Synthesis antimicrobial and anticancer activity of N′- arylmethylidene-piperazine-1-carbothiohydrazide

Article abstract of DOI:10.1007/s00044-012-0279-4

Ten newly synthesized thiosemicarbazones of piperazine (3a-3j) were evaluated for their antibacterial and antifungal activity against non-pathogenic strains of Escherichia coli (NCIM 2068), Klebsiella pneumonia (NCIM 2957), Staphylococcus aureus (NCIM 2079), and Bacillus subtilis (NCIM 2921); pathogenic strains of Vibrio cholerae, protease, Candida albicans and Aspergillus niger. All the 10 compounds (3a-3j) were found to be better than Ciprofloxacin against B. subtilis and four molecules (3c, 3d, 3e, and 3h) against S. aureus. Compound 3j, a derivative of benzophenone, has been identified as a potent and promising candidate against C. albicans. The compounds were also evaluated for their anticancer activity against HBL-100 and HL60 cell lines. Compound 3a, a p-hydroxy benzaldehyde derivative, has been identified as a potent and promising candidate.

Full text of DOI:10.1007/s00044-012-0279-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2802–2808
DOI 10.1007/s00044-012-0279-4
ORIGINAL RESEARCH
Synthesis antimicrobial and anticancer activity of N0-
arylmethylidene-piperazine-1-carbothiohydrazide
•
Umasankar Kulandaivelu Boyapati Shireesha Chidara Mahesh
•
•
•
•
Jannu Vincent Vidyasagar Tadikonda Rama Rao K. N. Jayaveera
•
•
Philipp Saiko Geraldine Graser Thomas Szekeres Venkatesan Jayaprakash
•
•
Received: 25 July 2012 / Accepted: 9 October 2012 / Published online: 23 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Ten newly synthesized thiosemicarbazones of
piperazine (3a–3j) were evaluated for their antibacterial
and antifungal activity against non-pathogenic strains of
Escherichia coli (NCIM 2068), Klebsiella pneumonia
(NCIM 2957), Staphylococcus aureus (NCIM 2079), and
Bacillus subtilis (NCIM 2921); pathogenic strains of Vibrio
cholerae, protease, Candida albicans and Aspergillus
niger. All the 10 compounds (3a–3j) were found to be
better than Ciprofloxacin against B. subtilis and four mol-
ecules (3c, 3d, 3e, and 3h) against S. aureus. Compound 3j,
a derivative of benzophenone, has been identified as a
potent and promising candidate against C. albicans. The
compounds were also evaluated for their anticancer activity
against HBL-100 and HL60 cell lines. Compound 3a,
a p-hydroxy benzaldehyde derivative, has been identified
as a potent and promising candidate.
Keywords Thiosemicarbazones ꢀ Piperazine ꢀ
Antibacterial ꢀ Antifungal ꢀ Anticancer
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0279-4) contains supplementary
material, which is available to authorized users.
U. Kulandaivelu (&) ꢀ B. Shireesha ꢀ C. Mahesh ꢀ
J. V. Vidyasagar
Introduction
Medicinal Chemistry Research Division, Vaagdevi College
of Pharmacy, Hanamkonda, Andhra Pradesh, India
e-mail: youmasankar@gmail.com
Many major pathogenic bacteria and parasites have acquired
resistance toward chemotherapeutic agents available in the
market during the last decade. This led to the adoption of a
resolution on antimicrobial resistance in World Health
Assembly during 1998 (Surveillance, 1999). Recent reports
on emergence of superbugs have raised the fear that infec-
tious diseases may once again become major cause of death
worldwide. Now, there is a need to give serious consider-
ation toward development of novel chemotherapeutic agents
that are structurally different from the existing molecules
and acting on newer targets to efficiently combat Multi-Drug
Resistant (MDR) strains and to prevent development of
quick resistance by the pathogens. Thiosemicarbazones were
very well known for their antimicrobial and anticancer
property (Rollas and Ku¨c¸u¨kgu¨zel, 2007). Thiosemicarba-
zones with free primary N4 amino group were reported for
their anticancer property (Liu et al., 1995; Alvero et al.,
2006; Finch et al., 1999; Finch et al., 2000). We reported
anticancer thiosemicarbazones with secondary N4 amino
group (Chetan et al., 2010; Krishnan et al., 2008;
T. R. Rao
KLR Pharmacy College, Paloncha, Andhra Pradesh, India
K. N. Jayaveera
Department of Chemistry, Jawaharlal Nehru Technological
University, College of Engineering, Anantapur,
Andhra Pradesh, India
P. Saiko ꢀ G. Graser ꢀ T. Szekeres
Department of Medical and Chemical Laboratory Diagnostics,
General Hospital of Vienna-Medical University of Vienna,
Waehringer Guertel 18-20, A-1090 Vienna, Austria
V. Jayaprakash (&)
Department of Pharmaceutical Sciences, Birla Institute of
Technology, Mesra, Jharkahand 835 215, India
e-mail: venkatesanj@bitmesra.ac.in
V. Jayaprakash
Valens Pharma Services, Regus Citi Centre, Level 6, Chennai
Citi Centre, 10/11, Dr. Radhakrishnan Salai, Chennai 600 004,
Tamil Nadu, India
123

Med Chem Res (2013) 22:2802–2808
2803
Kulandaivelu et al., 2011), the present work elucidates the
antimicrobial and anticancer property of thiosemicarbazones
with tertiary N4 amino group.
Table 1 Physico-chemical and spectral data of compounds 3a–3j
Materials and methods
Chemistry
Code
R
R1
H
MF
MW MP
%
yield
*Rf
Melting points were determined using Thermonik Melting
Point Apparatus (Campbell electronics, India) by capillary
method and are uncorrected. Infrared (IR) spectra were
taken on a Fourier Transform Infrared Spectrophotometer
IR-Prestige 21 (Shimatzu Corporation, Japan) from 4000 to
400 cm-1 using KBr disks. 1H-NMR spectra were recorded
at 400 MHz in DMSO-d6 using a Bruker Avance 400
instrument (Bruker Instruments Inc., USA). Chemical
shifts were measured at d units (ppm) relative to Tetra-
methylsilane (TMS). Fast-atom bombardment (FAB) mass
spectra were recorded on a Jeol SX 102/DA-6000 mass
spectrometer (Jeol Ltd. Akishima, Tokyo, Japan) using
argon/xenon (6 kV, 10 mA) as FAB gas, m-nitrobenzyl
alcohol as matrix, and 10 kV as accelerating voltage at
room temperature. Elemental analysis was performed on a
Vario EL III Elemental Analyser (Elementar, Germany)
using sulfanilamide as standard. All chemicals were pur-
chased 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
3a
3b
3c
3d
3e
3f
p-OH
p-OH
C12H16N4OS 264 210–212 76.4 0.67
CH3 C13H18N4OS 278 222–224 67.2 0.72
m-NO2 CH3 C13H17N5O2S 307 186–188 72.6 0.77
p-OCH3
p-Cl
H
H
H
C13H18N4OS 278 216–218 69.6 0.68
12H15ClN4S 282 196–198 70.2 0.74
C
CH3 C13H18N4S
12H16N4S
262 212–214 72.6 0.56
248 198–200 73.2 0.65
3g
3h
3i
H
H
C
p-Cl
p-CH3
H
CH3 C13H17ClN4S 297 220–222 67.6 0.66
H
C13H18N4S
262 198–200 69.3 0.68
324 200–202 70.0 0.74
3j
C6H5 C18H20N4S
* n-Hexane:ethyl acetate; 1:1
General procedure for synthesis of Schiff bases
methylhydrazine carbodithioate (2a-2j)
Methyl hydrazinecarbodithioate 1 (0.01 M, 1.22 g) and
(un)-substituted aromatic aldehydes/ketone (0.012 M) were
dissolved in methanol (10 mL). To this mixture, catalytic
amount of concentrated sulfuric 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.
(Klayman et al., 1979).
1
were characterized by H-NMR and EI-MS. The percent-
age yields and the physicochemical data of final com-
pounds 3a-3j are presented in Table 1.
General procedure for synthesis of methyl
hydrazinecarbodithioate (1)
General procedure for synthesis of N0-arylmethylidene-
piperazine-1-carbothio-hydrazide (3a–3j)
To a cooled solution of potassium hydroxide (0.1 M, 6.6 g/
7 mL) in 2-propanol (7 mL), hydrazine hydrate (85 %
solution, 0.1 M, 6 mL) was added with stirring. Ice-cooled
carbondisulfide (0.1 M, 10 mL) was added drop wise to the
above stirred solution that was maintained below 10 °C
over 1.5 h. The bright yellow mixture obtained was further
stirred for 1 h, and then, ice-cooled iodomethane (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. Yield: 43 %;
m.p.: 90–92 °C (Klayman et al., 1979).
Piperazine (0.005 M, 0.685 g) was added to appropriate
Schiff’s base (2a–2j, 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 recrystal-
lization from suitable solvent (physico-chemical and spectral
data in supplementary materials). (Kulandaivelu et al., 2011).
N0-[(4-hydroxyphenyl)-methylidene]-piperazine-1-carbothio-
hydrazide (3a) 1H-NMR (DMSO-d6, dppm): 1.9 (s, 1H,
pip-NH), 2.6 (t, 4H, pip-CH2), 4.15 (m, 4H, pip-CH2),
123

2804
Med Chem Res (2013) 22:2802–2808
N0-[1-(4-chlorophenyl)ethylidene]piperazine-1-carbothio-
hydrazide (3h) 1H-NMR (DMSO-d6, dppm): 1.65 (s, 3H
–CH3), 1.95 (s, 1H, pip-NH), 2.85 (t, 4H, pip-CH2), 4.1 (m,
4H, pip-CH2), 7.60–7.7 (m, 4H, Ar–H), 11.15 (s, 1H,
CS–N–H); EI-MS (m/z): 298[M?1]?; Elemental analyses
Found (Calcd.): C, 52.16 (52.60); H, 6.02 (5.77); N, 19.22
(18.88); S, 10.24 (10.80).
7.4–7.7 (m, 4H, Ar–H), 8.17 (s, 1H, =C–H), 9.7 (s, 1H,
Ar–OH), 11.5 (s, 1H, CS–N–H); EI-MS (m/z): 265[M?1]?;
Elemental analyses Found (Calcd.): C, 54.48 (54.52);
H, 6.14 (6.10); N, 21.32 (21.19); S, 12.25 (12.13).
N0-[1-(4-hydroxyphenyl)ethylidene]piperazine-1-carbothiohy-
drazide (3b) 1H-NMR (DMSO-d6, dppm): 1.7 (s, 3H,
–CH3), 1.9 (s, 1H, pip-NH), 2.7 (t, 4H, pip-CH2), 4.15 (m,
4H, pip-CH2), 6.8–7.3 (m, 4H, Ar–H), 9.7 (s, 1H, Ar–OH),
11.3 (s, 1H, CS–N–H); EI-MS (m/z): 279[M?1]?; Ele-
mental analyses Found (Calcd.): C, 55.86 (56.09); H, 6.60
(6.52); N, 21.06 (20.13); S, 11.60 (11.52).
N0-[(4-methylphenyl)methylidene]piperazine-1-carbothio
hydrazide (3i) 1H-NMR (DMSO-d6, dppm): 2.5 (s, 1H,
pip-NH), 3.1 (t, 4H, pip-CH2), 3.7 (s, 3H, CH3), 4.6 (m, 4H,
pip-CH2), 7.1–7.5 (m, 4H, Ar–H), 8.10 (s, 1H, =C–H), 10.8
(s, 1H, CS–N–H); EI-MS (m/z): 263[M?1]?; Elemental
analyses Found (Calcd.): C, 60.04 (59.51); H, 5.88 (6.91);
N, 21.68 (21.35); S, 12.04 (12.22).
N0-[1-(3-nitrophenyl)ethylidene]piperazine-1-carbothio-
hydrazide (3c) 1H-NMR (DMSO-d6, dppm): 1.7 (s, 3H,
–CH3), 1.95 (s, 1H, pip-NH), 2.7 (t, 4H, pip-CH2), 4.1
(m, 4H, pip-CH2), 7.4–7.8 (m, 4H, Ar–H), 11.5 (s,1H,
CS–N–H); EI-MS (m/z): 308[M?1]?; Elemental analyses
Found (Calcd.): C, 51.24 (50.80); H, 5.62 (5.57); N, 21.98
(22.78); S, 11.08 (10.43).
N0-(diphenylmethylidene)piperazine-1-carbothiohydrazide
(3j) 1H-NMR (DMSO-d6, dppm): 2.4 (s, 1H, pip-NH),
3.15(t, 4H, pip-CH2), 3.7(m, 4H, pip-CH2), 7.5–8.1 (m,
10H, Ar–H), 10.5 (s, 1H, N–H); EI-MS (m/z): 325[M?1]?;
Elemental analyses Found (Calcd.) C, 66.16 (66.63); H,
5.82 (6.21); N 16.68 (17.27); S, 10.02 (9.88).
N0-[(4-methoxyphenyl)methylidene]piperazine-1-carbothio-
hydrazide (3d) 1H-NMR (DMSO-d6, dppm): 1.98 (s, 1H,
pip-NH), 3.75 (t, 4H, pip-CH2), 2.7 (m, 4H, –OCH3), 4.15
(m, 4H, pip-CH2), 7.4–7.65 (m, 4H, Ar–H), 8.19 (s, 1H,
=C–H), 11.18 (s, 1H, CS–N–H); EI-MS (m/z): 279[M?1]?;
Elemental analyses Found (Calcd.): C, 55.98 (56.09); H,
6.78 (6.52); N, 20.88 (20.13); S, 11. 90 (11.52).
Antimicrobial study
Antibacterial studies
The antibacterial activities of the newly synthesized com-
pounds (3a–3j) were tested using serial double dilution
method against non-pathogenic strains of E. coli (NCIM
2068), P. aeuroginosa (NCIM 2967), S. aureus (NCIM
2079), and B. subtilis (NCIM 2921) and pathogenic strains
of V. cholera and protease in nutrient agar medium by
Cup-plate method. Sterilized media was cooled to 40 °C
and 0.5 mL of inoculum for 100 mL of media was added.
The flasks were shaken gently to avoid formation of air
bubbles. This medium was transferred to Petri dishes of
9-cm diameter in 25 mL portions, 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, four cups of suitable diameter were made with a
sterile borer. All these procedures were conducted asepti-
cally under laminar air flow workstation. The test com-
pounds and Ciprofloxacin (Symed Lab India Pvt Ltd.,
Hyderabad, India) were dissolved in DMSO (0.5 %) and
solution ranging between 0.1 and 100 lM were prepared.
DMSO control was also maintained. Test compounds
(40 lL) and standard (40 lL) were added into each cup
with the help of a micropipette. Plates were kept undis-
turbed for at least 2 h at room temperature to allow for
proper diffusion. Petri plates were then incubated at
37 1 °C for 24 h. Zone inhibitions (in mm) were mea-
sured after incubation, and IC50 values are calculated by
N0-[(4-chlorophenyl)methylidene]piperazine-1-carbothio-
hydrazide (3e) 1H-NMR (DMSO-d6, dppm): 2.55 (s, 1H,
pip-NH), 3.4 (m, 4H, pip-CH2), 4.11 (m, 4H, pip-CH2),
7.39–7.6 (m, 4H, Ar–H), 8.16 (s, 1H, =C–H), 11.15 (s,1H,
CS–N–H); EI-MS (m/z): 281[M-1]?; Elemental analyses
Found (Calcd.): C, 51.24 (50.97); H, 5.42 (5.35); N, 20.12
(19.81); S, 11.08 (11.34).
N0-[1-phenylethylidene]piperazine-1-carbothiohydrazide
(3f) 1H-NMR (DMSO-d6, dppm): 2.3 (s, 3H –CH3), 2.7
(s, 1H, pip-NH), 3.4 (m, 4H, pip-CH2), 4.05 (m, 4H, pip-
CH2), 7.45–7.8 (m, 5H, Ar–H), 9.95 (s,1H, CS–N–H);
EI-MS (m/z): 261 [M-1]?; Elemental analyses Found
(Calcd.): C, 60.12 (59.51); H, 6.78 (6.91); N, 21.14 (21.35);
S, 12.54 (12.22).
N0-[phenylmethylidene]piperazine-1-carbothiohydrazide
(3g) 1H-NMR (DMSO-d6, dppm): 1.95 (s, 1H, pip-NH),
2.7 (t, 4H, pip-CH2), 4.1 (m, 4H, pip-CH2), 7.4–7.7 (m, 5H,
Ar–H), 8.15 (s, 1H, =C–H), 11.15 (s, 1H, CS–N–H);
EI-MS (m/z): 249[M?1]?; Elemental analyses Found
(Calcd.): C, 57.78 (58.04); H, 6.58 (6.49); N, 22.90 (22.56);
S, 12.64 (12.91).
123

Med Chem Res (2013) 22:2802–2808
2805
Table 2 Antibacterial and antifungal activity of compounds 3a–3j
Code
R
R1
IC50 (lM)*
E. coli
P. aeuroginosa
S. aureus
B. subtilis
V. cholera
Protease
C. albicans
A. niger
3a
3b
3c
p-OH
p-OH
m-NO2
p-OCH3
p-Cl
H
H
1.64
3.48
0.57
1.59
1.52
1.76
3.91
0.88
1.74
3.03
0.03
1.63
3.51
0.60
1.57
0.60
2.11
1.97
0.71
1.70
1.44
0.05
1.70
1.58
0.86
0.79
0.54
2.07
1.36
1.04
1.66
1.33
1.20
1.62
3.49
0.32
0.66
0.13
1.78
0.93
0.98
1.70
1.38
4.70
1.66
1.59
0.86
1.53
0.60
1.17
2.23
0.78
3.57
1.38
0.20
1.67
1.21
0.73
2.41
0.65
0.84
1.78
0.98
1.26
4.44
0.20
1.94
2.56
2.00
2.15
1.85
3.75
1.85
3.37
3.72
1.47
1.97
3.55
1.56
2.09
2.14
3.76
2.21
3.32
3.79
1.62
CH3
CH3
H
3d
3e
H
3f
CH3
H
3g
3h
3i
H
p-Cl
p-CH3
H
CH3
H
3j
C6H5
CIP
FLU
0.98
[10.00
* Mean value of triplicate
CIP Ciprofloxacin; FLU Fluconazole
Anticancer studies (MTT assay)
plotting a graph between log concentrations and percentage
inhibition values. All the studies were performed in trip-
licate and results were presented in Table 2.
The compounds 3a-3j were evaluated for their anticancer
activities on HBL-100 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 lM) were made with growth
medium in such a way that the final DMSO concentration
is around 0.5 %. 100 lL of cell suspension and 100 lL of
test and standard were transferred aseptically to each
well. The plate was then incubated at 37 °C for 72 h in
CO2 incubator. After incubation, 20 lL 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 lL 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 with that of DMSO control to get the percent-
age inhibition and IC50 values are calculated by plotting a
graph between log concentrations and percentage inhibi-
tion value. All the studies were performed in duplicate
and results were presented in Table 3.
Antifungal studies
The antifungal activities of the test compounds were 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. Four cups of suitable diam-
eter were made on the solidified media. The test compounds
and Fluconazole (Symed Lab India Pvt Ltd, Hyderabad,
India) were dissolved in DMSO (0.5 %) and solution rang-
ing between 0.1 and 100 lM were prepared. DMSO control
was also maintained. Test compounds (40 lL) and standard
(40 lL) were added into each cup with the help of a
micropipette. Zones of inhibition (in mm) were measured
after 24 h of incubation 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.
123

2806
Med Chem Res (2013) 22:2802–2808
the logarithmic phase of growth were used for all experiments
described below. HL-60 cells (0.1 9 106 per mL) were seeded
in 25 cm2 Nunc tissue culture flasks and incubated with
increasing concentrations of drugs at 37 °C under cell culture
conditions. Cell counts and IC50 values were determined after
24, 48, and 72 h using the microcell counter CC-108. Viability
of cells was determined by trypan blue exclusion. Results were
calculated as number of viable cells. All the studies were
performed in triplicate and results were presented in Table 3.
Table 3 Anti-cancer activity of compounds 3a–3j
Code
R
R1
IC50 (lM)*
HBL-100
HL-60 (48 h)
Results and discussion
3a
3b
3c
p-OH
p-OH
m-NO2
p-OCH3
p-Cl
H
H
0.18
0.54
0.99
1.62
1.6
50
CH3
CH3
H
60
Chemistry
62
3d
3e
NT
69
The final compounds 3a–3j were synthesized following the
synthetic route outlined in Scheme 1. Methyl hydrazine
carbodithioate (1) was prepared by the reaction of hydra-
zine hydrate (85 %) with carbon disulfide in the presence
of potassium hydroxide (Chetan et al., 2010; Klayman
et al., 1979). Condensation of 1 with aromatic aldehydes/
ketones in the presence of catalytic amount of sulfuric acid
in methanol provided 2a–2j (Klayman et al., 1979; Chetan
et al., 2010). The final compounds 3a–3j was synthesized
by the reaction of piperazine with 2a–2j in ethanol. The
reaction comes to completion when evaluation of methyl
mercaptan ceases (Chetan et al., 2010; Klayman et al.,
1979). Intermediates were characterized by their IR-spec-
tral and elemental analysis data. CHNS microanalysis
revealed that variation in experimental values compared
with calculated values is within 0.4 %. Final compounds
H
3f
CH3
H
0.29
2.41
0.26
0.8
NT
63
3g
3h
3i
H
p-Cl
p-CH3
H
CH3
H
NT
70
3j
C6H5
0.34
0.04
NT
NT
MTX
* Mean value of triplicate
MTX methotrexate, NT not tested
HL-60 cell line assay
The HL-60 human promyelocytic leukemia cell line was pur-
chased from ATCC (American Type Culture Collection,
Manassas, VA, USA). Cells were grown in RPMI 1640 med-
ium supplemented with 10 % heat inactivated fetal calf serum
(FCS), 1 % L-Glutamine and 1 % Penicillin–streptomycin in a
humidified atmosphere containing 5 % CO2. All media and
supplements were obtained from Life Technologies (Paisley,
Scotland, UK). Cell counts were determined using a microcell
counter CC-108 (SYSMEX, Kobe, Japan). Cells growing in
1
3a–3j were characterized by their H-NMR and ES-MS
spectral data. All the thiosemicarbazone derivatives (3a–3j)
showed a characteristic peak for the aldehydic proton (=C–H)
between d 8.10–8.19 ppm as a singlet, ketonic methyl
proton (–CH3) between d 1.65–2.3 ppm as a singlet and
piperazine NH proton between d 1.90–2.70 ppm as singlet.
Scheme 1 Reagents and conditions: a KOH/i-PrOH, CS, stirring \10 °C, 2.5 h; CH3I, stirring, \10 °C, 3.5 h; b R-C6H4–CO-R/MeOH, H2SO4
[cat], reflux, 6–7 h; c Piperazine/EtOH, reflux
123

Med Chem Res (2013) 22:2802–2808
2807
Eight protons of piperazine displayed a triplet or multiplet
between d 2.60 and 3.75 ppm and a multiplet between d
3.70 and 4.15 ppm. The EI-MS spectra of all the com-
pounds displayed (M?1)? peak. The structure, physico-
chemical characterization of compounds 3a–3j is presented
in Table 1.
Conclusion
The present investigation provided ten molecules (3a–3j)
better than Ciprofloxacin against B. subtilis and four mol-
ecules (3c, 3d, 3e, and 3h) better than Ciprofloxacin against
S. aureus. Compounds 3j was identified as lead molecules
for the development of novel antifungal agent and thio-
semicarbazones of substituted benzophenone derivatives
may provide a promising candidate. Compound 3a exhib-
ited a potent anticancer activity against HBL-100 cell lines
and none of the compounds exhibited an appreciable
activity against HL-60 cell lines.
Antimicrobial and anticancer studies
All ten thiosemicarbazone derivatives (3a–3j) were evalu-
ated for their antibacterial/antifungal activity in serial dou-
ble dilution method (Barry, 1986) against non-pathogenic
strains of Escherichia coli (NCIM 2068), Klebsiella pneu-
monia (NCIM 2957), Staphylococcus aureus (NCIM 2079),
and Bacillus subtilis (NCIM 2921) and pathogenic strains of
Vibrio cholerae and protease. Similarly, they were also
evaluated for their antifungal activity against Candida
albicans and Aspergillus niger. The results are presented in
Table 2. Compound 3c, 3d, 3e, and 3h were found to be
more potent than the standard (Ciprofloxacin) used in the
study against S. aureus. Thiosemicarbazones derived from
benzaldehydes (3g and 3e) were found to be better than those
derived from acetophenones (3f and 3h) except hydroxyl
derivative (3b). Compound with chloro substitution at para
position (3e) was found to be the most potent in this series.
All the ten compounds (3a–3j) were found to be more
effective than standard against B. subtilis. Surprisingly, in
this case, also the thiosemicarbazones derived from benz-
aldehydes (3g, 3e and 3a) were more effective than those
derived from acetophenones (3f, 3h and 3b). Compound 3e
with chloro substitution at para position was found to be
potent within this series. In both the cases, antimicrobial
activities were in the following order: p-Cl (3e) [ p-OCH3
(3d) [ unsub. (3g) [ p-CH3 (3i). All the compounds were
found to inhibit C. albicans at concentration between 1.47
and 3.75 lM. Thiosemicarbazone derived from benzophe-
none (3j) was found to be the potent within this series and
almost equipotent to that of the standard (Fluconazole) used
in this study. Against A. niger, all the ten compounds were
found to be active at concentrations between 1.56 and
3.79 lM and better than the standard.
Acknowledgments Authors are thankful to Secretary, Viswabhara
Educational Society, Warangal for providing the facilities, Symed
Lab India Pvt Ltd, Hyderabad, India for providing gift sample of
Ciprofloxacin & Fluconazole and Director, IICT, Hyderabad for
providing spectral data.
Conflict of interest The authors have declared no conflict of
interest.
References
Alvero AB, Chen W, Sartorelli AC, Schwartz P, Rutherford T, Mor G
(2006) Triapine (3-aminopyridine-2-carboxaldehyde thiosemi-
carbazone) induces apoptosis in ovarian cancer cells. J Soc
Gynecol Investig 13(2):145–152. doi:10.1016/j.jsgi.2005.11.004
Barry A (1986) Procedure for testing antimicrobial agents in agar
media: theoretical considerations. Antibiotics in laboratory
Medicine Edition 2:1–26
Boyd MR, Paull KD (1995) Some practical considerations and
applications of the National Cancer Institute in vitro anticancer
drug discovery screen. Drug Dev Res 34(2):91–109
Chetan B, Bunha M, Jagrat M, Sinha BN, Saiko P, Graser G, Szekeres
T, Raman G, Rajendran P, Moorthy D, Basu A, Jayaprakash V
(2010) Design, synthesis and anticancer activity of piperazine
hydroxamates and their histone deacetylase (HDAC) inhibitory
activity. Bioorg Med Chem Lett 20(13):3906–3910. doi:
10.1016/j.bmcl.2010.05.020
Finch RA, Liu MC, Cory AH, Cory JG, Sartorelli AC (1999) Triapine
(3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP):
an inhibitor of ribonucleotide reductase with antineoplastic
activity. Adv Enzyme Regul 39:3–12
Finch RA, Liu M, Grill SP, Rose WC, Loomis R, Vasquez KM,
Cheng Y, Sartorelli AC (2000) Triapine (3-aminopyridine-
2-carboxaldehyde- thiosemicarbazone): a potent inhibitor of
ribonucleotide reductase activity with broad spectrum antitumor
activity. Biochem Pharmacol 59(8):983–991
Grever MR, Schepartz S, Chabner B The National Cancer Institute:
cancer drug discovery and development program. In, 1992. vol 6.
p 622
Klayman DL, Bartosevich JF, Griffin TS, Mason CJ, Scovill JP
(1979) 2-Acetylpyridine thiosemicarbazones. 1. A new class of
potential antimalarial agents. J Med Chem 22(7):855–862
Krishnan K, Prathiba K, Jayaprakash V, Basu A, Mishra N, Zhou B,
Hu S, Yen Y (2008) Synthesis and ribonucleotide reductase
inhibitory activity of thiosemicarbazones. Bioorg Med Chem
Lett 18(23):6248–6250
The compounds were also evaluated for their possible
anticancer activity by MTT assay against HBL-100 cell
lines (Grever et al., 1992; Boyd and Paull, 1995; Monks
et al., 1991). The reports are presented in Table 3. All the
compounds exhibited cytotoxic activity against HBL-100
cell lines at concentration between 0.18 and 2.21 lM.
Compound 3a was found to be potent within this series and
was 4.5-fold less potent than standard (Methotrexate) used
in the study. Thiosemicarbazones derived from acetophe-
nones were found to be better than their benzaldehyde
counterpart (except 3a). The anticancer activity was in the
following order: p-Cl (3h) C unsub. (3f) [ p-OH (3b).
Kulandaivelu U, Padmini VG, Suneetha K, Shireesha B, Vidyasagar
JV, Rao TR, K N J, Basu A, Jayaprakash J (2011) Synthesis,
123

2808
Med Chem Res (2013) 22:2802–2808
antimicrobial and anticancer activity of new thiosemicarbazone
derivatives. Arch Pharm (Weinheim) 344(2):84–90
Rollas S, Ku¨c¸u¨kgu¨zel SG (2007) Biological activities of hydrazone
derivatives. Molecules 12(8):1910–1939
Liu MC, Lin TS, Sartorelli AC (1995) 1 chemical and biological
properties of cytotoxic a-N-Heterocyclic carboxaldehyde thio-
semicarbazones. Prog Med Chem 32:1–35
Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D,
Hose C, Langley J, Cronise P, Vaigro-Wolff A (1991) Feasibility
of a high-flux anticancer drug screen using a diverse panel of
cultured human tumor cell lines. J Natl Cancer Inst 83(11):
757–766
Surveillance WHODoCD (1999) Containing antimicrobial resistance:
review of the literature and report of a WHO Workshop on the
Development of a Global Strategy for the Containment of
Antimicrobial Resistance; Geneva, Switzerland, 4–5 February
1999. World Health Organization, Dept. of Communicable
Disease Surveillance and Response, Geneva
123