Microwave assisted synthesis and evaluation of N-cinnamoyl aryl hydrazones for cytotoxic and antioxidant activities

Article abstract of DOI:10.13005/ojc/320350

A series of N-cinnamoyl aryl hydrazones 2a-2i were synthesized in good yields by microwave irradiation technique. The title compounds were formed by nucleophilic condensation of various N¹- substituted benzylidene-2-cyano aceto hydrazides with

Full text of DOI:10.13005/ojc/320350

ISSN: 0970-020 X
CODEN: OJCHEG
2016, Vol. 32, No. (3):
Pg. 1703-1709
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Microwave Assisted Synthesis and Evaluation of
N-cinnamoyl aryl hydrazones for Cytotoxic and Antioxidant
Activities
T.SARALA DEVI1* and G.RAJITHA2
1Department of Pharmaceutical Chemistry, KVSR Siddhartha College of Pharmaceutical
Sciences,Vijayawada-520010, Andhra Pradesh, India.
2Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam
(Women's University), Tirupati, India.
*Corresponding author E-mail:saralaratnakar@yahoo.com
http://dx.doi.org/10.13005/ojc/320350
(Received: April 08, 2016; Accepted: May 28, 2016)
ABSTRACT
A series of N-cinnamoyl aryl hydrazones 2a-2i were synthesized in good yields by microwave
irradiation technique. The title compounds were formed by nucleophilic condensation of various
N1- substituted benzylidene-2-cyano aceto hydrazides with N,N-dimethyl amino benzaldehyde.The
intermediate N1- substituted benzylidene-2-cyano aceto hydrazide was obtained by condensing
various substituted benzaldehydes with cyanoacetohydrazide.The structures of the compounds were
characterized by IR, 1H NMR and Mass spectra.The antioxidant activity was studied by reduction of
DPPH, scavenging of nitric oxide and hydrogen peroxide methods with ascorbic acid as the standard
drug. The compounds were evaluated for cytotoxic activity by BSLT method and their ED50 values
were compared the standard podophyllotoxin.Among the compounds evaluated, N1- benzylidene-2-
cyano-3-(4-dimethylamino) phenyl acrylo hydrazide (2a) and N1- (4-methoxy-benzylidene)-2-cyano-
3-(4-dimethylamino) phenyl acrylohydrazide (2e) showed good antioxidant activity towards all the
three models .The compounds 2a and 2e showed ED50 values 3.07 µg/ml and 3.7 µg/ml respectively
which were compared against the standard podophyllotoxin (1.64 µg/ml).
Keywords: Cinnamoyl hydrazones, Cyanoacetohydrazide, ED50,
BSLT, Cytotoxic,DPPH, Nitric Oxide, Hydrogen Peroxide.
INTRODUCTION
antioxidant10-12, antiinflammatory13, analgesic14,
antimalarial15, anti platelet activities16-17 etc . It
was reported that the presence of styryl ketone
moiety in the titled compounds were found to
be potent scavengers of free radicals. In view of
these observations it was considered of interest
to synthesise a new class of acyl hydrazones by
Acyl hydrazone derivatives possessing
an azometine -NHN=CH- proton are emerging
as a novel class of compounds for new lead
development1-4.They were reported to possess,
antimicrobial5-6, antitubercular7, antitumour8-9
,

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DEVI & RAJITHA, Orient. J. Chem., Vol. 32(3), 1703-1709 (2016)
incorporating the styryl carbonyl moiety along with by TLC and the solid formed was collected and
aryl hydrazone unit.The aim of present research was recrystallized from methanol.
to synthesize various N1-(substituted benzylidene)-2-
cyano-3-(4-dimethylamino ) phenyl acrylohydrazides General method of synthesis of compounds
and to evaluate for their in-vitro antioxidant and (2a-2i)
cytotoxic activities.
To 0.01 mol of various N1- substituted
benzylidene-2-cyanoacetohydrazides, 0.01mol of
N,N-di methylamino benzaldehyde was added in
few ml of ethanol followed by few drops of pyridine
MATERIALS AND METHODS
All the chemicals and solvents used in the and irradiated in microoven for 1 -3 minutes at 140
present study were purchased from Merck, Hi media, watts. The reaction was monitored by TLC and the
S.D. fine Chemicals limited, Mumbai and Sigma solid formed is collected and recrystallized from
Aldrich, USA. Melting points were determined in an methanol.
open capillary tube in Thermonik precision melting
point cum boiling point (C-PMB) apparatus and are
The physical data of compounds 2a-2i was
uncorrected. Silica gel G coated on laboratory micro tabulated in table-1.
slides prepared by dipping method were used. IR
spectra (KBr discs) were confirmed by Shimadzu FT- N1-benzylidene-2-cyano-3-(4-dimethylamino)
IR spectrophotometer using KBr pellets technique, 1H phenyl acrylo hydrazide (2a)
NMRspectrawererecordedonBruker300MHzNMR
Mol. Formula C19H18ON4,Yield :80 % ;m.p.:
spectrometer using DMSO as solvent.Mass spectra 161-162 0 C ;IR (KBr) cm-1:3209 (N-H), 3075 (Ar-H),
were recorded on Apex mass spectrophotometer.
2258 (C=N), 1671(C=O), 1551 (C=C) .1H NMR (300
MHz, DMSO-d6); d 3.1 (s,6H ,N(CH3)2), 6.8-7.77
(m,9H, Ar-H) , 8.0(s,IH,C=CH), 8.2(s,IH,N=CH),9.7(s
Chemistry
General method of synthesis of compounds 1H,-CONH); Mass: m/z ( M 1) 318, (M+H)+ 319
(1a-1i)
To 0.01 mol of various substituted N1- (3,4-dimethoxy-benzylidene)-2-cyano-3-(4-
benzaldehyde , 0.01 mol of cyanoacetohydrazide dimethylamino) phenyl acrylohydrazide (2b)
was added in few ml of ethanol followed by few drops Mol. Formula C21H22N4O3, Yield : 95 %; m.p.: 173-
of glacial acetic acid and irradiated in microoven for 1760 C ; IR (KBr) cm-1 : 3307 (N-H) , 3068 (Ar-H),
1-3 minutes at 140 watts .The reaction was monitored 2253 (C=N) , 1665(C=O), 1566 (C=C).1H NMR (300
Table 1: Physical data of N1-benzylidene-2-cyano-3-(4-dimethylamino)phenyl
acrylohydrazides (2a-2i)
Compound R1
code
R2
R3
M.P(OC)
Yield (%) Molecular
Formula
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
OCH3
OCH3
OCH3
H
H
H
H
3-NO2
H
OCH3
OH
OCH3
OCH3
OH
4-CH3
N(CH3)2
H
H
H
H
OCH3
H
H
H
H
H
161-162
173-176
178-179
161-166
167-175
160-162
178-179
158-160
178-179
80
85
84
82
89
60
50
49
76
C19H18N4 O
C21H22N4 O3
C20H18N4 O2
C22H24N4 O4
C20H20N4 O2
C19H18N4 O2
C20H20N4 O
C21H23N4 O
C19H17N5 O3

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DEVI & RAJITHA, Orient. J. Chem., Vol. 32(3), 1703-1709 (2016)
MHz,DMSO- d6); ä 3.0 (s,6H ,N(CH3)2), 3.7-3.8 C=CH), 8.1(s,IH,N=CH) , 9.6(s ,1H,-CONH)
(s,6H,-OCH3), 6.7-7.7 (m,7H, Ar-H), 7.9(s,IH,C=CH),
8.0 (s,IH,N=CH) , 9.6(s 1H,-CONH)
N1- (4 - dimethylamino -benzylidene)-2-cyano-3-
(4-dimethylamino) phenyl acrylohydrazide (2h)
N1-(3-methoxy,4-hydroxy-benzylidene)-2-cyano-
3-(4-dimethylamino) phenyl acrylohydrazide( m.p.158-160 C ; IR (KBr) cm-1 : 3242(N-H), 3076
2c)
Mol. formula C21H23N4O , Yield :89 %;
0
1
(Ar-H), 2325 (C=N), 1683(C=O), 1543 (C=C). H
Mol. formula C19H18N4O2, Yield : 94%; NMR (300 MHz,DMSO- d6) ; d 2.9-3.0 (s,12H,
m.p.:178-1790C ; IR (KBr) cm-1 : 3442 (N-H) , 3078 N(CH3)2), 6.7-7.5(m,8H, Ar-H), 7.9(s,IH,C=CH),
1
(Ar-H), 2239 (C=N), 1653(C=O), 1530 (C=C). H 8.0(s,IH,N=CH) , 9.6(s ,1H,-CONH)
NMR (300 MHz,DMSO- d6) ; d 3.0 (s,6H ,N(CH3)2), 3.7
(s,3H,OCH3), 6.7-6.8 (m,7H,Ar-H) , 7.8 (s,IH,C=CH), N1- (3-nitro -benzylidene)-2-cyano-3-(4-
8.0(s,IH,N=CH), 9.6(s ,1H,-CON-H)
dimethylamino) phenyl acrylohydrazide (2i)
Mol. formula C19H17N5O3 , Yield :89 %;
N1- (3,4,5-trimethoxy -benzylidene)-2-cyano-3-(4- m.p.178-1790 C ; IR (KBr) cm-1 : 3222 (N-H) , 3086
dimethylamino) phenyl acrylohydrazide (2d)
(Ar-H), 2285 (C=N) , 1628(C=O), 1513 (C=C).
Mol. Formula C22H24N4O4, Yield :92 %; 1H- NMR (300 MHz,DMSO- d6) ; d 3.0 (s,6H,
0
m.p.161-166 C;IR-(KBr) cm-1 : 3253 (N-H), 3068 N(CH3)2), 6.7-7.7( m,8H,Ar-H), 7.9(s,1H,C=CH),
(Ar-H), 2319 (C=N),1660 (C=O) , 1521 (C=C). 8.1(s,1H,N=CH) , 9.6(s ,1H,-CONH)
1H NMR (300 MHz,DMSO- d6) ; d 3.0 (s,6H,
N(CH3)2) 3.7-3.8 (s,9H,OCH3), 6.7-8.0(m,6H,Ar-H), Cytotoxic activity
9.6(s, 1H,-CONH),MASS:m/z ( M 1) 408, Brine shrimp lethality test18
(M+H)+ 409
Brine Shrimp (Artemia salina) nauplii were
hatched in sterile brine solution (prepared using sea
N1- (4-methoxy-benzylidene)-2-cyano-3-(4- salt 38g/L and adjusted the pH to 8.5 using 1N NaOH)
dimethylamino) phenyl acrylohydrazide (2e)
under constant aeration for 48 hr. After hatching,10
Mol. formula C20H20N4O2 , Yield :89 %; nauplii were placed in each vial and added various
m.p.167-175 0 C ; IR (KBr) cm-1 : 3213 (N-H) , 3088 concentrations of drug solutions in a final volume of
(Ar-H), 2320 (C=N) , 1672 (C=O), 1564 (C=C) .1H 5 mL, maintained at 37°C for 24 h under the light
NMR (300 MHz,DMSO- d6) ; d 3.0 (s,6H ,N(CH3)2), of incandescent lamps and surviving larvae were
3.7 (s,3H,-OCH3), 6.7-7.6(m, Ar-H), 7.9(s,1H, counted .Each experiment was conducted along with
C=CH) , 8.1(s,1H,N=CH), 9.6(s, 1H,-CONH)
control (vehicle treated), at various concentrations
of the test substances. Percentage lethality was
N1- (4-hydroxy-benzylidene)-2-cyano-3-(4- determined by comparing the mean surviving larvae
dimethylamino) phenyl acrylohydrazide (2f)
of test and control tubes. ED50 values were obtained
Mol. formula C19H18N4O2 , Yield :60 %; by using Finney probed analysis software .The result
m.p.160-162 0 C ; IR (KBr) cm-1 : 3282 (N-H) , 3095 for test compound was compared with the positive
(Ar-H), 2232 (C=N), 1628(C=O), 1553 (C=C).1HNMR control podophyllotoxin.
(300 MHz,DMSO- d6) ;d 3.0 (s,6H ,N(CH3)2), 6.7-7.5
( m,8H,Ar-H), 7.9(s,1H,C=CH) , 8.1(s,1H,N=CH), Antioxidant activity
9.6(s ,1H,-CONH)
Determination of nitric oxide Scavenging
Activity19
Nitric oxide scavenging activity of samples
was determined by the following procedure. 2ml
N1- (4-methyl-benzylidene)-2-cyano-3-
(4-dimethylamino) phenyl acrylohydrazide (2g)
Mol. formula C20H20N4O, Yield : 50 %; (10mM) of sodium nitro prusside dissolved in 1.5ml
m.p.178-179 0 C ; IR (KBr) cm-1 : 3260 (N-H) , 3100 phosphate buffer saline (PH-7.4) and 1 ml of different
1
(Ar-H), 2321 (C=N) , 1688(C=O), 1511 (C=C). H test samples corresponding to 100µM concentration
NMR (300 MHz,DMSO- d6) ; d 1.9 (s,1H,CH 3 ), was added in different test tubes respectively and
3.0 (s,6H ,N(CH3)2), 6.7-7.7( m,8H,Ar-H), 7.9(s,IH, incubated at 25OC for about 150 min.From this 0.5ml

DEVI & RAJITHA, Orient. J. Chem., Vol. 32(3), 1703-1709 (2016)
1706
was taken and 1ml sulphanilic acid reagent (33% in
20% glacial acetic acid) was added and incubated
at room temperature for 5min. 1ml of naphthyl
ethylene diamine dihydro chloride (0.1% w/v) was
added and again incubated at room temperature for
30min, then measured the absorbance at 540 nm in
spectrophotometer.
Determination of the effect of samples on 1,
1-diphenyl-2-picrylhydrazyl (DPPH)
Radical 19
DPPH scavenging activity was assessed
according to the reported method. Solutions of
various test samples at 100µM concentration were
added to 100µM DPPH in 95 % ethanol and tubes
were kept at an ambient temperature for 20-30 min
R1
O
O
C H 3 C O O H ,
N H 2
R 1
N C
N C
C
2 H 5 O H
R2
C H O
N
H
N
N
H
R 2
C
cyanoacetohydrazide
H
R3
R 3
1 a -1 i
py r idine
C 2 H 5 O H
,
O
C H 3
C H 3
H 3
C
C H 3
N
H C
N
N,N dimethyl amino benzaldehyde
R 1
H C
H
H
R 2
C
N
N
N C
O
R 3
2 a -2 i
Scheme
80
60
40
20
0
NITRIC OXIDE
HYDROGEN PEROXIDE
DPPH
i
f
a
c
e
b
d
2
g
h
d
r
2
2
2
2
2
2
2
2
a
d
n
a
t
S
Treament
Fig.1 :

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DEVI & RAJITHA, Orient. J. Chem., Vol. 32(3), 1703-1709 (2016)
Table 2: Antioxidant activity and Cyotoxic activity of compounds (2a-2i)
Compound
% Inhibition
of NO
% Inhibition
of H2O2
% Inhibition
of DPPH
ED50
(µg/ml)
2a
2b
2c
2d
2e
2f
2g
2h
10
8
11
8
11
8
8
7
7
22
-
59
31
34
39
49
20
18
20
32
71
-
50
36
45
38
51
31
31
25
40
52
-
3.07
5.28
6.37
10.50
3.77
-
-
4.07
-
-
2i
Ascorbic acid
Podopyllotoxin
1.64
and absorbance was measured at 517 nm. Ethanol amino group of cyanoacetohydrazide was condensed
was used as blank and DPPH solution in ethanol with carbonyl group of aldehyde to form schiffs
served as the control.The effect of Ascorbic acid on linkage.In the second step the various N1- substituted
DPPH was also assessed for comparison with that benzylidene-2-cyanoacetohydrazides are condensed
of samples.
with N,N–dimethylamino benzaldehyde at the
electrophilic carbon of cyanoacetohydrazide . The
Determination of Hydrogen Peroxide Scavenging structures of these compounds were established by
Activity20 means of their TLC, IR, 1H NMR and Mass spectra.
4mM solution of H2O2 was prepared in The synthesized compounds were evaluated for in-
phosphate - buffered saline (PBS, pH 7.4). H2O2 vitro antioxidant activity.Among the nine compounds
concentrationwasdeterminedspectrophotometrically synthesized six were evaluated for in-vitro cytotoxic
from absorbance at 230 nm using molar absorptivity activity.
81 M-1 cm-1 . 1 ml of different samples corresponding
to 100µM concentration were added to 0.6ml Antioxidant activity
hydrogen peroxide- PBS solution respectively and
The in-vitro antioxidant activity was
control without sample. Absorbance of H2O2 at evaluated by the reported methods of DPPH,
230nm was determined 10 minutes later against a nitric oxide and hydrogen peroxide . The results of
blank solution .
RESULTS AND DISCUSSION
Chemistry
antioxidant activities of the synthesized compounds
were shown in table-2. The unsubstituted (2a ) and
4-methoxy derivatives (2e) showed good scavenging
activity towards all the three models at 100µM
concentration , when compared with the standard
A series of N1-benzylidene-2-cyano-3- ascorbic acid.From the structure activity relationship
(4-dimethylamino)phenyl acrylohydrazides (2a-2i) studies of the compounds 2a-2i , it was observed
were synthesized by two step procedure .In the that the nature of the substitution on the benzylidene
first step various N1-substituted benzylidene- moiety affects the activity. Almost all the compounds
2-cyanoacetohydrazides were synthesized by showed good to moderate percentage scavenging
taking various substituted aromatic aldehydes and activity towards DPPH and NO radicals . The
cyanoacetohydrazide in few ml of ethanol by adding moderately electron releasing methoxy derivatives
a few drops of glacial acetic acid and irradiated in showed good to moderate activity towards H2O2
microoven for 1 -3 minutes at 140 watts . The free

DEVI & RAJITHA, Orient. J. Chem., Vol. 32(3), 1703-1709 (2016)
model. The electron releasing like 4-CH3 and N,N- CONCLUSION
1708
dimethylamino derivatives showed less scavenging
activity towards hydrogen peroxide radical when
compared with the standard.
In the present study we have described
the synthesis, invitro cytotoxicity screening and
antioxidant study of various N1- (substituted
benzylidene)-2-cyano-3-(4-dimethylamino) phenyl
acrylohydrazides. From the results it was evident
that the further substitution and modification on the
benzylidene moiety brings a new lead molecule.
Cytotoxic activity
The brine shrimp lethality test was
performed in order to evaluate the cytotoxic nature
of the compounds. ED50 values were calculated,
based on the percentage of larvae survived at
different concentrations of test and standard
drugs. Compounds 2a (3.07 µg/ml) and 2e (3.7 µg/
ml) showed good ED50 .The results are given in
table-2. It was observed that the compounds with
unsubstituted and 4-methoxy derivatives showed
good activity.
ACKNOwLEDGEMENTS
The authors are thankful to the Siddhartha
Academy of General and Technical Education
for providing necessary facilities to carry out this
research work.The authors are also thankful to laila
impex ,vijayawada for providing spectral analysis
and laila neutraceuticals for screening of cytotoxic
activity.
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