Synthesis, characterization and biological activity of some new carbostyril bearing 1H-pyrazole moiety

Article abstract of DOI:10.1007/s00044-011-9693-2

In this study, 16 new 4-pyrazolyl-N-arylquinoline-2,5-dione 4 have been synthesized by the multicomponent reaction of pyrazole-4-carbaldehyde 1, Meldrum's acid 2 and 3-aryl-5,5-disubstitutedcyclohex-2-enone 3. The structures of compounds 4 were established by the combined use of ¹HNMR, ¹³C NMR, FT-IR and mass spectra. All the 16 compounds were tested in vitro for their bacterial and fungal activity against a list of human pathogens, namely, Bacillus subtilis, Clostridium tetani, Streptococcus pneumoniae, Salmonella typhi, Vibrio cholerae, Escherichia coli, Aspergillus fumigatus and Candida albicans, using broth microdilution MIC (minimum inhibitory concentration) method. Some of the compounds were found to be the most effective analogs against the tested bacterial and fungal strains. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9693-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1751–1761
DOI 10.1007/s00044-011-9693-2
ORIGINAL RESEARCH
Synthesis, characterization and biological activity of some new
carbostyril bearing 1H-pyrazole moiety
•
Nilesh J. Thumar Manish P. Patel
Received: 6 May 2010 / Accepted: 1 June 2011 / Published online: 11 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, 16 new 4-pyrazolyl-N-arylquino-
line-2,5-dione 4 have been synthesized by the multicompo-
nent reaction of pyrazole-4-carbaldehyde 1, Meldrum’s acid
2 and 3-aryl-5,5-disubstitutedcyclohex-2-enone 3. The
structures of compounds 4 were established by the combined
use of 1H NMR, 13C NMR, FT-IR and mass spectra. All the
16 compounds were tested in vitro for their bacterial and
fungal activity against a list of human pathogens, namely,
Bacillus subtilis, Clostridium tetani, Streptococcus pneu-
moniae, Salmonella typhi, Vibrio cholerae, Escherichia coli,
Aspergillus fumigatus and Candida albicans, using broth
microdilution MIC (minimum inhibitory concentration)
method. Some of the compounds were found to be the most
effective analogs against the tested bacterial and fungal
strains.
decades constitutes a substantial need for new classes of
antibacterial agents (Chopra et al., 2008).
Carbostyril (2-quinolone) ring system represents an
important class of compounds not only for their theoretical
interest but also for their antimicrobial (Jayashree et al.,
2010; Creaven et al., 2010), antiproliferative (Chen et al.,
2007), antithrombotic (Buchanan et al., 1989), antihepatitis
B (Cheng et al., 2008), anti-HIV (Zhang et al., 2003),
antitumor (Joseph et al., 2002), antioxidative activity
(Chung and Woo, 2001) and cytotoxicity against human
tumor cell lines (He et al., 2005). Several carbostyril
derivatives are also nitric oxide production inhibitors (Ito
et al., 2004), 5-HT1B/5-HT2A receptor antagonists (McCort
et al., 2001), Rho-kinase inhibitors (Letellier et al., 2008),
androgen receptor (Oeveren et al., 2007), p38aMAP kinase
inhibitors (Peifer et al., 2008) and induce mitochondrial
dysfunction (Chilin et al., 2005).
Keywords Carbostyril ꢀ 1,3-Diarylpyrazole ꢀ
Antibacterial activity ꢀ Antifungal activity ꢀ
Broth-microdilution method
Recently, there has been considerable amount of pro-
gress in 1,3-diaryl pyrazole chemistry because of the rec-
ognition of importance of the pyrazole structure in
biological processes as antimicrobial (Damljanovic et al.,
2009), antiinflammatory (Bekhit et al., 2008), antituber-
cular (Chovatia et al., 2007), antitumor (Joksovic et al.,
2009), antiangiogenesis (Abadi et al., 2003), antiparasitic
(Rathelot et al., 2002), antiviral (Hashem et al., 2007) and
also possesses analgesic and anxiolytic activity (Shetty and
Bhagat, 2008).
Introduction
The treatment of microbial infections still remains an
important and challenging therapeutic problem because of
factors that include emerging infectious diseases and the
increasing number of multidrug-resistant microbial patho-
gens. In spite of the large number of antibiotics and che-
motherapeutics available for medical use, the emergence of
old and new antibiotic resistant bacterial strains in the last
The classic MCR approach for N-substituted-2-quino-
lones involves base-catalyzed cyclization of aldehyde,
Meldrum’s acid and b-enaminone. Literature survey (Wang
et al., 2007; Tu et al., 2001, 2006; Suarez et al., 1999) reveals
few studies concerning N-substituted quinolin-2-one deriv-
atives of aromatic aldehyde, but not a single reference where
1H-1,3-diaryl pyrazole-4-carbaldehydes are used and eval-
uated for their antimicrobial potency.
N. J. Thumar ꢀ M. P. Patel (&)
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar 388120, Gujarat, India
e-mail: patelmanish1069@yahoo.com
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Med Chem Res (2012) 21:1751–1761
It must be emphasized that combination of carbostyril
microdilution MIC (minimum inhibitory concentration)
method (NCCLS 2002, 2007a, b).
template with other heterocycles is a well-known approach
for drug-like molecules’ build-up, which allows achieving
new pharmacological profile, action strengthening or tox-
icity lowering. As part of our ongoing research in discovery
of new active antimicrobial compounds (Makawana et al.,
2011; Ladani et al., 2009a, b, 2010; Mungra et al., 2009,
2011; Nirmal et al., 2009, 2010; Shah et al., 2009a, b; Shah
and Patel, 2011; Thakor et al., 2008; Thumar and Patel,
2009a, b, 2011), in this article, we try to study the influence
of carbostyril moiety and pyrazole scaffold combination on
the antimicrobial effect. So we report herein the synthesis
of some new derivatives of 4-pyrazolyl-N-arylquinoline-
2,5-dione 4a–p through MCR approach. Newly synthe-
sized compounds were characterized by elemental analysis,
Results and discussion
Chemistry
The target molecules 4-pyrazolyl-N-(4-fluorophenyl)-quino-
line-2,5-dione 4 were synthesized in moderate to good yield
(50–77%)bytheone-pot three-componentcyclocondensation
reaction of 1H-1-phenyl-3-(het)aryl-pyrazole-4-carbalde-
hyde 1, Meldrum’s acid 2 and 3-(4-fluorophenylamino)-5,
5-disubstitutedcyclohex-2-enone 3 in the presence of piperi-
dine as catalyst using acetonitrile as solvent (Scheme 1). A
mechanism for the formation of the quinoline derivatives
4a–p is outlined in Scheme 2. The reaction occurs through an
in situ initial formation of the 5-heterylidene-2,2-dimethyl-
1,3-dioxane-4,6-dione, containing the electron-poor C=C
double bond, from the Knoevenagel condensation between
1H-pyrazole-4-carbaldehyde and Meldrum’s acid by loss of
water molecules. Finally, Michael addition of 3 to the initially
formed unsaturated dione, i.e., nucleophilic attack of
1
FT-IR, H NMR, 13C NMR and mass spectrometry. All
compounds were screened for in vitro antimicrobial
activity against eight human pathogens of which three
Gram-positive bacterial pathogens Streptococcus pneumo-
niae, Clostridium tetani and Bacillus subtilis, three Gram-
negative bacterial pathogens Salmonella typhi, Vibrio
cholerae and Escherichia coli and two fungal pathogens
Aspergillus fumigatus and Candida albicans, using broth
Scheme 1 Synthetic pathway for the synthesis of carbostyril derivatives of 1H-pyrazole 4a–p
Scheme 2 Plausible
mechanistic pathway of the
synthesis of quinolone
derivatives
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Med Chem Res (2012) 21:1751–1761
1753
enaminone to the heterylidene olefins gives intermediate
which on intramolecular cyclization losses acetone and CO2
to afford cyclized quinoline-2,5-dione derivatives 4a–p.
The structures of all the new compounds were estab-
lished by 1H NMR, 13C NMR, mass and FT-IR spec-
(Ar=4-CH3–C6H4, R=CH3) were found to exhibit compa-
rable activity to ampicillin (100 lg/ml). The compounds
4b (Ar=4-Br–C6H4, R=H), 4h (Ar=2-Thienyl, R=H), 4l
(Ar=4-F–C6H4, R=CH3), 4m (Ar=4-OCH3–C6H4, R=CH3)
and 4n (Ar=4-CH3–C6H4, R=CH3) found to be more effi-
cient, whereas 4c (Ar=4-Br–C6H4, R=H) was found equally
potent to ampicillin (250 lg/ml) towards C. tetani. The
compounds 4d (Ar=4-F–C6H4, R=H), 4g (Ar=4-NO2–
C6H4, R=H), 4i (Ar=C6H5, R=CH3), 4k (Ar=4-Cl–C6H4,
R=CH3) and 4m (Ar=4-OCH3–C6H4, R=CH3) show better
activity, whereas 4c (Ar=4-Br–C6H4, R=H), 4f (Ar=4-
CH3–C6H4, R=H), 4h (Ar=2-Thienyl, R=H), 4o (Ar=4-
NO2–C6H4, R=CH3) and 4p (Ar=2-Thienyl, R=CH3) found
equally potent to ampicillin (250 lg/ml), against B. sub-
tilis. Towards Gram-negative strain S. typhi, compounds 4b
(Ar=4-Br–C6H4, R=H), 4g (Ar=4-NO2–C6H4, R=H) and 4h
(Ar=2-Thienyl, R=H) were equally active to ampicillin
(100 lg/ml), whereas compound 4k (Ar=4-Cl–C6H4,
R=CH3) was found to be potent than ampicillin (100 lg/ml).
The compounds 4d (Ar=4-F–C6H4, R=H) and 4k (Ar=4-
Cl–C6H4, R=CH3) are found to exhibit better activity,
whereas 4h (Ar=2-Thienyl, R=H) and 4p (Ar=2-Thienyl,
R=CH3) found equipotent to ampicillin (100 lg/ml) against
V. cholerae. The compounds 4b (Ar=4-Br–C6H4, R=H),
4g (Ar=4-NO2–C6H4, R=H) and 4k (Ar=4-Cl–C6H4,
R=CH3) show better and 4c (Ar=4-Br–C6H4, R=H),
4e (Ar=4-OCH3–C6H4, R=H), 4h (Ar=2-Thienyl, R=H)
and 4o (Ar=4-NO2-C6H4, R=CH3) were found to exhibit
comparable activity to ampicillin (100 lg/ml) towards
E. coli.
1
trometry. H NMR (DMSO-d6) spectrum of 4a, molecule
of interest, exhibited doublet at d 4.50 for methine (H4) and
doublet at d 2.78 and doublet of doublets at d 6.03 ppm for
methylene (CH2) of the quinolone ring, respectively.
Aromatic protons resonate as multiplets appeared at around
d 7.02–7.93 ppm and a deshielded singlet, apart from
aromatic protons, at d 8.31 ppm is of H5 of pyrazole ring.
Moreover, multiplets of three methylene groups at d
1.72–2.23 ppm of the cyclohexanone ring, respectively.
The 13C NMR spectrum of all the compounds was in good
agreement with the structure assigned. The peaks at d
22.59, 28.02 and 35.97 ppm are assigned to three methy-
lene carbons of the cyclohexanone ring. A signal at d
25.86 ppm stands for methine carbon C4 of quinoline ring.
The distinctive peaks at d 169.62 (C2) and 195.57 (C5)
ppm are assigned to carbonyl carbons of quinolone ring.
All the aromatic carbons showed signals around d
114.93–156.41 ppm in the 13C NMR spectra confirms the
structure 4a. The IR spectrum of compound 4a exhibited
characteristic absorption band at 1,710 and 1,645 cm-1 for
both carbonyl (C=O) functional group of carbostyril skel-
eton, respectively. The mass spectra of compounds 4l and
4m, molecules of interest, detected the expected molecular
ion signals corresponding to respective molecular formula,
i.e., mass spectra of compound 4l (Ar=4-F–C6H4, R=CH3)
and 4m (Ar=4-OCH3–C6H4, R=CH3) gave molecular ion
peak at m/z 524.0 (M??H) and m/z 536.1 (M??H) cor-
responding to molecular formula C32H27F2N3O2 and
C33H30FN3O3, respectively. The obtained elemental anal-
ysis values are in good agreement with theoretical data.
Similarly, all the compounds were characterized on the
basis of spectral studies. All spectroscopic data have been
given in experimental section. All the compounds were
screened for their antibacterial and antifungal activity.
Against fungal pathogen C. albicans, compound 4j
found better active, whereas 4b–d (Ar=4-Br/Cl/F–C6H4,
R=H), 4g–i (Ar=4-NO2–C6H4/2-thienyl, R=H and Ar=
C6H5, R=CH3), 4l (Ar=4-F–C6H4, R=CH3), 4n (Ar=4-
CH3–C6H4, R=CH3) and 4p (Ar=2-Thienyl, R=CH3) were
found to be equipotent compared with the standard gris-
eofulvin (500 lg/ml). Only one of the tested compounds,
4j (Ar=4-Br–C6H4, R=CH3), found to be potent against
fungal strains, C. albicans and A. fumigatus, compared with
the standard drug nystatin (100 lg/ml). The remaining
compounds showed moderate to good activity to inhibit the
growth of bacterial pathogens and are all less effective than
standard drugs.
Antimicrobial screening
The examination of the data (Table 1) reveals that most of
the compounds showed antibacterial and antifungal activity
when compared with standard drugs ampicillin and gris-
eofulvin. Compounds 4g (Ar=4-NO2–C6H4, R=H), 4h
(Ar=2-Thienyl, R=H) and 4k (Ar=4-Cl–C6H4, R=CH3)
were found to be highly potent against most of the
employed strains to inhibit the growth of organism. In
particular, against Gram-positive pathogen S. pneumoniae,
compounds 4f (Ar=4-CH3–C6H4, R=H), 4g (Ar=4-NO2–
C6H4, R=H), 4m (Ar=4-OCH3–C6H4, R=CH3) and 4n
The investigation of the structure–activity relationship
(SAR) of antibacterial screening revealed that the com-
pounds with p-bromo, methoxy or methyl phenyl at the
3-position of the pyrazole nucleus gave equivalent results
against S. pneumoniae. Whereas towards C. tetani, com-
pounds with thienyl or p-bromo, fluoro or methyl phenyl
substituents were found to be highly active. Against B.
subtilis, p-chloro or fluoro phenyl containing compounds
were found to be more potent. Compounds containing
thienyl, p-bromo, chloro or nitro substituents have
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Med Chem Res (2012) 21:1751–1761
Table 1 Antimicrobial activity of the compounds 4a–p
Compds
Minimum inhibitory concentration (MIC, lg l-1
)
Gram-positive bacteria
Gram-negative bacteria
Fungi
Streptococcus
pneumoniae
MTCC 1936
Clostridium
tetani
MTCC 449
Bacillus
subtilis
MTCC 441
Salmonella
typhi
MTCC 98
Vibrio
cholerae
MTCC 3906
Escherichia
coli
MTCC 443
Aspergillus
fumigatus
MTCC 3008
Candida
albicans
MTCC 227
4a
500
250
250
200
250
100
100
200
150
250
250
500
100
100
250
500
100
50
500
100
250
500
1,000
500
500
100
1,000
1,000
500
50
500
500
250
100
500
250
200
250
200
500
150
500
200
500
250
250
250
50
500
100
250
500
250
250
100
100
200
250
50
1,000
250
250
25
500
62.5
100
250
100
250
62.5
100
500
500
50
[1,000
[1,000
[1,000
1,000
500
[1,000
500
500
500
1,000
1,000
500
500
500
100
1,000
500
1,000
500
1,000
500
–
4b
4c
4d
4e
500
1,000
1,000
100
1,000
500
50
4f
500
4g
1,000
1,000
500
4h
4i
4j
100
4k
500
4l
250
250
500
250
250
100
50
500
500
200
500
100
100
50
150
250
500
100
200
100
50
1,000
1,000
500
4m
4n
100
200
500
500
250
50
4o
1,000
1,000
–
4p
Amp.
Chlor.
Cipro.
Genta.
Gris.
Nys.
–
–
50
100
5
50
25
25
25
–
–
0.5
–
1
5
5
0.05
–
–
–
–
–
–
–
100
500
100
–
–
–
–
–
–
100
Amp. ampicillin, chlor. chloramphenicol, cipro. ciprofloxacin, genta. gentamicin, gris. Griseofulvin, nys. nystatin
exhibited better activity against S. typhi. Moreover, against
V. cholerae, compounds containing p-chloro or fluoro
phenyl substituents revealed potent inhibitory action.
Whereas towards E. coli, compounds with p-bromo, chloro
or nitro phenyl substituents were found to be more active.
Antifungal evaluation shown that p-bromophenyl ring
containing compound is found to be most potent among all
against fungal species both C. albicans and A. fumigatus.
From the SAR study of the title derivatives, it is interesting
to note that a minor alteration in the molecular configura-
tion of the investigated compounds may have a pronounced
effect on antimicrobial activity.
MCR approach and characterized through elemental and
spectral analysis. This synthetic strategy allows the con-
struction of relatively complicated nitrogen-containing
fused heterocyclic system as well as the introduction of
various (hetero)aromatic substitutions into 1- and 4-posi-
tion of quinoline system. It can be concluded from anti-
microbial screening (Table 1), against panel of human
pathogens, that most of the synthesized quinoline deriva-
tives were found to be highly active, compared with the
standard drugs against bacterial pathogens. It is worth
mentioning that minor changes in molecular configuration
of these compounds profoundly influence the activity.
Furthermore, synthetic work to intensify the potency of
carbostyril derivatives by changing molecular configura-
tion is in progress at our laboratory. This study throws light
on the identification of this new structural class as anti-
microbials that can be of interest for further detailed pre-
clinical investigations.
Conclusion
A new series of substituted 4-pyrazolyl-N-arylquinoline-
2,5-dione 4a–p derivatives has been synthesized through
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Med Chem Res (2012) 21:1751–1761
Experimental
1755
Switzerland) using solvent peak as internal standard at
400 MHz and 100 MHz, respectively. Chemical shifts are
reported in parts per million (ppm). Splitting patterns were
designated as follows: s singlet, d doublet, dd doublet of
doublet and m multiplet. Mass spectra were scanned on a
Shimadzu LCMS 2010 spectrometer (Shimadzu, Tokyo,
Japan). The compound of interest 1H-3-aryl-1-phenylpyr-
azole-4-carbaldehyde 1a–h was prepared according to liter-
ature procedure Kira et al., (1969), by Vilsmeier–Haack
reaction of acetophenone (het)arylhydrazones (Scheme 3).
Meldrum’s acid (2,2-dimethyl-1,3-dioxane-4,6-dione) was
synthesized according to literature method (Davidson and
Bernhard, 1948), by a condensation reaction of malonic acid
with acetone in acetic anhydride with sulfuric acid at 0°C
(Scheme 3). 3-(4-Fluorophenylamino)-5,5-(un)substitutedc-
yclohex-2-enones were synthesized according to literature
procedure (Chanda et al., 2004) by the solid-phase reaction of
1,3-dicarbonyl compound and 4-fluoroaniline under micro-
wave irradiation (Scheme 3).
Chemistry
General procedures
Required acetophenone, acetic anhydride, aniline, malonic
acid, phenyl hydrazine and phosphorous oxychloride were
obtained from S. D. Fine Chem Ltd., Vadodara, Gujarat,
India. Moreover, cyclohexanedione and dimedone were
obtained from Sigma–Aldrich and were used without further
purification. Solvents were purified and dried before being
used. The microwave-assisted reactions are conducted in a
‘‘RAGA’s Modified Electromagnetic Microwave System’’
whereby microwaves are generated by magnetron at a fre-
quency of 2,450 MHz having an adjustable output power
levels, i.e., 10 levels from 140 to 700 W and with an indi-
vidual sensor for temperature control (fiber optic is used as a
individual sensor for temperature control) with attachment of
reflux condenser with constant stirring (thus avoiding the risk
of high pressure development). All melting points were taken
in open capillaries and are uncorrected. Thin-layer chroma-
General procedure for 4-(3-(het)aryl-1-phenyl-1H-
pyrazol-4-yl)-1-(4-fluorophenyl)-7,7-disubstituted-
3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-dione (4a–p)
tography (TLC, on aluminium plates precoated with silica
60
gel,
F , 0.25-mm thickness) (Merck, Darmstadt, Ger-
254
many) was used for monitoring the progress of all reactions,
purity and homogeneity of the synthesized compounds; elu-
ent-toluene:ethyl acetate::7:3. UV radiation and/or iodine
were used as the visualizing agents. Elemental analysis (% C,
H, N) was carried out by Perkin-Elmer 2400 series-II ele-
mental analyzer (Perkin-Elmer, USA) and all compounds are
within 0.4% of theory specified. The IR spectra were
recorded in KBr on a Perkin-Elmer Spectrum GX FT-IR
Spectrophotometer (Perkin-Elmer, USA) and only the char-
acteristic peaks are reported in cm-1. 1H NMR and 13C NMR
spectra were recorded in DMSO-d6 on a Bruker Avance 400F
(MHz) spectrometer (Bruker Scientific Corporation Ltd.,
A mixture of 1H-3-aryl-1-phenylpyrazole-4-carbaldehyde
1a–h (30 mmol), Meldrum’s acid 2 (30 mmol) and
appropriate b-enaminone 3a–b (30 mmol) in acetonitrile
(20 ml) containing three drops of piperidine was slowly
heated and refluxed with stirring for 6 h. On completion
of reaction, monitored by TLC (ethyl acetate:tolu-
ene = 3:7), the reaction mixture was cooled to room
temperature and the solid separated was filtered and
washed with mixture of chloroform and methanol (1:1) to
obtain the pure compounds 4a–p. Physical, analytical and
spectroscopic characterization data of the compounds 4a–
p are given below:
Scheme 3 Synthetic pathway
for the intermediates 1a–h, 2
and 3a–b
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Med Chem Res (2012) 21:1751–1761
4-(1,3-Diphenyl-1H-pyrazol-4-yl)-1-(4-fluorophenyl)-
3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-dione (4a)
1-(4-Fluorophenyl)-4-[3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl]-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4d)
Yield: 71%; melting range: 238–240°C; IR (KBr, m, cm-1):
3,020 (ArC–H str.), 1,710 (C=O str.), 1,645 (C=O str.); H
Yield: 75%; melting range: 218–220°C; IR (KBr, m, cm-1):
3,010 (ArC–H str.), 1,705 (C=O str.), 1,655 (C=O str.); H
1
1
NMR (400 MHz, DMSO-d6) dH (ppm): 1.72–2.23 (m, 6H,
3 9 CH2), 2.78 (d, J = 16.4 Hz, 1H, H3), 3.13 (dd,
J = 15.6 Hz, J0 = 7.2 Hz, 1H, H3), 4.50 (d, J = 8.0 Hz,
1H, H4), 7.02–7.93 (m, 14H, Ar–H), 8.31 (s, 1H, pyrazole
H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm): 22.59
(C8), 25.86 (C4), 28.02 (C7), 35.97 (C3), 37.90 (C6),
114.93, 116.34, 117.66, 118.85, 125.40, 126.01, 126.19,
128.73, 128.82, 129.99, 130.05, 131.67, 136.82, 140.16,
145.18, 150.04, 156.41 (Ar–C), 169.62 (C2), 196.26 (C5);
Anal. Calcd. for C30H24FN3O2 (477.53 g/mol): C, 75.46;
H, 5.07; N, 8.80%. Found: C, 75.63; H, 4.81; N, 9.02%.
NMR (400 MHz, DMSO-d6) dH (ppm): 1.95–2.39 (m, 6H,
3 9 CH2), 2.72 (d, J = 15.2 Hz, 1H, H3), 3.17 (dd,
J = 16.0 Hz, J0 = 8.4 Hz, 1H, H3), 4.45 (d, J = 8.8 Hz,
1H, H4), 7.05–7.95 (m, 13H, Ar–H), 8.34 (s, 1H, pyrazole
H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm): 22.15
(C8), 25.95 (C4), 27.85 (C7), 36.53 (C3), 38.51 (C6),
114.13, 115.62, 116.15, 118.38, 124.95, 125.04, 126.80,
127.19, 127.97, 128.53, 128.64, 130.16, 136.79, 140.29,
146.24, 150.92, 155.89 (Ar–C), 170.02 (C2), 196.11 (C5);
Anal. Calcd. for C30H23F2N3O2 (495.52 g/mol): C, 72.72;
H, 4.68; N, 8.48%. Found: C, 72.51; H, 4.88; N, 8.65%.
4-[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-(4-
fluorophenyl)-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4b)
1-(4-Fluorophenyl)-4-[3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl]-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4e)
Yield: 65%; melting range: 224–226°C; IR (KBr, m, cm-1):
2985 (ArC–H str.), 1,700 (C=O str.), 1,640 (C=O str.); H
Yield: 69%; melting range: 265–267°C; IR (KBr, m, cm-1):
3,015 (ArC–H str.), 1,710 (C=O str.), 1,645 (C=O str.); H
1
1
NMR (400 MHz, DMSO-d6) dH (ppm): 1.78–2.15 (m, 6H,
3 9 CH2), 2.75 (d, J = 16.0 Hz, 1H, H3), 2.97 (dd,
J = 16.4 Hz, J0 = 8.4 Hz, 1H, H3), 4.39 (d, J = 7.2 Hz,
1H, H4), 6.97–7.88 (m, 13H, Ar–H), 8.23 (s, 1H, pyrazole
H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm): 21.76
(C8), 26.47 (C4), 28.67 (C7), 37.25 (C3), 38.72 (C6),
113.02, 115.40, 118.65, 118.71, 124.02, 125.70, 125.81,
126.59, 127.13, 127.17, 130.30, 130.98, 135.31, 139.15,
147.90, 151.33, 155.12 (Ar–C), 168.94 (C2), 195.88 (C5);
Anal. Calcd. for C30H23BrFN3O2 (556.42 g/mol): C, 64.76;
H, 4.17; N, 7.55%. Found: C, 64.43; H, 3.94; N, 7.77%.
NMR (400 MHz, DMSO-d6) dH (ppm): 1.87–2.26 (m, 6H,
3 9 CH2), 2.75 (d, J = 16.4 Hz, 1H, H3), 3.01 (dd,
J = 16.8 Hz, J0 = 8.8 Hz, 1H, H3), 3.89 (s, 3H, OCH3),
4.42 (d, J = 8.0 Hz, 1H, H4), 7.10–7.96 (m, 13H, Ar–H),
8.30 (s, 1H, pyrazole H5). 13C NMR (100 MHz, DMSO-
d6) dC (ppm): 21.88 (C8), 24.84 (C4), 28.33 (C7), 36.34
(C3), 38.40 (C6), 55.84 (OCH3), 113.31, 114.96, 116.05,
117.42, 124.46, 125.37, 126.54, 127.16, 127.23, 128.35,
131.55, 137.61, 141.77, 144.88, 150.28, 155.91 (Ar–C),
159.63 (ArC–OCH3), 169.72 (C2), 196.37 (C5); Anal.
Calcd. for C31H26FN3O3 (507.55 g/mol): C, 73.36; H, 5.16;
N, 8.28%. Found: C, 73.51; H, 4.93; N, 8.49%.
4-[3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-(4-
fluorophenyl)-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4c)
1-(4-Fluorophenyl)-4-(1-phenyl-3-p-tolyl-1H-pyrazol-4-
yl)-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-dione (4f)
Yield: 68%; melting range: 200–201°C; IR (KBr, m, cm-1):
2,975 (ArC–H str.), 1,695 (C=O str.), 1,630 (C=O str.); H
Yield: 65%; melting range: 231–232°C; IR (KBr, m, cm-1):
2,995 (ArC–H str.), 1,705 (C=O str.), 1,640 (C=O str.); H
1
1
NMR (400 MHz, DMSO-d6) dH (ppm): 1.88–2.30 (m, 6H,
3 9 CH2), 2.65 (d, J = 16.0 Hz, 1H, H3), 3.05 (dd,
J = 15.2 Hz, J0 = 8.0 Hz, 1H, H3), 4.54 (d, J = 8.0 Hz,
1H, H4), 6.93–7.92 (m, 13H, Ar–H), 8.20 (s, 1H, pyrazole
H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm): 21.17
(C8), 25.81 (C4), 28.26 (C7), 36.19 (C3), 38.66 (C6),
114.72, 115.30, 117.20, 119.41, 123.94, 125.80, 126.61,
126.74, 127.52, 128.06, 128.93, 129.03, 134.69, 140.14,
145.84, 151.42, 157.30 (Ar–C), 169.57 (C2), 195.94
(C5); Anal. Calcd. for C30H23ClFN3O2 (511.97 g/mol): C,
70.38; H, 4.53; N, 8.21%. Found: C, 70.50; H, 4.72; N,
7.99%.
NMR (400 MHz, DMSO-d6) dH (ppm): 1.79–2.31 (m, 6H,
3 9 CH2), 2.45 (s, 3H, CH3), 2.71 (d, J = 15.6 Hz, 1H, H3),
3.13 (dd, J = 16.4 Hz, J0 = 7.6 Hz, 1H, H3), 4.51 (d,
J = 7.6 Hz, 1H, H4), 7.06–7.83 (m, 13H, Ar–H), 8.24 (s, 1H,
pyrazole H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm):
21.33 (CH3), 22.90 (C8), 26.32 (C4), 28.69 (C7), 36.42 (C3),
37.85 (C6), 113.10, 115.56, 117.95, 118.79, 123.08, 125.20,
125.56, 126.60, 127.67, 128.07, 128.21, 130.18, 137.44,
141.76, 145.17, 151.02, 157.14 (Ar–C), 168.97 (C2), 195.79
(C5); Anal. Calcd. for C31H26FN3O2 (491.56 g/mol): C,
75.75; H, 5.33; N, 8.55%. Found: C, 76.04; H, 5.51; N, 8.34%.
123

Med Chem Res (2012) 21:1751–1761
1757
1-(4-Fluorophenyl)-4-[3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl]-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4g)
C32H28FN3O2 (505.58 g/mol): C, 76.02; H, 5.58; N, 8.31%.
Found: C, 75.87; H, 5.75; N, 8.59%.
4-[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-(4-
fluorophenyl)-7,7-dimethyl-3,4,7,8-tetrahydroquinoline-
2,5(1H,6H)-dione (4j)
Yield: 70%; melting range: 277–280°C; IR (KBr, m, cm-1):
3,000 (ArC–H str.), 1,715 (C=O str.), 1,650 (C=O str.); H
1
NMR (400 MHz, DMSO-d6) dH (ppm): 1.74–2.17 (m, 6H,
3 9 CH2), 2.67 (d, J = 16.4 Hz, 1H, H3), 2.92 (dd,
J = 15.6 Hz, J0 = 8.0 Hz, 1H, H3), 4.48 (d, J = 7.2 Hz,
1H, H4), 7.04–7.93 (m, 13H, Ar–H), 8.25 (s, 1H, pyrazole
H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm): 21.82
(C8), 25.78 (C4), 27.01 (C7), 36.21 (C3), 37.79 (C6),
114.93, 115.38, 116.32, 119.43, 126.21, 126.83, 127.36,
127.58, 128.09, 129.15, 129.32, 130.78, 134.27, 140.84,
146.87, 152.75, 154.09 (Ar–C), 169.44 (C2), 196.32 (C5);
Anal. Calcd. for C30H23FN4O4 (522.53 g/mol): C, 68.96;
H, 4.44; N, 10.72%. Found: C, 69.10; H, 4.20; N, 10.96%.
Yield: 50%; melting range: 242–244°C; IR (KBr, m, cm-1):
3,015 (ArC-H str.), 1,700 (C=O str.), 1,635 (C=O str.); H
1
NMR (400 MHz, DMSO-d6) dH (ppm): 0.89 (s, 3H, CH3),
0.95 (s, 3H, CH3), 2.08–2.39 (m, 4H, 2 9 CH2), 2.74 (d,
J = 15.2 Hz, 1H, H3), 3.12 (dd, J = 16.8 Hz, J0 = 8.4 Hz,
1H, H3), 4.34 (d, J = 7.2 Hz, 1H, H4), 7.01–7.85 (m, 13H,
Ar–H), 8.32 (s, 1H, pyrazole H5). 13C NMR (100 MHz,
DMSO-d6) dC (ppm): 26.34, 26.47 (CH3), 29.15 (C4), 33.61
(C7), 38.45 (C6), 41.93 (C8), 51.66 (C3), 113.98, 115.64,
116.24, 119.58, 125.23, 126.24, 126.47, 128.59, 128.71,
129.34, 129.85, 131.29, 137.53, 140.10, 146.05, 151.35,
156.90 (Ar–C), 170.06 (C2), 195.86 (C5); Anal. Calcd. for
C32H27BrFN3O2 (584.48 g/mol): C, 65.76; H, 4.66; N,
7.19%. Found: C, 65.93; H, 4.85; N, 6.93%.
1-(4-Fluorophenyl)-4-[1-phenyl-3-(thiophen-2-yl)-1H-
pyrazol-4-yl]-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4h)
Yield: 75%; melting range: 196–198°C; IR (KBr, m, cm-1):
3,010 (ArC–H str.), 1,705 (C=O str.), 1,645 (C=O str.); H
4-[3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-(4-
fluorophenyl)-7,7-dimethyl-3,4,7,8-tetrahydroquinoline-
2,5(1H,6H)-dione (4k)
1
NMR (400 MHz, DMSO-d6) dH (ppm): 1.81–2.25 (m,
6H, 3 9 CH2), 2.79 (d, J = 16.8 Hz, 1H, H3), 3.14 (dd,
J = 15.6 Hz, J0 = 7.6 Hz, 1H, H3), 4.36 (d, J = 8.0 Hz,
1H, H4), 6.94–7.87 (m, 12H, Ar–H), 8.23 (s, 1H, pyrazole
H5). 13C NMR (100 MHz, DMSO-d6) dC (ppm): 22.44
(C8), 26.17 (C4), 28.54 (C7), 36.88 (C3), 38.63 (C6),
114.39, 115.94, 116.08, 111.18, 123.22, 125.01, 125.49,
126.85, 127.08, 127.57, 130.45, 130.74, 135.36, 139.26,
147.23, 153.37, 157.49 (Ar–C), 169.71 (C2), 196.15 (C5);
Anal. Calcd. for C28H22FN3O2S (483.56 g/mol): C,
69.55; H, 4.59; N, 8.69%. Found: C, 69.37; H, 4.32;
N, 8.82%.
Yield: 72%; melting range: 186–187°C; IR (KBr, m, cm-1):
2,980 (ArC–H str.), 1,700 (C=O str.), 1,640 (C=O str.); 1H
NMR (400 MHz, DMSO-d6) dH (ppm): 0.87 (s, 3H, CH3),
0.94 (s, 3H, CH3), 2.15–2.41 (m, 4H, 2 9 CH2), 2.76 (d,
J = 16.8 Hz, 1H, H3), 2.96 (dd, J = 16.4 Hz, J0 = 7.2 Hz,
1H, H3), 4.44 (d, J = 8.4 Hz, 1H, H4), 7.00–7.92 (m, 13H,
Ar–H), 8.21 (s, 1H, pyrazole H5). 13C NMR (100 MHz,
DMSO-d6) dC (ppm): 27.03, 27.89 (CH3), 28.72 (C4), 31.73
(C7), 38.63 (C6), 40.15 (C8), 51.84 (C3), 114.45, 116.03,
117.64, 118.51, 125.25, 126.11, 127.27, 127.84, 128.23,
128.50, 128.75, 131.60, 135.33, 138.76, 145.69, 151.89,
155.36 (Ar–C), 169.38 (C2), 196.23 (C5); Anal. Calcd. for
C32H27ClFN3O2 (539.18 g/mol): C, 71.17; H, 5.04; N,
7.78%. Found: C, 70.94; H, 4.86; N, 7.96%.
4-(1,3-Diphenyl-1H-pyrazol-4-yl)-1-(4-fluorophenyl)-
7,7-dimethyl-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4i)
Yield: 63%; melting range: 207–210°C; IR (KBr, m, cm-1):
2,985 (ArC-H str.), 1,695 (C=O str.), 1,635 (C=O str.); H
1-(4-Fluorophenyl)-4-[3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl]-7,7-dimethyl-3,4,7,8-tetrahydroquinoline-
2,5(1H,6H)-dione (4l)
1
NMR (400 MHz, DMSO-d6) dH (ppm): 1.03 (s, 3H, CH3),
1.09 (s, 3H, CH3), 2.18–2.45 (m, 4H, 2 9 CH2), 2.66 (d,
J = 16.0 Hz, 1H, H3), 3.04 (dd, J = 15.2 Hz, J0 = 8.0 Hz,
1H, H3), 4.37 (d, J = 8.4 Hz, 1H, H4), 6.99–7.90 (m, 14H,
Ar–H), 8.29 (s, 1H, pyrazole H5). 13C NMR (100 MHz,
DMSO-d6) dC (ppm): 25.48, 26.96 (CH3), 28.20 (C4), 31.89
(C7), 37.87 (C6), 39.98 (C8), 50.82 (C3), 113.12, 115.26,
116.96, 118.06, 124.17, 125.48, 126.35, 126.62, 128.07,
128.78, 129.37, 130.59, 137.64, 140.43, 146.37, 150.18,
156.49 (Ar–C), 170.11 (C2), 195.83 (C5); Anal. Calcd. for
Yield: 77%; melting range: 234–235°C; IR (KBr, m, cm-1):
3,070 (ArC-H str.), 1,690 (C=O str.), 1,635 (C=O str.); H
1
NMR (400 MHz, DMSO-d6) dH (ppm): 0.91 (s, 3H, CH3),
0.95 (s, 3H, CH3), 2.12–2.36 (m, 4H, 2 9 CH2), 2.68 (d,
J = 15.6 Hz, 1H, H3), 3.15 (dd, J = 16.0 Hz, J0 = 7.2 Hz,
1H, H3), 4.42 (d, J = 8.8 Hz, 1H, H4), 6.95–7.84 (m, 13H,
Ar–H), 8.34 (s, 1H, pyrazole H5). 13C NMR (100 MHz,
DMSO-d6) dC (ppm): 25.61, 26.78 (CH3), 28.39 (C4), 31.18
123

1758
Med Chem Res (2012) 21:1751–1761
(C7), 38.28 (C6), 41.89 (C8), 50.37 (C3), 114.44, 116.19,
117.23, 118.86, 124.72, 125.56, 126.02, 126.17, 127.25,
128.09, 128.46, 131.48. 135.75, 140.50, 143.13, 150.93,
155.27 (Ar–C), 168.92 (C2), 196.20 (C5); Anal. Calcd. for
C32H27F2N3O2 (523.57 g/mol): C, 73.41; H, 5.20; N, 8.03%.
Found: C, 73.57; H, 4.98; N, 7.83%. MS: (M??H) 524.0.
0.94 (s, 3H, CH3), 2.08–2.31 (m, 4H, 2 9 CH2), 2.74 (d,
J = 15.6 Hz, 1H, H3), 3.06 (dd, J = 16.0 Hz, J0 = 8.0 Hz,
1H, H3), 4.38 (d, J = 8.4 Hz, 1H, H4), 6.90–8.23 (m, 13H,
Ar–H), 8.47 (s, 1H, pyrazole H5). 13C NMR (100 MHz,
DMSO-d6) dC (ppm): 26.57, 27.32 (CH3), 28.87 (C4), 33.32
(C7), 37.94 (C6), 41.72 (C8), 51.11 (C3), 114.24, 115.57,
117.96, 119.58, 124.22, 125.48, 125.54, 126.00, 127.15,
128.08, 129.91, 130.03, 136.51, 141.86, 145.09, 151.73,
156.07 (Ar–C), 169.62 (C2), 196.22 (C5); Anal. Calcd. for
C32H27FN4O4 (550.58 g/mol): C, 69.81; H, 4.94; N, 10.18%.
Found: C, 70.07; H, 5.19; N, 9.90%.
1-(4-Fluorophenyl)-4-[3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl]-7,7-dimethyl-3,4,7,8-tetrahydroquinoline-
2,5(1H,6H)-dione (4m)
Yield: 71%; melting range: 258–261°C; IR (KBr, m, cm-1):
3,050 (ArC–H str.), 1,705 (C=O str.), 1,640 (C=O str.); H
1
1-(4-Fluorophenyl)-7,7-dimethyl-4-[1-phenyl-3-(thiophen-
2-yl)-1H-pyrazol-4-yl]-3,4,7,8-tetrahydroquinoline-
2,5(1H,6H)-dione (4p)
NMR (400 MHz, DMSO-d6) dH (ppm): 0.89 (s, 3H, CH3),
0.97 (s, 3H, CH3), 2.06–2.32 (m, 4H, 2 9 CH2), 2.73 (d,
J = 16.0 Hz, 1H, H3), 2.98 (dd, J = 15.2 Hz, J0 = 8.8 Hz,
1H, H3), 3.92 (s, 3H, OCH3), 4.41 (d, J = 7.6 Hz, 1H, H4),
7.08–7.94 (m, 13H, Ar–H), 8.33 (s, 1H, pyrazole H5). 13C
NMR (100 MHz, DMSO-d6) dC (ppm): 25.16, 26.25 (CH3),
28.71 (C4), 32.80 (C7), 38.42 (C6), 39.76 (C8), 52.35 (C3),
53.29 (OCH3), 114.01, 115.22, 116.83, 118.46, 123.67,
125.12, 125.52, 126.49, 127.28, 128.31, 130.67, 137.30,
140.00, 147.32, 150.13, 154.50 (Ar–C), 159.85 (ArC-OCH3),
170.08 (C2), 195.91 (C5); Anal. Calcd. for C33H30FN3O3
(535.61 g/mol):C, 74.00;H, 5.65; N, 7.85%. Found:C, 73.84;
H, 5.45; N, 8.06%. MS: (M??H) 536.1.
Yield: 69%; melting range: 190–92°C; IR (KBr, m, cm-1):
3,000 (ArC–H str.), 1,695 (C=O str.), 1,630 (C=O str.); 1H
NMR (400 MHz, DMSO-d6) dH (ppm): 0.95 (s, 3H, CH3),
1.02 (s, 3H, CH3), 2.13–2.37 (m, 4H, 2 9 CH2), 2.67 (d,
J = 16.8 Hz, 1H, H3), 3.12 (dd, J = 15.6 Hz, J0 = 7.6 Hz,
1H, H3), 4.43 (d, J = 8.8 Hz, 1H, H4), 6.98–7.87 (m, 12H,
Ar–H), 8.26 (s, 1H, pyrazole H5). 13C NMR (100 MHz,
DMSO-d6) dC (ppm): 26.29, 27.83 (CH3), 29.74 (C4), 32.05
(C7), 37.84 (C6), 41.67 (C8), 52.06 (C3), 114.25, 115.21,
117.53, 119.11, 124.26, 125.66, 126.02, 126.33, 127.48,
128.68, 128.91, 130.77, 136.04, 139.52, 145.12, 150.88,
154.04 (Ar–C), 170.12 (C2), 196.15 (C5); Anal. Calcd. for
C30H26FN3O2S (511.61 g/mol): C, 70.43; H, 5.12; N, 8.21%.
Found: C, 74.21; H, 4.88; N, 13.92%.
1-(4-Fluorophenyl)-7,7-dimethyl-4-(1-phenyl-3-p-tolyl-
1H-pyrazol-4-yl)-3,4,7,8-tetrahydroquinoline-2,5(1H,6H)-
dione (4n)
Yield: 75%; melting range: 250–252°C; IR (KBr, m, cm-1):
2,990 (ArC–H str.), 1,710 (C=O str.), 1,650 (C=O str.); H
Methodology for in vitro antimicrobial screening
or minimal inhibitory concentration (MIC)
measurement
1
NMR (400 MHz, DMSO-d6)dH (ppm): 0.95 (s, 3H, CH3), 1.00
(s, 3H, CH3), 2.10–2.38 (m, 4H, 2 9 CH2), 2.50 (s, 3H, tolyl-
CH3), 2.77 (d, J = 15.2 Hz, 1H, H3), 3.16 (dd, J = 16.8 Hz,
J0 = 8.8 Hz, 1H, H3), 4.46 (d, J = 7.6 Hz, 1H, H4), 7.05–7.97
(m, 13H, Ar–H), 8.26 (s, 1H, pyrazole H5). 13C NMR
(100 MHz, DMSO-d6) dC (ppm): 21.27 (tolyl-CH3), 25.15,
26.41 (CH3), 29.13 (C4), 32.74 (C7), 38.31 (C6), 42.10 (C8),
49.98 (C3), 113.98, 114.87, 116.03, 118.20, 124.55, 124.74,
126.34, 127.04, 128.10, 128.24, 128.47, 130.70, 136.14,
141.65, 147.92, 150.26, 157.26 (Ar–C), 169.55 (C2), 196.14
(C5); Anal. Calcd. for C33H30FN3O2 (519.61 g/mol): C, 76.28;
H, 5.82; N, 8.09%. Found: C, 75.98; H, 6.05; N, 7.96%.
The in vitro antimicrobial activity of all the compounds
and standard drugs was assessed against three representa-
tive of Gram-positive bacteria, namely, Streptococcus
pneumoniae (MTCC 1936), Clostridium tetani (MTCC
449) and Bacillus subtilis (MTCC 441), three Gram-neg-
ative bacteria, namely, Salmonella typhi (MTCC 98),
Vibrio cholerae (MTCC 3906) and Escherichia coli
(MTCC 443) and two fungi, namely, Aspergillus fumigatus
(MTCC 3008) and Candida albicans (MTCC 227) by the
broth microdilution MIC method recommended by
National Committee for Clinical Laboratory Standards
(NCCLS). The strains employed for the activity were
procured from (MTCC—Micro Type Culture Collection)
Institute of Microbial Technology, Chandigarh. Inoculum
size for test strain was adjusted to 108 CFU ml-1 (colony
forming unit per milliliter) by comparing the turbidity
(turbidimetric method). Mueller–Hinton broth was used as
nutrient medium to grow and dilute the compound
1-(4-Fluorophenyl)-7,7-dimethyl-4-[3-(4-nitrophenyl)-1-
phenyl-1H-pyrazol-4-yl]-3,4,7,8-tetrahydroquinoline-
2,5(1H,6H)-dione (4o)
Yield: 67%; melting range: 244–246°C; IR (KBr, m, cm-1):
3,015 (ArC–H str.), 1,715 (C=O str.), 1,655 (C=O str.); 1H
NMR (400 MHz, DMSO-d6) dH (ppm): 0.89 (s, 3H, CH3),
123

Med Chem Res (2012) 21:1751–1761
1759
antimicrobial agents. Eur J Med Chem 43(3):456–463. doi:
10.1016/j.ejmech.2007.03.030
suspension for the test bacteria and Sabouraud Dextrose
Broth used for fungal nutrition. Ampicillin, chloramphen-
icol, ciprofloxacin, gentamicin and norfloxacin were used
as standard antibacterial drugs, whereas griseofulvin and
nystatin were used as standard antifungal drugs. DMSO
was used as diluents/vehicle to get desired concentration of
synthesized compounds and standard drugs to test on
standard microbial strains. Serial dilutions were prepared in
primary and secondary screening. Each compound and
standard drugs were diluted obtaining 2,000 lg l-1 con-
centration, as a stock solution. In primary screening, 1,000,
500 and 250 lg l-1 concentrations of the synthesized
compounds were taken. The active compounds found in
this primary screening were further diluted to obtain 200,
100, 62.5, 50, 25, 12.5 and 6.250 lg l-1 concentrations for
secondary screening to test in a second set of dilution
against all microorganisms. The control tube containing no
antibiotic is immediately subcultured (before inoculation)
by spreading a loopful evenly over a quarter of plate of
medium suitable for the growth of the test organism. The
tubes are then put for incubation at 37°C for 24 h for
bacteria and 48 h for fungi. The highest dilution (lowest
concentration) preventing appearance of turbidity is con-
sidered as minimal inhibitory concentration (MIC, lg l-1),
i.e., the amount of growth from the control tube before
incubation (which represents the original inoculum) is
compared. A set of tubes containing only seeded broth and
the solvent controls were maintained under identical con-
ditions so as to make sure that the solvent had no influence
on strain growth. The result of this is much affected by the
size of the inoculum. The test mixture should contain
108 CFU ml-1 organisms. The protocols were summarized
in Table 1 as the minimal inhibitory concentration (MIC,
lg l-1).
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Acknowledgments The authors are thankful to the Head, Depart-
ment of Chemistry, Sardar Patel University for providing 1H NMR,
13C NMR spectroscopy and research facilities. We are also thankful
to Oxygen Healthcare Research Pvt. Ltd., Ahmedabad, for providing
mass spectroscopy facilities, SICART, Vallabh Vidyanagar, for the
FT-IR and elemental analysis and Dhanji P. Rajani, PhD, Microcare
Laboratory, Surat, for antimicrobial screening of the compounds
reported herein. One of the authors is grateful to UGC, New Delhi for
research fellowship in Sciences for Meritorious Students.
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