Synthesis, analgesic and anti-inflammatory activities of chalconyl-incorporated hydrazone derivatives of mefenamic acid

Article abstract of DOI:10.1007/s00044-015-1318-8

A series of chalconyl-incorporated hydrazone derivatives of mefenamic acid was synthesized in order to obtain new compounds with potential analgesic and anti-inflammatory activity having lesser side effects. The structures of all synthesized compounds were confirmed by means of elemental analysis, IR, ¹H NMR, ¹³C NMR and mass spectra. All compounds were evaluated for their analgesic and anti-inflammatory activities by tail-flick method and carrageenan-induced rat paw edema test, respectively. Among all the synthesized compounds, compounds (4a) and (4j) exhibited the most prominent and consistent anti-inflammatory activity. In acute ulcerogenicity study, it can be concluded that compounds (4a) and (4j) are devoid of the deadlier gastrointestinal toxicities.

Full text of DOI:10.1007/s00044-015-1318-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1318-8
ORIGINAL RESEARCH
Synthesis, analgesic and anti-inflammatory activities of chalconyl-
incorporated hydrazone derivatives of mefenamic acid
•
Neeraj Kumar Lalit Singh Chauhan
•
•
•
Chandra Shekhar Sharma Nipun Dashora
Rajendra Bera
Received: 2 July 2014 / Accepted: 29 December 2014
Ó Springer Science+Business Media New York 2015
Abstract A series of chalconyl-incorporated hydrazone
derivatives of mefenamic acid was synthesized in order to
obtain new compounds with potential analgesic and anti-
inflammatory activity having lesser side effects. The
structures of all synthesized compounds were confirmed by
and other professionals and represent important therapeutic
and economic cost for the community. According to the
World Health Organization, 90 % of diseases are associated
with pain (Gokc¸e et al., 2009). Humans have been using
nonsteroidal anti-inflammatory drugs (NSAIDs) in various
forms for more than 3,500 years and have been among the
most widely used drugs for the treatment of pain and
inflammation (Vane, 2000). NSAIDs develop their mode of
action by blocking the cyclooxygenase (COX) enzyme and
thus the biosynthesis of Prostaglandins (PGs). Cyclooxy-
genase (COX) is the key enzyme in the rate-limiting step that
converts arachidonic acid to prostaglandins and thrombox-
ane (Vane, 1971; O’Banion et al., 1991). Three isoforms of
the COX enzyme have been characterized: cyclooxygenase-
1 (COX-1), cyclooxygenase-2 (COX-2) and cyclooxygen-
ase-3 (COX-3) (Willoughby et al., 2000).
1
means of elemental analysis, IR, H NMR, 13C NMR and
mass spectra. All compounds were evaluated for their
analgesic and anti-inflammatory activities by tail-flick
method and carrageenan-induced rat paw edema test,
respectively. Among all the synthesized compounds,
compounds (4a) and (4j) exhibited the most prominent and
consistent anti-inflammatory activity. In acute ulceroge-
nicity study, it can be concluded that compounds (4a) and
(4j) are devoid of the deadlier gastrointestinal toxicities.
Keywords Mefenamic acid ꢀ Hydrazones ꢀ Chalcone ꢀ
Analgesic ꢀ Anti-inflammatory
COX-1 is the constitutional isoenzyme that is important
in maintaining renal function, platelet function and the
gastrointestinal epithelium (Mitchell and Warner, 1999).
The isoenzyme COX-2 is inducible isoenzyme and pri-
marily associated with inflammation. Cytokines and
growth factors increase the expression of COX-2, mainly at
inflammatory sites, producing prostaglandins that mediate
inflammation, pain and fever (Crofford, 1997). A splice
variant of COX-1 mRNA isolated that was found in high
concentrations in the heart and cerebral cortices of the dog,
which they reported to be COX-3 (Chandrasekharan et al.,
2002). Expression of COX-3 mRNA is particularly marked
in the hypothalamus, pituitary and choroid plexus, sites
which are targets of antipyretic action of paracetamol.
COX-3 mRNA accounts for 30 % of COX-1 mRNA levels.
Inducible COX-3 may have a role in remission periods in
chronic inflammatory disease (Kam and So, 2009).
Introduction
Pain is a problem not solved by medicine and is the most
common reason that patients seek advice from pharmacists
N. Kumar (&) ꢀ N. Dashora
Department of Pharmaceutical Chemistry, B. N. Institute of
Pharmaceutical Sciences, Udaipur 313001, India
e-mail: neerajkumarkamra@gmail.com
L. S. Chauhan
Department of Pharmaceutical Sciences, Mohanlal Sukhadia
University, Udaipur 313001, India
C. S. Sharma ꢀ R. Bera
The major adverse effects of NSAIDs are gastrotoxic
(e.g., damage of gastric mucosa may provoke gastric
Department of Pharmaceutical Chemistry, B. N. College of
Pharmacy, Udaipur 313001, India
123

Med Chem Res
bleeding and gastroduodenal ulcers), increased bleeding
tendency and delay of the birth process. Selective COX-2
inhibitors fail to inhibit constitutive COX-1 isotherm and
have no gastrointestinal adverse effects. However, it has
been suggested that COX-2 inhibitors, like rofecoxib and
celecoxib, may be prothrombic and increase the risk of
Various acetophenones were reacted with substituted
aromatic aldehydes and gave chalcones by via Claisen
Schmidt reaction (3a–3j). The proposed structures of the
compounds were confirmed on the basis of elemental
analysis and spectroscopic data (IR, 1H NMR and 13C
NMR). The IR spectra showed C=C absorption bands at
1,613–1,576 cm-1. The 1H NMR spectra of the compounds
3a–3j showed the two doublets of CH=CH in the region
d = 7.69–7.83 and 8.06–8.26 ppm. The remaining protons
appeared at the expected chemical shifts.
´
myocardial infarction (Savic et al., 2011). Therefore, a
synthesis of novel NSAIDs, with potent anti-inflammatory,
analgesic and antipyretic action, with no adverse effects is
highly desired.
Chalcones belong to the flavonoid family from plant
origin and some of them possess anti-inflammatory activity
(Kim et al., 2007; Bano et al., 2013; Bonifait et al., 2014).
A new pharmacophore ‘Chalconesemicarbazone’ was
designed by pharmacophore hybridization approach of
drug design. A series of novel ‘Chalconylsemicarbazide’
derivatives was synthesized and evaluated for their anal-
gesic and anti-inflammatory activity. Most of the com-
pounds were found to be more or comparable potent than
the reference standard drugs (Singh et al., 2010).
In the last step, chalcones (3a–3j) were reacted with
2-(2,3-dimethylphenylamino)benzohydrazide (2) and gave
chalconyl-incorporated hydrazone derivatives of mefe-
namic acid (4a–4j). The proposed structures of 4a–4j were
confirmed by elemental analysis and spectroscopic data
(IR, 1H NMR and 13C NMR). The IR spectra showed C=N
absorption bands at 1,639–1,611 cm-1. The 1H NMR
spectra of the compounds 4a–4j showed the one singlet of
–NH–N=C– at the region d = 8.97–9.23 ppm. For the
compounds (4a–4j), the signals belonging to benzylidene
group were observed at aromatic region, while the signals
belonging to –NHNH2 disappeared, indicating functional-
ization of hydrazide to hydrazone with substituted chal-
cones. The remaining protons appeared at the expected
chemical shifts. The physical data, IR, 1H NMR, 13C NMR
and mass spectral data for all the synthesized compounds
are reported in experimental protocols.
Mefenamic acid is a well-known nonsteroidal anti-
inflammatory drug. 1,3,4-oxadiazole derivatives of mefe-
namic acid were synthesized and evaluated for analgesic
and anti-inflammatory activity. The oxadiazole ring being
weak acidic in nature reduces the ulcerogenicity of mefe-
namic acid and retains its anti-inflammatory potential
(Somani and Bhanushali, 2011). A series of N-arylhyd-
razone derivatives of mefenamic acid was synthesized and
evaluated for analgesic and anti-inflammatory activity.
From the results, it was concluded that replacement of the
acidic moiety of mefenamic acid with N-arylhydrazone
moiety can create potent analgesic anti-inflammatory
compounds (Almasirad et al., 2005).
Pharmacology
In the pharmacological study, we have investigated in vivo
analgesic and anti-inflammatory activity as well as the
acute ulcerogenicity of chalconyl-incorporated hydrazone
derivatives of mefenamic acid (4a–4j). Mefenamic acid,
the parent compound, was used as a reference standard.
The experiments were performed on albino rats of Wistar
strain of either sex, weighing 200–250 g (from animal
house of BN College of Pharmacy, Udaipur). The animals
were maintained at 25 2 °C, 50 5 % relative humid-
ity and 12 h light/dark cycles. The animals were fasted for
24 h prior to the experiments, and water provided ad libi-
tum. The test compounds were suspended in 1 % aqueous
carboxy methyl cellulose (CMC) solution and administered
orally to experimental animals (Table 1).
In the present work, we planned to incorporate the
substituted chalcones to the hydrazide derivative of mefe-
namic acid moiety as powerful and novel non-ulcerogenic
analgesic, anti-inflammatory lead-candidates.
Results and discussion
Chemistry
The synthetic route used to synthesize title compounds is
outlined in Scheme 1. Methyl-2-(2,3-dimethylphenylami-
no)benzoate (1), the starting material, was prepared
according to the method reported in the literature, using
2-(2,3-dimethylphenylamino)benzoic acid (mefenamic
acid) (Chandrasekhar et al., 2012). The 2-(2,3-dimethyl-
phenylamino)benzohydrazide (2) was prepared by esteri-
fication of 2-(2,3-dimethylphenylamino)benzoic acid
followed by treatment with hydrazine hydrate in absolute
ethanol.
Analgesic activity
The analgesic activity was determined in vivo by the
abdominal constriction test induced by tail-flick method.
All the compounds (4a–4j) were screened for analgesic
activity. The analgesic activity was evaluated at equimolar
doses equivalent to 25 mg/kg (mefenamic acid) body
weight (Khan and Akhter, 2005). From the results, it was
123

Med Chem Res
Scheme 1 Synthesis of
chalconyl-incorporated
hydrazone derivatives of
mefenamic acid: (i) CH3OH,
Conc. HCl; (ii) NH2NH2.H2O;
(iii) EtOH, NaOH; (iv) CHCl3,
CH3COOH
i
ii
NH
NH
NH
O
O
O
OCH3
OH
Mefanamic Acid
NHNH2
(2)
(1)
O
O
O
iii
iv
R1
R2
R2
(3a-3j)
R1
Comp.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
R1
R2
H
H
H
H
m-NO2
p-Cl
p-Cl
o-Cl
p-N(CH3)2 p-OH
m-NO2 p-Cl
p-N(CH3)2 p-F
p-Cl
p-NO2
NH
O
p-OH
p-OH
NHN
p-F
p-F
R1
R2
(4a-4j)
Table 1 Results of anti-inflammatory activity of entitled compounds (4a–4j) against carrageenan-induced rat paw edema model in rats
Compounds
Volume of edema (mean) SEM (ml)
Anti-inflammatory activity (% inhibition)
1 h
2 h
3 h
4 h
1 h
2 h
3 h
4 h
Control
0.030 0.001
0.035 0.001
0.050 0.006
0.033 0.005
–
–
–
–
Mefanamic acid 0.021 0.003ns 0.013 0.001** 0.007 0.001** 0.003 0.000** 29.16
60.71
55.34
23.20
51.77
57.14
23.20
32.14
23.70
39.28
39.28
40.05
85.00
87.50
42.50
85.00
90.00
40.00
72.50
58.70
71.20
67.50
78.70
90.56
86.79
18.86
84.90
92.45
60.36
71.70
60.36
75.47
71.70
83.02
4a
4b
4c
4d
4e
4f
0.023 0.003ns 0.015 0.002** 0.006 0.006** 0.004 0.001** 20.83
0.012 0.001** 0.026 0.003ns 0.028 0.003* 0.026 0.002ns 58.33
0.024 0.002ns 0.016 0.002** 0.007 0.002** 0.005 0.001** 18.73
0.025 0.002ns 0.015 0.001** 0.005 0.001** 0.002 0.001** 14.56
0.019 0.004*
0.019 0.001*
0.026 0.002ns 0.030 0.001ns 0.028 0.002ns 35.40
0.023 0.004** 0.013 0.013** 0.009 0.004** 35.40
4g
4h
4i
0.024 0.002ns 0.026 0.002ns 0.020 0.006** 0.013 0.004** 18.70
0.028 0.004ns 0.021 0.003** 0.014 0.003** 0.008 0.003** 06.20
0.015 0.003** 0.021 0.004** 0.016 0.003** 0.009 0.002** 47.90
0.026 0.003ns 0.020 0.002** 0.010 0.001** 0.005 0.000** 12.50
4j
Data analyzed by one-way ANOVA followed by Dunnett’s ‘t’ test, (n = 6)
ns not significant
* P \ 0.05; ** P \ 0.01 significant from control
123

Med Chem Res
Table 2 Results of analgesic activity of entitled compounds (4a–4j) against tail-flick method
Compounds
Reaction time (mean SEM) s
Relative analgesic activity (%)
1 h
2 h
3 h
4 h
1 h
2 h
3 h
4 h
Control
2.23 0.110
2.41 0.081
2.79 0.143
3.50 0.091
–
–
–
–
Mefanamic acid
3.99 0.208**
3.36 0.163**
2.71 0.087ns
2.87 0.204ns
3.60 0.242**
2.62 0.182ns
3.44 0.283*
2.89 0.206**
3.14 0.243ns
2.79 0.090*
3.40 0.322**
5.28 0.153**
4.30 0.128**
3.51 0.143**
4.04 0.046**
4.72 0.232**
3.49 0.128**
4.71 0.162**
3.07 0.292*
4.21 0.120**
3.00 0.065ns
4.18 0.088**
5.94 0.118**
5.11 0.249**
3.45 0.407ns
4.74 0.142**
5.01 0.334**
3.81 0.329ns
5.33 0.258**
3.53 0.234ns
4.45 0.106**
3.40 0.358ns
5.33 0.185**
6.81 0.183**
6.15 0.144**
4.30 0.362ns
5.86 0.224**
5.71 0.377**
4.80 0.348**
5.69 0.257**
4.26 0.314ns
5.84 0.238**
4.24 0.102ns
6.38 0.151**
100
100
100
100
4a
4b
4c
4d
4e
4f
63.93
27.06
36.29
77.89
21.76
68.94
37.43
51.39
31.62
66.66
65.95
38.50
56.89
80.76
37.80
80.24
23.27
62.82
20.55
61.67
73.53
20.96
62.03
70.36
32.40
80.74
23.44
52.57
19.31
80.78
80.13
24.10
71.36
66.88
39.21
66.13
22.89
70.91
22.28
86.99
4g
4h
4i
4j
Data analyzed by one-way ANOVA followed by Dunnett’s ‘t’ test, (n = 6)
ns not significant
* P \ 0.05, ** P \ 0.01 significant from control
with different therapeutic efficacy. In this series, com-
pounds 4a, 4c, 4d and 4j exhibited very significant anti-
inflammatory activity compared to standard drug mefe-
namic acid, among them 4d (p-chloro and p-hydroxy
substituted) showed 92.45 % inhibition, whereas standard
mefenamic acid showed 90.56 % inhibition.
Table 3 Ulcerogenic effects of compounds 4a and 4j in comparison
with mefanamic acid
Compounds
Ulcer score (mean SEM)
Ulcer index
Control
0.333 0.105
2.167 0.401**
0.750 0.112ns
1.167 0.279ns
–
Mefanamic acid
1.834
0.417
0.834
4a
4j
Based on the findings of these preclinical results, further
studies need to be carried out to investigate the other
specifications, such as in vitro assays, chronic ulceroge-
nicity studies and toxicological studies.
Data analyzed by one-way ANOVA followed by Dunnett’s ‘t’ test,
(n = 6)
ns not significant
From the analgesic and anti-inflammatory activity pro-
file, compounds 4a and 4j were subjected to ulcerogenicity
potential test at four times the therapeutic doses with
additional physical (cold) stress.
* P \ 0.05; ** P \ 0.01 significant from control
noticed that most of the compounds possess significant
analgesic activity (Table 2). Compounds 4a and 4j showed
comparable analgesic activity to standard mefenamic acid.
Acute ulcerogenicity studies
Ulcerogenic effect of most active chalconyl-incorporated
hydrazone derivatives of mefenamic acid (4a and 4j) was
evaluated for gastric ulcerogenic potential in the rat stress
model at four times the therapeutic doses. When compared
with standard, these compounds showed less ulceration
than the standard drugs. The results are shown in Table 3.
Anti-inflammatory activity
Anti-inflammatory activity of the synthesized compounds
was evaluated by carrageenan-induced rat paw edema
model, that is, equivalent to 25 mg/kg, of mefenamic acid
subplanter injection of 0.1 ml, 1 % carrageenan produced
an increase in paw volume (edema) of all the animals of
various groups. The onset of action was evident from 1 h in
various test groups. The significant (P \ 0.01) reduction in
rat paw edema was observed by most of the test com-
pounds at 4 h compared to control group (Table 1).
Computational studies
The TPSA and molecular volume of synthesized com-
pounds (4a–4j) were calculated as shown in Table 4.
Topological polar surface area (TPSA), i.e., surface
belonging to polar atoms, is a descriptor that was shown to
correlate with passive molecular transport through
From close inspection of the results of in vivo experi-
ments, we can conclude that substituted chalconyl hydra-
zone derivatives of mefenamic acid yielded compounds
123

Med Chem Res
Experimental protocols
Table 4 Molecular properties calculated by Molinspiration impor-
tant for good oral bioavailability
Synthetic procedures
Compounds
MV
TPSA
%ABS
Mefanamic acid
227.97
425.85
449.19
439.39
447.41
447.41
479.78
462.72
476.69
444.32
454.12
49.32
90.54
74.73
90.54
83.56
83.56
82.44
83.56
89.42
90.54
74.73
91.98
77.76
83.21
77.76
80.17
80.17
80.55
80.17
78.15
77.76
83.21
Melting points of the synthesized compounds were deter-
mined in open capillary tubes and are uncorrected. Ele-
mental analysis (C, H and N) was undertaken with a
Perkin-Elmer model 240C analyzer, and all analyses were
consistent with theoretical values (within 0.4 %) unless
indicated.
4a
4b
4c
4d
4e
4f
IR absorption spectra were recorded on Bruker alpha,
1
KBr diffuse reflectance. H NMR and 13C NMR spectra
4g
4h
4i
were recorded on the Bruker DPX-400 instrument at 400
and 100 MHz, respectively. The 1H and 13C chemical
shifts are reported as parts per million (ppm) downfield
4j
1
from TMS (Me4Si). H NMR and IR spectra were con-
MV molecular volume, TPSA topological polar surface area, %ABS
percentage of absorption
sistent with the assigned structures. The LC mass spectra of
the compounds were recorded on Shimadzu 8201PC
spectrometer. The homogeneity of the compounds was
monitored by ascending thin-layer chromatography (TLC)
on silica gel G (Merck)-coated aluminum plates, visualized
by iodine vapor.
membranes and, therefore, allows prediction of transport
properties of drugs in the intestines and blood–brain barrier
crossing (Ertl et al., 2000). The percentage of absorption
(%ABS) was calculated using TPSA. From all these
parameters, it can be observed that all synthesized com-
pounds exhibited a great %ABS ranging from 77.76 to
83.21 % (Table 4).
Methyl 2-(2,3-dimethylphenylamino)benzoate (1)
A solution of the appropriate mefenamic acid (10 mmol),
absolute methanol (10 ml) and concentrated sulfuric acid
(1 ml) was heated under reflux for the appropriate time
35–42 h. The solvent was evaporated under reduced pres-
sure, the remaining contents cooled to room temperature
and neutralized with a concentrated solution of sodium
carbonate, and then the aqueous solution extracted with
ether. The combined ether extracts were dried, and the
solvent is removed under reduced pressure to yield the
corresponding ester. Yield, 82 %; mp 176–180 °C; IR
Conclusion
In conclusion, we described analog-based design of chal-
conyl-incorporated hydrazone derivatives of mefenamic
acid for in vivo analgesic and anti-inflammatory activity as
COX inhibitor. Most of the entitled compounds exhibited
promising analgesic and anti-inflammatory activity.
Among all the synthesized compounds, compounds (4a)
and (4j) exhibited most prominent and consistent anti-
inflammatory activity. From acute ulcerogenicity studies, it
can be concluded that compounds (4a) and (4j) are devoid
of the deadlier gastrointestinal toxicities.
(KBr) tmax 3,260 (–OH), 1,692 (C=O) 1,589 (C=C) cm-1
;
1H NMR (DMSO-d6, 400 MHz,): d = 8.89 (s, 1H, NH),
6.89–7.44 (m, 7H, Ar–H), 3.86 (s, 3H, CH3), 2.38 (s, 3H,
CH3), 2.19 (s, 3H, CH3); 13C NMR (DMSO-d6,
100 MHz,): d = 169.7 (C=O), 148.3 (C1–Ar2), 148.2 (C2–
Ar1), 139.9 (C3–Ar2), 134.7 (C4–Ar1), 133.8 (C6–Ar1),
127.5 (C2–Ar2), 124.8 (C5–Ar2), 124.4 (C4–Ar2), 121.7
(C5–Ar1), 121.2 (C3–Ar1), 120.1 (C1–Ar1), 119.5 (C6–Ar2),
54.8 (–OCH3), 18.5 (–3–CH3), 15.7(–2–CH3); LCMS m/z
[M]? 255.1; Anal. Calcd. for C16H17NO2 (255.31): C,
75.27; H, 6.71; N, 5.49. Found: C, 75.36; H, 6.61; N, 5.44.
Topological polar surface area (TPSA) and molecular
volume were calculated using the web-based program
Molinspiration. The percentage of absorption (%ABS) was
calculated using TPSA. From all these parameters, it can be
observed that all synthesized compounds exhibited a great
%ABS ranging from 77.76 to 83.21 %.
The present investigation suggests that chalcone-incor-
porated hydrazone derivatives of conventional NSAIDs are
promising template for the design of new gastric safe anti-
inflammatory agents. Further, QSAR investigations are in
progress to examine possible descriptors influences on
various classes of COX inhibitors.
2-(2,3-Dimethylphenylamino)benzohydrazide (2)
A solution of hydrazine hydrate (99.9 %, 5 mmol) and the
appropriate ethyl ester (1 mmol) was brought to a gentle
reflux for the appropriate time 22 h and then cooled to
123

Med Chem Res
room temperature. The solid formed was filtered (ice/water
mixture was added in some cases to complete precipitation)
and washed with several portions of water. The filtrate was
dried by suction. Crystallization of crude product from
EtOH afforded the corresponding mefenamic acid hydra-
zides. Yield, 84 %; mp 202–204 °C; IR (KBr) tmax 3,306
(NH), 2,909, 2,857 (CH Str.), 1,645 (C=O), 1,573
135.0 (C4–Ar1), 130.3 (C5–Ar3), 129.6 (C2,6–Ar1), 128.7
(C3,5–Ar1), 126.5 (C4–Ar3), 124.3 (C2–Ar3), 123.2 (C2);
LCMS m/z [M]? 253.1; Anal. Calcd. for C15H11NO3
(253.25): C, 71.14; H, 4.38; N, 5.53. Found: C, 71.23; H,
4.31; N, 5.49.
3-(4-Chlorophenyl)-1-phenylprop-2-en-1-one (3c)
(CH=CH), 1,443, 1,423 (Ar. C=C) cm-1
;
1H NMR
(DMSO-d6, 400 MHz,): d = 8.73 (s, 1H, NH), 8.27 (s, 1H,
NH), 6.93–7.57 (m, 7H, Ar–H), 3.14 (s, 2H, NH2), 2.35 (s,
3H, CH3), 2.23 (s, 3H, CH3); 13C NMR (DMSO-d6,
100 MHz,) d = 174.7 (C=O), 147.8 (C1–Ar2), 147.3 (C2–
Ar1), 138.9 (C3–Ar2), 134.5 (C4–Ar1), 128.4 (C6–Ar1),
127.8 (C2–Ar2), 125.6 (C5–Ar2), 124.9 (C5–Ar2), 122.3
(C4–Ar2), 121.6 (C3–Ar1), 120.2 (C1–Ar1), 119.3 (C6–Ar2),
18.9 (–CH3), 13.7 (–CH3); LCMS m/z [M]? 255.1; Anal.
Calcd. for C15H17N3O (255.31): C, 70.56; H, 6.71; N,
16.46. Found: C, 70.43; H, 6.63; N, 16.54.
Yield, 64 %; mp 100–102 °C; IR (KBr) tmax 2,898 (CH
Str.), 1,672 (C=O), 1,591 (CH=CH), 1,445 (Ar. C=C)
1
cm-1; H NMR (DMSO-d6, 400 MHz,): d = 8.17 (d, 1H,
CH), 7.83 (d, 1H, CH), 7.04–7.39 (m, 9H, Ar–H); 13C
NMR (DMSO-d6, 100 MHz,): d = 189.3 (C=O), 144.3
(C1), 138.2 (C1–Ar1), 134.2 (C4–Ar3), 133.1 (C1–Ar3),
132.1 (C4–Ar1), 130.3 (C3,5–Ar3), 129.1 (C2,6–Ar3), 128.6
(C2,6–Ar1), 127.7 (C3,5–Ar1), 122.9 (C2); LCMS m/z [M]?
242.0; Anal. Calcd. for C15H11ClO (242.70): C, 74.23; H,
4.57. Found: C, 74.26; H, 4.63.
1,3-Diphenyl-2-propen-1-one (3a)
3-(4-Chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (3d)
A solution of benzaldehyde (12 g, 11.7 ml, 0.1 mol) in
ethanol was mixed with acetophenone (10.60 g, 10.2 ml,
0.1 mol), and an aqueous solution of sodium hydroxide
(10 %) was added to it till no turbidity occurs. The reaction
mixture was stirred for 20 h and kept overnight at room
temperature. The mixture was poured into crushed ice and
acidified with dilute hydrochloric acid. The crude product
so obtained was filtered and recrystallized from methanol
and dried at room temperature. The completion of the
reaction was monitored by running TLC (Singh et al.,
2010). Yield, 78 %; mp 58–60 °C; IR (KBr) tmax 1,671
Yield, 61 %; mp 142–146 °C; IR (KBr) tmax 3,627 (OH
Str), 2,922 (CH Str.), 1,644 (C=O), 1,576 (CH=CH), 1,519
(ArꢀC=C) cm-1
;
1H NMR (DMSO-d6, 400 MHz,):
d = 8.39 (s, 1H, OH), 8.08 (d, 1H, CH), 7.78 (d, 1H, CH),
7.03–7.32 (m, 8H, Ar–H); 13C NMR (DMSO-d6,
100 MHz,): d = 191.8 (C=O), 164.2 (C4–Ar1), 145.4 (C3),
138.2 (C1–Ar3), 134.1 (C4–Ar3), 133.3 (C2,6–Ar1), 131.2
(C2,6–Ar3), 129.3 (C3,5–Ar3), 128.1 (C1–Ar1), 123.9 (C2),
116.8 (C3,5–Ar1); LCMS m/z [M]? 258.0; Anal. Calcd. for
C15H11ClO2 (258.70): C, 69.64; H, 4.29. Found: C, 69.69;
H, 4.21.
1
(C=O), 1,598 (CH=CH), 1,493 (Ar. C=C) cm-1; H NMR
(DMSO-d6, 400 MHz,): d = 8.06 (d, 1H, CH), 7.79 (d, 1H,
CH), 6.96–7.33 (m, 10H, Ar–H); 13C NMR (DMSO-d6,
100 MHz,) d = 193.9 (C=O), 147.3 (C3), 141.2 (C1–Ar1),
137.8 (C1–Ar3), 136.0 (C4–Ar1), 132.4 (C4–Ar2), 131.2
(C3,5–Ar3), 130.6 (C3,5–Ar1), 129.5 (C2,6–Ar1), 128.0
(C2,6–Ar3), 124.9 (C1); LCMS m/z [M]? 208.1; Anal.
Calcd. for C15H12O (208.25): C, 86.51; H, 5.81. Found: C,
86.48; H, 5.88.
3-(2-Chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (3e)
Yield, 65 %; mp 166–168 °C; IR (KBr) tmax 3,219 (OH),
1,650 (C=O), 1,588 (CH=CH), 1,517 (Ar. C=C), 755 (C–
1
Cl) cm-1; H NMR (DMSO-d6, 400 MHz,): d = 8.32 (s,
1H, OH), 8.19 (d, 1H, CH), 7.69 (d, 1H, CH), 6.91–7.39
(m, 8H, Ar–H); 13C NMR (DMSO-d6, 100 MHz,):
d = 191.3 (C=O), 163.8 (C4–Ar1), 144.1 (C3), 133.2 (C2–
Ar3), 132.7 (C1–Ar3), 131.4 (C2,6–Ar1), 131.0 (C3–Ar3),
130.1 (C4–Ar3), 129.4 (C1–Ar1), 128.6 (C6–Ar3), 127.3
(C5–Ar3), 126.1 (C2), 115.5 (C3,5–Ar1); LCMS m/z [M]?
258.0; Anal. Calcd. for C15H11ClO2 (258.70): C, 69.64; H,
4.29. Found: C, 69.68; H, 4.19.
The other compounds 3b to 3j were prepared by the
same procedure using the corresponding aldehydes and
ketones.
3-(3-Nitrophenyl)-1-phenylprop-2-en-1-one (3b)
Yield, 81 %; mp 134–136 °C; IR (KBr) tmax 1,661 (C=O),
1,608 (CH=CH), 1,349 (–NO2), 1,349 (Ar. C=C) cm-1; 1H
NMR (DMSO-d6, 400 MHz,): d = 8.12 (d, 1H, CH), 7.72
(d, 1H, CH), 6.98–7.52 (m, 9H, Ar–H); 13C NMR (DMSO-
d6, 100 MHz,): d = 192.8 (C=O), 146.1 (C3–Ar3), 145.3
(C3), 140.7 (C1–Ar1), 139.9 (C1–Ar3), 136.4 (C6–Ar3),
3-(4-(Dimethylamino)phenyl)-1-(4-hydroxyphenyl)prop-2-
en-1-one (3f)
Yield, 74 %; mp 84–88 °C; IR (KBr) tmax 3,648 (OH Str.),
2,933, 2,899 (CH Str.), 1,612 (CH=CH), 1,653 (C=O),
1
1,507 (ArꢀC=C), 1,362 (C–N) cm-1; H NMR (DMSO-d6,
123

Med Chem Res
3-(4-Chlorophenyl)-1-(4-fluorophenyl)prop-2-en-1-one (3i)
Yield, 68 %; mp 136–138 °C; IR (KBr) tmax 1,662 (C=O),
400 MHz,): d = 8.53 (s, 1H, OH), 8.16 (d, 1H, CH), 7.59
(d, 1H, CH), 6.84–7.46 (m, 8H, Ar–H), 2.63 (s, 6H, CH3);
13C NMR (DMSO-d6, 100 MHz,): d = 192.1 (C=O), 163.4
(C4–Ar1), 151.1 (C4–Ar3), 144.3 (C3), 131.2 (C2,6–Ar1),
129.9 (C2,6–Ar3), 129.3 (C1–Ar1), 123.8 (C1–Ar3), 122.9
(C2), 115.9 (C3,5–Ar1), 112.7 (C3,5–Ar3), 41.9 (–N(CH3)2);
LCMS m/z [M]? 267.1; Anal. Calcd. for C17H17NO2
(267.32): C, 76.38; H, 6.41; N, 5.24. Found: C, 76.32; H,
6.46; N, 5.31.
1
1,602 (CH=CH), 1,488 (Ar. C=C), 1,012 (C–F) cm-1; H
NMR (DMSO-d6, 400 MHz,): d = 8.26 (d, 1H, CH), 7.76
(d, 1H, CH), 6.85–7.48 (m, 8H, Ar–H); 13C NMR (DMSO-
d6, 100 MHz,): d = 192.4 (C=O), 165.3 (C4–Ar1), 145.5
(C3), 136.8 (C4–Ar3), 135.1 (C1–Ar3), 134.8 (C1–Ar1),
133.1 (C2,6–Ar1), 130.3 (C3,5–Ar3), 129.5 (C2,6–Ar3), 122.9
(C2), 115.4 (C3,5–Ar1); LCMS m/z [M]? 260.0; Anal.
Calcd. for C15H10ClFO (260.69): C, 69.11; H, 3.87. Found:
C, 69.24; H, 3.78.
1-(4-Chlorophenyl)-3-(3-nitrophenyl)prop-2-en-1-one (3g)
Yield, 76 %; mp 132–134 °C; IR (KBr) tmax 1,667 (C=O),
1,588 (CH=CH), 1,522 (Ar. C=C), 1,350 (–NO2), 741 (C–
1-(4-Fluorophenyl)-3-(3-nitrophenyl)prop-2-en-1-one (3j)
1
Cl) cm-1; H NMR (DMSO-d6, 400 MHz,): d = 8.23 (d,
1H, CH), 7.79 (d, 1H, CH), 6.74–7.59 (m, 8H, Ar–H); 13C
NMR (DMSO-d6, 100 MHz,): d = 191.7 (C=O), 148.1
(C3–Ar3), 145.3 (C3), 139.6 (C4–Ar1), 138.9 (C1–Ar3),
137.3 (C1–Ar1), 135.2 (C6–Ar3), 130.6 (C2,6–Ar1), 129.2
(C5–Ar3), 128.8 (C3,5–Ar1), 125.5 (C4–Ar3), 123.9 (C2–
Ar3), 121.2 (C2); LCMS m/z [M]? 287.0; Anal. Calcd. for
C15H10ClNO3 (287.69): C, 62.62; H, 3.50; N, 4.87. Found:
C, 62.73; H, 3.44; N, 4.92.
Yield, 72 %; mp 164–166 °C; IR (KBr) tmax 1,669 (C=O),
1,613 (CH=CH), 1,507 (ArꢀC=C), 1,339 (–NO2), 1,031 (C–
1
F) cm-1; H NMR (DMSO-d6, 400 MHz,): d = 8.19 (d,
1H, CH), 7.83 (d, 1H, CH), 6.79–7.53 (m, 8H, Ar–H); 13C
NMR (DMSO-d6, 100 MHz,): d = 188.7 (C=O), 166. 4
(C4–Ar1), 151.1 (C3–Ar3), 147.3 (C3), 137.9 (C1–Ar3),
136.4 (C6–Ar3), 135.7 (C1–Ar1), 133.01 (C2,6–Ar1), 130.2
(C5–Ar3), 127.5 (C4–Ar3), 124.9(C2–Ar3), 121.4 (C2),
114.9 (C3,5–Ar1); LCMS m/z [M]? 271.0; Anal. Calcd. for
C15H10FNO3 (271.24): C, 66.42; H, 3.72; N, 5.16. Found:
C, 66.57; H, 3.64; N, 5.10 (Fig. 1).
3-(4-(Dimethylamino)phenyl)-1-(4-fluorophenyl)prop-2-
en-1-one (3h)
2-(2,3-Dimethylphenylamino)-N0-(1,3-
Yield, 70 %; mp 96–98 °C; IR (KBr) tmax 2,899 (CH Str.),
1,648 (C=O), 1,592 (CH=CH), 1,519 (ArꢀC=C), 1,027 (C–
diphenylallylidene)benzohydrazide (4a)
1
F) cm-1; H NMR (DMSO-d6, 400 MHz,): d = 8.21 (d,
A mixture of 2-(2,3-dimethylphenylamino)benzohydrazide
(2.55 g, 0.01 mol) and chalcone (2.08 g, 0.01 mol) in
chloroform was stirred at 60–70 °C for 6 h in the presence
of 2–3 ml of glacial acetic acid. The reaction mixture was
poured into a beaker containing crushed ice and allowed to
stand for 2 h. The precipitate so formed was filtered and
washed with ice-cold water. The crude product was dried
and recrystallized from chloroform. The completion of the
reaction was monitored by running TLC. Yield, 65 %; mp
176–180 °C; IR (KBr) tmax 3,312 (NH), 3,033 (NH), 1,686
(C=O), 1,639 (C=N), 1,574 (CH=CH), 14 61 (ArꢀC=C)
cm-1; 1H NMR (DMSO-d6, 400 MHz,): d = 9.48 (bs, 1H,
NH), 9.08 (bs, 1H, NH), 8.11 (d, 1H, CH), 7.68 (d, 1H,
CH), 6.65–7.53 (m, 17H, Ar–H), 2.34 (s, 3H, CH3), 2.21 (s,
3H, CH3); 13C NMR (DMSO-d6, 100 MHz,): d = 171.3
(C=O), 151.2 (C=N), 148.4 (C2–Ar), 146.3 (C1–NAr),
138.9 (C1–Ar1), 137.9 (C3–NAr), 136.6 (C3, CH), 135.8
1H, CH), 7.73 (d, 1H, CH), 6.91–7.56 (m, 9H, Ar–H). 2.71
(s, 6H, CH3); 13C NMR (DMSO-d6, 100 MHz,): d = 190.9
(C=O), 164.4 (C4–Ar1), 152.1 (C4–Ar3), 147.6 (C3), 136.8
(C1–Ar1), 134.0 (C2,6–Ar1), 130.9 (C2,6–Ar3), 124.8 (C1–
Ar3), 122.3 (C2), 115.8 (C3,5–Ar1), 112.7 (C3,5–Ar3), 42.9
(-N(CH3)2); LCMS m/z [M]? 269.1; Anal. Calcd. for
C17H16FNO (269.31): C, 75.82; H, 5.99; N, 5.20. Found: C,
75.69; H, 6.12; N, 5.28.
Ar3
R1
2-CH3
3-CH3
2
H3C
CH3
1
3
N
O
NAr
HN
HN
(C1–Ar3), 134.7 (C4–Ar), 129.8 (C4–Ar3), 129.0 (C3,5
–
Ar1
Ar3), 128.5 (C3,5–Ar1), 128.4 (C6–Ar), 128.1 (C4–Ar1),
127.6 (C2,6–Ar3), 127.3 (C2–NAr), 127.2 (C2,6–Ar1), 126.8
(C2), 124.9 (C4–NAr), 123.6 (C5–NAr), 122.0 (C5–Ar),
120.8 (C3–Ar), 120.1 (C1–Ar), 118.8 (C6–NAr), 19.7 (–3–
CH3), 14.1 (–2–CH3); LCMS m/z [M]? 445.2; Anal. Calcd.
Ar
R2
Fig. 1 Entitled compounds labeled for 13C NMR
123

Med Chem Res
for C30H27N3O (445.55): C, 80.87; H, 6.11; N, 9.43.
Found: C, 80.74; H, 6.19; N, 9.51.
cm-1
;
1H NMR (DMSO-d6, 400 MHz,): d = 9.72 (bs,
1H, NH), 9.13 (bs, 1H, NH), 8.48 (s, 1H, OH), 8.18 (d,
1H, CH), 7.81 (d, 1H, CH), 6.82–7.62 (m, 15H, Ar–H),
2.37 (s, 3H, CH3), 2.26 (s, 3H, CH3); 13C NMR (DMSO-
d6, 100 MHz,): d = 172.6 (C=O), 159.6 (C4–Ar1), 153.2
(C=N), 147.4 (C2–Ar), 145.3 (C1–NAr), 139.2 (C3–NAr),
136.5 (C3), 135.2 (C4–Ar3), 134.9 (C1–Ar3), 134.1 (C4–
The other compounds 4b to 4j were prepared by the
same procedure using the corresponding chalcones.
2-(2,3-Dimethylphenylamino)-N0-(-3-(3-nitrophenyl)-1-
phenylallylidene)benzohydrazide (4b)
Ar), 132.2 (C2,6–Ar1), 130.4 (C3,5–Ar3), 129.8 (C2,6
–
Yield, 68 %; mp 140–142 °C; IR (KBr) tmax 3,326 (NH),
3,063 (NH) 2,974 (CH Ar.), 1,660 (C=O), 1,616 (C=O),
Ar3), 129.2 (C6–Ar), 128.6 (C1–Ar1), 127.5 (C2–NAr),
126.6 (C2), 124.9 (C4–NAr), 124.3 (C5–NAr), 123.2 (C5–
Ar), 121.3 (C3–Ar), 120.6 (C1–Ar), 119.5 (C6–NAr),
117.3 (C2,5–Ar1), 18.6 (–3–CH3), 13.2 (–2–CH3); LCMS
m/z [M]? 495.2; Anal. Calcd. for C30H26ClN3O2
(495.99): C, 72.65; H, 5.28; N, 8.47. Found: C, 72.54; H,
5.23; N, 8.59.
1
1,576 (CH=CH), 1,495 (ArꢀC=C), 1,346 (–NO2) cm-1; H
NMR (DMSO-d6, 400 MHz,): d = 9.62 (bs, 1H, NH), 8.97
(bs, 1H, NH), 8.03 (d, 1H, CH), 7.71 (d, 1H, CH),
6.71–7.64 (m, 16H, Ar–H), 2.38 (s, 3H, CH3), 2.18 (s, 3H,
CH3); 13C NMR (DMSO-d6, 100 MHz,): d = 170.2
(C=O), 151.5 (C=N), 149.1 (C3–Ar3), 148.9 (C2–Ar), 146.5
(C1–NAr), 137.7 (C1–Ar3, C3–NAr), 136.4 (C1–Ar1), 135.4
(C6–Ar3), 134.2 (C3), 134.0 (C4–Ar), 131.2 (C5–Ar3),
129.5 (C3,5–Ar1), 128.6 (C6–Ar), 128.2 (C4–Ar1), 127.6
(C2–NAr), 127.5 (C2,6–Ar1), 126.4 (C2), 125.5 (C4–Ar3),
124.6 (C4–NAr), 124.2 (C5–NAr), 123.9 (C2–Ar3), 122.7
(C5–Ar), 121.6 (C3–Ar), 120.1 (C1–Ar), 119.0 (C6–NAr),
21.6 (–3–CH3), 15.8 (–2–CH3); LCMS m/z [M]? 490.2;
Anal. Calcd. for C30H26N4O3 (490.55): C, 73.45; H, 5.34;
N, 11.42. Found: C, 73.28; H, 5.46; N, 11.38.
N0-(3-(2-Chlorophenyl)-1-(4-hydroxyphenyl)allylidene)-2-
(2,3-dimethylphenylamino)benzohydrazide (4e)
Yield, 67 %; mp 194–196 °C; IR (KBr) tmax 3,309 (NH),
2,923 (OH), 1,646 (C=O), 1,633 (C=N), 1,575 (CH=CH),
1
1,436 (ArꢀC=C), 763 (C–Cl) cm-1; H NMR (DMSO-d6,
400 MHz,): d = 9.69 (bs, 1H, NH), 9.18 (bs, 1H, NH),
8.54 (s, 1H, OH), 8.08 (d, 1H, CH), 7.79 (d, 1H, CH),
6.78–7.53 (m, 15H, Ar–H), 2.32 (s, 3H, CH3), 2.28 (s, 3H,
CH3); 13C NMR (DMSO-d6, 100 MHz,): d = 170.1
(C=O), 160.2 (C4–Ar1), 152.5 (C=N), 148.9 (C2–Ar),
148.3 (C1–NAr), 139.9 (C3–NAr), 137.10 (C3), 136.10
(C4–Ar), 134.2 (C2–Ar3), 133.7 (C1–Ar3), 132.06 (C3–
Ar3), 131.3 (C2,6–Ar1), 130.1 (C4–Ar3), 129.6 (C6–Ar3),
128.4 (C6–Ar), 127.6 (C1–Ar1), 127.5 (C1–NAr), 127.3
(C2), 127.2 (C5–Ar3), 125.6 (C4–NAr), 124.8 (C5–NAr),
123.4 (C5–Ar), 122.8 (C3–Ar), 121.9 (C1–Ar), 119.0 (C6–
NAr), 116.3 (C3,5–Ar1), 20.2 (-3–CH3), 14.5 (–2–CH3);
LCMS m/z [M]? 495.2; Anal. Calcd. for C30H26ClN3O2
(495.99): C, 72.65; H, 5.28; N, 8.47. Found: C, 72.57; H,
5.19; N, 8.54.
N0-(3-(4-Chlorophenyl)-1-phenylallylidene)-2-(2,3-
dimethylphenylamino)benzohydrazide (4c)
Yield, 59 %; mp 184–186 °C; IR (KBr) tmax 3,308 (NH),
2,914 (CH Ar.), 1,645 (C=O), 1,624 (C=N), 1,574
1
(CH=CH), 1,436 (Ar. C=C), 751 (C–Cl) cm-1; H NMR
(DMSO-d6, 400 MHz,): d = 9.57 (bs, 1H, NH), 9.05 (bs,
1H, NH), 8.13 (d, 1H, CH), 7.76 (d, 1H, CH), 6.78–7.58
(m, 16H, Ar–H), 2.29 (s, 3H, CH3), 2.22 (s, 3H, CH3); 13
C
NMR (DMSO-d6, 100 MHz,): d = 168.1 (C=O), 152.5
(C=N), 149.6 (C2–Ar), 147.4 (C1–NAr), 139.6 (C3–NAr),
138.8 (C1–Ar1), 137.7 (C3), 136.4 (C4–Ar3), 135.2 (C1–
Ar3), 134.1 (C4–Ar), 132.3 (C3,5–Ar3), 130.1 (C2,6–Ar3),
129.4 (C3,5–Ar1), 128.6 (C6–Ar), 128.3 (C4–Ar1), 127.6
(C2–NAr), 127.5 (C2,6–Ar1), 126.6 (C2), 125.6 (C4–NAr),
124.9 (C5–NAr), 122.6 (C5–Ar), 121.5 (C3–Ar), 120.3 (C1–
Ar), 119.4 (C6–NAr), 20.4 (–3–CH3), 14.5 (–2–CH3);
LCMS m/z [M]? 479.2; Anal. Calcd. for C30H26ClN3O
(479.99): C, 75.07; H, 5.46; N, 8.75. Found: C, 75.17; H,
5.53; N, 8.69.
N0-(3-(4-(Dimethylamino)phenyl)-1-(4-
hydroxyphenyl)allylidene)-2-(2,3-
dimethylphenylamino)benzohydrazide (4f)
Yield, 56 %; mp 144–146 °C; IR (KBr) tmax 3,396 (NH),
3,308 (NH), 3,063 (CH Ar.), 1,652 (C=O), 1,618 (C=N),
1
1,575 (CH=CH), 1,436 (ArꢀC=C), 1,362 (C–N) cm-1; H
NMR (DMSO-d6, 400 MHz,): d = 9.64 (bs, 1H, NH), 9.21
(bs, 1H, NH), 8.47 (s, 1H, OH), 8.09 (d, 1H, CH), 7.83 (d,
1H, CH), 6.79–7.58 (m, 15H, Ar–H), 2.87 (s, 6H, CH3),
2.32 (s, 3H, CH3), 2.19 (s, 3H, CH3); 13C NMR (DMSO-d6,
100 MHz,): d = 169.3 (C=O), 159.8 (C4–Ar1), 153.1
(C=N), 152.4 (C4–Ar3), 148.4 (C2–Ar), 146.5 (C1–NAr),
N0-(3-(4-Chlorophenyl)-1-(4-hydroxyphenyl)allylidene)-2-
(2,3-dimethylphenylamino)benzohydrazide (4d)
Yield, 62 %; mp 162–166 °C; IR (KBr) tmax 3,145
(OH), 3,023 (NH), 1,648 (C=O), 1,621 (C=N), 1,557
(CH=CH), 1,522 (ArꢀC=C), 1,354 (C–N), 725 (C–Cl)
138.9 (C3–NAr), 137.6 (C3), 135.1 (C4–Ar), 133.4 (C2,6
–
Ar1), 131.9 (C2,6–Ar3), 129.4 (C6–Ar), 128.6 (C1–Ar1),
123

Med Chem Res
N0-(3-(4-Chlorophenyl)-1-(4-fluorophenyl)allylidene)-2-
127.8 (C2–NAr), 126.9 (C2), 125.6 (C4–NAr), 124.7 (C5–
NAr), 123.7 (C1–Ar3), 122.0 (C5–Ar), 121.5 (C3–Ar),
120.5 (C1–Ar), 119.3 (C6–NAr), 115.3 (C3,5–Ar1), 113.7
(C3,5–Ar3), 42.9 (–N(CH3)2), 19.5 (–3–CH3), 15.3 (–2–
CH3); LCMS m/z [M]? 504.3; Anal. Calcd. for
C32H32N4O2 (504.62): C, 76.16; H, 6.39; N, 11.10. Found:
C, 76.28; H, 6.31; N, 11.02.
(2,3-dimethylphenylamino)benzohydrazide (4i)
Yield, 58 %; mp 134–136 °C; IR (KBr) tmax 3,343 (NH),
3,064 (CH Ar.), 1,652 (C=O), 1,623 (C=N), 1,567
1
(CH=CH), 1,489 (ArꢀC=C), 1,011 (C–F) cm-1; H NMR
(DMSO-d6, 400 MHz,): d = 9.63 (bs, 1H, NH), 9.18 (bs,
1H, NH), 8.49 (s, 1H, OH), 8.22 (d, 1H, CH), 7.83 (d, 1H,
CH), 6.67–7.59 (m, 15H, Ar–H), 2.34 (s, 3H, CH3), 2.17 (s,
3H, CH3); 13C NMR (DMSO-d6, 100 MHz,): d = 169.8
(C=O), 160.0 (C4–Ar1), 150.5 (C=N), 146.4 (C2–Ar), 145.3
(C1–NAr), 137.9 (C3–NAr), 136.6 (C3), 135.8 (C4–Ar3),
135.1 (C1–Ar3), 134.2 (C1–Ar1), 134.1 (C4–Ar), 131.0
(C2,6–Ar1), 129.3 (C3,5–Ar1), 129.1 (C2,6–Ar1), 128.4 (C6–
Ar), 127.5 (C2–NAr), 126.6 (C2), 124.9 (C4–NAr), 124.6
(C5–NAr), 122.0 (C5–Ar), 121.6 (C3–Ar), 120.1 (C1–Ar),
118.7 (C6–NAr), 115.2 (C3,5–Ar1), 19.9 (–3–CH3), 16.2
(–2–CH3); LCMS m/z [M]? 497.2; Anal. Calcd. for
C30H25ClFN3O (497.16): C, 72.36; H, 5.06; N, 8.44.
Found: C, 72.51; H, 4.97; N, 8.37.
2-(2,3-Dimethylphenylamino)-N0-(1-(4-fluorophenyl)-3-(4-
hydroxyphenyl)allylidene)benzohydrazide (4g)
Yield, 63 %; mp 156–158 °C; IR (KBr) tmax 3,312 (NH),
3,033 (NH), 1,686 (C=O), 1,626 (C=N), 1,574 (CH=CH),
1
1,461 (ArꢀC=C) cm-1; H NMR (DMSO-d6, 400 MHz,):
d = 9.76 (bs, 1H, NH), 9.18 (bs, 1H, NH), 8.62 (s, 1H,
OH), 8.23 (d, 1H, CH), 7.84 (d, 1H, CH), 6.75–7.58 (m,
15H, Ar–H), 2.33 (s, 3H, CH3), 2.18 (s, 3H, CH3); 13C
NMR (DMSO-d6, 100 MHz,): d = 171.8 (C=O), 161.0
(C4–Ar1), 160.3 (C4–Ar3), 154.2 (C=N), 149.9 (C2–Ar),
145.3 (C1–NAr), 139.8 (C3–NAr), 138.6 (C3), 135.1 (C1–
Ar1), 134.1 (C4–Ar), 132.3 (C2,6–Ar1), 130.9 (C2,6–Ar3),
129.6 (C6–Ar), 128.6 (C1–Ar3), 128.5 (C2–NAr), 126.9
(C2), 125.3 (C4–NAr), 124.2 (C5–NAr), 122.0 (C5–Ar),
121.3 (C3–Ar), 120.6 (C1–Ar), 119.9 (C6–NAr), 117.7
(C3,5–Ar3), 116.3 (C3,5–Ar1), 21.4 (–3–CH3), 13.8 (–2–
CH3); LCMS m/z [M]? 469.2; Anal. Calcd. for
C30H26FN3O2 (479.54): C, 75.14; H, 5.46; N, 8.76. Found:
C, 75.03; H, 5.57; N, 8.69.
2-(2,3-Dimethylphenylamino)-N0-(1-(4-fluorophenyl)-3-(4-
nitrophenyl)allylidene)benzohydrazide (4j)
Yield, 66 %; mp 126–128 °C; IR (KBr) tmax 3,308 (NH),
1,628 (C=N), 1,660 (C=O), 1,575 (CH=CH), 1,506 (–NO2),
1
1,436 (ArꢀC=C) cm-1; H NMR (DMSO-d6, 400 MHz,):
d = 9.82 (bs, 1H, NH), 9.23 (bs, 1H, NH), 8.22 (d, 1H,
CH), 7.78 (d, 1H, CH), 6.69–7.54 (m, 15H, Ar–H), 2.38 (s,
3H, CH3), 2.23 (s, 3H, CH3); 13C NMR (DMSO-d6,
100 MHz,): d = 170.4 (C=O), 159.6 (C4–Ar1), 151.2
(C=N), 148.8 (C4–Ar3), 146.9 (C2–Ar), 146.2 (C1–NAr),
143.4 (C1–Ar3), 138.8 (C3–NAr), 137.5 (C3), 135.1 (C1–
Ar1), 134.9 (C4–Ar), 130.8 (C2,6–Ar1), 129.7 (C2,6–Ar3),
128.8 (C6–Ar), 127.6 (C2–NAr), 126.8 (C2), 125.3 (C4–
NAr), 124.9 (C5–NAr), 124.4 (C3,5–Ar3), 123.7 (C5–Ar),
121.3 (C3–Ar), 120.8 (C1–Ar), 119.1 (C6–NAr), 116.2
(C3,5–Ar1), 20.6 (–3–CH3), 15.6 (–2–CH3); LCMS m/z
[M]? 508.2; Anal. Calcd. for C30H25ClFN4O3 (508.54): C,
70.85; H, 4.96; N, 11.02. Found: C, 70.93; H, 4.89; N,
10.89.
N0-(3-(4-(Dimethylamino)phenyl)-1-(4-fluorophenyl)
allylidene)-2-(2,3-dimethylphenylamino)benzohydrazide
(4h)
Yield, 62 %; mp 152–156 °C; IR (KBr) tmax 3,308 (NH),
3,063 (CH Ar.), 1,645 (C=O), 1,611 (C=N), 1,573
1
(CH=CH), 1,504 (ArꢀC=C), 1,328 (C–N) cm-1; H NMR
(DMSO-d6, 400 MHz,): d = 9.76 (bs, 1H, NH), 9.23 (bs,
1H, NH), 8.57 (s, 1H, OH), 8.13 (d, 1H, CH), 7.79 (d, 1H,
CH), 6.82–7.65 (m, 15H, Ar–H), 2.82 (s, 6H, CH3), 2.39 (s,
3H, CH3), 2.21 (s, 3H, CH3); 13C NMR (DMSO-d6,
100 MHz,): d = 170.3 (C=O), 160.8 (C4–Ar1), 153.1 (C4–
Ar3), 152.5(C=N), 146.4 (C2–Ar), 146.1 (C1–NAr), 139.2
(C3–NAr), 138.5 (C3), 135.4 (C1–Ar1), 134.8 (C4–Ar),
131.3 (C2,6–Ar1), 130.2 (C3,5–Ar3), 129.4 (C6–Ar), 127.5
(C2–NAr), 126.6 (C2), 125.9 (C4–NAr), 124.6 (C5–NAr),
123.9 (C1–Ar3), 122.5 (C5–Ar), 120.9 (C3–Ar), 120.2 (C1–
Ar), 119.3 (C6–NAr), 115.8 (C3,5–Ar1), 112.6 (C3,5–Ar3),
42.9 (–N(CH3)2), 19.77 (–3–CH3), 14.2 (–2–CH3); LCMS
m/z [M]? 506.2; Anal. Calcd. for C32H31FN4O (506.61):
C, 75.87; H, 6.17; N, 11.06. Found: C, 75.73; H, 6.24; N,
11.14.
Pharmacology
Animals
Wistar rats weighing in the range 200–250 g were obtained
from BN College of Pharmacy, Udaipur, India. All the
animals were housed under standard ambient conditions of
temperature (25 2 °C) and relative humidity of
50 5 %. A 12:12 h light/dark cycle was maintained. All
the animals were allowed to have free access to water and
123

Med Chem Res
standard palletized laboratory animal diet 24 h prior to
pharmacological studies. All the experimental procedures
and protocols used in this study were reviewed and
approved by the Institutional Animal Ethical Committee
(IAEC) of College, constituted in accordance with the
guidelines of the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), Gov-
ernment of India. The protocol of the study was approved
by the Institutional Animal Ethical Committee (Ref. No.
99/LSC/BNCP-012/IAEC).
animals of the other group were pretreated with the test
drugs, 1 h before the carrageenan treatment. The volume
was measured before and after carrageenan treatment at the
1, 2, 3 and 4 h intervals with the help of plethysmometer.
Acute ulcerogenesis
The acute ulcerogenesis test was performed according to
the literature method (Cioli et al., 1979). Albino rats have
been divided into different groups consisting of six animals
in each group. Ulcerogenic activity was evaluated after p.o.
administration of test compounds or standard at the dose of
30 mg/kg. Control rats received p.o. administration of
vehicle (suspension of 1 % carboxy methyl cellulose).
Food but not water was removed 24 h before administra-
tion of the test compounds. After the drug treatment, the
rats were fed a normal diet for 17 h and then killed. The
stomach was removed and opened along the greater cur-
vature, washed with distilled water, and cleaned gently by
dipping in saline. The mucosal damage was examined by
means of a magnifying glass. For each stomach, the
mucosal damage was assessed according to the following
scoring system:
Preparation of test compounds
After suspending the test compounds in 1.0 % aqueous
solution of sodium carboxymethyl cellulose (CMC), test
samples were administered to test animals orally. The
positive and negative control group animals received the
same experimental handling as those of the test groups
except that the drug treatment, and control group animals
received only appropriate volumes of the vehicle and of the
reference drug, and mefenamic acid, respectively.
Analgesic activity
0.0 scores were given to normal stomach (no injury,
bleeding and latent injury).
Prescreened animals (reaction time: 3–4 s) of either sex
were assigned to seven groups of six each. Mefenamic acid
was used as a standard; 1 % CMC was used as a control.
Tail-flick latency was assessed with an analgesiometer. The
strength of the current passing through the naked nichrome
wire was kept constant at 6 amps. The reaction time was
recorded at 30 min, 1 and 2 h after the treatment, and the
cutoff time was set at 10 s to avoid tissue damage. The
difference in reaction time (sec) was calculated by com-
paring the test compounds/standard drug and the normal
controls. The % relative analgesic activity was calculated
by using the following formula:
0.5 scores were to latent injury or widespread bleeding
([2 mm).
1.0 was to slight injury (2–3 dotted lines).
2.0 for severe injury (continuous lined injury or 5–6
dotted injuries).
3.0 for very severe injury (several continuous lined
injuries) and
4.0 for widespread lined injury or widened injury.
The mean score of each treated group minus the mean
score of the control group was regarded as severity index of
gastric mucosal damage.
% Relative analgesic activity ¼ ðDRTtest=DRTstdÞ ꢁ 100
Data are expressed as mean SEM; Data analyzed by
one-way ANOVA followed by Dunnett’s test, the signifi-
cance of the difference between the control group and rats
treated with the test compounds. The difference in results
was considered significant when P \ 0.01.
where DRTtest is the difference in reaction time of the test
compound with respect to (w.r.t.) the control, and DRTstd is
the difference in reaction time of the standard drug used
w.r.t control (Sharma et al., 2009).
Statistical analysis
Anti-inflammatory activity
The results are expressed as the mean SEM per group,
and the data were statistically analyzed by one-way ana-
lysis of variance (ANOVA) followed by Dunnett’s test as a
post hoc test. P value \0.05 was considered statistically
significant. All statistical calculations were performed
using the evaluation version of Graph PadÒ Prism 3.0
(USA) statistical software.
Anti-inflammatory activity was evaluated using the well-
known carrageenan-induced rat paw edema model of
Winter et al. 30 using groups of six animals each. A freshly
prepared aqueous suspension of carrageenan (1.0 % w/v,
0.1 ml) was injected in the subplanter region of right hind
paw of each rat. One group was kept as control, and the
123

Med Chem Res
application to the prediction of drug transport properties. J Med
Chem 43:3714–3717
Computational studies
Gokc¸e M, Utku S, Kupeli E (2009) Synthesis and analgesic and anti-
inflammatory activities 6-substituted-3(2H)-pyridazinone-2-
acetyl-2-(p-substituted/nonsubstituted benzal) hydrazone deriva-
tives. Eur J Med Chem 44:3760–3764
Kam PCA, So A (2009) COX-3: uncertainties and controversies.
Current Anaesth Crit Care 20:50–53
Khan MSY, Akhter M (2005) Glyceride derivatives as potential
prodrugs: synthesis, biological activity and kinetic studies of
glyceride derivatives of mefenamic acid. Die Pharmazie Int J
Pharm Sci 60(2):110–114
Calculation of physicochemical parameters
In-silico study of synthesized compounds (4a–4j) was per-
formed for prediction of ADME properties. Polar surface
area (TPSA) and molecular volume were calculated online
using Molinspiration. Absorption (%ABS) was calculated
by: %ABS = 109 - (0.345 9 TPSA) (Zhao et al., 2002).
Kim YH, Kim J, Park H, Kim HP (2007) Anti-inflammatory activity
of the synthetic chalcone derivatives: inhibition of inducible
nitric oxide synthase-catalyzed nitric oxide production from
lipopolysaccharide-treated RAW 264.7 cells. Biol Pharm Bull
30:1450–1455
Mitchell J, Warner T (1999) Cyclo-oxygenase-2: pharmacology,
physiology, biochemistry and relevance to NSAID therapy. Br J
Pharmacol 128:1121–1132
Acknowledgments The authors deeply appreciate the assistance of
the Department of Pharmacology, B N College of Pharmacy, Udaipur,
India, in the biological screening of the compounds. The authors also
deeply appreciate the Punjab University, Punjab, India, for providing
the spectral studies.
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