Antibacterial and free radical scavenging potential of synthesized 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chromen-4-ones

Article abstract of DOI:10.1007/s00044-014-0938-8

A series of 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chromen-4-one derivatives (6a-m) was synthesized in quantitative yields and their structures were corroborated on the basis of FT-IR, ¹H, ¹³C NMR, ESI-MS, and elemental analysis data. The synthesized compounds were screened for in vitro antibacterial activities against a representative panel of Gram-positive and Gram-negative bacteria, as well as to evaluate their antioxidant potential using DPPH assay method. Bio-evaluation studies revealed that compounds 6c, 6d, 6e, 6j, and 6l exhibited moderate to good antibacterial activity against all the tested bacterial strains. Furthermore, from the antioxidant screening results, it has been observed that compounds 6c, 6e, and 6g manifested profound antioxidant potential (IC₅₀ <7.68 μM) in comparison to the standard antioxidant ascorbic acid.

Full text of DOI:10.1007/s00044-014-0938-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0938-8
ORIGINAL RESEARCH
Antibacterial and free radical scavenging potential of synthesized
7-hydroxy-2-aryl-6-aryldiazenyl-4H-chromen-4-ones
•
Pawan Kumar Sharma Prabal Bandyopadhyay
•
•
Pratibha Sharma Ashok Kumar
Received: 22 July 2013 / Accepted: 3 February 2014
ꢀ Springer Science+Business Media New York 2014
Abstract A series of 7-hydroxy-2-aryl-6-aryldiazenyl-
4H-chromen-4-one derivatives (6a–m) was synthesized in
quantitative yields and their structures were corroborated on
diseases. Some free radicals and reactive oxygen species
(ROS), such as superoxide anions, hydrogen peroxide,
hydroxyl, and nitric oxide radicals have been found to be
responsible for diabetes, neurodegenerative disorders,
aging, cancer causing ailments, cardiovascular, autoim-
mune, and inflammatory diseases (Beckman and Ames,
1998; Halliwell and Gutteridge, 1989). Therefore, mini-
mization of oxidative damage may be an important
approach to the primary prevention or treatment of these
diseases. In this regard, the role of antioxidants is very well
reported in the literature because they may stop the free-
radical formation, or interrupt an oxidizing chain reaction
(Block, 1992; Rice-Evans et al., 1996). Whence, the
development of synthetic compounds, capable of scav-
enging free radicals, has been a great success in the recent
past. Now-a-days, antioxidants that exhibit DPPH radical
scavenging activity are increasingly receiving attention
(Hossain and Bhattacharya, 2007). It is revealed from the
literature survey that flavonoids, being of natural origin
(Silva et al., 2000; Teixeira et al., 2005; Siquet et al., 2006)
and owing to their inherent antioxidative properties, have
been considered as attractive potential antioxidants.
1
the basis of FT-IR, H, 13C NMR, ESI–MS, and elemental
analysis data. The synthesized compounds were screened for
in vitro antibacterial activities against a representative panel
of Gram-positive and Gram-negative bacteria, as well as to
evaluate their antioxidant potential using DPPH assay
method. Bio-evaluation studies revealed that compounds 6c,
6d, 6e, 6j, and 6l exhibited moderate to good antibacterial
activity against all the tested bacterial strains. Furthermore,
from the antioxidant screening results, it has been observed
that compounds 6c, 6e, and 6g manifested profound anti-
oxidant potential (IC50 \7.68 lM) in comparison to the
standard antioxidant ascorbic acid.
Keywords Flavone ꢀ Antibacterial activity ꢀ
Antioxidant activity ꢀ DPPH radical scavenging method
Introduction
Antioxidants have gained a lot of importance due to their
potential to act as prophylactic and therapeutic agents in
many diseases and also to provide major defense against
radical mediated toxicity by protecting the damages caused
by free radicals (Valko et al., 2006). The generation of free
radicals which is observed during the metabolic process is
responsible for wide range of human conditions such as
viral infection, myocardial infarction, and neurogenerative
Flavones are privileged structural units that are
encountered in a wide range of biologically active and
medicinally significant compounds. Recent medicinal
chemistry applications of flavones derivatives have been
found to be associated with antioxidant (Kang et al., 2008;
Manthey et al., 2001), anti-inflammatory (Laupattarakasem
et al., 2004), antibacterial (Xu and Lee, 2001; Hamil-
tonmiller, 1995; Iinuma et al., 1994), antitumor (Han,
1997; Birt et al., 2001), anticancer (Kohen et al., 2007;
Blank et al., 2004), antifungal (Li et al., 2002), cardio-
vascular (Qin et al., 2008; Wang et al., 1996; Tzeng et al.,
1997; Liou et al., 1994), anti-osteoporotic (Delcanale et al.,
2001; Wang et al., 2005) effect and they are also effective
P. K. Sharma ꢀ P. Bandyopadhyay ꢀ P. Sharma ꢀ A. Kumar (&)
School of Chemical Sciences, Devi Ahilya University,
Takshashila Campus, Khandwa Road, Indore 452001, MP, India
e-mail: drashoksharma2001@yahoo.com
123

Med Chem Res
Scheme 1 Synthetic pathway for 7-hydroxy-2-aryl-6-aryldiazenyl-
4H-chromen-4-one derivatives (6a–m). Reagents and conditions:
aldehyde, KOH/EtOH, Room temp. for 72 h, neutralized with 1:1
cold HCl; (iv) 15 % HCl/H2O reflux, 30 min; and (v) NBS
(10 mol %)/CH3CN, Reflux, 20–30 min
(i)
NaNO2/HCl,
0–5 ꢁC,
1-(2,4-dihydroxyphenyl)ethanone,
140–150 ꢁC; (ii) C6H5COCl, NaOH, stirring for 30 min; (iii) aromatic
in the prevention and treatment of complex diseases such
as atherosclerosis, stroke, diabetes, and Alzheimer’s
disease. Flavones are polyphenolic compounds belonging
to the flavonoid group that occur naturally in fruits,
123

Med Chem Res
vegetables, nuts, seeds, flowers, and barks (Malikov and
Yuldashev, 2002; Huck et al., 2000; Nagao et al., 2002).
They are an integral part of the human diet and also the
core structure of flavones is also found in several natural
products (Awuah and Capretta, 2009; Valla at el., 2008).
Development of efficient methods for the construction of
the flavones structural framework has thus been an
important challenge in organic synthesis. Although the
skeleton of phenyl benzopyran-4-one pharmacophore
contributed an important role in biological effects of fla-
vonoid derivatives, however the peripheral substituents
also elicits their significant role in imparting enhanced
antioxidative potential of pharmacophoric entity. It is evi-
dent from a number of studies that their antioxidant activity
is strongly dependent on their structural features and
intrinsically related to the presence of hydroxyl func-
tion(s) in the aromatic core structure. A number of methods
are available for preparing flavones, chromones, and their
analogs (Banerji and Goomer, 1980; Khanna et al., 1992;
Looker and Hanneman, 1962; Baker, 1933), including the
Allan and Robinson (1924) synthesis. The most common
method, however, involves the Baker–Venkataraman
rearrangement, wherein an ortho-hydroxy acetophenone is
benzoylated and the ester is treated with base (pyridine/
KOH) to effect an acyl group migration, forming a 1,3-
diketone (Mahal and Venkataraman, 1934; Jain et al.,
1982; Saxena et al., 1985; Hirao et al., 1984). The ensuing
diketone is then cyclized under strong acidic conditions
using acetic acid and sulfuric acid to furnish flavones.
Buoyed from these findings and in continuation of our
studies on bioactive compounds (Sharma et al., 2012a, b;
Sahu et al., 2013; Bandyopadhyay et al., 2011), herein, we
have reported the synthesis of a series of 7-hydroxy-2-aryl-
6-aryldiazenyl-4H-chromen-4-one derivatives (6a–m)
(Scheme 1) followed by the evaluation of their antibacte-
(benzoyloxy)-4-(aryldiazenyl)phenyl benzoate 3. Further,
reaction has been accomplished in ethanolic solution of
aromatic aldehyde to obtain 5-(benzoyloxy)-4-(aryldiaze-
nyl)-2-(3-phenylprop-2-enoyl)phenyl benzoate 4. It was
then treated with dilute solution of hydrochloric acid to
hydrolyze the ester group in order to yield the compound
1-{2,4-dihydroxy-5-(aryldiazenyl)phenyl}-3-phenylprop-
2-en-1-one 5. The final step of this strategy involves oxi-
dative cyclization of compound using N-bromo succini-
mide (NBS) as catalyst in acetonitrile to give the desired
flavones 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chromen-4-
one 6 in appreciable yield. It is evident from Table 1 that,
the proposed synthetic procedure works well with different
substituents. As shown in synthetic pathway (Scheme 1), a
variety of substituted anilines and aromatic aldehydes
bearing electron-donating and electron-withdrawing sub-
stituents were successfully employed to prepare the cor-
responding flavone derivatives in quantitative yields
(66–84 %).
Results and discussion
Spectroscopic characterizations of compounds
All the synthesized compounds were characterized on the
basis of their physical, analytical, and spectral data. In
general, the FT-IR spectra of compounds (6a–m) have
given confirmation about the vibrational modes of syn-
1
thesized compounds. The H and 13C NMR spectra of all
the compounds displayed pertinent signals in their due
region. The mass spectra (ESI–MS) of the compounds
showed (M?H)? peaks, in agreement with their molecular
weight. Elemental analysis results for C, H, and N elements
were satisfactory within 0.4 % calculated values of the
compounds.
`
rial vis-a-vis free radical scavenging potential. Owing to
the presence of azo functionality in these compounds, they
can be used as chemotherapeutic agents for the treatment of
diseases caused by different microorganisms.
In vitro antibacterial activity
All the synthesized compounds were evaluated in vitro
against an assortment of three Gram-positive bacteria
Staphylococcus aureus MTCC 3160, Bacillus cereus
MTCC 430, and Arthobacter globiformis MTCC 4299 and
three Gram-negative bacteria Vibrio cholerae MTCC 3904,
Escherichia coli MTCC 1610, and Shigella dysenteriae
NICED. The antibacterial activity (zone of growth inhibi-
tion) of the tested 7-hydroxy-2-aryl-6-aryldiazenyl-4H-
chromen-4-one derivatives (6a–m) in comparison with that
of control drugs ampicillin and ciprofloxacin was deter-
mined by Kirby–Bauer disk-diffusion method on Mueller–
Hinton agar according to the guidelines of the Clinical
Laboratory Standards Institute, 2007, USA (Table 2).
Chemistry
A series of these compounds was synthesized containing
aryldiazenyl entity, coupled to flavone system. Moreover, it
was considered worthy of interest to investigate the influ-
ence of substituents as the structural variants on the
anticipated biological activities. As per the synthetic
pathway depicted in Scheme 1, substituted aniline 1 was
diazotized and coupled with 1-(2,4-dihydroxyphenyl)etha-
none (prepared by the acylation of resorcinol) to obtain
1-[2,4-dihydroxy-5-(aryldiazenyl)phenyl]ethanone 2.
Compound 2 was then benzoylated to yield 2-acetyl-5-
123

Med Chem Res
Table 1 List of synthesized 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chromen-4-one derivatives (6a–m)
Entry
R
R0
Product (6)
Yield (%)a
68
6a
4-OH
H
H
O
O
O
N
N
H
O
H
O
6b
6c
6d
6e
6f
H
OH
OCH3
H
70
67
76
75
73
66
79
H
O
O
O
N
N
O
2-OH
2-OCH3
3-OCH3
H
H
O
O
O
N
N
H
O
H
O
O
O
N
O
N
H
O
OH
OCH3
OH
H
H
O
O
O
N
O
N
O
H
O
O
O
N
N
H
O
6g
6h
2-OH
H
O
O
N
N
O
H
O
4-Cl
H
O
O
O
N
N
l
C
123

Med Chem Res
Table 1 continued
Entry
R
R0
Product (6)
Yield (%)a
74
H
O
6i
2-Cl
OH
H
O
O
O
N
N
l
C
O
6j
4-Cl
3-NO2
4-NO2
H
OCH3
77
84
81
78
H
O
O
O
N
N
l
C
6k
H
H
O
O
N
O2
N
N
O
H
O
6l
OH
H
O
O
O
N
N
N
O2
6m
H
H
O
O
O
N
N
a
Isolated yield
Among all the tested compounds, most of the com-
pounds exhibited moderate to excellent activity. The data
also revealed that derivatization of the parent molecule had
produced the marked enhancement in the potency of the
synthesized analogs as antibacterial agents.
and compound 6l bearing para-nitro group along with
para-hydroxyl group exhibited excellent efficacy (zone of
growth inhibition 33.47, 34.38, 33.25 and 32.38, 33.24, and
32.01 mm) against tested S. aureus MTCC 3160, B. cereus
MTCC 430, and A. globiformis MTCC 4299, respectively.
The zone of inhibition for compound 6g (24.85 and
25.97 mm for S. aureus MTCC 3160, B. cereus MTCC
430) was nearly equal to the control drug ampicillin (25.0
and 26.0 mm), whereas it exhibited slightly lesser activity
for A. globiformis (20.45 mm) compared to standard drug
ampicillin. Interestingly, in case of S. aureus MTCC 3160,
B. cereus MTCC 430, and A. globiformis MTCC 4299, a
number of compounds (6c, 6d, 6e, 6f, 6h, 6i, 6j, and 6l)
displayed inhibitory activity (26.92–39.55 mm) better than
that of ampicillin (25.0–29.0 mm) while compounds 6c,
6d, 6e, 6j, and 6l have shown better activity than standard
drug ciprofloxacin (28.0–31.0 mm). It is evident from
Table 2 that compound 6h displayed effective activity
From the data presented in Table 2, it is inferred that the
substitution in aryl ring exerted significant influence on the
antibacterial profile of the synthesized flavones. Interest-
ingly, compounds 6j (substituted by para-chloro and para-
methoxy groups) and 6e (substituted by meta-methoxy and
para-hydroxyl groups) are found to be more active mole-
cules of the series compared to the compounds bearing
other electron-donating or withdrawing groups. These
compounds (6j and 6e) showed better activity profile (zone
of growth inhibition 39.55 and 35.41 mm) against B. cer-
eus MTCC 430 as compared to the standard drugs ampi-
cillin (26.0 mm) and ciprofloxacin (28.0 mm). Compound
6c substituted by ortho-hydroxy and para-methoxy groups
123

Med Chem Res
Table 2 Antibacterial screening data for 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chromen-4-one derivatives (6a–m)
Compounds
R
R0
Zone of Inhibition (mm)
Gram-positive
Gram-negative
ECe
SAb
BCc
AGd
VCf
SDg
6a
4-OH
H
H
20.90 0.20
21.52 0.28
33.47 0.08
30.61 0.36
34.36 0.22
31.20 0.06
24.85 0.18
26.92 0.24
29.45 0.46
38.76 0.48
–
–
–
23.15 0.12
22.05 0.27
33.25 0.13
30.16 0.08
34.11 0.14
30.87 0.42
20.35 0.11
24.15 0.04
–
20.13 0.02
20.50 0.16
32.54 0.11
29.82 0.05
33.23 0.21
30.36 0.52
–
19.24 0.17
19.83 0.20
31.79 0.06
28.60 0.13
32.17 0.42
–
20.01 0.06
19.97 0.11
32.0 0.21
27.67 0.18
33.13 0.24
28.18 0.35
–
6b
OH
OCH3
H
6c
2-OH
2-OCH3
3-OCH3
H
34.38 0.19
31.62 0.27
35.41 0.09
32.15 0.46
25.97 0.12
27.82 0.23
30.20 0.04
39.55 0.14
29.91 0.07
33.24 0.26
19.58 0.23
26.0 0.10
28.0 0.04
6d
6e
OH
OCH3
OH
H
6f
6g
2-OH
4-Cl
2-Cl
4-Cl
3-NO2
4-NO2
H
23.0 0.07
–
6h
–
23.02 0.34
–
6i
OH
OCH3
H
28.57 0.40
38.0 0.29
28.32 0.14
31.48 0.22
15.34 0.16
37.0 0.13
33.0 0.11
27.72 0.16
36.96 0.26
–
6j
37.14 0.22
–
37.32 0.18
32.47 0.23
29.09 0.42
14.02 0.25
35.0 0.05
32.0 0.14
6k
6l
OH
H
32.38 0.35
18.0 0.14
25.0 0.07
30.0 0.13
32.01 0.03
16.21 0.47
29.0 0.15
31.0 0.09
30.68 0.39
13.25 0.31
32.0 0.18
30.0 0.16
6m
Ampicillina
Ciprofloxacina
–
–
–
–
The tests were repeated in triplicate and the average values along with standard deviation ( SD) were taken to show the inhibitory activity of
the synthesized flavone derivatives against the selected sets of bacteria
a
Antibacterial activity of the synthesized compounds was compared with the standard antibacterial drugs Ampicillin and Ciprofloxacin
b
SA: Staphylococcus aureus MTCC 3160
c
BC: Bacillus cereus MTCC 430
d
AG: Arthobacter globiformis MTCC 4299
e
EC: Escherichia coli MTCC 1610
f
VC: Vibrio cholerae MTCC 3904
g
SD: Shigella dysenteriae NICED
profile (zone of growth inhibition up to 24.15–27.82 mm)
against all the three Gram-positive bacteria. Likewise,
compound 6k (substituted by meta-nitro group) exhibited
antibacterial potency against Gram-negative E. coli MTCC
1610 and S. dysenteriae NICED and Gram-positive B.
cereus MTCC 430 with zone of growth inhibition 28.32,
32.47, and 29.91 mm, respectively compared to standard
drugs ampicillin (37.0, 35.0, and 26.0 mm) and ciproflox-
acin (33.0, 32.0, and 28.0 mm). Compounds 6e and 6j
demonstrated better activity profile (32.17–36.96 mm)
against V. cholerae MTCC 3904 as compared to the stan-
dard drugs ampicillin (32.0 mm) and ciprofloxacin
(30.0 mm). Furthermore, compound 6j displayed higher
inhibitory activity (38 mm) against E. coli MTCC 1610
over to the ampicillin (37 mm) and ciprofloxacin
(33.0 mm). Compound 6m exhibited minimum activity
profile (zone of growth inhibition 18.0, 19.58, 16.21, 13.25,
14.02, and 15.34 mm) compared to standard drugs ampi-
cillin (25.0, 26.0, 29.0, 32.0, 35.0, and 37.0 mm) and cip-
rofloxacin (30.0, 28.0, 31.0, 30.0, 32.0, and 33.0 mm)
against all Gram-positive bacteria S. aureus MTCC 3160,
B. cereus MTCC 430, and A. globiformis MTCC 4299 and
Gram-negative bacteria V. cholerae MTCC 3904, S. dy-
senteriae NICED, and E. coli MTCC 1610, respectively.
Compound 6f displayed better activity (31.20, 32.15, and
30.87 mm) than both standard drugs ampicillin and cip-
rofloxacin against Gram-positive bacteria S. aureus MTCC
3160, B. cereus MTCC 430, and A. globiformis MTCC
4299. It is also evident from the Table 2 that, compounds
6c, 6d, 6e, 6j, and 6l exhibited moderate to good antibac-
terial activity against all the Gram-positive as well as
Gram-negative bacteria.
Further, upon close inspection of results depicted in
Table 2, it has also been observed that Gram-positive
bacteria were more susceptible toward the newly synthe-
sized series of compounds (6a–m) as compared to the
Gram-negative bacteria. This may be due to the absence of
a unique outer membrane in Gram-positive bacteria, and
hence, the wall of Gram-positive bacteria is permeable to
these derivatives. Generally, the Gram-positive bacteria are
more susceptible having only an outer peptidoglycan layer
which is not an effective permeability barrier (Scherrer and
123

Med Chem Res
Fig. 1 Percent inhibition
(% inhibition) of antibacterial
activity for different derivatives
of 7-hydroxy-2-aryl-6-
aryldiazenyl-4H-chromen-4-one
(6a–m) as compared to the
a
standard drug Ampicillin. SA:
Staphylococcus aureus MTCC
b
3160; BC: Bacillus cereus
c
MTCC 430; AG: Arthobacter
d
globiformis MTCC 4299; EC:
Escherichia coli MTCC 1610;
eVC: Vibrio cholerae MTCC
f
3904; SD: Shigella dysenteriae
NICED
Fig. 2 Percent inhibition
(% inhibition) of antibacterial
activity for different derivatives
of 7-hydroxy-2-aryl-6-
aryldiazenyl-4H-chromen-4-one
(6a–m) as compared to the
standard drug Ciprofloxacin.
aSA: Staphylococcus aureus
b
MTCC 3160; BC: Bacillus
c
cereus MTCC 430; AG:
Arthobacter globiformis MTCC
d
4299; EC: Escherichia coli
e
MTCC 1610; VC: Vibrio
f
cholerae MTCC 3904; SD:
Shigella dysenteriae NICED
Gerhardt, 1971), whereas the Gram-negative bacteria pos-
sess an outer phospholipidic membrane carrying the
structural lipopolysaccharide components. This makes the
cell wall impermeable to drug constituents (Betoni et al.,
2006). Percent inhibition (% inhibition) of antibacterial
activity for different derivatives over control drugs was
calculated using following formula:
The negative % inhibition values of 6c, 6d, 6e, 6f, 6j, and
6l for various bacterial strains are suggestive of the better
activity profile of these derivatives as compared to the
standard drugs, whereas, the % inhibition ?100 indicates
that there was no antibacterial activity. The potencies for
antibacterial activity were observed in order of 6j [ 6e [
6c [ 6l [ 6d [ 6f [ 6i [ 6k [ 6h [ 6g [ 6b [ 6a [
6m.
% inhibition ¼ ða ꢁ bÞ=a ꢂ 100;
where a and b stands for zone of inhibition of control drugs
and synthesized compounds, respectively. The results for
the antibacterial activity of the selected synthesized com-
pounds against S. aureus MTCC 3160, B. cereus MTCC
430, A. globiformis MTCC 4299, V. cholerae MTCC 3904,
E. coli MTCC 1610, and S. dysenteriae NICED are
graphically represented in Figs. 1 and 2.
In vitro antioxidant activity
In order to explore the antioxidant potential of newly
synthesized compounds, the free radical scavenging
activity measurements were performed using 1,1-diphenyl-
2-picryl-hydrazyl (DPPH) assay method (Mensor et al.,
2001; Blois, 1958; Bondet et al., 1997). various methods
are available for the determination of free radical scav-
enging activity, but the assay employing the stable DPPH
has received the maximum attention owing to its accuracy
and precision. DPPH is a stable free radical which has an
Further, it is revealed from the results depicted in Figs. 1
and 2 that most of the synthesized compounds gave
promising results compared to the reference drugs ampi-
cillin and ciprofloxacin against the tested bacterial strains.
123

Med Chem Res
unpaired valence electron present on nitrogen bridge.
Because of this odd electron, DPPH gives a strong
absorption band at k = 517 nm in visible spectroscopy
(deep violet color). As this electron becomes paired off in
the presence of a free radical scavenger, the absorption
vanishes, and the resulting decolorization is stoichiometric
with respect to the number of electrons taken up. Thus,
antioxidant molecule can quench DPPH free radical (i.e.,
by providing hydrogen atoms or by electron-donating) and
converting them to a colorless/bleached product (i.e., 1,1-
diphenyl-2-picrylhydrazine), thereby resulted in a decrease
in absorbance. The antioxidant activity was expressed as
the 50 % inhibitory concentration (IC50) based on the
amount of compound required for a 50 % decrease of the
initial DPPH radical concentration. The values of IC50, the
effective concentration at which 50 % of the radicals were
scavenged, were calculated to evaluate the antioxidant
activities. A lower IC50 value indicated greater antioxidant
activity. The radical-scavenging activity of the tested
samples, expressed as percentage inhibition of DPPH, was
calculated according to the formula:
2.0 mL of compound and ascorbic acid (AA) solutions of
different concentrations taken in separate test tubes and
allowed to react at room temperature. After 15 min, the
absorbance values were measured at 517 nm and converted
into the percentage antioxidant activity. The experiments
were done in triplicate. The inhibitory concentration (IC50)
value, representing the concentration required to exhibit
50 % antioxidant activity, was extrapolated from the graph
plotted with percentage DPPH radical scavenging activity
on the y-axis and concentration on the x-axis (Fig. 3).
Radical-scavenging activity increases with increasing the
concentration of solutions while the absorbance decreases.
DPPH scavenging activity of phenolics is positively cor-
related with the number of hydroxyl groups. Compound 6g
with three hydroxyl groups has lower IC50 value as com-
pared to other compounds and ascorbic acid also.
The radical-scavenging activity of synthesized com-
pounds and ascorbic acid was increased with the increase
of time (Fig. 4) and the absorbance continued to decrease
till a period of 120 min of observation. For the sake of
uniformity, a time interval of 15 min was taken for syn-
thesized compounds and ascorbic acid radical scavenging
capacity measurements.
% DPPH radical scavenging activity
¼ ½ðAo ꢁ AtÞ=Aoꢃ ꢂ 100;
It can be seen from Table 3 that, in DPPH assay, com-
pounds 6c, 6e, and 6g displayed better radical-scavenging
activities as compared to the commercially available
standard antioxidant viz., ascorbic acid.
where At is the absorbance value of the tested compound
and Ao is the absorbance value of blank solution without
compound, at a particular time. Percentage inhibition after
15, 30, 45, 60, 75, 90, 105, and 120 min was plotted against
concentration, to obtain the IC50 value. The entire syn-
thesized compounds scavenged DPPH radical (DPPH•)
significantly in a concentration-dependent manner. We
determined the DPPH radical scavenging activity of all the
synthesized compounds (6a–m), and common antioxidant
ascorbic acid was used as the standard for comparing their
antioxidant potential. The order of absorbance was good in
methanolic solution for all the compounds (6a–m). Higher
absorbance in methanolic solution of DPPH implies better
sensitivity. We used a DPPH concentration of 100 lM, in
accordance with the requirement of the accuracy of spec-
trophotometric measurements. The absorbance of DPPH
without any additions was stable over 15 min. The DPPH•
absorbance decreases due to hydrogen transfer from the
antioxidant, thus forming the DPPH-H stable compound.
Methanolic DPPH• solutions used at different concentra-
tions were stable (checked over a 120 min period). A sig-
nificant decrease in the concentration of DPPH• radical due
to the scavenging ability of some of the antioxidants was
observed.
The structural features of synthesized flavones revealed
that as the number of hydroxyl groups increases, the
derivatives have a tendency to show better scavenging
effects. Thus, compound 6g exhibited highest radical-
scavenging activity as compared to other synthesized
compounds due to the presence of three hydroxy groups
with minimum IC50 value i.e., 1.76 lM. Compounds 6c
and 6e with IC50 values 5.60 and 4.64 lM, respectively,
demonstrated better antioxidant profile compared to
ascorbic acid (IC50 value 7.68 lM) as both these com-
pounds possessed two hydroxy groups which preferred the
radical-scavenging activity. Moreover, these compounds
also showed better antioxidant activity compared to 6a and
6b due to presence of methoxy group (i.e., the presence of
the electron-donating OCH3 group enhanced the stabiliza-
tion of the resulting nitrogen-centered radical as the num-
ber of conjugating structure is more than that without the
OCH3 group) which is absent in the latter cases (com-
pounds 6a and 6b). Compounds 6a and 6b displayed
effective DPPH radical-scavenging activity with IC50 val-
ues 11.03 and 10.0 lM, respectively. Compounds 6i and
6l, which have more than one hydroxy groups, have IC50
values 16.66 and 18.32 lM. Also, compound 6l has
stronger electron-withdrawing para-nitro group with
respect to aryldiazenyl ring which destabilizes the ring with
The stock solution (150 lM) of ascorbic acid and syn-
thesized compounds was diluted to final concentrations of
1, 2, 4, 8, 12, 16, 20, 24, and 30 lM in methanol. Meth-
anolic DPPH solution (1 mL, 100 lM) was added to
123

Med Chem Res
Table 3 Inhibition of DPPH radical by synthesized compounds
(6a–m)
R'
H
O
O
O
N
N
R
Compound
aIC50 (lM)
Compound
aIC50 (lM)
6a
6b
6c
6d
6e
6f
11.03
10.00
5.60
6g
6h
6i
1.76
27.74
16.66
23.75
26.77
18.32
25.52
21.00
4.64
6j
6k
6l
Fig. 3 Graphical representation of percentage (%) DPPH radical
scavenging activity for 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chro-
men-4-one derivatives (6a–m) and ascorbic acid (AA) at different
concentration (lM) after 60 min
21.68
7.68
Ascorbic acidb
6m
a
IC50 values were determined by linear regression analysis using at
least five different concentrations in triplicate
b
Antioxidant potential of the synthesized flavones was compared
with the standard antioxidant ascorbic acid
scavenging effect with IC50 of 27.74 and 26.77 lM,
respectively, in this assay. Compound 6k having nitro
group at meta position with respect to the aryldiazenyl ring,
while in compound 6h para-chloro group was present.
Therefore, in view of the aforementioned findings, the
order of antioxidant potencies was found to be 6g [
6e [6c [6b [6a [6i [6l [6d [6f [6j [6m [ 6k [
6h. All the synthesized compounds (6a–m) with IC50 val-
ues in the range of 1.76–27.74 lM showed moderate to good
radical-scavenging activities in comparison to ascorbic acid
as standard which was attributed to the presence of different
substituted functional groups attached to aryl diazenyl ring
and aryl ring of synthesized flavone derivatives (that can
donate hydrogen atoms) and governs the main factor behind
their ability to be scavenged by DPPH. After donating a
hydrogen atom, compounds exist in its radical form, and the
electron conjugation effect in the structure stabilizes the
radical which favors the reaction to occur. In essence,
hydroxyl group substitution in synthesized flavones deriva-
tives, as well as azo functionality attached to the aryl ring of
flavonoid structure, appeared to be major contributors to the
DPPH radical scavenging activities.
Fig. 4 Graphical representation of percentage (%) DPPH radical
scavenging activity for 7-hydroxy-2-aryl-6-aryldiazenyl-4H-chro-
men-4-one derivatives (6a–m) and ascorbic acid (AA) at different
time interval (minutes) at a concentration of 4 lM
interruption to give more favorable condition for scav-
enging effect, while in compound 6i, comparatively less
electron-withdrawing ortho-chloro group is present. IC50 of
6d and 6f was found to be 21 and 21.68 lM, respectively,
which exhibited lower radical-scavenging activity. Both of
these compounds were substituted by methoxy group
which preferred radical-scavenging activity, but only single
hydroxyl group was present in these compounds. Com-
pound 6j substituted by chloro and methoxy groups and
unsubstituted compound 6m displayed very low-antioxi-
dant activity-profile with IC50 of 23.75 and 25.52 lM,
whereas compounds 6h and 6k showed minimum
Conclusion
Keeping in view the antibiotic resistance to Gram-positive/
Gram-negative pathogens as a problem of major thrust in
hospitals, a number of substituted flavones derivatives
123

Med Chem Res
(6a–m) were synthesized as potential antibacterial agents.
Conclusively, the antibacterial results based on disk-dif-
fusion method showed that compounds 6c, 6d, 6e, 6j, and
6l exhibited moderate to good antibacterial activity against
all the tested bacterial strains. The compound 6j has shown
enhanced inhibitory activity compared with standard anti-
bacterial drugs ampicillin and ciprofloxacin against all the
Gram-positive and Gram-negative bacteria. It is concluded
from the results that substituents on the flavones skeleton
are responsible for the enhancement of the antibacterial
activity. It has been further deduced that when a methoxy
group was present in flavonoid ring, it enhances the activity
against S. aureus MTCC 3160, B. cereus MTCC 430, A.
globiformis MTCC 4299, V. cholerae MTCC 3904, E. coli
MTCC 1610S. dysenteriae NICED, and S. dysenteriae
NICED. These antibacterial results indicated that structural
factors, such as type of substitution in the ring and water
solubility of the target compounds, could further enhance
their inhibitory activities.
described as s (singlet), t (triplet), q (quartet), and m
(multiplet). Mass analysis was performed on quadruple-
time of flight (Q-Tof) mass spectrometer (Micromass,
USA) using electrospray ionization (ESI) in positive mode.
Elemental analyses (C, H, and N) were carried out on a
Vario Micro Cube Elemental Analyzer. TLC is performed
using precoated aluminium sheets with silica gel 60 F254
.
Column chromatography was performed on Merck silica
gel 60 (100–200 mesh).
General procedure for the synthesis of 7-hydroxy-2-
aryl-6-aryldiazenyl-4H-chromen-4-ones (6a–m)
Pertinent aniline derivatives (0.036 M) 1 were diazotized
and allowed to couple with 1-(2,4-dihydroxyphenyl)etha-
none (prepared by the acylation of resorcinol) at 0–5 ꢁC
under stirring to obtain 1-[2,4-dihydroxy-5-(aryldiaze-
nyl)phenyl]ethanone 2. Compound 2 (0.023 M, 6.56 g)
was treated using 10 % sodium hydroxide (10 mL) and
benzoyl chloride (0.023 M, 3.8 mL) under mechanical
stirring and resulted in the formation of benzoylated
Furthermore, evaluation of antioxidant potentials of
these compounds suggested that compound 6g has been
found to be a very good antioxidant due to the presence of
three hydroxyl groups in its structural armamentarium. The
results obtained from this study highlighted that three
substituted flavone derivatives (6c, 6e, and 6g) exhibited
potent antioxidant activities (IC50 \7.68 lM) as com-
pared to standard antioxidant ascorbic acid, which could
be the new addition in list of synthetic antioxidants.
Hence, in view to cater the needs associated with ever
increasing demand of newer antibacterial and antioxidant
agents, exploration of these findings can envisage these
compounds as powerful antibacterial and antioxidative
agents.
product
2-acetyl-5-(benzoyloxy)-4-(aryldiazenyl)phenyl
benzoate 3. Further reaction has been accomplished in
ethanolic solution of aromatic aldehyde to obtain 5-(ben-
zoyloxy)-4-(aryldiazenyl)-2-(3-phenylprop-2-enoyl)phenyl
benzoate 4. Synthesized compound 4 (0.007 M, 3.45 g)
was refluxed with 15 % HCl (30 mL) solution for half an
hour to hydrolyze the ester groups. The contents were
poured into ice-cold water and extracted with ethyl acetate
(30 mL). The ester layer was dried over anhydrous MgSO4
and the solvent was stripped off by distillation to obtained
1-[2,4-dihydroxy-5-(aryldiazenyl)phenyl]-3-phenylprop-2-
en-1-one 5. The final step of this strategy involves oxida-
tive cyclization of compound 5 using N-bromo succinimide
(NBS) (10 mol %) as catalyst in acetonitrile solvent to give
the desired compound 7-hydroxy-2-aryl-6-aryldiazenyl-
4H-chromen-4-one 6 in appreciable yield.
Experimental
Spectral characterization data of representative inter-
mediates (2, 3, 4, and 5m) and of all newly synthesized
substituted flavones derivatives (6a–m) are given below:
General
Reagents were obtained from commercial supplier and
used without further purification. Melting points were
determined with open capillary tube on a Gallenkamp
(variable heater) melting point apparatus and were uncor-
rected. FT-IR spectra were recorded as KBr pellets on a
Bruker Tensor 27 spectrometer with Opus 5.5 software.
1-{2,4-Dihydroxy-5-[(E)-2-phenyldiazen-1-
yl]phenyl}ethan-1-one (2m)
Yellowish brown solid (EtOH) (this compound was pre-
pared by coupling of diazotized aniline (0.05 M, 7.02 mL)
with 1-(2,4-dihydroxyphenyl)ethanone (0.05 M, 7.60 g)
(prepared by the acylation of resorcinol) at 0–5 ꢁC under
stirring. After separation and purification of the residue of
the reaction, the product 2m was obtained as a yellowish
brown solid); Yield: 78 %; m.p. 116–118 ꢁC; 1H NMR
1
The H and 13C NMR spectra of the synthesized com-
pounds were recorded at 400 and 100 MHz, respectively,
using Bruker AVANCE 400 MHz NMR spectrometer in
DMSO-d6 and CDCl3 solvents. The chemical shifts were
expressed in d relative to TMS as internal standard and
coupling constants (J) in Hz. Spin multiplicities are
123

Med Chem Res
(DMSO-d6, 400 MHz): d = 2.64 (s, 3H, CH3), 6.89 (s, 1H,
H-3), 7.16–7.20 (t, 1H, J = 7.4 Hz, H-40), 7.37–7.39 (d,
2H, J = 7.4 Hz, H-20, H-60), 7.40–7.44 (t, 2H, J = 7.4 Hz,
H-30, H-50), 7.91 (s, 1H, H-6), 9.86 (s, bs, 1H, OH), 9.95 (s,
bs, 1H, OH); 13C NMR (DMSO-d6, 100 MHz): d = 29.2
(CH3, COCH3), 102.8 (CH, C-3), 113.6 (C, C-1), 116.1 (C,
C-5), 122.3 (CH, C-20, C-60), 123.5 (CH, C-6), 128.4 (CH,
C-30, C-50), 130.0 (CH, C-40), 151.7 (C, C-10), 157.2 (C,
C-4), 163.6 (C, C-2), 198.4 (CO, COCH3); ESI–MS for
C14H12N2O3: 257 [M?H]?.
H ꢁ 60000, H ꢁ 80000), 7.13–7.15 (d, 2H, J = 7.9 Hz, H ꢁ 50000,
H ꢁ 90000), 7.16 (s, 1H, H-6), 7.18–7.20 (d, 2H, J = 7.8 Hz,
H-20, H-60), 7.32–7.36 (t, 2H, J = 7.8 Hz, H-30, H-50),
7.38–7.42 (t, 4H, J = 7.4 Hz, H-400, H-600, H-4¢¢¢, H-6¢¢¢),
7.45–7.49 (t, 2H, J = 7.4 Hz, H-500, H-5¢¢¢), 7.56–7.60 (d,
br, 1H, J = 16.4 Hz, H ꢁ 30000), 7.78–7.80 (d, 4H,
J = 7.7 Hz, H-300, H-700, H-3¢¢¢, H-7¢¢¢), 7.94 (s, 1H, H-3);
13C NMR (DMSO-d6, 100 MHz): d = 114.3 (CH, C-6),
120.5 (CH, C ꢁ 20000), 122.0 (CH, C-20, C-60), 124.5 (CH, C-
3), 125.7 (C, C-2), 127.6 (CH, C ꢁ 70000), 128.1 (C, C-4),
129.3 (CH, C-30), 130.8 (CH, C-40), 133.6 (CH, C-500, C-
5¢¢¢), 134.2 (C, C ꢁ 40000), 140.7 (C, C-5), 144.1 (CH,
C ꢁ 30000), 151.9 (C, C-10), 155.3 (C, C-1), 164.6 (C, C-100,
C-1¢¢¢), 188.1 (C, C ꢁ 10000 ); ESI–MS for C35H24N2O5: 553
[M?H]?.
2-Acetyl-5-(benzoyloxy)-4-[(E)-2-phenyldiazen-1-
yl]phenyl benzoate (3m)
Reddish brown solid (compound 2m (0.023 M, 5.89 g) was
treated with alkaline 10 % NaOH (10 mL) and benzoyl
chloride (0.023 M, 3.41 mL) under mechanical stirring for
half an hour in an ice bath. The reaction mixture is then
poured in ice-cold water. The solid thus generated was
filtered, dried, and recrystallized from methanol and
resulted in the formation of benzoylated product 3m as a
dark reddish brown solid); Yield: 64 %; m.p. 122–126 ꢁC;
1H NMR (DMSO-d6, 400 MHz): d = 2.62 (s, 3H, CH3),
7.07–7.11 (t, 1H, J = 7.1 Hz, H-40), 7.14 (s, 1H, H-6),
7.17–7.19 (d, 2H, J = 7.1 Hz, H-20, H-60), 7.33–7.37 (t,
2H, J = 7.4 Hz, H-500, H-500’), 7.39–7.41 (d, 4H,
J = 7.4 Hz, H-300, H-700, H-300’, H-700’), 7.46–7.50 (t, 2H,
J = 7.1 Hz, H-30, H-50), 7.85–7.89 (t, 4H, J = 7.4 Hz,
H-400, H-600, H-400’, H-600’), 7.92 (s, 1H, H-3); 13C NMR
(DMSO-d6, 100 MHz): d = 28.7 (CH3, COCH3), 114.2
(CH, C-6), 121.4 (CH, C-400, C-40’’), 122.6 (CH, C-3),
123.5 (CH, C-20, C-60), 126.4 (C, C-2), 127.7 (C, C-4),
130.3 (CH, C-300, C-30’’), 133.0 (CH, C-500, C-50’’), 139.1
(C, C-5), 151.8 (C, C-10), 154.2 (C, C-1), 164.4 (C, C-100,
C-10’’), 198.6 (CO, COCH3); ESI–MS for C28H20N2O5:
465 [M?H]?.
(2E)-1-{2,4-dihydroxy-5-[(E)-2-phenyldiazen-1-
yl]phenyl}-3-phenylprop-2-en-1-one (5m)
Reddish brown solid (synthesized compound 4m
(0.0046 M, 2.54 g) was refluxed with 15 % HCl (30 mL)
solution for half an hour to hydrolyze the ester group. The
contents were poured in ice-cold water and extracted with
ethyl acetate (30 mL). The ester layer was dried over
anhydrous MgSO4 and the solvent was stripped off by
distillation. The obtained solid product was recrystallized
from methanol to give compound 5m as a reddish brown
1
solid.); Yield: 74 %; m.p. 126–128 ꢁC; H NMR (DMSO-
d6, 400 MHz): d = 6.83 (s, 1H, H-3), 6.89–6.91 (t, 2H,
J = 8.1 Hz, H-600, H-800), 6.91–6.95 (d, br, 1H,
J = 16.4 Hz, H-200), 7.11–7.15 (t, 1H, J = 8.1 Hz, H-700),
7.15–7.19 (t, 1H, J = 7.5 Hz, H-40), 7.22–7.24 (d, 2H,
J = 7.5 Hz, H-20, H-60), 7.38–7.40 (d, 2H, J = 8.1 Hz,
H-500, H-900), 7.41–7.45 (t, 2H, J = 7.5 Hz, H-20,H-60),
7.56-7.60 (d, br, 1H, J = 16.4 Hz, H-300), 7.97 (s, 1H, H-6),
9.89 (s, bs, 1H, OH), 10.03 (s, bs, 1H, OH); 13C NMR
(DMSO-d6, 100 MHz): d = 103.7 (CH, C-3), 114.6 (C,
C-1), 117.3 (C, C-5), 121.4 (CH, C-200), 122.6 (CH, C-20,
C-60), 124.8 (CH, C-6), 125.4 (CH, C-700), 127.0 (CH, C-500,
C-900), 128.6 (CH, C-600, C-800), 130.8 (CH, C-40), 134.9 (C,
C-400), 144.2 (CH, C-300), 151.7 (C, C-10), 158.4 (C, C-4),
164.5 (C, C-2), 188.3 (C, C-100); ESI–MS for C21H16N2O3:
345 [M?H]?.
5-(Benzoyloxy)-4-[(E)-2-phenyldiazen-1-yl]-2-[(2E)-3-
phenylprop-2-enoyl]phenyl benzoate (4m)
Dark brown solid (EtOH) (a mixture of synthesized com-
pound 3m (0.0078 M, 3.63 g) and benzaldehyde
(0.0078 M, 0.89 mL) in 5 % potassium hydroxide in eth-
anol (21 mL) was kept at room temperature for 72 h. This
was neutralized with 1:1 cold HCl in cold conditions and
extracted with ether. The ethereal layer was dried over
anhydrous Na2SO4 and evaporation of the solvent gave
solid product. The obtained solid product was recrystal-
lized from methanol to give compound 4m as a dark brown
7-Hydroxy-6-[(E)-2-(4-hydroxyphenyl)diazen-1-yl]-2-
phenyl-4H-chromen-4-one (6a)
Reddish brown solid (this compound was prepared by
oxidative cyclization of synthesized compound (2E)-1-
{2,4-dihydroxy-5-[(E)-2-(4-hydroxyphenyl)diazen-1-yl]
phenyl}-3-phenylprop-2-en-1-one 5a (0.0054 M, 1.88 g)
using N-bromo succinimide (NBS) (10 mol %) as a cata-
lyst in acetonitrile solvent. The mixture of compounds was
1
solid.); Yield: 71 %; m.p. 121–122 ꢁC; H NMR (DMSO-
d6, 400 MHz): d = 6.88–6.90 (t, 1H, J = 7.8 Hz, H-40),
6.90–6.94 (d, br, 1H, J = 16.4 Hz, H ꢁ 20000), 6.97–7.01 (t,
1H, J = 7.9 Hz, H ꢁ 70000), 7.06–7.10 (t, 2H, J = 7.9 Hz,
123

Med Chem Res
refluxed for about 20–30 min, and then after cooling, it was
poured into ice-cold water. The precipitated solid was
collected and recrystallized from methanol was done to
achieve the desired compound 7-hydroxy-6-[(E)-2-(4-
hydroxyphenyl)diazen-1-yl]-2-phenyl-4H-chromen-4-one
6a as a dark reddish brown solid); Yield: 68 %; m.p.
130–132 ꢁC; FT-IR (KBr) (tmax, cm-1) 3,420, 3,052,
1,693, 1,605, 1,495, 1,453; 1H NMR (DMSO-d6,
400 MHz): d = 6.78 (s, 1H, H-3), 6.87–6.89 (d, 2H,
J = 8.1 Hz, H-20, H-60), 7.11 (s, 1H, H-8), 7.19–7.23 (t,
3H, J = 7.3 Hz, H-300, H-400, H-500), 7.30–7.32 (d, 2H,
J = 7.3 Hz, H-200, H-600), 7.37–7.39 (d, 2H, J = 8.1 Hz,
H-30, H-50), 8.36 (s, 1H, H-5), 9.47 (s, bs, 1H, OH), 10.02
(s, bs, 1H, OH); 13C NMR (DMSO-d6, 100 MHz):
d = 110.6 (CH, C-8), 118.6 (C, C-10), 121.8 (C, C-6),
126.5 (CH, C-5), 128.9 (CH, C-200, C-400, C-600), 131.7 (CH,
C-300, C-500), 133.3 (CH, C-20, C-60), 138.4 (C, C-100), 147.6
(C, C-10), 155.4 (C, C-7), 158.7 (C, C-40), 160.9 (C, C-9),
164.5 (C, C-2), 179.3 (C, C-4); ESI–MS: 359 [M?H]?;
Anal. Calcd. for C21H14N2O4: C, 70.39; H, 3.94; N,
7.82 %. Found: C, 70.46; H, 4.04; N, 7.71 %.
J = 8.4 Hz, H-200, H-600), 8.37 (s, 1H, H-5), 9.62 (s, bs, 1H,
OH), 10.18 (s, bs, 1H, OH); 13C NMR (DMSO-d6,
100 MHz): d = 62.7 (CH3, OCH3), 110.5 (CH, C-8), 118.2
(CH, C-30), 120.6 (C, C-6), 125.8 (CH, C-5), 128.4 (C,
C-100), 132.2 (CH, C-200, C-600), 133.7 (C, C-10), 138.5 (CH,
C-40), 145.5 (C, C-20), 155.7 (C, C-7), 158.8 (C, C-400),
160.2 (C, C-9), 164.6 (C, C-2), 180.1 (C, C-4); ESI–MS:
389 [M?H]?; Anal. Calcd. for C22H16N2O5: C, 68.04; H,
4.15; N, 7.21 %. Found: C, 68.12; H, 4.06; N, 7.29 %.
7-Hydroxy-6-[(E)-2-(2-methoxyphenyl)diazen-1-yl]-2-
phenyl-4H-chromen-4-one (6d)
Dark brown solid; Yield: 76 %; m.p. 135–137 ꢁC; FT-IR
(KBr) (tmax, cm-1) 3,447, 3,045, 2,976, 1,702, 1,603,
1,505, 1,460; 1H NMR (DMSO-d6, 400 MHz): d = 3.81 (s,
3H, OCH3), 6.76 (s, 1H, H-3), 7.07–7.11 (t, 1H,
J = 7.4 Hz, H-50), 7.12 (s, 1H, H-8), 7.17–7.21 (t, 1H,
J = 7.7 Hz, H-400), 7.34–7.36 (d, 2H, J = 7.7 Hz, H-200,
H-600), 7.41–7.45 (t, 2H, J = 7.7 Hz, H-300, H-500),
7.45–7.47 (d, 1H, J = 7.4 Hz, H-30), 7.49–7.53 (t, 1H,
J = 7.4 Hz, H-40), 7.82–7.84 (d, 1H, J = 7.4 Hz, H-60),
8.35 (s, 1H, H-5), 9.57 (s, bs, 1H, OH); 13C NMR (DMSO-
d6, 100 MHz): d = 63.5 (CH3, OCH3), 111.2 (CH, C-8),
116.7 (C, C-10), 118.3 (C, C-6), 121.8 (CH, C-50), 126.2
(CH, C-5, C-60), 128.7 (CH, C-300, C-500), 133.6 (C, C-100),
136.5 (CH, C-40), 154.7 (C, C-20), 158.8 (C, C-7), 160.6 (C,
C-9), 164.5 (C, C-2), 179.7 (C, C-4); ESI–MS: 373
[M?H]?; Anal. Calcd. for C22H16N2O4: C, 70.96; H, 4.33;
N, 7.52 %. Found: C, 71.05; H, 4.29; N, 7.64 %.
7-Hydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-phenyldiazen-1-
yl]–4H-chromen-4-one (6b)
Dark reddish brown solid; Yield: 70 %; m.p. 130–131 ꢁC;
FT-IR (KBr) (tmax, cm-1) 3,425, 3,060, 1,690, 1,600,
1,505, 1,465; 1H NMR (DMSO-d6, 400 MHz): d = 6.79 (s,
1H, H-3), 6.89–6.91 (d, 2H, J = 8.2 Hz, H-300, H-500), 7.11
(s, 1H, H-8), 7.16–7.20 (t, 1H, J = 7.5 Hz, H-40),
7.34–7.36 (d, 2H, J = 8.2 Hz, H-200, H-600), 7.37–7.39 (d,
2H, J = 7.5 Hz, H-20, H-60), 7.40–7.44 (t, 2H, J = 7.5 Hz,
H-30, H-50), 8.38 (s, 1H, H-5), 9.85 (s, bs, 1H, OH), 10.9 (s,
bs, 1H, OH); 13C NMR (DMSO-d6, 100 MHz): d = 109.7
(CH, C-8), 117.8 (C, C-10), 120.9 (C, C-6), 125.7 (CH,
C-5), 128.2 (CH, C-30, C-50), 131.5 (CH, C-200, C-600),
132.8 (CH, C-40), 154.5 (C, C-10), 158.3 (C, C-7), 160.5 (C,
C-9), 164.4 (C, C-2), 178.9 (C, C-4); ESI–MS: 359
[M?H]?; Anal. Calcd. for C21H14N2O4: C, 70.39; H, 3.94;
N, 7.82 %. Found: C, 70.47; H, 3.99; N, 7.75 %.
7-Hydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-(3-
methoxyphenyl)diazen-1-yl]-4H-chromen-4-one (6e)
Yellowish brown solid; Yield: 75 %; m.p. 136–138 ꢁC;
FT-IR (KBr) (tmax, cm-1) 3,428, 3,049, 2,964, 1,696,
1,608, 1,496, 1,453; 1H NMR (DMSO-d6, 400 MHz):
d = 3.79 (s, 3H, OCH3), 6.81 (s, 1H, H-3), 6.91–6.93 (d,
2H, J = 7.9 Hz, H-300, H-500), 7.14 (s, 1H, H-8), 7.19–7.21
(d, 1H, J = 6.8 Hz, H-40), 7.34–7.36 (d, 2H, J = 7.9 Hz,
H-200, H-600), 7.45–7.47 (t, 1H, J = 6.8 Hz, H-30),
7.51–7.53 (d, 1H, J = 6.8 Hz, H-20), 7.72 (s, 1H, H-60),
8.40 (s, 1H, H-5), 9.76 (s, bs, 1H, OH), 10.05 (s, bs, 1H,
OH); 13C NMR (DMSO-d6, 100 MHz): d = 63.7 (CH3,
OCH3), 110.9 (CH, C-40), 116.3 (C, C-10), 118.8 (C, C-6),
121.6 (CH, C-20), 126.4 (CH, C-5), 128.7 (C, C-100), 133.5
(CH, C-200, C-600), 137.9 (CH, C-30), 154.7 (C, C-10), 158.7
(C, C-7), 160.9 (C, C-50), 165.2 (C, C-2), 180.3 (C, C-4);
ESI–MS: 389 [M?H]?; Anal. Calcd. for C22H16N2O5: C,
68.04; H, 4.15; N, 7.21 %. Found: C, 68.13; H, 4.21; N,
7.16 %.
7-Hydroxy-6-[(E)-2-(2-hydroxyphenyl)diazen-1-yl]-2-(4-
methoxyphenyl)-4H-chromen-4-one (6c)
Yellowish brown solid; Yield: 67 %; m.p. 143–144 ꢁC;
FT-IR (KBr) (tmax, cm-1) 3,442, 3,062, 2,962, 1,699,
1,608, 1,515, 1,462; 1H NMR (DMSO-d6, 400 MHz):
d = 3.83 (s, 3H, OCH3), 6.78 (s, 1H, H-3), 7.07–7.09 (d,
2H, J = 8.4 Hz, H-300, H-500), 7.09–7.13 (t, 1H,
J = 7.2 Hz, H-50), 7.15 (s, 1H, H-8), 7.17–7.21 (t, 1H,
J = 7.2 Hz, H-40), 7.49–7.51 (d, 1H, J = 7.2 Hz, H-30),
7.72–7.74 (d, 1H, J = 7.2 Hz, H-60), 7.75–7.77 (d, 2H,
123

Med Chem Res
C-20, C-60), 128.8 (CH, C-300, C-500), 131.9 (C, C-100), 133.8
(CH, C-30, C-50), 137.5 (C, C-40), 155.8 (C, C-10), 159.7 (C,
C-7), 161.3 (C, C-9), 165.6 (C, C-2), 180.5 (C, C-4); ESI–
MS: 377.5 [M?H]?; Anal. Calcd. for C21H13ClN2O3: C,
66.94; H, 3.48; N, 7.43 %. Found: C, 66.87; H, 3.59; N,
7.51 %.
7-Hydroxy-2-(4-methoxyphenyl)-6-[(E)-2-phenyldiazen-1-
yl]-4H-chromen-4-one (6f)
Dark brown solid; Yield: 73 %; m.p. 134–135 ꢁC; FT-IR
(KBr) (tmax, cm-1) 3,452, 3,056, 2,983, 1,690, 1,609,
1,513, 1,464; 1H NMR (DMSO-d6, 400 MHz): d = 3.82 (s,
3H, OCH3), 6.77 (s, 1H, H-3), 6.96–6.98 (d, 2H,
J = 8.1 Hz, H-300, H-500), 7.14 (s, 1H, H-8), 7.15–7.19 (t,
1H, J = 7.6 Hz, H-40), 7.34–7.36 (d, 2H, J = 8.1 Hz,
H-200, H-600), 7.37–7.41 (t, 2H, J = 7.6 Hz, H-30, H-50),
7.51–7.53 (d, 2H, J = 7.6 Hz, H-20, H-60), 8.39 (s, 1H,
H-5), 9.83 (s, bs, 1H, OH); 13C NMR (DMSO-d6,
100 MHz): d = 63.2 (CH3, OCH3), 110.5 (CH, C-300,
C-500), 116.7 (C, C-10), 121.3 (C, C-100), 126.5 (CH, C-20,
C-60), 128.2 (CH, C-200, C-600), 133.8 (CH, C-30, C-50),
138.3 (CH, C-40), 154.5 (C, C-10), 158.7 (C, C-7), 160.7 (C,
C-9), 164.7 (C, C-2), 179.3 (C, C-4); ESI–MS: 373
[M?H]?; Anal. Calcd. for C22H16N2O4: C, 70.96; H, 4.33;
N, 7.52 %. Found: C, 71.07; H, 4.39; N, 7.44 %.
6-[(E)-2-(2-chlorophenyl)diazen-1-yl]-7-hydroxy-2-(4-
hydroxyphenyl)-4H-chromen-4-one (6i)
Reddish brown solid; Yield: 74 %; m.p. 144–145 ꢁC; FT-
IR (KBr) (tmax, cm-1) 3,466, 3,049, 1,695, 1,612, 1,508,
1
1,457, 768; H NMR (DMSO-d6, 400 MHz): d = 6.81 (s,
1H, H-3), 6.91–6.93 (d, 2H, J = 8.2 Hz, H-300, H-500), 7.16
(s, 1H, H-8), 7.34–7.36 (d, 2H, J = 8.2 Hz, H-200, H-600),
7.41–7.45 (t, 1H, J = 7.8 Hz, H-50), 7.47–7.51 (t, 1H,
J = 7.8 Hz, H-40), 7.71–7.73 (d, 1H, J = 7.8 Hz, H-30),
8.12–8.14 (d, 1H, J = 7.8 Hz, H-60), 8.42 (s, 1H, H-5),
9.84 (s, bs, 1H, OH), 10.01 (s, bs, 1H, OH); 13C NMR
(DMSO-d6, 100 MHz): d = 111.2 (CH, C-8), 117.6 (C,
C-10), 120.8 (C, C-6), 124.5 (CH, C-60), 126.7 (CH, C-50),
128.7 (C, C-20), 131.5 (CH, C-200, C-600), 136.2 (CH, C-40),
155.7 (C, C-400), 159.5 (C, C-7), 161.9 (C, C-9), 165.3 (C,
C-2), 179.9 (C, C-4); ESI–MS: 393.5 [M?H]?; Anal.
Calcd. for C21H13ClN2O4: C, 64.21; H, 3.34; N, 7.13 %.
Found: C, 64.27; H, 3.23; N, 7.24 %.
7-Hydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-(2-
hydroxyphenyl)diazen-1-yl]-4H-chromen-4-one (6g)
Reddish brown solid; Yield: 66 %; m.p. 139–140 ꢁC; FT-
IR (KBr) (tmax, cm-1) 3,443, 3,042, 1,696, 1,613, 1,518,
1
1,469; H NMR (DMSO-d6, 400 MHz): d = 6.79 (s, 1H,
H-3), 6.91–6.93 (d, 2H, J = 8.0 Hz, H-300, H-500), 7.11-7.15
(t, 1H, J = 7.5 Hz, H-50), 7.16 (s, 1H, H-8), 7.17–7.21 (t,
1H, J = 7.5 Hz, H-40), 7.34–7.36 (d, 2H, J = 8.0 Hz,
H-200, H-600), 7.51–7.53 (d, 1H, J = 7.5 Hz, H-30),
7.72–7.74 (d, 1H, J = 7.5 Hz, H-50), 8.41 (s, 1H, H-5),
9.52 (s, bs, 1H, OH), 10.07 (s, bs, 1H, OH), 10.16 (s, bs,
1H, OH); 13C NMR (DMSO-d6, 100 MHz): d = 110.8
(CH, C-300, C-500), 116.5 (C, C-10), 118.3 (CH, C-30), 121.8
(CH, C-50), 124.6 (CH, C-60), 128.3 (CH, C-200, C-600),
133.6 (CH, C-40), 154.8 (CH, C-20), 158.7 (C, C-7), 160.2
(C, C-9), 164.5 (C, C-2), 178.8 (C, C-4); ESI–MS: 358
[M?H]?; Anal. Calcd. for C21H14N2O4: C, 70.58; H, 3.67;
N, 7.84 %. Found: C, 70.66; H, 3.56; N, 7.78 %.
6-[(E)-2-(4-chlorophenyl)diazen-1-yl]-7-hydroxy-2-(4-
methoxyphenyl)-4H-chromen-4-one (6j)
Reddish brown solid; Yield: 77 %; m.p. 140–141 ꢁC; FT-
IR (KBr) (tmax, cm-1) 3,455, 3,043, 2,982, 1,703, 1,610,
1,518, 1,467, 750; 1H NMR (DMSO-d6, 400 MHz):
d = 3.83 (s, 3H, OCH3), 6.79 (s, 1H, H-3), 6.97–6.99 (d,
2H, J = 8.5 Hz, H-300, H-500), 7.16 (s, 1H, H-8), 7.32–7.34
(d, 2H, J = 8.5 Hz, H-200, H-600), 7.93–7.95 (d, 2H,
J = 7.9 Hz, H-30, H-50), 8.12–8.14 (d, 2H, J = 7.9 Hz,
H-20, H-60), 8.40 (s, 1H, H-5), 9.77 (s, bs, 1H, OH); 13C
NMR (DMSO-d6, 100 MHz): d = 63.9 (CH3, OCH3),
111.5 (CH, C-300, C-500), 117.3 (C, C-10), 120.8 (C, C-6),
124.6 (CH, C-20, C-60), 126.7 (CH, C-200, C-600), 129.8 (CH,
C-30), 137.1 (C, C-40), 154.3 (C, C-7), 159.4 (C, C-4),
161.3 (C, C-9), 165.7 (C, C-2), 180.1 (C, C-4); ESI–MS:
407.5 [M?H]?; Anal. Calcd. for C22H15ClN2O4: C,64.95;
H, 3.72; N, 6.89 %. Found: C,64.99; H, 3.66; N, 6.97 %.
6-[(E)-2-(4-chlorophenyl)diazen-1-yl]-7-hydroxy-2-
phenyl-4H-chromen-4-one (6h)
Dark reddish brown solid; Yield: 79 %; m.p. 137–138 ꢁC;
FT-IR (KBr) (tmax, cm-1) 3,459, 3,038, 1,691, 1,607,
1,509, 1,460, 776; 1H NMR (DMSO-d6, 400 MHz):
d = 6.78 (s, 1H, H-3), 7.15 (s, 1H, H-8), 7.17–7.21 (t, 1H,
J = 7.4 Hz, H-400), 7.34–7.36 (d, 2H, J = 8.2 Hz, H-30,
H-50), 7.41–7.45 (t, 2H, J = 7.4 Hz, H-300, H-500),
7.93–7.95 (d, 2H, J = 7.4 Hz, H-200, H-600), 8.12–8.14 (d,
2H, J = 8.2 Hz, H-20, H-60), 8.38 (s, 1H, H-5), 9.81 (s, bs,
1H, OH); 13C NMR (DMSO-d6, 100 MHz): d = 110.3
(CH, C-8), 117.8 (C, C-10), 120.5 (C, C-6), 124.3 (CH,
7-Hydroxy-6-[(E)-2-(3-nitrophenyl)diazen-1-yl]-2-phenyl-
4H-chromen-4-one (6k)
Dark reddish brown solid; Yield: 84 %; m.p. 153–154 ꢁC;
FT-IR (KBr) (tmax, cm-1) 3,420, 3,025, 1,692, 1,605,
1,500, 1,453, 1,520, 1,370; 1H NMR (DMSO-d6,
400 MHz): d = 6.81 (s, 1H, H-3), 7.16 (s, 1H, H-8),
123

Med Chem Res
7.38–7.40 (d, 2H, J = 7.1 Hz, H-200, H-600), 7.43–7.47 (t,
1H, J = 7.1 Hz, H-300, H-500), 7.47–7.49 (t, 1H,
J = 7.1 Hz, H-400), 7.91–7.93 (d, 1H, J = 6.9 Hz, H-40),
8.17–8.19 (d, 1H, J = 6.9 Hz, H-20), 8.29–8.33 (t, 1H,
J = 6.9 Hz, H-30), 8.41 (s, 1H, H-5), 8.48 (s, 1H, H-60),
9.69 (s, bs, 1H, OH); 13C NMR (DMSO-d6, 100 MHz):
d = 111.6 (CH, C-8), 118.7 (CH, C-60), 121.8 (C, C-6),
126.6 (CH, C-40), 128.3 (CH, C-300, C-500), 129.9 (CH,
C-20), 131.7 (C, C-100), 133.3 (CH, C-30), 151.3 (C, C-50),
154.8 (C, C-7), 160.6 (C, C-9), 164.5 (C, C-2), 168.7 (C,
C-10), 180.9 (C, C-4); ESI–MS: 388 [M?H]?; Anal. Calcd.
for C21H13N3O5: C, 65.12; H, 3.38; N,10.85 %. Found: C,
65.06; H, 3.29; N,10.98 %.
Microbiological method
The following bacterial cultures S. aureus MTCC 3,160, B.
cereus MTCC 430, A. globiformis MTCC 4299, V. cholerae
MTCC 3904, E. coli MTCC 1610, and S. dysenteriae
NICED were used for the present study to evaluate the
antibacterial activity of the synthesized compounds. The
antibacterial activity of newly synthesized compounds was
evaluated according to the guidelines of the Clinical Lab-
oratory Standards Institute (CLSI, 2007), USA using the
agar disk-diffusion method as well as liquid broth assay.
Kirby–Bauer agar diffusion method (antibacterial
assay)
7-Hydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-(4-
In Kirby–Bauer agar diffusion method, the Mueller–Hinton
agar (Hi-Media, Mumbai) was prepared according to the
manufacturer’s instructions; the media was sterilized at
121 ꢁC for 15–20 min and cooled to 50 ꢁC in heating water
bath. Approximately, 20–25 mL of the media was poured
on to the 90 mm sterile Petri plates, and it was spreaded in
anticlockwise motion. It was then kept for 10–15 min at
room temperature for solidification. Plates were inverted
and left for overnight in incubator to check the sterility of
the plates. Overnight grown cultures (100 lL) of S. aureus
MTCC 3160, B. cereus MTCC 430, A. globiformis MTCC
4299, V. cholerae MTCC 3904, E. coli MTCC 1610, and S.
dysenteriae NICED were spreaded over the agar surface of
the MHA plates. Whatman no. 1, filter paper disk
(approximately 6 mm in diameter) impregnated with dif-
ferent synthesized compounds (0.2 mg/mL in DMSO) were
gently placed on the surface of plates using sterile forceps.
The plates were inverted and incubated at 37 ꢁC for
18–24 h. The appearance of clear zone of growth inhibition
surrounding the disk was measured using antibiotic zone
measurement scale (Hi-media) and tabulated. All the tests
were performed in duplicate, and the average was taken as
the final reading. The standard antibiotics, ampicillin and
ciprofloxacin, were used as the positive control whereas
DMSO alone as the negative control for all the selected
bacteria.
nitrophenyl)diazen-1-yl]-4H-chromen-4-one (6l)
Dark reddish brown solid; Yield: 81 %; m.p. 150–152 ꢁC;
FT-IR (KBr) (tmax, cm-1) 3,426, 3,032, 1,698, 1,612,
1,506, 1,463, 1,531, 1,379; 1H NMR (DMSO-d6,
400 MHz): d = 6.81 (s, 1H, H-3), 6.91–6.93 (d, 2H,
J = 8.3 Hz, H-300, H-500), 7.15 (s, 1H, H-8), 7.34–7.36 (d,
2H, J = 8.3 Hz, H-200, H-600), 7.40–7.42 (d, 2H,
J = 8.0 Hz, H-20, H-60), 8.26–8.28 (d, 2H, J = 8.0 Hz,
H-30, H-50), 8.39 (s, 1H, H-5), 9.72 (s, bs, 1H, OH), 10.6 (s,
bs, 1H, OH); 13C NMR (DMSO-d6, 100 MHz): d = 111.8
(CH, C-300, C-500), 118.9 (C, C-10), 120.9 (C, C-6), 121.7
(CH, C-20, C-60), 123.9 (C, C-100), 126.7 (CH, C-30, C-50),
128.4 (CH, C-5), 131.5 (CH, C-200, C-600), 151.5 (C, C-40),
155.2 (C, C-10), 160.7 (C, C-9), 164.3 (C, C-2), 168.5 (C,
C-400), 180.6 (C, C-4); ESI–MS: 404 [M?H]?; Anal.
Calcd. for C21H13N3O6: C, 62.53; H, 3.25; N, 10.42 %.
Found: C, 62.67; H, 3.33; N, 10.31 %.
7-Hydroxy-2-phenyl-6-[(E)-2-phenyldiazen-1-yl]-4H-
chromen-4-one (6m)
Reddish brown solid; Yield: 78 %; m.p. 127–128 ꢁC; FT-
IR (KBr) (tmax, cm-1) 3,437, 3,036, 1,696, 1,606, 1,501,
1
1,457; H NMR (DMSO-d6, 400 MHz): d = 6.78 (s, 1H,
H-3), 6.89–6.91 (d, 2H, J = 7.6 Hz, H-200, H-600),
7.09–7.13 (t, 2H, J = 7.6 Hz, H-300, H-500), 7.14 (s, 1H,
H-8), 7.17–7.21 (t, 1H, J = 7.2 Hz, H-40), 7.32–7.34 (d,
2H, J = 7.2 Hz, H-20, H-60), 7.47–7.51 (t, 2H, J = 7.2 Hz,
H-30, H-50), 7.68–7.72 (t, 1H, J = 7.6 Hz, H-400), 8.38 (s,
1H, H-5), 9.65 (s, bs, 1H, OH); 13C NMR (DMSO-d6,
100 MHz): d = 108.8 (CH, C-8), 118.6 (C, C-10), 120.7
(C, C-6), 126.3 (CH, C-5), 128.6 (CH, C-200,C-400, C-600),
131.3 (CH, C-300), 133.1 (C, C-100), 138.3 (CH, C-40), 153.7
(C, C-10), 159.3 (C, C-7), 161.6 (C, C-9), 165.8 (C, C-2),
178.5 (C, C-4); ESI–MS: 343 [M?H]?; Anal. Calcd. for
C21H14N2O3: C, 73.68; H, 4.12; N, 8.18 %. Found: C,
73.66; H, 3.96; N, 8.28 %.
Antioxidant assay
DPPH (0.004 g) was dissolved in MeOH (100 mL) to
obtain a concentration of 100 lM. Serial dilutions were
carried out with stock solutions (150 lM) of the com-
pounds in methanol to obtain final concentrations of 1, 2, 4,
8, 12, 16, 20, 24, and 30 lM in cuvette. Diluted solutions
of compound and ascorbic acid (AA) (2 mL each) were
mixed with methanolic DPPH solution (1 mL, 100 lM)
and allowed to stand for 120 min for any reaction to occur.
The absorbance was recorded at 517 nm on Shimadzu
123

Med Chem Res
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Pharmaspec UV–Vis spectrophotometer (model-1700).
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age absorbance was noted for each concentration. The IC50
value, which is the concentration of the test compound that
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Acknowledgments We are grateful to the SAIF, Chandigarh and
Director, Defence R&D Establishment, Gwalior for providing facil-
ities for spectro-analytical studies and antibacterial evaluation of the
synthesized compounds.
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