Arabian Journal of Chemistry (2013) xxx, xxxxxx

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Study of physicochemical properties-inducible nitric

oxide synthase relationship of substituted quinazolinamines
analogs: Pharmacophore identication and QSAR studies
Mukesh C. Sharma a, Smita Sharma
a
b
c

b,*

, Pratibha Sharma c, Ashok Kumar

School of Pharmacy, Devi Ahilya University, Takshila Campus, Khandwa Road, Indore 452 017, India
Department of Chemistry, Chodhary Dilip Singh Kanya Mahavidyalya Bhind 477001, India
School of Chemical Sciences, Devi Ahilya University, Takshila Campus, Khandwa Road, Indore 452 017, India

Received 25 September 2012; accepted 20 January 2013

KEYWORDS
1,2-Dihydro-4-quinazolinamines;
Nitric oxide synthase;
Group based QSAR;
K nearest neighbor (KNN);
Partial least square;
Stepwise (SW);
Genetic algorithm (GA);
Simulated annealing (SA)

Abstract A series of substituted quinazolinamine derivatives with potent highly selective inhibitors
of inducible nitric oxide synthase (i-NOS) were subjected to two dimensional, Group-based QSAR,
k-nearest neighbor molecular eld analysis, and pharmacophore approach. Structural features
responsible for the activity of the compounds were characterized by using physicochemical, topological, and electrotopological descriptors and calculated from the Molecular Design Suite Software
(V-life MDS 3.5). The partial least square (PLS) regression methods coupled with various feature
selection methods, viz., stepwise (SW), genetic algorithm (GA) and simulated annealing (SA) were
applied to derive QSAR models. 2D-QSAR study provides details on the ne relationship linking
structure and activity and offers clues for structural modications that can improve the activity.
Statistically signicant equations with high correlation coefcient (R2 = 0.8604) and low standard
deviation (SD = 0.281) were developed by GA-PLS and best Group based QSAR (GQSAR) model
high correlation coefcient (R2 = 0.779) and low standard deviation (SD = 0.326) were developed
by GA-PLS method. k nearest neighbor molecular eld analysis combined with various selection
procedures was performed. The 3D-QSAR model built with the selected variables by GA method
resulted in better prediction incase of 3D-QSAR modeling as compared to other two methods. The
best GA-PLS model with good external and internal predictivity for the training and test set has
shown cross validation (q2) and external validation (pred_r2) values of 0.7966 and 0.7587, respectively. The steric and electrostatic descriptors at the grid points S_479, S_402, E_285 and E_636
play an important role in the design of new molecule. It also suggests the importance of electronegative (electron-donating) and electropositive groups (electron-withdrawing) substituent at position

* Corresponding author.
E-mail addresses: mukeshcsharma@yahoo.com (M.C. Sharma),
drsmita.sharma@rediffmail.com (S. Sharma).
Peer review under responsibility of King Saud University.

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http://dx.doi.org/10.1016/j.arabjc.2013.01.018
Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018

M.C. Sharma et al.

R1 to increase the activity. The 3D-QSAR study indicates less bulky (at R4 and R5) and more
hydrophobic substituent groups (at R3) can improve the activity. The hydrogen bond accepter,
hydrogen bond donor, and aromatic carbon, parameter are the important features which are contributing toward the activity. The QSAR models may lead to a better understanding of the structural requirements of quinazolinamines compounds and also help in the design of novel molecules.
2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction
Nitric oxide synthases (NOS) (Moncada et al., 1991) are a class
of enzymes found in mammals and other species that utilize Larginine to generate nitric oxide (NO). NO is an important
signaling molecule involved in a wide range of physiological
functions, as well as pathophysiological states (Nathan,
1997).Three isforms of the enzyme have been characterized.
Two of these, endothelial NOS (e-NOS) and neuronal NOS
(n-NOS), are constitutive and calcium dependent. The third isoform, inducible NOS (i-NOS), is formed in response to pathological challenges. It is not dependent on calcium and
produces much higher concentrations of nitric oxide than the
others. Over expression of i-NOS has been implicated in a number of inammatory diseases, for example, septic shock and
rheumatoid arthritis. Inhibition of i-NOS should be a useful approach to treatment of these conditions (Cochran et al., 1996).
Nitric oxide acts as a second messenger molecule through the
activation of its main target, soluble guanylatecyclase. In the
cardiovascular system, it is implicated in the regulation of vascular tone, platelet aggregation, and leukocyte adhesion on the
endothelial surface. In the central nervous system and in the
peripheral non-adrenergic non-cholinergic nerves, NO is a
potent neurotransmitter involved in long-term potentiation, migraine, and gastric motility. Elsewhere, Nitric oxide is a cytotoxic and cytostatic agent associated with phagocytic cells in
the immune system (Kerwin et al., 1995; Pfeiffer et al., 1999;
Davis et al., 2001). Nitric oxide is synthesized by oxidation of
the amino acid L-arginine (L-arg) catalyzed by three distinct isoforms of hemeproteins called NO synthases (NOS). The neuronal (nNOS) and endothelial NOS (eNOS) are constitutively
expressed and are Ca++ and calmodulin (CaM)-dependent enzymes, whereas inducible NOS (i-NOS) is expressed in response
to an immune challenge (Alderton et al., 2001; Li and Poulos,
2005; Masters et al., 1996). Quantitative structure activity relationships (QSAR) are the most important applications of
chemometrics giving useful information for the design of new
compounds acting on a specic target. A good QSAR model
both enhances our understanding of the specics of drug action
and provides a theoretical foundation for lead optimization
(Kubinyi, 1997).The pharmacophore modeling is a well-established approach to quantitatively explore common chemical features among a considerable number of structures, and a
qualied pharmacophore model could also be used as a query
for searching chemical databases to nd new chemical entities.
Pharmacophore modeling correlates activities with the spatial
arrangement of various chemical features (Liedl et al., 1996).
The present group of authors has developed a few QSAR models
to predict the biological activity of different groups of compounds such as anti-tubercular agents (Sharma and Sharma,
2011), antimalarial agents (Sahu et al., 2010a, 2011b, 2012c),
TRPV1 antagonists, anti-Alzheimers agents (Bhadoriya et al.,

2012a,b,2013), antimicrobial activity, antibacterial activity,

COX inhibitors (Dhakad et al., 2009; Sharma et al.,
2009a,b,c,d), and angiotensin II receptor antagonists (Sharma
et al., 2009e; Sharma and Kohli, 2011ac; Sharma et al.,
2011d,e; Sharma and Kohli, 2011fk; Sharma, 2012; Sharma
and Kohli, 2012a; Sharma et al., 2012al; Sharma et al.,
2013a), Protein Kinase CK2 Inhibitors (Sharma et al., 2013b)
and 5-lipoxygenase inhibitors (Sharma et al., 2013c), antitumor
activity (Sharma et al.,2013d). In the literature, there are QSAR
studies addressed to inducible nitric oxide synthase (Nagpal and
Tiwari, 2005).
The aim of the present work is to derive some statistically
signicant QSAR methods two dimensional, Group basedQSAR, k-nearest neighbor and pharmacophore approach
allowing exibility to the study of molecular substitution sites
of interest and statistically signicant QSAR models for 2substituted 1,2-dihydro-4-quinazolinamines for their nitric
oxide synthase (i-NOS) inhibitors. The developed model provides insight into the inuence of various interactive elds on
the activity and, thus, can help in designing and forecasting
the nitric oxide synthase (i-NOS) inhibitors activities of 2substituted 1,2-dihydro-4-quinazolinamines. In combination
with QSAR studies, Group based, k-nearest neighbor and Pharmacophore studies were done using VLife MDS molecular
modeling suite (VLife MDS 3.5, 2008).
2. Materials and method
All computations and molecular modeling studies (3D-QSAR)
were carried out on a Windows XP workstation using the
molecular modeling software package VLife Molecular Design
Suite (V-Life MDS) version 3.5.
2.1. Dataset and biological activity for analysis
A series of twenty-two compounds substituted 1,2-dihydro-4quinazolinamines potent highly selective inhibitors of inducible nitric oxide synthase (i-NOS) which show anti-inammatory activity in vivo and described in the literature were
considered to perform the QSAR study (Tinker et al., 2003).
The molar inhibitory concentrations of the compounds were
converted into pIC50 before being correlated with the structural features (physicochemical descriptors). Table 1 shows
the structure of Twenty-two such compounds along with their
biological activity values.
2.2. Dataset division into training and test sets
Compounds were sketched using the 2D drawing application
and converted to 3D structures. Energy minimization and
geometry optimization were conducted using Merck molecular
force eld (Halgren and Nachbar, 1996) and atomic charges,

Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018

Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs:
Table 1

Structures of substituted 1,2-dihydro-4-quinazolinamine derivatives with activities.

O
R2

R2

H
N
N

R1

H
N

R3
R4

R5

NH2

R1

Compound 1-14

NH2

Compound 15-21

Comp. NO

R1

R2

R3

R4

R5

IC50

pIC50a

QSAR setb

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
Cl
F
F
F
F
F

H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
F
F

H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H

C6H5
H
CH3
C2H5
c-propyl
c-butyl
c-pentyl
2-furanyl
2-thienyl
CH3
C2H5
c-butyl
2-furanyl
FPh
H
H
H
H
H
H
H

H
H
H
H
H
H
H
H
H
H
H
H
H
H
OEt
OEt
O(CH2)2SMe
3-thienyl
4-CNPh
4-CNPh
6-CN3-pyridyl

2.5
40
9.8
0.9
1.1
1.1
4.5
0.2
0.4
52
32
0.02
0.002
0.05
0.035
0.047
0.039
0.067
0.027
0.048
0.037
0.3

0.397
1.602
0.991
0.045
0.041
0.041
0.653
0.698
0.397
1.716
1.505
1.698
2.698
1.301
1.455
1.327
1.408
1.173
1.568
1.318
1.431
0.522

Training
Training
Test
Training
Training
Test
Training
Training
Training
Test
Training
Training
Training
Training
Test
Training
Training
Training
Test
Training
Training
Training

O
N

H
N
S

OEt

N
NH2

a
b

pIC50 to generate equation.

Indicates the compounds considered in the training and test set for QSAR study.

maximum number of cycles were 1000, convergence criteria

(RMS gradient) was 0.01 and mediums dielectric constant of
1 by batch energy minimization method. Conformational
search of each energy-minimized structure was performed
using stochastic approach. Stochastic conformational search
method is similar to the RIPS method, which generates new
molecular conformation by randomly perturbing the position
of each coordinate of each atom in the molecule followed by
energy minimization. Selection of the training and test set
for the QSAR model was done by considering the fact that
the test set compounds should represent structural diversity
and a range of biological activities similar to those of the training set. The sphere exclusion method (Golbraikh and Tropsha,
2003) was adopted for the division of training and test data set

comprising 22 molecules, with a dissimilarity value of 3.8

where the dissimilarity value gives the sphere exclusion radius
Table 1. The dataset was split randomly into a training set (17
compounds) for generation of models and a test set (ve compounds) for validation of the developed model.(see Table 2)
2.3. Molecular modeling
2.3.1. 2D-QSAR studies and calculation of 2D descriptor
2D-QSAR study requires the calculation of molecular descriptors; almost 239 physicochemical descriptors were calculated
by QSAR Plus module within VLife MDS. The invariable
descriptors (descriptors that are constant for all the molecules)
were removed, as they do not contribute to the QSAR. Variable

Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018

M.C. Sharma et al.

Table 2

Selected physico-chemical parameters of substituted 1,2-dihydro-4-quinazolinamine derivatives.

SsClE

T_C_O_1

SdsNE

E_636

E_285

S_479

H_183

0
2
2
3
2
3
1
3
1
0
3
1
3
0
3
2
2
3
2
2
3
2

1
2
2
3
0
2
2
2
0
1
2
2
3
2
0
3
0
1
0
1
3
1

9.474691
10.1818
10.8365
12.69831
13.40541
10.8365
10.8365
13.17336
11.82806
13.40541
11.66493
14.27566
17.69831
7.681798
8.552042
9.259149
10.8365
10.67336
12.25071
11.75915
10.95782
9.36987

0.063675
0.06146
0.146555
0.042375
0.143579
0.146483
0.055602
0.148693
0.214171
0.107656
0.048229
0.00453
0.019475
0.085608
0.092907
0.059964
0.066097
0.069174
0.064502
0.064017
0.103632
0.218741

3.73928
3.36961
0.240786
7.3999
2.189978
0.450164
0.495674
2.609555
0.418825
2.510523
1.90702
7.161841
8.109874
3.35415
4.83301
2.9282
2.6743
4.12694
2.37852
2.96916
4.42591
4.14896

0.08157
0.0892
0.06873
0.09992
0.08432
0.07769
0.07258
0.09124
0.08233
0.09233
0.08265
0.10938
0.22703
0.05987
0.08557
0.08128
0.08076
0.06942
0.08765
0.09315
0.08004
0.04128

0.123411
0.147843
0.151393
0.161064
0.171109
0.107057
0.133444
0.144078
0.149463
0.139411
0.157616
0.154781
0.220459
0.073433
0.099096
0.122915
0.155867
0.165446
0.201123
0.182863
0.141683
0.265874

Table 3

Correlation matrix between descriptors present in the 2D QSAR model.

Parameter

SsClE-index

SdsNE-index

HOMO energy

T_C_O_1

SsClE-index
SdsNE-index
HOMO energy
T_C_O_1

1.0000
0.7622
0.4116
0.4992

1.0000
0.6398
0.7941

1.0000
0.8163

1.0000

selection was performed using Genetic algorithm methodology.

2D descriptors were calculated which encoded different aspects
of molecular structure consisting of electronic, thermodynamic,
spatial and structural descriptors, e.g., retention index (chi),
atomic valence connectivity index (chiV), path count, chain path
count, cluster, path cluster, element count, estate number, semiempirical, molecular weight, molecular refractivity, slogP, and
topological index. The various alignment-independent (AI)
descriptors (Baumann, 2002) were also calculated. In this study
to calculate AI descriptors, we have used the following attributes, 2 (double bonded atom), 3 (triple bonded atom), C, N,
O, S, H, F, Cl, Br and I and the distance range of 07.
The QSAR models are considered acceptable (Golbraikh
and Tropsha, 2002) if they satisfy all of the following conditions: (i) Q2 > 0.5, (ii) R2 > 0.6. The Q2 value provided the
statistical signicance and predictability of the models, being
used as a criterion for both robustness and predictive ability
of the model. The high Q2 value (for instance Q2 > 0.5) suggests that the models will be appropriate for meaningful predictions (Gramatica, 2007; Moorthy et al., 2011). Standard
error of estimate (smaller is better) indicates how well the
regression function predicts observed data. The low standard
error of pred_r2se, q2se and r2se shows the absolute quality
of tness of the model. The generated QSAR model was validated for predictive ability inside the model by using cross val-

idation (LOO) for q2 and external validation pred_r2, which is

a more robust alternative method used by dividing the data
into training set and test set and calculating pred_r2. The high
pred_r2 and low pred_r2se showed the high predictive ability of
the model. The values (pred_r2 and pred_r2se) related to external validation are shown in all developed models. Further
analysis shows that for all compounds the error (residual value) is very small. Residuals values (difference between actual
and predicted activities) were found to be minimal. Smaller
values of residuals show the high predictive power of QSAR
models.
2.3.2. Group based (G-QSAR) QSAR
Group based QSAR allows establishing a correlation of chemical group/fragment variation at different molecular sites of
interest with the biological activity. Fragmentation can be
done by applying specic chemical rules for breaking the molecules along the specic bonds and/or bonds on ring fusion
and/or any Pharmacophoric feature such as hydrogen bond
acceptor, hydrogen bond donor, hydrophobic group, charged
group etc. The suggested important fragments can be used as
building blocks to design novel molecules (Ajmani et al.,
2009; Ajmani et al., 2010).
For Group based QSAR method, various 2D descriptors (as
discussed in 2D QSAR), were calculated for various groups

Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018

Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs:

Figure 1a

Quinazolinamines (template).

2006) using the most active molecule as reference Quinazolinamines as a template. Fig. 1b shows alignment of training set
molecules used in 3D-QSAR models.
For the purpose of 3D QSAR analysis, chemical structures
of all the compounds have been drawn using the builder module in Molecular Design Suite 3.5 software package. Molecular
descriptors such as steric, electrostatic and hydrophobic elds
have been calculated utilizing MDS 3.5 software which allows
the user to choose probe, grid size, and grid interval for the
generation of descriptors. In this study, we have generated a
common rectangular grid around the molecules, interaction
energies are computed at the lattice points of the grid using
a methyl probe of charge +1.
For calculation of 3D eld descriptor values, using Tripos
force eld (Clark et al.,1989) and Gasteiger and Marsili charge
type (Gasteiger and Marsili, 1980), electrostatic, steric and
hydrophobic eld descriptors were calculated. The dielectric
constant was set to 1.0 considering the distance dependent
dielectric function. The software produces more than 6000
descriptors and prior to model development descriptors having
zero values or same values are removed.
2.3.4. Pharmacophore approach

Figure 1b

3D view of aligned Quinazolinamines molecules.

present at different substitution sites of the molecules (i.e.

Fragment R1 and R2) and applied on a selected set of 360
descriptors which include group-based and interaction term
descriptors. Various 2D descriptors (as discussed in 2D
QSAR), like element counts, molecular weight, molecular
refractivity, topological index, electro-topological index, Baumann alignment independent topological descriptors, etc. were
calculated for various groups present at different substitution
sites of the molecules. Since the same descriptors are calculated
for various groups at different sites the following nomenclature
is used for naming a descriptor at a particular position, for
example R1_smr represents the molar refractivity of the group
present at substitution site R1(Sahu et al., 2011).
2.3.3. Three dimensional QSAR
The molecular alignment utility can be used to study the shape
variation with respect to the base structure selected for alignment. The alignments dene the putative pharmacophore for
the series of ligands. Alignment of all 22 compounds was done
using the template-based alignment. In the template-based
alignment, a template structure was dened and used as a basis
for the alignment of a set of molecules. The reference molecule
is chosen in such a way that it is the most active among the series of molecules considered. Compound 13 possessed very
highly selective inhibitors of inducible nitric oxide synthase
(i-NOS) which made it a valid lead molecule and, therefore,
was chosen as a reference molecule. Optimized molecules were
aligned Fig. 1a, by template based method (Ajmani et al.,

3D pharmacophore modeling is a technique for designing the

interaction of a small molecule ligand with a macromolecular
target. VLife MDS Mol Sign Module is used for the identication, generation and analysis of pharmacophore by aligning
small organic molecules based on their 3D pharmacophore
features.
In the present study, we have generated pharmacophore
model using mol sign software for a diverse set of molecules
as 2-substituted 1,2 dihydroquinazolinamines with an aim to
obtain pharmacophore model that would provide a hypothetical picture of the chemical features responsible for activity. All
22 aligned molecules were taken for pharmacophore development and the most active molecule was selected to set it as reference. The reference molecule is the molecule on which the
other molecules of the align dataset get aligned. For ve point
pharmacophore identication tolerance limit was set up to
30 A and max distance allowed between two features, setting
the value to 5 A. This abstract model, containing chemical
functionalities (such as hydrogen bond donor, hydrogen bond
acceptor and aromatic carbon center) can serve as an effective
search lter for virtual screening.
2.3.5. Model validation
Internal validation was carried out using leave-one-out (q2)
method. To calculate q2, each molecule in the training set
was sequentially removed, the model ret using same descriptors, and the biological activity of the removed molecule predicted using the ret model.
Models generated by 2D and 3D-QSAR studies were cross
validated using the standard LOO procedure (Cramer et al.,
1988; Assefa et al., 2003). The cross validated r2 (q2) value
was calculated using equation-1, where yi and y i are the actual and predicted activities of the ith molecule, respectively,
and ymean is the average activity of all molecules in the training set. Because of the calculation of the pair-wise molecular
similarities, predictions were based upon the current trial
solution, the q2 obtained is indicative of the predictive power

Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018

M.C. Sharma et al.

of the current kNNMFA model. The q2 was calculated

using Eq. (1).
P
yi  y^i 2
q2 1  P
1
yi  ymean 2
All cross-validation studies were performed by considering the
fact that a value of q2 is above 0.5 indicating high predictive
power of generated QSAR models.External validation of generated models was carried out by predicting the activity of test
set of compounds. The predicted r2 (pred_r2) value was calculated using Eq. (2), where yi and y i are the actual and predicted
activities of the ith molecule in test set, respectively, and ymean
is the average activity of all molecules in the training set. The
pred_r2 value is indicative of the predictive power of the current kNNMFA model for external test set. The pred_r2 value
is calculated as follows (Eq. (2))
P
yi  y^i 2
Pred r2 1  P
2
yi  ymean 2
A model is built using this random descriptor selection with
LOO cross-validation (Oloff et al., 2005), where each compound is eliminated from the training set and its biological
activity is predicted as the average activity of its k most similar
molecules (usually k = 1  4). The value k is optimized during
the model building process to give the best prediction for the
training set. The similarity is characterized by the Euclidean
distance between compounds in the multidimensional space
of selected descriptors.

k
X
aj wij

y^i

j1
k
X
wij

j1

where aj was the observed activity of the jth compound, and

weights wij are dened as
0
1
B
C
B
dij C
C
Wij B
1

B
C
k
X
@
A
dij
j1

,and dij was the distance between compound i and its jth nearest neighbor.
3. Results and discussion
Molecular modeling and Pharmacophore studies of the 1,2dihydro-4-quinazolinamines series resulted in several QSAR
equations. Some statistically signicant QSAR models were
chosen for discussion. The predicted (LOO) activities of the
compounds by the above models are shown in Table 4. The
QSAR models for inducible nitric oxide synthase which show
anti-inammatory activity are the following.
3.1. Interpretation of 2D QSAR model
pIC50 = +0.2917(0.0286) SsClE-index +0.8346(0.0734)
SdsNE-index
0.4067(0.1753)
HOMO
energy
+0.4997(0.0631) T_C_O_1 + 2.8631 [2D QSAR Model-1]

Table 4

Comparative observed and predicted activities of substituted 1,2-dihydro-4-quinazolinamine derivatives.

Com

pIC50

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

0.397
1.602
0.991
0.045
0.041
0.041
0.653
0.698
0.397
1.716
1.505
1.698
2.698
1.301
1.455
1.327
1.408
1.173
1.568
1.318
1.431
0.522

2D based QSAR model

Group based QSAR model

Genetic algorithm-PLS

Pred.

Res.

Pred.

Res.

Pred.

Res.

0.447
1.545
1.046
0.115
0.014
0.111
0.599
0.822
0.314
1.792
1.433
1.747
2.748
1.248
1.375
1.266
1.374
1.070
1.485
1.256
1.362
0.489

0.05
0.057
0.055
0.07
0.027
0.07
0.054
0.124
0.083
0.076
0.072
0.049
0.05
0.053
0.08
0.061
0.034
0.103
0.083
0.062
0.069
0.33

0.423
1.531
1.016
0.078
0.062
0.097
0.701
0.582
0.358
1.632
1.478
1.612
2.608
1.272
1.394
1.311
1.354
1.093
1.503
1.303
1.341
0.504

0.026
0.071
0.025
0.033
0.021
0.056
0.048
0.116
0.039
0.084
0.027
0.086
0.09
0.029
0.061
0.016
0.054
0.08
0.065
0.015
0.09
0.018

0.363
1.549
0.887
0.118
0.083
0.157
0.748
0.575
0.276
1.696
1.461
1.629
2.632
1.216
1.385
1.308
1.297
1. 112
1.589
1.293
1.334
0.474

0.034
0.053
0.104
0.073
0.042
0.116
0.095
0.123
0.121
0.02
0.044
0.069
0.066
0.085
0.07
0.019
0.111
0.141
0.021
0.025
0.097
0.048

Res. = Obs. pIC50Pred. pIC50.

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Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs:
Ntraining = 17, Ntest = 5, r2 = 0.8604, q2 = 0.7617, F
test = 48.526, r2_se = 0.4281, q2_se = 0.3784, pred_r2 =
0.8128, pred_r2se = 0.3578.
Substantial tetra parametric expression is presented in
Eq. (1).The statistically signicant 2D-QSAR model using
the GA-PLS analysis method having 0.8604 as the coefcient of determination (r2) was considered. This equation
shows a correlation coefcient (r2 = 0.8604) with the overall
internal statistically signicant level being better than 95%
as the calculated variance ratio, i.e., the Fischer value (F)
exceeded the tabulated value F = 48.526. It shows an internal predictive power (q2 = 0.761) of 76% and a predictivity
for the external test set (pred_r2 = 0.812) of about 81%.
The correlation matrix is shown in Table 3 which shows
good correlation of selected parameters with biological
activity. Model-1 reveals that the descriptor T_C_O_1 plays
the most important role (26%) in determining the potent
inducible nitric oxide synthase. Descriptor T_C_O_1 having
positive contribution in the QSAR model reveals that for
maximal activity, O-Me, hydroxy group should be directly
attached with substituted quinazolinamines and an increase
in R3 position on quinazolinamines moiety and O-Me, hydroxy group will increase the activity. The next descriptor
SsClE-index (10.3%), contribution which represents electrotopological state indices for a number of chlorine atoms
connect with one single bond. The positive coefcient of the
descriptor suggests that the inducible nitric oxide synthase
activity of quinazolinamine derivatives may be increased
by increasing the number of chlorine atoms present in the
nucleus. The above results are in close agreement with the
experimental observations where compounds 16 substituentCl group at the R1 produce a high activity. Another estate contribution descriptor SdsNE-index (17.8%) which
represents electro-topological state indices for a number of
nitrogen atom connected at two double bond and one single
bond is directly proportional to the activity. It shows that
the nitro group in substituted quinazolinamine derivatives
is essential for the activity. This type of descriptor shows
the importance of the presence of electron environment on
substituted quinazolinamine derivatives increasing the activity. Lastly HOMO energy contributes negatively to the
activity. An electron-donating substituent, such as hydroxy,
or methoxy group, on the ring increases the energy of the
HOMO orbital. Electron-withdrawing substituents, such as
halogens, lower the energy of HOMO. The contribution
chart for 2D-QSAR model is shown in Fig. 2a and plots
of observed vs. predicted values of pIC50 are shown in

Figure 2a

Plot of contribution chart 2D QSAR model.

Figure 2b Graph of observed versus predicted activity of QSAR

model ( -training set, -test set).

Fig. 2b. The predicted activities of the compounds by the

above model are shown in Table 4.
3.2. Interpretation of group based QSAR model
pIC50 = 0.8411(0.3761) R1-SssOE-index +0.3621(0.0380)
R2-SsCH3E-index 0.6864(0.0973) R2-StNE-index +0.4216
(0.0651) R1-T_C_F_1 [Group based QSAR Model-2]
Ntraining = 17, Ntest = 5, r2 = 0.7795, q2 = 0.6916, F
test = 27.5431, r2 se = 0.2172, q2 se = 0.3758, pred_r2 =
0.7281, pred_r2se = 0.4173, ZScore Q2 = 1.3271, Best Rand
Q2 = 0.6328.
The statistically best signicant Group based QSAR model
using the GA-PLS method having 0.779 as the coefcient of
determination (r2) was considered. Model 2 can explain
77.95% of the variance in the observed activity values. It
shows good internal predictive power (q2 = 0.6916) of 69%
and a predictivity for the external test set (pred_r2 = 0.7281)
of about 72%.The conventional QSAR model indicates the
signicance of basic molecular properties such as SssOE-index,
SsCH3E-index, StNE-index and T_C_F_1. The most contributing descriptor in part-R1 is the positive coefcient of R1SssOE-index, and R1-T_C_F_1showed that increases in the
values of these descriptors is benecial for the activity. The
presence of descriptor R1-SssOE-index which is the electro
topological state index for the number of oxygen atoms connected with two single bonds showed a positive contribution
of 27% activity. Such positive effect indicated that the potent
inducible nitric oxide synthase was increased with the presence
of methoxy groups in compounds. This indicates that increased SssOE-index of fragment R1 shows the role and suggests the methoxy group to be attached to the
quinazolinamine moiety. Moreover of additional descriptors,
one is R1-T_C_F_1 which is the count of the number of carbon atoms (single, double or triple bonded) separated from
uorine by a single bond and showed positive contribution
with a value of 35% revealing the increases in activity with
the presence of uorine atoms on quinazolinamine fragment
R1 as indicated in compounds 1215 and 1721. The most
contributing descriptor in part-R2 is R2-SsCH3E-index
(20%) which signies the number of eCH3 group connected
with one single bond. This descriptor showing positive contribution in the selected model revealed the increase of Inducible
Nitric Oxide Synthase of quinazolinamines with the presence
of CH3 group such as in compounds 3, 10 and 11.The descriptors R2-StNE-indexare directly contributing to the activity,

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M.C. Sharma et al.

suggested that an increase in these descriptors 4-cyanophenylgroup at R5 position (like in compounds 19 and 20) of fragment R2 may lead to an increase in the activity. The
contribution chart for 2DQSAR model is shown in Fig. 2c
and plots of observed vs. predicted values of pIC50 are shown
in Fig. 2d. The predicted activities of the compounds by the
above model are shown in Table 4.
3.3. Interpretation of 3D QSAR models
For a better understanding of the QSAR models of these quinazolinamine compounds, an attempt to generate 3D-QSAR
models has also been made. Comparing the three different feature selection methods, it is implicit that the model built with
the selected variables by genetic algorithm (GA) method gives
better prediction in the case of 3D QSAR modeling. This superiority of the genetic algorithm based QSAR model leads us to
explain the effect of steric, electrostatic and hydrophobic elds
on different substituents of quinazolinamine moiety. The training and test sets selected for such study are the same as has
been considered in 2D QSAR models for an effective comparison between genetic algorithms (GA), simulated annealing
(SA), and stepwise (SW) selection methodologies.
pIC50 = 0.0875  S_479 (0.7168, 0.0415)  S_402
(0.1229, 0.1069)  E_285 (1.6053, 1.1781) + E_636
(0.0430, 0.1476). [Genetic Algorithms-PLS 3]
k Nearest Neighbor = 4, Ntraining = 17, Ntest = 5, q2 =
0.7966, q2 se = 0.2176, pred_r2 = 0.7587, pred_r2se = 0.2437,
ZScore Q2 = 1.1764, Best Rand Q2 = 1.762

Figure 2c

Plot of contribution chart Group based QSAR model.

Figure 2d Graph of observed versus predicted activity of

GQSAR model ( -training set, -test set).

Here, n represents the number of observations, df is the

degree of freedom, r is the square root of the multiple Rsquared for regression, q2 is the cross-validated r2, and F is
the F-statistic for the regression model. The statistically best
signicant model (Eq. (3) using the GA-PLS analysis method
with 0.8208 as the coefcient of determination (r2) and standard error of 0.2276 was considered. The variance in the observed activity values is 82.08%. The best q2 of PLS analysis
was found to be 0.7966, which suggests that the model could
be useful for predicting inducible nitric oxide synthase activity for such quinazolinamine derivatives. S_479, S_402, E_285
and E_636 are the steric and electrostatic eld energy of
interactions between probe (CH3) and compounds at their
corresponding spatial grid points of 479, 402, 285 and 636.
3D data points were generated that contribute to GA-PLS
3D-QSAR model, and are shown in Fig. 3a. The range of
property values for the generated data points helped in the
design of potent inducible nitric oxide synthase molecules.
The range was based on the variation of the eld values at
the chosen points using the most active molecule and its nearest neighbor set. The plot of observed versus predicted activities for the test compounds is represented in Fig. 3b. From
Table 4 it is evident that the predicted activities of all the
compounds in the test set are in good agreement with their
corresponding experimental activities and optimal t is
obtained.
Similarly, 3D-QSAR model using PLS regression by simulated annealing (Eq. (4)) and stepwise (Eq. (5) based descriptor

Figure 3a Contribution plot for steric and electrostatic interactions of GA-PLS model.

Figure 3b Graph of observed versus predicted activity of GAPLS model ( -training set, -test set).

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Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs:
selection method was developed with the following statistical
parameters:
pIC50 = 0.87643  E_175 (0.2826, 0.9240)  S_475
(1.0128, 0.4945) + H_414 (0.1720, 0.3115) + E_456
(0.2364, 0.5047) [Simulated Annealing -PLS -4 ]
k Nearest Neighbor = 4, Ntraining = 17, Ntest = 5,
q2 = 0.7215, q2 se = 0.1865, pred_r2 = 0.6821, pred_r2se =
0.4653,ZScore Q2 = 0.9762, Best Rand Q2 = 0.7632.
pIC50 = S_560 (0.0635, 0.0537)  E_269 (10.0000,
10.0000) + H_183 (0.2788, 0.3994)  E_227 (0.2333,
0.3586) [Stepwise Selection -PLS -5 ]
k Nearest Neighbor = 4, Ntraining = 17, Ntest = 5,
q2 = 0.6934, q2 se = 0.2652, pred_r2 = 0.6327, pred_r2se = 0.4372, ZScore Q2 = 1.0431, Best Rand Q2 =1.2872
For simulated annealing Fig. 3c predictive R2 is found to be
0.6821 with an estimated standard error of 0.4163 whereas
these parameters are estimated as 0.6327 and 0.2418 respectively in the case of model developed by stepwise based model
Fig. 3d. The contribution plot of steric and electrostatic eld
interactions indicates relative regions of the local elds (steric
and electrostatic) around the aligned molecules, leading to
activity variation in the model (Kubinyi, 1993). Green, blue
and yellow balls represent steric, electrostatic and hydrophobic
eld effects, respectively. In the QSAR model, steric descriptors with positive coefcients represent regions of high steric
tolerance; bulky substituent is favorable in this region. Steric
descriptors with negative coefcients indicate that negative steric potential is favorable for increase in the activity and hence a
relatively less bulky substituent group is preferred in that region. Electrostatic eld descriptors with negative coefcients
indicate that negative electrostatic potential is favorable for

Figure 3c Contribution plot for steric, electrostatic and hydrophobic interactions of SA-kNN model.

the increase in the activity and hence a relatively more electronegative substitution is preferred in that region. Electrostatic
eld descriptors with positive coefcients representing regions
indicate that positive electrostatic potential is favorable for an
increase in the activity and hence a relatively less electronegative substitution is preferred in that region (Bhatia et al.,2012;
Choudhari and Bhatia, 2012; Sharma and Kohli, 2011acd,e).
From 3D-QSAR model Eq. (3) and Fig. 3a it is observed that
electrostatic descriptors like E_285 with negative coefcient are
near from the R1 position of the quinazolinamine moiety ring.
This indicates that electronegative groups are favorable on this
site and the presence of electronegative (electron-donating)
groups increases the activity of quinazolinamine compounds
(Sahu et al., 2011; Nandi and Bagchi, 2010).These results are
in close agreement with the experimental observations that compounds 1220 and 21 have chlorine, and uorine groups at R1,
R2, R3 and R4 -positions. These compounds produce a greater
activity due to electronegative substituents on the R1, R2, R3
and R4 -positions of the quinazolinamine ring (Tinker et al.,
2003). Presence of electrostatic eld descriptors like E_636 with
positive values is near from the R1 position of the quinazolinamine ring. This indicates that electropositive groups are favorable on this site and the presence of electropositive groups
increases the activity of quinazolinamine derivatives. Most of
the compounds (compounds 3, 10, 11, 19 and 20) with a higher
activity having electropositive substitution (CH3) at the R3 and
R4 positions and 4-cyanophenyl group of the quinazolinamine
ring strongly support the above statement. The positive values
of electrostatic descriptors suggested the requirement of electropositive groups like methyl, ethyl, propyl, isopropyl and butyl at
the position of the generated data point E_636 around Quinazolinamine analogs for maximum activity. This is also well supported by group based QSAR study. The presence of steric
descriptors S_479, and S_402 with negative values is also near
from the R4 and R5 positions of the quinazolinamine moiety
which indicates that less steric or less bulky substituents are
favorable on this site and the presence of less steric substituents
increases the inducible nitric oxide synthase activity of quinazolinamine compounds. Negative values in the steric eld descriptor indicated the requirement of negative steric potential for
enhancing the biological activity of quinazolinamine analog.
Furthermore, Fig. 3c and d compares the most signicant
favorable and unfavorable features at the position near R3
which indicated that electronegative groups at position-3 enhance the activity, while the presence of electron-withdrawing
groups decreases the activity. From 3D-QSAR model Eq. (5)
and Fig. 3d it is observed that the presence of hydrophobic
descriptor H_183 with positive values indicates that less hydrophobic substituents such as CH3, C2H5, C6H5 etc. are favorable on this site and the presence of less hydrophobic
substituents increases the activity of quinazolinamine
molecules.
3.3.1. Interpretation of pharmacophore models

Figure 3d Contribution plot for steric, electrostatic and hydrophobic interactions of SW-kNN model.

Pharmacophore model for inducible nitric oxide synthase

activity was generated using the mole sign module of Vlife
3.5 choosing the biophore with the lowest RMSD and generating the pharmacophore. It starts generating properties of molecules and nds the common three Dimensional map of ve to
maximum common properties Fig. 4a. The best aligned Fig. 4b
model includes ve features viz. three hydrogen bond donor

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10

M.C. Sharma et al.

Figure 4c
Figure 4a

Best Pharmacophoric features with distance A.

Pharmacophore features.

are favorable contour sites for both the activities. Distance

(33HDr- 31HAc) = 3.4259 A, 31HAc Atom selected- 1C
Atom selected, Distance (31HAc-1C) = 4.9099 A; 31HAc
Atom selected 32-HDr Atom selected Distance (31HAc
32HDr) = 5.1672 A, 31HAc Atom selected-34HDr Atom selected Distance (31HAc 34HDr) = 4.8481 A.
Hence, these results reveal the requirements on the structural properties and the distances between the pharmacophore
contour sites of the molecules responsible for their inducible
nitric oxide activities.
4. Conclusion

Figure 4b

Aligned Pharmacophoric features.

(HBD-magenta color), one hydrogen bond acceptor (HBAbuff color), and one aromatic region of the structure (orange
color). In hydrogen bond acceptor eld, the magenta (favorable) is seen near quinazolinamine ring suggests that acceptor
groups in this area are preferred for activity. Compounds with
one or more acceptor groups in this area result in higher inducible nitric oxide synthase activity. Furthermore, comparing the
most signicant favorable and unfavorable features at R1, R3
positions- near hydrogen bond acceptor indicated that electron
donating groups at positions-R3 and R5 enhance the activity,
while the presence of electron-withdrawing groups increases
the activity. Hydrogen bond donor shows that hydrogen bond
donor makes the highest contribution in the model. The average RMSD of the pharmacophore alignment of each of two
molecules is 0.8361 A. The distances between the pharmacophore sites Fig. 4c were measured in order to conrm their signicance to the activities. The results reveal that the acceptor
(acc), donor (don), and aromatic pharmacophore properties

The present studies were aimed at deriving predictive QSAR

model and pharmacophore studies capable of elucidating the
structural requirements for novel 1,2-dihydro-4-quinazolinamines as inducible nitric oxide synthase which show anti-inammatory activity. The best 2DQSAR model (model 1) resulted
in r2 = 0.8604 and pred_r2 = 0.8128 by GA-PLS conrms a
positive contribution of SsClE-index, SdsNE-index and
T_C_O_1 to the inducible nitric oxide synthase activity. The
3D-QSAR model built with the selected variables by the GA
method resulted in better prediction in the case of 3D-QSAR
modeling as compared to other two methods. This superiority
of the GA-based QSAR model leads us to explain the effect of
steric and electrostatic elds on different substituents of quinazolinamine moiety. The contribution plot of steric, electrostatic and hydrophobic eld interactions generated by 3DQSAR shows that electronegative groups at quinazolinamines
moiety are favorable. This nding is in close agreement with
the structures of these compounds, where the presence of electropositive groups is found in the quinazolinamine moiety. It
also suggests that bulky electronegative (electron-donating)
groups are favorable at R1-position of the quinazolinamine
template. This nding supports the experimental observations,
where the presence of bulky electronegative groups at R1 and
R2-position signies an increase in activities of compounds.
Three-dimensional features, steric, electrostatic and hydrophobic elds, can be easily identied from the map developed for
the best model. Signicant predictive ability of the model observed for the external test set molecules supports that the derived model can be used for the designing of the novel

Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018

Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs:
inhibitors. Furthermore, we hope that the current study provides better insight into the designing of more potent 1,2-dihydro-4-quinazolinamines as inducible nitric oxide synthase
agent in the future before their synthesis.

Acknowledgments
The author wishes to express gratitude to V-life Science Technologies Pvt. Ltd Pune (M.H) India for providing the trial version software for the study.
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Please cite this article in press as: Sharma, M.C. et al., Study of physicochemical properties-inducible nitric oxide synthase relationship of substituted quinazolinamines analogs: Pharmacophore identication and QSAR studies. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.01.018