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Combinatorial Chemistry & High Throughput Screening, 2013, 16, 57-72 57

ANN-QSAR Model for Virtual Screening of Androstenedione C-Skeleton

Containing Phytomolecules and Analogues for Cytotoxic Activity Against
Human Breast Cancer Cell Line MCF-7
Om Prakash1, Feroz Khan*,1, Rajender Singh Sangwan1 and Laxminarain Misra2

1
Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal & Aromatic Plants, Lucknow-
226015, India
2
Department of Phytochemistry, CSIR-Central Institute of Medicinal & Aromatic Plants, Lucknow-226015, India

Abstract: The present study deals with the development of an artificial neural network based quantitative structure
activity relationship (QSAR) model for virtual screening of active compounds which contain androstenedione carbon-
skeleton or their similar skeleton at the core. An empirical data modeling (with fitted data mapping) has been performed
on the basis of bioassay record for human breast cancer cell line MCF7. The whole experimental data set was considered
as test set. Standard feed-forward back-propagation neural network technique was applied to build the model. Leave-One-
Out (LOO) cross-validation was performed to evaluate the performance of the model. The mapped model became the
basis for selection best mapped compounds followed by development of Pharmacophore specific secondary QSAR model.
In the present study, two best mapped molecules ‘4beta-hydroxy Withanolide-E’ and ‘7, 8-Dehydrocalotropin’ were used
for development of the secondary QSAR model. These secondary-QSAR models were resulted with R2LOOCV value 0.9845
and 0.9666 respectively. Docking studies, in silico phamacokinetic and toxicity analysis was also done for selected
compounds. The screened compounds CID_73621, CID_16757497, CID_301751, CID_390666 and CID_46830222 were
found with promising binding affinity value with aromatase with reference to the co-crystallized control compound
androstenedione. Due to excellent extent of variance coverage in ANN based QSAR map model, it can be used as a robust
non-linear QSAR model for androstenedione carbon-skeleton containing molecules and the protocol can be used to derive
secondary QSAR models for other compounds set.
Keywords: Androstenedione, ANN, MCF-7, QSAR, withanolide.

INTRODUCTION 18
12 O
Androstenedione Fig. (1) bound with androgen receptor CH3

has been crystallized and proved to be cytotoxic for human 11

19 13 17
breast cancer cell line MCF-7. Androgen has also found to 16
1 CH3 9 H 14
be involved in the anti-breast-cancer activities at the
2
hormonal level control mechanism. Androstenedione acts as 10 8 15
competitive inhibitor, thus leading to anti-cancer activities. 5 H H
Chemotherapy is usually limited by the presence of 3
7
multidrug resistance (MDR). MDR is the cross-resistance of HO 4 6
tumor cell lines to several structurally and functionally H

unrelated chemotherapeutic agents after exposure to a single Fig. (1). Androstenedione skeleton (co-crystallized with
cytotoxic drug [1, 2]. Therefore, it becomes necessary to Aromatase).
identify a new generation of anti-MDR taxoids. Fast
Accurate models of QSAR are critical for any rational
identification of new cytotoxic agents is only possible
molecular design and for understanding the structuring of
through in-silico means. In-silico means involve virtual
molecular information. The data analysis techniques which
screening through QSAR models. QSAR model is the
can be applied at this level to find such relationships are the
function of a set of independent molecular descriptors.
multiple linear regressions (MLRs), partial least square
Molecular descriptors encode the most useful
(PLS), and methods based on machine learning approaches
physicochemical information on structural features that is
as artificial neural network (ANN), support vector machine
relative to the activities to be modeled. 2D & 3D descriptors
(SVM) and hidden markov model (HMM) etc. The artificial
are widely used in QSAR modeling, including recent cancer-
neural network (ANN), used as a modeling technique, has
related research [3, 4]. The large amount of descriptors
recently become a popular and powerful chemometric tool
represents the structural characteristics of molecules.
[5-7]. Compared with classical statistical methods, ANN-
based approaches do not require preliminary knowledge of
the mathematical form of the relationship between the
*Address correspondence to this author at the Department of Metabolic and variables [8], which make the ANN suitable for
Structural Biology, CSIR-Central Institute of Medicinal & Aromatic Plants, extrapolating the complex and unsure relationships between
Lucknow-226015, India; Tel: 0522-2718668; E-mail: f.khan@cimap.res.in

1875-5402/13 $58.00+.00 © 2013 Bentham Science Publishers

58 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1
the biological phenomenon and the structure of the
compounds. ANNs is one of the best methods which
establish the relationship among the non-linear features.
ANN consists of formal neurons or nodes and connections
(weights) among them. In ANN architecture, the neurons are
arranged in layers (input, hidden and output), and
unidirectional connections. The adjacent layers are fully
connected but no connections exist between the neurons
within the same layer. The architecture computes a
numerical output value for a given numerical input vector. A
formal neuron sums up incoming signals multiplied by the
connection weights, subtracts a threshold value (or bias) and
calculates an output signal by using a transfer function. A
neuron may differ in the transfer function. Input neurons
simply distribute data to the hidden layer neurons without
any further computation. Hidden layer neurons typically
have a sigmoid transfer function:
tf = 1
(1 + e
input )
That limits the neuron's output signal with values
between 0 and 1. The output layer neurons usually have
sigmoidal or linear transfer functions, depending on the
application. The whole network represents a non-linear
relationship which can be written for each output as:
   the lead compounds. Therefore, it is extremely important to


y =
Prakash et al.
core of Withanolides. Withanolide is a class of phytosteroid
named after W. somnifera. Withanolides, chemical
nomenclature as 22-hydroxy ergostane-26-oic acid 26, 22-
-
lactones are C28-steroidal lactones based on an intact or
rearranged ergostane frame through appropriate oxidations at
C-22 and C-26 to form a
-lactones ring. Aromatase is
reported for tumourogenesis mechanism in MCF-7 cell line.
QSAR analysis can be explained in two basic steps, first
is the development of highly predictive model and second
mechanisms related to specific properties of chemicals used
in model development. The explanation of the mechanism
involvement for chemical properties (descriptors) depends
on model development. The descriptors strongly depend on
selection of molecule in the training set. Alternatively, the
consensus ranking protocol has also been proposed for
descriptor selection [13]. Virtual screening is a tool to search
out the novel drug like compounds. A wide spectrum of
(1) methodology protocols is available for screening of lead
compounds. The number of methods and software targeting
the virtual screening is increasing day by day. However, the
expert understanding on the implacability of methodology is
not evolving as fast as the development of methodology.
Considering the diversity of targets for lead action, it is
necessary to develop QSAR models in a structured way by
understanding the experimental and the real applicability of
(x) =  tf   xi wih
i   wh
 k (2)
h  i
 [14]. In vitro effects of an in silico-modelled 17-estradiol
where wih is the connection weight between the input node i
with the hidden node h and wh are the connection weight
between each hidden node h with the final output considered,
y.
i and
h are the biases corresponding to the input and
hidden layers. The difference between y and y is the target
error, which is subsequently back-propagated to modify the
weights in order to attain the best fit [9].
Extensive research has been conducted to better
understand the various aspects of screening out the lead
molecules with anti-cancer activities. Until now, several
targets/cell lines specific in-silico models have been
developed to virtually screen out the various
derivatives/analogues of the known potential lead molecule.
When we deal with screening of cell line specific lead
molecules, it becomes necessary to identify those molecular
patterns which are concerned with the viability of that
particular cell line. In case of MCF-7 cell line, the
participation of androstenedione against androgen receptor
has been proved experimentally. Therefore, it becomes
logical that the molecules with a similar molecular descriptor
pattern can act as potential anti-cancerous lead molecule.
Pharmacophore models show a better ability to select active
ligands [10]. Pharmacophore analysis of consensus models
provides a comprehensive and efficient approach for
structure activity relationship study [11]. Pharmacophore
consensus provides a basic pattern, which should be
available in the core of the structures being used for
development of any Structure-Activity Relationship (SAR)
model. Experimentally known pharmacophore can be
considered as an efficient base for mapping and selection of
new pharmacophore(s) for QSAR model development [12].
The basic skeleton of ergostane or androstenedione exist
in crystallised protein Aromatase (PDBID: 3EQM), which is
develop robust protocols for development of specific models
derivative in combination with dichloroacetic acid has
reported two novel derivatives acting against MCF-7 cell
line of human breast cancer [15]. Non-linear QSAR model is
the relationship between ‘a series of diversified patterns
developed from an empirical compounds’ and their
respective bioactivity value in a high dimensional vector
space [16]. The pattern of core pharmacophore derivatives
can be accessed from the pharmacophore consensus. These
patterns will be unique for specific human target/cancer cell
line. The best mapped compound can be considered as the
consisting the basic pharmacophore pattern. Now the SAR
model on the basis of best mapped compound will be
considered as the best model for SAR.
In the present work an ANN-map-Model for
androstenedione skeleton containing lead molecules has been
proposed for prediction of logGI50 value for screening of
their derivatives and analogues against human breast cancer
cell line MCF-7.
EXPERIMENTAL
Dataset: Bioassay record (NCBI Assay ID: 83) for
inhibition of human breast cancer cell line (MCF7) with
compounds containing androstenedione similar skeleton was
extracted. Only 37 compounds with their molar
concentration of growth inhibitory concentration (logGI50)
were selected to map the pharmacophore. The crystal
structure (PDBID: 3EQM) of Aromatase bounded
androstenedione was collected from Protein Data Bank.
Descriptor generation: We used the PaDEL descriptor
software (National University of Singapore) to calculate
molecular structure descriptors, which contain CDK-library
to calculate descriptors [17]. In total, 404 standard
ANN-QSAR Model for Virtual Screening of Androstenedione Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 59
descriptors were calculated to include in 2D & 3D
descriptors of 45 classes, which can sufficiently represent the
structural characters of molecules.
Feature reduction: Initially 404 descriptors were
calculated for all compounds. Since, not all of the 404
descriptors contribute to the bioactivity; some measures were
taken to eliminate the noise (uninformative descriptors):
eliminating the descriptors with constant values and more
than 90% zero values because they offered little
discriminating information for the development of the
model. After this procedure, while selecting the best subset
of chemical descriptors, highly correlated descriptors were
excluded by using the correlation matrix approach.
Correlation matrix based feature reduction was used to
reduce the variable space and the chance of correlation
between the descriptors, has been done. Removal of
correlated descriptors removes the noise from the data.
Data mapping: The molecular properties and their
respective bioactivity values have been fitted into the
polynomial equational map. A series data generated from the
equations has been used as input and output value for
analysis in the back propagation neural network (BPNN)
model. Here point of attention is that, no any sample point
has been directly used in ANN based QSAR-map- model.
ANN-map-model: In order to build reliable and
predictive QSAR models, we adopted the ANN technique,
which has been proven to have outstanding non-linear
approximation ability [6, 7, 18]. A typical ANN consists of
an input layer, a hidden layer, and an output layer. In the
ANN, signals are propagated from the input neurons through
the hidden layer to the output neuron, and then the error is
calculated and back propagated to iteratively adjust weights
and biases in order to minimize the error in prediction; this is
the most distinct character of typical back propagation (BP)
algorithm. The ANN program used was the neural network
software package of MATLAB v2012 developed by the
MathWorks, USA (http://www.mathworks.com/). Some
fully connected 3-layer BP neural networks with sigmoid
transfer function were constructed. The number of neurons
in the input layer equaled the number of selected descriptors.
Before the net training, all of the input and output values
were normalized to between 0.1 and 0.9, and the outputs
were transferred back to the same units as the original
outputs for comparison purpose. The Levenberg–Marquardt
algorithm was adopted to optimize weight and biases
because it was significantly faster than other algorithms
based on gradient descent [19]. The four descriptors and
their respective bioactivity have been mapped in the form of
equation [20]. The equation derived training sets were used
to determine the architecture of the ANN model, and the
experimental datasets as well as independent external testing
set was used to evaluate the predictive ability of the models.
Evaluation of ANN-map-Model: The following
parameters were calculated to evaluate the performance of
the ANN and the predictive ability of the model:
1. R2 for training data
2. R2 for Leave One Out (LOO) cross validation
Secondary QSAR model and docking studies: The best
mapped compounds have been selected through ANN-map-
model. The secondary QSAR models were developed on the
basis of compounds containing the 95% fragments similarity
with the best mapped compounds. The selected compounds
were further screened by docking studies (using AutoDock
4.2) with aromatase receptor [21].
Virtual screening by in-silico bioactivity & toxicity
predictions: Absorption, Distribution, Metabolism,
Excretion and Toxicity (ADMET) evaluation was done by
Molinspiration & T.E.S.T softwares [22-25]. Bioactivity
prediction and toxicity testing were performed in support of
screening results of the model. The Pharmacokinetic
parameter screen shows the prioritization of predicted active
compounds screened out through the developed QSAR
model, so that to assist the Chemist while planning the
chemical synthesis. These analyses will also be a good
reference for prioritizing the compounds for in vitro or in
vivo experiments.
RESULTS & DISCUSSION
Compound Selection
As a cytochrome P450, aromatase utilizes electrons from
NADPH-cytochrome P450 reductase (CPR) to produce
estrogen from androgen. That’s why aromatase inhibitors
block estrogen synthesis and are widely used for treatment of
estrogen-dependent breast cancer. The binding features differ
for steroidal and non-steroidal inhibitors. Steroidal inhibitors
as exemestane and ardrostenedione have been proved active
against aromatase [26]. Signaling pathways that regulate the
expression and enzyme activity of aromatase in stromal and
epithelial breast cells has been studied with human breast
cell lines MCF-10F, MCF-7 and MDA-MB-231 for
identifying novel targets for regulation. A record of bioassay
was found for a diverse compound set tested against MCF-7
and is available at NCBI-PubChem bioassay database AID:
83. androstenedione skeleton, which is present in the core of
aromatase binding. The carbon-skeleton of ardrostenedione
matches with the core part of Withanolides. 4beta-hydroxy
Withanolide-E has found active against MCF-7. By
considering the above information, ergostane skeleton
containing 37 compounds were selected for development of
ANN-map-Model against human breast cancer cell line
MCF-7.
Descriptor Selection
Feature / descriptors are the basic requirement for
development of any model. Here molecular properties have
been taken as a feature vector, which provide pattern in high
dimension feature space for mapping of consensus
diversification of active compounds androstenedione
skeleton against MCF-7. 404 molecular descriptors (two &
three dimensional) were calculated for observational data
compounds. Such a huge descriptor set has high redundancy
as well as noise in the dataset. To select the independent set
of features, descriptor selection procedure has been
performed. The descriptor selection has done on the basis of
correlation matrix analysis, where descriptors with higher
correlation coefficient were removed from the data set.
Second filter step up include selection of those descriptors
which have correlation coefficient >0.5 (positively or
negatively) with bioactivity vector of available datasets. As a
60 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1
result only four descriptors came into existence for further
processing. The descriptors used are: nAtomP (Largest Pi
system), Weta2.unity (WHIM), BCUTp-1l (BCUT
topological descriptor) and WTPT-2 (weighted path). The
detail description about descriptors are available at the
PaDEL descriptors site [17]. The selected descriptors define
the cytotoxic activity of androstenedione carbon-skeleton
containing compounds against the MCF7 cell line.
Correlation coefficient based descriptor selection is a core
part of principal component analysis (PCA) based descriptor
selection, where independent principal component is being
arranged in order of decreasing variance. Besides, genetic
algorithm (GA) was also used in descriptor selection which
were used later to help in improving the back-propagation
training algorithm [27].
ANN-Map-Model Development
Random availability of experimental data is a hurdle for
development of a robust simulation model. So it becomes
necessary to fit the data in an equational map, which will
provide a good degree of data distribution. Here all four
descriptors and bioactivity vectors have been converted into
five degree polynomial equational map to achieve a good
data distribution (equations 3-7). Application of equational
mapping has also done for development of the ANN model
for prediction of in-vitro culture parameters [20].
Equations for Fitted Data Map
logGI50 = (6.9421e-007)*x^5 + (-8.0997e-005)*x^4 +
(0.0034333)*x^3 + (-0.063835)*x^2 + (0.55809)*x +
(-9.5425);
nAtomP = (9.1691e-007)*x^5 + (-4.5264e-005)*x^4 +
(4.1314e-006)*x^3 + (0.017173)*x^2 + (-0.11373)*x +
(5.6606);
Weta2.unit = (7.1015e-008)*x^5 + (-5.7449e-006)*x^4 +
(0.00015518)*x^3 + (-0.0015897)*x^2 + (0.0031797)*x +
(0.48224); (5)
BCUTp-1l = (-1.7124e-007)*x^5 + (1.4483e-005)*x^4 +
(-0.00045411)*x^3 + (0.0069282)*x^2 + (-0.044735)*x +
(4.2583); (6)
WTPT-2 = (-2.0598e-008)*x^5 + (2.2551e-006)*x^4 +
(-8.5248e-005)*x^3 + (0.0013186)*x^2 + (-0.0081033)*x +
(2.0762); (7)
where, ‘x’ is a sequence number ranging from ‘1’ to ‘N’. ‘N’
is the number of observations, worker want to put in the
training data.
Map based ANN model does not involve a single set of
empirical data in the training set. This totally depends on the
patterns available in molecular descriptors data distribution
on the available compound set. Map based model contains
the global information about the diversification of consensus
of molecular properties for available datasets. ANN-map-
Model has been implemented on standard feed-forward
back-propagation neural network by following architecture
with sigmoid function ‘logsig’ at hidden and output layer
and ‘purelin’ at the input layer:
Prakash et al.
1. Input layer: 04
2. Hidden layer: 03
3. Output layer: 01
4. Max Epochs (Back-propagation): 1000
5. Momentum: 0.2
6. Learning rate: 0.3
7. Initial weight range: (-0.5) to (+0.5)
8. Data normalization: 0.1-0.9
The developed ANN-map-Model has been evaluated on
two bases (Table 1):
1. Prediction for equational map data: R2 value
0.999172
2. Cross validation by LOO method: R2LOOCV 0.998456
External Validation of Model
The literature based external validation of ANN-map-
Model has been done with two ergostane derivatives 2-
methoxyestradiol and 17beta-estradiol derivative, which has
been found active against human breast cancer cell line
MCF-7. The wet lab experiment was performed to
investigate the anti-proliferative properties of in silico
modelled 17
-oestradiol derivative (C9), in combination
with dichloroacetic acid (DCA), on MCF-7 and MCF-12A
cells. The derivatives were found selective towards
tumourigenic cell and it was concluded to be used as
chemotherapeutic agent for further cancer cell lines [14].
(3) Predictions have been made for 2-methoxyestradiol
(Predicted activity (nlogGI50): -6.31 M) and 17beta
derivative (Predicted activity (nlogGI50): -7.89 M), both
compounds were found active, which validates the efficiency
(4) of the model.
Selection of Best Mapped Compound
Selection of best mapped compound available in
empirical data is on the basis of their bioactivity predicted
value. A ‘map value’ (mv) has been derived on the basis of
difference of empirical and predicted value. It was assumed
that the compounds with ‘mv’ >=2.0 will be considered as
well mapped.
Mapping value (mv) = nlog (1/ abs(e – p))
where ‘e’ is ‘Empirical_value’ and ‘p’ is ‘Predicted_value’
Mapping value >= 2.0 is taken as the threshold for selecting
significant mapping
The result was drawn that the ANN-map-Model will
work best for ‘4beta-hydroxy Withanolide-E’ and ‘7, 8-
Dehydrocalotropin’ or ‘ergostane’ skeleton having
derivatives and analogs. As a result two compounds have
been found well mapped. The compounds are:
The best mapped compounds can be used as parent
compound for the development of secondary QSAR
model(s) (Fig. 2).
ANN-QSAR Model for Virtual Screening of Androstenedione Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 61

Table 1. Predicted Bioactivity (logGI50M) Against Empirical Data

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

O O

CH3
H3C
1 O -9.119 -8.68635 0.43265
CH3
HO OH
OH
H3C O O

HO

O O

O CH3
O CH3
OH
HO CH3
2 -8.75 -9.33685 0.58685

CH3

O O

CH3
H3C
3 O -8.112 -8.85327 0.74127
CH3
HO OH
OH
H3C O O

CH3
CH3
O O
O O
O O

CH3
4 CH3 -8.02 -6.95354 1.06646
O
CH3

O O

CH3
H3C
5 O -8 -9.13202 1.13202
CH3
HO
OH
H3C O O

O O

CH3
OH CH3 OH
6 CH3 -7.886 -6.58109 1.30491
HO O HO O
O
OH
O O
HO O O H3C O O
CH3
HO O CH3
62 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 Prakash et al.

(Table 1) contd…..

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

O
O

CH3
7 -7.823 -9.38762 1.56462
CH3

OH
HO

O O

CH3
HO
8
CH3 HOHO -7.788 -7.68113 0.10687
HO
O
OH
HO O
OH
OH
O
O

CH3
9 -7.785 -9.05166 1.26666
CH3 O
O CH3
OH O
H3C O

O OH

H3C
O

10 -7.725 -7.73458 0.00958

O O
O
O
HO
HO CH3

O O

CH3
O
HO
11 -7.624 -7.71224 0.08824
OH
O
O
O

HO CH3

O
OH O
HO OH
CH3
12 HO -7.602 -7.16195 0.44005
O O
CH3
HO OH
OH
H3C O O
ANN-QSAR Model for Virtual Screening of Androstenedione Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 63

(Table 1) contd…..

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

O
O

CH3
13 -7.523 -9.10941 1.58641
OH
CH3
HO OH
OH
H3C O O

O OH

H3C
O

14 -7.475 -6.14317 1.33183

O O
O
O
HO
HO CH3

O
O

CH3

15 CH3 -7.426 -7.57763 0.15163

OH
OH
HO
O O

CH3

O
O

CH3
16 OH CH3 -7.426 -9.20606 1.78006
O
HO O
O
OH
O
HO HO O
OH
OH
HO

O O

CH3
OH OH
17 CH3 -7.384 -6.97196 0.41204
HO HO O
OH
O
H3C O O H3C O O
CH3

O
O

CH3
18 -7.301 -9.38977 2.08877
O

OH
HO
64 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 Prakash et al.

(Table 1) contd…..

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

OH O O
HO
OH OH
CH3
O
HO CH3 CH3
19 CH3 -7.292 -7.3969 0.1049
O HO O
O O
OH
O
O O HO O
CH3
O CH3

O O

CH3
OH CH3 O
HO O
O
OH
20 O -7.263 -6.65382 0.60918
HO O O
OH
CH3
O
HO
O

HO
OH OH

OH
OH
O CH3
O
O H3C
21 O -7.258 -7.2083 0.0497
H3C
OH
O
O
O

CH3
22 -7.112 -8.63552 1.52352
O OH

OH

OH

O O

CH3
H3C
23 O -6.796 -7.6739 0.8779
CH3 O
HO OH CH3
OH O
H3C O O

O CH3
H3C
HO CH3
24 OH -6.73 -6.72924 0.00076
OH
O CH3
HO
CH3
O
ANN-QSAR Model for Virtual Screening of Androstenedione Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 65

(Table 1) contd…..

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

H3C
O OH
OH
O
O
OH
O
H 3C
CH3
H3C CH3
H 3C
CH
25 CH3 3 -6.726 -6.49035 0.23565

OH O OH
O
HO O
O
O
O HO
HO CH3 OH
O

HO
OH

CH3
O
O
O O
H3C
CH3
O CH3
26 CH3 -6.632 -7.48586 0.85386

O
O O

CH3

O O

CH3
27 OH O -6.566 -6.41142 0.15458
HO
OH
H3C O O
OH

H3C CH3

OH
CH3
HO
O O
28 CH3
CH -6.337 -6.32129 0.01571
3
HO O
H3C O O CH3
OH
HO OH
OH

O O

CH3
H3C
29 O -6.334 -6.31571 0.01829
O
HO
OH
H3C O O
OH
66 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 Prakash et al.

(Table 1) contd…..

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

O O

CH3
CH3
OH O O CH3
30 CH3 OH -6.284 -7.76291 1.47891
HO O HO O
O
OH
O O
HO H 3C O O HO O
CH3
HO

OH
HO OH

CH3 O O
HO HO OH
O OH
O
HO O
31 OH O -6.231 -5.97782 0.25318
H3C

H3C
H3C
O
O
H3C

H3C
CH3

H3C
CH3 OH
HO
32 -6.183 -6.52446 0.34146
CH3 O OH

O OH
HO
H3C OH

H 3C
O OH
OH
O
O
OH
O
H3C
CH 3
H3C CH3
H3C
CH3
CH3
33 -6.161 -6.43981 0.27881
OH O OH
O
HO O
O
O
O HO
HO CH3 OH
O

HO OH
O OH

O
OH

H3C O

O HO
O O
34 CH3 -6.139 -6.65714 0.51814
O
CH3

H3C CH3
ANN-QSAR Model for Virtual Screening of Androstenedione Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 67

(Table 1) contd…..

Empirical Predicted Absolute

S. No.
nlogGI50M nlogGI50M Residual

OH
HO

CH3 O O OH
HO HO
O OH
O
HO O
35 OH O -6.111 -5.96685 0.14415
H3C

H3 C
H3C
O
O
H3C

H3C CH3
O O
CH3
HO OH
36 O OH -6.062 -5.99486 0.06714
OH
OH
CH3
H3C CH3
O
CH3

OH O O
OH
HO
CH3
O
OH O
37 -6.027 -6.62467 0.59767
O
O
OH
HO O
OH

Development of Predictive QSAR Model for Cytotoxic based on 23 compounds, which were also 95% similar to 7,
Activity 8-dehydrocalotropin. The two sec-QSAR models showed
R2LOOCV value 0.9845 and 0.9666 respectively Fig. (3). The
First sec-QSAR model was developed on the basis of 58 ANN-map-model covers as broad spectrum of molecular
compounds, which were 95% structurally similar to 4beta-
diversity, while the secondary QSAR model becomes more
hydroxy Withanolide E, while second sec-QSAR model was
3.5 NCBI CID: 73621
(mv = 3.119186408)
3

2.5
NCBI CID: 390666
Mapping value (mv)

(mv = 2.018634491)
2

1.5

0.5

0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37
-0.5
Sample number

Fig. (2). Selection of best mapped compound by ‘ANN-map-Model’.

68 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 Prakash et al.

-6
O -8.5 -8 -7.5 -7 -6.5 -6

R2cv = 0.9845 -6.5

O CH3
H3C

Training Bioactivity
-7
HO
CH3
OHOH -7.5
O CH3
HO
-8
CH3

O Predicted Bioactivity by LOOCV (Taking 73621 as mother compound)

-8.5

4beta-hydroxy Withanolide E Regression plot of QSAR model developed for 4beta-hydroxy Withanolide E

-5
-10 -9.5 -9 -8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5
-5.5

-6

OH -6.5
R2cv = 0.9666
O
Training Bioactivity
OH -7
H3C O O -7.5
O
O CH3 -8
O -8.5

-9
HO -9.5

-10
Predcited Bioactivity by LOOCV (Taking 390666 as mother compound)

7,8-Dehydrocalotropin Regression plot of QSAR model developed for 7, 8-Dehydrocalotropin

Fig. (3). Best mapped compounds and their respective secondary QSAR models.

specific for prediction of accurate bioactivity. In this way, and basic amino acid residue namely: HIS-171 (Histidine).
these two types of model can be used for refined virtual Similarly ‘7, 8-Dehydrocalotropin’ binding site at aromatase
screening of lead molecules. contains polar amino acid residues TRY-361 (Tyrosine),
GLN-428 (Glutamine), & TYR-441 (Tyrosine) and
hydrophobic amino acid ILE-350 (Isoleucine) (Figs. 4-6).
Virtual Screening by Docking Studies
The objective of docking studies was to explore the
Blind docking was performed with Aromatase and 81 binding affinity of studied compounds against selected
compounds involved in QSAR model development. It was aromatase enzyme (PDB ID: 3EQM) from human, and to
found that only two database molecules showed study their possible mechanisms of action. The results of
superimposition along with ‘4beta-hydroxy Withanolide-E’ docking studies suggest that studied compounds inhibit the
on target enzyme aromatase. However only one database activity of aromatase by binding on it strongly, as indicated
molecule showed superimposition along with best predicted by docking energy. In the studied work, we explored the
compound ‘7, 8-Dehydrocalotropin’ on target enzyme orientations and binding affinities (in terms of the docking
aromatase. Co-crystallized compound Androstenedione energy in kcal mol
1) of Androstenedione, 4beta-hydroxy
binding site at aromatase enzyme contains conserved Withanolide-E, and 7, 8-Dehydrocalotropin towards anti-
hydrophobic amino acid residues namely: MET-374 cancer target aromatase from human (PDB: 3EQM).
(Methionine), LEU-372 (Leucine), LEU-477 (Leucine),
VAL-370 (Valine), ALA-306 (Alanine); acidic residue When we compared how the binding pocket residues of
namely: ASP-309 (Aspartate) and basic residue namely: aromatase interacted with these compounds, we found that
ARG-115 (Arginine). ‘4beta-hydroxy Withanolide-E’ compounds androstenedione, CID_73621, CID_16757497,
binding site at aromatase contains polar amino acid residues CID_301751, CID_390666 and CID_46830222, showed
namely: SER-167 (Serine), THR-170 (Threonine) and THR- interaction with conserved amino acid residues thus lead to
20 (Threonine); acidic amino acid residues namely: ASP-197 more stability and potency in these cases. The docking
(Aspartate), GLU-210 (Glutamate), GLU-177 (Glutamate); results for the CID_73621, CID_16757497, CID_301751,
ANN-QSAR Model for Virtual Screening of Androstenedione
CID_390666 and CID_46830222 docked onto aromatase
enzyme of human significant docking energy of -8.7, -8.5, -
8.9, -9.0 and -9.5 kcal mol
1 respectively, comparable to co-
crystallized androstenedione, bound to aromatase target
enzyme (PDB: 3EQM) with the standard docking energy of -
4.9 kcal mol
1.
The binding site of androstenedione at aromatase
contains ARG-115, ALA-306, ASP-309, VAL-370, LEU-
372, MET-374, LEU-477 amino acid residues. Site specific
docking resulted into binding affinity with -4.9, -5.0, -5.0, -
4.9, -4.7, -4.9 kcal mol-1 for Androstenedione, CID_73621,
CID_390666, CID_16757497, CID_301751, CID_46830222
respectively (Tables 2 and 3).
Fig. (4). Blind docking results for ‘4beta-hydroxy Withanolide E’,
CID_16757497 and CID_301751 with aromatase (04 H-bonds).
Fig. (5). Blind docking results for ‘7, 8-Dehydrocalotropin’ and
CID_46830222 docked with aromatase (02 H-bonds).
In Silico Toxicity & Bioactivity Screening for Selected
Molecules
The resultant three molecules from secondary data and
two molecules (control) from experimental data were
screened out from three steps of virtual screening as ‘ANN-
map-model + sec-QSAR model + docking with aromatase’.
Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 69
The selected five compounds were evaluated on the basis of
ADMET parameters. All five compounds showed 0-1
violation of Lipinski’s rule of five, developmental toxicant,
non-mutagen activities and strong enzyme inhibition
properties. The predicted ‘oral rat LD50’ mol/kg was found
in the range of 3.89 to 4.58 (Tables 4-6).
Fig. (6). Docking results at androstenedione binding site (receptor:
Aromatase; ligands: Androstenedione, CID_73621, CID_16757497,
CID_301751, CID_390666, CID_46830222).
The ideal oral drug is one that is rapidly and completely
absorbed from the gastrointestinal tract, distributed
specifically to its site of action in the body, metabolized in a
way that does not instantly remove its activity, and
eliminated in a suitable manner without causing any harm. It
has been reported that around half of all drugs in
development fail to make it to the market because of poor
pharmacokinetics (PK) [26]. The PK properties depend on
the chemical properties of the molecule. PK properties such
as absorption, distribution, metabolism, excretion, and
toxicity (ADMET) are important determinants of the success
of the compound for human therapeutic use [28-30]. Some
important chemical descriptors correlate well with PK
properties, such as the polar surface area (PSA; a primary
determinant of fractional absorption) and low molecular
weight (MW; for oral absorption) [29]. The distribution of
the compound in the human body depends on factors such as
the blood – brain barrier (log BB), permeability (such as the
apparent Caco-2 permeability, apparent MDCK
permeability, logK p for skin permeability), the volume of
distribution, and plasma protein binding (logK hsa for serum
protein binding) [31], so these parameters were calculated
and checked for compliance with their standard ranges. The
octanol – water partition coefficient (logP) has been
implicated in BBB penetration and permeability prediction,
as has PSA. It has been reported that the process of excreting
the compound from the human body depends on the MW
and logP.
70 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 Prakash et al.

Table 2. BLIND Docking Results

S. No. NCBI CID/Name Binding Affinity (kcal/mol) BLIND Docking Binding Site Amino Acids

1 Androstenedione -4.9 ARG-115, ALA-306, ASP-309, VAL-370, LEU-372, MET-374, LEU-477

2 73621 -8.7
3 16757497 -8.5 SER-167, ASP-197, HIS-171, GLU-210, GLU-177, THR-170, THR-201

4 301751 -8.9

5 390666 -9.0
TRY-361, GLN-428, TYR-441, ILE-350
6 46830222 -9.5

Table 3. Androstenedione Binding Site Docking Results

S. No. NCBI CID/Name Binding Affinity (kcal/mol) Specified for the Binding Site of Androstenedione Binding Site Amino Acids

1 Androstenedione -4.9
2 73621 -5.0
3 390666 -5.0 ARG-115, ALA-306, ASP-309,
VAL-370, LEU-372, MET-374, LEU-477
4 16757497 -4.9
5 301751 -4.7
6 46830222 -4.9

Table 4. ADME Properties of QSAR Based Screened Out Molecules

Calculated Properties (Using Molinspiration)

S. No. NCBI CID
miLogP TPSA natoms MW nON nOHNH nviolations nrotb Volume

1 16757497 1.348 157.044 37 518.603 9 5 1 2 465.245

2 301751 3.179 116.588 35 486.605 7 3 0 2 449.158
3 73621 2.263 136.816 36 502.604 8 4 1 2 457.201
3 390666 0.966 131.762 38 530.614 9 3 1 2 474.509
4 46830222 0.452 151.99 39 548.629 10 4 1 2 488.766
Footnote: miLogP = octanol/water partition coefficient, TPSA= Topological Polar Surface Area, natoms = number of atoms, MW = Molecular weight, nON = Number of hydrogen
acceptor, nOHNH = Number of hydrogen donor, nviolations = Violations from Lipinski's rule, nrotb = Number of rotatable bonds, volume: Volume of molecule.

Table 5. Bioactivities Prediction of QSAR Based Screened Out Molecules

Predicted Bioactivities (Using Molinspiration)

S. No. NCBI CID
GPCR Ion Channel Kinase Nuclear Receptor Protease Enzyme
Ligand Modulator Inhibitor Ligand Inhibitor Inhibitor

1 16757497 0.0616 0.2492 -0.4092 0.5495 0.1412 0.8574

2 301751 -0.0656 0.158 -0.5031 0.6153 0.0634 0.8918
3 73621 -0.0052 0.1896 -0.4521 0.5855 0.1129 0.9517
3 390666 0.1334 -0.008 -0.1992 0.6575 0.2056 0.8768
4 46830222 0.041 -0.1119 -0.3351 0.3499 0.0732 0.8035

Likewise, rapid renal clearance is associated with small
and hydrophilic compounds. On the other hand, the
metabolism of most drugs, which takes place in the liver, is
associated with large and hydrophobic compounds [32].
Higher compound lipophilicity leads to increased
metabolism and poor absorption, along with an increased
probability of binding to unwanted hydrophobic
macromolecules, thereby increasing the potential for
toxicity. In spite of some observed exceptions to Lipinski’s
rule, the property values of the vast majority (90%) of orally
ANN-QSAR Model for Virtual Screening of Androstenedione
Table 6. Toxicity Estimation of Predicted Active Molecules Through Derived QSAR Model
Oral Rat LD50 (Method Used:
S. No. NCBI CID
Consensus) –Log10 Value mol/kg
1 16757497 3.98
2 301751 4.05
3 73621 3.89
3 390666 4.56
4 46830222 4.58
active compounds are within their cut-off limits [33].
Molecules that violate more than one of these rules may not
be sufficiently bioavailable. When studying PK properties,
screening based on the Lipinski ’ s rule of five (which is
used to assess drug-likeness) was applied to the
androstenedione analogues/derivatives. In addition, the oral
bioavailability of each lead molecule was assessed through
its topological polar surface area (TPSA) using
Molinspiration online software. This descriptor has been
shown to correlate well with passive molecular transport
through membranes, thus allowing the prediction of drug
transport properties, and it has been linked to drug
bioavailability (the percentage of the dose of the drug that
reaches the blood circulation). Also, the number of rotatable
bonds is a simple topological parameter used by researchers
as part of an extended Lipinski s rule of five as a measure of
molecular flexibility. This is a very good chemical descriptor
for oral bioavailability [34]. A rotatable bond is defined as
any single non-ring bond bound to a non-terminal heavy (i.e.
non-hydrogen) atom. Amide C – N bonds are not considered
in this context because of their high rotational energy barrier.
Moreover, some researchers have also included the sum of
H-bond donors and H-bond acceptors as a secondary
determinant of fractional absorption. The primary
determinant of fractional absorption is PSA [35]. According
to the extended Lipinski’s rule of five, the sum of H-bond
donors and acceptors should be
12 or the PSA should be

140Å 2 [35], and the number of rotatable bonds should be

10 [33].
CONCLUSION
The study has been concluded as; it was found that the
equational data fitting map can be directly used for
development of ANN-map-Model. The developed model can
be used as a non-linear QSAR model for virtual screening of
androstenedione skeleton containing compounds, for
Example, Withanolides which indicate cytotoxic activity
against human breast cancer cell line MCF-7 and thus may
lead to potential anticancer molecules by lead optimization.
The protocol of the ANN-map-Model development can be
used for other pharmacophore mapping. The excellent
mapped model can also be used for the selection of new
compounds to develop a robust secondary QSAR model.
Molecular docking studies and in silico bioactivity screening
results assists the reliability of the QSAR modelling process.
Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 1 71
Developmental Toxicity
Mutagenicity (Method Used: Consensus)
(Method Used: Consensus)
Developmental toxicant (0.78) Mutagenicity Negative (0.03)
Developmental toxicant (0.80) Mutagenicity Negative (0.17)
Developmental toxicant (0.61) Mutagenicity Negative (0.17)
Developmental toxicant (0.79) Mutagenicity Negative (0.04)
Developmental toxicant (0.69) Mutagenicity Negative (-0.04)
CONFLICT OF INTEREST
Authors declare that they have no conflict of interest by
any means with respect to the instant research manuscript.
ACKNOWLEDGEMENTS
We acknowledge the Council of Scientific and Industrial
Research, New Delhi, India for financial support as Senior
Research Fellowship at CSIR-Central Institute of Medicinal
and Aromatic Plants, Lucknow, Uttar Pradesh, India.
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