ABSTRACT
A series of novel (Z)-3-(2-(4-(2-oxo-2H-chromen-3-yl) thiazol-2-yl-)hydrazono)indolin-2-one (8a-8d, 9) were synthesized with various substituted indole derivatives. Structures of the newly synthesized compounds were elucidated by FT-IR, 1H-NMR, 13C-NMR and API-ES Mass spectral data. The in vitro cytotoxic activities of the complexes measurement against the human cancer T-lymphocyte cell lines. In vitro evaluation of these title complexes revealed cytotoxicity from 6.8-18µg/mL against CEM, 9.2-21µg/mL against L1210, 10-19µg/mL against Molt4/C8, 8-12µg/mL against HL60 and 8-16µg/mL against BEL7402. Coumarin derivatives 8c and 8d showed that quite significant anticancer activities. The antioxidant activity of the synthesized compounds was evaluated by DPPH scavenging method. Compounds 8c, 8d and 9 showed significant antioxidant activity compared with that of standard drug, ascorbic acid.
Key words: Coumarin, DPPH, Cytotoxic activity.
Synthesis, characterization, in vitro cytotoxic and antioxidant activities of novel coumarin thazolyl derivatives
1. 29
International Journal of Pharmacy Education and Research
Jan-Mar 2014; 1(1): 29-35.
Available online: www.ijper.net
Research Article
Synthesis, characterization, in vitro cytotoxic and antioxidant
activities of novel coumarin thiazolyl derivatives
Thota SREEKANTH*1
, Nadipelly KAVITHA1
, Anchuri SHYAMSUNDER1
, Yerra
RAJESHWAR1
, Karki SUBHAS SOMALINGAPPA2
, Jan BALZARINI3
, Erik DE CLERCQ3
1
Department of Pharmaceutical Chemistry, S. R. College of Pharmacy, Ananthasagar, Hasanparthy,
Warangal – 506 371, Andhra Pradesh, INDIA.
2
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Rajaji Nagar,
Bangalore – 560 010, Karnataka, INDIA.
3
Rega Institute for Medical Research, Katholieke, Universitteit Leuven, Minderbroedersstraat 10, B-
3000 Leuven, BELGIUM.
INTRODUCTION
Coumarin derivatives, an important group of
heterocyclic compounds, have been the subject of
extensive study in the recent past. Coumarin
derivatives are known to be a very interesting class of
synthetic or natural compounds. Coumarins (2H-1-
benzopyran-2-ones, 2H-chromen-2-ones) are very
attractive targets for combinatorial library synthesis
due to their wide range of valuable biological
activities. Many natural and synthetic coumarins have
occupied an important place in drug research, as a one
of the so-called privileged drug scaffold[1]
. They are
widely used as additives in food, perfumes,
cosmetics[2]
, pharmaceuticals and optical
brighteners[3]
and dispersed fluorescent and laser
dyes[4]
. Coumarin and its derivatives are biologically
interesting compounds known for their antimicrobial
and antifungal[5]
, antileishmanial[6]
, antioxidant[7]
,
antitumor[8]
, antiproliferation[9]
, hypotensive[10]
,
antiallergic[11,12]
, local anaesthetic[13]
, central nervous
Received on: 18 March, 2014
Revised on: 24 March, 2014
Accepted on: 26 March, 2014
*Corresponding author
T. Sreekanth
S. R. College of Pharmacy,
Ananthasagar, Hasanparthy,
Warangal – 506 371,
Andhra Pradesh, INDIA.
Mobile #: +91-99893-24804
Email id : stsreekanththota@gmail.com
ABSTRACT
A series of novel (Z)-3-(2-(4-(2-oxo-2H-chromen-3-yl)
thiazol-2-yl-)hydrazono)indolin-2-one (8a-8d, 9) were
synthesized with various substituted indole derivatives.
Structures of the newly synthesized compounds were elucidated
by FT-IR, 1
H-NMR, 13
C-NMR and API-ES Mass spectral data.
The in vitro cytotoxic activities of the complexes measurement
against the human cancer T-lymphocyte cell lines. In vitro
evaluation of these title complexes revealed cytotoxicity from 6.8-
18µg/mL against CEM, 9.2-21µg/mL against L1210, 10-19µg/mL
against Molt4/C8, 8-12µg/mL against HL60 and 8-16µg/mL
against BEL7402. Coumarin derivatives 8c and 8d showed that
quite significant anticancer activities. The antioxidant activity of
the synthesized compounds was evaluated by DPPH scavenging
method. Compounds 8c, 8d and 9 showed significant antioxidant
activity compared with that of standard drug, ascorbic acid.
Key words: Coumarin, DPPH, Cytotoxic activity.
2. Sreekanth et al. IJPER | Jan-Mar, 2014; 1(1): 29-35.
30
system (CNS) activities and effects[14]
, as well as
treatment of Alzheimer’s disease[15]
and
Schizophrenia disorder[16]
. Fused chromene ring
systems have platelet anti aggregating, local
anesthetic[17-19]
and antihistaminic activities[20]
. They
also exhibit antidepressant effects[21]
, inhibitory effect
on influenza virus sialidases[22,23]
, DNA breaking
activities and mutagenicity[24]
, antiviral activities[25]
and act as sex pheromonehomologes[26]
. The current
literature is enriched with progressive findings about
the synthesis and pharmacological action of fused
heterocycles. On the basis of all of this evidence, we
set out to prepare a new series of biologically active
agents containing both of these indole and thiazole
substituted coumarin derivatives. This study reports
the synthesis, characterization, and in vitro cytotoxic
and antioxidant activities of these new thiazolyl indole
coumarin derivatives.
MATERIALS AND METHODS
All the chemicals used were of Analytical
Range grade and procured from Sigma–Aldrich and
E.Merck and were used as received. Melting points
were recorded using a digital Gallenkamp (SANYO)
model MPD BM 3.5 apparatus and are uncorrected.
1
H-NMR and 13
C-NMR spectra were determined in
DMSO solution at 400 MHz using Bruker AM-400
spectrophotometer. FT-IR spectra were recorded using
an FTS 3000 MX spectrophotometer; Mass spectra
were recorded on a API-Electro spray (API-ES) Mass
spectrophotometer and elemental analyses with a
LECO-183 CHNS analyzer.
SYNTHESIS OF ACETYL COUMARIN (3) AND 3-BROMO
ACETYL COUMARIN (4)
Acetyl coumarin and 3-bromo acetyl
coumarin was prepared according to the previously
reported literature methods[27]
.
SYNTHESIS OF SUBSTITUTED ISATIN
THIOSEMICARBAZONES (6A-6D) AND COUMARIN
THIOSEMICARBAZONES (7)
Substituted isatin thiosemicarbazones (6a-
6d) and coumarin thiosemicarbazones (7) was
prepared according to the literature with some
modifications[28]
.
Scheme-1: Synthesis of Acetyl coumarin (3) and
3-bromo acetyl coumarin (4)
Scheme-2: Synthesis of Substituted isatin
thiosemicarbazones (6a-6d)
5a, R=H, R1=H; 5b, R=CH2-C6H5, R1=H;
5c, R=H, R1=Br; 5d, R=H, R1=Cl.
6a, R=H, R1=H; 6b, R=CH2-C6H5, R1=H;
6c, R=H, R1=Br; 6d, R=H, R1=Cl.
Scheme-3: Synthesis of Coumarin
thiosemicarbazone (7)
SYNTHESIS OF INDOLE THIAZOLYL COUMARIN
DERIVATIVES
Final coumarin derivatives (8a-8d & 9) were
obtained by the cyclocondensation of substituted isatin
thiosemicarbazone compounds (6a-6d) or coumarin
thiosemicarbazone compounds (7) with 3-
bromoacetylcoumarin (4). A solution of 3-
bromoacetylcoumarin (5mmol) and substituted
thiosemicarbazones (5mmol) in ethanol-chlorofrom
(1:2) mixture was refluxed for 2-3 h at 60°C to obtain
yellow or brown precipitates (Scheme-4). The reaction
mixture was cooled in ice bath and basified with
ammonium hydroxide to pH 7-8. The title compounds
were recrystallized from ethanol-ethyl acetate
mixture.
4. Sreekanth et al. IJPER | Jan-Mar, 2014; 1(1): 29-35.
32
the bleaching of the purple colored methanol solution
of 1,1-diphenyl-1-picrylhydrazyl (DPPH). The
spectrophotometric assay uses the stable radical DPPH
as a reagent. 1ml of various concentrations of the test
compounds (5, 10, 25, 50 and 100mg/ml) in methanol
was added to 4ml of 0.004% (w/v) methanol solution
of DPPH. After a 30 minutes incubation period at
room temperature, the absorbance was read against
blank at 517nm. The percent of inhibition (%I) of free
radical production from DPPH was calculated by the
following equation.
(Acontrol – Asample)
% of scavenging = ----------------------- × 100
Ablank
Where Acontrol is the absorbance of the control reaction
(containing all reagents except the test compound) and
Asample is the absorbance of the test compounds. Tests
were carried in triplicate.
IN VITRO CYTOTOXIC ACTIVITY
The in vitro cytotoxic activities of the title
compounds against the human cancer T-lymphocyte
cell lines Molt 4/C8 and CEM and the murine tumor
leukemia cell lines L1210, human oral epidermoid
carcinoma KBcells, human promyelocytic leukemia
cells (HL60) and Bel-7402 liver cancer cells were
evaluated by the standard MTT assay method[29,30]
.
RESULTS AND DISCUSSION
Five new series of coumarin-indole-thiazole
derivatives, (8a-8d & 9), which incorporated four
important pharmacophores (coumarin ring, indole
heterocycle, thiazole heterocycle and imine group),
were synthesized by the condensation of Isatin
thiosemicarbazones (6a-6d) coumarin thiosemi-
carbazone (7) with 3-bromoacetyl coumarin (4). The
first step of the synthesis involved the preparation of
acetyl coumarin (3), and 3-bromo acetyl coumarin (4)
as shown in (Scheme-1). In the first series, a
substituted isatin thiosemicarbzone (6a-6d) and
coumarin thiosemicarbazone (7) was synthesized by
reacting thiosemicarbazide with a substituted isatins
(5a-5d) and acetyl coumarin (3) in the presence of
catalytic amount of glacial aceticacid. Purification by
recrystallization technique gave the substituted isatin
thiosemicarbazones (6a-6d) (Scheme-2) and coumarin
thiosemicarbazones (7) (Scheme-3) in good yields.
Condensation of these substituted isatin thiosemi-
carbazones (6a-6d) and coumarin thiosemi-
carbazones (7) with 3-bromoacetyl coumarin (4) in a
CHCl3-EtOH solvent mixture (2:1) (Scheme-4),
followed by alkalization with ammonium hydroxide
gave the first library of coumarin derivatives (8a-8d &
9) in good yield after purification by recrystallization.
Structure elucidation was confirmed by spectroscopic
and elemental analysis, for 9 (Fig. 1).
Infrared spectroscopic analysis of coumarin
8a-8d & 9 revealed the presence of lactone (C=O) and
imine (C=N) functional group with absorption at
1708-1719 & 1612-1604 cm-1
respectively along with
the characteristic absorption bands.
1
H-NMR spectroscopic analysis revealed
diagnostic resonances for the coumarin (4-H) and
thiazole methane portion at 8.54-8.74 and 7.76-7.94
ppm, respectively.
Fig. 1: 1
H-NMR Spectrum of 3-(2-(2-(1-(2-oxo-
2H-chromen-3-yl)ethylidine) hydrazinyl)thiazole-
4-yl)-2H-chromen-2-one
13
C-NMR spectroscopic analysis also
confirmed structural identity with resonance observed
at 168.7-172.4 (thiazole 2-C), 159.1-162.2 (C=O),
and 153.2-156.4 (C=N) ppm. Elemental analysis of
coumarin (8a-8d & 9) were within the range 0.3%
and fully supported structural assignment. 3D diagram
of the coumarin derivative (8c) given in Fig. 2.
Fig. 2: 3D diagram of (Z)-5-chloro-3-(2-(4-(2-oxo-
2H-chromen-3-yl)thiazol-2-yl)hydrazono)indolin-
2-one
In the present work, the antioxidant activity
of the synthesized coumarin compounds was assessed
in vitro by the 1,1-diphenyl-2- picrylhydrazyl (DPPH)
radical scavenging assay[31-35]
. These methods was
based on measuring the continual absorbance decrease
of the methanolic solution of the DPPH at 517 nm, in
the presence of antioxidant compound. The DPPH has
an odd electron so it can accept an electron or
hydrogen free radical. In the presence of antioxidant,
this odd electron becomes paired due to H transfer
from antioxidant and hence DPPH absorbance
decreases. The ability of newly synthesized
compounds (8a-8d & 9) to act as hydrogen donors or
5. Sreekanth et al. IJPER | Jan-Mar, 2014; 1(1): 29-35.
33
free radical scavengers was tested by in vitro
antioxidant assays involving DPPH radical, and the
results were compared with that of standard
antioxidant Ascorbic acid.
All the synthesized compounds showed
significant antioxidant activity compared to the
standard, Ascorbic acid. Among the synthesized
compounds (8c, 8d and 9) were found to be the most
potent antioxidant activity compared with that of the
standard (AA, 12.270.86) with the least values of
IC50 12.160.28, 11.320.22 and 11.240.4g/ml,
inhibition concentration respectively (Table 1). These
results gives an account for this antioxidant evolution
and indicates that compounds were capable with
significant scavenging properties towards DPPH. The
reason would be the presence of halogens (Chlorine
and bromine) in the indole ring which is linked with
thiazole along with coumarin ring. Almost, all the
tested compounds (8a-8d & 9) exhibited optimistic
efficacy for scavenging DPPH free radical. The next
promising antioxidant activity was showed by
compounds 8b and 8a were found to be moderate to
strong. This may be due to the presence of benzyl ring
on the nitrogen of the indole ring which linked to the
thiazole and coumarin rings.
Table 1: Antioxidant activity of the compounds
Compound
% inhibition
(IC50 µg/ml)
8a 12.68 0.24*
8b 12.28 0.16*
8c 11.32 0.22**
8d 11.24 0.14**
9 12.16 0.28**
AA 12.27 0.86**
AA – Ascorbic acid;
All the values were expressed as Mean SEM, n=3,
statistical analysis was done by one way ANOVA
using Graph Pad Prism 5.
**Represents statistical significance P<0.01,
*Represents statistical significance P<0.05.
The in vitro antineoplastic activities of the
synthesized compounds against the human cancer T-
lymphocyte cell lines Molt 4/C8 and CEM and the
murine tumor leukemia cell lines L1210, human oral
epidermoid carcinoma KB cells, human promyelo-
cytic leukemia cells (HL60) and Bel-7402 liver cancer
cells were evaluated by the standard MTT assay[29,30]
As described in Table 2, compounds 8c & 8d exhibit
very potent cytotoxic activity against all the cell lines.
On comparison to standard drug cisplatin, the
coumarin derivatives displayed the cytotoxicity at
higher concentration.
CONCLUSION
In the present work, a series of novel
thiazolyl coumarin derivatives were synthesized and
characterized by spectral studies. All the synthesized
compounds were evaluated fort heir in vitro cytotoxic
activities against Molt 4/C8, CEM, L1210, BEL7402
& HL60 by MTT assay method. Compounds 8c and
8d has exhibited potent anticancer activity against all
the cell lines. The antioxidant activity of the
synthesized compounds was evaluated by DPPH
scavenging method. Compounds 8c, 8d & 9 showed
significant antioxidant activity compared with that of
standard drug ascorbic acid. This research presented
many advantages such as novel block buster
compounds would helpful to develop new
chemotherapeutic agents, potential antioxidants and
some new therapeutic reagents for some diseases.
ACKNOWLEDGEMENTS
The authors are thankful to Mr. A. Madhukar
Reddy, Secretary and Correspondent, S. R. College of
Pharmacy, Warangal, Andhra Pradesh, India for
providing the laboratory facilities for carrying out the
research work.
Table 2: Cytotoxic studies of Coumarin thiazolyl compounds
Compound
IC50 (µg/L)
CEM L1210 Molt 4/C8 HL60 BEL7402
8a 120.4 211.2 160.4 9.21.4 140.6
8b 9.60.6 140.8 182.6 110.6 81.2
8c 81.2 125.2 100.2 81.4 91.2
8d 6.80.4 9.20.8 120.6 91.8 100.6
9 184.2 218.2 190.5 120.4 161.2
Cisplatin 0.510.06 1.20.02 0.870.06 0.980.02 0.780.04
6. Sreekanth et al. IJPER | Jan-Mar, 2014; 1(1): 29-35.
34
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![29
International Journal of Pharmacy Education and Research
Jan-Mar 2014; 1(1): 29-35.
Available online: www.ijper.net
Research Article
Synthesis, characterization, in vitro cytotoxic and antioxidant
activities of novel coumarin thiazolyl derivatives
Thota SREEKANTH*1
, Nadipelly KAVITHA1
, Anchuri SHYAMSUNDER1
, Yerra
RAJESHWAR1
, Karki SUBHAS SOMALINGAPPA2
, Jan BALZARINI3
, Erik DE CLERCQ3
1
Department of Pharmaceutical Chemistry, S. R. College of Pharmacy, Ananthasagar, Hasanparthy,
Warangal – 506 371, Andhra Pradesh, INDIA.
2
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Rajaji Nagar,
Bangalore – 560 010, Karnataka, INDIA.
3
Rega Institute for Medical Research, Katholieke, Universitteit Leuven, Minderbroedersstraat 10, B-
3000 Leuven, BELGIUM.
INTRODUCTION
Coumarin derivatives, an important group of
heterocyclic compounds, have been the subject of
extensive study in the recent past. Coumarin
derivatives are known to be a very interesting class of
synthetic or natural compounds. Coumarins (2H-1-
benzopyran-2-ones, 2H-chromen-2-ones) are very
attractive targets for combinatorial library synthesis
due to their wide range of valuable biological
activities. Many natural and synthetic coumarins have
occupied an important place in drug research, as a one
of the so-called privileged drug scaffold[1]
. They are
widely used as additives in food, perfumes,
cosmetics[2]
, pharmaceuticals and optical
brighteners[3]
and dispersed fluorescent and laser
dyes[4]
. Coumarin and its derivatives are biologically
interesting compounds known for their antimicrobial
and antifungal[5]
, antileishmanial[6]
, antioxidant[7]
,
antitumor[8]
, antiproliferation[9]
, hypotensive[10]
,
antiallergic[11,12]
, local anaesthetic[13]
, central nervous
Received on: 18 March, 2014
Revised on: 24 March, 2014
Accepted on: 26 March, 2014
*Corresponding author
T. Sreekanth
S. R. College of Pharmacy,
Ananthasagar, Hasanparthy,
Warangal – 506 371,
Andhra Pradesh, INDIA.
Mobile #: +91-99893-24804
Email id : stsreekanththota@gmail.com
ABSTRACT
A series of novel (Z)-3-(2-(4-(2-oxo-2H-chromen-3-yl)
thiazol-2-yl-)hydrazono)indolin-2-one (8a-8d, 9) were
synthesized with various substituted indole derivatives.
Structures of the newly synthesized compounds were elucidated
by FT-IR, 1
H-NMR, 13
C-NMR and API-ES Mass spectral data.
The in vitro cytotoxic activities of the complexes measurement
against the human cancer T-lymphocyte cell lines. In vitro
evaluation of these title complexes revealed cytotoxicity from 6.8-
18µg/mL against CEM, 9.2-21µg/mL against L1210, 10-19µg/mL
against Molt4/C8, 8-12µg/mL against HL60 and 8-16µg/mL
against BEL7402. Coumarin derivatives 8c and 8d showed that
quite significant anticancer activities. The antioxidant activity of
the synthesized compounds was evaluated by DPPH scavenging
method. Compounds 8c, 8d and 9 showed significant antioxidant
activity compared with that of standard drug, ascorbic acid.
Key words: Coumarin, DPPH, Cytotoxic activity.](https://image.slidesharecdn.com/05dr-160414064001/85/Synthesis-characterization-in-vitro-cytotoxic-and-antioxidant-activities-of-novel-coumarin-thazolyl-derivatives-1-320.jpg)
![Sreekanth et al. IJPER | Jan-Mar, 2014; 1(1): 29-35.
30
system (CNS) activities and effects[14]
, as well as
treatment of Alzheimer’s disease[15]
and
Schizophrenia disorder[16]
. Fused chromene ring
systems have platelet anti aggregating, local
anesthetic[17-19]
and antihistaminic activities[20]
. They
also exhibit antidepressant effects[21]
, inhibitory effect
on influenza virus sialidases[22,23]
, DNA breaking
activities and mutagenicity[24]
, antiviral activities[25]
and act as sex pheromonehomologes[26]
. The current
literature is enriched with progressive findings about
the synthesis and pharmacological action of fused
heterocycles. On the basis of all of this evidence, we
set out to prepare a new series of biologically active
agents containing both of these indole and thiazole
substituted coumarin derivatives. This study reports
the synthesis, characterization, and in vitro cytotoxic
and antioxidant activities of these new thiazolyl indole
coumarin derivatives.
MATERIALS AND METHODS
All the chemicals used were of Analytical
Range grade and procured from Sigma–Aldrich and
E.Merck and were used as received. Melting points
were recorded using a digital Gallenkamp (SANYO)
model MPD BM 3.5 apparatus and are uncorrected.
1
H-NMR and 13
C-NMR spectra were determined in
DMSO solution at 400 MHz using Bruker AM-400
spectrophotometer. FT-IR spectra were recorded using
an FTS 3000 MX spectrophotometer; Mass spectra
were recorded on a API-Electro spray (API-ES) Mass
spectrophotometer and elemental analyses with a
LECO-183 CHNS analyzer.
SYNTHESIS OF ACETYL COUMARIN (3) AND 3-BROMO
ACETYL COUMARIN (4)
Acetyl coumarin and 3-bromo acetyl
coumarin was prepared according to the previously
reported literature methods[27]
.
SYNTHESIS OF SUBSTITUTED ISATIN
THIOSEMICARBAZONES (6A-6D) AND COUMARIN
THIOSEMICARBAZONES (7)
Substituted isatin thiosemicarbazones (6a-
6d) and coumarin thiosemicarbazones (7) was
prepared according to the literature with some
modifications[28]
.
Scheme-1: Synthesis of Acetyl coumarin (3) and
3-bromo acetyl coumarin (4)
Scheme-2: Synthesis of Substituted isatin
thiosemicarbazones (6a-6d)
5a, R=H, R1=H; 5b, R=CH2-C6H5, R1=H;
5c, R=H, R1=Br; 5d, R=H, R1=Cl.
6a, R=H, R1=H; 6b, R=CH2-C6H5, R1=H;
6c, R=H, R1=Br; 6d, R=H, R1=Cl.
Scheme-3: Synthesis of Coumarin
thiosemicarbazone (7)
SYNTHESIS OF INDOLE THIAZOLYL COUMARIN
DERIVATIVES
Final coumarin derivatives (8a-8d & 9) were
obtained by the cyclocondensation of substituted isatin
thiosemicarbazone compounds (6a-6d) or coumarin
thiosemicarbazone compounds (7) with 3-
bromoacetylcoumarin (4). A solution of 3-
bromoacetylcoumarin (5mmol) and substituted
thiosemicarbazones (5mmol) in ethanol-chlorofrom
(1:2) mixture was refluxed for 2-3 h at 60°C to obtain
yellow or brown precipitates (Scheme-4). The reaction
mixture was cooled in ice bath and basified with
ammonium hydroxide to pH 7-8. The title compounds
were recrystallized from ethanol-ethyl acetate
mixture.](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)