Synthesis and cytotoxicity of bis-1,3,4-oxadiazoles and bis-pyrazoles derived from 1,4-bis[5-thio-4-substituted-1,2,4-triazol-3-yl]-butane and their DNA binding studies

Article abstract of DOI:10.1002/ardp.201000177

A new series of 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4- substituted-1,2,4-triazol-3-yl]-butane 7-12 and 1,4-bis[5-(1-oxo-1-(3,5 dimethyl pyrazol-1-yl)-methyl)-thio-4-substitued-1,2,4-triazol-3-yl]-butane 13-18 were prepared from 1,4-bis

Full text of DOI:10.1002/ardp.201000177

248
Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
Full Paper
Synthesis and Cytotoxicity of Bis-1,3,4-oxadiazoles and
Bis-pyrazoles Derived from 1,4-Bis[5-thio-4-substituted-1,2,4-
triazol-3-Yl]-butane and Their DNA Binding Studies
Madhusudan Purohit1, V.V.S. Rajendra Prasad4, and Yergeri C. Mayur2,3
1 Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS University, Mysore, Karnataka,
India
2 Medicinal Chemistry Research Division, V.L. College of Pharmacy, Raichur, India
3 Center for Drug Design and Discovery, SVERI’s College of Pharmacy, Pandharpur, India
4 Department of Pharmaceutical Chemistry, Sita Institute of Pharmaceutical Sciences, Hyderabad, India
A new series of 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-substituted-1,2,4-triazol-3-yl]-
butane 7-12 and 1,4-bis[5-(1-oxo-1-(3,5 dimethyl pyrazol-1-yl)-methyl)-thio-4-substitued-1,2,4-triazol-3-
yl]-butane 13-18 were prepared from 1,4-bis(5[hydrazinocarbonylmethylthio]-4-substituted-1,2,4-
triazol-3-yl) butane based derivativess were synthesized 1-6. All the synthesized compounds were
characterized by IR, NMR and Mass spectral studies. The synthesized compounds 7-18 were screened
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. DNA binding studies
were conducted for three potent molecules by absorption titration method.
Keywords: Bis-1,2,4-triazole / Bis-1,3,4-oxadiazole / Bis-pyrazole / Cytotoxic activity / MTT assay
Received: June 10, 2010; Revised: July 26, 2010; Accepted: August 13, 2010
DOI 10.1002/ardp.201000177
Introduction
Many of the bis-1, 2, 4-triazoles [13–16], bis-oxadiazoles [17],
and bis-pyrazoles [18, 19] have also been reported to possess
wide spectrum of biological activity. Keeping these obser-
vations in mind and in continuation of our work on the
synthesis of bis-heterocyclic compounds [20]; we report
herein the synthesis and in-vitro cytotoxic activity of certain
novel oxadiazole and pyrazole incorporated bis-1,2,4-
triazoles separated by a butyl side chain. Then the most
potent three molecules were subjected to DNA intercalation
studies by UV-absorption method.
Dimeric analogues of various heterocyclic compounds are
drawing much attention in the recent past. This is largely due
to the wide spectrum of activity exhibited by these molecules.
Many dimeric compounds designed as bis-DNA intercalators
have been evaluated as anticancer agents. Dimers of more
lipophilic compounds have shown potent and broad spec-
trum 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 [1, 2]. The broad spectrum
antifungal agent fluconazole is a bis-triazole derivative. The
bis heterocyclic molecules have also shown to exhibit several
diverse pharmacological activity such as antimicrobial [3],
anti-protozoal [4], anti-inflammatory [5], anti-HIV [6], and
cytotoxicity [7–12].
Results and discussion
Chemistry
Synthesis of the intermediate and target compounds was
accomplished according to the steps depicted in Scheme 1.
The carbohydrazides 1–6 which served as key intermediates
were prepared starting from adipoyldihydrazide according
to the previously reported reaction conditions [21]. Briefly,
the adipoyldihydrazide was treated with six different
substituted isothiocyantes namely phenyl, p-tolyl, m-tolyl,
p-ethoxyphenyl, cyclohexyl and n-butyl under reflux condition
Correspondence: Professor Dr. Yergeri C. Mayur, SVERI’s College of
Pharmacy, Pandharpur, India
E-mail: mayuryc@rediff.com, mayuryc@hotmail.com
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Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
Cytotoxicity Study of Bis-1,3,4-oxadiazoles and Bis-pyrazoles
249
Table 1. Physical and lipophilicity data of synthesized compounds.
N
R
N
N
N
N
MP (8C)a
clog Pb
S
Compound
R
Yield
SH
O
N
O
HS
S
7
8
9
Phenyl
p-Tolyl
m-Tolyl
55
62
60
45
50
45
60
65
62
60
55
60
203
212
225
245
207
198
232
247
263
271
217
220
3.53
4.52
4.52
4.59
3.42
2.53
5.98
6.97
6.97
7.04
5.87
4.98
N
N
N
7-12
R
N
10
11
12
13
14
15
16
17
18
p-Ethoxy phenyl
Cyclohexyl
n-butyl
CS /KOH, reflux
2
Phenyl
p-Tolyl
m-Tolyl
NH2
HN
R
p-Ethoxy phenyl
Cyclohexyl
n-butyl
N
N
N
O
S
S
O
N
N
N
NH
H2N
R
a
Average of three trialsb Determined by using software Bioloom
(BioByte corp, USA)
1-6
CH COCH COCH / reflux
3
2
3
disulfide and potassium hydroxide showed absence of strong
–
absorptions due to C O stretching and N–H stretching indi-
–
cating the cyclization of hydrazide group. The 1H-NMR spec-
trum of the compound showed a broad hump corresponding
to two protons between 5.5–5.9 ppm which was assigned to
the mercapto group of the 1,3,4-oxadiazole. Further
the absence CONH and NH2 signals corresponding to the
carbohydrazide moiety of the starting material 1 confirms
the formation of oxadiazole. The molecular ion peak at
m/z 637 (M þ H) also is in agreement with the proposed
structure. The bis-1,3,4-oxadiazoles 8-12 were similarly
characterized.
R
N
O
N N
N
S
N
S
N
N N
N
R
O
N
13-18
R= phenyl/p-tolyl/m-tolyl/p-ethoxy phenyl/cyclohexyl/n-butyl
Scheme 1. Synthesis of the intermediate and target compounds.
to obtain corresponding thiosemicarbazides in quantitative
yield, which in turn were further converted to respective bis-
(5-mercapto or 4-substituted)-1,2,4 triazoles by heating under
strong alkaline conditions. Stirring these triazoles with ethyl
bromoacetate in dry acetone under reflux temperature
yielded corresponding thioesters in moderate to good yield.
Heating these thioesters with excess of hydrazine hydrate in
absolute ethanol afforded acid hydrazides 1–6 in good yield.
The spectral data and physical characteristics of 1–6 is pre-
sented else where. The bis-1,3,4-oxadiazole-5-thiones 7–12
were synthesized from intermediate acid hydrazides by
refluxing with excess of carbon disulfide and potassium
hydroxide. Similarly the acid hydrazides on heating with
excess of acetyl acetone yielded 1,4-bis[5-(1-oxo-1-(3,5
dimethyl pyrazol-1-yl)-methyl)-thio-4-substitued-1,2,4-triazol-
3-yl]-butane 13–18. The list of compounds synthesized is given
In the 1H-NMR spectrum of the compound 13 the aromatic
protons resonated as multiplet between 7.3–7.6 ppm. The
proton of C4 of pyrazole appeared as a singlet 4.6 ppm.
The singlet signal at 3.8 ppm was assigned to -SCH2 protons.
The two methyl protons of the pyrazole resonated as singlet
at 2.8 and 2.6 ppm respectively. The alkyl protons of the butyl
chain resonated at 2.3 and 1.2 ppm. The 13C-NMR spectrum
shows a signal due to carbonyl group at 195.23 ppm. The
signals at 153.25 and 148.65 ppm were assigned to the car-
bon atoms of the triazole moiety (C3 and C5). The carbons of
pyrazole ring appeared at 145.23, 142.75, and 112.64 ppm.
The six aromatic carbon atoms of the phenyl ring were
assigned to the signal at 131.42, 129.75, 128.82, and
127.39 ppm. The thiomethylene group resonated at 34.92,
17.43, and 15.83 ppm, respectively. The signals at 32.33 and
25.12 ppm were attributed to the methylene carbon atoms of
the butyl chain. 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 þ H. Most of the compounds yield abundant molecular
ions in the form of M þ H peaks.
in Table
1 along with corresponding melting points
and lipophilicity values. The structures of all compounds
were elucidated on the basis of elemental analysis, IR,
1H-NMR, 13C-NMR and mass spectral data.
The IR spectrum of the compound 7, which was prepared
by refluxing the carbohydrazide 1 with excess of carbon
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M. Purohit et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
Table 2. In vitro anticancer activity of synthesized compounds.
Biological activity
Lipophilicity
Compound
IC50 (mM)a)
The lipophilic character plays a major role in the cytotoxic
effect of the compounds since the efficiency of the cytotox-
icity of the drug depends on the accumulation of the com-
pound into the cell. The partition coefficient (log10P) of the
compounds which is a measure of lipophilicity was calcu-
lated using the software Bioloom (version 1) from Biobyte
Corp. (Claremont, CA). The lipophilicity data of all the com-
A-549
HT-29
MDA-MB-231
7
8
9
49.91 ꢀ 2.11
61.23 ꢀ 2.98
55.25 ꢀ 1.84
63.06 ꢀ 3.63
69.94 ꢀ 1.98
52.30 ꢀ 3.34
35.25 ꢀ 5.92
5.09 ꢀ 1.68
9.23 ꢀ 3.36
4.73 ꢀ 2.98
63.37 ꢀ 5.66
58.86 ꢀ 4.57
45.81 ꢀ 2.53
48.18 ꢀ 5.75
65.31 ꢀ 4.72
69.82 ꢀ 2.15
65.31 ꢀ 1.48
31.18 ꢀ 2.49
38.25 ꢀ 2.66
9.24 ꢀ 2.43
12.90 ꢀ 3.16
6.90 ꢀ 1.24
51.87 ꢀ 1.10
61.50 ꢀ 2.78
71.63 ꢀ 3.90
66.17 ꢀ 1.74
70.34 ꢀ 2.44
42.17 ꢀ 1.13
24.31 ꢀ 2.03
52.19 ꢀ 3.51
40.17 ꢀ 1.48
12.83 ꢀ 3.68
15.10 ꢀ 2.61
6.34 ꢀ 2.91
70.20 ꢀ 4.16
59.82 ꢀ 3.83
10
11
12
13
14
15
16
17
18
pounds expressed in log10P, are given in Table 1. The log10
P
values range from 2.53 to 7.04. The compounds having
phenyl and substituted phenyl groups have higher log10P
values. The compounds 7–12 which contain tautomeric
5-mercapto-1,3,4-oxadiazole substitution were found to have
lower lipophilicity (2.53–4.52) than the compounds substi-
tuted with dimethyl pyrazole 13–18 which showed higher
log10P values (4.98–7.04). Presence of a methyl group or
ethoxy group on the phenyl ring markedly increased the
log10P values, while the replacement of aryl ring with cyclo-
hexyl moiety slightly decreased the log10P values. The
triazoles 8, 9, 14 and 15 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, 16 > 15 > 14 > 13 > 17 > 18 > 10 >
9 > 8 > 7 > 11 > 12. The analysis of the relationship
between log10P values and the efficiency of the compounds
cytotoxicity in cancer cells showed a poor correlation. The
bis-triazole dimethyl pyrazole derivative 16 with ethoxy phe-
nyl substitution having a log10P value (7.04) showed signifi-
cant cytotoxicity while the bis-triazole 5-mercapto-1,3,4-
oxadiazole derivative 12 having n-butyl substitution with
lower log10P value was not effective against cancer cell lines.
It is also speculated that the bis-triazole nucleus with substi-
tution 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 anticancer
activity of the triazoles.
a)
The drug concentration that causes 50% cell growth inhibition.
A-549: Lung carcinoma; HT-29: Colon adenocarcinoma; MDA-MB-
231: Breast carcinoma.SEM: average of three experiments.
triazoles substituted with pyrazole moiety 14, 15, and 16
exhibited higher cytotoxic activity against all the tested cell
lines as indicated by lower IC50 values. The presence of elec-
tron releasing substituent such as methyl and ethoxy group
seems to be contributing factor for higher activity.
Apparently the high lipophilic nature of these compounds
also contributes for their higher cytotoxic activity. The
ethoxy substituted triazole 16 exhibited higher cytotoxicity
than other molecules. Among three cell lines tested A-549
lung carcinoma cell line was found to be more susceptible
against the compounds.
DNA binding studies
The DNA-binding properties of the three potent compounds
were evaluated based on their affinity or intercalation with
CT-DNA measured with absorption titration method [25]. The
DNA-binding properties of the compounds were studied by
monitoring the changes in the UV-VIS absorption spectra of
the triazole derivatives upon addition of CT-DNA. In the
range from 330 to 350 all the three derivatives exhibited
strong absorption peaks with maxima near 340–345 nm.
Progressive addition of DNA led to strong hypochromism
in the absorption intensities in all the compounds studied
(Fig. 1). The percentage hypochromism were found to be 50.2,
55.3 and 54.1. The half-reciprocal plots for binding of
triazoles with CT DNA were presented (Fig. 2). The com-
pounds exhibited the similar absorption spectra pertaining
to the chromophore. The selection of ionic strength (150 mM
NaCl) in the absorption titration experiment was mainly
based on the avoidance of DNA deposition in all drug
solution (20 mm). The DNA-binding constants and related
properties of derivatives were calculated after intercalation
Cytotoxicity studies
The cytotoxicity of all the compounds was evaluated by
in-vitro method 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 contin-
ued exposure to the test compounds and the mean of the
results obtained from triplicate assays are shown in Table 2
[22–24]. The IC50 data reported in Table 2 indicate that the bis-
triazoles substituted with 1,3,4-oxadiazole moiety 7–12 did
not show significant anticancer activity where as the bis-
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Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
Cytotoxicity Study of Bis-1,3,4-oxadiazoles and Bis-pyrazoles
251
NMR, and LCMS data. The yields of triazoles were between
45-65%. The in-vitro cytotoxicity potential of the compounds
was tested against the panel of three human cancer cell lines:
A-549 lung carcinoma, HT-29 colon adenocarcinoma and
MDA-MB-231 breast carcinoma by using standard MTT assay
method. The bis-triazoles 14, 15, and 16 possessing the
pyrazole moiety with substituents like p-tolyl, m-tolyl and
p-ethoxy phenyl respectively were found to be more active
than other compounds. Further studies are underway to
understand the possible role of DNA affinity in cytotoxic
activity of these compounds. DNA binding studies indicate
that compound 14 has strong binding affinity among the
derivatives studied.
Experimental
Materials and methods
The melting points were determined in open glass capillaries
and are uncorrected. The follow-up of the reactions and checking
the purity of the synthesized compounds was made by thin layer
chromatography (TLC) on silica gel precoated aluminium sheets
(Type 60GF254; Merck, Germany) and the spots were detected by
exposure to UV lamp at l254 for a few seconds. Elemental analysis
was performed and found values are ꢀ0.4% of theoretical values
unless otherwise noted. IR spectra were recorded on Shimadzu
FT-IR 8400-S spectrophotometer by KBr pellet technique. 1H-NMR
and 13C-NMR spectra were recorded on AMX-400 NMR spectro-
photometer at 400 MHz using DMSO-d6 as the solvent and tetra
methyl silane (TMS) as internal standard. The chemical shifts are
expressed in d ppm. The splitting patterns were designated as
follows; b: broad; s: singlet; d: doublet; m: multiplet. LCMS were
recorded by using Shimadzu LCMS-2010A instrument by ESI.
Figure 1. Absorption titration of compound 14 at 25 mM in 30 mM
sodium phosphate buffer (pH 6.5) with 150 mM NaCl at increasing
CT DNA concentration.
y = 3.1443x + 0.1195
3.5
R2 = 0.9944
3
2.5
2
1.5
1
General procedure for the preparation of 1,4-bis[5-(5-
mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
(substituted)-1,2,4-triazol-3-yl]-butane (7–12)
0.5
0
A suspension of 1, 4-bis-(5[hydrazinocarbonylmethylthio]- 4-(sub-
stituted)-1,2,4-triazol-3-yl)-butane derivatives 1–6 (0.01 mol) in
15 mL of absolute ethanol was added to a mixture of carbon
disulfide (2.28 mL, 0.03 mol) and potassium hydroxide (1.68 g,
0.03 mol) in 15 mL of absolute ethanol with continuous stirring
during 10minutes. The resultant reaction mixture was heated
under reflux till the evolution of hydrogen sulfide ceased. After
the completion of reaction, the excess of the solvent was
removed under reduced pressure and the residue was dissolved
in 30 mL cold water. The resulting solution was acidified with
acetic acid. The white solid that separated was filtered and
crystallized from ethyl acetate. The physical data of the synthe-
sized compounds is presented in Table 1.
0
0.2
0.4
0.6
0.8
1
1.2
D x 10
Figure 2. Half-reciprocal plot for binding of compound 14 with CT
DNA.
with CT-DNA. One compound 14 containing p-tolyl among
the derivatives studied showed strong DNA-binding affinities
like hypochromicity and isobastic points.
1,4-Bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
Conclusion
(phenyl)-1,2,4-triazol-3-yl]-butane (7)
Yield: 55%, M.p. 203–2058C, IR (n cmꢁ1, KBr): 3045, 2965, 1225,
1H-NMR (DMSO-d6, d ppm): 7.2–7.6 (m, Ar–H, 10H), 5.5–5.9
(b, 2H,SH), 4.0 (s, 4H, SCH2), 2.45 (t, 4H, C1 and C4 methylene
protons of butyl chain), 1.4 (t, 4H, C2 and C3 methylene protons of
Twelve new bis 1,2,4-triazoles substituted with oxadiazole
and pyrazole moiety were synthesized starting from 1,4-
bis-(5[hydrazinocarbonylmethylthio]-4-(substituted)-1,2,4-
triazol-3-yl)-butane derivatives and characterized using IR,
butyl chain); 13C-NMR (DMSO-d , d ppm): 158.3 (C S), 155.42 (C of
–
–
6
2
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Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
oxadiazole), 153.20 (C3 of triazole), 148.56 (C5 of triazole), 131.46
(C1 of phenyl ring), 129.85 (C2 and C6 of phenyl ring), 128.92 (C3
and C5 of phenyl ring), 127.35 (C4 of phenyl ring), 34.92 (SCH2),
32.31 (C2 and C3 of butyl chain), 25.18 (C1 and C4 of butyl chain);
LCMS m/z: 637 (M þ H, 40), 609 (25), 495 (45), 409 (100). Anal.
calcd. for C26H24N10O2S4: C, 49.04; H, 3.80; N, 22.00. Found: C,
49.14; H, 3.92; N, 22.06.
(m, 4H, C4 protons of cyclohexyl), 1.32 (t, 4H, C2 and C3 meth-
ylene protons of butyl chain); 13C-NMR (DMSO-d6, d ppm): 157.52
–
(C S), 155.05 (C of oxadiazole), 153.39 (C of triazole), 148.65 (C
5
–
2
3
of triazole), 40.25 (C1 of cyclohexyl), 34.50 (SCH2), 34.05 (C2 and C6
of cyclohexyl), 32.38 (C2 and C3 of butyl chain), 28.45 (C4 of
cyclohexyl), 25.20 (C1 and C4 of butyl chain), 22.11 (C3 and C5
of cyclohexyl); LCMS m/z: 649 (M þ H, 35)þ, 535 (60), 421 (100).
Anal. calcd. for C26H36N10O2S4: C, 48.12; H, 5.59; N, 21.59. Found:
C, 48.32; H, 5.48; N, 21.51.
1,4-Bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
(p-tolyl)-1,2,4-triazol-3-yl]-butane (8)
Yield: 62%, M.p. 212–2158C, IR (n cmꢁ1, KBr): 3035, 2972, 1232;
1H-NMR (DMSO-d6, d ppm): 7.2–7.4 (m, Ar-H, 8H), 5.5–6.0
(b, 2H,SH), 3.9 (s, 4H, SCH2), 2.5 (t, 4H, C1 and C4 methylene
protons of butyl chain), 2.2 (s, 6H, p-tolyl CH3), 1.3 (t, 4H, C2
and C3 methylene protons of butyl chain); 13C-NMR (DMSO-d6, d
1,4-Bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
(n-butyl)-1,2,4-triazol-3-yl]-butane (12)
Yield: 45%, M.p. 198–2028C IR (n cmꢁ1, KBr): 2975, 1237; 1H-NMR
(DMSO-d6, d ppm): 5.45–5.80 (b, 2H, SH), 3.90 (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
–
ppm): 158.22 (C S), 155.56 (C of oxadiazole), 153.24 (C3 of
2
–
triazole), 148.55 (C5 of triazole), 137.35 (C4 of phenyl ring),
131.66 (C1 of phenyl ring), 129.85 (C2 and C6 of phenyl ring),
128.92 (C3 and C5 of phenyl ring), 34.59 (SCH2), 32.35 (C2 and C3 of
butyl chain), 25.20 (C1 and C4 of butyl chain), 20.32 (p-tolyl CH3);
LCMS m/z: 665 (M þ H) þ. Anal. calcd. for C28H28N10O2S4: C, 50.58;
H, 4.24; N, 21.07. Found: C, 50.40; H, 4.21; N, 21.16.
(m, 4H, C3 protons of n-butyl), 0.91 (t, 6H, C4 protons of n-butyl);
13
–
C-NMR (DMSO-d , d ppm): 157.42 (C S), 155.15 (C of oxadia-
6
–
2
zole), 153.42 (C3 of triazole), 148.55 (C5 of triazole), 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), 12.32
(C4 of butyl); LCMS m/z: 597 (M þ H, 15)þ, Anal. calcd.
for C22H32N10O2S4: C, 44.27; H, 5.40; N, 23.47. Found: C, 44.32;
H, 5.48; N, 23.51.
1,4-Bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
(m-tolyl)-1,2,4-triazol-3-yl]-butane (9)
Yield: 60%, M.p. 221–2258C IR (n cmꢁ1, KBr): 3043, 2972, 1224;
1H-NMR (DMSO-d6, d ppm): 7.1–7.3 (m, Ar-H, 8H), 5.6–6.0 (b, 2H,
SH), 4.0 (s, 4H, SCH2), 2.54 (t, 4H, C1 and C4 methylene protons of
butyl chain), 2.25 (s, 6H, p-tolyl CH3), 1.34 (t, 4H, C2 and C3
methylene protons of butyl chain); 13C-NMR (DMSO-d6, d ppm):
General procedure for the preparation of 1,4-bis[5-(1-oxo-
1-(3,5-dimethyl pyrazol-1-yl)-methyl)-thio-4-substituted-
1,2,4-triazol-3-yl]-butane (13–18)
The intermediate hydrazide derivative 1,4-bis-(5[hydrazinocarbo-
nylmethylthio]-4-(substituted)-1,2,4-triazol-3-yl)butane derivatives
1–6 (0.01 mol) was suspended in 10 mL acetyl acetone in pres-
ence of a drop of conc. HCl and refluxed for 18–22 h (reaction
monitored by TLC). The resulting solution was concentrated and
poured in to crushed ice to get the solid which was further
washed with petroleum ether to obtain pyrazoles derivative.
Recrystallization was done by using mixture of DMF.
–
158.25 (C S), 155.65 (C of oxadiazole), 153.31 (C3 of triazole),
2
–
148.45 (C5 of triazole), 137.38 (C4 of phenyl ring), 131.62 (C1 of
phenyl ring), 129.85 (C2 and C6 of phenyl ring), 128.92 (C3 and C5
of phenyl ring), 34.79 (SCH2), 32.35 (C2 and C3 of butyl chain),
25.20 (C1 and C4 of butyl chain), 20.45 (p-tolyl CH3); LCMS m/z: 665
(M þ H) þ. Anal. calcd. for C28H28N10O2S4: C, 50.58; H, 4.24; N,
21.07. Found: C, 50.51; H, 4.28; N, 21.06.
1,4-Bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
1,4-Bis[5-(1-oxo-1-(3,5-dimethyl pyrazol-1-yl)-methyl)-
(p-ethoxyphenyl)-1,2,4-triazol-3-yl]-butane (10)
thio-4-(phenyl)-1,2,4-triazol-3-yl]-butane (13)
Yield: 45%, M.p. 245–2488C IR (n cmꢁ1, KBr): 3048, 2970, 1233;
1H-NMR (DMSO-d6, d ppm): 7.1–7.6 (m, Ar-H, 8H), 5.5–6.0 (b, 2H,
SH), 4.3 (q, 4H, OCH2), 4.0 (s, 4H, SCH2), 2.55 (t, 4H, C1 and C4
methylene protons of butyl chain), 1.32 (t, 4H, C2 and C3 meth-
Yield: 60%, M.p. 230–2348C, IR (n cmꢁ1, KBr): 3045, 2965, 1652,
1225, 1H-NMR (DMSO-d6, d ppm): 7.3–7.6 (m, 10H, Ar-H), 4.6
(s, 2H, C4 of pyrazole), 3.8 (s, 4H, SCH2), 2.8 (s, 6H, CH3), 2.6
(s, 6H, CH3), 2.3 (t, 4H, C1 and C4 methylene protons of butyl
ylene protons of butyl chain), 1.13 (t, 6H, CH3 of ethoxy group);
13
chain), 1.2 (t, 4H, C2 and C3 methylene protons of butyl chain);
13
–
C-NMR (DMSO-d , d ppm): 158.52 (C S), 156.05 (C of oxadia-
6
–
–
C-NMR (DMSO-d , d ppm): 195.23 (C O), 153.25 (C of triazole),
6 3
2
–
zole), 153.30 (C3 of triazole), 148.45 (C5 of triazole), 137.40 (C4 of
phenyl ring), 131.65 (C1 of phenyl ring), 129.86 (C2 and C6 of
phenyl ring), 128.90 (C3 and C5 of phenyl ring), 62.37 (OCH2),
34.90 (SCH2), 32.33 (C2 and C3 of butyl chain), 25.20 (C1 and C4 of
butyl chain), 15.11 (methyl of ethoxy group); LCMS m/z: 725
(M þ H) þ. Anal. calcd. for C30H32N10O4S4: C, 49.71; H, 4.45; N,
19.32. Found: C, 49.52; H, 4.34; N, 19.51.
148.65 (C5 of triazole), 145.23 (C3 of pyrazole), 142.75 (C5 of
pyrazole), 131.42 (C1 of phenyl ring), 129.75 (C2 and C6 of phenyl
ring), 128.82 (C3 and C5 of phenyl ring), 127.39 (C4 of phenyl
ring), 112.64 (C4 of pyrazole), 34.92 (SCH2), 32.33 (C2 and C3 of
butyl chain), 25.12 (C1 and C4 of butyl chain), 17.43 (CH3), 15.83
(CH3); LCMS m/z: 682 (M þ H, 20). Anal. calcd. for C34H36N10O2S2:
C, 59.98; H, 5.33; N, 20.57. Found: C, 59.64; H, 5.12; N, 20.46.
1,4-Bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-
1,4-Bis[5-(1-oxo-1-(3,5-dimethyl pyrazol-1-yl)-methyl)-
(cyclohexyl)-1,2,4-triazol-3-yl]-butane (11)
thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (14)
Yield: 50%, M.p. 205–2078C IR (n cmꢁ1, KBr): 2984, 1239; 1H-NMR
(DMSO-d6, d ppm): 5.5–6.0 (b, 2H, SH), 4.0 (s, 4H, SCH2), 3.69
(m, 2H, C1 protons of cyclohexyl), 2.44 (t, 4H, C1 and C4 meth-
ylene 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
Yield: 65%, M.p. 245–2488C, IR (n cmꢁ1, KBr): 3048, 2960, 1662,
1225, 1H-NMR (DMSO-d6, d ppm): 7.2–7.5 (m, 8H, Ar-H), 4.5
(s, 2H, C4 of pyrazole), 3.82 (s, 4H, SCH2), 2.7 (s, 6H, CH3), 2.5
(s, 6H, CH3), 2.35 (t, 4H, C1 and C4 methylene protons of butyl
chain), 2.2 (s, 6H, p-tolyl CH3), 1.3 (t, 4H, C2 and C3 methylene
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Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
Cytotoxicity Study of Bis-1,3,4-oxadiazoles and Bis-pyrazoles
253
protons of butyl chain); 13C-NMR (DMSO-d , d ppm): 197.30 (C O),
SCH2), 3.75 (t, 4H, C1 protons of n-butyl), 2.7 (s, 6H, CH3), 2.5 (s, 6H,
CH3), 2.36 (t, 4H, C1 and C4 methylene protons of butyl chain),
1.75 (m, 4H, C2 protons of n-butyl), 1.44 (t, 4H, C2 and C3 methy-
lene protons of butyl chain), 1.30 (m, 4H, C3 protons of n-butyl),
0.91 (t, 6H, C4 protons of n-butyl); LCMS m/z: 641(M þ H, 20)þ,
Anal. calcd. for C30H44N10O2S2: C, 56.22; H, 6.92; N, 21.86. Found:
C, 56.32; H, 6.88; N, 21.71.
–
–
6
153.32 (C3 of triazole), 148.69 (C5 of triazole), 145.35 (C3 of
pyrazole), 142.70 (C5 of pyrazole), 137.32 (C4 of phenyl ring),
131.60 (C1 of phenyl ring), 129.75 (C2 and C6 of phenyl ring),
128.72 (C3 and C5 of phenyl ring), 112.61 (C4 of pyrazole), 34.88
(SCH2), 32.23 (C2 and C3 of butyl chain), 25.32 (C1 and C4 of butyl
chain), 20.70 (p-tolyl CH3), 17.43 (CH3), 15.83 (CH3); LCMS m/z: 709
(M þ H, 20). Anal. calcd. for C36H40N10O2S2: C, 60.99; H, 5.69; N,
19.76. Found: C, 60.84; H, 5.72; N, 20.06.
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 mg/mL
gentamicin sulphate in a CO2 incubator in a humidified atmo-
sphere of 5% CO2 and 95% air. The in-vitro cytotoxicity was
determined using a standard MTT assay [22–24]. Briefly, the
exponentially growing cells were plated in 96- well plates
(104 cells/well in 100 mL 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, 50 and 100 mM) in a volume of 100 mL/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 mL of MTT reagent (0.1%) for 4 h at 378C. the
pink colored formazan was dissolved in 100 mL 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 cytoxicity was calculated using the following formula.
1,4-Bis[5-(1-oxo-1-(3,5-dimethyl pyrazol-1-yl)-methyl)-
thio-4-(m-tolyl)-1,2,4-triazol-3-yl]-butane (15)
Yield: 62%, M.p. 250–2548C, IR (n cmꢁ1,KBr): 3114, 2950, 1668,
1229, 1H-NMR (DMSO-d6, d ppm): 7.0–7.5 (m, 8H, Ar-H), 4.5
(s, 2H, C4 of pyrazole), 3.85 (s, 4H, SCH2), 2.6 (s, 6H, CH3), 2.5
(s, 6H, CH3), 2.38 (t, 4H, C1 and C4 methylene protons of butyl
chain), 2.1 (s, 6H, m-tolyl CH3), 1.3 (t, 4H, C2 and C3 methylene
protons of butyl chain); 13C-NMR (DMSO-d , d ppm): 196.58 (C O),
–
–
6
153.50 (C3 of triazole), 148.67 (C5 of triazole), 145.35 (C3 of
pyrazole), 142.74 (C5 of pyrazole), 137.33 (C4 of phenyl ring),
131.60 (C1 of phenyl ring), 129.79 (C2 and C6 of phenyl ring),
128.62 (C3 and C5 of phenyl ring), 112.69 (C4 of pyrazole), 34.98
(SCH2), 32.27 (C2 and C3 of butyl chain), 25.36 (C1 and C4 of butyl
chain), 20.73 (m-tolyl CH3), 17.40 (CH3), 15.87 (CH3); LCMS m/z: 709
(M þ H, 20). Anal. calcd. for C36H40N10O2S2: C, 60.99; H, 5.69; N,
19.76. Found: C, 60.94; H, 5.62; N, 19.85.
1,4-Bis[5-(1-oxo-1-(3,5-dimethyl pyrazol-1-yl)-methyl)-
thio-4-(p-ethoxyphenyl)-1,2,4-triazol-3-yl]-butane (16)
Yield: 60%, M.p. 270–2738C, IR (n cmꢁ1, KBr): 3058, 2970, 1674,
1233; 1H-NMR (DMSO-d6, d ppm): 7.1–7.4 (m, Ar-H, 8H), 4.6
(s, 2H, C4 of pyrazole), 4.1 (q, 4H, OCH2), 3.75 (s, 4H, SCH2), 2.7
(s, 6H, CH3), 2.5 (s, 6H, CH3), 2.3 (t, 4H, C1 and C4 methylene
protons of butyl chain), 1.4 (t, 4H, C2 and C3 methylene protons of
butyl chain), 1.1 (t, 6H, CH3 of ethoxy group); 13C-NMR (DMSO-d6,
% Cytotoxicity ¼ ðControl abs ꢁ Test absÞ
ꢂ 100 = Control abs:
The drug concentration that causes 50% cell growth inhibition
–
d ppm): 196.50 (C O), 152.50 (C of triazole), 148.63 (C5 of
3
–
triazole), 145.33 (C3 of pyrazole), 142.75 (C5 of pyrazole),
137.43 (C4 of phenyl ring), 131.49 (C1 of phenyl ring), 129.65
(C2 and C6 of phenyl ring), 128.62 (C3 and C5 of phenyl ring),
112.69 (C4 of pyrazole), 62.37 (OCH2), 34.96 (SCH2), 32.22 (C2
and C3 of butyl chain), 25.36 (C1 and C4 of butyl chain), 17.40
(CH3), 15.87 (CH3) 15.10 (methyl of ethoxy group); LCMS m/z: 769
(M þ H, 40). Anal. calcd. for C38H44N10O4S2: C, 59.35; H, 5.77; N,
18.22. Found: C, 59.54; H, 5.72; N, 18.36.
after 72 h of continuous exposure to the test compounds (IC50
)
was determined by plotting the graph of concentration of the
drug against the percent cytotoxicity and performing the
regression analysis. The IC50 values of the test compounds are
shown in Table 2.
DNA binding assay by absorption titration
The spectrometric titration was conducted by Nano Drop ND-
1000 UV Spectrophotometer at room temperature (ꢃ308C). The
CT DNA (Sigma, St. Louis, MO) was dissolved in double distilled
de-ionized water with 50 mM NaCl, and dialyzed against a buffer
solution for 2 days. Its concentration was determined by absorp-
tion spectrometry at 340 nm using a molar extinction coefficient
6600 Mꢁ1 cmꢁ1. The ratio A325/A355 > 1.80 was used as an
indication of a protein-free DNA. Absorption titration was per-
formed at a fixed concentration of drugs (25 mM) in a sodium
phosphate buffer (20 mM sodium phosphate, 150 mM NaCl, pH
6.5). Small aliquots of concentrated CT DNA (5 mM) were added
into the solution at final concentrations from 0 to 100 mM, and
1,4-Bis[5-(1-oxo-1-(3,5-dimethyl pyrazol-1-yl)-methyl)-
thio-4- (cyclohexyl)-1,2,4-triazol-3-yl]-butane (17)
Yield: 55%, M.p. 215–2178C, IR (n cmꢁ1, KBr): 2984, 1675, 1233;
1H-NMR (DMSO-d6, d ppm): 4.5 (s, 2H, C4 of pyrazole), 3.8 (s, 4H,
SCH2), 3.66 (m, 2H, C1 protons of cyclohexyl), 2.6 (s, 6H, CH3), 2.5
(s, 6H, CH3), 2.4 (t, 4H, C1 and C4 methylene protons of butyl chain),
2.10 (m, 8H, C2 and C6 protons of cyclohexyl), 1.77 (m, 8H, C3
and C5 protons of cyclohexyl), 1.60 (m, 4H, C4 protons of cyclo-
hexyl), 1.34 (t, 4H, C2 and C3 methylene protons of butyl chain);
LCMS m/z: 693 (M þ H, 15)þ. Anal. calcd. for C34H48N10O2S2:
C, 58.93; H, 6.98; N, 20.21. Found: C, 58.82; H, 6.85; N, 20.32.
stirred for 5 min before measurement. The parameters, lmax
,
hypochromicity, isobastic point and binding constant (Table 3)
were found from the absorption spectra. The intrinsic binding
constant (Ki) for a given complex with DNA was obtained from a
plot of D/Deapp versus D according to the following equation, D/
Deapp ¼ D/De þ 1/De ꢂ K, where D ¼ concentration of DNA in
1,4-Bis[5-(1-oxo-1-(3,5-dimethyl pyrazol-1-yl)-methyl)-
thio-4- (n-butyl)-1,2,4-triazol-3-yl]-butane (18)
Yield: 55%, M.p. 219–2228C, IR (n cmꢁ1, KBr): 2985, 1680, 1238;
1H-NMR (DMSO-d6, d ppm): 4.4 (s, 2H, C4 of pyrazole), 3.90 (s, 4H,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

254
M. Purohit et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 248–254
Table 3. Binding constant (Ki) and photometric properties of
triazoles in contact with CT-DNA
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Compound
Ki
lmax Hypochromicity Isobastic
point
(T10 TMS1
)
(nm)
(%)
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14
15
16
3.92
3.13
3.52
341
338
336
57.2
51.3
53.1
391
327
345
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base molarities, Deapp ¼ jea ꢁ efj and De ¼ jeb ꢁ efj, where ea and
ef are respective extinction coefficient of the complex in the
presence and absence of DNA. The apparent extinction coeffi-
cient ea is obtained by calculating Aobs/ [bis compounds]. The data
were fitted to the equation with a slope equal to 1/De and
Y-intercept equal to 1/(De ꢂ K).The intrinsic binding constant
(Ki) is determined from the slope of Y-intercept.
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Authors are thankful to The Principal, JSS College of Pharmacy, Mysore,
India for providing necessary facilities. 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 anticancer activity.
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The authors have declared no conflict of interest.
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www.archpharm.com