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179
diaminophenol·2HCl with 0.01 mol p-substituted ben-
zoic acid in 24 g polyphosphoric acid and stirring for
2.5 h. At the end of the reaction period, the residue was
poured into ice-water mixture and neutralized with
excess of % 10 NaOH solution extracted with benzene;
the benzene solution was dried over anhydrous sodium
sulfate and evaporated under diminished pressure. The
residue was boiled with 200 mg charcoal in ethanol and
filtered. After the evaporation of the solvent in vacuo,
the crude product was obtained and recrystallized.
Ampicillin, amoxycillin, tetracycline, streptomycin,
ketoconazole and fluconazole were used as control
drugs. The observed data on the antimicrobial activity
of the compounds and the control drugs are given in
Table 2.
3.5. Antibacterial and antifungal assay
The cultures were obtained from Mueller–Hinton
broth (Difco) for all the bacterial strains after 24 h of
incubation at 3791 °C. The yeast C. albicans was
maintained in Sabouraud dextrose broth (Difco) after
incubation for 24 h at 2591 °C. Testing was carried
out in Mueller–Hinton broth and Sabouraud dextrose
broth (Difco) at pH 7.4 and the two-fold serial dilution
technique was applied. The final inoculum size was 105
CFU/ml for the antibacterial assay and 104 CFU/ml for
the antifungal assay. A set of tubes containing only
inoculated broth was kept as controls. For the antibac-
terial assay after incubation for 24 h at 3791 °C and
after incubation for 48 h at 2591 °C for the antifungal
assay, the last tube with no growth of microorganism
and/or yeast was recorded to represent the MIC ex-
pressed in mg/ml. Every experiment in the antibacterial
and antifungal assays was replicated twice in order to
define the MIC values.
3.3. General procedure for amide deri6ati6es (5–22)
Appropriate carboxylic acid (0.5 mmol) and thionyl
chloride (1.5 ml) were refluxed in benzene (5 ml) at 80
°C for 3 h. Excess thionyl chloride was then removed in
vacuo. The residue was dissolved in ether (10 ml) and
solution added during 1 h to a stirred, ice-cold mixture
of 5-amino-2-(p-substituted-phenyl)benzoxazoles (0.5
mmol), sodium bicarbonate (0.5 mmol), diethyl ether
(10 ml) and water (10 ml). The mixture was kept stirred
overnight at room temperature and filtered. The precipi-
tate was washed with water, 2 N HCl and water,
respectively and finally with ether to give 5–22. The
products were recrystallized from ethanol–water mix-
ture and needles are dried in vacuo. The chemical,
physical and spectral data of the compounds 5–22 are
reported in Table 1.
4. Results and discussion
3.4. Microbiology
The synthesized compounds 5–22 exhibited in vitro
antimicrobial activity showing MIC values between 100
and 12.5 mg/ml and their potencies were compared to
some control drugs as given in Table 2.
For both the antibacterial and antimycotic assays,
the compounds were dissolved in absolute ethanol (0.8
mg/ml). Further dilutions of the compounds and stan-
dard drugs in the test medium were prepared at the
required quantities of 400, 200, 100, 50, 25, 12.5, 6.25,
3.12, 1.56, and 0.78 mg/ml concentrations with
Mueller–Hinton broth and Sabouraud dextrose broth.
The minimum inhibitory concentrations (MIC), were
determined using the method of two-fold serial dilution
technique [11,16,17]. In order to ensure that the solvent
per se had no effect on bacterial growth, a control test
was also performed containing inoculated broth supple-
mented with only ethanol at the same dilutions used in
our experiments and found inactive in culture medium.
All the compounds were tested for their in vitro growth
inhibitory activity against different bacteria and the
yeast C. albicans RSKK 628. Origin of bacterial strains
are Staphylococcus aureus ATCC 6538, Streptococcus
faecalis ATCC 10541 and Bacillus subtilis ATCC 6033
as Gram-positive and Escherichia coli ATCC 10536,
and P. aeruginosa RSKK 355 as Gram-negative bacte-
ria. RSKK strains of the microorganisms used in this
study were obtained from the culture collection of
Refik Saydam Health Institution of Health Ministry,
Ankara and maintained at the Microbiology Depart-
ment of Faculty of Pharmacy of Ankara University.
Table 2 shows that the synthesized compounds 5–22
exhibited antibacterial activity against S. aureus and S.
faecalis with MIC values generally between 100 and
12.5 mg/ml, 9 and 11 being the most active compounds
of the series. However, all the synthesized compounds
exhibited lower activity than the control drugs against
Gram-positive bacterial strains and E. coli. Compounds
6 and 9 were found more active the other tested com-
pounds against P. aeruginosa providing a MIC value of
12.5 mg/ml.
All the compounds showed antifungal potencies at a
MIC values of 50–12.5 mg/ml. The compounds 9, 10,
11, 15 and 21 were more found active than the other
compounds at a MIC value 12.5 mg/ml. However the
control drugs ketoconazole and fluconazole exhibited
better antifungal activity than any of the synthesized
compounds.
Qualitatively, the importance of the substitution pat-
tern of the 2-phenyl and of the 5-arylalkyl or 5-ary-
loxyalkyl groups both for the antifungal and
antibacterial activity is evident. The explored set, how-
ever, is too small to allow any significant structure–ac-
tivity relationship.