Synthesis, characterization and in vitro cytotoxic properties of some new Schiff and Mannich bases in Hep G2 cells

Article abstract of DOI:10.1007/s00044-010-9433-z

A series of 5-substituted-4-amino-3-mercapto- 1,2,4-triazoles were synthesized and were treated with various 3-substituted pyrazole aldehydes to obtain a series of new Schiff bases (3a-l). Few of the selected Schiff bases were converted into Mannich bases by reaction with diphenylamine/morpholine in presence of formaldehyde in ethanol media (4a-e, 5a-e). These newly synthesized compounds were characterized by elemental analysis, IR, NMR and mass spectrometry studies. A comparative study on the cytotoxic activities of few selected Schiff and Mannich bases was done in HepG2 cells using MTT assay. Few of the screened Schiff bases, 3a, 3d, 3e, 3g and 3h showed dose dependent cytotoxic activity, 3a being the most potent with an IC₅₀ value of 0.018 g/l comparable to the standard drug doxorubicin. Among the Mannich bases, 5b was the most active with an IC₅₀ value of 0.034 g/l. The Schiff bases were found to be more active, when compared to Mannich bases derived from them. The morpholine derived Mannich bases were more potent than those obtained from diphenyl amine. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9433-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1024–1032
DOI 10.1007/s00044-010-9433-z
ORIGINAL RESEARCH
Synthesis, characterization and in vitro cytotoxic properties
of some new Schiff and Mannich bases in Hep G2 cells
•
Dhanya Sunil Arun M. Isloor Prakash Shetty
•
•
•
B. Chandrakantha K. Satyamoorthy
Received: 14 January 2010 / Accepted: 13 September 2010 / Published online: 29 September 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of 5-substituted-4-amino-3-mercapto-
1,2,4-triazoles were synthesized and were treated with
various 3-substituted pyrazole aldehydes to obtain a series
of new Schiff bases (3a–l). Few of the selected Schiff bases
were converted into Mannich bases by reaction with
diphenylamine/morpholine in presence of formaldehyde in
ethanol media (4a–e, 5a–e). These newly synthesized
compounds were characterized by elemental analysis, IR,
NMR and mass spectrometry studies. A comparative study
on the cytotoxic activities of few selected Schiff and
Mannich bases was done in HepG2 cells using MTT assay.
Few of the screened Schiff bases, 3a, 3d, 3e, 3g and 3h
showed dose dependent cytotoxic activity, 3a being the
most potent with an IC50 value of 0.018 g/l comparable to
the standard drug doxorubicin. Among the Mannich bases,
5b was the most active with an IC50 value of 0.034 g/l. The
Schiff bases were found to be more active, when compared
to Mannich bases derived from them. The morpholine
derived Mannich bases were more potent than those
obtained from diphenyl amine.
Keywords 1,2,4-Triazoles Á Schiff bases Á
Mannich bases Á HepG2 cell line Á MTT assay Á
Cytotoxic activity
Introduction
The term cancer refers to more than hundred different
diseases that share the common biologic characteristic of
abnormal cell growth. These malignant cells, if untreated
metastasize to other parts of the body and cause death
of the patient. Cancer remains a major public health
problem throughout the world with the most feared
diagnosis.
Human liver carcinoma is the fifth most common cancer
in the world and is responsible for more than 600,000
deaths annually. The majority of patients with hepatocel-
lular carcinoma die within 1 year after diagnosis. At
present, the treatment mainly includes surgery and che-
motherapy, but the curative effects of the existing che-
motherapeutic drugs are not good enough and they have
numerous side effects. Our understanding of the biological
processes which govern carcinogenesis is growing rapidly
and provides the basis for identifying novel cellular targets
for anticancer drug development. Therefore, searching for
highly efficient antitumor drug remains a hot research area.
Synthetic organic chemistry has always been a vital part
of the highly integrated and multidisciplinary process of
various drug developments. There has been a hiatus in the
momentum of research and discovery of novel antineo-
plastic drugs. In last few decades, triazoles have received
D. Sunil Á B. Chandrakantha
Department of Chemistry, Manipal Institute of Technology,
Manipal University, Manipal 576104, India
A. M. Isloor (&)
Department of Chemistry, National Institute of Technology,
Surathkal, Mangalore 575025, India
e-mail: isloor@yahoo.com
P. Shetty
Department of Printing and Media Engineering,
Manipal Institute of Technology, Manipal University,
Manipal 576104, India
K. Satyamoorthy
Department of Biotechnology, Life Sciences Centre,
Manipal University, Manipal 576104, India
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Med Chem Res (2011) 20:1024–1032
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Characterization
much significant attention in the field of medicinal chem-
istry because of their diversified biological properties like
antibacterial (Isloor et al., 2009, 2010; Prakash et al.,
2004), antifungal (Hirpara et al., 2003), antiviral (Tarmas
et al., 2006), anti-inflammatory (Udupi et al., 2003, 2004),
analgesic (Turan et al., 1999), antimicrobial (Chandra-
kantha et al., 2010; Swamy et al., 2006), anti-hypertensive
(Press et al., 1986) and antitubular (Priyadarsini et al.,
2004) properties. The amino and mercapto groups in tria-
zoles are readily accessible nucleophilic centres. In recent
years, various 1,2,4-triazole derivatives have been found to
be associated with anticancer (Al-Soud et al., 2006; Holla
et al., 2002; Yassen et al., 2004) properties. Several Schiff
and Mannich bases were reported to possess potential
anticancer properties (Holla et al., 2003). Further, current
literature indicates 1,2-pyrazole derivatives to possess
various biological activities (Mathew et al., 2007; Konagai
et al., 2002). It is also observed that incorporation of aryl
substituents and halogen atoms into the heterocyclic ring
systems augments the biological activities considerably
(Mohan et al., 1985). In view of the wide spectrum of
medicinal applications, especially anticancer properties of
various triazole derivatives and in continuation of our
research on biologically active new molecules, we hereby
report the synthesis of some new Schiff and Mannich
bases. The newly synthesized compounds were character-
ized by elemental analysis, IR, NMR and mass spectra. The
cytotoxicity screening of these compounds was done in
various concentrations in HepG2 cell lines and compared
with the standard drug doxorubicin. From the results
obtained, a comparative study on the extent of cytotoxic
activity of the Schiff and Mannich bases was done.
All the compounds were recrystallized and purity was
checked by thin layer chromatography. Melting points
were determined by open capillary method. The IR spectra
in KBr pellets were recorded on a Schimadzu FTIR 8400S
spectrophotometer. 1H-NMR spectra were recorded in
CDCl3 on an AV500 NMR spectrometer using TMS as an
internal standard. The mass spectra were recorded on a
Schimadzu GCMS-QP5050 mass spectrometer. The purity
of the compounds was checked by thin layer chromatog-
raphy on 0.25 mm silica gel plates. A 1:1 mixture of ethyl
acetate and petroleum ether solution was used as the eluent.
Visualization was made by using iodine vapours.
IR spectrum of Schiff bases showed an absorption band
at 1600–1610 cm-1 characteristic of –C=N– group in the
molecule. Also the absence of carbonyl stretching band at
1700 cm-1 clearly indicated amino condensation and
1
hence the formation of Schiff bases. The H-NMR spec-
trum of Schiff bases showed a singlet at d 9.95 due to the
presence of proton in –N=CH group in the molecule,
confirming the formation of Schiff base. In the IR spectra
of Mannich bases, the presence of –C=S functional group,
evident from the absorption band at 1284 cm-1 and
absence of broad absorption band at 2550 cm-1 due to S–H
str., provided a clear proof for formation of N-mannich
bases and not S-mannich bases. The absorption band
observed at around 1600 cm-1 can be attributed to the
–C=N– functional group. The regio-specificity of the
reaction was clear in 1H-NMR spectra as the peak at d 11.5
due to S–H proton was absent. The singlet at d 5.6 clearly
indicated the formation of [N–CH2–N\ which confirmed
the formation of Mannich base from Schiff base. Mass
spectrum of all Schiff and Mannich bases showed molec-
ular ion peaks which were in agreement with their
molecular formula. The characterization data of Schiff and
Mannich bases are shown in Tables 1 and 2.
Results and discussion
Synthesis
Cytotoxicity assay
5-Substituted 4-amino-3-mercapto-1,2,4-triazoles 1 were
synthesized from substituted phenols by multistep reactions
(Isloor et al., 2000). 3-Substituted pyrazole aldehydes 2
were prepared from substituted semicarbazones by Vils-
meier Haack reaction (Kalluraya et al., 2001, 2004; Singh
et al., 2005) using phosphorous oxychloride and dimethyl
formamide followed by hydrolysis. Reaction of 5-substi-
tuted 1,2,4-triazoles with different 3-substituted pyrazole
aldehydes in ethanol medium in presence of catalytic
amount of sulphuric acid yielded new Schiff bases 3a–
l. Mannich bases 4a–e and 5a–e were synthesized from the
Schiff bases by reacting with secondary amines, morpho-
line and diphenyl amine, respectively, in ethanol medium
in presence of formaldehyde. The reaction sequence is
outlined in Scheme 1.
Unless otherwise mentioned, all the chemicals used in this
study were from Sigma-Aldrich, USA. The hepatocellular
carcinoma cell line, HepG2 was used for the study. HepG2
cell lines were purchased from National Center for Cell
Science, Pune, India. The cell line was cultured in DMEM
containing 10% FBS at 37°C in an atmosphere containing
5% CO2.
Few of the selected newly synthesized Schiff and
Mannich bases were screened for their cytotoxic activities
at Life Sciences Centre, Manipal University, India.
Thirteen compounds containing mainly halogen substitu-
ents were selected for anticancer studies, eight Schiff
bases and five Mannich bases. Cytotoxic effect of the
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Med Chem Res (2011) 20:1024–1032
N
N
R1
Scheme 1 Synthesis route of
Schiff bases (3a–l) and Mannich
bases (4a–e and 5a–e)
O
+
R
SH
N
N
N
H
H2N
1
2
Conc.H 2SO4, Et-OH
N
NH
R
S
N
N
R1
N
3a-l
N
H
HCHO, EtOH
Diphenyl amine
Morpholine
C6H5
C6H5
N
N
O
N
N
N
N
R
S
S
R
N
N
N
N
R1
R1
5a-e
4a-e
N
N
N
H
N
H
3a : R = C6H5OCH2 , R1= 4-ClC6H4
3c : R = C6H5OCH2, R1= C2H5
3b : R = C6H5OCH2 R1= 4-FC6H4
3d : R = 2-CH3C6H4-OCH2 R1= 4-ClC6H4
3e : R = 2-CH3C6H4-OCH2, R1= 4-FC6H4 3f : R = 2-CH3C6H4-OCH2 R1= C2H5
3g : R = 4- CH3C6H4-OCH2, R1= 4-ClC6H4 3h :R = 4- CH3C6H4-OCH2 R1= 4-FC6H4
3i : R = 4- CH3C6H4-OCH2, R1= C2H5
3k : R = 2-ClC6H4-OCH2, R1= 4-FC6H4
3j : R = 2-ClC6H4-OCH2
3l : R = 2-ClC6H4-OCH2,
R1= 4-ClC6H4
R1= C2H5
4a : R = 4- CH3C6H4-OCH2, R1= 4-ClC6H4 4b : R = 4- CH3C6H4-OCH2, R1= 4-FC6H4
4c : R = 2-CH3C6H4-OCH2, R1= 4-FC6H4 4d : R = 2-CH3C6H4-OCH2, R1= 4-ClC6H4
4e : R = C6H5OCH2,
R1= 4-ClC6H4 5a : R = 4- CH3C6H4-OCH2, R1= 4-ClC6H4
5b : R = 4- CH3C6H4-OCH2, R1= 4-FC6H4 5c : R = 2-CH3C6H4-OCH2, R1= 4-FC6H4
5d : R = 2-CH3C6H4-OCH2, R1= 4-ClC6H4 5e : R = C6H5OCH2,
R1= 4-ClC6H4
compounds was assessed using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
(Mosmann, 1983). Cells were seeded in duplicates in
96-well plates at 1 9 104 cells/well. After 24 h, the
compounds were added at a concentration of 0.0001,
0.001, 0.01 and 0.1 g/l and incubated for another 24 h.
5 9 10-3 mol of MTT reagent was added and incubated
for additional 4 h. The purple formazan crystals formed
were then dissolved in 100 9 10-6 l of hydrochloric acid
(0.4 N):isopropanol (1:24). Cells grown in culture media
alone and with appropriate concentration of DMSO were
used as control and vehicle control, respectively. The
standard drug used was doxorubicin. The optical density
of each well was measured at 570 nm in an ELISA plate
reader. The results were reported as percentage survival
( S.D) of the cells when compared to that of the
untreated control cells for each concentration. The IC50
for each compound screened was also calculated. Results
of cytotoxicity studies of Schiff bases and Mannich bases
are given in Table 3.
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Med Chem Res (2011) 20:1024–1032
Experimental
Preparation of 4-({3-substituted-1H-pyrazol-4-
methylene}amino)-5-(substituted methyl)-2-hydro-
1,2,4-triazole-3-thione (3a–l)
0.01 mol of 5-substituted 4-amino-3-mercapto-1,2,4-tria-
zole 1, dissolved in minimum amount of hot ethanol (5 ml)
and 0.01 mol of 3-substituted pyrazole aldehyde, 2 dis-
solved in minimum amount of hot ethanol (4 ml) were
mixed well. Catalytic amount of conc. sulphuric acid was
added to the above mixture and the contents were refluxed
on water bath for 10 h. The resulted solution was allowed
to stand overnight and the precipitated solid was filtered,
dried and recrystallized from ethanol to yield the title
compounds 3a–l.
Preparation of 4-({3-substituted-1H-pyrazol-4-
methylene}amino)-2,5-(substituted methyl)-1,2,4-
triazole-3-thione (4a–e and 5a–e)
0.001 mol Schiff base was dissolved in minimum amount
of hot ethanol (4 ml). Then 1 ml of 40% formaldehyde was
added. To the above solution, 0.001 mol of secondary
amine (diphenyl amine/morpholine) dissolved in minimum
ethanol (1 ml) was introduced. The mixture was stirred for
24 h at room temperature. The solid separated was filtered,
dried and recrystallized from ethanol–dioxane mixture, to
yield the title compounds 4a–e and 5a–e.
Conclusions
We have successfully synthesized new Schiff and Mannich
bases derived from 1,2,4-triazoles. In the MTT assay, the
IC50 of doxorubicin was found to be 0.017 g/l. Interest-
ingly, Schiff bases 3a, 3d, 3e, 3g and 3h containing fluoro
and chloro substituents were found to be active with low
IC50 values. The halogen atoms might have added to the
potency of the compounds as a halogen substituent pro-
duces simultaneously an increase in lipophilicity and an
electron attracting effect. 3a was found to be the most
potent among all the Schiff bases with IC50 value of
0.018 g/l, comparable to doxorubicin. Schiff base 3i which
had ethyl substituent was found to be the least active
among all the Schiff bases. The replacement of aromatic
phenyl group connected to pyrazole ring by ethyl sub-
stituent must have been the cause for the reduced activity.
The Mannich bases 5a, 5b and 5d which were derived
from morpholine showed better activity with lower IC50
compared to that derived from diphenyl amine. Among the
Mannich bases 5b was the most potent. In conclusion, the
Schiff bases showed better results when compared to
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Med Chem Res (2011) 20:1024–1032
1031
Table 3 Cytotoxicity data of Schiff and Mannich bases
Comp. no.
Vehicle control
(% survival)
0.0001 g/l
(% survival)
0.001 g/l
(% survival)
0.01 g/l
(% survival)
0.1 g/l
(% survival)
IC50 g/l
3a
3b
3d
3e
80.75 2.04
80.75 1.94
94.64 1.06
78.07 2.12
80.75 4.03
80.75 0.36
78.07 1.89
78.07 2.09
95.31 0.56
95.31 3.92
95.31 3.09
94.64 2.54
94.64 0.98
80.69 1.97
90.92 0.69
82.94 2.45
86.27 3.24
71.18 0.96
66.82 2.09
77.15 2.13
99.60 0.89
99.00 1.85
92.60 1.32
97.53 3.40
94.82 2.10
81.90 1.89
85.74 3.67
90.02 0.89
74.33 2.39
88.26 4.07
76.92 2.45
85.38 1.36
69.48 2.20
77.15 1.07
99.00 0.57
98.00 1.01
89.64 3.29
96.35 4.51
90.58 2.03
76.84 1.57
82.57 1.29
74.21 1.36
51.64 2.30
92.00 1.06
67.57 2.21
77.03 1.96
61.03 2.45
66.51 3.05
93.94 2.46
97.00 1.98
85.00 3.98
84.61 2.21
80.25 1.38
66.44 2.76
70.28 0.54
51.22 1.09
36.46 1.09
30.04 2.34
23.57 1.76
13.15 0.88
7.66 2.36
24.88 1.87
75.78 5.08
28.18 1.01
49.21 1.57
45.71 3.28
18.88 2.01
5.65 0.98
29.48 1.07
35.87 0.95
0.018
0.071
0.044
0.048
0.028
0.045
[0.10
0.071
0.097
0.090
0.054
0.034
0.054
0.017
3g
3h
3i
3k
4a
4b
5a
5b
5d
Std.
Standard deviation. Doxorubicin is used as the standard (std.)
pyrazolidene hydrazine-4-thiazolyl) sydnones as possible
analgesic and anticonvulsant agents. J Saudi Chem Soc 4:265–
270
the corresponding Mannich bases. The Mannich bases
derived from morpholine were found to be more effective
than those derived from diphenyl amine. Since myocardial
toxicity is one of the side effects of doxorubicin, there is
ample scope of further study on the mode of action of 3a.
Isloor AM, Kalluraya B, Shetty P (2009) Regioselective reaction:
synthesis, characterization and pharmacological studies of some
new Mannich bases derived from 1,2,4-triazoles. Eur J Med
Chem 44:3784–3787
Isloor AM, Kalluraya B, Pai SK (2010) Synthesis, characterization
and biological activities of some new benzo[b]thiophene deriv-
atives. Eur J Med Chem 45:825–830
Kalluraya B, Isloor AM, Shenoy S (2001) Synthesis and biological
activity of 6-substituted-3-[4-(3-substituted pyrazolidene) hydra-
Acknowledgments The authors are thankful to the Head-SIF,
Indian Institute of Science, Bangalore for providing NMR and mass
spectral data. One of the author (D.S.) thankful to Director and Head
Department of Chemistry, Manipal Institute of Technology, Manipal
University for rendering us the necessary laboratory facilities. AMI is
thankful to Director, National Institute of Technology Karnataka,
India for the research facilities and encouragement.
zino-4-thiazolyl] coumarins. Indian
11:159–162
J
Heterocycl Chem
Kalluraya B, Isloor AM, Priya FV, Jagadeesha RL (2004) Synthesis
and pharmacological activity of some 4-(substituted)-2-[4-
arylhydrazono-3-methyl-5-oxo-2-pyrazolin-1-yl]thiazoles. J Het-
erocycl Chem 13:245–248
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