Synthesis, spectroscopy and electrochemistry of new 4-(4-acetyl-5- substituted-4, 5-dihydro-1, 3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-ones as a novel class of potential antibacterial and antioxidant derivatives

Article abstract of DOI:10.1016/j.crci.2010.11.001

The synthesis of the new 4-(4-acetyl-5- substituted -4, 5-dihydro-1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-ones derivatives 5 was accomplished by the use of 4-hydroxycoumarine as a starting material. The structures of the compounds were confirmed by analytical UV, IR, ¹H, ¹³C-NMR, NOESY and HMBC NMR spectra to elucidate the different positions of protons and carbons. All the compounds exhibited one quasireversible redox process. The UV absorption spectra of the obtained compounds showed strong absorption bands between 264 and 291 nm assigned to π-π* transitions of the oxadiazole group. All the newly synthesized compounds were screened for their antibacterial and antioxidant activities. Antimicrobial studies revealed that compounds 5a and 5b showed significant antibacterial activity against Escherichia coli and Pseudomonas Aeruginosa 27853. Furthermore these compounds showed antioxidant activities of different extents with respect to individual compounds as well as to the antioxidant methods. The compounds 5a-d was found to be the most active antioxidant in the series then Trolox, which makes the investigated complexes promising a new class of antibacterial compounds.

Full text of DOI:10.1016/j.crci.2010.11.001

C. R. Chimie 14 (2011) 548–555
Contents lists available at ScienceDirect
Comptes Rendus Chimie
www.sciencedirect.com
´
Full paper/Memoire
Synthesis, spectroscopy and electrochemistry of new 4-(4-acetyl-5-
substituted-4, 5-dihydro-1, 3,4-oxodiazol-2-yl)methoxy)-2H-chromen-
2-ones as a novel class of potential antibacterial and antioxidant
derivatives
b
b
N. Hamdi a, , V. Passarelli , A. Romerosa
*
a Heterocyclic and Organometallic Chemistry Laboratory, High Institute of Environmental Science and Technologies (HIEST-Borj Cedria, Tunisia), ‘‘University of 7th
november at Carthage, Tunisia’’, Touristic road of Soliman, BP 95, 2050 Hammam-Lif, Tunisia
b
´
´
´
´
Area de Quı´mica Inorganica, Universidad de Almerıa, 04120 Almerıa, Spain
A R T I C L E I N F O
A B S T R A C T
Article history:
The synthesis of the new 4-(4-acetyl-5- substituted -4, 5-dihydro-1,3,4-oxodiazol-2-
Received 16 July 2010
yl)methoxy)-2H-chromen-2-ones derivatives 5 was accomplished by the use of 4-
Accepted after revision 2 November 2010
Available online 31 December 2010
hydroxycoumarine as
a starting material. The structures of the compounds were
confirmed by analytical UV, IR, 1H, 13C-NMR, NOESY and HMBC NMR spectra to elucidate
the different positions of protons and carbons. All the compounds exhibited one
quasireversible redox process. The UV absorption spectra of the obtained compounds
Keywords:
4-hydroxycoumarin
1,3,4-Oxodiazole
Carbohydrazide
showed strong absorption bands between 264 and 291 nm assigned to
p-p * transitions of
the oxadiazole group. All the newly synthesized compounds were screened for their
antibacterial and antioxidant activities. Antimicrobial studies revealed that compounds 5a
and 5b showed significant antibacterial activity against Escherichia coli and Pseudomonas
Aeruginosa 27853. Furthermore these compounds showed antioxidant activities of
different extents with respect to individual compounds as well as to the antioxidant
methods. The compounds 5a-d was found to be the most active antioxidant in the series
Antibacterial activity
Antioxydant activity
then Trolox, which makes the investigated complexes promising
antibacterial compounds.
a new class of
´
ß 2010 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
rings have been used as electron transporting materials and
emitters in organic EL devices [3–5]. Recently 1,3,4-
1,3,4-Oxodiazole derivatives are highly attractive com-
pounds for the development of materials for organic
electroluminescent (EL) devices since they possess high
electron-accepting properties and display strong fluores-
cence with high quantum yield [1]. This behavior is
exemplified by 2,5-diphenyl-1,3,4-oxodiazole and 2,5-di-
2-naphthyl-1,3,4-oxodiazole, for which quantum yields of
0.80 and 0.85 in cyclohexane solution, respectively, were
reported [2]. Thus, compounds involving 1, 3,4-oxadiazole
oxodiazole derivatives have aroused considerable interest
in the area of organic light-emitting diodes (OLEDs) [6–9].
Oxadiazole fragments have also been connected to classical
chelating ligands (such as bipyridines) in luminescent
complexes, to obtain multifunctional (emitting and charge
transporting) molecular species [10–17]. Furthermore, it
has been reported that substituted 1, 3,4-oxadiazole
derivatives show a broad spectrum of biological activity
including anticancer effects [18,19]. In recent years, the
application of 1,3,4-oxodiazole consisting of five membered
heterocyclic ring have been described [20–24].
On the other hand, coumarins and structurally related
compounds have been shown to inhibit replication of HIV
* Corresponding author.
E-mail address: naceur.hamdi@isste.rnu.tn (N. Hamdi).
1631-0748/$ – see front matter ß 2010 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.crci.2010.11.001

N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
549
and thus exhibit a therapeutic potential [25]. A large
number of structurally novel coumarin derivatives have
been reported to show substantial cytotoxic and anti-HIV
activity in vitro and in vivo [26,27]. A variety of synthetic
coumarins have unique mechanisms of action referring to
the different stages of HIV replication [28]. Thus, coumar-
ins are important lead compounds for the development of
antiviral and/or virucidal drugs against HIV [29–31].
Keeping in view of the properties of 1,3,4 oxodiazole
derivatives and in prolongation of our research on
biologically active molecules [32,33], we have carried
out the present study to describe new convenient and
general procedures to afford novel 2-[(coumarin-4-oxy)-
by refluxing with hydrazine hydrate in absolute ethanol,
generating colorless crystalline product for which
structure 3 was assigned.
a
The resulting of carbohydrazide 3 was reacted with
arylaldehydes to give rise the (E)2-(coumarin-4-oxyace-
tic)-N-benzylideneacetohydrazide 4, which precipitated
by mixing the carbohydrazide 3 and the corresponding
ArCHO in hot ethanol.
The obtained compounds 4 were then refluxed with
acetic anhydride to give the corresponding 4-(4-acetyl-5-
substituted -4, 5-dihydro-1,3,4-oxodiazol-2-yl)methoxy)-
2H-chromen-2-ones
Scheme 1.
5 in good yields according to
methyl]-4-acetyl-5-substitued–1,3,4-oxodiazole
5
con-
All the new 4-(4-acetyl-5- substituted -4, 5-dihydro-
1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-ones 5,
taining 4-hydroxycoumarine moieties and investigate
both their electrochemistry and antimicrobial properties
against Staphylococcus aureus (CIP 7625), S. aureus,
Escherichia coli ATCC 25922, Klebsiella pneumonia CIP
104727 and Pseudomonas aeruginosa 27853 (CIP 76110)
using the agar disk diffusion assay. The antioxidant
properties of these compounds have been studied using
two different test methods, namely 2,2-diphenyl-1-picryl-
hydrazyl and ABTS radicals, respectively. The differences of
radical scavenging and antioxidant properties of 2-
[(coumarin-4-oxy) methyl]-4-acetyl-5-substitued–1,3,4-
oxodiazole were compared with similar doses of Trolox,
a standard antioxidant commonly used in food and
pharmaceutical industries.
have been characterized by UV, IR, 1H, 13C-NMR spectra
as well as by NOESY and HMBC NMR experiments to
elucidate their structures and assign completely the
structural network of both protons and carbons. The
obtained spectral data were in accordance with the
proposed structures.
As example, the IR spectra of compound 5c showed the
characteristic absorption bands for 1612(C C), 1472(N N)
and 1289(C–O–C). In addition, the detection of a strong
C
N stretching band at 1555 cmꢀ1 evidenced the forma-
tion of the 1,3,4-oxodiazol ring. The 1H NMR spectra of 5c
displays a signal at 6.07 ppm that ascribable to the proton
H-3 from the coumarine moiety. A characteristic singlet
proton signal at 8.53 ppm was assigned to H-5’ proton
d
d
from the oxodiazole fragment. In addition, the aromatic
protons (both coumarinic and oxodiazolinic) are observed
2. Results and discussion
between
d 7.08 and d 7.91 ppm (see experimental), and the
Compound 2 was prepared by reaction of 4-hydro-
xycoumarine with ethylbromoacetate in the presence of
anhydrous potassium carbonate in dry acetone, followed
expected singlet for methylenic moiety is observed at
5.47 ppm. The methyl protons of the acyl group arose at
d
d
[()TD$FIG]
H
N
O
OH
O
O
NH2
O
O
O
O
N2H4
EtOH
BrCH2COOEt
acetone/K2CO3
O
O
O
O
1
2
3
H
O
N
N
O
N
Ar
O
O
N
O
O
O
ArCHO
EtOH
CH3COOCOCH3
Ar
O
O
5a-e
4a-e
Ar
phenyl
Compound
4a 5a
4-fluorophenyl
4-methoxy-phenyl
4-nitro-phenyl
3,4,5-trimethoxy-phenyl
4b 5b
4c 5c
4d 5d
4e 5e
Scheme 1. Synthesis of 4-(4-acetyl-5- substituted -4, 5-dihydro-1, 3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-ones5.

550
N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
2.39 ppm. The singlet at 3.84 ppm was assigned to the three
protons of the methoxy group. Full assignment of the 1H
NMR spectra of 5c was deduced from the NOESY spectrum.
An observed NOE cross peak between both H-5’ and
methylic proton CH3 placed these two units on the same
side of the oxodiazole ring while H-3 and CH2 are,
therefore, on the other side. The 13C{1H} NMR spectrum
3. Absorption spectral characteristics of the compounds
5a–e
The UV spectra of the compounds 5 measured in
methanol solutions are shown in Fig. 2. As shown in this
figure compounds 5 display similar absorptions ranging
from 200 to 350 nm that are attributed to
of conjugate system.
p- p* transition
of 5c in DMSO-d6 showed two downfield signals at
166.7 ppm (C2’) and 162.0 ppm (lactone C2 O) as well as
a signal to up field at 55.9 ppm (OCH3). The signals at
26.0, 91.9 and 168.1 ppm were assigned to the COCH3,
d
d
The lowest energy absorption bands are from the p- p*
d
d
transitions by virtue of their large molar extinction
coefficients. Two absorption bands were observed for 5a
and 5e respectively, while only one was observed for 5b.
Comparing compounds 5b and 5e, electron-withdraw-
ing group (F) result in a blue shift (6 nm) with contrast to
an electron-donating group (3.4.5 MeO). Compounds 5
have almost same maximum absorption (200–350 nm)
that means substituent R in para position does not
influence the absorption.
d
d
the C3 and C5’, respectively.
The structure of 5c was finally elucidated through the
analysis of the 1H, 13C HMBC spectrum (Fig. 1), which
indicates that the methylenic protons at
correlate with C2’ 166.7 ppm) and C4 ( 164.8 ppm). The
hydrogen atom H-5’, at 8.53 ppm, correlate with Ca (
125.5 ppm). Moreover, the proton H-3 6.07 ppm)
correlates with C4 164.8 ppm). Furthermore, methylic
protons CH3CO ( 2.39 ppm) and the CO carbon signal (
171.2 ppm) are also correlated.
d 5.47 ppm
(
d
d
d
(
d
(d
d
d
4. Electrochemistry
Assignment of the protons of 2-[(coumarin-4-oxy)
methyl]-4-acetyl-5-substitued–1,3,4-oxodiazole 5 was de-
duced from their NOESY spectra. An observed nOe cross
peak between H5’ and methylic proton CH3 places these two
units on the same side of the average oxadiazoline ring
plane. H3 and Hb are therefore on the other side of this plane.
Electrochemical studies of 4-(4-acetyl-5- substituted -
4, 5-dihydro-1, 3,4-oxodiazol-2-yl)methoxy)-2H-chro-
men-2-ones 5 are of interest due to the electron-deficient
nature of the oxadiazole unit.
Hence, the electrochemical properties of compounds
5a–e, were determined by cyclic voltammetry in CH3CN
(1 ꢁ 10ꢀ3 M) solutions, using 0.1 M tetrabutylammonium
bromide (C4H12BrN) as the supporting electrolyte. Both
platinum and gold were used as working electrodes, Ag/
AgCl (0.1 M) as the reference electrode, and platinum as
the counter electrode. Under these electrochemical con-
ditions, 5 show a quasi-reversible behavior for the first
reduction process. This can be deduced from the fact that
the cathodic–anodic peak separations (Epc–Epa) are ca.
100 mV. The ratio of the peak current intensity for the
cathodic and anodic processes is about 0.5–0.7. As
expected, the reduction peak potential of the 2-[(couma-
A
mechanistic rationalization for this reaction is
provided in Scheme 2.
The formation of the 4-(4-acetyl-5- substituted -4, 5-
dihydro-1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-
ones 5 occurred via two steps: the carbohydrazide 3
initially formed rearranges to generate a negative oxygen
which then attacks on the C N bond, followed by attack of
the nitrogen atom to the C O bond of the acetic anhydride
accompanied by the loss of acetic acid yielding the 4-(4-
acetyl-5- substituted -4, 5-dihydro-1,3,4-oxodiazol-2-
yl)methoxy)-2H-chromen-2-ones 5.
[()TD$FIG]
100
110
120
130
140
150
160
O
2'
N
170
O
N
5
4
9
O
3
6
7
5'
a
b
180
2
c
ppm
10
O
1
O
8
d
9.0
ppm
8.0
7.0
6.0
5.0
4.0
3.0
2.0
OMe
5c
Fig. 1. 1H 13C HMBC spectrum of compound 5c.

[()TD$FIG]
N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
551
O
O
O
N
O
H
2'
N
O
O
N
N
O
N
O
N
Ar
O
O
5
4
- CH3COO-
9
O
5'
Ar
3
- H+
6
7
O
O
a
b
2
c
O
1
O
10
O
8
d
4
Ar
5
Ar
Compound
phenyl
5a
5b
5c
5d
5e
4-fluorophenyl
4-methoxy-phenyl
4-nitro-phenyl
3,4,5-trimethoxy-phenyl
Scheme 2. Proposed mechanism for the synthesis of 5a-e.
rin-4-oxy) methyl]-4-acetyl-5-substitued–1,3,4-oxodia-
zole 5 are strongly influenced by the para substitutent
on the phenylene ring. Compared to the unsubstituted
compound 5, the methoxy group, having a slight electron
donor behavior, shifts the reduction peak potential of 5 to
more negative values. The redox behaviors of all the 1,3,4-
oxadiazolines are summarized in Fig. 3. All the compounds
exhibited one quasireversible redox processes.
For example, compounds 5d showed one quasirever-
sible reduction at ꢀ0.8 and ꢀ1.15 V, respectively. We
assume that the curve at lower reduction potential may be
due to the more electron-deficient dications in the ring
system, and the curve at higher reduction potential can be
attributed to the redox behavior of the oxadiazole unit.
[35]. The reduction capability of DPPH radical was
determined by the decrease in absorbance induced by 4-
(4-acetyl-5- substituted -4, 5-dihydro-1,3,4-oxodiazol-2-
yl)methoxy)-2H-chromen-2-ones 5. Briefly, 1.5 ml etha-
nolic solution of the synthesized compounds (0.2 mM) was
added to 1.5 ml (0.2 mM) solution of DPPH radical in
ethanol (final concentration of DPPH and synthesized
compounds was 0.1 mM). The mixture was shaken
vigorously and allowed to stand for 30 min. After this,
the absorbance at 534 nm was determined and the
percentage of scavenging activity was calculated using
the following formula:
Scavenging activity ¼ f½Ab þ As ꢀ Amꢂ=Abg ꢁ 100%
Ab: absorbance of 0.1 mM ethanolic solution of DPPH at
534 nm, As: absorbance of 0.1 mM ethanolic solution of
test compound at 534 nm, Am: absorbance of ethanolic
mixture of the drug and DPPH at 534 nm.
5. Antibacterial and antioxidant studies
5.1. Free radical scavenging activity assay
Trolox was used as reference compound. All tests and
analyses were undertaken and averaged on three samples.
The results are given in Scheme 3.
Among the compounds from the 4-(4-acetyl-5- substi-
tuted -4, 5-dihydro-1,3,4-oxodiazol-2-yl)methoxy)-2H-
The free radical scavenging activity of the new 4-(4-
acetyl-5- substituted -4, 5-dihydro-1,3,4-oxodiazol-2-
yl)methoxy)-2H-chromen-2-ones 5 was tested by utilizing
DPPH scavenging [34].
DPPH is a free radical and accepts one electron or one-
hydrogen radical to become a stable diamagnetic molecule
[)(TD$FIG]
[()TD$FIG]
100
80
Electrolyte support
5 b
5 d
5 e
60
40
20
0
-20
-40
-60
-80
-100
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
Potentiel, E / VECS
Fig. 3. Cyclic voltammograms of compounds 5d, 5b and 5e (1 ꢁ 10ꢀ3 M)
Fig. 2. The UV spectra of the compounds 5a–d and 2–4 in methanol (2
10ꢀ5 M).
in CH3CN, scanned at 100 mVsꢀ1
.

[
[()TD$FIG]
552
N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
Scheme 3. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical of compounds 5.
Scheme 4. Scavenging ability on ABTS radical of compounds 5.
chromen-2-ones 5 series 5b showed moderate antioxidant
activity.
5.2. ABTS radical cation decolorization Assay
The activity exhibited by the compound 5e was the
highest. In addition the experimental data show that
compound 5a has a stronger effect of scavenging free
radical than Trolox.
According to the experimental results, we can note that
any increase in the concentration of the obtained 1,3,4
oxodiazole in the medium involves an attenuation of
percentage of inhibition. Indeed, this last can reach 90% for
The potential of 4-(4-acetyl-5- substituted -4, 5-
dihydro-1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-
ones 5 to scavenge free radicals was also assessed by their
ability to quench ABTS+. Scheme 4 depicts the concentra-
tion-dependent decolourization of ABTS+.
ABTS radical-scavenging activity of 4-(4-acetyl-5-
substituted -4, 5-dihydro-1,3,4-oxodiazol-2-yl)methoxy)-
2H-chromen-2-ones 5 was determined according to Re et
al. [30]. The ABTS+.. Cation radical was produced by the
reaction between 5 ml of 14 mM ABTS solution and 5 ml of
4.9 mM potassium persulfate (K2S2O8) solution, stored in
the dark at room temperature for 16 h. Before use, this
solution was diluted with ethanol to get an absorbance of
0.700 ꢃ 0.020 at 734 nm. In a final volume of 1 ml, the
reaction mixture comprised 950 ml of ABTSꢃ solution and
50 ml of the 1,3,4 oxadiazoles 5 at various concentrations. The
reaction mixture was homogenized and its absorbance was
recorded at 734 nm. Ethanol blanks were run in each assay,
and all measurements were done after at least 6 min.
Similarly, the reaction mixture of standard group was
obtained by mixing 950 ll of ABTS+ solution and 50 ml of
Trolox. As for the antiradical activity, ABTS scavenging ability
was expressed as EC50 (<mu> g/ml). The inhibition percent-
age of ABTS radical was calculated using the following
formula:
a
concentration about 1 mM for all the synthesized
products. Thus, we can conclude that substituent’s on
the aryl group does not have an influence on the anti-
oxidant activity.
One parameter that has been introduced recently for
the interpretation of the results from the DPPH method is
the efficient concentration or EC50 value (otherwise called
the IC50 value); this is defined as the concentration of
substrate that causes 50% loss of the DPPH activity (color),
and it is corresponding to the endpoint of the titration. It
should be noted that in all cases, any residual (yellow)
color from the reduced form or any non specific absor-
bance from the sample has to be taken into account in
defining the ‘‘endpoint’’ of the titration, or the 50% point,
this IC50 parameter also has the drawback that the higher
the antioxidant activity, the lower is the value of EC50
.
The EC50 values exhibited by 4-(4-acetyl-5- substituted
5-dihydro-1,3,4-oxodiazol-2-yl)methoxy)-2H-chro-
-4,
men-2-ones 5 are summarized in Table 1.
ABTS scavenging effect% ¼ f½A0þA1 ꢀ A0ꢂg ꢁ 100%
It was found that oxodiazoles 5a–d possess higher
activity than Trolox with EC50 value of 0.00521, 0.005217,
0.00551and 0. 00506 mmol.l-1 respectively followed by 5e
with EC50 value of 0.00591. The low activity of the
compound 5e is attributed to the methoxy groups.
where A0 is the absorbance of the control at 30 min, and
A1 is the absorbance of the sample at 30 min. All samples
were analyzed in triplicate.
As shown for DPPH scavenging, these data indicate the
higher capacity of 4-(4-acetyl-5- substituted -4,5-dihydro-
1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-ones 5 to
quench ABTS+ as compared to the synthetic antioxidant
Trolox.
The variation of the percentage of inhibition (PI) is
almost constant starting from a value of the concentration
equal to 1.34 mM. In addition, the synthesized products 5
have an antioxidant activity better than Trolox. Indeed, the
antioxydant capacity seems to be attenuated when the
concentration increases in the medium. This can be
explained by the existence of the peroxides sites, which
are susceptible for oxidizing when the concentration
Table 1
The EC50 values exhibited by 4-(4-acetyl-5- substitut-
ed -4, 5-dihydro-1, 3, 4-oxodiazol-2-yl)methoxy)-2H-
chromen-2-ones 5.
Compounds 5a–e
EC50 (mmol.lꢀ1)
5a
0.00521
0.005217
0.00551
0.00506
0.00591
0.005879
5b
5c
5d
5e
Trolox

N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
553
Table 2
Table 3
The EC50 values exhibited by 4-(4-acetyl-5- substitut-
ed -4, 5-dihydro-1, 3, 4-oxodiazol-2-yl) methoxy)-2H-
chromen-2-ones 5.
Antibacterial activity spectrum of compounds 5a–e.
Indicator organism
Inhibition zone
(mm)
Compounds
Compounds 5a–e
CI50 (mmol.lꢀ1
)
Staphylococcus aureus
(CIP 7625)
34
5a
5a
0.073
0.072
0.07
27
5b
5b
28
5c
5c
33
5d
5d
0.056
0.06
34
5e
5e
24–28
Gentamycin
Trolox
0.109
Staphylococcus aureus*
26
28
27
33
34
24
5a
5b
5c
5d
5e
Gentamycin
increases. We have just shown that the synthesized
oxdiazole derivatives 5a–e have good antioxidant
activity under weak concentration, but it proves to be
necessary to determine the reaction time necessary to
highlight the antioxidant effect to be able to use these
derivatives in pharmay.
Escherichia coli ATCC 25922
30
5a
a
27
5b
35
5c
32
5d
31
5e
22–26
Gentamycin
The EC50 values exhibited by 4-(4-acetyl-5- substituted
-4, 5-dihydro-1,3,4-oxodiazol-2-yl) methoxy)-2H-chro-
men-2-ones 5 are summarized in Table 2.
Klebsiella pneumonia CIP
104727
25
27
31
30
28
21
5a
5b
5c
5d
The 4-(4-acetyl-5- substituted -4, 5-dihydro-1,3,4-
5e
oxodiazol-2-yl)methoxy)-2H-chromen-2-ones
5
were
Gentamycin
shown to be efficient antioxidants. They showed higher
free radical scavenging activity than Trolox scavenging
activities.
Pseudomonas aeruginosa
30
5a
27853 (CIP 76110)
26
5b
34
5c
These compounds have a remarkable capacity oxidizing
which explains their susceptibility to fix free radicals DPPH
and ABTS+.
33
5d
32
5e
15–22
Gentamycin
The residual antibacterial activity in the compounds was tested by disc
diffusion assay against the indicator strain in LB medium at 28 8C
ATCC: American Type Culture Collection, USA; CIP: collection de l’Institut
Pasteur, Paris, France; LM: laboratoire de microbiologie, Centre national
de Greffe de Moelle Osseuse, Tunis, Tunisia.
5.3. Antibacterial activity
The antibacterial activity of 4-(4-acetyl-5- substituted -
4, 5-dihydro-1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-
2-ones 5 was assessed by the agar disk diffusion assay [36]
against five human pathogenic bacteria: Gram-positive
including S. aureus (CIP 7625), S. aureus and Gram-negative
bacteria including E. coli (ATCC 25922), P. aeruginosa (ATCC
27853)(CIP 76110) and K. pneumonia CIP 104727. The
bacterial strains were first grown on Muller Hinton medium
at 37 8C for 24 h prior to seeding onto the nutrient agar. The
antibacterial activity was assessed by measuring the zone of
growth inhibition surrounding the discs and compared with
the known antibiotic gentamycin. Standard discs of genta-
mycin(10UI)servedaspositiveantibioticcontrolsaccording
6. Conclusions
A
new versatile synthetic route to 4-(4-acetyl-5-
substituted -4, 5-dihydro-1, 3,4-oxodiazol-2-yl)
methoxy)-2H-chromen-2-ones 5 by the treatment of 4-
hydroxycoumarine with different reagents is described.
The method is easy; rapid and produced the title
compounds 5 in good yields: the structures were verified
by, UV/IRj 1Dj 2D NMR. All the newly synthesized
compounds were screened for their antibacterial and
antioxidant activities. Among the screened samples,
compounds 5a and 5b showed excellent antibacterial
activity against E. coli. Compounds with 4-phenyl, 4-
methoxyphenyl, 4-fluorophenyl and 4-nitrophenyl sub-
stituents in the 1,3,4-oxodiazole ring exhibited highest
antioxidant activity than Trolox and 1,3,4- oxodiazole
bearing 3.4.5 trimethoxysubstituent in the phenyl ring
exhibited enhanced antioxidant activity from the respec-
tive series. Absorption spectral characteristics of the
compounds were investigated in methanol by UV absorp-
tion and they were correlated with substituent on benzene
rings.
to CASFM 2005 guidelines. Discs with 10 ml of pure
methanol were used as negative controls. The results are
given in Table 3.
5.3.1. Methicillin-resistant clinical isolates
An examination of the data reveals that all the
compounds showed antibacterial activity ranging from
25 to 100
m
g mlꢀ1. The compounds 5a and 5e were highly
active against all the five organisms employed. Compound
5c was highly active against E. coli. From the screened
results, it is observed that the presence of methoxy/NO2
group at the phenyl ring increases the antibacterial
activity. The activity is maximum in a compound with a
methoxy group at 4th position.

554
N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
7. Experimental
7.1. General
7.2.3. Preparation of (E)- N- arylidene coumarin-4-oxyacetic
hydrazones 4
Coumarin-4-oxyacetic hydrazide 3 (2.34 g, 0.01 mol)
and the substituted aromatic aldehyde (0.01 mol) were
dissolved in a boiling methanol–water mixture (50:6 v/v)
mL of methanol. The reaction mixture was refluxed for 5 h.
Then concentrated and cooled. The solid product was
filtered off, washed with the appropriate solvent.
All reactions were magnetically stirred. Commercially
available reagents were used without further purification.
All chemicals were supplied from Aldrich, Merck and Fluka
Co. Melting points were determined by open capillary
method and were uncorrected.
All reactions were monitored by thin layer chromatog-
raphy (TLC). Compounds were visualized with UV light at
254 and 365 nm. Melting points were measured on a
WRX-1S instrument. Infrared (IR) spectra were recorded
with a Perkin-Elmer spectrum one B spectrometer. 1H
NMR spectra were recorded on a Varian-Unity spectrom-
eter at 300 MHz using tetramethylsilane (TMS) as an
internal standard. UV absorption spectra were recorded
on a Lambda 20 UV spectrometer Perkin Elmer. Cyclic
voltammetry (CV) was performed on a BAS 100BW
electrochemical workstation. All CV measurements were
carried out in tetrabutylammonium bromide (C4H12BrN)
as a supporting electrolyte, purging with nitrogen prior to
conduct the experiment. Platinum wire (MF-2013) was
used as a working electrode, Ag/AgCl as a reference
electrode, and another platinum wire (MF-1032) as a
counter electrode.
7.2.4. (E)-N- benzylidene coumarin-4-oxyacetic hydrazone:
4a
Yield 75%. mp 244 8C. IR spectrum,
n
cm–1: 1692 (C O);
1430 (C N); 3118 (NH).
1H NMR spectrum (DMSO-d6).
d
ppm: 4.98(s,1H,H3);
5.47(s,2H,OCH2);
H
11.78(s,1H,NH); 7.38-8.03(m,8H,
arom); 7.89(s,1H, N CH). 13CNMR spectrum (DMSO-
d6).
d ppm: 66.4(OCH2); 91.6 (C3); 144.6 (N C);115.6–
134.2(Carom); 161.9(C2); 165.1(C4); 167.5(C1’).
7.2.5. (E)- N- (4-fluorobenzylidene) coumarin-4-oxyacetic
hydrazone: 4b
Yield 72%; mp 234 8C. IR spectrum,
1430 (C N); 3118 (NH). 1H NMR spectrum (DMSO-d6).
ppm: 4.98(s,1H,H3); 5.47(s,2H,OCH2); 11.79(s,1H,NH);
n
cm–1: 1692 (C=O);
d
7.26–8.02(m,8H,
Harom); 5.92(s,1H, N
CH). 19FNMR
(DMSO-d6) ꢀ110.55 ppm. 13CNMR spectrum (DMSO-
d6).
d ppm: 66.4(OCH2); 91.6 (C3); 115.6-133.3(Carom);
7.2. Preparation of compounds 2–5
147.5 (N C); 161.9(C2); 165.1(C4); 167.6(C1’).
7.2.1. Preparation of ethyl (coumarin-4-oxy) acetate 2
4-hydroxycoumarin (16.2 g, 0.1 mol), anhydrous po-
tassium carbonate (13.8 g, 0.1 mol), ethylbromoacetate
(11.1 ml, 0.1 mol) are mixed in round bottomed flask. The
latter is stirred at reflux in dry acetone for 10 h. The
reaction mixture was filtered, while hot and the residue
washed with boiling acetone. Yield (80%); mp 245 8C. IR
7.2.6. (E)- N- (4-methoxybenzylidene) coumarin-4-oxyacetic
hydrazone: 4c
Yield 73%; mp 265 8C. IR spectrum,
1686 (C N); 3106 (NH).
n
cm–1: 1684 (C O);
1H NMR spectrum (DMSO-d6).
d ppm: 3.79 (s,3H,
OCH3); 4.95(s,1H,H3); 5.44(s,2H,OCH2); 5.88(s,1H, N CH);
11.64(s,1H,NH); 6.98-8.24(m,8H, Harom). 13CNMR spec-
trum (DMSO-d6). d ppm: 55.7(OCH3); 66.4(OCH2); 91.7
(C3); 114.6–133.3(Carom); 144.5 (N C); 162.5(C2);
165.1(C4); 167.3(C1’).
spectrum,
NMR spectrum (DMSO-d6).
4.23(q,2H,CH2); 5.1(s,2H,OCH2); 5.93 (s,1H,H3); 7.36-
n
cm–1: 1732 (CO); 1525 (O–CO lactone). 1H
ppm: 1.23(t,3H,CH3);
d
7.82(m,4H,
H
arom). 13CNMR spectrum (DMSO-d6).
d
ppm: 14.2(CH3);
61.4(CO–O–CH2);
91.7(C3),
7.2.7. (E)- N- (4-nitrobenzylidene) coumarin-4-oxyacetic
65.7(OCH2); 115.2–153.0(Carom); 161.7(C2); 164.3(C4);
167.3(CO).
hydrazone: 4d
Yield75%; mp 248 8C. IR spectrum,
1628 (C N); 2962 (NH). 1H NMR spectrum (DMSO-d6).
n
cm–1: 1710 (C O);
d
7.2.2. Preparation of coumarin-4-oxyacetic hydrazide 3
Ethyl (coumarin-4-oxy) acetate (9.72 g, 0.037 mol)
obtained in previous reaction and hydrazine hydrate
(1.1 ml, 0.022 mol) in absolute ethanol. The reaction
mixture was then stirred for 7 h at room temperature.
The reaction is controlled by TLC (hexane: ethyl acetate
2:1) when the quantities of reagents are depleted, the
stirring was stopped. The residue from the reaction
mixture was filtered off and washed with ethanol to give
a white precipitate of coumarin-4-oxyacetic hydrazide 3,
which was crystallized to give colourless plates. Yield 80%;
ppm: 5.99(s,1H,H3); 5.48(s,2H,OCH2); 8.14(s,1H, N CH);
11.86(s,1H,NH); 7.38–8.27(m,8H, Harom). 13CNMR spec-
trum (DMSO-d6).
d ppm: 66.4(OCH2); 91.9 (C3); 115.2-
133.3(Carom); 142.2 (N C); 161.9(C2); 165.0(C4);
168.0(C1’).
7.2.8. (E)- N- (3,4,5- trimethoxybenzylidene) coumarin-4-
oxyacetic hydrazone: 4e
Yield75%; mp 216 8C. IR spectrum,
141 0 (C N); 3118 (NH).
n
cm–1: 1692 (C O);
1H NMR spectrum (DMSO-d6).
d
ppm: 3.69(s,3H,
5.50(s,2H,OCH2);
mp 265 8C. IR spectrum,
n
cm–1: 1720(CO). 1715(O–CO
ppm:
OCH3);
3.82(s,6H,
OCH3);
lactone). 1H NMR spectrum (DMSO-d6).
d
5.87(s,1H,H3); 7.02–7.94(m,6H, Harom); 7.97(s,1H, N CH);
CH); 11.80(s,1H,NH). 13CNMR spectrum (DMSO-d6).
5.71(s,3H,H3); 4.78 (s,2H,OCH2); 4.42(s,2H,NH2); 7.34–
8.03(m,4H, Harom); 9.51(s,1H,NH). 13CNMR spectrum
d
ppm: 56.3(OCH3); 60.5(OCH3); 66.5(OCH2); 91.6 (C3);
104.7–144.5(Carom); 144.5 (N C); 162.7(C2); 165.2(C4);
167.6(C1’).
(DMSO-d6).
d ppm: 67.4(OCH2); 91.6 (C3); 115.3–
133.2(Carom); 161.8(C2); 164.8(C4); 165.3(CO).

N. Hamdi et al. / C. R. Chimie 14 (2011) 548–555
555
7.2.9. Preparation of 2-[(coumarin-4-oxy) methyl]-4-acetyl-
Acknowledgments
5-substitued – 1, 3,4-oxodiazoline 5
The appropriate 2-[(coumarin-4-oxy) methyl]-4-ace-
tyl-5-substitued–1,3,4-oxadiazolines 5 (0.001 mol) were
refluxed with acetic anhydride (10 ml) for 8 h, unreacted
acetic anhydride was removed and the residue was
solidified by trituration with ether and crystallized from
the proper solvent.
This work was carried out with financial aid of both the
Tunisian Ministry of Higher Education and Scientific
Research and Technology and the Spanish Agency of
International Cooperation through projects (A/9549/07
and A/8302/07).
We are very grateful to Professor A. Romerosa,
University of Almeria, Spain, for his help and for recording
the 1D and 2D NMR spectra.
7.2.10. 4-(4-acetyl-5-phenyl-4,5-dihydro-1,3,4-oxodiazol-2-
yl)methoxy)-2H-chromen-2-one 5a
Yield 65%. mp 218 8C. IR spectrum,
1414 (N–N); 1284 (C–O–C).1H NMR spectrum (DMSO-d6).
ppm: 1.89(s,3H, CH3); 4.91(s,2H,OCH2); 5.88(s,1H,H3);
7.35–7.97(m,9H, Harom); 7.99(s,1H,H5’). 13CNMR spectrum
(DMSO-d6). ppm: 20.8(CH3); 65.8(OCH2); 91.6 (C3);
n
cm–1: 1624 (C N);
References
[1] G. Hughes, M.R. Bryce, J. Mater. Chem. 15 (2005) 94.
[2] N.I. Nijegorodov, W.S. Downey, Spectrochimica Acta Part A 51 (1995)
2335.
d
d
[3] Y. Shirota, H. Kageyama, Chem. Rev. 107 (4) (2007) 953.
[4] A.P. Kulkarni, C.J. Tonzola, A. Babel, S.A. Jenekhe, Chem. Mater. 16 (23)
(2004) 4556.
[5] K. Ono, H. Ito, A. Nakashima, M. Uemoto, M. Tomura, K. Saito, Tetrahe-
dron Lett. 49 (40) (2008) 5816.
115.4 (C5’);116.7–133.2(Carom); 153.1(C2); 161.8(C4);
165.3(C O); 168.8(C2’).
[6] Y. Tao, Q. Wang, Y. Shang, C. Yang, L. Ao, J. Qin, et al. Chem. Commun.
(2009) 77.
[7] Y. Tao, Q. Wang, C. Yang, Q. Wang, Z. Zhang, T. Zou, et al. Angew. Chem.
Int. Ed. 47 (2008) 8104.
[8] L. Chen, C. Yang, J. Qin, J. Gao, H. You, D. Ma, J. Organomet. Chem. 691
(2006) 3519.
[9] Z. Xu, Y. Li, X. Ma, X. Gao, H. Tian, Tetrahedron 64 (2008) 1860.
[10] Z. Si, J. Li, B. Li, F. Zhao, S. Liu, W. Li, Inorg. Chem. 46 (15) (2007) 6155.
[11] Y.P. Wong, W.F. Xie, B. Li, W.L. Li, Chin. Chem. Lett. 18 (12) (2007) 1501.
[12] C.B. Liu, J. Li, B. Li, Z.R. Hong, F.F. Zhao, S.Y. Liu, et al. Chem. Phys. Lett.
435 (1–3) (2007) 54.
7.2.11. 4-(4-acetyl-5-(4-fluorophenyl)-4,5-dihydro-1,3,4-
oxodiazol-2-yl)methoxy)-2H-chromen-2-one 5b
Yield 65%, mp 222 8C. IR spectrum,
1404 (N–N); 1246 (C–O–C).1H NMR spectrum (DMSO-d6).
ppm: 2.41(s,3H, CH3); 5.48(s,2H,OCH2); 6.0(s,1H,H3);
n
cm–1: 1630 (C N);
d
7.34-7.99(m,8H, Harom); 8.67(s,1H,H5’). 19F NMR (DMSO-
d6) ꢀ107.13 ppm.13CNMR spectrum (DMSO-d6).
d ppm:
26.0(CH3); 69.3(OCH2); 91.9 (C3); 115.5 (C5’);116.4-
133.2(Carom);
171.2(C2’).
162.0(C2);
164.8(C4);
166.8(C=O);
[13] N.J. Lundin, A.G. Blackman, K.C. Gordon, D.L. Officer, Angew. Chem. Int.
Ed. 45 (16) (2006) 2582.
[14] N.J. Xiang, L.M. Leung, S.K. So, J. Wang, Q. Su, M.L. Gong, Mater. Lett. 60
(23) (2006) 2909.
[15] Z. He, W.Y. Wong, X. Yu, H.S. Kwok, Z. Lin, Inorg. Chem. 45 (26) (2006)
10922.
[16] W.Y. Wong, Z. He, S.K. So, K.L. Tong, Z. Lin, Organometallics 24 (2005)
4079.
[17] M. Mauro, M. Panigati, D. Donghi, P. Mercandelli, P. Mussini, A. Sironi,
et al. Inorg. Chem. 47 (23) (2008) 11154.
[18] A. Kumar, S.S. D’Souza, S.L. Gaonkar, K.M.L. Rai, B.P. Salimath, Invest.
New Drugs 26 (2008) 425.
[19] K.M. Abdel, M.E. Mohga, S.A. Nasser, Molecules 8 (2003) 744.
[20] P. Rajakumar, S. Raja, Tetrahedron Lett. 50 (2) (2009) 223.
[21] A. Kudelko, W. Zielinski, Tetrahedron Lett. 65 (6) (2009) 1200.
7.2.12. 4-(4-acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1,3,4-
oxodiazol-2-yl)methoxy)-2H-chromen-2-one 5c
Yield 67%; mp 258 8C. IR spectrum,
1388 (N–N); 1246 (C–O–C).1H NMR spectrum (DMSO-d6).
ppm: 2.38(s,3H, CH3); 3.83(s,3H,OCH3);
5.46(s,2H,OCH2); 6.07(s,1H,H3); 7.07-7.85(m,8H, Harom);
8.53(s,1H,H5’).13CNMR spectrum (DMSO-d6).
ppm:
25.9(CH3); 55.9(OCH3); 69.3(OCH2); 91.9 (C3); 114.9
n
cm–1: 1624 (C N);
d
d
(C5’);115.5–153.1(Carom);
168.0(C O); 171.2(C2’).
162.0(C2);
164.8(C4);
´
´
[22] P. Gomez-Saiz, R. Gil-Garcıa, M.A. Maestro, F.J. Arnaiz, L. Lezama, T.
Rojo, et al. Eur. J. Inorg. Chem. 3 (2009) 373.
[23] H. Li, S. Kang, Z. Xing, H. Zeng, H. Wang, Dyes Pigments 80 (1) (2009) 163.
[24] Y.W. Ho, W.H. Yao, Dyes Pigments 82 (1) (2009) 6.
[25] N. Marquez, R. Sancho, L.M. Bedoya, J. Alcamy, J.L. Lopez-Perez, A.S.
Feliciano, B.L. Fiebich, E. Munoz, Antiviral Res. 66 (2005) 137.
[26] C. Spino, M. Dodier, S. Sotheeswaran, Bioorg. Med. Chem. Lett. 8 (1998)
3475.
[27] S. Thaisrivongs, K.D. Watenpaugh, W.J. Howe, P.K. Tomich, L.A. Dolak,
K.T. Chong, C.C. Tomich, A.G. Tomasselli, S.R. Turner, J.W. Strohbach,
A.M. Mulichak, M.N. Janakiraman, J.B. Moon, J.C. Lynn, M. Horng, R.R.
Hinshaw, K.A. Curry, D.J.J. Rothrack, Med. Chem. 38 (1995) 3624.
[28] D. Yu, M. Suzuki, L. Xie, S.L. Morris-Natschke, K.H. Lee, Med. Res. Rev. 23
(2003) 322.
7.2.13. 4-(4-acetyl-5-(4-nitrophenyl)-4,5-dihydro-1,3,4-
oxodiazol-2-yl)methoxy)-2H-chromen-2-one 5d
Yield 70%. mp 216 8C. IR spectrum,
1410 (N–N); 1242 (C–O–C).1H NMR spectrum (DMSO-d6).
ppm: 2.18(s,3H, CH3); 5.40(s,2H,OCH2); 5.92(s,1H,H3);
7.18-8.29(m,8H, Harom); 7.80(s,1H,H5’). 13CNMR spectrum
(DMSO-d6). ppm: 20.8(CH3); 66.4(OCH2); 91.9 (C3);
n
cm–1: 1628 (C N);
d
d
115.4 (C5’);115.4–153.1(Carom); 157.6(C2); 161.8(C4);
163.0(C O); 164.8(C2’).
[29] J.W. Erickson, S.K. Burt, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 545–
571.
[30] I. Kostova, S. Raleva, P. Genova, R. Argirova, Bioinorg. Chem. Appl.
(2005–2006) 1.
[31] A.J. Vlietinck, T. DeBruyne, S. Apers, L.A. Pieters, PlantaMed. 64(1998) 97.
[32] N. Hamdi, Carmen Puerta and Pedro Valerga, Eur. J. Med. Chem. 43
(2008) 2541.
[33] N. Hamdi, Mustapha Saoud and Antonio Romerosa, J. Het. Chem. 45
(2008) 1835.
[34] S. Kirkiachiarian, R. Bakhchinian, H. Chidiak, M. Mazmanian, C. Planche,
Ann. Pharm. Fr. 57 (1999) 251.
[35] J.R. Soares, T.C.P. Dins, A.P. Cunha, L.M. Ameida, Free Radical Res. 26
(1997) 469.
[36] J. Oszmianski, A. Wojdylo, E. Lamer-Zarawska, K. Swiader, Food Chem.
100 (2007) 579.
7.2.14. (4-acetyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
1,3,4-oxodiazol-2-yl)methoxy)-2H-chromen-2-one 5e
Yield 72%; mp 248 8C. IR spectrum,
1408 (N–N); 1284 (C–O–C).1H NMR spectrum (DMSO-d6).
ppm: 2.41(s,3H, CH3); 3.75(s,3H,OCH3);
3.78(s,6H,OCH3); 4.99(s,2H,OCH2); 5.88(s,1H,H3); 7.21–
n
cm–1: 1634 (C N);
d
7.67(m,6H,
(DMSO-d6).
H
d
arom); 8.43(s,1H,H5’).13CNMR spectrum
ppm: 20.8(CH3); 56.3(OCH3); 56.4(OCH3);
60.6(OCH2); 91.9 (C3); 116.8 (C5’);123.3–153.7(Carom);
162.0(C2); 164.8(C4); 172.4(C O); 192.2(C2’).