Synthesis, biological evaluation and 2D-QSAR analysis of benzoxazoles as antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2008.04.001

A new series of 5(or 6)-nitro/amino-2-(substituted phenyl/benzyl)benzoxazole derivatives (1a-1m, 2a-2l) were synthesized and evaluated for antibacterial and antifungal activities against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pse

Full text of DOI:10.1016/j.ejmech.2008.04.001

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 501e510
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and 2D-QSAR analysis
of benzoxazoles as antimicrobial agents
a
a
Tugba Ertan a, Ilkay Yildiz a, , Betul Tekiner-Gulbas , Kayhan Bolelli ,
*
Ozlem Temiz-Arpaci a, Semiha Ozkan b, Fatma Kaynak b, Ismail Yalcin a, Esin Aki a
a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, TR-06100 Tandogan, Ankara, Turkey
b Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06330 Etiler, Ankara, Turkey
Received 25 July 2007; received in revised form 28 March 2008; accepted 8 April 2008
Available online 27 April 2008
Abstract
A new series of 5(or 6)-nitro/amino-2-(substituted phenyl/benzyl)benzoxazole derivatives (1ae1m, 2ae2l) were synthesized and evaluated
for antibacterial and antifungal activities against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Escherichia coli, Candida albicans and their drug-resistant isolate. Microbiological results indicated that the synthesized compounds possessed
a broad spectrum of activity against the tested microorganisms at MIC values between >400 and 12.5 mg/ml. The results against B. subtilis,
P. aeruginosa, drug-resistant B. subtilis, drug-resistant E. coli, and C. albicans isolate for these kinds of structures are quite encouraging. The
2D-QSAR analysis of a set of newly and previously synthesized benzoxazoles tested for growth inhibitory activity against B. subtilis ATCC 6633
was performed by using the multivariable regression analysis. The activity contributions for substituent effects of these compounds were deter-
mined from the correlation equation for predictions of the lead optimization.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Antibacterial activity; Antifungal activity; Benzoxazoles; 2D-QSAR
1. Introduction
activities [6e33]. In the last few years, we reported some
derivatives of benzoxazoles, which exhibited antimicrobial
[14e16], antiviral [23,24], multidrug-resistance cancer cell
activities [33] with inhibiting activity on eukaryotic topoiso-
merase II enzyme in cell-free system [25e27].
The goal of outset of this research was to develop new
effective antimicrobial agents having benzoxazole nuclei.
Herein, we have described the synthesis of a series of 2-
(substituted phenyl and benzyl)benzoxazole derivatives which
have a nitro group attached on position 5 or 6 of heterocylic
nuclei binding them as a new class of synthetic antimicrobial
agents along with their in vitro antimicrobial activity.
Additionally, we also put an electron-donating group such as
amine instead of nitro which is an electron-withdrawing group
for the same position in order to be able to discuss the effect of
substituent for biological activity.
The dramatically rising prevalence of multidrug-resistant
microbial infections in the past few decades has become
a serious health care problem. In particular, the emergence
of multidrug-resistant strains of Gram-positive bacterial
pathogens such as methicillin-resistant Staphylococcus aureus
and Staphylococcus epidermis and vancomycin-resistant
Enterococcus is a problem of ever-increasing significance
[1e5]. In order to prevent this serious medical problem, the
elaboration of the new types of the previously known drugs
is a very actual task.
The benzoxazoles have been the aim of many researches
for many years because they constitute an important class of
heterocyclic compounds exhibiting substantial chemotherapeutic
In the drug design area, quantitative structureeactivity rela-
tionship (QSAR) modelling is an area of research pioneered by
Hansch and Leo [34] and Hansch and Fujita [35]. The QSAR
* Corresponding author. Tel.: þ90 312 203 3069x2308; fax: þ90 312 223
69 40.
E-mail address: iyildiz@pharmacy.ankara.edu.tr (I. Yildiz).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.001

502
T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
method assumes that differences in the structural or physical
properties measured experimentally account for differences
in the observed biological or chemical properties [34e37].
A QSAR study usually leads to a predictive formula and
attempts to model the activity of a series of compounds using
measured or computed properties of the compounds.
The other aim of this study is to derive quantitative struc-
tureeactivity relationships (QSARs) from multivariable re-
gression analysis (MRA) in order to investigate the
quantitative effect of structural properties of the previously
[10,38] and newly synthesized 5(or 6)-nitro/amino/methyl-2-
(substituted phenyl/benzyl)benzoxazoles on their antibacterial
activity against Gram-positive bacteria Bacillus subtilis ATCC
6633.
2. Results and discussion
2.1. Chemistry
The synthetic pathways for preparation of the target
compounds listed in Table 1 are shown in Scheme 1. The
synthesis of compounds (1ae1m) was performed by con-
densing of appropriate aminophenols and suitable acids in
polyphoshoric acid. Reduction of the nitro group of
1ae1m afforded 2ae2l. Compounds 2b, 2c, 2g, 2he2j,
and 2l were obtained from 1c, 1d, 1he1l, respectively, by
using NiCl2$6H2O and Zn in methanol for reduction. Ten
percent PdeC was used to synthesize the other amines
(2a, 2de2f, 2k).
Table 1
The antimicrobial and antifungal activities of the synthesized compounds (1ae1m, 2ae2l) and the control drugs (MIC in mg/ml)
R3
O
X
R1
N
R2
Compound
number
R1
R2
R3
X
Microorganismsa
Gram negative
Gram positive
Fungi
Kp
Kpb
Ec
Ecb
Pa
50
Bs
Bsb
Sa
Sab
100
Ca
Cab
1a
1b
C(CH3)3
H
H
NO2
NO2
NO2
NO2
NO2
H
e
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
16
2
100
100
100
100
100
100
100
100
100
100
100
100
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
8
100
25
50
25
100
25
25
50
25
25
25
25
25
25
100
50
50
25
50
25
100
50
50
50
50
25
50
25
50
256
8
100
200
100
100
100
200
100
100
100
200
100
100
50
50
100
50
100
50
100
50
100
50
100
100
100
50
50
50
25
50
50
50
50
50
50
50
100
50
50
50
50
e
H
e
e
50
50
50
50
25
50
50
25
25
50
25
50
25
50
50
50
50
50
50
50
25
25
50
50
32
4096
2
100
100
>400
100
>400
100
100
100
100
100
100
50
1c
1d
F
Br
H
100
25
50
H
e
12. 5
50
100
50
1e
C2H5
H
H
e
100
50
1f
1g
NO2
NO2
NO2
H
e
e
12. 5
50
100
50
C2H5
F
H
100
100
100
100
50
1h
1i
H
e
50
25
50
50
Br
Cl
NO2
NO2
NO2
H
CH2
CH2
CH2
CH2
CH2
e
1j
1k
H
12. 5
12. 5
12. 5
50
100
100
100
50
F
H
1l
1m
F
NO2
NO2
H
50
50
CH3
C(CH3)3
F
H
2a
2b
NH2
NH2
NH2
NH2
H
100
100
100
100
100
100
100
100
100
100
100
100
256
256
64
100
100
50
25
50
100
100
100
100
100
25
100
100
100
100
100
100
100
100
100
100
100
100
8
50
50
H
e
2c
2d
Br
C2H5
H
H
e
25
50
100
50
H
e
e
100
50
2e
NH2
NH2
NH2
H
12. 5
12. 5
25
100
50
2f
2g
C2H5
F
H
e
100
100
100
100
50
H
e
50
50
50
2h
2i
Br
Cl
NH2
NH2
NH2
H
CH2
CH2
CH2
CH2
CH2
25
100
100
100
100
200
H
12.5
50
50
50
2j
2k
F
H
CH3
F
NH2
NH2
50
25
12. 5
50
100
100
e
2l
H
Rifampicin
Ampicillin trihydrate
Gentamycin sulphate
Ofloxacin
64
64
0.5
8
256
1
0.25
1
0.03
0.06
0.25
2
e
e
8
0.25
0.5
0.125
512
32
e
1024
2
e
e
e
e
64
32
8
0.125
e
Fluconazole
Amphotericin B
a
e
e
e
e
e
e
e
e
e
1
64
1
e
e
e
e
e
e
e
1
Abbreviations: Kp, Klebsiella pneumoniae RSHM 574; Ec, Escherichia coli ATCC 25922; Pa, Pseudomonas aeruginosa ATCC 25853; Bs, Bacillus subtilis
ATCC 6633; Sa, Staphylococcus aureus ATCC 25923; Ca, Candida albicans ATCC 10231.
b
Kp, K. pneumoniae isolate (resistant to trimethoprim sulfamethoxazole, amoxicilin clavulonat, ceftriaxone, cephepim, aztreonam); Ec, E. coli isolate (resistant
to trimethoprim sulfamethoxazole, cephepim, tazobactam); Bs, B. subtilis isolate (resistant to ceftriaxone); Sa, S. aureus isolate (resistant to oxacillin, gentamycin,
aztreonam, trimethoprim sulfamethoxazole); Ca, C. albicans isolate.

T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
503
OH
R
O
N
PPA
+
O2N
O2N
X
R
HOOC
NH2
X
1a- 1m
X = -, CH2
R = F, Cl, Br, CH3,C2H5, C(CH3)3
A or B
O
H2N
X
R
N
2a-2l
Scheme 1. Synthesis of the target benzoxazoles. Reagents: (A) 10% PdeC, H2, EtOH for compounds 2a, 2de2f, 2k; (B) NiCl2$6H2O, Zn, MeOH for compounds
2b, 2c, 2ge2j, 2l.
2.2. In vitro antibacterial and antifungal activities
had more potent activity than the tested compounds in drug-
resistant K. pneumoniae isolate except 1m that indicated
comparable inhibitory effect, while none of the synthesized
structures showed a good inhibitory activity against Escheri-
chia coli strains than the standard drugs, all of them were
more active than ampicillin against drug-resistant E. coli. In
particular, compounds 1b, 1d, and 2l hopefully had a compara-
ble inhibitory effect with ofloxacin. We could consider that
switching the nitro group to the amine for 5th position of 2-
benzylbenzoxazole structure or using a nitro group instead
of amine at position 6 of 2-phenylbenzoxazole caused an
increase in the antibacterial activity against drug-resistant E.
coli. The tested compounds showed very important inhibitory
effect against Pseudomonas aeruginosa with MIC values of
25e50 mg/ml as well. Among the tested compounds, deriva-
tives 1f, 1i, 1j, 1l, 2a, 2i, and 2j displayed very significant
antibacterial activity against the Gram-negative enterobacter
P. aeruginosa, which is effective in nosocomial infections
and often resistant to antibiotic therapy, comparable to that
of rifampicin. Additionally, all of the derivatives were found
to be more potent than ampicillin.
On the other hand, although most of the tested compounds
were found to be noticeably active against C. albicans, they
exhibited less effect than the standard drugs. Surprisingly, all
derivatives except 1a, 1c, 1e, 1ge1i, 2h had comparable activ-
ity with fluconazole for C. albicans isolate. Structureeactivity
relationships suggested that substitution with the amine
instead of the nitro at position R2 or R3 enhanced the potency.
It could be pointed out that there has no any considerable
result achieved about the preference of the phenyl or benzyl
ring at position 2 of benzoxazole.
All the newly synthesized 5(or 6)-nitro/amino-2-( p-
substituted phenyl/benzyl)benzoxazole derivatives (1ae1m,
2ae2l) were assayed in vitro for antimicrobial activity against
several Gram-positive, Gram-negative bacteria strains with its
drug-resistant isolate and Candida albicans and its clinical
isolate. The standard agents, rifampicin, ampicillin trihydrate,
gentamycin, and ofloxacin for antibacterial activity and fluco-
nazole and amphotericin B for antifungal activity were also
screened under identical conditions for comparison. The
MIC values were determined by the twofold serial dilution
technique in MuellereHinton broth and Sabouraud dextrose
agar for the antibacterial and antifungal assays, respectively
[39].
According to the obtained data (Table 1), most of the syn-
thesized compounds showed better antibacterial activity
against Gram-positive bacteria B. subtilis and its drug-resistant
isolate than the other tested microorganisms. Compounds 1a,
1c, 1e, 1g, 1h, 1m, 2b, 2d, 2j, 2l had comparable results
with rifampicin, the other derivatives indicated one or more di-
lution better inhibitory effect against B. subtilis ATCC 6633.
On the other hand, all of the compounds had more potent ac-
tivity than rifampicin in drug-resistant B. subtilis. It should be
noted that an H-acceptor group was enough for enhancing the
activity of these series against B. subtilis. Besides, a halogen
atom in the position of R1 played an important role for increas-
ing the potency as well. Having phenyl or benzyl group on
second position of benzoxazole wasn’t change the activity.
None of the compounds exhibited more activity than the all
tested standard drugs against S. aureus and its drug-resistant
isolate. In particular, 5-amino-2-( p-ethylphenyl)benzoxazole,
2f, came out with very significant activity at MIC value
25 mg/ml against S. aureus. Derivatives 1m, 2-( p-methylben-
zyl)-5-nitrobenzoxazole, and 2g, 5-amino-2-( p-fluorophenyl)-
benzoxazole (50 mg/ml) had a good inhibitory effect as well. It
could be pointed out that there is no considerable difference
for results about the preference of the phenyl or benzyl ring
at position 2 of benzoxazole.
2.3. Structureeactivity relationship of B. subtilis
inhibitors with representative QSAR equation
The QSAR analysis in this study was performed using the
extra-thermodynamic method, correlating the antibacterial
activity against B. subtilis ATCC 6633 with various physico-
chemical parameters in order to reveal predictions for the
lead optimization in the training set of compounds of newly
synthesized 5(or 6)-nitro/amino-2-(substituted phenyl/benzyl)-
The antibacterial activity against Gram-negative bacteria
Klebsiella pneumoniae and its isolate of tested compounds
was found to be moderately active. Gentamycin and ofloxacin

504
T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
and previously synthesized [10,38] 5(or 6)-methyl-2-
(substituted phenyl/benzyl)benzoxazoles.
2h, 2i, 2k, 4ke4m [10] that are not included in the developed
model. The observed, calculated log 1/C values and residuals
of the test set molecules obtained by Eq. (1) are given in Table
3. Fig. 2 represents the graph of the obtained vs. calculated
log 1/C values of the test set molecules for the used model,
which has an r value of 0.604.
The derived QSAR analysis revealed that the compounds
possessing a methyl group at the positions 5 and 6 of benzox-
azole moiety play a role in increasing the activity against
B. subtilis. In addition, Eq. (1) demonstrates that substituent
effects on the position R4 is also important for the activity
and holding a substituent possessing a minimum width prop-
erty on this position like as alkyl groups enhance the potency.
Furthermore, substituting position R1 with a group enhancing
the electron-withdrawing capability of the phenyl ring system
plays a role in increasing the activity. Moreover, the best equa-
tion of these series structures has a molecular descriptor,
which is superdelocalizability (Sr). Sr was originally derived
as an index of reactivity of aromatic hydrocarbons [41]. The
index is based on the idea that early interaction of the
Results of QSAR analysis obtained by the MRA of the
training set of compounds given in Table 2 demonstrate that
the best equation (Eq. (1)) is statistically significant.
As can be deduced from Fig. 1, the goodness-of-fit of equa-
tion is very significant, possessing a high r (92.3%) and a small
s (0.108) with an overall F test value of 31.028 at the signifi-
cant level of p < 0.05. From a statistical point of view, equa-
tion has a sufficient number of DF (degrees of freedom,
DF ¼ 27) that can be judged significant for overall F statistics
at the 5% level of probability.
In order to avoid the risk of chance correlation, some cir-
cumstances, which were pointed out by Kubinyi [40], have
been taken into consideration in the study. Cross-validation
was applied to the original data set and the resulting PRESS
was calculated. The calculated overall PRESS values and for
Eq. (1) is 0.131.
The obtained correlation equation was screened by using
a test set (Table 3) concerning compounds 1b, 1d, 1f, 1j, 2f,
Table 2
Training set of compounds, biological activity against B. subtilis and parameters used in Eq. (1)
R4
R3
O
X
R1
N
R2
Compound
number
R1
R2
R3
R4
X
MIC
(mg/ml)
IR2
IR3
sR1
B1R4
Sr
1a
1c
C(CH3)3
F
H
NO2
NO2
NO2
H
e
50
50
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
1
1
1
1
ꢀ0.20
0.06
ꢀ0.15
ꢀ0.15
0.06
0.23
0.06
0.06
ꢀ0.17
ꢀ0.20
0.06
0.23
ꢀ0.15
0
1
1.8749
1.5396
1.5075
1.4924
1.5427
1.5427
0.4866
0.4866
1.5779
0.6891
1.6413
1.6413
1.2621
0.6697
1.6455
1.7619
1.7459
1.5847
1.5263
1.5263
1.2646
1.5241
1.5241
1.6075
1.4705
1.6304
1.4785
1.4470
1.6070
1.5077
1.6292
1.4471
1.8861
H
e
1
1e
1g
C2H5
C2H5
F
H
e
e
50
50
1
NO2
NO2
H
1
1h
H
e
50
25
1
1i
1k
Br
F
NO2
NO2
H
CH2
CH2
CH2
CH2
e
1
H
12.5
12.5
50
1
1l
1m
F
NO2
NO2
H
1
CH3
C(CH3)3
F
H
1
2a
2b
NH2
NH2
NH2
NH2
H
25
50
1
H
e
e
1
2c
2d
Br
C2H5
H
H
25
50
1
H
e
1
2e
2g
NH2
NH2
H
e
12.5
25
1
F
H
e
0.06
0.06
0.06
0
1
2j
F
NH2
H
CH2
CH2
e
50
50
1
2l
F
NH2
CH3
CH3
CH3
CH3
1
3a [38]
3b [38]
3c [38]
3d [38]
3e [38]
3f [38]
4a [10]
4b [10]
4c [10]
4d [10]
4e [10]
4f [10]
4g [10]
4h [10]
4i [10]
4j [10]
H
Cl
H
12.5
12.5
12.5
6.25
12.5
12.5
25
1
H
e
0.23
0.23
0.78
0.23
0.23
0
1
Br
NO2
Cl
H
e
1
H
e
1
CH3
CH3
H
e
e
1
Br
H
H
Cl
1
CH3
CH3
CH3
CH3
CH3
e
1.8
1.35
1.35
1.7
1.8
1.8
1.35
1.35
1.8
1.52
H
OCH3
F
e
25
25
0
H
e
e
0
H
Cl
NO2
Cl
25
25
0
e
0.23
0
H
CH3
CH3
CH3
CH3
CH3
Cl
e
e
25
25
H
OCH3
F
Cl
0
H
Cl
e
25
25
0
e
0.23
ꢀ0.17
CH3
CH3
e
50

T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
505
4,8
4,6
4,4
4,2
4,0
3,8
3,6
3,4
4,0
3,9
3,8
3,7
3,6
R = 0.604
3,6
3,8
4,0
Observed log 1/C values
4,2
4,4
4,6
3,9
4,0
4,1
4,2
Observed log 1/C values
4,3
4,4
Fig. 1. Plot of observed vs. predicted log 1/C values of the training set com-
pounds obtained from Eq. (1).
Fig. 2. Plot of observed vs. calculated log 1/C values of the test set compounds
obtained by using Eq. (1).
benzoxazole moiety plays a role in increasing the activity.
Moreover, substituting position R1 with a group enhancing
the electron-withdrawing capability of the phenyl ring system
increases the potency. These observations could be guided to
the lead optimization of the new candidate antibacterial
agents.
molecular orbitals of reactants can be regarded as a mutual
perturbation, the relative energies of orbitals changing to-
gether and maintaining a similar degree of overlap as reactants
approach each other. The index is calculated by the sum
2
m
X
Cjr
Sr ¼ 2
ej
j¼1
4. Experimental
Here the term Sr is the superdelocalizability at position r, ej is
the bonding energy coefficient in the jth molecular orbital (ei-
genvalue), c is the molecular orbital coefficient at position r in
the HOMO (highest occupied molecular orbital) and m is the
index of the HOMO. This descriptor has been found to corre-
late highly to the activity of these sets of compounds.
The chemicals were purchased from the commercial
venders and were used without purification. The reactions
were monitored and the purity of the products was checked
by thin layer chromatography (TLC). Silica gel 60 F254 chro-
matoplates were used for TLC. All the melting points were
measured with a capillary melting point apparatus (Buchi
SMP 20 and Electrothermal 9100) and are uncorrected. Yields
were calculated after recrystallization. The IR spectra were
recorded on a Jasco FT/IR-420 spectrometer as KBr discs.
3. Conclusion
In conclusion, the results against B. subtilis, P. aeruginosa,
drug-resistant B. subtilis, drug-resistant E. coli, and C. albi-
cans isolate for these kinds of structures are quite encouraging.
According to 2D-QSAR study against B. subtilis, methyl
group instead of nitro or amine at the positions 5 and 6 of
1
The H NMR spectra were recorded employing a VARIAN
Mercury 400 MHz FT spectrometer, chemical shifts (d) are
in parts per million relative to TMS, and coupling constants
(J ) are reported in hertz. Mass spectra for compounds 1a,
Table 3
Compounds, parameters, MIC values (mg/ml), observed, and calculated log 1/C values of the test set by using Eq. (1)
Compound
number
R1
R2
R3
R4
X
MIC
(mg/ml)
IR2
IR3
sR1
B1R4
Sr
Observed
log 1/C
Calculated
log 1/C
Residuals
1b
1d
H
Br
H
NO2
NO2
H
e
25
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0.23
1
2.0546
1.5396
2.0472
1.4203
1.3394
1.6455
1.8378
1.1520
1.8453
1.9043
2.2047
3.983
4.407
4.284
4.364
4.280
4.084
4.316
4.280
3.977
4.032
4.032
3.610
3.954
3.613
4.003
3.817
3.911
3.832
3.882
3.761
3.744
3.607
0.373
0.453
0.671
0.360
0.463
0.173
0.484
0.398
0.217
0.289
0.425
H
e
e
12.5
12.5
12.5
12.5
25
1
1f
H
Cl
NO2
H
0
0.23
1
1j
NO2
H
CH2
e
1
2f
2h
C2H5
Br
NH2
H
ꢀ0.15
0.23
0.23
1
NH2
NH2
H
CH2
CH2
CH2
e
1
2i
2k
Cl
H
12.5
12.5
25
1
CH3
CH3
OCH3
OCH3
NH2
CH3
CH3
ꢀ0.17
ꢀ0.17
ꢀ0.27
ꢀ0.27
1
4k [10]
4l [10]
4m [10]
CH3
OCH3
OCH3
1.52
1.35
1.35
e
25
25
CH3
e

506
T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
1c, 1e, 1g, 2b, 2d, 2he2j were taken on a Waters Micromass
ZQ by using ESI(þ) method. Elemental analyses of com-
pounds 1d, 1ie1m, which were not ionized on Waters Micro-
mass ZQ, were taken on a Leco 932 CHNS-O analyzer. The
results of the elemental analyses (C, H, N) were within
ꢁ0.4% of the calculated amounts.
1H, J 8.4 Hz), 8.28e8.35 (m, 3H), 8.49 (d, 1H, J 2.0 Hz).
ESI(þ) m/e 259.11 (M þ 1, 100).
4.2.3. 2-( p-Bromophenyl)-6-nitrobenzoxazole (1d)
Yield: 51%, mp 162e164 ꢂC. IR (cmꢀ1): 3099, 1593e
1618, 1480, 1548, 1345, 1125e1280, 1073, 604e921. 1H
NMR (400 MHz, CDCl3): d 7.72e7.74 (m, 2H), 7.85 (d,
1H, J 9.2 Hz), 8.15e8.18 (m, 2H), 8.35 (dd, 1H, J 8.8,
2.4 Hz), 8.50 (d, 1H, J 2.0 Hz). Anal. Found: C, 48.12; H,
2.196; N, 8.673. Calcd for C13H7N2O3Br: C, 48.93; H, 2.21;
N, 8.78.
4.1. General procedure for the preparation
of 2-( p-substituted phenyl/benzyl)-5(or 6)
-nitrobenzoxazoles (1ae1m)
The derivatives were synthesized by heating 0.01 mol
appropriate o-aminophenol with 0.01 mol suitable acid in
24 g polyphosphoric acid (PPA) and stirring for 2e3 h. At
the end of the reaction period, the residue was poured into
an ice-water mixture and neutralized with an excess of 10 M
NaOH solution extracted with ethyl acetate. Then, this solu-
tion was dried over anhydrous sodium sulphate and evaporated
under diminished pressure. The residue was boiled with
200 mg charcoal in ethanol and filtered. After the evaporation
of solvent in vacuo, the crude product was obtained and
recrystallized from ethanol. In the present study, all the
compounds except 1b [42], 1f [42], and 1h [43] are new.
4.2.4. 2-( p-Ethylphenyl)-6-nitrobenzoxazole (1e)
Yield: 66%, mp 116e118 ꢂC. IR (cmꢀ1): 3103, 2969,
1518e1607, 1497, 1551, 1343, 1124e1270, 733e921. 1H
NMR (400 MHz, CDCl3): d 1.29e1.33 (m, 3H), 2.77 (q,
2H, J 7.6 Hz), 7.41 (d, 2H, J 8.4 Hz), 7.83 (d, 1H, J 8.8 Hz),
8.2 (d, 2H, J 8.4 Hz), 8.31e8.34 (m, 1H), 8.49 (d, 1H, J
2.0 Hz). ESI(þ) m/e 269.17 (M þ 1, 100).
4.2.5. 2-( p-Ethylphenyl)-5-nitrobenzoxazole (1g)
Yield: 70%, mp 128e130 ꢂC. IR (cmꢀ1): 3036, 2969,
1526e1616, 1498, 1526, 1347, 1126e1267, 660e944. 1H
NMR (400 MHz, CDCl3): d 1.31 (t, 3H, J 7.6 Hz), 2.77 (q,
2H, J 7.6 Hz), 7.40 (d, 2H, J 8.0 Hz), 7.67 (d, 1H, J 8.8 Hz),
8.18 (d, 2H, J 8.0 Hz), 8.31 (dd, 1H, J 9.2, 2.4 Hz), 8.63 (d,
1H, J 2.0 Hz). ESI(þ) m/e 269.17 (M þ 1, 100).
4.2. General procedure for the preparation
of 5(or 6)-amino-2-( p-substituted phenyl/
benzyl)benzoxazoles (2ae2l)
4.2.6. 2-( p-Bromobenzyl)-6-nitrobenzoxazole (1i)
Compounds 2b, 2c, 2ge2j, and 2l were obtained from 1c,
1d, 1he1l, respectively, which (5 mmol) were treated with
NiCl2$6H2O (15 mmol) and Zn (40 mmol) in methanol
(25 ml) and refluxing the mixture at 60 ꢂC for 2e4 h. The
precipitate was filtered. The crude product was purified by
recrystallization from methanol. The crystals were dried in
vacuo. Compounds 1a, 1e, 1f, 1g, and 1m (5 mmol) in ethanol
(50 ml) were reduced by hydrogenation using 40 psi of H2 and
10% PdeC (40 mg) until cessation of H2 uptake to obtain
compounds 2a, 2de2f, and 2k, respectively. The catalyst
was filtered on a bed of Celite, washed with ethanol, and the
filtrate was concentrated in vacuo. The crude product was
purified by recrystallization from ethanol. The crystals were
dried in vacuo. In the present study, all the compounds except
2c [44], 2ee2g [45], 2k [37], and 2l [37] are new.
Yield: 52%, mp 173e175 ꢂC. IR (cmꢀ1): 3107, 1520e
1616, 1485, 1520, 1344, 1145e1272, 690e941, 1011. 1H
NMR (400 MHz, CDCl3): d 4.61 (s, 2H), 7.39 (dd, 2H, J
8.0, 2.4 Hz), 7.58 (dd, 2H, J 8.4, 2.8 Hz), 7.92 (dd, 1H,
J 8.8, 2.8 Hz), 8.27 (dd, 1H, J 8.8, 2.4 Hz), 8.67 (d, 1H, J
2.0 Hz). Anal. Found: C, 50.12; H, 2.649; N, 8.357. Calcd
for C14H9N2O3Br: C, 50.48; H, 2.72; N, 8.41.
4.2.7. 2-( p-Chlorobenzyl)-6-nitrobenzoxazole (1j)
Yield: 75%, mp 119e121 ꢂC. IR (cmꢀ1): 3107, 1524e
1617, 1488, 1524, 1343, 1090e1273, 1090, 622e942. 1H
NMR (400 MHz, CDCl3): d 4.31 (s, 2H), 7.35 (s, 4H), 7.78
(dd, 1H, J 8.8, 2.8 Hz), 8.29 (dd, 1H, J 8.0, 2.0 Hz), 8.40 (d,
1H, J 2.8 Hz). Anal. Found: C, 57.77; H, 2.972; N, 9.625.
Calcd for C14H9N2O3Cl: C, 58.25; H, 3.14; N, 9.70.
4.2.1. 2-( p-t-Butylphenyl)-6-nitrobenzoxazole (1a)
Yield: 53%, mp 164e165 ꢂC. IR (cmꢀ1): 3092, 2964,
1576e1608, 1497, 1528, 1342, 1119e1266, 661e916. 1H
NMR (400 MHz, CDCl3): d 1.39 (s, 9H), 7.57 (d, 2H, J
8.4 Hz), 7.81 (d, 1H, J 8.4 Hz), 8.19 (d, 2H, J 8.8 Hz), 8.29
(dd, 1H, J 8.8, 2.4 Hz), 8.46 (d, 1H, J 2.0 Hz). ESI(þ) m/e
297.25 (M þ 1, 100).
4.2.8. 2-( p-Fluorobenzyl)-6-nitrobenzoxazole (1k)
Yield: 60%, mp 109e110 ꢂC. IR (cmꢀ1): 3105, 1510e
1605, 1462, 1095e1307, 1344, 1213, 664e950. 1H NMR
(400 MHz, CDCl3): d 4.31 (s, 2H), 7.06 (m, 2H), 7.37 (m,
2H), 7.77 (dd, 1H, J 8.8, 1.2 Hz), 8.27 (m, 1H), 8.39 (d, 1H,
J 1.6 Hz). Anal. Found: C, 61.19; H, 3.20; N, 10.18. Calcd
for C14H9N2O3F: C, 61.77; H, 3.33; N, 10.29.
4.2.2. 2-( p-Fluorophenyl)-6-nitrobenzoxazole (1c)
4.2.9. 2-( p-Fluorobenzyl)-5-nitrobenzoxazole (1l)
Yield: 54%, mp 158e159 ꢂC. IR (cmꢀ1): 3086, 1520e
1600, 1496, 1520, 1343, 1123e1288, 1226, 627e922. 1H
NMR (400 MHz, CDCl3): d 7.24e7.30 (m, 2H), 7.84 (d,
Yield: 81%, mp 88e90 ꢂC. IR (cmꢀ1): 3104, 1507e1618,
1459, 1531, 1349, 1217, 1092e1276, 657e958. 1H NMR
(400 MHz, CDCl3): d 4.31 (s, 2H), 7.31 (m, 2H), 7.37 (m,

T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
507
2H), 7.59 (d, 1H, J 8.0 Hz), 8.29 (dd, 1H, J 8.8, 2.4 Hz), 8.59
(d, 1H, J 2.0 Hz). Anal. Found: C, 60.95; H, 3.225; N, 10.20.
Calcd for C14H9N2O3F: C, 61.77; H, 3.33; N, 10.29.
7.00e7.10 (t, 2H, J 8.65 Hz), 7.30e7.40 (q, 2H, J 8.40 Hz),
7.40e7.45 (d, 1H, J 8.43 Hz). ESI(þ) m/e 243 (M þ 1, 100),
147 (100%).
4.2.10. 2-( p-Methylbenzyl)-5-nitrobenzoxazole (1m)
Yield: 64%, mp 98e100 ꢂC. IR (cmꢀ1): 2919, 1535e1617,
1456, 1535, 1345, 1131e1257, 668e955. 1H NMR (400 MHz,
CDCl3): d 2.36 (s, 3H), 4.29 (s, 2H), 7.20 (d, 2H, J 8.0 Hz),
7.28 (dd, 2H, J 8.0, 2.8 Hz), 7.57 (d, 1H, J 9.2 Hz), 8.28
(dd, 1H, J 9.2, 2.4 Hz), 8.50 (d, 1H, J 2.0 Hz). Anal. Found:
C, 66.72; H, 4.365; N, 10.31. Calcd for C15H12N2O3: C,
67.16; H, 4.51; N, 10.44.
4.3. Microbiology
Standard strains of K. pneumoniae RSHM 574, P. aerugi-
nosa ATCC 25853, E. coli ATCC 25922, B. subtilis ATCC
6633, S. aureus ATCC 25923, C. albicans ATCC 10231 and
clinical isolates of these microorganisms that are known to
be resistant to various antimicrobial agents were included in
the study. Resistance was determined by KirbyeBauer Disk
Diffusion method according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI) [46] in the clinical
isolates.
Standard powders of rifampicin, ampicillin trihydrate,
gentamycin sulphate, ofloxacin, fluconazole and amphotericin
B were obtained from the manufacturers. Stock solutions were
dissolved in dimethylsulphoxide (ofloxacin), methanol (rifam-
picin), pH 8 phosphate-buffered saline (PBS) (ampicillin trihy-
drate) and distilled water (gentamycin sulphate, fluconazole
and amphotericin B).
4.2.11. 6-Amino-2-( p-t-butylphenyl)benzoxazole (2a)
Yield: 61%, mp 188e190 ꢂC. IR (cmꢀ1): 3411, 3202, 2951,
1624, 1449, 1111e1267, 699e948. 1H NMR (400 MHz,
CDCl3): d 1.32 (s, 9H), 5.51 (s, 2H), 6.64 (dd, 1H, J 8.4,
2.0 Hz), 6.82 (s, 1H), 7.39 (d, 1H, J 8.4 Hz), 7.57 (d, 2H, J
8.4 Hz), 8.01 (d, 2H, J 8.0 Hz). ESI(þ) m/e 269.17 (M þ 1,
100).
4.2.12. 6-Amino-2-( p-fluorophenyl)benzoxazole (2b)
Yield: 45%, mp 224e225 ꢂC. IR (cmꢀ1): 3400e3309,
3060, 1557e1633, 1497, 1130e1293, 1120, 651e948. 1H
NMR (400 MHz, CDCl3): d 5.49 (s, 2H), 6.65 (dd, 1H, J
8.4, 2.0 Hz), 6.83 (d, 1H, J 1.6 Hz), 7.40 (m, 3H), 8.11e
8.15 (m, 2H). ESI(þ) m/e 229.18 (M þ 1, 100).
All bacterial isolates were subcultured in MHA plates and
incubated overnight at 37 ꢂC and all Candida isolates were
subcultured in SDA plates at 35 ꢂC for 24e48 h. The microor-
ganisms were passaged at least twice to ensure purity and
viability.
The solution of the synthesized compounds and standard
drugs were prepared at 400, 200, 100, 50, 25, 12.5, 6.25,
3.125, 1.56, 0.78, 0.39 mg/ml concentrations, at 4096, 2048,
1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125,
0.0625 mg/ml concentrations in the wells of microplates by
diluting in MHB, respectively. Bacterial susceptibility testing
was performed according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI) M100-S16 [39,47].
The bacterial suspensions used for inoculation were prepared
at 105 cfu/ml by diluting fresh cultures at MacFarland 0.5
density (107 cfu/ml). Suspensions of the bacteria at 105 cfu/
ml concentration were inoculated to the twofold diluted
solution of the compounds. There were 104 cfu/ml bacteria
in the wells after inoculations. MHB was used for diluting
the bacterial suspension and for twofold dilution of the com-
pound. DMSO (80%), EtOH (20%), methanol, DMSO, PBS,
pure microorganisms and pure media were used in control
wells. A 10 ml bacteria inoculum was added to each well of
the microdilution trays. The trays were incubated at 37 ꢂC in
a humid chamber and MIC endpoints were read after 24 h of
incubation. All organisms were tested in triplicate in each
run of the experiments. The lowest concentration of the com-
pound that completely inhibits macroscopic growth was deter-
mined and minimum inhibitory concentrations (MICs) were
reported. All Candida isolates were subcultured in SDA plates,
and incubated at 35 ꢂC for 24e48 h prior to antifungal suscep-
tibility testing, and passaged at least twice to ensure purity and
viability. Susceptibility testing was performed in RPMI-1640
medium with L-glutamine buffered to pH 7 with MOPS and
culture suspensions were prepared through the guideline of
4.2.13. 6-Amino-2-( p-ethylphenyl)benzoxazole (2d)
Yield: 62%, mp 198e200 ꢂC. IR (cmꢀ1): 3401, 2958, 1634,
1495, 1143, 824e840. 1H NMR (400 MHz, CDCl3): d 1.21 (t,
3H, J 7.6 Hz), 2.68 (q, 2H, J 8.0 Hz), 5.44 (s, 2H), 6.64 (dd,
1H, J 8.4, 2.0 Hz), 6.81 (d, 1H, J 2.0 Hz), 7.39 (d, 3H, J
8.4 Hz), 7.99 (dd, 2H, J 8.4, 1.6 Hz). ESI(þ) m/e 239.22
(M þ 1, 100).
4.2.14. 6-Amino-2-( p-bromobenzyl)benzoxazole (2h)
Yield: 59%, mp 274e275 ꢂC (decompose). IR (cmꢀ1):
3060, 2820, 1621, 1495, 1118e1259, 1089, 704e955. 1H
NMR (400 MHz, CDCl3): d 4.35 (s, 2H), 7.27e7.37 (m,
5H), 7.63 (d, 1H, J 2.0 Hz), 7.77 (d, 1H, J 8.4 Hz). Anal.
Found: C, 54.86; H, 4.142; N, 9.289. Calcd for C14H11N2OBr:
C, 55.47; H, 3.66; N, 9.24.
4.2.15. 6-Amino-2-( p-chlorobenzyl)benzoxazole (2i)
Yield: 58%, mp 210e212 ꢂC. IR (cmꢀ1): 3024, 2827, 2606,
2544, 1624, 1569, 1487, 1261e1119, 1092, 954e705, 600. 1H
NMR (400 MHz, DMSO þ CDCl3): d 4.20 (s, 2H), 7.15e7.35
(m, 4H), 7.30e7.40 (dd, 1H, J 1.92, 8.42 Hz), 7.53 (s, 2H),
7.55e7.65 (d, 1H, J 8.43 Hz), 7.65e7.775 (d, 1H, J
1.77 Hz). ESI(þ) m/e 259 (M þ 1), 225 (100%), 147 (100%).
4.2.16. 6-Amino-2-( p-fluorobenzyl)benzoxazole (2j)
Yield: 45%, mp 115 ꢂC. IR (cmꢀ1): 3343, 1631, 1567,
1509, 1494, 1272e1018, 957e736. 1H NMR (400 MHz,
CDCl3): d 3.70e3.85 (s, 2H), 4.20e4.30 (s, 2H), 6.60e6.70
(dd, 1H, J 8.43, 2.02 Hz), 6.75e6.80 (d, 1H, J 1,99 Hz),

508
T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
Table 4
Correlation matrix of the variables used in Eq. (1)
CLSI M27-A [48]. The yeast suspensions used for inoculation
were prepared at 104 cfu/ml by diluting fresh cultures at
MacFarland 0.5 density (106 cfu/ml). Suspensions of the yeast
at 104 cfu/ml concentration were inoculated to the twofold
diluted solution of the compounds. There were 103 cfu/ml
bacteria in the wells after inoculations. A 10 ml yeast inoculum
was added to each well of the microdilution trays. The trays
were incubated at 35 ꢂC in a humid chamber and MIC
endpoints were read after 48 h of incubation. All organisms
were tested in triplicate in each run of the experiments. The
lowest concentration of the compound that completely inhibits
macroscopic growth was determined and minimum inhibitory
concentrations (MICs) were reported.
IR2
IR3
sR1
B1R4
Sr
IR2
IR3
sR1
B1R4
Sr
1.000
0.090
1.000
0.137
0.000
1.000
0.104
0.223
0.000
1.000
0.011
0.045
0.001
0.042
1.000
log 1=C ¼ þ0:329ðꢁ0:12ÞIR2c þ 0:315ðꢁ0:13ÞIR3c
þ 0:577ðꢁ0:24ÞsR1 ꢀ 0:287ðꢁ0:18ÞB1R4
ꢀ 0:410ðꢁ0:12ÞSr þ 4:740ðꢁ0:25Þ
ð1Þ
4.4. QSAR analysis
n ¼ 33; r ¼ 0.923; s ¼ 0.108; F ¼ 31.028; Q2 ¼ 0.783;
s-PRESS ¼ 0.131.
Chemical structures and antibacterial activity against
Gram-positive bacteria B. subtilis ATCC 6033 strains of the
previously [10,38] and newly synthesized compounds shown
as MIC values are given in Tables 2 and 3. The potency was
defined as log 1/C where C was the MIC value expressed in
molar concentration units. A training set including compounds
1a, 1c, 1e, 1ge1i, 1ke1m, 2ae2e, 2g, 2j, 2l, 3ae3f [38], 4ae
4j [10] and a test set consisting in compounds 1b, 1d, 1f, 1j,
2f, 2h, 2i, 2k, 4ke4m [10] were considered. The variables
used as independent descriptors in the QSAR analysis were
hydrophobic, electronic, steric, and structural parameters.
The structural variable Ix expresses a value of 1 for the pres-
ence of CH2 group as a bridge element and 0 for the absence
of it between phenyl and benzoxazole moiety. The screened
physicochemical parameters in this QSAR study are log P,
p for the hydrophobic effects, Sr, s, F (field effect), R (reso-
nance effect), as the electronic influences and Verloop’s
STERIMOL descriptors (L, B1, B4) for the steric interactions
of the substituents R1, R2, R3 and R4. Additionally, H-bond
donor, H-bond acceptor and the other FreeeWilson type struc-
tural indicator variables were also used for positions R2 and R3
such as IR2a (IR3a), IR2b (IR3b), IR2c (IR3c) represented
a value of 1 for the presence of NO2, NH2, CH3 and 0 for
the absence, respectively. Values of the physicochemical
parameters used in this QSAR study were taken from the
Table of Hansch and Leo [49] except log P and Sr which
were calculated by using the Acclerys’s Cerius2 [50] program.
The values of the parameters used in the correlation equation
related to the activity among the candidate set of variables in
the training set are shown in Table 2. Multivariable regression
analysis (MRA) of the QSAR study was run on a PC using the
BILIN [51].
In the equation, the figures in parentheses are the standard
errors of the regression coefficients, n is the number of com-
pounds, r is the correlation coefficient, F is the significance
test and s is the standard error of estimate.
In order to judge the validity of the predictive power of the
QSAR analysis, the cross-validation method was also applied
Table 5
Observed and predicted log 1/C values with residuals obtained from Eq. (1)
Compound
number
Observed
log 1/C
Predicted
log 1/C
Residuals
1a
1c
3.773
3.713
3.730
3.730
3.713
4.125
4.338
4.338
3.730
4.027
3.659
4.063
3.678
4.226
3.960
3.685
3.685
4.224
4.290
4.363
4.609
4.290
4.363
3.989
3.981
3.959
4.007
4.046
3.989
3.981
3.959
4.046
3.676
3.568
3.856
3.748
3.754
3.855
3.953
4.288
4.288
3.707
4.055
3.814
3.912
3.849
4.178
3.812
3.765
3.771
4.131
4.288
4.288
4.713
4.276
4.276
3.892
4.078
4.012
3.974
4.091
3.879
4.049
3.999
4.078
3.747
0.205
ꢀ0.143
ꢀ0.018
ꢀ0.024
ꢀ0.142
0.172
1e
1g
1h
1i
1k
0.050
0.050
0.022
1l
1m
2a
2b
ꢀ0.027
ꢀ0.155
0.151
2c
2d
ꢀ0.170
0.048
0.148
2e
2g
2j
ꢀ0.079
ꢀ0.086
0.092
2l
3a [38]
3b [38]
3c [38]
3d [38]
3e [38]
3f [38]
4a [10]
4b [10]
4c [10]
4d [10]
4e [10]
4f [10]
4g [10]
4h [10]
4i [10]
4j [10]
0.002
0.075
ꢀ0.104
0.014
0.087
MRA that involves finding the best fit of dependent variable
(antibacterial activity) to a combination of independent vari-
ables (descriptors) is used by the least squares method. The
tabulated F(5, 30, 0.95) and F(5, 25, 0.95) are 2.53, 2.60, respec-
tively, whereas the overall F test values for the obtained equa-
tion was 31.028 which is statistically significant at the 5%
level of probability [52]. The statistically significant correla-
tion equation (Eq. (1)) obtained from MRA to describe the
QSAR analysis is given below.
0.097
ꢀ0.097
ꢀ0.054
0.033
ꢀ0.045
0.110
ꢀ0.068
ꢀ0.041
ꢀ0.031
ꢀ0.071

T. Ertan et al. / European Journal of Medicinal Chemistry 44 (2009) 501e510
509
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J.A. Kovacs, J. Falloon, R.E. Walker, H. Masur, S.E. Haniwich,
D.G. O’Neill, M.R. Decker, J.A. Metcalf, M.A. Deloria, O.L. Laskin,
N. Salzman, H.C. Lane, Proc. Natl. Acad. Sci. U S A 90 (12) (1993)
5608.
to the original data set by removing a compound from the data
in such a way that each observation (compound) is deleted
only once. For each reduced data set a model was developed
and the response values of the deleted observations were
predicted from this model and finally the resulting PRESS
(predictive residual sum of squares) and Q2 (the square of
predictive power of coefficient) were calculated for the equa-
tion [53,54]. The search for the simple correlation coefficients
which are given in Table 4 also reveals that there is no inter-
correlation between the independent variables in any case
entered in the correlation equations. The predicted log 1/C
values with residuals of the training set determined from equa-
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This work was supported by Ankara University Research
Fund (Grant no. 2005-0803049). The Central Lab of the
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