I2 catalyzed Friedel-Crafts alkylation reaction of substituted anilines with ninhydrin: Formation of novel products and their antimicrobial evaluation

Article abstract of DOI:10.1007/s00044-012-0202-z

Friedel-Crafts reaction of differently substituted anilines with ninhydrin in the presence of molecular iodine at ambient temperature constitutes a facile, cost effective, and regioselective synthesis of a series of 2-mono/2,2-bis-(amino-phenyl)-indane-1,3-dione derivatives. Under identical conditions, use of different substituents in aniline ring led to the formation of different products, emphasizing the pivotal role of the nature and position of the substituents in product formation. In vitro antimicrobial activity evaluation of the synthesized molecules against eight bacterial and four fungal strains revealed that four of them possess duel efficacies (bactericidal as well as fungicidal). The activities are attributed to the disruption of architecture of the microbes as revealed from ultrastructural studies (SEM). Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0202-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2023–2037
DOI 10.1007/s00044-012-0202-z
ORIGINAL RESEARCH
I2 catalyzed Friedel–Crafts alkylation reaction of substituted
anilines with ninhydrin: formation of novel products
and their antimicrobial evaluation
•
Krishnendu B. Sahu Maitreyee Banerjee Soma Ghosh
•
•
•
Arindam Maity Shymal Mondal Rupankar Paira Abhijit Hazra
•
•
•
•
Sanmoy Karmakar Amalesh Samanta Nirup B. Mondal
•
Received: 24 May 2012 / Accepted: 9 August 2012 / Published online: 26 August 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Friedel–Crafts reaction of differently substi-
tuted anilines with ninhydrin in the presence of molecular
iodine at ambient temperature constitutes a facile, cost
effective, and regioselective synthesis of a series of
2-mono/2,2-bis-(amino-phenyl)-indane-1,3-dione deriva-
tives. Under identical conditions, use of different substit-
uents in aniline ring led to the formation of different
products, emphasizing the pivotal role of the nature and
position of the substituents in product formation. In vitro
antimicrobial activity evaluation of the synthesized mole-
cules against eight bacterial and four fungal strains
revealed that four of them possess duel efficacies (bacte-
ricidal as well as fungicidal). The activities are attributed to
the disruption of architecture of the microbes as revealed
from ultrastructural studies (SEM).
due to their interesting biological activities, namely, anti-
bacterial, antiprotozoal, anti-inflammatory, anticonvulsant,
anticancer, laxative, and diuretic properties (Garrido et al.,
1975; Poupelin et al., 1980; Shoichet et al., 1993).
In most of the cases, the derivatives were prepared by
Friedel–Crafts type reactions employed on different arenes,
catalyzed by an inorganic acid or a Lewis acid, where
excess or stoichiometric amounts of catalyst and prolonged
heating were required for the completion of the reactions
(Klumpp et al., 1998, 1999; Song et al., 1998). The haz-
ardous reaction conditions and the use of excess amount of
Lewis or inorganic acids meant that these reactions were
neither eco-friendly nor atom economic. The demanding
environmental legislation and public/corporate pressure
toward cleaner technology (Anastas and Warner, 1998;
Dallinger and Kappe, 2007; Polshettiwar and Varma, 2007)
has driven organic chemists to search for alternatives to
acid-dependent organic reactions. Great efforts have been
made to discover and optimize new reactions/methodolo-
gies which would be operationally simple, cost effective,
atom economic as well as environmentally benign. As a
part of this effort, very recently we have developed a
protocol for the synthesis of novel bis-indolylindane-1,3-
diones from ninhydrin and 3-substituted/unsubstituted in-
doles (Naskar et al., 2010). Ninhydrin and electron-rich
anilines/aromatic amines are versatile reagents, the former
possessing several potential electrophilic sites and the latter
Keywords Friedel–Crafts reaction ꢀ Ninhydrin ꢀ
Molecular iodine ꢀ 2,2-bis-(amino-phenyl)-indane-1,
3-dione ꢀ Regioselectivity
Introduction
Diaryl derivatives of cyclic ketones like ninhydrin, alloxan,
isatin, and parabanic acid have been studied extensively
K. B. Sahu ꢀ M. Banerjee ꢀ A. Maity ꢀ S. Mondal ꢀ R. Paira ꢀ
A. Hazra ꢀ N. B. Mondal (&)
´
multiple nucleophilic sites (Taylor and Joulie, 1998). The
acid-catalyzed reactions of ninhydrin with electron-rich
aromatic amines have yielded various products (Fig. 1), the
regiochemistry of which remains to be well explained
(Bullington and Dodd, 1993; Black et al., 1994; Friedman,
1967). Chatterjee and Shapiro reported (Shapiro and
Chatterjie, 1970) that anilines with an electron-releasing
group at the meta-position react with ninhydrin, initially
Department Of Chemistry, Indian Institute of Chemical Biology,
Council of Scientific & Industrial Research, 4 Raja S. C. Mullick
Road, Jadavpur, Kolkata 700032, India
e-mail: nirup@iicb.res.in
S. Ghosh ꢀ S. Karmakar ꢀ A. Samanta
Division of Microbiology, Department of Pharmaceutical
Technology, Jadavpur University, Kolkata 700032, India
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Med Chem Res (2013) 22:2023–2037
Fig. 1 Products of ninhydrin
with aromatic amines
O
H
N
HO
OH
HO
NH
OH
OH
O
O
NH2
O
1a
1b
1c
through nitrogen, to give tetrahydroindeno[2,1-b]indolones
(la). Bullington and Dodd first demonstrated that the
reaction of ninhydrin with substituted anilines could afford
tetrahydroindeno[1,2-b]indolones (1b), and suggested that
the initial attack was not by the amino function but by the
activated carbon atom at the ortho-position (Bullington and
Dodd, 1993). Black et al. reported that treatment of nin-
hydrin with a number of substituted electron-rich anilines
under anhydrous condition provided o-substituted indol-
ones (1b) as the major products. However, the p-substituted
derivatives (1c) were the predominant products in aqueous
solvent (Black et al., 1994). These interesting results
intrigued us to pursue a thorough study of the reaction of
ninhydrin with substituted anilines.
followed by chromatographic separation afforded a brown-
colored crystalline product in very good yield. Evaluation
of H and 13C NMR spectral data in DMSO-d6 and mass
spectral analysis clearly revealed that two aniline units
1
1
have coupled at the C2 of ninhydrin to form 3a. In H
NMR, the presence of a distinct two proton singlet at d 5.14
clearly indicated that the coupling did not involve the NH2
group of aniline. No shifting of equilibrium toward nin-
hydrin and aniline on D2O addition also indicative that the
reaction had indeed taken place on the aromatic ring
(Bullington and Dodd, 1993). For unambiguous determi-
nation of the structure, single crystal X-ray analysis was
performed, which confirmed the structure of the product as
2,2-bis-(4-amino-phenyl)-indan-1,3-dione, 3a (Fig. 2). The
formation of such a diarylated derivative of ninhydrin with
aniline is unprecedented under mild reaction conditions as
employed by us, although in super acid medium ninhydrin
produced similar products with substituted benzenes
(Klumpp et al., 1998 and Song et al., 1998).
We intended to carry out reactions in the presence of
molecular iodine, because the reagent has received con-
siderable attention as an inexpensive, nontoxic, and readily
available catalyst for various organic transformations with
excellent yields and high degree of selectivity (Yadav
et al., 2007; Kidwai et al., 2007; Togo and Iida, 2006). We
also investigated the effect of the substitution in the for-
mation of the products and the suitability of I2 over other
Lewis acid catalysts for this reaction. In this communica-
tion, we wish to report that reacting ninhydrin with anilines
in the presence of molecular iodine at ambient temperature
constitutes an easy and straight forward synthetic route to
2-monosubstituted/2,2-disubstituted-1,3-indanediones.
Finally, the microbicidal efficacy of the synthesized ana-
logs has also been studied.
The reactions were also performed using different Lewis
acids like AlCl3, ZnCl2, CAN, FeCl3, SnCl4, and mont-
morollonite K10 in CH3CN (Table 1). Molecular iodine
Results and discussion
Chemistry
In our initial experiment, ninhydrin (1) and aniline (2a)
were chosen as the model reaction partners to study the
reactions under various catalytic conditions. In each case,
two equivalents of aniline were added to the mixture of one
equivalent of ninhydrin and 5 mol% of the catalyst. Iodine,
various other Lewis acid catalysts, and different solvents
were screened for the standardization of the reaction. With
molecular iodine as catalyst in acetonitrile, the reaction
was found to be complete (TLC) within 6 h. Usual workup
Fig. 2 ORTEP representations of the title compound 3a the dis-
placement ellipsoids are drawn at a probability of 50 %
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Med Chem Res (2013) 22:2023–2037
2025
appears to produce the best result. However, no significant
increase in yield was observed with the increase of catalyst
loading (from 5 mol%) or of the reaction time, though
reaction carried out in the absence of iodine did not yield
any product even after 24 h.
Table 1 Reaction of ninhydrin (1) and aniline (2a) using different
Lewis acid catalysts
Entry Catalyst
Time (h)a Isolated yieldb (%)
1
2
3
4
5
6
7
8
I2
6
6
90
35
45
45
20
40
55
90
FeCl3
Following the same reaction protocol, the study was
carried out in other solvents, viz. CH2Cl2, CH3NO2,
i
CH3OH, DMF, THF, and PrOH. The results (Table 2)
SnCl4
8
ZnCl2
7
InCl3
9
reveal that the reactions were more effective in polar sol-
vents compared to non polar ones with respect to yield of
the product as well as the reaction time. Acetonitrile
emerged to be the most effective solvent and therefore all
the remaining studies were performed in this medium.
We then performed reactions using differently substi-
tuted anilines both for the generalization of this method-
ology and for studying the effect of substitutions (electron
donating or withdrawing) at different positions of the ani-
line ring (Scheme 1). The reactions performed with
N-substituted anilines revealed that N-methyl aniline (2j)
produced an analogous product 3j (Table 3,entry 10) in
higher yield, but N,N-dimethyl (2n) and diethyl aniline (2o)
produced only monoalkylated products (3n and 3o) even
after stirring for 24 h at room temperature (entry 14,15).
Similar type of product [2-(5-amino-quinolin-8-yl)-2-
hydroxy-indan-1,3-dione), 3q] was also obtained when
5-aminoquinoline (2q) was used as the nucleophile (entry
17). It may be mentioned that monoarylations at C-2
position of ninhydrin have been reported in the literature
with substituted anilines, though under different reaction
Ceric (IV) ammonium nitrate
Montmorillonite K-10 clay
I2
8
10
10
Reaction conditions: All the reactions were performed in acetonitrile
solvent at rt
a
Extension of the reaction time did not improve yield of the product
b
Isolated yield of 3a
Table 2 Standardization of the reaction conditions for the reaction of
ninhydrin (1) and aniline (2a) using different solvents
Entry No
Solvent
Time (h)a
Isolated yieldb (%)
1
2
3
4
6
7
8
a
CH3CN
CH2Cl2
Toluene
CH3NO2
THF
1
6
8
6
5
4
6
90
35
15
25
58
86
75
iPrOH
MeOH
´
conditions (Taylor and Joulie, 1998; Bullington and Dodd,
1993; Black et al., 1994).
Next, we attempted reactions with anilines substituted at
different positions of the ring. In case of electron-rich
Reaction Conditions: Ninhydrin (1.68 m mol), aniline (3.36 m mol),
iodine (5 m mol% w.r.t ninhydrin), rt
b
Isolated yield
Scheme 1 Plausible pathway
for the formation of
NH2
NH2
I2
symmetrical 2,2-bis-(4-amino-
phenyl)-indane-1,3-diones
H2N
H2N
OH
H
H2N
O
OH
H
NH2
H
O
H
O
O
2
O
O
3
O
O
4
NH2
NH2
H2N
NH2
I2
H
2a
O
OH
O
O
O
O
3a
1
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Med Chem Res (2013) 22:2023–2037
Table 3 Reaction of
ninhydrine (1) and differently
substituted aniline (2a–q)a
Productb
O
Yieldc (%)
Alkylating
Agent
Entry No.
1
Time (h)
6
NH2
NH2
80
75
3a
3b
O
O
NH2
NH2
CH3
CH3
2
NH2
CH3
7
O
O
NH2
NH2
OCH3
OCH3
NH2
OCH3
3
4
5
3c
80
84
O
O
NH2
NH2
Cl
Cl
3d
9
NH2
Cl
Br
O
O
NH2
NH2
Br
Br
NH2
5
6
3e
3f
75
78
O
O
NH2
NH2
I
I
6
5
NH2
NH2
O
O
I
NH2
NH2
NO2
NO2
7
3g
80
7
O
NO2
NH2
anilines, diarylated products were isolated with higher
yields than with deactivated anilines. But 2,6-dimethyl
aniline (2h), which is electron rich, also afforded slightly
lower yield of the product, ostensibly due to steric inhibi-
tion of resonance by the two methyl groups (entry 8).
Finally, we studied the effect of substituents located at
two different positions of the aniline ring.
The results permit the following conclusions to be
drawn: (i) N-dialkylated anilines (entries 14, 15) react
through the para-position (both had a free para carbon
atom) to form monosubstituted products. (ii) N-unsubsti-
tuted or monosubstituted derivatives with free para-posi-
tion (entries 1–11) afford disubstituted products. It seems
likely that the intermediate monosubstituted product
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Med Chem Res (2013) 22:2023–2037
2027
Table 3 continued
Alkylating
Agent
Products
Yield(%)
Entry No.
8
Time
6
H3C
CH3
NH2
CH3
O
NH2
CH3
3h
CH3
70
O
NH2
H3C
F
9
NH2
NH2
CH3
7
F
O
CH3
3i
65
90
CH3
O
NH2
F
H
N
Me
O
10
NH
Me
3j
6
Me
O
N
H
NH2
OCH3
Cl
O
Cl
11
NH2
OCH3
7
3k
84
75
NH OCH3
O
Cl
Cl
H2N
O
Cl
Cl
NH Cl
12
6
Cl
NH2
3l
O
H2N
O
Cl
CH3
NH
3m
CH3
80
H3C
H3C
O
5
13
NH2
CH3
CH3
suffers dehydration to generate a quinone imine type
compound, which readily undergoes nucleophilic addition
to form the final disubstituted product. If the meta carbon
in the aniline ring carries a bulky group (entry 11), the
second aniline molecule prefers to attack through the
nitrogen atom to avoid a sterically encumbered product,
leading to an unsymmetrically disubstituted derivative (3k)
(Scheme 2). The only exceptions are m-methoxyaniline
(entry 16) and the quinoline derivative (entry 17), where
the reaction proceeds only to monosubstitution despite the
presence of an NH2 group. Perhaps the m-substituent
(methoxy or ring residue) in these cases is large enough to
123

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Med Chem Res (2013) 22:2023–2037
Table 3 continued
Products
Alkylating
Agent
Entry No.
Yield(%)
Time
O
CH3
N
CH3
OH
OH
CH3
CH3
9
14
3n
3o
78
N
O
O
C2H5
C2H5
C2H5
C2H5
7
N
N
15
80
O
O
H3CO
OH
O
NH2
72
NH2
3p
6
16
17
O
CH3
a
All the reactions were carried
out in acetonitrile using 5 mol%
of iodine as catalyst at rt
NH2
O
O
OH
N
8
68
N
NH2
3q
b
All the products were
characterized by mass,
1H, 13C NMR
c
Isolated yield
discourage the dehydration step. (iii) Anilines having a
substituted para-position (entries 12 and 13) react through
the ortho carbon, and through the less crowded one if there
be a choice. The second aniline attacks via the nitrogen
atom thereafter, as the symmetrically substituted product
would have been sterically crowded (3l, m) (Scheme 2).
Attempts were also made to isolate the intermediate to
the unsymmetrically disubstituted products to ascertain
which of the groups, the amino or activated carbon, gets
coupled first with ninhydrin. A series of reactions were
performed following the aforesaid protocols and monitor-
ing at different time intervals (30 min and 60 min). How-
ever, in all cases the products isolated were the
disubstituted ones. Reactions performed with one equiva-
lent of aniline and monitored at different time intervals also
failed to furnish any monosubstituted product. Instead, we
could isolate only the disubstituted product (yields pro-
portional to the time) and the unutilized ninhydrin.
monoarylated products as reported in the literature
(Bullington and Dodd, 1993, Kok and Roth, 1975; Roth
and Kok, 1976. The monoarylated products (3n–q) were
further reacted with unhindered anilines but failed to pro-
duce the corresponding biarylated product.
We next tried to prepare mixed type biarylated products
using different aniline units. For this purpose, a mixture of
two differently substituted anilines, viz. 2-methoxyaniline
(2c) and 2-bromoaniline (2e), one equivalent each with
respect to ninhydrin, were used. Interestingly, the products
were the symmetrically diarylated ones (3c, 3e) without
any cross product. Similar result was also obtained when
we used the anilines one after another.
All the products obtained from the reactions were
purified by chromatography and are quite stable. It is
noteworthy that our attempts to rearrange the monoarylated
product (3n–q) in acetic acid medium (Bullington and
Dodd, 1993) also failed.
In order to make the methodology an environmentally
benign one, we tried to perform few reactions with dif-
ferently substituted anilines and ninhydrin in water in the
presence of molecular iodine with KI. It is noteworthy that
within 15 min of the reaction, the solid products precipitate
out of the solution. These were characterized as the
Thestructuresweredeterminedwiththehelp of1H and13
C
NMR data along with extensive 2D NMR spectrum analysis.
Important correlations of COSY, HMBC, and NOESY of a
representative compound 3k are being presented in Fig. 3.
The proposed structure of 3k was supported by an unam-
biguous single crystal X-ray diffraction analysis (Fig. 4).
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Med Chem Res (2013) 22:2023–2037
2029
Scheme 2 Plausible pathway
for the formation of
unsymmetrical 2-(2-amino-4,5-
dimethyl-phenyl)-2-(3,4-
dimethyl-phenylamino)-indan-
1,3-dione
R1
R1
R2
NH2
OH
R1
O
NH2
O
I2
H
H2N
NH2
R2
H
R2
O
O
O
O
R1
OH
R2
O
H2N
R2
7
8
6
R1
NH2
NH2
R1
R1
OH
R1
I2
H
R2
H2N
R2
R2
O
OH
R2
HN
O
O
O
O
R1
9
1
H3
C
O
Antimicrobial activity
NH2
H
H
The synthesized compounds were tested in vitro for their
antibacterial and antifungal effects. The microorganisms
used in this study consisted of eight strains of bacteria,
namely, Bacillus pumilus 11778, Staphylococcus aureus
29737, Micrococcus luteus ATCC 9341, Escherichia coli
319, Klebsiella pneumoniae J/I/4, Pseudomonas aerugin-
osa 71, Vibrio cholerae 759, and Shigella dysenteriae 15.
Antifungal studies were carried out on four strains of fungi,
viz. Aspergillus niger, Candida albicans, C. tropicalis, and
Cryptpcoccus neoformans. The results of the antimicrobial
activity study given in Table 4 demonstrate that among the
tested compounds, nine possess a good degree of antibac-
terial activity against various bacterial strains. The MIC
tests revealed that 3k had MIC value 32 lg/ml against two
bacterial strains (Micrococcus luteus ATCC 9341 and
Vibrio cholerae 759) and their zone diameter were found
within 8–9 mm; 3d also had very good activity against
three stains (S. dysenteriae 15, E. coli 319, K. pneumoniae
J/I/4) with MIC value between 32 and 128 lg/ml and zone
diameter within 6–8 mm. Similarly, both 3i and 3j are
effective against three bacterial stain with MIC value
128 lg/ml and the bacterial stains for 3i were E. coli 319,
B. pumilus 11778, K. pneumoniae J/I/4 and that for 3j
E. coli 319, V. cholerae 759, K. pneumoniae J/I/4; 3e
active against four bacterial stains (E. coli 319, B. pumilus
11778, V. cholerae 759, K. pneumoniae J/I/4) with MIC
value 128 lg/ml and zone of diameter 6–9 mm. The
derivatives 3f, 3o, and 3b showed activity against three
bacterial stains with MIC value 64–128 lg/ml where 3f
active against S. aureus 29737, M. luteus ATCC 9341, and
O
H
H
Cl
H
H
N
O
Cl
H
H
O
H
H3
C
H
Fig. 3 Important correlations COSY (
) of 3k (Color figure online)
) HMBC (
) NOESY
(
P. aeruginosa 71 with zone diameter of 8 mm, and 3o and
3b active against S. dysenteriae 15, E. coli 319, and
B. pumilus 11778 with zone diameter of 7–9 mm. 3b is also
active against V. cholerae 759. Likewise, 3a and 3l, showed
good activity against four bacterial stains with MIC value
100–128 lg/ml. 3a showed activity against S. dysenteriae
15, E. coli 319, B. pumilus 11778, and K. pneumoniae J/I/4
with zone diameter 6–8 mm and 3l showed activity against
S. dysenteriae 15, E. coli 319, V. cholerae 759, and
K. pneumoniae J/I/4 with zone diameter 7–8 mm (Table 5).
Among the identified nine effective derivatives, seven
are diarylated ones. The diarylated product with aniline
(3a) or o-anisidine (3b) emerged effective against all
eight bacterial strains with varying degree of efficacy.
Products derived from ortho-substituted anilines displayed
efficacy against three to eight bacterial strains, while those
obtained from meta-or para-substituted ones showed
insignificant effect. Interestingly, products with a methyl
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Med Chem Res (2013) 22:2023–2037
and bactericidal effect was noticed against Gram-negative
organisms, i.e., E. coli 319 and Vibrio cholerae 759.
After treatment with the test compounds, bacterial cell
structure was observed by SEM micrographs (Fig. 7),
which demonstrated that some morphological changes
had occurred in bacteria after the treatments. In case of
E. coli 319, the original rod shape was swollen to
assume an irregular shape with high agglomeration of the
cells. It is presumed that antimicrobial activity of the
synthesized product may be due to damage of microbial
enzyme or cell wall permeability. The antimicrobial
activity of test product is also related to its ability to
modify the DNA replication mechanism as well as to
cause abnormalities in size, cytoplasmic contents,
cell membrane, and outer cell layers of sensitive cells
(Russell and Hugo, 1994). The study revealed that
Gram-negative bacteria were more susceptible to the
antimicrobial effects of samples than Gram-positive ones,
presumably due to the thinner nature of their peptido-
glycan cell wall network, which may allow more rapid
absorption of the drug into the cell.
Fig. 4 ORTEP representations of the title compound 3k, the
displacement ellipsoids are drawn at a probability of 50 %
Among all the tested compounds, only four (3a, 3b, 3e,
3l) appeared effective against three fungi, i.e., Aspergillus
niger, Candida albicans, and C. tropicalis, which had MIC
values ranging between 1,000 and 2,500 lg/ml, while 3e
showed recognizable antifungal activity against Crypto-
coccus neoformans also (Table 6). The zone of inhibition
of the compounds is shown in Table 7.
group at ortho-position showed activity against greater
number of bacterial strains than halo-substituted analogs,
possibly due to the electronegativity of the halogens, which
might have led to unfavorable hydrophilic interaction.
Likewise, substitution on NH2 with hydrophobic group
(methyl or ethyl) as in 3n or 3o generated sign ificant effect
toward few bacterial strains, likely due to favorable
hydrophobic interaction at the binding site.
Conclusion
A viable colony count method was performed to confirm
the antimicrobial test result of the compounds and the
results are shown in Figs. 5 and 6. Significantly lower
levels of viable CFU count compared to control (without
compound) indicated test compound (3b, 3k) incorporation
In summary, we have developed an easy, simple, and mild
reaction condition for the construction of 2,2-diarylindan-
ediones in neutral medium by the reaction of anilines with
Table 4 Antibacterial activity of test compounds against different bacterial strains
Name of the organisms
Minimum inhibitory concentration (mg/ml) of test compounds and standard antibiotics
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q GT AM
[1,000 [500
Staphylococcus aureus
29737
_
_
_
64 64
_
_
_
_
_
_
128
_
128
_
1
0.5
Shigella dysenteriae 15
Micrococcus luteus
ATCC 9341
128
128
_
_
32
_
_
_
_
_
_
_
_
_
_
_
_
_
128
_
_
_
_
_
128
_
_
_
_
2
10
1
[1,000 [500
128
32
128
24
Escherichia coli 319
Bacillus pumilus 11778
128
100
64
_
100 128
_
_
_
_
_
_
_
128 128
_
_
_
100 128 100 64
_
_
_
_
_
100 0.5
4
128 250
_
_
_
128
_
_
250 128
_
_
_
_
_
_
_
_
_
_
_
_
128
_
_
_
_
_
10
4
0.5
256
256
Pseudomonas aeruginosa 71 [500 [1,000
_
128
_
_
_
_
_
_
Vibrio cholerae 759
[500
128
128 250
128
128 32 128
128
_
2
Klebsiella pneumoniae J/I/4
500 250 128 128
_
128 128
_
_
0.25 256
GT Gentamicine, AM Amoxicilline
123

Med Chem Res (2013) 22:2023–2037
2031
Fig. 5 Time-dependent in vitro growth curve of E. coli 319 at their
MIC values against test sample 3b
Fig. 6 Time-dependent in vitro growth curve of Vibrio cholerae 759
at their MIC values against test sample 3k
ninhydrin using 5 mol % of molecular iodine in acetoni-
trile at room temperature. The novelty of this versatile
methodology lies in the operational simplicity, environ-
ment friendly reaction conditions, and the use of cheap and
readily available starting materials.
Some of the products were identified to possess potential
antimicrobial activity, especially the efficacy of the com-
pounds against Vibrio choleare 759 and Shigella dysente-
riae 15 demand further study as both the diseases are
prevalent in our geographic region.
Experimental section
Chemistry
General methods
Melting points were determined with a capillary melting
point apparatus and are uncorrected. IR spectra were
recorded on a JASCO FTIR (model 410) in KBr pellets.
ESI-MS (positive) was conducted using LC-ESI-Q-TOF
1
Micromass spectrometer. H and 13C NMR spectra were
123

2032
Med Chem Res (2013) 22:2023–2037
Fig. 7 SEM micrographs of microbes: (a1) before and (b1) after treatment with corresponding MIC concentration of test sample 3b against
E. Coli 319
Table 6 Antifungal activities of chemically synthetic compound
against different fungi strains
Table 7 Zone of inhibition of the tested compounds
Sample no. Name of the of organism and the diameter of inhibition
zones in mm
Sample
drugsa
Name of the of organism and minimum inhibitory
concentration (lg/ml) of test
Aspergillus Candida
Niger albicans
Candida Cryptococcus
Nigertropicalis neoformans
Aspergillus Candida Candida Cryptococcus
Niger albicans Nigertropicalis neoformans
3a
3b
3c
3d
3e
3f
10 0.18 9.0 0.23 8.2 0.15
7.6 0.4 10 0.15 8.0 0.05
–
–
3a
3b
3c
3d
3e
3f
1,000
1,000
1,000
–
1,000
1,000
2,000
–
–
–
–
–
–
–
–
–
–
–
–
–
12 0.17 10 0.18 8..0 0.12
8
0.15
1,000
2,500
2,000
1,500
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4
–
–
–
–
–
–
–
–
–
–
–
–
–
8
3g
3h
3i
–
3g
3h
3i
–
–
–
–
–
–
–
–
3j
–
3j
–
–
3k
3l
–
3k
3l
–
–
8.0 0.10
–
8.0 0.20
1,000
1,000
3m
3n
3o
3p
3q
–
–
–
–
–
–
–
–
–
–
3m
3n
3o
3p
3q
–
–
–
–
–
–
–
–
–
–
8
Fluconazole 10
performed with neutral alumina obtained from SRL (India).
Thin-layer chromatography was performed on pre-coated
silica gel 60 F254 aluminum sheets (E. Merck, Germany)
using 30 % ethyl acetate-hexane as solvent and the spots
were developed using iodine or Liebermann-Burchard
reagent.
a
With respect to Fluconazole; a standard antifungal antibiotic
taken on a Bruker 300 MHz DPX spectrometer at 300 and
74.99 MHz, respectively, with tetramethylsilane as internal
standard and the chemical shifts are reported in d units.
Ninhydrin, molecular iodine, and aniline derivatives were
purchased from Aldrich Chemical Ltd (USA). Organic
solvents used for the chemical synthesis and for chroma-
tography were acquired from E. Merck (India) and were of
analytical grade. All chromatographic purifications were
General method of preparation of 2-mono/2,
2-bis-(4-amino-phenyl)-indan-1,3-diones (3a–q)
Appropriate amount (1.68 mmol) of ninhydrin (1) was
dissolved in 15 ml of acetonitrile in a 50 ml RB flask,
123

Med Chem Res (2013) 22:2023–2037
2033
2,2-Bis-(4-amino-3-methyl-phenyl)-indan-1,3-dione (3b)
catalytic amount (5 mol %) of molecular iodine was added,
and the mixture was stirred at room temperature for about
10 min. Then the aniline derivative 2a (3.36 mmol) was
added to the solution and the reaction mixture was stirred at
ambient temperature for 2 h. After completion of the
reaction (checked by TLC), the contents of the reaction
mixture were evaporated to dryness. The residue was
digested with ethyl acetate and poured into a separating
funnel, and then 30 ml of 10 % aqueous Na2S2O4 solution
was added. The organic layer was separated followed by
extraction of the aqueous layer once with 25 ml of ethyl
acetate. All the organic layers were mixed together, washed
thoroughly with water, dried over sodium sulfate, and
evaporated to dryness in a rotary evaporator under reduced
pressure. The crude product was chromatographed over
neutral alumina, which on eluting with a mixture of
petroleum ether-ethyl acetate in different ratios yielded the
respective products.
Brown solid, Mp 230–232 °C; Rf (30 % Ethyl acetate-
hexane) 0.70; IR (KBr, cm-1) m 3462, 3378, 1707, 1626,
1
1505, 1257, 810; H NMR (300 MHz, CDCl3) d 2.07 (6H,
s), 3.59 (4H, s), 6.58 (2H, d, J = 8.4 Hz), 6.90–6.93 (4H,
m), 7.83–7.86 (2H, m), 8.03–8.06 (2H, m); 13C NMR
(75 MHz, DMSO-d6) d 17.6 (2xCH3), 66.3 (C), 113.7
(2xCH), 120.9 (2xC), 123.7 (2xCH), 125.3 (2xC), 126.8
(2xCH), 129.9 (2xCH), 136.7 (2xCH), 140.9 (2xC), 146.0
(2xC) 200.6 (2xC). HRMS [ESI], m/z calcd for
C23H20N2O2Na: [M ? Na]? 379.1422; found: 379.1426.
2,2-Bis-(4-amino-3-methoxy-phenyl)-indan-1,3-dione (3c)
Brown solid, Mp 198–200 °C; Rf (30 % Ethyl acetate-
hexane) 0.70; IR (KBr, cm-1) m 3430, 3346, 1702, 1618,
1498, 1254, 813; 1H NMR (300 MHz, CDCl3) d 3.73
(6H, s), 3.79 (4H, s), 6.58–6.66 (4H, m), 6.72 (2H, s),
7.84–7.87 (2H, m), 8.04–8.06 (2H, m); 13C NMR
(75 MHz, DMSO-d6) d 55.2 (2x-OCH3), 66.6 (C), 110.9
(2xCH), 113.2 (2xCH), 121.3 (2xCH), 123.8 (2xCH), 125.3
(2xC), 136.7 (2xCH), 137.4 (2xC), 140.9 (2xC), 146.1
(2xC) 200.2 (2xC). HRMS [ESI], m/z calcd for
C23H20N2O4Na: [M ? Na]? 411.1321; found: 411.1320.
Experimental protocol in aqueous solution In a 50 ml RB
flask, 1.68 mmol of Ninhydrin was dissolved in 10 ml
distilled water, a pinch of iodine and potassium iodide were
added subsequently followed by the addition of
2-Methoxyaniline (3.36 mmol). The reaction mixture was
stirred at ambient temperature and within 15 min, the
product precipitates out of the solution. The content of the
reaction mixture was filtered, washed with water, and dried
under vacuum. The residue was further recrystallized from
chloroform.
2,2-Bis-(4-amino-3-chloro-phenyl)-indan-1,3-dione (3d)
Brown solid, Mp 220–222 °C; Rf (30 % Ethyl acetate-hex-
ane) 0.70; IR (KBr, cm-1) m 3235, 2945, 1704, 1456, 1324,
1117, 776; 1H NMR (300 MHz, DMSO-d6) d 5.51 (4H, s),
Protocol for cross-over experiment Cross-over experi-
ment was performed with 2.0 mmol of ninhydrine dis-
solved in a 50 ml RB flask containing 20 ml acetonitrile.
Catalytic amount of molecular iodine (5 mol%) was added
to the mixture and stirred for 10 min at room temperature.
A mixture of 2-methoxyaniline and 2-bromoaniline one
equivalent each was added and stirred for 2 h. After
completion of the reaction (checked by TLC), the contents
of the reaction mixture were evaporated to dryness. The
residue was then purified as stated earlier.
13
6.72–6.80 (4H, m), 6.86 (2H, s), 8.07 (4H, s); C NMR
(75 MHz, DMSO-d6) d 65.0 (C), 115.3 (2xCH), 116.8 (2xC),
124.1 (2xCH), 125.6 (2xC), 127.8 (2xCH), 128.7 (2xCH),
137.1 (2xCH), 140.6 (2xC), 144.4 (2xC), 199.5 (2xC).
HRMS [ESI], m/z calcd for C21H14Cl2N2O2Na: [M ? Na]?
419.0330; found: 419.0365.
2,2-Bis-(4-amino-3-bromo-phenyl)-indan-1,3-dione (3e)
2,2-Bis-(4-amino-phenyl)-indan-1,3-dione (3a)
Brown solid, Mp 190–192 °C; Rf (30 % Ethyl acetate-
hexane) 0.75; IR (KBr, cm-1) m 3457, 3362, 1696, 1618,
1491, 1261, 807,771; H NMR (300 MHz, CDCl3) d 4.10
1
Brown solid, Mp 215–217 °C; Rf (30 % Ethyl acetate-
hexane) 0.65; IR (KBr, cm-1) m 3472, 3376, 1698, 1619,
(4H, s), 6.80 (2H, d J = 8.4 Hz), 7.02 (2H, dd J = 8.4,
1.8 Hz), 7.28 (2H, d J = 1.8 Hz), 7.88–7.90 (2H, m),
8.05–8.08 (2H, m); 13C NMR (75 MHz, DMSO-d6) d 64.8
(C), 107.1 (2xC), 115.2 (2xCH), 124.1 (2xCH), 126.1
(2xC), 128.4 (2xCH), 131.7 (2xCH), 137.1 (2xCH), 140.6
(2xC), 145.5 (2xC), 199.4 (2xC). HRMS [ESI], m/z calcd
for C21H14Br2N2O2Na: [M ? Na]? 506.9320; found:
506.9310.
1
1511, 1258, 814; H NMR (300 MHz, DMSO-d6) d 5.14
(4H, s), 6.48 (4H, d, J = 8.4 Hz), 6.75 (4H, d,
J = 8.4 Hz), 8.04 (4H, s); 13C NMR (75 MHz, DMSO-d6)
d 66.3 (C), 113.8 (4xCH), 123.8 (2xCH), 125.0 (2xC),
129.2 (4xCH), 136.9 (2xCH), 140.9 (2xC), 148.1 (2xC),
200.7 (2xC). HRMS [ESI]: m/z calcd for C21H16N2O2Na:
[M ? Na]? 351.1109; found: 351.1085.
123

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Med Chem Res (2013) 22:2023–2037
2,2-Bis-(4-amino-3-iodo-phenyl)-indan-1,3-dione (3f)
2,2-Bis-(4-methylamino-phenyl)-indan-
1,3-dione (3j)
Brown solid, Mp 180–182 °C; Rf (30 % Ethyl acetate-
hexane) 0.78; IR (KBr, cm-1) m 3488, 3370, 1699, 1632,
1510, 1341, 1258, 774; 1H NMR (300 MHz, CDCl3) d 4.12
(4H, s), 6.66 (2H, d J = 8.4 Hz), 7.04 (2H, dd J = 8.4,
2.1 Hz), 7.47 (2H, d J = 2.1 Hz), 7.88–7.90 (2H, m),
8.05–8.07 (2H, m); 13C NMR (75 MHz, DMSO-d6) d 64.5
(C), 82.8 (2xC), 114.1 (2xCH), 124.0 (2xCH), 126.7 (2xC),
129.2 (2xCH), 137.1 (2xCH), 137.9 (2xCH), 140.5 (2xC),
148.2 (2xC), 199.5 (2xC). HRMS [ESI], m/z calcd for
C21H14I2N2O2Na: [M ? Na]? 602.9042; found: 602.9019.
Brown solid, Mp 195–197 °C; Rf (30 % Ethyl acetate-
hexane) 0.72; IR (KBr, cm-1) m 3406, 1700, 1610, 1519,
1325, 1256, 816, 528; H NMR (300 MHz, CDCl3) d 2.79
(6H, s), 3.71 (2H, bs), 6.50–6.53 (4H, m), 7.06–7.09 (4H,
m) 7.83–7.87 (2H, m), 8.03–8.07 (2H, m); 13C NMR
(75 MHz, CDCl3) d 30.1 (2xCH3), 66.3 (C), 111.9 (4xCH),
123.5 (2xCH), 126.2 (2xC), 129.3 (4xCH), 135.7 (2xCH),
141.2 (2xC), 148.4 (2xC), 200.8 (2xC). HRMS [ESI],
m/z calcd for C23H20N2O2Na: [M ? Na]? 379.1422;
found: 379.1443.
1
2,2-Bis-(4-amino-3-nitro-phenyl)-indan-1,3-dione (3g)
2-(4-Amino-2-chloro-5-methoxy-phenyl)-2-(5-chloro-2-
methoxy-phenylamino)-indan-1,3-dione (3k)
Brown solid, Mp 198–200 °C; Rf (30 % Ethyl acetate-
hexane) 0.65; IR (KBr, cm-1) m 3459, 3370, 1701, 1619,
1499, 1255, 812; H NMR (300 MHz, DMSO-d6) d 7.03
(2H, d J = 9.0 Hz), 7.21–7.25 (2H, m), 7.71 (4H, s), 7.71
13
(2H, s), 8.08–8.17 (4H, m); C NMR (75 MHz, DMSO-
1
Brown solid, Mp 210–212 °C; Rf (30 % Ethyl acetate-
hexane) 0.80; IR (KBr, cm-1) m 3479, 3385, 1744, 1709,
1593, 1509, 1247, 1173, 959, 742; H NMR (600 MHz,
1
d6) d 64.3 (C), 120.4 (2xCH), 123.8 (2xC), 124.3 (2xCH),
124.8 (2xCH), 129.5 (2xC), 135.6 (2xCH), 137.5 (2xCH),
140.5 (2xC), 145.8 (2xC), 198.8 (2xC). HRMS [ESI], m/
z calcd for C21H14N4O6Na: [M ? Na]? 441.0811; found:
441.0810.
DMSO-d6) d 3.49 (3H, s), 3.70 (3H, s), 5.27 (2H, s), 5.77
(1H, s), 6.60 (1H, s), 6.69 (1H, dd J = 2.4, 9.0 Hz), 6.78
(1H, s), 6.83 (1H d J = 8.4 Hz), 6.87 (1H, d J = 2.4 Hz),
8.01–8.05 (4H, m); 13C NMR (75 MHz, DMSO-d6) d 55.4
(CH3), 55.8 (CH3), 69.1 (C), 111.3 (CH), 113.3 (CH),
113.8 (CH), 115.3 (CH), 117.7 (CH), 117.9 (C), 122.5 (C),
123.5 (C), 124.3 (2xCH), 134.8 (C), 136.7 (2xCH), 139.9
(3xC), 144.8 (C), 146.1 (C) 195.1 (2xC). HRMS [ESI],
m/z calcd for C23H18Cl2N2O4Na: [M ? Na]? 479.0541;
found: 479.0540.
2,2-Bis-(4-amino-3,5-dimethyl-phenyl)-indan-1,3-dione
(3h)
Brown solid, Mp 224–226 °C; Rf (30 % Ethyl acetate-
hexane) 0.70; IR (KBr, cm-1) m 3496, 3400, 1703, 1631,
1508, 1244, 1051, 850, 764; 1H NMR (300 MHz, CDCl3) d
2.09 (12H, s), 3.56 (4H, s), 6.81 (4H, s), 7.83–7.85 (2H, m),
8.03–8.06 (2H, s); 13C NMR (75 MHz, DMSO-d6) d 18.1
(4xCH3), 66.4 (C), 120.5 (4xC), 123.7 (2xCH), 125.0
(2xC), 127.9 (4xCH), 136.7 (2xCH), 140.9 (2xC), 143.8
(2xC), 200.6 (2xC). HRMS [ESI], m/z calcd for
C25H24N2O2Na: [M ? Na]? 407.1735; found: 407.1754.
2-(2-Amino-3,5-dichloro-phenyl)-2-(2,4-dichloro-
phenylamino)-indan-1,3-dione (3l)
Brown solid, Mp 216–218 °C; Rf (30 % Ethyl acetate-hex-
ane) 0.78; IR (KBr, cm-1) m 3478, 3380, 1709, 1616, 1589,
1
1497, 1246, 948, 742; H NMR (300 MHz, DMSO-d6) d
5.93 (2H, s), 6.31 (1H, s), 6.80 (2H, s), 6.98 (1H, s), 7.18 (1H,
s), 7.32 (1H, d J = 8.4 Hz), 8.06 (4H, s); 13C NMR
(75 MHz, DMSO-d6) d 68.8 (C), 115.6 (CH), 115.9 (C),
116.9 (CH), 118.5 (C), 119.0 (C), 119.3 (CH), 124.4 (C),
124.4 (CH), 129.7 (C), 130.4 (CH), 130.9 (C), 131.2 (CH),
136.9 (2xCH), 139.9 (2xC), 141.4 (C), 146.4 (C), 194.4
(2xC). HRMS [ESI], m/z calcd for C21H12Cl4N2O2Na:
[M ? Na]? 486.9551; found: 486.9552.
2,2-Bis-(4-amino-2-fluoro-5-methyl-phenyl)-indan-1,3-
dione (3i)
Brown solid, Mp 206–208 °C; Rf (30 % Ethyl acetate-
hexane) 0.75; IR (KBr, cm-1) m 3474, 3393, 1706, 1627,
1
1487, 1240, 779; H NMR (300 MHz, CDCl3) d 2.04 (6H,
s), 3.71 (4H, s), 6.31 (2H, d J = 12.6 Hz), 6.83 (2H, d
J = 8.1 Hz), 7.81–7.84 (2H, m), 8.02–8.05 (2H, m); 13C
NMR (75 MHz, DMSO-d6) d 16.9 (2xCH3), 62.7 (C),
100.0 (CH), 100.4 (CH), 109.0 (C), 109.2 (C), 117.0 (2xC),
123.7 (2xCH), 131.6 (CH), 131.7 (CH), 136.4 (2xCH),
140.1(2xC), 148.4 (C), 148.5 (C), 157.5 (C), 160.7 (C),
197.9 (2xC). HRMS [ESI], m/z calcd for C23H18F2N2O2Na:
[M ? Na]? 415.1234; found: 415.1227.
2-(2-Amino-4,5-dimethyl-phenyl)-2-(3,4-dimethyl-
phenylamino)-indan-1,3-dione (3m)
Yellow solid, Mp 225–227 °C; Rf (30 % Ethyl acetate-
hexane) 0.80; IR (KBr, cm-1) m 3368, 3336, 1712, 1614,
1502, 1288, 1046, 777; 1H NMR (300 MHz, CDCl3) d 2.05
(3H, s), 2.09–2.10 (9H, s), 4.04 (1H, s), 4.54 (1H, s), 5.07
123

Med Chem Res (2013) 22:2023–2037
2035
(1H, s), 6.33–6.37 (1H, m), 6.43 (1H, s), 6.54 (1H, d
J = 1.5 Hz), 6.80 (1H, d J = 8.1 Hz), 7.02 (1H, s),
7.45–7.50 (1H, m), 7.75–7.80 (2H, m), 7.89 (1H, d
J = 7.5 Hz); 13C NMR (75 MHz, DMSO-d6) d 18.3
(CH3), 19.0 (CH3), 19.7 (CH3), 19.9 (CH3), 74.6 (C), 93.3
(C), 109.7 (CH), 112.3 (CH), 116.7 (CH), 121.3 (CH),
122.3 (CH), 123.4 (C), 125.2 (CH), 125.8 (CH), 128.9
(CH), 129.3 (CH), 133.8 (C), 135.0 (C), 136.2 (CH), 138.5
(C), 144.7 (C), 145.8 (C), 152.8 (C), 200.5 (2xC). HRMS
[ESI], m/z calcd for C25H24N2O2Na: [M ? Na]? 407.1735;
found: 407.1746.
(C), 200.7 (2xC). HRMS [ESI], m/z calcd for
C16H13NO4Na: [M ? Na]? 306.0742; found: 306.0723.
2-(5-Amino-quinolin-8-yl)-2-hydroxy-indan-
1,3-dione (3q)
Brown solid, Mp 201–203 °C; Rf (30 % Ethyl acetate-
hexane) 0.62; IR (KBr, cm-1) m 3374, 3071, 2787, 1739,
1707, 1589, 1472, 1232, 855, 714; H NMR (300 MHz,
1
DMSO-d6) d 6.08 (2H, s), 6.80 (1H, d J = 7.8 Hz), 7.06
(1H, s), 7.17–7.21 (1H, m), 7.84 (1H, d J = 7.8 Hz), 8.00
(4H, bs), 8.08 (1H, s), 8.48 (1H, d J = 8.4 Hz); 13C NMR
(75 MHz, DMSO-d6) d, 77.1 (C), 107.1 (CH), 117.4 (C),
119.2 (CH), 123.4 (C), 123.6 (2xCH), 130.0 (CH), 131.6
(CH), 136.0 (2xCH), 140.8 (C), 144.7 (C), 145.6 (2xC),
148.4 (CH), 200.2 (2xC). HRMS [ESI], m/z calcd for
C18H12N2O3Na: [M ? Na]? 327.0746; found: 327.07424.
2-(4-Dimethylamino-phenyl)-2-hydroxy-indan-
1,3-dione (3n)
Brown solid, Mp 200–202 °C; Rf (30 % Ethyl acetate-
hexane) 0.80; IR (KBr, cm-1) m 3469, 1745, 1610, 1529,
1367, 1180, 979, 801; H NMR (300 MHz, CDCl3) 2.91
1
(6H,s), 3.17–3.18 (1H, m), 6.62 (2H, d J = 8.7 Hz), 7.31
(2H, d J = 9.0 Hz), 7.86–7.89 (2H, m), 8.01–8.04 (2H, m);
13C NMR (75 MHz, DMSO-d6) d 39.8 (2xCH3), 79.1 (C),
112.0 (2xCH), 123.6 (2xCH), 124.0 (C), 127.0 (2xCH),
136.9 (2xCH), 140.2 (2xC), 150.1 (C), 199.9 (2xC). HRMS
[ESI], m/z calcd for C17H15NO3Na: [M ? Na]? 304.9050;
found: 304.9010.
Crystal data for 3a
Brown rectangular shaped crystals were grown from chlo-
roform-hexane. C21H16N2O2, Mr = 328.36. Space group
˚
monoclinic C2/c. Lattice constants (A): a = 12.7349(9),
b = 17.3728(13), c = 14.8084(11), a = 90, b = 93.
443(3), c = 90, cell volume V = 3270.3(4) A , formula
3
˚
units/cell Z = 8, number of independent reflections 2878,
after convergence R1 = 0.0383, wR2 = 0.1499.
2-(4-Diethylamino-phenyl)-2-hydroxy-indan-
1,3-dione (3o)
Crystal data for 3k
Brown solid, Mp 195–197 °C; Rf (30 % Ethyl acetate-
hexane) 0.78; IR (KBr, cm-1) m 3452, 2972, 1747, 1708,
1603, 1523, 1349, 1197, 1135, 783, 733; 1H NMR
(300 MHz, DMSO-d6) d 1.00–1.03 (6H, m), 3.25–3.29
(4H, m), 6.55–6.60 (2H, m), 7.01 (2H, d, J = 7.8 Hz), 8.03
Brown rectangular shaped crystals were grown from chlo-
roform-hexane. C23H18Cl2N2O4, Mr = 457.29. Space
˚
group triclinic P-1. Lattice constants (A): a = 8.9748(9),
13
b = 8.9748(9), c = 8.9748(9), a = 8.9748(9), b = 99.
469(4), c = 99.469(4), cell volume V = 1061.54(18) A ,
formula units/cell Z = 2, Number of independent reflections
9539, after convergence R1 = 0.0519, wR2 = 0.1967.
(4H, s); C NMR (75 MHz, DMSO-d6) d 12.4 (2xCH3),
3
˚
43.6 (2xCH2), 79.2 (C), 111.2 (2xCH), 122.7 (C), 123.6
(2xCH), 127.5 (2xCH), 137.0 (2xCH), 140.2 (2xC), 147.2
(C), 200.8 (2xC). HRMS [ESI], m/z calcd for
C19H19NO3Na: [M ? Na]? 332.1263; found: 332.1243.
[The –OH signal is incorporated within the solvent pick]
Pharmacological studies
Materials and methods
2-(4-Amino-2-methoxy-phenyl)-2-hydroxy-indan-
1,3-dione (3p)
Microorganisms The microorganisms used in this study
consisted of eight strains of bacteria: Staphylococcus aureus
29737, Shigella dysenteriae 15, Micrococcus luteus ATCC
9341, Escherichia coli 319, Bacillus pumilus 11778, Pseu-
domonas aeruginosa 71, Vibrio cholerae 759, and Klebsiella
pneumoniae J/I/4, and four fungi: Aspergillus niger, Can-
dida albicans, C. tropicalis, and Cryptococcus neoformans.
All the strains were clinical isolates from human beings. The
strains were identified using Barrow and Feltham’s method
(Barrow and Feltham, 1993). These were obtained from the
Yellow solid, Mp 210–212 °C; Rf (30 % Ethyl acetate-
hexane) 0.80; IR (KBr, cm-1) m 3391, 3324, 1709, 1608,
1
1248, 1027, 850, 727; H NMR (300 MHz, DMSO-d6) d
3.11 (3H, s), 5.18 (2H, s), 6.04 (1H, s), 6.23 (1H, d
J = 8.1 Hz), 6.08 (1H, s), 7.30 (1H, d J = 8.1 Hz), 7.99
(4H, s); 13C NMR (75 MHz, DMSO-d6) d 54.7 (CH3), 75.5
(C), 97.0 (CH), 106.0 (CH), 113.6 (C), 123.1 (2xCH),
128.4 (CH), 136.2 (2xCH), 139.9 (2xC), 150.2 (C), 155.6
123

2036
Med Chem Res (2013) 22:2023–2037
Division of Microbiology, Dept. of Pharmaceutical Tech-
nology, Jadavpur University, Kolkata-700 032, India. The
bacterial strains were grown in Mueller–Hinton Agar
(Merck India Ltd.) at 37 °C for 24 h, while the fungi were
grown on Sabouraud dextrose agar at 28 °C for 3–5 days.
sterile paper disks (5 mm diameter) impregnated with
different sample concentrations were placed aseptically on
sensitivity plates. The plates were then incubated at 37 °C
overnight for bacteria and 28 °C for 96 h for fungi. The
sensitivity was recorded by measuring the clear zone of
inhibition on agar surface around the disks.
Preparation of inoculums The desired bacteria and fungi
were cultured in Mueller–Hinton Broth (MHB) at 37 °C
for 24 h and in Sabouraud dextrose broth (SDB) at 28 °C
for 72 h, respectively. The microorganism suspension
was prepared by matching a 0.5 McFarland standard
(McFarland, 1907).
Determination of the killing rate The quantitative anti-
microbial activity of the synthesized product was measured
by viable cell count experiments (Rhim et al., 2009). The
microorganism suspension was diluted by sterile distilled
water to 2 9 106 cfu/ml. The standardized suspension
(1 ml) of bacteria was added into Mueller–Hinton broth
containing test sample (3b, 3k) at their MIC ranges, while
another test tube without drug sample served as control.
The mixtures were then incubated at 37 °C with shaking on
a platform shaker at 200 rpm. Aliquots (1 ml each) of
sample were withdrawn at 0, 1, 2, 4, 6, 8 h till 48 h, spread
on nutrient agar plates, and incubated at 37 °C overnight.
The numbers of colonies were counted and reduction in
plate colonies was calculated by comparing with control
plates.
Preparation of stock solution All the test samples,
screened for their antimicrobial activity, were dissolved up
to 4 % DMSO to get the concentration of 1 mg/ml and
were used as stock solution.
Determination of minimum inhibitory concentration
(MIC) The antimicrobial activities of the test samples
were evaluated by finding the minimum inhibitory con-
centration (MIC) by agar dilution and broth dilution
methods (Chattopadhyay et al., 2002). For broth dilution
assay, 0.1 ml standardized suspensions of bacteria
(2 9 106 cfu/ml) were added to Mueller–Hinton broth
containing test sample at the concentration 1–1,000 lg/ml
with appropriate antibiotic control and incubated at 37 °C
overnight for bacteria. For agar dilution assay, previously
prepared dilutions of the test sample, with appropriate
antibiotic control, were prepared in Mueller–Hinton Agar
or Sabouraud dextrose agar. The mixtures were added into
sterile petri-dishes after complete mixing. A loop of each
standardized suspension (2 9 106 cfu/spot for bacteria and
2 9 105 spores/spot for fungi) of microorganism was
inoculated on prepared agar plates by drawing a stripe. The
inoculated plates were then incubated at 37 °C for 24 h for
bacteria and at 28 °C for 96 h for fungi. The experiments
were done in triplicate. The lowest concentration required
to inhibit the growth of microorganism, i.e., the concen-
tration at which no microorganism colony or less than 5
colonies were visible within 19–38 h, was considered as
the MIC (Wang et al., 2006).
Scanning electron microscopic (SEM) study Scanning
electron microscopic observations were carried out on
bacterial cells after 12 h incubation in Mueller–Hinton
broth at 37 °C. The suspension was divided into two por-
tions and test sample (3b) was added to one portion so as to
achieve a concentration at MIC range. The other portion
was left untreated as a control. After 24 h incubation, the
cells from both tubes were harvested by centrifugation,
washed twice, and fixed in 3 % (v/v) glutaraldehyde buf-
fered with 0.1 M sodium phosphate buffer (pH 7.2) for an
hour at room temperature. They were dehydrated in a
graded alcohol series. The specimens were dried and
mounted onto stubs using double-sided carbon tape, and
then coated with a thin layer of gold using a microscope
sputter coater for 1 min at 20 mA. The coated sample was
then examined in a JEOL JSM 6360 Scanning Electron
Microscope (Hayat, 1981).
Supplementary data
Antimicrobial assay (evaluation of zone of inhibi-
tion) Qualitative antimicrobial property of test samples
was evaluated by disk diffusion method, as per NCCLS
protocol (NCCLS 1993). For sensitivity testing, 0.1 ml of
2 9 106 cfu/ml of bacterial suspension or 0.1 ml of
2 9 105 spores/ml of fungal spore suspension was trans-
ferred to a freshly prepared Mueller–Hinton Agar plate or
Sabouraud dextrose plate. A sterile bent glass rod was used
to spread the suspension to achieve uniform growth. Then,
1H and 13CNMR spectra of all new compounds associated
with this article can be found in the online version. Crys-
tallographic data in CIF format available free of charge via
the Internet at CCDC 816861-816863 contains the sup-
plementary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the Cambridge Crystallo-
graphic Data Centre, 12, Union Road, Cambridge CB2
123

Med Chem Res (2013) 22:2023–2037
2037
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64:5152–5155
Kok W, Roth HJ (1975) Zur Kenntnis der Ninhydrin-Reaktion, 2.
Mitt.: Das farbige Reaktions produkt aus Ninhydrin und
2-Methoxyanilin. Arch Phrm 308:301–307
McFarland J (1907) An instrument for estimating the number of
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Montmorillonite K-10 clay catalyzed solvent-free synthesis of
bis-indolylindane-1,3-dione,2-(10,30 dihydro-1H-[2,30]biindolyl-
20-ylidene)-indan-1,3-dione and bisindolylindeno[1,2-b]quinoxa-
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J
Org Chem
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ac.uk).
Acknowledgments The authors express their gratitude to the
Director, IICB for laboratory facilities, and the Council of Scientific
and Industrial Research (CSIR) for providing the funding and fel-
lowships to K.B.S., M. B., A.M., S.K., and A.S. We are indebted to
Dr. R. Mukherjee and Mr. K. Sarkar for recording the spectra and
Dr. B. Achari, Emeritus Scientist, CSIR, for his valuable suggestions.
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