International

Shaheen Begum et al. : 3D QSAR Journaland

Studies Benzoxazoles of Pharmaceutical Sciences
Oxazolo-(4, 5-b)pyridines and Nanotechnology
as Anti-fungal agents 557
Volume 2 Issue 2 July September 2009

Research Paper
3D QSAR Studies on Benzoxazoles and Oxazolo-(4, 5-b)pyridines as
Anti-fungal agents

Shaheen Begum1, Satya Parameshwar K2,*, Ravindra G K3, Achaiah G4,*

1
Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam, Tirupathi, Andhra Pradesh.
2
L.B. College, Warangal.
3
National institute of Pharmaceutical Education and Research, Hyderabad.
4
University College of Pharmaceutical Sciences, Kakatiya University, Warangal.

ABSTRACT: Benzoxazoles and Oxazolo-[4,5-b]pyridines have been reported as potent anti-fungal agents. 3D QSAR
tools including CoMFA and CoMSIA have been known to be a promising approaches is to correlate structures and activity
which further enable the medicinal chemists to design more potent molecules thus curtailing the cost and time in drug
research. CoMFA and CoMSIA studies have been carried out on 31 molecules of benzoxazole and oxazolopyridines in
order to determine the structural properties required for effective antifungal activity. 26 compounds were evaluated for
establishing QSAR model, which was then validated by predicting the activities of five test set molecules. All the molecules
were aligned by SYBYL database alignment which led to a best model with q2 value of 0.835, r2=0.976 and r2pred=0.773.
This model was further employed to derive CoMSIA models, a best model with steric, electrostatic, hydrophobic and
hydrogen bond acceptor indices exhibited q2 = 0.812, r2=0.971 and r2pred=0.81. The models thus obtained from this study
can be useful for the design and development of new potential anti-fungal agents.

Introduction activity. It is imperative to improve predictability of QSAR

Azoles have been one of the extensively studied scaffolds
in medicinal chemistry as anti-microbial agents in
particular as anti-fungal agents [Cavelleri et al., 1978,
Cuckler et al., 1966, Elamine et al., 1981]. In the last two
decades there has been shift in the development of anti-
fungal drugs. Inspite of expansion in anti-fungal drug
discovery there remains a wide gap in the range and scope
of current anti-fungal chemotherapy (Graybill et al., 1996).
Benzoxazoles have been extensively studied for their
antibacterial and anti-fungal activity [Oren et al., 1999,
Temiz et al., 1998], anticancer activity [Kumar D et al.,
2002], and also as new non-nucleoside topoisomerase I
poisons [Kim et al., 1996] and HIV-1 reverse transcriptase
inhibitors [Perrin et al., 1996].
Quantitative structureactivity relationships
(QSARs) are computational statistical methods which
reveal explainable difference in the observed biological
* For correspondence: Satya Parameshwar K, Achaiah G,
E-mail: spkonda@yahoo.co.in ; achaiah_g@yahoo.co.in
model of test compounds by considering the structural and
physicochemical features. Three dimensional quantitative
structureactivity relationship (3D-QSAR) techniques
including comparative molecular field analysis (CoMFA)
and comparative molecular similarity indices analysis
(CoMSIA) could facilitate in designing unsynthesized
agents for therapeutic purpose [Lepper et al., 2004,
Ravindra et al., 2007, Sperandio da Silva et al., 2004].
Both the techniques predict the activities based on the
existing set of molecules, CoMFA develops the 3D QSAR
model based on the relationship between the
steric/electrostatic properties and biological activities,
while CoMSIA develops the models using steric,
electrostatic, hydrophobic and hydrogen bonding
properties. The addition of other fields in CoMSIA resulted
in augmented significance and predictive power of model
and also interpretation of the resultant correlation of the
model. CoMSIA is known to be less sensitive to molecular
alignments/ conformations than CoMFA [Bandyopadhyaya
et al., 2005].
Inumerable reports are available containing 2D QSAR
on various benzoxazoles as anti-fungal agents and very
few 3D QSAR on the same scaffolds are available. In this
connection, it is aimed is to analyze 3D structural

557
558 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009

requirements of benzoxazoles and Oxazolo-(4,5-
b)pyridines as anti-fungal agents and also to understand the
structural basis for their affinity for the activity and to
design still better potent molecules.
Materials and methods
A series, containing 31 benzoxazole and oxazolopyridines
have been obtained from the literature to establish CoMFA
and CoMSIA models [Oren et al., 2004, Yalcin et al.,
2000]. The structures and their anti-fungal activities have
been mentioned in Table 1. The anti-fungal activity of the
data set has been converted into pIC50 using the formula
pIC50 = -log IC50 and which was in the range of 3.8-4.4.
The inhibitors were randomly sorted into training set and
test set containing 26 and 5 molecules respectively.

Table1 Structure and in vitro anti-fungal activity of training set compounds

R1 X N
R
O

Comp. No X R R1 pIC50
1 CH -H -H 3.892
2 CH -C(CH3)3 -H 4.001
3 CH -NH2 -H 3.924
4* CH -NHCH3 -H 3.952
5 CH -CH2CH3 -Cl 4.013
6 CH -NHCOCH3 -Cl 4.059
7 CH -NHCH3 -Cl 4.015
8 CH -Cl -Cl 4.024
9 CH -NO2 -Cl 4.04
10 CH -H -Cl 4.282
11 CH -CH3 -NO2 4.308
12 CH -C(CH3)3 -NO2 4.375
13* CH -NH2 -NO2 4.31
14 CH -Cl -NO2 4.342
15 CH -Br -NO2 4.406
16 CH -CH2CH3 -NH2 3.976
17 CH -F -NH2 3.96
18 CH -N(CH3)2 -NH2 4.005
19* CH -CH3 -CH3 3.95
20 CH -CH2CH3 -CH3 3.977
21 CH -CH2CH3 -CH3 3.98
22 CH -F -CH3 3.958
23 CH -NHCOCH3 -CH3 4.027
24* CH -NHCH3 -CH3 3.979
25 CH -N(CH3)2 -CH3 4.004
26 N -CH3 -H 4.225
27 N -CH2CH3 -H 4.253
28 N -OCH3 -H 4.257
29 N -OCH2CH3 -H 4.283
30* N -NH2 -H 4.227
31 N -NO2 -H 4.285
 Shaheen Begum et al. : 3D QSAR Studies Benzoxazoles and Oxazolo-(4, 5-b)pyridines as Anti-fungal agents
All the molecular modeling studies were performed
using Sybyl 6.8 version [Sybyl 6.8, Tripos Inc.] on silicon
graphics O2 workstation with IRIX 6.5 operating system.
All the structures were constructed using sketch module of
SYBYL using standard library models. The modeled
structure was minimized using Tripos force field with
distance dependent dielectric function and applying
Gasteiger-Hckel partial atomic charges. These structures
were further optimized by semi-empirical MOPAC
calculations using AM1 method. These structures were
considered for generating CoMFA and CoMSIA
calculations.
Fig.1 Database alignment of anti-fungal agents.
Methodology of CoMFA and CoMSIA
A 3D cubic lattice was defined in CoMFA automatically
by extending at least 4 beyond all the aligned molecules
in all the three axes (X, Y, Z directions) with a grid
spacing of 2.0 . Lennard-Jones and Columbic potentials
were used to calculate the steric and electrostatic
interaction fields respectively at each lattice intersection.
Tripos force field with distant dependent dielectric
constant e = e0 Rij with e0 =1.0 was considered for the
calculation of interaction energies considering an sp3
carbon atom with a radius of 1.52 bearing +1 charge as a
probe atom. Steric and electrostatic cutoffs were applied to
truncate the contribution up to 30 kcal/mol [Cramer et al.,
1988].
Klebe et al introduced CoMSIA analysis which
calculates five similarity descriptors namely steric,
electrostatic, hydrophobic, hydrogen bond donor and
acceptor [Klebe et al., 1994, Klebe et al., 1999)]. Gaussian
type function was employed to gauge similarity indices at
all the grid points, a default attenuation factor () of 0.3
was employed as a smoothening function. A probe atom
559
Alignment
The important aspect of 3DQSAR is the alignment of the
inhibitors that defines the presumed pharmacophore of the
series under the study. As no structural information is
available about ligand-target complex, the lowest energy
conformation obtained after MOPAC of most active
molecule has been employed as templet of alignment using
SYBYL database alignment (Fig.1).
having charge +1, hydrophobicity +1, and hydrogen bond
donor and acceptor property of +1 was assigned to
calculate similarity indices. Atom based parameters were
employed to compute hydrophobic descriptors
[Viswanadhan et al., 1989] and rule based method was
used to get the hydrogen bond donor and acceptor fields.
Partial least square analysis
Partial least square analysis (PLS) is the best statistical tool
to derive the correlation between biological activity and
descriptors calculated by CoMFA and CoMSIA
[Ciubotariu et al., 1993]. Leave one out (LOO) method
was carried out as a rudimentary step to ascertain the
predictivity of the model and also to determine the
optimum number of components with minimum standard
error of prediction (SEP). The optimum number of
components thus obtained from the previous LOO method
was further applied to derive the final non-cross validated
correlation r2. Group cross validation and bootstrapping
were performed to get the confidence intervals (mean and
standard deviation) and also to assess the robustness of the
model. In the group cross validation, which was conducted
for 50 iterations in which the training set molecules were
560 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009

randomly divided into groups and subjected to the
correlation. The mean of 50 cross validation runs was
considered as correlation coefficient, rcv2. The
bootstrapping (rbs2) analysis for 100 runs was performed
with optimum number of components, which provides an
estimate of the variability of the parameters in a final
model [Bunce D et al., 1988].
Result and discussions
CoMFA model resulted from SYBYL database alignment
(Fig.1) has yielded a crossvalidated q2 value of 0.835, non-
cross validated r2 value of 0.976 with six components, and
standard error of estimation (SEE) of 0.106 as listed in
Table 2. The contributions of steric and electrostatic were
60.5% and 39.5%, respectively. Group cross validation
was carried out for 50 iterations to prove for the
confidence of the model that resulted in a mean group
cross-validated r2 value of 0.81. Bootstrapping analysis of
100 runs was led to an r2bs of 0.943 with deviation of 0.03
(Table 3) and the CoMSIA predicted activities of both
training and test sets are presented in Fig 2.

Table 2 CoMFA and CoMSIA statistics.

CoMFA CoMSIA
Parameters
I II
2
r 0.976 0.971

q2 0.835 0.812

SEE 0.029 0.023

SEP 0.075 0.032

Number of components 6 6

F value 129.92 105.45

% Contribution

Steric 60.5 38.7

Electrostatic 39.5 14.3

Hydrophobic -- 34.4

H acceptor -- 12.6

r2pred 0.773 0.81

Table 3 Bootstrapping results of CoMFA and CoMSIA.

r2bsa r2cvb

CoMFAc CoMSIAd CoMFAc CoMSIAd

Mean 0.945 0.98 0.81 0.785

SD 0.03 0.019 0.059 0.07

a
From 100 run boot strapping
b
From mean of 50 run group cross validation
c
CoMFA with atom fit based alignment
d
CoMSIA model generated by the combination of steric, hydrophobic and hydrogen bond
acceptor fields
 Shaheen Begum et al. : 3D QSAR Studies Benzoxazoles and Oxazolo-(4, 5-b)pyridines as Anti-fungal agents 561

Fig. 2 Predicted and observed activities of training
and test sets using CoMFA.
CoMSIA results
CoMFA model obtained as explained in the previous
section, has been considered to derive CoMSIA model and
five different models with different CoMSIA field
combination were generated. One best model containing
steric, electrostatic, hydrophobic and hydrogen bond
acceptor fields demonstrated q2 and r2 value of 0.812 and
0.971, respectively with acceptable F-value of 105.52 from
six components. The contributions of all fields were found
to be 32.4 % Steric, 12.3 % Electrostatic, 28.2 %
Hydrophobic, 16.5 % Hydrogen bond acceptor and 10.6 %
Hydrogen bond donor. The aforementioned model was
further subjected to group cross validation for 50 runs and
correlation of 0.785 was obtained and the boot strapping
for 100 runs was found to be 0.98 with 0.19 SD as
summarized in Table 3. The predicted activity of both test
set and training set molecules by CoMFA and CoMSIA
models are listed in Table 5 and the graphs of experimental
activity against the CoMSIA predicted activities of both
training and test sets are presented in Fig 3.

Table 5 actual and predicted (CoMFA and CoMSIA) activities of test set molecules.

Comp. No Actual pIC50 Predicted Residuals Predicted pIC50 Residuals

pIC50 CoMFA CoMFA CoMSIA CoMSIA
4 3.952 4.027 -0.077 3.988 -0.038
13 4.31 4.252 0.058 4.315 -0.005
19 3.95 3.962 -0.012 3.952 -0.002
24 3.979 4.004 -0.024 3.965 0.015
30 4.227 4.157 0.073 4.165 0.065

Fig. 3 Predicted and observed activities of training and test sets using CoMSI.
562 International Journal of Pharmaceutical Sciences and Nanotechnology
CoMFA and CoMSIA contour map analysis
The steric and electrostatic contour plots of CoMFA
revealed that three green colored plots at the 5th position of
benzoxazole, 3 or 4 of 2-phenyl infer the sterically
favorable groups required for anti-fungal activity (Fig. 4).
A small red contour plot was also noticed at 5th position
along with green. From the SAR it is found that
unsubstituted molecule 1 exhibited least anti-fungal
activity against Candida albicans. Substitution at 5th
position by chlorine, nitro, amino and methyl groups has
led to molecules with greater potency, however nitro group
Fig. 4 CoMFA steric and electrostatic contour maps. Green contour favors steric bulk, whereas yellow color
disfavors steric bulk. Blue contour indicates electropositive charge and red contour electronegative charge.
Two more green colored plots have been spotted at the 4
of 2-phenyl ring which suggests that substitution with
bulky groups at this position could influence positively for
the antifungal activity. Activity of compounds was found
increasing from fluorine to bromine as in molecules 22, 14
and 15. Compound 12 has also exhibited potent activity
having t-butyl group at the same place. Moderate increase
in the potency of the compounds was also observed in the
molecules bearing substituted amino groups.
CoMSIA steric and electrostatic contour plots are quite
similar however one big yellow plot was found at the 5th
position of heterocyclic ring (Fig 5). A careful observation
of the data set considered for the model development
revealed that a few molecules (26-29, 31) which are
unsubstituted found to be potent This indicates that
Volume 2 Issue 2 July - September 2009
was found to be the most influential for the antifungal
activity. Further insight into the contour plots at this
position led to the conclusion that not only steric property
but also electronic effect guides the activity.
Comparatively, electron releasing groups demonstrated
reduced activity, whereas groups with electron
withdrawing nature such as chlorine and nitro groups
showed better activity as noticed in molecule 10 and 15
respectively. A big blue contour map which covered the
benzoxazole was found which indicates that the electron
withdrawing groups would create lesser electron cloud
over the aromatic ring by withdrawing the electrons.
substitution at 5th position might not be essential for the
activity however bulky groups alone at the 4 position on
2-phenyl ring is vital for the activity.
Four yellow isopleths could be sighted one at the 5th
position, one on the 2-phenyl ring and two across the 4
position of 2-phenyl ring infer about the requirement of
hydrophobicity (Fig. 6). From the previous explanation,
substitution at the 5th position might not be key
requirement for potent activity, hence yellow isopleth at
this position can not be explained. However two yellow
contours across the 4-position indicates that the sustituents
that increase the lipophilicity contribute positively for the
activity. Compounds like 11 and 12 bearing methyl and t-
butyl demonstrated potent anti-fungal activity.
 Shaheen Begum et al. : 3D QSAR Studies Benzoxazoles and Oxazolo-(4, 5-b)pyridines as Anti-fungal agents 563

Fig. 5 CoMSIA steric and electrostatic fields are represented by green, yellow, blue and red colour regions.

Fig. 6 CoMSIA hydrophobic contour map.

Validation of CoMFA and CoMSIA models
The most critical and important task of 3D-QSAR is model
validation in which the predictive ability of the model
established by reproducing the activity of test set
compounds which are not included in training set. The best
CoMFA and CoMSIA models thus resulted were subjected
to external validation by predicting the activity of test set
compounds. All the test set compounds were constructed
and aligned similarly. Table 4 contains the predicted
activity of test set compounds which are found to be in
greater agreement with the observed activities with
marginal error range with predictive correlation coefficient
of r2pred of 0.771 and 0.81 for CoMFA and CoMSIA
models respectively. The predicted activities of both test
set and training set compounds by both CoMFA and
CoMSIA verses actual activities has been shown in Fig.
These results indicate that the CoMFA and CoMSIA
models could be consistently employed in the design for
developing inhibitors against fungal infections caused by
candida albicans.
564 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009

Table 4 actual and predicted activities of training set molecules.

Comp. Actual pIC50 Predicted Residuals Predicted pIC50 Residuals

No pIC50 CoMFA CoMFA CoMSIA CoMSIA

1 3.892 3.977 -0.087 3.882 0.010

2 4.001 4.016 -0.016 3.970 0.030
3 3.924 3.912 0.008 3.934 -0.010
5 4.013 3.996 0.014 4.049 -0.036
6 4.059 4.043 0.017 4.071 -0.012
7 4.015 4.018 -0.008 4.020 -0.005
8 4.024 3.980 0.040 4.013 0.011
9 4.04 4.016 0.024 4.081 -0.040
10 4.282 4.317 -0.037 4.263 0.019
11 4.308 4.334 -0.024 4.349 -0.041
12 4.375 4.356 0.014 4.377 -0.002
14 4.342 4.341 -0.001 4.346 -0.004
15 4.406 4.345 0.065 4.356 0.050
16 3.976 3.977 0.003 3.960 0.19
17 3.96 3.957 0.003 3.919 0.040
18 4.005 3.983 0.027 3.978 0.027
20 3.977 3.983 -0.003 3.972 0.005
21 3.98 3.984 -0.004 4.001 -0.021
22 3.958 3.990 -0.030 3.992 -0.034
23 4.027 4.026 0.004 4.039 -0.012
25 4.004 3.983 0.017 4.030 -0.027
26 4.225 4.240 -0.010 4.202 0.024
27 4.253 4.263 -0.013 4.238 0.016
28 4.257 4.263 -0.003 4.266 -0.008
29 4.283 4.286 -0.006 4.295 -0.012
31 4.285 4.284 -0.004 4.165 0.065

Conclusion
CoMFA and CoMSIA methods could derive best models
employing twenty six anti-fungal agents. These models
were successfully validated by predicting the anti-fungal
activity of five test set molecules. In comparison, the
predictivity of the CoMFA model was found to be superior
to that of the CoMSIA model. However, a combined use of
both the CoMFA and the CoMSIA methods would further
enhance the predictivity of the derived models.
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