Synthesis and evaluation of 2,5-di(4-aryloylaryloxymethyl)-1,3,4- oxadiazoles as anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.02.040

A series of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles 9a-j were obtained via multistep synthesis from hydroxybenzophenones 4a-e. The cytotoxicity of compounds 9a-j was evaluated against human leukemia cell lines (K562 and CEM). The compounds exhibited moderate to good anti-cancer activity with compounds 9b and 9i having a chloro group exhibiting the best activity (IC₅₀ = 10 μM). Compound 9i exhibited activity against both the cell lines and 9b only exhibited activity against CEM. Further, a lactate dehydrogenase (LDH) assay and DNA fragmentation studies of the compounds 9a-j were also performed.

Full text of DOI:10.1016/j.ejmech.2013.02.040

European Journal of Medicinal Chemistry 63 (2013) 536e543
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of 2,5-di(4-aryloylaryloxymethyl)-
1,3,4-oxadiazoles as anti-cancer agents
,
H.D. Gurupadaswamy a, V. Girish a, C.V. Kavitha b c, Sathees C. Raghavan c,
,
Shaukath Ara Khanum a
*
a Department of Chemistry, Yuvaraja’s College Mysore, University of Mysore, Mysore 570 006, India
b Department of Chemistry, Government College for Women Mandya, Mandya 571 401, India
c Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles 9aej were obtained via multistep synthesis
from hydroxybenzophenones 4aee. The cytotoxicity of compounds 9aej was evaluated against human
leukemia cell lines (K562 and CEM). The compounds exhibited moderate to good anti-cancer activity
Received 4 March 2012
Received in revised form
21 January 2013
Accepted 28 February 2013
Available online 14 March 2013
with compounds 9b and 9i having a chloro group exhibiting the best activity (IC50 ¼ 10
mM). Compound
9i exhibited activity against both the cell lines and 9b only exhibited activity against CEM. Further, a
lactate dehydrogenase (LDH) assay and DNA fragmentation studies of the compounds 9aej were also
performed.
Keywords:
Ó 2013 Elsevier Masson SAS. All rights reserved.
1,3,4-Oxadiazoles
Cytotoxicity
Apoptosis
1. Introduction
analogs like 5-fluorouracil and enzyme inhibitors like methotrexate
[8,9]. Other than that imatinib, a drug developed against the acti-
Cancer is a disease in which a group of cells display uncontrolled
growth in a body. Under normal conditions, cell death and cell
proliferation are balanced throughout the life of multicellular or-
ganisms [1]. The various types of mature cells in the body have a
specific life span. After the death of these cells, new cells are
generated by proliferation and differentiation into different types of
cells. Sometimes cells begin to divide in an uncontrolled manner
and they no longer respond to the growth regulatory mechanisms.
These cells divide and form a clump of cells producing a tumor or
neoplasm [2]. About 200 types of cancers affecting major organs like
lungs, brain, kidneys, colon, breasts and stomach have been iden-
tified. According to the National Cancer Institute, most can fit into
categories of carcinoma, sarcoma, leukemia, lymphoma, and central
nervous system cancers. The development of efficient, selective and
less toxic anti-cancer agents is a challenge in cancer research [3].
Studies have shown that modification in DNA and RNA through
mutation leads to altered protein production which is the main
cause for the development of cancer [4e7]. Nowadays the field of
drug discovery for cancer has expanded in such a way as to target
the disease in regulating DNA replication by using nucleotide
vated tyrosine kinases in chronic myelogenous leukemia is yet
another development in the field of cancer [10].
Heterocyclic compounds play an important role as anti-cancer
agents because of their excellent inhibitory activity against recep-
tor tyrosine kinases [11], raf kinases [12], protein tyrosine kinases
[13] and NADH oxidase [14], which play critical roles in many as-
pects of tumorigenesis. Among heterocyclic compounds, nitrogen
containing compounds like 1,3,4 oxadiazoles have exhibited broad
spectrum anti-tumor activity with an IC50 concentration range
from 7.21
mM to 25.87
mM against the HeLa cancer cell lines [15] and
with an IC50 of 9.3
mM against the DU145 cancer cell lines [16].
Oxadiazole analogs also inhibit bacterial growth by inhibiting DNA
replication or DNA transcription [17].
In continuation of our research work on anti-cancer agents [18,19]
and with the goal of discovering new anti-cancer agents, we have
focused on the synthesis of a novel 2,5-di(4-aryloylaryloxymethyl)-
1,3,4-oxadiazoles 9aej which possess anti-proliferative property by
inhibiting cell cycle and promoting apoptosis.
2. Chemistry
The synthesis of the hitherto unreported title compounds is as
outlined in Scheme 1. (4-Hydroxyaryl)aryl methanones commonly
* Corresponding author. Tel./fax: þ91 9901888755.
E-mail address: shaukathara@yahoo.co.in (S.A. Khanum).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.040

H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
537
known as hydroxybenzophenones 4aee were achieved in excellent
yield using benzoylation of compound 1 with benzoyl chloride
derivatives 2aee followed by Fries rearrangement of substituted
arylbenzoates 3aee. Compounds 4aee on reaction with ethyl
bromoacetate afforded ethyl 4-aryloylaryloxyacetates 5aee which
on treatment with sodium hydroxide in presence of THF gave 4-
aryloylaryloxyethanoic acids 6aee. Further, compounds 5aee on
treatment with hydrazine hydrate in the presence of ethanol yield
4-aryloylaryloxyacethydrazides 7aee. Condensation of 6aee with
7aee in the presence of 2,6 lutidine, O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU) and
dichloromethane (DCM) afforded N,N-di(2-(4-aryloylaryloxy)
acetyl)hydrazines 8aej. Finally, title compounds 9aej were
achieved by intramolecular cyclization of 8aej in the presence of
triflic anhydride, pyridine and DCM.
3. Pharmacology
The human leukemia cells, K562 and CEM were selected for the
purpose of preliminary anti-cancer screening of newly synthesized
compounds 9aej. To assess the cytotoxicity, trypan blue dye
exclusion assay, MTT assay and LDH assay were employed. For this,
cells growing in log phase were treated with different concentra-
tions (10, 50, 100 and 250 mM) of the title compounds 9aej. Besides,
cytotoxicity of compounds 9aej on the growth of normal cells was
assessed using MTT assay.
R
OH
O
Cl
OH
F
Cl
F
Cl
DCM, TEA
RT, 3h
AlCl3
+
150°C, 1 h
O
F
O
O
R
1
Cl
2a-e
R
4a-e
3a-e
O
O
F
O
F
OH
R
O
R
O
BrCH2COOC2H5
10% NaOH, THF
RT, 1 h
6a-e
DMF, K2CO3
60° C, 3 h
Cl
Cl
N
H
O
O
N
H
O
.
H
+
O
5a-e
NH2
F
N
H
a: R=F
b: R=Cl
c: R=Br
d: R=I
R
O
7a-e
Cl
e: R=Me
O
2,6 Lutidine, TBTU,
DCM, RT, 12 h
O
F
F
O
H
N
R1
O
N
H
O
R2
a: R1=R2=F
O
Cl
b: R1=F, R2=Cl
c: R1=F, R2=Br
d: R1=F, R2=I
e: R1=F, R2=Me
f: R1=R2=Cl
Cl
O
Triflic anhyride, Pyridine
DCM, 0°C, 3 h
8a-j
g: R1=Cl, R2=Me
h: R1=Cl, R2=I
i: R1=Cl, R2=Br
j: R1=R2=Br
F
N
N
F
O
O
O
O
R1
Cl
Cl
9a-j
O
R2
Scheme 1. Synthesis of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles.

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H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
Table 1
4. Results and discussion
IC50 values of compounds 9aej as determined based on MTT assay.
4.1. Chemistry
Compound
IC50
(mM)
K562
CEM
1,3,4-Oxadiazole ring in the title compounds 9aej was con-
structed by cyclizing N,N-di(2-(4-aryloylaryloxy)acetyl)hydrazines
8aej in the presence of pyridine and triflic anhydride in DCM. The
structures of the compounds were elucidated by IR, 1H NMR and
mass spectral studies and microanalyses.
9a
9b
9c
9d
9e
9f
9g
9h
190 ꢀ 10.0
16 ꢀ 2.8
60 ꢀ 5.5
75 ꢀ 7.2
22 ꢀ 4.4
73 ꢀ 7.0
122 ꢀ 10.0
73 ꢀ 6.0
10 ꢀ 2.1
20 ꢀ 2.8
28 ꢀ 3.8
195 ꢀ 12.2
10 ꢀ 3.5
62 ꢀ 6.1
78 ꢀ 7.6
24 ꢀ 4.8
75 ꢀ 7.2
125 ꢀ 10.4
74 ꢀ 6.5
12 ꢀ 2.2
20 ꢀ 3.0
32 ꢀ 4.1
4.2. Biology
9i
9j
Newly synthesized 1,3,4-oxadiazole analogs 9aej were assessed
for cytotoxicity against two human leukemia cell lines, K562
(Chronic myelogenous leukemia) and CEM (T-cell leukemia). The
effective concentrations of compounds 9aej required to inhibit
K562 and CEM cell growth and survival were determined by car-
rying out dose response experiments using a trypan blue dye
exclusion assay. The cell viability was further assessed by MTT assay
and 5-fluorouracil treated cells were used as a positive control. The
5-Fluorouracil
with increasing concentrations of compounds 9b and 9i chromo-
somal DNA was extracted and used for agarose gel electrophoresis.
The result of this assay showed that DNA fragmentation leads to a
smear formation in gel lanes in which cells treated with com-
pounds 9b and 9i (Fig. 2). Number of breaks in the chromosomal
DNA of K562 cells resulted in smearing. The intensity of smear
cells with DMSO (equivalent to DMSO used in 250 mM) were used as
vehicle control, since the compounds 9aej were dissolved in
DMSO. The number of viable cells decreased at different points of
time and concentrations on exposure to compounds 9aej (Fig. 1).
Compounds 9aej did not exhibit any significant inhibition of cell
increased with the dose, 50 mM showing moderate and 100 mM
showing maximum smearing (Fig. 2). This suggests that com-
pounds 9b and 9i induce fragmentation of chromosomal DNA
leading to apoptosis.
proliferation at a concentration of 10
mM against both cell lines. The
cell viability at both 48 and 72 h were affected at 50 and 100
m
M
5. Conclusion
concentrations for compounds 9bef and 9hej. Compounds 9b with
fluoro and chloro groups, 9e with fluoro and methyl groups, 9i with
chloro and bromo groups and 9j with two bromo groups in the two
different benzophenone moieties showed complete inhibition at 48
In summary, a series of 1,3,4 oxadiazoles 9aej were synthesized
and evaluated for anti-proliferative activity against human
leukemic cell lines. From the current investigation, structural ac-
tivity relationship of these compounds suggests that the position
and the type of substituent on the aromatic ring in 9aej are
important for activity. Compounds 9b and 9i with chloro group play
a dominant role in inhibiting the leukemic cell proliferation.
Further, a detailed investigation on the structural activity rela-
tionship should entail structural modification on the aromatic ring
for the discovery of more potent cytotoxic compounds. Studies on
the mechanism of action of the title compounds and modification
are under progress.
and 72 h treatment at a 250
line (Fig. 1). Compound 9i was most effective at 50
m
M concentration against the CEM cell
m
M exhibiting
complete inhibition. However the DMSO control did not show any
significant toxic effect. Compounds 9a with two fluoro groups in
the two different benzophenone moieties and 9g with chloro and
methyl groups in the two different benzophenone moieties showed
moderate inhibitory activity. The electron withdrawing halo groups
at the para position in the benzophenone moieties are important
for enhancing the inhibitory activity while the electron releasing
methyl group at the para position decreases the activity as seen in
9e and 9g (Table 1).
Release of LDH serves as a marker for membrane integrity.
Hence, LDH release assay was performed to test the cell damage by
compounds 9b, 9e, 9i and 9j. Results showed a dose- and time-
dependent increase in LDH (Fig. 1), which further confirmed our
results.
Oligonucleosomal DNA fragmentation and nuclear condensa-
tion are the criteria to analyze the DNA damage consequent upon
treatment with compounds 9b and 9i. From the K562 cells treated
6. Experimental section
Chemicals were purchased from Sigma Aldrich Chemical Co. TLC
was performed on aluminum-backed silica plates and visualized by
UV-light. Melting points were determined on a Thomas Hoover
capillary melting point apparatus with a digital thermometer. IR
spectra were recorded on FT-IR Shimadzu 8300 spectrophotometer,
1H NMR spectra were recorded on a Bruker 400 MHz NMR
Fig. 1. Time-and dose dependent LDH release in K562 cells treated with compounds 9b, 9e, 9i and 9j. Cells were incubated for 24 and 48 h with different concentrations of
compounds 9b, 9e, 9i and 9j. Release of LDH in the medium was measured at 490 nm. Results are presented as percentage of LDH release.

H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
539
Compounds 3bee were synthesized analogously starting with
2bee respectively.
3b: Yield: 95%; m.p.: 52.1e53.5 ꢁC; IR (KBr) nmax (cmꢃ1): 1750
(ester, C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 7.39e7.51
(m, 3H, AreH), 7.69e7.71 (d, 2H, AreH), 8.16e8.17 (d, 2H, AreH).
MS (EI): m/z (72%) Mþ 285; Anal. Calcd. for C13H7Cl2FO2 (285): C,
54.77; H, 2.47; Cl, 24.87; F, 6.66. Found: C, 54.57; H, 2.33; Cl, 24.64;
F, 6.42%.
3c: Yield: 97%; m.p.: 67.2e68.7 ꢁC; IR (KBr) nmax (cmꢃ1): 1710
(ester, C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 7.40e7.53
(m, 3H, AreH), 7.85e7.87 (d, 2H, AreH), 8.08e8.10 (d, 2H, AreH).
MS (EI): m/z (70%) Mþ 329.5; Anal. Calcd. for C13H7BrClFO2
(329.5): C, 47.38; H, 2.14; Br, 24.25; Cl, 10.76; F, 5.76. Found: C, 47.18;
H, 2.29; Br, 24.36; Cl, 10.51; F, 5.58%.
3d: Yield: 93%; m.p.: 78.4e79.2 ꢁC; IR (KBr) nmax (cmꢃ1): 1765
(ester, C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 7.39e7.54
(m, 3H, AreH), 7.66e7.97 (d, 2H, AreH), 8.03e8.06 (d, 2H, AreH).
MS (EI): m/z (74%) Mþ 376.5; Anal. Calcd. for C13H7ClFIO2 (376.5):
C, 41.47; H,1.87; Cl, 9.42; F, 5.05; I, 33.70. Found: C, 41.29; H,1.68; Cl,
9.31; F, 5.23; I, 33.53%.
3e: Yield: 96%; m.p.: 62.0e63.1 ꢁC; IR (KBr) nmax (cmꢃ1): 1780
(ester, C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 7.38e7.51
Fig. 2. Detection of DNA damage induced by 9ior 9j in K562 cells. The chromosomal
DNA was extracted from K562 cells treated with different concentrations of 9i (A) and
9j (B). The purified DNA was then resolved on a 1% agarose gel at 30 V for 6 h. In both
(m, 3H, AreH), 7.6e7.9 (d, 2H, AreH), 8.05e8.07 (d, 2H, AreH).
MS (EI): m/z (78%) Mþ 264.5; Anal. Calcd. for C14H10ClFO2 (264.5):
C, 63.53; H, 3.81; Cl, 13.39; F, 7.18. Found: C, 63.69; H, 3.71; Cl, 13.09;
F, 7.30%.
panels, lane 1: DMSO; lane 2e4: K562 cells treated with 10, 50 and 100
tively. “M” is marker.
mM, respec-
6.1.2. General procedure for (4-hydroxyaryl)aryl methanones (4aee)
Compound 3aee (0.1903 mol) and aluminum chloride
(0.5388 mol) were blended and the mixture was heated to 150 ꢁC
and this temperature was maintained for 1 h. Then the reaction
mixture was cooled to 0 ꢁC and quenched with 6 N hydrochloric
acid (200 mL) and extracted with DCM (3 ꢂ 100 mL). The organic
layer was washed with water (3 ꢂ 40 mL), brine (3 ꢂ 30 mL) and
again with water (3 ꢂ 40 mL). The organic layer was dried over
anhydrous sodium sulfate and the solvent was evaporated to afford
compounds 4aee.
spectrophotometer in DMSO-d6 and the chemical shifts were
recorded in parts per million down field from tetramethylsilane.
Mass spectra were obtained with a VG70-70H spectrophotometer
and important fragments are given with the relative intensities
in brackets. Results of elemental analysis are within 0.4% of the
calculated value.
6.1. Synthesis
6.1.1. General procedure for substituted arylbenzoates (3aee)
2-Chloro-6-fluoro phenol (1, 0.2054 mol) was dissolved in DCM,
triethylamine (TEA, 0.4519 mol) was added and the reaction
mixture was cooled to 0 ꢁC. A solution of benzoyl chloride de-
rivatives (2aee, 0.2157 mol) in DCM was added slowly to the above
mixture and stirred for 3 h. Then the reaction mass was diluted
with DCM (200 mL), washed with 10% sodium hydroxide solution
(3 ꢂ 30 mL), water (3 ꢂ 30 mL), brine (2 ꢂ 60 mL), and again with
water (3 ꢂ 30 mL). The organic layer was dried over sodium sulfate
and the solvent was evaporated to achieve compounds 3aee.
6.1.2.1. Synthesis of (3-chloro-5-fluoro-4-hydroxyphenyl)4-
fluorophenyl methanone (4a). Compound 3a (51 g, 0.1903 mol)
and aluminum chloride (71.05 g, 0.5388 mol) were blended and
the mixture was heated to 150 ꢁC and this temperature was
maintained for 1 h. Then the reaction mixture was cooled to 0 ꢁC
and quenched with 6 N hydrochloric acid (200 mL) and extracted
with DCM (3 ꢂ 100 mL). The combined organic layer was washed
with water (3 ꢂ 40 mL), brine (3 ꢂ 30 mL) and again with water
(3 ꢂ 40 mL). The organic layer was dried over anhydrous sodium
sulfate and the solvent was evaporated to afford compound 4a as
pale yellow solid.
6.1.1.1. Synthesis of 2-chloro-6-fluorophenyl-4-fluorobenzoate (3a).
2-Chloro-6-fluoro phenol (1, 30 g, 0.2054 mol) was dissolved in
DCM, triethylamine (TEA, 45.73 g, 0.4519 mol) was added and the
reaction mixture was cooled to 0 ꢁC. A solution of 4-fluorobenzoyl
chloride (2a, 33.9 g, 0.2157 mol) in DCM was added slowly to the
above mixture and internal temperature was maintained to 0e
10 ꢁC. Finally the reaction mixture was stirred at ambient temper-
ature for 3 h. Then the reaction mass was diluted with DCM
(200 mL), washed with 10% sodium hydroxide solution (3 ꢂ 30 mL),
water (3 ꢂ 30 mL), brine (2 ꢂ 60 mL), and again with water
(3 ꢂ 30 mL). The organic layer was dried over sodium sulfate and
the solvent was evaporated to achieve compound 3a as white solid.
Yield: 94%; m.p.: 52.6e54.1 ꢁC; IR (KBr) nmax (cmꢃ1): 1738 (ester,
Yield: 61%; m.p.: 146.3e147.7 ꢁC; IR (KBr) nmax (cmꢃ1): 1671 (C]
O), 3545e3635 (OH); 1H NMR (400 MHz) (DMSO-d6)
d (ppm):
7.36e7.82 (m, 6H, AreH), 11.64 (bs, 1H, OH). MS (EI): m/z (83%): Mþ
268.5; Anal. Calcd. for C13H7ClF2O2 (268.5): C, 58.12; H, 2.63; Cl,
13.20; F, 14.14. Found: C, 58.21; H, 2.52; Cl, 13.20; F, 14.25%.
Compounds 4bee were synthesized analogously starting with
3bee respectively.
4b: Yield: 68.6%; m.p.: 167.8e169.1 ꢁC; IR (KBr) nmax
(cmꢃ1):1660 (C]O), 3525e3625 (OH); 1H NMR (400 MHz) (DMSO-
d6) d (ppm): 7.52e7.94 (m, 6H, AreH), 11.60 (bs,1H, OH). MS (EI): m/
z (80%): Mþ 285; Anal. Calcd. for C13H7Cl2FO2 (285): C, 54.77; H,
2.47; Cl, 24.87; F, 6.66. Found: C, 54.65; H, 2.32; Cl, 24.711; F, 6.53%.
4c: Yield: 71%; m.p.: 172.1e173.3 ꢁC; IR (KBr) nmax (cmꢃ1): 1610
C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 7.42e7.53 (m, 4H,
AreH), 8.25e8.28 (m, 3H, AreH); MS (EI): m/z (75%) Mþ 268.5;
Anal. Calcd. for C13H7ClF2O2 (268.5): C, 58.12; H, 2.63; Cl, 13.20; F,
14.14. Found: C, 58.22; H, 2.43; Cl, 13.30; F, 14.29%.
(C]O), 3495e3580 (OH); 1H NMR (400 MHz) (DMSO-d6)
d (ppm):
7.54e7.79 (m, 6H, AreH), 11.63 (bs, 1H, OH). MS (EI): m/z (81%): Mþ
329.5; Anal. Calcd. for C13H7BrClFO2 (329.5): C, 47.38; H, 2.14; Br,

540
H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
24.25; Cl, 10.76; F, 5.76. Found: C, 47.46; H, 2.29; Br, 24.41; Cl, 10.59;
F, 5.62%.
6.1.4. General procedure for 4-aryloylaryloxyethanoic acids (6aee)
A mixture of compounds 5aee (0.0532 mol), 10% aqueous so-
dium hydroxide solution (100 mL) and THF (100 mL) was stirred at
room temperature for 1 h. The reaction mass was acidified with 6 N
hydrochloric acid (150 mL) and the aqueous layer was extracted
with ethyl acetate (3 ꢂ 100 mL). The organic layer was washed with
brine (3 ꢂ 60 mL), dried over anhydrous sodium sulfate and
concentrated to achieve compounds 6aee.
4d: Yield: 65%; m.p.: 182.1e183.2 ꢁC; IR (KBr) nmax (cmꢃ1): 1635
(C]O), 3430e3590 (OH); 1H NMR (400 MHz) (DMSO-d6)
d (ppm):
7.46e7.8 (m, 6H, AreH), 11.60 (bs, 1H, OH). MS (EI): m/z (79%): Mþ
376.5; Anal. Calcd. for C13H7ClFIO2 (376.5): C, 41.47; H, 1.87; Cl, 9.42;
F, 5.05; I, 33.70. Found: C, 41.32; H, 1.71; Cl, 9.58; F, 5.21; I, 33.61%.
4e: Yield: 68%; m.p.: 198.1e199.5 ꢁC; IR (KBr) nmax (cmꢃ1): 1640
(C]O), 3580e3685 (OH); 1H NMR (400 MHz) (DMSO-d6)
d (ppm):
3.03 (s, 3H, CH3), 7.2e7.6 (m, 6H, AreH), 11.52 (bs, 1H, OH). MS (EI):
m/z (85%): Mþ 264.5; Anal. Calcd. for C14H10ClFO2 (264.5): C, 63.53;
H, 3.81; Cl, 13.39; F, 7.18. Found: C, 63.41; H, 3.72; Cl, 13.22; F, 7.29%.
6.1.4.1. Synthesis of [4-(4-fluorobenzoyl)-2-chloro-6-fluorophenoxy]
ethanoic acid (6a). A mixture of compound 5a (18 g, 0.0532 mol),
10% aqueous sodium hydroxide solution (100 mL) and THF
(100 mL) was stirred at room temperature for 1 h. The reaction
mass was acidified with 6 N hydrochloric acid (150 mL) and the
aqueous layer was extracted with ethyl acetate (3 ꢂ 100 mL). The
organic layer was washed with brine (3 ꢂ 60 mL), dried over
anhydrous sodium sulfate and concentrated to achieve compound
6a as white solid.
6.1.3. General procedure for ethyl 4-aryloylaryloxyacetates (5aee)
To a solution of compounds 4aee (0.1156 mol) in dry DMF
(175 mL), potassium carbonate (0.3468 mol) and ethyl bromoace-
tate (0.1273 mol) were added and the reaction mass was heated to
60 ꢁC for 3 h. The reaction mass was diluted with ethyl acetate
(200 mL), potassium carbonate was filtered off and the bed was
washed with ethyl acetate (100 mL). The organic layer was washed
with water (3 ꢂ 30 mL), brine (2 ꢂ 40 mL), dried over sodium
sulfate and concentrated to yield compounds 5aee.
Yield: 92%; m.p.: 127.3e128.6 ꢁC; IR (KBr) nmax (cmꢃ1): 1660
(C]O), 1738 (acid C]O), 3470e3575 (acid OH); 1H NMR (400 MHz)
(DMSO-d6)
(s, 1H, COOH). MS (EI): m/z (55%): Mþ 326.5; Anal. Calcd. for
15H9ClF2O4 (326.5): C, 55.15; H, 2.78; Cl, 10.85; F, 11.63. Found: C,
d (ppm): 4.9 (s, 2H, OCH2), 7.3e7.87 (m, 6H, AreH), 13.1
C
6.1.3.1. Synthesis of ethyl [2-(4-fluorobenzoyl)-2-chloro-6-
fluorophenoxy]acetate (5a). To a solution of compound 4a (31 g,
0.1156 mol) in dry DMF (175 mL), potassium carbonate (47.83 g,
0.3468 mol) and ethyl bromoacetate (21.11 g, 0.1273 mol) were
added and the reaction mass was heated to 60 ꢁC and maintained
for 3 h. The reaction mass was diluted with ethyl acetate (200 mL),
potassium carbonate was filtered off and the bed was washed with
ethyl acetate (100 mL). The organic layer was washed with water
(3 ꢂ 30 mL), brine (2 ꢂ 40 mL), dried over sodium sulfate and
concentrated to yield compound 5a as brown pasty mass.
55.25; H, 2.61; Cl, 10.72; F, 11.49%.
Compounds 6bee were synthesized analogously starting with
5bee respectively.
6b: Yield: 90.6%; m.p.: 137.7e139.1 ꢁC; IR (KBr) nmax (cmꢃ1):
1640 (C]O), 1750 (acid C]O), 3480e3590 (acid OH); 1H NMR
(400 MHz) (DMSO-d6)
d (ppm): 4.85 (s, 2H, OCH2), 7.15e7.77 (m,
6H, AreH), 12.9 (s, 1H, COOH). MS (EI): m/z (53%): Mþ 343; Anal.
Calcd. for C15H9Cl2FO4 (343): C, 52.50; H, 2.64; Cl, 20.66; F, 5.54.
Found: C, 52.59; H, 2.54; Cl, 20.75; F, 5.39%.
6c: Yield: 92%; m.p.: 153.8e155.3 ꢁC; IR (KBr) nmax (cmꢃ1): 1655
Yield: 97%; IR (KBr) nmax (cmꢃ1): 1660 (C]O),1730 (ester, C]O);
(C]O),1760 (acid C]O), 3490e3595 (acid OH); 1H NMR (400 MHz)
1H NMR (400 MHz) (DMSO-d6)
d
(ppm): 1.16e1.22 (t, 3H, CH3),
(DMSO-d6)
(s, 1H, COOH). MS (EI): m/z (51%): Mþ 387.5; Anal. Calcd. for
15H9BrClFO4 (387.5): C, 46.48; H, 2.34; Br, 20.62; Cl, 9.15; F, 4.90.
d (ppm): 4.9 (s, 2H, OCH2), 7.25e7.88 (m, 6H, AreH),12.8
4.14e4.21 (q, 2H, CH2), 5.02 (s, 2H, OCH2), 7.64e7.8 (m, 6H, AreH).
MS (EI): m/z (59%): Mþ 354.5; Anal. Calcd. for C17H13ClF2O4 (354.5):
C, 57.56; H, 3.69; Cl, 9.99; F, 10.71. Found: C, 57.41; H, 3.52; Cl, 9.79;
F, 10.88%.
C
Found: C, 46.33; H, 2.46; Br, 20.52; Cl, 9.33; F, 4.79%.
6d: Yield: 88%; m.p.: 139.7e140.8 ꢁC; IR (KBr) nmax (cmꢃ1): 1615
Compounds 5bee were synthesized analogously starting with
4bee respectively.
(C]O),1770 (acid C]O), 3465e3575 (acid OH); 1H NMR (400 MHz)
(DMSO-d6)
(s, 1H, COOH). MS (EI): m/z (54%): Mþ 434.5; Anal. Calcd. for
15H9ClFIO4 (434.5): C, 41.46; H, 2.09; Cl, 8.16; F, 4.37; I, 29.20.
d (ppm): 4.8 (s, 2H, OCH2), 7.1e7.65 (m, 6H, AreH), 11.9
5b: Yield: 92%; IR (KBr) nmax (cmꢃ1): 1650 (C]O), 1740 (ester,
C]O); 1H NMR (400 MHz) (DMSO-d6)
d
(ppm): 1.18e1.22 (t, 3H,
C
CH3), 4.12e4.19 (q, 2H, CH2), 5.02 (s, 2H, OCH2), 7.59e7.75 (m, 6H,
AreH). MS (EI): m/z (58%): Mþ 371; Anal. Calcd. for C17H13Cl2FO4
(371): C, 55.01; H, 3.53; Cl, 19.10; F, 5.12. Found: C, 55.19; H, 3.41; Cl,
19.18; F, 5.23%.
Found: C, 41.33; H, 2.19; Cl, 8.29; F, 4.21; I, 29.41%.
6e: Yield: 89%; m.p.: 119.2e120.5 ꢁC; IR (KBr) nmax (cmꢃ1): 1630
(C]O), 1765 (acid C]O), 3415e3545 (acid OH); 1H NMR (400 MHz)
(DMSO-d6)
d (ppm): 2.4 (s, 3H, CH3), 4.96 (s, 2H, OCH2), 7.16e7.77
5c: Yield: 91%; IR (KBr) nmax (cmꢃ1): 1660 (C]O), 1765 (ester,
(m, 6H, AreH), 12.65 (s, 1H, COOH). MS (EI): m/z (53%): Mþ 322.5;
Anal. Calcd. for C16H12ClFO4 (322.5): C, 59.55; H, 3.75; Cl, 10.99; F,
5.89. Found: C, 59.63; H, 3.61; Cl, 10.78; F, 5.72%.
C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 1.18e1.22 (t, 3H,
CH3), 4.21e4.12 (q, 2H, CH2), 5.0 (s, 2H, OCH2), 7.38e7.85 (m, 6H,
AreH). MS (EI): m/z (57%): Mþ 415.5; Anal. Calcd. for C17H13BrClFO4
(415.5): C, 49.12; H, 3.15; Br, 19.22; Cl, 8.53; F, 4.57. Found: C, 49.28;
H, 3.01; Br, 19.13; Cl, 8.62; F, 4.47%.
6.1.5. General procedure for 4-aryloylaryloxyacethydrazides (7aee)
Hydrazine hydrate (0.3372 mol) was added to a solution of
compounds 6aee (0.0562 mol) in ethanol (100 mL) at 0 ꢁC and
stirred the reaction mixture at the same temperature for 1 h. A
white solid was separated out, which was quenched with water
(100 mL), filtered and washed with water (50 mL). Finally, solid was
dried under vacuum to obtain compounds 7aee.
5d: Yield: 94%; IR (KBr) nmax (cmꢃ1): 1605 (C]O), 1750 (ester,
C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 1.16e1.22 (t, 3H,
CH3), 4.14e4.21 (q, 2H, CH2), 5.0 (s, 2H, OCH2), 7.21e7.9 (m, 6H, Are
H). MS (EI): m/z (57%): Mþ 462.5; Anal. Calcd. for C17H13ClFIO4
(462.5): C, 44.13; H, 2.83; Cl, 7.66; F, 4.11; I, 27.43. Found: C, 44.23;
H, 2.72; Cl, 7.57; F, 4.23; I, 27.31%.
5e: Yield: 96%; IR (KBr) nmax (cmꢃ1): 1610 (C]O), 17,665 (ester,
6.1.6. Synthesis of 2-[4-(4-fluorobenzoyl)-2-chloro-6-fluoropheno-
xy]acethydrazide (7a)
C]O); 1H NMR (400 MHz) (DMSO-d6)
d (ppm): 1.19e1.23 (t, 3H,
CH3), 2.4 (s, 3H, AreCH3), 4.2e4.3 (q, 2H, CH2), 5.1 (s, 2H, OCH2),
7.1e7.55 (m, 6H, AreH). MS (EI): m/z (57%): Mþ 350.5; Anal. Calcd.
for C18H16ClFO4 (350.5): C, 61.63; H, 4.60; Cl, 10.11; F, 5.42. Found: C,
61.51; H, 4.52; Cl, 10.19; F, 5.29%.
Hydrazine hydrate (16.90 g, 0.3372 mol) was added to a solu-
tion of compound 6a (19 g, 0.0562 mol) in ethanol (100 mL) at
0 ꢁC and stirred the reaction mixture at the same temperature for
1 h. A white solid was separated out, which was quenched with

H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
541
water (100 mL), filtered and washed with water (50 mL). Finally,
solid was dried under vacuum to obtain compound 7a as white
needle.
for C30H18Cl2F4N2O6 (649): C, 55.49; H, 2.79; Cl, 10.92; F, 11.70; N,
4.31. Found: C, 55.37; H, 2.88; Cl, 10.83; F, 11.77; N, 4.43%.
Similarly compounds 8bej were synthesized starting from
compounds 6bee and 7aee.
Yield: 79%; m.p.: 107.5e109.1 ꢁC; IR (KBr) nmax (cmꢃ1): 1610 (C]
O), 1645 (amide, C]O), 3100e3205 (NHeNH2); 1H NMR (400 MHz)
8b: Yield: 78%; m.p.: 189.2e190.5 ꢁC; IR (KBr) nmax (cmꢃ1): 1680
(C]O), 1605 (amide, C]O), 3710e3510 (NHeNH); 1H NMR
(DMSO-d6) d (ppm): 4.35 (bs, 2H, NH2), 4.69 (s, 2H, OCH2), 7.2e7.86
(m, 6H, AreH), 9.32 (bs, 1H, CONH). MS (EI): m/z (42%): Mþ 340.5;
Anal. Calcd. for C15H11ClF2N2O3 (340.5): C, 52.88; H, 3.25; Cl, 10.41;
F, 11.15; N, 8.22. Found: C, 52.75; H, 3.38; Cl, 10.29; F, 11.24; N, 8.11%.
Compounds 7bee were synthesized analogously starting with
6bee respectively.
(400 MHz) (DMSO-d6) d (ppm): 4.85 (s, 4H, 2CH2), 7.15e7.85 (m,
12H, AreH),10.38 (bs, 2H, 2NH). MS (EI): m/z (60%): Mþ 665.5; Anal.
Calcd. for C30H18Cl3F3N2O6 (665.5): C, 54.12; H, 2.72; Cl, 15.97; F,
8.56; N, 4.21. Found: C, 54. 21; H, 2.63; Cl, 15.84; F, 8.42; N, 4.13%.
8c: Yield: 79%; m.p.: 209.1e210.4 ꢁC; IR (KBr) nmax (cmꢃ1): 1685
(C]O), 1610 (amide, C]O), 3720e3520 (NHeNH); 1H NMR
7b: Yield: 75.66%; m.p.: 141.4e142.9 ꢁC; IR (KBr) nmax (cmꢃ1):
1625 (C]O), 1655 (amide, C]O), 3150e3255 (NHeNH2); 1H NMR
(400 MHz) (DMSO-d6) d (ppm): 4.86 (s, 4H, 2CH2), 7.0e7.79 (m,12H,
(400 MHz) (DMSO-d6)
d
(ppm): 4.25 (bs, 2H, NH2), 4.70 (s, 2H,
AreH), 10.4 (bs, 2H, 2NH). MS (EI): m/z (61%): Mþ 710; Anal. Calcd.
for C30H18BrCl2F3N2O6 (710): C, 50.73; H, 2.55; Br, 11.25; Cl, 9.98; F,
8.02; N, 3.94. Found: C, 50.61; H, 2.47; Br, 11.13; Cl, 9.86; F, 8.16; N,
3.82%.
OCH2), 7.1e7.75 (m, 6H, AreH), 9.30 (bs, 1H, CONH). MS (EI): m/z
(43%): Mþ 357; Anal. Calcd. for C15H11Cl2FN2O3 (357): C, 50.44; H,
3.10; Cl, 19.85; F, 5.32; N, 7.84. Found: C, 50.31; H, 3.22; Cl, 19.71; F,
5.21; N, 7.72%.
8d: Yield: 78%; m.p.: 189.3e190.7 ꢁC; IR (KBr) nmax (cmꢃ1): 1675
7c: Yield: 66%; m.p.: 137.2e138.6 ꢁC; IR (KBr) nmax (cmꢃ1): 1635
(C]O), 1615 (amide, C]O), 3725e3525 (NHeNH); 1H NMR
(C]O), 1660 (amide, C]O), 3155e3270 (NHeNH2); 1H NMR
(400 MHz) (DMSO-d6) d (ppm): 4.85 (s, 4H, 2CH2), 7.1e7.75 (m, 12H,
(400 MHz) (DMSO-d6)
d
(ppm): 4.35 (bs, 2H, NH2), 4.70 (s, 2H,
AreH), 10.8 (bs, 2H, 2NH). MS (EI): m/z (59%): Mþ 757; Anal. Calcd.
for C30H18Cl2F3N2O6 (757): C, 47.58; H, 2.40; Cl, 9.36; F, 7.53; I,
16.76; N, 3.70. Found: C, 47.46; H, 2.49; Cl, 9.25; F, 7.61; I, 16.66; N,
3.79%.
OCH2), 7.2e7.65 (m, 6H, AreH), 9.32 (bs, 1H, CONH). MS (EI): m/z
(41%): Mþ 401.5; Anal. Calcd. for C15H11BrClFN2O3 (401.5): C, 44.86;
H, 2.76; Br, 19.90; Cl, 8.83; F, 4.73; N, 6.98. Found: C, 44.74; H, 2.62;
Br, 19.78; Cl, 8.71; F, 4.61; N, 6.85%.
8e: Yield: 84%; m.p.: 178.5e180.1 ꢁC; IR (KBr) nmax (cmꢃ1): 1690
7d: Yield: 78%; m.p.: 120.1e123.2 ꢁC; IR (KBr) nmax (cmꢃ1): 1615
(C]O), 1620 (amide, C]O), 3735e3530 (NHeNH); 1H NMR
(C]O), 1615 (amide, C]O), 3120e3250 (NHeNH2); 1H NMR
(400 MHz) (DMSO-d6)
d (ppm): 2.4 (s, 3H, CH3), 4.8 (s, 4H, 2CH2),
(400 MHz) (DMSO-d6)
d
(ppm): 4.38 (bs, 2H, NH2), 4.72 (s, 2H,
7.2e7.72 (m, 12H, AreH), 10.36 (bs, 2H, 2NH). MS (EI): m/z (58%):
Mþ 645; Anal. Calcd. for C31H21Cl2F3N2O6 (645): C, 57.69; H, 3.28;
Cl, 10.99; F, 8.83; N, 4.34. Found: C, 57.58; H, 3.36; Cl, 10.88; F, 8.74;
N, 4.25%.
OCH2), 7.15e7.55 (m, 6H, AreH), 9.32 (bs, 1H, CONH). MS (EI): m/z
(40%): Mþ 448.5; Anal. Calcd. for C15H11ClFIN2O3 (448.5): C, 40.16;
H, 2.47; Cl, 7.90; F, 4.23; I, 28.29; N, 6.24. Found: C, 40.03; H, 2.33; Cl,
7.79; F, 4.15; I, 28.18; N, 6.14%.
8f: Yield: 79%; m.p.: 193.2e194.4 ꢁC; IR (KBr) nmax (cmꢃ1): 1695
7e: Yield: 80%; m.p.: 78.3e79.7 ꢁC; IR (KBr) nmax (cmꢃ1): 1605
(C]O), 1605 (amide, C]O), 3720e3520 (NHeNH); 1H NMR
(C]O), 1610 (amide, C]O), 3135e3270 (NHeNH2); 1H NMR
(400 MHz) (DMSO-d6)
d (ppm): 4.88 (s, 4H, 2CH2), 7.12e7.73 (m,
(400 MHz) (DMSO-d6)
d
(ppm): 2.40 (s, 2H, CH3), 4.37 (bs, 2H, NH2),
12H, AreH), 10.38 (bs, 2H, 2NH). MS (EI): m/z (59%): Mþ 682; Anal.
Calcd. for C30H18Cl4F2N2O6 (682): C, 52.81; H, 2.66; Cl, 20.78; F,
5.57; N, 4.11. Found: C, 52.72; H, 2.55; Cl, 20.69; F, 5.66; N, 4.21%.
8g: Yield: 84%; m.p.: 178.3e179.8 ꢁC; IR (KBr) nmax (cmꢃ1): 1680
(C]O), 1615 (amide, C]O), 3735e3525 (NHeNH); 1H NMR
4.69 (s, 2H, OCH2), 7.05e7.69 (m, 6H, AreH), 9.31 (bs, 1H, CONH).
MS (EI): m/z (43%): Mþ 336.5; Anal. Calcd. for C16H14ClFN2O3
(336.5): C, 57.07; H, 4.19; Cl, 10.53; F, 5.64; N, 8.32. Found: C, 57.17;
H, 4.11; Cl, 10.41; F, 5.78; N, 8.47%.
(400 MHz) (DMSO-d6)
d (ppm): 2.3 (s, 3H, CH3), 4.86 (s, 4H, 2CH2),
6.1.7. General procedure for N,N-di(2-(4-aryloylaryloxy)acetyl)
hydrazines (8aej)
7.05e7.75 (m, 12H, AreH), 10.35 (bs, 2H, 2NH). MS (EI): m/z (61%):
Mþ 661.5; Anal. Calcd. for C31H21Cl3F2N2O6 (661.5): C, 56.26; H,
3.20; Cl, 16.07; F, 5.74; N, 4.23. Found: C, 56.35; H, 3.28; Cl, 16.15; F,
5.65; N, 4.31%.
To a solution of compounds 6aee (0.0032 mol) in DCM (20 mL),
2,6-dimethylpyridine (0.0107 mol) and TBTU (0.00323 mol) were
added at room temperature. Finally, compounds 7aee
(0.00294 mol) were added to the reaction mixture and stirred at
room temperature for 12 h. The reaction mixture was quenched
with 10% sodium bicarbonate solution (20 mL) and stirred for
30 min. The solid precipitate was filtered, washed with water
(20 mL) and dried to yield compounds 8aej.
8h: Yield: 77%; m.p.: 180.3e181.6 ꢁC; IR (KBr) nmax (cmꢃ1): 1685
(C]O), 1610 (amide, C]O), 3745e3530 (NHeNH); 1H NMR
(400 MHz) (DMSO-d6) d (ppm): 4.75 (s, 4H, 2CH2), 7.05e7.8 (m,12H,
AreH),10.6 (bs, 2H, 2NH). MS (EI): m/z (60%): Mþ 773.5; Anal. Calcd.
for C30H18Cl3F2IN2O6 (773.5): C, 46.57; H, 2.34; Cl, 13.75; F, 4.91; I,
16.40; N, 3.62. Found: C, 46.64; H, 2.26; Cl, 13.83; F, 4.84; I, 16.47; N,
3.55%.
6.1.7.1. Synthesis of N,N-di[di(2-chloro-6-fluoro-4-(4-fluoro-benzoyl)
phenoxy)]acetyl hydrazide (8a). To a solution of compound 6a
(1.05 g, 0.0032 mol) in DCM (20 mL), 2,6-dimethylpyridine (1 g,
0.0107 mol) and TBTU (1.04 g, 0.00323 mol) were added at room
temperature. Finally, compound 7a (1 g, 0.00294 mol) was added to
the reaction mixture and stirred at room temperature for 12 h. The
reaction mixture was quenched with 10% sodium bicarbonate so-
lution (20 mL) and stirred for 30 min. The solid precipitate was
filtered, washed with water (20 mL) and dried to yield compound
8a as white solid.
8i: Yield: 80%; m.p.: 182.4e183.8 ꢁC; IR (KBr) nmax (cmꢃ1): 1670
(C]O), 1600 (amide, C]O), 3715e3520 (NHeNH); 1H NMR
(400 MHz) (DMSO-d6) d (ppm): 4.85 (s, 4H, 2CH2), 7.0e7.79 (m,12H,
AreH), 11.0 (bs, 2H, 2NH). MS (EI): m/z (62%): Mþ 726.5; Anal. Calcd.
for C30H18BrCl3F2N2O6 (726.5): C, 49.58; H, 2.50; Br, 10.99; Cl, 14.64;
F, 5.23; N, 3.85. Found: C, 49.49; H, 2.56; Br, 10.89; Cl, 14.77; F, 5.31;
N, 3.78%.
8j: Yield: 82%; m.p.: 201.7e203.1 ꢁC; IR (KBr) nmax (cmꢃ1): 1685
(C]O), 1605 (amide, C]O), 3720e3535 (NHeNH); 1H NMR
(400 MHz) (DMSO-d6)
d (ppm): 4.88 (s, 4H, 2CH2), 6.9e7.71 (m,
Yield: 81%; m.p.: 194.8e196.2 ꢁC; IR (KBr) nmax (cmꢃ1): 1690
(C]O), 1610 (amide, C]O), 3700e3500 (NHeNH); 1H NMR
12H, AreH), 10.35 (bs, 2H, 2NH). MS (EI): m/z (61%): Mþ 771; Anal.
Calcd. for C30H18Br2Cl2F2N2O6 (771): C, 46.72; H, 2.35; Br, 20.72; Cl,
9.19; F, 4.93; N, 3.63. Found: C, 46.64; H, 2.42; Br, 20.65; Cl, 9.28; F,
4.82; N, 3.71%.
(400 MHz) (DMSO-d6)
d (ppm): 4.90 (s, 4H, 2CH2), 7.1e7.87 (m, 12H,
AreH), 10.36 (bs, 2H, 2NH). MS (EI): m/z (61%): Mþ 649; Anal. Calcd.

542
H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
6.1.8. General procedure for 2,5-di(4-aryloylaryloxymethyl)-1,3,4-
oxadiazoles (9aej)
C31H19Cl3F2N2O5 (643.5): C, 57.83; H, 2.97; Cl, 16.52; F, 5.90; N, 4.35.
Found: C, 57.74; H, 2.88; Cl, 16.41; F, 5.81; N, 4.26%.
To a solution of compounds 8aej (0.0023 mol) in DCM (20 mL),
pyridine (0.0069 mol) and triflic anhydride (0.0051 mol) were added
at 0 ꢁC and the reaction mixture was stirred at 0 ꢁC for 3 h. The re-
action mass was diluted with DCM (20 mL), the organic layer was
washed with 10% sodium bicarbonate (3 ꢂ 10 mL), water (3 ꢂ 10 mL)
and brine (3 ꢂ 10 mL). Finally, the organic layer was dried over so-
dium sulfate and concentrated to yield compounds 9aej.
9h: Yield: 71%; m.p.: 118.3e119.6 ꢁC; IR (Nujol) nmax (cmꢃ1): 1105
(CeOeC linkage), 1610 (C]O), 1690 (C]N); 1H NMR (400 MHz)
(DMSO-d6)
d (ppm): 5.55 (s, 4H, 2CH2), 7.12e7.72 (m, 12H, AreH).
MS (EI): m/z (69%): Mþ 755.5; Anal. Calcd. for C30H16Cl3F2IN2O5
(755.5): C, 47.68; H, 2.13; Cl, 14.07; F, 5.03; I, 16.79; N, 3.71. Found: C,
47.61; H, 2.22; Cl, 14.15; F, 5.14; I, 16.68; N, 3.63%.
9i: Yield: 73%; m.p.: 130.6e131.8 ꢁC; IR (Nujol) nmax (cmꢃ1): 1110
(CeOeC linkage), 1615 (C]O), 1685 (C]N); 1H NMR (400 MHz)
6.1.8.1. Synthesis of 2,5-di[2-fluoro-4-(4-fluoro)benzoyl-6-
chlorophenoxymethyl]1,3,4-oxadiazole (9a). To a solution of com-
pound 8a (1.5 g, 0.0023 mol) in DCM (20 mL), pyridine (0.56 g,
0.0069 mol) and triflic anhydride (1.44 g, 0.0051 mol) were added
at 0 ꢁC and the reaction mixture was stirred at 0 ꢁC for 3 h. After the
completion of the reaction monitored by TLC, the reaction mass
was diluted with DCM (20 mL), the organic layer was washed with
10% sodium bicarbonate (3 ꢂ 10 mL), water (3 ꢂ 10 mL) and brine
(3 ꢂ 10 mL). Finally, the organic layer was dried over sodium sulfate
and concentrated to yield a brown gummy mass. The crude product
was purified by column chromatography using silica gel as sta-
tionary phase and hexane:ethyl acetate as mobile phase to achieve
compound 9a as white solid.
(DMSO-d6) d (ppm): 5.6 (s, 4H, 2CH2), 7.12e7.79 (m, 12H, AreH). MS
(EI): m/z (67%): Mþ 708.5; Anal. Calcd. for C30H16BrCl3F2N2O5
(708.5): C, 50.84; H, 2.28; Br, 11.27; Cl, 15.01; F, 5.36; N, 3.95. Found:
C, 50.77; H, 2.35; Br, 11.37; Cl, 15.11; F, 5.27; N, 3.88%.
9j: Yield: 71%; m.p.: 132.3e133.6 ꢁC; IR (Nujol) nmax (cmꢃ1): 1106
(CeOeC linkage), 1610 (C]O), 1675 (C]N); 1H NMR (400 MHz)
(DMSO-d6)
d (ppm): 5.57 (s, 4H, 2CH2), 7.05e7.78 (m, 12H, AreH).
MS (EI): m/z (68%): Mþ 753; Anal. Calcd. for C30H16Br2Cl2F2N2O5
(753): C, 47.84; H, 2.14; Br, 21.22; Cl, 9.41; F, 5.04; N, 3.72. Found: C,
47.75; H, 2.22; Br, 21.31; Cl, 9.48; F, 5.12; N, 3.65%.
6.2. Biological study
Yield; 73%; m.p.: 129.5e130.2 ꢁC; IR (Nujol) nmax (cmꢃ1): 1153
(CeOeC linkage), 1658 (C]O), 1683 cm-1 (C]N); 1H NMR
The human chronic myelogenous leukemia (CML) K562 and
CEM were selected for the purpose of preliminary anti-cancer
screening of newly synthesized compounds. To assess the cyto-
toxicity, trypan blue dye exclusion assay, MTT assay and LDH assay
were employed. A DNA fragmentation assay, which is an indicator
of apoptosis, was also performed.
(400 MHz) (DMSO-d6)
AreH). MS (EI): m/z (70%): Mþ 631; Anal. Calcd. for
30H16Cl2F4N2O5 (631): C, 57.07; H, 2.55; Cl, 11.23; F, 12.04; N, 4.44.
d (ppm): 5.6 (s, 4H, 2CH2), 7.05e7.81 (m,12H,
C
Found: C, 57.17; H, 2.47; Cl, 11.31; F, 12.13; N, 4.36%.
Similarly compounds 9bej were synthesized starting from
compounds 8bej.
6.2.1. Cell lines and culture
9b: Yield; 76%: m.p.: 88.9e90.2 ꢁC; IR (Nujol) nmax (cmꢃ1): 1158
(CeOeC linkage), 1660 (C]O), 1685 (C]N); 1H NMR (400 MHz)
Human cell lines, K562 and CEM (T-cell leukemia) were pur-
chased from National Center for Cell Science, Pune, India. Cells were
grown in RPMI 1640 supplemented with 10% heat-inactivated fetal
(DMSO-d6)
d (ppm): 5.6 (s, 4H, 2CH2), 7.1e7.79 (m, 12H, AreH). MS
(EI): m/z (71%): Mþ 647.5; Anal. Calcd. for C30H16Cl3F3N2O5 (647.5):
C, 55.62; H, 2.49; Cl, 16.42; F, 8.80; N, 4.32. Found: C, 55.71; H, 2.41;
Cl, 16.51; F, 8.72; N, 4.23%.
bovine serum (FBS), 100 U/mL of Penicillin, and 100 mg of strepto-
mycin/mL and incubated at 37 ꢁC in a humidified atmosphere
containing 5% CO2.
9c: Yield: 71%; m.p.: 88.9e90.2 ꢁC; IR (Nujol) nmax (cmꢃ1): 1155
(CeOeC linkage), 1665 (C]O), 1680 (C]N); 1H NMR (400 MHz)
6.2.2. In vitro cell viability and cell proliferation assay: trypan blue
exclusion assay
(DMSO-d6)
d (ppm): 5.55 (s, 4H, 2CH2), 7.05e7.78 (m, 12H, AreH).
MS (EI): m/z (69%): Mþ 692; Anal. Calcd. for C30H16 BrCl2F3N2O5
(692): C, 52.05; H, 2.33; Br,11.54; Cl,10.24; F, 8.23; N, 4.05. Found: C,
52.15; H, 2.26; Br, 11.46; Cl, 10.17; F, 8.31; N, 4.11%.
Cell viability was monitored by the trypan blue exclusion assay
as reported earlier [20]. To determine the effect of compounds 9aej
on viability of K562 or CEM cells, approximately 0.75 ꢂ 105 cells/mL
were seeded in a 6-well tissue culture plate for 24 h and com-
pounds 9aej were added at a concentration of 10, 50, 100 and
9d: Yield: 75.2%; m.p.: 88.9e90.6 ꢁC; IR (Nujol) nmax (cmꢃ1):
1160 (CeOeC linkage), 1650 (C]O), 1685 (C]N); 1H NMR
(400 MHz) (DMSO-d6)
AreH). MS (EI): m/z (68%): Mþ 739; Anal. Calcd. for
30H16Cl2F3IN2O5 (739): C, 48.74; H, 2.18; Cl, 9.59; F, 7.71; I, 17.17; N,
3.79. Found: C, 48.66; H, 2.24; Cl, 9.51; F, 7.63; I, 17.27; N, 3.69%.
9e: Yield: 68.5%; m.p.: 72.0e73.4 ꢁC; IR (Nujol) nmax (cmꢃ1):
1150 (CeOeC linkage), 1635 (C]O), 1690 (C]N); 1H NMR
d
(ppm): 5.6 (s, 4H, 2CH2), 7.1e7.75 (m, 12H,
250 mM. 5-Fluorouracil treated cells were used as positive control.
Cells were collected at intervals of 24 h and resuspended in 0.4%
trypan blue (viable-unstained and non viable-blue). The number of
viable cells were counted using hemocytometer chamber.
C
6.2.3. MTT assay
(400 MHz) (DMSO-d6)
d
(ppm): 2.40 (s, 3H, CH3), 5.59 (s, 4H, 2CH2),
Cell proliferationwas further assessed by 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which is based
on the ability of viable cells to metabolize a yellow tetrazolium salt to
violet formazan. Exponentially growing K562, or CEM cells
(1 ꢂ104 cells/well) were plated in triplicates and incubated with 10,
7.05e7.8 (m, 12H, AreH). MS (EI): m/z (69%): Mþ 627; Anal. Calcd.
for C31H19Cl2F3N2O5 (627): C, 59.35; H, 3.05; Cl, 11.30; F, 9.08; N,
4.47. Found: C, 59.43; H, 3.14; Cl, 11.21; F, 9.17; N, 4.55%.
9f: Yield: 75.3%; m.p.: 110.4e111.7 ꢁC; IR (Nujol) nmax (cmꢃ1):
1152 (CeOeC linkage), 1627 (C]O), 1685 (C]N); 1H NMR
50, 100 and 250 mM of compounds 9aej. Cells were harvested after
(400 MHz) (DMSO-d6)
AreH). MS (EI): m/z (66%): Mþ 664; Anal. Calcd. for
30H16Cl4F2N2O5 (664): C, 54.24; H, 2.43; Cl, 21.35; F, 5.72; N, 4.22.
d
(ppm): 5.6 (s, 4H, 2CH2), 7.1e7.77 (m, 12H,
48 and 72 h of treatment and incubated with MTT (0.5 mg/mL) at
37 ꢁC. The blue MTT formazan precipitate was then solubilized in
detergent (50% final concentration of N,N-dimethylformamide and
10% of sodium dodecyl sulfate). Absorbance was measured at 570 nm
using ELISA plate reader. The mean absorbance of culture medium
was used as the blank and was subtracted. IC50 values (concentration
of compound causing 50% inhibition of cell growth) were estimated
after 72 h of title compounds treatment (Table 1).
C
Found: C, 54.31; H, 2.35; Cl, 21.26; F, 5.61; N, 4.32%.
9g: Yield: 72%; m.p.: 87.6e89.9 ꢁC; IR (Nujol) nmax (cmꢃ1): 1100
(CeOeC linkage), 1600 (C]O), 1675 (C]N); 1H NMR (400 MHz)
(DMSO-d6)
d (ppm): 2.3 (s, 3H, CH3), 5.6 (s, 4H, 2CH2), 7.05e7.79 (m,
12H, AreH). MS (EI): m/z (67%): Mþ 643.5; Anal. Calcd. for

H.D. Gurupadaswamy et al. / European Journal of Medicinal Chemistry 63 (2013) 536e543
543
6.2.4. LDH release assay
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per standard protocols [21] to assess the LDH release in the culture
media following the treatment with compounds 9b, 9e, 9i and 9j (10,
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6.2.5. DNA fragmentation assay
DNA fragmentation was performed for elucidating the mode of
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induction of oligonucleosomal DNA fragmentation (DNA ladder),
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mM for
m
Acknowledgments
We, the authors, express our sincere gratitude to Yuvaraja’s
College and University of Mysore, Mysore, for the laboratory facil-
ities provided to conduct this research.
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