Synthesis, antimicrobial, and antioxidant activity of benzofuran barbitone and benzofuran thiobarbitone derivatives

Article abstract of DOI:10.1007/s00044-013-0892-x

The synthesis of novel series of benzofuran derivatives, containing barbitone moiety, 5-[(2/4-substitutedphenyl)(5-substituted-1-benzofuran-2-yl) methylidene]pyrimidin-2,4,6(1H,3H,5H)-trione (4a-i) and thiobarbitone moiety, 5-[(2/4-substitutedphenyl)(5-su

Full text of DOI:10.1007/s00044-013-0892-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:3065–3081
DOI 10.1007/s00044-013-0892-x
ORIGINAL RESEARCH
Synthesis, antimicrobial, and antioxidant activity of benzofuran
barbitone and benzofuran thiobarbitone derivatives
•
R. Kenchappa Yadav D. Bodke B. Asha
•
•
•
Sandeep Telkar M. Aruna Sindhe
Received: 5 October 2013 / Accepted: 12 December 2013 / Published online: 27 December 2013
Ó Springer Science+Business Media New York 2013
Abstract The synthesis of novel series of benzofuran
derivatives, containing barbitone moiety, 5-[(2/4-substi-
tutedphenyl)(5-substituted-1-benzofuran-2-yl) methylidene]
pyrimidin-2,4,6(1H,3H,5H)-trione (4a–i) and thiobarbitone
moiety, 5-[(2/4-substitutedphenyl)(5-substituted-1-benzo-
furan-2-yl)methylidene]-2-thioxodihydropyrimidin-4,6(1H,
5H)-dione (5a–i) have been reported. The target com-
pounds (4a–i) and (5a–i) were synthesized by the Knoe-
venagel condensation of (5-substituted-1-benzofuran-2-yl)
(2/4-substitutedphenyl) methanone (3a–i) with barbituric
acid and thiobarbituric acid, respectively, in acid medium.
These compounds were screened for the antimicrobial and
antioxidant activities. From antimicrobial activity results it
was found that compounds 4a, 5a, 4c, and 5c displayed
good antibacterial and antifungal activity against all tested
strains. Further, the synthesized compounds were studied
for docking on the enzyme, Glucosamine-6-phosphate
synthase and the compounds 4c and 5c have emerged has
an active antimicrobial agent with least binding energy
(-5.27 and -4.85 kJ mol-1). Compounds 4e, 4f, 5e, and
5f showed promising free radical scavenging activity and
compounds 5a and 5b showed good chelating ability with
Fe2? ions.
Keywords Benzofuran ꢀ Barbiturates ꢀ
Knoevenagel condensation ꢀ Antimicrobial ꢀ Antioxidant ꢀ
Docking
Introduction
The resistance of pathogenic bacteria toward available
antibiotics is rapidly becoming a major worldwide prob-
lem. On the other hand, primary and opportunistic fungal
infections continue to grow rapidly because of the
increased number of immune compromised patients
(Grange and Zumla, 2002; Guzeldemirci and Kucukb-
asmaci, 2010). In order to combat this new problem, novel,
structurally diverse antibiotic compounds are required
(Francesca, 2011).
Benzofuran heterocycles exhibit a broad range of bio-
logical activities. Recently, compounds containing metha-
none linkage between the benzofuran at C-2 position and
aromatic rings have been investigated and reported as
antimicrobials (Koca et al., 2005; Kirilmis et al., 2008;
Bondock et al., 2011; Jiang et al., 2011; Liu et al., 2012;
Rajanarendar et al., 2013), novel SIRT1 inhibitors (Wu
et al., 2013), anticancer (Parekh et al., 2011), and antiox-
idant agents (Karatas et al., 2006). Also, benzofuran-2-
yl(phenyl)methanone analogs displayed anti-tumor (Agat-
suma et al., 2004), antitubercular (Manna and Agrawal,
2010, 2011), anti-inflammatory, analgesic (Rajanarendar
et al., 2013), CYP19 inhibitory activity, molecular docking
(Saberi et al., 2006), and cytotoxicity properties (Bigler
et al., 2007; Saberi et al., 2006).Cloridarol, a 2-substituted
benzofuran is used for treatment of lipidemia and has
R. Kenchappa ꢀ Y. D. Bodke (&) ꢀ M. Aruna Sindhe
Department of P.G. Studies and Research in Industrial
Chemistry, Jnana Sahyadri, Kuvempu University,
Shankaraghatta, Shivamogga 577451, Karnataka, India
e-mail: yd_bodke@yahoo.co.in; ydbodke@gmail.in
B. Asha
Department of P.G. Studies and Research in Applied Botany,
Jnana Sahyadri, Kuvempu University, Shankaraghatta,
Shivamogga 577451, Karnataka, India
S. Telkar
Department of P.G. Studies and Research in Biotechnology,
Jnana Sahyadri, Kuvempu University, Shankaraghatta,
Shivamogga 577451, Karnataka, India
123

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Med Chem Res (2014) 23:3065–3081
anticoagulant, antifungal and aromatase inhibiting activi-
ties (Ghelardoni et al., 1981). Amiodarone, a benzofuran
derivative was shown to have antiarrhythmic property
(Singh and Vaughan, 1970).
(Basavaraja et al., 2010; Vijaya Laxmi et al., 2011), anti-
inflammatory (Holy et al., 2002), antiviral activity(Andres
and Marhold, 1996) as well as in agrochemical and vet-
erinary products (El-Gaby et al., 2006; Aly and Kamal,
2012; Dhorajiya et al., 2012).
Barbituric acid (BA) derivatives are versatile reagents,
capable of condensing with a wide range of carbonyl
compounds (Vijaya Laxmi et al., 2012; Mathew et al.,
2012; Moskvin et al., 2002) and the reaction of BA with
carbonyl compounds was studied nearly 40 years ago
(Rezende et al., 2001; Reddy and Nagaraj, 2007). The
condensed barbiturates and thiobarbiturates possesses dif-
ferent pharmacological profile such as antimicrobial,
selective cell adhesion inhibitors, antioxidant, and DNA
cleavage activities (Sangani et al., 2006; Harriman et al.,
2008; Biradar et al., 2010; Mathew et al., 2012). They also
possess chemical and pharmacological importance due to
their anti-neoplastic, anticancer, antitumor, antitubercular
Therefore, it is envisaged that chemical entities with
benzofuran and barbituric/thiobarbituric moieties would
result in compounds of interesting biological activities. In
view of these findings, we have attempted to incorporate
these two biologically active components together and to
investigate a possible additive effect of these rings on
biological activity.
Earlier, our research group has synthesized different
derivatives of benzofuran bearing aryl substituents at the
C-2 position through a methanone linkage (Kenchappa
et al., 2013a, b; Sheelavanth et al., 2013; Chandrashekar
et al., 2013; Venkatesh et al., 2010a, b). These results
Scheme 1 Synthesis of
benzofuran barbitone (4a–i)
and benzofuran thiobarbitone
(5a–i) derivatives
O
Br
R
CHO
OH
R2
+
R1
2(a-c)
1(a-c)
CH3CN/Reflux
O
K2CO3
R
O
R2
R1
3(a-i)
O
O
O
O
Reflux
AcOH
Reflux
AcOH
HN
NH
HN
NH
O
R1
S
R1
R2
O
R2
O
R
R
O
O
O
O
NH
NH
S
N
H
N
H
O
5(a-i)
4(a-i)
R2
H
H
H
H
H
R2
H
Comp
4a/5a
4b/5b
4c/5c
4d/5d
4e/5e
R
R1
Br
Comp
4f/5f
R
R1
H
OH
H
OCH3
H
H
OCH3
Br
Br
Br
Br
4g/5g
4h/5h
4i/5i
Br
Br
OH
Br
OH
H
OCH3
Br
H
123

Med Chem Res (2014) 23:3065–3081
3067
R
CHO
O
R1
O
Br
R
CHO
OH
R2
K2CO3
+
O
R2
R1
R2
OH
O
H
O
R
R1
X
-H2O
HN
NH
H
O
O
R
R
O:
O
R
OH
R2
AcOH
O
O
O
R2
R2
CH3COO-
R1
R1
H H
O
O
R1
+
HN
NH
-H2O
X
H
N
X
O
NH
O
R
O
R2
R1
- O, S
X
Comp
4a/5a
4b/5b
4c/5c
4d/5d
4e/5e
R
R1
R2
H
H
H
H
H
Comp
4f/5f
R
R1
R2
H
Br
OH
H
OCH3
H
H
H
OCH3
Br
Br
Br
Br
4g/5g
4h/5h
4i/5i
Br
Br
OH
Br
H
OCH3
Br
OH
H
Fig. 1 Proposed reaction mechanism of synthesized compounds (4a–i) and (5a–i)
encouraged us to extend the scope of this methodology to
build new systems for improving the spectrum of activity
of these scaffolds.
The present study described the Knoevenagel condensa-
tion of (5-substituted-1-benzofuran-2-yl) (2/4-substituted-
phenyl) methanone (3a–i) with barbituric/thiobarbituric acid.
Initially, (5-substituted-1-benzofuran-2-yl) (2/4-substituted-
phenyl) methanone undergoes protonation by acetic acid. The
protonated form of the methanone then facilitates the addition
reaction toward a nucleophile. The acetate ion which was
formed in the former step can accept a proton from the
methylene unitof barbituric/thiobarbituric acid and generate a
carbanion. The electron-rich carbanion attacks on the electron
deficient carbonyl carbon of (5-substituted-1-benzofuran-2-
yl) (2/4-substitutedphenyl) methanone to form an adduct,
which upon dehydration furnished the target compounds. The
proposed mechanism has been described in Fig. 1.
Result and discussion
Chemistry
The reaction pathway used for the synthesis of title com-
pounds (4a–i) and (5a–i) have been shown in Scheme 1. The
key intermediates, 5-substituted-1-benzofuran-2-yl) (2/4-
substitutedphenyl) methanone (3a–i) were synthesized by a
known method (Stille et al., 1996; Mahboobi et al., 2007).
123

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Med Chem Res (2014) 23:3065–3081
Pharmacology
Table 1 Characterization data of synthesized compounds 4a–i and
5a–i
R1
R2
Yield (%)
Mol. wt.
In vitro antibacterial and antifungal activity
Compd.
R
4a
4b
4c
4d
4e
4f
H
Br
H
79
90
86
83
85
76
91
75
83
79
89
77
84
76
81
75
87
85
410
362
488
440
426
378
410
488
426
426
378
504
456
442
394
426
504
442
Though, we have many synthetic drugs in the market, the
bacterial mutations are making them resistance. To over-
come this problem, there is an urgent need to discover the
new and specific enzyme targeting novel synthetic mole-
cules. In view of this, the compounds synthesized in the
present investigation (4a–i) and (5a–i) were evaluated for
their antimicrobial activity as primary screening at three
different concentrations and the results have been dis-
played in Tables 2 and 3. The antimicrobial activity was
carried out against five bacterial strains Pseudomonas sy-
ringae, Salmonella tyhphi, Bacillus subtilis, Klebsiella
pneumonia, Escherichia coli, and four fungal strains As-
perigillus flavous, Candida albicans, Microspora griseus,
and Asperigillus terus.
H
OCH3
Br
H
Br
Br
OH
OH
H
H
OCH3
Br
H
H
OCH3
H
H
4g
4h
4i
Br
Br
Br
H
Br
OH
H
H
H
5a
5b
5c
5d
5e
5f
Br
H
OCH3
Br
H
Br
Br
OH
OH
H
H
OCH3
Br
H
H
The investigation of antimicrobial screening revealed
that, test compounds showed varying degree of activity
against all the tested micro-organisms. The zone of inhi-
bition shown by the test compounds against bacterial and
fungal strains has shown in Fig. 2a, b.
OCH3
H
H
5g
5h
5i
Br
Br
Br
Br
OH
H
H
Further, the compounds which showed good activity in
primary screening were assessed by minimum inhibitory
concentration (MIC) at different concentrations to quantify
the antimicrobial potency of the compounds. The results of
MIC values of antimicrobial activity are given in Table 4.
From literature survey it is clear that, the structural and
electronic parameters may have greater impact in varying
the efficacy of antimicrobial activity (Tomasz et al., 2011;
Stefania et al., 2012; Stefania et al., 2012; Lopez et al.,
2001; Sharma et al., 2004; Dhorajiya et al., 2012).
A close investigation of the MIC values indicates that all
the compounds exhibited a varied degree of MIC
(11.38–199.10 lmol/L) of antibacterial activity against all
the tested bacterial strains. The compounds 4c, 5c having
two bromo substituents on C-5 of benzofuran and C-4 of
phenyl ring, respectively were found to exhibit excellent
antibacterial activity against all the tested bacterial strains
with MIC value 29.76–31.96 lmol/L. Compounds 4a, 5a
having –Br substituent on C-4 position of the aryl ring
showed very good ability to inhibit S. tyhphi at MIC
36.61–37.92 lmol/L; the same compounds showed good
activity against P. syringae with MIC 37.20–38.50 lmol/L.
In case of compounds 4e and 5e, which are having hydroxyl
and bromo-substituent, exhibited moderate to good activity
against S. tyhphi with MIC value 36.08–36.73 lmol/L.
Compounds 4d and 5d were inactive against K. pneumoniae,
P. syringae, and E. coli.
The structures of synthesized compounds were estab-
lished on the basis of spectral data. In IR spectra of com-
pounds, the characteristic absorption bands between 1670
and 1710 cm-1 confirmed the presence of C=O group of
BA. In thiobarbiturates (TBA), C=S stretching frequency
was observed between 1395 and 1452 cm-1. Bands in the
region 3230–3345 cm-1 attributed to NH group of the BA
and TBA. The broad absorption band observed at
3425–3465 cm-1 corresponds to OH group. 1H NMR
spectrum of compound 5d showed two singlets at d 10.03
and 10.61 ppm due to two NH protons of TBA, the mul-
tiplet between d 7.13 and 8.07 ppm were assigned to aro-
matic protons. The singlet observed at d 3.88 ppm
corresponds to OCH3 protons. In compound 5f, the –OH
proton on the benzofuran ring exhibited as broad singlet at
d 5.8 ppm and a singlet at d 3.72 ppm corresponding to
OCH3 protons. Further, 13C NMR spectrum of compound
5d confirmed the proposed structure by appearance of
signal at d 178.2 ppm due to the C=S carbon and another
signal at d 167.1 ppm correspond to C=O carbon of TBA
ring. Another signal at d 55.9 ppm attributed to OCH3
carbon. In compound 4d, the signal at d 165.0 ppm was
assignable to C=O carbon and the other signals are in well
agreement with the suggested structures. Mass spectrum of
compound 5d displayed a molecular ion peak at m/z 457
[M?] corresponding to the molecular mass of the com-
pound along with an isotopic peak at m/z 459 [M?2]. The
physical and analytical data of synthesized compounds
(4a–i) and (5a–i) have been given in Table 1.
The MIC of antifungal activity of title compounds indi-
cated that, the compounds 4c and 5c exhibited equipotent
activity against all the tested fungal strains with MIC value
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Med Chem Res (2014) 23:3065–3081
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Table 2 Antibacterial activity data of synthesized compounds 4a–i and 5a–i
Comp. no.
Conc. M (Molar)
Zone of inhibition in mm (mean SD) n = 3
P.s SD
S.t SD
B.s SD
K.p SD
E.c SD
4a
3.9
1.5
0.6
4.4
1.7
0.7
3.2
1.3
0.5
3.6
1.4
0.5
3.7
1.5
0.5
4.2
1.7
0.6
3.9
1.5
0.6
3.2
1.3
0.5
3.7
1.5
0.5
3.7
1.5
0.5
4.2
1.7
0.6
3.1
1.2
0.5
3.5
1.4
0.5
3.6
1.4
0.5
4.0
1.6
0.6
14 0.2
11 0.3
09 0.1
09 0.2
08 0.3
–
13 0.1
12 0.2
10 0.3
10 0.1
08 0.2
–
16 0.2
14 0.1
11 0.3
10 0.2
06 0.2
–
16 0.3
14 0.1
13 0.1
11 0.3
05 0.1
02 0.1
17 0.3
15 0.1
14 0.1
13 0.3
12 0.1
10 0.1
15 0.3
14 0.1
12 0.1
11 0.3
05 0.1
02 0.1
09 0.3
05 0.1
02 0.1
12 0.3
08 0.1
05 0.1
08 0.3
05 0.1
02 0.1
17 0.3
15 0.1
13 0.1
11 0.3
09 0.1
05 0.1
17 0.3
16 0.1
13 0.1
15 0.3
13 0.1
12 0.1
16 0.3
14 0.1
13 0.1
–
13 0.2
12 0.3
10 0.1
–
4b
4c
4d
4e
4f
–
–
14 0.2
11 0.3
10 0.1
11 0.2
10 0.3
08 0.1
13 0.2
10 0.3
09 0.1
09 0.2
06 0.3
–
14 0.1
13 0.2
11 0.3
13 0.1
11 0.2
06 0.3
13 0.1
11 0.2
10 0.3
–
16 0.2
14 0.1
12 0.3
12 0.2
10 0.1
09 0.3
15 0.2
13 0.1
10 0.3
09 0.2
06 0.2
–
14 0.2
13 0.3
10 0.1
12 0.2
10 0.3
08 0.1
13 0.2
11 0.3
10 0.1
–
–
–
–
–
4g
4h
4i
11 0.2
08 0.3
05 0.1
11 0.2
09 0.3
06 0.1
10 0.2
08 0.3
–
11 0.1
08 0.2
06 0.3
12 0.1
09 0.2
06 0.3
09 0.1
08 0.2
06 0.3
14 0.1
13 0.2
11 0.3
10 0.1
04 0.2
–
11 0.2
07 0.1
03 0.3
11 0.2
10 0.1
03 0.3
10 0.2
–
08 0.2
07 0.3
02 0.1
10 0.2
10 0.3
02 0.1
07 0.2
07 0.3
-1
–
5a
5b
5c
5d
5e
5f
13 0.2
11 0.3
09 0.1
09 0.2
07 0.3
04 0.3
13 0.2
12 0.3
10 0.1
12 0.2
11 0.3
09 0.1
13 0.2
10 0.3
09 0.1
08 0.2
07 0.3
05 0.3
17 0.2
14 0.1
12 0.3
09 0.2
08 0.1
04 0.1
17 0.2
14 0.1
11 0.3
16 0.2
13 0.1
10 0.3
16 0.2
14 0.1
11 0.3
09 0.2
07 0.1
03 0.1
14 0.2
12 0.3
09 0.1
10 0.2
04 0.3
03 0.1
15 0.2
12 0.3
11 0.1
13 0.2
11 0.3
09 0.1
14 0.2
13 0.3
10 0.1
09 0.2
04 0.3
01 0.1
15 0.1
13 0.2
12 0.3
13 0.1
12 0.2
11 0.3
14 0.1
12 0.2
10 0.3
10 0.1
–
–
–
–
123

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Med Chem Res (2014) 23:3065–3081
Table 2 continued
Comp. no.
Conc. M (Molar)
Zone of inhibition in mm (mean SD) n = 3
P.s SD
S.t SD
B.s SD
K.p SD
E.c SD
5g
3.7
1.5
0.5
3.1
1.2
0.5
3.6
1.4
0.5
2.7
1.1
0.4
–
10 0.2
09 0.3
–
12 0.1
09 0.2
06 0.3
12 0.1
11 0.2
09 0.3
10 0.1
08 0.2
07 0.3
16 0.1
15 0.2
13 0.3
00
13 0.2
10 0.1
05 0.3
15 0.2
11 0.1
–
13 0.3
09 0.1
–
12 0.2
09 0.3
06 0.1
12 0.2
10 0.3
08 0.1
10 0.2
05 0.3
03 0.1
16 0.2
14 0.3
12 0.1
00
5h
12 0.2
10 0.3
–
14 0.3
10 0.1
09 0.1
12 0.3
08 0.1
04 0.1
19 0.3
17 0.1
16 0.1
00
5i
10 0.2
06 0.3
03 0.3
15 0.2
13 0.3
11 0.1
00
11 0.2
09 0.1
–
Streptomycin
18 0.2
16 0.1
13 0.3
00
Control (DMF)
Each value is expressed as mean SD of three replicates for zone of inhibition
P.s Psedomonas syringae, S.t Salmonella typhi, B.s Bacillus subtilis, K.p Klebsiella pneumoniae, E.c Escherichia coli, SD standard deviation
12.50–66.49 lmol/L. Compounds 4a and 5a showed
excellent activity against the tested organisms with MIC
value 14.90–29.92 lmol/L. In case of compounds 4e and 5e
which are having hydroxyl and bromo substituent exhibited
moderate activity against the tested fungal strains with MIC
value 15.37–31.22 lmol/L.
hexamine biosynthesis, converting D-fructose-6-phosphate
(Fru-6-P) into D-glucosamine 6-phosphate (GlcN-6-P) using
glutamine as the ammonia source.
Automated docking was used to assess the orientation of
inhibitors bound in the active pockets of GlcN-6-P synthase.
The molecular docking of ligand molecules 4a, 4c, 4e, 5a,
5c, and 5e with GlcN-6-P synthase revealed that all the tested
ligand molecules showed encouraging binding energy and
the compounds have exhibited the bonding with one or the
other amino acids in the active pockets as shown in Fig. 3.
Among the six molecules, 4a, 4c, 4e, 5a, 5c, and 5e, the
docking of GlcN-6-P synthase with compounds 4c and 5c was
found with least binding energy (-5.27 and -4.85 kJ mol-1).
Compound 4c establishes three hydrogen bonds between
barbitone oxygen and with gln 348, ser 349 and thr 352 amino
acids in the active site of the target protein with minimum bond
From the structure–antimicrobial activity relationship of
the synthesized compounds, it revealed that, to evaluate the
SAR studies, the effect of structural changes in the target
compounds (4a–i) and (5a–i) play an important role.
Regarding the impact of substituents on the benzofuran ring
and substituents on ortho and para position of aryl ring, it was
observed that electron withdrawing groups in the ortho
position of benzofuran ring and in the para position of aryl
ring have a tendency to increase the potency. Nevertheless,
when the –Br group is in o-position of aryl ring (4g, 4i, 5g, and
5i), wefound decreaseinactivity, suggestingthatthe presence
of a group in the o-position of the aryl ring could introduce
important steric effects. Interestingly, compounds 4h and 5h
exhibited moderate activity even though the substituent
present on the o-position, this could be due to the influence of
electron withdrawing group present on the C-5 of benzofuran
ring. Compounds containing electron donating groups (4f, 4i,
5f and 5i) found to weaken the antimicrobial activity. How-
ever, when –Br group is introduced to o-position of aryl ring
(4i and 5i), the activity did not change significantly.
˚
length (2.118, 2.207, and 2.195 A). Compound 5c establishes
two hydrogen bonds between thiobarbitone oxygen with gln
348 and thr 352 amino acids with bond length (2.149, 2.105,
˚
and 2.195 A) with the highest affinity and hence is the best
dock conformation. In in vitro studies too, compound 4c and
5c have emerged has an active antimicrobial agent against all
tested organisms. Molecular docking results of synthesized
compounds are given in Table 5.
Antioxidant activity
In correlation to in vitro antimicrobial activity, it thought
worthwhile to carryout in silico studies to predict the binding
affinity and orientation at the active site of the receptor.
Glucosamine-6-phosphate synthase (L-glutamine: D-fructose-
6-phosphate amino transferase) catalyze the first step in
Free radical scavenging activity by DPPH method
All the synthesized compounds were screened for their
radical scavenging activity by DPPH method. The freshly
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Med Chem Res (2014) 23:3065–3081
3071
Table 3 Antifungal activity data of synthesized compounds 4a–i and
5a–i
Table 3 continued
Comp. no. Conc.
Zone of inhibition in mm (mean SD)
Comp. no. Conc.
Zone of inhibition in mm (mean SD)
(mg/mL) n = 3
(mg/mL) n = 3
A.f SD C.a SD M.g SD A.t SD
A.f SD C.a SD M.g SD A.t SD
1.5
0.5
08 0.3 09 0.2 09 0.1 10 0.1
4a
4b
4c
4d
4e
4f
3.9
1.5
0.6
4.4
1.7
0.7
3.2
1.3
0.5
3.6
1.4
0.5
3.7
1.5
0.5
4.2
1.7
0.6
3.9
1.5
0.6
3.2
1.3
0.5
3.7
1.5
0.5
0.37
1.5
0.5
4.2
1.7
0.6
3.1
1.2
0.5
3.5
1.4
0.5
3.6
1.4
0.5
4.0
1.6
0.6
3.7
12 0.2 17 0.1 16 0.2 18 0.3
10 0.3 16 0.2 15 0.1 16 0.1
09 0.1 14 0.3 14 0.3 15 0.1
07 0.2 09 0.1 09 0.2 10 0.3
–
–
06 0.3 09 0.1
5h
5i
3.1
1.2
0.5
3.6
1.4
0.5
10 0.2 14 0.1 14 0.2 14 0.3
08 0.3 12 0.2 13 0.1 13 0.1
06 0.1 11 0.3
–
10 0.1
06 0.3 06 0.2 06 0.1
02 0.2
–
–
09 0.2 12 0.1 10 0.2 12 0.3
06 0.3 08 0.2 08 0.1 08 0.1
–
–
12 0.2 18 0.1 17 0.2 19 0.3
11 0.3 16 0.2 16 0.1 17 0.1
08 0.1 15 0.3 14 0.3 16 0.1
12 0.2 17 0.1 16 0.2 17 0.3
10 0.3 15 0.2 15 0.1 16 0.1
09 0.1 12 0.3 13 0.3 15 0.1
12 0.2 17 0.1 17 0.2 18 0.3
11 0.3 16 0.2 15 0.1 17 0.1
09 0.1 13 0.3 14 0.3 15 0.1
07 0.2 08 0.1 07 0.2 06 0.3
–
03 0.3 06 0.3 04 0.1
Fluconazole 5.2
14 0.2 20 0.1 19 0.2 22 0.3
12 0.3 19 0.2 17 0.1 19 0.1
10 0.1 18 0.3 16 0.3 18 0.1
2.0
0.8
Control
(DMF)
–
00
00
00
00
Each value is expressed as mean SD of three replicates for zone of
inhibition
A.f Asperigillus flavous, C.a Candida albicans, M.g Microspora
griseus, A.t Asperigillus terus, SD standard deviation
–
–
–
04 0.1
–
–
–
prepared solution exhibits a deep blue color with the
absorption maximum at 517 mm. This purple color gen-
erally fades when antioxidant is present in the solution. All
compounds have exhibited varied free radial scavenging
capacity by comparison with the standard butylated
hydroxytoluene (BHT). The variation exhibited in DPPH
scavenging activity could be attributed to the effect of
different substituents. It has been reported that, the com-
pounds substituted with phenolic hydroxyl group has the
high potential for scavenging radicals (Hatano et al., 1989;
Sawa et al., 1999; Babita et al., 2011). Phenolic com-
pounds donate hydrogen to reactive radicals and break the
chain reaction of lipid oxidation at the initiation step
(Gu¨lcin et al., 2004). Thus, the number and configuration
of hydrogen donating hydroxyl groups are the main
structural features that influence the radical scavenging
activity (Cao et al., 1997; Pannala et al., 2001). On the
other hand, presence of two amino groups can also donate
their free electrons to the reactive ions, making them
stable.
02 0.2
4g
4h
4i
11 0.2 14 0.1 13 0.2 14 0.3
09 0.3 12 0.2 10 0.1 12 0.1
06 0.1
–
–
–
11 0.2 15 0.1 13 0.2 15 0.3
09 0.3 13 0.2 10 0.1 12 0.1
07 0.1 06 0.3
–
10 0.1
10 0.2 10 0.1 11 0.2 12 0.3
07 0.3 09 0.2 09 0.1 10 0.1
–
–
–
03 0.1
5a
5b
5c
5d
5e
5f
13 0.2 18 0.1 17 0.2 19 0.3
11 0.3 16 0.2 15 0.1 17 0.1
08 0.1 15 0.3 14 0.3 15 0.1
07 0.2 10 0.1 08 0.2 10 0.3
05 0.3 05 0.2 06 0.1 06 0.1
01 0.3 02 0.3 01 0.3 02 0.1
13 0.2 18 0.1 18 0.2 20 0.3
10 0.3 17 0.2 16 0.1 17 0.1
09 0.1 16 0.3 14 0.3 16 0.1
12 0.2 16 0.1 15 0.2 16 0.3
09 0.3 15 0.2 14 0.1 15 0.1
08 0.1 14 0.3 13 0.3 14 0.1
13 0.2 17 0.1 17 0.2 18 0.3
10 0.3 16 0.2 15 0.1 17 0.1
08 0.1 14 0.3 13 0.3 16 0.1
The activity results of the newly synthesized compounds
are represented in Fig. 4. Among the tested compounds,
compounds substituted with hydroxyl group (4e, 4f, 4i, 5e,
5f, and 5i) at C-5 position of benzofuran ring displayed
potent DPPH free radical scavenging activity with the lowest
IC50 value (128.87–153.89 lmol/L). The lone pair of elec-
trons present on the ring heteroatom along with electron
donating group (–OH) may be responsible for blocking the
DPPH free radical by donating electron pair, and hence
fading the color. We also observed that, absence of hydroxyl
07 0.2
05 0.3
–
–
–
–
09 0.2 11 0.3
06 0.1 07 0.1
–
03 0.1
5g
10 0.2 12 0.1 12 0.2 12 0.3
123

3072
Med Chem Res (2014) 23:3065–3081
Fig. 2 a. Zone of inhibition of compound 4c against Salmonella typhi (bacteria). b. Zone of inhibition of compound 4c against Microspora
griseus (fungus)
Table 4 Minimum inhibitory concentration (MIC) of synthesized compounds 4a–i and 5a–i
Comp. no.
Minimum inhibitory concentration (lmol/L)
P.s
S.t
B.s
K.p
E.c
38.90
A.f
C.a
M.g
A.t
4a
38.50
30.22
68.97
38.14
12.34
37.20
29.76
–
37.92
39.14
38.90
30.90
25.81
–
30.85
25.81
55.68
30.04
66.49
29.81
25.09
53.72
31.22
–
15.48
12.90
37.50
15.84
52.66
14.90
12.50
40.57
16.96
50.99
–
15.85
13.01
42.15
15.37
50.61
15.37
12.59
45.06
16.96
49.00
–
4c
30.73
57.95
36.73
11.38
36.61
29.76
121.16
36.08
199.10
24.95
–
31.96
41.47
38.61
31.76
31.96
46.59
38.73
63.21
37.79
31.05
4d
–
4e
38.84
63.21
38.14
31.54
30.16
–
4h
–
5a
37.79
31.64
99.12
36.42
29.92
25.00
51.86
31.10
66.07
–
5c
5d
101.64
36.31
189.18
26.24
–
–
5e
36.99
–
36.31
5h
188.98
26.67
–
169.34
26.24
–
Streptomycin
Flucanazole
24.95
–
–
40.84
40.84
20.42
20.42
P.s Psedomonas syringae, S.t Salmonella typhi, B.s Bacillus subtilis, K.p Klebsiella pneumoniae, E.c Escherichia coli, A.f Asperigillus flavous,
C.a Candida albicans, M.g Microspora griseus, A.t Asperigillus terus
group and incorporation of –Br group at C-5 position of
benzofuran ring drastically reduced the activity (as observed
in compounds 4a, 5c, 4h, and 5h).On the other hand, com-
pounds 4d and 5d displayed moderate to good activity with
IC50 value 162.36 and 160.85 lmol/L, respectively.
revealed that, among the tested barbiturate series, com-
pounds 4a, 4b, and 4g showed very good chelating ability
with IC50 value 146.63–175.93 lmol/L. In thiobarbiturates
series, compounds 5a, 5b, and 5g exhibit excellent activity
with IC50 value 126.57–147.67 lmol/L. Furthermore, it
was observed that the presence of OH group at C-5 of
benzofuran ring and OCH3/Br group on C-4 of aryl ring
(4e, 4f, 5e, and 5f) showed good chelating ability. Further,
declining trend in chelating ability was found in 4d, 4i, 5d,
and 5i. Compounds with halogen substituents (4c, 5c, 4h,
and 5h) lowered the activity with higher IC50 value
(284.01–458.75 lmol/L).
Iron chelating ability
The iron chelating study measures the ability of antioxi-
dants to compete with ferrozine in chelating ferrous ion
(Elmastas et al., 2006). The ferrous ion-chelating activity
of the newly synthesized compounds is represented in
Fig. 5. The Fe2? chelating capacities varied significantly
among different compounds. From the activity results it
IC50 values of DPPH radical scavenging and ferrous ion
chelating activity of test compounds is given in Table 6.
123

Med Chem Res (2014) 23:3065–3081
3073
Fig. 3 Interaction of ligand molecules 4a, 4c, 4e, 5a, 5c, and 5e with
GlcN-6-P. A: interaction of 4a with GlcN-6-P, B: interaction of 4c
with GlcN-6-P, C: interaction of 4e with GlcN-6-P, D: interaction of
5a with GlcN-6-P, E: interaction of 5c with GlcN-6-P, F: interaction
of 5e with GlcN-6-P, G: interaction of Streptomycin with GlcN-6-P,
H: interaction of Fluconazole with GlcN-6-P
Experimental protocols
taken in KBr pellets (100 mg) using Shimadzu FT-Infrared
1
spectrophotometer. H NMR and 13C NMR spectra were
The BA, thiobarbituric acid, and phenacyl bromides with
98 % purity were purchased from Sigma Aldrich Company.
Melting points were recorded on electro thermal melting
point apparatus and are uncorrected. Column chromatogra-
phy was performed using silica gel (230–400 mesh), silica
gel GF254 plates from Merck were used for TLC and spots
were identified under ultraviolet radiation. Ethyl acetate:pet
ether (1:4) is used as a mobile phase. The FT-IR spectra were
recorded on Bruker 400 MHz spectrometer and chemical
shifts are shown in d values (ppm) with tetramethylsilane
(TMS) as internal standard. LC–MS were obtained using
C-18 column on Shimadzu, LCMS 2010A, Japan.
The results of biological studies have been expressed in
molar unit. In antimicrobial activity the zone of inhibition
and in antioxidant activity the IC50 values are expressed as
mean SD of three replicates.
123

3074
Med Chem Res (2014) 23:3065–3081
Table 5 Molecular docking results of synthesized compounds with Glucosamine-6-phosphate synthase
Comp. no.
Binding energy
(kJ mol-1
Inhibition
constant (lM)
RMSd Ligand
efficiency
No. of hydrogen Bonding residues
bonds
Bond
length (A)
˚
)
4a
-4.71
295.65
229.07
0.0
0.0
-0.19
2
2VF5: GLN348: HE22: Ligands/ 2.204
4a:: : O
2VF5: THR352: HG1: Ligands/4a:: 1.919
: O
4c
4e
-5.27
-4.53
-0.18
-0.17
3
3
2VF5: GLN348: HE22: Ligands/ 2.118
4c:: : O
2VF5:SER349: HG: Ligands/4c:: : 2.207
O
2VF5:THR352: HG1: Ligands/4c:: 2.195
: O
476.51
0.0
2VF5:GLN348: HE22: Ligands/
4e:: : O
2.074
2VF5:SER349: HG: Ligands/4e:: : 2.231
O
2VF5:THR352:HG1: Ligands/4c:: 2.215
: O
5a
5c
5e
-4.66
-4.85
-4.33
353.32
280.61
0.0
0.0
0.0
0.0
0.0
-0.18
-0.18
-0.16
-0.17
-0.26
2
2
2
2
4
2VF5:GLN348:HE22: Ligands/5a:: 2.067
: O
2VF5:THR352:HG1: Ligands/5a:: 2.104
: O
2VF5:GLN348:HE22: Ligands/5c:: 2.149
: O
2VF5:THR352:HG1: Ligands/5c:: 2.105
: O
673.95
2VF5:GLN348:HE22: Ligands/5c:: 2.187
: O
2VF5:THR352:HG1: Ligands/5c:: 2.140
: O
Streptomycin -6.72
Fluconazole -5.65
-181.49
-197.44
2VF5:SER349:HG: Ligands/
Streptomycin:: : O
1.922
1.894
2.167
2.161
2.09
2VF5:THR352: OG1: Ligands/
Streptomycin:: : H
2VF5:GLN348:HG: Ligands/
Fluconazole:: : O
2VF5:THR352:OG1: Ligands/
Fluconazole:: : H
2VF5:SER349:OG: Ligands/
Fluconazole:: : H
2VF5: SER303:OG: Ligands/
Fluconazole:: : H
1.962
Experimental procedure
mass was cooled to room temperature, poured into crushed
ice and neutralized with NaHCO3 solution. The product
was filtered, dried and recrystallized using ethyl acetate/
ethanol, further purified by silica gel column chromatog-
raphy eluting with petroleum ether, ethyl acetate mixture
(80:20, v/v).
Chemistry
General procedure for the synthesis of benzofuran
barbitone derivatives (4a–i)
The mixture of (2/4-substitutedphenyl) methanone (3a–
i) (0.01 mol) and BA (0.01 mol, 1.44 g) was taken in
acetic acid and refluxed in an oil bath for about 8–10 h at
110–115 °C. After the completion of reaction, the reaction
5-[(Benzofuran-2-yl)(4-bromophenyl)methylidene]pyrimi-
din-2,4,6(1H,3H,5H)-trione (4a) Yellow solid (EtOH);
m.p. 161 °C; IR (KBr, t cm-1): 3317 (N–H), 3155 (Ar–
CH), 1715 (C=O), 1675 (–NHC=O), 684 (C–Br); 1H NMR
123

Med Chem Res (2014) 23:3065–3081
3075
Fig. 4 DPPH free radial
scavenging activity of test
compounds (4a–i) and (5a–i)
DPPH free radical scavenging assay
100
90
80
70
60
50
40
30
20
10
0
25 µmol
50 µmol
75 µmol
100 µmol
4a 4b 4c 4d 4e 4f 4g 4h 4i 5a 5b 5c 5d 5e 5f 5g 5h 5i Std
Test compounds
Fig. 5 Ferrous ion chelating
assay of test compounds
(4a–i) and (5a–i)
Ferrous ion chelating assay
90
80
70
60
50
40
30
20
10
0
25 µmol
50 µmol
75 µmol
100 µmol
4a 4b 4c 4d 4e 4f 4g 4h 4i 5a 5b 5c 5d 5e 5f 5g 5h 5i Std
Test compounds
(400 MHz, DMSO-d6, d ppm): 10.68 (s, 1H –NH), 10.21
(s, 1H –NH), 7.2-8.3 (m, 9H Ar–H); 13C NMR (400 MHz,
DMSO-d6, d ppm): 165.0 (C=O, C-11, C-110), 159.9 (C-9),
158.3 (C-4), 157.3 (C-1), 150.5 (C=O, C-12), 139.0 (C-13),
131.6 (CH, C-15, C-150), 130.2 (C-3), 128.6 (CH, C-14,
C-140), 124.7 (CH, C-6), 123.3 (CH, C-7), 122.0 (C-10,
C-16), 121.0 (CH, C-8), 111.6 (CH, C-5), 106.9 (CH, C-2);
MS (LCMS): m/z 410 [M], 412 [M?2].
1H NMR (400 MHz, DMSO-d6, d ppm): 10.80 (s, 1H
–NH), 10.21 (s, 1H –NH), 7.4–8.0 (m, 8H Ar–H); 13C
NMR (400 MHz, DMSO-d6, d ppm): 165.0 (C=O, C-11,
C-110), 159.9 (C-9), 157.3 (C-1), 150.5 (C=O, C-12), 139.0
(C-13), 133.7 (C-4), 131.6 (CH, C-15, C-150), 129.0 (CH,
C-6), 128.6 (CH, C-14, C-140), 124.2 (CH, C-8), 122.3 (C-
10), 116.5 (C-7), 113.8 (C-5), 106.9 (CH, C-2); MS
(LCMS): m/z 488 [M], 490 [M?2], 492 [M?4].
5-[(Benzofuran-2-yl) (4-methoxyphenyl) methylidene] pyr-
imidin-2,4,6(1H,3H,5H)-trione (4b) Off-white solid
(EtOH); m.p. 163–165 °C; IR (KBr, t cm-1): 3310 (N–H),
1710 (C=O), 1648 (–NHC=O); 1H NMR (400 MHz,
DMSO-d6, d ppm): 10.71 (s, 1H –NH), 10.14 (s, 1H –NH),
7.0–8.1 (m, 9H Ar–H), 3.85 (s, 3H –OCH3); 13C NMR
(400 MHz, DMSO-d6, d ppm): 167.1 (C=O, C-11, C-110),
160.0 (C-9, C-16), 158.3 (C-4), 157.3 (C-1), 150.5 (C=O,
C-12), 132.3 (C-13), 131.0 (C-3), 127.4 (CH, C-14, C-140),
124.7 (CH, C-6), 123.3 (CH, C-7), 122.0 (C-10), 121.0
(CH, C-8), 114.5 (CH, C-15, C-150), 111.6 (CH, C-5), 106.
9 (CH, C-2), 55.8 (O–CH3); MS (LCMS): m/z 362 [M].
5-[(5-Bromo-1-benzofuran-2-yl) (4-methoxyphenyl) meth-
ylidene] pyrimidin-2,4,6(1H, 3H, 5H)-trione (4d) Color-
less solid (ethyl acetate); m.p. 161–163 °C; IR (KBr, t
cm-1): 3308 (N–H), 1705 (C=O), 1669 (–NHC=O), 659
1
(C–Br); H NMR (400 MHz, DMSO-d6, d ppm): 10.65 (s,
1H –NH), 10.02 (s, 1H –NH), 7.2-8.0 (m, 8H Ar–H), 3.86
(s, 3H –OCH3); 13C NMR (400 MHz, DMSO-d6, d ppm):
165.0 (C=O, C-11, C-110), 159.9 (C-9, C-16), 157.3 (C-4),
150.5 (C=O, C-12), 133.7 (C-3), 132.3 (C-13), 129.0 (CH,
C-6), 127.4 (CH, C-14, C-140), 124.2 (CH, C-8), 122.3 (C-
10), 116.5 (C-7), 114.2 (CH, C-15, C-150), 112.9 (C-5),
106.9 (CH, C-2), 55.6 (O–CH3); MS (LCMS): m/z 440
[M], 442 [M?2].
5-[(5-Bromo-1-benzofuran-2-yl)(4-bromophenyl) methyli-
dene] pyrimidin-2,4,6(1H,3H,5H)-trione (4c) Off-white
solid (ethyl acetate); m.p. 160–162 °C; IR (KBr, t cm-1):
3419 (N–H), 1708 (C=O), 1654 (–NHC=O), 669 (C–Br);
5-[(4-Bromophenyl) (5-hydroxy-1-benzofuran-2-yl) meth-
ylidene] pyrimidin-2,4,6(1H,3H,5H)-trione (4e) Off-
white solid (EtOH); m.p. 168–169 °C; IR (KBr, t cm-1):
123

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Med Chem Res (2014) 23:3065–3081
5-[1-Benzofuran-2-yl (2-bromophenyl) methylidene] pyr-
Table 6 Half-maximum inhibition concentrations (IC50) for of
DPPH radical scavenging and ferrous ion chelating activity of test
compounds
imidin-2,4,6(1H,3H,5H)-trione
(4g) Colorless
solid
(ethyl acetate); m.p. 78–80 °C; IR (KBr, t cm-1): 3318
(N–H), 1731 (C=O), 1709 (–NHC=O), 688 (C–Br); 1H
NMR (400 MHz, DMSO-d6, d ppm): 10.69 (s, 1H –NH),
10.12 (s, 1H –NH), 7.2–8.3 (m, 9H Ar–H); 13C NMR
(400 MHz, DMSO-d6, d ppm): 165.0 (C=O, C-11, C-110),
159.9 (C-9), 158.3 (C-4), 157.3 (C-1), 150.1 (C=O, C-12),
138.2 (C-13), 132.0 (CH, C-15), 131.2 (C-3), 130.2 (CH,
C-16), 128.6 (CH, C-140), 127.1 (CH, C-150), 124.8 (CH,
C-6), 123.3 (CH, C-7), 122.0 (C, C-10), 121.0 (CH, C-8),
118.9 (C–Br, C-14), 111.6 (CH, C-5), 106.9 (CH, C-2); MS
(LCMS): m/z 410 [M], 412 [M?2].
Compd.
DPPH (IC50
lmol/L)
Fe2? ion Chelating
activity (IC50 lmol/L)
4a
4b
4c
4d
4e
4f
304.87 0.19
256.74 0.29
341.78 0.15
162.36 0.14
128.87 0.12
148.09 0.22
269.48 0.40
399.95 0.25
135.51 0.14
193.02 0.11
208.54 0.32
284.28 0.10
160.85 0.15
147.78 0.18
150.91 0.21
238.05 0.13
458.75 0.17
153.89 0.13
203.82 0.23
146.63 0.33
175.93 0.18
284.01 0.15
269.38 0.19
256.24 0.14
268.28 0.10
160.95 0.15
355.10 0.25
299.90 0.24
126.57 0.11
147.67 0.31
299.78 0.29
245.28 0.21
104.46 0.15
236.33 0.20
135.56 0.20
402.46 0.17
262.08 0.13
150.93 0.15
4g
4h
4i
5-[(5-Bromo-1-benzofuran-2-yl)(2-bromophenyl) methyli-
dene] pyrimidin-2,4,6(1H,3H,5H)-trione (4h) Brown
solid (ethyl acetate); m.p. 155–157 °C; IR (KBr, t cm-1):
3412 (N–H), 1718 (C=O), 1675 (–NHC=O), 669 (C–Br);
1H NMR (400 MHz, DMSO-d6, d ppm): 10.80 (s, 1H
–NH), 10.22 (s, 1H –NH), 7.5–8.1 (m, 8H Ar–H); 13C
NMR (400 MHz, DMSO-d6, d ppm): 165.2 (C=O, C-11,
C-110), 159.9 (C-9), 157.3 (C-1), 150.5 (C=O, C-12), 138.2
(C-13), 133.7 (C-3), 131.6 (CH, C-15), 130.1 (CH, C-16),
128.5 (CH, C-6), 127.8 (CH, C-140), 126.6 (CH, C-150),
124.2 (CH, C-8), 122.3 (C-10), 118.9 (C–Br, C-14), 116.5
(C–Br, C-7), 113.8 (CH, C-5), 106.5 (CH, C-2); MS
(LCMS): m/z 488 [M], 490 [M?2], 492 [M?4].
5a
5b
5c
5d
5e
5f
5g
5h
5i
Stda,b
Each value is expressed as mean SD of three replicates
Stda BHT used as standard for DPPH radical scavenging activity; Stdb
EDTA is used as a standard for Fe2? ion chelating activity
5-[(2-Bromophenyl) (5-hydroxy-1-benzofuran-2-yl) meth-
ylidene] pyrimidin-2,4,6(1H,3H,5H)-trione (4i) Green
solid (EtOH); m.p. 96–98 °C; IR (KBr, t cm-1): 3421 (O–
H), 3395 (N–H), 1721 (C=O), 1668 (–NHC=O), 694 (C–
3421 (O–H), 3391 (N–H), 1702 (C=O), (–NHC=O), 696
1
(C–Br); H NMR (400 MHz, DMSO-d6, d ppm): 10.55 (s,
1H –NH), 10.12 (s, 1H –NH), 7.2–8.1 (m, 8H Ar–H), 5.2
(s, 1H –OH); 13C NMR (400 MHz, DMSO-d6, d ppm):
165.4 (C=O, C-11, C-110), 159.9 (C-10), 157.6 (C-1), 153.6
(C–OH, C-7), 151.1 (C-4), 150.5 (C=O, C-12), 139.0 (C-
13), 132.9 (C-3), 131.2 (CH, C-15, C-150), 128.6 (CH,
C-14, C-140), 122.3 (C-10, C-16), 114.3 (CH, C-6), 113.0
(CH, C-5), 106.9 (CH, C-2), 106.1 (CH, C-8); MS
(LCMS): m/z 426 [M], 428 [M?2].
1
Br); H NMR (400 MHz, DMSO-d6, d ppm): 10.58 (s, 1H
–NH), 10.12 (s, 1H –NH), 7.2–8.0 (m, 8H Ar–H), 5.4 (s,
1H -OH); 13C NMR (400 MHz, DMSO-d6, d ppm): 166.0
(C=O, C-11, C-110), 159.9 (C-9), 157.3 (C-1), 153.6 (C–
OH, C-7), 151.3 (C-4), 150.5 (C=O, C-12), 138.2 (C-13),
132.9 (C-3), 131.6 (CH, C-15), 130.2 (CH, C-16), 128.6
(CH, C-140), 127.0 (CH, C-150), 122.3 (C-10), 118.9 (C–
Br, C-14), 114.3 (CH, C-6), 113.0 (CH, C-5), 106.9 (CH,
C-2), 106.0 (CH, C-8); MS (LCMS): m/z 426 [M], 428
[M?2].
5-[(5-Hydroxy-1-benzofuran-2-yl) (4-methoxyphenyl) meth-
ylidene] pyrimidin-2,4,6(1H,3H,5H)-trione (4f) Light yel-
low solid (EtOH); m.p. 161–163 °C; IR (KBr, t cm-1):
3380 (N–H), 1703 (C=O), 1670 (–NHC=O), 1H NMR
(400 MHz, DMSO-d6, d ppm): 10.60 (s, 1H –NH), 10.12
(s, 1H –NH), 7.2–8.2 (m, 8H Ar–H), 5.5 (s, 1H -OH); 13C
NMR (400 MHz, DMSO-d6, d ppm): 165.0 (C=O, C-11,
C-110), 160.1 (C-9, C-16), 157.3 (C-1), 153.6 (C–Br, C-7),
151.8 (C-4), 150.1 (C=O, C-12), 133.0 (C-3), 131.6 (C-13),
127.4 (CH, C– C-14, C-140), 122.3 (C-10), 114.3 (CH,
C-6), 113.0 (CH, C-15, C-150), 112.0 (CH, C-5), 107.0
(CH, C-2), 106.1 (CH, C-8), 55.9 (O–CH3); MS (LCMS):
m/z 378 [M].
General procedure for the Synthesis of benzofuran
thiobarbitone derivatives (5a–i)
The mixture of (2/4-substituted-phenyl) methanone (3a–
i) (0.01 mol) and thiobarbituric acid (0.01 mol, 1.44 g) was
taken in acetic acid and refluxed in an oil bath for about
8–10 h at 110–115 °C. After the completion of reaction,
the reaction mass was cooled to room temperature, poured
into crushed ice and neutralized with NaHCO3 solution.
The product was filtered, dried, and recrystallized using
123

Med Chem Res (2014) 23:3065–3081
3077
ethyl acetate/ethanol, further purified by silica gel column
chromatography eluting with petroleum ether, ethyl acetate
mixture (80:20, v/v).
C-16), 157.3 (C, C-1, C-4), 133.7 (C, C-3), 132.3 (C,
C-13), 129.0 (CH, C-6), 127.4 (CH, C-14, C-140), 124.2
(CH, C-8), 122.3 (C-10), 116.5 (C–Br, C-7), 114.2 (CH,
C-15, C-150), 112.9 (CH, C-5), 106.9 (CH, C-2), 55.9 (O–
CH3); MS (LCMS): m/z 456 [M], 458 [M?2].
5-[1-Benzofuran-2-yl (4-bromophenyl) methylidene]-2-
thioxodihydropyrimidin-4,6(1H,5H)-dione (5a) Brown
solid (EtOH); m.p. 102–104 °C; IR (KBr, t cm-1): 3317
5-[(4-Bromophenyl) (5-hydroxy-1-benzofuran-2-yl) methyli-
dene]-2-thioxodihydropyrimidin-4,6(1H,5H)-dione (5e) Col-
orless solid (EtOH); m.p. 109 °C; IR (KBr, t cm-1): 3419
(O–H), 3394 (N–H), 1279 (C=S), 1641 (C=O), 694 (C–Br);
1H NMR (400 MHz, DMSO-d6, d ppm): 10.13 (s, 1H –NH),
10.51 (s, 1H –NH), 7.2-8.1 (m, 8H Ar–H), 5.6 (s, 1H –OH);
13C NMR (400 MHz, DMSO-d6, d ppm): 178.3 (C=S, C-12),
167.5 (C=O, C-11, C-110), 159.3 (C-9), 157.6 (C-1), 153.6
(C–OH, C-7), 150.9 (C-4), 139.2 (C-13), 132.8 (C-3), 131.5
(CH, C-15, C-150), 128.6 (CH, C-14, C-140), 122.6 (C, C-10,
C-16), 114.3 (CH, C-6), 113.0 (CH, C-5), 106.9 (CH, C-2),
106.1 (CH, C-8); MS (LCMS): m/z 442 [M], 444 [M?2].
1
(N–H), 1229 (C=S), 1735 (C=O), 686 (C–Br); H NMR
(400 MHz, DMSO-d6, d ppm): 10.67 (s, 1H –NH), 10.24
(s, 1H –NH), 7.1-8.2 (m, 9H Ar–H); 13C NMR (400 MHz,
DMSO-d6, d ppm): 178.1 (C=S, C-12), 167.0 (C=O, C-11,
C-110), 159.9 (C-10), 158.3 (C-4), 156.3 (C-1), 139.0 (C-
13), 131.6 (CH, C-15, C-150), 130.1 (C-3), 128.6 (CH,
C-14, C-140), 124.7 (CH, C-6), 123.3 (CH, C-7), 122.0 (C–
Br, C-16), 121.0 (CH, C-8), 111.6 (CH, C-5), 106.9 (CH,
C-2); MS (LCMS): m/z 426 [M], 428 [M?2].
5-[1-Benzofuran-2-yl (4-methoxyphenyl) methylidene]-2-
thioxodihydropyrimidin-4,6(1H, 5H)-dione (5b) Yellow
solid (EtOH); m.p. 92–94 °C; IR (KBr, t cm-1): 3304 (N–
H), 1329 (C=S), 1633 (C=O); 1H NMR (400 MHz, DMSO-
d6, d ppm): 10.13 (s, 1H –NH), 10.70 (s, 1H –NH), 7.1–8.0
(m, 9H Ar–H), 3.88 (s, 3H –OCH3); 13C NMR (400 MHz,
DMSO-d6, d ppm): 179.1 (C=S, C-12), 167.8 (C=O, C-11,
C-110), 159.9 (C-9, C-16), 158.2 (C-4), 155.7 (C-1), 135.0
(C-13), 130.9 (C-3), 127.4 (CH, C-14, C-140), 124.4 (CH,
C-6), 123.0 (CH, C-7), 122.3 (C-10), 117.9 (CH, C-8), 114.
5 (CH, C-15, C-150), 111.6 (CH, C-5), 106.9 (CH, C-2), 55.
8 (O–CH3); MS (LCMS): m/z 378 [M].
5-[(5-Hydroxy-1-benzofuran-2-yl)
(4-methoxyphenyl)
methylidene]-2-thioxodihydropyrimidin-4,6(1H,5H)-dione
(5f) Brown solid(EtOH); m.p. 103–106 °C; IR (KBr, t
1
cm-1): 3384 (N–H), 1310 (C=S), 1653 (C=O); H NMR
(400 MHz, DMSO-d6, d ppm): 10.36 (s, 1H –NH), 10.52
(s, 1H –NH), 7.1-8.2 (m, 8H Ar–H), 5.8 (s, 1H –OH) 3.72
(s, 3H –OCH3); 13C NMR (400 MHz, DMSO-d6, d ppm):
178.5 (C=S, C-12), 167.5 (C=O, C-11, C-110), 160.1 (C-9,
C-16), 157.3 (C-1), 153.2 (C–OH, C-7), 150.8 (C-4), 133.2
(C-3), 131.1 (C-13), 128.4 (CH, C-14, C-140), 122.3 (C-
10), 115.3 (CH, C-15, C-150), 113.1 (CH, C-6), 112.3 (CH,
C-5), 107.0 (CH, C-1), 106.1 (CH, C-8), 55.9 (OCH3); MS
(LCMS): m/z 394 [M].
5-[(5-Bromo-1-benzofuran-2-yl)(4-bromophenyl)methyl-
idene]-2-thioxodihydropyrimidin-4,6(1H,5H)-dione (5c)
Light yellow solid (ethyl acetate); m.p. 100–102 °C; IR
(KBr, t cm-1): 3417 (N–H), 1293 (C=S), 1735 (C=O), 666
5-[1-Benzofuran-2-yl (2-bromophenyl) methylidene]-2-
thioxodihydropyrimidin-4,6(1H,5H)-dione (5g) Pale yel-
low solid(ethyl acetate); m.p. 105–108 °C; IR (KBr, t
cm-1): 3317 (N–H), 1290 (C=S), 1735 (C=O), 686 (C–Br);
1H NMR (400 MHz, DMSO-d6, d ppm): 10.24 (s, 1H
–NH), 10.67 (s, 1H –NH), 7.1–8.2 (m, 9H Ar–H); 13C
NMR (400 MHz, DMSO-d6, d ppm): 178.1 (C=S, C-12),
167.0 (C=O, C-11, C-110), 159.9 (C-9), 158.3 (C-4), 157.3
(C-1), 139.2 (C-13), 131.8 (CH, C-14), 131.1 (C-3), 130.2
(CH, C-16), 129.0 (CH, C-140), 127.6 (CH, C-150), 124.8
(CH, C-6), 123.5 (CH, C-7), 122.0 (C-10), 121.0 (CH,
C-8), 118.9 (C–Br, C-14), 111.6 (CH, C-5), 106.9 (CH,
C-2); MS (LCMS): m/z 426 [M], 428 [M?2].
1
(C–Br); H NMR (400 MHz, DMSO-d6, d ppm): 10.22 (s,
1H –NH), 10.79 (s, 1H –NH), 7.5-8.0 (m, 8H Ar–H); 13C
NMR (400 MHz, DMSO-d6, d ppm): 178.1 (C=S, C-12),
167.0 (C=O, C-11, C-110), 160.0 (C-9), 157.3 (C-1, C-4),
139.2 (C-13), 133.7 (C-3), 131.6 (CH, C-15, C-150), 129.0
(CH, C-14, C-140), 128.1 (CH, C-6), 124.2 (CH, C-8), 122.
3 (C-10, C-16), 116.5 (C–Br, C-7), 113.8 (CH, C-5), 106.9
(CH, C-2); MS (LCMS): m/z 504 [M] 506 [M?2], 508
[M?4].
5-[(5-Bromo-1-benzofuran-2-yl)(4-methoxyphenyl)methyli-
dene]-2-thioxodihydropyrimidin-4,6(1H,5H)-dione
(5d)
Brown solid (ethyl acetate); m.p. 110–112 °C; IR (KBr, t
cm-1): 3302 (N–H), 1286 (C=S), 1641 (C=O), 650 (C–Br);
1H NMR (400 MHz, DMSO-d6, d ppm): 10.03 (s, 1H
–NH), 10.61 (s, 1H –NH), 7.1–8.0 (m, 8H Ar–H), 3.88 (s,
3H -OCH3); 13C NMR (400 MHz, DMSO-d6, d ppm): 178.
2 (C=S, C-12), 167.1 (C=O, C-11, C-110), 159.9 (C, C-9,
5-[(5-Bromo-1-benzofuran-2-yl)(2-bromophenyl)methyli-
dene]-2-thioxodihydropyrimidin-4,6(1H,5H)-dione (5h)
Brown solid (ethyl acetate); m.p. 110–114 °C; IR (KBr, t
cm-1): 3417 (N–H), 1285 (C=S), 1735 (C=O), 666 (C–Br);
123

3078
Med Chem Res (2014) 23:3065–3081
1H NMR (400 MHz, DMSO-d6, d ppm): 10.22 (s, 1H
–NH), 10.79 (s, 1H –NH), 7.5-8.0 (m, 8H Ar–H); 13C NMR
(400 MHz, DMSO-d6, d ppm): 178.3 (C=S, C-12), 167.2
(C=O, C-11, C-110), 159.9 (C, C-9), 157.3 (C-1, C-4), 138.
2 (C-13), 133.7 (C-3), 131.6 (CH, C-15), 129.0 (CH, C-6)
128.5 (CH, C-140), 127.2 (CH, C-150), 124.2 (CH, C-8),
122.3 (C-10), 118.9 (C–Br, C-14), 116.5 (C–Br, C-7), 113.
8 (CH, C-5), 106.5 (CH, C-2); MS (LCMS): m/z 504
[M] 506 [M?2], 508 [M?4].
4.2) with Lamarckian genetic algorithm. The synthesized
ligand molecules having 2D structure were converted to
energy minimized 3D structures and were further used for
in silico protein–ligand docking. The docking of receptor
GlcN-6-P with newly synthesized ligands exhibited well
established bonds with one or more amino acids in the
receptor active pocket. The active pocket was considered to
be the site, where glucosamine-6-phosphate complexes
with GlcN-6-P of 2VF5. The active pocket consisted of 12
amino acid residues as Ala602, Val399, Ala400, Gly301,
Thr302, Ser303, Cys300, Gln348, Ser349, Thr352, Ser347,
and Lys603 (Wallace et al., 1995).
5-[(2-Bromophenyl) (5-hydroxy-1-benzofuran-2-yl) meth-
ylidene]-2-thioxodihydropyrimidin-4,6(1H,5H)-dione (5i)
Brown solid(EtOH); m.p. 114 °C; IR (KBr, t cm-1): 3419
(O–H), 3394 (N–H), 1260 (C=S), 1641 (C=O), 694 (C–Br);
1H NMR (400 MHz, DMSO-d6, d ppm): 10.13 (s, 1H
–NH), 10.51 (s, 1H –NH), 7.2-8.1 (m, 8H Ar–H), 5.6 (s, 1H
–OH); 13C NMR (400 MHz, DMSO-d6, d ppm): 178.1 (C=
S, C-12), 167.0 (C=O, C-11, C-110), 159.9 (C, C-9), 157.3
(C-1), 153.6 (C–OH, C-7), 150.9 (C, C-4), 138.2 (C-13),
132.9 (C-3), 131.6 (CH, C-14), 130.2 (CH, C-16), 128.6
(CH, C-140), 127.0 (CH, C-150), 122.3 (C-10), 118.9 (C–
Br, C-14), 114.3 (CH, C-6), 113.0 (CH, C-5), 106.9 (CH,
C-2), 106.1 (CH, C-8); MS (LCMS): m/z 442 [M], 444
[M?2].
The crystal structure of GlcN-6-P synthase (PDB ID
2VF5) from the PDB (http://www.pdb.org/pdb/home/
home.do) was selected and edited by removing the het-
eroatoms and adding C-terminal oxygen (Binkowski et al.,
2003). The Graphical User Interface program ‘‘AutoDock
Tools’’ was used to prepare, run, and analyze the docking
simulations. Kollman united atom charges, solvation
parameters, and polar hydrogens were added to the receptor
for the preparation of protein in docking simulation. Since
ligands are not peptides, Gasteiger charge was assigned and
then non-polar hydrogens were merged.
Antimicrobial activity
Antioxidant activity
Antimicrobial activity of the synthesized compounds was
tested against five bacterial strains using agar well diffu-
sion method (Nasser Khalil, 2010). Dimethylsulfoxide
(DMSO) was used as solvent control. The bacterial cultures
were inoculated on nutrient agar (Merck) and fungal cul-
ture was inoculated on potato dextrose agar media
(20 mL). The test compounds were dissolved in DMSO to
get a concentration of 12.79 M and 100 lL of this sample
was loaded into the wells of agar plates directly. Plates
Free radical scavenging activity by DPPH method
Free radical-scavenging capacities of synthesized com-
pounds were determined according to the reported proce-
dure (Braca et al., 2001). The newly synthesized
compounds at different concentrations (25–100 lmol/L)
were added to each test tube and volume was made up to
4 mL using methanol. To this, 3 mL of 0.004 % DPPH in
methanol was added and the mixtures were incubated at
room temperature under dark condition for 30 min. The
absorbance was recorded at 517 nm using UV–Visible
spectrophotometer (Shimadzu UV-1800, Japan). Butylated
hydroxytoluene (BHT) dissolved in distilled water was
used as a reference. Control sample was prepared using the
same volume without any compound and BHT, 95 %
methanol served as blank. Test was performed in triplicate
and the results were averaged. Radical scavenging activity
was calculated using the formula:
°
inoculated with the bacteria were incubated at 37 C for
°
24 h and the fungal culture was incubated at 25 C for
72 h. All determinations were done in triplicates. The
Streptomycin (1.71 and 0.85 M) and Fluconazole (3.26 and
1.6 M) were used as standard drugs for antibacterial and
antifungal activities, respectively.
The MIC was performed by serial broth-dilution method
(National Committee for Clinical Laboratory Standards
(NCCLS), 1982) at different concentrations like 1, 10, 25,
50, and 100 mol/L. After the incubation period, the mini-
mum inhibition zone at which the micro-organism growth
was inhibited was measured in M.
% of radical scavenging activity
¼ ½ðAcontrol ꢁ AtestÞ=Acontrolꢂ ꢃ 100
where Acontrol is the absorbance of the control sample
(DPPH solution without test sample) and Atest is the
absorbance of the test sample (DPPH solution ? test
compound).
In silico molecular docking studies
The compounds in the present investigation were subjected
for molecular docking studies using Auto Dock (version
123

Med Chem Res (2014) 23:3065–3081
3079
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Iron chelating ability
The chelating effect was determined according to the lit-
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(2 mL) of different concentrations (25–100 lmol/L) in
methanol was added to a solution of 2 mM FeCl2
(0.05 mL), the reaction was initiated by adding 5 mM
ferrozine (0.2 mL) and total volume was adjusted to 5 mL
with methanol. Then, the mixture was shaken vigorously
and left at room temperature for 10 min. Absorbance of the
solution was measured spectrophotometrically at 562 nm.
EDTA was used as a standard. The inhibition percentage of
ferrozine–Fe2? complex formations was calculated using
the formula:
ÂÀ
Á
Ã
Metal chelating effect ð%Þ ¼ Acontrol ꢁ Asample =Acontrol
ꢃ 100
Bondock S, Khalifa W, Fadda AA (2011) Synthesis and antimicrobial
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derivatives. Eur J Med Chem 46:2555–2561
where Acontrol is the absorbance of control (control contains
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performed in triplicate and the results were averaged.
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Conclusion
We synthesized novel series of benzofuran derivatives
containing barbiturate and thiobarbiturates nucleus. The
structure of synthesized compounds was confirmed by FT-
IR, NMR, and mass spectroscopic methods. From the
antimicrobial study results it revealed that, compounds
containing two bromo substituents are responsible for
increased antimicrobial activity than those bearing a single
halogen/other substituent group. In case of antioxidant
screening, compounds containing hydroxyl groups showed
very good DPPH radical scavenging activity. In metal
chelating assay, compounds having thiobarbiturate nucleus
showed comparatively predominant activity than barbitu-
rate series, this could be due to the presence of thione
group in thiobarbiturates.
¨
¨
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Acknowledgments The authors are thankful to the Chairman,
Department of Industrial Chemistry, Kuvempu University, Shankar-
aghatta for providing the laboratory facilities. One of the authors
(Kenchappa. R) is thankful to the UGC for the award of Rajiv Gandhi
National Fellowship (RGNF).
˘
Gu¨lcin I, Beydemir S, Alici HA, Elmastas M, Bu¨yu¨kokurolu ME
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