Synthesis, in-vitro antimicrobial and cytotoxic studies of novel azetidinone derivatives

Article abstract of DOI:10.1002/ardp.200900188

Developing novel antimicrobial drugs is increasingly important in the modern pharmaceutical industry. A series of novel 3-chloro-4-[4-(2-oxo-2H- chromen-4-ylmethoxy)phenyl]-1-phenylazetidin-2-ones 5a-o have been synthesized from 4-bromomethylcoumarins 1a-

Full text of DOI:10.1002/ardp.200900188

Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
R. S. Keri et al.
237
Full Paper
Synthesis, in-vitro Antimicrobial and Cytotoxic Studies of
Novel Azetidinone Derivatives
Rangappa S. Keri, Kallappa M. Hosamani, Harisha S. Reddy, and Ramya V. Shingalapur
P. G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad, India
Developing novel antimicrobial drugs is increasingly important in the modern pharmaceutical
industry. A series of novel 3-chloro-4-[4-(2-oxo-2H-chromen-4-ylmethoxy)phenyl]-1-phenylazetidin-
2-ones 5a–o have been synthesized from 4-bromomethylcoumarins 1a–e and 4-arylimino-
methyl-phenols 3a–c. These compounds were screened for their in-vitro antibacterial activity
against two Gram-positive (Staphylococcus aureus and Vancomycin resistant enteroccoccus) and two
Gram-negative (Escherichia coli and Shigella dysentery) bacterial strains and antifungal activity
against Aspergillus fumigatus, Candida albicans, and Penicillium. Results revealed that compounds
5c, 5f, 5h, 5j, and 5m showed excellent activity against a panel of microorganisms. The brine-
shrimp bioassay was also carried out to study their in-vitro cytotoxic properties and two com-
pounds, 5h and 5m, possessing LD50 = 7.154610– 4 M and 5.782610– 4 M, respectively, displayed
potent cytotoxic activity against Artemia salina. The presence of a chlorine group in the cou-
marin moiety, its effect on their antibacterial, antifungal, and cytotoxic activities is discussed.
1
All newly synthesized compounds were characterized by elemental analysis, IR, H-NMR, 13C-
NMR, and MS.
Keywords: Antibacterial / Antifungal / Azetidinone / Coumarin / Cytotoxicity /
Received: August 7, 2009; accepted: November 25, 2009
DOI 10.1002/ardp.200900188
many clinically relevant pathogens are now resistant.
Through various molecules designed and synthesized for
this aim, it was demonstrated that azetidinone and its
derivatives could be considered as possible antimicrobial
agents [1, 2].
Introduction
The treatment of infectious diseases still remains an
important and challenging problem caused by a combi-
nation of factors; among them emerging infectious dis-
eases and the increasing number of multi-drug resistant
microbial pathogens. In spite of a large number of antibi-
otics and chemotherapeutics available for medical use,
the emergence of old and new antibiotic resistance cre-
ated in the last decades revealed a substantial medical
need for new classes of antimicrobial agents. There is a
real perceived need for the discovery of new compounds
endowed with antimicrobial activity, possibly acting
through mechanisms, which are distinct from those of
well-known classes of antibacterial agents, to which
2-Azetidinones, commonly known as b-lactams, are
heterocyclic compounds well-known among organic and
medicinal chemists [3]. The activity of the famous antibi-
otics such as penicillins, cephalosporins, thienamycine,
nocardicins, aztreonam, and carbapenems are attributed
to the presence of the 2-azetidinone ring in them. Azetidi-
nones are a very important class of compounds possess-
ing a wide range of biological activities such as antimi-
crobial [4], anti-inflammatory [5], anticonvulsant [6], anti-
biotic [7], anticancer [8], anti-elastase [9], antiviral [10],
antitumor [4], and anti-HCMV [11] activities. Recently, it
has been reported that b-lactams have novel biological
properties such as acting as cytomegalovirus protease
inhibitors [12], thrombin and tryptase inhibitors [13],
cholesterol absorption inhibitors [14], human leukocyte
Correspondence: Kallappa M. Hosamani, P .G. Department of Studies
in Chemistry, Karnatak University, Pavate Nagar, Dharwad-580 003, In-
dia.
E-mail: dr_hosamani@yahoo.com
Fax: +91 836 274-7884 and +91 836 277-1275
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

238
R. S. Keri et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
tate. 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 cm– 1, whereas the aldehydic-carbonyl stretching
appeared at 1690 cm– 1. In the 1H-NMR spectrum of com-
pound 2a, a singlet was observed at d = 2.38 ppm due to C6-
CH3 protons. The C4-CH2 protons were observed downfield
as a singlet at d = 5.31 ppm. The C3-H of coumarin appeared
atd=6.64ppm. The aldehydic protonappeared asasinglet
in the downfield at d = 9.90 ppm. The 13C-NMR spectral
data of compound 2a is given in the experimental section.
In view of the poor reactivity of the p-carbonyl group
observed in 4-aryloxy-methyl coumarins, pre-functional-
ized phenols 3a–c (obtained by the reaction of p-formyl
phenol and aromatic amines) were used for room-temper-
ature allylic SN reaction under standard acetone potas-
sium carbonate conditions generating the required pre-
cursors 4a–o in high yields. The IR spectrum of 6-methyl-
4-(4-phenyliminomethyl-phenoxymethyl)-chromen-2-one
4a (R = 6-CH3, R1 = H) showed a lactone-carbonyl stretching
frequency at 1718 cm– 1 and the stretching frequency for
the C=N group at 1605 cm– 1. The PMR spectrum of 4a
exhibited a singlet due to the CH3 protons at d = 2.32 ppm,
C4-CH2 at d = 5.31 ppm, and C3-H at d = 6.43 ppm. The aro-
matic protons resonated in the range of d = 7.10–8.32
ppm. The azomethine proton appeared downfield as a sin-
glet at d =9.83ppm. The azomethine group in 4a–o under-
going cycloaddition with chloroacetylchloride in the
presence of triethylamine [Et3N] as a catalyst in DMF/ben-
zene afforded 3-chloro-4-[4-(2-oxo-2H-chromen-4-ylmeth-
oxy)-phenyl]-1-phenyl-azetidin-2-ones 5a–o (Scheme 1).
The IR spectrum of 3-chloro-4-[4-(6-methyl-2-oxo-2H-chro-
Scheme 1. Schematic representation for the synthesis of azeti-
dinone derivatives from 4-bromo-methyl coumarin.
elastease (HLE) inhibitors [15], porcine pancreatic elas-
tease (PPE) inhibitors [16], HIV-1 protease [17], and peroxi-
some proliferator-activated receptors (PPARs) [18]. Besides
their biological activities, the importance of b-lactams as
synthetic intermediates has been widely recognized in
organic synthesis [19] for example in the semi-synthesis
of Taxol [20]. Like azetidinone, coumarin also exhibits
diverse biological properties [21].
In the design of new drugs, the development of hybrid
molecules through the combination of different pharma-
cophores in one frame may lead to compounds with
interesting biological profiles. Owing to the importance
and in continuation of our work of the synthesis of anti-
microbially active compounds [22–24], we now wish to
describe the synthesis of new azetidinone derivatives
from 4-aryloxy-methyl coumarins (Scheme 1) and the in-
vitro screening results of their antibacterial, antifungal,
and cytotoxic activities.
men-4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-one
5a
showed bands at 1768 cm– 1 (AC=O of b-lactam) and the
coumarin-carbonyl stretching at 1722 cm– 1. In the PMR
spectra of compound 5a, the peak was observed at d = 5.32
ppm due to the ACH–Cl in the b-lactam ring; in the 13C-
NMR spectrum of compound 5a, a peak at d = 53.3 ppm
was observed due to ACH–Cl, at 184.4 ppm (cyclic, AC=O),
and at 158.2 ppm (hetero-aromatics) in the b-lactam moi-
ety. The mass spectrum of compound 5a showed the
molecular ion peak at 446[M+ +1].
Biological evaluation
Antibacterial bioassay
Results and discussion
The synthesized compounds 5a–o were screened in vitro
for their antibacterial activity against two Gram-positive
(Staphylococcus aureus, Vancomycin-resistant enteroccoccus)
and two Gram-negative (Escherichia coli, Shigella dysentery)
bacterial strains by agar well diffusion method [25]. The
Chemistry
The present synthetic strategy begins with the generation
of the required 4-bromo-methyl coumarins 1a–e by Pech-
mann cyclization of phenols with 4-bromo-ethyl acetoace-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Azetidinone Derivatives from Coumarin Moiety
239
Table 1. Physical and analytical data of the 2-azetidinone derivatives.
Entry Producta)
R
Ar
Yield
(%)
M. p.b)
(8C)
Mol. Formula /
Mol. Wt.
Elemental Analysis (Calcd./Found)
C
H
N
1
2
5a
5b
5c
5d
5e
5f
6-CH3
Ph
88.00
71.00
62.80
74.10
65.12
64.20
71.80
82.4
138–140
114–116
118–120
198–200
232–234
132–134
208–210
183–185
220–222
110–112
170–172
98–100
C26H20ClNO4
446
C26H20ClNO4
446
C25H17Cl2NO4
466
C29H20ClNO4
482
C29H20ClNO4
482
C27H22ClNO4
460
C27H22ClNO4
460
C26H19Cl2NO4
481
C30H22ClNO4
496
C30H22ClNO4
496
C27H22ClNO5
477
C27H22ClNO5
477
C26H19Cl2NO5
497
C30H22ClNO5
512
C30H22ClNO5
512
70.03
70.08
70.03
70.06
64.39
64.43
72.27
72.29
72.27
72.26
70.51
70.50
70.51
70.55
65.01
65.05
72.65
72.61
72.65
72.67
68.14
68.17
68.14
68.12
62.92
62.95
70.38
70.40
70.38
70.36
4.52
4.56
4.52
4.55
3.67
3.69
4.18
4.23
4.18
4.20
4.82
4.84
4.82
4.82
3.99
4.00
4.47
4.50
4.47
4.45
4.66
4.63
4.66
4.69
3.86
3.83
4.33
4.35
4.33
4.32
3.14
3.17
3.14
3.18
3.00
2.98
2.92
2.96
2.92
2.90
3.05
3.04
3.05
3.01
2.92
2.90
2.82
2.84
2.82
2.80
2.94
2.97
2.94
2.93
2.82
2.80
2.74
2.70
2.74
2.77
7-CH3
Ph
3
6-Cl
Ph
4
5,6-Benzo
7,8-Benzo
6-CH3
Ph
5
Ph
6
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeOC6H4
4-MeOC6H4
4-MeOC6H4
4-MeOC6H4
4-MeOC6H4
7
5g
5h
5i
7-CH3
8
6-Cl
9
5,6-Benzo
7,8-Benzo
6-CH3
71.12
65.20
63.8
10
11
12
13
14
15
5j
5k
5l
7-CH3
70.8
5m
5n
5o
6-Cl
63.4
130–132
282–284
195–197
5,6-Benzo
7,8-Benzo
61.02
70.13
a)
Products were characterized by IR, NMR, MS, and elemental analysis.
Yields of isolated compounds.
Melting points are uncorrected.
b)
c)
results were compared with those of the standard drug tion in the coumarin ring have shown good activity for
Ciprofloxacin. The zone of inhibition and percentage both the bacterial and fungal strains at 25 lg/mL concen-
inhibition of the compounds are given in (Table 2). The tration.
results indicate that all the synthesized compounds are
good to moderately active against a panel of microorgan- Antifungal bioassay
isms. Compounds 5c, 5f, 5h, 5j, and 5m exhibited excel- The antifungal screening of all compounds was carried
lent activity against S. aureus and V.-r. enteroccoccus. Out of out against Aspergillus fumigatus, Candida albicans, and
these, 5m was found to be the most active. However, com- Penicillium fungal strains. The results were compared
pound 5a, 5b, 5d, and 5e showed moderate activity with those of standard drug Gentamycin. These results
against S. aureus and V.-r. enteroccoccus. Compound 5n and (see Table 3) indicate that the compounds 5c, 5f, 5h, 5j,
5o were found to be inactive against S. aureus and S. dysen- and 5m exhibited excellent activity against all the fungal
tery. The compounds 5c, 5h, and 5m with 6-Cl substitu- strains. The compounds 5f and 5h showed l 90% activity
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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240
R. S. Keri et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Table 2. In-vitro antibacterial activities of synthesized compounds 5a–o in terms of zone of inhibition and percentage inhibition.
Comp.
Zone of inhibition% (mm)
V.-r. enteroccoccus Conc. (lg/mL) E. coli Conc. (lg/mL)
S. aureus Conc. (lg/mL)
S. dysentery Conc. (lg/mL)
100
50
25
100
50
25
100
50
25
100
50
25
5a
5b
5c
5d
5e
5f
34a)
61b)
37
66
49
87
30
54
32
57
45
80
38
68
46
82
34
61
44
79
36
64
35
62
45
80
29
52
34
51
17
57
16
53
23
77
16
53
16
53
19
63
15
50
24
80
18
60
22
73
19
63
20
66
22
73
14
47
15
50
30
–
10
29
10
55
14
78
Nil
–
45
72
39
63
54
87
42
68
46
74
52
84
43
69
56
90
44
71
52
84
38
61
39
63
49
79
37
60
39
63
62
–
17
57
16
53
26
87
20
67
18
60
23
77
17
57
22
73
19
63
25
83
17
57
15
50
21
70
18
60
16
53
30
–
13
59
11
50
17
77
14
64
13
59
16
73
13
59
17
77
Nil
–
19
86
12
55
Nil
–
15
68
Nil
–
40
67
37
62
52
87
44
73
40
67
56
93
39
65
52
87
41
68
50
83
47
78
49
82
54
90
46
77
44
73
60
–
23
64
20
56
31
86
20
56
20
56
26
72
21
58
30
83
20
56
29
81
19
53
20
56
31
86
23
64
22
61
36
–
12
60
Nil
–
16
80
11
55
Nil
–
15
70
12
60
16
80
10
50
14
70
Nil
–
11
55
16
80
Nil
–
39
67
34
58
46
79
35
60
31
53
34
59
35
60
52
90
45
78
52
90
46
79
49
84
54
93
34
59
36
62
58
–
18
56
15
47
26
81
17
53
20
62
17
53
15
46
24
75
17
53
26
81
22
69
19
59
25
78
15
47
20
62
32
–
12
55
Nil
–
17
77
12
54
13
59
11
50
Nil
–
16
73
13
59
18
82
14
64
13
59
16
73
Nil
–
10
55
12
55
08
44
13
72
10
56
13
72
10
56
11
61
14
67
Nil
–
5g
5h
5i
5j
5k
5l
5m
5n
5o
09
50
18
–
13
59
22
–
12
60
20
–
11
50
22
–
Standard 56
Control
–
Standard used: Ciprofloxacin; control: DMSO.
a)
Zone of inhibition (first row).
Percentage inhibition (second row).
b)
against C. albicans and A. fumigatus, respectively. How- inhibitory concentration was determined using the agar
ever, compounds 5a, 5b, 5d, 5n, and 5o exhibited low to diffusion technique. Each of the test compounds and
moderate activity against the fungal strains.
standards, Ciprofloxacin and Gentamycin, were dis-
In general, the introduction of chlorine substituents in solved in DMSO at an initial concentration of 250 lg/mL,
the coumarin ring have elevated the antibacterial and and then, were serially diluted in culture medium as fol-
antifungal activities to 90% as compared to other sub- lows: 125, 62.5, 31.25, 16, 8, 4, 2, 1 lg/mL concentrations.
stituents (-CH3, -OCH3). The graphical representation of The minimum inhibitory concentrations (MIC) were
antibacterial and antifungal activities of azetidinone defined as the lowest concentrations of the compounds
derivatives at a concentration of 100 lg/mL is depicted in that prevented visible growth.
the Figs. 1 and 2, respectively.
Cytotoxic activity
Minimum inhibitory concentration (MIC)
All the synthesized compounds 5a–o were screened for
Compounds showing antibacterial and antifungal activ- their cytotoxicity (brine-shrimp bioassay) using the pro-
ity above 80% were selected for minimum inhibitory con- tocol of Meyer et al. [26]. From the data recorded in Table
centration (MIC) studies (Tables 4 and 5). The minimum 6, it is evident that the compounds 5c, 5f, 5h, 5j, and 5m
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Azetidinone Derivatives from Coumarin Moiety
241
Table 3. In-vitro antifungal activities of synthesized compounds 5a–o in terms of zone of inhibition and percentage inhibition.
Comp.
Zone of inhibition% (mm)
Aspergillus fumigates Conc. (lg/mL)
Candida albicans Conc. (lg/mL)
Penicillium Conc. (lg/mL)
100
50
25
100
50
25
100
50
25
5a
5b
5c
5d
5e
5f
54a)
77b)
48
69
62
89
52
74
41
59
62
89
42
60
64
91
37
53
57
81
38
54
45
64
57
81
46
66
45
64
70
–
28
67
25
59
36
85
27
64
25
50
36
85
28
67
34
81
19
45
31
74
19
45
22
52
34
81
26
62
23
55
42
–
16
57
15
54
23
82
16
57
Nil
–
22
78
17
61
22
78
Nil
–
20
71
Nil
–
13
46
19
67
16
57
Nil
–
45
70
42
66
57
89
46
71
40
62
58
90
38
59
57
89
32
50
52
81
41
64
38
59
48
75
39
61
45
70
64
–
26
65
26
65
32
80
26
65
26
65
33
82
22
55
34
85
18
40
29
72
22
55
20
50
28
70
23
57
26
65
40
–
16
57
15
54
21
75
15
54
17
61
22
79
Nil
–
21
75
Nil
–
20
71
Nil
–
38
59
42
66
56
87
42
66
38
59
39
61
38
59
48
75
42
66
52
81
40
62
44
69
56
87
40
62
46
72
64
–
18
50
20
56
28
78
22
61
22
61
20
56
20
56
25
69
22
61
20
56
19
53
21
58
27
75
17
47
24
67
36
–
Nil
–
12
46
19
73
11
42
14
54
Nil
–
12
46
17
65
14
54
Nil
–
14
54
13
50
18
69
Nil
–
14
54
26
–
5g
5h
5i
5j
5k
5l
Nil
–
5m
5n
5o
19
68
14
50
16
57
28
–
Standard
Control
28
–
Standard used: Gentamycin; control: DMSO.
a)
Zone of inhibition (first row).
Percentage inhibition (second row).
b)
Table 4. In-vitro antibacterial activities of the selected com-
pounds in MIC (lg/mL).
ity in this assay. Compound 5h showed maximum activ-
ity (LD50 = 7.154610– 4 M) in the present series of com-
pounds, whereas the other active compounds 5c, 5f, 5j,
Compound
MIC (lg/mL)
and 5m demonstrated slightly lower activity (LD50
=
5.231610– 4 M, 4.854610– 4 M, 5.616610– 4 M, and
5.782610– 4 M, respectively) than compound 5h.
S. aureus
V.-r.
enteroccoccus
E. coli
S. dysentery
5c
04
04
04
08
5f
08
08
01
16
5h
5j
5m
04
02
04
01
16
02
08
16
01
01
01
01
Conclusion
In conclusion, a series of 3-chloro-4-[4-(2-oxo-2H-chromen-
4-ylmethoxy)-phenyl]-1-phenyl-azetidin-2-one derivatives
5a–o were prepared and screened for their in-vitro anti-
bacterial activity against four strains of bacteria and anti-
fungal activity against three fungi strains, as well as cyto-
toxic studies. The bioassays indicated that the com-
Standard
0.78
0.70
0.19
0.19
Standard used: Ciprofloxacin.
displayed potent cytotoxic activity against Artemia salina,
while the other compounds have shown moderate activ-
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

242
R. S. Keri et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Table 6. Brine-shrimp bioassay data for the compounds 5a–o.
Compound
LD50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
3.753
4.104
0.5231
2.967
3.737
0.4854
2.749
0.7154
2.745
0.5616
2.531
3.0564
0.5782
2.023
2.349
Figure 1. The antibacterial activities of selected azetidinone
derivatives: 5c, 5f, 5h, 5j, and 5m and Ciprofloxacin (Standard)
at 100 lg/mL concentration.
pounds 5c, 5f, 5h, 5j, and 5m are excellently active (up to
90%) against all the strains and the remaining com-
pounds showed good to moderate activity comparable
with the standard drugs. Compounds 5f, 5h, and 5m dis-
played potent cytotoxic activity against Artemia salina.
On the basis of structure-activity relationship study of
the compounds, it can be concluded that, the presence of
a chlorine group in the coumarin moiety enhanced the
activity of the compounds. Henceforth, our findings may
have a good impact on chemists and biochemists for fur-
ther investigations in this field in search of chlorine-con-
taining antimicrobial and cytotoxic agents.
Experimental
Materials and methods
All reagents and solvents were used as obtained from the sup-
plier or recrystalized/redistilled as necessary. The melting points
of the products were determined by open capillaries on a Bꢀchi
apparatus (Bꢀchi Labortechnik, Flawil, Switzerland) and are
uncorrected. The IR spectra were recorded on a Nicolet Impact-
410 FT-IR spectrophotometer (Nicolet, Madison, WI, USA), using
KBr pellets. 1H-NMR and 13C-NMR spectra were recorded on a
Bruker AC-300F 300 MHZ spectrometer (Bruker Bioscience, USA)
Figure 2. The antifungal activities of selected azetidinone deriv-
atives: 5c, 5f, 5h, 5j, and 5m and Gentamycin (Standard) at 100
lg/mL concentration.
Table 5. In vitro antifungal activity of the selected compounds in
MIC (lg/mL).
1
in DMSO-d6/CDCl3 using TMSi as an internal standard with H-
resonant frequency of 300 MHZ and13C resonant frequency of 75
MHZ. D2O exchange was applied to confirm the assignment of
the signals of NH protons. The mass spectra were recorded on an
Autospec EI-MS (Micromass, Waters). The elemental analysis was
carried out using Heraeus CHN rapid analyzer (Heraeus,
Gemany). All the compounds gave C, H, and N analysis within €
0.4% of the theoretical values. The homogeneity of the com-
pounds was described by TLC on aluminium silica gel 60 F254
(Merck, Germany) detected by UV light (254 nm) and iodine
vapors. Compound's nomenclature was made using Chem. Draw
Ultra version 6.0. In-vitro antibacterial and antifungal properties
were studied at Luqman Pharmacy College, Gulbraga, Rajiv Gan-
dhi University, India.
Compound
MIC (lg/mL)
Aspergillus fumigates Candida albicans Penicillium
5c
5f
5h
5j
5m
Standard
04
08
01
02
08
2.0
04
08
04
16
08
2.0
08
16.125
08
16
16.125
2.0
Standard used: Gentamycin.
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Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Azetidinone Derivatives from Coumarin Moiety
243
5.75 (s, 2H, CH2O), 6.94 (s, 1H,C3-H), 7.13–8.15 (m, 10H, Ar-H),
9.96 (s,1H, CHO); 13C-NMR (75 MHz, CDCl3, ppm) d: 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 [M+]. Anal. calcd. for C21H14O4: C, 76.38; H, 4.27. Found: C,
76.42; H, 4.32.
Chemistry
General procedure for the preparation of 4-bromo-methyl
coumarins 1a–e
The present synthetic strategy begins with the generation of the
required 4-bromo-methyl coumarins 1a–c. These compounds
were prepared by Pechmann cyclization of phenols with 4-bro-
moethyl acetoacetate [27, 28].
4-(2-Oxo-2H-benzochromen-4-ylmethoxy)benzaldehyde
2e
Light redish solid, m. p.: 248–2498C; IR (KBr, cm– 1) m: 3052
General procedure preparation of 2-oxo-2H-chromen-4-
ylmethoxy)benzaldehydes 2a–e
(=CH-), 2842 (-CH of CHO), 1718 (C=O of coumarin), 1707 (C=O of
aldehyde), 1569 (C=C), 1028 (C-O-C); H-NMR (300 MHz, CDCl3,
1
p-Hydroxy benzaldehyde (10 mmol) and anhydrous K2CO3 (1.38
g, 10 mmol) were stirred in dry acetone 25 mL for 30 min. 4-
Bromo-methyl coumarins (2.52 g, 10 mmol) were added and stir-
ring was continued for 24 h. The reaction mixture was concen-
trated to one-fourth of the original volume and poured onto ice-
cold water. The solid which separated was filtered and washed
with 5% HCl (10 mL) to neutralize the excess of potassium carbo-
nate, then washed with 100 mL of cold water and with dilute
ethanol. The crude product was dried and recrystallized from
DMF.
ppm) d: 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, CDCl3, ppm) d: 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 [M+]. Anal. calcd. for C21H14O4: C, 76.39; H, 4.26.
Found: C, 76.45; H, 4.30.
Preparation of 4-(4-phenyliminomethyl-
phenoxymethyl)chromen-2-ones 4a–o
4-Phenyliminomethyl-phenols 3a–c (10 mmol) and anhydrous
K2CO3 (1.38 g, 10 mmol) were stirred in dry acetone (25 mL) for
30 min. 4-Bromo-methyl coumarins 1a–e (2.52 g, 10 mmol) were
added and stirring was continued for 24 h. The reaction mixture
was concentrated and poured into crushed ice (100 g). The solid
which separated was filtered and washed with 5% HCl (10 mL) to
neutralize the 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.
4-(6-Methyl-2-oxo-2H-chromen-4-
ylmethoxy)benzaldehyde 2a
Colorless solid, m. p.: 218–2198C; IR (KBr, cm– 1) m: 3046 (=CH-),
2778 (CH of CHO), 1718 (C=O of coumarin), 1690 (C=O of alde-
hyde), 1534 (C=C), 1022 (C-O-C);1H-NMR (300 MHz, CDCl3, ppm) d:
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, CDCl3, ppm) d:
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 [M+]; Anal. calcd. for C18H14O4: C, 73.45; H, 4.80.
Found: C, 73.51; H, 4.87.
6-Methyl-4-(4-phenyliminomethyl-
phenoxymethyl)chromen-2-one 4a
Yield: 81%, colorless crystals from DMF, m. p.: 215–2178C; IR
(KBr, cm– 1) m: 1718 (C=O of coumarin), 1605 (C=N); 1H-NMR (300
MHz, DMSO-d6, ppm) d: 2.32 (s, 3H, C6-CH3), 5.31 (s, 2H, CH2-O),
6.43 (s, 1H, C3-H), 7.10–8.32 (m, 12H, Ar-H), 9.83 (s, 1H, -CH-N);13C-
NMR (75 MHz, DMSO-d6, ppm) d: 18.8, 80.03, 105.06, 112.20,
120.12, 122.20, 125.6, 127.56, 129.40, 132.10, 135.8, 149.12,
155.0, 162.02, 164.12, 167.42; ESI-MS: 370 [M+ + 1]. Anal. calcd. for
C24H19NO3: C, 78.03; H, 5.18; N, 3.79. Found: C, 78.04; H, 5.21; N,
3.81.
4-(7-Methyl-2-oxo-2H-chromen-4-
ylmethoxy)benzaldehyde 2b
Colorless solid, m. p.: 223–2258C; IR (KBr, cm– 1) m: 3038 (=CH-),
2788 (CH of CHO), 1707 (C=O of coumarin), 1697 (C=O of alde-
hyde), 1562 (C=C), 1016 (C-O-C);1H-NMR (300 MHZ, CDCl3, ppm) d:
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, CDCl3, ppm) d:
20.6, 78.0, 113.5, 117.8.0, 121.3, 134.1, 148.2, 155.4, 167.6, 188.0;
ESI-MS: 294 [M+]. Anal. calcd. for C18H14O4: C, 73.44; H, 4.81.
Found: C, 73.48; H, 4.86.
7-Methyl-4-(4-phenyliminomethyl-
phenoxymethyl)chromen-2-one 4b
4-(6-Chloro-2-oxo-2H-chromen-4-
ylmethoxy)benzaldehyde 2c
Yield: 77%, colorless crystals from DMF, m. p.: 187–1888C; IR
(KBr, cm– 1) m: 1710 (C=O of coumarin), 1598 (C=N); 1H-NMR (300
MHz, DMSO-d6, ppm) d: 1.91 (s, 3H, C7-CH3), 4.99 (s, 2H, CH2-O),
6.31 (s, 1H, C3-H), 6.89–7.79 (m, 12H, Ar-H), 9.71 (s, 1H, -CH-N);13C-
NMR (75 MHz, DMSO-d6, ppm) d: 21.31, 77.12, 108.12, 111.00,
119.91, 121.71, 123.43, 126.71, 130.02, 134.31, 138.82, 146.54,
153.07, 157.76, 160.01, 162.17; ESI-MS: 370 [M+ + 1]. Anal. calcd.
for C24H19NO3: C, 78.03; H, 5.18; N, 3.79. Found: C, 78.06; H, 5.19;
N, 3.77.
Colorless solid, m. p.: 220–2228C; IR (KBr, cm– 1) m: 3062 (=CH-),
2808 (CH of CHO), 1722 (C=O of coumarin), 1698 (C=O of alde-
hyde), 1571 (C=C), 1021 (C-O-C); 1H-NMR (300 MHz, CDCl3, ppm) d:
5.25 (s, 2H, CH2O), 6.73 (s, 1H, C3-H), 7.21–7.91 (m, 7H, Ar-H), 9.90
(s,1H, CHO); 13C-NMR (75 MHz, CDCl3, ppm) d: 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+ + 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-Chloro-4-(4-phenyliminomethyl-
phenoxymethyl)chromen-2-one 4c
Yield: 59%, light yellow crystals from DMF, m. p.: 232–2348C; IR
(KBr, cm– 1) m: 1722 (C=O of coumarin), 1612 (C=N); 1H-NMR (300
MHz, DMSO-d6, ppm) d: 4.83 (s, 2H, CH2-O), 6.36 (s, 1H, C3-H),
6.88–7.49 (m, 12H, Ar-H), 9.05 (s, 1H, -CH-N); 13C-NMR (75 MHz,
4-(3-Oxo-3H-benzochromen-1-ylmethoxy)benzaldehyde
2d
Light yellow solid, m. p.: 222–2248C; IR (KBr, cm– 1) m: 3080 (=CH-
), 2836 (CH of CHO), 1712 (C=O of coumarin), 1695 (C=O of alde-
hyde), 1562 (C=C), 1031 (C-O-C); 1H-NMR (300 MHz, CDCl3, ppm) d:
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244
R. S. Keri et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
DMSO-d6, ppm) d: 77.56, 104.97, 115.10, 119.03, 121.78, 123.86,
126.1, 127.23, 129.55, 130.02, 146.87, 153.56, 158.23, 161.23,
163.83, 164.17; ESI-MS: 390 [M+ + 1]. Anal. calcd. for C23H16ClNO3:
C, 70.86; H, 4.14; N, 3.59. Found: C, 70.85; H, 4.17; N, 3.56.
6-Chloro-4-(4-(p-tolylimino-methyl)phenoxymethyl]-
chromen-2-one 4h
Yield: 66%, colorless crystals from DMF, m. p.: 201–2028C; IR
(KBr, cm– 1) m: 1705 (C=O of coumarin), 1603 (C=N); 1H-NMR (300
MHz, DMSO-d6, ppm) d: 2.12 (s, 1H, CH3), 4.91 (s, 2H, CH2-O), 6.27
(s, 1H, C3-H), 6.92–7.79 (m, 11H, Ar-H), 9.57 (s, 1H, -CH=N); 13C-
NMR (75 MHz, DMSO-d6, ppm) d: 17.83, 80.13, 106.79, 112.72,
117.92, 120.29, 124.31, 125.68, 126.79, 129.87, 133.46, 144.65,
151.23, 155.65, 159.01, 160.43, 167.15; ESI-MS: 404 [M+ + 1]. Anal.
calcd. for C24H18ClNO3: C, 71.38; H, 4.49; N, 3.47. Found: C, 71.40;
H, 4.53; N, 3.50.
1-(4-Phenyliminomethyl-phenoxymethyl)benzo[f]-
chromen-3-one 4d
Yield: 72%, reddish-colored crystals from DMF, m. p.: 232–
2338C; IR (KBr, cm– 1) m: 3013 (=CH) 1724 (C=O of coumarin), 1612
(C=N), 1506 (C=C), 1115 (C-O-C); 1H-NMR (300 MHz, DMSO-d6,
ppm) d: 4.10 (s, 2H, CH2-O), 6.31 (s, 1H, C3-H), 6.98–7.99 (m, 15H,
Ar-H), 9.91 (s, 1H, -CH=N); 13C-NMR (75 MHz, DMSO-d6, ppm) d:
73.56, 103.65, 112.36, 114.76, 118.78, 120.03, 121.87, 122.68,
123.01, 124.26, 125.87, 127.98, 129.76, 132.65, 137.87, 143.76,
151.21, 154.65, 155.54, 158.41, 161.21; ESI-MS: 406 [M+ + 1]. Anal.
calcd. for C27H19NO3: C, 79.97; H, 4.68; N, 3.45. Found: C, 80.00; H,
4.69; N, 3.44.
1-[4-(p-Tolylimino-methyl)phenoxymethylbenzo[f]-
chromen-3-one 4i
Yield: 72%, reddish crystals from DMF, m. p.: 283–2848C; IR (KBr,
cm– 1) m: 1735 (C=O of coumarin), 1615 (C=N); 1H-NMR (300 MHz,
DMSO-d6, ppm) d: 2.28 (s, 3H, CH3), 4.72 (s, 2H, CH2-O), 6.56 (s, 1H,
C3-H), 6.88–7.49 (m, 14H, Ar-H), 8.89 (s, 1H, -CH=N); 13C-NMR (75
MHz, DMSO-d6, ppm) d: 20.22, 80.13, 106.23, 113.43, 117.32,
120.76, 122.76, 123.97, 126.32, 129.34, 130.67, 137.12, 151.03,
156.97, 162.34, 165.10, 166.54; ESI-MS: 420 [M+ + 1]. Anal. calcd.
for C28H21NO3: C, 80.16; H, 5.05; N, 3.34. Found: C, 80.17; H, 5.02;
N, 3.32.
1-(4-Phenyliminomethyl-phenoxymethyl)benzo[h]-
chromen-2-one 4e
Yield: 83%, yellow-colored crystals from DMF, m. p.: 203–2048C;
IR (KBr, cm– 1) m: 3034 (=CH), 1719 (C=O of coumarin), 1607 (C=N),
1
1478 (C=C), 1122 (C-O-C); H-NMR (300 MHz, DMSO-d6, ppm) d:
4-[4-(p-Tolylimino-methyl)phenoxymethylbenzo[h]-
chromen-2-one 4j
4.94 (s, 2H, CH2-O), 6.19 (s, 1H, C3-H), 6.88–8.23 (m, 15H, Ar-H),
8.76 (s, 1H, -CH=N); 13C-NMR (75 MHz, DMSO-d6, ppm) d: 70.21,
100.95, 110.20, 116.07, 119.67, 121.05, 121.37, 122.18, 123.32,
124.00, 125.65, 126.54, 128.54, 131.45, 133.65, 144.45, 152.31,
157.43, 160.06, 163. 23, 167.18; ESI-MS: 406 [M+ + 1]. Anal. calcd.
for C27H19NO3: C, 79.97; H, 4.68; N, 3.45. Found: C, 79.99; H, 4.71;
N, 3.47.
Yield: 57%, yellow crystals from DMF, m. p.: 256–2578C; IR (KBr,
cm– 1) m: 1727 (C=O of coumarin), 1600 (C=N); 1H-NMR (300 MHz,
DMSO-d6, ppm) d: 2.07 (s, 3H, CH3), 4.41 (s, 2H, CH2-O), 6.41 (s, 1H,
C3-H), 6.67–7.87(m, 14H, Ar-H), 9.87 (s, 1H, -CH=N); 13C-NMR (75
MHz, DMSO-d6, ppm) d: 17.65, 77.92, 103.80, 111.09, 114.78,
119.71, 121.32, 124.05, 127.46, 128.91, 132.54, 139.07, 145.67,
154.43, 157.43, 159.92, 162.03; ESI-MS: 420 [M+ + 1]. Anal. calcd.
for C28H21NO3: C, 80.16; H, 5.05; N, 3.34. Found: C, 80.19; H, 5.06;
N, 3.36.
6-Methyl-4-(4-(p-tolylimino-methyl)phenoxymethyl)-
chromen-2-one 4f
Yield: 71%, colorless crystals from DMF, m. p.: 177–1788C; IR
(KBr, cm– 1) m: 3019 (=CH), 1715 (C=O coumarin), 1589 (C=N), 1501
(C=C), 1099 (C-O-C); 1H-NMR (300 MHz, DMSO-d6, ppm) d: 2.31 (s,
3H, C6-CH3), 2.41 (s, 3H, CH3), 5.22 (s, 2H, CH2-O), 6.32 (s, 1H, C3-H),
6.76–7.98 (m, 11H, Ar-H), 9.78 (s, 1H, -CH=N); 13C-NMR (75 MHz,
DMSO-d6, ppm) d: 17.08, 21.87, 76.19, 107.89, 111.87, 120.92,
122.76, 123.59, 124.12, 125.07, 127.87,128.91, 133.57, 135.67,
139.91, 144.02, 149.94, 155.61, 158.52, 160.02, 163.01; ESI-MS:
384 [M+ + 1]. Anal. calcd. for C25H21NO3: C, 78.29; H, 5.48; N, 3.65.
Found: C, 78.27; H, 5.52; N, 3.63.
4-{4-[(4-Methoxy-phenylimino)methyl]phenoxymethyl)-6-
methylchromen-2-one 4k
Yield: 69%, colorless crystals from DMF, m. p.: 205–2068C; IR
(KBr, cm– 1) m: 3024 (=CH), 1712 (C=O coumarin), 1613 (C=N), 1512
(C=C), 1114 (C-O-C); 1H-NMR (300 MHz, DMSO-d6, ppm) d: 2.32 (s,
3H, C6-CH3), 3.13 (s, 3H, OCH3), 4.87 (s, 2H, CH2-O), 6.23 (s, 1H, C3-
H), 6.83–7.65 (m, 11H, Ar-H), 8.65 (s, 1H, -CH=N); 13C-NMR (75
MHz, DMSO-d6, ppm) d: 19.14, 51.23, 79.34, 101.32, 111.43,
115.55, 119.23, 121.32, 123.13, 125.78, 126.84, 128.76, 130.32,
133.13, 143.23, 146.67, 156.32, 159.54, 161.23, 165.31, 166.43;
ESI-MS: 400 [M+ + 1]. Anal. calcd. for C25H21NO4: C, 75.15; H, 5.26;
N, 3.50. Found: C, 75.18; H, 5.29; N, 3.53.
7-Methyl-4-(4-(p-tolylimino-methyl)phenoxymethyl)-
chromen-2-one 4g
4-{4-[(4-Methoxy-phenylimino)methyl]phenoxymethyl)-7-
methylchromen-2-one 4l
Yield: 78%, colorless crystals from DMF, m. p.: 233–2358C; IR
(KBr, cm– 1) m: 3067 (=CH), 1709 (C=O coumarin), 1611 (C=N), 1507
(C=C), 1109 (C-O-C); 1H-NMR (300 MHz, DMSO-d6, ppm) d: 1.87 (s,
3H, C7-CH3), 2.34 (s, 3H, CH3), 5.07 (s, 2H, CH2-O), 6.23 (s, 1H, C3-H),
6.89–7.66 (m, 11H, Ar-H), 9.03 (s, 1H, -CH=N); 13C-NMR (75 MHz,
DMSO-d6, ppm) d: 15.9, 22.5, 78.12, 104.65, 113.45, 119.65,
121.45, 122.32, 124.56, 125.32, 126.54,129.32, 130.5, 136.65,
137.54, 149.32, 150.12, 156.45, 159.65, 162.32, 165.43; ESI-MS:
384 [M+ + 1]. Anal. calcd. for C25H21NO3: C, 78.29; H, 5.48; N, 3.65.
Found: C, 78.32; H, 5.50; N, 3.67.
Yield: 74%, colorless crystals from DMF, m. p.: 231–2328C; IR
(KBr, cm– 1) m: 3109 (=CH), 1709 (C=O coumarin), 1621 (C=N), 1501
(C=C), 1109 (C-O-C); 1H-NMR (300 MHz, DMSO-d6, ppm) d: 1.85 (s,
3H, C7-CH3), 3.68 (s, 3H, OCH3), 4.54 (s, 2H, CH2-O), 6.09 (s, 1H, C3-
H), 6.77–8.09 (m, 11H, Ar-H), 9.92 (s, 1H, -CH=N); 13C-NMR (75
MHz, DMSO-d6, ppm) d: 22.61, 59.04, 81.73, 103.09, 112.00,
117.03, 118.07, 122.05, 124.10, 126.90, 127.23, 129.03, 131.68,
137.57, 144.04, 148.59, 155.56, 157.05, 159.45, 160.89, 163.12;
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Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Azetidinone Derivatives from Coumarin Moiety
245
ESI-MS: 400 [M+ + 1]. Anal. calcd. for C25H21NO4: C, 75.15; H, 5.26;
N, 3.50. Found: C, 75.13; H, 5.25; N, 3.52.
4.98 (s, 2H, CH2-O), 5.05 (s, 1H, -N-CH), 5.34 (s, 1H, -CH-Cl), 6.65 (s,
1H, C3-H), 6.88–7.96 (m, 12H, Ar-H); 13C-NMR (75 MHz, CDCl3,
ppm) d: 21.0, 53.3, 62.0, 80.8, 108.4, 115.2, 121.2, 125.2, 128.1,
134.4, 140.2, 148.4, 158.2, 162.1, 184.4; ESI-MS: 446 [M+ + 1].
6-Chloro-4-{4-[(4-methoxy-phenyliminomethyl]-
phenoxymethyl]chromen-2-one 4m
Yield: 71%, colorless crystals from DMF, m. p.: 167–1688C; IR
(KBr, cm– 1) m: 1721 (C=O of coumarin), 1609 (C=N); 1H-NMR (300
MHz, DMSO-d6, ppm) d: 3.45 (s, 1H, OCH3), 4.39 (s, 2H, CH2-O), 6.26
(s, 1H, C3-H), 6.67–7.89 (m, 11H, Ar-H), 9.73 (s, 1H, -CH=N); 13C-
NMR (75 MHz, DMSO-d6, ppm) d: 56.32, 77.52, 104.99, 110.59,
114.36, 119.17, 122.80, 124.24, 127.89, 130.09, 137.89, 142.89,
148.17, 156.32, 157.03, 159.04, 161.38; ESI-MS: 420 [M+ + 1]. Anal.
calcd. for C24H18ClNO4: C, 68.66; H, 4.32; N, 3.34. Found: C, 68.69;
H, 4.35; N, 3.32.
3-Chloro-4-[4-(7-methyl-2-oxo-2H-chromen-4-
ylmethoxy)phenyl]-1-phenyl-azetidin-2-one 5b
Colorless crystals from DMF; IR (KBr, cm– 1) m: 3109 (=CH-), 1772
(AC=O of b-lactam), 1718 (>C=O of coumarin), 1506 (C=C), 778
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.26 (s, 3H, C7-CH3), 4.67
(s, 2H, CH2-O), 4.98 (s, 1H, -N-CH), 5.40 (s, 1H, -CH-Cl), 6.56 (s, 1H,
C3-H), 6.70-8.02 (m, 12H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d:
23.4, 56.7, 60.4, 82.0, 106.7, 114.0, 122.0, 126.8, 128.8, 132.8,
142.0, 149.0, 157.7, 160.6, 173.7; ESI-MS: 446 [M+ + 1].
1-{4-[(4-Methoxy-phenylimino)methyl]phenoxymethyl)-
benzo[f]chromen-3-one 4n
3-Chloro-4-[4-(6-chloro-2-oxo-2H-chromen-4-
Yield: 59%, reddish crystals from DMF, m. p.: 232–2348C; IR (KBr,
cm– 1) m: 1721 (C=O of coumarin), 1617 (C=N); 1H-NMR (300 MHz,
DMSO-d6, ppm) d: 3.34 (s, 3H, OCH3), 4.94 (s, 2H, CH2-O), 6.26 (s,
1H, C3-H), 6.89–7.87 (m, 14H, Ar-H), 9.69 (s, 1H, -CH=N); 13C-NMR
(75 MHz, DMSO-d6, ppm) d: 59.76, 75.69, 107.82, 112.69, 114.70,
118.32, 119.07, 120.29, 123.06, 125.99, 128.73, 132.18, 138.79,
143.77, 148.67, 150.78, 155.09, 158.89, 160.03, 162.43; ESI-MS:
436 [M+ + 1]. Anal. calcd. for C28H21NO4: C, 77.23; H, 4.86; N, 3.21.
Found: C, 77.20; H, 4.87; N, 3.22.
ylmethoxy)phenyl]-1-phenyl-azetidin-2-one 5c
Colorless crystals from DMF; IR (KBr, cm– 1) m: 3088 (=CH-), 1765
(AC=O of b-lactam), 1722 (AC=O of coumarin), 1500 (C=C), 782
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 4.79 (s, 2H, CH2-O), 5.20
(s, 1H, -N-CH), 5.47 (s, 1H, -CH-Cl), 6.40 (s, 1H, C3-H), 7.12–7.92 (m,
12H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d: 60.2, 63.6, 79.6,
108.3, 113.2, 121.1, 123.6, 126.4, 136.6, 145.8, 150.3, 155.9,
164.4, 178.9; ESI-MS: 466 [M+ + 1].
4-{4-[(4-Methoxy-phenylimino)methyl]phenoxymethyl)-
benzo[h]chromen-2-one 4o
3-Chloro-4-[4-(3-oxo-3H-benzo[f]chromen-1-
ylmethoxy)phenyl]-1-phenyl-azetidin-2-one 5d
Yield: 65%, colorless crystals from DMF, m. p.: 203–2048C; IR
(KBr, cm– 1) m: 1728 (C=O of coumarin), 1603 (C=N); 1H-NMR (300
MHz, DMSO-d6, ppm) d: 3.59 (s, 3H, OCH3), 4.81 (s, 2H, CH2-O), 6.51
(s, 1H, C3-H), 6.72–7.83 (m, 14H, Ar-H), 8.97 (s, 1H, -CH=N); 13C-
NMR (75 MHz, DMSO-d6, ppm) d: 54.04, 77.19, 104.55, 114.24,
115.54, 117.88, 120.45, 121.46, 122.92, 124.58, 127.43, 130.05,
134.65, 145.65, 150.54, 157.33, 159.63, 160.04, 163.45, 164.23;
ESI-MS: 436 [M+ + 1]. Anal. calcd. for C28H21NO4: C, 77.23; H, 4.86;
N, 3.21. Found: C, 77.26; H, 4.88; N, 3.24.
Light yellow crystals from DMF; IR (KBr, cm– 1) m: 3122 (=CH-),
1782 (AC=O of b-lactam), 1730 (AC=O of coumarin), 1521 (C=C),
792 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 4.57 (s, 2H, CH2-O),
5.08 (s, 1H, -N-CH), 5.40 (s, 1H, -CH-Cl), 6.36 (s, 1H, C3-H), 6.86–
8.35 (m, 15H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d: 56.7, 64.9,
82.1, 102.6, 110.8, 116.7, 120.0, 122.6, 126.4, 129.7, 134.0, 140.2,
149.6, 157.3, 160.1, 170.2; ESI-MS: 482 [M+ + 1].
3-Chloro-4-[4-(3-oxo-2H-benzo[h]chromen-4-
ylmethoxy)phenyl]-1-phenyl-azetidin-2-one 5e
Preparation of 3-chloro-4-[4-(2-oxo-2H-chromen-4-
Reddish crystals from DMF; IR (KBr, cm– 1) m: 3122 (=CH-), 1777
(AC=O of b-lactam), 1726 (AC=O of coumarin), 1507 (C=C), 781
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 4.62 (s, 2H, CH2-O), 5.32
(s, 1H, -N-CH), 5.44 (s, 1H, -CH-Cl), 6.20 (s, 1H, C3-H), 6.70–7.97 (m,
15H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d: 60.0, 63.5, 80.0,
99.8, 107.2, 113.1, 122.4, 125.0, 127.7, 130.3, 135.3, 142.0, 150.8,
158.7, 163.2, 172.0; ESI-MS: 482 [M+ + 1].
ylmethoxy)phenyl]-1-phenylazetidin-2-ones 5a–o
The mixture of 4-(4-phenyliminomethyl-phenoxymethyl)-chro-
men-2-ones (0.01 mol) and triethyl amine (0.01 mol) was dis-
solved in DMF/benzene (50 mL), cooled and stirred. To this well-
stirred cold solution of chloro acetyl chloride (0.01 mmol) was
added dropwise within a period of 20 min. The reaction mixture
was then stirred for an additional 3 h and left at room temper-
ature 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 a suitable solvent which yielded
the 2-azetidinones 5a–o.
3-Chloro-4-[4-(6-methyl-2-oxo-2H-chromen-4-
ylmethoxy)phenyl]-1-p-tolyl-azetidin-2-one 5f
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 3089 (=CH-),
1768 (AC=O of b-lactam), 1722 (AC=O of coumarin), 1520 (C=C),
782 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.17 (s, 3H, C6-CH3),
2.38 (s, 3H, CH3), 4.57 (s, 2H, CH2-O), 5.12 (s, 1H, -N-CH), 5.49 (s, 1H,
-CH-Cl), 6.55 (s, 1H, C3-H), 6.78–7.75 (m, 11H, Ar-H); 13C-NMR (75
MHz, CDCl3, ppm) d: 19.2, 23.2, 59.1, 63.6, 82.5, 103.6, 110.0,
121.2, 125.2, 128.1, 134.4, 140.2, 148.4, 158.2, 162.1, 184.4; ESI-
MS: 460 [M+ + 1].
3-Chloro-4-[4-(6-methyl-2-oxo-2H-chromen-4-
ylmethoxy)phenyl]-1-phenyl-azetidin-2-one 5a
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 3089 (=CH-),
1768 (AC=O of b-lactam), 1722 (AC=O of coumarin), 1520 (C=C),
782 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.38 (s, 3H, C6-CH3),
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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246
R. S. Keri et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
782 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.21 (s, 3H, C7-CH3),
3.71 (s, 3H, OCH3), 4.53 (s, 2H, CH2-O), 4.93 (s, 1H, -N-CH), 5.33 (s,
1H, -CH-Cl), 6.30 (s, 1H, C3-H), 6.66–7.70 (m, 11H, Ar-H); 13C-NMR
(75 MHz, CDCl3, ppm) d: 21.0, 47.7, 59.1, 62.0, 83.1, 105.9, 112.1,
115.2, 121.1, 122.5, 124.2, 126.3, 127.2, 128.8,132.1, 134.7, 138.3,
150.1, 156.0, 159.0, 160.7, 162.3, 169.3; ESI-MS: 477 [M+ + 1].
3-Chloro-4-[4-(7-methyl-2-oxo-2H-chromen-4-
ylmethoxy)phenyl]-1-p-tolyl-azetidin-2-one 5g
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 3102 (=CH-),
1772 (AC=O of b-lactam), 1717 (AC=O of coumarin), 1501 (C=C),
766 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.26 (s, 3H, C7-CH3),
2.41 (s, 3H, CH3), 4.62 (s, 2H, CH2-O), 5.06 (s, 1H, -N-CH), 5.51 (s, 1H,
-CH-Cl), 6.40 (s, 1H, C3-H), 6.68–7.56 (m, 11H, Ar-H); 13C-NMR (75
MHz, CDCl3, ppm) d: 20.0 22.7, 60.3, 63.9, 80.8, 106.0, 112.3,
119.0, 122.2, 124.5, 125.5, 126.5, 133.0, 136.5, 138.3, 150.3,
154.5, 157.7, 160.0, 171.3; ESI-MS: 460 [M+ + 1].
3-Chloro-4-[4-(6-chloro-2-oxo-2H-chromen-4-
ylmethoxy)phenyl]-1-(4-methoxyphenyl)azetidin-2-one
5m
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 3091 (=CH-),
1777 (AC=O of b-lactam), 1716 (AC=O of coumarin), 1487 (C=C),
779 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 3.50 (s, 3H, OCH3),
4.61 (s, 2H, CH2-O), 5.04 (s, 1H, -N-CH), 5.41 (s, 1H, -CH-Cl), 6.51(s,
1H, C3-H), 6.76–7.88 (m, 11H, Ar-H); 13C-NMR (75 MHz, CDCl3,
ppm) d: 50.3, 60.2, 63.6, 80.6, 105.9, 113.5, 116.2, 120.4, 121.9,
127.1, 128.5, 130.1,133.0, 135.1, 145.7, 155.3, 159.2, 160.0, 163.1,
170.7; ESI-MS: 497 [M+ + 1].
3-Chloro-4-[4-(6-chloro-2-oxo-2H-chromen-4-
ylmethoxy)phenyl]-1-p-tolyl-azetidin-2-one 5h
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 2993 (=CH-),
1768 (AC=O of b-lactam), 1709 (AC=O of coumarin), 1487 (C=C),
1
804 (-C-Cl); H-NMR (300 MHz, CDCl3, ppm) d: 2.20 (s, 3H, CH3),
4.32 (s, 2H, CH2-O), 4.78 (s, 1H, -N-CH), 5.37 (s, 1H, -CH-Cl), 6.26 (s,
1H, C3-H), 6.82–7.68 (m, 11H, Ar-H); 13C-NMR (75 MHz, CDCl3,
ppm) d: 17.9, 56.0, 61.6, 77.9, 109.1, 113.0, 120.2, 126.0, 128.5,
130.0, 134.6, 138.0, 150.0, 158.8, 163.3, 169.7; ESI-MS: 481 [M+ +
1].
3-Chloro-1-(4-methoxyphenyl)-4-[4-(3-oxo-3H-
benzo[f]chromen-1-ylmethoxy)phenyl]-1-p-tolyl-azetidin-
2-one 5n
3-Chloro-4-[4-(3-oxo-3H-benzo[f]chromen-1-
Yellow crystals from DMF; IR (KBr, cm– 1) m: 3118 (=CH-), 1770
(AC=O of b-lactam), 1731 (AC=O of coumarin), 1515 (C=C), 792
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 3.41 (s, 3H, OCH3), 4.53
(s, 2H, CH2-O), 5.16(s, 1H, -N-CH), 5.51 (s, 1H, -CH-Cl), 6.30 (s, 1H,
C3-H), 6.85–7.91 (m, 14H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d:
53.1, 60.0, 63.3, 80.4, 108.0, 112.7, 114.7, 121.2, 123.0, 126.6,
127.3, 128.9, 133.4, 137.0, 145.3, 150.2, 157.1, 159.7, 163.7,
168.2; ESI-MS: 512 [M+ + 1].
ylmethoxy)phenyl]-1-p-tolyl-azetidin-2-one 5i
Yellow crystals from DMF; IR (KBr, cm– 1) m: 3102 (=CH-), 1777
(AC=O of b-lactam), 1726 (AC=O of coumarin), 1507 (C=C), 779
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.17 (s, 3H, CH3), 4.47 (s,
2H, CH2-O), 5.07 (s, 1H, -N-CH), 5.45 (s, 1H, -CH-Cl), 6.42 (s, 1H, C3-
H), 6.65–8.06 (m, 14H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d:
19.4, 52.3, 59.2, 78.6, 106.9, 114.2, 117.4, 120.0, 123.3, 127.0,
129.9, 136.2, 143.4, 148.2, 153.1, 158.7, 160.5, 165.3, 173.0; ESI-
MS: 496 [M+ + 1].
3-Chloro-1-(4-methoxyphenyl)-4-[4-(2-oxo-2H-
benzo[h]chromen-4-ylmethoxy)phenyl]azetidin-2-one 5o
Pale red crystals from DMF; IR (KBr, cm– 1) m: 3104 (=CH-), 1782
(AC=O of b-lactam), 1722 (AC=O of coumarin), 1506 (C=C), 786
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 3.63 (s, 3H, OCH3), 4.67
(s, 2H, CH2-O), 4.78 (s, 1H, -N-CH), 5.23 (s, 1H, -CH-Cl), 6.23 (s, 1H,
C3-H), 6.76–7.83 (m, 14H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm):
47.7, 56.3, 61.2, 82.1, 107.3, 113.0, 115.8, 118.2, 121.6, 123.4,
126.3, 127.7, 129.2, 133.7, 135.3, 148.3, 156.0, 159.1, 162.3,
170.3; ESI-MS: 512 [M+ + 1].
3-Chloro-4-[4-(2-oxo-2H-benzo[h]chromen-4-
ylmethoxy)phenyl]-1-p-tolyl-azetidin-2-one 5j
Reddish crystals from DMF; IR (KBr, cm– 1) m: 3115 (=CH-), 1782
(AC=O of b-lactam), 1722 (AC=O of coumarin), 1495 (C=C), 787
(-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.25 (s, 3H, CH3), 4.55 (s,
2H, CH2-O), 5.12 (s, 1H, -N-CH), 5.52 (s, 1H, -CH-Cl), 6.30 (s, 1H, C3-
H), 6.70–7.91 (m, 14H, Ar-H); 13C-NMR (75 MHz, CDCl3, ppm) d:
21.0, 60.1, 64.1, 80.2, 107.0, 112.9, 120.1, 121.4, 122.8, 124.5,
127.7, 130.2, 133.2, 135.0,138.6, 150.2, 155.3, 159.1, 160.7, 163.8,
169.6; ESI-MS: 496 [M+ + 1].
Biological evaluation
Antibacterial assay
3-Chloro-1-(4-methoxyphenyl)-4-[4-(6-methyl-2-oxo-2H-
The synthesized compounds 5a–o were screened in vitro for
their antibacterial activity against two Gram-positive [Staphylo-
coccus aureus (ATCC-29213) and Vancomycin-resistant enteroccoccus
(ATCC-51299)] and two Gram-negative [Escherichia coli (ATCC-
25922) and Shigella dysentery (natural isolates)] bacterial strains
by the agar-well diffusion method [25]. Ciprofloxacin was used
as reference standards to compare antibacterial activities. The
wells (6 mm in diameter) were dug in the media with the help of
a sterile metallic borer with centers at least 24 mm apart. Two to
eight hours old bacterial inocula containing approximately
104 –106 colony-forming units (CFU/mL) were spread on the sur-
face of the nutrient agar with the help of a sterile cotton swab.
The tests were carried out at a concentration of 100 lg/mL, 50
lg/mL, and 25 lg/mL. After 48 h of incubation at 378C, the zone
of inhibition was measured in millimeters. The percentage
chromen-4-ylmethoxy)phenyl]azetidin-2-one 5k
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 3089 (=CH-),
1773 (AC=O of b-lactam), 1716 (AC=O of coumarin), 1479 (C=C),
779 (-C-Cl); 1H-NMR (300 MHz, CDCl3, ppm) d: 2.09 (s, 3H, C6-CH3),
3.56 (s, 3H, OCH3), 4.74 (s, 2H, CH2-O), 5.05 (s, 1H, -N-CH), 5.51 (s,
1H, -CH-Cl), 6.37 (s, 1H, C3-H), 6.70–7.88 (m, 11H, Ar-H); 13C-NMR
(75 MHz, CDCl3, ppm) d: 20.3, 51.9, 60.2, 63.7, 80.8, 107.0, 111.3,
113.8, 120.7, 127.0, 128.6, 140.4, 146.9, 155.3, 158.1, 160.5,
163.0, 171.0; ESI-MS: 477 [M+ + 1].
3-Chloro-1-(4-methoxyphenyl)-4-[4-(7-methyl-2-oxo-2H-
chromen-4-ylmethoxy)phenyl]azetidin-2-one 5l
Colorless shiny crystals from DMF; IR (KBr, cm– 1) m: 3105 (=CH-),
1766 (AC=O of b-lactam), 1710 (AC=O of coumarin), 1501 (C=C),
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 343, 237–247
Azetidinone Derivatives from Coumarin Moiety
247
inhibition of test compounds was related to the standard whose
zone of inhibition was taken as 100%. In order to clarify any par-
ticipating role of DMSO in the biological screening, separate
studies were carried out with the solutions alone of DMSO and
they showed no activity against any bacterial strains.
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Aspergillus fumigatus (36607), Candida albicans (10231), and Peni-
cillium (natural isolates). Sabouraud dextrose agar (Oxoid, Hamp-
shire, England) was seeded with 105 (CFU/mL) fungal spore sus-
pensions and transferred to petri plates. Discs soaked in 20 mL
(100 mg/mL in DMSO) of all compounds were placed at different
positions on the agar surface. The plates were incubated at 378C
for seven days. Dimethyl sulphoxide (DMSO) was used as a sol-
vent control and Gentamycin used as a standard drug to com-
pare antifungal activity. The tests were carried out at a concen-
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Brine shrimp (Artemia salina leach) eggs were hatched in a shal-
low rectangular plastic dish (22 6 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 pip-
ette 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, and 5 lg/mL were transferred
to nine 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 lar-
vae were ready, 1 mL of sea water and ten 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 survi-
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This research work is financially supported by the Council of
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The authors have declared no conflict of interest.
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i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com