T. Ertan et al. / Bioorg. Med. Chem. 15 (2007) 2032–2044
2043
(5 mmol) were treated with NiCl2Æ6H2O (15 mmol) and
Zn (40 mmol) in methanol (25 ml) refluxing the mixture
at 60 °C for 4 h. The precipitate was filtered. The crude
product was purified by recrystallization from methanol.
The crystals were dried in vacuo (Scheme 2).
All bacterial isolates were subcultured in MHA plates
and incubated overnight at 37 °C and all Candida iso-
lates were subcultured in SDA plates at 35 °C for 24–
48 h. The microorganisms were passaged at least twice
to ensure purity and viability.
Compounds 1a, 1b, 1e–1g, 1k, 1m (5 mmol) in ethanol
(50 ml) were reduced by hydrogenation using 40 psi of
H2 and 10% Pd–C (40 mg) until cessation of H2 uptake
to obtain compounds 2a, 2b, 2e–2g, 2k, 2m, 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 eth-
anol. The crystals were dried in vacuo.
The solution of the newly synthesized compounds (1a–1n,
2a–2n) and standard drugs was prepared at 1000, 500,
250, 125, 62.5, 31.25, 15.625, 7.8, 3.9, 1.95, 0.98 lg/ml
concentrations, at 4096, 2048, 1024, 512, 256, 128, 64,
32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 lg/ml concentra-
tions 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-S16.21 The bacterial suspensions
used for inoculation were prepared at 105 cfu/ml by dilut-
ing fresh cultures at MacFarland 0.5 density (107 cfu/ml).
Suspensions of the bacteria at 105 cfu/ml concentration
were inoculated to the 2-fold diluted solution of the com-
pounds. There were 104 cfu/ml bacteria in the wells after
inoculations. MHB was used for diluting the bacterial sus-
pension and for 2-fold dilution of the compound. 80%
DMSO, 20% EtOH, methanol, DMSO, PBS, pure micro-
organisms, and pure media were used as control wells. A
10 ll bacteria inoculum was added to each well of the mic-
rodilution trays. The trays were incubated 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 concentration
of the compound that completely inhibits macroscopic
growth was determined and minimum inhibitory concen-
trations (MICs) were reported.
4.3. Microbiology
4.3.1. Materials. Mueller–Hinton Agar (MHA) (Merck),
Mueller–Hinton Broth (MHB) (Merck), Sabouraud Dex-
trose Agar (SDA) (Merck), RPMI-1640 medium with L-
glutamine (Sigma), 3-[N-morpholino]-propansulfonic
acid (MOPS) (Sigma), 96-well microplates (Fal-
conÒ),Transfer pipette (Biohit) Rifampicin (Sifar Ilac
Sanayii), Ampicillin trihydrate (Paninkret Chem.
Pharm.), Gentamycin sulfate (Deva Ilac Sanayii), Oflox-
acin (Zhejiang Huangyan East Asia Chemical CO.),
Fluconazole (Nobel), Amphotericin B (Bristol Myers
Squibb), Ethanol (Riedel de HaenÒ), Dimethylsulfoxide
(DMSO) (Riedel de HaenÒ), Dimethylformamide (Riedel
de HaenÒ).
Microorganisms. Klebsiella pneumoniae isolate (Resistant
to Trimethoprim sulfamethoxazole, Amoxicilin clavulo-
nat, Ceftriaxon, Cephepim, Aztreonam), Pseudomonas
aeruginosa isolate (Resistant to Amoxicilin clavulonat),
E.coli isolate (Resistant to Trimethoprim sulfamethoxa-
zole, Cephepim, Tazobactam), Bacillus subtilis isolate
(Resistant to Ceftriaxon), Staphylococcus aureus isolate
(Resistant to Oxacilin, Gentamycin, Aztreonam, Tri-
methoprim sulfamethoxazole), C. albicans isolate (Bio-
film positive), K. pneumoniae RSHM 574 (Refik Saydam
Hıfzısıhha Merkezi 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.
All Candida isolates were subcultured in SDA plates,
incubated at 35 °C for 24–48 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, pH 7,
with MOPS and culture suspensions were prepared
through the guideline of CLSI M27-A.22 The yeast sus-
pensions 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 con-
centration were inoculated to the 2-fold diluted solution
of the compounds. There were 103 cfu/ml bacteria in the
wells after inoculations. A 10 ll yeast inoculum was add-
ed to each well of the microdilution trays. The trays were
incubated at 35 °C 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) was
reported.
4.3.2. Method. Standard strains of K. pneumoniae RSHM
574, P. aeruginosa ATCC 25853, E. coli ATCC 25922, B.
subtilis ATCC 6633, S. aureus ATCC 25923, C. albicans
ATCC 10231, and clinical isolates of these microorgan-
isms that are known to be resistant to various antimicro-
bial 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)20 in the clinical isolates.
Standard powders of rifampicin, ampicillin trihydrate,
gentamycin sulfate, ofloxacin, fluconazole, and ampho-
tericin B were obtained from the manufacturers. Stock
solutions were dissolved in dimethylsulfoxide (ofloxa-
cin), methanol (rifampicin), pH 8 phosphate-buffered
saline (PBS) (ampicillin trihydrate), and distilled water
(gentamycin sulfate, fluconazole, and amphotericin B).
Acknowledgments
This work was supported by Ankara University Re-
search Fund (Grant No. 2005-0803049). The Central
Lab. of the Faculty of Pharmacy of Ankara University
Ertan, Tugba
