Synthesis of some novel 3,5-disubstituted 1,3,4-oxadiazole derivatives and anticancer activity on EAC animal model

Article abstract of DOI:10.1007/s00044-010-9455-6

A series of novel 3,5-disubstituted 1,3,4-oxadiazole- 2-thione derivatives (1e, 2e, 3e, 4e, and 5e) have been synthesized from different substituted aromatic acids. The structure determination of these compounds have been made on the basis of IR, 1H NMR, and Elemental analysis. The effect of all the compounds on tumor growth inhibition was evaluated by studying the parameters-tumor volume, percentage of the tumor cell count (viable and nonviable), hematological values, and the mean survival status of the treated animals on eight groups of Swiss albino mice. Compounds were given at the dose of 50 mg/kg body weight intraperitoneally and all exhibited the significant (P0.001) anticancer activity compared to control. 5-Fluorouracil was used as a standard drug (20 mg/kg body weight i.p.) in the study. All the compounds demonstrated a prominent anticancer activity. The study supported the derivatives of oxadiazoles for the development as potent anticancer molecules. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9455-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1206–1213
DOI 10.1007/s00044-010-9455-6
ORIGINAL RESEARCH
Synthesis of some novel 3,5-disubstituted 1,3,4-oxadiazole
derivatives and anticancer activity on EAC animal model
•
Sasmita Dash B. Ashok Kumar Jagadish Singh
•
•
•
B. C. Maiti T. K. Maity
Received: 6 May 2010 / Accepted: 25 September 2010 / Published online: 13 October 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of novel 3,5-disubstituted 1,3,4-oxa-
diazole-2-thione derivatives (1e, 2e, 3e, 4e, and 5e) have
been synthesized from different substituted aromatic acids.
The structure determination of these compounds have been
made on the basis of IR, 1H NMR, and Elemental analysis.
The effect of all the compounds on tumor growth inhibition
was evaluated by studying the parameters—tumor volume,
percentage of the tumor cell count (viable and nonviable),
hematological values, and the mean survival status of the
treated animals on eight groups of Swiss albino mice.
Compounds were given at the dose of 50 mg/kg body
weight intraperitoneally and all exhibited the significant
(P \ 0.001) anticancer activity compared to control.
5-Fluorouracil was used as a standard drug (20 mg/kg body
weight i.p.) in the study. All the compounds demonstrated a
prominent anticancer activity. The study supported the
derivatives of oxadiazoles for the development as potent
anticancer molecules.
Introduction
1,3,4-Oxadiazole ring is associated with number of bio-
logical properties like anti-inflammatory (Raman et al.,
1993; Sahin et al., 2001), hypoglycemic (Husain et al.,
1986), antifungal (Giri et al., 1976), antibacterial (Misra,
1983; Oludotun et al., 2009) and anticancer activities
(Sengupta et al., 2008). It also has gained a significant
interest in medicinal chemistry for a number of biological
targets including benzodiazepine receptor agonists (Trully
et al., 1991), 5-HT receptor agonists (Cehn et al., 1994),
muscarinic agonists (Swan et al., 1991), 5-HT antagonists
(Ladduwahetty et al., 1996), antirhinoviral compounds
(Diana et al., 1994), and anti-inflammatory agents (Omar
et al., 1996). Certain 1,3,4-oxadiazole derivatives and
their Mannich bases are reported to possess anti-inflam-
matory, antitubercular (Mamolo et al., 2005), antifungal
(Kucukguzel et al., 1999), and anticancer activities (Loet-
chutinat et al., 2003). It has also been reported that
incorporation of various chemical moieties with Mannich
base of 1,3,4-oxadiazoles give the compounds of compact
structure and expected anticancer activities (Ladduwahetty
et al., 1996). Therefore, the development of novel and
simple synthetic routes to achieve the generation of the
molecules with potential anticancer activity are of great
interest in the field of pharmaceutical industries.
Keywords 1,3,4-Oxadiazoles Á Synthesis Á EAC cell Á
Anti cancer activity
Thus the aim was to synthesize some 3,5-disubstituted
1,3,4-oxadiazole-2-thiones derivatives using the synthetic
procedure based on the ring closure reactions of appro-
priate acid hydrazides with carbon disulfide and potassium
hydroxide solution to form the 1,3,4-oxadiazole deriva-
tives. Among the compounds, thione is the preferred form
because when it is transferred to its thiolic structure it
possesses an aromatic units as well as an acidic function.
The attachment of an acidic moiety at the thiol equivalent
S. Dash Á B. A. Kumar Á J. Singh Á B. C. Maiti Á
T. K. Maity (&)
Division of Pharmaceutical Chemistry, Department
of Pharmaceutical Technology, Jadavpur University,
Jadavpur, Kolkata 700032, India
e-mail: jutkmaity@yahoo.com
S. Dash
e-mail: sasju369@gmail.com
J. Singh
e-mail: jagu_pharm@yahoo.co.in
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Med Chem Res (2011) 20:1206–1213
1207
side results in necessary aromaticity in the ring. Keeping
this fact in mind, the parent 1,3,4-oxadiazole thiones are
converted to their acetic acid derivatives to achieve better
results.
temperature. Then it was kept overnight in refrigerator and
filtered to give the desired product. The structure of these
compounds have been elucidated by spectral and elemental
1
(IR, H NMR, and C, H, N analysis) analysis.
Chemistry
Experimental protocols
Aromatic acid 1a–5a was esterified with methanol using
sulfuric acid according to the procedure depicted in
Scheme 1, and the resulting compounds 1b–5b were
refluxed with hydrazine hydrate in ethanol to give aroyl
hydrazine 1c–5c. Aroyl hydrazines were refluxed with CS2
and KOH in ethanol to get cyclized to form aryl derivatives
of 1,3,4-oxadiazole-2-thione (1d–5d) in good yield. Then
the aryl derivatives of 1,3,4-oxadiazoles were reacted with
formaldehyde (40%) and a primary amine (i.e., 2-amino-
pyridine or aniline) to form 3,5-substituted aryl derivative
of 1,3,4-oxadiazole-2-thione by continuous stirring at room
General
All the chemicals used in synthesis were supplied from
Merck and SRL fine Chemicals, Mumbai, and all are of
synthetic grade. Melting point of the synthesized com-
pounds was determined using a Digital Electrothermal
Melting point apparatus (VEEGO, VMP-DS) and are
uncorrected. For monitoring the chemical reactions and
purity of the intermediates and final compounds, thin layer
chromatography (TLC) was carried out using Silica gel G
coated plates with the appropriate solvent system of
Scheme 1 Synthetic pathway
for compounds 1e–5e
A
COOCH3
R
COOH
R
R'
1b-5b
R'
1a-5a
B
N
O
N
C
CONHNH2
R
R
SH
R'
R'
1d-5d
1c-5c
CH2
H
N
N
D
N
N
R''
R
R
O
S
O
S
R'
R'
1e-5e
Where,
A=CH3OH / H+
C=CS2 / KOH
B = NH2NH2. H2O
D = Formalin (HCHO) 40%, 2-Aminopyridine, Aniline
While,
For 1e, R= NO2 For 2e,
R'= H,
R= NO2
R'= H
R''= 2- Amino pyridine
R''= Aniline
For 3e, R= Cl
R'= Cl
For 4e, R= OH
R'= H
R''= 2- Amino pyridine R'= 2- Amino pyridine
R=CH
R'=H
For 5e,
3
R=
2-Amino pyridine
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Med Chem Res (2011) 20:1206–1213
5-Aryl-3-substituted 2,3-dihydro-1,3,4-oxadiazole-2-thione
[1e–5e]
different ratios. Then the plates were visualized under
ultraviolet light at a wavelength (k) of 254 nm to determine
the Rf value. IR spectra of all the synthesized compounds
were performed as KBr discs on a Shimazdzu 470 IR
spectrophotometer. The structure of all the compounds was
confirmed by 1H NMR spectra with Brucker DPX
300 MHz Instrument (DMSO-d6, CDCl3 and TMS) and
Elemental analysis using CHN analyzer 2400 ser II (Perkin
Elmer).
A 40% formalin (1.5 ml, 0.02 mol) was added to a stirred
solution of 5-aryl-2,3-dihydro-1,3,4-oxadiazole-2-thione
(0.02 mol) in absolute ethanol (40 ml). An ethanolic
solution (10 ml) of the appropriate amine (0.02 mol) was
added to a little portion at a time to the above reaction
mixture, stirred for 3 h at room temperature, and left
overnight in a refrigerator. The precipitate formed was
filtered, washed with cold ethanol, dried, and recrystallized
from ethanol.
Synthesis
Aryl substituted methyl benzoate [1b–5b]
Compound 1e: 5-(4-nitrophenyl)-3-[(2-pyridylamino)
methyl]-2,3-dihydro-1,3,4-oxadiazole-2-thione M.P.: 190°C
(61%); Rf = 0.43; solvents used for TLC: Ethanol:Benzene
(1:2); IR (KBr, Vmax cm-1) 3297, 3105 (N–H and C–H),
1622 (C=N), 1611 (C=C), 1549 (C–NO2), 1511 (C=C
aromatic stretch), 1381 (C=S), 1343 (C=N, aromatic 20
amine, stretch), 1144 (C–O–C). 1H NMR (DMSO-d6),
d ppm: 8.38–8.36 [d, 2H, J = 3 Hz, (5,6)nitrophenyl],
8.06–8. 04 [d, 2H, J = 3 Hz, (2,3)nitro-phenyl], 7.89, 7.91
[d, 1H (6) pyridine], 7.65, 7.68, 7.71 [t, NH, hydrazide],
6.60, 6.62 [d, 1H, (3) pyridine], 6.67, 6.67 [m, 2H,
(4,5)pyridine]. 5. 68–5.70 [d, 2H, N–CH2] Anal. Calcd. for
C14H11O3N5S: C, 51.06; H, 3.37; N, 21.27; found: C,
50.79; H, 3.35; N, 21.80.
In a clean, dried 250-ml round bottomed flask, methanol
(36 g, 45 ml, and 1.13 mol) and concentrated H2SO4
(1 ml) were taken. To this, the aryl substituted aromatic
acid (0.06 mol) was added and refluxed for 6 h. The
reaction mixture was then cooled to room temperature.
After cooling, crystals of the aryl substituted methyl ben-
zoate were filtered and washed successively with saturated
solution of sodium bicarbonate and water. It was then re-
crystallized from aqueous ethanol (Ladduwahetty et al.,
1996).
Aryl substituted benzoic acid hydrazide [1c–5c]
Hydrazine hydrate (6.18 g, 6 ml, and 0.12 mol) was placed
in a small, round bottomed flask fitted with a reflux con-
denser. Aryl substituted methyl benzoate (0.04 mol) was
added to this and gently heated under reflux for 10 min.
Sufficient quantity of absolute alcohol was added through
the condenser to get a clear solution (about 10 ml), then the
reaction mixture was refluxed further for 3 h. The product
obtained was concentrated and cooled to room temperature.
The concentrated solution was filtered to get the crystals of
acid hydrazide and recrystallized from aqueous ethanol.
Compound 2e: 5-(4-nitrophenyl)-3-[anilinomethyl]-2,3-
dihydro-1,3,4-oxadiazole-2-thione M.P.: 209°C (88%);
Rf = 0.62; solvents used for TLC: Ethanol:Benzene (1:4);
IR (KBr, Vmax cm-1) 3342–3061 (N–H and aromatic C–H
stretching), 1523 (C=N), 1597 (C=C), 1346 (C–NO2 aro-
matic), 1241 (C=S) and 1253 (C–O–C). 1H NMR (CDCl3),
d ppm: 8.08–8.05 [d, 2H, J = 3 Hz, (2,3)nitro-phenyl],
8.32, 8.34 [d, 2H, J = 2 Hz, (5,6)nitro-phenyl], 7.23,7.25
[d, 2H, (2,6) phenyl], 6.09, 6.92 [d, 2H, (3,5) phenyl], 6.81,
6.83 and 6.86 [t, 1H, (4) phenyl], 6.99, 7.02, 7.05, [t, NH,
hydrazide], 5.58–5.56 [d, 2H, methylene (N-CH2)]. Anal.
Calcd. for C15H12O3N5S: C, 54.87; H, 3.68; N, 17.06;
found: C, 54.84; H, 3.61; N, 16.61.
5-Aryl-2,3-dihydro-1,3,4-oxadiazole-2-thione [1d–5d]
In a clean, dried 250-ml round bottomed flask, ethanol
(120 ml) and potassium hydroxide (1.68 g, 0.03 mol, and
dissolved in 6 ml of water) were taken. To this, the aryl
substituted benzoic acid hydrazide (0.03 mol) was added.
Then to the above clear solution, carbon disulfide (22.8 g,
18 ml, and 0.6 mol) was added and refluxed for 4 h. A
solid appeared initially dissolved on heating. Then 60 ml of
ethanol was distilled off and cooled to room temperature.
The content was poured into water (*25 ml) and acidified
with ice cold concentrated HCl (3 ml, 0.03 mol). A solid
separated was filtered and washed with ice cold water. The
resulting compound 5-aryl-2,3-dihydro-1,3,4-oxadiazole-2-
thione was recrystallized with aqueous ethanol.
Compounds 3e: 5-(2,4-dichlorophenyl)-3-[(2-pyridylami-
no) methyl]-2,3-dihydro-1,3,4-oxadiazole-2-thione M.P.:
199°C (60%); Rf = 0.56; solvents used for TLC: Etha-
nol:Chloroform (1:2); IR (KBr, Vmax cm-1) 3450 (N–H and
C–H). 3060 (C–H, methylene). 2920 (C–H). 1645 (C=N).
1590 (C–C=C, stretch, aromatic). 1481 (C=S). 1247 and
1150 (C–O–C). 839 and 769 (C–Cl, aromatic).1H NMR
(DMSO-d6), d ppm: 7.59 [S, aromatic peak of proton
associated with C3 proton due to Cl] 8.04 [S, 1H, (6)
pyridine], 7.46, 7.49, 7.52 [t, 1H, hydrazide], 6.69, 6.72 [d,
1H, (3) pyridine], 6.59, 6.55, 6.59 [t, 2H, (4,5)pyridine].
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Med Chem Res (2011) 20:1206–1213
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Anal. Calcd. for C15H11ON3 CI2 S: C, 47.60; H, 2.85; N,
15.86; found: C, 6.71; H, 2.76; N, 15.08.
administered intraperitoneally (i.p.) to groups (III–VII) and
VIII, respectively, for 7 days at 24 h interval. On 8th day,
Ehrlich cells were harvested, washed five times with 0.9%
saline and resuspended in Dulbecco’s phosphate-buffered
saline (PBS). Ehrlich cells were counted microscopically at
100X magnification. Cell viability was confirmed by
exclusion of 0.25% trypan blue dye.
Compound 4e: 5-(4-hydroxyphenyl)-3-[(2-pyridylamino)
methyl]-2,3-dihydro-1,3,4-oxadiazole-2-thione M.P.: 168°C
(75%); Rf = 0.69; solvents used for TLC: Ethylace-
tate:Benzene (1:2); IR (KBr, Vmax cm-1): 3320–3236 (O–H
and N–H). 3007 (C–H, alkyl, methylene, stretch). 1603
(C=N). 1508 (C–C=C, aromatic, stretch). 1443 (C=S).
The evaluation of the test drug was made by comparing
the cell count of the test with that of control. The per-
centage inhibition of cell count was obtained by the for-
mula TCI = (1 - T/C) 9 100, where T is the average
number of Ascitic cells/ml in test animals and C is the
average number of the ascitic cells/ml in control animals.
The anticancer activity of the compounds was measured in
EAC-treated animals with respect to the following
parameters (Qureshi et al., 1993; Zarafoenetis, 1969;
Qureshi et al., 2001; Al-Harbi et al., 1995) such as,
1
1253 and 1163 (C–O–C, stretch). H NMR (DMSO-d6),
d ppm: 10.44 [s, 1H, OH], 8.04 [S, 1H, (6) pyridine],
7.66, 7.69 [d, 2H, (2,6)phenyl], 7.45, 7.48, 7.50 [t, 1H,
hydrazide], 6.91, 6.93 [d, 2H, (3,5)phenyl], 6.71, 6.73 [d,
1H, (3) pyridine], 6.52, 6.55, 6.58 [t, 2H, (4,5)pyridine],
5.61,5.64 [d, 2H, methylene].Anal. calcd. For C14H13
O2N4S: C, 55.99; H, 4.03; N, 18.65; found: C, 55.83; H,
3.78; N, 18.49.
Compound 5e: 5-(4-methylphenyl)-3-[(2-pyridylamino)
methyl]-2,3-dihydro-1,3,4-oxadiazole-2-thione M.P.: 205°C
(69%); Rf = 0.81; solvents used for TLC: Ethylacetate:
Benzene (1:2); IR (KBr, Vmax, cm-1): 3339 (N–H and
C–H, stretch). 1598, 1503 (C=C and C=N, stretch, pyri-
dine). 1426 (C=S) and 1253 (C–O–C, asymmetric stretch).
1H NMR (CDCl3), d ppm: 8.18 [s, 1H, (6) pyridine], 7.78,
7.80 [d, 2H, (2,6)phenyl], 7.26, 7.28 [d, 2H, (3,5)phenyl],
7.47, 7.50, 7.52 [t, 1H, NH, hydrazide], 5.83, 5.85 [d, 1H,
(3) pyridine], 5.83–5.85 [d, 2H, methylene]. 2.59 (S, 3H,
Ar–CH3). Anal. Calcd. for C15H13ON4S: C, 60.38; H, 4.73;
N, 18.7: found: C, 59.66; H, 4.52; N, 18.78.
Tumor volume and tumor weight
The mice were dissected, the ascitic fluid was collected
from the peritoneal cavity, and the tumor volume was
noted. The tumor weight was calculated from the differ-
ence in weight of mice before dissection and after collec-
tion of ascitic fluid.
Cancer is a pathological state where uncontrolled pro-
liferation of the cancer cells is found. The synthesized
compounds 1e–5e have reduced the growth of the Ehrlich
ascites carcinoma cells in mouse model. The efficacy of
the compounds was expressed in terms of percentage
inhibition of tumor formation and other relevant parame-
ters described based on anticancer activity. Among the test
compounds, compound 4e exhibited highest inhibition
(73.9%) of tumor at the dose of 50 mg/kg body weight
(i.p.) when compared with control. The standard drug
showed 93% inhibition. The rest of the compounds 1e, 2e,
3e, and 5e inhibited the tumor formation 60.8, 56.5, 52.2,
and 56.5%, respectively.
Biological testing
Anticancer activity
Determination of anticancer activity
The 10-week-old Swiss albino mice with an average body
weight of 18–20 g were used in the study. All the animals
were kept on basal metabolic diet after dividing into eight
groups of six animals each. The groups except normal
control (group I) were injected with 0.2 ml of 2 9 106
viable EAC cells in ice cold normal saline (0.9%) intra-
peritoneally (Aspirated from EAC bearing mice at the log
phase 7–8th day). Animals were allowed for 18 h incuba-
tion to establish the disease in the body before starting the
drug administration. On first day, 5 ml/kg body weight of
normal saline (0.9% W/V NaCl) and phosphate buffer (pH
7.2) was administered to group I (Normal) and group II
(EAC control), respectively. The synthesized compounds
[1e–5e] (50 mg/kg body weight/day) and the standard
drug (5-Fluorouracil) (20 mg/kg body weight/day) were
It was also observed that the volume of EAC was sig-
nificantly (P \ 0.001) reduced when the EAC implanted
animals treated with compounds (1e–5e) and standard drug
(5-Fluorouracil) for 7 days.
Viable and non-viable tumor cell count
The ascitic fluid was diluted 100 times, and the cell sus-
pension was mixed with the trypan blue and allowed to
stand for few minutes at room temperature. The cell sus-
pension was then placed over the Neubeauer counting
chamber, and the stained (Non viable) and unstained
(Viable) cells were counted in the 64 small squares.
The number of viable EAC cells was significantly
(P \ 0.001) reduced, whereas the nonviable cells were
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Med Chem Res (2011) 20:1206–1213
found to be increased in drug and standard treated animal
groups on comparison with the EAC control animals.
Treatment of synthesized compounds for the period of
7 days increased the survival time of the EAC-treated
animals significantly (P \ 0.01) as shown in Table 2.
Hematological parameters
Statistical analysis
The blood was collected by retro-orbital puncture under
anesthesia and performed the estimation of hematological
parameters like hemoglobin content (Hb), red blood cells
(RBC) count, and white blood cells (WBC) count.
The count of red blood cells and the content of hemo-
globin were observed to be restored significantly (P \
0.001) in all the drug-treated animals except group 1e
which was less significant (P \ 0.01) than that of the EAC
control. The reliable criteria for judging the value of any
anticancer drug is prolongation of life span and decrease of
WBC from blood (Latha and Panikkar, 1998). This fact
was supported from the result that the count of WBC was
also decreased significantly (P \ 0.001) than that of EAC
control.
The values were recorded as means SD. The data were
analyzed by using ANOVA; differences below the 0.001
level (P \ 0.05) were considered as statistically significant.
Discussion
It was clearly understood from the result that all final
[1e–5e] compounds and the standard drug exhibited a
prominent anticancer activity against the EAC implanted
mice models. According to the standard of National Cancer
Institute, a substance is considered active if it exhibits the
tumor growth inhibition by 50%. All the tested compounds
were found to show inhibition of tumor growth above 50%
which supports the efficacy of the oxadiazole derivatives to
serve as potent anticancer agents against EAC cells.
The tumorigenesis and its progression have been
reported to be accompanied by the following changes
compared with normal cytogenesis: (1) gradual decrease in
hemoglobin content and erythrocyte count, (2) gradual
increase in leukocytes, thrombocytes, and splenic cellu-
larity, and (3) reversal of the lymphoid–myeloid ratio in the
differential WBC count. It was observed in the study that
there was notable decrease in the hemoglobin level and
erythrocyte count in animals of EAC control group; how-
ever, the level was effectively improved and restored
toward the normal group by treatment with the synthesized
compounds [1e–5e]. The leukocytes count was drastically
increased in the tumor bearing mice which was signifi-
cantly brought back toward normal level on the treatment
(Tables 1, 2)
Survival time
The sets of animal groups allotted for observations on body
weight changes and survival were maintained separately.
These animals were observed for their weight changes and
mortality day-to-day until their death or up to a maximum
of 62 days. The effect of the compounds on life span was
measured by calculating median survival time (MST) and
average survival time (AST). The MST and AST were
calculated as follows: MST = (first death ? last death of
animal)/2; AST = sum of the animal death in different
days/N, where N is number of animals. The mortality rate
was calculated in all the groups of animals, and the
percentage increase in life span was calculated using the
following formula. Percentage Increase in Life Span
(%ILS) = Mean survival of treated group/Mean survival
of control group 9 100 and Mean Survival = (Day of first
death ? Day of last death)/2.
Table 1 Anticancer activity of oxadiazole derivatives against EAC bearing mice
Group
Compounds
RBC (106 cells)
WBC (103 cells)
Tumor volume (ml)
% of Viable cell
% of Non-viable cell
I
Normal Saline
11.04 0.33
4.47 0.15
–
–
–
II
Induce control
2.77 0.13
10.31 0.23
3.35 0.07
85.17 0.95
36.83 1.49***
33.50 1.15***
27.33 0.88***
22.50 0.99***
24.83 1.19***
14.83 1.19***
14.83 0.95
63.17 1.49
66.50 1.15
72.67 0.88
77.50 0.99
75.17 1.19
85.17 1.19
III
IV
V
1e
3.57 0.20*
7.73 0.21***
5.57 0.14***
4.47 0.12***
4.97 0.10***
4.62 0.14***
4.81 0.14***
1.60 0.06***
1.57 0.03***
0.92 0.08***
0.88 0.09***
0.60 0.04***
0.25 0.04***
2e
5.68 0.30***
7.23 0.07***
6.25 0.16***
8.27 0.16***
8.32 0.15***
3e
VI
VII
VIII
4e
5e
5-Fluorouracil
The results given are mean SEM; number of animal used (n = 6)
Experimental groups were compared with Induce control; * P \ 0.05, ** P \ 0.01, *** P \ 0.01
123

Med Chem Res (2011) 20:1206–1213
1211
The present study clearly demonstrated the tumor
inhibitory activity of the oxadiazole derivatives against
transplantable tumor cell line (Table 1). In the EAC bear-
ing mice, cells were present in the peritoneal cavity, and
the compounds were administered directly into the perito-
neum. Thus, tumor inhibition might be due to the direct
effect of the compounds on the tumor cells. The standard
drug 5-fluorouracil acts cytostatically by interfering with
nucleotide metabolism in S phase of the cell cycle. The
reduced tumor cell count and increased percentage of
nonviable cell count in the study led us to the assumption
that the test compounds might be acting by interfering with
S phase of cell cycle through apoptosis. However, the exact
molecular mechanism behind the action of these com-
pounds is yet to be ascertained.
Table 2 Anticancer activity of oxadiazole derivatives against EAC
bearing mice
Group
Compounds
HB
%IALS
%IMLS
I
Normal Saline
13.92 0.10
4.97 0.19
7.717 0.10
11.75 0.08
12.28 0.15
12.08 0.16
13.77 0.17
13.35 0.15
–
–
II
Induce control
–
–
III
IV
V
1e
38.54
46.35
53.64
80.72
73.95
92.70
36.92
44.61
47.69
76.92
73.84
90.76
2e
3e
VI
VII
VIII
4e
5e
5-Fluorouracil
P \ 0.001, when compare all treated groups with phosphate buffer
control in HB parameter. Other parameters are no need analysis by
ANOVA
In general, the anticancer activity seemed to be depen-
dent on the nature of the substituent rather the basic skel-
eton of the molecule (Adnan et al., 2007). Within the
oxadiazole series, it was noticed that the substituent at the
position 5 has great influence on the anticancer activity.
Substitution of various pharmacopores at this position may
give rise to the novel molecules with enhanced anticancer
properties. The activity of the compounds increased due to
the attachment of the phenyl ring with the substitution of
the electron withdrawing/donating group at 5th position of
the oxadiazole group and also due to the presence of the
Mannich side chain. Among the synthesized compounds,
those substituted with hydroxyl, methyl, and 2,4-dichloro
phenyl group showed significant anticancer activity com-
pared to other substituents. Another reason for the
increasing activity might be due to the chlorine atoms
which are expected to have more receptor binding capacity.
Compound 4e substituted with 2-amino pyridine
through a methylene bridge at 3rd position and by
p-hydroxy phenyl group at 5th position of the 1,3,4-oxa-
diazoles exhibited marked cytotoxicity to the EAC cells
which was confirmed from the percentage of non viable
Though all the compounds exhibited prominent anti-
cancer activity, the efficacy of the compounds against
different biochemical parameters studied varied from each
other. For example, the count of RBC, WBC, and hemo-
globin content levels in drug-treated animals have been
restored significantly when compared to the EAC control
values which is near to saline control value (Fig. 1).
However, compound 5e having substituted with o-amino
pyridine and p-methyl phenyl groups at 3rd and 5th posi-
tions of oxadiazole and 5-Flurouracil exhibited prominent
recovery in the RBC level and hemoglobin content than
those of other compounds, whereas the WBC level was
found to be decreased significantly in compounds 3e, 4e,
5e, and 5-Flurouracil-treated groups. One of the common
side effects associated with most of the treatment of anti-
cancer drugs is anemia and elevation of WBC level. The
study indicates that compounds [1e–5e] did not adversely
affect the process of haematopoiesis instead it was found to
be restored. This exhibits their efficacy as a potent source
for development of anticancer molecule with decreased or
negligible adverse effects.
Fig. 1 Anticancer activity of
oxadiazole derivatives against
EAC bearing mice
Effect of Compounds on heamatological parameters in EAC treated mice
16.00
14.00
12.00
10.00
8.00
I (Normal Saline)
II (Phosphate Buffer)
III (Compound (1e))
IV (Compound (2e))
V (Compound (3e))
VI (Compound (4e))
VII (Compound (5e))
VIII (5-fluorouracil)
6.00
4.00
2.00
0.00
RBC (million WBC (thousand
cells/µl) cells/µl)
HB (g/dl)
123

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(77.50 0.99) and viable cell (22.50 0.99) counts.
Replacement of p-hydroxyl group of phenyl ring at 5th
position with methyl group (5e) decreased the ratio of
percentage of non viable (75.17 1.19) and viable cell
(24.83 1.19) counts. It exhibited reduced cytotoxic
efficacy of the electron donating methyl group in the
molecule. Substitution of p-hydroxyl group of phenyl ring
with the electron withdrawing nitro group showed 63.17
1.49% and 36.83 1.14% of nonviable and viable cells
count. They were found to be less significant than those of
other compounds in the study. Inclusion of aniline group at
3rd position of oxadiazole ring through methylene linkage
and the p-nitro group of phenyl ring at 5th position pro-
duced 2e, and it showed nonviable (66.50 1.15%) and
viable (33.50 1.15%) cells count. Substitution of 2,
4-dichloro phenyl at 5th position of the compound 3e
showed 72.67 0.88% of nonviable and 27.33 0.88%
of viable cells.
The anticancer efficacy of the compounds was also
supported by the enhanced survival time of the EAC
bearing mouse (Table 2). Compounds 4e and 5e were
found to increase % IALS and % IMLS more significantly.
Hence, it can be concluded that the Mannich base deriva-
tives of oxadiazoles are effective source of novel drug
development for cancer.
Conclusion
A series of 3,5-disubstituted 1,3,4-oxadiazole-2-thione
derivatives possessing Mannich base have been synthe-
sized using simple synthetic procedures, and their anti-
cancer activity against EAC cancer cell has been evaluated.
All the final compounds exhibited good anticancer activity.
Compound 4e with the 73.9% of tumor inhibition was
found to be the most active among all. This might be due to
the hydrophilic p-hydroxyl group of phenyl ring substituted
at position 5. Further exploration of derivatives of this class
is on the progress. From the above experiments and
observations, it can be concluded that the 1,3,4-oxadiazole
derivatives are potential molecule to be developed as sig-
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Acknowledgments One of the authors is thankful to the All India
Council for Technical Education (AICTE) and University Grand
Commission (UGC) for providing financial support for the comple-
tion of this project and the authority of Jadavpur University for
providing the laboratory facility.
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