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Arch. Pharm. Chem. Life Sci. 2011, 2, 84–90
analysis was performed on a Vario EL III Elemental Analyser
(Elementar, Germany) using sulfanilamide as standard. All
chemicals were purchased from Merck, Spectrochem or CDH,
India. Solvents were of reagent grade and were purified and dried
by standard procedure. Reactions were monitored by thin-layer
chromatography on silica gel plates in either iodine or UV
chambers. Intermediates were characterized by IR spectroscopic
analysis and elemental analysis for CHNS. In the elemental
analysis, the observed values were within ꢁ0.4% of the calculated
values. Final compounds were characterized by 1H-NMR and FAB-
MS. The final yields and the physicochemical and spectral data of
intermediates 3a–3l and final compounds 4a–4l are presented in
Table 1.
Antimicrobial study [21]
Antibacterial studies
The antibacterial activity of the test compounds was assayed
using serial double dilution method against non-pathogenic
strains of E. coli (NCIM 2068), K. pneumoniae (NCIM 2957),
S. aureus (NCIM 2079), and B. subtilis (NCIM 2921) and pathogenic
strains of V. cholera and protease in nutrient agar medium by the
Cup-plate method. Sterilized media was cooled to 408C and
0.5 mL of inoculum for 100 mL of media was added. The flasks
were shaken gently to avoid formation of air bubbles. 25 mL
portions of this media were transferred to Petri dishes of 9 cm
diameter so as to obtain 4–5 mm thickness of the media layer.
The plates were left at room temperature to allow solidification
of the media. In each Petri plate, 4 cups of suitable diameter were
made with a sterile borer. All these procedures were conducted
aseptically under laminar air flow workstation. The test com-
pounds and Ciprofloxacin (standard) were dissolved in DMSO
(0.5%) and solution ranging between 0.1 and 100 mM were pre-
pared. DMSO control was also maintained. 40 mL of the test
compounds and standard were added into each cup with the
help of a micropipette. Plates were kept undisturbed for at least
2 h at room temperature to allow for proper diffusion. Petri
plates were then incubated at 37 ꢁ 18C for 24 h. Zone inhi-
bitions (in mm) were measured after incubation and IC50 values
are calculated by plotting a graph between log concentrations
and percentage inhibition values. All the studies were performed
in triplicate and results were presented in Table 2.
General procedure for the synthesis of methyl
hydrazinecarbodithioate (1)
To a cooled solution of potassium hydroxide (0.1 M, 6.6 g/7 mL)
was added 2-propanol (7 mL) hydrazine hydrate (85% solution,
0.1 M, 6 mL) with stirring. Ice-cooled carbondisulfide (0.1 M,
10 mL) was added drop wise to the above stirred solution that
was maintained <108C over 1.5 h. The bright yellow mixture
obtained was further stirred for 1 h and then ice-cooled iodo-
methane (0.1 M, 7 mL) was added drop wise over a period of 2 h.
Stirring was continued for an additional 1.5 h to obtain a white
precipitate of 1. Filtered, washed with ice-cooled water and
recrystallized from dichloromethane.
General procedure for the synthesis of Schiff bases
methylhydrazine carbodithioate (2a–2l)
Antifungal studies
The antibacterial activity of the test compounds was assayed
using serial double dilution method against C. albicans and
A. niger in Sabouraud dextrose agar medium by cup plate
method. The sterile medium was inoculated using 24 h slant
cultures of test organisms and transferred into sterile Petri
dishes and allowed to solidify. 4 cups of suitable diameter were
made on the solidified media. The test compounds and flucona-
zole (standard) were dissolved in DMSO (0.5%) and solution
ranging between 0.1 and 100 mM were prepared. DMSO control
was also maintained. 40 mL of test compounds and standard
were added into each cup with the help of a micropipette.
Zones of inhibition (in mm) were measured after 24 h of incu-
bation and IC50 values are calculated by plotting a graph between
log concentrations and percentage inhibition value. All the
studies were performed in triplicate and results were presented
in Table 2.
Methyl hydrazinecarbodithioate 1 (0.01 M, 1.22 g) and (un)sub-
stituted aromatic aldehydes/ketone (0.012 M) were dissolved in
methanol. To this mixture catalytic amount of concentrated
sulphuric acid was added and refluxed for 6–7 h. The reaction
mixture turned yellow as the methylhydrazine carbodithioate
dissolved and the yellow product began to precipitate. The solid
obtained was filtered, dried and recrystallized from suitable
solvent.
General procedure for the synthesis of 4-[(2-
arylidenehydrazyl)-carbothionyl]-aminobenzoic acid
(3a–3l)
PABA (0.005 M, 0.685 g) was added to appropriate Schiff
base (2a–2l, 0.005 M) in ethanol (25 mL) and refluxed until
the evolution of methyl mercaptane almost completely ceased.
Solvent present in the reaction mixture was evaporated
under vacuum and the solid was collected and washed with cold
ethanol, further purified by recrystallization from suitable
solvent.
Anticancer studies (MTT assay) [22–24]
Compounds 4a–4l were evaluated for their anticancer activity on
HT-29 cell lines using MTT assay by serial double dilution method
in 96-well plate. Cells seeded in plate at 5000 cells/well. Different
dilutions of test and standard (0.1–100 mM) were made with
growth medium in such a way that the final DMSO concen-
tration is around 0.5%. 100 mL of cell suspension and 100 mL
of test and standard were transferred aseptically to each well. The
plate was then incubated at 378C for 72 h in CO2 incubator. After
incubation, 20 mL of MTT was added to each well and plate was
wrapped in aluminum foil to prevent the oxidation of the dye.
The plate was again incubated for 2 h. 80 mL of lysis buffer was
added to each well and the plate was placed on a shaker over-
night. The absorbance was recorded on the ELISA reader at
562 nm wavelength. The absorbance of the test was compared
General procedure for the synthesis of N-hydroxy-4-[(2-
arylidenehydrazinyl)-carbothionyl]-aminobenzamide
(4a–4l)
Phenyl chloroformate (0.001 M) and triethylamine (0.001 M)
were added to an ice-cooled solution of appropriate benzoic acid
derivative (3a–3l, 0.001 M) in dry THF and the mixture was
stirred for 1 h. The solid obtained was filtered off and to the
filtrate was added freshly prepared solution of hydroxylamine.
After stirring at room temperature for 3 h, the solid obtained
was filtered, dried and recrystallized from suitable solvent.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
Kulandaivelu, Umasankar
