2,5-Disubstituted-1,3,4-oxadiazoles/thiadiazole as surface recognition moiety: Design and synthesis of novel hydroxamic acid based histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.08.022

The enzymatic inhibition of histone deacetylase activity has come out as a novel and effectual means for the treatment of cancer. Two novel series of 2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/thiadiazol-2-ylamino] -pyrimidine-5-carboxylic acid (tetrahydro-pyran-2-yloxy)-amides were designed and synthesized as novel hydroxamic acid based histone deacetylase inhibitors. The antiproliferative activities of the compounds were investigated in vitro using histone deacetylase inhibitory assay and MTT assay. The synthesized compounds were also tested for antitumor activity against Ehrlich ascites carcinoma cells in Swiss albino mice. The efforts were also made to establish structure-activity relationships among synthesized compounds. The results of the present studying indicates 2,5-disubstituted 1,3,4-oxadiazole/thiadiazole as promising surface recognition moiety for development of newer hydroxamic acid based histone deacetylase inhibitor.

Full text of DOI:10.1016/j.bmcl.2011.08.022

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5735–5738
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,5-Disubstituted-1,3,4-oxadiazoles/thiadiazole as surface recognition
moiety: Design and synthesis of novel hydroxamic acid based histone
deacetylase inhibitors
Harish Rajak a, , Avantika Agarawal b, Poonam Parmar a, Bhupendra Singh Thakur a,
⇑
Ravichandran Veerasamy c, Prabodh Chander Sharma d, Murli Dhar Kharya e
a Medicinal Chemistry Research Laboratory, SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur 495 009, CG, India
b Chhattisgarh Institute of Medical Sciences, Bilaspur 495 009, CG, India
c Faculty of Pharmacy, AIMST University, Semeling, Bedong 08100, Kedah Darul Aman, Malaysia
d Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136 119, Haryana, India
e Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, MP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The enzymatic inhibition of histone deacetylase activity has come out as a novel and effectual means for
the treatment of cancer. Two novel series of 2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/thiadiazol-2-
ylamino]-pyrimidine-5-carboxylic acid (tetrahydro-pyran-2-yloxy)-amides were designed and synthe-
sized as novel hydroxamic acid based histone deacetylase inhibitors. The antiproliferative activities of
the compounds were investigated in vitro using histone deacetylase inhibitory assay and MTT assay.
The synthesized compounds were also tested for antitumor activity against Ehrlich ascites carcinoma
cells in Swiss albino mice. The efforts were also made to establish structure–activity relationships among
synthesized compounds. The results of the present studying indicates 2,5-disubstituted 1,3,4-oxadiazole/
thiadiazole as promising surface recognition moiety for development of newer hydroxamic acid based
histone deacetylase inhibitor.
Received 25 May 2011
Revised 31 July 2011
Accepted 3 August 2011
Available online 8 August 2011
Keywords:
1,3,4-Oxadiazoles
1,3,4-Thiadiazoles
Anticancer activity
Histone deacetylase inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Epigenetics indicates to a phenomenon in which gene expres-
sion can be changed by reversible chemical modifications to DNA
or nucleosomal core histones. The acetylation of lysine side chains
of histone tails is one of the most studied epigenetic activity. It is
carried out by a family of histone acetyltransferases (HATs) and re-
versed by histone deacetylases (HDACs).1–4
The alterations in the structure or expression of HDACs have
been clearly linked to the pathogenesis of cancer and successively
HDAC inhibitors (HDACi) have emerged as a novel class of antican-
cer agents.5–7 HDACi selectively induce growth arrest, differentia-
tion, and apoptosis in tumor cells via transcriptional activation of
a small set of genes that control cell proliferation and cell cycle
development like p21.8–10
In 2006, Zolinza™ (SAHA, vorinostat) became the first HDACi to
acquire FDA approval and is used for the management of the cuta-
neous manifestations of T-cell lymphoma. HDACi generally con-
form to a broadly accepted pharmacophore. The crystallographic
studies for the design of valuable HDACi have pointed out three
structural requirements11: a cap group (A) that provides interac-
tion with the pocket entrance, a terminal group (B) that can bind
to the zinc ion at the bottom of the active site, also known as zinc
binding group (ZBG), and between both, a linker (C) fitting the
tube-like portion of the binding pocket12 (Fig. 1).
The common ZBG of HDACi is the hydroxamate moiety. Its
structural modifications have been successful with isosteres such
as benzamide, electrophilic ketones, mercaptoamide, a-ketoesters,
and phosphonates.13–15 Hence, the cap group offers an alternative
prospect to discover potent and more selective HDACi. In the pres-
ent study, 2,5-disubstituted-1,3,4-oxadiazole/thiadiazole has been
employed as cap group in hydroxamate based HDACi possessing
pyrimidine as a cyclic linker. This cap group was selected with
two major concepts, that is, it could serve as an isostere to amide,
ketone, and other groups possessing troublesome pharmacokinetic
and toxic properties. Secondly, it could result in better cap group
interactions with the amino acid side chains at the opening of
the HDAC active site.
The 1,3,4-oxadiazole/thiadiazole nucleus represents an impor-
tant core structure found in biologically active small molecules re-
ported to possess anticancer activity.16–18 Like many other
heterocyclic aromatic structures, 2,5-disubstituted 1,3,4-oxadiaz-
ole/thiadiazole core provides numerous avenues for further chem-
ical modifications. Thus, it was interesting to design hydroxamic
acid based HDACi containing 1,3,4-oxadiazole/thiadiazole as sur-
face recognition moiety.
⇑
Corresponding author. Tel.: +91 9827911824.
E-mail address: harishdops@yahoo.co.in (H. Rajak).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.022

5736
H. Rajak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5735–5738
O
O
H
HN
OH
N
NH
NH
N
H
OH
O
B
C
B
C
A
SAHA
LBH
A
O
N
O
S
O
O
N
N
OH
N
H
OH
NH
N
N
H
NH
X
B
C
C
R
B
A
VII (a-v)
PXD101
A
Figure 1. Presence of pharmacophoric elements in clinically established drugs and VII (a–v). A, surface recognition moiety (green color); B, linker (black color); and C, zinc
binding group (red color).
The title compounds were prepared using the synthetic strategy
described in Scheme 1. The required semicarbazones II (a–k) and
thiosemicarbazones II (l–v) were synthesized from various aro-
matic aldehyde (I) according to reported procedure.19,20 The com-
pound II (a–k) were reacted with bromine (oxidative cyclization)
in the presence of sodium acetate resulted in the formation of cor-
responding 2-amino-5-Aryl-1,3,4-oxadiazoles III (a–k).21 The com-
pound II (l–v) were cyclized to 2-amino-5-Aryl-1,3,4-thiadiazoles
III (l–v) using ferric chloride.20 Compound III (a–v) were reacted
with 2-methylsulfonyl-5-pyrimidinecarboxylic acid ethyl ester to
yield 2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/thiadiazol-2-
ylamino]-pyrimidine-5-carboxylic acid ethyl ester IV (a–v). The
2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/thiadiazol-2-ylami-
no]-pyrimidine-5-carboxylic acid V (a–v) were synthesized from
IV (a–v) on saponification with sodium hydroxide. The compound
V (a–v) on reaction with O-(tetrahydro-2H-pyran-2-yl)hydroxyl-
amine yielded 2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/
thiadiazol-2-ylamino]-pyrimidine-5-carboxylic acid (tetrahydro-
pyran-2-yloxy)-amides VI (a–v). In the last step, title compounds
VII (a–v) were prepared by removal of the tetrahydropyranyl
(THP) protecting moiety in the presence of trifluoroacetic acid.22
All the synthesized compounds were tested for their ability to
inhibit HDAC-1 activity and antiproliferative activity by MTT as-
says. The HDAC-1 was chosen since it is widely implicated in both
transcriptional repression and chromatin remodeling. Test com-
pounds exhibited significant activity in HDAC-1 inhibitory assay
indicating that their anticancer property is due to inhibition of his-
tone deacetylase-1. Inhibition of HDAC-1 causes apoptosis leading
to anticancer activity.23 The Mitomycin-C was chosen as standard
drug in vivo anticancer studies as its mechanism of action involves
apoptosis.24 The assay of HDAC-inhibitory activity was assessed
using the commercially available HDAC fluorescence activity as-
say/drug discovery (Fluor de Lys) kit (AK-500, BIOMOL Research
Laboratories) according to the supplier’s protocol. The IC50 of SAHA
they have a high level of HDAC expression. The test compounds
were evaluated in vivo for their anticancer activity against Ehrlich
ascites carcinoma cells in Swiss albino mice as per reported proce-
dure.27,28 The tumor weight inhibition (TWI) and tumor cell inhibi-
tion (TCI) were measured as parameter for anticancer evaluation.
The results of in vitro anticancer studies shows that in oxadiaz-
ole series VII (a–k), compound VII (e) displayed maximum HDAC
inhibitory activity with an IC50 = 0.017
an IC50 = 0.28 M in HCT-116 cell proliferation assay, while in thia-
diazole series VII (l–v), compound VII (p) exhibited maximum
HDAC inhibitory activity with an IC50 = 0.018 M against HDAC-1
and an IC50 = 0.31 M in HCT-116 cell proliferation assay (Table 1).
lM against HDAC-1 and
l
l
l
In terms of potency of synthesized compounds, almost similar re-
sults were obtained during in vivo anticancer studies against Ehr-
lich ascites carcinoma cells in Swiss albino mice. The compound VII
(e) (%TWI = 85.7; %TCI = 77.7) and VII (p) (%TWI = 75.0;
%TCI = 76.8) were found most potent in oxadiazole and thiadiazole
series of compounds, respectively (Table 2).
On correlating the structures of the synthesized compounds
with their biological activity, it has been observed that compounds
bearing the groups like hydroxyl, methoxy on phenyl ring attached
to 1,3,4-oxadiazole/thiadiazole possess high potency in in vitro and
in vivo anticancer evaluation. On the other hand, replacement of
these groups with fluoro or chloro groups resulted in compounds
with lesser antiproliferative activity. On comparison of results, it
has been found that antitumor activity of test compounds changes
on varying para-substituted group on aryl moiety attached to
1,3,4-oxadiazole/thiadiazole as follows: hydroxy > methoxy >
methyl > amino > dimethylamino > nitro > chloro > fluoro > no
substitution. It is to be noted that para-chloro analogs, that is, VII
(b) and VII (m) showed greater potency than parent compound,
that is, VII (a) and VII (l) in HDAC inhibition and MTT assay. The
position of the substitution had dramatic effects on the HDAC
activity. Ortho- and meta-chloro analogs, that is, VII (h), VII (i),
VII (s), and VII (t) was found to be lesser active than para-chloro
analogs. This might be attributed to unfavorable steric effect
of meta- and ortho-substitution or to exceeding the optimum
and TSA was reported to be 0.290 and 0.016 l
M, respectively.25
The antiproliferative activity of test compounds was evaluated by
MTT assays.26 HCT-116 cells were chosen for MTT assays because

H. Rajak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5735–5738
5737
N
N
H
COOC2H5
O
NH2
a
b
N
O
N
NH2
X
N
R
CH
O
+
S
N
X
R
R
III (a-v)
H3C
II (a-v)
I
c
O
N
O
N
N
N
O
N
NH
N
CH3
d
H
O
N
X
NH
N
X
IV (a-v)
R
V (a-v)
R
e
3'
O
2'
N
3
N
4
O
N
OH
N
N
4'
5'
N
6''
3''
f
N
NH
1'
O
1''
H
N
2
N
6'
O
X
1
5''
NH
5
H
N
X
2''
VII (a-v)
R
4''
VI (a-v)
R
Compound Code
VII(a)
VII(b)
VII(c)
VII(d)
VII(e)
VII(f)
VII(g)
VII(h)
X
R
H
4-Cl
4-NO2
4-CH3
4-OH
4-F
4-OCH3
2-Cl
Compound Code
VII(l)
VII(m)
VII(n)
VII(o)
VII(p)
VII(q)
VII(r)
VII(s)
VII(t)
X
R
H
4-Cl
4-NO2
4-CH3
4-OH
4-F
4-OCH3
2-Cl
O
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
S
S
S
VII(i)
VII(j)
VII(k)
3-Cl
4-NH2
4-N(CH3)2 VII(v)
3-Cl
4-NH2
4-N(CH3)2
VII(u)
Scheme 1. Synthesis of 2,5-disubstituted-1,3,4-oxadiazoles/thiadiazole analogs. Reaction conditions: (a) semicarbazide hydrochloride/thiosemicarbazide hydrochloride,
CH3COONa, distilled water, rt, 67–85%; (b) FeCl3/Br2, CH3COONa, CH3COOH, 30 min stirring, rt, 57–81%; (c) NaH (60%), THF, RT, 12 h, 53–68%; (d) NaOH, C2H5OH, 80 °C, 2 h,
48–66%; (e) H2NAOATHP, EDC, HOBT, THF, RT, 18 h, 46–64%; (f) CF3COOH, CH3OH, RT, 45–65%.
lipophilic value of the substituents. An overview of the results of
anticancer evaluation shows that thiadiazole analogs were found
to possess more active than corresponding oxadiazole analogs.
Table 1
HDAC-1 inhibition and antiproliferative assay results
The structure of the title compounds fulfilled all the three phar-
S. No.
Compound
HDAC Inhibitory
MTT assaya HCT-116
(IC50 (lM))
macophoric structural requirements for HDAC inhibitors, that is,
hydroxamate group as zinc binding group, aminopyrimidine moi-
ety as linker and 2,5-disubstituted-1,3,4-oxadiazole/thiadiazole
moiety as surface recognition cap group. All the title compounds
exhibited varied degree of anticancer activity. It is the surface rec-
ognition moiety that is responsible for variation in anticancer
activity among different test compounds. The presence of different
substitutions in 2,5-disubstituted-1,3,4-oxadiazole/thiadiazole
might have facilitated stronger cap group interactions with the
amino acid side chains at the entrance of the HDAC active site.
In conclusion, we have designed and synthesized two novel ser-
ies of compounds bearing 2,5-disubstituted 1,3,4-oxadiazole/thia-
diazole moiety as a surface recognition moiety for development
of newer hydroxamic acid based HDACi. These results represent a
noteworthy step regarding the discovery of novel HDAC small mol-
ecule inhibitors.
activitya (IC50
(
lM))
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
VII (a)
VII (b)
VII (c)
VII (d)
VII (e)
VII (f)
VII (g)
VII (h)
VII (i)
VII (j)
VII (k)
VII (l)
VII (m)
VII (n)
VII (o)
VII (p)
VII (q)
VII (r)
VII (s)
VII (t)
VII (u)
VII (v)
SAHA
0.006
0.009
0.010
0.014
0.017
0.009
0.016
0.008
0.006
0.011
0.009
0.008
0.010
0.010
0.015
0.018
0.007
0.017
0.008
0.007
0.013
0.011
0.290
0.016
0.09
0.18
0.22
0.25
0.28
0.06
0.26
0.15
0.11
0.22
0.20
0.08
0.16
0.20
0.26
0.31
0.08
0.28
0.16
0.12
0.24
0.20
—
Acknowledgments
Two of the authors, Poonam Parmar and Bhupendra Singh
Thakur, are thankful to AICTE New Delhi, India for awarding Junior
Research Fellowship and financial assistance. The help rendered by
SAIF, CDRI Lucknow for elemental and spectral analysis is
gratefully acknowledged.
TSA
—
—, Not tested.
a
Values are means of observation recorded in two experiments.

5738
H. Rajak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5735–5738
Table 2
Anticancer activities of 1,3,4-oxadiazole/thiadiazole analogs in Swiss albino mice
Compound Average wt of Ascitic
fluid (control)
Average wt of Ascitic
fluid (test)a
% Inhibition of Ascitic Average no of cells/mL in
fluid (TWI)
Average no. of cells/mL % inhibition of
controla ꢁ105
testa ꢁ105
Ascitic cell (TCI)
VII (a)
VII (b)
VII (c)
VII (d)
VII (e)
VII (f)
VII (g)
VII (h)
VII (i)
VII (j)
VII (k)
VII (l)
VII (m)
VII (n)
VII (o)
VII (p)
VII (q)
VII (r)
VII (s)
VII (t)
VII (u)
VII (v)
MMCb
2.8 ( 0.3)
3.0 ( 0.7)
3.2 ( 0.4)
3.7 ( 0.3)
4.2 ( 0.2)
2.9 ( 0.5)
4.0 ( 0.5)
3.0 ( 0.3)
2.9 ( 0.5)
3.4 ( 0.2)
3.6 ( 0.4)
2.8 ( 0.7)
3.1 ( 0.5)
3.4 ( 0.5)
3.8 ( 0.2)
4.4 ( 0.3)
3.1 ( 0.5)
4.1 ( 0.3)
2.9 ( 0.4)
2.8 ( 0.2)
3.7 ( 0.5)
3.6 ( 0.3)
3.9 ( 0.2)
1.8 ( 0.5)
1.8 ( 0.2)
1.4 ( 0.5)
1.0 ( 0.3)
0.6 ( 0.3)
2.2 ( 0.2)
0.8 ( 0.5)
1.8 ( 0.4)
2.0 ( 0.5)
1.2 ( 0.5)
1.6 ( 0.5)
2.4 ( 0.2)
1.6 ( 0.3)
1.2 ( 0.5)
0.8 ( 0.5)
1.1 ( 0.4)
2.8 ( 0.2)
1.0 ( 0.4)
1.7 ( 0.5)
2.1 ( 0.5)
1.3 ( 0.3)
1.4 ( 0.2)
0.0 ( 0.0)
35.7
40.0
56.0
72.0
85.7
24.1
80.0
40.0
31.0
64.7
55.5
14.2
48.3
64.7
78.9
75.0
09.6
75.6
58.6
25.0
64.8
61.1
100
348.2 ( 3.2)
455.7 ( 5.2)
440.5 ( 5.6)
475.4 ( 6.7)
504.0 ( 3.5)
308.2 ( 5.2)
501.8 ( 5.0)
360.7 ( 6.3)
378.9 ( 5.2)
460.2 ( 3.2)
445.7 ( 6.6)
290.1 ( 6.7)
440.3 ( 3.5)
447.9 ( 3.5)
492.2 ( 6.7)
518.6 ( 5.6)
321.8 ( 3.2)
510.5 ( 5.6)
365.3 ( 3.2)
355.4 ( 5.6)
472.6 ( 5.0)
462.7 ( 6.3)
463.2 ( 8.4)
311.3 ( 3.6)
292.4 ( 3.2)
215.1 ( 5.6)
152.8 ( 4.8)
112.2 ( 2.2)
257.6 ( 5.5)
128.3 ( 3.4)
270.5 ( 7.4)
297.6 ( 5.1)
178.2 ( 4.8)
192.1 ( 5.1)
204.5 ( 5.1)
299.1 ( 4.8)
262.5 ( 7.4)
168.0 ( 2.2)
120.1 ( 3.4)
260.3 ( 2.2)
137.7 ( 4.5)
298.0 ( 4.8)
310.2 ( 4.5)
172.1 ( 6.8)
225.9 ( 5.5)
0.0 ( 0.0)
10.5
35.8
51.1
67.8
77.7
16.4
74.4
25.0
21.4
61.2
56.8
29.5
32.0
41.3
65.8
76.8
19.1
73.0
18.4
12.7
63.5
51.1
100.0
Abbreviations: TWI, tumor weight inhibition; TCI, tumor cell inhibition; MMC, Mitomycin-C.
a
Observation shown are mean of six readings and data in parenthesis indicates standard error of mean.
Mitomycin-C at a dose level of 1 mg/kg body weight was employed as standard, which showed 100% inhibition.
b
Compound VII (p) mp (°C) 246–247 °C; yield 61%; IR (cmꢀ1) (KBr) 3028.3
(Aromatic CAH str), 1601.4 and 1505.1 (Aromatic CAC str), 746.2 (CAS of 1,3,4-
thiadiazole nucleus), 1644.2 (C@N of 1,3,4-thiadiazole nucleus), 1690.5 (C@O
str of amide), 3327.7 (NAH str of amide), 3442.8 (OAH str); 1H NMR (300 MHz,
DMSO-d6, TMS, d ppm): 6.72–7.41 (m, 4H, ArH), 5.40 (s, 1H, NH), 8.74 (s, 2H,
CH of Pyridine), 11.04 (s, 1H, NHOH), 9.18 (s, 1H, NHOH), 5.19 (s, 1H, ArOH);
13C NMR (75 MHz, DMSO-d6, TMS, d ppm): 164.5 (C-2), 156.4 (C-5), 166.8 (C-
20), 158.6 (C-4’ and C-60), 115.7 (C-50), 129.4 (C-100), 128.3 (C-200 and C-600),
116.7 (C-300 and C-500), 156.3 (C-400), 163.3 (CONHOH); ESI-MS (Methanol) m/z
331.0 ([M+H]+).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.022.
References and notes
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Compound VII (g) mp (°C) 230–231 °C; yield 59%; IR (cmꢀ1) (KBr) 3040.4
(Aromatic CAH str), 1602.4 and 1502.3 (Aromatic CAC str), 1092.4 (CAO of
1,3,4-oxadiazole nucleus), 1669.3 (C@N of 1,3,4-oxadiazole nucleus), 1692.9
(C@O str of amide), 3329.5 (NAH str of amide), 3446.2 (OAH str), 1251.2 (CAO
of OCH3 group); 1H NMR (300 MHz, DMSO-d6, TMS, d ppm): 6.88–7.65 (m, 4H,
ArH), 5.49 (s, 1H, NH), 8.77 (s, 2H, CH of Pyridine), 11.06 (s, 1H, NHOH), 9.19 (s,
1H, NHOH), 3.71 (s, 3H, ArOCH3); 13C NMR (75 MHz, DMSO-d6, TMS, d ppm):
172.2 (C-2), 169.4 (C-5), 166.6 (C-20), 158.9 (C-40 and C-60), 115.5 (C-50), 129.2
(C-100), 128.4 (C-200 and C-600), 115.7 (C-300 and C-500), 162.1 (C-400), 163.2
(CONHOH), 56.2 (OCH3C6H5); ESI-MS (Methanol) m/z 329.1 ([M+H]+).
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passage after every 10 days. EAC cells 9 days old were employed for anticancer
evaluation of test compounds. Mitomycin-C at a dose level of 1 mg/kg body
weight was used as standard, which showed 100% inhibition. The compounds
were dissolved/suspended in phosphate buffered saline with pH 7.2. EAC cells
were collected from the donor mouse and suspended in sterile isotonic saline.
The EAC cells were counted under the microscope and adjusted to
10 ꢁ 106 cells/mL. On day zero, 0.1 mL of EAC cells per 10-g body weight of
the animal was injected (ip) followed by a day for incubation to permit
proliferation of the cells. Seven doses of compound (0.2 mmol/kg, 0.1 mL per
10-g body weight) were injected ip from the first day up to the seventh day
with 24-h intervals. Control group of animals received only vehicle. On eighth
day, food and water were withdrawn 6 h before sacrificing the animals. All the
animals were sacrificed; peritoneal fluid was collected for cell count. All the
fluid in the peritoneal cavity was wiped off with absorbent cotton. The weight
of the animals was measured before sacrificing and after removing the fluid
from their peritoneal cavity. The difference in weight is considered as tumor
weight. The percentage inhibition of cell count was calculated by equation as
follows: percentage inhibition of Ascitic cell (TCI) = [(1 ꢀ T/C) ꢁ 100];
percentage inhibition of Ascitic fluid (TWI) = [(1 ꢀ T/C) ꢁ 100].
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22. Compound VII (e) mp (°C) 234–236 °C; yield 48%; IR (cmꢀ1) (KBr) 3044.3
(Aromatic CAH str), 1604.8 and 1502.2 (Aromatic CCAC str), 1093.8 (CCAO of
1,3,4-oxadiazole nucleus), 1668.6 (C@N of 1,3,4-oxadiazole nucleus), 1692.8
(C@O str of amide), 3328.1 (NAH str of amide), 3440.9 (OAH str); 1H NMR
(300 MHz, DMSO-d6, TMS, d ppm): 6.78–7.51 (m, 4H, ArH), 5.42 (s, 1H, NH),
8.69 (s, 2H, CH of Pyridine), 11.13 (s, 1H, NHOH), 9.16 (s, 1H, NHOH), 5.16 (s,
1H, ArOH); 13C NMR (75 MHz, DMSO-d6, TMS, d ppm): 172.0 (C-2), 169.3 (C-5),
166.7 (C-20), 158.5 (C-40 and C-60), 115.9 (C-50), 129.5 (C-100), 128.6 (C-200 and C-
600), 117.3 (C-300 and C-500), 157.1 (C-400), 163.3 (CONHOH); ESI-MS (Methanol)
m/z 315.1 ([M+H]+).
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