Nonsteroidal anti-inflammatory drug-based N-allylidene benzohydrazides and 1-acyl-2-pyrazolines: Their synthesis as potential cytotoxic agents in vitro

Article abstract of DOI:10.1002/jhet.1993

A series of 1-acyl-2-pyrazoline derivatives derived from nonsteroidal anti-inflammatory drugs was designed as potential anticancer agents. Synthesis of these compounds was carried out via the condensation reaction of chalcones and acid hydrazides under he

Full text of DOI:10.1002/jhet.1993

Month 2014
Nonsteroidal Anti-inflammatory Drug-based N-Allylidene
Benzohydrazides and 1-Acyl-2-pyrazolines: Their Synthesis as Potential
Cytotoxic Agents In Vitro
Kavitha Kankanala,a,b V. Ranga Reddy,c Yumnam Priyadarshini Devi,d Lakshmi Narasu Mangamoori,d D. Rambabu,e
a
b
*
K. Mukkanti, and Sarbani Pal
aJNTUH, Kukatpally, Hyderabad-500085, Andhra Pradesh, India
bDepartment of Chemistry, MNR Degree and PG College, Kukatpally, Hyderabad-500085, Andhra Pradesh, India
cDr. Reddy’s Laboratories Limited, Integrated Product Development, Bachupally, Hyderabad-500055, Andhra Pradesh, India
dCentre for Biotechnology, IST, JNTUH, Kukatpally, Hyderabad-500085, Andhra Pradesh, India
eInstitute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, Andhra Pradesh, India
*
E-mail: sarbani277@yahoo.com
Received November 21, 2012
DOI 10.1002/jhet.1993
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
A series of 1-acyl-2-pyrazoline derivatives derived from nonsteroidal anti-inflammatory drugs was
designed as potential anticancer agents. Synthesis of these compounds was carried out via the condensation
reaction of chalcones and acid hydrazides under heating. The methodology did not require the use of any
costly reagents or catalysts, and the acid hydrazide reactants were readily prepared from mefenamic acid
or ibuprofen. A variety of 1-acyl-2-pyrazolines was prepared in good to excellent yields. An N-allylidene
benzohydrazide intermediate was isolated during the reaction optimization study, the structure of which
was confirmed unambiguously by X-ray single crystal data. A range of N-allylidene benzohydrazides were
also prepared in good yields. Some of the compounds synthesized showed promising cytotoxic activities
when tested against HCT-15 human colon cancer cell line in vitro.
J. Heterocyclic Chem., 00, 00 (2014).
features of known NSAIDs, for example, mefenamic acid
and ibuprofen. Because the free carboxylic acid moiety of
these NSAIDs are known to be partially responsible for their
increased risk of stomach ulcers and gastrointestinal bleeding
[20], hence, new compounds were designed by linking the
carboxylic acid moiety of mefenamic acid/ibuprofen to the
NH group of 2-pyrazoline in the form of an amide bond.
To the best of our knowledge, the design, synthesis, and
in vitro evaluation of this class of compounds as potential
anticancer agents have not been reported earlier.
In spite of their medicinal and other importance,
only few methods have been reported for the synthesis
of 1-acyl-2-pyrazolines. These include synthesis of
N1-propanoyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole deriv-
atives via the reaction of chalcone with hydrazine hydrate and
propionic acid in the same pot [9]. Alternatively, 1-benzoyl-2-
pyrazolines were prepared via a linear synthesis involving
the reaction of chalcone and hydrazine hydrate followed by
treatment with benzoyl chloride in pyridine [10]. The synthe-
sis of isoniazide-based 1-acyl-2-pyrazolines has been carried
out via the reaction of chalcone with isonicotinohydrazide in
the presence of acetic acid in EtOH under conventional con-
ditions [21]. We adopted a similar strategy to prepare our
INTRODUCTION
The 2-pyrazoline (4,5-dihydro-1H-pyrazole, A) (Figure 1)
framework plays an important role in medicinal/heterocyclic
chemistry and has attracted particular attention in drug dis-
covery. Derivatives of A have shown diverse pharmacologi-
cal activities [1–10]. For example, 1-acyl-2-pyrazoline
derivatives B, C, and D have shown antibacterial [2], amino
oxidase inhibitory [3], and anticancer [7] properties. Several
nonsteroidal anti-inflammatory drugs (NSAIDs), on the other
hand, have shown promising cytotoxic activities [11]. For
example, well-known anti-inflammatory drugs piroxicam
and mefenamic acid have shown selective in vitro cytotoxic
effects on several cancer cell lines [12]. Thus, a combination
of A and an appropriate NSAID in a single molecule is
expected to provide a new template E (Figure 2) for the
design and identification of novel anticancer agents.
Prompted by this idea and because of our longstanding
interest in the chemical modification of NSAIDs [13–19],
we became interested in the synthesis and pharmacological
evaluation of a library of compounds based on E. Herein,
we report in vitro cytotoxic activities of novel 1-acyl-2-
pyrazoline derivatives based on E incorporating the structural
© 2014 HeteroCorporation

K. Kankanala, V. R. Reddy, Y. P. Devi, L. N. Mangamoori, D. Rambabu, K. Mukkanti, and S. Pal Vol 000
Figure 1. 2-Pyrazoline (A) and its 1-acyl derivatives (B–D) possessing pharmacological activities.
Figure 2. Design of novel nonsteroidal anti-inflammatory drug-based 1-acyl-2-pyrazolines E.
1
target molecules based on E. Thus, appropriate chalcones (1)
were reacted with acid hydrazides (2) prepared from
mefenamic acid or ibuprofen in glacial acetic acid under re-
flux to give the desired 1-acyl-2-pyrazolines (4) (Scheme 1).
compound was elucidated by MS, IR, H, and 13C NMR
spectroscopic measurements as well as single crystal X-ray
data. To our surprise, the product isolated in 96% yield was
identified as an open chain intermediate, that is, (1E)-2-
(2,3-dimethylphenylamino)-N0-((Z)-3-phenyl-1-(4-
propoxyphenyl)allylidene) benzohydrazide 3a (Scheme 1)
instead of the desired pyrazoline 4a. The MS spectrum of
3a showed a protonated molecular ion peak at m/z 504,
whereas its IR spectrum displayed a strong absorption at
1657, 1604, and 1500cmꢀ1 due to C═O, C═N, and C═C
stretching, respectively. The 1H NMR spectrum of 3a
displayed two singlets at d 9.20 and 9.14 due to two NH
groups that disappeared during D2O exchange experiments.
Signals in the range of d 7.41–6.45 were due to the aromatic
and olefinic protons, whereas the protons of OCH2 moiety
RESULTS AND DISCUSSION
Initially, the synthesis of a representative compound,
for example, (2-(2,3-dimethylphenylamino)phenyl)(4,5-
dihydro-5-phenyl-3-(4-propoxyphenyl)pyrazo-1-yl)methanone
(4a), was examined via the condensation of 2-(2,3-
dimethylphenylamino)benzohydrazide (2a) and (E)-3-
phenyl-1-(4-propoxyphenyl)prop-2-en-1-one (1a) in glacial
acetic acid at room temperature for 25 h (Scheme 2). After
usual work up and purification, the structure of the
Scheme 1. Synthesis of nonsteroidal anti-inflammatory drug-based 1-acyl-2-pyrazolines 4.
Scheme 2. Preparation of 4a via the condensation of 1a and 2a.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Nonsteroidal Anti-inflammatory Drug-based N-Allylidene Benzohydrazides and
1-Acyl-2-pyrazolines: Their Synthesis as Potential Cytotoxic Agents In Vitro
Month 2014
were observed at d 4.02 ppm as a triplet having J = 6.0 Hz.
The 13C NMR spectrum of 3a showed a signal at
168.4 ppm corresponding to carbonyl carbon (C═O) along
with signals at 22.5 and 20.6 ppm for the methyl groups.
Finally, the structure of 3a was confirmed unambiguously
by single crystal X-ray analysis [22] (Figure 3) that showed
E and Z-geometry of the C═C and C═N, respectively.
It was evident that the acid hydrazide 2a underwent a
simple Schiff-base formation reaction (via nucleophilic
attack on the carbonyl group of 1a followed by elimination
of water molecule) instead of 1,4-addition with the chalcone
1a in a regioselective manner to give the open chain product
3a. In a separate experiment, a solution of compound 3a in
acetic acid was refluxed for 18 h when 3a underwent cycliza-
tion via an intramolecular nucleophilic attack by the NH
moiety of hydrazide to the C═C (cf a Michael type reaction)
to afford the 5-membered aza heterocycle pyrazoline 4a. The
direct formation of 4a from the reaction of 1a and 2a at room
temperature was not observed; perhaps, the intramolecular
cyclization of the intermediate 3a was not favored at room
temperature. The poor nucleophilicity of the NH moiety
(i.e., the less availability of lone pair due to the adjacent
carbonyl group) of hydrazide and the bulky phenyl group
(that may cause some steric crowding) attached to the double
bond could be the reason for this observation. The intramo-
lecular cyclization of 3a therefore required additional energy
that was supplied by increasing the reaction temperature in a
separate pot. Nevertheless, the structure of 4a was
1
confirmed by MS, IR, H, and 13C NMR spectra. The MS
spectrum of 4a showed a protonated molecular ion peak
at m/z 504, whereas the IR spectrum displayed strong
absorptions at 1745cmꢀ1 due to C═O stretching and at
1
1627 cmꢀ1 due to C═N stretching. The H NMR spectra
of 4a (Figure 4) showed the two diastereotopic protons
Figure 3. ORTEP representation of compound 3a. (Thermal ellipsoids are drawn at 50 % probability level).
Figure 4. 1H NMR (CDCl3, 400 MHz) spectrum of 4a.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

K. Kankanala, V. R. Reddy, Y. P. Devi, L. N. Mangamoori, D. Rambabu, K. Mukkanti, and S. Pal Vol 000
attached to C-4 and one proton at C-5 of the pyrazoline ring
that appeared as three characteristic doublet of doublets
(ABX spin system) at d 5.83 (dd, J = 11.7, 4.9 Hz, 1H),
3.77 (dd, J = 17.1, 11.7 Hz, 1H), and 3.17 (dd, J = 15.6,
4.9 Hz, 1H) for HX, HA, and HB, respectively. This is incon-
sistent with the reported 1H NMR data for HA, HB, and HX
protons of a pyrazoline ring (of a range of compounds) that
appeared as doublets of doublets at 3.10–3.30, 4.00–4.10,
and 5.60–5.70ppm (JAB = 17.07, JAX = 6.30, JBX = 11.05
Hz), respectively [23]. A D2O exchangeable singlet
appeared near d 8.14 indicated the presence of an NH group,
whereas signals due to the methyl and propoxy substituents
were appeared at appropriate places (Figure 4). The 1-acyl-
2-pyrazoline structure was further supported by the 13C
NMR chemical shift values at 171.5 (CO), 167.3 (C-3),
and 69.6 (C-5) ppm.
We then examined the reaction of various chalcones 1 and
the acid hydrazide 2a at room temperature results of which
are summarized in Table 1. A range of N-allylidene
benzohydrazides 3 were obtained in these cases in good to
excellent yields.
On the basis of results presented in Scheme 2, we exam-
ined the reaction of chalcones 1 with hydrazides 2 for lon-
ger duration when 1-acyl-2-pyrazolines (4) were obtained
in good yields. Results of these studies are summarized
in Table 2. The hydrazide reactants were readily prepared
from mefenamic acid or ibuprofen. Thus, a number of
chalcones and hydrazides were employed to afford a vari-
ety of 1-acyl-2-pyrazolines (4). In an alternative method,
refluxing of an appropriate N-allylidene benzohydrazide
(3a–e) in glacial acetic acid for 10–18 h depending on the
nature of the reactant employed also provided the corre-
sponding 1-acyl-2-pyrazoline (4) in good yield.
Having synthesized a range of N-allylidene benzohydrazides
(3) and 1-acyl-2-pyrazolines (4), the in vitro cytotoxic evalu-
ation of some of these compounds was performed against
human HCT-15 for colon cancer cells. Cancer, according
to WHO, is the second leading cause of death worldwide af-
ter cardiovascular disease [24], whereas colon (or colorectal)
cancer is widespread in the Western world. Although various
therapies are available to treat different types of cancer
including colon cancer, the worldwide deaths, however,
due to cancer has been projected to increase over 11 million
in 2030 according to WHO. Thus, identification and devel-
opment of new and suitable agents to treat various types of
cancer is of particular interest. Colon cancer being our major
focus, we have used human colon cancer cell line HCT-15
for our in vitro assay [25]. Doxorubicin, a known
anthracycline antibiotic, was used as a reference compound.
All the test compounds were examined for their ability to
inhibit the cancerous cells (HCT-15) based on a 3,4,5-
dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT)
assay. All these compounds were tested at five different con-
centrations, for example, 1, 2, 5, 10, and 25mg/mL, and the
percentage of cell death measured for each compound along
with IC50 values of active compounds are summarized in
Table 3. It is evident from Table 3 that N-allylidene
benzohydrazides, for example, 3b, 3c, and 3d, and 1-acyl-
2-pyrazolines, for example, 4c, 4e, and 4f, showed good
activities especially at higher dose. All these compounds
showed IC50 values in the range 13–22 mg/mL (Table 3)
compared with doxorubicin’s IC50 value of 50 mg/mL
(0.09 mM). Thus, the present N-allylidene benzohydrazides
and 1-acyl-2-pyrazoline class of compounds have potential
for the development of new anticancer agents especially for
colon cancer.
CONCLUSIONS
In conclusion, 1-acyl-2-pyrazoline derivatives derived
from NSAIDs were designed as potential anticancer
agents. Synthesis of these compounds was carried out via
the condensation reaction of chalcones and acid hydrazides
under heating. The methodology did not require any costly
reagents or catalysts, and the acid hydrazide reactants were
readily prepared from mefenamic acid or ibuprofen. A
variety of 1-acyl-2-pyrazolines was prepared in good to ex-
cellent yields. We were also able to isolate an N-allylidene
benzohydrazide intermediate (during the reaction optimi-
zation of a chalcone and acid hydrazide), the structure of
which was confirmed unambiguously by X-ray single
crystal data. This not only helped us to establish the 1,2-
nucleophilic addition pathway of this reaction but also
allowed us to prepare a range of N-allylidene benzohydrazides
in good yields. A number of compounds synthesized were
tested for their cytotoxicity in vitro against HCT-15 human
colon cancer cell line, and some of them showed promising
activities compared with doxorubicin. Thus, the present
N-allylidene benzohydrazides and 1-acyl-2-pyrazoline class
of compounds represent new templates for the identification
and development of novel anticancer agents.
EXPERIMENTAL
Chemistry
General methods. Melting points were determined by open
glass capillary method on a Cintex melting point apparatus and
are uncorrected. IR spectra were recorded on a Perkin-Elmer
1
spectrometer using KBr pellets. H NMR spectra were recorded
on a Varian 400 MHz spectrometer using CDCl3 or DMSO-d6,
with reference to tetramethylsilane as an internal reference. 13C
NMR spectra were recorded on a 100 MHz spectrometer. Mass
spectra were recorded on a Jeol JMC D-300 instrument by
using electron ionization at 70 ev. All reactions were monitored
by TLC on precoated silica gel plates. Column chromatography
was performed on 100–200 mesh silica gel (SRL, India) using
10–20 times (by weight) of the crude product.
Mefenamic acid and ibuprofen are commercially available.
Methyl esters of mefenamic acid and ibuprofen were prepared
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Nonsteroidal Anti-inflammatory Drug-based N-Allylidene Benzohydrazides and
1-Acyl-2-pyrazolines: Their Synthesis as Potential Cytotoxic Agents In Vitro
Month 2014
Table 1
Preparation of N-allylidene benzohydrazides 3 from 1 and 2a.a
Entry
1
Chalcone (1)
Product (3)
Time (h)
25
Yieldb (%)
96
2
3
15
15
98
84
4
12
85
5
15
89
aAll the reactions were performed using chalcone 1 (5 mmol) and acid hydrazide 2a (10 mmol) in glacial acetic acid (10 mL).
bIsolated yield.
according to the known methods [26]. Acid hydrazides of these
acids from esters were prepared following the methods available
in literature [27].
General methods for synthesis of N-allylidene benzohydrazides
A mixture of chalcone 1 (5 mmol) and acid hydrazide 2a
(2.5g, 10 mmol) in glacial acetic acid (10 mL) was stirred at room
3.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

K. Kankanala, V. R. Reddy, Y. P. Devi, L. N. Mangamoori, D. Rambabu, K. Mukkanti, and S. Pal Vol 000
Table 2
Preparation of 1-acyl-2-pyrazolines 4 from chalcones 1 and hydrazides 2.a
Entry
1
Chalcone (1)
Hydrazide (2)
Product (4)
Time
18 h
Yieldb (%)
90
1a
2a
2
3
1b
1c
2a
2a
15 h
98
97
4 h
4
1d
1b
2a
15 h
96
5
2 h
89
6
1c
35 min
88
(Continues)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Nonsteroidal Anti-inflammatory Drug-based N-Allylidene Benzohydrazides and
1-Acyl-2-pyrazolines: Their Synthesis as Potential Cytotoxic Agents In Vitro
Month 2014
Table 2
(Continued)
Entry
7
Chalcone (1)
Hydrazide (2)
Product (4)
Time
Yieldb (%)
93
1d
15 min
aAll the reactions were performed using chalcone 1 (5 mmol) and an hydrazide 2 (10 mmol) in glacial acetic acid (10 mL) under reflux.
bIsolated yield.
Table 3
The in vitro anticancer properties of some of the N-allylidene benzohydrazides (3) and 1-acyl-2-pyrazolines (4) synthesized.
% of cell death at various concentrations
Entry
Compounds
1 mg/mL
2 mg/mL
5 mg/mL
10 mg/mL
25 mg/mL
IC50 (mg/mL)
1
2
3
4
5
6
3b
3c
3d
4c
4e
4f
16.8
25.6
15.0
24.8
18.5
29.1
27.2
30.3
32.2
29.1
27.7
33.7
33.0
35.1
33.6
30.3
32.4
46.0
45.7
39.3
43.6
38.2
35.8
46.5
61.8
63.6
64.0
52.9
54.0
57.0
13.9
16.6
14.7
22.0
21.8
15.0
All the values are the average of the experiments performed in triplicates. The cell line used is HCT-15 human colon cancer cell line. Doxorubicin
[IC50 = 50 mg/mL (0.09 mM)] was used as a reference compound.
temperature for the time indicated in Table 1 (the progress of the
reaction was monitored by TLC). After completion of the
reaction, the mixture was filtered off, washed with petroleum ether
(2ꢁ 10mL), and dried to afford the pure compound.
127.5, 127.3, 127.2, 126.4, 125.3, 125.1, 119.9, 117.3, 116.1,
115.1, 20.1, 13.3; MS (m/z): 446 [M+ H]+, (100%).
(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-3-(4-methoxyphenyl)-
1-phenylallylidene)benzohydrazide (3c). Off white solid; mp
140–141ꢂC; Rf 0.27 (petroleum ether/EtOAc = 8.5:1.5); IR (KBr)
(1E)-2-(2,3-Dimethylphenylamino)-N0-((Z)-3-phenyl-1-(4-
propoxyphenyl)allylidene)benzohydrazide (3a).
Off white
n
max/cmꢀ1: 3283, 3238, 1655, 1505; 1H NMR (400MHz,
solid; mp 159–160ꢂC; Rf 0.38 (petroleum ether/EtOAc= 8.5:1.5),
CDCl3): d 9.25 (bs, 1H, NH, D2O exchangeable), 8.98 (bs, 1H,
NH, D2O exchangeable), 7.64–7.55 (m, 3H), 7.36–7.13 (m, 7H),
7.06 (t, J = 7.4 Hz, 1H), 6.96–6.85 (m, 5H), 6.54 (t, J = 7.3 Hz,
1H), 6.37 (d, J = 16.1Hz, 1H), 3.81 (s, 3H), 2.32 (s, 3H), 2.19 (s,
3H); 13C NMR (100MHz, DMSO-d6): d 167.3, 158.8, 154.3,
146.1, 139.4, 137.8, 133.8, 132.0, 131.4, 130.0, 129.4, 129.1,
128.6, 128.4, 128.2, 127.9, 125.9, 125.0, 120.1, 118.2, 116.2,
114.8, 114.3, 114.2, 55.3, 20.3, 13.5; MS (m/z): 476 [M + H]+,
(100%).
IR (KBr) nmax/cmꢀ1: 3230, 2959, 1657, 1604, 1500, 1248; 1H
NMR (400 MHz, CDCl3)
d
9.20 (bs, 1H, NH, D2O
exchangeable), 9.14 (bs, 1H, NH, D2O exchangeable), 741–6.91
(m, 16H), 6.58 (t, J = 7.4 Hz, 1H), 6.45 (d, J = 16.4Hz, 1H), 4.02
(t, J = 6.0Hz, 2H), 2.32 (s, 3H), 2.19 (s, 3H), 1.90 (m, 2H), 1.10
(t, J = 7.5 Hz, 3H); 13C NMR (100MHz, CDCl3): d 168.3, 160.2,
154.5, 148.0, 139.1, 138.1, 137.8, 136.2, 132.8, 130.2, 129.7,
128.8, 128.7, 128.6, 127.1, 125.8, 125.6, 122.2, 120.9, 116.6,
115.7, 114.9, 69.7, 22.5, 20.6, 13.8, 10.5; MS (m/z): 504
[M+ H]+, (100%).
(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-3-(4-chlorophenyl)-
1-phenylallylidene)benzohydrazide (3d).
Brownish yellow
(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-1,3-diphenyl
allylidene)benzohydrazide (3b). White solid; mp 166–167 C;
solid; mp 146–147ꢂC; Rf 0.47 (petroleum ether/EtOAc= 8.5:1.5);
IR (KBr) nmax/cmꢀ1: 3254, 2922, 1661; 1H NMR (400MHz,
CDCl3): d 9.20 (bs, 1H, NH, D2O exchangeable), 9.03 (bs, 1H,
NH, D2O exchangeable), 7.63–7.58 (m, 3H), 7.40–7.28 (m,
6H) 7.17–6.89 (m, 7H), 6.54 (t, J = 7.3 Hz, 1H), 6.36 (d,
J = 16.5 Hz, 1H), 2.31 (s, 3H), 2.18 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 167.1, 158.5, 154.5, 148.5, 139.0, 138.1,
136.4, 134.6, 132.9, 130.6, 130.2, 129.9, 129.4, 129.0, 128.2,
125.9, 125.7, 121.0, 116.6, 115.0, 29.7, 20.6; MS (m/z): 480
[M + H]+, (100%).
Rf 0.45 (petroleum ether/EtOAc= 8.5:1.5). IR (KBr) nmax/cmꢀ1
:
1
3286, 3242, 1660; H NMR (400MHz, CDCl3): d 9.25 (bs, 1H,
NH, D2O exchangeable), 9.03 (bs, 1H, NH, D2O exchangeable),
7.66–7.56 (m, 3H), 7.45–7.27 (m, 6H), 7.19–7.14 (m, 3H), 7.13–
7.04 (m, 3H), 6.96–6.90 (m, 3H), 6.55 (t, J = 7.4 Hz, 1H), 6.42
(d, J = 16.1 Hz, 1H), 2.32 (s, 3H), 2.19 (s, 3H); 13C NMR
(100MHz, DMSO + CDCl3): d 174.4, 154.1, 145.5, 140.0, 139.4,
137.2, 135.4, 132.2, 130.2, 129.4, 129.1, 128.8, 128.3, 128.1,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

K. Kankanala, V. R. Reddy, Y. P. Devi, L. N. Mangamoori, D. Rambabu, K. Mukkanti, and S. Pal Vol 000
(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-3-(3-nitrophenyl)-
167.6, 159.0, 154.6, 146.4, 139.9, 137.8, 134.1, 132.1, 131.5,
1-phenylallylidene)benzohydrazide (3e).
Pale yellow solid;
131.4, 130.4, 130.1, 128.7, 126.8, 126.7, 125.6, 125.0, 120.3,
118.4, 116.6, 115.1, 114.3, 60.4, 55.3, 41.7, 20.6, 13.9; MS (m/z):
476 [M+ H]+, (100%).
mp 168–170 ꢂC; Rf 0.37 (petroleum ether/EtOAc= 8:2); IR (KBr)
1
n
max/cmꢀ1: 2920, 1650; H NMR (400 MHz, CDCl3): d 9.32 (bs,
1H, NH, D2O exchangeable), 9.10 (bs, 1H, NH, D2O
exchangeable), 8.18 (s, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.78–6.87
(m, 14H), 6.58 (t, J = 7.4 Hz, 1H), 6.46 (d, J = 16.2Hz, 1H), 2.32
(s, 3H), 2.19 (s, 3H); MS (m/z): 491 [M+ H]+, (100%).
(2-(2,3-Dimethylphenylamino)phenyl)(5-(4-chlorophenyl)-
4,5-dihydro-3-phenylpyrazo-1-yl)methanone (4d).
Pale
yellow solid; mp 108–110ꢂC; Rf 0.61 (petroleum ether/
EtOAc= 8.5:1.5); IR (KBr) nmax/cmꢀ1: 2921, 1627, 1585; 1H
General procedure for the synthesis of 1-acyl-2-pyrazolines 4
NMR (400 MHz, CDCl3):
d 8.13 (bs, 1H, NH, D2O
Method A.
A mixture of chalcone 1 (5 mmol) and an
exchangeable), 7.91 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 7.4 Hz, 2H),
7.42–7.41 (m, 3H), 7.28–7.21 (m, 5H), 7.12–7.10 (m, 1H), 7.05
(t, J = 7.4 Hz, 1H), 6.90 (d, J = 7.8 Hz, 2H), 6.81 (t,
J = 7.4 Hz, 1H), 5.81 (dd, J = 12.1, 5.1 Hz, 1H), 3.80 (dd,
J = 17.9, 11.8 Hz, 1H), 3.17 (dd, J = 17.6, 5.0 Hz, 1H), 2.29
(s, 3H), 2.10 (s, 3H); 13C NMR (100 MHz, CDCl3): d 167.6,
154.5, 146.5, 140.4, 139.9, 137.9, 133.4, 132.0, 131.8,
131.1, 130.5, 130.1, 129.2, 128.7, 126.9, 126.8, 125.7,
125.1, 120.2, 118.0, 116.6, 115.2, 60.4, 41.5, 20.6, 13.8.
MS (m/z): 480 [M + H]+, (100%).
appropriate hydrazide 2 (10 mmol) in glacial acetic acid (10 mL)
was refluxed for the time indicated in Table 2 (the progress of
the reaction was monitored by TLC). After completion of the
reaction, the mixture was cooled and poured into crushed ice
(25 g). The separated solid was filtered and dried. The crude
product was purified by column chromatography on silica using
petroleum ether–EtOAc as eluent.
Method B. Appropriate N-allylidene benzohydrazide (3a–e)
(10 mmol) in glacial acetic acid was refluxed for 10–18 h
depending on the nature of the reactant employed. The
progress of the reaction was monitored by TLC. After
completion of the reaction, the mixture was poured into
crushed ice (25 g). The separated solid was filtered, dried, and
purified by column chromatography on silica using petroleum
ether–EtOAc as eluent.
1-(4,5-Dihydro-3,5-diphenylpyrazol-1-yl)-2-(4-isobutyl phenyl)
propan-1-one (4e).
White solid; mp 151–152ꢂC; Rf 0.71
(petroleum ether/EtOAc = 8:2); IR (KBr) nmax/cmꢀ1: 2930,
1660, 1625; 1H NMR (400 MHz, CDCl3): d 7.48–7.22 (m,
12H), 7.15 (d, J = 8.6 Hz, 2H), 5.43 (dd, J = 11.5, 4.6 Hz,
1H), 4.78 (q, 1H), 3.60 (dd, J = 17.5, 11.8 Hz, 1H), 3.15
(dd, J = 17.7, 4.8 Hz, 1H), 2.44 (d, J = 7.0 Hz, 2H), 1.80–1.90
(m, 1H), 1.58 (d, J = 7.2 Hz, 3H), 0.86 (d, J = 7.9 Hz, 6H);
13C NMR (100 MHz, CDCl3): d 176.7, 1 48.6, 140.8, 138.6,
135.1, 129.6, 129.4, 129.2, 128.9, 128.7, 128.5, 128.4,
128.3, 127.7, 127.2, 127.1, 116.4, 45.1, 41.3, 30.2, 22.4,
18.5; MS (m/z): 411 [M + H]+, (100%).
(2-(2,3-Dimethylphenylamino)phenyl)(4,5-dihydro-5-phenyl-
3-(4-propoxyphenyl)pyrazo-1-yl)methanone (4a).
Off white
solid; mp 85–88ꢂC; Rf 0.55 (petroleum ether/EtOAc = 8.5:1.5); IR
(KBr) nmax/cmꢀ1: 2923, 1745, 1627, 1607, 1582; 1H NMR
(400MHz, CDCl3): d 8.14 (bs, 1H, NH, D2O exchangeable), 7.93
(d, J = 7.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.33–6.79 (m, 13H),
5.83 (dd, J = 11.7, 4.9 Hz, 1H), 3.95 (t, J = 6.3 Hz, 2H), 3.77 (dd,
J = 17.1, 11.7 Hz, 1H), 3.17 (dd, J = 15.6, 4.9 Hz, 1H), 2.28 (s,
3H), 2.10 (s, 3H), 1.82 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H); 13C
NMR (100 MHz, CDCl3): d 171.5, 167.3, 161.0, 146.3, 142.0,
140.0, 131.4, 130.1, 128.9, 128.4, 127.6, 125.7, 125.4, 124.9,
123.7, 120.2, 116.6, 115.1, 114.6, 69.6, 60.8, 41.8, 22.5, 20.6,
13.9, 10.5; MS (m/z): 504 [M+ H]+, (100%).
1-(4,5-Dihydro-5-(4-methoxyphenyl)-3-phenylpyrazol-1-yl)-
2-(4-isobutyl phenyl)propan-1-one (4f).
116–118ꢂC; Rf 0.54 (petroleum ether/EtOAc = 8:2); IR (KBr)
max/cmꢀ1: 3282, 1713, 1589, 1508; 1H NMR (400 MHz,
White solid; mp
n
CDCl3): d 7.72 (m, 2H), 7.44–7.42 (m, 3H), 7.33 (d, J = 8.3 Hz,
2H), 7.17 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 6.85 (d,
J = 8.6 Hz, 2H), 5.45 (dd, J = 11.6, 4.6 Hz, 1H), 4.77 (q,
J = 7.4 Hz, 1H), 3.78 (s, 3H), 3.61 (dd, J = 17.7, 11.9 Hz, 1H),
3.12 (dd, J = 17.7, 4.9 Hz, 1H), 2.40 (d, J = 7.0 Hz, 2H), 1.81
(q, 1H), 1.47 (d, J = 7.3 Hz, 3H), 0.86 (d, J = 7.9 Hz, 6H); 13C
NMR (100 MHz, CDCl3): d 172.3, 159.0, 153.4, 139.9, 138.9,
134.4, 131.6, 130.1, 129.0, 128.6, 127.6, 126.9, 126.6, 114.3,
59.9, 55.2, 45.1, 42.7, 42.0, 30.1, 22.4, 18.8; MS (m/z): 441
[M + H]+, (100%).
(2-(2,3-Dimethylphenylamino)phenyl)(4,5-dihydro-3,5-
diphenylpyrazo-1-yl)methanone (4b).
80–82ꢂC; Rf 0.61 (petroleum ether/EtOAc = 8.5:1.5); IR (KBr)
max/cmꢀ1: 3338, 2922, 1627, 1579; 1H NMR (400 MHz,
White solid; mp
n
CDCl3): d 8.16 (bs, 1H, NH, D2O exchangeable), 7.93 (d,
J = 7.4 Hz, 1H), 7.71 (d, J = 3.0 Hz, 2H), 7.42–7.33 (m, 7H), 7.24–
7.20 (m, 2H), 7.11 (d, J = 7.8 Hz, 1H), 7.02 (t, J = 7.8Hz, 1H),
6.89 (d, J = 8.3 Hz, 2H), 6.82 (t, J = 8.3 Hz, 1H), 5.86 (dd,
J = 11.7, 4.9 Hz, 1H), 3.80 (dd, J = 17.6, 11.7Hz, 1H), 3.20 (dd,
J = 17.6, 4.9 Hz, 1H), 2.29 (s, 3H), 2.11 (s, 3H); MS (m/z): 446
[M+ H]+, (100%).
(4-Chlorophenyl)(5-(4-chlorophenyl)-4,5-dihydro-3-phenyl
pyrazo-1-yl)methanone (4g). Off white solid; mp 172–174ꢂC;
Rf 0.50 (petroleum ether/EtOAc = 8:2); IR (KBr) nmax/cmꢀ1
:
2925, 1640, 1580; 1H NMR (400 MHz, CDCl3): d 7.9 (d,
J = 8.4 Hz, 2H), 7.70 (d, J = 7.3 Hz, 2H), 7.45–7.41 (m, 5H),
7.33–7.28 (m, 4H), 5.76 (dd, J = 11.5, 5.1 Hz, 1H), 3.80 (dd,
J = 17.6, 11.7 Hz, 1H), 3.19 (dd, J = 17.6, 5.1 Hz, 1H); MS
(m/z): 395 [M + H]+, (100%).
(2-(2,3-Dimethylphenylamino)phenyl)(4,5-dihydro-5-(4-
methoxyphenyl)-3-phenylpyrazo-1-yl)methanone (4c).
Off
white solid; mp 95–98ꢂC; Rf 0.45 (petroleum ether/
EtOAc = 8.5:1.5); IR (KBr) nmax/cmꢀ1: 3559, 3418, 2923, 1628;
1H NMR (400 MHz, CDCl3): d 8.14 (bs, 1H, NH, D2O
exchangeable), 7.89 (d, J = 7.9 Hz, 1H), 7.73 (d, J = 6.7 Hz, 1H),
7.41 (d, J = 1.8 Hz, 2H), 7.40 (d, J = 1.8 Hz, 1H), 7.27–7.25 (m,
3H), 7.23–7.19 (m, 1H), 7.11 (d, J = 7.9 Hz, 1H), 7.02 (t,
J = 7.9 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H),
6.80 (t, J = 8.0Hz, 2H), 5.81 (dd, J = 11.6, 4.9 Hz, 1H), 3.78 (dd,
J = 11.6, 17.7 Hz, 1H), 3.77 (s, 3H), 3.20 (dd, J = 17.7, 4.9 Hz,
1H), 2.29 (s, 3H), 2.11 (s, 3H); 13C NMR (100 MHz, CDCl3): d
Single crystal X-ray data for compound 3a. Single crystal
suitable for X-ray diffraction of 3a was grown from methanol.
The crystals were carefully chosen using a stereo zoom
microscope supported by a rotatable polarizing stage. The data
were collected at room temperature on Bruker Kappa APEX- ΙΙ
CCD DUO diffractometer with graphite monochromated
Mo-Ka radiation (0.71073 Å). The crystals were glued to a thin
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Nonsteroidal Anti-inflammatory Drug-based N-Allylidene Benzohydrazides and
1-Acyl-2-pyrazolines: Their Synthesis as Potential Cytotoxic Agents In Vitro
Month 2014
Acknowledgments. The authors (S. P. and K. K.) thank Mr. M.
N. Raju, the chairman of M.N.R. Educational Trust for his
constant encouragement and G. R. Krishna and Dr. C. M.
Reddy of IISER, Kolkata, India for X-ray single crystal data.
glass fiber using FOMBLIN immersion oil and mounted on a
diffractometer. The intensity data were processed using Broker’s
suite of data processing programs (SAINT), and absorption
corrections were applied using SADABS [28]. The crystal
structure was solved by direct methods using SHELXS-97,
and the data was refined by full matrix least-squares
refinement on F2 with anisotropic displacement parameters
for non-H atoms, using SHELXL-97 [29].
Crystal data of 3a: molecular formula = C33H33N3O2, formula
weight = 503.62, crystal system = monoclinic, space group = P21/
c, a = 17.386(3) Å, b = 20.968(5) Å, c = 15.394(3) Å, V = 5586
(2) Å3, T = 100(2) K, Z = 8, Dc = 1.198 Mg mꢀ3, m(Mo-Ka) = 0.08
mmꢀ1, 21063 reflections measured, 4057 independent reflec-
tions, 2850 observed reflections [I > 2.0 s (I)], R1_obs = 0.078,
goodness of fit = 1.133.
REFERENCES AND NOTES
[1] Suresh, K.; Sandhya, B.; Sushma, D.; Rajiv, K.; Himanshu, G.
Recent Pat Anti-Infect Drug Disc 2009, 4, 154.
[2] Mamolo, M. G.; Zampieri, D.; Falagiani, V.; Vio, L.; Banfi, E.
IL Farmaco 2001, 56, 593.
[3] Manna, F.; Chimenti, F.; Bolasco, A.; Secci, D.; Bizzarri, B.;
Befani, O.; Turini, P.; Mondovi, B.; Alcaroc, S.; Tafid, A. Bioorg Med
Chem Lett 2002, 12, 3629.
[4] Godoy, M. C. M.; Fighera, M. R.; Souza, F. R.; Flores, A. E.;
Rubin, M. A.; Oliveira, M. R.; Zannata, N.; Martins, M. A. P.; Mello, C.
F. Eur J Pharmacol 2004, 496, 93.
Biological activity
Chemical and reagents.
[5] Camacho M. E.; Leon, J.; Entrena, A.; Velasco, G.; Carrion,
M. D.; Escames, G.; Vivo, A.; Acun, C. D.; Gallo, M. A.; Espinosa, A.
J. Med Chem 2004, 47, 5641.
[6] Rani, P.; Srivastava, V. K.; Kumar, A. Eur J Med Chem 2004,
39, 449.
[7] Manna, F.; Chimenti, F.; Fioravanti, R.; Bolasco, A.; Secci, D.;
Chimenti, P.; Ferlini, C.; Scambi, G. Bioorg Med Chem Lett 2005, 15, 4632.
[8] Francesc, P. B.; Mark, T.; Brett, M. F.; Sean, M. P.; Noel, G.
E.; David, E. W.; Scott, J. G.; Paul, S. M.; Barry, F.; Ping, R.; David,
M. F.; Pei, Y. S. Antimicrob Agents Chemother 2006, 50, 1320.
[9] Chimenti, F.; Fioravanti, R.; Bolasco, A.; Manna, F.;
Chimenti, P.; Secci, D.; Rossi, F.; Turini, P.; Ortuso, F.; Alcaro, S.;
Cardia, M. C. Eur J Med Chem 2008, 43, 2262.
[10] Amir, M.; Kumar, H.; Khan, S. A. Bioorg Med Chem Lett
2008, 18, 918.
[11] Kugawa, F.; Ide, K.; Aoki, M. J. Health Sci 2004, 50, 271.
[12] Somchit, M. M.; Faizah, S.; Zuraini, A.; Khairi, H. M.; Hasiah,
A. H.; Zakaria, Z. A. Res J Pharmcol 2009, 3, 15.
[13] Pericherla, S.; Mareddy, J.; Rani, D. P. G.; Gollapudi, P. V.;
Pal, S. J. Braz Chem Soc 2007, 18, 384.
[14] Reddy, L. V.; Nakka, M., Suman, A.; Ghosh, S.; Helliwell, M.;
Mukkanti, K.; Mukherjee, A. K.; Pal, S. J. Heterocycl. Chem. 2011, 48, 555.
[15] Durgadas, S.; Chatare, V. K.; Mukkanti, K.; Pal, S. Appl
Organomet Chem 2010, 24, 680.
RPMI-1640, L-glutamine,
streptomycin, and penicillin were obtained from Sigma-Aldrich,
USA. Trypsin-versene, fetal 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.
Preparation of test compounds.
Test compounds were
dissolved in DMSO and were diluted appropriately with
culture media before treatment of cells. The final
concentration of DMSO used in the culture medium was less
than 0.2%.
Cell lines and culture conditions. The cell line used in this
work was obtained from the National Centre for Cell Science,
Pune, India. The cell line is human colon cancer cell line,
HCT15. The cells were grown in RPMI 1640 culture
medium supplemented with 2 mM L-glutamine, 10% fetal
bovine serum, penicillin (50 IU/mL), and streptomycin
(50 mg/mL) at 37ꢂC in a humidified incubator with a 5%
CO2 atmosphere and passage thrice weekly to maintain a
subconfluent state.
MTT assay for cytotoxicity. The effect of test compounds
on cell viability was measured using an MTT assay, which is
based on the reduction of MTT by the mitochondrial
dehydrogenase of intact cells to a purple formazan product [25].
Doxorubicin, a known anticancer drug, was used as a reference
compound in this assay. Cells (1 ꢁ 104) were seeded in a 96-
well plate. After 24 h, they were treated with different
concentration (1–25 mg/mL) of various test compounds diluted
appropriately with culture media for 48h. 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, medium-containing
compounds were carefully removed by aspiration. An amount of
100 mL of 0.4 mgmLꢀ1 MTT in phosphate-buffered solution was
added to each well and incubated in the dark for 4 h. An amount
of 100 mL of DMSO was added to each well and kept in an
incubator for 4 h for dissolution of the formed formazan
crystals. An amount of formazan was determined by
measuring the absorbance at 540 nm using an enzyme-linked
immuno sorbent assay plate reader. The data were presented as
percent dead cells, whereas the absorbance from nontreated
control cells was defined as 100% live cells. The percent dead
cells was plotted (Y-axis) against concentration (X-axis) of
compounds, when IC50 values could be interpolated from
the graph.
[16] Reddy, L. V.; Kethavath, M.; Nakka, M.; Beevi, S. S.;
Mangamoori, L. N.; Mukkanti, K.; Pal, S. J Heterocycl Chem 2012, 49, 80.
[17] Kankanala, K.; Reddy, V. R.; Mukkanti, K.; Pal, S. J Braz
Chem Soc 2010, 21, 1060.
[18] Ghosh, A. B.; Kankanala, K.; Reddy, V. R.; Mukkanti, K.; Pal, S.;
Mukherjee, A. K. Chem Phys Lett 2010, 493, 151.
[19] Kankanala, K.; Varsha, P.; Mukkanti, K.; Reddy, V. R.; Pal, S.
Molbank 2011, M740; doi: 10.3390/M740.
[20] Traversa, G.; Walker, A. M.; Ippolito, F. M.; Caffari, B.;
Capurso, L.; Dezi, A.; Koch, M.; Maggini, M.; Alegiani, S. S.; Raschetti, R.
Epidemiology 1995, 6, 49.
[21] Shaharyar, M.; Siddiqui, A. A.; Ali, M. A.; Sriram, D.;
Yogeeswari, P. Bioorg Med Chem Lett 2006, 16, 3947.
[22] Crystallographic data (excluding structure factors) for 3a has
been deposited with the Cambridge Crystallographic Data Center as
supplementary publication number CCDC 899091.
[23] Turan-Zitouni, G.; Chevallet, P.; Kiliç, F. S.; Erol, K. Eur J
Med Chem 2000, 35, 635.
[24] Parkin, D. M.; Bray, F.; Ferlay, J.; Pisani, P. CA Cancer J.
Clin. 2005, 55, 74.
[25] Mosmann, T. J. Immunol Methods 1983, 65, 55.
[26] Zmitek, J.; Verhnjak, K.; Temeljotov, D. F.; Kovacic, M.;
Lavric, A.; Vunduk, B. B. US US5840714, November 24, 1998.
[27] Kansara, S. G.; Pandit, R. D.; Bhawe, V. G. Rasayan J Chem
2009, 2, 699.
[28] Bruker SADABS V2008-1, Bruker AXS.: Madison, WI, USA, 2008.
[29] Sheldrick, G. M.; SHELX-97, Program for Crystal Structure
Determination, University of Göttingen, 1997.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet