Design and evaluation of novel oxadiazole derivatives as potential prostate cancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.04.058

Various 1,3,4-oxadiazole derivatives have been synthesized and their antiproliferative properties have been studied. The in vitro screening was performed against androgen dependent (LNCaP) and androgen independent (PC-3) prostate cancer cell lines. Most o

Full text of DOI:10.1016/j.bmcl.2016.04.058

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and evaluation of novel oxadiazole derivatives as potential
prostate cancer agents
b
c
d
a
c
Bereket Mochona a, , Xin Qi , Suresh Euynni , Donald Sikazwi , Nelly Mateeva , Karam F. Soliman
⇑
a Department of Chemistry, Florida A&M University, Tallahassee, FL 32307, United States
b Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32603, United States
c College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, United States
d Feik School of Pharmacy, University of the Incarnate Word, San Antonio, TX 78209, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Various 1,3,4-oxadiazole derivatives have been synthesized and their antiproliferative properties have
been studied. The in vitro screening was performed against androgen dependent (LNCaP) and androgen
independent (PC-3) prostate cancer cell lines. Most of the compounds showed promising activity. Among
Received 12 March 2016
Accepted 20 April 2016
Available online xxxx
them, compounds 2d (IC50 = 0.22 and 1.3 lM) and 2a (IC50 = 8.34 and 2,5 lM) have shown significant
activities on PC-3 and LNCaP cell lines respectively. To investigate the mechanism of cell death we per-
formed cell apoptosis staining and cell cycle arrest assay on more sensitive PC-3 cell lines on 2d. The
results demonstrated that 2d induced apoptosis and shifted the cells to the sub G0/G1 and S phase.
Our study evidently identified the potency of compound 2d as potential anti-prostate cancer agent.
Published by Elsevier Ltd.
Keywords:
Azoles
1,3,4-Oxadiazole
Prostate cancer
Apoptosis
Cell cycle
The American Cancer Society estimates that in 2016 in the Uni-
ted States there will be an estimated 1,685,210 new cancer cases
and 595,690 cancer deaths.1 Carcinoma of the prostate (CaP) is
the second leading cause of cancer related death in men after lung
cancer in the United States. If the cancer is confined to the prostate,
it is often curable by surgery or radiation treatment. However, in
many cases, it has already spread to other tissues, particularly
the bone, at diagnosis and usually requires some form of hormone
therapy to keep it in check.
Growth of CaP is primarily stimulated by androgens. Androgens
bind to the androgen receptor (AR) and support the development
and maintenance of normal prostate tissue. The antiandrogens
such as Bicalutamide and Enzalutamide (Fig. 1) target the AR,
which is responsible for growth and progression of the disease.
However, the emergence of recurrent, metastatic forms of castra-
tion-resistant/androgen-independent PCa continues to be a major
challenge.2 The mechanism of resistance to current antiandrogens
is unclear,3 but it is known that certain mutations in the hormone
binding site (HBS) of the AR will convert the drugs from receptor’s
antagonists to agonists.4–6 Part of the problem is that all known
antiandrogens share the same structural motif responsible for
effective AR HBS binding which may attribute to occurring
resistance. Therefore, more efforts are expected to discover AR
antagonists possessing novel chemical scaffolds that may partially
address the problem of resistance in PCa.
Several reports recently revealed the essential features of 1,3,4-
oxadiazole scaffold in medicinal chemistry to attribute various
pharmacological properties. Molecular modeling and pharmacoki-
netic studies have also demonstrated that incorporating 1,3,4-oxa-
diazole moieties to drug-like molecules change their polarity,
flexibility as well as metabolic profile and ability to engage in
hydrogen bonding. Moreover, oxadiazoles have been widely
employed as bioesters of amide and esters for a number of biolog-
ical targets.7,8
Derivatives of naphthalene such as naphthalimides are impor-
tant aromatic heterocycles with immense pharmacological signifi-
cance as they serve as core scaffold in drug designing.
Naphthalimide based anticancer drugs constitute an indispensable
part in the development of anticancer drugs. Naphthalimides have
been reported as potent apoptosis inducers, P-glycoprotein inhibi-
tors, microtubule inhibitors or glutamamide analogues. Taking into
account the importance of the above considerations we investi-
gated a new series of compounds with 1,3,4-oxadiazole scaffold
as potential anticancer agents.9–12
Chemistry: The 2-amino-1,3,4-oxadiazole 1 was prepared from
the corresponding hydrazides by reported procedure.13 The amide
or sulfonamide derivatives 2a–e were synthesized by using acyl or
sulfonyl chlorides in pyridine following procedure reported in liter-
ature.14,15 (Scheme 1). The structures of the newly synthesized com-
pounds were confirmed using IR, 1H NMR and 13C NMR spectral data.
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.04.058
0960-894X/Published by Elsevier Ltd.
Please cite this article in press as: Mochona, B.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.058

2
B. Mochona et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
NC
NC
F
NC
NC
O
O
O
O
O
OH
F3C
N
H
S
O
F3C
N
F3C
N
H
F3C
N
H
OH
NH
O
(III)
Hydroxyflutamide
O
R-Bicalutamide (I)
Nilutamide
(IV)
Flutamide
(II)
O
O
N
O
N
O
N
N
O
N
S
N
N
S
S
N
2bH
O
F3C
N
H
O
O
2a
2c H
O
O
F3C
N
N
O
N
N
N
N
N
H
N
H
O
2f
N
CF3
O
O
H
2e
2d
Figure 1. Known nonsteroidal androgen receptor antagonists (I–IV) and designed prototypes (2a–f).
O
N
N
F3C
N
N
O
N
H
O
2c
i
O
O
S
ii
N
H
2d
N
N
NH2
i
O
N
N
O
O
N
O
N
S
ii
N
H
X
(X = H, CF3)
O
N
H
O
CF3
2e
2b
ii
i
O
O
O
N
N
N
N
S
N
H
N
O
O
H
2a
2f
F3C
Scheme 1. Reagents and conditions: (i) pyridine, acid chloride, 0 °C, 1 h. (ii) Pyridine, sulfonyl chloride, 0 °C, 1 h.
Figure 2. The effect of 2d on cell cycle was analyzed with a flow cytometry: M1: Sub G0/G1 phase; M2: G0/G1 phase; M3: S phase; M4: G2/M phase. FACS analysis revealed
that treatment of PC-3 cells with 2d led to arrest at the subG0/G1 and S phase of the cell cycle. The sub-G1 population increased indicating that some cells underwent
apoptosis. The drug shifted the PC3 cells to the sub G0/G1 and S phase after exposure to drugs for 48 h.
Biology: In vitro cytotoxic activity: Androgen receptor dependent
(LNCaP) and androgen independent (PC-3) cell lines were selected
to evaluate compounds 2a–f for their capability to inhibit
androgen mediated growth in vitro. The cytotoxic activity of the
synthesized compounds was evaluated using the sulfo-rhodamine
B (SRB) colorimetric assay as described previously.14 The cytotoxic
activities are expressed as the median growth inhibitory concen-
tration (IC50) and are provided in Table 1. From the results, it is
evident that some of the tested compounds displayed moderate
growth inhibitory activity. Compound 2d (IC50 = 0.22 lM) the most
potent against PC-3 cell line.
Mechanism of cell death (cell cycle arrest and apoptosis induction):
Regulation of the cell cycle and apoptosis are considered to be
effective cancer therapeutic methods and most of the cytotoxic
compounds exert their growth inhibitory effect either by arresting
the cell cycle at a particular checkpoint of cell cycle or induction of
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B. Mochona et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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Figure 3. Flow cytometric apoptosis staining analysis of PC-3 cells: viable (lower left), early apoptotic (lower right), late apoptotic (upper right), and necrotic cells (upper
left). The population of early apoptotic, late apoptotic, and necrotic cells increase with the drug treatment. Cells were exposed to 0.2 lM drug 2d for 48 h treatments.
Figure 4. (A) Compound 2d. (B) Compound 2a docked in active site of androgen receptor.
apoptosis. Therefore, it was considered of interest to understand
whether the inhibition of cell growth of 2d was on account of cell
cycle arrest. In this study PC-3 cells were treated with 2d at IC50
concentration for 48 h (Fig. 2).
Apoptosis is one of the major pathways that leads to the process
of cell death and is associated with chromatin condensation,
nuclear shrinking and fragmented nuclei. Tumor cells often have
irregular apoptotic pathways and induction of tumor cell apoptosis
by natural or synthetic compounds is considered an effective ther-
apy for cancer.15,16 Hence it was of interest to investigate the
effects of 2d on apoptosis inducing effect in PC-3 cancer cells
(Fig. 3).
Molecular modeling studies: Molecular modeling studies were
also carried out for compound 2d which has been proved to be
most active (on androgen independent cell line) and 2a (on andro-
gen dependent cell line). To investigate possible interactions of 2d
and 2a with androgen receptor, we have carried out the docking
studies of compound 2d and 2a in the active site of androgen
receptor. Compounds were constructed and docked with builder
tool kit of software package (Fig. 4).
Both compounds are structurally distinct from the current AR
antagonists but compound 2a still demonstrated sufficient binding
to the human androgen receptor ligand binding domain (hARLBD).
To understand the essential features responsible for the potency of
these compounds, the receptor–ligand interaction at the binding
Table 1
Cytotoxic activity of the new compounds against LNCaP and PC-3 cancer cell lines
Compound
LNCaP
2.5 0.11
3.2 0.39
3.9 0.26
1.33 0.40
6.55 0.13
32.33 0.83
0.03 0.39
PC-3
8.34 0.20
47.8 0.19
22.2 0.46
0.22 0.10
11.06 0.32
26.00 0.46
0.03 0.39
site was examined. The hARLBD is well-characterized as
a
2a
2b
2c
2d
2e
2f
hydrophobic cavity that forms strong hydrophobic interactions
with a steroidal core of androgens. In that prospective, both com-
pounds possess hydrophobic scaffolds with one or two polar
groups attached, and the docked poses of these compounds adopt
similar orientations in the hARLBD compared to crystallographic
ligands. As observed from the figure, no polar groups of both
5-FU
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B. Mochona et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
compounds form hydrogen bonds. The binding modes of these
compounds suggest that hydrophobic interactions may be essen-
tial for the ligand coordination, while hydrogen bonding may not
play a determining role.
A series of 1,3,4-oxadiazole derivatives carrying aromatic scaf-
folds were synthesized, characterized and evaluated for their
in vitro cytotoxic effect on PC3 and LNCaP cancer cell lines. Com-
pound 2d bearing phenyl-1,3,4-oxadiazolyl showed cytotoxic
moiety. The ligand set comprised of our synthesized investiga-
tional oxadiazoles, hydroxyflutamide, R-bicalutamide and cypro-
terone acetate (CYP) which is co-crystallized with the protein in
2OZ7.pdb. Since the protein complex in the present study has
bound ligand (CYP), HYBRID v3.0.1 of OEDocking16 was chosen as
the appropriate docking method for our studies.
To begin with, the docking method in our current study,
HYBRID was used to validate the correctness of the bound ligand
(CA4) poses upon re-docking into the receptor structure. The top
ten docking poses of the bound ligand were examined and super-
imposed with its original bound conformation in the crystal struc-
ture. The poses were identical to the original pose of the cognate
ligand (C4A) in 2OZ7 crystal structure with root mean square devi-
ation (rmsd) less than 2 Å for all poses indicating the OEDocking
application’s reliability as a docking tool in our modeling studies.
The ligands in the present study were sketched using Sybyl
sketch Sybyl-X 1.3 Modeling suite.17 Energy was minimized for
every ligand under study and was stored together as a molecule
(.sdf) file. The conformer ensembles of these compounds were gen-
erated using OMEGA v2.5.1.418,19 prior to docking. OMEGA ensures
that low strain energy conformations were retained in the ensemble.
By using Structure Preparation tool of the biopolymer module with
in Sybyl-X 1.3 modeling suite, Chain A of the crystal structure 2OZ7.
pdb was extracted, hydrogen atoms were added potential bumps
were corrected and water molecules were removed prior to docking.
Finally the structure was energy minimized using MMFF94s force
fields and MMFF94 charges assigned. The water molecules in the
crystal structure were removed. The resulting refined mutated
Androgen receptor was used for docking the prepared ligands.
Chemistry: Reagents and solvents were purchased from Sigma–
Aldrich Chemical Company Inc. and used as received. Melting
points were determined in open capillaries on a Gallenkamp digital
melting point apparatus and were uncorrected. The Infrared spec-
tra were recorded in KBr discs using Shimadzu FT-IR 8000 spec-
trometer. 1H NMR (DMSO-d6) and 13C NMR spectra were
recorded using Bruker 300 MHz spectrometer using tetramethylsi-
lane (TMS) as internal standard. Peak multiplicities are expressed
as: s, singlet; d, doublet, t, triplet; q, quartet; dd, doublet of dou-
blet; br, broad; br s, broad singlet; m, multiplet. Thin layer chro-
matography was performed using precoated silica gel plates
(silica gel 0.25 mm, 60G F254).
effect in low lM range compared to 5-FU standard. The cytotoxic
effect of 2d is associated with apoptosis induction and cell cycle
arrest in sub G0/G1 and S phases of the cell cycle. The present inves-
tigation revealed that compound 2d could serve as a lead in the
future development of new agents that are more potent and selec-
tive in prostate cancer chemotherapy.
Biology: Chemicals: F12K medium, RPMI medium, penicillin–
streptomycin antibiotic solution (100x) fetal bovine serum (FBS),
trypsin–EDTA solution, phosphate-buffer solution (PBS), 50% glu-
taraldehyde, crystal violet, propidium iodide, rhodamine-123,
IGPAL CA-630, paclitaxel were obtained from Sigma–Aldrich Com-
pany (St. Louis, MO, USA). Potassium phosphate, EDTA, D-glucose
and ethanol were obtained from Thomas Scientific Company
(Swedesboro, NJ, USA).
Cell line maintenance: The LNCaP and PC-3 cancer cells were
grown in RPMI-1460 supplemented with 10% heat inactivated
FBS, medium containing 50 units of penicillin/mL, 50 g/mL of
streptomycin/mL, in T-75 cm2 flasks at 37 °C in a humidified atmo-
sphere containing 5% CO2 incubator. The cells were maintained as
‘monolayer culture’ by serial sub-culturing.
Treatment of cells: The cells were plated at density of 1000–200
cells per well in DMEM supplemented medium, allowed to stabi-
lize overnight in a CO2 incubator at 37 °C. Next, the cells were trea-
ted with compounds (2a–f) as well as 5-Fluorouracil (5-FU) as
reference compound at various concentrations in a final volume
of 1 mL per well in triplicate wells for each treatment for 24 h at
37 °C in a 5% CO2 incubator. All studies were repeated at least three
times.
Cell viability and proliferation: At the end of incubation period,
the cell viability was evaluated using Cell Glo assay according to
previously reported method. The cells were fixed with 10% tri-
chloroacetic acid for 1 h at 4 °C. Wells were stained for 10 min at
room temperature with 0.4% SRB dissolved in 1% acetic acid. The
plates were air dried for 24 h and the dye was solubilized with
Tris–HCl for 5 min on a shaker at 1600 rpm. The optical density
of each well was measured spectrophotometrically at 564 nm with
an ELSA microplate reader (ChroMate-4300, FL, USA). The IC50 val-
ues were calculated according to the equation for Boltzman sig-
moidal concentration–response curve using the nonlinear
regression fitting models. The results reported are mean of at least
three separate experiments. Significant differences were analyzed
according the ANOVA wherein the differences were considered to
be significant at p <0.05.
N-(5-Phenyl-1,3,4-oxadiazol-2-yl)-benzamide (2d): Benzoyl chlo-
ride (3.68 mL, 0.032 mol) was added dropwise into the stirred
slurry of compound 2-amino-5-phenyl-1,3,4-oxadiazole (5.64 g,
0.035 mol) in 50 mL pyridine. After 2 h, the solution was poured
into ice-water; and the white precipitate formed was collected
and dried under vacuum. Pure product was obtained by re-crystal-
lization from ethanol, mp 203–205 °C. Yield: 6.28 g, 74%. 1H NMR
(DMSO, 300 MHz) d/ppm: 7.55–8.05 (m, 10 H), 12.14 (s, 1H). 13C
NMR (DMSO, 300 MHz): d/ppm: 165.18, 161.43, 158.14, 133.20,
132.39, 132.00, 129.70, 128.84, 128.45, 126.29, 123.59. IR (KBr):
1713, 1618, 1582, 1391, 1293, 1245, 1023, 694 cmÀ1. HRMS
(ESI+) calcd. for C15H12N3O2 [M+H]+ 266.0929, found 266.0923.
Cell cycle analysis (flow cytometry): The PC-3 cells were treated
with 0.2 lM of 2d for 48 h. After treatment, the cells were washed
twice with ice-cold PBS, collected by centrifugation, and fixed in
ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with
0.1 mg/mL RNase, stained with 40 mg/mL PI, and analyzed by flow
cytometry using FACScalibur (Becton Dickinson). The cell cycle dis-
tributions were calculated using Cell Quest software (Becton
Dickinson).
Docking: Docking of 2d and 2a into the human androgen recep-
tor ligand binding domain (hARLBD): for our docking studies,
hARLBD (PDB: 20Z7) was considered as it correlates very well
structurally with the T877A mutant version of hAR as expressed
in LNCaP cell. This mutation produces a more promiscuous binding
site able to accommodate a broader range of ligands, and removal
of the threonine 877 residue removes a key hydrogen bonding
Acknowledgments
Research reported in this publication was supported by the
National Institute on Minority Health and Health Disparities
(NIMHD) of the National Institutes of Health (NIH) under Award
Number G12MD007582.
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