4-Fluorophenylhydrazones as potential COX-2 inhibitors: A novel, efficient, one pot solid phase synthesis, docking study and pharmacological evaluation

Article abstract of DOI:10.1007/s00044-013-0566-8

In search of a new class of organic compounds as potential COX-2 inhibitors, various 4-Fluorophenylhydrazones (3a-3i) have been synthesized and molecular docking study was conducted. All the synthesized compounds were also evaluated for their in vivo anti-inflammatory potential using carrageenan-induced rat paw odema method. In the present manuscript, a novel, simple, and greener protocol has been developed for the first time to prepare the hydrazo compounds by a one pot solid phase reaction between various active methylene compounds and p-fluoroaniline in the presence of p-toluene sulfonic acid as a new solid phase organocatalyst. The catalyst dramatically facilitates the reaction under solvent-free condition at moderate temperature (10-15 C). The present protocol not only provides an expeditious route to prepare hydrazo compounds in excellent yields (with in 3-5 min) but also avoids the use of two step conventional methods, and formation of side products. The results obtained from in vivo anti-inflammatory activity through carrageenan-induced rat paw odema assay showed that compounds 3a-3b, and 3d displayed excellent level of activity which was further supported by molecular docking study. A cyclooxygenase-II inhibitory molecular docking study has been carried out using (pdb: 1CX2) via Molegro Virtual Docker version 4.2.1. All the compounds were found to exhibit good level of inhibition and binding in the enzyme active site. Compounds 3a-3b, 3d, and 3e have been found to display high moldock scores -118.333, -118.778, -118.422, and -111.13, respectively, and are strongly bound with Arg120, Tyr355, His90, and Arg513 amino acids, which are responsible for COX-2 inhibition within the active site. In the present investigation, it can be concluded that the best scored inhibitors with good in vivo anti-inflammtory activity will have better chances to be used as anti-inflammatory leads.

Full text of DOI:10.1007/s00044-013-0566-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0566-8
ORIGINAL RESEARCH
4-Fluorophenylhydrazones as potential COX-2 inhibitors: a novel,
efficient, one pot solid phase synthesis, docking study
and pharmacological evaluation
•
Vinod Kumar Girish Kumar Gupta
•
•
Kamalneet Kaur Randhir Singh
Received: 27 November 2012 / Accepted: 27 February 2013
Ó Springer Science+Business Media New York 2013
Abstract In search of a new class of organic compounds as
potential COX-2 inhibitors, various 4-Fluorophenylhydraz-
ones (3a–3i) have been synthesized and molecular docking
study was conducted. All the synthesized compounds were
also evaluated for their in vivo anti-inflammatory potential
using carrageenan-induced rat paw odema method. In the
present manuscript, a novel, simple, and greener protocol has
been developed for the first time to prepare the hydrazo
compounds by a one pot solid phase reaction between vari-
ous active methylene compounds and p-fluoroaniline in the
presence of p-toluene sulfonic acid as a new solid phase
organocatalyst. The catalyst dramatically facilitates the
reaction under solvent-free condition at moderate tempera-
ture (10–15 °C). The present protocol not only provides an
expeditious route to prepare hydrazo compounds in excellent
yields (with in 3–5 min) but also avoids the use of two step
conventional methods, and formation of side products. The
results obtained from in vivo anti-inflammatory activity
through carrageenan-induced rat paw odema assay showed
that compounds 3a–3b, and 3d displayed excellent level of
activity which was further supported by molecular docking
study. A cyclooxygenase-II inhibitory molecular docking
study has been carried out using (pdb: 1CX2) via Molegro
Virtual Docker version 4.2.1. All the compounds were found
to exhibit good level of inhibition and binding in the enzyme
active site. Compounds 3a–3b, 3d, and 3e have been found to
display high moldock scores -118.333, -118.778,
-118.422, and -111.13, respectively, and are strongly
bound with Arg120, Tyr355, His90, and Arg513 amino
acids, which are responsible for COX-2 inhibition within the
active site. In the present investigation, it can be concluded
that the best scored inhibitors with good in vivo anti-
inflammtory activity will have better chances to be used as
anti-inflammatory leads.
Keywords COX-2 inhibitors ꢀ Docking study ꢀ
Anti-inflammatory activity ꢀ 4-Fluorophenylhydrazones ꢀ
Solid phase synthesis ꢀ p-Toluene sulfonic acid
Introduction
Fast and effective relief from pain and inflammation in the
human being has continued to be a major challenge for the
medicinal chemists. Various inflammatory diseases are
currently treated with steroidal and non-steroidal anti
inflammatory drugs (NSAIDs) (Moeser, 1991). The mech-
anism of action of NSAIDs is to decrease prostaglandin (PG)
synthesis through the inhibition of cyclooxygenase (COX), a
key enzyme in PG biosynthesis that catalyzes the conversion
of arachidonic acid into PG and thromboxane (Khanna et al.,
2000). Conventional NSAIDs that non-selectively inhibit the
major cyclooxygenase isoforms, COX-1 and COX-2, are
widely used to treat symptoms of inflammation, particularly
arthritic pain (Dannhardt and Laufer, 2000; Carter, 2000;
Tally, 1999; Hla and Neilson, 1992). COX-1, the constitutive
isoform, is mainly responsible for the synthesis of
V. Kumar (&) ꢀ K. Kaur
Department of Chemistry, Maharishi Markandeshwar
University, Mullana, Ambala 133203, India
e-mail: vinodbatan@gmail.com
G. K. Gupta
Department of Pharmaceutical Chemistry, Maharishi
Markandeshwar College of Pharmacy, Maharishi
Markandeshwar University, Mullana, Ambala 133203, India
R. Singh
Department of Pharmacology, Maharishi Markandeshwar
College of Pharmacy, Maharishi Markandeshwar University,
Mullana, Ambala 133203, India
123

Med Chem Res
cytoprotective PGs in gastrointestinal (GI) tract, whereas
COX-2 is inducible and plays a major role in PG biosynthesis
in inflammatory cells (Marnett and Kalgutkar, 1999; Dann-
hardt and Kiefer, 2001; Almansa et al., 2003). It is believed
that the inhibition of COX-1 causes unfavorable GI side
effects (Mitchell et al., 1993). Though the selective COX-2
inhibitors (coxibs) with better safety profile have been
marketed as a new generation of NSAIDs, a careful pro-
spective evaluation of coxibs has revealed some unexpected
adverse effects (Kalgutkar, 1999; Tally et al., 2000). The two
main adverse drug reactions (ADRs) associated with NSA-
IDs are related to the GI and renal effects (kidney failure) of
the agents (Kulkarni and Varghese, 1998; Kulkarni et al.,
2000). Moreover, they have also shown some unexpected
cardiovascular adverse effects (Donge et al., 2005). There-
fore, development of some novel compounds having anti
inflammatory activity with an improved safety profile is still
a necessity.
harmful solvents. Solvent-free reactions represent an ideal
green chemical technological procedure from both eco-
nomical and environmental point of view. Also, in recent
times, solid phase acid-catalyzed organic reactions are
gaining much popularity due to proven advantage of het-
erogeneous catalyst such as simplified product isolation,
mild reaction conditions, high selectivity, and reduction in
generation of byproducts (Clark, 2002).
In search of new potential COX-2 inhibitors and in
continuation of our work related to the new synthetic
approaches for important organic intermediate and het-
erocycles, herein we report the study which was undertaken
to explore the usefulness of p-toluene sulfonic acid (PTSA)
as a solid phase organocatalyst for diazotization of
p-fluoroaniline and coupling of various active methylene
compounds under one pot solvent-free conditions and to
explore the potential of 4-fluorophenylhydrazones (3a–3i)
to inhibit COX-2 activity via molecular docking study and
in vivo anti-inflammatory activity. The present study not
only provides a simple and efficient protocol in terms of
mild reaction conditions, clean reaction profile, and easy
work up procedure but also discloses a new class of anti-
inflammatory agents.
As an important carbon–nitrogen bond forming reaction
in organic synthesis, diazotization of anilines (Bercean et al.,
2010) with active methylene compounds (unsaturated
b-diketones, malononitrile, etc.) has been commonly
employed in the synthesis of numerous speciality chemicals
and heterocycles such as pyrazoles, isoxazoles, aminopy-
razoles, pyrazolopyridines, pyrazolopyrimidines, and thia-
zoles, which, themselves, have significant biological
activities (Singh et al., 2004; Heller and Natarajan, 2006;
Kumar et al., 2006; Emelina et al., 2007; Deng and Mani,
2008). The chemistry of aliphatic and aromatic diazo com-
pounds has been examined in a number of reviews; however,
considerably less attention has been directed to their fluori-
nated heterocyclic analogs. These heterocyclic compounds
exhibit antimicrobial, anti-inflammatory, anticancer, cyto-
toxic, analgesic, anti-HIV, anti-malarial, and antimycobac-
terial activity (Kucukguzel et al., 1999; Cunha et al., 2002;
Savini et al., 2004; Sondhi et al., 2006; Tonelli et al., 2008).
A liquid phase diazotization in which nitrous acid is gener-
ated in situ at 0–5 °C is a well-known process (Furniss et al.,
2008), but its main limitation is environmental compatibil-
ity, because nowadays, the prohibition of environmental
pollution is the main issue in the global world (Clark, 1995).
Recently, hydrazo compounds have been prepared by diaz-
otization using NO2 (Kaupp et al., 2003), ferric nitrate as an
oxidizing agent (Wang et al., 2000) via resin-supported
diazonium salts (Merrington et al.,2002), clay as catalyst
(Dabbagh et al., 2007), coupling in supercritical carbon
dioxide (Hooker et al., 2002), and ionic liquids (Lycka et al.,
2007) also. Most of these methods suffer from several
drawbacks such as a very low temperature range, use of
hazardous organic solvents, and expensive catalysts.
Results and discussion
Chemistry
As discussed earlier, hydrazo compounds are generally
synthesized in two steps, involving preparation of diazo-
nium ion and subsequent coupling reaction with active
methylene compounds. It is a well-known fact that unsat-
urated b-diketones in the presence of weak bases like
sodium acetate react readily with diazonium ion in water–
alcohol mixture to yield the desired products which are
formulated as monohydrazones along with the formation of
corresponding formazans, as side products (Kumar et al.,
2006). Herein, regioselective formation of hydrazo com-
pounds using PTSA at a moderate temperature range has
been described. The reaction proceeds smoothly without
the formation of any byproduct or rearranged product and
affords hydrazo compounds in excellent yields. PTSA is
commercially available, cheap, environmentally benign,
readily biodegradable, easy to handle, and is removed
easily from reaction mixture by washing with water. All
products were characterized on the basis of combined use
1
of IR, H & 13C NMR spectroscopy, mass spectrometry,
and by the comparison of their melting points with those
reported in previous reports (Sharma et al., 2005; Kumar
et al., 2006). Spectroscopic NMR data ensure the existence
of azo-hydrazone tautomerism in these compounds as
shown in Scheme 1.
As far as environmental safety is concerned, waste
should be either eliminated or minimized, the bulk wastes
in the manufacturing of chemicalsare often organic and
123

Med Chem Res
Molecular modeling and docking studies
Ar
N
Ar
N
To pre-access the anti-inflammatory behavior of the syn-
thesized 4-fluorophenylhydrazones (3a–3i) on structural
basis, automated docking studies were carried out using
Molegro Virtual Docker version 4.2.1 program. The scoring
function and a number of hydrogen bondings formed with
the surrounding amino acids are used to predict their
binding modes, binding affinities; the scoring function also
depends on the orientation of the compounds in the active
sites of COX-2 enzyme. For this study, the protein–ligand
complex was constructed on the X-ray structure (PDB entry
1CX2) of COX-2 with its bound inhibitor SC558. The
scoring functions for all the compounds were calculated
from minimized ligand–protein complexes to compare the
binding affinities of synthesized compounds (3a–3i) from
docked values which are in conformity with the results
obtained from in vivo anti-inflammatory activity.
N
H
N
R'
R''
R'
R''
H
3
4
Scheme 1 Azo-hydrazone tautomerism
In a proposed mechanism as depicted in Scheme 2a,
PTSA, in the presence of sodium nitrite, is converted into
sodium p-toluene sulfonate and nitrous acid. The nitrous
acid formed in the reaction mixture subsequently reacts
with aniline, which leads to the formation of diazonium ion
which is further stabilized to a greater extent by the pres-
ence of sodium p-toluene sulfonate in reaction mixture
which helps the coupling reaction to occur easily at
10–15 °C (Scheme 2b). PTSA is also responsible for the
activation of methylene carbon in active methylene com-
pounds. Finally, nucleophilic addition of active methylene
compounds to diazonium salt forms the hydrazone (3).
Furthermore, appearance of moist nature of the reaction
mixture indicates the elimination of water while grinding.
The reaction is very rapid and the end point may be judged
by color change of reaction mixture.
The crystallographic and molecular surface visualization
studies revealed that the active site of COX-2 is composed
mostly of hydrophobic residues. There are three major
pockets (S1, S2, S3) and one shallow pocket (S4) on the
COX-2 active site wherein Pocket S1 consists of the amino
acid residues Ala516, Arg513, His90, Gln192, and the
backbone of Phe518, whereas Pocket S2 is localized at the
top of the channel and consists of several hydrophobic
amino acid residues: Tyr348, Tyt385, Phe518, Met522, and
Trp387. Pocket S3 is localized in the mouth of the active
site and is delimited by Leu359, Tyr355, Val116, and
Arg120. Pocket S4 shallow pocket is localized in a
It has been observed that each compound shows a
common mass fragment B and fragment A (Scheme 3) in
mass spectrum, which further confirms the formation of
3a–3i.
Scheme 2 a Proposed
mechanism of PTSA-catalyzed
R'
F
R''
F
a
H2N
F
C
diazotization followed by
NaNO2
H2
coupling. b Synthesis of
hydrazo compounds (3) using
-2 H2O
R'
O
+
OH
ONa
PTSA under solvent-free
conditions
N
N
N N
N
+
R''
OH
Reaction Mixture
R''
b
PTSA, NaNO2
R'
+
N
H2N
F
10-15 oC, 3-5 min.
R'
R''
1
N
F
2
3
Scheme 3 Common mass
fragmentation patterns observed
for compounds 3a–3i
R''
R'
+
+
N
N
N
N
F
R''
F
R'
-
N
N
C6H4-p-F
-R', -R''
CH
B
3a-i
c
A
a
b
c
d
e
a
b
d
e
+
m/z = 136
m/z = 293, 245, 260, 233, 221
m/z = M = 416, 368, 383, 356, 344
f
g
h
i
f
g
h
i
206, 99, 103, 65
329, 222, 226, 188
123

Med Chem Res
hydrophobic area close to the hydrophobic pocket S2
which has a lesser number of residues, including Leu534,
Leu531, and Ile345. It has previously been shown that
Arg513 is involved in a key hydrogen bond interaction
with most selective COX-2 inhibitors (Bauer et al., 2007;
Sanda et al., 2008; Zebardast et al., 2009; Bouaziz-Terra-
chet et al., 2010). It is important to note here that analysis
of crystal structure of COX-2 inhibitor complexes (Picot
et al., 1994; Loung et al., 1996; Chakraborti and Thilag-
avathi, 2004) reveals a network of hydrogen bonds
involving Arg120, Tyr355, His90, Arg513, Val523,
Ser353, Glu524, which are believed to act as gates for
ligand entrance to the COX active sites. This cavity also
contains a larger number of non-conserved residues
between the two isoenzymes; Thr89 (Val), Leu92 (Ile),
Val119 (Ser), and Leu357 (Phe). SC558, like other highly
selective inhibitors (Celecoxib, Indomethacin, Diclofenac),
exhibits a time-dependent mechanism in COX-2 and a
time-independent mechanism in COX-1. This ligand forms
a stable complex in the cavity of COX-2 with hydrogen
bonds pointing toward the side chains of Tyr355 and
Arg120 (Ermondi et al., 2004).
often provides optimum COX-2 selectivity and potency
(Tally, 1999). Recently, it has also been reported that
hydrazo and diones could also serve as useful probes to
study the function and catalytic activity of the COX-2
isoenzyme. (Magda et al., 2011; Moldovan et al., 2011).
In the present investigation, all compounds (3a–3i) were
embedded in the hydrophobic pocket formed by the amino
acids, Leu359, Arg120, His90, Arg513, Phe518, Val523,
Tyr385, Ala516, Gln192, Leu352, Ile517, and Ser353
(Table 1; Figs. 1, 2, 3, 4). A detailed study explains that the
compounds, 3a and 3b, form two hydrogen bonds each and
have Moldock scores -118.33 and -118.77, respectively.
Compound 3a showed one hydrogen bond between O of 1,
3-dione moiety and Arg120 and another between O of 1,
3-dione moiety and Tyr 355. However, 3b showed one
hydrogen bond between O of OCH3 moiety and His90 and
another between O of OCH3 moieties and Arg513. In case
of 3d, the Moldock score has been found to be -111.13
Extensive structural–activity relationship (SAR) studies
for a diarylheterocyclic class have been reported and it has
been disclosed that the presence of SO2NH2 or SO2CH3 or
F substituent at the para position of one of the aryl rings
Table 1 Results of docking analysis
Compounds MolDock No. of
score
No. of
H-bonding amino
interaction acids
Structural features
Fig. 1 1-(p-Bromophenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluo-
robutane-1,3-dione (3a) (Color figure online)
involved
3a
3b
3c
-118.333
-118.778
2
2
Arg120 1,3-dione
Tyr355 1,3-dione
His90
p-OCH3C6H4
Arg513 p-OCH3C6H4
Arg120 O of p-NO2C6H4
Arg513 O of p-NO2C6H4
Tyr355 1,3-dione
-64.2355 2
3d
3e
3f
-111.135
-118.422
3
1
Tyr355 1,3-dione
-86.8422 4
Arg513 O of 2-OCH3C6H4
Tyr355 N of
3-oxobutanamide
Arg120 O of
3-oxobutanamide
3g
3h
-101.875
-94.676
2
5
Tyr355 1,3-dione
Tyr355 1,3-dione
Phe518 OH of COOH
Ile517
Arg513 OH of COOH
His90 N of Diazo
Tyr355 N of Diazo
OH of COOH
Fig. 2 1-(p-Methoxyphenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluo-
robutane-1,3-dione (3b) (Color figure online)
3i
-87.1034 2
123

Med Chem Res
3-dione moiety further leads to increase in Moldock scores.
It is evident from the results that incorporation of an
electron-withdrawing (NO2) group as in 3c leads to
decrease of -64.24 in value of Moldock score, which in
turn, may lead to decrease in the anti-inflammatory activ-
ity; on the other hand, insertion of an electron-donating
group (OCH3) as in 3b leads to an increase in the value of
Moldock score (-118.78), which may increase anti-
inflammatory effect. It has also been noted that wherever
the methoxy group is present in the molecule, it plays an
important role to make interactions particularly with
Arg513 of the enzyme.
On the basis of the docking results obtained, it has been
concluded that all the compounds show some common
binding mode, i.e., hydrogen bonding in the binding
pockets of COX-2 enzyme (PDB Code 1CX2) when
compared with standard Celecoxib and Indomethacin and
SC558. Upon docking, Celecoxib shows hydrogen binding
with Arg120 and His90, whereas Indomethacin was docked
by showing binding with Arg120 (Llorens et al. 1999).
SC558 was docked by showing binding with His90,
Arg513, Gln192, and Ser353 Fig. 5. Compounds 3a–3i
expressed comparable docking results which, in turn,
indicate the excellent binding affinity of compounds to
COX-2 enzyme, particularly at those sites which are
responsible for the binding of three active compounds
namely, Celecoxib, Indomethacin, and SC558.
Fig. 3 1-(p-Fluorophenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluorobu-
tane-1,3-dione (3d) (Color figure online)
On the basis of these results, all compounds were sub-
jected to evaluate their anti-inflammatory activity in vivo.
Biological evaluation
In vivo anti-inflammatory activity
Fig. 4 1-(Thien-20-yl)-2-(p-fluorophenyldiazo)-4,4,4-trifluorobutane-
1,3-dione (3e) (Color figure online)
All the synthesized compounds were evaluated for their
in vivo anti-inflammatory activity in carrageenan-induced
paw edema method (El-gazzar et al., 2007; Rao et al.,
2007). Subcutaneous administration of carrageenan in sub-
plantar region of paw was used to induce inflammation in
rats and the effect was measured at the interval of 1, 2, and
3 h. The percentage inhibition of edema was measured in
context to control group, as depicted in Table 2. Carra-
geenan produced inflammation by acting as chemo attrac-
tant factor. In the 1 h of carrageenan administration,
histamine is released, whereas in 2 and 3 h, bradykinin and
prostaglandin are released, respectively. Test compounds
produced maximum percentage inhibition in the 3 h by
attenuating prostaglandin release.
which corresponds to the formation of three hydrogen
bonds between O of 1, 3-dione moiety and Tyr355 of
enzyme. Compound 3e formed only one hydrogen bond
with Tyr355. Moreover 3c, 3f–3i were also found to dis-
play good Moldock scores ranging from -64 to -101, and
it has been observed that most of the compounds form
strong hydrogen bondings with Tyr355, Arg120, His90,
Arg513, Phe518, and Ile517 Table 1.
It is quite worth mention here that most of the com-
pounds having 1, 3-diketone group possess high Moldock
scores as compared to others having nitrile, amide, and
carboxylic acid groups. From the molecular docking study,
it has also been concluded that dione moiety has very high
tendency to bind with active sites of an enzyme through
carbonyl oxygen specifically with Tyr355 and Arg120
amino acids. The presence of trifluoromethyl group in 1,
Most of the tested compounds have shown good results
as compared to the control. Among all, compounds 3a, 3b,
and 3d have shown potent anti-inflammatory activity
(Table 2).
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Med Chem Res
Fig. 5 Binding mode of
compound (SC558) into the
binding site of cyclooxygenase
2. It forms five hydrogen bonds
shown as green dotted lines
(Table 1) and has Moldock
score -153.93, showing two
hydrogen bonds between O of
SO2NH2 moiety and His90, and
Arg513 of distance 2.70 and
˚
3.53 A, respectively, and three
other hydrogen bonds between
N of SO2NH2 moiety and
Gln192, Leu352, and Ser353 of
distances 3.18, 3.09, and
˚
2.98 A, respectively (Color
figure online)
Table 2 Anti-inflammatory activity of compounds (3a–3i) against carrageenan-induced rat paw edema over 3 h
Compounda
Volume of odema(ml)b
1 h
2 h
3 h
Control
1.06
1.07
1.12
Standard
3a
0.91 0.04 (20.15)*
0.94 0.02 (17.32)*
0.96 0.04(15.43)*
1.07 0.02(5.05)
0.93 0.05(18.26)*
1.08 0.07(4.11)
1.12 0.05(0.33)
1.09 0.03(3.16)
1.11 0.02(1.28)
1.08 0.01(4.11)
0.82 0.02(30.36)**
0.82 0.05(30.36)*
0.86 0.04(26.62)*
1.05 0.03(8.86)
0.84 0.04(28.49)*
1.06 0.07(7.93)
1.07 0.03(7)
0.73 0.04(46.82)**
0.77 0.01(43.25)**
0.8 0.02(40.57)**
1.05 0.03(18.25)
0.78 0.05(42.35)**
1.06 0.06(17.35)*
1.09 0.02(14.67)*
1.07 0.01(16.46)*
1.1 0.01(13.78)
3b
3c
3d
3e
3f
3g
1.08 0.04(6.06)
1.09 0.04 (5.13)
1.06 0.03(7.93)
3h
3i
1.11 0.03(12.89)*
Values in parentheses (percentage anti-inflammatory activity, AI%)
** p \ 0.01(significant); * p \ 0.05. Values are compared with control group
a
Dose levels: test compounds (50 mg/kg bw), Diclofenac sodium (50 mg/kg bw)
b
N = 8, values are expressed as mean SEM and analyzed by ANOVA
Conclusion
present protocol not only furnishes the hydrazo com-
pounds exclusively but also avoids the formation of other
possible impurities. Results of molecular docking, reported
in the present study, indicate that some compounds show
common binding modes, i.e., hydrogen bonding in the
binding pockets of COX-2 enzyme (PDB Code 1CX2) as
compared to standard Celecoxib, Indomethacin, and
SC558. Most of the compounds in the series form strong
hydrogen bondings with Tyr355, Arg120, His90, Arg513,
Phe518, and Ile517 but compounds 3a, 3b, 3d, and 3e have
particularly been found to display high moldock scores -
118.333, -118.778, -118.422, and -111.13, respectively,
and have been shown tobe strongly bound with Arg120,
In conclusion, we have developed, for the first time, a
highly simple, expeditious, and facile method for the syn-
thesis of various hydrazo compounds, and all synthesized
compounds were evaluated for their anti-inflammatory
activity on the basis of molecular docking study and in vivo
anti-inflammatory activity was carried out by using carra-
geenan-induced paw odema assay. In this one pot protocol,
p-fluoroaniline was allowed to treat with various active
methylene compounds and sodium nitrite in the presence of
p-toluene sulfonic acid as a new organocatalyst under
solvent-free conditions at moderate temperature. The
123

Med Chem Res
4
Tyr355, His90, Arg513 amino acids, which are responsible
for COX-2 inhibition within the active site of the men-
tioned enzyme. So the compounds 3a, 3b, and 3d with
good in vivo activity can have better potential to be used as
anti-inflammatory leads in future.
(d, C-100, JCF = 3 Hz), 135.75 (C-10), 131.25 (C-2),
130.95 (C-20, 60), 129.16 (C-30, 50), 127.25 (C-40), 118.08
(d, C-200, 600, JCF = 8.25 Hz), 116.61 (q, C-4, JCF
290.25 Hz), 116.40 (d, C-300, 500, JCF = 23.25 Hz); 19F
NMR (CDCl3) dF: -71.2 (s, 3F, CF3), -114.1 (s, 1F, 400-
F); MS (m/z): 416 (M?) and 418 (M??2) in the ratio
showing typical bromine isotope profile (1:1), C16H9BrF4
N2O2 requires 416. Anal. calc.: N, 6.73 % (found: 6.54).
3
1
=
2
Experimental
1-(p-Methoxyphenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluo-
robutane-1,3-dione (3b) M.p. 120 °C [Lit. m.p. 120–
121 °C, Kumar et al., 2006]; IR (KBr, mmax, cm-1): 1690
(CO str.), 3214 (OH str.), 3319 (NH str.); 1H NMR
(CDCl3) dH: 3.88 (s, 3H, OCH3), 6.93 (d, 2H, 30, 50-H,
Jo = 8.7 Hz), 7.13–7.19 (m, 2H, 300, 500-H), 7.43–.48 (m,
2H, 200, 600-H), 7.65 (d, 2H, 20, 60-H, Jo = 8.7 Hz), 13.62
(bs, 1H, NH or OH, D2O exchangeable); 13C NMR
Melting points were determined in open capillaries in
electrical apparatus and are uncorrected. IR spectra were
recorded on a Buck Scientific IR M500 instrument, and 1H
and 13C NMR spectra were run on a Brucker instrument
at 300 and 75 MHz, respectively. Chemical shifts are
expressed in d-scale downfield from TMS as an internal
standard. 19F NMR spectra were run on DRX 300 and DPX
400 at 282 and 376 MHz, respectively, using CDCl3 as
solvent. The internal standard for 19F spectra was CFCl3,
setting the CFCl3 signal at d 0.00 ppm. Mass spectra were
recoded on orthogonal accelerating time of flight (TOF)
mass spectrometer. Compounds 1g–1i and 2 are commer-
cially available and were purchased from Aldrich. Fluori-
nated b-diketones 1a–1f were synthesized according to the
literature procedures (Reid and Calvin, 1950; Charles and
Reidel, 1967; Dilli and Robards, 1984; Uddin et al., 2003).
2
(CDCl3) dc: 189.92 (C-1), 176.35 (q, C-3, JCF = 33 Hz),
1
163.98 (C-40), 161.11(d, C-400, JCF = 246.75 Hz), 137.11
4
(d, C-100, JCF = 3 Hz), 131.32 (C-2), 131.21 (C-20, 60),
3
128.80 (C-10), 118.26 (d, C-200, 600, JCF = 8.25 Hz),
1
117.27 (q, C-4, JCF = 290.25 Hz), 116.45 (d, C-300, 500,
2JCF = 23.25 Hz), 113.61(C-30, 50), 55.51 (OCH3); 19F
NMR (CDCl3) dF: -71.2 (s, 3F, CF3), -114.4 (s, 1F, 400-
F); MS (m/z): 368 (M?), C17H12F4N2O3 requires 368.
Anal. calc.: N, 7.61 % (found: 7.49).
1-(p-Nitrophenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluorobu-
tane-1,3-dione (3c) M.p. 108–109 °C [Lit. m.p. 110 °C,
Kumar et al., 2006]; IR (KBr, mmax, cm-1): 1690 (CO str.),
One pot solid phase synthesis of hydrazo compounds
(3a–3i)
1
3214 (OH str.), and 3319 (NH str.); H NMR (CDCl3) dH:
General procedure
7.14–7.28 (m, 2H, 300, 500-H), 7.54–7.59 (m, 2H, 200, 600-H),
7.70 (d, 2H, 20, 60-H, Jo = 8.7 Hz), 8.31 (d, 2H, 30, 50-H,
Jo = 8.7 Hz), 13.62 (bs, 1H, NH or OH, D2O exchange-
able); 13C NMR (CDCl3) dc: 191.09 (C-1), 176.71 (q, C-3,
2JCF = 33 Hz), 161.52 (d, C-400, 1JCF = 247.5 Hz), 149.68
To a mixture of 4-fluoroaniline (10 mmol) and active
methylene compound (10 mmol) added p-toluene sulfonic
acid (20 mmol) in a pestle. The reaction mixture was
cooled to 10–15 °C and added sodium nitrite (12 mmol).
Resulting mixture immediately was allowed to grind vig-
orously with the help of mortar for appropriate time
(Table 3) at appropriate temperature. Added water (50 ml)
and the crystals separated were filtered, washed with water,
and dried. Crude product was recrystallized from ethanol if
necessary. M.p. and spectral data of compounds are given
below.
4
(C-40), 143.19 (C-10), 136.60 (d, C-100, JCF = 3 Hz),
131.14 (C-2), 128.82 (C-20, 60), 123.56 (C-30, 50), 119.18
3
(d, C-200, 600, JCF = 8.25 Hz), 117.25 (d, C-300, 500,
2JCF = 23.25 Hz), 117.21 (q, C-4, 1JCF = 290.25 Hz); 19
F
NMR (CDCl3) dF: -71 (s, 3F, CF3), -114.1 (s, 1F, 400-F).
MS (m/z): 383 (M?), C16H9F4N3O4 requires 383. Ana-
l.Calc.: N, 10.96 % (Found: 10.72).
1-(p-Fluorophenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluorobu-
tane-1,3-dione (3d) M.p. 97–98 °C [Lit. m.p. 98–99 °C,
Kumar et al., 2006]; IR (KBr, mmax, cm-1): 1690 (CO str.),
1-(p-Bromophenyl)-2-(p-fluorophenyldiazo)-4,4,4-trifluorobu-
tane-1,3-dione (3a) M.p. 102 °C [Lit. m.p. 101–102 °C,
Kumar et al., 2006]; IR (KBr, mmax, cm-1): 1690 cm-1
1
3214 (OH str.), and 3319 (NH str.); H NMR (CDCl3) dH:
1
(CO), 3214 cm-1 (OH str.), and 3319 cm-1 (NH str.); H
6.97–7.56 (m, 8H, p-FPh’-H, p-FPh’’-H), 14.02 (bs, 1H,
NH or OH, D2O exchangeable); 13C NMR (CDCl3) dc:
190.57 (C-1), 176.75 (q, C-3, JCF = 33 Hz), 165.80
NMR (CDCl3) dH: 7.16–7.68 (m, 8H, p-FPh00-H, p-BrPh0-
H), 14.19 (bs, 1H, NH or OH, D2O exchangeable); 13C
NMR (CDCl3) dc: 190.59 (C-1), 176.08 (q, C-3,
2
1
1
(d, C-40, JCF = 253.5 Hz), 161.39 (d, C-400, JCF
=
1
2JCF = 33 Hz), 160.91(d, C-400, JCF = 247.5 Hz), 136.18
4
246.75 Hz), 136.89 (d, C-100, JCF = 3 Hz), 133.75 (d,
123

Med Chem Res
Table 3 Reaction time and % yields
For compounds 1, 3
R00
R00
Reaction time (min.)
4
Yield (%)a
90
a
COCF3
O
Br
O
b
c
COCF3
COCF3
COCF3
4
4
4
88
92
91
H CO
3
O
O2N
O
d
F
O
e
f
COCF3
COCH3
5
5
89
92
S
O
NH
OCH
3
g
h
i
COCH3
COCH3
COOH
CN
3
5
4
94
87
92
COOH
CN
a
Isolated yields
4
C-10, JCF = 3.75 Hz), 132.46 (C-2), 131.16 (d, C-20, 60,
117.04 (d, C-200, 600, 3JCF = 8.35 Hz), 116.92 (d, C-300, 500,
2JCF = 23.25 Hz); 19F NMR (CDCl3) dF: -71 (s, 3F, CF3),
-114.1 (s, 1F, 400-F); MS (m/z): 344 (M?); C14H8F4N2O2S
requires 344. Anal.Calc.: N, 8.14 % (Found: 8.18).
3JCF = 9.0 Hz), 118.62 (d, C-200, 600, JCF = 8.25 Hz),
3
1
117.19 (q, C-4, JCF = 290.25 Hz), 116.94 (d, C-300, 500,
2JCF = 23.25 Hz), 115.46 (d, C-30, 50, JCF = 21.75 Hz);
2
19F NMR (CDCl3) dF: -71 (s, 3F, CF3), -106 (s, 1F, 40-F),
-115 (s, 1F, 400-F); MS (m/z): 356 (M?); C16H9F5N2O2
requires 356. Anal.Calc.: N, 7.86 % (Found: 7.76)
2-(p-Fluorophenyldiazo)-N-(2-methoxyphenyl)-3-oxobutana-
mide (3f) M.p. 134–135 °C, IR (KBr, mmax, cm-1): 1688
(CO str., amide), 1723 (CO str.), 3217 (OH str.), and 3316
(NH str.); 1H NMR (CDCl3) dH: 2.64 (s, 3H, CH3), 3.99 (s,
3H, OCH3), 6.99–7.49 (m, 8H), 11.75 (s, 1H), 14.85 (s,
1-(Thien-2-yl)-2-(p-fluorophenyldiazo)-4,4,4-trifluorobutane-
1,3-dione (3e) M.p. 90–92 °C, IR (KBr, mmax, cm-1): 1698
1
13
(CO str.), 3215 (OH str.), and 3319 (NH str.); H NMR
1H); C NMR (CDCl3) dc: 194.03 (C-3), 162.91(C-20),
1
(CDCl3) dH: 7.15–7.18 (m, 1H, 40H), 7.47–7.50 (m, 2H, 300,
500-H), 7.73–7.79 (m, 2H, 200, 600-H), 7.92–7.99 (m, 2H, 30, 50-
H), 13.85 (bs, 1H, NH or OH, D2O exchangeable); 13C NMR
161.55 (d, C-400, JCF = 246.25 Hz), 156.40 (C-1), 138.20
(C-100), 127.15 (C-2), 126.59 (C-40), 124.63 (C-10), 120.78
3
(C-60), 117.19 (d, C-200, 600, JCF = 8.35 Hz), 116.60 (C-
2
2
(CDCl3) dc: 181.61 (C-1), 177.04 (q, C-3, JCF = 33 Hz),
50), 114.29 (d, C-300, 500, JCF = 23.25 Hz), 110.39 (C-30),
1
161.23 (d, C-400, JCF = 246.75 Hz), 140.06 (C-20), 142.11
55.96 (OCH3), 26.02 (C-4); 19F NMR (CDCl3) dF: -114.1
(s, 1F, 400-F); MS (m/z): 329 (M?); C17H16FN3O3 requires
329. Anal.Calc.: N, 12.76 % (Found: 12.72).
4
(d, C-100, JCF = 3 Hz), 135.35 (C-2), [129.98, 128.40,
127.05 (C-30, 40 & 50)], 119.12 (q, C-4, 1JCF = 290.25 Hz),
123

Med Chem Res
3-(p-Fluorophenyldiazo)pentane-2,4-dione (3g) M.p. 105
–107 °C, IR (KBr, mmax, cm-1): 1722 (CO str.), 3211 (OH
str.), and 3313 (NH str.); 1H NMR (CDCl3) dH: 2.61 (s, 3H),
2.49 (s, 3H), 7.15–7.28 (m, 2H, 30, 50-H), 7.47–7.55 (m, 2H,
20, 60-H), 13.92 (bs, 1H); 13C NMR (CDCl3) dc: 198.01* (C-
paw volume. Paw volume was measured using digital ple-
thysmometer (model no. PTH707, Medicaid System, India).
Thirty minutes after administration of test compounds and
standard drug, 0.1 ml of 1 % w/v of carrageenan suspension
in normal saline was injected into subplanter region of the
left hind paw of all the rats. The initial paw volume was
measured within 30 s of the injection and remeasured again
1, 2, and 3 h after administration of carrageenan. The edema
was expressed as an increase in the volume of paw, and the
percentage of edema inhibition for each rat and each group
was obtained as follows:
1
2), 196.90* (C-4), 161.25 (d, C-40, JCF = 246.25 Hz),
3
137.87 (C-10), 133.26 (C-3), 117.70 (d, C-20, 60, JCF
=
8.25 Hz), 116.73 (d, C-30, 50, 2JCF = 23.25 Hz), 31.59* (C-1),
26.57* (C-2); 19F NMR (CDCl3) dF: -114.1 (s, 1F, 40-F); MS
(m/z): 222 (M?); C11H11FN2O2 requires 222. Anal.Calc.: N,
12.61 % (Found: 12.56). (Note-* indicates that signals are
interchangeable).
% Inhibition ¼ ðVt ꢁ V0Þ control ꢁ ðVt
ꢁ V0Þ tested compound=ðVt ꢁ V0Þ control
ꢂ 100;
2-(p-Fluorophenyldiazo)malonic acid (3h) M.p. 88–90 °C,
IR (KBr, mmax, cm-1): 1705 (CO), 3010 (b, OH), 3317
1
(NH); H NMR (CDCl3) dH: 7.13–7.27 (m, 2H, 30, 50-H),
where Vt is the volume of edema at specific time interval
and V0 is the volume of edema at zero time interval.
7.49–7.58 (m, 2H, 20, 60-H), 13.72 (bs, 1H), 10.96 (bs, 1H),
8.52 (bs, 1H); 13C NMR (CDCl3) dc: 163.29 (C-1, 3),
1
161.52 (d, C-40, JCF = 246.25 Hz), 140.25 (C-10), 120.04
Drug modeling studies
(C-2), 117.1 (d, C-20, 60, 3JCF = 8.25 Hz), 116.41 (d, C-30,
50, 2JCF = 23.25 Hz); 19F NMR (CDCl3) dF: -114.1 (s, 1F,
40-F); MS (m/z): 226 (M?); C9H7FN2O4 requires 226.
Anal.Calc.: N, 12.38 % (Found: 11.94).
All docking studies were performed using Molegro Virtual
Docker 4.2.1 Program; MVD docking is probably the most
accurate predictive tool of binding geometry today.
2-(p-Fluorophenyldiazo)malononitrile (3i) M.p. 174–
176 °C (Krystof et al., 2006), IR (KBr, mmax, cm-1): 3217
(NH), 2222 (CN); 1H NMR (CDCl3) dH: 7.11–7.23 (m, 2H,
30, 50-H), 7.49–7.53 (m, 2H, 20, 60-H), 14.02 (bs, 1H, NH or
OH, D2O exchangeable); 13C NMR (CDCl3) dc: 161.55 (d,
C-40, 1JCF = 246.25 Hz), 136.12 (C-10), 117.79 (d, C-20, 60,
Preparation of molecules
A set of nine derivatives to inhibit cyclooxygenase-2 was
compiled by us earlier. The molecules were built using
Maestro 8.5.207 or converted to 3D structure from the 2D
structure using LigPrep.Version 2.2. Chemdraw 3D struc-
tures were energetically minimized using Schrodinger
Macromodel Module and saved as MDL MolFile (H.mol2).
2
3JCF = 8.25 Hz), 117.08 (d, C-30, 50, JCF = 23.25 Hz),
112.09 (C-1, 3), 108.38 (C-2); 19F NMR (CDCl3) dF:
-114.1 (s, 1F, 40-F); MS (m/z): 188 (M?); C9H5FN4
requires 188. Anal.Calc.: N, 29.78 % (Found: 30.01).
Generation of ligand and enzyme structures
The crystal structure of target protein cyclooxygenase
(1CX2) is a COX-2 complexed with SC558 which was
Pharmacological assay
Carrageenan-induced rat paw edema assay
Male Wistar albino rats weighing 150–175 g were used for
the study. Animals were kept in the animal house under
12 h light and dark cycle and had free access to food and
ad-libitum. Food was withdrawn 16 h before the experi-
ment. Animals were randomly divided into different groups,
each consisting of eight rats. Group 1 was kept as control
and received tween 80 (95:5) only. Group 2 received the
standard drug diclofenac sodium at a dose of 50 mg/kg
body weight i.p., whereas other groups of rats were
administered the test compounds at a dose of 50 mg/kg
body weight orally. A mark was made on the left hind paw
just beyond the tibiotarsal articulation, so that, every time
the paw was dipped up to the fixed mark, to ensure constant
Fig. 6 Binding pocket of COX-2 (Color figure online)
123

Med Chem Res
Dannhardt G, Kiefer W (2001) Cyclooxygenase inhibitors—current
status and future prospect. Eur J Med Chem 36:109–126
Dannhardt G, Laufer S (2000) Structural approaches to explain the
selectivity of COX-2 inhibitors: is there a common pharmaco-
phore. Curr Med Chem 7:1101–1112
retrieved from the Protein Data Bank (http://www.rscb.
org/pdb/explore.do). All bound waters ligands and cofac-
tors were removed from the protein.
Deng X, Mani NS (2008) Base mediated reaction of hydrazones and
nitroolefins with a reversed regioselectivity: a novel synthesis of
1, 3, 4-trisubstituted pyrazoles. Org Lett 10:1307–1310
Docking using Molegro Virtual Docker 4.2.1 Program
For the COX-2 complex, pdb code 1CX2 was selected and
used for the docking study. The motif of the complex is shown
in Fig. 6. Three types of ligands were clarified as HEM (pro-
toporphyrin IX Fe [Heme], N-acetyl-D-glucosamine (NAG),
and SC558 (1-phenylsulfonamide-3-trifluoromethyl-5-bro-
mophenylpyrazole). Since SC558 seems to play a role of
inhibitor, the vicinity where the SC558 is situated should be an
active site. Therefore, SC558 was taken out of the pdb file of
this COX-2 complex and treated as the so-called model
inhibitor for the docking study.
Dilli S, Robards
K (1984) Spectroscopic properties and gas
chromatographic behavior of aryl b-diketones and selected
chelates. J Chromatogr A 312:109–120
Donge JM, Supuran CT, Pratico D (2005) Advesre cardiovascular
effects of the coxibs. J Med Chem 48:2251–2257
El-gazzar ARBA, Hussein HAR, Hafez HN (2007) Synthesis and
biological evaluation of thieno[2,3-d] pyrimidine derivatives for
anti-inflammatory, analgesic and ulcerogenic activity. Acta
Pharm 57:395–411
Emelina EE, Petrov AA, Firsov AV (2007) a-Aminoazoles in
syntheses of heterocycles. 3(5)-aminopyrazole-4-carbonitriles
in the synthesis of pyrazolo[1,5-a]pyrimidines. Russ J Org
Chem 43:471–473
Ermondi G, Caron G, Lawrence R, Longo D (2004) Docking studies
on NSAID/COX-2 isozyme complexes using contact statistics
analysis. J Comput Aided Mol Des 18:683–696
Furniss BS, Hannaford AJ, Smith PWG, Tatchell AR (2008) Vogel’s
textbook of practical organic chemistry. Pearson Education, New
Delhi
Acknowledgments Authors are thankful to Maharishi Markan-
deshwar University, Mullana, India for financial support. Authors also
thank DST, New Delhi, India for awarding INSPIRE Fellowship to
Ms. Kamalneet Kaur.
Heller ST, Natarajan SR (2006) 1, 3-diketones from acid chlorides
and ketones: a rapid and general one pot synthesis of pyrazoles.
Org Lett 8:2675–2678
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