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1429
buffer saline (PBS) (ampicilin trihydrate) and distilled water
(gentamicin sulfate, fluconazole and amphotericin B).
All bacterial isolates were subcultured in MHA plates and
incubated over night at 37 ꢁC and all Candida isolates were
subcultured in SDA plates at 35 ꢁC for 24e48 h. The microor-
ganisms were passaged at least twice to ensure purity and
viability.
acids (2ae2t) with 4-ethylsulphonyl-2-aminophenol (1) in
PPA (polyphosphoric acid) as the cyclodehydration reagent
in a one step procedure [9]. All of these syntheses are shown
in Scheme 1.
All the compounds 3ae3t were prepared as new products
except 3a, 3g, 3h, 3k [1]. Their structures were supported
1
by spectral data. The IR, H, 13C NMR and Mass spectra
The solution of the newly synthesized compounds (3ae3t)
and standard drugs were prepared at 1000, 500, 250, 125, 62.5,
31.25, 15.625, 7.8, 3.9, 1.95, 0.98 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 mi-
croplates by diluting in MHB, respectively.
are in agreement with the proposed structures. Physical and
spectral data of the compounds are reported in Table 1.
All the synthesized derivatives (3ae3t) were assayed in vitro
for antibacterial activity against K. pneumoniae RSHM 574, P.
aeruginosa ATCC 25853, E. coli ATCC 25922, K. pneumoniae
isolate (resistant to trimethoprim sulfamethoxazole, amoxicillin
clavulonat, ceftriaxon, cephepim, aztreonam), E. coli isolate (re-
sistant to trimethoprim sulfamethoxazole, cephepim, tazobac-
tam) as Gram-negative bacteria, B. subtilis ATCC 6633,
S. aureus ATCC 25923, B. subtilis isolate (resistant to cef-
triaxon), S. aureus isolate (resistant to oxacilin, gentamicin, az-
treonam, trimethoprim sulfamethoxazole) as Gram-positive
bacteria and the antifungal activity was evaluated against
C. albicans ATCC 10231 and its isolate. The MIC values were
determined by twofold serial dilution technique in Mueller Hin-
ton Broth and Sabouraud Dextrose Agar for the antibacterial and
antifungal assay, respectively. For comparison of the antimicro-
bial activity, rifampicin, ampicillin trihydrate, gentamicin
sulphate, ofloxacin were used as the reference antibacterial
agents and fluconazole, amphotericin B were employed as the
reference antifungal agents. All the biological results of the
tested compounds are given in Table 2.
In this study, our goal was to investigate the role of efficient
substitution on the position 2 of 5-ethylsulphonyl-benzoxazole
ring for antimicrobial activity. Therefore, we put a methylene,
an ethylene groups as a bridge between benzoxazole moiety
and phenyl ring or did not place any component between them.
According to Table 2, the synthesized compounds showed
a broad spectrum of activity with MIC values 125e7.81 mg/ml
against some Gram-positive bacteria such as S. aureus, B. sub-
tilis and their isolates. The compounds 3ae3t displayed lower
antibacterial activity against S. aureus and its isolate than the
compared control drugs, ampicillin trihydrate, and ofloxacin.
It can be considered that structurally differences on the substi-
tution at position 2 of benzoxazole nucleus did not change
their activity against S. aureus and its isolate.
Bacterial susceptibility testing was performed according to
the guidelines of Clinical and Laboratory Standards Institute
(CLSI) M100-S16 [25]. The bacterial suspensions used for in-
oculation were prepared at 105 cfu/ml by diluting fresh cul-
tures 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 di-
lution of the compound. 80% DMSO, 20% EtOH, methanol,
DMSO, PBS, pure microorganisms and pure media were
used as control wells. A 10 ml bacteria inoculum was added
to each well of the microdilution trays. The trays were incu-
bated at 37 ꢁC 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 concen-
tration of the compound that completely inhibits macroscopic
growth was determined and minimum inhibitory concentra-
tions (MICs) were reported.
All Candida isolates were subcultured in SDA plates, and
incubated at 35 ꢁC for 24e48 h prior to antifungal susceptibil-
ity testing, and passaged at least twice to ensure purity and vi-
ability. Susceptibility testing was performed in RPMI-1640
medium with L-glutamine buffered pH 7 with MOPS and cul-
ture suspensions were prepared through the guideline of CLSI
M27-A [26]. The yeast suspensions used for inoculation were
prepared at 104 cfu/ml by diluting fresh cultures at MacFar-
land 0.5 density (106 cfu/ml). Suspensions of the yeast at
104 cfu/ml concentration were inoculated to the twofold di-
luted solution of the compounds. There were 103 cfu/ml bacte-
ria 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 ꢁC 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 con-
centration of the compound that completely inhibits macro-
scopic growth was determined and minimum inhibitory
concentrations (MICs) were reported in Table 2.
Although derivatives 3d and 3e showed only significant ac-
tivity with MIC values of 7.81 mg/ml but less active than tested
standard drug ampicillin trihydrate against B. subtilis. They in-
dicated two- or onefold less potency against drug-resistant
B. subtilis, respectively. On the other hand, compounds 3e, 3h,
3o and 3r possessed the highest activity with MIC values of
15.625 mg/ml. Structure-activity relationships revealed that
compounds possessing a substituent such as eCl, et-C4H9 of
para position of phenyl improved the potency against B. subtilis.
All of the new compounds 3ae3t showed a broad antibac-
terial activity against some E. coli, K. pneumoniae, P. aerugi-
nosa as Gram-negative bacteria possessing MIC values
between 125 and 31.25 mg/ml and they showed with MIC
values 250e31.25 mg/ml against their isolates. All compounds
4. Results and discussion
The synthesis of the 5-ethylsulphonyl-2-(substituted-phenyl/
substituted-benzyl and/or 2-phenylethyl)benzoxazole deriva-
tives (3ae3t) was obtained by heating appropriate carboxylic
Temiz-Arpaci, Oezlem
