Design and synthesis of novel benzopyran-2-one derivatives of expected antimicrobial activity through DNA gyrase-B inhibition

Article abstract of DOI:10.1002/ardp.200700266

In an attempt to find a new class of antibacterial agents, we have synthesized thirty new coumarin (2H-benzopyran-2-one) analogues. These derivatives include substituted azetidin-2-ones (β-lactam) 3a-f, pyrrolidin-2-ones 4a-f, 2H-1,3,4-oxadiazoles 5a-f, and thiazolidin-4-ones 6a-f attached to 4-phenyl-2H-benzopyran-2-one through an oxyacetamido or an oxymethyl bridge. The target compounds were synthesized starting from 2-oxo-4-phenyl-2H-benzo[b]pyran-7-yl-oxyacetic acid hydrazides 2a-f. The new compounds were evaluated as DNA gyrase-B inhibitors through molecular modeling and docking techniques using the Molsoft ICM 3.4-8C program. The synthesized compounds were also screened for antibacterial activity against four different species of Gram-positive and Gram-negative bacteria; as well as screening against C. albicans for antifungal activity. The molecular modeling data were in accordance with the antimicrobial screening results.

Full text of DOI:10.1002/ardp.200700266

Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
G. S. Hassan et al.
725
Full Paper
Design and Synthesis of Novel Benzopyran-2-one Derivatives
of Expected Antimicrobial Activity through DNA Gyrase-B
Inhibition
Ghaneya S. Hassan, Nahla A. Farag, Gehan H. Hegazy, and Reem K. Arafa
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
In an attempt to find a new class of antibacterial agents, we have synthesized thirty new cou-
marin (2H-benzopyran-2-one) analogues. These derivatives include substituted azetidin-2-ones (b-
lactam) 3a–f, pyrrolidin-2-ones 4a–f, 2H-1,3,4-oxadiazoles 5a–f, and thiazolidin-4-ones 6a–f
attached to 4-phenyl-2H-benzopyran-2-one through an oxyacetamido or an oxymethyl bridge.
The target compounds were synthesized starting from 2-oxo-4-phenyl-2H-benzo[b]pyran-7-yl-oxy-
acetic acid hydrazides 2a–f. The new compounds were evaluated as DNA gyrase-B inhibitors
through molecular modeling and docking techniques using the Molsoft ICM 3.4-8C program.
The synthesized compounds were also screened for antibacterial activity against four different
species of Gram-positive and Gram-negative bacteria; as well as screening against C. albicans for
antifungal activity. The molecular modeling data were in accordance with the antimicrobial
screening results.
Keywords: Antimicrobial activity / Coumarin / DNA gyrase-B inhibitor / 2H-benzopyran-2-one / Molecular modeling /
Received: December 18, 2007; accepted: April 2, 2008
DOI 10.1002/ardp.200700266
plex, cleavage of this DNA on both strands, and the pas-
sage of a segment of DNA through the double strand
break. Re-ligation of the break results in the introduction
of two negative supercoils. These processes require the
binding and hydrolysis of ATP [4]. Inhibition of DNA
gyrase blocks relaxation of supercoiled DNA, relaxation
being a requirement for transcription and replication.
DNA gyrase is a selective target for antibacterial agents,
such as the most studied quinolone and coumarin antibi-
otics. Quinolone drugs (e.g. ciprofloxacin) affect the pro-
tein subunit GyrA, and coumarins (e.g. novobiocin) act on
GyrB [5, 6].
The synthesis and antibacterial activity of azetidin-2-
ones as new antimicrobial agents against multidrug-
resistant pathogens were reported [7, 8]. Furthermore, a
number of substituted pyrrolidinone, 1,3,4-oxadiazoles,
and thiazolidin-4-ones were found to exhibit appreciable
antimicrobial and antifungal activities [9–14]. These
observations prompted us to incorporate the b-lactam
(azetidin-2-one), pyrrolidin-2-one, 2H-1,3,4-oxadiazole, or
thiazolidin-4-one moieties with the coumarin nucleus to
synthesize new derivatives of expected antimicrobial
Introduction
Antibiotic resistance is a major problem worldwide and,
therefore, it is essential to develop new antibacterial
drugs. In DNA replication, one group of enzymes has
proved to be an effective target for therapeutic agents,
which are topoisomerase enzymes. DNA gyrase – a type II
topoisomerase – is found in all bacteria and controls the
topological state of DNA [1]. DNA gyrase consists of two
subunits GyrA (875 amino acids) and GyrB (804 amino
acids) with the active species being a heterotetramer A2B2
[2]. Mechanistic studies have revealed the steps that are
involved in the gyrase-supercoiling reaction [3]. This proc-
ess involves the wrapping of DNA around the A2B2 com-
Correspondence: Gehan Hegazy Hegazy, Pharmaceutical Chemistry
Department, Faculty of Pharmacy, Cairo University, El-Kasr El-Eini
Street, Cairo 11562, Egypt.
E-mail: gehan_hegazy@yahoo.com
Fax: +20 2 362 8426
Abbreviation: Internal Coordinate Mechanics (ICM)
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activity potentially through inhibition of DNA gyrase-B atives 3a–f and pyrrolidin-2-one derivatives 4a–f, respec-
enzyme. Molecular docking represents one of the grow- tively. Studies on the mechanism of azetidin-2-one (b-lac-
ing applications in computational biology, where in tam) formation [19] concluded that the salt (I) is formed
molecular modeling techniques are used to predict how as an intermediate which undergoes dehydrohalogena-
macromolecules (typically a protein) interact with other tion in the presence of triethylamine to produce 3a–f
molecules (may be other proteins, nucleic acids, or small (see Equation 1 and Scheme 1). In the intermediate (I), a
drug-like molecules). In many cases, molecular modeling partial positive charge should reside on the carbon atom
results give a reasonable prediction of the biological of the imine which becomes later C-4 of the azetidin-2-
activity a drug molecule may possess. So, to understand one (b-lactam) 3a–f. It is reasonable to expect that the
the obtained microbiological data on a structural basis, atom carrying a free pair of electrons would form a loose
we evaluated the scoring functions of the gyrase-B inhibi- bond with this carbon and generate a transition state.
tors (Novobiocin, and the newly synthesized compounds) This would assure the ease of formation of b-lactam by
through molecular modeling and docking techniques bringing the appropriate carbon atoms C-3 and C-4 close
using Molsoft ICM 3.4-8C program. ICM stands for “Inter- to bond forming distance. Similarly, pyrrolidin-2-one
nal Coordinate Mechanics” – a program originally aimed derivatives 4a–f were formed. In another approach, treat-
at energy optimization of several biopolymers with ment of arylidene derivatives 2a–f with acetic anhydride
respect to an arbitrary subset of internal coordinates and anhydrous sodium acetate gave 2H-1,3,4-oxadiazoles
such as bond lengths, bond angles, torsion angles, and 5a–f through acetylation with concomitant cyclization
phase angles [15–17].
(Scheme 1). Finally, the reaction of 2a–f with mercapto-
acetic acid gave the corresponding 2,3-diaryl-1,3-thiazoli-
din-4-ones 6a–f (see Equation 2 and Scheme 1). Both ana-
Results and discussion
Chemistry
The key starting materials (2-oxo-4-phenyl-2H-benzo[b]-
pyran-7-yl) oxyacetic acid arylidene hydrazides 2a–f were
obtained in good yields upon condensation of (2-oxo-4-
phenyl-2H-benzo[b]pyran-7-yl) oxyacetic acid hydrazide 1
with different aromatic aldehydes in acetic acid [18].
Reaction of the arylidene derivatives 2a–f with two
different acid chlorides namely chloroacetyl chloride
and 3-chloropropionyl chloride in presence of triethyl-
amine gave azetidin-2-one (monocyclic b-lactam) deriv-
lytical and spectroscopic data of all the synthesized com-
pounds are in full agreement with the proposed struc-
tures.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
Benzopyran-2-one Derivatives and Antimicrobial Activity
727
Scheme 1. Mechanisms of formation of compounds 3a–f, 4a–f, and 6a–f.
Antimicrobial screening
the other hand, compound 2c showed only slight activity
All the 30 newly synthesized compounds were tested in against Bacillus subtilis (8 mm). As for the b-lactam deriv-
vitro for their antibacterial activity (10 mg/mL) using the atives 3a–f, compound 3f gave good activity against
agar diffusion method [20] against Bacillus subtilis, Sarcina Staphylococcus aureus (20 mm) and slight activity against
lutea, and Staphylococcus aureus as representatives of Escherichia coli (9 mm), while compound 3c was only
Gram-positive bacteria, Escherichia coli as representative slightly active against Bacillus subtilis (9 mm). On the
of Gram-negative bacteria, and the fungus Candida albi- other hand, screening of pyrrolidin-2-ones 4a–f revealed
cans. The antimicrobial activity of the newly synthesized four derivatives possessing antimicrobial activity. Com-
compounds is reflected as zone of growth inhibition of pound 4f gave moderate activity against Bacillus subtilis
the tested microorganisms (measured in mm) (Table 1). (15 mm), while 4a and 4c showed also moderate activity
Among the arylidene hydrazides 2a–f, compound 2f was against Staphylococcus aureus (15 and 12 mm, respec-
the most active derivative giving a significant activity tively). Finally, 4c and 4e showed slight activity against
against Bacillus subtilis (20 mm), Sarcina lutea (15 mm), Sarcina lutea (8 mm for both). Of the 2H-1,3,4-oxadiazoles
and Staphylococcus aureus (20 mm) as Gram-positive bacte- 5a–f, 5a, and 5d showed slight activity against Staphylo-
ria, Escherichia coli (15 mm) as Gram-negative bacteria coccus aureus (9 mm) and Sarcina lutea (9 mm), respec-
and also against the fungus Candida albicans (15 mm). On tively. Finally, 6a was the only 1,3-thiazolidin-4-one deriv-
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G. S. Hassan et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
Table 1. Antimicrobial activity of the tested benzopyran-2-one
analogues.
Com-
Bacillus
Sarcina
lutea
Staph.
aureus
(G+ve)
E.coli
(G-ve)
C. albicans
(fungus)
pounda) subtilis
(G+ve)b, c) (G+ve)c)
2c
2f
3c
8
20
9
–
15
–
–
20
–
–
15
–
–
15
–
3f
4a
4c
4e
–
7
–
–
–
–
8
8
20
15
12
–
9
–
–
–
–
–
–
–
4f
5a
15
–
–
9
–
–
–
–
–
–
Figure 1. The docking solution of Novobiocin within gyrase-B
binding pocket. (ICM score –123).
5d
6a
Tetra-
cycline
Ampho-
tercin B
–
8
30
–
10
40
9
–
36
–
8
34
–
–
–
–
–
–
–
30
a)
All the newly prepared compounds were subjected to micro-
biological evaluation. Yet, only those compounds that
showed antimicrobial activity are enlisted in the table.
The antimicrobial activity of the newly synthesized com-
pounds against test organisms is expressed in terms of zone
of growth inhibition (measured in mm).
b)
c)
Test solutions of the benzopyran-2-one analogues were pre-
pared in a concentration of 10 mg/ml.
Figure 2. The docking solution of 2f within gyrase-B binding
pocket. (ICM score –80.44).
ative possessing antimicrobial efficacy with slight activ-
ity against Bacillus subtilis (8 mm), Sarcina lutea (10 mm)
and Escherichia coli (8 mm).
Drug-modeling studies
To pre-assess the potential microbiological behavior of
our new coumarin derivatives on structural basis, the
scoring functions are used to estimate or predict the
binding affinity / biological activity of the DNA gyrase-B
inhibitor (Novobiocin, coumarin derivative antibiotic),
and the newly synthesized compounds (five different
classes of coumarin derivatives) through molecular mod-
eling and docking techniques using Molsoft ICM 3.4-8C
program [21–23] in order to predict the binding geome-
try for each binder.
Figure 3. The docking solution of 5d within gyrase-B binding
pocket. (ICM score –93.36).
The goal is to have an adequate 3D-model of the recep-
tor pocket to dock a ligand. Usually, a good start is to try
to dock the known ligand to the receptor model, then to inhibitor novobiocin. In these simulations, the ligand is
dock the tested compounds to determine ICM scores. ICM fully flexible (free translation, rotation of functional
scores of –32 and lower are generally considered good groups about single bonds). Figures 1–3 show the dock-
scores but this usually depends on the receptor.
In order to compare the binding affinity of the newly for gyrase B with the different coumarin derivatives.
synthesized coumarin analogues, we docked all the deriv-
As shown in Tables 1 and 2 and Figs. 1–3 the following
atives 2–6 (a–f) into the empty binding site of the experi- results can be drawn:
ing solutions with the highest predicted binding affinity
mentally-known bacterial topoisomerase with its bound
ICM score of novobiocin is –123.72 (Fig. 1).
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Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
Benzopyran-2-one Derivatives and Antimicrobial Activity
729
Table 2. ICM Scores of novobiocin and the tested benzopyran-
2-one analogues.
ishes antifungal and antibacterial activity against Gram-
negative bacteria and decreases antibacterial activity
against Gram-positive bacteria. Reaction of 2 with mer-
captoacetic acid to give 1,3-thiazolidin-4-ones 6a–f abol-
ishes antifungal activity and decreases antibacterial
activity against Gram-positive and Gram-negative bacte-
ria. Substitution with p-nitro phenyl moiety gives good
activity, while substitution with phenyl moiety shows
moderate activity as reflected in the screening data of 2f
versus 2c, 3f versus 3c, and 4f versus 4c.
In many cases, the experimental findings are in good
agreement with predicted binding affinities obtained by
molecular docking studies. Yet, there are some deriv-
atives (5a and 6a) that showed promising in-vitro antibac-
terial activity not typically coinciding with their overall
ICM scores among other members of the chemical series.
The aforementioned infers that the antibacterial activity
can not merely be attributed to those parameters meas-
ured during the molecular modeling study which
reflects the necessity of performing the biological screen-
ing.
N8
ICM
scores
N8
ICM
scores
N8
ICM
scores
Novobiocin –123.72 3e
–87.16 5d
–89.96 5e
–89.07 5f
–85.93 6a
–86.78 6b
–83.42 6c
–87.22 6d
–86.94 6e
–87.29 6f
–88.91
–93.36
–91.08
–92.68
–77.30
–79.97
–78.91
–86.37
–84.20
–83.20
2a
2b
2c
2d
2e
2f
–73.48 3f
–75.61 4a
–78.19 4b
–79.07 4c
–76.52 4d
–80.44 4e
–85.69 4f
–84.08 5a
–84.53 5b
–87.08 5c
3a
3b
3c
3d
–87.90
ICM scores of oxyacetic acid arylidene hydrazide 2a–f
range from –73.48 to –80.44. Compound 2f, the biologi-
cally most active with antimicrobial efficacy against all
the five tested microorganisms (Table 1), possessed the
lowest ICM score -80.44 (Fig. 2).
ICM scores of azetidin-2-one 3a–f range from –84.08
to –89.96. Within this series, the biologically most active
compound was 3f which showed the lowest ICM score –
89.96.
ICM scores of pyrrolidin-2-one 4a–f range from –83.42
to –89.07. The biologically most active compound 4a dis-
played the lowest ICM score –89.07, while compounds 4f
and 4c, with good activity, possessed ICM score of –86.90
and –86.78, respectively.
The authors wish to offer their deep gratitude to Prof. Dr. Abd
El-Haliem Ramadan and Dr. Yossra Mohammed, Department
of Microbiology, Faculty of Pharmacy, Cairo University for
their carrying-out the microbiological screening.
The authors have declared no conflict of interest.
ICM scores of 2H-1,3,4-oxadiazole 5a–f range from –
87.29 to –93.36 where the biologically most active com-
pounds 5d (Fig. 3, lowest ICM score of all the new com-
pounds) and 5a possessed the lowest and highest ICM
scores, respectively (–93.36 and –87.29).
ICM scores of 1,3-thiazolidin-4-one 6a–f range from –
77.30 to –86.37, but the antibacterially most active com-
pound 6a had the highest ICM score (–77.30).
Experimental
Chemistry
All melting points are uncorrected and determined by the open
capillary method using a Gallenkamp melting point apparatus
(MFB-595-010M; Weiss-Gallenkamp, London, UK). Microanalyses
were carried out at the microanalytical unit, Faculty of Science,
Cairo University. Infrared spectra were determined (KBr) using
Schimadzu Infrared Spectrometer (IR-435; Shimadzu, Japan) and
FT-IR 1650 (Perkin Elmer, USA). 1H-NMR Spectra were carried out
using Joel, FX 90Q, NMR Spectrometer at 200 MHz and Fourier
transform EM-390, 300 MHz NMR Spectrometer (JEOL, Tokyo,
Japan). Mass spectra were carried out using Finnigan SSQ 7000
Gas Chromatograph-Mass spectrometer (Thermo Electron Cor-
poration, Bremen, Germany). TLC was carried out using Art.
5735, DC-Plastikfolien, Kieselgel 60 F254 sheets (Merck, Darm-
stadt, Germany), the developing solvents were CCl4 :CH3OH
(9 : 1) and the spots were visualized by UV 366 and 254 nm.
Conclusions
From the literature and our own studies we knew the
importance of the coumarin structure for inhibition of
gyrase B [5]. In our study, we focused on substitution in
position 7 of the coumarin moiety. Reaction of 2 with
chloroacetyl chloride or 3-chloropropionyl chloride to
give aztidin-2-ones 3a–f and pyrrolidin-2-ones 4a–f abol-
ishes antifungal activity and decreases antibacterial
activity against Gram-negative bacteria, while retaining
the antibacterial activity against Gram-positive bacteria.
Cyclization of 2 to give 2H-1,3,4-oxadiazoles 5a–f abol-
General method for preparation of (2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy) acetic acid arylidene hydrazides
2a–f
A mixture of 1 (3.10 g, 10 mmol) and the appropriate aromatic
aldehyde (10 mmol) in acetic acid (20 mL) was refluxed for 6 h.
The excess solvent was then removed under reduced pressure.
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G. S. Hassan et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
The precipitate formed after cooling was collected by filtration
and crystallized from ethanol to give 2a–f.
C24H17N3O6 (443.42): C, 65.01; H, 3.86; N, 9.48. Found: C, 64.96; H,
3.90; N, 9.60.
1-[(Furan-2-yl)methylene]-2-(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetohydrazide 2a
General method for preparation of 4-aryl-3-chloro-1-[(2-
oxo-4-phenyl-2H-benzo[b]pyran-7-yloxy)acetamido]
The general method was adopted to prepare 2a employing fur-
furaldehyde. Yield: 85%; mp.: 254–2568C. IR (cm– 1): 3150 (NH),
1731, 1683 (2 C=O). 1H-NMR (DMSO-d6) d ppm: 5.22 (s, 2H, OCH2),
6.23 (s, 1H, H-3 coumarin), 6.60–7.90 (m, 11H, furan and ArH),
8.20 (s, 1H, CH=N), 11.20 (s, 1H, NH exch.). Anal. Calcd. for
C22H16N2O5 (388.37): C, 68.04; H, 4.15; N, 7.21. Found: C, 68.02; H,
4.16; N, 6.99.
azetidin-2-ones 3a–f
To a mixture of the appropriate arylidene derivative 2a–f
(10 mmol) and triethylamine (2 mL, 20 mmol) in dry benzene
(50 mL), monochloroacetyl chloride (2.2 mL, 20 mmol) was
added dropwise with cooling. The reaction mixture was stirred
at room temperature for 30 min and then refluxed for 3 h. The
solid which precipitated upon cooling was filtered off and crys-
tallized from chloroform / methanol.
1-[(Thiophen-2-yl)methylene]-2-(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)aceto-hydrazide 2b
3-Chloro-4-(furan-2-yl)-1-[(2-oxo-4-phenyl-2H-
The general method was adopted to prepare 2b employing thio-
phene-2-carbaldehyde. Yield: 86%; mp.: 241–2438C. IR (cm– 1):
3200 (NH), 1730, 1680 (2 C=O). 1H-NMR (DMSO-d6) d ppm: 4.94 (s,
2H, OCH2), 6.25 (s, 1H, H-3), 6.07–7.73 (m, 11H, thiophene and
Ar), 8.00 (s, 1H, CH=N), 11.20 (s, 1H, NH exch.). Anal. Calcd. for
C22H16N2O4S (404.44): C, 56.34; H, 3.99; N, 6.92. Found: C, 65.33;
H, 4.04; N, 7.18.
benzo[b]pyran-7-yloxy)acetamido] azetidin-2-one 3a
Yield: 45%; mp.: 116–1188C. IR (cm– 1): 3200 (NH), 1750, 1700,
1650 (3 C=O). 1H-NMR (DMSO-d6) d ppm: 4.16 (s, 2H, OCH2), 4.90
(d, J = 7 Hz ,1H, Ar-CH b-lactam), 5.10 (d, J = 7 Hz, 1H, Cl-CH b-lac-
tam), 6.25 (s, 1H, H-3), 7.00–7.95 (m, 11H, furan and Ar), 10.30 (s,
1H, NH exch.). Anal. Calcd. for C24H17ClN2O6 (464.86): C, 62.01; H,
3.69; N, 6.03. Found: C, 62.09; H, 3.57; N 6.11.
1-(Benzylidene)-2-(2-oxo-4-phenyl-2H-benzo[b]pyran-7-
3-Chloro-4-(thiophen-2-yl)-1-[(2-oxo-4-phenyl-2H-
yloxy)acetohydrazide 2c
The general method was adopted to prepare 2c employing ben-
zaldehyde. Yield: 85%; mp.: 222–2238C. IR (cm– 1): 3150 (NH),
1756, 1708 (2 C=O). 1H-NMR (CDCl3) d ppm: 4.69 (s, 2H, OCH2),
6.24 (s, 1H, H-3), 6.75–7.65 (m, 13H, Ar), 8.95 (s, 1H, CH=N), 10.20
(s, 1H, NH exch.). Anal. Calcd. for C24H18N2O4 (398.41): C, 72.35; H,
4.55; N, 7.03. Found: C, 72.71; H, 4.56; N, 6.93.
benzo[b]pyran-7-yloxy)acetamido] azetidin-2-one 3b
Yield: 60%; mp.: 120–1228C. IR (cm– 1): 3200 (NH), 1750, 1700,
1
1650 (3 C=O). H-NMR (DMSO-d6) d ppm: 4.24 (s, 2H, OCH2), 4.94
(d, J = 7 Hz, 1H, Ar-CH b-lactam), 5.20 (d, J = 7 Hz, 1H, Cl-CH b-lac-
tam), 6.24 (s, 1H, H-3), 7.07-7.72 (m, 11H, thiophene and Ar),
10.00 (s, 1H, NH exch.). Anal. Calcd. for C24H17ClN2O5S (480.92): C,
59.94; H, 3.56; N, 5.82. Found: C, 59.90; H, 3.30; N, 5.80.
1-(4-Methoxybenzylidene)-2-(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetohydrazide 2d
3-Chloro-4-phenyl-1-[(2-oxo-4-phenyl-2H-benzo[b]pyran-
The general method was adopted to prepare 2d employing 4-
methoxybenzaldehyde. Yield: 90%; mp.: 228–2308C. IR (cm– 1):
3200 (NH), 1730, 1680 (2 C=O). 1H-NMR (DMSO-d6) d ppm: 3.80 (s,
3H, OCH3), 5.20 (s, 2H, OCH2), 6.22 (s, 1H, H-3), 6.80–7.90 (m, 12H,
Ar), 8.20 (s, 1H, CH=N), 11.20 (s, 1H, NH exch.). MS: m/z (%) = 428
(49) [M+]. Anal. Calcd. for C25H20N2O5 (428.25): C, 70.08; H, 4.71; N,
6.54. Found: C, 70.11; H, 5.20; N, 6.39.
7-yloxy)acetamido]azetidin-2-one 3c
Yield: 50%; mp.: 128–1308C. IR (cm– 1): 3300 (NH), 1750, 1710,
1610 (3 C=O). 1H-NMR (CHCl3-d6) d ppm: 4.77 (s, 2H, OCH2), 5.10 (d,
J = 7 Hz, 1H, Ar-CH b-lactam), 5.30 (d, J = 7 Hz, 1H, Cl-CH b-lactam),
6.30 (s, 1H, H-3), 6.90–7.95 (m, 13H, Ar), 10.03 (s, 1H, NH exch.).
Anal. Calcd. for C26H19ClN2O5 (474.90): C, 65.76; H, 4.03; N, 5.90.
Found: C, 65.63; H, 4.19; N, 5.70.
1-(4-Chlorobenzylidene)-2-(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetohydrazide 2e
3-Chloro-4-(4-methoxyphenyl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetamido] azetidin-2-one 3d
Yield: 50%; mp.: 187–1888C. IR (cm– 1): 3200 (NH), 1750, 1700,
1650 (3 C=O). 1H-NMR (CHCl3-d6) d ppm: 3.80 (s, 3H, OCH3), 4.26 (s,
2H, OCH2), 4.95 (d, J = 7 Hz, 1H, Ar-CH b-lactam), 5.40 (d, J = 7 Hz,
1H, Cl-CH b-lactam), 6.30 (s, 1H, H-3), 6.85–7.90 (m, 12H, Ar),
10.00 (s, 1H, NH exch.). Anal. Calcd. for C27H21ClN2O6 (504.93): C,
64.23; H, 4.19; N, 5.55. Found: C, 63.98; H, 4.40; N, 5.62.
The general method was adopted to prepare 2e employing 4-
chlorobenzaldehyde. Yield: 90%; mp.: 242–2448C. IR (cm– 1):
3150 (NH), 1729, 1685 (2 C=O). 1H-NMR (CDCl3) d ppm: 5.23 (s, 2H,
OCH2), 6.24 (s, 1H, H-3), 6.80–7.80 (m, 12H, Ar), 8.80 (s, 1H,
CH=N), 10.00 (s, 1H, NH exch.). MS: m/z (%) = 434 (16) [M+ +2], 432
(41) [M+]. Anal. Calcd. for C24H17ClN2O4 (432.86): C, 66.59; H, 3.96;
N, 6.47. Found: C, 66.60; H, 4.00; N, 6.42.
1-(4-Nitrobenzylidene)-2-(2-oxo-4-phenyl-2H-
3-Chloro-4-(4-chlorophenyl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetohydrazide 2f
benzo[b]pyran-7-yloxy)acetamido] azetidin-2-one 3e
The general method was adopted to prepare 2f employing 4-
nitrobenzaldehyde. Yield: 95%; mp.: 225–2288C. IR (cm– 1): 3200
(NH), 1756, 1685 (2 C=O), 1609 (NH bending). 1H-NMR (DMSO-d6) d
ppm: 4.93 (s, 2H, OCH2), 6.24 (s, 1H, H-3), 6.80–7.95 (m, 12H, Ar),
8.30 (s, 1H, CH=N), 10.20 (s, 1H, NH exch.). Anal. Calcd. for
Yield: 55%; mp.: 194–1958C. IR (cm– 1): 3224 (NH), 1754, 1708,
1
1650 (3 C=O). H-NMR (DMSO-d6) d ppm: 4.17 (s, 2H, OCH2), 4.79
(d, J = 7.2 Hz, 1H, Ar-CH b-lactam), 4.95 (d, J = 7.2 Hz, 1H, Cl-CH b-
lactam), 6.27 (s, 1H, H-3), 6.99–7.57 (m, 12H, Ar), 10.39 (s, 1H, NH
exch.). MS: m/z (%) = 509 (0.98) [M+]. Anal. Calcd. for C26H18Cl2N2O5
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Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
Benzopyran-2-one Derivatives and Antimicrobial Activity
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(509.35): C, 61.31; H, 3.56; N, 5.50. Found: C, 61.10; H, 3.60; N,
5.45.
4-Chloro-5-(4-chlorophenyl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetamido] pyrrolidin-2-one 4e
Yield: 55%; mp.: 130–1328C. IR (cm– 1): 3200 (NH), 1756, 1708,
1650 (3 C=O).1H-NMR (CDCl3) d ppm: 3.49 (d, J = 2 Hz, 2H, CH2 pyr-
rol.), 4.33 (q, 1H, Cl-CH pyrrol.), 4.69 (s, 2H, OCH2), 4.81 (d, J =
4 Hz, 1H, Ar-CH pyrrol.), 6.24 (s, 1H, H-3), 6.83–6.95 (m, 3H, Ar),
7.42–7.52 (m, 9H, Ar), 10.10 (s, 1H, NH exch.). MS: m/z (%) = 509
(0.98) [M+]. Anal. Calcd. for C27H20Cl2N2O5 (523.37): C, 61.96; H,
3.85; N, 5.35. Found: C, 61.93; H, 3.91; N, 5.15.
3-Chloro-4-(4-nitrophenyl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetamido] azetidin-2-one 3f
Yield: 65%; mp.: 156–1578C. IR (cm– 1): 3200 (NH), 1750, 1703,
1
1624 (3 C=O). H-NMR (DMSO-d6) d ppm: 4.93 (s, 2H, OCH2), 5.20
(d, J = 7 Hz, 1H, Ar-CH b-lactam), 5.90 (d, J = 7 Hz, 1H, Cl-CH b-lac-
tam), 6.24 (s, 1H, H-3), 6.98–7.90 (m, 12H, Ar), 10.10 (s, 1H, NH
exch.). MS: m/z (%) = 521 (0.33) [M++2], 519 (0.25) [M+]. Anal. Calcd.
for C26H18ClN3O7 (519.90): C, 60.07; H, 3.49; N, 8.08. Found: C,
60.01; H, 3.37; N, 8.10.
4-Chloro-5-(4-nitrophenyl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetamido] pyrrolidin-2-one 4f
Yield: 55%; mp.: 140–1428C. IR (cm– 1): 3275 (NH), 1750, 1710,
1640 (3 C=O). 1H-NMR (CDCl3) d ppm: 3.80 (d, J = 2.2 Hz, 2H, CH2
pyrrol.), 4.30 (q, 1H, Cl-CH pyrrol.), 4.68 (s, 2H, OCH2), 4.90 (d, J =
4 Hz, 1H, Ar-CH pyrrol.), 6.21 (s, 1H, H-3), 7.40–7.80 (m, 8H, Ar),
8.10 (d, J = 8 Hz, 2H, Ar), 8.40 (d, J = 8 Hz, 2H, Ar), 10.20 (s, 1H, NH
exch.). Anal. Calcd. for C27H20ClN3O7 (533.37): C, 60.74; H, 3.78; N,
7.87. Found: C, 60.60; H, 3.60; N 7.44.
General method for preparation of 5-aryl-4-chloro-1-[(2-
oxo-4-phenyl-2H-benzo[b]pyran-7-yloxy)acetamido]
pyrrolidin-2-one 4a–f
As described above for 3a–f, using 3-chloropropionyl chloride.
The solid obtained was crystallized from chloroform / ether.
4-Chloro-5-(furan-2-yl)-1-[(2-oxo-4-phenyl-2H-
General method for preparation of 3-Acetyl-2-aryl-5-(2-
oxo-4-phenyl-2H-benzo[b]pyran-7-yloxymethyl)-1,3,4-
(2H)-oxadiazole 5a–f
A mixture of 2a–f (10 mmol), acetic anhydride (15 mL), and
anhydrous sodium acetate (0.1 g) was refluxed for 1 h. The solu-
tion was cooled and poured onto ice cold water and stirred for
1 h. The solid was filtered off, washed with water, dried, and
crystallized from methanol.
benzo[b]pyran-7-yloxy)acetamido] pyrrolidin-2-one 4a
Yield: 50%; mp.: 124–1268C. IR (cm– 1): 3200 (NH), 1756, 1700,
1
1650 (3 C=O). H-NMR (DMSO-d6) d ppm: 3.91 (d, J = 2.2 Hz, 2H,
CH2 pyrrol.), 4.24 (q, 1H, Cl-CH pyrrol.), 4.94 (s, 2H, OCH2), 5.10 (d,
J = 4 Hz, 1H, Ar-CH pyrrol.), 6.25 (s, 1H, H-3), 7.07–7.90 (m, 11H,
furan and Ar), 9.60 (s, 1H, NH exch.). Anal. Calcd. for
C25H19ClN2O6 (478.89): C, 62.70; H, 4.00; N, 5.85. Found: C, 62.90;
H, 3.94; N, 5.90.
3-Acetyl-2-(furan-2-yl)-5-(2-oxo-4-phenyl-2H-
4-Chloro-5-(thiophen-2-yl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxymethyl)-1,3,4-(2H)-oxadiazole 5a
benzo[b]pyran-7-yloxy)acetamido] pyrrolidin-2-one 4b
Yield: 55%; mp.: 132–1348C. IR (cm– 1): 3200 (NH), 1750, 1700,
1650 (3 C=O). 1H-NMR (DMSO-d6) d ppm: 3.90 (d, J = 2.2 Hz, 2H,
CH2 pyrrol.), 4.24 (q, 1H, Cl-CH pyrrol.), 4.95–5.20 (m, 3H, OCH2
and Ar-CH pyrrol.), 6.23 (s, 1H, H-3), 7.06–7.90 (m, 11H, thio-
phene and Ar), 9.60 (s, 1H, NH exch.). Anal. Calcd. for
C25H19ClN2O5S (494.95): C, 60.67; H, 3.87; N, 5.66. Found: C, 60.89;
H, 4.10; N, 5.62.
Yield: 75%; mp.: 127–1288C. IR (cm– 1): 1739, 1693 (2 C=O). H-
1
NMR (DMSO-d6) d ppm: 2.20 (s, 3H, COCH3), 4.81 (s, 2H, OCH2),
6.22 (s, 1H, H-3), 6.87–7.02 (m, 4H, oxadiazole and furan), 7.30–
7.80 (m, 8H, Ar). Anal. Calcd. for C24H18N2O6 (430.42): C, 66.97; H,
4.22; N, 6.51. Found: C, 66.47; H, 3.80; N, 6.80.
3-Acetyl-2-(thiophen-2-yl)-5-(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxymethyl)-1,3,4-(2H)-oxadiazole 5b
Yield: 70%; mp.: 120–1228C. IR (cm– 1): 1750, 1708 (2 C=O). 1H-
NMR (CDCl3) d ppm: 2.25 (s, 3H, COCH3), 4.75 (s, 2H, OCH2), 6.24
(s, 1H, H-3), 6.82–6.95 (m, 4H, oxadiazole and thiophene), 7.39–
7.60 (m, 8H, Ar). Anal. Calcd. for C24H18N2O5S (446.48): C, 64.56;
H, 4.03; N, 6.27. Found: C, 64.59; H, 3.80; N, 6.13.
4-Chloro-5-phenyl-1-[(2-oxo-4-phenyl-2H-benzo[b]pyran-
7-yloxy)acetamido]pyrrolidin-2-one 4c
Yield: 50%; mp.: 78–808C. IR (cm– 1): 3275 (NH), 1756, 1708, 1640
1
(3 C=O). H-NMR (CDCl3) d ppm: 3.75 (d, J = 2.2 Hz, 2H, CH2 pyr-
rol.), 4.25 (q, 1H, Cl-CH pyrrol.), 4.68 (s, 2H, OCH2), 4.80 (d, J =
4.4 Hz, 1H, Ar-CH pyrrol.), 6.21 (s, 1H, H-3), 6.74–6.91 (m, 4H, Ar),
7.33-7.56 (m, 9H, Ar), 10.10 (s, 1H, NH exch.). Anal. Calcd. for
C27H21ClN2O5 (488.93): C, 66.33; H, 4.33; N, 5.73. Found: C, 66.39;
H, 4.36; N, 5.53.
3-Acetyl-2-phenyl-5-(2-oxo-4-phenyl-2H-benzo[b]pyran-
7-yloxymethyl)-1,3,4-(2H)-oxadiazole 5c
Yield: 70%; mp.: 182–1848C. IR (cm– 1): 1750, 1700 (2 C=O). 1H-
NMR (CDCl3) d ppm: 2.40 (s, 3H, COCH3), 4.82 (s, 2H, OCH2), 6.22
(s, 1H, H-3), 6.99 (s, 1H, oxadiazole), 7.31–7.50 (m, 13H, Ar). Anal.
Calcd. for C26H20N2O5 (440.46): C, 70.90; H, 4.58; N, 6.36. Found: C,
71.15; H, 4.69; N, 6.39.
4-Chloro-5-(4-methoxyphenyl)-1-[(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxy)acetamido] pyrrolidin-2-one 4d
Yield: 50%; mp.: 169–1708C. IR (cm– 1): 3275 (NH), 1756, 1708,
1
1640 (3 C=O). H-NMR (DMSO-d6) d ppm: 3.53 (d, J = 2.0 Hz, 2H,
3-Acetyl-2-(4-methoxyphenyl)-5-(2-oxo-4-phenyl-2H-
CH2 pyrrol.), 3.87 (s, 3H, OCH3), 4.20 (q, 1H, Cl-CH pyrrol.), 4.80–
5.20 (m, 3H, OCH2 and Ar-CH pyrrol.), 6.24 (s, 1H, H-3), 6.90–7.75
(m, 12H, Ar), 10.30 (s, 1H, NH exch.). Anal. Calcd. for C28H23ClN2O6
(518.95): C, 64.80; H, 4.47; N, 5.40. Found: C, 64.68; H, 4.55; N,
5.66.
benzo[b]pyran-7-yloxymethyl)-1,3,4-(2H)-oxadiazole 5d
Yield: 75%; mp.: 185–1878C. IR (cm– 1): 1754, 1708 (2 C=O). 1H-
NMR (DMSO-d6) d ppm: 2.25 (s, 3H, COCH3), 3.70 (s, 3H, OCH3),
4.95 (s, 2H, OCH2), 6.26 (s, 1H, H-3), 6.89 (s, 1H, oxadiazole), 7.09–
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Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
7.83 (m, 12H, Ar). Anal. Calcd. for C27H22N2O6 (470.48): C, 68.93; H,
4.71; N, 5.95. Found: C, 68.84; H, 4.80; N, 5.84.
3-[2-Oxo-4-phenyl-2H-benzo[b]pyran-7-yloxyacetamido]-
2-phenyl-thiazolidin-4-one 6c
Yield: 60%; mp.: 152–1568C. IR (cm– 1): 3286 (NH), 1754–1712,
1
1660 (3 C=O). H-NMR(CDCl3) d ppm: 3.92 (s, 2H, CH2 thiazol.),
3-Acetyl-2-(4-chlorophenyl)-5-(2-oxo-4-phenyl-2H-
4.62 (s, 2H, CH2O), 5.95 (s, 1H, H-3), 6.18 (s, 1H, CH thiazol.), 6.77–
8.10 (m, 13H, Ar), 10.00 (s, 1H, NH exch.). Anal. Calcd. for
C26H20N2O5S (472.52): C, 66.09; H, 4.27; N, 5.93. Found: C, 66.00;
H, 4.20; N, 6.11.
benzo[b]pyran-7-yloxymethyl)-1,3,4-(2H)-oxadiazole 5e
Yield: 75%; mp.: 138–1408C. IR (cm– 1): 1754, 1712 (2 C=O). H-
1
NMR (CDCl3) d ppm: 2.40 (s, 3H, COCH3), 4.93 (s, 2H, OCH2), 6.25
(s, 1H, H-3), 6.90 (d, J = 13.4Hz, 2H, Ar), 7.08 (s, 1H, oxadiazole),
7.40 (d, J = 13.4Hz, 2H, Ar), 7.55 (broad, 8H, Ar). MS: m/z (%) = 476
(0.99) [M++2], 474 (1.13) [M+]. Anal. Calcd. for C26H19ClN2O5
(474.90): C, 65.76; H, 4.03; N, 5.90. Found: C, 65.80; H, 4.10; N
5.76.
2-(4-Methoxyphenyl)-3-[2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxyacetamido]-thiazolidin-4-one 6d
Yield: 60%; mp.: 114–1168C. IR (cm– 1): 3200 (NH), 1750–1718,
1660 (3 C=O). 1H-NMR(CDCl3) d ppm: 3.80 (s, 3H, OCH3), 3.91 (s,
2H, CH2 thiazol.), 5.01 (s, 2H, CH2O), 6.24 (s, 2H, H-3 and CH thia-
zol.), 7.20–7.90 (m, 12H, Ar), 9.90 (s, 1H, NH exch.). Anal. Calcd.
for C27H22N2O6S (502.54): C, 64.53; H, 4.41; N, 5.57. Found: C,
64.20; H, 4.53; N, 5.16.
3-Acetyl-2-(4-nitrophenyl)-5-(2-oxo-4-phenyl-2H-
benzo[b]pyran-7-yloxymethyl)-1,3,4-(2H)-oxadiazole 5f
Yield: 70%; mp.: 143–1458C. IR (cm– 1): 1754, 1708 (2 C=O). 1H-
NMR (DMSO-d6) d ppm: 2.20 (s, 3H, COCH3), 4.94 (s, 2H, OCH2),
6.25 (s, 1H, H-3), 6.90 (s, 1H, oxadiazole), 7.20–7.95 (m, 12H, Ar).
Anal. Calcd. for C26H19N3O7 (485.45): C, 64.33; H, 3.95; N, 8.66.
Found: C, 64.20; H, 3.92; N 8.78.
2-(4-Chlorophenyl-3-[2-oxo-4-phenyl-2H-benzo[b]pyran-
7-yloxyacetamido]-thiazolidin-4-one 6e
Yield: 70%; mp.: 128–1308C. IR (cm– 1): 3150 (NH), 1750, 1700,
1660 (3 C=O). 1H-NMR(DMSO-d6) d ppm: 4.13 (s, 2H, CH2 thiazol.),
4.83 (s, 2H, CH2O), 6.23 (s, 2H, H-3 and CH thiazol.), 6.96 (d, J =
8.8 Hz, 2H, Ar), 7.36 (d, J = 8.0 Hz, 2H, Ar), 7.54 (m, 8H, Ar), 10.00
(s, 1H, NH exch.). Anal. Calcd. for C26H19ClN2O5S (506.96): C,
61.60; H, 3.78; N, 5.53. Found: C, 61.82; H, 3.99; N, 5.76.
General method for preparation of 2-aryl-3-[2-oxo-4-
phenyl-2H-benzo[b]pyran-7-yloxyacetamido]-thiazolidin-
4-one 6a–f
Method A: A mixture of 2a–f (10 mmol) and thioglycolic acid
(2.76 g, 2.1 mL, 30 mmol) in dry benzene (100 mL) was refluxed
for 72 h using a Dean–Stark apparatus. The solution was evapo-
rated under reduced pressure and the residue washed with 5%
NaHCO3 solution followed by water, dried and crystallized from
CHCl3 / ether.
Method B: To a solution of 2a–f (10 mmol) in dry dioxane
(15 mL), mercaptoacetic acid (0.91 g, 0.7 mL, 10 mmol) and anhy-
drous ZnCl2 (0.2 g) was added and the mixture heated to reflux
for 10 h. The solvent was removed under reduced pressure and
the residue washed with 5% NaHCO3 (3625 mL) followed by
water, dried and crystallized from CHCl3 / ether.
2-(4-Nitrophenyl)-3-[2-oxo-4-phenyl-2H-benzo[b]pyran-7-
yloxyacetamido]-thiazolidin-4-one 6f
Yield: 70%; mp.: 132–1348C. IR (cm– 1): 3200 (NH), 1754, 1708,
1660 (3 C=O). 1H-NMR(DMSO-d6) d ppm: 4.12 (s, 2H, CH2 thiazol.),
4.79 (s, 2H, CH2O), 6.24 (s, 1H, H-3), 6.40 (s, 1H, CH thiazol.), 7.03
(d, J = 8.0 Hz, 2H, Ar), 7.33 (d, J = 8.0 Hz, 2H, Ar), 7.56 (m, 8H, Ar),
10.39 (s, 1H, NH exch.). Anal. Calcd. for C26H19N3O7S (517.51): C,
60.34; H, 3.70; N, 8.12. Found: C, 60.30; H, 3.80; N, 7.76.
Antimicrobial activity
Test organisms
The antimicrobial activity of 30 novel compounds 2–6a–f was
evaluated in vitro against Bacillus subtilis, Sarcina lutea, and Staph-
ylococcus aureus (as representative examples of Gram-positive bac-
teria), Escherichia coli (as representative of Gram-negative bacte-
ria), and the fungus Candida albicans (Table 1).
2-(Furan-2-yl)-3-[2-oxo-4-phenyl-2H-benzo[b]pyran-7-
yloxyacetamido]-thiazolidin-4-one 6a
Yield: 60%; mp.: 125–1278C. IR (cm– 1): 3200 (NH), 1730–1720,
1681 (3 C=O). 1H-NMR (DMSO-d6) d ppm: 3.84 (s, 2H, CH2 thiazol.),
5.22 (s, 2H, CH2O), 6.24 (s, 1H, H-3), 6.60–6.90 (m, 3H, furan), 7.00
(s, 1H, CH thiazol.), 7.30-7.92 (m, 8H, Ar), 11.60 (s, 1H, NH exch.).
Anal. Calcd. for C24H18N2O6S (462.48): C, 62.33; H, 3.92; N, 6.06.
Found: C, 62.54; H, 3.72; N, 6.31.
Method – Agar plate disc diffusion technique
Agar plate disc diffusion technique has been used as general
method [20].
Test materials of 6 mm in diameter sterilized Whatman filter
paper discs were impregnated with 10 mg/mL solution of the
test compound and standard (tetracycline and amphotercin B)
dissolved in DMF and allowed to air-dry. The discs were applied
to the surface of nutrient agar plates seeded with the test organ-
ism (each plate contains 15 mL of the agar medium previously
seeded with 0.2 mL of 18 h growth culture in liquid media for
each organism). The incubated plates were incubated at 378C for
48 h and the inhibition zone was measured in mm around each
disc. Each experiment was repeated three times and the results
are the average of the three runs. Discs impregnated with DMF
were used as a control.
3-[2-Oxo-4-phenyl-2H-benzo[b]pyran-7-yloxyacetamido]-
2(thiophen-2-yl)-thiazolidin-4-one 6b
Yield: 65%; mp.: 122–1238C. IR (cm– 1): 3200 (NH), 1756, 1708,
1
1660 (3 C=O). H-NMR(CDCl3) d ppm: 4.28 (s, 2H, CH2 thiazol.),
4.69 (s, 2H, CH2O), 6.24 (s, 2H, H-3 and CH thiazol.), 6.80–6.95 (m,
3H, thiophene), 7.32–7.52 (m, 8H, Ar), 10.00 (s, 1H, NH exch.).
MS: m/z (%) = 480 (10.28) [M++ 2]. Anal. Calcd. for C24H18N2O5S2
(478.54): C, 60.24; H, 3.79; N, 5.85. Found: C, 60.54; H, 3.89; N,
5.75.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 725–733
Benzopyran-2-one Derivatives and Antimicrobial Activity
733
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The crystal structure of enzyme (DNA GYRASE-B) with its bound
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(3-methylbut-2-enyl)phenyl]carbonylamino)-8-methyl-2-oxo-
chromen-7-yl]oxy-3-methoxy-2,2-dimethyl-oxan-4-yl) carbamate)
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The novel energy-minimized benzopyran-2-one analogues were
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using ICM-Pro software version 3.4-8C. ICM-Pro scores the bind-
ing of a ligand to a receptor based upon the comparison of a ser-
ies of small molecule / protein interactions that have been
reported in the PDB database. A rigid receptor / flexible ligand
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