A "green" synthesis of N-(quinoline-3-ylmethylene)benzohydrazide derivatives and their cytotoxicity activities

Article abstract of DOI:10.1590/S0103-50532011000900017

We report some hybrid molecules based on N-(quinoline-3-ylmethylene) benzohydrazide template the synthesis of which was carried out using 2-chloroquinoline-3-carbaldehyde and a variety of substituted hydrazides in PEG 400. The "green" solvent PEG 400 was recovered and reused for several times in the present reaction and was found to be effective in terms of product yield. Some of the compounds synthesized showed significant cytotoxic activity when tested in vitro. ?2011 Sociedade Brasileira de Qui?mica.

Full text of DOI:10.1590/S0103-50532011000900017

J. Braz. Chem. Soc., Vol. 22, No. 9, 1742-1749, 2011.
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Article
A
A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
and their Cytotoxicity Activities
LingamVenkata Reddy,a Suresh Babu Nallapati,b Syed Sultan Beevi,c
Lakshmi Narasu Mangamoori,c Khagga Mukkantia and Sarbani Pal*,b
aCenter for Chemical Sciences and Technology and cCenter for Biotechnology,
Institute for Sciences and Technology, JNT University, Kukatpally, 500072 Hyderabad, India
bDepartment of Chemistry, MNR Degree and PG College, Kukatpally, 500072 Hyderabad, India
Relatamos algumas moléculas híbridas baseadas na N-(quinolina-3-ilmetileno)benzoidrazida,
cuja síntese foi realizada usando-se 2-cloroquinolina-3-carbaldeído e uma variedade de hidrazidas
substituídas, em PEG 400. O solvente PEG 400 foi recuperado e reutilizado diversas vezes na
presente reação, mostrando-se eficiente em termos de rendimento dos produtos. Alguns dos
compostos sintetizados mostraram atividade citotóxica significativa quando testados in vitro.
We report some hybrid molecules based on N-(quinoline-3-ylmethylene)benzohydrazide
template the synthesis of which was carried out using 2-chloroquinoline-3-carbaldehyde and a
variety of substituted hydrazides in PEG 400. The “green” solvent PEG 400 was recovered and
reused for several times in the present reaction and was found to be effective in terms of product
yield. Some of the compounds synthesized showed significant cytotoxic activity when testedin vitro.
Keywords: 2-chloroquinoline-3-carboxaldehyde, acylhydrazone, PEG 400, cytotoxicity
Introduction
In the light of these observations, we became interested
in the synthesis, characterization and evaluation of
pharmacological activities of hybrid molecules related
to N-acylhydrazones and quinolines. It was thought
worthwhile to combine these two potential pharmacophores
to generate a new hybrid class of molecules and evaluate
them for their synergistic cytotoxic activities. The hybrid
molecule A (Figure 1) was expected to be cytotoxic due
to the presence of hydrazone moiety as well as quinoline
structure. Indeed, the fragment –CO−NH−N=CH−
(2-hydroxyphenyl) was found to be responsible for potent
antiproliferative activities of benzo[d]isothiazole-3-
carboxylic acid (4-methoxy-benzylidene)-hydrazide (B,
Figure 1).6,7 This compound showed the most marked
effects on the ovarian cancer cell line (OVCAR log10
GI50 value −5.51) when tested in vivo. Thus, the common
structural features between A and B prompted us to evaluate
the cytotoxic potential of A in vitro. While the synthesis of
compounds related to A have been reported earlier,8,9 their
utility as potential cytotoxic agents has not been explored.
In performing the majority of organic transformations,
solvents play an important role in mixing the ingredients
to make the system homogeneous and allow molecular
A current rational approach of drug design characterized
as ‘‘covalent bitherapy’’ involves linking of two molecules
possessing individual inherent activity into a single agent,
thusincorporatingdualactivityintoasinglehybridmolecule.
Contemporary research in this area seems to endorse hybrid
molecules as the next-generation drug candidates.1
The quinoline scaffold is prevalent in a variety of
pharmacologically active compounds.2 They also occur
widely in nature indicating nature’s preference for this
fragment and identifying it as one of the so-called privileged
structures. Compounds based on the quinoline moiety
joined to other pharmacophore are of considerable interest
because of their potential pharmacological properties.
Hydrazones and substituted hydrazones on the other hand,
because of their distinctive structural features and presence
of azomethine group, continue to attract the attention of the
medical researchers.3-5 This class of compounds show an
extensive range of pharmacological properties especially
antitumor activities.
*e-mail: sarbani277@yahoo.com

Vol. 22, No. 9, 2011
Reddy et al.
1743
H
Results and Discussion
N
Ar
O
N
R
N
O
Ar
NHNH2
N
X
Chemistry
A
H3C
N
N
H
Responsible for
antiproliferative
activities
S
N
The key step for the synthesis of N-(quinoline-3-
ylmethylene)benzohydrazides (3) involved the reaction
of 2-chloro-3-quinoline carboxaldehyde (1) with an
appropriate hydrazide (2). Thus, the key intermediate 1
required for our synthesis was prepared from acetanilide
according to a similar procedure reported in the literature
(Scheme 1). Cyclization of acetanilide in the presence
of POCl3 and N,N-dimethyl formamide (DMF) leads to
the formation of the desired intermediate 1. The other
reactant, i.e., hydrazide (2), was prepared via esterification
of the corresponding carboxylic acids with methanol in
the presence of catalytic amount of concentrated H2SO4
followed by treating the resulting ester with hydrazine
hydrate in methanol. The compound 3 was finally prepared
successfully by condensation of 1 and 2 in PEG 400.
The earlier method for the preparation of this type of
compounds, e.g., N’-((2-chloroquinolin-3-yl)methylene)
benzohydrazide, involved heating the appropriate reactants
in ethanol for several hours.8,9 However, to establish a milder
and environmentally friendly condition, we examined the
reaction of 2-chloro-3-quinoline carboxaldehyde (1) with
2-methoxybenzohydrazide (2a) in various solvents at room
temperature. The results of this study are summarized in
Table 1.As indicated in Table 1 that no desired product (3a)
was isolated when the reaction was performed in solvents
such as benzene, chloroform and dichloromethane (entries
1, 2 and 3, Table 1), even after 48 h whereas only 37% of 3a
was formed when iso-propanol was used as solvent (entry 4,
Table 1). While the reaction time was reduced from 48 to
7 h when EtOH was used as a solvent (entry 5, Table 1),
the best yield of 3a, however, was obtained in PEG 400
or 1,4-dioxane (entry 6 and 7, Table 1). Interestingly, the
work-up procedure was found to be simple when PEG 400
was used. After completion of the reaction, the mixture
was extracted with diethyl ether and the pure product 3a
was isolated simply after purification via crystallization.
The PEG 400 recovered was recycled without further
purification. We have reused the recovered PEG 400 in
the reaction of 1 with 2 at least for three times and 3a was
isolated each time almost in similar yield. Thus, we decided
N
N
O
CH3
OH
B
Figure 1. Quinoline and acylhydrazone based hybrid molecule A and a
known antiproliferative agent B.
interactions to be more efficient. Recently, control of
environmental pollution caused by chemical reactions has
become a challenging as well as inspiring issue and many
strategies and techniques have been explored to overcome
these harmful events, for example, conducting reaction
in dry medium or the use of micro wave irradiation, solid
support, ionic liquid, water etc. In case of catalysis it
is customary to measure the efficiency of a catalyst by
its environmental impact, how easily it can be exposed,
how many times it can be recycled in addition to its low
volatility, non flammability, solubility etc.
The use of a simple and widely available polymer, e.g.,
polyethylene glycol (PEG) as a nontoxic, inexpensive,
non-ionic liquid solvent of low volatility has been explored
in several reactions. In general, PEG being a biologically
acceptable polymer has been used extensively in drug
delivery and in bioconjugates as tool for diagnostics.
It was already used successfully in different reactions
like oxidation,10 reduction,11 Michael reaction,12 Baylis-
Hillman reaction,13 Heck coupling,14 Suzuki-Miyaura
cross coupling,15 synthesis of bezimidazole,16 etc. It was
also used in enantioselective asymmetric synthesis like
asymmetric dihydroxylation,17 asymmetric aldol reaction,18
etc. In addition, aqueous PEG solutions may often be used
as substitute for expensive and often toxic phase transfer
catalysts (PTCs). All these attractive “green” advantages
encouraged us to study the PEG as alternative reaction
medium for our purpose. While the synthesis of similar
compounds represented by general formula “A” has been
reported earlier, their preparation using PEG 400 is not
known. Herein, we report the use of PEG 400as a recyclable
reaction medium for the synthesis of compound A (or 3,
Scheme 1) at room temperature without using any acid or
base catalysts.
H
NHCOCH3
N
Ar
ArCONHNH2 (2)
CHO
Cl
N
POCl3, DMF
O
PEG-400
0 oC, 30 min
75 oC; 8 h
68%
N
Cl
N
Ar = substituted arene or
aryloxy methylene
3
1
Scheme 1. Synthesis of N-(quinoline-3-ylmethylene)benzohydrazide derivatives (3).

1744
A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
J. Braz. Chem. Soc.
to choose PEG 400 as a “green” and recyclable solvent for
our further studies. Notably, no reaction was observed when
water was used as a solvent (entry 8, Table 1) perhaps due
to the poor solubility of reactants in water.
of compounds 3. Moreover, based on the earlier report
that N-acylhydrazones derived from aromatic aldehydes in
solution remained in the E form, because of the hindered
rotation on the imine bond,20 we considered E-geometry
in our cases. However, in few cases, e.g., for compounds
3e and 3g, the presence of a mixture of rotameric forms21
was detected (see the experimental section).
Table 1. Effect of solvent on the reaction of 2-chloro-3-quinoline
carboxaldehyde (1) with benzohydrazide (2a)a
H
N
Pharmacology
H
solvent
N
N
H2N
+
1
O
OMe
N
Cl
O
OMe
To assess the cytotoxic activity potential of this class
of compounds some of the compounds synthesized were
tested against human lung adenocarcinoma cell line (A549)
in vitro.Adenocarcinoma is the most common type of lung
cancer22 that contains certain distinct malignant tissue
architectural, cytological, or molecular features, including
gland and/or duct formation and/or production of significant
amounts of mucus. Adenocarcinomas account for
approximately 40% of lung cancers.Adenocarcinoma is not
as responsive to radiation therapy and is rather treated by
surgically, for example by pneumonectomy or lobectomy.
Thus small molecules that are active against human lung
adenocarcinoma cell line may be useful for the development
of potential agents to treat lung cancer. With this objective
we conducted in vitro screen of compounds 3a-i and the
results are summarized in Table 3. Since acylhydrazones
may undergo hydrolysis to release toxic entities hence
blank experiments were also carried out to confirm that
the observed activity was due to the parent compounds
but not their hydrolyzed products.All the compounds were
tested at five different concentrations ranging from 1.0
to 25 µg mL-1. At the concentration of 25 µg mL-1 all the
compounds showed significant inhibition and most of them
showed reasonable dose responses across all the doses. The
compounds 3b, 3c, 3d and 3h showed good inhibition at
25 µg whereas low to moderate inhibition was observed for
compounds 3d, 3g, 3h and 3i at the lowest concentration,
i.e., 1.0 µg tested. The other compounds were found to be
either inactive or less effective at low concentrations. A
known compound, i.e., etoposide (IC50 = 9.85 µmol L-1)
was used as a reference compound in this assay that showed
100% inhibition when tested at 25 µg mL-1.
2a
3a
entry
solvent
time / h
48
yieldb / (%)
1
2
3
4
5
6
7
8
C6H6
CHCl3
DCM
0
0
48
48
0
i-PrOH
EtOH
48
37
64
7
PEG 400
1,4-Dioxane
Water
6
85 (83, 80, 79)c
6.5
17
88
0
aAll the reactions were performed using 1 (1.0 mmol) and 2a (1.0 mmol)
in a solvent at room temperature under an open air condition. Isolated
yield. cYields of 3a in recycled PEG 400 for 1st, 2nd and 3rd time.
b
Having identified PEG 400 as a suitable and green
solvent for the efficient preparation of 3a via the reaction
of 1 and benzohydrazide (2a) at room temperature, we
decided to examine the generality of this process in
the preparation of other N-(quinoline-3-ylmethylene)
benzohydrazide derivatives. Accordingly, a variety of
functionalized benzohydrazides were reacted with 1
in PEG 400 at room temperature (Table 2). As evident
from Table 2, the reactions proceeded well in all these
cases, providing the desired products in good yields. The
presence of electron donating, e.g., OMe (entry 1, Table 2),
phenolic OH (entry 3, Table 2) or chloro (entry 8, Table 2),
and electron withdrawing group, e.g., NO2 (entries 2 and
6, Table 2) present at the aryl ring of hydrazide 2, were
well tolerated. A hydrazide 2d (entry 4, Table 2) prepared
from a well known anti-inflammatory drug, mefenamic
acid, also participated well in the present reaction and
provided the expected product 3d in good yield.Apart from
benzohydrides, we examined the reactivity of two 2-aryloxy
acetohydrazides in the present reaction both of which
provided the corresponding products in good yields (entries
5 and 7, Table 2).All the compounds synthesized were well
characterized by spectral (1H NMR, IR and MS) data.8,9 The
presence of C=O and C=N groups19 were indicated by the
appearance of stretching frequencies in the range of 1700-
1650 and 1650-1590 cm-1, respectively, in the IR spectra
It is known that a number of acylhydrazone derivatives
that showed activities in standard growth inhibition assays,
were found to inhibit tubulin polymerization23 (which is the
most probable primary mechanism of the action of these
compounds). Tubulin-containing structures are important
for diverse cellular functions, including chromosome
segregation during cell division, intracellular transport,
development and maintenance of cell shape, cell motility,
and possibly distribution of molecules on cell membranes.

Vol. 22, No. 9, 2011
Reddy et al.
1745
Table 2. Preparation of N-(quinoline-3-ylmethylene)benzohydrazide derivatives in PEG 400a
entry
1
benzohydrazide (2)
products (3)
time / h
yieldb/%
CONHNH2
OMe
H
N
2a
2b
2c
3a
3b
3c
6
85
80
82
N
O
OMe
N
Cl
CONHNH2
NO2
H
N
2
2
4
N
O
N
Cl
NO2
CONHNH2
OH
H
N
3
N
O
OH
N
Cl
O
H
N
NHNH2
N
NH
Me
O
HN
Me
4
2d
3d
4
78
N
Cl
Me
Cl
Me
CONHNH2
O
H
5
2e
3e
2
79
N
N
O
Cl
O
Cl
Cl
N
Cl
CONHNH2
NO2
NO2
H
N
6
7
8
9
2f
3f
3
76
87
77
75
N
O
NO2
N
Cl
NO2
OCH2CONHNH2
H
N
2g
2h
2i
3g
3h
3i
4.5
N
O
O
N
Cl
CONHNH2
Cl
H
N
6
N
O
N
Cl
Cl
CONHNH2
H
N
2.5
N
O
N
Cl
aAll the reactions were performed using 1 (1.0 mmol) and 2 (1.0 mmol) in PEG 400 at room temperature under an open air condition. bIsolated yield.

1746
A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
J. Braz. Chem. Soc.
Table 3. In vitro MTT assay results of some of the N-(quinoline-3-ylmethylene)benzohydrazide derivatives
% of inhibition at various concentrationsa
entry
compounds
1 µg mL-1
8.00
2 µg mL-1
5 µg mL-1
27.38
46.27
30.12
56.83
18.46
12.61
27.38
45.56
32.61
10 µg mL-1
25 µg mL-1
43.38
78.90
89.32
88.72
20.92
53.53
43.38
79.55
66.76
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
12.61
23.85
23.52
36.54
16.30
9.53
31.69
54.78
67.89
68.90
19.07
23.07
31.69
67.13
38.15
14.56
17.86
20.78
10.30
0
3g
3h
3i
21.53
22.56
26.15
22.61
37.83
29.83
aThe data presented are the average of three separate experiments. Etoposide (IC50 = 9.85 µmol L-1) was used as a reference compound.
The drugs that interact with tubulin cause its precipitation
and sequestration to interrupt many important biologic
functionsthatdependonthemicrotubularclassofsubcellular
organelles. Thus, cytotoxicity shown by the present series
of compounds (3) could be due to their covalent binding
with b-tubulin thereby inhibiting the tubulin polymerization.
(TMS, d = 0.00). 13C NMR spectra were recorded on a
BrukerACF-300 machine using either TFA (trifluoroacetic
acid) or DMSO as a solvent. Chemical shift values of
rotameric hydrogens whenever identified are presented
within the parenthesis by assigning asterisk (*) mark along
with that of other form. Elemental analyses were performed
byVarian 3LV analyzer series CHN analyzer. Mass spectra
were recorded on a Jeol JMCD-300 instrument.All solvents
used were commercially available and distilled before use.
All reactions were monitored by TLC on pre-coated silica
gel plates (60 F 254; Merck). Column chromatography
was performed on 100-200 mesh silica gel (SRL, India)
using 10-20 fold excess (by weight) of the crude product.
The organic extracts were dried over anhydrous Na2SO4.
The percentage of ap rotamer present was calculated
by using the following formula
Conclusions
In conclusion, we have described the synthesis, and
in vitro pharmacological properties of a number of novel
N-(quinoline-3-ylmethylene)benzohydrazide derivatives.
Syntheses of these compounds were carried out using
2-chloroquinoline-3-carbaldehyde and a variety of
substituted hydrazides under a mild reaction conditions.
PEG 400 was identified as a green, effective and recyclable
solvent for our synthesis. Some of the compounds
synthesized showed significant cytotoxicity when tested
against human lung adenocarcinoma cell line (A549) in
vitro. Since quinoline24 and benzohydrazide derivatives25,26
have medicinal value, hence we believe that the present
class of hydrazide derivatives represents an interesting
profile for further experimental investigations especially
in the area of anticancer research.
% of ap rotamer = (Iap/ Iap + Isp) × 100
Iap = intensity of a singlet appeared in the 1H NMR of ap
rotamer
Isp = intensity of the corresponding singlet appeared in the
1H NMR of sp rotamer
Preparation of 2-chloro-3-quinoline carboxaldehyde27 (1)
To an ice-cold solution of dimethylformamide
(7.20 mL, 93.0 mmol) was added phosphorus oxychloride
(24.0 mL, 260 mmol) dropwise and the mixture was stirred
at 0 ºC for 45 min. To this mixture was added acetanilide
(5.0 g, 37.0 mmol). The mixture was stirred initially at 0 ºC
for 30 min and then heated at 75 ºC for 8 h. The mixture
was cooled, poured into ice-water (300 mL) and stirred at
0-5 ºC for 45 min. The solid separated was filtered, washed
with cold water (150 mL), dried and recrystallized from
ethyl acetate to give the title compound as a light yellow
solid (4.8 g, 68%); mp 146-147 ºC (lit27 148-149 ºC).
Experimental
General methods
Melting points were all determined by open glass
capillary method on a Cintex melting point apparatus
and are uncorrected. IR spectra were recorded on a
1
Perkin Elmer spectrometer in KBr pellets. H NMR
spectra were recorded on a Bruker ACF-300 machine
or a Varian 300 MHz spectrometer using DMSO-d6 as
a solvent with tetramethylsilane as internal reference

Vol. 22, No. 9, 2011
Reddy et al.
1747
General procedure for the synthesis of 3a-k
4-H), 8.45 (d,1H, J 7.5 Hz, ArH), 8.00 (d,1H, J 8.4 Hz,
ArH), 7.90 (t, 2H, J 7.2 Hz, ArH), 7.70 (t,1H, J 7.5 Hz,
ArH), 7.50 (t, 1H, J 7.2 Hz, ArH), 7.00 (t, 2H, J 7.8 Hz,
ArH). Elemental analysis found: C 62.45, H 3.67, N 12.66;
C17H12ClN3O2 requires C 62.68, H 3.71, N 12.90%.
To a cold solution of 2-chloroquinoline-3-
carboxaldehyde (2.07 g, 0.011 mol) in PEG 400 (5.0 mL)
was added an appropriate hydrazide (0.01 mol) and the
solution was stirred vigorously at room temp for the time
indicted in Table 2. The progress of the reaction was
monitored by TLC. After completion of the reaction the
mixture was diluted with diethyl ether (25 mL), stirred for
10 min and then allowed to settle. The ether layer separated
was collected, washed with cold water (2 × 15 mL), dried
over anhydrous Na2SO4, filtered and concentrated. The
crude compound isolated was purified by re-crystallization
(EtOH) to furnish the desired product.
N’-((2-Chloroquinolin-3-yl)methylene)-2-(2,3-
dimethylphenylamino)benzohydrazide (3d)
o
Yellow solid; yield 3.34 g (78%); mp 218-220 C
(EtOH); Rf 0.40 (ethyl acetate:hexane, 2:3); MS m/z 428
(M+100%), 430 (M+2) 3:1 ratio; IR (KBr) νmax/cm-1:
3309, 3213, 3039, 2920, 1674, 1629, 1616, 1577;
1H NMR (DMSO-d6, 300 MHz) d 12.30 (s, 1H, NH, D2O
exchangable), 9.40 (s,1H, NH, D2O exchangable), 8.95
(s,1H, -CH=N-), 8.90 (1H, s, 4-H), 8.25 (d,1H, J 8.4 Hz,
ArH), 8.08-7.70 (4H, ArH, m), 7.35 (t,1H, J 7.2 Hz),
7.10-6.85 (m, 5H, ArH), 2.30 (s, 3H, CH3), 2.10 (s, 3H,
CH3); 13C NMR (TFA, 75 MHz) d 168.3, 148.8, 148.1,
145.5, 143.1, 141.1, 140.3, 137.8, 137.6, 133.3, 133.2,
132.8, 131.9, 131.6, 130.6, 129.2, 128.9, 128.8, 127.9,
126.5, 126.4, 123.4, 120.7, 19.9, 13.5. Elemental analysis
found: C 70.25, H 4.74, N 13.14; C25H21ClN4O requires
C 70.01, H 4.93, N 13.06%.
The PEG 400 layer was collected and recycled in the
next reaction.
N’-((2-Chloroquinolin-3-yl)methylene)-2-methoxybenzo-
hydrazide (3a)
Light orange solid; yield: 2.88 g (85%); mp 142-144 oC
(EtOH); Rf = 0.45 (ethyl acetate:hexane, 2:3); MS m/z 339
(M+100%), 341 (M+2) 3:1 ratio; IR (KBr) νmax/cm-1: 3432,
3266, 2956, 1676, 1649, 1607, 1578; 1H NMR (DMSO-d6,
300 MHz) d 12.10 (1H, NH, s, D2O exchangable), 9.00 (s,
1H, -CH=N-), 8.90 (s, 1H, 4-H), 8.25 (d, 1H, J 8.1 Hz,
ArH), 8.00 (m, 3H, ArH), 7.90 (t,1H, J 8.1Hz, ArH,), 7.70
(t, 2H, J 7.8Hz, ArH), 7.10 (d,1H, J 9.0 Hz, ArH), 3.06 (s,
3H, OMe). Elemental analysis found: C 63.39, H 4.11, N
12.52; C18H14ClN3O2 requires C 63.63, H 4.15, N 12.37%.
N’-((2-Chloroquinolin-3-yl)methylene)-2-(3,5-dichloro-
phenoxy)acetohydrazide (3e)
o
White solid; yield 3.22 g (79%); mp 198-200 C
(EtOH); Rf = 0.62 (ethyl acetate:hexane, 2:3); MS m/z 408
(M+100%); IR (KBr) νmax/cm-1: 3192, 3113, 2987, 1685,
1635, 1616, 1583; 1H NMR (DMSO-d6,300 MHz) d 12.10
(12.00*, s, 1H, NH, D2O exchangable), 9.03 (8.98*, s, 1H,
-CH=N-), 8.70 (8.46*, s,1H, ArH), 8.22 (8.13*, d,1H,
J 12 Hz,ArH), 7.98 (d,1H, J 8 Hz,ArH), 7.88 (t,1H, J 8 Hz,
ArH), 7.70-7.34 (m, 3H, ArH), 7.20 (m,1H, ArH), 5.20 (s,
2H, OCH2) (ap and sp rotameric ratio 75:25, I9.03 = 0.67,
I8.98 = 0.22, % of ap = (0.67/0.67 + 0.22) × 100 = 75);
13C NMR (DMSO-d6, 75 MHz) d 165.8, 162.0, 161.4,
158.1, 145.3, 133.2, 132.0, 130.7, 129.7, 128.7 (2C), 125.4,
121.1, 118.5, 113.7 (2C), 113.6, 55.3. Elemental analysis
found: C 52.67, H 2.71, N, 10.41; C18H12Cl3N3O2 requires
C 52.90, H 2.96, N 10.28%.
N’-((2-Chloroquinolin-3-yl)methylene)-4-nitrobenzo-
hydrazide (3b)
o
Yellow solid; yield 2.83g (80%); mp 258-260 C
(EtOH); Rf = 0.65 (ethyl acetate:hexane, 2:3); MS m/z 354
(M+100%), 356 (M+2) 3:1 ratio; IR (KBr) νmax/cm-1: 3437,
3181, 2853, 1670, 1617, 1597, 1523; 1H NMR (DMSO-d6,
300 MHz) d 12.50 (s,1H, NH, D2O exchangable), 9.05
(s,1H, -CH=N-), 8.95 (s,1H, 4-H), 8.40 (d, 2H, J 8.7 Hz,
ArH), 8.30 (m, 3H, ArH), 8.00 (d,1H, J 8.4 Hz, ArH), 7.90
(t,1H, J 6.9 Hz,ArH), 7.70 (t,1H, J 7.5 Hz,ArH). Elemental
analysis found: C 57.33, H 3.19, N 15.58; C17H11ClN4O3
requires C 57.56, H 3.13, N 15.79%.
N’-((2-Chloroquinolin-3-yl)methylene)-2,4-dinitrobenzo-
N’-((2-Chloroquinolin-3-yl)methylene)-2-hydroxybenzo-
hydrazide (3f)
o
hydrazide (3c)
Yellow solid; yield 3.03g (76%); mp 142-144 C
o
Off white solid; yield 2.67 g (82%); mp 158-160 C
(EtOH); Rf = 0.6 (ethyl acetate:hexane, 2:3); MS m/z 399
(M+100%), 401 (M+2) 3:1 ratio; IR (KBr) νmax/cm-1:
3414, 3250, 2916, 1665, 1630, 1619, 1582, 1563;
1H NMR (DMSO-d6, 300 MHz ) d 12.40 (1H, NH, s, D2O
exchangable), 8.69 (1H, s, -CH=N-), 8.68 (s,1H, 4-H),
8.26 (d,1H, J 8 Hz, ArH), 8.00-7.86 (m, 2H, ArH), 7.72
(EtOH); Rf = 0.36 (ethyl acetate:hexane, 2:3); MS m/z 325
(M+100%), 327 (M+2) 3:1 ratio; IR (KBr) νmax/cm-1: 3196,
3051, 1669, 1590, 1556; 1H NMR (DMSO-d6, 300 MHz)
d 12.20 (s, 1H, NH, D2O exchangable), 11.75 (s,1H, OH,
D2O exchangable), 9.00 (s,1H, -CH=N-), 8.95 (1H, s,

1748
A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
J. Braz. Chem. Soc.
(t,1H, J 8 Hz, ArH), 7.57-7.56 (m,1H, ArH), 6.19-6.10 (m,
2H, ArH); 13C NMR (DMSO-d6, 75 MHz) d 161.8, 150.3,
148.7, 148.5, 147.2, 143.4, 135.8, 134.7, 131.8, 129.0,
127.8, 127.6, 126.8, 126.0, 118.9, 109.9, 108.3. Elemental
analysis found: C 51.26, H 2.29, N 17.39; C17H10ClN5O5
requires C 51.08, H 2.52, N 17.52%.
penicillin were obtained from Sigma-Aldrich, USA. Foetal
bovine serum was procured from PAA Biotech, Germany.
All other fine chemicals/reagents used in this study were
of cell culture grade and obtained from Sigma-Aldrich
and/or Merck.
Cell line and culture conditions: A549 (human lung
adenocarcinoma cell line) was obtained from National
Centre for Cell Science, Pune, India. The cells were grown
in DMEM culture medium supplemented with 2 mmol L-1
L-glutamine, 10% FBS, penicillin (50 IU per mL) and
streptomycin (50 µg mL-1) at a temperature of 37 °C in a
humidified incubator with a 5% CO2 atmosphere.
MTT assay for cytotoxicity: The viability of the cells
was assessed by MTT (3, 4, 5-dimethylthiazol-2-yl)-2-5-
diphenyltetrazolium bromide) assay, which is based on the
reduction of MTT by the mitochondrial dehydrogenase
of intact cells to a purple formazan product. Fifteen cells
(1 × 104) were plated in a 96-well plate. After 24 h, they
were treated with different concentration (0-10 µg mL-1)
of different test compounds diluted appropriately with
culture media for 48 h. Cells grown in media containing
equivalent amount of DMSO served as positive control and
cells in medium without any supplementation were used
as negative control. After the treatment, media containing
compound were carefully removed. 100 µL of 0.4 mg mL-1
MTT in PBS was added to each well and incubated in the
dark for 4 h. 100 µL of DMSO was added to each well and
kept in an incubator for 4 h for dissolution of the formed
formazan crystals.Amount of formazan was determined by
measuring the absorbance at 540 nm using an ELISA plate
reader. The data were presented as percent post treatment
recovery (% of live cells), whereas the absorbance from
non-treated control cells was defined as 100% live cells.
N’-((2-Chloroquinolin-3-yl)methylene)-2-phenoxy-
acetohydrazide (3g)
o
Off white solid; yield 2.96 g (87%); mp 168-170 C
(EtOH); Rf = 0.86 (ethyl acetate:hexane, 2:3); MS m/z 340
(M+100%); IR (KBr) νmax/cm-1: 3279, 3194, 2939, 1681,
1614, 1597, 1585; 1H NMR (DMSO-d6,300 MHz) d 12.10
(12.00*, s, 1H, NH, D2O exchangable), 9.02 (8.99*, s,
1H, =CHN), 8.85 (8.46*, s, 1H, ArH), 8.22 (8.18*, m,
1H, ArH), 7.98 (d, 1H, J 8 Hz, ArH), 7.84 (m, 1H, ArH),
7.70 (m, 1H, ArH), 7.32 (m, 2H, ArH), 7.00 (m, 3H, ArH),
5.23 (4.70*, s, 2H, CH2) (ap and sp rotameric ratio 54:46,
I5.23 = 0.60, I4.70 = 0.50, % of ap = (0.60/0.60 + 0.50) ×
100 = 54). Elemental analysis found: C 63.34, H 4.08,
N 12.46; C18H14ClN3O2 requires C 63.63, H 4.15, N 12.37%.
4-Chloro-N’-((2-chloroquinolin-3-yl)methylene)
benzohydrazide (3h)
o
Off white solid; yield 2.65 g (77%); mp 268-270 C
(EtOH); Rf = 0.52 (ethyl acetate:hexane, 2:3); MS m/z 344
(M+100%) 346 (M+2) 3:1 ratio; IR (KBr) νmax/cm-1: 3174,
3053, 2902, 1651, 1618, 1593, 1552; 1H NMR (DMSO-d6,
300 MHz) d 12.40 (s, 1H, NH, D2O exchangable), 8.95
(s,1H, -CH=N-), 8.94 (s,1H, 4-H), 8.25 (d,1H, J 8.1 Hz,
ArH), 8.00 (m, 3H, ArH), 7.85 (t, 1H, J 6.9 Hz, ArH),
7.76-7.65 (m, 3H). Elemental analysis found: C 59.51, H
3.29; C17H11Cl2N3O requires C 59.32, H 3.22, N 12.21%.
N’-((2-Chloroquinolin-3-yl)methylene)benzohydrazide8
(3i)
Supplementary Information
White solid; yield 2.32 g (75%); mp 208-210 oC (EtOH)
Supplementary information (copies of 1H NMR spectra
of all the compounds synthesized) is available free of charge
at http://jbcs.sbq.org.br as a PDF file.
o
(lit8 202-205 C); Rf = 0.71 (ethyl acetate:hexane, 2:3);
MS m/z 309 (M+100%) 311 (M+2) 3:1 ratio; IR (KBr)
ν
max/cm-1: 3748, 3182, 2923, 1698, 1618, 1600, 1563;
1H NMR (DMSO-d6, 300 MHz ) d 12.30 (s,1H, NH, D2O
exchangable), 8.98 (s, 1H, -CH=N-), 8.97 (s,1H, 4-H),
8.25 (d,1H, J 8.1 Hz, ArH), 7.98 (m, 3H, ArH), 7.86 (m,
1H, ArH), 7.74-7.57 (m, 4H, ArH). Elemental analysis
found: C 65.72, H 3.84; C17H12ClN3O requires C 65.92,
H 3.90, N 13.57%.
Acknowledgments
The author (S. Pal) thanks Mr. M. N. Raju, the
chairman of M. N. R. Educational Trust for his constant
encouragement.
References
Biological assay
1. Muregi, F. W.; Ishih, A.; Drug Dev. Res. 2010, 71, 20.
2. Kaur, K.; Jain, M.; Reddy, R. P.; Jain, R.; Eur. J. Med. Chem.
2010, 45, 3245.
Chemical and reagents: Dulbecco’s modified eagle
medium (DMEM), L-glutamine, streptomycin and

Vol. 22, No. 9, 2011
Reddy et al.
1749
3. Tributino, J. L. M.; Duarte, C. D.; Corrêa, R. S.; Doriguetto, A.
C.; Ellena, J.; Romeiro, N. C.; Castro, N. G.; Miranda, A. L.
P.; Barreiro, E. J.; Fraga, C. A. M.; Bioorg. Med. Chem. 2009,
17, 1125.
16. Kidwai, M.; Jahan, A.; Bhatnagar, D.; J. Chem. Sci. 2010, 122,
607.
17. Chandrasekhar, S.; Narsihmulu, Ch.; Sultana, S. S.; Reddy, N.
R.; Chem. Commun. 2003, 1716.
4. Zheng, L.-W.; Wu, L.-L.; Zhao, B.-X.; Dong, W.-L.; Miao,
J.-Y.; Bioorg. Med. Chem. 2009, 17, 1957.
18. Chandrasekhar, S.; Narsihmulu, C.; Reddy, N. R.; Sultana, S.
S.; Tetrahedron Lett. 2004, 45, 4581.
5. Vera-DiVaio, M. A. F.; Freitas, A. C. C.; Castro, H. C.; de
Albuquerque, S.; Cabral, L. M.; Rodrigues, C. R.;Albuquerque,
M. G.; Martins, R. C. A.; Henriques, M. G. M. O.; Dias, L. R.
S.; Bioorg. Med. Chem. 2009, 17, 295.
19. Sondhi, S. M.; Dinodia, M.; Jain, S.; Kumar,A.; Indian J. Chem.,
Sect. B: Org. Chem. Incl. Med. Chem. 2009, 48, 1128.
20. Palla, G.; Predieri, G.; Domiano, P.; Tetrahedron 1986, 42, 3649.
21. Palmer, R. B.;Andersen, N. H.; Bioorg. Med. Chem. Lett. 1996,
6, 2173.
6. Vicini, P.; Incerti, M.; Doytchinova, I.A.; Colla, P. L.; Busonera,
B.; Loddo, R.; Eur. J. Med. Chem. 2006, 41, 624.
7. Terzio lu, N.; Gürsoy, A.; Eur. J. Med. Chem. 2003, 38, 781.
8. Dubey, P. K.; Rao, S. S.; Reddy, P. V. P.; Heterocycl. Commun.
2003, 9, 411.
22. Subramanian, J.; Govindan, R.; J. Clin. Oncol. 2007, 25, 561.
23. Zhang, H.; Drewe, J.; Tseng, B.; Kasibhatla, S.; Cai, S. X.;
Bioorg. Med. Chem. 2004, 12, 3649.
24. Datta, N. J.; Khunt, R. C.; Parikh, A. R.; J. Inst. Chem. 2000,
72, 133.
9. Khalil, M. A.; El-Sayed, O. A.; El-Shamy, H. A.; Archiv der
Pharmazie 1993, 326, 489.
25. Borchhardt, D. M.; Mascarello,A.; Chiaradia, L. D.; Nunes, R.
J.; Oliva, G.;Yunes, R. A.; Andricopulo, A. D.; J. Braz. Chem.
Soc. 2010, 21, 142.
10. Haimov, A.; Neumann, R.; Chem. Commun. 2002, 876.
11. Santaniello, E.; Fiecchi, A.; Manzocchi, A.; Ferraboschi, P.;
J. Org. Chem. 1983, 48, 3074.
26. Reddy, L. V.; Suman, A.; Beevi, S. S.; Mangamoori, L. N.;
Mukkanti, K.; Pal, S.; J. Braz. Chem. Soc. 2010, 21, 98.
27. Meth-Cohn, O.; Narhe, B.; Tarnowski, B.; J. Chem. Soc., Perkin
Trans. I 1981, 1520.
12. Kumar, R.; Chaudhary, P.; Nimesh, S.; Chandra, R.; Green
Chem. 2006, 8, 356.
13. Chandrasekhar, S.; Narsihmulu, ch.; Saritha, B.; Sultana, S. S.;
Tetrahedron Lett. 2004, 45, 5865.
14. Chandrasekhar, S.; Narsihmulu, C.; Sultana, S. S.; Reddy, N.
R.; Org. Lett. 2002, 4, 4399.
Submitted: January 14, 2011
Published online: June 21, 2011
15. Liu, L.; Zhang, Y.; Wang, Y.; J. Org. Chem. 2005, 70, 6122.

J. Braz. Chem. Soc., Vol. 22, No. 9, S1-S5, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Supplementary Information
SI
A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
and their Cytotoxicity Activities
LingamVenkata Reddy,a Suresh Babu Nallapati,b Syed Sultan Beevi,c
Lakshmi Narasu Mangamoori,c Khagga Mukkantia and Sarbani Pal*,b
aCenter for Chemical Sciences and Technology and cCenter for Biotechnology,
Institute for Sciences and Technology, JNT University, Kukatpally, 500072 Hyderabad, India
bDepartment of Chemistry, MNR Degree and PG College, Kukatpally, 500072 Hyderabad, India
1HNMR spectra of compounds 3a-i
Figure S1. 1H NMR of compound 3a in DMSO-d6.
*e-mail: sarbani277@yahoo.com

S2
A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
J. Braz. Chem. Soc.
Figure S2. 1H NMR of compound 3b in DMSO-d6.
Figure S3. 1H NMR of compound 3c in DMSO-d6.

Vol. 22, No. 9, 2011
Reddy et al.
S3
Figure S4. 1H NMR of compound 3d in DMSO-d6.
Figure S5. 1H NMR of compound 3e in DMSO-d6.

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A “Green” Synthesis of N-(Quinoline-3-ylmethylene)benzohydrazide Derivatives
J. Braz. Chem. Soc.
Figure S6. 1H NMR of compound 3f in DMSO-d6.
Figure S7. 1H NMR of compound 3g in DMSO-d6.

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Reddy et al.
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Figure S8. 1H NMR of compound 3h in DMSO-d6.
Figure S9. 1H NMR of compound 3i in DMSO-d6.