyl CH3), 13.85
(CH3); MS m/z: 609 [M ? H]? (100).
1,4-bis(5[hydrazinocarbonylmethylthio]-4-phenyl-1,2,
4 triazol-3-yl)butane 6a
1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxyphenyl)-
1,2,4-triazol-3-yl]-butane 5d
A mixture of 5a (0.58 g, 0.001 mol) and hydrazine hydrate
(0.15 g, 0.14 ml, 0.003 mol) in absolute ethanol (25 ml)
was heated under reflux for 8 h on boiling water bath. The
resulting solution was concentrated and cooled to room
temperature during which a crystalline white precipitate
separated out. The solid obtained was filtered, washed with
cold aqueous ethanol, and recrystallized from a mixture of
DMF and methanol.
The method described in the synthesis of 5a was repeated
using 3d (0.5 g, 0.001 mol), ethyl bromoacetate (0.67 g,
0.5 ml, 0.004 mol), and anhydrous K2CO3 (0.5 g,
0.0036 mol).
1
IR: 1680 cm-1 (C=O stretching); H-NMR d: 7.0–7.3
(m, 8H, Ar–H), 4.2(q, 4H, OCH2), 4.1(q, 4H, OCH2), 3.9
(s, 4H, SCH2), 2.4 (t, 4H, C1 and C4 methylene protons of
butyl chain), 1.45 (t, 4H, C2 and C3 methylene protons of
butyl chain), 1.3 (t, 6H, CH3 of ester group), 1.1 (t, 6H,
CH3 of ethoxy group); MS m/z: 669 [M ? H]? (75).
1
IR: 1663 cm-1 (C=O stretching); H-NMR d: 9.3 (bs,
2H, CONH), 7.2–7.4 (m, 10H, Ar–H), 4.0 (s, 4H, NH2), 3.8
(s, 4H, SCH2), 2.5 (t, 4H, C1 and C4 methylene protons of
butyl chain), 1.47 (t, 4H, C2 and C3 methylene protons of
butyl chain); 13C-NMR d: 166.01 (C=O), 155.18 (C3 of
triazole), 149.40 (C5 of triazole), 132.12 (C1 of phenyl),
129.16 (C2 and C6 of phenyl), 128.31 (C3 and C5 of phe-
nyl), 126,87 (C4 of phenyl), 34.24 (S-CH2), 32.90 (C2 and
C3 of butyl chain), 25.33 (C1 and C4 of butyl chain); MS m/
z: 553 [M ? H]? (100).
Synthesis of 1,4-bis[5-(carbethoxy-methyl)-thio-4-
cyclohexyl-1,2,4-triazol-3-yl]-butane 5e
The method described in the synthesis of 5a was repeated
using 3e (0.5 g, 0.0012 mol), ethyl bromoacetate (0.67 g,
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1,4-bis(5[hydrazinocarbonylmethylthio]-4-p-tolyl-1,2,4
triazol-3-yl)butane 6b
1,4-bis(5[hydrazinocarbonylmethylthio]-4-cyclohexyl-
1,2,4 triazol-3-yl)butane 6e
The experimental procedure was repeated as with 6a using
5b (0.61 g, 0.001 mol) and hydrazine hydrate (0.15 g,
0.14 ml, 0.003 mol) in absolute ethanol (25 ml).
The experimental procedure described as in the preparation
of 6a was followed using 5e (0.59 g, 0.001 mol) and
hydrazine hydrate (0.15 g, 0.14 ml, 0.003 mol) in absolute
ethanol (25 ml).
1
IR: 1667 cm-1 (C=O stretching); H-NMR d: 9.33 (bs,
2H, CONH), 7.1–7.5 (m, 8H, Ar–H), 4.3 (s, 4H, NH2), 3.8
(s, 4H, SCH2), 2.52 (t, 4H, C1 and C4 methylene protons of
butyl chain), 2.32 (s, 6H, tolyl CH3), 1.47 (t, 4H, C2 and C3
methylene protons of butyl chain); 13C-NMR d: 166.20
(C=O), 155.28 (C3 of triazole), 149.50 (C5 of triazole),
126.9 (C1 of phenyl), 128.24 (C2 and C6 of phenyl), 130.31
(C3 and C5 of phenyl), 139.73 (C4 of phenyl), 34.20 (S-
CH2), 32.85 (C2 and C3 of butyl chain), 25.35 (C1 and C4
of butyl chain), 20.7 (tolyl CH3); MS m/z: 581 [M ? H]?
(100).
IR: 1650 cm-1 (C=O stretching); 1H-NMR d: 9.3 (s, 2H,
CONH), 4.3 (s, 4H, NH2), 3.9 (s, 4H, SCH2), 3.60 (m, 2H,
protons of C1 of cyclohexyl), 2.44 (t, 4H, C1 and C4
methylene protons of butyl chain), 2.16 (m, 8H, C2 and C6
protons of cyclohexyl), 1.85 (m, 8H, C3 and C5 protons of
cyclohexyl), 1.69 (m, 4H, C4 protons of cyclohexyl), 1.4 (t,
4H, C2 and C3 methylene protons of butyl chain); 13C-
NMR d: 166.12 (C=O), 155.05 (C3 of triazole), 147.49 (C5
of triazole), 55.16 (C1 of cyclohexyl), 35.09 (S-CH2), 33.23
(C2 and C3 of butyl chain), 30.71 (C2 and C6 of cyclo-
hexyl), 26.11 (C4 of cyclohexyl), 25.18 (C1 and C4 of butyl
chain), 24.49 (C3 and C5 of cyclohexyl); MS m/z: 565
[M ? H]? (76).
1,4-bis(5[hydrazinocarbonylmethylthio]-4-m-tolyl-1,2,4
triazol-3-yl)butane 6c
1,4-bis(5[hydrazinocarbonylmethylthio]-4-butyl-1,2,
4-triazol-3-yl)butane 6f
The method described as in the preparation of 6a was
followed using 5c (0.61 g, 0.001 mol) and hydrazine
hydrate (0.15 g, 0.14 ml, 0.003 mol) in absolute ethanol
(25 ml).
The method described as in the preparation of 6a was
followed using 5f (0.54 g, 0.001 mol) and hydrazine
hydrate (0.15 g, 0.14 ml, 0.003 mol) in absolute ethanol
(25 ml).
1
IR: 1677 cm-1 (C=O stretching); H-NMR d: 9.34 (s,
2H, CONH), 7.1–7.5 (m, 8H, Ar–H), 4.32 (s, 4H, NH2),
3.78 (s, 4H, SCH2), 2.51 (t, 4H, C1 and C4 methylene
protons of butyl chain), 2.30 (s, 6H, tolyl CH3), 1.45
(t, 4H, C2 and C3 methylene protons of butyl chain);
13C-NMR d: 166.26 (C=O), 153.15 (C3 of triazole),
148.55 (C5 of triazole), 126.9 (C1 of phenyl), 124.24 (C2
of phenyl), 138.31 (C3 of phenyl), 129.23 (C4 of phenyl),
128.12 (C5 of phenyl), 126.72 (C6 of phenyl), 34.25
(S-CH2), 32.81 (C2 and C3 of butyl chain), 25.33 (C1
and C4 of butyl chain), 20.67 (tolyl CH3); MS m/z: 581
[M ? H]? (100).
1
IR: 1676 cm-1 (C=O stretching); H-NMR d: 9.3 (bs,
2H, CONH), 4.3 (s, 4H, NH2), 3.9 (t, 4H, C1 protons of
n-butyl), 3.8 (s, 4H, SCH2), 2.65 (t, 4H, C1 and C4 methy-
lene protons of butyl chain), 1.7 (t, 4H, C2 and C3 methylene
protons of butyl chain), 1.5 (m, 4H, C2 protons of n-butyl),
1.25 (m, 4H, C3 protons of n-butyl), 0.91 (t, 6H, C4 protons
of n-butyl); 13C-NMR d: 166.14 (C=O), 155.17 (C3 of tri-
azole), 148.15 (C5 of triazole), 42.85 (C1 of butyl), 35.03
(S-CH2), 31.43 (C2 and C3 of butyl chain), 25.81 (C2 of
butyl), 23.76 (C1 and C4 of butyl chain), 19.13 (C3 of butyl),
13.29 (C4 of butyl); MS m/z: 513 [M ? H]? (75).
The physical data of the compounds prepared is pre-
sented in Table 1.
1,4-bis(5[hydrazinocarbonylmethylthio]-4-p-ethoxyphenyl-
1,2,4-triazol-3-yl)butane 6d
Biological activity
A mixture of 5d (0.7 g, 0.001 mol) and hydrazine hydrate
(0.15 g, 0.14 ml, 0.003 mol) in absolute ethanol (25 ml)
was treated similar to the method explained as in the
preparation of 6a.
IR: 1680 cm-1 (C=O stretching); 1H-NMR d: 9.2 (s, 2H,
CONH), 7.0–7.3 (m, 8H, Ar–H), 4.3 (s, 4H, NH2), 4.0 (q,
4H, OCH2), 3.8 (s, 4H, SCH2), 2.3 (t, 4H, C1 and C4
methylene protons of butyl chain), 1.4 (t, 4H, C2 and C3
methylene protons of butyl chain), 1.3 (t, 6H, CH3 of
ethoxy group); MS m/z: 641[M ? H]? (100).
In vitro cytotoxicity activity
The cytotoxicity of the compounds was evaluated in vitro
against the following human cancer cell lines: A-549 lung
carcinoma, HT-29 colon adenocarcinoma and MDA-MB-
231 breast carcinoma. The cell lines were procured from
National Centre for Cell Sciences, Pune, India, and were
cultured in DMEM medium supplemented with 10% FBS,
1% L-glutamine, and 50 lg/ml gentamicin sulfate in a CO2
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Med Chem Res (2012) 21:174–184
183
incubator in a humidified atmosphere of 5%CO2 and 95%
air. The in vitro cytotoxicity was determined using a
standard MTT assay (Molinari et al., 2009, Manjula et al.,
2009). Briefly, the exponentially growing cells were plated
in 96-well plates (104 cells/well in 100 ll of medium) and
incubated for 24 h for attachment. The test compounds
were prepared prior to the study by dissolving in 0.1%
DMSO and diluted with medium. The cells were then
exposed to different concentration of test compounds (10,
20, and 50 lM) in a volume of 100 ll/well. The cells in the
growth control wells received only the same volume of
medium containing 0.1%DMSO. After 72 h of exposure,
the medium was removed and the cell cultures were
incubated with 100 ll of MTT reagent (0.1%) for 4 h at
37°C. The pink colored formazan was dissolved in 100 ll
of DMSO and absorbance of each well was read in an
ELISA micro plate reader at 570 nm. The experiment was
performed in triplicate and the percentage cytotoxicity was
calculated using the following formula.
resulted in no visible growth on the plate. A control test was
also performed with test medium supplemented with
DMSO at the same dilutions as used in the experiment to
ensure that the solvent had no influence on bacterial growth.
Ampicillin was used as standard drug for the comparison.
The mean of the values of MIC obtained from three inde-
pendent measurements is presented in Table 3.
Acknowledgments Authors are thankful to The Principal, JSS
College of Pharmacy, Mysore, India for providing necessary facili-
ties. Authors also thankful to The Director, NMR research centre,
Indian Institute of Science, Bangalore for spectral data. Thanks are
due to Mr. V.M. Chandrashekhar, Asst. Professor, Department of
Pharmacology, HSK college of Pharmacy, Bagalkot, India for car-
rying out anti-cancer activity.
Conflict of interest The authors have declared no conflict of
interest.
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