1
722 Alotaibi
Asian J. Chem.
(
(
1H, d, J = 5.4 Hz, H-11), 6.80-8.11 (11H, m, Ar-H), 8.39
1H, s, CH), 8.54 (1H, s, CH); MS m/z (%) 400 (M ). Anal.
Treatment of staph strains with compounds 4 and 7 on
DNA fragmentation and DNA cleavage:A purified colonies
of S. aureus strains were grown in trypticase soya broth for 24 h
at 37 ºC. For DNA cleavage assay, bacterial colonies were
incubated with either compounds 4 and 7 at a dose of 100 mg/
mL DMSO for 4, 8, 24, 48 and 72 h. Bacterial broth was preci-
pitated after centrifugation at 10000 rpm for 10 min. The pellets
of colonies (200 colonies) were suspended in 500 µL DEPC
water and heated at 100 ºC in vortex well for 2 min. Supernatant
clear fluid was taken and a double volume of ice cold absolute
ethanol were added and incubated for overnight at -20 ºC.
Contents were centrifuged at 12000 rpm for 15 min and then
discard supernatant. A DNA pellet were washed with 70 %
ethanol, dried in air and then dissolved in double distilled water.
A DNA concentration was measured in BIORAD spectrophoto-
meter at O.D. 260 nm. Then 250 ng of extracted and purified
DNAwas loaded in 1.2 agarose gel stained with ethidium bromide
and photocopied using gel documentation system (Bio-Rad,
Co., USA).
Computational study: Target compounds were built and
minimized their energy with PM3 through MOPAC then DFT
using B3LYP/6-311G.All the quantum chemical computations
were performed using the PM3 semi-empirical Hamiltonian
molecular orbital calculation MOPAC16 package [20], then
employed DFT in Gaussian 09 W program package [21] with
Becke3-Lee-Yang-parr (B3LYP) level using 6-311G* basis as
implemented in MOE 2015 package [22].
+
calcd. (found) % for C23
3.95); N, 7.00 (6.88).
-(Furan-2-methylene)amino)-3-(3-(thiophene-2-
yl)acryloyl)quinolone-2(1H)-one (9d): Brown powder in 88
yield, m.p.: 276-278 ºC; R : 0.45 (3 % methanol:methylene
chloride). IR spectra (KBr, νmax, cm ): 3075 (Ar-H), 2880 (CH
H
16
2 3
N O S: C, 68.98 (69.00); H, 4.03
(
1
%
f
–1
1
aliphatic), 1700 (CO), 1710 (CO); H NMR (DMSO-d
7
1
6
): δ ppm:
.01 (1H, d, J = 5.4 Hz, H-10), 7.62 (1H, d, J = 5.4 Hz, H-
1), 6.50-8.16 (10H, m, Ar-H), 8.11 (1H, s, CH), 8.37 (1H, s,
+
CH); MS m/z (%) 375 (M +H). Anal. calcd. (found) % for
S: C, 67.37 (67.49); H, 3.77 (3.85); N, 7.48 (7.21).
-(Pyridin-3-methylene)amino)-3(3-(thiophene-2-
yl)acryloyl)quinolone-2(1H)-one (9e): Brown powder in 85 %
yield, m.p.: 296-298 ºC; R : 0.55 (3 % methanol:methylene
C
21
H
14
N O
2 3
1
f
–1
chloride). IR (KBr, νmax, cm ): 3070 (Ar-H), 2870 (CH aliphatic),
1
1
(
7
695 (CO), 1705 (CO); H NMR (DMSO-d
1H, d, J = 5.4 Hz, H-10), 7.68 (1H, d, J = 5.4 Hz, H-11),
.37-9.00 (11H, m, Ar-H), 8.22 (1H, s, CH), 8.42 (1H, s, CH);
6
): δ (ppm): 7.00
+
MS m/z (%) 385 (M ). Anal. calcd. (found) for C22
C, 68.55 (68.63); H, 3.92 (3.77); N, 10.90 (10.83).
H
15
N
3
O S:
2
Bacterial isolates: Microorganisms (Staphylococcus aureus,
Escherichia coli and pseudomonas aeruginosa) used in this
study were obtained from King Faisal Hospital after receiving
the ethical approval from Taif Directorate of Health Affairs,
Taif, Saudi Arabia.
Antimicrobial assay: The antibacterial activity of five
synthesized compounds (9c, 6a, 6b, 4 and 7) were carried out
using agar well diffusion test with minor modifications [18].
Agar poured into sterile petri-dishes and allowed to harden,
then 1 mL of each tested bacterial isolates inoculum equal to
Selection of proteins structure: Docking experiment was
carried out for the target active site into DNA Gyrase B (ID: 2EX6)
using MOE 2015 . The errors of active sites were corrected by
the structure preparation process in MOE. After correction,
hydrogens were added and then partial charges were calculated.
Energy minimization (AMBER12:EHT, root mean square
gradient: 0.100) was also performed.
Binding site analysis: The binding site of each receptors
were identified through the MOE Site Finder program, which
uses a geometric approach to calculate putative binding sites
in a protein, starting from its tridimensional structure. This
method is based on alpha spheres, which are generalization of
convex hulls. The prediction of binding sites, performed by
the MOE Site Finder module, confirmed the binding sites defined
by co-crystallized ligands in the holo-forms of the investigated
proteins.
0
.5 McFarland standard was inoculated into the Mueller agar
plates. A 30 µL of stock concentration (100 mg/mL) diluted
in DMSO from each synthesized compounds was added into
the agar wells. The plates were incubated at 37 ºC for 24 h.
Positive and negative control discs for each bacterial isolate
including ciprofloxacin (5 µg/mL) and amoxicillin (10 µg/mL)
for Escherichia coli and Pseudomonas auroginosa; and amoxi-
cillin (10 µg/mL) and vancomycin (10 µg/mL) for S. aureus
were prepared.
Determination of minimum inhibitory concentration
(
MIC): The MIC values were determined using broth micro-
diltion test in 96 well microtiter plate with triplicates and with
minor modifications [19]. A 100 µL of DMSO was added to
the wells and 100 µL of stock concentration (100 mg/mL
DMSO) of each synthesized compound was added and thus
double fold serial dilution was achieved followed by a addition
of 100 µL of sterile double strength Mueller-Hinton broth was
to each well. Enrichment of all bacterial isolates were done on
Mueller Hinton broth and incubated over night at 37 ºC with
agitation.A 100 µL of 0.5 McFarland standards of each inoculum
was added to all tested wells incubated and finally the plates
were again incubated at 37 ºC for 24 h. Turbidity was measured
at 600 nm using microtiter plate reader. A negative control
RESULTS AND DISCUSSION
In this work, salicylaldehyde (1) was reacted with ethylaceto-
acetate (2) in the presence of pyridine as base under reflux to
give 3-acetyl coumarin (3) in 95% yield. Structural analyses
1
were done with H NMR showed that a singlet at 1.98 ppm for
COCH and multiplet at 7.33-7.91 ppm for CH aromatic. The
3
mass analysis showed that m/z % 188 (M+). 3-Acetyl coumarin
(3) was reacted with hydrazine hydrate under reflux to give 3-
acetyl-1-aminoquinolin-2(1H)-one (4) in 80% yield. Structural
-1
analyses were done with FT-IR, showed that peak at 3442 cm
-1
1
for NH
showed that a singlet at 2.10 ppm for COCH
ppm for NH , a multiplet at 6.80-7.29 ppm for aromatic system
2
, peak at 1720 cm for COCH
3
and done with H NMR
(
blank) was taken as Mueller Hinton broth without any agents
3
, abroad at 2.65
and positive control as medium with agents.
2
Alotaibi, Saad H.
