H. Vijay Kumar, N. Naik / European Journal of Medicinal Chemistry 45 (2010) 2–10
9
5.1.5. 1-(5H-dibenz[b,f]azepin-5-yl)-3-(2-hydroxy-5-nitrophenyl-
amino)propan-1-one (Compound 2d)
(4 mL) was transferred to different stopper test tubes containing
compound (50 and 100 M/mL) in distilled ethanol. Control was
m
Light yellow solid, Yield (61%), M.p. 145–147 ꢂC. IR (KBr) nmax
(cmꢀ1): 3047.5–2967.5 (Ar C–H), 1662.4 (C]O), 3313.4 (N–H),
3217.4–3510.2 (phenolic-OH), 2971.0–3026.1 (CH2); 1H NMR
prepared with distilled ethanol (1 mL) and emulsion (4 mL). BHA
and ascorbic acid solution as internal standards of the same
concentration were also analyzed for comparison. Zero adjustment
was done using distilled water. As soon as the emulsion was added
to each test tube, the zero time (t ¼ 0) absorbance was measured at
470 nm using spectrophotometer (Shimadzu 160A) and subse-
quently absorbance was measured for every 30 min up to 3 h
(t ¼ 180) time interval. The tubes were placed in a water bath at
50 ꢂC between the readings. Percentage antioxidant activities of
each compound were evaluated in triplicates in terms of photo-
(250 mHz) (CDCl3)
d (ppm): d 7.19–7.18 (m, 8H, Ar–H), 6.9 (s, 2H,
Seven membered Ar–H), 2.68 (t, 2H, CH2, C]O), 3.55 (t, 2H, CH2,
CH2N–H), 5.82 (s, 1H, N–H), 6.71–6.93 (m, 3H, Ar–H of amino-
phenol), 10.0 (s, 1H, OH); Mass (m/z %): Mþ 401.15 (100.0%), 402.16
(25.2%), 403.14 (4.1%), 402.13 (1.1%); Anal. Calcd. for C23H19N3O4: C,
68.82; H, 4.77; N, 10.47; O, 8.94%; Found: C,68.84; H, 4.75; N, 10.46;
O, 8.95%.
oxidation of b-carotene using the following formula:
5.1.6. 1-(5H-dibenz[b,f]azepin-5-yl)-3-(3-hydroxy-4-methoxy-
phenylamino)propan-1-one(Compound 2e)
Brownish solid, Yield (71%), M.p. 134–137 ꢂC. IR (KBr) nmax
(cmꢀ1): 3049.7–2834.4 (Ar C–H), 1662.1 (C]O), 3364.8 (N–H),
3196.4–3524.7 (phenolic-OH), 2971.0–3026.1 (CH2); 1H NMR
h
ꢀ
ꢁi
% Antioxidant activity ¼ 100 1 ꢀ Ao ꢀ At=Aoo ꢀ Aot
where,
Ao ¼ Initial absorbance of the sample. (t ¼ 0 min)
(250 mHz) (CDCl3)
d (ppm): d 7.19–7.18 (m, 8H, Ar–H), 6.9 (s, 2H,
At ¼ Absorbance of the sample after time‘t’. (t ¼ 180 min)
Aoo ¼ Initial absorbance of the control. (t ¼ 0 min)
Ato ¼ Absorbance of control after time‘t’. (t ¼ 180 min)
Seven membered Ar–H), 2.68 (t, 2H, CH2, C]O), 3.55 (t, 2H, CH2,
CH2N–H), 5.82 (s, 1H, N–H), 6.0–6.6 (m, 3H, Ar–H of aminophenol),
9.4 (s, 1H, OH), 3.8 (t, 3H, OCH3); Mass (m/z %): Mþ 386.36 (100.0%),
387.16 (26.3%), 388.16 (3.9%); Anal. Calcd. for C24H22N2O3: C, 74.59;
H, 5.74; N, 7.25; O, 12.42%; Found: C, 74.55; H, 5.73; N, 7.27; O, 12.4%
5.2.3. Reducing power assay (iron reducing activity)
The reducing power of synthesized compounds was determined
according to the method of Oyaizu [28]. The compounds having 50
and 100 mM were mixed with 2.5 mL of phosphate buffer (0.2 M, pH
5.2. Antioxidant activity
6.6) and 2.5 mL of 1% potassium ferric cyanide, and then incubated
at 50 ꢂC for 20 min. To this mixture 2.5 mL of 10% trichloroacetic
acid was added and the mixture was centrifuged at 3000 rpm for
20 min. The upper layer (2.5 mL) was mixed with 2.5 mL of
deionised water and 0.5 mL of 0.1% ferric chloride and the absor-
bance was measured at 700 nm using a spectrophotometer (Shi-
madzu 160A). Increases of absorbance of the reaction mixture
indicate higher reducing power. Mean values from three indepen-
dent samples were calculated for each compound and standard
deviations were less than 5%.
5.2.1. DPPH radical scavenging activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging
effect was carried out according to the method first employed by
Blois [26]. Compounds of different concentrations were prepared in
distilled ethanol, 1 mL of each compound solutions having different
concentrations (10, 25, 50, 100, 200 and 500 mM) were taken in
different test tubes, 4 mL of 0.1 mM ethanol solution of DPPH was
added and shaken vigorously. The tubes were then incubated in the
dark room at RT for 20 min. A DPPH blank was prepared without
compound, and ethanol was used for the baseline correction.
Changes (decrease) in the absorbance at 517 nm were measured
using a UV–visible spectrophotometer (Shimadzu 160A). The
radical scavenging activities were expressed as the inhibition
percentage and were calculated using the formula:
5.2.4. Human LDL oxidation
Fresh blood was obtained from fasting adult human volunteers
and plasma was immediately separated by centrifugation at
1500 rpm for 10 min at 4 ꢂC. LDL (0.1 mg LDL protein/mL) was
isolated from freshly separated plasma by preparative ultra
centrifugation using a Beckman L8-55 ultra centrifuge. The LDL was
prepared from the plasma according to the method of [29]. The
isolated LDL was extensively dialyzed against phosphate buffered
Radical scavenging activityð%Þ ¼ ½ðA0 ꢀ A1=A0Þ ꢃ 100ꢄ
where A0 is absorbance of the control (blank, without compound)
and A1 is absorbance of the compound. The radical scavenging
activity of BHA and ascorbic acid was also measured and compared
with that of the different synthesized compound. The compound
concentration providing 50% inhibition (IC50) was calculated from
the graph of RSA percentage against compound concentrations.
saline (PBS) pH 7.4 and sterilized by filtration (0.2
membrane system, USA) and stored at 4 ꢂC under nitrogen. 1 mL of
various concentrations (10, and 25 M) of compounds were taken
in test tubes, 40 L of copper sulphate (2 mM) was added and the
mm Millipore
m
m
volume was made up to 1.5 mL with phosphate buffer (50 mM, pH
7.4). A tube without compound and with copper sulphate served as
a negative control, and another tube without copper sulphate with
compound served as a positive control. All of the tubes were
incubated at 37 ꢂC for 45 min. To the aliquots of 0.5 mL drawn at 2,
4 and 6 h intervals from each tube, 0.25 mL of thiobarbutaric acid
(TBA, 1% in 50 mM NaOH) and 0.25 mL of trichloro acetic acid (TCA,
2.8%) were added. The tubes were incubated again at 95 ꢂC for
45 min and cooled to room temperature and centrifuged at
2500 rpm for 15 min. A pink chromogen was extracted after the
mixture was cooled to room temperature by further centrifugation
at 2000 rpm for 10 min. Thiobarbituric acid reactive species in the
pink chromogen were detected at 532 nm by a spectrophotometer
against an appropriate blank. Data were expressed in terms of
malondialdehyde (MDA) equivalent, estimated by comparison with
5.2.2. Antioxidant activity by
Each compound at the final concentrations of 50 and 100
were incorporated into -carotene-linoleic acid model system
independently and the activity was monitored spectrophotomet-
rically at 470 nm [27].
b-carotene-linoleic acid assay
m
M/mL
b
5.2.2.1. Preparation of the suspension. The substrate suspension
was prepared by addition of b-carotene (4 mg dissolved in 5 mL
chloroform) into a covered round bottomed flask containing
Tween-40 (600 mg) followed by the addition of linoleic acid
(60 mL). The chloroform was removed completely under vaccum
using rotary evaporator at 40 ꢂC. The resulting solution was diluted
with triple distilled water (30 mL) and the emulsion was mixed
well and diluted with oxygenated water (120 mL). The aliquots
Vijay Kumar
