Synthesis, antimicrobial activity, pharmacophore analysis of some new 2-(substitutedphenyl/benzyl)-5-[(2-benzofuryl)carboxamido]benzoxazoles

Article abstract of DOI:10.1016/j.ejmech.2007.12.019

The synthesis and antimicrobial activity of a new series of 2-(substitutedphenyl/benzyl)-5-[(2-benzofuryl)carboxamido]benzoxazole derivatives 3-12 were described. The in vitro antimicrobial activity of the compounds was determined against some Gram-positi

Full text of DOI:10.1016/j.ejmech.2007.12.019

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2568e2578
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antimicrobial activity, pharmacophore analysis
of some new 2-(substitutedphenyl/benzyl)-5-
[(2-benzofuryl)carboxamido]benzoxazoles
a
Sabiha Alper-Hayta , Mustafa Arisoy , Ozlem Temiz-Arpaci , Ilkay Yildiz
a
a
a,
¨
*
,
a
b
Esin Aki , Semiha Ozkan , Fatma Kaynak
b
¨
a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Tandogan, Ankara, Turkey
b Gazi University, Faculty of Pharmacy, Department of Microbiology, Etiler, Ankara, Turkey
Received 23 May 2007; received in revised form 7 December 2007; accepted 12 December 2007
Available online 31 December 2007
Abstract
The synthesis and antimicrobial activity of a new series of 2-(substitutedphenyl/benzyl)-5-[(2-benzofuryl)carboxamido]benzoxazole deriva-
tives 3e12 were described. The in vitro antimicrobial activity of the compounds was determined against some Gram-positive, Gram-negative
bacteria and fungi and their drug-resistant isolates in comparison with standard drugs. Antimicrobial results indicated that the synthesized com-
pounds possessed a broad spectrum of activity with MIC values 500e15.625 mg/ml. In the series, the most active compound against Candida
krusei and Candida albicans isolate is 8 with MIC value 31.25 mg/ml. However, it is one dilution less potent than the compared fluconazole.
Some of the screened compounds exhibit significant activity, having MIC value as 31.25 mg/ml in Pseudomonas aeruginosa having same activity
as Rifampicin. Furthermore, considering the worth of developing new antibacterial agents against drug-resistant P. aeruginosa the present study
explores the structureeactivity relationship analysis of 2-(substitutedphenyl/benzyl)-5-[(2-benzofuryl)carboxamido]benzoxazoles using 3D-
common features pharmacophore hypotheses approach.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Benzoxazole; Benzofuran; Antifungal activity; Antibacterial activity; Pharmacophore analysis
1. Introduction
caused significant nosocomial infections and were the cause
of major morbidity and mortality in hospitalized patients,
more recently they have spread to community, causing severe
illnesses in previously healthy and otherwise non-vulnerable
patients [1e6]. Consequently, to prevent the emergence and
dissemination of resistant bacteria, continuing efforts to de-
velop new antibacterial agents are needed.
Benzofurans have drawn considerable attention over the
last few years due to their profound physiological and chemo-
therapeutic properties as well as their widespread occurrences
in nature. The antimicrobial activity of some 2-arylbenzo-
furans was investigated against vancomycin-resistant entero-
cocci and methicillin-resistant S. aureus [7e12]. Besides,
substituted-benzoxazoles possess diverse chemotherapeutic
activities including antibiotic [13,14], antimicrobial [15e26],
antiviral [27e31], topoisomerase I and II inhibitors [32e34],
The rising prevalance of multi-drug resistant microbial in-
fections in the past few decades has become a serious health
care problem. In particular, the emergence of multi-drug resis-
tant strains of Gram-positive bacterial pathogens such as meth-
icillin-resistant Staphylococcus aureus and Staphylococcus
epidermis and vancomycin-resistant Enterococcus is a problem
of ever-increasing significance. In fact every day more com-
mon and uncommon bacteria previously susceptible to com-
mon antimicrobials are reported to have developed resistance
to different antibiotics. Although these bacteria initially
* Corresponding author. Tel.: þ90(312)203 30 69; fax: þ90(312)223 69 40.
E-mail address: iyildiz@pharmacy.ankara.edu.tr (I. Yildiz).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.12.019

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 43 (2008) 2568e2578
2569
O
N
2. Results and discussion
X
R1
O
2.1. Chemistry
NH
R
For the synthesis of compounds 3e12 firstly, 5-amino-2-
[p-substitutedphenyl/benzyl]benzoxazoles and 5-amino-2-
[o-bromophenyl]benzoxazole (1) were obtained by heating
p-substitutedbenzoic acid and/or p-substitutedphenylacetic
acid and/or o-bromobenzoic acid with 2,4-diaminophenol in
PPA (polyphosphoric acid).
O
X= -, CH2
R1= H, Br
R= H, CH3, F, Cl, Br, C2H5, C(CH3)3
Fig. 1.
Compounds 3e12 were obtained from 5-amino-2-[p-sub
stitutedphenyl/benzyl]benzoxazoles and 5-amino-2-[o-bro-
mophenyl]benzoxazole with benzofuran-2-carboxylic acid
chloride (2) obtained by treating benzofuran-2-carboxylic
acid with thionyl chloride as given in Scheme 1 [20].
All the compounds 3e12 were prepared as new products.
The structures of 3e12 were supported by spectral data. The
antitumor activities [35e38]. In our previous studies, various
2-(substitutedphenyl or benzyl)benzoxazole derivatives have
been reported to be microbiologically active [15e24,20,39].
Because of the need of new antimicrobial drugs, we de-
signed and synthesized new compounds having both benzoxa-
zole and benzofuran binding via an amide bridge as a new
class antimicrobial agents (Fig. 1). In this study, the strategy
employed was to examine the effect of two isosteric heterocy-
clic nucleus connected with a carboxamide bridge together
with the role of substituent on the second position of benzox-
azole against some Gram-positive, Gram-negative bacteria,
fungi and their drug-resistant isolates in comparison with the
control drugs. The other goal at the outset of this research,
was to compare how activity changed by attaching 2-benzo-
furyl ring instead of 2-furyl to carboxamido moiety of
benzoxazole.
1
IR, H NMR, mass spectra and elemental analysis results are
in agreement with the proposed structures. Additionally, 13C
NMR spectral results are given only for the compound 8 at
the end of the experimental part. Physical and spectral data
of the compounds are reported in Table 1.
2.2. In vitro antibacterial and antifungal activity
All the synthesized 2-(substitutedphenyl/benzyl)-5-[(2-ben-
zofuryl)carboxamido]benzoxazole derivatives 3e12 were as-
sayed in vitro for antibacterial activity against Klebsiella
pneumoniae RSHM 574, Pseudomonas aeruginosa ATCC
COOH
X
R1
O
OH
R1
X
PPA
2 HCl
+
N
H2N
NH2
H2N
R
R
1
O
O
SOCl2
COCl
COOH
2
O
N
NaHCO3/ ETHER/ H2O
R1
R
X
1
2
+
O
NH
O
3-12
X= -, CH2
R1= H, Br
R= H, CH3, F, Cl, Br, C2H5
, C(CH3)3
Scheme 1. Synthetic pathway of the target compounds 3e12.

Table 1
Physical properties and spectral data of the compounds 3e12
O
N
R1
O
X
N
H
O
R
Compound
no.
R1
Br
R
H
X
Formula: calculated/
found
Mp
Yield (%)
IR (cmꢀ1
)
1H NMR (d ppm) J ¼ Hz
MS (ESIþ)
m/z (% X)
3
e
C22H13BrN2O3: C:
60.99, H: 3.02, N: 6.47/
C: 59.60, H: 3.02, N:
6.09
168e170
90
3509, 1690, 1623, 1553,
1481, 1345e1308, 1258,
1188e1109, 962e472
8.551 (1H, s), 8.247e8.242 (1H, d,
J ¼ 2.0), 8.084e8.060 (1H, dd,
J ¼ 8.0, J0 ¼ 1.6), 7.773e7.680 (3H,
m), 7.617e7.547 (3H, m), 7.467e
7.428 (2H, m), 7.381e7.258 (2H, m)
8.94 (1H, s), 8.270e8.235 (2H, dd,
J ¼ 6.8, J0 ¼ 7.2), 8.161e8.156 (1H,
d, J ¼ 2.0), 7.723e7.697 (2H, dd,
J ¼ 1.6, J0 ¼ 8.8), 7.624 (1H, s),
7.587e7.556 (2H, m), 7.483e7.441
(1H, dd, J ¼ 8.0, J0 ¼ 8.8), 7.347e
7.310 (1H, dd, J ¼ 7.6, J0 ¼ 7.2),
7.257e7.193 (2H, dd, J ¼ 8.4,
J0 ¼ 8,8)
433 (% 100)
(M þ H), 435 (%
100) (M þ Hþ2)
4
H
H
e
C22H14N2O3$0.65HCl:
C: 70.00, H: 3.88, N:
7.42/C: 70.00, H: 3.70,
N: 7.42
204e208
76
3400, 3077, 1671, 1621,
1563, 1487, 1354e1312,
1256, 1177e1111,
961e479
355 (% 20)
(M þ H)
5
6
H
H
C2H5
e
e
C24H18N2O3$0.6H2O:
C: 73.30, H: 4.92, N:
7.12/C: 73.30, H: 4.65,
N: 6.77
202e206
237e240
60
94
3402, 2956, 1670, 1612,
1558, 1483, 1353e1291,
1255, 1175e1111,
960e475
8.504 (1H, s), 8.173e8.134 (2H, dd,
J ¼ 8.4, J0 ¼ 7.2), 7.727e7.547 (5H,
m), 7.475e7.436 (1H, dd, J ¼ 7.2,
J0 ¼ 8.4), 7.364e7.303 (3H, m),
2.761e2.705 (2H, q), 1.307e1.269
(3H, t)
383 (% 100)
(M þ H)
F
C22H13FN2O3$0.9H2O:
C: 68.00, H: 3.84, N:
7.21/C: 67.98, H: 3.57,
N: 6.99
3400, 1671, 1621, 1559,
1487, 1354e1292, 1256,
1177e1111, 949e472
8.438 (1H, s), 8.183e8.148 (2H, m),
8.081 (1H, s), 7.647e7.609 (2H, dd,
J ¼ J0 ¼ 7.6), 7.540 (1H, s), 7.490e
7.469 (2H, m), 7.396e7.357 (1H, dd,
J ¼ 7.6, J0 ¼ 8.0), 7.262e7.225 (1H,
dd, J ¼ 7.6, J0 ¼ 7.2), 7.152e7.110
(2H, dd, J ¼ J0 ¼ 8.4)
373 (% 100)
(M þ H)
7
8
H
H
C(CH3)3
e
C26H22N2O3: C: 76.08,
H: 5.40, N: 6.82/C:
75.99, H: 5.32, N: 6.81
223e226
177e180
37
54
3403, 2961, 1671, 1620,
1580, 1485, 1352e1290,
1255, 1176e1110,
962e476
10.73 (1H, s), 8.283e8.279(1H, d,
J ¼ 1.6), 8.121e8.100 (2H, d,
J ¼ 8.4), 7.828e7.713 (5H,m),
7.623e7.601 (2H, d, J ¼ 8.8),
7.516e7.477 (1H, dd, J ¼ 8.0,
J0 ¼ 7.6), 7.377e7.339 (1H, dd,
J ¼ 8.0, J0 ¼ 7.2), 1.307 (9H, s)
10.650 (1H, s), 8.185 (1H, s), 7.812e
7.695 (4H, m), 7.643e7.621 (1H, d,
J ¼ 8.8), 7.501e7.463 (1H, dd,
J ¼ J0 ¼ 7.6), 7.350e7.260 (6H, m),
4.313 (2H, s)
411 (% 100)
(M þ H)
H
CH2
C23H16N2O3: C: 74.99,
H: 4.38, N: 7.60/C:
74.91, H: 4.37, N: 7.60
3270, 3078, 1669, 1625,
1569, 1482, 1354e1297,
1258, 1174e1144,
949e471
369 (% 100)
(M þ H)
(continued on next page)

Table 1 (continued)
Compound
no.
R1
R
X
Formula: calculated/
found
Mp
Yield (%)
89
IR (cmꢀ1
)
1H NMR (d ppm) J ¼ Hz
MS (ESIþ)
m/z (% X)
9
H
Br
CH2
C23H15BrN2O3: C:
61.76, H: 3.39, N: 6.26/
C: 61.61, H: 3.39, N:
6.26
202e205
3268, 3078, 1683, 1669,
1625, 1589, 1486, 1354e
1298, 1257, 1172e1108,
971e474
10.713 (1H, s), 8.201e8.197 (1H, d,
J ¼ 1.6), 7.854e7.834 (1H, d,
J ¼ 8.0), 7.790e7.733 (3H, m),
7.691e7.669 (1H, d, J ¼ 8.8),
7.586e7.505 (3H, m), 7.405e7.361
(3H, m), 4.357 (2H, s)
447 (% 80) (Mþ),
449 (% 100)
(M þ 2)
10
H
CH3
CH2
C24H18N2O3: C: 75.38,
H: 4.74, N: 7.32, C:
75.21, H: 4.38, N: 7.30
172e175
54
3077, 1670, 1626, 1567,
1482, 1354e1277, 1258,
1183e1144, 971e471
10.672 (1H, s), 8.173e8.168 (1H, d,
J ¼ 2.0), 7.819e7.800 (1H, d,
J ¼ 7.6), 7.760e7.699 (3H, m),
7.639e7.618 (1H, d, J ¼ 8.4),
7.510e7.468 (1H, dd, J ¼ J0 ¼ 8.4),
7.371e7.332 (1H, dd, J ¼ 8.0,
J0 ¼ 7.6), 7.247e7.226 (2H, d,
J ¼ 8.4), 7.148e7.129 (2H, d,
J ¼ 7.60), 4.256 (2H, s), 2.255
(3H, s)
383 (% 100)
(M þ H)
11
H
F
CH2
C23H15FN2O3: C:
71.50, H: 3.91, N: 7.25/
C: 71.21, H: 3.63, N:
7.23
200e203
82
3270, 3077, 1670, 1625,
1571, 1482, 1355e1299,
1258, 1185e1109, 972e471
10.679 (1H, s), 8.176e8.171 (1H, d,
J ¼ 2.0), 7.820e7.801 (1H, d,
J ¼ 7.6), 7.759e7.700 (3H, m),
7.656e7.635 (1H, d, J ¼ 8.4),
7.508e7.471 (1H, dd, J ¼ 7.2,
J0 ¼ 7.6), 7.433e7.398 (2H, m),
7.371e7.333 (1H, dd, J ¼ 8.0,
J0 ¼ 7.2), 7.194e7.151 (2H, dd,
J ¼ 8.4, J0 ¼ 8.8), 4.327 (2H, s)
10.682 (1H, s), 8.177e8.172 (1H, d,
J ¼ 2.0), 7.817e7.799 (1H, d,
J ¼ 7.2), 7.761e7.698 (3H, m),
7.655e7.634 (1H, d, J ¼ 8.4),
7.507e7.468 (1H, dd, J ¼ 7.6,
J0 ¼ 8.0), 7.403e7.331 (5H, m), 4.40
(2H, s)
387 (% 100)
(M þ H)
12
H
Cl
CH2
C23H15ClN2O3: C:
68.58, H: 3.75, N: 6.95/
C: 68.58, H: 3.67, N:
6.48
182e185
82
3268, 3077, 1670, 1625,
1569, 1482, 1354e1299,
1257, 1185e1109, 971e487
403 (% 100)
(M þ H)

Table 2
Antimicrobial activity results (MIC mg/ml) of newly synthesized compounds (3e12) with the standard drugs
O
N
R1
O
X
N
H
O
R
Compound no
X
R1
R
Kp*
Kp
Pa*
62.5
Pa
Ec*
Ec
Bs*
Bs
Sa*
31.25
Sa
Ca*
Ca
Ck
3
e
Br
H
H
H
H
H
H
H
H
H
H
125
125
125
125
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
125
125
125
125
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
3.9
125
250
125
125
125
125
125
125
125
125
500
125
500
500
125
125
125
125
125
125
125
62.5
62.5
62.5
62.5
62.5
31.25
62.5
62.5
62.5
62.5
e
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
e
250
4
e
H
C2H5
62.5
62.5
62.5
62.5
62.5
31.25
62.5
62.5
62.5
62.5
62.5
62.5
62.5
5
e
62.5
31.25
62.5
125
125
125
125
125
125
125
125
125
125
125
6
e
e
F
C(CH3)3
7
125
8
CH2
CH2
CH2
CH2
CH2
H
62.5
62.5
125
62.5
15.625
31.25
31.25
62.5
62.5
31.25
62.5
250
9
Br
CH3
F
125
125
125
125
10
11
12
125
125
125
125
125
62.5
31.25
250
125
e
Cl
125
Ampicillin trihydrate
Gentamicin
Rifampicin
15.625
0.48
>500
>500
31.25
>500
62.5
>15.625
15.625
3.9
0.48
0.48
0.24
0,12
0.12
1.9
7.8
0.9
1.9
0.48
7.8
7.8
3.9
0.24
1.9
62.5
>500
62.5
0.48
1.9
0.12
3.9
0.48
0.06
0.12
e
e
e
e
e
e
Ofloxacin
Amphotericin B
Fluconazole
0.12
7.8
0.12
3.9
0.24
0.48
0.48
1.9
1.9
15.625
Kp*: Klebsiella pneumoniae isolate; Kp: K. pneumoniae RSHM 574; Pa*: Pseudomonas aeruginosa isolate; Pa: P. aeruginosa ATCC 25853; Ec*: Escherichia coli isolate; Ec: E. coli ATCC 25922; Bs*: Bacillus
subtilis isolate; Bs: B.subtilis ATCC 6633; Sa*: Staphylococcus aureus isolate; Sa: S.aureus ATCC 25923; Ca*: Candida albicans isolate; Ca: C. albicans ATCC 10231; Ck: C. krusei ATCC 6258.

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 43 (2008) 2568e2578
2573
25853, Escherichia coli ATCC 25922, K. pneumoniae isolate
[has Extended Spectrum b Lactamase (ESBL) enzyme], E.
coli isolate [has Extended Spectrum b Lactamase (ESBL) en-
zyme] as Gram-negative bacteria, Bacillus subtillis ATCC
6633, S. aureus ATCC 25923, B. subtillis isolate (resistant to
ceftriaxon), S. aureus isolate [resistant to meticillen
(MRSA)] as Gram-positive bacteria and the antifungal activity
was evaluated against Candida krusei ATCC 6258, Candida
albicans ATCC 10231, C. albicans isolate (biofilm positive).
The MIC values were determined by two-fold serial dilution
technique in MuellereHinton broth and Sabouraud Dextrose
agar for the antibacterial and antifungal assay, respectively.
For comparison of the antimicrobial activity, rifampicin, ampi-
cillin trihydrate, gentamycin sulfate, ofloxacine were used as
the reference antibacterial agents and fluconazole, amphoteri-
cin B were employed as the reference antifungal agents. All
the biological results of the tested compounds are given in
Table 2.
According to Table 2, the synthesized compounds showed
a broad spectrum of activity with MIC values 500e62.5 mg/
ml against some Gram-positive bacteria such as S. aureus
ATCC 25923, B. subtillis ATCC 6633. Interestingly, the anti-
bacterial activity against meticillen resistant S. aureus
(MRSA) of the tested compounds 3e12 was found to have
more potent than against S. aureus strains. However, they
were less active than the compared control drugs, rifampicin,
gentamicin, ampicillin trihydrate and ofloxacin.
It could be concluded that if the hydrophobicity is higher,
the potency gets lower. In contrast, attaching 2-benzofuryl in-
stead of 2-furyl group at carboxamido moiety causes impor-
tant enhancing inhibitory effect against Gram-negative
bacterium P. aeruginosa. At this time, highly hydrophobic
molecule could be needed for increasing activity.
In the past 10 years there has been a major expansion in the
development of antifungal drugs, but there are still weaknesses
in the range and scope of current antifungal chemotherapy
[40]. New developments have included the modification of ex-
isting drug molecules to eliminate toxicity and improve activ-
ity. Therefore, we also tried to screen the antifungal activity
besides their antibacterial activity to be able to discover new
lead compounds.
All the newly synthesized derivatives showed the least ac-
tivity than amphotericin B and fluconazole. The MIC range
(250e31.25 mg/ml) of this series for C. krusei ATCC 6258 is
larger than the MIC range (62.5e31.25 mg/ml) of C. albicans
and its isolates. In one of our previous studies [39], 22-[p-
substituted-benzyl]-5-[substitutedcarbonylamino]benzoxazole
derivatives showed antifungal activity with MIC value of
6.25e100 mg/ml against C. albicans and C. krusei. The results
of the previous study revealed that the p-substituted-benzyl
moiety at position two of the benzoxazole ring slightly im-
proved the antifungal activity. Therefore in this study, we
got the best antifungal activity result with compound 8 carry-
ing benzyl group at the second position of the benzoxazole
ring. In addition, attaching a hydrogen or bromine on position
R played a role for getting a good inhibitory potency. How-
ever, substituting position R1 with the bromine atom, while
the main structure was 2-phenylbenzoxazole decreased the in-
hibitory effect.
It seemed to be some more modifications on the benzofuran
moiety are needed for the further study to get lead compounds.
Moreover, the yielded result against C. krusei for derivative 8
was also quite encouraging due to its activity caused by only
one-fold less dilution than fluconazole. As a result of our
studies we could say that the derivatives with a phenyl ring
at the fifth position of the benzoxazole ring, are more potent
than the ones having a benzofuran ring at the same position
against the Candida species.
All of the new compounds 3e12 showed a narrow antibac-
terial activity against some E. coli, K. pneumoniae, P. aerugi-
nosa as Gram-negative bacteria possessing MIC values
between 62.5 and 31.25 mg/ml and they were found to have in-
hibitory effect with MIC values 125e15.625 mg/ml against
their isolates.
All compounds were less active than standard drugs against
K. pneumoniae and its isolate. Structurally differences on the
substitution at position two of benzoxazole ring did not change
the activity against K. pneumoniae. Newly synthesized com-
pounds 6, 8, 9, 12 showed higher activity against P. aerugi-
nosa and its isolate when compared with all the standard
drugs. Among the tested derivatives only 9 had a pretty
good activity with a MIC value 15.625 mg/ml against drug-re-
sistant P. aeruginosa even more potent than all standard drugs.
Additionally, derivatives 6, 8, 9, and 12 showed higher activity
against P. aeruginosa than ampicillin, rifampicin and ofloxa-
cin and had the same potency with gentamycin. The other
compounds had a comparable effect with ofloxacin. It could
be concluded that, this kind of structures might be a lead for
P. aeruginosa. On the other hand, although the screened deriv-
atives against E. coli were found to have a moderate potency,
they showed very weak activity than the standard drugs.
Among the molecules, only 2-benzyl-5-[(2-benzofuryl)car-
boxamido]benzoxazole 8, has been found to be significantly
active with a MIC value 62.5 mg/ml against drug-resistant E.
coli.
For the 3D-common feature pharmacophore hypothesis ap-
proach, the hypothesis 1 that consists three HpArs and three
HBAs as shown in Fig. 2 was generated as the common-fea-
ture functions to explain the specification of the antibacterial
agents against drug-resistant P. aureuginosa. Fig. 3 depicted
that the most active molecule is compound 9 showing
a good fit with all the generated features than the rest of the
compounds.
When the training compounds were mapped onto the com-
mon-feature functions generated by hypothesis 1, one of the
HBA features slightly maps onto ‘‘N’’ of benzoxazole at the
compounds 3, 4, 5, 6, showed a better match for derivative
8. However, one of the HBA features slightly fitted into
‘‘O’’ of benzofuran at compound 8. It could be concluded
that ‘‘N’’ was more important than ‘‘O’’ of benzoxazole for in-
creasing the potency.
Furthermore, bearing 2-furyl [22] instead of 2-benzofuryl
on the carboxamido moiety caused slightly better activity
against Gram-positive bacteria S. aureus and B. subtilis.

2574
S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 43 (2008) 2568e2578
Fig. 2. Mapping of hypothesis 1, which contains three HpArs (blue), three HBAs (green) pharmacophore features (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.).
On the other side, when the less active compounds given in
Table 1 mapped onto the generated hypothesis 1, it has been
observed that they only showed a maximum match on five
common features. While the ‘‘O’’ of carbonyl group at the
compounds 10, 11, 12, possessed a fit onto the HBA feature,
the same group at the compound 7 did not show a match on
this feature. The ‘‘O’’ of benzofuran of structure 7 fit into the
HBA in contrast derivatives 10, 11, and 12. It was noticed
that, if the ‘‘O’’ of either benzofuran or benzoxazole placed
at the same side played very important role for increasing the
activity against drug-resistant P. aeruginosa.
the substituents at position two of the benzoxazole nucleus
did not constitute a major role at their antimicrobial activity
against Gram-positive bacteria. Consequently, this kind of
structures might be guides for Gram-negative bacterium
P. aeruginosa. In particular, compound 9 having 2-( p-bromo-
benzyl)benzoxazole group exhibited the highest activity with
MIC value of 15.625 mg/ml against drug-resistant P. aerugi-
nosa. Additionally, the result against C. krusei for 8 is quite
encouraging due to its activity caused by only one-fold less di-
lution than fluconazole. 3D-common features pharmacophore
hypothesis generation revealed that the conformational proper-
ties of the compounds are important for the activity against
drug-resistant P. aureuginosa and the HBA features are
more significant than the HpAr for increasing the activity.
These observations provided us noteworthy predictions in or-
der to design further antimicrobial active compounds prior to
their synthesis.
3. Conclusion
As a result of this study, some novel benzoxazole deriva-
tives including 2-benzofuryl as antimicrobial agents have
been discovered. It was found that structurally differences of
Fig. 3. Mapping of 9 onto hypothesis 1, which contain three HpArs (blue) and three HBAs (green) (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.).

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 43 (2008) 2568e2578
2575
4. Experimental
129.081, 129.233, 134.087, 134.857, 142.142, 148.482,
148.634, 155.035, 156.910, 166.549.
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 choromatography TLC. Kieselgel HF 254 chro-
matoplates (0.3 mm) were used for TLC and the solvent sys-
tems were ethylacetateen-hexane (5:5). All the melting
points were taken on a Buchi SMP 20 capillary apparatus
and are uncorrected. IR spectra were recorded on a Jasco
4.3. Microbiology
4.3.1. Materials
MuellereHinton Agar (MHA) (Merck), MuellereHinton
Broth (MHB) (Merck), Sabouraud Dextrose Agar (SDA)
(Merck), RPMI-1640 medium with L-glutamine (Sigma), 3-
[N-morpholino]-propan-sulfonic acid (MOPS) (Sigma), 96-
well microplates (Falcon), Transfer pipette (Biohit), Rifampi-
cin (Kocak), Ampicillin trihydrate (Paninkret Chem. Pharm.),
Gentamicin sulfate (Deva Ilac Sanayii), Ofloxacin (Zhejiang
Huangyan East Asia Chemical Co.), Fluconazole (Nobel),
Amphotericin B (Bristol Myers Squibb), Ethanol (Riedel de
Haen), Dimethylsulphoxide (DMSO) (Riedel de Haen).
1
FT/IR-420 spectrometer as KBr discs. H NMR spectra were
obtained with a Varian 400 MHz spectrometer in chloro-
form-d (CDCl3) or dimethylsulfoxide-d6 (DMSO-d6) and tet-
ramethylsilane (TMS) was used as an internal standard.
Mass analyses were carried out with a Waters Micromass
ZQ by using ESIþ method. Elemental analysis were performed
on LECO 932 CHNS (Leco 932, St. Joseph, MI, USA) instru-
ment and were within ꢁ0.4% of the theoretical values. All
chemicals and solvents were purchased from Aldrich Chemi-
cal Co. or Fischer Scientific.
4.3.2. Microorganisms
4.3.2.1. Isolates. K. pneumoniae isolate [has Extended Spec-
trum b Lactamase (ESBL) enzyme], P. aeruginosa isolate (re-
sistant to gentamicin), E. coli isolate [has Extended Spectrum
b Lactamase (ESBL) enzyme], B. subtilis isolate (resistant to
ceftriaxon), S. aureus isolate [resistant to meticillen
(MRSA)] and C. albicans isolate (biofilm positive).
4.1. General procedure for the compounds 1
5-Amino-2-[p-substitutedphenyl/benzyl]benzoxazoles and
5-amino-2-[o-bromophenyl]benzoxazole were synthesized by
heating 0.01 mol 2,4-diaminophenol$2HCl with 0.01 mol p-
substitutedphenyl acetic acid and/or p-substitutedbenzoic
acid and/or o-bromobenzoic acid in 12.5 g polyphosphoric
acid (PPA) and stirring for 1.5e2.5 h. At the end of the reac-
tion period, the residue was poured into iceewater mixture
and neutralized with excess of 10 M NaOH solution extracted
with benzene and then this solution was dried over anhydrous
sodium sulfate and evaporated under diminished pressure. The
residue was boiled with 200 mg charcoal in ethanol and fil-
tered. After the evaporation of solvent in vacuo, the crude
product was obtained and recrystallized from ethanol [20].
4.3.2.2. Standard strains. K. pneumoniae RSHM 574 (Refik
Saydam Hygiene Center Culture Collection), P. aeruginosa
ATCC 25853 (American Type Culture Collection), E. coli
ATCC 25922, B. subtilis ATCC 6633, S. aureus ATCC
25923, C. albicans ATCC 10231, C. krusei ATCC 6258.
4.3.3. Method
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 Kirby Bauer Disk
Diffusion method according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI) in the clinical isolates
[41].
Standard powders of rifampicin, ampicillin trihydrate, gen-
tamicin sulfate, 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 (gentamicin sulfate, fluconazole,
and amphotericin B).
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 newly synthesized compounds
and standard drugs were prepared at 1000, 500, 250, 125,
62.5, 31.25, 15.63, 7.8, 3.9, 1.95, 0.98, 0.48, 0.24, 0.12,
0.06 mg/ml concentrations in the wells of microplates by
4.2. General procedure for the compounds 2 and 3e12
Appropriate carboxylic acid (0.005 mol) and thionyl chlo-
ride (1.5 ml) were refluxed in benzene (5 ml) at 80 ꢂC for
3 h. Excess thionyl chloride was removed in vacuo for the
compound 2. The residue was dissolved in ether (10 ml) and
this solution was added during 1 h to a stirred ice-cold mixture
of 5-amino-2-[p-substitutedphenyl/benzyl]benzoxazoles and
5-amino-2-[o-bromophenyl]benzoxazole 1 (0.005 mol), so-
dium bicarbonate (0.005 mol), diethyl ether (10 ml) and water
(10 ml). The mixture was kept stirred overnight at room tem-
perature and filtered. The precipitate was washed with water,
2 N HCl and water, respectively, and finally with ether to
give 3e12. The products were recrystallized from ethanole
water as needles which are dried in vacuo [20]. The chemical,
physical and spectral data of the compounds 3e12 are re-
ported in Table 1. The result of the C-13 spectra of compound
8 is obtained as: 35.543, 110.770, 111.722, 112.027, 112.088,
118.146, 123.091, 124.143, 127.481, 127.603, 127.915,

2576
S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 43 (2008) 2568e2578
diluting in the liquid media. Bacterial susceptibility testing
was performed according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI) M100-S16 [42]. 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 concen-
tration were inoculated to the two-fold diluted solution of the
compounds. There were 104 cfu/ml bacteria in the wells after
inoculations. MHB was used for diluting the bacterial suspen-
sion and for two-fold dilution of the compound. DMSO (80%)
and EtOH (20%), methanol, DMSO, PBS, pure microorgan-
isms, and pure media were used as control wells. A 10 ml bac-
teria 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 or-
ganisms were tested in triplicate in each run of the experi-
ments. The lowest concentration of the compound that
completely inhibits macroscopic growth was determined and
minimum inhibitory concentrations (MICs) were reported.
All Candida isolates were subcultured in SDA plates, incu-
bated at 35 ꢂC for 24e48 h prior to antifungal susceptibility
testing, and passaged at least twice to ensure purity and viabil-
ity. Susceptibility testing was performed in RPMI-1640 me-
dium with L-glutamine buffered, pH 7, with MOPS and
culture suspensions were prepared through the guideline of
CLSI M27-A [43]. The yeast suspensions used for inoculation
were prepared at 104 cfu/ml by diluting fresh cultures at Mac-
Farland 0.5 density (106 cfu/ml). Suspensions of the yeast at
104 cfu/ml concentration were inoculated to the two-fold di-
luted solution of the compounds. There were 103 cfu/ml yeasts
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 end-
points 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.
A pharmacophoric hypothesis was then generated from these
aligned structures.
HipHop provides feature-based alignment of a collection of
compounds without considering the activity. It matches the
chemical features of a molecule, against drug candidate mol-
ecules. HipHop takes a collection of conformational models
of molecules and a selection of chemical features, and pro-
duces a series of molecular alignments in a variety of standard
file formats. HipHop begins by identifying configurations of
features common to a set of molecules. A configuration con-
sists of a set of relative locations in 3D space and associated
feature types. A molecule matches the configurations if it pos-
sesses conformations and structural features that can be super-
imposed within a certain tolerance from the corresponding
ideal locations. HipHop also maps partial features of mole-
cules in the alignment set. This provision gives the option to
use partial mapping during the alignment. Partial mapping al-
lows to identify larger, more diverse, more significant hypoth-
eses and alignment models without the risk of missing
compounds that do not have to map to all of the pharmaco-
phore features.
In this research, HipHop common feature hypotheses were
generated to explain the specification of the antibacterial
agents against drug-resistant P. aureuginosa, which are the
best significant results among the screened microorganisms.
This tool builds hypotheses (overlays common features) for
which the fit of individual molecules to a hypothesis can be
correlated with activity of the molecule. A set of six active
compounds from Table 2 was selected as the target training
set (Table 3). Among the selected six molecules, the most ac-
tive molecule, compound 9, was chosen as the reference,
which should allow to map all features on the generated hy-
potheses for the antibacterial activity against drug-resistant
P. aureuginosa.
The geometry of each compound was built with a visualizer
and optimized by using the generalized CHARMm-like force
Table 3
Selected active compounds and characteristics for the common feature hypoth-
esis run
4.4. Common features pharmacophore
hypotheses generation
Compounds
Confsa
Features/confsa
Principalb
MaxOmitFeatc
3
4
5
6
8
9
a
32
27
36
13
49
71
9.75
9.85
9.83
9.85
9.82
9.41
1
1
1
1
1
2
2
2
2
2
2
0
All computational experiments were conducted on a Silicon
Graphics O2, running under the IRIX 6.5 operating system.
Hypotheses generation was applied against previously de-
scribed data sets by using Catalyst/HipHop (version 4.9)
from Accelrys [44]. Molecules were edited using the Catalyst
2D/3D visualizer. Catalyst automatically generated conforma-
tional models for each compound using the Poling Algorithm
[45e47]. The ‘‘best conformer generation’’ procedure was ap-
plied to provide the best conformational coverage for a maxi-
mum number of conformers generated defaulted to 250 in
a 0e25 kcal/mol range from the global minimum. The confor-
mations generated were used to align common molecular fea-
tures and generate pharmacophore hypothesis. HipHop is used
to the conformations generated to align chemically important
functional groups common to the molecules in the study set.
Confs, number of conformers; Features/confs, total number of features di-
vided by the number of conformers (summed over the entire family of
conformers).
b
Principal ¼ 1 means that this molecule must map onto the hypothese gen-
erated by the search procedure. Partial mapping is allowed. Principal ¼ 2
means that this is a reference compound. The chemical feature space of the
conformers of such a compound is used to define the initial set of potential
hypotheses.
c
The MaxOmitFeat column specifies how many hypothesis features must
map to the chemical features in each compound. A 0 in this column forces
mapping of all features, a 2 allows hypotheses to which no compound features
map.

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 43 (2008) 2568e2578
2577
Table 4
Results of the common feature hypothesis run
Hypotheses
Featurea
Rank score
Direct hitb
Partial hitb
1
2
3
4
5
6
7
8
9
10
a
HpAr HpAr HpAr HBA HBA HBA
HpAr HpAr HpAr HBA HBA HBA
HpAr HpAr HpAr HBA HBA HBA
HpAr HpAr HpAr HBA HBA HBA
HpAr HpAr HBD HBA HBA
HpAr HpAr HBD HBA HBA
HpAr HpAr HpAr HBA HBA
HpAr HpAr HBA HBA HBA
HpAr HpAr HBD HBA HBA
HpAr HpAr HBD HBA HBA
102.164
102.028
100.904
98.9037
91.3093
90.4743
88.8775
88.4734
87.7719
87.2921
111111
111111
111111
111111
111111
111111
111111
111111
111111
111111
000000
000000
000000
000000
000000
000000
000000
000000
000000
000000
HpAr, Hydrophobic aromatic; HBA, Hydrogen-bond acceptor; HBD, Hydrogen-bond donor.
Direct hit, all the features of the hypothesis are mapped. Direct hit ¼ 1 means yes; partial hit, partial mapping of the hypothesis. Partial hit ¼ 0 means no. Each
b
number refers to a molecule in Table 5 (same order).
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This work was supported by Ankara University Research
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