Synthesis and in vitro antibacterial activity of schiff bases of N-substituted isatins as effective scaffolds

Article abstract of DOI:10.1007/s00044-012-0087-x

Novel Schiff bases of N-substituted isatin, 1-13, were synthesized starting from isatin and N-(4-amino-2-methylphenyl)-4-chlorophthalimide and their structures were confirmed by spectral and elemental analyses. All new compounds were tested for their in v

Full text of DOI:10.1007/s00044-012-0087-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0087-x
ORIGINAL RESEARCH
Synthesis and in vitro antibacterial activity of schiff bases
of N-substituted isatins as effective scaffolds
•
Garima Kumari Ramendra K. Singh
Received: 26 November 2011 / Accepted: 10 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Novel Schiff bases of N-substituted isatin, 1–13,
were synthesized starting from isatin and N-(4-amino-2-
methylphenyl)-4-chlorophthalimide and their structures
were confirmed by spectral and elemental analyses. All new
compounds were tested for their in vitro antibacterial activity
against a range of Gram ?ve bacterial strains, like Bacillus
subtilis (NCIM-2156), Staphylococcus aureus (NCIM-
2079) and Staphylococcus epidermis (NCIM-2493) and
Gram -ve bacterial strains, like Pseudomonas aeruginosa
(NCIM-2036), Escherichia coli (NCIM-2065) and Proteus
vulgaris (NCIM-2027) following broth dilution method as
recommended by the National Committee for clinical labo-
ratory standards using ciprofloxacin as reference. Determi-
nation of minimum inhibitory concentration (MIC) and zone
of inhibition showed that the molecules were more active
against Gram -ve bacteria than Gram ?ve bacteria. The
compounds showed promising antibacterial properties with
MIC ranging between 10 and 30 lg/mL.
problem because of development of resistance to a
number of antimicrobial agents, like b-lactam antibiotics,
macrolides, quinolines and vancomycin and this pattern is
shown by a variety of clinically significant species of
microorganisms. Hence, there is always a vital need to
discover new chemotherapeutic agents to avert the
emergence of resistance and ideally shorten the duration
of therapy.
Isatin (indole-2,3-dione) is an important pharmaco-
phore and a privileged structure in medicinal chemistry.
Isatin and its various derivatives are reported to show a
variety of biological activities. The isatin moiety is also
present in a range of compounds, which can act as
inhibitors of apoptosis (Chapman et al., 2002; Lee et al.,
2001), anticonvulsants (Verma et al., 2004), antiviral
(Sriram et al., 2004; Pirrung et al., 2005), antibacterial
and antifungal (Chohan et al., 2004) agents. Besides
these, it also has antitumor, antiangiogenic, antitubercular
and anxyolytic effects. Isoindolinediones are also reported
to have a wide range of biological activities (Al-Soud and
AI-Masoudi, 2001; Hashimoto, 2002; Bailleux et al.,
1995; Usifoh et al., 2001; Sou et al., 2000; Takahashi
et al., 2000; Shimazawa et al., 1999; Hashimoto, 1998;
Chapman et al., 1979; Ando et al., 1989; Spallarossa
et al., 2009; Ranise et al., 2005), especially the anti-
bacterial activity (Bansode et al., 2009). In view of these
facts, different Schiff bases of isatin, viz. 5-chloro-2-[2-
methyl-4-(5-nitro-2-oxo-1,2-dihydro-indol-3-ylideneamino)
-phenyl]-isoindole-1,3-dione (1); 5-chloro-2-[2-methyl-4-
(2-oxo-5-trifluoromethoxy-1,2-dihydro-indol-3-ylidenea-
mino)-phenyl]-isoindole-1,3-dione (2); 5-chloro-2-[2-
methyl-4-(5-methyl-2-oxo-1,2-dihydro-indol-3-ylidenea-
mino)-phenyl]-isoindole-1,3-dione (3) and their deriva-
tives 4–13 have been synthesized and subjected to their
antimicrobial screening.
Keywords Schiff base ꢀ Isatin ꢀ Antibacterials ꢀ
MIC ꢀ Zone of inhibition
Introduction
Infectious microbial diseases, despite being treated suc-
cessfully in the most of cases, remain a global health
G. Kumari ꢀ R. K. Singh (&)
Nucleic Acids Research Laboratory, Department of Chemistry,
University of Allahabad, Allahabad 211002, India
e-mail: singhramk@rediffmail.com
123

Med Chem Res
Results and discussion
of NH stretching peaks for NH of isatin thus confirming the
formation of products. In 13C NMR spectra, several singlets
were obtained and have been summarized. Elemental anal-
ysis also confirmed formation of the desired products.
Chemistry
For the synthesis of Schiff bases, 1–3, substituted isatins
(nitro, trifluoromethoxy or methyl) were treated with a stir-
red solution of N-(4-amino-2-methylphenyl)-4-chloroph-
thalimide in ethanol. The completion of the reaction was
assessed by the appearance of a yellow colored precipitate.
The precipitates were filtered, air-dried and recrystallized
with ethanol–chloroform mixture to get compounds 1–3.
Similarly, compound 1 or 2 on treatment with different sul-
fonyl chlorides in dry pyridine gave compounds 4–8. Com-
pound 1 or 2 on treatment with different alkyl/aryl halides in
the presence of anhydrous potassium carbonate (K2CO3)
gave compounds 9–13 (Scheme 1). All compounds have
been characterized by different spectral techniques and ele-
mental analyses. The IR data of compound 1 showed the
presence of NH stretching peak at 3,417 cm-1 for NH of
isatin. The IR data of compound 4 and 9 showed the absence
Microbiology
The compounds were tested against a range of Gram ?ve
and Gram -ve bacteria using broth dilution method for
determining the minimum inhibitory concentration (MIC)
and disc diffusion method for zone of inhibition. Different
concentrations (80, 40, 20, 10, 5, 2.5, 1.25 and 0.65 lg/
mL) of all compounds were prepared using dimethyl-
sulphoxide (DMSO) as solvent. Muller Hinton broth
(M391) was used to prepare nutrient broth. The tubes were
incubated at 37 °C for 48 h and checked for appearance of
any turbidity, which showed the effect of the test molecules
against the pathogens studied. Similarly, Whatman No. 1
sterilized discs were used for studying zone of inhibition
using nutrient agar medium seeded with fresh bacteria
Scheme 1 Synthetic route for
compounds 1–13. Reagents and
conditions: (i) ethanol, pyridine,
rt, 6 h; (ii) pyridine, 0 °C; (iii)
alkyl halide, K2CO3, reflux
O
O
R
+
H2N
N
O
N
Cl
H
CH
3O
ethanol, stirring
O
R
N
N
NO2
OCF3
CH3
1
2
3
R
Cl
CH
O
3 O
a
N
H
1-3
O
S
pyridine, 0 oC
R''X
K2CO3, reflux
Cl
R'
O
O
O
N
N
N
N
R
R
Cl
Cl
CH
3O
O
CH
O
N
3O
N
R''
O
S
O
9-13
R'
4-8
R
R''
C6H4-CH2-
9
NO2
R
R'
C
CH2-
H2
HC
10 NO2
11 NO2
12 OCF3
4
5
6
7
NO2
NO2
OCF3
OCF3
OCF3
p-C6H4 NO2
CH3
CF3
C
CH2-
H3C
C
CH2-
HC
CH3
8
p-C6H4NO2
13 OCF3
C6H4-CH2-
123

Med Chem Res
Table 1 Antibacterial activity and log P values of compounds 1–13
Compound
Log P
Antibacterial activity
MIC in lg/mL (zone of inhibition in mm)
Gram ?ve bacterial strains
Gram -ve bacterial strains
S. aureus
B. subtilis
P. aeruginosa
E. coli
S. epidermis
P. vulgaris
1
2.75
5.65
4.61
2.36
0.47
7.09
4.91
3.22
3.87
2.70
3.31
6.10
7.62
1.32
10 (20)
10 (18)
10 (20)
10 (18)
15 (16)
15 (18)
15 (16)
10 (20)
30 (12)
25 (13)
30 (10)
30 (10)
25 (14)
10 (26)
15 (16)
15 (16)
25 (12)
15 (16)
15 (16)
10 (20)
15 (18)
15 (14)
25 (10)
30 (8)
10 (18)
10 (20)
15 (16)
10 (18)
10 (18)
15 (12)
15 (14)
10 (18)
15 (14)
15 (14)
20 (10)
15 (12)
20 (10)
10 (24)
10 (22)
10 (24)
20 (12)
10 (22)
10 (24)
10 (23)
10 (22)
10 (20)
15 (16)
15 (18)
20 (12)
20 (10)
15 (17)
10 (30)
10 (20)
10 (18)
15 (10)
10 (20)
10 (18)
15 (10)
10 (18)
10 (18)
15 (10)
20 (8)
15 (11)
15 (14)
20 (10)
15 (14)
15 (14)
10 (20)
15 (16)
10 (22)
15 (14)
20 (10)
20 (12)
15 (14)
20 (12)
10 (28)
2
3
4
5
6
7
8
9
10
11
30 (10)
25 (11)
25 (12)
10 (22)
15 (12)
15 (10)
20 (8)
12
13
Ciprofloxacin (14)
10 (28)
MIC minimum inhibitory concentration
E.coli
S.aureus
B.subtilis
Compounds 9–13 bearing alkyl or aryl substituents at
nitrogen on isatin moiety, were not as potent as derivatives
1–8 bearing H or sulfonyl group at the same position on
isatin. Thus, the compounds bearing a sulfonyl group
(hydrogen acceptor) attached to nitrogen or free NH group
(hydrogen donor) on isatin, were more potent than other
molecules studied. The plausible cause for variation in
antibacterial profile of substituted derivatives may be that
these atoms (electron withdrawing groups) are very useful
to modulate the steric effect on phenyl ring of drug and
which, in turn, alters the ease of penetration of molecule in
bacterial cell wall. These groups are further expected to
alter the hydrophobic–hydrophilic balance of the target
molecule and thus, affect their cell permeation, which
remains the topic of interest of SAR studies.
35
30
25
20
15
10
5
P.vulgaris
P.aeruginosa S.epidermis
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Compounds
Fig. 1 Graphical presentation of antibacterial activity of compounds
1–13
separately. The incubation was carried out at 37 °C for
24 h. Ciprofloxacin was used as a standard drug for
determining the MIC and zone of inhibition. Zone of
inhibition and MICs have been shown in Table 1.
It is also believed that the lipophilic nature of com-
pounds plays a key role for generation of antimicrobial
effect by increasing the membrane permeation and hence, a
strong correlation exists between lipophilicity and activity.
In this context, lipophilicity of compound expressed as
logP (octanol/water partition coefficient) value, the main
predictor of activity, was calculated using the methodology
developed by Molinspiration as a sum of fragment-based
contributions and correction factors as shown in Table 1. It
is evident that compounds having low logP values have
shown better biological activity as is evident from their
zone of inhibition than those having higher logP values.
We have noticed that a preferred logP for optimum activity
The antibacterial studies showed that all compounds had
moderate to good antibacterial activity against Gram ?ve
as well as Gram -ve bacteria. Almost all compounds were
active against different bacterial strains at 10 lg/mL con-
centration. However, better activity was found in the case
of Gram -ve bacteria than Gram ?ve bacteria as this trend
was substantially supported by their respective MIC values.
This may be because of structural differences in the cell
wall of Gram ?ve and Gram -ve bacteria. Out of 13
compounds studied, seven compounds showed MIC in the
range of 10–15 lg/mL (Fig. 1).
123

Med Chem Res
always advocated for an optimum amphiphilicity of pos-
sible drug molecules.
followed by water (H2O), dried, and recrystallized with
ethanol–chloroform mixture to get the products 1, 2 or 3.
5-Chloro-2-[2-methyl-4-(5-nitro-2-oxo-1,2-dihydro-
indol-3-ylideneamino)-phenyl]-isoindole-1,3-dione (1)
Conclusion
In conclusion, we have developed a simple and efficient
method for synthesis of Schiff bases of N-substituted isa-
tins and screened them for antibacterial activity. All com-
pounds showed moderate to good antibacterial activity.
The molecules were found to be more active against Gram
-ve bacteria than Gram ?ve bacteria on the basis of their
MIC values and zone of inhibition. The presence of elec-
tron withdrawing groups and hydrogen donor–acceptor
sites on the test molecules produced better results. Most of
these compounds showed bacterial inhibition comparable
to the standard drug—ciprofloxacin.
Dark yellow solid Yield 79 % Rf: 0.46 (DCM:MeOH
9.8:0.2) mp: 234 °C. UV (MeOH) kmax 224 nm. IR (KBr,
cm-1): 3417 (NH of Isatin), 1779, 1729 (C=O imide), 1703
(C=O of isatin), 1603 (C=N), 1470 (C=C), 1310 (N=O),
1
806, 740 (Ar–H), 630 (C–Cl). H NMR dH (DMSO-d6):
2.34 (s, 3H, CH3), 7.1–7.5 (m, 3H, ArH), 7.70–8.14 (m,
3H, ArH), 8.04 (s, 1H, =NH), 7.90–8.53 (m, 3H, ArH). 13
C
NMR d (ppm): 177.2 (–C=N), 176.6 (C=O), 176.2 (C=O),
175.4 (NH–C=O), 151.7 (C–N), 148.9 (C–NH), 146.3 (C–
NO2), 139.1 (ArC–N imide), 138.4 (ArC–Cl), 134.8, 133.6,
131.9, 131.6, 129.8, 128.9, 128.4, 126.3, 125.4, 124.8,
123.4, 123, 121.2 (Aryl carbons), 12.9 (–CH3). MS (EI?)
m/z 460.06 (100.0 %), 461.03 (26.0 %), 462.04 (32.0 %).
Anal. Calcd for C23H13ClN4O5: C 59.95, H 2.84, N 12.16.
Found: C 59.98, H 2.81, N 12.12.
Materials and methods
Chemistry
5-Chloro-2-[2-methyl-4-(2-oxo-5-trifluoromethoxy-1,2-
dihydro-indol-3-ylideneamino)-phenyl]-isoindole-1,3-
dione (2)
Silica gel G for TLC was obtained from E. Merck India Ltd.
Substituted isatins, sulfonyl chlorides and alkyl halides were
purchased from Aldrich Chemical Co., USA. N-(4-amino-
2-methylphenyl)-4-chlorophthalimide was prepared from
4-chlorophthalimide and 4-chloro-3-methylaniline. All reac-
tions were performed in oven-dried flasks and solvents used
were anhydrous. Melting points were determined on an
electrothermal apparatus and are uncorrected. Mass spectra
were recorded on Micromass Quattro II. UV spectra were
recorded on a Perkin-Elmer k25 spectrophotometer. 1H NMR
spectra were recorded on a Bruker AVANCE 400 MHz
Fourier transform spectrometer and using an internal deute-
rium lock. Chemical shifts are quoted in parts per million
downfield from tetramethylsilane. 13C-NMR spectra were
recorded at 100 MHz on Bruker AMX 400 MHz spectrom-
eterin CDCl3. IRspectra wererecorded in AVATAR-330FT-
IR spectrophotometer and only noteworthy absorption levels
are listed. Mass spectra were recorded at PESCIEX API 3000
series mass spectrometer. Elemental analysis was carried out
using Heraeus Vario EL III Carlo Erba 1108.
Yellow solid Yield 82 % Rf: 0.49 (DCM:MeOH 9.8:0.2)
mp: 245 °C. UV (MeOH) kmax 225 nm. 1H NMR dH
(DMSO-d6): 2.34 (s, 3H, CH3), 7.1–7.5 (m, 3H, ArH),
6.78–7.56 (m, 3H, ArH), 7.70–8.14 (m, 3H, ArH), 8.04 (s,
1H, NH). MS (EI?) m/z 499.05 (100 %), 500.08 (26.0 %),
501.05 (32.0 %). Anal. Calcd for C24H13ClF3N3O4: C
57.67, H 2.62, N 8.41. Found: C 57.62, H 2.64, N 8.38.
5-Chloro-2-[2-methyl-4-(5-methyl-2-oxo-1,2-dihydro-
indol-3-ylideneamino)-phenyl]-isoindole-1,3-dione (3)
Orange solid Yield 81 % Rf: 0.47 (DCM:MeOH 9.8:0.2)
mp: 143 °C. UV (MeOH) kmax 225 nm. 1H NMR dH
(DMSO-d6): 2.28 (s, 3H, CH3), 2.34 (s, 3H, CH3), 7.0–7.5
(m, 3H, ArH), 7.07–7.55 (m, 3H, ArH), 7.70–8.14 (m, 3H,
ArH), 8.02 (s, 1H, NH). MS (EI?) m/z 429.09 (100.0 %),
430.05 (28.0 %), 431.05 (36.2 %). Anal. Calcd for
C24H16ClN3O3: C 67.06, H 3.75, N 9.78. Found: C 67.10,
H 3.71, N 9.72.
General procedure for synthesis of compounds 1–3
General procedure for synthesis of compounds 4–8
To a stirred solution of N-(4-amino-2-methylphenyl)-4-
chlorophthalimide (0.5 mmol) in ethanol (10 mL) was
added isatin (0.5 mmol). The reaction mixture was stirred
overnight. Completion of the reaction was assessed by the
appearance of a yellow coloured precipitate. The precipi-
tate was filtered off, washed first with aqueous ethanol
To a solution of 1 or 2 (0.1 mmol) in dry pyridine (10 mL)
at 0 °C sulfonyl chloride (0.1 mmol) was added drop wise.
The reaction mixture was stirred overnight and monitored
on TLC. Completion of the reaction was assessed by
consumption of the starting material. The reaction mixture
123

Med Chem Res
5-Chloro-2-[4-(1-methanesulfonyl-2-oxo-5-
trifluoromethoxy-1,2-dihydro-indol-3-ylideneamino)-2-
methyl-phenyl]-isoindole-1,3-dione (7)
was partitioned between dichloromethane (DCM) and H2O.
The organic layer was collected, dried over anhydrous
sodium sulphate (Na2SO4), filtered, concentrated in vacuo
and the product was crystallized with hexane.
Light brown solid Yield 73 % Rf: 0.6 (DCM:MeOH
1
9.6:0.4) mp: 130 °C. UV (MeOH) kmax 399 nm. H NMR
5-Chloro-2-{2-methyl-4-[5-nitro-1-(4-nitro-
benzenesulfonyl)-2-oxo-1,2-dihydro-indol-3-
ylideneamino]-phenyl}-isoindole-1,3-dione (4)
dH (DMSO-d6): 2.34 (s, 3H, CH3), 2.82 (s, 3H, CH3),
7.1–7.5 (m, 3H, ArH), 6.78–7.56 (m, 3H, ArH), 7.70–8.14
(m, 3H, ArH). MS (EI?) m/z 577.03 (100.0 %), 578.07
(28.0 %),
579.06
(37.0 %).
Anal.
Calcd
for
Light brown solid Yield 72 % Rf: 0.48 (DCM:MeOH
9.8:0.2) mp: 84 °C. UV (MeOH) kmax 398 nm. IR (KBr,
cm-1): 1781, 1727 (C=O imide), 1705 (C=O of isatin),
1604 (C=N), 1468 (C=C), 1314 (N=O), 1180 (SO2N), 809,
744 (Ar–H), 634 (C–Cl). 1H NMR dH (DMSO-d6): 2.34 (s,
3H, CH3), 7.1–7.5 (m, 3H, ArH), 7.70–8.14 (m, 3H, ArH),
8.19–8.47 (m, 4H, ArH), 7.90–8.53 (m, 3H, ArH). MS
(EI?) m/z 645.04 (100.0 %), 646.02 (34.0 %), 647.06
(36.0 %). 13C NMR d (ppm): 176.2 (–C=N), 175.6 (C=O),
175.2 (C=O), 174.4 (NH–C=O), 152.6 (C–NO2), 150.7 (C–
N), 148.5 (C–NH), 146.3 (C–SO2N), 145.3 (C–NO2),
138.8 (ArC–N imide), 137.4 (ArC–Cl), 134.8, 133.4,
131.8, 131.6, 129.4, 129.1, 128.9, 128.4, 127.9, 125.9,
125.4, 124.8, 124.1, 123.9, 123.2, 123, 122.2 (Aryl car-
bons), 12.6 (–CH3). Anal. Calcd for C29H16ClN5O9S: C
53.92, H 2.50, N 10.84, S 4.96. Found: C 53.92, H 2.50, N
10.84, S 4.96.
C25H15ClF3N3O6S: C 51.96, H 2.62, N 7.27, S 5.55.
Found: C 51.96, H 2.68, N 7.22, S 5.57.
5-Chloro-2-{2-methyl-4-[1-(4-nitro-benzenesulfonyl)-
2-oxo-5-trifluoromethoxy-1,2-dihydro-indol-3-
ylideneamino]-phenyl}-isoindole-1,3-dione (8)
Brown solid Yield 72 % Rf: 0.43 (DCM) mp: 1958C. UV
(MeOH) kmax 402 nm. H NMR dH (DMSO-d6): 2.34 (s,
1
3H, CH3), 7.1–7.5 (m, 3H, ArH), 6.78–7.56 (m, 3H, ArH),
7.70–8.14 (m, 3H, ArH), 8.19–8.47 (m, 4H, ArH). MS
(EI?) m/z 684.03 (100.0 %), 685.03 (34.0 %), 686.07
(37.0 %). Anal. Calcd for C30H16ClF3N4O8S: C 52.60, H
2.35, N 8.18, S 4.68. Found: C 52.65, H 2.33, N 8.13, S
4.69.
General procedure for synthesis of compounds 9–13
5-Chloro-2-[4-(1-methanesulfonyl-5-nitro-2-oxo-1,2-
dihydro-indol-3-ylideneamino)-2-methyl-phenyl]-
isoindole-1,3-dione (5)
1 or 2 (0.1 mmol) and anhydrous K2CO3 (0.55 mmol) were
taken together and crushed with a glass rod. The respective
alkyl/aryl halide, R00X, was added in excess and the reac-
tion mixture was refluxed for 4–5 h and monitored on TLC.
Completion of the reaction was assessed by consumption of
the starting materials. The reaction mixture was partitioned
between DCM and H2O. The organic layer was collected,
dried over anhydrous Na2SO4, filtered, concentrated in
vacuo and the product was crystallized with hexane.
Dark brown solid Yield 75 % Rf: 0.58 (DCM:MeOH
9.6:0.4) mp: 94 °C. UV (MeOH) kmax 392 nm. 1H NMR dH
(DMSO-d6): 2.34 (s, 3H, CH3), 2.92 (s, 3H, CH3), 7.1–7.5
(m, 3H, ArH), 7.70–8.14 (m, 3H, ArH), 7.90–8.53 (m, 3H,
ArH). MS (EI?) m/z 538.03 (100.0 %), 539.06 (27.0 %),
540.01 (36.0 %). Anal. Calcd for C24H15ClN4O7S: C
53.49, H 2.81, N 10.40, S 5.95. Found: C 53.41, H 2.814, N
10.43, S 5.92.
2-[4-(1-Benzyl-5-nitro-2-oxo-1,2-dihydro-indol-3-
ylideneamino)-2-methyl-phenyl]-5-chloro-isoindole-
1,3-dione (9)
5-Chloro-2-[2-methyl-4-(2-oxo-1-
trifluoromethanesulfonyl-5-trifluoromethoxy-1,2-dihydro-
indol-3-ylideneamino)-phenyl]-isoindole-1,3-dione (6)
Dark brown solid Yield 77 % Rf: 0.8 (DCM:MeOH
9.9:0.1) mp: 65 °C. UV (MeOH) kmax 407 nm. IR (KBr,
cm-1): 2926 (N–CH2), 1789, 1724 (C=O imide), 1709
(C=O of isatin), 1600 (C=N), 1472 (C=C), 1317 (N=O),
805,740 (Ar–H), 637(C–Cl). 1H NMR dH (DMSO-d6): 2.34
(s, 3H, CH3), 4.22 (s, 2H, CH2), 7.06–7.14 (m, 5H, ArH),
7.1–7.5 (m, 3H, ArH), 7.70–8.14 (m, 3H, ArH), 7.90–8.53
(m, 3H, ArH). 13C NMR d (ppm): 177.2 (–C=N), 176.6
(C=O), 176.2 (C=O), 175.4 (NH–C=O), 151.7 (C–N),
148.9 (C–NH), 146.3 (C–NO2), 139.1 (ArC–N imide),
138.4 (ArC–Cl), 137.9, 134.8, 133.6, 131.9, 131.6, 129.8,
Brown solid Yield 71 % Rf: 0.7 (DCM:MeOH 9.6:0.4)
mp: 160 °C. UV (MeOH) kmax 407 nm. 1H NMR dH
(DMSO-d6): 2.34 (s, 3H, CH3), 7.1–7.5 (m, 3H, ArH),
6.78–7.56 (m, 3H, ArH), 7.70–8.14 (m, 3H, ArH). MS
(EI?) m/z 631.00 (100.0 %), 632.01 (28.0 %), 633.05
(37.0 %). Anal. Calcd for C25H12ClF6N3O6S: C 47.52, H
1.91, N 6.65, S 5.07. Found: C 47.56, H 1.89, N 6.63, S
5.02.
123

Med Chem Res
128.9, 128.4, 127.9, 127.8, 127.7, 127.1, 126.7, 126.3,
125.4, 124.8, 123.4, 123, 121.2 (Aryl carbons), 54.1 (N–
CH2–Ph), 12.9 (–CH3). MS (EI?) m/z 550.10 (100.0 %),
551.7 (34.0 %), 552.18 (33.0 %). Anal. Calcd for
C30H19ClN4O5: C 65.40, H 3.48, N 10.17. Found: C 65.46,
H 3.43, N 10.14.
(m, 3H, ArH), 7.70–8.14 (m, 3H, ArH). MS (EI?) m/
z 589.10 (100.0 %), 59.3 (36.0 %), 591.5 (33.0 %). Anal.
Calcd for C31H19ClF3N3O4: C 63.11, H 3.25, N 7.12.
Found: C 63.15, H 3.23, N 7.15.
Antibacterial studies
5-Chloro-2-[2-methyl-4-(5-nitro-2-oxo-1-prop-2-ynyl-1,
2-dihydro-indol-3-ylideneamino)-phenyl]-isoindole-1,3-
dione (10)
All compounds were screened for their antibacterial
activity against a range of Gram ?ve and Gram –ve bac-
teria, viz. Escherichia coli, Pseudomonas aeruginosa,
Proteus vulgaris, Staphylococcus aureus, Bacillus subtilis
and Staphylococcus epidermis. The MIC was determined
by broth dilution method and zone of inhibition by disc
diffusion method (Cruickshank et al., 1975; Collins, 1976;
Singh et al., 2010; Srivastava et al., 2007).
Reddish brown solid Yield 75 % Rf: 0.7 (DCM) mp:
1
162 °C. UV (MeOH) kmax 392 nm. H NMR dH (DMSO-
d6): 2.25 (s, 1H, CH), 2.34 (s, 3H, CH3), 3.72 (s, 2H, CH2),
7.1–7.5 (m, 3H, ArH), 7.70–8.14 (m, 3H, ArH), 7.90–
8.53 (m, 3H, ArH). MS (EI?) m/z 498.07 (100.0 %),
499.05 (30.0 %), 500.05 (33.0 %). Anal. Calcd for
C26H15ClN4O5:C 62.60, H 3.03, N 11.23. Found: C 62.64,
H 3.06, N 11.18.
Determination of MIC by the broth dilution method
Different concentrations (80, 40, 20, 10, 5, 2.5, 1.25 and
0.65 lg/mL) of all the compounds were prepared using
dimethylsulphoxide (DMSO) as solvent in sterile dry test
tubes to determine MIC by the broth dilution method.
Nutrient broth was prepared using Muller Hinton broth
(M391) and 4.9 mL of it was taken in each test tube and
was sterilized after plugging. After cooling, 0.1 mL of each
dilution was added to the test tube and the final volume was
made up to 5.0 mL. The test tubes were shaken to uni-
formly mix the inocula with the broth. The tubes were
incubated at 37 °C for 48 h. Appearance of any turbidity
showed that the compound was not able to inhibit the
growth of bacteria, while no turbidity indicated the inhi-
bition of microorganism by the sample.
5-Chloro-2-[2-methyl-4-(5-nitro-2-oxo-1-propyl-1,2-
dihydro-indol-3-ylideneamino)-phenyl]-isoindole-1,3-
dione (11)
Brown solid Yield 72 % Rf: 0.8 (DCM:MeOH 9.8:0.2).
mp: 104 °C. UV (MeOH) kmax 405 nm. 1H NMR dH
(DMSO-d6): 0.96 (t, 3H, CH3), 1.59 (m, 2H, CH2), 2.34 (s,
3H, CH3), 2.96 (t, 2H, CH2), 7.1–7.5 (m, 3H, ArH),
7.70–8.14 (m, 3H, ArH), 7.90–8.53 (m, 3H, ArH). MS
(EI?) m/z 502.10 (100.0 %), 503.2 (30.0 %), 504.5
(33.0 %). Anal. Calcd for C26H19ClN4O5: C 62.09, H 3.81,
N 11.14. Found: C 62.02, H 3.78, N 11.18.
5-Chloro-2-[2-methyl-4-(2-oxo-1-prop-2-ynyl-5-
trifluoromethoxy-1,2-dihydro-indol-3-ylideneamino)-
phenyl]-isoindole-1,3-dione (12)
Determination of zone of inhibition by the disc diffusion
method
Orange solid Yield 76 % Rf: 0.42 (DCM) mp: 136 °C. UV
(MeOH) kmax 413 nm. H NMR dH (DMSO-d6): 2.25 (s,
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 contained 100
discs. The discs of each concentration were placed in
triplicate in nutrient agar medium seeded with fresh bac-
teria separately. The incubation was carried out at 37 °C
for 24 h. Ciprofloxacin was used as a standard drug.
1
1H, CH), 2.34 (s, 3H, CH3), 3.72 (s, 2H, CH2), 7.1–7.5 (m,
3H, ArH), 6.78–7.56 (m, 3H, ArH), 7.70–8.14 (m, 3H,
ArH). MS (EI?) m/z 537.07 (100.0 %), 538.04 (32.0 %),
539.05 (33.0 %). Anal. Calcd for C27H15ClF3N3O4: C
60.29, H 2.81, N 7.81. Found: C 60.22, H 2.80, N 7.78.
2-[4-(1-Benzyl-2-oxo-5-trifluoromethoxy-1,2-dihydro-
indol-3-ylideneamino)-2-methyl-phenyl]-5-chloro-
isoindole-1,3-dione (13)
Light brown solid Yield 75 % Rf: 0.8 (DCM:MeOH
1
9.6:0.4) mp: 101 °C. UV (MeOH) kmax 415 nm. H NMR
Determination of logP
dH (DMSO-d6): 2.34 (s, 3H, CH3), 4.22 (s, 2H, CH2),
7.14–7.16 (m, 5H, ArH), 7.1–7.5 (m, 3H, ArH), 6.78–7.56
logP was calculated using Molinspiration and ChemDraw
softwares.
123

Med Chem Res
Acknowledgments Financial assistance from DBT-ICMR, Gov-
ernment of India, is sincerely acknowledged. GK thanks CSIR-UGC
for award of JRF and SRF. The authors are grateful to Prof. K.V.R.
Chary, TIFR, Mumbai, for 1H-NMR spectra of newly synthesized
compounds.
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