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373
2.4. Characterization data
molecular weight of 7326.84. All hydrogen atoms were added to
the structure with their standard geometry followed by their en-
ergy optimization tool using MOPAC 7.0. The resulting DNA model
was subjected to systematic conformational search at default pa-
rameters with RMS gradient of 0.01 kcal molꢁ1 using Site Finder. A
number of runs were carried out to get a final binding docking
pose as accurate as possible. The best conformation was selected
based on energetic ground and the minimum Final Docking En-
2.4.1. (E)-N0-(4-bromobenzylidene)isonicotinohydrazide (SF 1)
Light blue Solid (85%) m.p 142 ꢂC; Rf: 0.78; IR; (KBr, cmꢁ1): 3190
(NeH), 1610 (CN), 1585(Ar-C]C), 1095; 1H NMR (Acetone-
d6,300 MHz) d: 11.54 (bs NH), 8.45 (t, 2H) 7.57 (t, 2H); 7.55 (d, 2H,
J ¼ 7.5 Hz, ArH), 7.41 (d, 2H, J ¼ 7.5 Hz, ArH) 7.34 (s, 1H); 13C NMR
(75 MHz) d: 173(C]O),157(C]N), 144(Ar), 140 (Ar), 137 (Ar), 129
(Ar), 128(Ar), 123(Ar), GC-MS (EI, 70 eV): m/z (%): 303(21), 154 (65),
ergy (DG) [36,37].
106(100).
2.5.3. Potato disc antitumor assay
Agrobacterium tumefaciens strain (AT-10) was used in this assay
as inoculums with the final concentrations of 1000, 100, 10 mg/mL
2.4.2. (E)-N0-(4-methoxybenzylidene)isonicotinohydrazide (SF 2)
yellow Solid (88%) m.p 165 ꢂC; Rf: 0.73; IR; (KBr, cmꢁ1): 3245
(NeH), 1618 (CN), 1585(Ar-C]C), 1077; 1H NMR (Acetone-
of each test compound respectively [38]. The surface of red skinned
potato was sterilized with 0.1% HgCl2 and their cylinders were
prepared with the help of sterilized cork borer. These cylinders
were cut in to 5 ꢀ 8 mm discs and ten discs were placed on each
d6,300 MHz)
J ¼ 7.5 Hz, ArH), 7.49 (d, 2H, J ¼ 7.5 Hz, ArH) 7.38 (s,1H); 3.68 (s, 3H);
13C NMR (75 MHz)
: 189(C]O),169(C]N), 148(Ar), 144 (Ar), 139
d: 11.96 (bs NH), 8.65 (t, 2H) 7.58 (t, 2H); 7.53 (d, 2H,
d
(Ar), 137 (Ar), 131(Ar), 129(Ar), 59(0Me), GC-MS (EI, 70 eV): m/z (%):
255(16), 107(65), 106(100).
solidified agar plate. Then 50 mL of inoculums was added to the top
of each disc and each petriplate was wrapped with parafilm strips
to avoid contamination and loss of moisture during incubation
period. These petriplates were then incubated at 28 ꢂC. After 21
days number of tumors was counted after staining with the Lugol's
solution (10% KI and 5% I2). Vincristine sulphate and DMSO were
used as positive and negative control respectively. Each experiment
was repeated three times and IC50 values for each compound were
evaluated. Percentage inhibition was calculated by the following
formula;
2.4.3. (E)-N0-(2-hydroxybenzylidene)isonicotinohydrazide (SF 3)
yellow Solid (70%) m.p 238 ꢂC; Rf: 0.83; IR; (KBr, cmꢁ1): 3443
(OH), 3243 (NeH), 1615 (CN), 1588(Ar-C]C), 1074; 1H NMR
(Acetone-d6,300 MHz)
8.68 (s, 1H); 7.82e7.85 (t, 2H), 7.55e7.61 (d, 1H) 7.38 (s, 1H);
7.27e7.33 (t, 1H); 6.89e6.89 (m, 2H); 13C NMR (75 MHz)
: 179(C]
d: 12.27 (bs 1H), 11.08(bs 1H), 8.77 (t, 2H)
d
O),169(C]CeOH), 155(C]N), 145(Ar), 140 (Ar), 137 (Ar), 134 (Ar),
129(Ar), 124(Ar), 122(Ar), 120(Ar). GC-MS (EI, 70 eV): m/z (%):
241(26), 93(55), 106(100).
ꢀ
ꢁ
Number of tumors in test compound
Number of tumors in negative control
% inhibition ¼ 100 ꢁ
ꢀ 100
2.4.4. (E)-N0-(4-fluorobenzylidene)isonicotinohydrazide (SF 4)
yellow Solid (78%) m.p 145 ꢂC; Rf: 0.65; IR; (KBr, cmꢁ1): 3230
(NeH), 1600 (CN), 1575(Ar-C]C), 1075; 1H NMR (Acetone-
d6,300 MHz) d: 11.66 (bs NH), 8.55 (t, 2H) 7.62 (t, 2H); 7.57 (d, 2H,
J ¼ 7.5 Hz, ArH), 7.50 (d, 2H, J ¼ 7.5 Hz, ArH) 7.44 (s, 1H); 13C NMR
(75 MHz)
d
: 179(C]O),165(C]N), 146(Ar), 141 (Ar), 136 (Ar), 132
2.5.4. Antimicrobial assay
(Ar), 129(Ar), 127(Ar), GC-MS (EI, 70 eV): m/z (%): 243(16), 94 (65),
106(100).
Antibacterial and antifungal activities of the test compounds
were studied by employing disc diffusion method. Four bacterial
strains; 2 g positive Staphylococcus aureus (ATCC 6538) and
Micrococcus luteus (ATCC 10240) and 2 g negative Escherichia coli
(ATCC 15224) and Salmonella typhimurium(ATCC 14028) for anti-
bacterial while four fungal strains; Mucor species (FCBP 0300),
Aspergillusniger (FCBP 0198), Aspergillusfumigatus (FCBP 66) and
Fusariumsolani (FCBP 0291) were used in the this method [39,40].
In antibacterial assay, organisms were cultured in nutrient
broth at 37 ꢂC for 24 h. 106 colony-forming units (CFU/mL) of test
strain was added to nutrient agar medium at 45 ꢂC and poured
2.5. Procedure for analysis
2.5.1. UVevisible spectroscopic titrations
DNA concentration at 260 nm was evaluated 6.5 ꢀ 10ꢁ5 M.
Spectroscopic titrations were done at 37 ꢂC (human body temper-
ature). The concentration of each synthesized compound (SF 1 e SF
4) was optimized and prepared as 1.14 ꢀ 10ꢁ4 M. Absorbance
measurements were performed by keeping the concentration of
synthesized compounds (SF 1 e SF 4) constant (1.14 ꢀ 10ꢁ4 M) in
the sample cuvette, while varying the concentration of ds.DNA
into sterile petri plates. The medium was allowed to solidify. 5
mL
of each test compound with 200 g/mL final concentration were
m
from 10
m
M to 70
m
M. Before running the spectra for absorbance
poured on 4 mm sterile paper discs separately and placed on
nutrient ager plates. In each plate DMSO served as negative con-
trol and Kanamycin served as positive control. Plates were incu-
bated at 37 ꢂC for 24 h. The antibacterial activity was determined
by measuring the diameter of zones showing complete inhibition
(mm).
measurement, each solution was allowed to stay for few minutes so
that equilibrium could be achieved between compound and DNA
during complex formation. Sample solutions were further kept for
few seconds within the cell cavity to assure the required temper-
ature (37 ꢂC).
In antifungal assay, organisms were cultured on sabouraud
dextrose agar (SDA) at 28 ꢂC for 24 h. Autoclaved broth culture
(3 mL) was allowed to cool down to 45 ꢂC and poured into sterile
2.5.2. Molecular docking method
Ligand molecules (SF 1 e SF 4), were drawn and minimized on
MOE window using MOE builder and entered into MOE database.
The starting point of the docking simulation was the X-ray
structure of the DNA with PDB ID 1BNA obtained through the
protein data bank (PDB) and imported to MOE window. All water
molecules were removed from the complex with 12 base pairs
running in 50-30 direction. The base pair sequence was (50-
petri plates. 5
mL of each test compound with 200 mg/mL final
concentration were poured on 4 mm sterile paper discs separately
and placed on SDA plates. The discs supplemented with DMSO and
Terbinafine were used as negative and positive control, respec-
tively. Plates were incubated at 28 ꢂC for seven days and fungal
growth was determined by measuring growth diameter (mm) and
growth inhibition was calculated with reference to the controls.
D(CGCGAATTCGCG)-30):
(50-D(CGCGAATTCGCG)-30)
with