Amberlite-IRA-402 (OH) ion exchange resin mediated synthesis of indolizines, pyrrolo [1,2-a] quinolines and isoquinolines: Antibacterial and antifungal evaluation of the products

Article abstract of DOI:10.1016/j.ejmech.2011.02.066

A number of indolizines and pyrrolo[1,2-a]quinolines/isoquinolines were prepared from phenacyl pyridinium, quinolinium and isoquinolinium salts derived fromthe reaction of the heterocycles with 2-bromo acetophenone with alkynes and alkenes using amberlite-IRA-402 (OH) ion exchange resin as the base. Antibacterial and antifungal studies were carried out against thirteen bacterial and four fungal strains, which revealed that three derivatives (4a, 4b, 7a) out of fifteen are effective against all the thirteen strains and one derivative, 10, showed dual antibactericidal and antifungal efficacy.

Full text of DOI:10.1016/j.ejmech.2011.02.066

European Journal of Medicinal Chemistry 46 (2011) 2132e2140
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
AmberliteeIRA-402 (OH) ion exchange resin mediated synthesis of indolizines,
pyrrolo [1,2-a] quinolines and isoquinolines: Antibacterial and antifungal
evaluation of the products
Abhijit Hazra a, Shyamal Mondal a, Arindam Maitya, Subhendu Naskar a, Pritam Saha a, Rupankar Paira a,
Krishnendu B. Sahu a, Priyankar Paira a, Soma Ghosh b, Chandrima Sinha b, Amalesh Samanta b,
,
Sukdeb Banerjee a, Nirup B. Mondal a
*
a Department Of Chemistry, Indian Institute of Chemical Biology, Council of Scientific & Industrial Research, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India
b Division of Microbiology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata-700032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of indolizines and pyrrolo[1,2-a]quinolines/isoquinolines were prepared from phenacyl pyr-
idinium, quinolinium and isoquinolinium salts derived fromthe reaction of the heterocycles with
2-bromo acetophenone with alkynes and alkenes using amberliteeIRA-402 (OH) ion exchange resin as
the base. Antibacterial and antifungal studies were carried out against thirteen bacterial and four fungal
strains, which revealed that three derivatives (4a, 4b, 7a) out of fifteen are effective against all the
thirteen strains and one derivative, 10, showed dual antibactericidal and antifungal efficacy.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 1 July 2010
Received in revised form
22 February 2011
Accepted 26 February 2011
Available online 5 March 2011
Keywords:
Indolizines
Pyrrolo quinolines/isoquinolines
AmberliteeIRA-402 (OH)
Dipolar cycloaddition
Antibacterial
Antifungal
1. Introduction
It is small wonder therefore that great efforts have been made
to discover and optimize new reactions that facilitate the
A great deal of research in heterocyclic chemistry is concerned
with the discovery of new methods of ring formation since more
than half of the biologically active compounds produced by nature
contain a heterocyclic moiety as a fundamental unit in their struc-
ture [1].
The heterocyclic aromatic compound indolizine (isomer of
indole) forms the structural core of a variety of alkaloids such as
swainsonine and monomorine [2,3], which were prepared for both
practical and theoretical reasons [4]. Indolizine derivatives con-
taining a variety of functional groups are being used due to their
interesting biological activities like antibacterial [5], antiviral and
antileishmanial [6], antiinflamatory [7], analgesic [8], antitumor [9],
antioxidant [10], aromatase inhibitory [11], calcium entry blocking
[12], and histamine H3 receptor antagonist [13] and also evaluated as
agrochemical [14]. As such, the indolizines are important synthetic
targets for developing new pharmaceuticals or agrochemicals.
construction of indolizine derivatives. One of the most important
methods for the preparation of indolizines (and benzoindolizines)
is 1,3 dipolar cycloaddition of pyridinium and related hetero-
aromatic ylides, e.g. quinolinium or isoquinolinium ylides, with
alkynes [15]. Recently various metal catalyzed reactions of heter-
oaryl halide with propargyl amine or of heteroaromatic aldehydes
with amines and alkynes followed by cyclization have been
developed [16]. The scope of all these procedures has been limited
to the use of alkynes, very few of which are commercially available.
As an alternative to the alkynes, the olefinic dipolarophiles have
been utilized to play the pivotal role in this strategy, although
there remain some difficulties, as the intermediate tetrahy-
droindolizines formed are unstable and reversibly transform into
a betaine intermediate followed by decomposition [17]. To over-
come this problem fluoroalkenes, fluoroiodo alkenes, and fluori-
nated vinyl tosylates are being used in the cycloaddition where
hydrogen fluoride or TSOH is eliminated to prepare fluorinated or
unfluorinated indolizines [18e20]. Also the tetrahydroindolizines
can be aromatized into indolizines in situ via dehydrogenative
* Corresponding author. Tel.: þ91 33 2499 5721; fax: þ91 33 2473 5197.
E-mail address: nirup@iicb.res.in (N.B. Mondal).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.066

A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
2133
aromatization in a one-pot sequence [21] using a mild dehydro-
genative oxidant like TPCD or MnO2.
yields (Scheme 2, Table 1, entry 7e11), though with isoquinolinium
salts it took 6e7 h producing a convincing yield (Table 1, entry
12e14). Using TPCD as the oxidizing agent, pyrrolo[1,2-a]quinolines
were not formed from quinoliniums, rather the indolizine deriva-
tives were produced due to oxidation of the quinoline ring.
However, using MnO2 as the oxidizing agent the expected pyrrolo
[1,2-a]quinolines were isolated in low yields [25], on the other
hand, replacing MnO2 with DDQ, in presence of Amberlite resin the
pyrrolo [1,2-a] quinolines yield was quite satisfactory.
In case of isoquinolinium salt the yield with alkynic or olefinic
dipolarophile is higher than in case of quinolinium or pyridinium
salt (Table 1, entry 5e6, 12e14). This might be due to the fact that
the C-1 of isoquinoline is more electrophilic in nature than C-2 of
quinoline or pyridine where electrophilicity is distributed in
between C-2 and C-4. The compounds were characterized on the
basis of spectral analysis like IR, 1H and 13C NMR and also by MS.
Besides the spectral studies, the structure of 9c was unambiguously
established by single crystal X-ray crystallography (Fig. 1).
Very recently, we reported the synthesis of bioactive heterocy-
cles via azomethine ylide cycloaddition [22]. We felt that this
approach can be judiciously extended for the syntheses of indoli-
zines and pyrrolo[1,2-a]quinolines/isoquinolines. We also intended
to utilize the advantages of AmberliteeIRA-402(OH) ion-exchange
resin [23] as a base cum catalyst in this biphasic system in lieu of
bases like Et3N, DBU etc. Since newer antibiotics and chemother-
apeutic agents are being steadily synthesized in controlling many
infections but the success depends on non-development of drug
resistance [24]. In this endeavor our approach is centered with the
synthesis of new chemical entities for the search of newer anti-
microbial agents. Herein, we wish to report in detail the syntheses
of indolizines, pyrrolo[1,2-a]quinolines/isoquinolines using amb-
erlite resin along with the results of investigation on their bacte-
ricidal and fungicidal properties.
It is noteworthy that the reaction of isoquinolinium salt with
trans-beta-nitrostyrene yielded no expected cycloaddition product
when carried out following similar reaction protocol rather we
were able to isolate a product 10 whose structure was established
with the help of spectroscopic analysis and also confirmed by single
crystal X-ray crystallography (Fig. 2). It is a cyclized product and
appears to have formed by the self condensation of the salt with its
enol form (Scheme 3) (Table 1, entry 15). Although there appears
a similar report on self condensation of pyridinium salt with its
enol form [20b], however, we were unable isolate similar product
from the reaction of quinolinium salt. The reaction of iso-
quinolinium salt in the presence of amberlite and DDQ without
a dipolarrophile, the yield of 10 was quite good.
To establish the effectiveness of Amberlite resin we also per-
formed reactions in presence of other bases like Et3N, DBU etc in
homogenous as well as biphasic conditions. In each case it took
8e10 h to complete the reaction and the yield was quite good. It is
noteworthy that in these cases some colored material was also
produced. On the other hand, using resin, which is reusable after
simple washing with solvent and NaOH solution, the formation of
colored material was checked.
2. Results and discussion
2.1. Chemistry
Isoquinolinium, quinolinium or pyridinium salts were prepared
by the reaction of isoquinoline or quinoline or pyridine with
2-bromo acetophenone in diethylether at room temperature. For
the cycloaddition reaction, the salts were dissolved in water and
a solution of dipolarophile in chloroform was added. Then amber-
liteeIRA-402 (OH) ion exchange resin (as the base) and DDQ (as the
oxidizing agent) were added to the reaction mixture. Initially we
reacted with pyridinium, quinolinium and isoquinolinium salts
with alkynic dipolarophile. The reactions proceeded smoothly and
became complete within 5 h with 65e70% yield. However, addition
of DDQ was not mandatory for oxidation in case of alkynic dipo-
larophiles like DMAD/DEAD (Scheme 1). Without DDQ, the reaction
was complete within 5e6 h and the yield of the products was quite
similar (Table 1, entry 1e6). But the reaction of olefinic dipolar-
ophiles with pyridinium or quinolinium salts in presence of DDQ
took almost 9e10 h to complete and generated only moderate
H2O
2.2. Pharmacology
R
R
in chloroform
IRA-402 (OH)
Br
The synthesized compounds were tested for their antimicrobial
and antifungal studies in vitro. The microorganisms used in this
study consisted of 13 strains of bacteria namely: Bacillus subtilis
UC564, Staphylococcus aureus 25923, Streptococcus faecalis 29212,
Micrococcus luteus AGD1, Escherichia coli ATCC25938, Klebsiella
pneumoniae J/I/4, Pseudomonas aeruginosa ATCC27853, Vibrio cholera
7201, Vibrio parahaemolyticus 72016, Shigella dysenteriae 3, Shigella
flexneri DN13, Salmonella typhi DIRW, and Salmonella typhimurium
11. Antifungal studies were carried out on four strains of fungi, viz.
Aspergillus niger, Candida albicans, Candida tropicalis, and Cryptp-
coccus neoformans. The results of the antimicrobial activity testing
are shown in Table 2, which demonstrates that out of the tested
compound six (4a, 4b, 7a, 9a, 9c and 10) possess significant anti-
bacterial activity. Compounds 4a, 4b, and 7a demonstrated activity
against all the 13 Gram-positive and Gram-negative strains.
N
R
N
O
O
R
Ph
Ph
1
2a-b
H2O
R
R
in chloroform
IRA-402 (OH)
Br
N
R
N
O
O
R
Ph
Ph
4a-b
3
H2O
R
R
O
in chloroform
The MIC tests revealed that 7a had MIC between 16 and 128
mg/
O
N
N
IRA-402 (OH)
ml against five bacterial strains (V. cholera 7201, B. subtilis UC 564, S.
aureus ATCC 25923, K. pneumoniae J/1/4, and S. dysenteriae 3) and
their zone diameters were found within 8e12 mm. Similarly, 4a and
Ph
Br
Ph
R
R
4b had MIC between 16 and 128 mg/ml and the bacterial strains for
5
6a-b
4a were S. dysenteriae 3, B. subtilis UC 564, S. faecalis 29212, K.
pneumoniae J/1/4, S. typhimurium 11 and that for 4b were B. subtilis
UC 564, S. dysenteriae 3, P. aeruginosa ATCC 27853, S. aureus ATCC
Scheme 1. Synthesis of indolizines, pyrrolo [1,2-a] quinolines, pyrrolo [1,2-a]
isoquinolines using alkynes.

2134
A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
Table 1
Structure and yield of the products.
Entry
Products
Structure
Yields
65
Entry
8.
Products
Structure
Yields
53
CO2Me
CO2Me
N
N
1.
2a
7b
O
O
O
O
CO2Me
Ph
N
Ph
CO2Et
CO2Et
N
2.
3.
2b
4a
63
65
9.
7c
8a
52
50
CO2Et
Ph
Ph
CO2Me
CO2Me
N
N
10.
O
O
O
O
CO2Me
Ph
Ph
CO2Et
CO2Et
N
N
4.
5.
4b
6a
62
84
11.
12.
8b
9a
51
75
CO2Et
Ph
Ph
O
O
N
N
Ph
Ph
MeO2C
CO2Me
NC
O
O
N
N
6.
7.
6b
7a
85
54
13.
9b
73
Ph
CO2Et
Ph
EtO2C
N
MeO2C
EtO2C
O
N
14.
15.
9c
10
75
75
Ph
O
CN
Ph
O
N
Ph
Ph
25923, V. parahaemolyticus 72016, V. cholera 7201. Likewise,
compound 10 had MIC value 32e128 g/ml against seven bacterial
rather than the pyrrolo isoquinoline where the presence of one
extra ester group diminishes its activity (6a, 6b).
m
strains (B. subtilis UC 564, E. coli ATCC 25938, K. pneumoniae J/1/4, V.
cholera 7201, S. aureus ATCC 25923, P. aeruginosa ATCC 27853, S.
dysenteriae 3) and their zone diameters were within 8e12 mm
(Table 3).
Among all the compounds, the compound 10 shown sensitivity
towards three fungi i.e A. niger, C. albicans, C. tropicalis which had
MIC value 500e1000
mg/ml (Table 4) and the zone diameter of
inhibition are 12, 30 and 10 mm respectively. However, it showed
no recognizable antifungal activity against C. neoformans.
The data presented in Table-2 summarizes the effect of all types
of ring system along with their substituent. The parent indolizine or
benzo-fused indolizines are more or less active against 4e5 types of
bacterial strain. The presence of the ester group (methyl/ethyl)
seemed to be quite beneficial and also the nitrile (7a, 9a) and
phenyl ring (10) was found to be effective. However, structural
features of the active compounds revealed that the compounds
with ester or nitrile substituent adjacent to the benzoyl group is
somehow more active than the compounds where the substituent
is one carbon away from the benzoyl group. In case of pyrrolo
quinoline the presence of two ester groups is beneficial (4a, 4b)
Further study was conducted to determine minimum bactericidal
concentration (MBC)/minimum fungicidal concentration (MFC) of
the effective compounds (4a, 4b, 7a and 10) against four susceptible
bacterial and two fungal strains at different concentrations.
The MIC of compound 7a against S. aureus ATCC25923, B. subtilis
UC564, V. cholera 7201 was found to be 16e32 mg/ml. So double
folded drug dilutions were added to the nutrient broth culture of
S. aureus, B. subtilis, and V. cholera at the logarithmic growth phase.
Similarly, for compounds 4a and 4b, double folded dilutions were
added to the broth culture of B. subtilis UC564, S. dysenteriae 3, E. coli

A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
2135
H2O
R
in chloroform
IRA-402 (OH)
DDQ
Br
N
N
O
O
R
Ph
R= -CO2Me/-CO2Et/-CN
Ph
1
7a-c
H2O
R
in chloroform
Br
N
IRA-402 (OH)
DDQ
N
O
O
R
Ph
R= -CO2Me/-CO2Et
H2O
3
Fig. 2. ORTEP diagram of compound 10, displacement ellipsoids are drawn at 50%
probability level.
Ph
8a-b
The minimum bactericidal concentration (MBC) was always found to
be 2e4 folds higher than MIC values. At this stage we are not certain
about the exact mechanism of bactericidal/fungicidal activity of the
compounds at ultrastructural level, which needs further concerted
investigation on this aspect. However, we believe that the
compounds should have inhibitory effect on the growth of biofilm-
associated microorganisms and that may be by their penetration and
chemical reaction into biofilm matrix, the extracellular polymeric
material.
R
O
in chloroform
O
N
N
Ph
IRA-402 (OH)
DDQ
Br
Ph
R
9a-c
5
R= -CO2Me/-CO2Et/-CN
Scheme 2. Synthesis of indolizines, pyrrolo [1,2-a] quinolines, pyrrolo [1,2-a]
isoquinolines using alkenes.
Newer antibiotics and chemotherapeutic agents are being
steadily synthesized in controlling many infections but the success
depends on non-development of drug resistance. So, the search for
antimicrobials using different approaches has been done with
worthwhile antimicrobial action in few compounds.
ATCC 25938, and P. aerugenosa ATCC27853 and then colony count
was done on nutrient agar plates, this did not show any colony at
the highest concentrations. So, the compounds 4a, 4b and 7a were
concluded to show bactericidal action against respective Gram-
positive and Gram-negative bacterial strains (Fig. 3a,b and c).
Among all the compounds tested, compound 10 showed
significant sensitivity to A. niger and C. albicans. So dilutions of 10
were added to the fungi suspension and the growth of the fungi
was determined by dry weight calculation. The results showed that
the growth of these organisms decreased on increasing the
concentration of the drug, and complete inhibition occurred at the
highest concentrations (Fig. 4).
3. Conclusion
In summary, we have developed a novel methodology for the
syntheses of indolizines and pyrrolo[1,2-a]quinolines/isoquinolines
using amberliteeIRA-402 (OH) ion exchange resin as the base and
DDQ as the oxidizing agent in one-pot sequence. Of the 15
compounds three showed significant antibacterial actions and one
showed very significant antifungal activity. The identified four
compounds (4a, 4b, 7a and 10) exhibited bacteriostatic or fungi-
static activity and may be developed as newer antimicrobial agents.
Thus, the drugs 4a, 4b, 7a and 10 exhibited bacteriostatic or
fungistatic activity at lower but bactericidal at higherconcentrations.
4. Experimental section
4.1. Chemistry
4.1.1. 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
O
N
Ph
O
H
N
O
O
H
N
Ph
OH
Ph
N
N
Ph
Ph
Br
Ph
10
7
OH
Ph
N
Fig. 1. ORTEP diagram of compound 9c, displacement ellipsoids are drawn at 50%
probability level.
Scheme 3. Self cycloaddition of isoquinolinium salt.

2136
A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
Table 2
Antimicrobial activity of the test compounds.
Name of bacteria
Minimum inhibitory concentration (
2a 2b 4a 4b 6a 6b
>500 >500 >500 >500 256 128 >500 >500 >500 >500 >500 256 500
m
g/ml) of test compounds and standard antibiotics
7a 7b 7c 8a 8b 9a 9b
9c
10
Gentamycin Amoxicillin
32 0.25 0.50
128
0.50
256
256
0.50
64
0.25
Escherichia coli
64
Klebsiella pneumoniae J/1/4
Staphylococcus aureus ATCC 25923
Pseudomonas aeruginosae ATCC 27853 500 >500 >500
Vibrio cholera 7201
Bacillus subtilis UC 564
Shigella dysenteriae 3
Streptococcus faecalis 29212
Shigella flexneri DN13
Salmonella typhi DIRW
Vibrio parahaemolyticus 72016
Micrococcus luteus AGD1
Salmonella typhimurium 11
>500 >500 64 256 500 128 64 >500 >500 >500 >500 32 256 256
>500 >500 500 64 256 500 32 >500 >500 >500 >500 256 128 256 128
64
2
1
2
32 500 500 >500 >500 >500 >500 >500
16 >500 >500 >500 >500
64 128
32 256
64 128
>500 >500 500 128 500 500
32
64
64 0.5
32
>500 >500
>500 >500
>500 >500
32
16
16 >500 256
16 >500 >500 >500 >500 128 256
4
1
32 >500 >500 128 >500 >500 >500 >500 >500 256 128 128
64 500 >500 >500 500 >500 >500 >500 >500 >500 256 256 500 0.50
>500 >500 500 >500 >500 256 500 >500 >500 >500 >500 >500 128 256 500
>500 >500 128 500 >500 128 500 >500 >500 >500 >500 >500 256 >500 >500
64 256 128 256 >500 >500 >500 >500 >500 256 256 500
>500 >500 >500 500 >500 >500 256 >500 >500 >500 >500 >500 >500 500 500
>500 >500 500 >500 256 >500 500 >500 >500 >500 >500 >500 500 256 >500
1
1
1
8
1
128
128
256
0.5
>1000
>500 >500 >500
conducted using LC-ESI-Q-TOF micro Mass spectrometer. 1H and
13C NMR spectra were taken on a Bruker 300 MHz DPX spec-
trometer at 300 and 74.99 MHz, respectively, with tetramethylsi-
lane as internal standard and the chemical shifts are reported in
4.1.3. 3-Benzoyl-indolizine-1,2-dicarboxylic acid dimethyl ester (2a)
Yellowish solid, mp 160e162 ꢀC; IR (KBr, nmax) 1739, 1698, 1624,
1451, 1217 cmꢁ1 1H NMR (CDCl3)
; d 3.31 (3H, s, CH3), 3.88 (3H, s,
CH3), 7.11 (1H, m), 7.45 (3H, m), 7.56 (1H, t, J ¼ 7.5 Hz), 7.69 (2H, d,
d
units. Pyridine, quinoline, isoquinoline and the alkynes and
J ¼ 7.2 Hz), 8.39 (1H, d, J ¼ 8.7 Hz), 9.64 (1H, d, J ¼ 6.9 Hz); 13C NMR
alkenes were purchased from Aldrich Chemical Ltd (USA). Organic
solvents used for the chemical synthesis and for chromatography
were acquired from E. Merck (India) were of analytical grade. All
chromatographic purifications were performed with silica gel
(60e120 mesh) obtained from SRL (India). Thin layer chromatog-
raphy was performed on pre-coated silica gel 60 F254 aluminum
sheets (E. Merck, Germany) using 10e30 % ethyl acetate in petro-
leumeether (60e80 ꢀC) as solvent and the spots were developed
using iodine or LiebermanneBurchard reagent.
(CDCl3) d 51.6 (OMe), 52.1 (OMe), 103.9 (C), 115.9 (CH), 119.8 (CH),
120.7 (C), 127.9 (CH), 128.0 (2ꢂ CH), 128.4 (CH), 128.5 (2ꢂ CH), 131.5
(C), 131.7 (CH), 138.1 (C), 139.5 (C), 163.2 (C]O), 165.1 (C]O), 186.6
(C]O); MS (ESI-MS, positive ion) m/z 360 [M þ Na]þ.
4.1.4. 3-Benzoyl-indolizine-1,2-dicarboxylic acid diethyl ester (2b)
Yellowish solid, mp 91e93 ꢀC; IR (KBr, nmax) 1739, 1705, 1607,
1450 cmꢁ1; 1H NMR (CDCl3)
d
1.06 (3H, t, J ¼ 7.2 Hz, CH3),1.34 (3H, t,
J ¼ 7.2 Hz, CH3), 3.65 (2H, q, J ¼ 7.2 Hz, CH2), 4.34 (2H, q, J ¼ 7.2 Hz,
CH2), 7.10 (1H, t, J ¼ 6.9 Hz), 7.45 (3H, m), 7.55 (1H, t, J ¼ 7.5 Hz), 7.70
(2H, m), 8.42 (1H, d, J ¼ 9 Hz), 9.63 (1H, d, J ¼ 7.2 Hz); 13C NMR (CDCl3)
4.1.2. General method of preparation
Compound 1 (0.9 mmol, 250 mg) or 3 (0.76 mmol, 250 mg) or 5
(0.76 mmol, 250 mg) was dissolved in 20 ml of water. To this 20 ml
of chloroform was added. Then methyl acrylate (0.9 mmol, 0.08 ml/
0.76 mmol, 0.07 ml) or ethyl acrylate (0.9 mmol, 0.10 ml/0.76 mmol,
0.08 ml) was added to this mixture followed by 300 mg of
AmberliteeIRA-402 (OH) ion exchange resin and DDQ (0.9 mmol,
191 mg/0.76 mmol, 161 mg). The mixture was stirred vigorously at
room temperature for about 8e10 h. After completion of the
reaction as evident from TLC, the chloroform layer was separated
and evaporated in vacuum. The crude product was subjected to
column chromatography and eluted with 15% ethyl acetate-petro-
leumeether mixture. The product was crystallized from chloro-
form-hexane mixture.
d 13.4 (Me), 14.1 (Me), 60.3 (CH2), 61.5 (CH2), 104.1 (C), 115.8 (CH),
119.8 (CH),120.5 (C),127.8 (CH),127.9 (2ꢂ CH),128.4 (CH),128.6 (2ꢂ
CH),131.6 (C),131.8 (CH),138.3 (C),139.5 (C),162.8 (C]O),164.8 (C]
O), 186.6 (C]O); MS (ESI-MS, positive ion) m/z 388 [M þ Na]þ.
4.1.5. Dimethyl-1-benzoyl-pyrrolo[1,2-a]quinoline-2,3-
dicarboxylate (4a)
Yellow solid, mp 188e190 ꢀC; IR (KBr, nmax) 1737, 1698, 1632,
1445, 1258 cmꢁ1 1H NMR (CDCl3)
; d 3.43 (3H, s, CH3), 3.91 (3H, s,
CH3), 7.44 (2H, m), 7.50 (2H, t, J ¼ 7.5 Hz), 7.62 (3H, m), 7.79 (1H, m),
7.97 (2H, d, J ¼ 7.2 Hz), 8.26 (1H, d, J ¼ 9.3 Hz); 13C NMR (CDCl3)
d
51.7 (OMe), 52.2 (OMe), 105.4 (C), 117.8 (CH), 118.9 (CH), 125.2 (2ꢂ
C), 125.6 (CH), 126.3 (C), 127.9 (CH), 128.6 (2ꢂ CH), 128.9 (CH), 129.1
Table 3
Zone of inhibition of the tested active compounds.
Name of bacteria
Diameter of zone inhibition in mm
4a
4b
8.1 ꢃ 0.12
7a
ea
9a
9c
10
Escherichia coli ATCC 25938
Klebsiella pneumonia J/1/4
Staphylococcus aureus ATCC25923
Pseudomonas aerugenosa ATCC27853
Vibrio cholera 7201
Vibrio cholera 720
Bacillus subtilis UC564
Shigella dysenteriae 3
Streptococcus faecalis 29212
Shigella flexneriDN13
12.1 ꢃ 0.1
8.0 ꢃ 0.1
11.2 ꢃ 0.4
8.5 ꢃ 0.17
8.3 ꢃ 0.1
11 ꢃ 0.3
10.2 ꢃ 0.2
10.2 ꢃ 0.2
11.4 ꢃ 0.2
9.7 ꢃ 0.3
7 ꢃ 0.25
7.1 ꢃ 0.2
e
10.0 ꢃ 0.01
10.0 ꢃ 0.06
8.6 ꢃ 0.06
10.2 ꢃ 0.01
9.7 ꢃ 0.2
7.7 ꢃ 0.1
7.7 ꢃ 0.1
10 ꢃ 0.15
11.1 ꢃ 0.2
e
8.9 ꢃ 0.1
11.0 ꢃ 0.25
10.5 ꢃ 0.12
e
12.1 ꢃ 0.7
e
7.5 ꢃ 0.3
8.5 ꢃ 0.2
e
9.5 ꢃ 0.5
8.1 ꢃ 0.25
e
10.0 ꢃ 0.2
10.0 ꢃ 0.2
9.7 ꢃ 0.2
9.0 ꢃ 0.2
e
11.0 ꢃ 0.4
9.0 ꢃ 0.02
e
11.0 ꢃ 0.4
9.0 ꢃ 0.02
10.2 ꢃ 0.17
7.5 ꢃ 0.4
10.2 ꢃ 0.1
11.5 ꢃ 0.12
e
e
e
e
e
e
e
8.0 ꢃ 0.17
12 ꢃ 0.14
e
e
e
e
e
e
e
e
e
Salmonella typhi DI RW
7.5 ꢃ 0.03
e
e
Vibrio parahaemolyticus 72016
Micrococcus luteus AGD1
Salmonella typhimurium 11
e
e
e
9.3 ꢃ 0.13
e
7.5 ꢃ 023
7.5 ꢃ 0.17
e
8.0 ꢃ 0.7
8.1 ꢃ 0.1
e
e
a
‘e’ shows no measurable zone of inhibion.

A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
2137
Table 4
(CH), 129.2 (CH), 130.0 (2ꢂ CH), 132.4 (C), 133.9 (CH), 137.2 (C), 137.7
(C), 163.2 (C]O), 164.8 (C]O), 187.7 (C]O); MS (ESI-MS, positive
ion) m/z 438 [M þ Na]þ.
Antifungal activity of compound 10 taking Fluconazole as a standard.
Name of organism
MIC
m
g/ml
Zone of inhibition
10
Fluconazole
10
4.1.7. Dimethyl- 3-benzoyl-pyrrolo[2,1-a]isoquinoline-1,2-
dicarboxylate (6a)
Aspergillus niger
Candida albicans
Candida tropicalis
Cryptpcoccus neoformans
500
10
4
8
12
30
10
e
500
1000
e
Orange solid, mp 155e157 ꢀC; IR (KBr, nmax) 1731, 1624, 1508,
1358 cmꢁ1 1H NMR (CDCl3)
; d 3.43 (3H, s, CH3), 3.91 (3H, s, CH3),
8
7.44 (2H, t, J ¼ 3.9 Hz), 7.50 (2H, t, J ¼ 7.5 Hz), 7.62 (3H, m), 7.79 (1H,
m), 7.97 (2H, d, J ¼ 7.2 Hz), 8.26 (1H, d, J ¼ 9.3 Hz); 13C NMR (CDCl3)
(CH), 129.8 (2ꢂ CH), 132.3 (C), 133.8 (CH), 137.0 (C), 137.5 (C), 163.5
(C]O), 165.1 (C]O), 187.6 (C]O); MS (ESI-MS, positive ion) m/z
410 [M þ Na]þ.
d
51.9 (OMe), 52.5 (OMe), 109.6 (C), 115.8 (CH), 122.9 (C), 123.6 (CH),
124.2 (C), 125.6 (CH), 127.1 (CH), 128.3 (CH), 128.4 (2ꢂ CH), 128.9
(2ꢂ CH, eC), 129.0 (CH), 129.5 (C), 132.3 (C), 132.6 (CH), 139.7 (C),
164.6 (C]O), 165.9 (C]O), 187.2 (C]O); MS (ESI-MS, positive ion)
m/z 410 [M þ Na]þ.
4.1.6. Diethyl-1-benzoyl-pyrrolo[1,2-a]quinoline-2,3-dicarboxylate
(4b)
Yellow solid, mp 158e160 ꢀC; IR (KBr, nmax) 1726, 1698, 1633,
4.1.8. Diethyl- 3-benzoyl-pyrrolo[2,1-a]isoquinoline-1,2-
dicarboxylate (6b)
1440, 1256 cmꢁ1; 1H NMR (CDCl3)
d
1.06 (3H, t, J ¼ 7.2 Hz, CH3), 1.37
(3H, t, J ¼ 7.2 Hz, CH3), 3.85 (2H, q, J ¼ 7.2 Hz, CH2), 4.37 (2H, q,
J ¼ 7.2 Hz, CH2), 7.48 (4H, m), 7.61 (3H, m), 7.78 (1H, m), 8.00 (2H, d,
Orange solid, mp 118e120 ꢀC; IR (KBr, nmax) 1726, 1622, 1356,
1225 cmꢁ1; 1H NMR (CDCl3)
J ¼ 6.9 Hz, CH3), 3.66 (2H, q, J ¼ 6.9,14.1 Hz, CH2), 4.44 (2H, q, J ¼ 6.9,
14.1 Hz, CH2), 7.16 (1H, d, J ¼ 7.5 Hz), 7.47 (2H, t, J ¼ 7.2 Hz), 7.59 (3H,
d
0.95 (3H, t, J ¼ 7.2 Hz, CH3),1.37 (3H, t,
J ¼ 6.9 Hz), 8.29 (1H, d, J ¼ 9.3 Hz); 13C NMR (CDCl3)
d 13.6 (Me),14.2
(Me), 60.5 (CH2), 61.5 (CH2), 105.7 (C), 117.9 (CH), 119.0 (CH), 125.2
(C), 125.6 (CH), 126.1 (C), 127.8 (CH), 128.2 (C), 128.6 (2ꢂ CH), 128.9
Fig. 3. a) Mode of action of drug 7a on three different bacteria S. aureus ATCC25923, B. subtilis UC564, V. cholera 7201. b) Mode of action of drug 4a on three different bacteria
B. subtilis UC564, S. dysenteriae 3 and E. coli ATCC 25938. c) Mode of action of drug 4b on three different bacteria B. subtilis UC564, S. dysenteriae 3 and P. aerugenosa.

2138
A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
(2H, q, J ¼ 6.6 Hz), 7.53 (4H, m), 7.68 (3H, t, J ¼ 9.6 Hz), 7.82 (1H, d,
J ¼ 7.5 Hz), 8.08 (3H, m), 8.34 (1H, d, J ¼ 9.3 Hz); 13C NMR (CDCl3)
d
14.5 (Me), 60.1 (CH2), 107.6 (C), 117.6 (CH), 120.1 (CH), 125.0 (C),
125.3 (CH), 128.0 (C), 128.4 (2ꢂ CH), 128.6 (CH), 128.8 (CH), 128.9
(CH),129.4 (CH), 130.1 (2ꢂ CH),132.8 (CH), 133.1 (C),138.3 (C), 140.2
(C), 164.0 (C]O), 184.8 (C]O); MS (ESI-MS, positive ion) m/z 366
[M þ Na]þ.
4.1.14. 3-Benzoyl-pyrrolo[2,1-a]isoquinoline-1-carbonitrile (9a)
White solid, mp 198e200 ꢀC; IR (KBr, nmax) 2221, 1628,
1344 cmꢁ1; 1H NMR (CDCl3)
d 7.29 (1H, m), 7.55 (3H, m), 7.63 (1H,
m), 7.71 (2H, m), 7.84 (3H, m), 8.97 (1H, d, J ¼ 4.5 Hz), 9.56 (1H, d,
J ¼ 7.2 Hz); 13C NMR (CDCl3)
d 85.7 (C), 115.7 (CH), 117.0 (C), 123.5
(C),123.9 (CH), 124.3 (C), 125.1 (CH),127.1 (CH), 128.5 (2ꢂ CH), 128.7
(CH), 128.8 (CH), 129.1 (2ꢂ CH), 129.7 (C), 129.9 (CH), 132.2 (CH),
137.7 (C), 139.0 (C), 185.4 (C]O); MS (ESI-MS, positive ion) m/z 319
[M þ Na]þ.
Fig. 4. Mode of action of drug 10 on in vitro growth of two fungi A. niger and C. albicans.
m), 7.72 (1H, m), 7.81 (2H, d, J ¼ 7.5 Hz), 8.88 (2H, m); 13C NMR
4.1.15. 3-Benzoyl-pyrrolo[2,1-a]isoquinoline-1-carboxylic acid
methyl ester (9b)
(CDCl3) d 13.5 (Me), 13.9 (Me), 61.4 (CH2), 61.6 (CH2), 110.0 (C), 115.7
(CH), 122.7 (C), 123.6 (CH), 124.3 (C), 125.6 (CH), 127.1 (CH), 127.2
(C), 128.2 (CH), 128.4 (2ꢂ CH), 128.9 (CH), 129.2 (2ꢂ CH), 129.5 (C),
132.2 (C),132.7 (CH),139.7 (C),164.3 (C]O),165.5 (C]O),187.1 (C]
O); MS (ESI-MS, positive ion) m/z 438 [M þ Na]þ.
Greenish white solid, mp 183e185 ꢀC; IR (KBr, nmax) 1704, 1617,
1455, 1187 cmꢁ1 1H NMR (CDCl3)
; d 3.92 (3H, s, CH3), 7.30 (1H, d,
J ¼ 7.5 Hz), 7.57 (3H, m), 7.68 (2H, m), 7.83 (4H, m), 9.67 (1H, d,
J ¼ 7.5 Hz), 9.86 (1H, m); 13C NMR (CDCl3)
d 51.8 (Me), 109.7 (eC),
115.5 (CH), 123.3 (C), 124.5 (C), 125.0 (CH), 126.7 (CH), 127.8 (CH),
128.1 (CH), 128.4 (2ꢂ CH), 129.2 (2ꢂ CH), 129.3 (CH), 130.1 (CH),
130.5 (C), 131.7 (CH), 137.0 (C), 139.8 (C), 164.9 (C]O), 185.9 (C]O);
MS (ESI-MS, positive ion) m/z 352 [M þ Na]þ.
4.1.9. 3-Benzoyl-indolizine-2-carbonitrile (7a)
White solid, mp 124e126 ꢀC; IR (KBr, nmax) 2222, 1621, 1479,
1342 cmꢁ1; 1H NMR (CDCl3)
d
7.17 (1H, t, J ¼ 6.6 Hz), 7.52 (3H, m), 7.61
(2H, m), 7.82 (3H, m), 9.96 (1H, d, J ¼ 6.9 Hz); 13C NMR (CDCl3)
d 84.7
(C), 115.1 (C), 115.8 (CH), 117.4 (CH), 122.8 (C), 127.8 (CH), 128.4 (2ꢂ
CH),128.8 (2ꢂ CH),129.2 (CH),129.4 (CH),131.9 (CH),139.1 (C),141.1
(C), 185.0 (C]O); MS (ESI-MS, positive ion) m/z 269 [M þ Na]þ.
4.1.16. 3-Benzoyl-pyrrolo[2,1-a]isoquinoline-1-carboxylic acid ethyl
ester (9c)
Yellowish white solid, mp 138e140 ꢀC; IR (KBr, nmax) 1704, 1623,
1525, 1176 cmꢁ1; 1H NMR (CDCl3)
d
1.40 (3H, t, J ¼ 7.2 Hz, CH3), 4.40
4.1.10. 3-Benzoyl-indolizine-2-carboxylic acid methyl ester (7b)
Greenish white solid, mp 157e159 ꢀC; IR (KBr, nmax) 1698, 1623,
(2H, q, J ¼ 6.9, 14.1 Hz, eCH2), 7.28 (1H, d, J ¼ 7.8 Hz), 7.61 (5H, m),
7.77 (1H, m), 7.82 (1H, s), 7.87 (2H, m), 9.66 (1H, d, J ¼ 7.5 Hz), 9.83
1341, 1209 cmꢁ1 1H NMR (CDCl3)
; d 3.90 (3H, s, CH3), 7.11 (1H, t,
(1H, m); 13C NMR (CDCl3)
d 14.4 (Me), 60.5 (CH2), 110.1 (C), 115.4
J ¼ 6.3 Hz), 7.53 (4H, m), 7.81 (3H, m), 8.40 (1H, d, J ¼ 8.7 Hz), 9.98
(CH), 123.2 (C), 124.4 (C), 124.9 (CH), 126.6 (CH), 127.6 (CH), 128.0
(CH), 128.3 (2ꢂ CH), 129.2 (3ꢂ CH), 129.9 (CH), 130.4 (C), 131.6 (CH),
136.8 (C), 139.8 (C), 164.5 (C]O),185.8 (C]O); MS (ESI-MS, positive
ion) m/z 366 [M þ Na]þ.
(1H, d, J ¼ 6.9 Hz); 13C NMR (CDCl3)
d 51.2 (OMe), 105.8 (C), 115.3
(CH), 119.3 (CH), 122.4 (C), 127.7 (CH), 128.3 (2ꢂ CH), 128.8 (2ꢂ CH),
128.9 (CH), 129.1 (CH), 131.4 (CH), 139.7 (C), 139.8 (C), 164.3 (C]O),
185. 5 (C]O); MS (ESI-MS, positive ion) m/z 302 [M þ Na]þ.
4.1.17. 3-Benzoyl- 2-phenyl-pyrrolo[2,1-a]isoquinoline (10)
4.1.11. 3-Benzoyl-indolizine-2-carboxylic acid ethyl ester (7c)
White solid, mp 81e83 ꢀC; IR (KBr, nmax) 1696, 1613, 1343,
Yellow solid, mp 191e193 ꢀC; IR (KBr, nmax) 1605, 1402,
1340 cmꢁ1 1H NMR (CDCl3)
; d 7.06 (6H, m), 7.17 (4H, m), 7.55 (4H,
1215 cmꢁ1; 1H NMR (CDCl3)
d
1.40 (3H, t, J ¼ 6.9 Hz, CH3), 4.38 (2H, q,
m), 7.71 (1H, m), 8.18 (1H, m), 9.30 (1H, d, J ¼ 7.5 Hz); 13C NMR
J ¼ 6.6, 13.8 Hz), 7.10 (1H, m), 7.51 (4H, m), 7.82 (3H, m), 8.40 (1H, d,
J ¼ 8.7 Hz), 9.98 (1H, d, J ¼ 6.9 Hz); 13C NMR (CDCl3)
d
14.4 (Me), 60.0
(CDCl3)
d 104.0 (CH), 113.4 (CH), 122.4 (C), 123.8 (CH), 125.1 (C),
125.7 (CH), 127.0 (CH), 127.3 (CH), 127.9 (2ꢂ CH), 128.0 (2ꢂ CH),
128.1 (CH), 128.3 (CH), 129.3 (C), 130.2 (2ꢂ CH), 130.5 (2ꢂ CH), 131.6
(CH), 135.2 (C), 136.2 (C), 138.5 (C), 140.1 (C), 187.9 (C]O); MS (ESI-
MS, positive ion) m/z 370 [M þ Na]þ.
(CH2),106.1 (C),115.1 (CH),119.3 (CH),122.3 (C),127.5 (CH),128.2 (2ꢂ
CH), 128.8 (3ꢂ CH), 129.0 (CH), 131.3 (CH), 139.7 (C), 139.8 (C), 163.9
(C]O), 185.4 (C]O); MS (ESI-MS, positive ion) m/z 316 [M þ Na]þ.
4.1.12. 1-Benzoyl-pyrrolo[1,2-a]quinoline-2-carboxylic acid methyl
ester (8a)
4.1.18. Crystal data for 9c
Yellowish white solid, mp 154e156 ꢀC; IR (KBr, nmax) 1705, 1624,
C22H17NO3, Mr ¼ 343.37, greenish rectangular shaped crystals
were grown from chloroforme-hexane. Space group Triclinic P-1.
Lattice constants (Aꢀ): a ¼ 8.4125(6), b ¼ 9.9415(7), c ¼ 11.0268(8),
1452, 1234 cmꢁ1; 1H NMR (CDCl3)
d 3.90 (3H, s, CH3), 7.54 (4H, m),
7.64 (1H, s), 7.69 (2H, m), 7.82 (1H, d, J ¼ 7.8 Hz), 8.09 (3H, m), 8.33
(1H, d, J ¼ 9.3 Hz); 13C NMR (CDCl3)
d 51.3 (OMe), 107.2 (eC), 117.5
a
¼ 65.643(4),
b
¼ 84.421(4),
g
¼ 87.463(4), cell volume V ¼ 836.13
(10) Aꢀ3, formula units/cell Z ¼ 2, Number of independent reflec-
(CH),120.1 (CH),125.0 (C),125.4 (CH),128.1 (C),128.4 (2ꢂ CH),128.6
(CH), 128.8 (CH), 129.0 (CH), 129.5 (CH), 130.1 (2ꢂCH), 132.8 (CH),
133.0 (C), 138.3 (C), 140.2 (C), 164.4 (C]O), 184.8 (C]O); MS (ESI-
MS, positive ion) m/z 352 [M þ Na]þ.
tions 2935, after convergence R1 ¼ 0.0430, wR2 ¼ 0.1219.
4.1.19. Crystal data for 10
C25H17NO, Mr ¼ 347.40, yellowish-green rambohedral shaped
4.1.13. 1-Benzoyl-pyrrolo[1,2-a]quinoline-2-carboxylic acid ethyl
ester (8b)
crystals were grown from chloroforme-hexane. Space group
Monoclinic P21/c. Lattice constants (Aꢀ):
a
¼
18.6732(15),
Yellowish white solid, mp 164e166 ꢀC; IR (KBr, nmax) 1690, 1629,
b ¼ 13.1118(12), c ¼ 7.4589(8),
a
¼ 90,
b
¼ 98.697(7),
g
¼ 90, cell
1453, 1210 cmꢁ1; 1H NMR (CDCl3)
d
1.39 (3H, t, J ¼ 6.9 Hz, CH3), 4.38
volume V ¼ 1805.2(3) Aꢀ3, formula units/cell Z ¼ 4, Number of

A. Hazra et al. / European Journal of Medicinal Chemistry 46 (2011) 2132e2140
2139
independent reflections 3181, after convergence R1 ¼ 0.0759,
wR2 ¼ 0.1942.
inoculated (2 ꢂ 106 cfu/spot for bacteria and 2 ꢂ 105 spores/spot for
fungi). The inoculated plates were then incubated at 37 ꢀC for 24 h
for bacteria and 28 ꢀC for 96 h for fungi. The lowest concentration of
tube or plate which did not show any visible growth after macro-
scopic evaluation was considered as the MIC.
4.2. Pharmacological studies
4.2.1. Materials and methods
4.2.1.1. Microorganisms. The microorganisms used in this study
consisted of 13 strains of bacteria namely: B. subtilis UC564,
S. aureus 25923, S. faecalis 29212, M. luteus AGD1, E. coli ATCC25938,
K. pneumoniae J/I/4, P. aeruginosa ATCC27853, V. cholera 7201, V.
parahaemolyticus 72016, S. dysenteriae 3, Shigella flexnarae DN13, S.
typhi DIRW, and S. typhimurium 11. Antifungal studies were also
carried out on four strains of fungi namely, A. niger, C. albicans,
C. tropicalis, and C. neoformans. All the strains were clinical isolates
from human beings. The strains were identified using Barrow and
Feltham’s method [26]. These were obtained from Division of
Microbiology, Department of Pharmaceutical Technology, Jadavpur
University, Kolkata-32, India. The bacterial strains were grown in
MuellereHinton Agar (Merck India Ltd.) at 37 ꢀC for 24 h while the
fungi were grown on Sabouraud dextrose agar at 28 ꢀC for 3e5 days.
4.2.1.6. Determination of minimal bactericidal concentration (MBC)
and minimum fungicidal concentration (MFC). The test drugs (4a,
4b, 7a and 10), which exhibited considerable antibacterial and
antifungal activity, were diluted double fold with MuellereHinton
broth for bacterial strains and Sabouraud dextrose broth for fungi in
a series of test tubes. An aliquot of 1 ml of the bacterial suspension
(2 ꢂ 106 cfu/ml) and fungal spores (2 ꢂ 105 spores/ml) were inoc-
ulated into each tube. The control tubes were inoculated with same
quantity of broth culture only. All tubes were incubated at 37 ꢀC for
24 h and 28 ꢀC for 96 h with shaking on a platform shaker at
200 rpm. The test drugs were added to the mid-logarithmic phase
of growth and aliquots of 1.0 ml were withdrawn for determination
of colony count [31] while the growth of the fungi was determined
by dry weight of the sample at 60ꢀc for 20 h for 3 days [32].
4.2.1.2. Preparation of inoculums. Active cultures for experiments
were prepared by transferring a loopful of inoculum from the stock
cultures to MuellereHinton Broth (MHB) for bacteria and Sabour-
aud dextrose broth (SDB) for fungi that were incubated without
stirring for 24 h at 37 ꢀC and for 72 h at 28 ꢀC respectively. The
cultures were diluted with sterile normal saline to achieve the
surface viable counting [27] corresponding to 2.0 ꢂ 106 colony
forming units (CFU/ml) for bacteria and 2.0 ꢂ 105 spore/ml for fungi
strains.
Acknowledgments
The authors express their gratitude to the Director, IICB for
laboratory facilities, the Council of Scientific and Industrial
Research, Government of India for providing fellowship to A.H.,
S.M., A.M., S.N., P.S., R.P., K.B.S. and P.P. Our thanks are also due to Dr.
B. Achari (Emeritus Scientist, CSIR) for helpful suggestions and Dr.
R. Mukherjee and Mr. K. Sarkar for recording the spectra.
Appendix. Supplementary data
4.2.1.3. Preparation of stock solution. The compounds, 2aeb, 4aeb,
6aeb, 7aec, 8aeb, 9aec and 10, screened for their antimicrobial
activity were dissolved either in Tween 80 or in propylene glycol.
6a, 9b, 6b, and 9c were dissolved in 4% Tween 80 and the remaining
ones were dissolved in 4% of propylene glycol to get the concen-
tration of 1 mg/ml and were used as stock solution.
1H and 13C NMR spectra of all compounds associated with this
article can be found in the online version. Crystallographic data in
CIF format are available free of charge via the Internate at CCDC
767293 and 767294. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ,
UK; fax: þ44 1223 336033; or deposit@ccdc.cam.ac.uk).
4.2.1.4. Antimicrobial assay. Antimicrobial sensitivity tests were
performed by disc diffusion method following the NCCLS protocol
[28]. For sensitivity testing 0.1 ml of bacterial suspension
(2 ꢂ 106 cfu/ml) and 0.1 ml of fungal spore suspension (2 ꢂ 105
spores/ml) were transferred to freshly prepared MuellereHinton
Agar plates and Sabouraud dextrose plates respectively. Then
sterile paper discs (6 mm diameter) impregnated into prepared
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.066.
solution of the compounds at concentrations of 1e1000
mg/ml for
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