2-Azetidinone derivatives: Design, synthesis, in vitro anti-microbial, cytotoxic activities and DNA cleavage study

Article abstract of DOI:10.1016/j.ejmech.2009.09.011

A novel series of 3-chloro-4-[4-(2-oxo-2H-chromen-4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-one derivatives (5a-j) have been synthesized from 4-aryloxymethylcoumarins (1a-e) and 4-aryliminomethyl-phenols (3a-b). The title compounds were screened for their

Full text of DOI:10.1016/j.ejmech.2009.09.011

European Journal of Medicinal Chemistry 44 (2009) 5123–5130
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
2-Azetidinone derivatives: Design, synthesis, in vitro anti-microbial, cytotoxic
activities and DNA cleavage study
*
Rangappa S. Keri, Kallappa M. Hosamani , Ramya V. Shingalapur, Harisha R. Seetharama Reddy
P.G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel series of 3-chloro-4-[4-(2-oxo-2H-chromen-4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-one
Received 3 March 2009
Received in revised form
3 June 2009
Accepted 7 September 2009
Available online 11 September 2009
derivatives (5a–j) have been synthesized from 4-aryloxymethylcoumarins (1a–e) and 4-aryliminomethyl-
phenols (3a–b). The title compounds were screened for their in vitro anti-bacterial and anti-fungal activ-
ities. Results revealed that, compounds (5c), (5f), (5h) and (5j) showed excellent anti-microbial activity
against a panel of microorganisms. Brine shrimp bioassay was also carried out to study their in vitro
cytotoxic properties among which (5h) and (5j) displayed potent cytotoxic activity against Artemia salina.
The DNA cleavage activity of some compounds was studied by agarose gel electrophoresis method. All
synthesized compounds were characterized using IR, 1H NMR, 13C NMR, MS and elemental analysis.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
2-Azetidinone
Coumarin
Anti-microbial activity
Cytotoxicity
DNA cleavage
1. Introduction
cephalosporins, thienamycin, nocardicins, aztreonam and carba-
penems are attributed to the presence of 2-azetidinone ring in
The discovery and development of new anti-microbial drugs in
the search for better treatment has been a main goal for scientists.
In recent decades, problems of multi-drug resistant microorgan-
isms have reached an alarming level in many countries around the
them. Azetidinones are a very important class of compounds pos-
sessing a wide range of biological activities such as anti-microbial
[5], anti-inflammatory [6], anti-convulsant [7], antibiotic [8], anti-
cancer [9], anti-elastase [10], anti-viral [11], anti-tumor [5] and
world. Resistance to a number of anti-microbial agents (
b
-lactam
anti-HCMV [12] activities. Recently, it has been reported that b-
antibiotics, macrolides, quinolones and vancomycin) among
a variety of clinically significant species of bacteria is becoming
increasingly important global problem. Also a number of recent
clinical reports describe the increasing occurrence of Methicillin
resistant Staphylococcus aureus (MRSA) which is most disturbing
cause of nosocomial infections in developed countries [1,2] and
other antibiotic-resistant human pathogenic microorganisms in US
and European Countries. Infections caused by these microorgan-
isms pose a serious challenge to the medical community and the
need for an effective therapy has led to the search for novel anti-
microbial agents. In particular, increasing drug resistance among
gram positive bacteria such as staphylococci, enterococci and
streptococci is a significant health matter [3].
lactams have novel biological activities such as cytomegalovirus
protease inhibitors [13], thrombin and tryptase inhibitors [14],
cholesterol absorption inhibitors [15], human leukocyte elastase
(HLE) inhibitors [16], porcine pancreatic elastase (PPE) inhibitors
[17], HIV-1 protease [18] and peroxisome proliferator-activated
receptors (PPARs) [19]. Besides their biological activities, the
importance of
b-lactams as synthetic intermediates has been
widely recognized in organic synthesis [20] for example in the
semisynthesis of Taxol [21]. Like azetidinone, coumarin also
exhibits diverse biological properties [22].
In the design of new drugs, the development of hybrid molecules
through the combination of different pharmacophores in one frame
may lead to compounds with interesting biological profiles. Owing to
the importance and in continuation of our work on to the synthesis of
biologically active compounds [23], now we wish to describe the
synthesis of new azetidinone derivatives from 4-aryloxymethyl-
coumarins (Scheme 1) and in vitro screening results of anti-bacterial,
anti-fungal, cytotoxic activities and DNA cleavage study. The other
biological activities are under progress.
2-Azetidinones, commonly known as b-lactams are well-known
heterocyclic compounds among organic and medicinal chemists
[4]. The activity of famous antibiotics such as penicillins,
* Corresponding author. Tel.: þ91 836 2215286; fax: þ91 836 2747884/2771275.
E-mail address: dr_hosamani@yahoo.com (K.M. Hosamani).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.011

5124
R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
Br
O
CHO
Acetone K CO
/
2
3
R
HO
CHO
+
R
24 Hrs
stirring
O
O
O
O
(1a-e)
(2a-e)
Ar-NH2
HO
CH
(3a-b)
Acetone /
N
R'
K CO
2
3
O
O
CH
N
R'
R
O
(4a-j)
ClCH2COCl
Dioxane/Et3N
N
R'
O
O
Cl
(5a-j)
O
R
O
Where R = 6-CH3, 7-CH3, 6-Cl,5,6-Benzo,7,8-Benzo
= -Cl, -Br
R'
Scheme 1. Schematic representation for the synthesis of azetidinone derivatives from 4-aryloxymethylcoumarin.
2. Chemistry
a singlet due to –CH3 protons at
and C3–H at 6.33 ppm. The aromatic protons resonated in the
range of 7.04–8.32 ppm. The azomethine proton appeared
downfield as a singlet at 9.85 ppm. The azomethine group in (4a–j)
underwent cycloaddition with chloroacetyl chloride in the presence
of triethylamine [Et3N] as a catalyst in dioxane afforded 3-chloro-4-
[4-(2-oxo-2H-chromen-4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-
ones (5a–j) (Scheme 1). The IR spectrum of 3-chloro-4-[4-(6-methyl-
2-oxo-2H-chromen-4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-one
d 2.24 ppm, C4–CH2 at d 5.16 ppm
d
The present synthetic strategy begins with the generation of the
required 4-bromomethylcoumarins (1a–e) by the Pechmann cyc-
lisation of phenols with 4-bromoethylacetoacetate. Condensation
of (1a) with 4-hydroxy benzaldehyde in presence of anhydrous
K2CO3 yielded (2-oxo-2H-chromen-4-ylmethoxy) benzaldehydes
(2a–e). In the IR Spectrum of 4-(2-oxo-2H-chromen-4-yl-
methoxy)-benzaldehyde (2a), the lactone carbonyl stretching
frequency was observed at 1718 cmꢀ1, whereas the aldehydic
carbonyl stretching appeared at 1690 cmꢀ1. In the 1H NMR spec-
d
d
(5a), the bands at 1764 cmꢀ1 (pC]O of
carbonyl stretching at 1723 cmꢀ1. In the PMR spectra of compound
(5a), the peak was observed at 5.29 ppm due to the pCH–Cl in the
-lactam ring; in 13C NMR spectra of compound (5a), peaks at
57.3 ppm was observed due to pCH–Cl, 173.2 ppm (cyclic, pC]O)
-lactam moiety. The mass
b-lactam) and coumarin
trum of compound (2a), a singlet was observed at
C6–CH3 protons. The C4–CH2 protons were observed downfield as
a singlet at 5.31 ppm. The C3–H of coumarin appeared at
6.64 ppm. The aldehydic proton appeared as singlet at
9.95 ppm. The 13C NMR spectral data of compound (2a) is given in
d
2.38 ppm due to
d
b
d
d
d
d
a
and 158.7 ppm (heteroaromatics) in the b
spectra of compound (5a) showed the molecular ion peak at 481
[M þ 1]. The various new compounds synthesized during the present
investigation are listed in Table 1.
the experimental section. In view of the poor reactivity of the p-
carbonyl group observed in 4-aryloxymethylcoumarins, pre-func-
tionalized phenols (3a–b) (obtained by the reaction of p-formyl
phenol and aromatic amines) were used for room temperature
allylic SN reaction under standard acetone, K2CO3 conditions
generating the required precursors (4a–j) in high yields. The IR
spectrum of 6-methyl-4-(4-phenyliminomethyl-phenoxymethyl)-
chromen-2-one (4a) (R ¼ 6-CH3, R1 ¼ H) showed lactone carbonyl
stretching frequency at 1709 cmꢀ1 and stretching frequency for
C]N group at 1615 cmꢀ1. The PMR spectrum of (4a) exhibited
3. Pharmacology
All the compounds (5a–j) prepared herein were screened for
their in vitro anti-bacterial activity against two gram positive [S.
aureus (ATCC-29213) and Vancomycin resistant enteroccus (ATCC-
51299)] and two gram-negative [Escherichia coli (ATCC-25922) and
Shigella dysentery (natural isolates)], Aspergillus fumigatus (ATCC-

R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
5125
Table 1
Physical and analytical data of the 2-azetidinone derivatives.
N
Ar
O
5
8
4
6
3
Cl
O
R
7
O
O
1
2
Entry
Producta
R
Ar
Yieldb (%)
m.p.c (ꢁC)
Mol. formula/mol. wt
Elem. analysis (Cal./found)
C
H
N
1
2
5a
5b
5c
5d
5e
5f
6-CH3
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
73.35
69.93
88.12
67.56
71.46
77.16
69.78
64.68
80.34
70.23
174–176
132–133
175–177
122–124
180–182
167–168
237–238
159–160
192–193
230–231
C26H19Cl2NO4
480
C26H19Cl2NO4
480
C25H16Cl3NO4
500
C29H19Cl2NO4
516
C29H19Cl2NO4
516
C26H19BrClNO4
524
C26H19BrClNO4
524
C25H16BrCl2NO4
545
65.01
65.03
65.01
64.98
59.96
60.00
67.45
67.41
67.45
67.43
59.51
59.55
59.51
59.49
55.07
55.05
62.11
62.07
62.11
62.14
3.99
4.02
3.99
4.00
3.22
3.25
3.71
3.74
3.71
3.68
3.65
3.63
3.65
3.68
2.96
2.97
3.41
3.44
3.41
3.40
2.92
2.90
2.92
2.93
2.80
2.78
2.71
2.69
2.71
2.73
2.67
2.65
2.67
2.70
2.57
2.60
2.50
2.53
2.50
2.47
7-CH3
3
6-Cl
4
5,6-Benzo
7,8-Benzo
6-CH3
5
6
7
5g
5h
5i
7-CH3
8
6-Cl
9
5,6-Benzo
7,8-Benzo
C29H19BrClNO4
560
C29H19BrClNO4
560
10
5j
a
Products were characterized by IR, NMR, MS and elemental analysis.
Isolated yields.
Melting points are uncorrected.
b
c
36607), Candida Albicans (ATCC-10231) and Penicillium (natural
isolates) as fungal strains were used for the in vitro study by the
tube dilution technique [24]. The strains used in this study were
maintained at the Department of Microbiology, Luqman Pharmacy
College, Gulbarga. Each of the test compounds and standards,
Ciprofloxacin and Gentamycin were dissolved in DMSO initially at
activity against any of the test microorganisms. The brine shrimp
bioassay was also carried out to study their in vitro cytotoxic
activity against A. salina. The DNA cleavage of some 2-azetidinone
derivatives was studied by agarose gel electrophoresis method.
4. Results and discussion
250
mg/ml and then were serially diluted in culture medium as
follows: 125, 62.5, 31.250, 16, 8, 4, 2, and 1
mg/ml concentrations.
4.1. Anti-bacterial bioassay
The minimum inhibitory concentrations (MIC) were defined as the
lowest concentrations of the compounds that prevented visible
growth. It was determined that the solvent had no anti-bacterial
MIC values for the in vitro anti-bacterial studies of the
compounds (5a–j) and the standard are represented in Table 2. The
Table 2
Anti-microbial activity of the synthesized compounds (5a–5j).
MIC(mg/ml)
Comp
Bacterial strains
Fungal strains
S. Aureus
V. r. enteroccoccus
(51299)
E. coli
(25922)
S. dysentery (natural
isolates)
Aspergillus fumigatus
(36607)
Candida albicans
Penicillium (natural
isolates)
(29213)a
(10231)a
5a
5b
5c
5d
5e
5f
08
08
01
16.0
31.250
04
16.0
08
02
16.0
62.5
08
04
31.250
01
31.250
62.5
08
08
31.250
01
31.250
62.5
02
16.0
31.250
08
31.250
16.0
08
16.0
31.250
08
16.0
16.0
08
16.0
16.0
16.0
16.0
16.0
08
5g
5h
5i
08
01
16.0
04
04
02
31.250
08
08
01
62.5
08
08
04
16
04
16.0
08
31.250
08
16.0
08
31.250
16
08
08
31.250
16
5j
Ciprofloxacin
Gentamycin
0.86
–
0.80
–
0.20
–
0.20
–
–
10
–
10
–
10
a
ATCC number.

5126
R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
Table 3
et al. [25]. From the data recorded in Table 3, it is evident that the
compounds (5c), (5f), (5h) and (5j) displayed potent cytotoxic
activity against A. salina, while the other compounds were almost
inactive in this assay. Compound (5h) showed maximum activity
(LD50 ¼ 7.421 ꢂ10ꢀ4 M) in the present series of compounds,
whereas the other active compounds (5c), (5f) and (5j) demon-
strated slightly less activity (LD50 ¼ 5.634 ꢂ10ꢀ4 M, 5.157 ꢂ 10ꢀ4 M
and 6.145 ꢂ10ꢀ4 M respectively) than compound (5h). The rela-
tionship between cytotoxicity and activity however, reveals that
cytotoxicity is approximately 100-fold greater than concentration
for the activity of the most active compound against the selected
bacterial strains.
Brine shrimp bioassay data for the compounds (5a–5j).
Compound
LD50 (M)
5a
5b
5c
5d
5e
5f
5g
5h
5i
3.164 ꢂ 10ꢀ3
3.934 ꢂ 10ꢀ3
5.634 ꢂ 10ꢀ4
3.362 ꢂ 10ꢀ3
2.994 ꢂ 10ꢀ3
5.157 ꢂ 10ꢀ4
1.948 ꢂ 10ꢀ3
7.421 ꢂ 10ꢀ4
3.014 ꢂ 10ꢀ3
6.145 ꢂ 10ꢀ4
5j
4.4. Electrophoretic analysis
anti-bacterial activity of all the compounds against S. aureus, Van-
comycin resistant enteroccus as gram (þ), E. coli, S. dysentery as gram
(ꢀ) bacteria showed good potencies compared to control drug
Ciprofloxacin. Among the synthesized compounds (5c), (5f), (5h)
The DNA cleavage of some 2-azetidinone derivatives was
studied by agarose gel electrophoresis method and is presented in
Fig. 1. The gel after electrophoresis clearly revealed that, the
compound (5i) did acted on the DNA, as the streak was seen. The
difference was observed in the bands of compounds (Lanes 1–5)
compared to the control DNA of E. coli. This shows that the control
DNA alone does not show any apparent cleavage as the
compounds did. However, the nature of reactive intermediates
involved in the DNA cleavage by the compounds has not been
clear. The results indicate the importance of 2-azetidinone
derivatives in these isolated DNA cleavage reactions. As the
compound (5i) was observed to cleave the DNA, it can be
concluded that the compound inhibits the growth of the patho-
genic organism by cleaving the genome.
and (5j) showed very good activity with MIC value of 8–1
Compound (5c) showed excellent activity with an MIC of 1
m
m
g/ml.
g/ml
against all bacterial strains which is comparable with Ciprofloxacin.
Compound (5h) exhibited MIC of 1 g/ml against S. Aureus and
g/ml against S. dysentery. Compound (5f) was also comparable
with standard against S. dysentery with an MIC of 2 g/ml. Partic-
m
4
m
m
ularly, compounds 5c & 5h with chloro substituent of coumarin
moiety and bromo/chloro substituent at 4-position of phenyl ring
of azetidinone moiety, have showed excellent activity with MIC
value of 4–1 mg/ml. The other compounds (5a), (5b), (5d), (5e), (5g)
and (5i) have exhibited moderate activity against all the bacterial
strains.
5. Conclusion
4.2. Anti-fungal bioassay
In conclusion, a series of 3-chloro-4-[4-(2-oxo-2H-chromen-
4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-one derivatives were
prepared (5a–j) and screened for their in vitro anti-bacterial
activity against four strains of bacteria, anti-fungal activity
against three strains of fungi and cytotoxic studies. Bioassay
indicated that the compounds (5c), (5f), (5h) and (5j) were
excellently active against all the strains and the remaining
compounds showed good to moderate activity comparable with
standard drugs. The compounds (5c), (5h) and (5g) displayed
potent cytotoxic activity against A. salina. The DNA cleavage
studies revealed that, 2-azetidinone derivative (5i) showed non-
specific cleavage of DNA. On the basis of structure–activity rela-
tionship study of the compounds, it can be concluded that the
presence of chlorine/bromine atom in the azetidinone moiety
enhanced the activity of the compounds. Henceforth, our find-
ings will have a good impact on chemists and biochemists for
further investigations in this field in search of chlorine containing
anti-microbial and cytotoxic agents.
The anti-fungal screening of all compounds were carried out
against A. fumigatus, C. albicans and Penicillium fungal strains. The
results were compared with that of standard drug Gentamycin.
These results (Table 2) indicate that, the compounds (5c) and (5f),
(5h) and (5j) exhibited promising activity against all the fungal
strains. Compound (5c) was promising with an MIC of 8 mg/ml
against both the fungi A. fumigatus and C. albicans. However,
compounds (5a), (5b), (5d), (5e) and (5g) exhibited low to
moderate activity against the fungal strains. In general, the high
activity is attributed to the presence of electron withdrawing chloro
and bromo functional groups as compared to other substituents.
4.3. Cytotoxic activity
All the synthesized compounds (5a–j) were screened for their
cytotoxicity (brine shrimp bioassay) using the protocol of Meyer
6. Experimental protocol
6.1. Chemistry
Melting points of the synthesized compounds were determined
in open capillaries and are uncorrected. Infrared spectra were
recorded using KBr pellets on Nicolet 5700 FT-IR instrument. The
1H NMR and 13C NMR spectra were recorded on Brucker Avance-
300 (300 MHz) model spectrophotometer in CDCl3 and DMSO as
solvent and TMSi as internal standard with 1H resonant frequency
of 300 MHz and 13C resonant frequency of 75 MHz. The chemical
shifts were measured in
TMSi at
¼0. The TLC was performed on alumina silica gel 60 F254
(Merck). The mobile phase was ethyl acetate and n-hexane (1:1)
d ppm downfield from internal standard
d
Fig. 1. DNA cleavage analysis of azetidinone derivatives- 5a, 5c, 5f, 5h and 5i.

R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
5127
and detection was made using UV light and iodine. The resulting
6.2.2.5. 4-(2-Oxo-2H-benzochromen-4-ylmethoxy)-benzaldehyde
(2e). Light reddish solid, m.p.248–249 ꢁC, IR (KBr, cmꢀ1): 3052
compounds were purified by column chromatography. For column
chromatography Merck silica gel (0.040–0.063 mm) was used. All
the compounds gave C, H and N analysis within ꢃ0.5% of the
theoretical values.
y
(]CH–), 2842 (–CH of CHO),1718 (C]O of coumarin),1707 (C]O of
aldehyde), 1569 cmꢀ1 (C]C), 1028 (C–O–C); 1H NMR (300 MHz,
d
ppm, CDCl3): 5.48 (s, 2H, CH2O), 6.80 (s, 1H, C3–H), 7.08–8.89 (m,
10H, Ar–H), 9.92 (s, 1H, CHO); 13C NMR (75 MHz,
d
ppm, CDCl3):
6.2. General procedure for the preparation of compounds
80.8, 107.5, 113.1, 122.1, 127.6, 129.8, 133.0, 146.6, 164.4, 168.8, 191.5.;
ESI-MS: 330 (Mþ); Anal. Calcd for C21H14O4: C, 76.39; H, 4.26; found
C, 76.45; H, 4.30%.
6.2.1. Preparation of 4-bromomethylcoumarins (1a–e)
The present synthetic strategy begins with the generation of the
required 4-bromomethylcoumarins (1a–e). These compounds were
prepared by the Pechmann cyclization of phenols with 4-bromoe-
thylacetoacetate [28,29].
6.2.3. Preparation of 4-(4-phenyliminomethyl-phenoxymethyl)-
chromen-2-ones 4(a–j)
4-Phenyliminomethyl-phenols (3a–b) (10 mmol) and anhy-
drous K2CO3 (1.38 g, 10 mmol) were stirred in dry acetone (25 ml)
for 30 min. 4-Bromomethylcoumarins (1a–e) (2.52 g, 10 mmol) was
added and stirring was continued for 24 h. The reaction mixture
was concentrated and poured into crushed ice (100 g). The solid
separated was filtered and washed with 5% HCl (10 ml) to
neutralize excess of potassium carbonate, then washed with 100 ml
of cold water and with dilute ethanol. The crude product was dried
and recrystallized from DMF.
6.2.2. Preparation of (2-oxo-2H-chromen-4-ylmethoxy)-
benzaldehydes (2a–e)
p-Hydroxy benzaldehyde (1.24 g, 10 mmol) and anhydrous
K2CO3 (1.38 g, 10 mmol) were stirred in dry acetone 25 ml for
30 min. 4-Bromomethylcoumarins (2.52 g, 10 mmol) were added
and stirring was continued for 24 h. The reaction mixture was
concentrated to one-fourth of the original volume and poured into
ice-cold water. The solid separated was filtered and washed with 5%
HCl (10 ml) to neutralize excess of potassium carbonate, then
washed with 100 ml of cold water and with dilute ethanol. The
crude product was dried and recrystallized from DMF.
6.2.3.1. 4-{4-[(4-Chloro-phenylimino)-methyl]-phenoxymethyl}-6-
methyl-chromen-2-one (4a). Colourless crystals from DMF; Yield
69%; m.p. 244–245 ꢁC; IR (KBr,
y
cmꢀ1): 1709 (C]O of coumarin),
d ppm, DMSO-d6): 2.24 (s, 3H, C6–
1615 (C]N); 1H NMR (300 MHz,
CH3), 5.16 (s, 2H, CH2–O), 6.33 (s, 1H, C3–H), 7.04–8.32 (m, 11H, Ar–
H), 9.85 (s, 1H, –CH]N) ppm; 13C NMR (75 MHz,
ppm, DMSO-d6):
6.2.2.1. 4-(6-Methyl-2-oxo-2H-chromen-4-ylmethoxy)-benzalde-
hyde (2a). Colourless solid, m.p.218–219 ꢁC, IR (KBr
y
cmꢀ1): 3046
d
(]CH–), 2778 (CH of CHO), 1718 (C]O of coumarin), 1690 (C]O of
18.8, 81.4, 106.0, 113.6, 119.4, 122.8, 126.9, 128.9, 130.9, 134.6, 135.8,
146.8, 150.7, 156.6, 160.1, 162.3, 164.1; ESI-MS: 404 (M þ 1); Anal.
Calcd for C24H18O3NCl: C, 71.38; H, 4.49, N, 3.47; found C, 71.40; H,
4.51, N, 3.50%.
aldehyde), 1534 (C]C) cmꢀ1, 1022 (C–O–C); 1H NMR (300 MHz,
d
ppm, CDCl3): 2.38 (s, 3H, CH3), 5.31 (s, 2H, CH2O), 6.64 (s, 1H,C3H),
7.12–7.97 (m, 7H, Ar–H), 9.95 (s, 1H, CHO); 13C NMR (75 MHz,
d
ppm, CDCl3): 21.1, 72.1, 114.0, 118.0, 123.0, 133.1, 133.3, 148.2, 152.4,
161.0, 192.0; ESI-MS: 294 (Mþ); Anal. Calcd for C18H14O4: C, 73.45;
H, 4.80; found C, 73.51; H, 4.87 %.
6.2.3.2. 4-{4-[(4-Chloro-phenylimino)-methyl]-phenoxymethyl}-7-
methyl-chromen-2-one (4b). Colourless crystals from DMF; Yield
69%; m.p. 257–258 ꢁC; IR (KBr,
y
cmꢀ1): 1715 (C]O of coumarin),
d ppm DMSO-d6): 2.31 (s, 3H, C7–
6.2.2.2. 4-(7-Methyl-2-oxo-2H-chromen-4-ylmethoxy)-benzalde-
1606 (C]N); 1H NMR (300 MHz,
CH3), 4.87 (s, 2H, CH2–O), 6.32 (s, 1H, C3–H), 6.88–7.87 (m, 11H, Ar–
H), 9.89 (s, 1H, –CH]N) ppm; 13C NMR (75 MHz,
ppm, DMSO-d6):
hyde (2b). Colourless solid, m.p.223–225 ꢁC, IR (KBr
y
cmꢀ1): 3038
(]CH–), 2788 (CH of CHO), 1707 (C]O of coumarin), 1697 (C]O of
d
aldehyde), 1562(C]C) cmꢀ1, 1016 (C–O–C); 1H NMR (300 MHz,
20.1, 79.8, 107.9, 112.8, 118.8, 121.9, 125.3, 127.3, 129.7, 133.5, 137.8,
144.3, 149.8, 155.9, 159.8, 160.8, 166.5; ESI-MS: 404 (M þ 1); Anal.
Calcd for C24H18O3NCl: C, 71.38; H, 4.49, N, 3.47; found C, 71.41; H,
4.52, N, 3.46%.
d
ppm, CDCl3): 2.48 (s, 3H, CH3), 5.33 (s, 2H, CH2O), 6.60 (s, 1H,C3H),
7.15–7.91 (m, 7H, Ar–H), 9.91 (s, 1H, CHO); 13C NMR (75 MHz,
d
ppm, CDCl3): 20.6, 78.0, 113.5, 117.8.0, 121.3, 134.1, 148.2, 155.4,
167.6, 188.0; ESI-MS: 294 (Mþ); Anal. Calcd for C18H14O4: C, 73.44;
H, 4.81; found C, 73.48; H, 4.86%.
6.2.3.3. 6-Chloro-4-{4-[(4-chloro-phenylimino)-methyl]-phenox-
ymethyl}-chromen-2-one (4c). Colourless crystals from DMF; Yield
6.2.2.3. 4-(6-Chloro-2-oxo-2H-chromen-4-ylmethoxy)-benzaldehyde
72 %; m.p. 214–216 ꢁC; IR (KBr,
y
cmꢀ1): 1721 (C]O of coumarin),
(2c). Colourless solid, m.p.220–222 ꢁC, IR (KBr
y
cmꢀ1): 3062
1612 (C]N); 1H NMR (300 MHz,
d ppm DMSO-d6): 4.59 (s, 2H, CH2–
(]CH–), 2808 (CH of CHO), 1722 (C]O of Coumarin), 1698 (C]O of
O), 6.38 (s, 1H, C3–H), 6.94–7.92 (m, 11H, Ar–H), 9.95 (s, 1H,
–CH]N) ppm; 13C NMR (75 MHz,
ppm, DMSO-d6): 75.5, 109.1,
aldehyde), 1571 cmꢀ1 (C]C), 1021 (C–O–C); 1H NMR (300 MHz,
d
d
ppm, CDCl3): 5.25 (s, 2H, CH2O), 6.73 (s, 1H, C3–H), 7.21–7.91 (m,
111.3, 116.9, 120.3, 124.7, 126.2, 129.3, 134.7, 136.3, 146.5, 147.5, 154.1,
157.4, 160.0, 162.3; ESI-MS: 425 (M þ 1); Anal. Calcd for
C23H15O3Cl2N: C, 65.11; H, 3.57; N, 3.30; found C, 65.13; H, 3.55, N,
3.33%.
7H, Ar–H), 9.90 (s, 1H, CHO); 13C NMR (75 MHz,
d
ppm, CDCl3): 83.4,
110.0, 116.0, 126.0, 135.1, 138.3, 148.2, 158.4, 161.0, 167.7, 192.0; ESI-
MS: 315 (M þ 1); Anal. Calcd for C17H11ClO4: C, 64.87; H, 3.57, Cl,
11.28; found C, 64.91; H, 3.62, Cl, 11.32%.
6.2.3.4. 4-{4-[(4-Chloro-phenylimino)-methyl]-phenoxymethyl}-
6.2.2.4. 4-(3-Oxo-3H-benzochromen-1-ylmethoxy)-benzaldehyde
benzo[f] chromen-3-one (4d). Yellow crystals from DMF; Yield 72 %;
(2d). Light yellow solid, m.p.222–224 ꢁC, IR (KBr
y
cmꢀ1): 3080
m.p. 246–247 ꢁC; IR (KBr, cmꢀ1): 1730 (C]O of coumarin), 1623
y
(]CH–), 2836 (CH of CHO), 1712 (C]O of coumarin), 1695(C]O of
(C]N); 1H NMR (300 MHz,
6.29 (s, 1H, C3–H), 6.78–7.85 (m, 14H, Ar–H), 9.08 (s, 1H, –CH]N)
ppm; 13C NMR (75 MHz,
ppm, DMSO-d6): 80.2, 105.6, 113.4, 115.5,
d ppm DMSO-d6): 4.56 (s, 2H, CH2–O),
aldehyde), 1562 cmꢀ1 (C]C), 1031 (C–O–C); 1H NMR (300 MHz,
d
ppm, CDCl3): 5.75 (s, 2H, CH2O), 6.94 (s, 1H, C3–H), 7.13–8.15 (m,
d
10H, Ar–H), 9.96 (s, 1H, CHO); 13C NMR (75 MHz,
d
ppm, CDCl3):
116.5, 121.4, 123.5, 125.4, 127.5, 128.3, 129.9, 131.3, 149.3, 150.8,
155.7, 158.7, 161.2, 164.4; ESI-MS: 441 (M þ 1); Anal. Calcd for
C27H18O3ClN: C, 73.72; H, 4.12; N, 3.18; found C, 73.74; H, 4.15, N,
3.15%.
81.4,106.0,114.0,123.0,126.8,129.1,130.3,148.7,163.5,169.3,193.2.;
ESI-MS: 330 (Mþ); Anal. Calcd for C21H14O4: C, 76.38; H, 4.27; found
C, 76.42; H, 4.32%.

5128
R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
6.2.3.5. 4-{4-[(4-Chloro-phenylimino)-methyl]-phenoxymethyl}-
6.2.4. Preparation of 3-chloro-4-[4-(2-oxo-2H-chromen-4-
benzo[h] chromen-2-one (4e). Reddish crystals from DMF; Yield 65
ylmethoxy)-phenyl]-1-phenyl-azetidin-2-ones (5a–j)
%; m.p. 203–204 ꢁC; IR (KBr,
y
cmꢀ1): 1723 (C]O of coumarin), 1614
ppm DMSO-d6): 4.39 (s, 2H, CH2–O),
The mixture of 4-(4-Phenyliminomethyl-phenoxymethyl)-
chromen-2-ones (0.01 mol) and triethylamine (0.01 mol) was dis-
solved in dioxane (50 ml), cooled and stirred. To this well-stirred
cold solution of chloroacetyl chloride (0.01 mmol) was added drop
wise within a period of 20 min. The reaction mixture was then
stirred for an additional 3 h and left at room temperature for 48 h.
The resultant mixture was concentrated, cooled, poured into ice
cold water, filtered and then dried. The product thus obtained was
purified by column chromatography over silica gel using 30% ethyl
acetate: 70% benzene as an eluent. Recrystallization was done from
suitable solvent which gave 2-azetidinones (5a–j).
(C]N); 1H NMR (300 MHz,
d
6.43 (s, 1H, C3–H), 6.86–7.81 (m, 14H, Ar–H), 9.32 (s, 1H, –CH]N)
ppm; 13C NMR (75 MHz,
d
ppm, DMSO-d6): 78.5, 106.1, 111.9, 114.8,
118.2, 120.9, 122.9, 126.1, 127.7, 128.6, 130.4, 136.4, 147.5, 148.9,
155.3, 157.9, 160.3, 163.9; ESI-MS: 441 (M þ 1); Anal. Calcd for
C27H18O3ClN: C, 73.72; H, 4.12; N, 3.18; found C, 73.74; H, 4.15, N,
3.15%.
6.2.3.6. 4-{4-[(4-Bromo-phenylimino)-methyl]-phenoxymethyl}-6-
methyl-chromen-2-one (4f). Colourless crystals from DMF; yield 72
%; m.p. 241–242 ꢁC; IR (KBr,
y
cmꢀ1): 1717 (C]O of coumarin), 1603
ppm DMSO-d6): 1.93 (s, 3H, C6–CH3),
(C]N); 1H NMR (300 MHz,
d
6.2.4.1. 3-Chloro-1-[(4-chloro-phenyl)-4-[4-(6-methyl-2-oxo-2H-
4.94 (s, 2H, CH2–O), 6.09 (s, 1H, C3–H), 6.87–7.95 (m, 11H, Ar–H),
chromen-4-ylmethoxy)-phenyl]-azetidin-2-one](5a). Colourless shiny
9.53 (s, 1H, –CH]N) ppm; 13C NMR (75 MHz,
d
ppm, DMSO-d6):
crystals from DMF, m.p. 174–176 ꢁC, IR (KBr, cmꢀ1): 3107 (]CH–),
y
20.2, 78.9, 108.4, 114.3, 118.3, 121.9, 125.3, 127.2, 129.8, 133.4, 137.6,
147.3, 151.3, 155.3, 158.5, 159.7, 162.0; ESI-MS: 449 (M þ 1); Anal.
Calcd for C24H18O3NBr: C, 64.30; H, 4.05, N, 3.11; Found C, 64.27; H,
4.07, N, 3.13%.
1764 (pC]O of
b
-lactam), 1723 (pC]O of coumarin), 1503 (C]C),
782 (–C–Cl) cmꢀ1
;
1H NMR (300 MHz,
d
ppm, CDCl3): 2.22 (s, 3H,
C6–CH3), 4.63 (s, 2H, CH2–O), 4.95 (s, 1H, –N–CH), 5.29 (s, 1H, –CH–
Cl), 6.21 (s, 1H, C3–H), 6.67–7.92 (m, 11H, Ar–H); 13C NMR (75 MHz,
d
ppm, CDCl3): 17.9, 57.3, 61.0, 63.7, 80.2, 104.2, 111.2, 119.3, 120.4,
6.2.3.7. 4-{4-[(4-Bromo-phenylimino)-methyl]-phenoxymethyl}-7-
126.3, 129.3, 130.5, 140.4, 147.1, 157.0, 158.7, 162.1, 173.2; ESI-MS: 481
methyl-chromen-2-one (4g). Colourless crystals from DMF; yield
(M þ 1).
65%; m.p. 245–247 ꢁC; IR (KBr,
y
cmꢀ1): 1724 (C]O of coumarin),
1616 (C]N); 1H NMR (300 MHz,
CH3), 5.18 (s, 2H, CH2–O), 6.42 (s, 1H, C3–H), 7.00–8.25 (m, 11H, Ar–
H), 9.17 (s, 1H, –CH]N) ppm; 13C NMR (75 MHz,
ppm, DMSO-d6):
d
ppm DMSO-d6): 2.21 (s, 3H, C6–
6.2.4.2. 3-Chloro-1-[(4-chloro-phenyl)-4-[4-(7-methyl-2-oxo-2H-
chromen-4-ylmethoxy)-phenyl]-azetidin-2-one](5b). Colourless shiny
d
crystals from DMF, m.p. 132–133 ꢁC, IR (KBr,
y
cmꢀ1):
y
3089 (]CH–),
-lactam), 1703 (pC]O of coumarin), 1489 (C]C),
780 (–C–Cl) cmꢀ1 1H NMR (300 MHz,
ppm, CDCl3): 2.03 (s, 3H,
19.7, 80.5, 106.5, 113.8, 117.6, 122.7, 126.7, 128.3, 130.7, 135.8, 138.9,
148.9, 153.2, 156.2, 158.8, 159.2, 161.4; ESI-MS: 449 (M þ 1); Anal.
Calcd for C24H18O3NBr: C, 64.30; H, 4.05, N, 3.11; Found C, 64.29; H,
4.09, N, 3.11%.
1758 (pC]O of b
;
d
C7–CH3), 4.44 (s, 2H, CH2–O), 5.12 (s, 1H, –N–CH), 5.42 (s, 1H, –CH–
Cl), 5.89 (s, 1H, C3–H), 6.76–7.79 (m, 11H, Ar–H); 13C NMR (75 MHz,
d
ppm, CDCl3): 21.0, 57.3, 63.7, 81.2, 102.3, 113.0, 116.8, 121.0, 125.7,
6.2.3.8. 4-{4-[(4-Bromo-phenylimino)-methyl]-phenoxymethyl]-6-
127.8, 132.6, 139.3, 145.9, 156.2, 157.5, 160.4, 167.5; ESI-MS: 481
chloro-chromen-2-one (4h). Colourless crystals from DMF; yield
(M þ 1).
68%; m.p. 231–232 ꢁC; IR (KBr,
y
cmꢀ1): 1718 (C]O of coumarin),
1608 (C]N); 1H NMR (300 MHz,
CH2–O), 6.29 (s, 1H, C3–H), 6.77–7.89 (m, 11H, Ar–H), 9.78 (s, 1H,
–CH]N) ppm; 13C NMR (75 MHz,
ppm, DMSO-d6): 77.9, 104.8,
d
ppm DMSO-d6): ¼ 4.67 (s, 2H,
6.2.4.3. 3-Chloro-4-[4-(6-chloro-2-oxo-2H–chromen-4-ylmethoxy)-
phenyl]-1-(4-chloro-phenyl)–azetidin-2-one (5c). Colourless shiny
d
crystals from DMF, m.p. 175–177 ꢁC, IR (KBr, cmꢀ1): 3089 (]CH–),
y
112.6, 114.8, 119.7, 123.8, 125.8, 130.5, 133.5, 138.6, 144.7, 148.9,
153.8, 156.9, 159.7, 160.9; ESI-MS: 425 (M þ 1); Anal. Calcd for
C23H15O3BrClN: C, 58.95; H, 3.25; N, 3.00; Found C, 58.97; H, 3.22,
N, 3.03%.
1771 (pC]O of
791 (–C–Cl) cmꢀ1
CH2–O), 4.79 (s, 1H, –N–CH), 5.19 (s, 1H, –CH–Cl), 6.09 (s, 1H, C3–H),
6.59–7.76 (m, 11H, Ar–H); 13C NMR (75 MHz,
ppm, CDCl3): 53.8,
b
-lactam), 1718 (pC]O of coumarin), 1497 (C]C),
;
1H NMR (300 MHz,
d
ppm, CDCl3): 4.12 (s, 2H,
d
61.2, 79.3, 107.3, 112.0, 120.3, 122.6, 125.7, 127.2, 129.9, 133.3, 137.3,
6.2.3.9. 1-{4-[(4-Bromo-phenylimino)-methyl]-phenoxymethyl}-
146.8, 155.7, 159.8, 163.6, 179.6; ESI-MS: 502 (M þ 2).
benzo[f] chromen-3-one (4i). Yellow crystals from DMF; Yield 81%;
m.p. 177–179 ꢁC IR (KBr,
y
cmꢀ1): 1726(C]O of coumarin), 1609
6.2.4.4. 3-Chloro-1-(4-chloro-phenyl)-4-[4-(3-oxo-3H-benzo[f]chro-
men-1-ylmethoxy)-phenyl]- azetidin-2-one (5d). Light brown crys-
(C]N); 1H NMR (300 MHz,
6.42 (s, 1H, C3–H), 6.89–7.99 (m, 14H, Ar–H), 9.76 (s, 1H, –CH]N)
ppm; 13C NMR (75 MHz,
ppm, DMSO-d6): 76.8, 107.2, 114.6, 116.9,
d
ppm DMSO-d6): 4.66 (s, 2H, CH2–O),
tals from DMF, m.p. 122–124 ꢁC, IR (KBr,
1773 (pC]O of b
y
cmꢀ1):
y
3133 (]CH–),
-lactam), 1725 (pC]O of coumarin), 1506 (C]C),
785 (–C–Cl) cmꢀ1 1H NMR (300 MHz,
ppm, CDCl3): 4.34 (s, 2H,
CH2–O), 4.78 (s, 1H, –N–CH), 5.30 (s, 1H, –CH–Cl), 6.19 (s, 1H, C3–H),
6.64–7.83 (m, 14H, Ar–H); 13C NMR (75 MHz,
ppm, CDCl3): 57.6,
d
118.9, 120.3, 122.7, 124.8, 126.9, 127.7, 130.0, 133.7, 147.5, 151.7, 153.9,
157.9, 158.0, 163.3; ESI-MS: 485 (M þ 1); Anal. Calcd for
C27H18O3BrN: C, 66.95; H, 3.75; N, 2.89; Found C, 66.98; H, 3.78, N,
2.92%.
;
d
d
64.1, 80.4, 104.2, 111.8, 115.2, 116.4, 120.1, 124.3, 126.4, 127.7, 130.5,
133.4, 134.6, 139.4, 150.9 156.7, 160.1, 165.7, 171.0; ESI-MS: 518
(M þ 2).
6.2.3.10. 4-{4-[(4-Bromo-phenylimino)-methyl]-phenoxymethyl}-
benzo[h] chromen-2-one (4j). Reddish crystals from DMF; yield
72%; m.p. 237–238 ꢁC; IR (KBr,
y
cmꢀ1): 1731 (C]O of coumarin),
6.2.4.5. 3-Chloro-1-(4-chloro-phenyl)-4-[4-(2-oxo-2H-benzo[h]-
1601 (C]N); 1H NMR (300 MHz,
d
ppm DMSO-d6): 4.61 (s, 2H,
chromen-4-ylmethoxy)-phenyl]- azetidin-2-one (5e). Light reddish
CH2–O), 6.32 (s, 1H, C3–H), 6.94–7.92 (m, 14H, Ar–H), 9.64 (s, 1H,
crystals from DMF, m.p. 180-182 ꢁC, IR (KBr, cmꢀ1): 3088 (]CH–),
y
–CH¼N) ppm; 13C NMR (75 MHz,
d
ppm, DMSO-d6): 80.3, 107.7,
1767 (pC]O of
779 (–C–Cl) cmꢀ1
CH2–O), 4.91 (s, 1H, –N–CH), 5.41 (s, 1H, –CH–Cl), 6.37 (s, 1H, C3–H),
6.71–7.92 (m, 14H, Ar–H); 13C NMR (75 MHz,
ppm, CDCl3): 53.9,
62.6, 78.3, 102.4, 110.1, 113.9, 115.3, 119.7, 123.5, 125.0, 127.0, 130.8,
b
-lactam), 1718 (pC]O of coumarin), 1478 (C]C),
110.1,113.3, 116.0,119.3, 123.3, 125.4, 126.8, 129.2, 131.9,138.2, 145.3,
151.3, 153.4, 154.7, 157.7, 162.5; ESI-MS: 485 (M þ 1); Anal. Calcd for
C27H18O3BrN: C, 66.95; H, 3.75; N, 2.89; Found C, 66.93; H, 3.77, N,
2.93%.
;
1H NMR (300 MHz,
d
ppm, CDCl3): 4.56 (s, 2H,
d

R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
5129
132.7, 135.7, 140.7, 147.7 154.5, 157.7, 160.1, 168.3; ESI-MS: 518
recorded to represent MIC expressed in
mg/ml. Ciprofloxacin was
(M þ 2).
used as standard drug.
6.2.4.6. 1-(4-Bromo-phenyl)-3-chloro-4-[4-(6-methyl-2-oxo-2H–
6.3.2. Anti-fungal assay
chromen-4-ylmethoxy)-phenyl]-azetidin-2-one (5f). Colourless shiny
The yeasts were maintained in Sabouraud Dextrose Broth
(Difco) after incubation for 48 h at 25 ꢃ1 ꢁC. Testing was performed
in Sabouraud Dextrose Broth at pH 7.4 and the twofold dilution
technique was applied. A set of tubes containing only inoculated
broth was kept as controls. After incubation for 48 h at 25 ꢃ1 ꢁC,
the last tube with no growth of yeast was recorded to represent MIC
crystals from DMF, m.p. 167–168 ꢁC, IR (KBr, cmꢀ1): 3089 (]CH–),
y
1756 (pC]O of
b
-lactam), 1713 (pC]O of coumarin), 1457 (C]C),
775 (–C–Cl) cmꢀ1; 1H NMR (300 MHz,
d
ppm, CDCl3): 1.78 (s, 3H, C6–
CH3), 4.57 (s, 2H, CH2–O), 4.88 (s, 1H, –N–CH), 5.38 (s, 1H, –CH–Cl),
6.09 (s, 1H, C3–H), 6.85–7.68 (m, 11H, Ar–H); 13C NMR (75 MHz,
d
ppm, CDCl3): 20.4, 60.2, 64.6, 77.5, 106.7, 113.9, 116.7, 123.7, 127.7,
expressed in mg/ml. Gentamycin was used as standard drug.
129.9, 133.6, 138.9, 147.7, 158.4, 160.1, 161.7, 167.6; ESI-MS: 525
(M þ 1).
6.3.3. Cytotoxic activity
Brine shrimp (A. salina leach) eggs were hatched in a shallow
rectangular plastic dish (22 ꢂ 32 cm), filled with artificial sea water,
which was prepared with commercial salt mixture and double
distilled water. An unequal partition was made in the plastic dish
with the help of a perforated device. Approximately 50 mg of eggs
were sprinkled into the large compartment, which was darkened
while the other compartment was opened to ordinary light. After
two days nauplii were collected by a pipette from the lighted side. A
sample of the test compound was prepared by dissolving 20 mg of
each compound in 2 ml of DMF. From this stock solutions 500, 50
6.2.4.7. 1-(4-Bromo-phenyl)-3-chloro-4-[4-(7-methyl-2-oxo-2H–
chromen-4-ylmethoxy)-phenyl]-azetidin-2-one (5g). Colourless shiny
crystals from DMF, m.p. 237–238 ꢁC, IR (KBr, cmꢀ1): 3105 (]CH–),
y
1771 (pC]O of
b
-lactam), 1722 (pC]O of coumarin), 1418 (C]C),
779 (–C–Cl) cmꢀ1; 1H NMR (300 MHz,
d
ppm, CDCl3): 1.92 (s, 3H, C7–
CH3), 4.39 (s, 2H, CH2–O), 5.06 (s, 1H, –N–CH), 5.27 (s, 1H, –CH–Cl),
6.22 (s, 1H, C3–H), 6.76–7.79 (m, 11H, Ar–H); 13C NMR (75 MHz,
d
ppm, CDCl3): 21.5, 59.7, 63.3, 82.0, 106.7, 110.7, 117.8, 121.6, 126.7,
128.8, 131.7, 141.6, 146.8, 155.8, 159.1, 160.0, 164.5; ESI-MS: 525
(M þ 1).
and 5 mg/ml were transferred to 9 vials (three for each dilutions
were used for each test sample and LD50 is the mean of three
values) and one vial was kept as control having 2 ml of DMF only.
The solvent was allowed to evaporate overnight. After two days,
when shrimp larvae were ready, 1 ml of sea water and 10 shrimps
were added to each vial (30 shrimps/dilution) and the volume was
adjusted with sea water to 5 ml per vial. After 24 h the number of
survivors was counted [25]. Data were analyzed by a Finney
computer program to determine the LD50 values [26].
6.2.4.8. 1-(4-Bromo-phenyl)-3-chloro-4-[4-(6-chloro-2-oxo-2H–
chromen-4-ylmethoxy)-phenyl]-azetidin-2-one (5h). Colourless shiny
crystals from DMF, m.p.159–160 ꢁC, IR (KBr,
1749 (pC]O of
789 (–C–Cl) cmꢀ1
CH2–O), 4.93 (s, 1H, –N–CH), 5.32 (s, 1H, –CH–Cl), 6.26 (s, 1H, C3–H),
6.76–7.78 (m, 11H, Ar–H); 13C NMR (75 MHz,
ppm, CDCl3): 57.9,
y
cmꢀ1): 3078 (]CH–),
-lactam), 1712 (pC]O of coumarin), 1444 (C]C),
b
;
1H NMR (300 MHz,
d
ppm, CDCl3): 4.34 (s, 2H,
d
60.9, 83.1, 106.8, 114.6, 119.6, 121.7, 124.6, 126.9, 130.5, 132.6, 138.2,
143.7, 153.9, 158.1, 160.4, 164.9; ESI-MS: 547 (M þ 2).
6.3.4. DNA cleavage experiment
6.3.4.1. Preparation of culture media. DNA cleavage experiments
were done according to the literature [27]. Nutrient broth [peptone,
10; Yeast extract, 5; NaCl,10; in (g/l)] was used for culturing of E. coli.
50 ml media was prepared, autoclaved for 15 min at 121 ꢁC under 15
lb pressure. The autoclaved media were inoculated for 24 h at 37 ꢁC.
6.2.4.9. 1-(4-Bromo-phenyl)-3-chloro–4-[4-(3-oxo-3H-benzo[f]chro-
men-1-ylmethoxy)-phenyl]-azetidin-2-one (5i). Light yellow crys-
tals from DMF, m.p.192–193 ꢁC, IR (KBr, cmꢀ1): 3110 (]CH–), 1766
y
(pC]O of
(–C–Cl) cmꢀ1; 1H NMR (300 MHz,
O), 5.13 (s,1H, –N–CH), 5.37 (s,1H, –CH–Cl), 6.35 (s,1H, C3–H), 6.61–
7.76 (m, 14H, Ar–H); 13C NMR (75 MHz,
ppm, CDCl3): 58.5, 61.9,
b
-lactam), 1720 (pC]O of coumarin), 1454 (C]C), 779
d
ppm, CDCl3): 4.60 (s, 2H, CH2–
6.3.4.2. Isolation of DNA. The fresh bacterial culture (1.5 ml) is
centrifuged to obtain the pellet which is then dissolved in 0.5 ml of
lysis buffer (100 mM tris pH 8.0, 50 mM EDTA, 10% SDS). To this
0.5 ml of saturated phenol was added and incubated at 55 ꢁC for
10 min, then centrifuged at 10,000 rpm for 10 min and to the
supernatant, equal volume of chloroform: isoamyl alcohol (24:1)
and 1/20th volume of 3 M sodium acetate (pH 4.8) was added.
Again centrifuging at 10,000 rpm for 10 min and to the superna-
tant, 3 volumes of chilled absolute alcohol were added. The
precipitated DNA was separated by centrifugation and the pellet
was dried and dissolved in TAE buffer (10 mM tris pH 8.0, 1 mM
EDTA) and stored in cold condition.
d
78.9, 106.5, 113.9, 114.6, 115.9, 119.6, 124.2, 125.9, 127.9, 131.6, 133.0,
134.9, 140.8, 150.3, 155.2, 159.5, 160.5, 164.3; ESI-MS: 562 (M þ 2).
6.2.4.10. 1-(4-Bromo-phenyl)-3-chloro–4-[4-(3-oxo-3H-benzo[h]-
chromen-1-ylmethoxy)-phenyl]-azetidin-2-one (5j). Light brown
crystals from DMF, m.p.230–231 ꢁC, IR (KBr, cmꢀ1): 3093 (]CH–),
y
1757 (pC]O of
787 (–C–Cl) cmꢀ1
CH2–O), 5.14 (s, 1H, –N–CH), 5.31 (s, 1H, –CH–Cl), 6.32 (s, 1H, C3–H),
6.73–7.88 (m, 14H, Ar–H); 13C NMR (75 MHz,
ppm, CDCl3): 55.9,
b
-lactam), 1722 (pC]O of coumarin), 1467 (C]C),
;
1H NMR (300 MHz,
d
ppm, CDCl3): 4.25 (s, 2H,
d
60.7, 79.9, 103.9, 112.0, 113.6, 115.8, 118.9, 122.1, 125.7, 126.9, 129.7,
132.5, 135.6, 138.6, 146.7 155.9, 159.6, 160.4, 163.2; ESI-MS: 562
(M þ 2).
6.3.4.3. Agarose gel electrophoresis. Cleavage products were
analyzed by agarose gel electrophoresis method [27]. Test samples
(1 mg/ml) were prepared in DMF. The samples (25 mg) were added
to the isolated DNA of E. coli. The samples were incubated for 2 h at
37 ꢁC and then 20 ml of DNA sample (mixed with bromophenol
blue dye at 1:1 ratio) was loaded carefully into the electrophoresis
chamber wells along with standard DNA marker containing TAE
buffer (4.84 g tris base, pH 8.0, 0.5 M EDTA/1 L) and finally loaded
on agarose gel and passed the constant 50 V of electricity for
around 30 min. Removing the gel and being stained with 10 .0 mg/
ml ethidium bromide for 10–15 min, the bands were observed
under Vilberlourmate Gel documentation system and then
6.3. Pharmacology
6.3.1. Anti-bacterial assay
The cultures were obtained in Mueller–Hinton Broth (Difco) for
all the bacteria after 18–24 h of incubation at 37 ꢃ 1 ꢁC. Testing was
carried out in Mueller–Hinton Broth at pH 7.4 and two-fold dilution
technique was applied. A set of tubes containing only inoculated
broth was kept as controls. After incubation for 18–24 h at
37 ꢃ 1 ꢁC, the last tube with no growth of microorganism was

5130
R.S. Keri et al. / European Journal of Medicinal Chemistry 44 (2009) 5123–5130
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the results were compared with standard DNA marker.
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Acknowledgments
This research work is financially supported by the Council of
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