Design, synthesis and biological evaluation of 2-(phenoxymethyl)-5-phenyl-1,3,4-oxadiazole derivatives as anti-breast cancer agents

Article abstract of DOI:10.1016/j.ejmech.2019.02.033

Structural based molecular docking approach revealed the findings of 2-(phenoxymethyl) -5-phenyl-1,3,4-oxadiazole derivatives. The compounds (7a-o) were synthesized and characterized well by using conventional methods. The compounds, 7b and 7m were reconfirmed through single crystal XRD analysis. The synthesized compounds (7a-o) were evaluated their antiproliferative activities against MCF-7 and MDA-MB-453. Furthermore, Lipinski's rule of five and pharmacokinetic properties were predicted for the test compounds. These results demonstrate that the compounds 7b and 7d exhibit more potent cytotoxicity and 7d exhibits dose-dependent activity and reduced cell viability. Further, the mechanism of action for the induced apoptosis was observed through morphological changes and western blotting analysis. These findings may furnish the lead for further development.

Full text of DOI:10.1016/j.ejmech.2019.02.033

Accepted Manuscript
Design, synthesis and biological evaluation of 2-(phenoxymethyl)-5-phenyl-1,3,4-
oxadiazole derivatives as anti-breast cancer agents
K. Lakshmithendral, K. Saravanan, R. Elancheran, K. Archana, N. Manikandan, H.A.
Arjun, M. Ramanathan, N.K. Lokanath, S. Kabilan
PII:
S0223-5234(19)30147-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.033
EJMECH 11125
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 December 2018
Revised Date: 9 February 2019
Accepted Date: 10 February 2019
Please cite this article as: K. Lakshmithendral, K. Saravanan, R. Elancheran, K. Archana, N.
Manikandan, H.A. Arjun, M. Ramanathan, N.K. Lokanath, S. Kabilan, Design, synthesis and biological
evaluation of 2-(phenoxymethyl)-5-phenyl-1,3,4-oxadiazole derivatives as anti-breast cancer agents,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.033.
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Graphical Abstract
O
N
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O
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N
O
H
O
S
N
O
O
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O
N
O
OH
O
O
O
H2N
N
N
O
Furamizole
(Anti-biotic)
Nesapidil
(Anti-hypertensive)
Zibotentan
(Anti-cancer)
Hydrophobic interactions with
Flexible linker
Bulkier C ring
Leu 349, Leu 384, Ala 350, Trp 383
Essential H-Bond with
Arg 394 and Hydrophobic
interactions with Leu 387,
Met 388, Leu 391
B ring
Hydrophobic interactions
R2
with
R3
R4
N
N
Met 528, Leu 525, Leu 346
O
O
R1
Essential for interaction
with helix-12
A ring
C ring
7a-o
Designed molecules

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Design, synthesis and biological evaluation of 2-(phenoxymethyl)-5-phenyl-1,3,4-
oxadiazole derivatives as anti-breast cancer agents
K. Lakshmithendrala, K. Saravanana, R. Elancherana, K. Archanaa, N. Manikandanb, H. A.
Arjuna, M. Ramanathanb, N. K. Lokanathc, S. Kabilana, *
a Drug Discovery Lab, Department of Chemistry, Annamalai University, Annamalai Nagar -
608002, Tamil Nadu, India
b
Department of Pharmacology, PSG College of Pharmacy, Coimbatore - 641004, Tamil
Nadu, India
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c Department of Studies in Physics, University of Mysore, Manasagangotri, Mysuru-570 006,
India
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*Corresponding author: Prof. S. Kabilan, Drug Discovery Lab, Department of Chemistry,
Annamalai University, Annamalai Nagar - 608002, Tamil Nadu, India Tel: + (91) -4144-
238796 (IC-7378)
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E-mail address: profdrskabilanau@gmail.com
Abstract
Structural based molecular docking approach revealed the findings of 2-(phenoxymethyl) -5-
phenyl-1,3,4-oxadiazole derivatives. The compounds (7a-o) were synthesized and
characterized well by using conventional methods. The compounds, 7b and 7m were
reconfirmed through single crystal XRD analysis. The synthesized compounds (7a-o) were
evaluated their antiproliferative activities against MCF-7 and MDA-MB-453. Furthermore,
Lipinski’s rule of five and pharmacokinetic properties were predicted for the test compounds.
These results demonstrate that the compounds 7b and 7d exhibit more potent cytotoxicity and
7d exhibits dose-dependent activity and reduced cell viability. Further, the mechanism of
action for the induced apoptosis was observed through morphological changes and western
blotting analysis. These findings may furnish the lead for further development.
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Keywords
Estrogen Receptor
Breast Cancer
1,3,4-oxadiazoles
Molecular docking
1. Introduction
Cancer is a group of heterogeneous diseases involving dysregulation of cell growth
and functions to proliferate to other parts of the body. Breast cancer (BCa) is the second
most common cancer worldwide. National Cancer Institute (NCI) has assessed that the
diagnosis of 266,120 new cases and 40,920 dying due to BCa in the United States in
2018.1,2 The global burden of BCa surpasses all other cancers, and the frequency is still
rising. Over the last decade, several drugs and monoclonal antibodies have been approved
and are in the advanced stages of clinical trials that target the receptors and signaling
pathways.3 ER-positive (ER+) breast cancer is estrogen-dependent including luminal types
A and B. ER-negative (ER-) breast cancer is estrogen independent including the subtypes
human epidermal growth factor receptor 2 (HER2) in which ErbB2 is overexpressed.3
Tamoxifen is a non-steroidal antiestrogen and widely used for the treatment of breast
cancer, which acts on estrogen receptor.4 Inhibition of cancerous inhibitor of protein
phosphatase 2A (CIP2A) determines the effects of tamoxifen-induced apoptosis in ER-
negative breast cancer cells.5 ER alpha, ER beta, and Progesterone receptor are not
expressed in Triple-negative breast cancer (TNBC) which still exhibits an extraordinary
clinical challenge due to the unavoidably ineluctable advancement of medical obstruction.6
DNMT3B (DNA Methyltransferase 3 Beta) is a Protein-Coding gene, which is related to
abnormal methylation of tumour suppressor and repair genes and its overexpression
contributes to oncogenic processes and tumorigenesis.7 OTUD1 (ovarian tumor
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deubiquitinase 1) represses breast cancer metastasis by mitigating Transforming growth
factor beta (TGF-β) induced pro-oncogenic responses via deubiquitination of SMAD7
(SMAD Family Member 7) which is a protein-coding gene.8 The five-membered
heterocyclic compound containing more than two heteroatoms like azole, thiazole,
oxadiazole, triazene, imidazole, purines, etc. have tremendous importance in human life
due to their assortment of medicinal applications against several maladies.9,10
In recent years, the structural activity relationship with target structures and their
mechanism used for the drug design and oxadiazole has been reported with various
biological activities such as antitubercular, antiviral, antifungal, antibacterial,
antimicrobial, antidiabetic and anticancer activities.11-14 In particular, 1,3,4-oxadiazole is
an important moiety that exhibits more potent and selective inhibitory activity against
various cancer cell lines. Compound 1 with p-methoxybenzenesulfonamido moiety showed
more than 80% mean percentage inhibition at 10 µM concentration against breast cancer
cell line, MDA-MB-468.15 Compound 2 exerted submicromolar IC50 values of 0.67, 0.80,
and 0.87 µM against prostate cancer cell line (PC-3), colon cancer cell line (HCT-116),
and kidney adenocarcinoma cell line (ACHN) respectively. Also, it exhibited anti-
proliferative potential against MDA-MB-231 breast cancer cell line.16,17 MCF-7 is a breast
cancer cell line which retained several characteristics of differentiated mammary
epithelium, including the ability to process estradiol via cytoplasmic estrogen receptors
and the capability of forming domes.18 Organotin compounds showed higher activity
against breast cancer cells MCF-7 (ER-positive) than against MDA-MB-231 (ER-
negative) indicating that estrogen receptors may also be involved in their antitumour
mechanism.19,20 A novel silver iodide metalo-drug exhibits equal activity against MDA-
MB-231 cells where estrogen receptors (ERs) are devoid with the one against MCF-7
where ERs are present.21 MDA-MB-453 is human breast cancer cell line that has an active
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glycerol 3-phosphate shuttle and expresses high amounts of functional androgen
receptor.22 Nimesulide, Aspirin and salicylic acid metal complexes have high binding
affinity against DNA and their stronger inhibitory activity against Lipooxygenase
(LOX).23,24 Taken together, the 1,3,4-oxadiazole derivatives were designed and depicted in
Fig.1. Herein, we reported the design, synthesis and biological evaluation of a series of
novel 2-(phenoxymethyl)-5-phenyl-1,3,4-oxadiazole derivatives.
O
N
O
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N
N
H
O
S
N
O
O
N
N
N
N
O
O
N
O
OH
O
O
O
H2N
N
N
O
Furamizole
(Anti-biotic)
Nesapidil
(Anti-hypertensive)
Zibotentan
(Anti-cancer)
CF3
F
F
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O
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O O
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CF3
N
Cl
Cl
H
H
H
O
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2
R2
R3
R4
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N
O
O
R1
7a-o
Figure 1. Structural modification of designed oxadiazole derivatives
2. Results and discussion
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2.1. Molecular Modeling
Molecular docking approach is followed for the designing of 1,3,4-oxadiazole
derivatives by using the Schrödinger (Maestro 11.2) software.25 The structure of human
estrogen receptor alpha ligand-binding domain in complex with 4-hydroxytamoxifen (PDB
ID: 3ERT, resolution 1.9 Å) was obtained from the protein data bank, refined the raw
structure and utilized for this study.26 We have found that the compounds (7a-o) depicted
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specific van der Waals (vdW) interactions with the surrounding hydrophobic residues LEU
346, ALA 350, LEU 384, LEU 387, MET 388, LEU 391, ILE 424 and form hydrogen
bonds through the cyano group with two residues (GLU 353,and ARG 394) in the Helix
12. Of these, the best scoring pose of the compounds (7b & 7d) from the docking studies
is shown in Fig. (2) along with receptor residues which interact with the ligands within
binding sites.
A
B
C
D
Figure 2. Predicted binding mode of the compounds (7b & 7d) in ER LBD (PDB: 3ERT)
highlighting the H-Bond and specific hydrophobic interactions (Fig. 2A & 2B: coloured by
atom types- C light gray, F green, N blue, O red, H white).
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From the docking studies, we found that the compounds, 7b & 7d showed the
highest Glide score (-9.968 and -9.982 kcal/mol respectively). The compounds (7a-o) have
shown binding affinity to ER, and their docking scores were related to the standards (Table
1). The compounds (7b, 7d, 7g-k) has highest binding energy in the range of -9.1 to -9.9
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kcal/mol and the glide energy vary from -46.364 to -35.322 kcal/mol. The standards,
Imatinib and Tamoxifen glide energy were -55.298 and -57.000 kcal/mol respectively.
These studies were well established, and the results were concertized with our previously
reported work.34-38
Table 1 Lowest binding energy for the compounds in the ER (PDB ID: 3ERT).
Interacting Residues
glide
glide
evdw
glide
glide
Compound
gscore
ecoul energy
(3ERT)
-6.944 -35.772 -0.115 -35.887
-9.968 -35.748 -3.738 -39.486
-7.401 -33.415 -3.753 -37.168
-
7a
ARG 394, GLU 353, LEU 387, HOH
7b
ARG 394
7c
-9.982
-36.18
0.857 -35.322
-
7d
-6.982 -37.661 -3.604 -41.265
-8.531 -40.919 -0.014 -40.933
-9.222 -37.286 -2.413 -39.699
CYS 530, HOH
7e
-
7f
ARG 394, HOH
7g
-9.72
-42.32
0.009 -42.311
-
7h
-9.353 -34.073 -2.701 -36.774
-9.466 -36.448 -2.75 -39.198
-9.171 -42.126 0.892 -41.234
ARG 394, LEU 387, GLU 353, HOH
7i
ARG 394, LEU 387, GLU 353, HOH
7j
-
7k
7l
-8.29
-36.226 -4.354 -40.58
ARG 394, GLU 353, LEU 387, HOH
-7.724 -38.491 0.204 -38.287
-8.044 -36.715 -2.809 -39.524
-8.325 -42.907 -3.457 -46.364
-9.178 -51.793 -3.505 -55.298
-13.042 -50,841 -6.158 -57.000
-
7m
ARG 394, GLU 353, LEU 387, HOH
ARG 394, GLU 353, LEU 387, HOH
-
7n
7o
Imatinib
Tamoxifen
H- Bond interaction with HOH
glide evdw = van der Waals interaction energies, glide ecoul = Coulomb interaction energies
2.2. Chemistry
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Molecular docking results intend the synthesis of the compounds (7a-o). The
compounds (6a-e) were synthesized from the commercially available benzoic acid by
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esterification, hydrazine hydrate then refluxed with chloroacetic acid and POCl3.27,28
Further, the compounds (7a-o) were obtained by the reaction of the compounds (6a-e) with
substituted phenol in the presence of K2CO3 and KI in DMF for 1-2 hrs, in moderate to
excellent yield, 70–90 %. The general synthesis of 2-(phenoxymethyl)-5-phenyl-1,3,4-
oxadiazole derivatives (7a-o) is depicted in Scheme 1.
ID
7a
7b
R1
H
R2
R3
H
R4
H
ID
7i
R1
F
R2
H
R3
F
R4
H
OCH3
OCH3
Cl
H
F
H
OCH3
H
H
7j
CH3
F
OCH3
OCH3
OCH3
H
H
H
H
F
H
H
H
H
H
OCH3
CH3
F
H
H
H
H
H
H
H
H
H
H
7c
7d
7e
7f
7k
7l
Cl
H
7m
7n
7o
OCH3
CH3
H
H
F
F
H
H
Cl
7g
7h
Scheme 1. Synthesis of oxadiazole derivatives
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The synthesized compounds (7a-o) were well characterized by IR, NMR, and
HRMS, and all the data are in accordance with the proposed structures. Single crystals of
the compounds, 7b and 7m were obtained from ethyl acetate/petroleum ether solvent by
slow evaporation at room temperature.29 The compound, 7b belongs to triclinic system
with space group, P-1, a = 6.0847(14) Å, b = 8.5048(19) Å, c = 17.286(4) Å, α = 102.668°
(7), β = 90.646° (6), γ = 109.813° (8) and Z=2.30 Similarly, the compound, 7m belongs to
triclinic system with space group, P-1, a = 8.45(6) Å, b = 10.79(8) Å, c = 11.03(8) Å, α =
113.63° (4), β = 96.51° (2), γ = 111.65° (8) and Z=2. The ORTEP structure of the
compounds, 7b and 7m were illustrated in Fig. 3. (Table S2).
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7b
113
114
7m
Figure 3. ORTEP diagram of 7b & 7m
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In compound 7b, the 4-methoxyphenyl and oxadiazole (r.m.s. deviation 0.007 A˚)
rings are almost coplanar with a dihedral angle of 1.4. The methoxy atoms O4 and C16 are
also coplanar with the rings, deviating by 0.080 and 0.020 A˚ from the mean plane of the
phenyl ring, respectively. The compound 7b is associated via C—H…O interactions into
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inversion dimers (C16—H16B…O3. In addition, offset π–π interactions are observed
between the centroids of inversion-related oxadiazole and 4-methoxyphenyl rings with a
centroid-centroid distance of 3.700 A˚ and slippage of 1.037 A˚. Similarly, in compound 7m,
the methyl phenyl ring is almost coplanar with the oxadiazole ring, the angle between their
mean planes being 4.0 and is associated via C—H…O interactions. Also, offset π–π
interactions are observed between the centroids of inversion-related oxadiazole and methyl
phenyl rings with a centroid-centroid distance of 3.720 A˚, and the shift distance is 1.078.
The distance between C2 and O3 is 15.660.
2.3. Biological evaluation
All the synthesized compounds (7a-o) were evaluated for the anticancer activities
against breast cancer cell lines (MCF-7 & MDA-MB-453) and their cytotoxicity using
non-cancerous cell line (MCF-10A) as compared with the standard, Imitanib by using
MTT assay. Cytotoxicity assays assess the integrity of the cell's plasma membrane, and it
was determined in the presence and absence of the compounds as described in the earlier
methods.31,32 The results demonstrated that the compounds (7a-o) were shown the
significant cytotoxic potential in MDA-MB-453 and MCF-7 cell lines in the IC50 range of
10-50 µM as compared with imatinib (IC50 12.84 ± 2.2, 6.33 ± 1.9 respectively) (Table 2).
The compounds, 7b and 7d, showed the highest activities against MCF-7 cell lines (IC50
12.95 ± 3.3 and 10.51 ± 1.9 respectively) and MDA-MB-453 (IC50 11.12 ± 2.1 and 10.25 ±
2.5 respectively). It suggested that the CN group at R4 and methoxy group at R2 has
influenced the anticancer activities. Also, the replace of H to F atom at R3 has slightly
decreased the IC50 values. Similarly, at R4, CN group has better activities that 1,3,4-
oxadiazole group. This comparison was beneficial for improving the efficacy of the
compounds. Also, the compounds, 7b and 7d have not shown the toxicity against MCF-
10A which reliably represent normal human mammary cells.
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Table 2
IC50 of test and standard compounds
MCF-7
MDA-MB-453
MCF-10A
ID
7a
R1
R2
R3 R4
(µM)a
(µM)a
(µM)a
H
OCH3
H
H
H
H
H
F
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
32.17 ± 2.7
11.12 ± 2.1
27.13 ± 3.1
10.25 ± 2.5
21.54 ± 1.9
>50
40.15 ± 1.9
12.95 ± 3.3
38.41 ± 2.7
10.51 ± 1.9
33.87 ± 1.1
>50
>100
>100
7b OCH3 OCH3
CH3 OCH3
>100
7c
7d
7e
7f
F
Cl
OCH3
>100
OCH3
H
89.5 ± 2.1
>100
H
OCH3
CH3
F
H
F
42.15 ± 1.8
>50
45.49 ± 2.9
>50
>100
7g
7h
7i
H
F
>100
H
F
20.45 ± 0.9
48.32 ± 2.3
25.13 ± 3.1
>50
84.2 ± 3.2
>100
Cl
H
F
7j
H
OCH3
CH3
F
H
H
H
H
H
H
H
H
H
>50
>50
>100
>100
>100
>100
7k
7l
31.16 ± 2.7
>50
36.18 ± 1.1
>50
7m
7n
7o
35.62 ± 3.2
38.19 ± 2.5
Cl
H
45.23 ± 3.1
6.33 ± 1.9
>50
>100
>100
Imitanibb
12.84 ± 2.2
a
The values are the mean ± standard deviation (SD) of three independent experiments
performed in triplicate
b Positive control
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From these series, the compounds, 7b and 7d showed the promising anti-breast
cancer activities in MDA-MB-453 and MCF-7 cell lines. It is manifest that the groups
especially OCH3, F in the R1 and R2 substitutions were determining the anticancer
activities in the cell proliferation assays. The compound, 7d exhibits dose-dependent
activity in triple negative breast cancer cell lines. The IC50 value was calculated as ~10 µM
for this active compound shown in Fig. 4. The compound, 7d reduced cell viability and
induced the apoptosis confirmed through AO/EtBr staining.33
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Figure 4. Morphological changes in 7d and imatinib treated breast cancer cell lines. The
treated and untreated cells were stained with Acridine orange and Ethidium bromide and
observed under an inverted fluorescent microscope. Live cells appear green, late apoptotic
cells are appearing orange in colour, and necrotic cells are coloured red. Abbreviations:
LC - Live Cells, CB - Cell membrane Blebbing, AC- Apoptotic Cells, NC-Necrotic Cells,
PL - Protrusion elongation, CC- Chromatin Condensation
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The western blotting analysis was carried out as described earlier shown in Fig.
5.34-36 The compound, 7d shows DNMT 3B enzyme inhibitory activity (approx. 33%) at
100µM concentration. Also, it exhibits only 10% activity in inhibiting the DNMT1
enzyme. Since it is more specific towards the DNMT 3B, it can be used DNMT 3B
isoform targeted therapies to treat breast cancer. Dose-Dependent expression of the p53
gene was found in the compound 7d incubated cancer cells. It was observed that DNA
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double-strand repair was retained with the low level of p53 in MDA-MB-453 cell line.37
So this compound may exhibit its beneficial effect at the dose of 0.1µM. High dose of 7d
(10 µM) increases the SMAD7 expression in the MDA-MB-453 cell line.
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Figure 5. Mechanism of action of the compound, 7d which causes p53 dependent
apoptotic cell death in breast cancer cells. Protein expression of SMAD7, Bcl2, Bax, pS2,
and p53 was determined by western blot. GAPDH was used as loading control.
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The absorption, distribution, metabolism, and elimination (ADME) properties of the
synthesized compounds (7a-o) were evaluated for the pharmacokinetic and pharmaceutical
relevant properties as described earlier.35,36 The qikpro v3.5 was used for the evaluation of
ADME parameters38, and its permissible ranges are listed in the supporting information
(Table S1). In this study, the test compounds expressed the recommended drug-like
properties from Lipinski’s rule of five indicating zero violation and providing orally active.
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There were no more than 5 hydrogen bond donors and no more than 10 hydrogen bond
acceptors. The molecular masses were less than 500 daltons (within 290-360 ranges) and
octanol-water partition coefficient was not greater than 5. In terms of Jorgensen’s rules, the
blockage of HERG K+ channels (-6.2 to -6.9), Caco-2 cell permeability (580 to 725 nm/s),
the logarithm of predicted binding constant to human serum albumin (-1.5 to 1.2) were
observed within the range of 95% of known drugs. There was no pharmacokinetic violation
for these molecules. Overall, the pharmacokinetic results are good agreement with our
previous reports.32,36
3. Conclusion
In conclusion, the molecular docking approach was used for the designing of
molecules with the estrogen receptor. The compounds, 7a-o were synthesized and
demonstrated significant anti-breast cancer activities. In particular, the compound, 7b & 7d
were shown as the most anticipating among the series against MCF-7 and MDA-MB-453
cell lines. Also, the compounds, 7a-o were screened in MCF-10A cell line for the toxicity.
Moreover, the compound 7d showed that it could reduce cell viability and induce apoptosis.
Further, the investigation of the target site and the in vivo anticancer activity of the active
compounds will be further examined.
4. Experimental section
4.1. Molecular Modeling
The ligands for docking studies were prepared using LigPrep, Schrödinger. The
ligands were geometrically refined and assigned appropriate protonation state at pH 7.0 ± 2.0.
The energy minimization was carried out by OPLS 2005 force field. The protein was
prepared using the protein preparation wizard in Maestro. The preprocessed protein was then
used to generate a grid for docking studies. The grid was assigned by picking the ligand as
the center of the grid and then the grid box was generated by applying default parameters.
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The docking was carried out using GLIDE, Schrödinger. GLIDE XP (extra precision) method
was followed for docking calculations.
4.2. Chemistry
4.2.1. General
The entire chemicals, reagents, and solvents were procured from mercantile companies like
Spectrochem, Merck, Alfa Aesar and Sigma-Aldrich, etc. The complete reactions were
monitored by thin layer chromatography (TLC) via precoated aluminum plates of Merck
silica gel 60 F254 furthermore visualized in UV light chamber. Column chromatography was
accomplished on Merck silica gel (100-200 mesh) for the purification of intermediates.
Melting points were determined by the open capillary method and are uncorrected.
Potassium bromide disk (KBr) was formulated for IR spectra attainment in the range of
4000–400 cm-1. IR spectra were recorded on Thermo Nicolet FT-IR model iS5
spectrophotometer. NMR spectra attainment was done using Bruker 400 MHz AMX (1H
NMR) and 100 MHz (13C NMR) in CDCl3.The spectra were recorded in δ (ppm) with
tetramethylsilane (TMS) as an internal standard. HRMS spectra were recorded in Thermo
Exactive Orbitrap ESI-MS/MS.
4.2.2. General procedure for preparation of substituted aromatic esters (4a-4e)
To a solution of substituted aromatic benzoic acid (3a-3e) (1mmol) in EtOH (15ml), a
catalytic amount of Conc. H2SO4 was added slowly at RT, then heated to reflux for 16 h with
stirring. After cooling, EtOH was removed and concentrated in vacuum; the reaction mixture
was diluted with ethyl acetate (30mL) and washed with sat.NaHCO3 and cold water, followed
by separating the organic layer. The combined organic layer was washed with brine, dried
over anhydrous Na2SO4, filtered, concentrated, and dried under vacuum for 12 h.
4.2.3. General procedure for preparation of aromatic acid hydrazides (5a-5e)
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Substituted aromatic ester (1mmol) was heated to reflux with hydrazine hydrate (2
mL) for 1 h. Upon completion of the reaction, the reaction mixture was quenched with water
and extracted with ethyl acetate. The combined organic layer was washed with brine solution,
dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure.
4.2.4. General procedure for preparation of 2-(chloromethyl)-5-aryl-1,3,4-oxadiazole
derivatives (6a-6e)
Substituted aromatic acid hydrazides (1mmol) were heated to reflux with chloroacetic acid
and phosphorus oxychloride for 1h. After completion, the reaction mixture was cooled to RT,
and about 40 mL of water was added. The mixture was involved sonication for 30- 40 min.
The solid product was collected by filtration and dried under vacuum. The crude was purified
by silica gel (100-200 mesh) column chromatography using 10-20 % ethyl acetate-petroleum
ether.
4.2.5. General procedure for preparation of substituted ethyl 4-hydroxybenzoate
To a solution of 4-hydroxybenzoic acid (1mmol) in EtOH (15ml), catalytic amount of Conc.
H2SO4 was added slowly at RT. It had been then heated to reflux for 16 h with stirring. After
cooling, removal of the EtOH and concentrated in vacuum, the reaction mixture was diluted
with ethyl acetate (30mL) and washed with sat.NaHCO3 and cold water, followed by
separating the organic layer. The combined organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered, concentrated, and dried under vacuum for 12 h.
4.2.6. General procedure for preparation of 4-hydroxybenzohydrazide
Ethyl 4-hydroxybenzoate (1mmol) was heated to reflux with hydrazine hydrate (2 mL) for 2-
3 h. The solid was washed with water and dried under vacuum for 12 h. The corresponding
aromatic acid hydrazides were used without further purification.
4.2.7. General procedure for preparation of 4-(1,3,4-oxadiazol-2-yl)phenol
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4-hydroxybenzohydrazide (1mmol) was heated to reflux with triethylorthoformate (6 mL) for
16 h. After removal of the triethylorthoformate and concentrated in vacuum, then solid was
dried under vacuum. The pale yellow solid was used without further purification.
4.2.8. General procedure for preparation of 2-(phenoxymethyl)-5-phenyl-1,3,4-oxadiazole
derivatives (7a-7o)
To a stirred solution of substituted phenol (1mmol) in DMF (4 mL), K2CO3 (3mmol) was
added. Furthermore 2-(chloromethyl)-5-aryl-1,3,4-oxadiazole derivatives (6a-6e) and KI (0.5
mmol) was added. The reaction mixture was stirred at RT for 1-2 hr. After completion of the
reaction, the reaction mixture was washed with cold water. The solid product was collected
by filtration and dried under the vacuum. It had been further crystallized in ethyl
acetate/petroleum ether.
4.2.9. 3-methoxy-4-((5-phenyl-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7a)
Yield:89 %; mp: 234-236°C, white powder; IR (KBr) ν max: 3005, 2940, 2843, 2221, 1599
cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 7.98 (d, J = 1.6Hz, 2H), 7.50-7.45 (m, 3H), 7.21-
7.19 (m, 2H), 7.06 (d, J = 1.6Hz, 1H), 4.72 (s, 2H), 3.84 (s, 3H); 13C NMR (100 MHz,
CDCl3, ppm): δ 166.1, 161.4, 150.5, 149.9, 132.2, 129.1, 127.1, 126.1, 123.2, 118.7, 114.9,
114.6, 106.2, 60.9, 56.2; HRMS (ESI): m/z calculated for C17H13N3O3 [M+H]+ 308.10297;
Found: 308.10269.
4.2.10. 3-methoxy-4-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7b)
Yield: 92 %; mp: 174-176 °C, white powder; IR (KBr) ν max: 3005, 2942, 2837, 2220, 1613,
1
837 cm-1; H NMR (CDCl3, 400 MHz, ppm): δ 7.90 (d, J = 8.4 Hz, 2H), 7.19 (d, J =7.6 Hz,
1H), 7.11 (d, J = 8Hz, 1H), 7.04 (s, 1H), 6.92 (d, J = 8 Hz, 2H), 5.34 (s, 2H), 3.81 (d, J = 11.2
Hz, 6H); 13C NMR (100 MHz, CDCl3, ppm): δ 164.9, 161.6, 159.8, 149.5, 148.8, 127.8,
125.1, 125.0, 117.7, 114.6, 113.7, 113.5, 105.0, 59.7, 55.1, 54.4; HRMS (ESI): m/z
calculated for C18H15N3O4 [M+H]+ 338.11353; Found: 338.11273.
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4.2.11. 3-methoxy-4-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7c)
Yield: 87 %; mp: 172-174 °C, white powder; IR (KBr) ν max: 3003, 2936, 2843, 2219, 1601,
847 cm-1;1H NMR (CDCl3, 400 MHz, ppm): δ 7.87(d, J = 7.6 Hz, 2H), 7.25-7.21(m, 3H),
7.11-7.05(m, 2H),5.36 (s, 2H), 3.83 (s, 3H), 2.35(s, 3H); 13C NMR (100 MHz, CDCl3, ppm):
δ 166.2, 161.2, 150.6, 149.9, 142.9, 129.8, 127.1, 126.2, 120.5, 118.7, 114.9, 114.6, 106.2,
61.01, 56.2, 21.7.; HRMS (ESI): m/z calculated for C18H15N3O3 [M+H]+ 322.11862; Found:
322.11954.
4.2.12. 4-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxybenzonitrile (7d)
Yield: 98 %; mp: 162-164 °C, white powder; IR (KBr) ν max: 2934, 2221, 1601, 1022,
1
845cm-1; H NMR (CDCl3, 400 MHz, ppm): δ 7.99-8.02 (m, 2H), 7.23-7.6 (m, 5H), 5.37 (s,
13
2H), 3.84 (s, 3H); C NMR (100 MHz, CDCl3, ppm): δ 164.2, 164.0 (d, J =253 Hz), 160.4,
149.4, 148.8, 128.5 (d, J =9 Hz), 125.1, 118.5, 117.6, 115.5 (d, J =22 Hz), 113.8, 113.4,
105.2, 59.7, 55.1; HRMS (ESI): m/z calculated for C17H12FN3O3 [M+H]+ 326.09355; Found:
326.09698.
4.2.13. 4-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxybenzonitrile (7e)
Yield: 83 %; mp: 164-166 °C, white powder; IR (KBr) ν max: 3086, 2999, 2938, 2221, 1599,
847, 730 cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 7.93 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.8
Hz, 2H), 7.2-7.19 (m, 1H), 7.11-7.06 (m, 2H), 5.37 (s, 2H), 3.84 (s, 3H); 13C NMR (100
MHz, CDCl3, ppm): δ 164.2, 160.5, 149.5, 148.8, 137.6, 128.5, 127.4, 125.1, 120.7, 117.6,
113.8, 113.5, 105.3, 59.8, 55.2; HRMS (ESI): m/z calculated for C17H12ClN3O3 [M+H]+:
342.06400; Found: 342.06351.
4.2.14. 2-fluoro-4-((5-phenyl-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7f)
Yield: 82 %; mp: 148-150 °C, white powder; IR (KBr) ν max: 3108, 2928, 2231, 1619, 1122
cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 7.99 (d, J = 7.2, 2H), 7.53-7.43 (m, 4H), 6.89-6.84
13
(m, 2H), 5.32 (s, 2H); C NMR (100 MHz, CDCl3, ppm): δ 165.1, 163.4 (d, J =257 Hz),
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161.2, 161.1, 159.7, 133.64, 133.62, 131.3, 128.1, 126.1, 122.0, 112.8, 110.6, 110.5, 102.7,
102.5, 93.9, 93.7, 59.1; HRMS (ESI): m/z calculated for C16H10FN3O2 [M+H]+: 296.08298;
Found: 296.08612.
4.2.15. 2-fluoro-4-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7g)
Yield: 92 %; mp: 150-152 °C, white powder; IR (KBr) ν max: 3090, 2936, 2845, 2231, 1619,
1029, 870 cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 7.92 (d, J = 8.8 Hz, 2H), 7.50-7.48 (m,
1H), 6.935 (d, J = 8.8 Hz, 2H), 6.87 -6.83(m, 2H), 5.29 (s, 2H), 3.81 (s, 3H); 13C NMR (100
MHz, CDCl3, ppm): δ 165.0, 163.4 (d, J =258 Hz), 161.7, 161.3, 161.2, 159.2, 133.6, 133.5,
127.9, 114.4, 113.5, 112.9, 110.6, 110.5, 102.7, 102.4, 93.8, 93.6, 59.1, 58.9, 54.4; HRMS
(ESI): m/z calculated for C17H12FN3O3 [M+H]+: 326.09335; Found: 326.09695.
4.2.16. 2-fluoro-4-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7h)
Yield: 85 %; mp: 132-134 °C, white powder; IR (KBr) ν max: 3106, 2879, 2229, 1616, 1017,
1
813 cm-1; H NMR (CDCl3, 400 MHz, ppm): δ 7.87(d, J = 7.6 Hz, 2H), 7.52-7.49(m, 1H),
13
7.24(d, J = 7.6 Hz, 2H), 6.89-6.83(m, 2H), 5.30 (s, 2H), 2.36 (s, 3H); C NMR (100 MHz,
CDCl3, ppm): δ 166.3, 164.4 (d, J =258 Hz), 162.3, 162.2, 160.5, 143.1, 134.6, 129.9, 127.1,
120.3, 113.8, 111.65, 111.62, 103.8, 103.5, 95.0, 94.8, 60.3, 21.7; HRMS (ESI): m/z
calculated for C17H12FN3O2 [M+H]+: 310.09863; Found: 310.10159.
4.2.17. 2-fluoro-4-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)benzonitrile (7i)
Yield: 81 %; mp: 140-142 °C, white powder; IR (KBr) ν max: 3108, 2822, 2233, 1609, 1017,
855 cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 8.03-7.99(m, 2H), 7.52-7.50(m, 1H), 7.19-
13
7.13(m, 2H), 6.90-6.83(m, 2H), 5.31 (s, 2H); C NMR (100 MHz, CDCl3, ppm): δ 165.4,
165.16 (d, J =252 Hz), 164.4 (d, J =258 Hz), 162.2, 162.1, 160.8, 134.72, 134.7, 129.6,
129.5, 119.5, 119.4, 116.7, 116.5, 113.8, 111.6, 111.5, 103.7, 103.5, 95.1, 94.9, 60.1; HRMS
(ESI): m/z calculated for C16H9F2N3O2 [M+H]+: 314.07356; Found: 314.07275.
4.2.18. 4-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)-2-fluorobenzonitrile (7j)
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Yield: 90 %; mp: 166-168 °C, white powder; IR (KBr) ν max: 3102, 2232, 1624, 1017, 839,
728cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 7.94 (d, J = 8.8 Hz, 2H), 7.54-7.50 (m, 1H),
13
7.44 (d, J = 8.4 Hz, 2H), 7.19 (s, 1H), 6.90-6.83(m, 1H), 5.31 (s, 2H); C NMR (100 MHz,
CDCl3, ppm): δ 164.4, 163.4 (d, J =258 Hz), 159.8, 137.7, 133.7, 133.6, 128.6, 127.4, 120.5,
112.8, 110.5, 110.4, 102.7, 102.5, 59.0; HRMS (ESI): m/z calculated for C16H9ClFN3O2
[M+H]+: 330.04401; Found: 330.04364.
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4.2.19. 2-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-5-phenyl-1,3,4-oxadiazole (7k)
Yield: 80 %; mp: 154-156°C; Appearance: white powder; IR (KBr) ν max: 3130, 1612,
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836cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 8.36 (s, 1H), 7.99 (s, 4H), 7.45 (d, J = 7.2 Hz,
3H), 7.12(d, J = 8Hz, 2H), 5.34 (s, 2H) ;13C NMR (CDCl3, 100 MHz, ppm): δ 166.0, 164.3,
161.6, 160.2, 152.4, 132.2, 129.2, 129.1, 127.1, 123.3, 117.5, 115.4, 59.9; HRMR (ESI): m/z
calculated for C17H12N4O3 [M+H] +: 321.09822; Found: 321.10147.
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(7l)
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Yield: 87%; mp: 150-152°C; Appearance: white powder; IR (KBr) ν max: 3136, 2918, 1617,
845cm-1; 1H NMR (CDCl3, 400 MHz, ppm): δ 8.36(s, 1H), 7.99-7.92(m, 4H), 7.11(d, J = 8.4
Hz, 2H), 6.93(d, J = 8Hz, 2H), 5.31 (s, 2H), 3.80 (s, 3H) ; 13C NMR (CDCl3, 100 MHz,
ppm): δ 165.9, 164.3, 162.7, 161.1, 160.3, 152.3, 129.1, 128.9, 117.5, 115.8, 115.4, 114.5,
59.9, 55.4; HRMR (ESI): m/z calculated for C18H14N4O4 [M+H]+: 351.10878; Found:
351.10818.
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4.2.21. 2-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-5-(p-tolyl)-1,3,4-oxadiazole (7m)
Yield:85%;mp:170-172°C;Appearance: white powder; IR (KBr) ν max: 3096, 2922, 2863,
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1612, 816 cm-1; H NMR (CDCl3, 400 MHz, ppm): δ 8.36(s, 1H), 7.98(d, J = 8.8 Hz, 2H),
7.88(d, J = 8Hz, 2H), 7.23(d, J = 8Hz, 2H), 7.11(d, J = 8.4Hz, 2H), 5.32(s, 2H), 2.35(s, 3H) ;
13C NMR (CDCl3, 100 MHz, ppm): δ166.2, 164.3, 161.3, 160.3, 152.3, 142.8, 129.8, 129.1,
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127.1, 120.5, 117.5, 115.4, 59.9, 21.6; HRMR (ESI): m/z calculated for C18H14N4O3 [M+H]
+: 335.11387; Found: 335.11353.
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4.2.22. 2-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole (7n)
Yield: 92%; mp:164-166°C; Appearance: white powder; IR (KBr) ν max: 3100, 2920, 1612,
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356
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1036, 845 cm-1; H NMR (CDCl3, 400 MHz, ppm): δ 8.37 (s, 1H), 8.02-7.98 (m, 4H), 7.14-
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7.10 (m, 4H), 5.34 (s, 2H) ; C NMR (CDCl3, 100 MHz, ppm): δ 165.2, 165.1 (d, J =252
Hz), 164.3, 161.6, 160.2, 152.3, 129.5, 129.4, 129.1, 119.7, 119.6, 117.6, 116.6, 116.4, 115.3,
59.8; HRMR (ESI): m/z calculated for C17H11FN4O3 [M+H] +: 339.08879; Found:
339.08829.
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(7o)
2-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole
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Yield: 89%; mp: 166-168°C; Appearance: white powder. IR (KBr) ν max: 3110, 2920, 1611,
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845 cm-1. H NMR (CDCl3, 400 MHz, ppm): δ 8.37 (s, 1H), 8.01-7.94 (m, 3H), 7.43 (d, J =
8.4 Hz, 2H), 7.19 (s, 1H), 7.12 (d, J = 8.8 Hz, 2H), 5.34 (s, 2H). 13C NMR (CDCl3, 100 MHz,
ppm): δ 165.2, 164.3, 161.7, 160.2, 152.3, 138.6, 129.6, 129.1, 128.4, 121.7, 117.6, 115.3,
59.8. HRMR (ESI): m/z calculated for C17H11ClN4O3 [M+H]+: 355.05924; Found:
355.05887.
4.3. Biological evaluation
4.3.1. Cytotoxicity assay
Breast cancer cell lines (MCF-7 & MDA-MB-453) and non-cancerous cell line
(MCF-10A) were obtained from NCCS Pune. The cells were cultured using DMEM with
10% FBS. The cells were incubated at 37°C with 5% CO2. After achievement of 70-80%
confluence, the cells were trypsinized and harvested. Approximately 5,000 cells/well were
seeded into a 96 well cell culture plate. The cells were incubated to recover from handling
for 24 hours. The Anti-cancer activity was measured by adding the test compounds of
concentrations up to 100 µM. Test compounds dissolved in DMSO was added to each well
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and control wells contain DMSO without test compounds. Then the plate was incubated for
48 hours to allow the test compound to take effect. The culture medium was changed at
the end of the treatment, and 10µL of MTT (5mg/ml) was added to each well and
incubated for an additional 4 hours. The medium was aspirated, and formazan crystals
were dissolved in 150µL DMSO/well, and optical density was measured at 560 nm. For
each concentration of test compounds, triplicates were performed, and the average value
was considered.
4.3.2. Acridine orange (AO)/ Ethidium bromide (EtBr) dual staining
For conventional AO/EtBr apoptosis assay 5X105 cells /well were seeded in a 6 well plate
and incubated (37°C and 5% CO2) for overnight. MDA-MB 453 cell lines were treated with
the test compound for 48 hrs. The concentration of dye was prepared as 1:1 ratio, each dye
concentration was 100 µg/ml. Cells were stained with 8µL of dye for 5 min, and images
were taken immediately by fluorescence microscopy.
4.3.3. DNAMethyl Transferase enzyme inhibitor assay
DNMT inhibition assay was carried out using EpiQuik DNA methyltransferase
activity/inhibition screening assay kit (Epigentek, Brooklyn, NY, USA) according to the
manufacturer’s instruction.
4.3.4. PCR
MDA-MB 453 cells (1X106) were cultured in 100mm Petri dishes. After attaining 70-80%
confluence, the cells were incubated with different concentration of test drug for 48 hrs. Total
RNA was isolated from the cells using TRI reagent (sigma life science) and quantified by
quick drop (Molecular Devices). The total RNA was converted to cDNA using High capacity
cDNA reverse transcription conversion kit (Applied Biosystems). Total RNA (3µg) from
each sample was subjected to reverse transcription according to the manufacturer protocol.
The PCR products were resolved by electrophoresis through a 2% agarose gel and stained
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with ethidium bromide. The intensities of PCR product in the agarose gel were scanned with
G: BOX (Syngene) image scanner.
Acknowledgements
This work was supported by Govt. of India funded by the Ministry of Science & Technology,
Department of Biotechnology (DBT) (Sanctioned No. BT/PR16268/NER/95/183/2015).
Dr. R. Elancheran thank DST-PURSE phase II for the support as Research Associate.
Conflict of interest: The Authors declare no competing interests.
Supplementary data
Supplementary data associated HRMS & NMR spectra of compounds, 7a-7o. The
Crystallographic data for compounds, 7b & 7m has been deposited with the Cambridge
Crystallographic Data Centre, CCDC No. 1881074; 1876400. Copies of the data can be
obtained free of charge on application to CCDC; E-mail: deposit@ccdc.cam.ac.uk).
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Research Highlights
•
•
•
15 new compounds with 1,3,4-oxadiazole scaffolds were designed and synthesized.
The compounds, 7b and 7m were reconfirmed through single crystal XRD analysis.
The morphological changes and induced apoptosis in triple negative breast cancer
were observed after the treatment with the compound, 7d.
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Research Highlights
•
•
•
15 new compounds with 1,3,4-oxadiazole scaffolds were designed and synthesized.
The compounds, 7b and 7m were reconfirmed through single crystal XRD analysis.
The morphological changes and induced apoptosis in triple negative breast cancer
were observed after the treatment with the compound, 7d.