Synthesis and antimicrobial activity of novel coumarin derivatives from 4-methylumbelliferone

Article abstract of DOI:10.1007/s00044-015-1368-y

Considering the potential interest of heterocyclic compounds, the aim of the present study is to synthesize new coumarin derivatives, to provide their full chemical characterization and to evaluate their antimicrobial activities. The reaction of ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate 2 with sodium hydroxide afforded the corresponding 2-(2-oxo-4-methyl-2H-chromen-7-yloxy) acetic acid 3 which was esterified using a series of alcohols in the presence of iodine to yield a new series of coumarin esters 4a-j. On the other hand, treatment of the key intermediate 2 with an aqueous solution of hydrazine in ethanol at reflux gave the corresponding hydrazide 5 which further converted into coumarin derivatives 6a-f and 7a-c by condensation with a series of aromatic aldehydes and cyclic anhydrides, respectively. The synthesized compounds were completely characterized by ¹H NMR, ¹³C NMR, IR and HRMS. The antibacterial and antifungal activities of the new synthesized compounds were evaluated using the disc diffusion method and seemed to be significant.

Full text of DOI:10.1007/s00044-015-1368-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1368-y
ORIGINAL RESEARCH
Synthesis and antimicrobial activity of novel coumarin derivatives
from 4-methylumbelliferone
Sined Medimagh-Saidana1 Anis Romdhane1 Mejda Daami-Remadi2
•
•
•
Haifa Jabnoun-Khiareddine2 David Touboul3 Hichem Ben Jannet1
M’hamed Ali Hamza1
•
•
•
Received: 2 October 2014 / Accepted: 15 April 2015
Ó Springer Science+Business Media New York 2015
Abstract Considering the potential interest of hetero-
cyclic compounds, the aim of the present study is to syn-
thesize new coumarin derivatives, to provide their full
chemical characterization and to evaluate their antimicro-
bial activities. The reaction of ethyl 2-(4-methyl-2-oxo-2H-
chromen-7-yloxy) acetate 2 with sodium hydroxide af-
forded the corresponding 2-(2-oxo-4-methyl-2H-chromen-
7-yloxy) acetic acid 3 which was esterified using a series of
alcohols in the presence of iodine to yield a new series of
coumarin esters 4a–j. On the other hand, treatment of the
key intermediate 2 with an aqueous solution of hydrazine
in ethanol at reflux gave the corresponding hydrazide 5
which further converted into coumarin derivatives 6a–f and
7a–c by condensation with a series of aromatic aldehydes
and cyclic anhydrides, respectively. The synthesized
Keywords
Ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate Á
Coumarin esters Á Coumarin hydrazides Á
Antifungal activity Á Antibacterial activity
Introduction
Heterocyclic compounds are indispensable structural units
that are useful in medicinal chemistry and valuable as
synthetic organic blocks. In various classes of natural
heterocyclic compounds of biological interest, the
coumarin moiety is commonly present (Wu et al., 2009).
They display interesting biological and pharmacological
profiles, such as the antibacterial (Al-Amiery et al., 2011a),
cytotoxic (Al-Amiery et al., 2011a), antioxidant (Al-Ami-
ery et al., 2011a), antifungal (Al-Amiery et al., 2012),
anticoagulant (Manolov and Danchev, 1995), anti-inflam-
matory (Emmanuel-Giota et al., 2001), antitumor and anti-
HIV (Harvey et al., 1988; Kostova et al., 2006) activities.
In addition, these compounds have varied bioactivities and
applications in cosmetics, pharmaceuticals, food, flavoring
and agrochemicals (Borges et al., 2005; Kabalka et al.,
2005; Morimoto et al., 2003; Srinivasan and Rampally,
2004).
1
compounds were completely characterized by H NMR,
13C NMR, IR and HRMS. The antibacterial and antifungal
activities of the new synthesized compounds were
evaluated using the disc diffusion method and seemed to be
significant.
& Hichem Ben Jannet
hich.benjannet@yahoo.fr
1
´ ´
Laboratoire de Chimie Heterocyclique, Produits Naturels et
Sincethe coumarinnucleus isassociatedwiththese diverse
biological and pharmacological activities, several methods
for the preparation of coumarin-based compounds have been
reported (Gammonn et al., 2005; Messaoudi et al., 2010;
Santana et al., 2006). Particularly, the use of 4-methylum-
belliferoneasa precursorrepresentsa usefulsynthetic method
for the preparation of such compounds (Ganesh et al., 2010;
Kumar et al., 2011a; Ramesh et al., 2008).
´
Reactivite (LR11SE39), Equipe: Chimie Medicinale et
Produits Naturels, Faculte des Sciences de Monastir,
´
Universite de Monastir, Avenue de l’Environnement,
5019 Monastir, Tunisia
´
´
´
2
3
´
UR13AGR09, Production Horticole Integree au Centre Est
Tunisien, Centre Regional des Recherches en Horticulture et
´
´
´
Agriculture Biologique de Chott-Mariem, Universite de
Sousse, 4042 Chott-Mariem, Tunisia
Centre de Recherche de Gif, Institut de Chimie des
Substances Naturelles, CNRS, Avenue de la Terrasse,
91198 Gif-Sur-Yvette Cedex, France
Our research has been devoted to the development of a
new class of heterocyclic systems which incorporate the
123

Med Chem Res
coumarin moiety with the hope that they may be
biologically active.
spectrum of 4a (as an example) gave a pseudo-molecular
ion peak [M ? H]? at m/z 305.1381, which is consistent
with the molecular formula (C17H21O5). In addition to the
signals corresponding to the protons introduced by
4-methylumbelliferone, new signals related to the alcohol
used were observed in the 1H-NMR spectrum. Examination
at 300 MHz showed a multiplet at dH 3.99 (2H, H-50) and
also a multiplet at dH 0.87 (6H, H-60, H-70). The 13C-NMR
spectrum confirmed the above spectral data by the obser-
vation of new signals at dC 11.2, 16.3, 25.9, 34.1 and
70.1 ppm relative to carbons of the alcohol used. The same
spectrum showed a signal at dC 168.1 ppm attributable to
the carbonyl of the ester group (C-20).
Herein, we report here the synthesis of ethyl 2-(4-methyl-
2-oxo-2H-chromen-7-yloxy) acetate 2 and its use as a
building block in the synthesis of some new coumarin-based
derivatives 4–7. Their antimicrobial activities were evaluat-
ed, and the structure–activity relationship was also discussed.
Our approach to the target coumarin derivatives was
firstly started by the synthesis of product 2 via condensa-
tion reaction of equimolar amount of 4-methylumbellifer-
one, ethyl chloroacetate and anhydrous K2CO3 in dry DMF
(Al-Amiery et al., 2011b). Then, compound 2 was con-
verted into the 2-(2-oxo-4-methyl-2H-chromen-7-yloxy)
acetic acid 3 (Abd El-Fattah et al., 2011) which was es-
terified using a series of alcohols in the presence of iodine
(Ramalinga et al., 2002; Jereb et al., 2009) to yield a new
series of coumarin esters 4a–j (Scheme 1; Table 1).
In the second step, we have subjected the key interme-
diate 2 to reaction with an aqueous solution of hydrazine in
ethanol at reflux (Al-Amiery et al., 2011b). The so-formed
hydrazide 5, which belongs to a class of intermediates,
known to be highly reactive and used as precursors for the
synthesis of new nitrogen’s compounds (Abdel-Aziz et al.,
2007) was further converted into coumarin derivatives 6
and 7 by treatment with a series of aromatic aldehydes
(Scheme 2; Table 2) and cyclic anhydrides, respectively
(Scheme 3; Table 3).
1
In the H-NMR spectrum of compound 2, a 3H triplet
was observed at dH 1.25 ppm (J = 7.2 Hz) due to the ester
CH3 protons and a quartet at dH 4.23 ppm (J = 7.2 Hz)
due to the ester CH2 protons. The isolated CH2 protons
were observed downfield as a singlet (2H) at dH 4.63 ppm.
The 13C-NMR spectrum analysis for compound 2, com-
bined with the information from 1H-NMR experiment, can
be considered enough to guide future synthetic work. The
13C NMR spectrum of this compound confirms the pro-
posal structure by the observation of the signals at dC 13.6
(C-40), 61.1 (C-30) and 167.4 (C-20) ppm. The 13C-NMR
spectrum analysis for compound 2, combined with the in-
1
The H-NMR spectrum of the intermediate 5 showed
characteristic signals at dH 5.02, 4.38 and 9.45 ppm cor-
responding to the methylene (C-10), NH2 and NH groups,
respectively. Its 13C-NMR spectrum reinforced this struc-
ture by the appearance of the signal of (C-20) at dC
169.6 ppm and the disappearance of the two signals at dC
13.6 (C-40) and 61.1 (C-30) ppm attributed to the ethoxy
carbons in compound 2.
1
formation from H-NMR experiments, can be considered
enough to guide future synthetic work.
In the 1H-NMR spectrum of compound 3, the CH2
protons were observed a singlet (2H) at dH 4.78 ppm and
the CH3 of the coumarin moiety appeared at dH 2.39 ppm.
On the same spectrum, we noticed the appearance of a new
signal at dH 13.07 ppm corresponding to the OH group of
the acetic acid fragment. The 13C-NMR spectrum of
compound 3 shows in particular a signal at dC 169.6 ppm
attributable to the carbonyl of the acid group (C-20).
The structure of these compounds (4a–j) was estab-
lished on the basis of their analytical data. Thus, mass
Compound 6b was obtained as a white powder. Its
ESHRMS gave pseudo-molecular ion peak [M ? H]? at
m/z 327.0975, which is consistent with the molecular for-
1
mula C17H14 N2O5. Thus, the H-NMR spectrum of 6b
showed the absence of the NH2 protons at dH 4.38 ppm,
and the appearance of a new characteristic singlet at dH
7.83 ppm, assignable to the proton –N = CH and the
presence of the NHCO proton resonating at dH 11.63 ppm.
Also the structure of compound 7 has been assigned
from their analytical data. In fact the ESHRMS of
9CH3
CH
CH3
3
5
8
4
4a
1/ NaOH (5 %)
2/ HCl
6
7
3
2
ClCH2COOC2H5/ K2CO3
4'
1'
1'
2'
H3C 3'
O
HO
2'
8a
DMF reflux
O
2
O1
O
O
O
O
HO
O
1
O
3
O
O
CH
3
I
2/ reflux
1'
3
R'-OH
+
R'O
2'
O
O
O
4a-j
O
Scheme 1 Synthesis of new coumarin esters 4a–j
123

Med Chem Res
Table 1 Synthesis of compounds 4a–j
Entry
Product
Entry
6
Product
9
9
CH3
4
CH3
4
5
5
7'
4a
8a
4a
8a
3
2
3
2
6
CH3
6
6'
4'
4'
H3C
1'
1'
2'
H3C
O
3'
5'
O
3'
2'
7
O
O1
O
7
O
O1
O
1
8
8
O
CH3
5'
O
4a
4f
a
9
CH3
4
CH3
4
5
5
4a
3
2
4a
8a
6
3
2
6
1'
4'
3'
5'
1'
2'
O
4'
2'
7
O
3'
8a
CH3
O
O1
O
7
8
HO
O
O1
O
2
3
7
8
O
O
4g
4b
9
9
CH3
4
CH3
4
5
5
5'
4a
8a
5'
CH3
4a
3
2
6
CH3
3
6
1'
1'
2
O
6'
CH3
2'
3'
4'
4'
O
7
2'
3'
O
O1
O
8a
7
HO
O
O1
O
8
8
O
O
8
9
4h
4c
9
9
CH3
CH3
4
5
4
5
4a
8a
4a
3
2
6
3
6
OH
4'
6'
CH3
1'
4'
1'
2
2'
O
5'
3'
O
2'
3'
7
O
O1
O
7
4
5
8a
5'
O
O1
O
8
8
O
OH
O
4i
4d
9
9
CH3
CH3
5
4
4a
8a
5
4
3
2
6
3
2
6
4'
6'
1'
C7H' 3
O
O1
O
O
2'
1'
3'
5'
4'
7
2'
O
3'
8
5'
6'
7
10
O
O1
O
O
8
8'
O
7'
4e
4j
Conditions: acid (0.1g, 0.4 mmol), alcohol (10 mL), iodine (5 mg), reflux 2-24 h
compound 7a gave pseudo-molecular ion peak [M ? H]?
at m/z 377.0777, which is consistent with the molecular
of the protons introduced by the hydrazide 5, we note the
presence of a new multiplet (4H) at dH 8.01 ppm relative to
the aromatic protons of the phthalic anhydride. The 13C-
NMR spectrum confirmed the above spectral data by the
observation of new signals at dC 129.3, 135.4, 154.5 and
1
formula C21H19O6N2. Furthermore, the H NMR spectrum
of this compound was compatible with the proposed
structure. In addition to the signals corresponding to most
123

Med Chem Res
CH3
O
CH3
CH3
O
NH2-NH2, H2O
Ethanol reflux
O
3'
1'
Ar-CHO
O
Ethanol reflux
1'
4'
NH
2'
H3C
O
NH 2'
H2N
O
O
Ar
N
O
O
O
2
O
O
5
O
6a-f
Scheme 2 Synthesis of hydrazone 6a–f
Table 2 Synthesis of compounds 6a–f
Entry
1
Product
Entry
4
Product
9CH3
4
9
CH3
5
5
4a
4
3
2
4a
8a
6
3
2
OH
3"
H
1"
6
H
1"
2'
2'
NH 1'
O
2"
S
7
8a
NH 1'
N
4"
5"
2"
3"
N
O
O1
O
8
7
O
O1
O
5"
4"
8
7"
O
6"
6a
6d
9
9
CH3
CH3
5
4
4a
5
4
3
6
4a
H
1"
3
6
H
1"
2
7"
2'
2
5
NH
2"
1'
8a
2
2'
7
6"
N
O
O
O
8
1
O
1'
NH
2"
7
8a
N
O
O1
O
5"
4"
8
3"
O
5"
HO
4"
3"
O
OCH3
8"
6b
6e
9
9
CH3
4
CH3
4
5
5
4a
4a
3
3
2
6
H
1"
6
H
1"
2
7"
2'
3
6
3"
2'
NH
4"
2"
1'
8a
NH
7
3"a
6"a
6"
1'
2"
8"
N
O
O1
O
7
8
5"
6"
8a
N
O
O
O
8
1
O
3"
5"
O
H3CO
9"
4"
N
7"
OCH3
8"
6f
6c
Conditions: hydrazide 5 (4 mmol), aromatic aldehydes (1eq), ethanol (20 mL), 2h, reflux
164.9 ppm relative to carbons of the phthalic anhydride
moiety.
increasing levels of resistance to classical antibiotics (Rice
et al., 1999; Al-Amiery et al., 2009; Kadhum et al., 2011),
and the discovery and development of effective antibac-
terial and antifungal drugs with novel mechanisms of ac-
tion have become urgent tasks for infectious disease
research programs (Kumar et al., 2011b). All the synthe-
sized compounds were analyzed on what concerns their
antimicrobial inhibition (Table 4). The antimicrobial inhi-
bition was determined using the disc diffusion method
Biological activity
Antimicrobial activity
The serious medical problem of bacterial and fungal re-
sistance and the rapid rate of its development has led to
123

Med Chem Res
CH3
O
Ethanol reflux
+
Cyclic anhydride
7a-c
NH
O
O
NH2
5
O
Scheme 3 Synthesis of novel coumarin derivatives 7a–c
Table 3 Synthesis of compounds 7a–c
Entry
1
Product
Entry
3
Product
9
7'
6'
CH3
8'
5'
4a
8a
O
C5'
6
3
4'a
3'
NH
9
7'
2'
8'a
2
4'
NH
HO
8'
CH3
6'
1'
O
7
O
O
O
1
O
9'
4'
5
8
4
8
N
4a
8a
O
O
6
3
2'
2
7c
NH
1'
3'
7
O
O1
O
O
7a
9
9'
CH3
4
5
8'
O
4a
8a
H
6
3
2
10'
7'
3'
NH
2
2'
8a'
4a'
N
1'
5'
7
O
O1
O
8
6'
4'
H
O
O
7b
Conditions: hydrazide 5 (4 mmol), cyclic anhydre (1eq), ethanol (20 mL), 8h, reflux
described in the literature (Marmonier, 1987; Barry and
Thornsberry, 1991).
exhibited an important antimicrobial activity (Lin et al.,
2012; Singh et al., 2010).
According to the results given in Table 4, most compounds
have displayed good antifungal activity against Botrytis
cinerea and Fusarium oxysporum f. sp. lycopersici. Com-
pounds 4a and 4g exhibited an important inhibitory activity
against B. cinerea (IZ = 36 and 39 mm, respectively), while
compound 4j was found to be the most active against F.
oxysporum f. sp. lycopersici (IZ = 16 mm). The compounds
4a, 4i and 7a showed moderate activity against Aspergilus
niger (IZ = 6–8 mm).
Moreover, it was indicated that the combination of the
coumarin skeleton with some nitrogen-containing hetero-
cyclic moieties could significantly increase their antimi-
crobial efficiency (Keri et al., 2009; Ronad et al., 2010).
The antimicrobial activity of the compounds 4a–j de-
pends on the structure of the fragment introduced by the
alcohol. Generally, the esterification does not seem
beneficial for such antibacterial activity. Secondly, the
behavior of the fungi used, such as B. cinerea, to com-
pounds 4a–j appears complex, but in all cases the hydroxyl
group or groups remained free in certain alcohols used
(cases of 4b, 4c and 4d) did not appear an interesting factor
that brings activity. The relatively high activity of com-
pound 6d against F. oxysporum (IZ = 15 mm) could be
explained by the presence of the free phenol function. The
ortho-position of the latter allows the formation of a pos-
sible hydrogen bond with the free electron pair of the sp2
hybridized nitrogen atom of the hydrazone function, thus
From the data of the antibacterial activity, few compounds
(4f, 4h, 4i, 6e, 7a and 7c) were found to be active against
Pseudomonas sp. (Pa 499) and Bacillus sp (Bp 420)
(IZ = 5–8 mm) (Table 4). However, compound 7b exhibited
an interesting antibacterial activity against Pseudomonas sp.
(IZ = 12 mm) which was comparable to that obtained with
the standard reference (ampicillin) used.
Some results prove consistent with several studies
showing that coumarins and some of their derivatives
123

Med Chem Res
Table 4 Antimicrobial activity of coumarin derivatives 4a–j, 6a–f and 7a–c
Bacteria
Fungi
Compound
Pseudomonas
sp. (Pa 499)
Bacillus
sp. (Bp 420)
Aspergillus
niger
Botrytis cinerea
Fusarium
oxysporum f.
sp. lycopersici
Inhibition zone (IZ) in mm
4a
–
–
–
–
–
5
–
5
–
–
–
–
–
–
8
–
6
12
5
10
–
–
–
–
–
–
–
–
–
6
–
–
–
–
–
–
–
–
6
–
10
–
7
–
–
–
–
–
–
–
8
–
–
–
–
–
–
–
6
–
–
–
10
36
–
–
4b
12
11
13
–
4c
–
4d
11
11
–
4e
4f
12
11
12
12
16
–
4g
39
–
4h
4i
–
4j
–
6a
10
–
6b
11
11
15
11
–
6c
–
6d
13
–
6e
6f
–
7a
12
15
–
11
13
12
–
7b
7c
Ampicillin
Carbendazim
–
30
10
– No inhibition zone observed
further stabilizing the molecule; this stability may be at the
origin of the activity of this derivative. On the other hand,
the para-position of this phenol function to the hydrazone
in compound 6e and the presence of a methoxyl group
certainly explain the attenuation of this activity against the
same strain (IZ = 11 mm). The relatively high activity of
compound 7b toward bacteria and fungi used except A.
niger compared to its analogue 7a could be partly ex-
plained by the non-aromatic bicyclic structure of the
moiety introduced by the anhydride used. The open
structure of the fragment introduced by the succinic an-
hydride used in the preparation of compound 7c and the
emergence of a carboxylic acid function were without
significant effect on its antimicrobial activity.
the synthetic compounds possessed good antibacterial and
antifungal activities. Most of the compounds might be
promising for any investigations to develop new effective
antibacterial and antifungal drugs.
Experimental section
Chemistry
Melting points were determined on a Bu¨chi 510 apparatus
using capillary tubes. Mass spectra were obtained with ESI-
TOF (LCT Premier XE, Waters) using the reflectron mode
in the positive ion mode. Leucine-enkephalin peptide was
1
employed as the LockSpray lockmass. H (300 MHz) and
13C (75 MHz) NMR spectra were recorded on a Bruker
AM-300 spectrometer, using CDCl3 and DMSO-d6 as sol-
vents and non-deuterated residual solvents as internal stan-
dards. Chemicals shifts (d) are given in parts per million
(ppm) and coupling constants (J) in Hertz. Commercial TLC
plates (Silica gel 60, F254, SDS) were used to monitor the
progress of the reaction. Column chromatography was
Conclusion
In conclusion, the present study provides a novel and ef-
ficient synthetic route to the preparation of new coumarin
esters, hydrazone and novel coumarin derivatives from
4-methylumbelliferone. The results showed that some of
123

Med Chem Res
performed with silica gel 60 (particle size 40–63 lm, SDS).
The starting materials 2, 3 and 5 were prepared according to
the literature (Ramesh et al., 2008; Al-Amiery et al., 2011).
(CH2-10), 70.1 (CH2-30), 101.7 (CH-8), 112.4 (CH-6),
114.3 (CH-3), 125.8 (CH-5), 152.5 (C-8a), 155.0 (C-4),
160.5 (C-7), 161.1 (C-2), 168.2 (C-20). ESI-HRMS m/z
[M ? H]? calcd. for (C17H21O5)?:305.1389 found:
305.1381.
Preparation of compounds 4a–j
A mixture of acid 3 (0.1 g, 0.4 mmol), alcohol (10 ml) and
iodine (5 mg,) was boiled under reflux for 2 to 24 h. The
progress of the reaction was monitored by TLC. The sol-
vent (excess of alcohol) was evaporated in vacuo. The
mixture was extracted twice with the appropriate solvent
and then dried over Na2SO4. After removing the solvent
under reduced pressure, the residue thus obtained was pu-
rified by column chromatography on silica gel eluted with a
mixture PE/EtOAc (8:2) to give the desired carboxylic
esters 4a–j.
2-Hydroxyethyl 2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetate (4b)
White solid, yield 70 %, mp 308 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 2.33 (s, 3H, CH3), 3.82 (t,
2H, J1 = 3.3 Hz, J2 = 1.2 Hz, H-40), 4.30 (t, 2H,
J1 = 3.3 Hz, J2 = 1.3 Hz, H-30), 4.68 (s, 2H, H-10), 6.10
(s, 1H, H-3), 6.72 (d, 1H, J = 2.4 Hz, H-8), 6.87 (dd, 1H,
J1 = 8.7 Hz, J2 = 2.4 Hz, H-6), 7.45 (d, 1H, J = 8.7 Hz,
H-5). 13C-NMR (75 MHz, CDCl3): dc 18.7 (CH3-9), 60.8
(CH2-40), 65.2 (CH2-10), 66.9 (CH2-30), 101.7 (CH-8),
112.6 (CH-6), 114.5 (CH-3); 125.9 (CH-5), 152.5 (C-8a),
155.1 (C-4), 160.5 (C-7), 161.2 (C-2), 168.3 (C-20). ESI-
HRMS [M ? H]? calcd. for (C14H15O6)?: 279.0790
found: 279.0796.
Ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate (2)
1
Yellow solid, yield 72 %, mp 102 °C (EtOH). H-NMR
(300 MHz, CDCl3): dH 1.25 (t, 3H, J = 7,2 Hz, CH3), 2.33
(s, 3H, CH3), 4.23 (q, 2H, J = 7,2 Hz, H-30), 4.63 (s, 2H,
H-10), 6.06 (s, 1H, H-3), 6.69 (d, 1H, J = 2,7 Hz, H-8),
6.83 (dd, 1H, J1 = 8,7 Hz, J2 = 2,7 Hz, H-6), 7.46 (d, 1H,
J = 8,7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): dc 13.6
(CH3-40), 18.0 (CH3-9), 61.1 (CH2-30), 64.8 (CH2-10),
101.2 (CH-8), 111.8 (CH-6 ? C-3), 113.8 (C-4a), 125.3
(CH-5), 151.9 (C-8a), 154.4 (C-4), 160.1 (C-7 ? C-2),
167.4 (C-20).
2-Hydroxypropyl 2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetate (4c)
White solid, yield 72 %, mp 219 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 1.22 (m, 3H, H-50), 2.38 (s,
3H, CH3), 3.64 (m, 1H, H-40), 4.12 (m, 2H, H-30), 4.76 (s,
2H, H-10), 6.14 (1H, s, H-3), 6.79 (d, 1H, J = 2.4 Hz, H-8),
6.87 (dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz, H-6), 7.50 (d, 1H,
J = 8.7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): dc 18.2
(CH3-9), 18.7 (CH3-50), 64.7 (CH-40), 69.3 (CH2-10), 73.0
(CH2-30), 101.3 (CH-8), 112.1 (CH-6), 114.0 (CH-3), 125.3
(CH-5), 151.9 (C-8a), 154.5 (C-4), 160.5 (C-7), 167.5 (C-
2), 167.6 (C-20). ESI-HRMS: m/z [M ? H]? calcd. for
(C15H16O6)?: 292.0947 found: 292.0950.
2-(4-Methyl-2-oxo-2H-chromen-7-yloxy) acetic acid (3)
White solid, yield 67 %, mp 206 °C (EtOH).1H-NMR
(300 MHz, DMSO-d6): dH 2.39 (s, 3H, CH3), 4.78 (s, 2H,
H-20), 6.21 (s, 1H, H-3), 6.98 (dd, 1H, J1 = 8,4 Hz,
J2 = 2,4 Hz, H-8), 7.69 (d, 1H, J = 8,7 Hz, H-5), 13.07 (s,
1H, OH). 13C-NMR (75 MHz, DMSO-d6): dc 18.1 (CH3-
9), 64.8 (CH2-10), 101.5 (CH-8), 111.4 (CH-6), 112.2 (CH-
3), 113.5 (C-4a), 126.5 (CH-5), 153.3 (C-8a), 154.5 (C-4),
160.7 (C-7 ? C-2), 169.6 (C-20).
2,3-Dihydroxypropyl 2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetate (4d)
White solid, yield 22 %, mp 208 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 2.40 (s, 3H, CH3), 3.70 (dd,
2H, J1 = 15.2 Hz, J2 = 6 Hz, H-50), 4.15 (dd, 2H,
J = 2.1 Hz, J = 11.4 Hz, H-30), 4.08 (m, 1H, H-40), 4.78
(s, 2H, H-10), 6.18 (s, 1H, H-3), 6.82 (d, 1H, J = 2.4 Hz,,
H-8), 6.95 (dd, 1H, J = 8.7 Hz, J = 2.4 Hz,, H-6), 7.55 (d,
1H, J = 8.7 Hz,, H-5) 13C-NMR (75 MHz, CDCl3): dc
20.2 (CH3-9), 62.4 (CH2-50), 64.1 (CH-40), 66.1 (CH2-30),
70.1 (CH2-10), 102.0 (CH-8), 110.9 (CH-6), 112.2 (CH-3),
115.3 (C-4a), 128.8 (CH-5), 152.0 (C-8a), 155.1 (C-4),
160.7 (C-7) 162.4 (C-2), 168.9 (C-20). ESI-HRMS: m/z
[M ? H]? calcd. for (C15H17O7)?: 309.0896 found:
309.0899.
2-Methylbutyl 2-(4-methyl-2-oxo-2H-chromen-7- yloxy)
acetate (4a)
White solid, yield 53 %, mp 301 °C (EtOH). 1H-NMR
(300 MHz, CDCl3): dH 2.37 (s, 3H, CH3), 0.87 (m, 6H,
H-60 ? H-70), 1.13 (m, 1H, H-30a), 1.35 (m, 1H, H-30b),
1.70 (m, 1H, H-40), 3.99 (m, 2H, H-50), 4.69 (s, 2H, H-10),
6.12 (s, 1H, H-3), 6.75 (d, 1H, J = 2.4 Hz, H-8), 6.83 (dd,
1H, J1 = 8.7 Hz, J2 = 2.7 Hz, H-6), 7.46 (d, 1H,
J = 8.7 Hz, H-5).
13C-NMR (75 MHz CDCl3): dc 11.2 (CH3-60), 16.3
(CH3-70), 18.7 (CH3-9), 25.9 (CH2-50), 34.1 (CH-40), 65.2
123

Med Chem Res
J = 6.9 Hz, H-40), 3.92 (d, 2H, J = 6.6 Hz, H-30), 4.63 (s,
2H, H-10), 6.07 (s, 1H, H-3), 6.71 (d, 1H, J = 2.7 Hz, H-8),
6.83 (dd, 1H, J1 = 8.4 Hz, J2 = 2.4 Hz, H-6), 7.43 (d,1H,
J = 8.4 Hz, H-5). 13C-NMR (75 MHz, CDCl3): dc 27.7
(CH3-50 ? 60), 18.6 (CH3-9), 65.3 (CH2-30), 71.6 (CH2-10),
101.7 (CH-8), 112,5 (CH-6), 114.4 (CH-3), 125.7 (CH-5),
152.3 (C-8a), 155.1 (C-4), 160.7 (C-7), 161.0 (C-2), 168.1
(C-20).ESI-HRMS: m/z [M ? H]? calcd. for (C16H19O5)?:
291.1154 found: 291.1151.
Pentyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate
(4e)
White solid, yield 72 %, mp 202 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 1.18 (m, 8H, H-30 ? H-
40 ? H-50 ? H-60), 1.59 (m, 3H, CH3), 2.31 (s, 3H, CH3),
4.61 (s, 2H, H-10), 6,05 (s, 1H, H-3), 6.69 (d, 1H,
J = 2.4 Hz, H-8), 6.82 (dd, 1H, J1 = 8.7 Hz, J2 = 2.7 Hz,
H-6), 7.44 (d, 1H, J = 8.7 Hz, H-5). 13C-NMR (75 MHz,
CDCl3): dc 18.7 (CH3-9), 18.6 (CH3-70), 22.2 (CH2-60),
29.4 (CH2-50), 29.6 (CH2-40), 65.7 (CH2-10), 67.7 (CH2-30),
101.7 (CH-8), 104.9 (CH-6), 114.3 (C-3), 125.7 (CH-5),
152.4 (C-8a), 155.1 (C-4), 160.6 (C-2), 160.9 (C-7), 168.1
(C-20). ESI-HRMS: m/z [M ? H]? calcd. for (C17H21O5)?:
305.1311 found: 305.1318.
Butyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4i)
White solid, yield 65 %, mp 221 °C (EtOH). 1H-
NMR (300 MHz CDCl3, 300 MHz): dH 0.85 (t, 3H,
J1 = 7.5 Hz, J2 = 7.2 Hz, CH3), 2.40 (s, 3H, CH3), 1.33
(m, 2H, H-50), 1.65 (m, 2H, H-40) 4.22 (t, 2H, J1 = 6.9 Hz,
J2 = 6.6 Hz, H-30), 4.69 (s, 2H, H-10), 6.15 (s, 1H, H-3),
6.78 (d, 1H, J1 = 2.7 Hz, H-8), 6.92 (dd, 1H, J1 = 8.4 Hz,
J2 = 2.4 Hz, H-6), 7.51 (d, 1H, J = 8.4 Hz, H-5).13C-
NMR (75 MHz, CDCl3): dc 13.6 (CH3-60), 18.7 (CH2-50),
19.0 (CH3-9), 29.4 (CH2-40), 65.3 (CH2-30), 65.5 (CH2-10),
101.7 (CH-8), 112.5 (CH-6), 114.4 (C-3), 125.8 (CH-5),
152.4 (C-8a), 155.1 (C-4), 160.6 (C-7), 161.1 (C-2), 168.1
(C-20). ESI-HRMS: m/z [M ? H]? calcd. for (C16H19O5)?:
291.1154 found: 291.1159.
Isopropyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate
(4f)
White solid, yield 78 %, mp 218 °C (EtOH).1H-
NMR (300 MHz, CDCl3): dH 1.20 (d, 6H, J = 6 Hz,
H-40 ? H-50), 2.31 (s, 3H, CH3), 4.55 (s, 2H, H-10), 5.07
(hept, 1H, J = 6 Hz, H-30), 6.07 (s, 1H, H-3), 6.71 (d, 1H,
J = 2.7 Hz, H-8), 6.83 (dd, 1H, J1 = 8.4 Hz, J2 = 2.4 Hz,
H-6), 7.42 (d, 1H, J = 8.4 Hz, H-5). 13C-NMR (75 MHz,
CDCl3): dc 21.2 (CH3-40 ? 50), 18.1 (CH3-9), 65.1 (CH2-
10), 69.2 (CH-30), 101.2 (CH), 112.0 (CH-6 ? C-3), 113.8
(C-4a), 125.2 (CH-5), 151.8 (C-8a), 154.6 (C-4), 160.2 (C-
2), 160.5 (C-7), 177.0 (C-20). ESI-HRMS: m/z [M ? H]?
calcd. for (C15H17O5)?: 277.0998 found: 277.0993.
Cyclohexyl 2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetate (4j)
White solid, yield 69 %, mp 210 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 1.33 (m, 2H, H-60), 1.42 (m,
2H, H-50), 1.65 (m, 2H, H-40), 1.89 (m, 2H, H-80), 2.40 (s,
3H, CH3), 4.65 (s, 2H, H-10), 4.90 (m, 1H, H-30), 6.15 (s,
1H, H-3), 6.78 (d, 1H, J = 2.7 Hz, H-8), 6.93 (dd, 1H,
J1 = 8.7 Hz, J2 = 2.7 Hz, H-6), 7.55 (d, 1H, J = 8.7 Hz,
H-5). 13C-NMR (75 MHz, CDCl3): dc 19.2 (CH3-9), 22.4
(CH2-80), 23,4 (CH2-60), 25.3 (CH2-50), 31.0 (CH2-40), 67.8
(CH2-10), 72,2 (CH2-70), 78.9 (CH-30), 103.2 (CH-8), 112.0
(CH-6), 112.1 (C-4a), 116.5 (CH-3), 128.7 (CH-5), 155.3
(C-8a), 158.0 (C-4), 160.3 (C-7), 161.8 (C-2), 169.2 (C-20).
ESI-HRMS: m/z [M ? H]? calcd. for (C18H21O5)?:
316.1311 found: 316.1316.
Propyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate
(4 g)
White solid, yield 71 %, mp 212 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 0.91 (t, 3H, J = 7.5 Hz,
CH3), 1.65 (m, 2H, H-40), 2.36 (s, 3H, CH3), 4.15 (t, 2H,
J = 6.6 Hz, H-30), 4.67 (s, 2H, H-10), 6.10 (s, 1H, H-3),
6.73 (d, 1H, J = 2.7 Hz, H-8), 6.88 (dd, 1H, J1 = 8.4 Hz,
J2 = 2.4 Hz, H-6), 7.47 (d, 1H, J = 8.4 Hz, H-5).13C-
NMR (75 MHz, CDCl3): dc 21.8 (CH3-9), 18.6 (CH3-50),
10.2 (CH2-40), 65.2 (CH2-30), 67.2 (CH2-10), 101.7 (CH-8),
112.4 (CH-6), 114.3 (C-3), 125.2 (CH-5), 152.5 (C-8a),
154.9 (C-4), 160.6 (C-7), 161.1 (C-2), 168.1 (C-20). ESI-
HRMS: m/z [M ? H]? calcd. for (C15H17O5)?: 277.1076
found: 277.1073.
2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (5)
White solid, yield 63 %, mp 212 °C (EtOH). 1H-NMR
(300 MHz, DMSO-d6): dH 2.39 (s, 3H, CH3), 4.38 (s, 2H,
NH2), 5.02 (s, 2H, H-10), 6.23 (s, 1H, H-3), 7.0 (m, 2H,
H-6 ? H-8), 7.68 (d, 1H, H-5), 9.45 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6): dc 18.1 (CH3-9), 64.9 (CH2-
10), 102.0 (CH-8), 111.6 (CH-6), 112.3 (CH-3), 113.5 (C-
4a), 126.4 (CH-5), 153.5 (C-8a), 154.5 (C-4), 160.8 (C-
7 ? C-2), 169.9 (C-20).
Isobutyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate
(4h)
White solid, yield 79 %, mp 228 °C (EtOH).1H-
NMR (300 MHz, CDCl3): dH 0.85 (d, 6H, J = 3.9 Hz,
H-50 ? H-60), 2.32 (s, 3H, CH3), 1.89 (hept, 1H,
123

Med Chem Res
Preparation of compounds 6a–f: A mixture of compound 5
(E)-N’-(2-hydroxybenzylidene)-2-(4-methyl-2-oxo-2H-
chromen-7-yloxy)acetohydrazide (6d)
(4 mmol) and the appropriate aromatic aldehyde (1 eq)
was refluxed in ethanol (20 mL) for 2 h. The excess of
solvent was then removed under reduced pressure, the
precipitate formed after cooling was collected by filtration
and recrystallized from ethanol to give compounds 6a–f.
White solid, yield 82 %, mp 212 °C (EtOH). 1H-
NMR (300 MHz, DMSO-d6): dH 2.19 (s, 3H, CH3), 5.28 (s,
2H, H-20), 6.21 (s, 1H, H-3), 7.0 (m, 2H, H-6 ? H-8), 7.05-
8.22 (m, 5H, H-100 ? H-400 ? H-500 ? H-600 ?H-700), 11.01
(s, 1H, OH), 11.85 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-
d6): dc 18.1 (CH3-9), 64.8 (CH2-20), 101.5 (CH-8), 111,4 (CH-
6), 112.2 (CH-3), 113.5 (C-4a), 120.2 (C-200); 121.5 (CH-400),
123.0 (CH-600), 127.2 (CH-700), 130.1 (CH-500), 126.5 (CH-5),
153.3 (C-8a), 154.5 (C-4), 156.0 (CH-100), 160,1 (C-300), 163,5
(C-2 ? 7), 168,1 (C-10). ESI-HRMS: m/z [M ? H]? calcd.
for (C19H17O5N2)?: 353.1059 found: 353.1063.
(E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N’-(thiophen-
2-ylmethylene)acetohydrazide (6a)
White solid, yield 66 %, mp 268 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 2.33 (s, 3H, CH3), 4.86 (s,
2H, H-20), 6.22 (s, 1H, H-3), 6.65 (s, 1H, H-40’), 6,94-7,09
(m, 3H, H-300 ? H-6 ? H-8), 7.78 (d, 1H, J = 8,7 Hz,
H-5), 7.92 (s, 1H, H-100), 7.98 (s, 1H, H-500), 11.63 (s, 1H,
NH). 13C-NMR (75 MHz, CDCl3): dc 18.1 (CH3-9), 66.7
(CH2-20), 101.6 (CH-8), 113.4 (CH-3 ? C-6), 113.7 (C-
4a), 125.0 (CH-100), 125.8 (CH-500), 127.2 (CH-400), 128.0
(CH-300),, 145.4 (C-8a), 153.3 (C-4), 144.5 (C-200),
160.6(C-7 ? 2), 168.8 (C-10). ESI-HRMS: m/z [M ? H]?
calcd. for (C17H15 N2O4S)?: 342.0674, found: 342.0677.
(E)-N’-(4-hydroxy-3-methoxybenzylidene)-2-(4-methyl-2-
oxo-2H-chromen-7-yloxy)acetohydrazide (6e)
White solid, yield 78 %, mp 229 °C (EtOH). 1H-
NMR (300 MHz, DMSO-d6): dH 2.19 (s, 3H, CH3), 3.85
(s, 3H, H-800), 5.28 (s, 2H, H-20), 6.21 (s, 1H, H-3), 7.02 (m,
2H, H-6 ? H-8), 7.11-7.22 (m, 4H, H-100 ? H-300 ? H-
500 ? H-700), 11.01 (s, 1H, OH), 11.85 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6): dc 18.1 (CH3-9), 56.2 (OCH3-
800), 66.5 (CH2-20), 113.0 (C-500), 126.5 (CH-5), 130.1
(CH), 131.4 (CH), 153,3 (C-8a), 154.5 (C-4), 156,1 (CH-
100); 160,6 (C-7); 160,1 (CH-700); 160,0 (CH-300); 163.5 (C-
2 ? 7), 168.1 (C-10). ESI-HRMS: m/z [M ? H]? calcd. for
(C20H19O6N2)?: 383.1165 found: 383.1169.
(E)-N’-(furan-2-ylmethylene)-2-(4-methyl-2-oxo-2H-
chromen-7-yloxy)acetohydrazide (6b)
White solid, yield 76 %, mp 248 °C (EtOH). 1H-
NMR (300 MHz, CDCl3): dH 2.39 (s, 3H, CH3), 4.80 (s,
2H, H-20), 6.23 (s, 1H, H-3), 6.62 (s, 1H, H-400), 6,92-7,06
(m, 3H, H-300 ? H-6 ? H-8), 7.71 (d, 1H, J = 8,7 Hz,
H-5), 7.83 (s, 1H, H-100), 7.91 (s, 1H, H-500), 11.63 (s, 1H,
NH). 13C-NMR (75 MHz, CDCl3): dc 18.1 (CH3-9), 65.8
(CH2-20), 101.8 (CH-8), 113.5 (CH-3 ? C-6), 114.2 (C-
4a), 125.4 (CH-100), 125.8 (CH-500), 127.2 (CH-400), 128.0
(CH-300),, 145.4 (C-8a), 153.3 (C-4), 144.5 (C-200),
160.6(C-7 ? 2), 168.8 (C-10). ESI-HRMS: m/z [M ? H]?
calcd. for (C17H15 N2O5)?: 327.0981, found: 327.0975.
(E)-N0-(3,4-dimethoxybenzylidene)-2-(4-methyl-2-oxo-2H-
chromen-7-yloxy)acetohydrazide (6f)
White solid, yield 81 mp 222 °C (EtOH). 1H-
NMR (300 MHz, DMSO-d6): dH 2.19 (s, 3H, CH3), 3.85
(s, 3H, H-800), 3.91 (s, 3H, H-900), 5.28 (s, 2H, H-20), 6.21
(d, 1H, J = 0.9 Hz, H-3), 7.02 (m, 2H, H-6 ? H-8), 7.10-
7.24 (m, 4H, H-100 ? H-300 ? H-600 ? H-700), 11.85 (s, 1H,
NH). 13C-NMR (75 MHz, DMSO-d6): dc 18.1 (CH3-9),
65.2 (OCH3-800), 66.5 (CH2-20), 163.5 (C-2), 160.8 (C-7),
160.0 (CH-100), 131.4 (C-200), 113.0 (CH-300), 155.0 (C-4a),
152.0 (C-400), 148.0 (C-500), 130.1 (CH-600), 121.0 (CH-700),
126.5 (CH-5), 152.3 (C-8a), 154.5 (C-4), 156.0 (CH-100),
168.5 (C-10).ESI-HRMS: m/z [M ? H]? calcd. for
(C21H23O6N2)?: 397.1321 found: 397.1325.
(E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N’-(quinolin-
6-ylmethylene)acetohydrazide (6c)
White solid, yield 84 %, mp 238 °C (EtOH). 1H-
NMR (300 MHz, DMSO-d6): dH 2.40 (s, 3H, CH3), 5.41
(s, 2H, H-20), 6.21 (s, 1H, H-3), 7.0 (m, 2H, H-6 ? H-8),
7.69-9.0 (m, 8H, H-5 ? H-100 ? H-300 ? H-400 ? H-
500 ? H-600 ? H-700 ? H-800), 12.03 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6): dc 18.1 (CH3-9), 66.7 (CH2-
20), 101.6 (CH-8), 113.4 (CH-3 ? C-6), 113.7 (C-4a),
119.7 (CH-800), 120.2 (C-200), 123.9 (CH-5), 124.5 (CH-500),
126.4-126.5 (CH-700 ?300a), 126.6 (CH-500), 145.4 (C-8a),
153.3 (C-4), 154.5 (CH-100), 160.6 (C-7 ? 2), 164.2 (CH-
600), 168.8 (C-10). ESI-HRMS: m/z [M ? H]? calcd. for
(C22H18O4N3)?: 388.1297 found: 388.1301.
Preparation of compounds 7a–c
A mixture of hydrazide 5 (100 mg, 4 mmol), cyclic anhy-
dride (1 eq) and 20 mL of ethanol was refluxed. The progress
of the reaction was monitored by TLC. Once the reaction is
completed, the precipitate was filtered and recrystallized
from ethanol to obtain the desired products, 7a–c.
123

Med Chem Res
N-(1,3-dioxoisoindolin-2-yl)-2-(4-methyl-2-oxo-2H-
three bacterial agents were selected as test microorganisms,
Pseudomonas sp. (Pa 499), Burkholderia sp. (Bg 35) and
Bacillus sp. (Bp 420). They were cultured at 25 °C on
Nutrient Agar (NA) medium for 48 h before use.
chromen-7-yloxy)acetamide (7a)
White solid, yield 26 %, mp 280 °C (EtOH). 1H-
NMR (DMSO-d6, 300 MHz): dH 2.42 (s, 3H, CH3), 5.0 (s,
2H, H-20), 6.27 (s, 1H, H-3), 7.10 (m, 2H, H-6 ? H-8),
7.76 (d, 1H, J = 8.4 Hz, H-5), 8.01 (m, 4H, H-50 ? H-
60 ? H-70 ? H-80), 11.10 (s, 1H, NH).
13C-NMR (75 MHz, DMSO-d6): dc 18.2 (CH3-9), 66.1
(CH2-20), 111.6 (CH-8), 112.6 (CH-3 ? C-6), 115.0 (C-
4a), 123.9 (CH-5), 129.3 (CH-50 ? C-80), 135.4 (CH-
60 ? C-70), 160.1-160.3 (C-2 ? C-7), 166.9 (C-10), 101.7
(CH-8), 153.4 (C-8a), 154.5 (C-40a ? C-90a), 164.9 (C-
90 ? C-40). ESI-HRMS: m/z [M ? H]? calcd. For
(C20H13O6N2)?: 377.0787 found: 377.0777.
Antifungal activity was performed against Aspergillus
niger, Botrytis cinerea and Fusarium oxysporum f. sp. ly-
copersici. These fungi were obtained from the Laboratory
of Phytopathology of the Regional Center of Research in
Horticulture and Organic Agriculture (CRRHAB) of Chott-
Mariem, Tunisia. They were cultured at 25 °C on potato
dextrose agar (PDA) medium 1 week before use. The
screening results were compared with those of ampicillin
and carbendazim used as standard references for antibac-
terial for antifungal activities, respectively.
Antibacterial activity
N0-(2,9-dioxo ([2,2.1] hept-5-en)-2-yl)-2-(4-methyl-2-oxo-
2H-chromen-7-yloxy) acetohydrazide (7b)
The purified products were screened for their antibacterial
activity by using the agar disc diffusion method (Mar-
monier, 1987). NA medium cooled at 45 °C was supple-
mented with a bacterial suspension (106 CFU/mL) and
poured into Petri plates. After solidification, sterile What-
man paper discs (diameter 6 mm) were placed at the sur-
face of the culture medium and 20 lL (1000 lg/mL) of the
product dissolved in DMSO was dropped onto each disc.
The negative control plates had no product added to the
filter paper, whereas in the positive control plates, discs
were impregnated with the same volume of ampicillin so-
lution (5 mg/mL). The treated Petri dishes were incubated
at 25 °C for 48 h. The antibacterial activity was evaluated
by measuring the diameter of the inhibitory zones formed
around the discs. The experiment was replicated twice.
White solid, yield 66 %, mp 270 °C (EtOH). 1H-NMR
(300 MHz, CDCl3): dH 1.71 (m, 2H, H-90), 2.41 (s, 3H, CH3),
3.41(m, 2H, H-40a ? H-80a), 3.44(m, 2H, H-80 ? H-50), 4.81
(s, 2H, H-20), 6.18 (s, 1H, H-3), 6.22 (s, 1H, H-60), 6.31 (s, 1H,
H-70), 7.02 (m, 2H, H-6 ? H-8), 7.80 (d, 1H, J = 8.7 Hz,
H-5), 8.53 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3) dc 18.7
(CH3-9), 22.1 (CH2-90), 44.4 (C-40a ? C-80a), 45.1 (CH-
80 ? C-50), 67.1 (CH2-20), 111.6 (CH-8), 112.6 (CH-3 ? C-
6), 155.0 (C-4a), 123.9 (CH-5), 134.8 (CH-60 ? C-70), 152.5
(C-8a), 160.1–160.6 (C-2 ? C-7), 160,9 (C-40), 161.1 (C-
100), 173.6 (C-10). ESI-HRMS: m/z [M ? H]? calcd. for
(C21H19O6N2)?: 395.1243 found: 395.1240.
4-(2-(2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetyl)hydrazinyl)-4-oxobutanoic acid (7c)
Antifungal activity
White solid, yield 76 %, mp 266 °C (EtOH). 1H-NMR
(300 MHz, DMSO-d6) dH 2.41 (s, 3H, CH3), 2.54 (m, 4H,
H-60 ? H-70), 4.81 (s, 2H, H-20), 6.22 (s, 1H, H-3), 7.02
(m, 2H, H-6 ? H-8), 7.80 (d, 1H, J = 8.7 Hz, H-5), 10.34
(s, 1H, NH), 10.53 (s, 1H, NH), 13 (s, 1H, OH). 13C-
NMR (75 ppm, DMSO-d6): dc 18.2 (CH3-9), 28.0 (CH2-
60 ? C-70), 66.1 (CH2-20), 111.0 (CH-8), 112.8 (CH-
3 ? C-6), 114.2 (C-4a), 155.1 (C-8a), 123.9 (CH-5),
160.1–160.6 (C-2 ? C-7), 165.9 (C-10), 170.1 (C-50), 173.5
(C-80). ESI-HRMS: m/z [M ? H]? calcd. for
(C16H17O7N2)?: 349.0958 found: 349.0964.
Aspergillus niger, Botrytis cinerea and Fusarium oxyspo-
rum f. sp. lycopersici were used for the screening of anti-
fungal activity of the products tested by using the disc
diffusion method (Barry and Thornsberry, 1991). A coni-
dial suspension of the tested fungi was prepared (104–105
CFU/mL) and added to PDA medium cooled at 45 °C and
poured uniformly into Petri plates (diameter 90 mm).
Sterilized paper discs (6 mm, Whatman No. 1 filter paper)
were impregnated with 20 lL (1000 lg/mL) of the product
dissolved in DMSO and placed on the culture plates,
whereas the negative control plates had no product added
to the filter paper. In the positive control plates, discs were
imbibed with the same volume of a carbendazim suspen-
sion (0.5 mg/mL). The diameter of the inhibition zone
(mm) around the disc was measured after incubation at
25 °C for 4 days and compared with control. The test was
performed in triplicate.
Biological activities
Antimicrobial evaluation
All the synthesized compounds have been evaluated for their
antibacterial and antifungal activities. For antibacterial test,
123

Med Chem Res
cytotoxic activities and DNA cleavage study. Eur J Med Chem
44(12):5123–5130
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