Design, synthesis and in vitro antibacterial and antifungal activities of some novel spiro[azetidine-2,30-indole]-2,4(10H)-dione

Article abstract of DOI:10.1007/s00044-010-9354-x

The present study deals with the synthesis of novel spiro[azetidine-2,30- indole]-20,4(10H)-dione derivative from the reactions of 3-(phenylimino)-1,3- dihydro-2Hindol- 2-one derivatives with chloracetyl chloride in the presence of triethylamine (TEA). All the compounds were characterized using IR, 1H-NMR, MS, and elemental analysis. They were screened for their antibacterial and antifungal activities. The bacterial strains used were Grampositive Staphylococcus aureus (MTCC-96) and Gramnegative Escherichia coli (MTCC-521) and Pseudomonas aeruginosa (MTCC-647). The antifungal screening was done on Candida albicans (MTCC-183) and Asperigillus niger (MTCC-343) fungal strains. Results revealed that, compounds (7a), (7b), (7c), (7d), and (7e) showed very good activity with MIC value of 6.25-12.5 lg/ml against three evaluated bacterial strains and the remaining compounds showed good to moderate activity comparable to standard drugs as antibacterial agents. Compounds (7c) and (7h) displayed equipotent antifungal activity in comparison to standard drugs. Amoxicillin, gentamycin, and streptomycin were used as standard drugs for antibacterial activity while fluconazole and itraconazole were used as standard drugs for antifungal activity. Structure-activity relationship study of the compounds showed that the presence of electron withdrawing group substitution at 50 and 70 positions of indoline ring and on ortho or para position of phenyl ring increases both antibacterial and antifungal activity of the compound. Henceforth, our findings will have a good impact on chemists and biochemists for further investigations in search of spiro-fused antimicrobial agents. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-010-9354-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:587–594
DOI 10.1007/s00044-010-9354-x
ORIGINAL RESEARCH
Design, synthesis and in vitro antibacterial and antifungal
activities of some novel spiro[azetidine-2,30-indole]-2,4(10H)-dione
•
Ravi J. Shah Neha R. Modi Manish J. Patel
•
•
•
•
Laxmanbhai J. Patel Bhupendrasinh F. Chauhan
Madhabhai M. Patel
Received: 28 December 2009 / Accepted: 21 April 2010 / Published online: 6 May 2010
Ó Springer Science+Business Media, LLC 2011
Abstract The present study deals with the synthesis of
novel spiro[azetidine-2,30-indole]-20,4(10H)-dione deriva-
tive from the reactions of 3-(phenylimino)-1,3-dihydro-2H-
indol-2-one derivatives with chloracetyl chloride in the
presence of triethylamine (TEA). All the compounds were
characterized using IR, 1H-NMR, MS, and elemental
analysis. They were screened for their antibacterial and
antifungal activities. The bacterial strains used were Gram-
positive Staphylococcus aureus (MTCC-96) and Gram-
negative Escherichia coli (MTCC-521) and Pseudomonas
aeruginosa (MTCC-647). The antifungal screening was
done on Candida albicans (MTCC-183) and Asperigillus
niger (MTCC-343) fungal strains. Results revealed that,
compounds (7a), (7b), (7c), (7d), and (7e) showed very
good activity with MIC value of 6.25–12.5 lg/ml against
three evaluated bacterial strains and the remaining com-
pounds showed good to moderate activity comparable to
standard drugs as antibacterial agents. Compounds (7c) and
(7h) displayed equipotent antifungal activity in comparison
to standard drugs. Amoxicillin, gentamycin, and strepto-
mycin were used as standard drugs for antibacterial activity
while fluconazole and itraconazole were used as standard
drugs for antifungal activity. Structure–activity relationship
study of the compounds showed that the presence of
electron withdrawing group substitution at 50 and 70 posi-
tions of indoline ring and on ortho or para position of
phenyl ring increases both antibacterial and antifungal
activity of the compound. Henceforth, our findings will
have a good impact on chemists and biochemists for further
investigations in search of spiro-fused antimicrobial agents.
Keywords Antibacterial activity ꢀ Antifungal activity ꢀ
2-Azetidinone ꢀ Indoline
Introduction
In recent decades, problems of multidrug resistant micro-
organisms have reached an alarming level in around the
world. Resistance to a number of antimicrobial agents
(b-lactam antibiotics, macrolides, quinolones, and vanco-
mycin) among a variety of clinically significant species of
bacteria is becoming increasingly major global problem.
These pose a serious challenge to the scientific community
hence emphasis has been laid on the development of new
antimicrobial agents (Francis et al., 2004; Kruszewska
et al., 2004).
Moreover, 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 dual biological profiles. 2-Azetidinone con-
taining antibiotics inhibit the transpeptidase enzyme which
is required for peptodoglycan synthesis, an essential com-
ponent of cell wall of bacteria, while isatin inhibits DNA
gyrase enzyme, required for DNA synthesis of bacteria. We
hypothesized that fusion of these two pharmacophore may
provides better antibacterial activity. Based on this
hypothesis, novel synthesis scheme was designed to
R. J. Shah (&) ꢀ B. F. Chauhan ꢀ M. M. Patel
Department of Pharmaceutical Chemistry,
Kalol Institute of Pharmacy, Kalol, Gujarat, India
e-mail: ravishah_pharma@yahoo.com
N. R. Modi ꢀ M. J. Patel ꢀ L. J. Patel
Department of Pharmaceutical Chemistry, S. K. Patel College
of Pharmaceutical Education and Research, Ganpat University,
Mehsana, Gujarat, India
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Med Chem Res (2011) 20:587–594
synthesize certain compounds containing both 2-azetidi-
none and isatin moieties in a single molecule.
The 2-azetidinones, commonly known as b-lactams, are
among the most useful azaheterocyclic compounds from
both synthetic and medicinal chemistry points of views.
Most of the researches up to early 1990s focused on syn-
thesis of 2-azetidinones and their antibacterial property
(De Kimpe and Rees, 1996; Brown, 1989; Isaacs, 1976).
Later researchers have shown some other useful biological
activities of these compounds, e.g., cholesterol absorption
inhibition, enzymes inhibition, hypoglycemic, and anti-
cancer activity (Singh, 2004a, b). Ezetimibe, a monocyclic
2-azetidinone, is now in clinical use as a cholesterol
absorption inhibitor (Clader, 2004). Extensive investigation
has been done on use of 2-azetidinones as synthons for
diverse types of medicinally important compounds such as
b-aminoacids, c-aminoalcohols, azetidines, pyrimidones,
etc. (Deshmukh et al., 2004; Alcaide and Almendros, 2004;
Singh, 2003; Singh and Pheko, 2008; Ojima and Delaloge,
1997). However, most of these studies are undertaken
either on monocyclic or fused bicyclic 2-azetidinones. The
spiro-fused 2-azetidinones constitute a scarce class of
compounds. There are only a few reports in the literature
on the synthesis, reactivity, and biological activity of
2-azetidinones (Manhas et al., 1969; Barba et al., 1999;
Moriconi and Kelly, 1966; Bose et al., 1963; Singh and
Mehrotra, 1982; Singh et al., 2008; Singh, 2000; Alonso
et al., 2002; Arjona et al., 2002; Nishikawa et al., 2004;
Zanobini et al., 2004; Basak and Ghosh, 2004; Khasanov
et al., 2004; Singh and Mrnolotsi, 2006). The diverse types
of biological activities associated with indoline-2,3-dione
(isatin) derivatives and their interesting chemistry (Singh
et al., 1992; Somogyi, 2001) prompted us to synthesize
novel spiro [azetidine-2,30-indole]-20,4(10H)-dione deriva-
tives. They were successfully screened for their antibac-
terial and antifungal activities.
Scheme 1 Schematic representation for the synthesis of spiro[azeti-
dine-2,30-indole]-2, 4(10H)-dione derivatives
frequency at 1718 cm-1, stretching frequency for amide
carbonyl group at 1665 cm-1, and –C–Br group stretching
frequency at 575 cm-1. The 1H-NMR spectrum of (4)
exhibited a singlet was observed at d 8.00 ppm due to N–H
proton. The aromatic protons resonated in the range of d
7.86–7.95 ppm. Schiff base (6a–j) of isatin was synthe-
sized by reaction between isatin (3) or bromo isatin (4)
with different primary aromatic amines (5a–e) (like p-tol-
uedine, o-toluedine, p-nitrobenzenamine, o-nitrobenzen-
amine and aniline, respectively), in the presence of
absolute methanol and the pH was adjusted to 4 with gla-
cial acetic acid. IR spectra of the Schiff base compounds
(6a–j) showed most prominent peak of imine function
group (–C=N–) at 1630 cm-1, secondary amino function
(–NH–) at 3200 cm-1 and carbonyl function (–C=O) of
indoline ring at 1720 cm-1. The 1H-NMR spectrum of 5,7-
dibromo-3-[(4-methylphenyl)imino]-1,3-dihydro-2H-indol-
2-one (6a) exhibited a singlet was observed at d 8.00 ppm
Chemistry
The present synthetic strategy begins with the synthesis of
isatin (3) (Marvel and Hiers, 1941) by the reaction of dry
isonitrosoacetanilide (2) with concentrated sulfuric acid. In
the IR spectrum of isatin (3), the carbonyl stretching fre-
quency was observed at 1718 cm-1, whereas the amide
carbonyl stretching appeared at 1660 cm-1. In the 1H-
NMR spectrum of compound (3), a singlet was observed at
d 8.00 ppm due to N–H proton. The aromatic protons
resonated in the range of d 7.52–7.83 ppm. The synthesis
of 5,7-dibromoisatin (4) was based on the method of
Lindwall (Vine et al., 2007). In this reaction isatin (3) react
with bromine in methanol and yield (4). The IR spectrum
of 5,7-dibromoisatin (4) showed carbonyl stretching
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Med Chem Res (2011) 20:587–594
589
due to N–H proton, d 2.35 ppm due to presence of –CH3
group at phenyl ring and aromatic protons resonated in the
range of d 7.10–7.71 ppm.
Results and discussion
Antibacterial bioassay
Final product (7a–j) was synthesized by staudinger
ketene–imine cycloaddition reaction (Holton and Liu,
1993) in high yields (Table 1). It involves reaction of
imines (schif bases) (6a–j) with acid chloride in the
presence of a tertiary base, usually triethylamine (TEA) at
temperatures ranging from 80°C to reflux temperature in
various solvents such as dichloromethane, acetonitrile,
toluene, etc. The IR spectrum of 50,70-dibromo-3-chloro-1-
(4-methylphenyl)-4H-spiro[azetidine-2,-30-indole]-20,4(10H)-
dione (7a) showed sharp peak near 1745 cm-1 indicates
the presence of ketone (–C=O) functional group of aze-
tidinone ring. It also showed prominent peak of –N–CO at
1785 cm-1. Chlorine functional group showed a peak at
Zone of inhibition and MIC values for the in vitro antibac-
terial studies of the compounds (7a–j) and the standard are
represented in Table 2. The antibacterial activity of all the
compounds against Staphylococcus aureus as Gram-posi-
tive, Escherichia coli and Pseudomonas aeruginosa as
Gram-negative bacteria showed good potencies which are
comparable to control drugs amoxicillin, gentamycine, and
streptomycin. Among the synthesized compounds (7a), (7b),
(7c), (7d), and (7e) with bromo substituent at 50 and 70
position of indoline showed very good activity with MIC
value of 6.25–12.5 lg/ml. Particularly, compounds (7c) and
(7d) with bromo substituent at 50 and 70 position of indoline
moiety and nitro substitution at ortho or para position of
phenyl ring of azetidinone moiety, have showed compara-
tively better activity with MIC value of 6.25 lg/ml. The
other compounds (7f), (7g), (7h), (7i), and (7j) have exhib-
ited moderate activity against all three bacterial strains.
1
760 cm-1. The H-NMR spectra of spiro-compound (7a)
was showed the signal at 5.80 ppm due to –CH–Cl on
azetidinone ring. Singlets was observed at d 7.96 ppm due
to N–H proton, d 2.24 ppm due to the presence of –CH3
group at phenyl ring and aromatic protons resonated in
the range of d 6.90–7.40 ppm. The mass spectra of these
compounds exhibited intense molecular ion peak at one
unit greater than molecular weight of the compound
(M ? 1). We report the synthesis of novel spiro[azeti-
dine-2,30-indole]-20,4(10H)-dione derivatives (Scheme 1)
and their antibacterial and antifungal potential.
Antifungal bioassay
The antifungal screening of all compounds were carried out
against Aspergillus Niger and Candida Albicans fungal
strains. The results were compared with that of standard drugs
fluconazole and itraconazole. These results (Table 3) indicate
that the compounds (7c) and (7h) exhibited promising anti-
fungal activity with an MIC of 6.25 lg/ml against both fungi
Aspergillus fumigatus and Candida albicans. While restof the
compounds (7a), (7b), (7d), (7e), (7f), (7g), (7i), and (7j)
exhibited low to moderate activity against both evaluated
fungal strains. In general, the high activity is attributed to the
presence of electron withdrawing para nitro functional group
on phenyl ring as compared to other substitution.
Pharmacology
All the synthesized compounds were screened for their
antibacterial activities by disc diffusion method (Cruick-
shank et al., 1975; Collins, 1976) and antifungal activities
by serial plate dilution method (Khan, 1997; Varma, 1998).
The bacterial strains used were Gram-positive Staphylo-
coccus aureus (MTCC-96) and Gram-negative Escherichia
coli (MTCC-521) and Pseudomonas aeruginosa (MTCC-
647). The antifungal screening was done on Candida
albicans (MTCC-183) and Asperigillus niger (MTCC-343)
fungal strains. The strains used in this study were main-
tained at the Department of Microbiology, S. K. Patel
College of Pharmaceutical Science and Research, Ganapat
University, Gujarat, India. Amoxicillin, gentamycin, and
streptomycin were used as standard drugs for antibacterial
activity while fluconazole and itraconazole were used as
standard drugs for antifungal activity. The test compounds
were prepared with different concentrations using DMSO.
Zones of inhibition and minimum inhibitory concentrations
(MICs) were determined. The MICs were defined as the
lowest concentrations of the compounds that prevented
visible growth.
Conclusion
In conclusion, a series of spiro[azetidine-2,30-indole]-
20,4(10H)-dione (7a–j) were synthesized and screened for
their in vitro antibacterial and antifungal activity. Result of
bioassay indicated that the compounds (7a), (7b), (7c),
(7d), and (7e) were almost equivalent antibacterial activity
against all three bacterial strains compare to standard drugs
and the remaining compounds showed good to moderate
activity. The compounds (7c) and (7h) displayed equipo-
tent antifungal activity compare to standard drugs.
Furthermore, the MIC values of synthesized compounds
(7a–j) (Tables 2, 3 and Fig. 1) showing their comparable
activity against bacterial and fungal strains than respective
standard drugs. On the basis of structure–activity
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Med Chem Res (2011) 20:587–594
Table 1 Physical and analytical data of the spiro[azetidine-2,30-indole]-20,4(10H)-dione derivatives
O
R4
Cl
N
R3
R1
O
N
H
R2
Entry
Producta
R1
R2
R3
R4
Yieldb
(%)
m.p.c
(°C)
Mol. formula/mol. wt.
Elem. analysis (%) (cal./found)
C
H
N
1
2
3
4
5
6
7
8
9
10
a
7a
7b
7c
7d
7e
7f
Br
Br
Br
Br
Br
H
Br
Br
Br
Br
Br
H
CH3
H
H
67.85
71.05
75.00
84.00
79.87
72.35
76.87
77.60
71.64
77.90
125–128
141–143
104–107
110–112
118–121
133–135
134–137
130–135
135–138
125–128
C17H11Br2ClN2O2
43.39
43.43
43.39
43.42
38.32
38.31
38.32
38.28
42.10
42.13
65.29
65.31
65.29
65.27
55.91
55.88
55.91
55.93
64.33
64.35
2.36
2.40
2.36
2.33
1.61
1.59
1.61
1.64
1.99
2.03
4.19
4.23
4.19
4.18
2.93
2.89
2.93
2.94
3.71
3.69
5.95
470
5.93
5.95
5.94
8.38
8.36
8.38
8.35
6.14
6.11
8.96
8.93
8.96
8.98
12.23
12.25
12.23
12.24
9.38
9.42
CH3
H
C17H11Br2ClN2O2
470
NO2
H
C16H8Br2ClN3O4
502
NO2
H
C16H8Br2ClN3O4
502
H
C16H9Br2ClN2O2
456
CH3
H
H
C17H13ClN2O2
312
7g
7h
7i
H
H
CH3
H
C17H13ClN2O2
312
H
H
NO2
H
C16H10ClN3O4
344
H
H
NO2
H
C16H10ClN3O4
344
7j
H
H
H
C
16H11ClN2O2
299
1
Products were characterized by IR, H-NMR, MS, and elemental analysis
b
c
Isolated yields
Melting points are uncorrected
relationship study of the compounds, it can be concluded
that the presence of electron withdrawing group substitute
at 50, 70 position of indoline ring and on ortho or para
position of phenyl ring increase the antimicrobial activity
of the compounds. Henceforth, our findings will have a
good impact on chemists and biochemists for further
investigations in search of spiro-fused antimicrobial agents.
Apparatus, Model VMP-D (Veego India Ltd., Mumbai,
India) and are uncorrected. Infrared spectra were recorded
using KBr pellets on SHIMADZU-FT-IR 8400S instru-
ment. Mass spectra were recorded on Perkin–Elmer
1
LC–MS PE Sciex API/65 Spectrophotometer. The H-NMR
spectra was recorded on Brucker Avance-300 (300 MHz)
model spectrophotometer in CDCl3 and DMSO as solvent
1
and TMSi as internal standard with H resonant frequency
of 300 MHz. The chemical shifts were measured in d ppm
downfield from internal standard (TMSi) at d = 0. The
TLC was performed on alumina silica gel 60 F254 (Merck).
The mobile phase was ethyl acetate and n-hexane (1:1) and
detection was made using UV light and iodine spotting.
The resulting compounds were purified by column chro-
matography. For column chromatography Merck silica gel
Experimental protocol
Chemistry
Melting points of the synthesized compounds were deter-
mined in open capillaries using Veego Melting Point
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591
Table 2 Antibacterial activity
of the synthesized compounds
(7a–j)
Comp. no.
Staphylococcus
aureus (MTCC-96)
Escherichia
coli (MTCC-521)
Pseudomonas
aeruginosa (MTCC-647)
7a
24 (6.25)
23 (6.25)
28 (6.25)
27 (6.25)
24 (6.25)
19 (25)
22 (12.5)
23 (6.25)
24 (6.25)
21 (6.25)
21 (12.5)
20 (25)
26 (6.25)
24 (12.5)
28 (6.25)
27 (6.25)
26 (12.5)
17 (25)
7b
7c
7d
7e
7f
7g
22 (12.5)
19 (25)
19 (25)
17 (25)
7h
20 (25)
17 (25)
MIC values are given in
brackets; MIC (lg/ml) 1/4
minimum inhibitory
concentration, i.e., lowest
concentration to completely
inhibit bacterial growth; zone of
inhibition is expressed in mm
7i
17 (12.5)
21 (12.5)
29 (6.25)
30 (6.25)
31 (6.25)
21 (12.5)
19 (25)
19 (25)
7j
18 (25)
Amoxicillin
Gentamycine
Streptomycin
25 (6.25)
26 (6.25)
26 (6.25)
30 (6.25)
29 (6.25)
31 (6.25)
General procedure for the preparation of compounds
Table 3 Antifungal activity of the synthesized compounds (7a–j)
Comp. no.
Candida albicans
(MTCC-183)
Asperigillus niger
(MTCC-343)
Preparation of isatin (3)
7a
16 (12.5)
19 (6.25)
26 (6.25)
19 (6.25)
17 (12.5)
16 (12.5)
11 (25)
–
It was done by two steps.
7b
17 (12.5)
23 (6.25)
14 (25)
Step 1: Chloral hydrate (9 g, 55 mmol) and 120 ml of
water was taken in 1,000 ml beaker. To this solution were
then added, in order: crystallized sodium sulfate (130 g,
920 mmol); a solution of aniline (4.6 g, 50 mmol) in 30 ml
of water to which concentrated hydrochloric acid (5.12 g,
142 mmol) has been added to dissolve the amine; and,
finally, a solution of hydroxylamine hydrochloride (11 g,
157 mmol) in 50 ml of water. The reaction mixture was
heated to 40–45 min. Once the reaction was completed,
cool the reaction mixture which gives crystals of isonitr-
osoacetanilide (2). The resulting crystals were filtered with
suction and dried which was used as such for further
reaction.
7c
7d
7e
14 (25)
7f
22 (6.25)
24 (6.25)
24 (6.25)
17 (12.5)
22 (12.5)
26 (6.25)
25 (6.25)
7g
7h
25 (6.25)
–
7i
7j
23 (6.25)
27 (6.25)
27 (6.25)
Fluconazole
Itraconazole
‘‘–’’ Indicates fungi are resistant to the compounds at concentration
[100 lg/ml; MIC values are given in brackets; MIC (lg/
ml) = minimum inhibitory concentration, i.e., lowest concentration
to completely inhibit fungal growth; zone of inhibition is expressed in
mm
Step 2: Concentrated sulfuric acid (60 g, 612 mmol) was
warmed to 50–60°C in a round-bottomed flask with con-
tinuous stirring. To this, dry isonitrosoacetanilide (2)
(7.5 g, 46 mmol) was added at such a rate as to keep the
temperature between 60 and 70°C. After the addition of the
isonitrosoacetanilide was finished, the solution was heated
to 80°C for 10 min to complete the reaction. Then the
reaction mixture was cooled to room temperature and
poured upon 10 times its volume of ice. After 0.5 h, the
isatin was filtered, washed three times with cold water to
remove the sulfuric acid, and then dried in the air to yield
bright red crystals of isatin (3).
(0.040–0.063 mm) was used. The elemental analyses were
done on Elementar Vario EL III Carlo Erba 1108 and were
in well accordance with the structures assigned to the
compounds. All the compounds gave C, H, and N analysis
within 0.4% of the theoretical values. Synthetic grade
chemicals procured from SD fine Chemicals, Baroda,
India, were used for the synthesis of the target compounds,
only after purification. All the starting materials were
prepared according to the literature procedures with some
minor modifications. General synthetic procedures used for
the preparation of the target compounds (7a–j) are as
follows:
Preparation of 5,7-dibromo isatin (4)
Isatin (3) (5.0 g, 34 mmol) was warmed in ethanol (95%,
100 ml) with continuous stirring until it dissolved.
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Med Chem Res (2011) 20:587–594
Fig. 1 Comparison of MIC values (in lg/mL) of synthesized compounds (7a–j) and standard drugs against different bacteria and fungi
Bromine (16.3 g, 206 mmol) was added drop wise to the
isatin solution by maintaining the temperature between 70
and 75°C. The solution was cooled to room temperature
and placed on ice for 30 min. The resulting precipitate was
washed with water and cold ethanol and then crystallized
from ethanol to yield bright orange-red crystals of 5,7-
dibromoisatin (4).
column chromatography over silica gel using 30% ethyl
acetate: 70% benzene as an eluent.
50,70-Dibromo-3-chloro-1-(4-methylphenyl)-4H-
spiro[azetidine-2,-30-indole]-20,4(10H)-dione (7a)
Colorless crystals from DMF; yield 67.85%; mp 125–128°C;
IR (KBr, t cm-1): 1726 ([C=O of indoline), 1745 ([C=O
of azetidine), 1785 (–NH–CO), 760 (–C–Cl); 1H-NMR
(300 MHz, d ppm, CDCl3): 2.24 (s, 3H, Ar–CH3), 5.80 (s, H,
HC–Cl), 7.96 (s, H, NH), 6.90–7.40 (m, 6H, Ar–H) ppm;
ESI–MS: 470 (M?), 471 (M ? 1).
General preparation method for Schiff base (6a–j)
Equimolar concentration of isatin (3) (1 g, 6.8 mmol) or
5,7-dibromo isatin (4) (1 g, 3.3 mmol) was allowed to react
with different primary aromatic amines (like p-toluedine,
o-toluedine, p-nitrobenzenamine, o-nitrobenzenamine, and
aniline (1 g), respectively), in the presence of absolute
methanol and the pH was adjusted to 4 with glacial acetic
acid to obtain 5,7-dibromo-3-[(4-methylphenyl)imino]-1,3-
dihydro-2H-indol-2-one derivatives (6a–e) and 3-(phe-
nylimino)-1,3-dihydro-2H-indol-2-one derivatives (6f–j).
The reaction mixture was refluxed for 7–11 h. It was
cooled to room temperature for overnight, the resulting
solid was separated by filtration and vacuum dried.
50,70-Dibromo-3-chloro-1-(2-methylphenyl)-4H-
spiro[azetidine-2,-30-indole]-20,4(10H)-dione (7b)
Colorless crystals from DMF; yield 71.05%; mp 141–143°C;
IR (KBr, t cm-1): 1731 ([C=O of indoline), 1758 ([C=O
of azetidine), 1790 (–NH–CO), 768 (–C–Cl); 1H-NMR
(300 MHz, d ppm, CDCl3): 2.30 (s,3H, Ar–CH3), 5.90 (s, H,
HC–Cl), 7.90 (s, H, NH), 7.10–7.90 (m, 6H, Ar–H) ppm;
ESI–MS: 470 (M?), 471 (M ? 1).
General method for synthesis of final products (7a–j)
50,70-Dibromo-3-chloro-1-(4-nitrophenyl)-4H-
spiro[azetidine-2,-30-indole]-20,4(10H)-dione (7c)
Solution of Schiff base of 5,7-dibromo isatin (6a–e) or
isatin (6f–j) (1 g) in DMF (13–15 ml) reacted with chlo-
racetyl chloride (1 ml, 8.8 mmol) in the presence of TEA
(1 ml, 9.9 mmol). The reaction mixture was refluxed for
7–9 h. The reaction mixture was cooled, then poured into
ice cold water. Precipitates were filtered by vacuum pump
and dried. The product thus obtained was purified by
Light red crystals from DMF; yield 75.00%; mp
104–107°C; IR (KBr, t cm-1): 1720 ([C=O of indoline),
1747 ([C=O of azetidine), 1794 (–NH–CO), 764 (–C–Cl),
1360 (NO2 of phenyl); 1H-NMR (300 MHz, d ppm,
CDCl3): 5.90 (s, H, HC–Cl), 7.94 (s, H, NH), 6.80–7.40 (m,
6H, Ar–H) ppm; ESI–MS: 502 (M?), 503 (M ? 1).
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Med Chem Res (2011) 20:587–594
593
50,70-Dibromo-3-chloro-1-(2-nitrophenyl)-4H-
spiro[azetidine-2,-30-indole]-20,4(10H)-dione (7d)
3-Chloro-1-(2-nitrophenyl)-4H-spiro [azetidine-2,-30-
indole]-20,4(10H)-dione (7i)
Light yellow crystals from DMF; yield 84.00%; mp
110–112°C; IR (KBr, t cm-1): 1723 ([C=O of indoline),
1759 ([C=O of azetidine), 1792 (–NH–CO), 767 (–C–Cl),
1355 (NO2 of phenyl); 1H-NMR (300 MHz, d ppm,
CDCl3): 5.66 (s, H, HC–Cl), 7.94 (s,H, NH), 6.82–7.40 (m,
6H, Ar–H) ppm; ESI–MS: 502 (M?), 503 (M ? 1).
Colorless crystals from DMF; yield 71.64%; mp 135–138°C;
IR (KBr, t cm-1): 1729 ([C=O of indoline), 1780 ([C=O of
azetidine), 1790 (–NH–CO), 767 (C–Cl), 1360 (NO2 of
phenyl); 1H-NMR (300 MHz, d ppm, CDCl3): 5.65–5.66 (m,
H, HC–Cl), 7.96 (s, H, NH), 6.82–7.59 (m, 8H, Ar–H) ppm;
ESI–MS: 344 (M?), 345 (M ? 1).
3-Chloro-1-phenyl-4H-spiro[azetidine-2,-30-indole]-
20,4(10H)-dione (7j)
50,70-Dibromo-3-chloro-1-phenyl-4H-spiro[azetidine-2,-30-
indole]-20,4(10H)-dione (7e)
Colorless crystals from DMF; yield 77.90%; mp
125–128°C; IR (KBr, t cm-1): 1739 ([C=O of indoline),
1788 ([C=O of azetidine), 1800 (–NH–CO), 764 (–C–Cl);
1H-NMR (300 MHz, d ppm, CDCl3): 5.80 (s, H, HC–Cl),
7.97(s, H, NH), 6.95–7.59 (m, 9H, Ar–H) ppm; ESI–MS:
299 (M?), 300 (M ? 1).
Light yellow crystals from DMF; yield 79.87%; mp
118–121°C; IR (KBr, t cm-1): 1728 ([C=O of indoline),
1746 ([C=O of azetidine), 1800 (–NH–CO), 762 (–C–Cl);
1H-NMR (300 MHz, d ppm, CDCl3): 5.90 (s, H, HC–Cl),
7.94 (s, H, NH), 7.10–7.40 (m, 7H, Ar–H) ppm; ESI–MS:
456 (M?), 457 (M ? 1).
Antimicrobial assay
3-Chloro-1-(4-methylphenyl)-4H-spiro[azetidine-2,-30-
indole]-20,4(10H)-dione (7f)
Antibacterial assay
Brown colored crystals from DMF; yield 72.35%; mp
133–135°C; IR (KBr, t cm-1): 1715 ([C=O of indoline),
1780 ([C=O of azetidine), 1804 (–NH–CO), 777 (–C–Cl);
1H-NMR (300 MHz, d ppm, CDCl3): 2.35 (s,3H, Ar–CH3),
5.66 (s, H, HC–Cl), 8.00 (s, H, NH), 6.95–7.59 (m, 8H,
Ar–H) ppm; ESI–MS: 312 (M?), 313 (M ? 1).
Antibacterial assay was done by disc diffusion method
(Cruickshank et al., 1975; Collins, 1976). Discs measuring
6.25 mm in diameter were punched from Whatman no. 1
filter paper. Batches of 100 discs were dispensed to each
screw capped bottles and sterilized by dry heat at 140°C for
an hour. The test compounds were prepared with different
concentrations using DMSO. One milliliter containing 100
times the amount of chemical in each disc was added to each
bottle, which contains 100 discs. The discs of each concen-
tration were placed in triplicate in nutrient agar medium
seeded with fresh bacteria separately. The incubation was
carried out at 37°C for 24 h. Amoxicillin, gentamycine, and
streptomycin were used as standard drugs.
3-Chloro-1-(2-methylphenyl)-4H-spiro[azetidine-2,-30-
indole]-20,4(10H)-dione (7g)
Light yellow crystals from DMF; yield 76.87%; mp
134–137°C; IR (KBr, t cm-1): 1731 ([C=O of indoline),
1758 ([C=O of azetidine), 1790 (–NH–CO), 768 (–C–Cl);
1H-NMR (300 MHz, d ppm, CDCl3): 2.37–2.38 (d, 3H,
Ar–CH3), 5.70 (s, H, HC–Cl), 7.80 (s, H, NH), 6.95–7.59
(m, 8H, Ar–H) ppm; ESI–MS: 312 (M?), 313 (M ? 1).
Antifungal assay
Antifungal assay were done by serial plate dilution method
(Khan, 1997; Varma, 1998). Sabourands agar media were
prepared by dissolving peptone (1 g), D-glucose (4 g), and
agar (2 g) in distilled water (100 ml) and adjusting pH to
5.7. Normal saline was used to make a suspension of spore
of fungal strain for lawning. A loopful of particular fungal
strain was transferred to 3 ml saline to get a suspension of
corresponding species. Agar media (20 ml) were poured
into each Petri dish. Excess of suspension was decanted
and the plates were dried by placing in an incubator at
37°C for 1 h. Using an agar punch, wells were made and
3-Chloro-1-(4-nitrophenyl)-4H-spiro[azetidine-2,-30-
indole]-20,4(10H)-dione (7h)
Light orange crystals from DMF; yield 77.60%; mp
130–135°C; IR (KBr, t cm-1): 1719 ([C=O of indoline),
1761 ([C=O of azetidine), 1810 (–NH–CO), 780 (–C–Cl),
1350 (NO2 of phenyl); 1H-NMR (300 MHz, d ppm,
CDCl3): 5.60 (s, H, HC–Cl), 8.25 (s, H, NH), 6.82–7.59 (m,
8H, Ar–H) ppm; ESI–MS: 344 (M?), 345 (M ? 1).
123

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Acknowledgments The authors are grateful to the Head, Zydus
Research Center, for providing 1H-NMR, mass spectral, elemental
analysis and Ganpat University for providing financial support.
Nishikawa T, Kajii S, Isobe M (2004) Synthesis of model compound
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