Design, synthesis, in silico, and in vitro evaluation of novel pyrimidine phosphonates with cytotoxicity against breast cancer cells

Article abstract of DOI:10.1007/s00044-013-0628-y

Pyrimidine derivatives are widely used for the treatment of breast cancer and rapid efforts are contributing to develop highly potential novel molecules. In view of this, in this study, novel pyrimidine phosphonate molecules were designed so as to inhibit

Full text of DOI:10.1007/s00044-013-0628-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0628-y
ORIGINAL RESEARCH
Design, synthesis, in silico, and in vitro evaluation of novel
pyrimidine phosphonates with cytotoxicity against
breast cancer cells
•
Nanda Kumar Yellapu Navya Atluri Kalpana Kandlapalli
•
•
•
Ravendra Babu Kilaru Jhansi Rani Vangavaragu Hariprasad Osuru
•
•
•
Nagaraju Chamarthi P. V. G. K. Sarma Bhaskar Matcha
•
Received: 3 February 2013 / Accepted: 14 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Pyrimidine derivatives are widely used for the
treatment of breast cancer and rapid efforts are contributing to
develop highly potential novel molecules. In view of this, in
this study, novel pyrimidine phosphonate molecules were
designed so as to inhibit aromatase, a potential target of breast
cancer. Quantitative structure activity relationship descriptors
were defined and they revealed that the molecules have an
effective and safer drug-like properties, which also supported
by absorption, distribution, metabolism, and excretion study.
Molecular docking of these compounds into the aromatase
active site revealed that they are showing strong interaction
forming H-bonds and arene cationic interactions. The ligand–
receptor complex of compound 6i showed a best docking
score of -15.776 kcal/mol among all. Hence, this compound
was synthesized and tested in vitro at different concentrations
of 25, 50, 100, and 200 lg/mL against MDA-MB-231 ade-
nocarcinoma breast cancer cells and it exhibited excellent
antiproliferative activity and also induced apoptosis. The
results from in silico structure activity relationships, molec-
ular docking study and in vitro assays suggesting the com-
pound as a potential source of chemotherapeutic drug for the
treatment and management of breast cancer.
Keywords Breast cancer ꢀ QSAR ꢀ MOE ꢀ
MDA-MB-231
Introduction
Breast cancer is the second leading cause of deaths in
women today and it is the most common cancer among
women (Parkin et al., 1999). One of eight women is affected
by breast cancer and one-third of affected women die from
breast cancer (http://www.cancer.org). Breast tumors are
comprised of phenotypically diverse populations of breast
cancer cells. Although there is a rapid advancement in the
Nanda Kumar Yellapu and Navya Atluri contributed equally to
this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0628-y) contains supplementary
material, which is available to authorized users.
N. K. Yellapu ꢀ B. Matcha (&)
J. R. Vangavaragu
Department of Pharmacology and Toxicology, University
of Kansas, Lawrence, KS 66047, USA
Division of Animal Biotechnology, Department of Zoology,
Sri Venkateswara University, Tirupati 517 502, India
e-mail: matchabhaskar2010@gmail.com
H. Osuru
Department of Anesthesiology, University of Virginia,
Charlottesville, Virginia, USA
N. Atluri
Department of Applied Microbiology, Sri Padmavati Mahila
Visvavidyalayam, Tirupati 517 502, Andhra Pradesh, India
P. V. G. K. Sarma
Department of Biotechnology, Sri Venkateswara Institute
of Medical Sciences, Tirupati 517 507, India
K. Kandlapalli
Department of Biochemistry, Sri Venkateswara Institute
of Medical Sciences, Tirupati 517 507, India
R. B. Kilaru ꢀ N. Chamarthi
Department of Chemistry, Sri Venkateswara University,
Tirupati 517 502, India
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Med Chem Res
diagnosis, treatment and preventive measures, mortality
rates are still remaining high due to limitations of current
therapies and the emergence of therapy-resistant cancerous
cells. These multiple factors make breast cancer as a prob-
lematic incurable disease by the current treatment strategies
available. Breast cancers are associated with multiple fac-
tors especially hormonal imbalance (Henderson and Fei-
gelson, 2000) and mutations (Al-Hajj and Clarke, 2004;
Clarke, 2005; Golub, 2001). Significant reports are there on
the implication of estrogen and its metabolites in the
development of breast cancer. Binding of estrogens to its
receptors results in proliferation of estrogen-sensitive epi-
thelial tissues (Yager, 2000). The rapid proliferation leads to
potential genetic errors and consequently malignant cellular
transformation. Moreover, estrogen metabolites have
intrinsic genotoxic effects causing DNA damage and cel-
lular transformation (Cavalieri et al., 2000). This estrogen
pathology is associated with aromatase which converts
androgens into estrogens by aromatization process. So
decreasing the production of estrogens by inhibiting aro-
matase by means of aromatase inhibitors will be an ideal
way of suppression of breast tumor proliferation (Bonfield
et al., 2012). Hence, in this study, we have chosen aromatase
as the target to predict the inhibitory action of the present
test compound. The structural organization and catalytic
mechanism of the aromatase have been studied and it was
in silico means. The insight into the intermolecular inter-
action of the compounds in the catalytic site provides much
essential and critical information for the better rationaliza-
tion and determining the strength of the compounds (Ric-
cardo, 2012; Ou-Yang et al., 2012; Therrien et al., 2012).
Hence, we carried out molecular docking study to find the
binding energy, strength of affinity, and the inhibitory action
of the compound. These studies suggesting the use of
phosphonate moiety as it is promoting the interaction with
aromatase active site and the hydrophobic interactions are
induced by the aromatic ring structures of the compounds
that are favored by the hydrophobic active site residues like
methionine, valine, leucine and isoleucine.
On the other hand, QSAR analysis always helps to
derive and screen large amount of highly reactive mole-
cules that can optimize the designing of novel leads. QSAR
is associated with the prediction of numerous physico-
chemical properties like molecular weight, energy levels,
polarizability, hydrogen bond donors and acceptors, molar
refractivity, surface area, volume and logP etc., which are
all calculated for the present novel compounds along with
the application of Lipinski filters. The absorption, distri-
bution, metabolism and excretion (ADME) profile will also
have a major impact on the likelihood of success of a drug
and is often the most difficult and challenging part of the
whole drug discovery process. Hence, ADME properties
were predicted for all the test compounds.
3
˚
found that the volume of binding pocket is less than 400 A
which limits the entry of the molecule with appropriate
volume only (Ghosh et al., 2009).
We here in most importantly reported the antiprolifer-
ative activity of the compound having best interaction with
aromatase against MDA-MB-231 adenocarcinoma breast
cancer cell lines to find out its anti-breast cancer activity.
These cell lines are widely accepted models for assessing
potent antiproliferative activity (Zhu et al., 2012a, b).
In vitro cell cytotoxicity of this novel compound was
evaluated against MDA-MB-231 by trypan blue dye
exclusion method, MTT assay, apoptosis assay and DNA
fragmentation assay. A well significant and potent inhibi-
tory activity was observed from these evaluations which
are discussed in detail in this article.
The aromatic rings of the lead can create the hydro-
phobicity so that it shows strong interaction with the
hydrophobic residues of the active site like leucine, valine
and tryptophan. The major novelty of the present approach
is the use of a phosphonate moiety which is strongly polar
and can readily interact with the polar residues like serine
and arginine. These are all the residues that are observed in
the aromatase active site. Based on these findings, the
compounds were designed and the one showing the best
interaction with aromatase was synthesized so as to bind
with the active site and inhibit the aromatase which in turn
control the proliferation of breast malignancy (Ghosh et al.,
2012; Mayhoub et al., 2012). The phosphonate moiety is
highly potent, which readily penetrates biological mem-
branes and enters the target organ. Phosphate esters are
frequently used as a prodrug strategy, especially for water-
insoluble compounds, since the phosphate group enables
following characteristic features to the xenobiotic-like
decreases the adverse effects of the drugs, increases water
solubility and thereby enables delivery of the drug paren-
terally (Somogyi et al., 1998a, b).
Materials and methods
In silico study
All in silico studies were performed using Molecular
Operating Environment (MOE), 2011.10.
QSAR descriptors
Based on these considerations, we have concentrated to
find out the binding efficiency and inhibitory activity of the
novel compounds as aromatase inhibitors initially through
The three dimensional structures of the compounds (6a–i) were
constructed using the builder interface of the MOE program
and subjected to energy minimization using the included
123

Med Chem Res
Merck molecular Force Field 94x (MMFF94x) (Halgren,
1996a, b; Halgren and Nachbar, 1996; Valasani et al., 2013).
The produced models were subjected to systematic confor-
mational search, where all items were set as default with RMS
ignoring all heteroatoms and water molecules. The struc-
ture was energy minimized in AMBER force filed to an
˚
RMSD of 0.05 A where the implicit solvated environment
was specified and the stabilized conformation was saved.
This stabilized conformation was subjected to molecular
dynamics simulation under generalized born implicit sol-
vated environment and the same AMBER force filed is
specified. The initial temperature is set to 30 K and
increased to a run time temperature of 300 K with a pH of
7 and a pressure of 101 kPa. The total run of simulations
was carried out for 10 ns and the final stabilized trajectory
was chosen for docking analysis. Active site was defined
with the residues Trp224, Asp309, Val370, Leu372,
Val373, Met374 and Leu477 that were found to be in the
surrounding of the ligand that was cocrystallized in the
aromatase PDB structure (http://www.ebi.ac.uk/thornton-
srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=3s7s&
template=ligands.html&l=2.1). Molecular docking was
carried out using MOE dock module to dock the com-
pounds into specified binding site along with the reference
compound Exemestane that was found in contact with
3S7S using alpha principle moments of inertia (PMI)
placement methodology where poses are generated by
aligning ligand conformations principal moments of iner-
tia to a randomly generated subset of alpha spheres in the
receptor site. 30 docked conformations were generated for
each ligand and ranked by alpha HB scoring function
which is a linear combination of the geometric fit of the
ligand to the binding site and hydrogen bonding effects.
From all the receptor–ligand complexes, the conformation
with the lowest docking score was chosen for the analysis.
˚
gradient of 0.01 kcal/mol and RMS distance of 0.1 A. In
order to explore the bioactivity of the compound, theoretical
calculations were carried out to predict some physicochemical
properties. Lipinski’s rule of five (Lipinski et al., 1997, 2001)
and the later addition of other parameters such as polar sur-
face area, well known in pharmaceutical industry that outline
the basic principles for orally active small molecules,
molecular weight should be under 500, lipophilicity which is
expressed as logarithm of partition coefficient between
n-octanol and water (LogP) should be less than five, the
number of hydrogen bond donors should be not more than
five, and the number of hydrogen bond acceptors should be
not more than ten. Among these parameters, LogP, the
measure of the compound’s solubility and permeability, is
believed to be very important. Very high lipophilicity and the
resulted large LogP values cause poor absorption or perme-
ation and they can be avoided (Hansch and Clayton, 1973;
Hansch and Dunn, 1972).
ADME study
ADME study describes the disposition of any compound
within an organism by defining the major pharmacokinetic
parameters like absorption, distribution, metabolism and
excretion. These four properties influence the drug levels
and kinetics of drug exposure to the tissues in the body and
in turn influence the performance and pharmacological
activity of the compound as a drug (Beresford et al., 2002).
Hence, in this study, the ADME properties were defined for
all the compounds as important conditions to choose them
as drug candidates. The structures were submitted to
PreADMET online software tool (http://preadmet.bmdrc.org/
index.php?option=com_content&view=frontpage&Itemid=1),
a web-based application server for predicting ADME. The
parameters such as colon adenocarcinoma (Caco-2) cell per-
meability, Madin-Darby canine kidney (MDCK) cell perme-
ability, blood–brain barrier (BBB) penetration, human
intestinal absorption (HIA), skin permeability and plasma
protein binding ability were predicted.
Chemistry
Based on the QSAR, ADME and molecular docking
studies, we have chosen the compound ethyl 3-(2-((dieth-
oxyphosphoryl) methoxy) ethyl)-4-(4-hydroxyphenyl)-6-
methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
for the synthesis (6i). This compound has shown the best
interaction with aromatase active site with lowest docking
score among all compounds and even it showed satisfactory
QSAR and ADME properties and hence considered for the
synthesis and evaluation of anticancer activity in vitro.
General
Molecular docking
Sigma-Aldrich, Merck and Lancaster chemicals were used
as such without further purification. All solvents used for
spectroscopic and other physical studies were reagent
grade and were further purified by literature methods.
Melting points were determined by Guna Digital Melting
Point apparatus using a calibrated centigrade thermometer.
IR spectra were recorded in KBr disks on a Perkin–Elmer
Molecular docking studies were carried out to provide the
understandable evidence with an insight into binding ori-
entations of the compounds in the aromatase active site.
The X-ray crystallographic structure of aromatase has been
retrieved from Protein Data Bank (Berman et al., 2000a, b)
(ID: 3S7S) and loaded into MOE working environment
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Med Chem Res
Model 281-B spectrophotometer in wave numbers (cm-1).
1H, 13C and 31P NMR spectra were recorded as solutions in
DMSO-d6 on a Bruker AVANCE III 500 MHz spectrom-
the presence of polyphosphoric acid (1 mol %) for 12 h
under nitrogen. The progress of the reaction was monitored
by TLC (hexanes: ethyl acetate, 5:5 v/v). The reaction
mixture, after being concentrated under vacuum at 50 °C
cooled to room temperature, it was poured into crushed ice
(10 g) and stirred for 5–10 min. The solid separated was
filtered under suction, washed with ice-cold water (20 mL),
and then recrystallized from hot ethanol to afford pure
product (2 g, 88 %).
1
eter operating at 500 MHz for H, 125 MHz for 13C and
202.4 MHz for 31P nuclei and their chemical shifts were
expressed in ppm with reference to tetramethylsilane and
85 % H3PO4, respectively. Mass spectra were recorded on
a JEOL GCMATE II Mass spectrometer. Elemental anal-
yses were performed by Central Drug Research Institute,
Lucknow, India.
Ethyl 3-(2-((diethoxyphosphoryl) methoxy)ethyl)-4-(4-
hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6i)
General synthetic procedure for the preparation
of compounds 4a-4i
A solution of ethyl acetoacetate (1 mol eq), aromatic alde-
hyde (1 mol eq), and urea (1 mol eq) in ethanol (5 mL) was
heated under reflux (78–80 °C) in the presence of poly-
phosphoric acid (1 mol eq). The progress of the reaction was
monitored by TLC (hexanes: ethyl acetate, 1:1 v/v). The
reaction mixture, after being concentrated under vacuum at
60 °C, cooled to room temperature, it was poured into cru-
shed ice (10 g) and stirred for 5–10 min. The solid separated
was filtered under suction, washed with ice-cold water
(20 mL) and then recrystallized from hot ethanol to afford
pure products (4a–i).
This compound was prepared by the reaction of ethyl 4-(4-
hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (1 mol eq), diethyl (2-chloroethoxy)methyl-
phosphonate (1 mol eq) in the presence of K2CO3 (2 mol eq)
in acetonitrile/DMF-10 mL at room temperature to slowly
increase the temperature to 80–85 °C and maintain for
20 h. The progress of the reaction was monitored by TLC
(1:2 ethyl acetate: hexane). The reaction mixture was fil-
tered to remove the salts and washed with DMF solvent
removed under vacuum residue taken into MDC/ethyl
acetate and washed with water (3 9 10 mL). The crude
product was purified by column chromatography on silica
gel (60–120 mesh) with chloroform:methanol (9:1) as
eluent to obtained as a thick liquid. Rf = 0. 22 (2:1 hexane/
EtOAc); FTIR (thin film): 3,342, 3,210, 3,020, 2,983,
General synthetic procedure for the preparation
of compounds 6a–i
1
Ethyl
4-(4-substituted)-6-methyl-2-thioxo-1,2,3,4-tetra-
2,856, 1,685, 1,603, 1,218 cm-1; H NMR (500 MHz, d6-
hydropyrimidine-5-carboxylate (1 mol eq) and K2CO3
(1.5 mol eq) were taken in acetonitrile (10 mL) at room
temperature. Then the contents were heated to 80–85 °C and
diethyl (2-chloroethoxy) methylphosphonate (2 mol eq) was
added slowly and stirred for appropriate time at 85 °C.
Progress of the reaction was monitored by TLC mobile
phase (n-hexane: ethyl acetate, 7:3). The salts were filtered
and washed with acetonitrile and the solvent was removed
under vacuum, the residue was taken into ethyl acetate and
washed with water (3 9 10 mL). Solvent was completely
removed under vacuum; the residue was purified by column
chromatography. Initially, the column was run with n-hex-
ane and ethyl acetate was slowly increased to 100 %. Fur-
ther, it was separated in ethyl acetate: methylene dichloride.
Solvent was evaporated under reduced pressure to obtain the
title products 6a–i.
DMSO) (ppm) 9.16 (d, J = 5.0 Hz, 1H), 7.69–7.68 (m,
1H), 7.16–7.13 (m, 2H), 6.90–6.87 (m, 2H), 5.09 (d,
J = 5.0 Hz, 1H), 4.09–3.96 (m, 8H), 3.90–3.88 (m, 3H),
2.24 (s, 3H), 1.22 (t, 6H), 1.10 (t, 3H). 13C NMR
(125 MHz, d6-DMSO): 165.3 (C-5), 157.5 (C-12), 152.1
(C-1), 148.0 (C-2), 137.2 (C-9), 127.4 (C-10 and C-14),
114.2 (C-11 and C-13), 99.4 (C-4), 70.8 (C-6), 66.8 (C-17),
64.7 (C-16), 63.4 (C-6), 61.7 (C-18 and 20), 53.2 (C-15),
17.7 (C-3), 16.2 (C-19 and C-21), 14.0 (C-7) ppm. 31P
NMR (162 MHz, d6-DMSO) (ppm) 21.91; HRMS cald for
C21H32N2O8P = 471.1896; found 471.1884 (TOF MS
ES?).
In vitro study
Screening for antiproliferative activity/cytotoxicity
Synthesis of ethyl 4-(4-hydroxyphenyl)-6-methyl-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
Cell line and cell culture media MDA-MB-231 human
breast adenocarcinoma cells were obtained from National
Cell Center for Science, Pune, India. The cell line was
routinely maintained as monolayer in Leibovitz’s L-15
medium supplemented with 2 mM/L glutamine (Himedia),
10 % fetal bovine serum (FBS, Himedia), and 10 lg/mL
To a stirred solution of ethyl acetoacetate (1 mmol),
4-hydroxybenzaldehyde (1 mmol) and urea (1 mmol) in
ethanol (10 mL) were heated under reflux (78–80 °C) in
123

Med Chem Res
ciprofloxacin in humidified incubator (Binder) containing
(5 lg/mL of Bisbenzimide H 33342 trihydrochloride in
L-15 media with 10 % FBS, Sigma) was added to cells and
incubated at room temperature for 1 min. Then the Hoechst
solution was removed and L-15 containing 10 % FBS was
added to cells. The nuclear changes were analyzed with
fluorescence microscopy (Olympus, USA) at 360 nm of
excitation and 485 nm of emission (Dieudonne et al.,
2006).
100 % air at 37 °C.
Cell viability assay The effect of compound 6i on MDA-
MB-231 cells was evaluated by trypan blue dye exclusion
method (Boiadjieva et al., 1984, 1983). In brief, the cells
were seeded at 2 9 105 cells per well in a 6-well tissue
culture plates in L-15 media supplemented with 10 % FBS,
incubated for 24 h at 37 °C. Later the medium was
replaced with fresh medium containing various concen-
trations (25, 50, 100, and 200 lg/mL) of 6i and 0.1 % of
DMSO (served as solvent control), incubated for 24–48 h
(37 °C). To assess the cell viability after incubation for
specified time, aliquots from both floating and trypsinized
adherent cells were mixed with a 0.4 % trypan blue (Hi-
media) solution (1:1) and loaded on to a haemocytometer,
observed under light microscope. The dead cells and total
cells were counted and the percentage of viable cells per
sample was calculated.
DNA fragmentation assay Cellular DNA of treated cells
was extracted according to Eastman (Eastman, 1995) with
slight modifications. In brief, MDA-MB-231 cells were
grown in 25-cm2 tissue culture flasks in L-15 media with
10 % FBS for 24 h at 37 °C in humidified incubator and
were replaced with fresh medium containing 6i (25, 50,
100, and 200 lg/mL) and 0.1 % of DMSO, incubated for
24 h at 37 °C. Both attached and detached cells were
scraped, washed with PBS and resuspended in ice-cold
lysis buffer (20 mM Tris–HCl, pH 7.5, 10 mM EDTA,
0.5 % Triton X-100, Himedia) for 30 min. After centrifu-
gation at 10,000 rpm for 5 min, supernatant was treated
with 10 % SDS (Himedia) and 50 mg/mL RNase A
(Bangalore Genie), incubated at 37 °C for 1 h. Then
2.5 mg/mL of Proteinase K (Bangalore Genie) was added
and incubated for 2 h at 4 °C. DNA was extracted using
phenol–chloroform and precipitated with double the vol-
ume of absolute ethanol. The pelleted DNA was dissolved
in Tris–EDTA (pH 8.0) and resolved electrophoretically on
1.8 % agarose gel prestained with 0.5 lg/mL ethidium
bromide.
Total cells ꢁ Dead cells
Viable cells ð%Þ ¼
ꢂ 100
Total cells
Cell proliferation (MTT) assay The MTT colorimetric
assay (Borenfreund et al., 1988) was performed to detect
the effect of compound on MDA-MB-231 breast cancer
cells proliferation. In brief, the cells were seeded at
1 9 104 cells per well in 96-well tissue culture plates in
L-15 media supplemented with 10 % FBS. After overnight
incubation (37 °C), the medium was replaced with fresh
medium containing various concentrations (25, 50, 100,
and 200 lg/mL) of 6i and 0.1 % of DMSO, incubated for
24 h at 37 °C. Then 20 lL of MTT (5 mg/mL in phosphate
buffered saline, Himedia), a tetrazolium dye [3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide]
was added to each well and incubated for 4 h. The medium
was replaced with 200 lL of DMSO to dissolve blue for-
mazan crystals (MTT metabolic product) and absorbance
was read at 570 nm using a microplate reader (Bio-Rad),
the percentage of proliferation inhibition was calculated.
Statistical analysis
All the experiments were done in triplicates and the data
expressed as mean standard deviation (SD) of three
independent assays. Statistical significance (P \ 0.05) was
assessed by t test.
Results and discussion
Inhibitory rate ð%Þ
Absorption of test ꢁ Absorption of control
¼
ꢂ 100
Chemistry
Absorption of test
Apoptosis assay The apoptotic effect of 6i on MDA-MB-
231 cells was analyzed by nuclear DNA Hoechst 33342
staining. For Hoechst 33342 staining, 1 9 105 cells/mL
was seeded in a 24-well tissue culture plates which was
adhered with cover slips. After overnight incubation at
37 °C in humidified incubator, cells were treated with
varying concentrations (25, 50, 100, and 200 lg/mL) of 6i
and 0.1 % of DMSO, incubated for 24 h at 37 °C. Then the
medium was discarded and cells were washed twice with
0.1 M PBS. 0.5 mL of Hoechst 33342 staining solution
Designing of pyrimidine phosphonate as breast cancer
inhibitor
The pyrimidine derivatives are widely used for the treat-
ment of breast cancer (Nunez et al., 2008; Yonekura et al.,
1999). A number of recent innovative research approaches,
targeting malignant abnormalities of breast cancer cells are
under improvement (Rao et al., 2011). However, new
derivatives of presently known ‘‘small molecule’’ cytotoxic
agents possessing improved properties, such as broader
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Med Chem Res
activity and lower toxicity, will continue to be an essential
part of cancer therapy in the near future. Therefore, a
strong need for new ‘‘small molecule’’ cytotoxic agents
with improved properties still exists. Pyrimidine deriva-
tives were built and screened virtually using the docking
method. However, there is no disclosure of utility of
pyrimidine phosphonate derivatives as breast cancer
inhibitor so far. Based on the above reports of utility of
pyrimidines in breast cancer, we have designed the present
phosphonate derivatives as anti-breast cancer agents.
The major novelty of the present approach is the use of a
phosphonate moiety which readily penetrates biological
membranes and enters the target organ. Accordingly,
phosphate esters are frequently used as a prodrug strategy,
especially for water-insoluble compounds since the phos-
phate group enables following characteristic features to the
Scheme 1 Synthetic route of
target molecule 6i
R
CHO
O
O
O
O
R1
PPA, Ethanol
Reflux, 12 h
O
NH
+
R1
O
H2N
NH2
+
R
N
O
H
(2)
(3)
(1a-i)
(4)
O
O
R
P
O
O
O
O
P
Cl
K2CO3, 20 h
O
(4)
+
O
R1
O
Acetonitrile/DMF
N
O
(5)
O
N
H
(6a-i)
6e = R, 3-NO2; R1, Methyl
6f = R, 4-NO2; R1, Methyl
6a, = R, 4-NO2; R1, Ethyl
6b = R, 3-NO2; R1, Ethyl
6c = R, 2-OH; R1, Ethyl
6d = R, 2-OH; R1, Methyl
6g = R, H; R1, Methyl
6h = R, H; R1, Ethyl
6i = R, 4-OH; R1, Ethyl
Table 1 Molecular descriptors defined for the pyrimidine phosphonate derivatives (6a–6i)
Ligand Molecular Hydrogen Hydrogen LogP Molar
Surface
˚
Volume Hydration
3
(A )
Polarizability Gradient
Total
energy
(K.cal/mol) (K.cal/mol)
2
3
(A )
˚
˚
weight bond
(Daltans) donors
bond
acceptors
refractivity area (A )
(A )
energy
(K.cal/mol)
energy
3
˚
6a
6b
6c
6d
6e
6f
471.40
471.40
442.41
428.38
457.38
457.38
430.37
444.40
466.00
1
1
2
2
1
1
1
1
2
7
7
6
6
7
7
6
6
6
2.55 78.88
712.43
667.66
695.68
615.52
726.49
639.93
672.77
685.76
769.35
1246.80 -18.67
1216.70 -14.96
1212.14 -16.26
1116.60 -12.71
1249.14 -18.82
1166.43 -17.22
1166.51 -13.22
1202.19 -13.13
1242.06 -15.27
41.42
41.42
40.34
38.50
39.58
39.58
37.78
39.61
40.36
0.084
0.091
0.092
0.092
0.098
0.095
0.099
0.098
0.095
143.802
139.705
142.039
128.187
135.890
135.336
126.186
135.892
129.654
0.55 78.88
1.34 74.27
3.00 69.52
0.21 74.14
0.21 74.14
1.43 68.04
3.77 72.79
2.21 92.00
6g
6h
6i
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Med Chem Res
Fig. 1 ADME descriptors of
pyrimidine phosphonate
derivatives (6a–6i) plotted as
graphs
Table 2 Molecular docking interaction of pyrimidine phosphonate
derivatives (6a–6i) in the aromatase active site
Compound Docking score
(Kcal/mol)
No. H-bonds
Interacting
residues
H-bonds
˚
length (A)
6a
6b
6c
6d
6e
6f
-12.162
-12.128
-11.208
-12.500
-13.092
-14.226
1
–
–
1
–
M374
–
2.1
–
–
–
R435
–
2.7
–
Arene–arene
interaction
W224
–
6g
6h
-13.486
-12.001
Arene cationic
interaction
W224
–
1
R115
2.8
–
Arene–arene
interaction
W224
6i
-15.776
1
1
R115
2.8
2.6
Exemestane -10.141
M374
xenobiotics: decreases the adverse effects of the drugs,
increases water solubility, and thereby enables delivery of
the drug parenterally. We have therefore focused on the
Fig. 2 The molecular interaction of compound 6i with aromatase
active site. Green color dotted line indicates the hydrogen bond
between the compound and active site residues (Color figure online)
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Med Chem Res
The chemical structure of the compounds 6i was char-
acterized by IR, 1H, 13C, 31P NMR, and HRMS studies and
their data are presented in the experimental section.
Characteristic IR stretching absorptions were observed in
the regions 1,218, 3,210, and 3,342 cm-1 for P=O, N–H,
O–H, respectively, (Rao et al., 2012, 2010a, b, 2011). In
1
the H NMR spectra of compounds 6–17, the chemical
shifts of aromatic hydrogens of the phenyl ring appeared as
multiplets in the region d 7.69–6.87 (Rao et al., 2009). In
13C NMR chemical shifts for compounds 6i were observed
in their expected regions. 31P NMR signals were observed
in the region 21.91 ppm (Rao et al., 2011).
Fig. 3 Cytotoxicity of 6i on MDA-MB-231 cells determined by
trypan blue dye exclusion method at 24 h. Each value represents the
mean SD from triplicate determinants. The P value is \0.0001
QSAR descriptors
development of more active and selective ligands
(Scheme 1) which are capable of inhibiting antitumor
activities of MDA MB 231 cell lines.
The activity/function of the compounds should be corre-
lated to their structures and associated descriptors that can
define them as suitable aromatase inhibitors. This
Fig. 4 Morphological changes
in MDA-MB-231 cells treated
with 6i at 24 h show rounded up
and detached cells from the
substratum. a DMSO control,
b 25 lg/mL, c 50 lg/mL,
d 100 lg/mL, and e 200 lg/mL
of compound treated cells
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Med Chem Res
investigation revealed that all the compounds are showing
significant molecular descriptors that are tabulated in
Table 1. Greater the significance of the descriptors greater
will be the potency for the compound to inhibit aromatase
and in turn breast cancer cells and even more likely to be
safer drugs. The stabilized conformation of the molecules
showed the energy values that are enough to interact with
the target under optimal conditions. They are all have
molecular weight less than 500 Da, logP value is below
five, hydrogen bond donors and acceptors are less than five
and ten, respectively and the molar refractivity is found to
be in the optimal range of 40–130. Finally, the molecular
surface area of the compounds is also defined that indicates
the extent of interacting region in the molecule to the
receptor. These values confirmed that the molecules are
obeying Lipinski rule which helps in distinguishing drug-
and nondrug-like molecules. It predicts high probability of
success or failure due to drug likeness for any molecules.
The compound whichever accepts the Lipinski rule is more
likely to be membrane permeable and easily absorbed by
the body. Hence, it can be concluded from this study that
all the molecule can be prompted as effective and safer
drugs with high solubility and stability.
diffusion or transport across cell membranes and also for
interaction with a pharmacological target. The plasma
protein binding capacity was observed to be weak for all
the compounds that are more desirable as their availability
and exposure to target is more.
Molecular docking study
The aromatase crystal structure was energy minimized and
the active site was defined in the stabilized conformation.
The ligands were effectively docked into aromatase active
site and from the 30 receptor–ligand conformations
ADME study
Fig. 5 Inhibition of MDA-MB-231 cells proliferation by 6i deter-
mined by MTT assay at 24 h. Each value represents the mean SD
from triplicate determinants. The P value is \0.0001
All the compounds were screened against PreADMET
server and the results are shown in Fig. 1. This study
revealed that the compounds 6a–6h showed low perme-
ability and compound 6i showed medium permeability for
Caco-2 cells. All the compounds showed low-permeability
values for MDCK permeability, whereas the compounds
6a, 6b, 6e and 6f showed moderate absorption and 6c, 6d,
6h and 6i showed well absorption to human intestine. The
BBB penetration is represented as BB = [Brain]/[Blood],
where [Brain] and [Blood] are the steady-state concentra-
tion of radio-labeled compound in brain and peripheral
blood. Predicting BBB penetration means predicting whe-
ther compounds pass across the BBB. This parameter tells
the absorption rate of compound to CNS that will define its
BBB penetration capacity. All the compounds were
observed to be having moderate absorption to CNS.
The skin permeability is a crucial parameter that can
define the transdermal delivery of the compound as the risk
assessment during accidental contact with the skin. The
skin permeability values are defined as logKp (cm/h) for all
the compounds, where Kp = Km 9 D/h. Km is distribution
coefficient between stratum corneum and vehicle, D is
average diffusion coefficient (cm2/h), and h is thickness of
skin (cm).
Fig. 6 DNA fragmentation analysis by 1.8 % agarose gel electro-
phoresis in genomic DNA isolated from MDA-MB-231 cells treated
with 6i at 24 h shows laddering pattern. M DNA marker, L1 DMSO
control, L2 25 lg/mL, L3 50 lg/mL, L4 100 lg/mL, and L5 200 lg/
mL of compound treated cells
The degree of plasma protein binding of a drug influ-
ences not only on the drug’s action, but also its disposition
and efficacy. In general, the unbound drug is available for
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Med Chem Res
generated for each compound, the conformation with
lowest docking score was chosen for analyzing the
interactions.
strength and stable those are favored by tryptophan residue
at 224th position in the aromatase.
It is observed from the docking conformations that the
compounds are showing interactions majorly with their
phosphonate moiety. This is because of the high-polarity
induced in the hydroxyl groups of the compounds which
made them to interact with the polar residues-like Arg 115.
The aromatase active site is highly hydrophobic and is
dominated by aliphatic amino acid residues such as
Val370, Leu372, Val373, Met374 and Leu477; hence these
compounds with aromatic groups are expected to bind with
high affinity. This enables the formation of hydrophobic
interactions around the ring structures in the active site. In
All the compounds were observed to be showing best
docking scores where a best least docking score of
-15.776 kcal/mol was observed for compounds 6i
(Table 2) (Fig. 2). Hydrogen bond interactions were
observed in the docking complexes of 6a, 6d, 6h, and 6i
compounds, whereas arene cationic interaction was
observed in the docking complex of 6g and arene–arene
interactions were observed for the compounds 6f and 6h.
Arene type interactions are the strong interactions than
hydrogen bonds that make the docking complex more
Fig. 7 Fluorescence
microscopy of MDA-MB-231
cells treated with compound
shows apoptotic cells
determined by Hoechst 33342
staining at 24 h. a DMSO
control, b 25 lg/mL, c 50 lg/
mL, d 100 lg/mL, and
e 200 lg/mL of compound
treated cells. Arrows represent
chromatin condensation and
nuclear fragmentation
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Med Chem Res
addition, the alkyl groups present along with phosphonate
moiety may be the responsible factors for the p–p stacking
interactions. This basic scaffold might be influencing the
inhibitory activity significantly. These described interac-
tions are typical of selective inhibition of aromatase, con-
firming the molecular design of the compounds studied is
reliable and much effective.
favored the binding orientations and its interaction within
the active site. In conclusion, our findings concerning
antitumor activities of the compound 6i as detected in
MDA MB 231 cell lines further corroborate its potential
usefulness as a safer and effective chemotherapeutic agent
used in the treatment of breast cancer. Orientation of 6i in
the aromatase active site is in such a way that the phos-
phonate moiety because of its charge is interacting with the
polar residue Arg115 by means of hydrogen bonding,
whereas the hydrophobic ring moieties were oriented
toward the hydrophobic residues like Ile133, Phe134,
Trp224, Ala306, and Val370 strengthening the interaction
and the overall strongly recommending 6i as the best aro-
matase inhibitor. In vivo studies are to be carried out in
animal models to prove its efficacy, which is our future
prospective to promote 6i for further clinical trials.
These interactions were compared with the binding
mode of reference compound exemestane, which showed a
higher docking score of -10.141 kcal/mol than the above
compounds and it is showing a single hydrogen bond
interaction with M374 of aromatase active site which is
also a hydrophobic residue (Table 2; Table S2). The higher
is the docking score the lower is the affinity of the ligand
with the receptor. This implies that the compounds studied
will be the effective inhibitors than exemestane.
Antiproliferative activity/cytotoxicity
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