Synthesis, Molecular Docking, and Biofilm Formation Inhibitory Activity
3,4-Dibromo-5-((3-nitrophenyl)methylene)-furan-
2(5H)-one (3g, dr = 57:43)
pounds were measured. It was performed on planktonic cultures
using the twofold dilution method according to clinical and labora-
tory standardsa. MICs were performed in 96-well sterile microplates
(cosmo), and the results were recorded after 18 h of incubation at
37 ꢀC.
Yield 60%. MS (ESI) [M)H]+ m ⁄ z 374; H NMR (500 MHz, CDCl3) d:
8.54(t, J1 = 20.0 Hz, J2 = 15.0 Hz, 0.57H), 8.17–8.34 (m, 1.71), 7.72
(dd, J1 = 60.0 Hz, J2 = 15.0 Hz, 0.43H), 7.57–7.68 (m, 1.29H), 7.03(s,
0.43H), 6.50 (s, 0.57H). 13C NMR (125 MHz, CDCl3) d: 162.0, 146.3,
136.2, 134.9, 131.5, 129.1, 128.3, 124.3, 123.2, 119.8, 113.9, 109.8.
High-resolution mass spectra (ESI) calcd for C11H4Br2NO4 373.84927
(M)H)), found 373.84989.
1
In vitro determination of biofilm formation
All the synthetic compounds as mentioned earlier were assayed for
their in vitro bacterial biofilm formation inhibitory activities against
P. aeruginosa ATCC 27853, ATCC 9027, and PAOA. Crystal violet
staining method was used. Pseudomonas aeruginosa was diluted
with LB broth at OD600 = 0.05. A concentrated compound solution
was transferred into each well, except those used as controls, to
achieve a final test concentration of 64 lg ⁄ mL, and each well was
filled to a final volume of 200 lL. After 20 h of incubation at
35 ꢀC and being washed twice with phosphate-buffered saline
(PBS) solution, the biofilm remained on the wells was fixed with
200 lL of 99% methanol for 15 min. The solution was discarded
and the microplate was put in super-clean bench. When the wells
were dried, each of them was added with 250 lL of 1% solution
of crystal violet (CV). After 15 min staining at room temperature,
the wells were washed twice carefully with distilled solution. Then,
250 lL of 95% ethanol was added to each well, as to dissolve the
stain and biofilm. Fifteen minutes later, the absorbance of plates
was determined at 570 nm in a spectrophotometer. The percentage
of biofilm inhibition was calculated using the following formula:
3,4-Dibromo-5-((4-nitrophenyl)methylene)-furan-
2(5H)-one (3h, dr = 82:18)
Yield 61%. 1H NMR (500 MHz, CDCl3) d: 8.26(m, 2H), 7.96(dd,
J1 = 7 Hz, J2 = 0.5 Hz, 1H), 7.58(dd, J1 = 7 Hz, J2 = 0.5 Hz, 1H),
7.02(s, 1H); 13C NMR (125 MHz, CDCl3) d: 161.9, 147.9, 147.1,
137.6, 131.4, 131.2, 124.1, 123.4, 120.9, 115.4, 110.8. High-resolu-
tion mass spectra (ESI) calcd for C11H4Br2NO4 373.84927 (M)H)),
found 373.84925.
Procedure for the synthesis of compound 3i
To
a solution of 3, 4-dibromo-2(5H)-furanone (1b) (0.173 g,
0.5 mmol) in anhydrous CH3OH (7 mL) was added piperidine
(0.009 g, 0.1 mmol) and 4-hydroxybenzaldehyde (0.073 g, 0.6 mmol).
The mixture was refluxed for 1.5 h under nitrogen, and then the
mixture was concentrated in vacuo. The residue was exacted with
CH2Cl2 (20 mL · 3), washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by column chroma-
tography on silica gel using ethyl acetate ⁄ petroleum ether (1:15) as
the eluent to give compound 3i (0.05 g, yield: 29%) as a yellow
solid.
inhibition percentage = [(ODnegative control)ODx) ⁄ ODnegative
con-
trol] · 100, where x refers to the tested halogenated compounds
(23–28).
To find out the effect of concentration on the QS system, some
of the synthesized compounds were chosen to determine the bio-
film inhibition percentages at concentrations of MIC, 1 ⁄ 2MIC,
1 ⁄ 4MIC, 1 ⁄ 8MIC, and 1 ⁄ 16MIC using the same method described
earlier.
3,4-Dibromo-5-((4-hydrophenyl)methylene)-
furan-2(5H)-one (3i)
Yield 29%. 1H NMR(400 MHz, CDCl3) d: 7.69(d, J = 8.7 Hz, 1H),
6.84(d, J = 8.7 Hz, 1H), 3.30(s, 1H); 13C NMR (125 MHz, CDCl3) d:
162.1, 159.2, 142.1, 140.9, 133.2, 132.5, 122.9, 116.2, 116.1, 112.9.
High-resolution mass spectra (ESI) calcd for C11H7Br2O3 346.8736
(M + H)+, found 346.8748.
Observation of Pseudomonas aeruginosa
biofilm formation with scanning electron
microscopy (SEM)
The P. aeruginosa inoculums were centrifuged (11 100g, 5 min) and
washed twice with saline to acquire planktonic cultures. It was
diluted with sterile saline at 0.5 MCF (Mcfarland standard), and
then diluted 25-fold with sterile saline. One milliliter of the pre-
pared cultures was added to each test tube, as well as 1 mL com-
pound solution (dissolved by LB broth, 128 lg ⁄ mL). The final
concentration of compounds was 64 lg ⁄ mL. After the polyvinyl
chloride catheter (0.5 cm · 0.5 cm) was set on the bottom of each
tube, cultures were incubated in shaking bath at 37 ꢀC for 7 days.
Following washing twice with PBS solution, the bacteria on the
catheter was fixed by placing in 2.5% glutaraldehyde ⁄ cacodylate
(v ⁄ v) buffer for 5 h. The samples were then dried with ethanol
steps – 50% aqueous ethanol (v ⁄ v) for 10 min, 75% aqueous etha-
nol (v ⁄ v) for 10 min, 85% aqueous ethanol (v ⁄ v) for 10 min, 95%
aqueous ethanol (v ⁄ v) for 10 min, 100% aqueous ethanol (v ⁄ v) for
10 min, and isoamyl acetate for 10 min. They were frozen in a free-
zer at )65 ꢀC and dried at the critical point of vacuum pressure at
Biological evaluation
Bacterial strains
Pseudomonas aeruginosa strains ATCC 27853, ATCC 9027, and
PAOA (clinical isolates) were used to study the effects of new com-
pounds to biofilm assay. A bacterial suspension made from fresh
culture and aliquots were stored at )20 ꢀC in glycerol and used
within 2 weeks. Before used, bacterial suspensions were spread
onto Mueller-Hinton solid medium and incubated at 37 ꢀC for 18 h.
MIC determination
To define the concentration of the biofilm inhibitory test under MIC
and make sure the effects of new compounds were via QS inhibi-
tion not inhibiting the bacteria themselves. The MICs of all com-
Chem Biol Drug Des 2012; 79: 628–638
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