Design, synthesis, computational and biological evaluation of some new hydrazino derivatives of DHA and pyranopyrazoles

Article abstract of DOI:10.1016/j.ejmech.2012.01.042

Two series of compounds namely, 4-aryl/heteroaryl hydrazino-3-acetyl-6- methyl-2H-pyran-2-ones (4a-4j) and pyrano[4,3-c]pyrazoles (6a-6e and 6g) were synthesized starting from 3-acetyl-4-chloro-6-methyl-2H-pyran-2-one (2). Estimation of pharmacotherapeuti

Full text of DOI:10.1016/j.ejmech.2012.01.042

European Journal of Medicinal Chemistry 50 (2012) 81e89
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, computational and biological evaluation of some new
hydrazino derivatives of DHA and pyranopyrazoles
Ajay Kumar a, Poonam Lohan b, Deepak K. Aneja b, Girish Kumar Gupta c, Dhirender Kaushik a,
,
Om Prakash a
*
a Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136 119, India
b Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India
c M.M. College of Pharmacy, M.M. University, Mullana, Ambala 133 203, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of compounds namely, 4-aryl/heteroaryl hydrazino-3-acetyl-6-methyl-2H-pyran-2-ones
(4ae4j) and pyrano[4,3-c]pyrazoles (6ae6e and 6g) were synthesized starting from 3-acetyl-4-chloro-
6-methyl-2H-pyran-2-one (2). Estimation of pharmacotherapeutic potential, possible molecular mech-
anism of action, toxic/side effects and interaction with drug-metabolizing enzymes were made for the
synthesized compounds on the basis of prediction of activity spectra for substances (PASS) prediction
results and their analysis by PharmaExpert software. COX inhibition predicted by PASS was confirmed by
experimental evaluation and validated via docking studies. Out of all the compounds, compounds 4h, 4j,
6e, 6g exhibited good anti-inflammatory activity, whereas compounds 4b, 4c, 4h, 4i, 4j, 6b, 6e, 6g
showed excellent analgesic activity compared with standard drug Diclofenac sodium.
Received 11 August 2011
Received in revised form
18 January 2012
Accepted 19 January 2012
Available online 1 February 2012
Keywords:
Dehydroacetic acid
Hydrazine
Pyranopyrazole
Docking study
Ó 2012 Elsevier Masson SAS. All rights reserved.
Analgesic activity
Anti-inflammatory activity
1. Introduction
such as harsh reaction conditions, formation of by-products,
unsatisfactorily yields, and long reaction time.
2-Pyrones and their fused derivatives have attracted a great deal
of interest due to their wide range of biological activities [1e6]. The
incorporation of another heterocyclic moiety in pyrones either in
the form of a substituent or as a fused component often leads to
incredible diverse biological activity. In addition, pyrazoles act as
core nucleus in various drugs due to their activities such as anti-
diabetic, anti-tumour, antipyretic, anti-inflammatory, anti-hyper-
tensive and antidepressant agents [7e12]. Considering the
importance of pyrone and pyrazole derivatives, it was thought
worthwhile to combine both pharmacophoric groups (pyran-2-
one þ pyrazole) in one molecular frame [13e20].
The present study illustrates a new preparative strategy for
synthesis of pyranopyrazoles by overcoming all the above
mentioned limitations.
2. Results and discussion
2.1. Chemistry
Our research work started with the preparation of 3-acetyl-4-
chloro-6-methyl-2H-pyran-2-one (Cl-DHA, 2) to be used as
a
substitute for 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one
These observations contemplated us to synthesize some new
pyranopyrazole derivatives of type 6 with a view to explore their
potency as good analgesic and anti-inflammatory agents.
(dehydroacetic acid, DHA, 1) and 4-chloro-3-(1-chlorovinyl)-6-
methyl-2H-pyran-2-one (dehydroacetchlorid, 8 ) for the synthesis
of pyrano[4,3-c]pyrazoles, which was prepared following the
literature method [22] (Scheme 1).
Then Cl-DHA was treated with phenylhydrazine using ethanol
as a solvent and progress of the reaction was monitored by thin
layer chromatography (TLC). The complete characterization (IR, 1H
NMR and 13C NMR) and elemental analysis data of the product
indicated the formation of new hydrazino pyrone 4a rather than
the expected hydrazone 5a. The scope of the reaction was studied
Earlier, the synthesis of pyranopyrazoles has been reported from
dehydroacetic acid (2-pyrone) following two different routes [21]
but the reported methods suffer from some serious drawbacks
* Corresponding author. Tel.: þ91 1744 239617 (0); fax: þ91 1744 238277/035/
628.
E-mail address: dromprakash50@rediffmail.com (O. Prakash).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.042

82
A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
treatment of dehydroacetic acid with hydrazines to form
N-substituted hydrazones which were further cyclised in the
presence of xylene or DME for long time refluxing. Major draw-
backs associated with this method were harsh reaction conditions,
long reaction time and more critical was low yields of product (33%)
because of the formation of mixture of by-products (10 and 11).
Although another route was somewhat better in terms of yields
(50%), it suffered from two additional drawbacks (i) poor yield of
starting material and (ii) excessive use of hydrazines.
OH
O
O
Cl
O
O
PCl5
O
ground, 6hrs
O
1
2
Scheme 1. Synthesis of chloro derivative of dehydroacetic acid (2) from dehydroacetic
acid (1).
From chemistry point of view, we have developed an alternative
route for the preparation of pyranopyrazoles 6 using milder
conditions, lesser reaction time with better yields than the reported
methods. In addition, it has also been established that the conver-
sion 2 / 6 proceeds via the intermediacy of hydrazine pyrones 4.
by making variation in hydrazines (3be3j) and similar results were
obtained in each case (Scheme 2). This is an interesting result as
such type of hydrazino pyrones are not reported so far and further
they seem to be the potential precursors for the synthesis of pyrano
[4,3-c]pyrazoles (6).
2.2. Biological evaluation
Encouraged by the results on the conversion of 2 / 4, we
became interested in the synthesis of pyranopyrazoles of type 6 by
cyclization of 4. Accordingly, we conducted the cyclization reaction
of 4a in acetic acid under reflux (method A, Scheme 3) and the
reaction indeed afforded the desired pyranopyrazole 6a, as evi-
denced by IR, 1H NMR and 13C NMR and elemental analysis data. In
the light of successful cyclization of 4a to 6a, we attempted the
synthesis of 6a directly from 2 without isolating intermediate 4a.
Fortunately, one-pot method (method B, Scheme 3) involving the
reaction between 2 and phenylhydrazine in acetic acid under reflux
6.30 h afforded the cyclised product 6a in 76% yield (Scheme 3).
Although both the methods (method A and method B) gave the
desired product without any difficulty, the one-pot procedure is
advantageous in terms of time over the two-step procedure. So, we
carried out the reaction of 2 with various aryl/heteroaryl hydra-
zines (3be3j) by adopting the one-pot procedure (method B,
Scheme 3). The results are summarized in Table 2. It can be seen
that the reaction is successful in the case of 3ae3e and 3g but in
other cases 4f and 4he4j intermediates hydrazino pyrones were
obtained. Even after making several attempts, either using more
harsh condition or starting from hydrazino pyrones 4, we were
unable to isolate any cyclised product. The poor nucleophilicity of
hydrazines and steric factor could be possible reasons for the failure
of these cases (Table 1).
2.2.1. Biological activity spectra prediction
The analysis of biological activity spectra prediction for the
synthesized compounds made in this publication is a good example
of in silico study of chemical compounds before their experimental
investigations. Anyone can do the same analysis using the free
available web-site with the internet version of PASS and Phar-
maExpert: http://www.ibmc.msk.ru/PASS/ [23e30].
A biological activity spectrum for a substance is a list of bio-
logical activity types for which the probability to be revealed (Pa)
and the probability not to be revealed (Pi) are calculated. Pa and Pi
values are independent and their values vary from 0 to 1. Biological
activity spectra were predicted for all the synthesized structures
with PASS 2005 version. The result of prediction is valuable at
planning of the experiment, but one should take into account some
additional factors: particular interest to some kinds of activity,
desirable novelty of a substance, available facilities for experi-
mental testing, etc. The more is Pa value, the less is the probability
of false positives in the set of compounds selected for biological
testing. By default in PASS, Pa ¼ Pi value is chosen as a threshold;
therefore all compounds with Pa > Pi are suggested to be active.
The other criterion for selection is the compound’s novelty. If Pa
value is high, sometimes one may find close analogues of known
biologically active substances among the tested compounds. If
Pa < 0.5 the chance to find the activity in experiment is even more
less, but if it will be confirmed the compound might occur to be
a New Chemical Entity (NCE).
It is noteworthy to mention here that the synthesis of pyrano
[4,3-c]pyrazoles is reported in literature from DHA and its deriva-
tives following two different routes (Scheme 4). First route involves
NHNHR
COCH3
H3C
O
O
Cl
O
O
4
EtOH
CH3
+
RNHNH2
H3C
O
stir, 15min
Cl
NNHR
CH3
O
2
3
H3C
O
5
where R = C6H5 (a); 4-MeC6H4 (b); 4-ClC6H4 (c); 4-BrC6H4 (d); 4-MeOC6H4 (e); 4-O2NC6H4 (f); 4-
phenyl-2-thiazolyl (g); 2-benzothiazolyl (h); 4,6-dimethyl-2-pyrimidinyl (i); 4-methyl-2-quinolinyl (j)
Scheme 2. Reaction of 2 with various aryl/heteroaryl hydrazines (3ae3j).

A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
NHNHR
83
COCH3
AcOH, reflux
Method A
R
N
H3C
O
O
N
4
CH3
H3C
O
O
Cl
O
RNHNH2
6
CH3
O
AcOH, reflux
H3C
O
Method B
2
Where R = C6H5 (a); 4-MeC6H4 (b); 4-ClC6H4 (c); 4-BrC6H4 (d); 4-MeOC6H4 (e) 4-phenyl-2-thiazolyl (g)
Scheme 3. Synthesis of pyrano[4,3-c]pyrazoles 6ae6g.
PharmaExpert was used for statistical and ‘activityeactivity’
relationship analysis of PASS prediction results for the set of
studied compounds. Analgesic activity was predicted for all
compounds with Pa value between 0.592 and 0.794 and anti-
inflammatory activity with Pa between 0.421 and 0.592. Besides,
all compounds were predicted as cyclooxygenase inhibitors with
Pa > 0.3. For example PASS prediction results of one of the
compound 6g are summarized here.
We have checked for the analgesic and anti-inflammatory
activity for the compounds which have Pa nearly equal to 0.5 and
experimentally they have shown good results which means they
are NCE.
Thus, our study showed that prediction of biological activity
spectra for synthesized compounds by PASS and its analysis made
by PharmaExpert may estimate pharmacotherapeutic potential,
possible molecular mechanisms of action, toxic/side effects and
interaction with drug-metabolizing enzymes. These results may be
quite helpful in further experimental studies.
Activity prediction
38 Substructure descriptors; 1 new.
104 Possible activities at Pa > Pi
2.2.2. Analgesic activity
The analgesic activity of the above mentioned derivatives was
evaluated by following tail immersion methods [31] and acetic acid
induced writhing assay [32,33] using diclofenac as a standard.
Results were expressed as mean ꢀ SEM differences between
control and treatment group and were tested using one-way
ANOVA followed by the least significant differences (LSD).
According to Tables 3A and 3B, compounds 4h, 4j, 6a, 6d, 6g, 6j
exhibited equipotent analgesic effect or slightly less than that of
standard after 1 h and 2 h post administration. While compounds
4a, 4b, 4h, 4i, 4j, 6a exhibited the analgesic effect after 1 h of
administration only. After 2 h compounds 4g, 4j, 6b also have
shown analgesic effect. Thus, it can be concluded that, compounds
4a, 4b, 4g, 4h, 4i, 6a, 6b, 6d, 6g have significant analgesic activity
according to tail immersion method. The results from acetic acid
Induced writhing assay revealed that all tested compounds
exhibited significant activity. Compounds 4b, 4c, 4f, 4h, 4i, 6b, 6e,
6g have nearly the same activity as the reference drug.
Pa
Pi
Activity
0.702
0.681
0.677
0.621
0.606
0.597
0.592
0.613
0.572
0.532
0.454
0.400
0.431
0.411
0.432
0.466
0.352
0.390
0.302
0.287
0.327
0.011
0.005
0.025
0.022
0.021
0.020
0.039
0.067
0.050
0.033
0.044
0.004
0.050
0.036
0.068
0.122
0.016
0.068
0.016
0.004
0.050
Analgesic, non-opioid
5 Hydroxytryptamine release inhibitor
Antineoplastic (ovarian cancer)
Analgesic
Antiarthritic
Cognition disorders treatment
Anti-inflammatory
Antiviral (Arbovirus)
Complement factor D inhibitor
Immunomodulator
Immunosuppressant
Cyclooxygenase inhibitor
HCV IRES inhibitor
Endothelial growth factor antagonist
Histamine release inhibitor
Antianaemic
Follicle-stimulating hormone agonist
Mediator release inhibitor
Antithrombocytopenic
Cyclooxygenase 2 inhibitor
GABA receptor agonist
2.2.3. Anti-inflammatory activity
All the newly synthesized compounds were evaluated for their
in vivo anti-inflammatory activity by carrageenan-induced paw
oedema method [34] and the results of tested compounds as well
Table 1
Table 2
Physical data of the synthesized compounds 4ae4j.
Physical data of the synthesized compounds 6ae6e and 6g.
R
Product
Mp (ꢁC)
Yields (%)
R
Product Mp (ꢁC)
Lit. mp (ꢁC) Yields (%)
C6H5
4-MeC6H4
4-ClC6H4
4-BrC6H4
4-MeOC6H4
4-O2NC6H4
4-Phenyl-2-thiazolyl
2-Benzothiazolyl
4,6-Dimethyl-2-pyrimidinyl
4-Methyl-2-quinolinyl
4a
4b
4c
4d
4e
4f
4g
4h
4i
267e268
209e210
207e208
185e186
225e226
285e286
>300
297e299
129e131
222e223
89
87
96
95
89
94
93
95
90
87
C6H5
4-MeC6H4
4-ClC6H4
4-BrC6H4
4-MeOC6H4
4-O2NC6H4
4-Phenyl-2-thiazolyl
2-Benzothiazolyl
4,6-Dimethyl-2-pyrimidinyl 4i
4-Methyl-2-quinolinyl 4j
6a
6b
6c
6d
6e
4f
206e207 209e210 76
170e172 172e173
180e182 179e182
82
85
87
79
86
80
93
90
87
289e290
153e154
285e286
248e249
297e299
129e131
222e223
e
e
e
e
e
e
e
6g
4h
4j

84
A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
OH
O
O
CH3
H3C
O
1
RNHNH2
POCl3
reflux, 4.5 h
MeOH, reflux
Cl
Cl
OH NNHR
CH3
CH2
O
H3C
O
H3C
O
O
8
7
RNHNH2
DME, reflux, 49 h
or
DME, reflux, 54 h
Xylene, reflux, 12 h
R
N
R
O
CH3
N
N
N
CH3
CH3
N
+
N
H3C
O
H3C
O
O
H3C
O
O
R
6
9
10
+
H3C
HO
H3C
N
N
N
N
R
R
11
Where R= C6H5 (a); 4-MeC6H4 (b); 4-ClC6H4 (c)
Scheme 4. Method by Cantos et al. [21] for the synthesis of pyranopyrazoles.
Table 3A
Table 3B
Analgesic activity of compounds 4ae4j and 6ae6e, 6g by tail immersion method.
Analgesic activity of compounds 6ae6g and 4ae4j by writhing method.
S. no Compound Time
S. no
Drug
treatment
Dose
(mg/kg)
No. of
animals
Change in no.
of wriths
Percentage
inhibition
30 min
1.010 ꢀ 0.07 1.388 ꢀ 0.03 1.730 ꢀ 0.10 1.714 ꢀ 0.09
Standard 5.576 ꢀ 0.17** 7.420 ꢀ 0.36** 8.934 ꢀ 0.18** 8.464 ꢀ 0.24**
60 min
90 min
120 min
1
2
3
Control
1
2
3
4
5
6
7
8
Control
Standard
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
6a
6b
6c
6d
6e
e
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
49.00 ꢀ 2.16**
11.00 ꢀ 0.70**
24.60 ꢀ 2.90**
14.60 ꢀ 2.61**
12.20 ꢀ 2.05**
23.00 ꢀ 3.03**
20.00 ꢀ 2.40**
25.80 ꢀ 2.31**
23.80 ꢀ 3.59**
17.20 ꢀ 2.91**
17.20 ꢀ 2.63**
12.40 ꢀ 1.07**
24.40 ꢀ 2.37**
15.00 ꢀ 1.67**
17.00 ꢀ 2.56**
27.00 ꢀ 4.82**
11.80 ꢀ 2.22**
14.60 ꢀ 3.29**
e
50
77.55
49.79
70.20
75.10
53.06
59.18
47.34
51.42
65.30
64.89
75.00
50.20
69.38
65.30
44.89
75.91
70.20
4a
4b
4c
4d
4e
4f
3.100 ꢀ 0.36 6.462 ꢀ 0.43** 3.656 ꢀ 0.22 2.744 ꢀ 0.25
2.390 ꢀ 0.21 3.502 ꢀ 0.14** 2.540 ꢀ 0.24* 2.520 ꢀ 0.36
3.262 ꢀ 0.43* 3.552 ꢀ 0.53 3.896 ꢀ 0.41 3.776 ꢀ 0.35
3.496 ꢀ 0.85* 3.534 ꢀ 0.56 3.230 ꢀ 0.27 2.630 ꢀ 0.34
2.924 ꢀ 0.30 3.174 ꢀ 0.19 3.006 ꢀ 0.20 2.840 ꢀ 0.81
3.512 ꢀ 0.67* 4.276 ꢀ 0.79* 3.536 ꢀ 0.28 3.544 ꢀ 0.61
3.094 ꢀ 0.41 4.114 ꢀ 0.58 5.234 ꢀ 1.07** 4.796 ꢀ 1.06**
3.918 ꢀ 0.32** 4.582 ꢀ 0.30** 3.930 ꢀ 0.58 3.854 ꢀ 0.31
4.136 ꢀ 0.61** 4.640 ꢀ 0.45** 4.562 ꢀ 0.56* 4.186 ꢀ 0.65*
4.896 ꢀ 0.52** 4.970 ꢀ 1.06** 6.380 ꢀ 1.16** 4.594 ꢀ 0.42**
3.664 ꢀ 0.35** 4.802 ꢀ 0.78** 5.516 ꢀ 1.02** 3.492 ꢀ 0.14
4.134 ꢀ 0.84* 4.300 ꢀ 0.03* 3.680 ꢀ 0.97 3.376 ꢀ 0.91
3.596 ꢀ 0.49* 3.972 ꢀ 0.85* 2.968 ꢀ 0.57 2.892 ꢀ 0.43
3.690 ꢀ 0.52** 3.770 ꢀ 0.50 3.728 ꢀ 0.37 3.690 ꢀ 0.28
3.578 ꢀ 0.19* 3.976 ꢀ 0.28* 5.078 ꢀ 1.13** 3.832 ꢀ 1.12
4.086 ꢀ 0.29** 4.034 ꢀ 0.27* 3.812 ꢀ 0.27 3.512 ꢀ 0.23
4
5
6
7
8
9
4g
4h
4i
9
10
11
12
13
14
15
16
17
18
10
11
12
13
14
15
16
17
18
4j
6a
6b
6c
6d
6e
6g
6g
N ¼ 5, values are expressed as mean ꢀ SEM and analyzed by ANOVA.
**P < 0.01(significant), *P < 0.05. Values are compared with control group.
N ¼ 5, values are expressed as mean ꢀ SEM and analyzed by ANOVA.
**P < 0.01(significant), *P < 0.05. Values are compared with control group.

A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
85
as reference standard were measured before administration of
carrageenan. After the administration of carrageenan inflammation
was developed in mice, the effect was measured in the interval of
1 h, 2 h, 3 h, and 4 h respectively. The percentage inhibition of
oedema was calculated as a regard to saline control group, as
depicted in Table 4.
Most of the tested compounds have shown good results in
comparison with standard drug. Amongst all the compounds,
compounds 4h, 4j, 6e, 6g, have shown potent anti-inflammatory
activity.
of the ligands to give a number of conformations from which the
best mode could be achieved. In the analysis of docking results we
tried to find a correlation between the biological results and docking
studies.
From Table 5 and Figs. 1e5 the following results can be drawn:
SC-558 (the original ligand) reveals docking score of ꢂ153.93 and
forms five interactions shown as green dotted lines (Fig. 1),
showing two hydrogen bonds between O of SO2NH2 moiety with
ꢀ
ꢀ
His90 and Arg513 of distances 2.70 A and 3.53 A respectively and
three hydrogen bonds between N of SO2NH2 moiety with Gln192,
ꢀ
ꢀ
ꢀ
Leu352 and Ser353 of distances 3.18 A, 3.09 A, and 2.98 A respec-
tively. Compound 4a exhibits relatively weak binding affinity with
docking score of ꢂ81.86 but forms two hydrogen bonds between
C]O of pyrone with Ser530 and Tyr385. Further an increase in
activity was observed in case of compound 4b, when eCH3 group
was introduced at para position. Compound 4b showed a docking
score of ꢂ87.90 and forms two hydrogen bonds between C]O of
pyrone with Ser530 and Tyr385 where interspatial distance
2.3. Docking studies
2.3.1. Aim of work
To pre-asses the anti-inflammatory behaviour of hydrazino
derivatives and pyrano[4,3-c]pyrazoles on a structural basis, auto-
mated docking studies were carried out using Molegro Virtual
Docker, the scoring functions and hydrogen bonds formed with the
surrounding amino acids are used to predict their binding modes,
their binding affinities and orientation of these compounds at the
active site of cyclooxygenase-II enzyme [35].
ꢀ
ꢀ
between these is about 2.97 A and 3.19 A respectively. Similar types
of interactions were observed in case of compounds 4c, 4d when
electron withdrawing groups (Cl and Br) were inserted at para
position. However, their cyclised analogues were found to be more
active as clear from their docking scores and compound 6g has the
best docking score (ꢂ123.978) being able to interact by its ring-N
and pyran-O with Ser353, Gln192, His90 and Asp515, respectively.
Also, some interesting results were obtained in case of compounds
4f, 4g, 4h, 4i, 4j as clear from their docking score which may be due
to insertion of heteroaryl moieties at the place of phenyl ring.
Hence, it can be concluded that relative COX-II inhibitory
potency order for this group follows the trend phenylthiazolyl >
benzothiazolyl > quinolinyl > pyrimidinyl > OCH3 > Br > CH3 > H.
2.3.2. Binding affinities of the synthesized compounds into COX-II
The crystal structure was downloaded (PDB code: 1CX2) and our
compounds were docked into the active sites using Molegro Virtual
Docker 2008.3.2. software [35]. Compounds were ranked after
docking according to their docking scores and were visualised inside
the pocket to view their fitting and closure to main residues. Molec-
ular docking studies were revealed further insight into the nature of
interactions between the compounds and the active site amino acids
to rationalize the obtained biological results. The next stage of protein
preparation is to optimize the H-bond network by reorienting
hydroxyl group, water molecules and amide groups of Arg120,
Tyr355, His90, Arg513, Val523, Ser353, Glu524 that are thought to act
as a gate for ligand entrance to the COX active sites [36e41]. It is clear
from Table 5 that compounds 4h, 4j, 6e, 6g have best docking score as
well and we can see the possible interactions (Figs. 1e5).
3. Conclusion
The present study offers:(i) Synthesis of new hydrazino deriv-
atives of dehydroacetic acid (4ae4j). (ii) Superior approach for the
synthesis of pyrano[4,3-c]pyrazoles (6ae6e and 6g). (iii) Prelimi-
nary prediction of biological activity spectra for title compounds by
PASS computer program and further confirmation by experimental
evaluation and validation via docking studies. (iv) Identification of
2.3.3. Results and discussion
Molegro Virtual Docker allows the flexible docking of ligands
into its site of action. It has the ability to use all the rotatable bonds
Table 4
Anti-inflammatory activity of compounds 4ae4j and 6ae6e, 6g against carrageenan-induced rat paw oedema over 4 h.
Compounda
Volume of oedema (ml)b
1 h
2 h
3 h
4 h
Control
Standard
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
6a
6b
6c
6d
6e
0.158 ꢀ 0.050
0.178 ꢀ 0.031
0.198 ꢀ 0.043
0.204 ꢀ 0.057
0.046 ꢀ 0.022(70.88)c
0.064 ꢀ 0.008(59.49)
0.063 ꢀ 0.014(60.12)
0.072 ꢀ 0.023(54.43)
0.084 ꢀ 0.012(46.84)
0.079 ꢀ 0.015(50.00)
0.074 ꢀ 0.015(53.16)
0.076 ꢀ 0.019(51.90)
0.076 ꢀ 0.023(64.56)
0.064 ꢀ 0.042(59.49)
0.076 ꢀ 0.015(51.90)
0.068 ꢀ 0.019(56.96)
0.058 ꢀ 0.015(63.29)
0.066 ꢀ 0.044(58.23)
0.064 ꢀ 0.025(59.49)
0.056 ꢀ 0.038(64.56)
0.052 ꢀ 0.016(67.09)
0.058 ꢀ 0.008**(67.41)
0.076 ꢀ 0.010*(57.30)
0.083 ꢀ 0.017(53.37)
0.076 ꢀ 0.016*(57.30)
0.094 ꢀ 0.015(47.19)
0.090 ꢀ 0.016(49.43)
0.080 ꢀ 0.025*(55.05)
0.092 ꢀ 0.024(48.31)
0.074 ꢀ 0.019*(58.42)
0.068 ꢀ 0.046*(61.79)
0.080 ꢀ 0.026*(55.05)
0.080 ꢀ 0.012*(55.05)
0.074 ꢀ 0.014(58.42)
0.072 ꢀ 0.014*(59.55)
0.088 ꢀ 0.012(50.56)
0.082 ꢀ 0.024(53.93)
0.072 ꢀ 0.024*(59.55)
0.060 ꢀ 0.020**(69.70)
0.088 ꢀ 0.020*(55.56)
0.080 ꢀ 0.024(59.59)
0.082 ꢀ 0.010*(58.59)
0.118 ꢀ 0.014(40.40)
0.110 ꢀ 0.018(44.44)
0.108 ꢀ 0.023(45.45)
0.108 ꢀ 0.024(45.45)
0.090 ꢀ 0.026*(54.55)
0.088 ꢀ 0.042(55.56)
0.088 ꢀ 0.026*(55.56)
0.094 ꢀ 0.016*(52.53)
0.091 ꢀ 0.015(54.04)
0.086 ꢀ 0.008*(56.57)
0.100 ꢀ 0.022(49.49)
0.084 ꢀ 0.024*(57.58)
0.074 ꢀ 0.020*(62.63)
0.068 ꢀ 0.022*(66.67)
0.098 ꢀ 0.014(51.96)
0.102 ꢀ 0.015(50.00)
0.146 ꢀ 0.014(28.43)
0.166 ꢀ 0.020(18.63)
0.154 ꢀ 0.019(24.50)
0.114 ꢀ 0.034(44.12)
0.156 ꢀ 0.021(23.53)
0.104 ꢀ 0.013(49.02)
0.118 ꢀ 0.075(42.16)
0.100 ꢀ 0.008(50.98)
0.116 ꢀ 0.028(43.14)
0.119 ꢀ 0.021(41.66)
0.126 ꢀ 0.024(38.24)
0.126 ꢀ 0.022(38.24)
0.118 ꢀ 0.020(42.16)
0.096 ꢀ 0.025(52.94)
6g
**P < 0.01(significant), *P < 0.05. Values are compared with control group.
a
Dose levels: test compounds (50 mg/kg b.w.), Diclofenac sodium (50 mg/kg b.w.).
b
N ¼ 5, values are expressed as mean ꢀ SEM and analyzed by ANOVA.
c
Values in parentheses (percentage anti-inflammatory activity, AI%).

86
A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
Table 5
Docking score with the hydrogen bond interactions with amino acids.
Compounds Docking
scores
No. of
H-bond
Amino acid Interaction
hydrogen distance (A0) involved
bonds
with compound
or structural
feature
SC-558
ꢂ153.933
5
2.70
3.53
3.18
3.09
2.98
3.13
3.09
2.97
3.19
2.97
3.19
2.97
3.19
3.41
2.84
2.48
2.94
2.60
3.27
2.63
3.05
2.92
2.82
3.56
2.47
3.06
3.43
3.42
3.01
3.01
2.67
2.72
2.93
3.10
3.20
3.46
3.36
3.43
3.14
3.05
2.80
2.80
3.05
2.99
2.94
3.26
3.31
3.07
3.00
3.32
2.77
3.07
3.50
3.30
His90
SO2NH2-O
SO2NH2-O
SO2NH2-N
SO2NH2-N
SO2NH2-N
C]O
C]O
C]O
C]O
C]O
Arg513
Gln192
Leu352
Ser353
Ser530
Tyr385
Ser530
Tyr385
Ser530
Tyr385
Ser530
Tyr385
Ser530
Arg120
Tyr355
Arg513
Tyr355
His90
4a
4b
4c
4d
4e
ꢂ81.8629 2
ꢂ87.9093 2
ꢂ87.8557 2
ꢂ87.8676 2
ꢂ86.4865 3
C]O
C]O
C]O
Fig. 1. Binding mode of compound SC-558 into cyclooxygenase II pocket. It has Mol-
dock score ꢂ153.93 and forms 5 hydrogen bonds shown as green dotted lines (Table 5),
showing two hydrogen bonds between O of SO2NH2 moiety with His90, and Arg513 of
Acetyl C]O
OCH3-O
C]O
NO2-O
NO2-O
NO2-N
NO2-O
NO2-N
C]O
ꢀ
ꢀ
distances 2.70 A and 3.53 A respectively and three other hydrogen bonds between N of
ꢀ
ꢀ
ꢀ
SO2NH2 moiety with Gln192, Leu352, and Ser353 of distances 3.18 A, 3.09 A, and 2.98 A
respectively. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
4f
ꢂ83.3152 8
His90
Tyr355
Ser530
Tyr385
Tyr385
Ser353
Thr94
His90
Gln192
His90
Tyr355
Tyr355
Tyr355
Tyr355
His90
Ser530
Gly526
Ala527
Ser530
Tyr385
Ser530
Tyr385
Ser530
Tyr385
Ser530
Tyr385
Ser530
Tyr385
Ser530
Tyr385
Arg120
Ser353
Gln192
His90
electrical apparatus Labindia visual melting range apparatus and
are uncorrected. IR spectra were recorded on a PerkineElmer 1800
FT-IR spectrophotometer. The 1H NMR and 13C NMR spectra were
recorded in CDCl3 on Bruker Nuclear Magnetic Resonance (NMR)
spectrometer at 300 and 75 MHz respectively, using tetrame-
thylsilane (TMS) as an internal standard. Chemical shifts are
C]O
Pyran-O
Thiaz-N
Acetyl-O
NHNH-2
NHNH-1
Pyran-O
Acetyl C]O
C]O
4g
ꢂ108.078
4
4h
4i
ꢂ108.721
ꢂ101.868
ꢂ106.178
3
3
3
expressed in
d ppm. Mass spectra were recorded on 2500 eV (ESI
source) using a Water’s Q-TOF micro instrument.
Acetyl-O
C]O
4.1.1. Synthesis of substituted hydrazino derivatives of
dehydroacetic acid (4ae4j)
Pyran-O
Pyran-O
Acetyl-O
Acetyl-O
C]O
C]O
C]O
C]O
C]O
4j
4.1.1.1. Procedure. 3-Acetyl-4-chloro-6-methyl-2H-pyran-2-one (2,
1.86 g, 10 mmol) was treated with phenylhydrazine (3a, 1.08 ml,
10 mmol) using ethanol (20 ml) as a solvent and stirred for
15e20 min at room temperature to give hydrazino derivative 4a in
fair to good yields.
6a
6b
6c
6c
6d
6e
ꢂ96.5692 2
ꢂ99.0714 2
ꢂ99.0692 2
ꢂ87.6278 2
Similar procedure was adopted for synthesis of other derivatives
(4be4j).
C]O
Pyran-O
Pyran-O
C]O
C]O
C]O
ꢂ99.787
2
3
ꢂ103.644
C]O
OCH3-O
Thiaz-N
Ring-N
N-Attach
Pyran-O
6g
ꢂ123.978
4
Asp515
six-membered lactone (pyran-2-one) ring as a suitable central ring
template to design selective COX-2 inhibitors.
The study revealed a close agreement between in vitro and
in vivo analysis with some compounds (4d, 4h, 4j, 6e) having dual
analgesic and anti-inflammatory profile and therefore become lead
molecules for further synthetic and biological evaluation.
4. Experimental protocols
Fig. 2. Binding mode of compound (4h) into the binding site of cyclooxygenase II
pocket. It has Moldock score ꢂ108.721 and forms 3 hydrogen bonds shown as green
dotted lines (Table 5), showing one hydrogen bond between eOe of pyran moiety with
4.1. Materials and methods
ꢀ
His90 of distance 3.01 A, one hydrogen bond between eOe of acetyl moiety with
ꢀ
Tyr355 of distance 3.01 A and one hydrogen bond between C]O of pyrone moiety
ꢀ
All reagents were purchased from commercial sources and were
with Tyr355 of distance 2.67 A respectively. (For interpretation of the references to
used without purification. Melting points were taken on slides in an
colour in this figure legend, the reader is referred to the web version of this article.)

A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
87
Fig. 3. Binding mode of compound (4j) into the binding site of cyclooxygenase II
pocket. It has Moldock score ꢂ106.178 and forms 3 hydrogen bonds shown as green
dotted lines (Table 5), showing one hydrogen bond between eOe of pyran moiety with
ꢀ
Ser530 of distance 3.20 A, two hydrogen bonds between eOe of acetyl moiety with
Fig. 5. Binding mode of compound (6g) into the binding site of cyclooxygenase II pocket.
It has Moldock score ꢂ123.978 and forms 4 hydrogen bonds shown as green dotted lines
(Table 5), showing one hydrogen bond between eNe of thiazole moiety with Ser353 of
ꢀ
ꢀ
Gly526 and Ala527 of distances 3.46 A and 3.36 A respectively. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
ꢀ
distance 2.77 A, one hydrogen bond between eNe of pyrazole moiety with Gln192 of
ꢀ
distance 3.07 A, one hydrogen bond between tert-Ne of pyrazole moiety with His90 of
ꢀ
distance 3.50 A and one hydrogen bond between eOe of pyran moiety with Asp515 of
ꢀ
distance 3.30 A respectively. (For interpretation of the references to colour in this figure
4.1.1.2. Characterization data of 4-(2-substitutedhydrazinyl)-3-
acetyl-6-methyl-2H-pyran-2-ones (4ae4j)
legend, the reader is referred to the web version of this article.)
4.1.1.2.1. 4-(2-Phenylhydrazinyl)-3-acetyl-6-methyl-2H-pyran-2-
one (4a). IR (nmax, cmꢂ1, KBr): 3402, 3356, 1705, 1660, 1566, 1466,
1396,1358; 1H NMR (CDCl3): 2.2 (s, 3H), 2.75 (s, 3H), 5.82 (s,1H), 6.5
(s, 1H, NH), 6.8e7.3 (m, 5H), 15.6 (s, 1H, NH); MS (ESI) m/z ¼ 259.17
(M þ Hþ). Anal. Calcd. for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85.
Found: C, 65.03; H, 5.41; N, 10.79.
4.1.1.2.2. 4-[2-(4-Methylphenylhydrazinyl)]-3-acetyl-6-methyl-
2H-pyran-2-one (4b). IR (nmax, cmꢂ1, KBr): 3413, 3358, 1705, 1649,
1551, 1450, 1350; 1H NMR (CDCl3): 2.21 (s, 3H), 2.30 (s, 3H), 2.72 (s,
3H), 5.86 (s, 1H), 6.54 (s, 1H, NH), 6.7e7.4 (m, 4H), 15.5 (s, 1H, NH).
Anal. Calcd. for C15H16N2O3: C, 66.16; H, 5.92; N, 10.29. Found: C,
66.13; H, 5.87; N, 10.22.
1H), 6.55 (s, 1H, NH), 6.8e7.34 (m, 4H), 15.5 (s, 1H, NH). Anal. Calcd.
for C14H13ClN2O3: C, 57.44; H, 4.48; N, 9.57. Found: C, 57.39; H, 4.41;
N, 9.53.
4.1.1.2.4. 4-[2-(4-Bromophenylhydrazinyl)]-3-acetyl-6-methyl-
2H-pyran-2-one (4d). IR (nmax, cmꢂ1, KBr): 3448, 3356, 1713, 1651,
1612, 1543, 1450, 1389; 1H NMR (CDCl3): 2.2 (s, 3H), 2.71 (s, 3H),
5.84 (s, 1H), 6.65 (s, 1H, NH), 6.75e7.42 (m, 4H), 15.6 (s, 1H, NH); MS
(ESI) m/z ¼ 337.11 (M þ Hþ). Anal. Calcd. for C14H13BrN2O3: C, 49.87;
H, 3.89; N, 8.31. Found: C, 49.83; H, 3.81; N, 8.29.
4.1.1.2.5. 4-[2-(4-Methoxyphenylhydrazinyl)]-3-acetyl-6-methyl-
2H-pyran-2-one (4e). IR (nmax, cmꢂ1, KBr): 3418, 3364, 1697, 1651,
1620, 1558, 1474, 1366; 1H NMR (CDCl3): 2.18 (s, 3H), 2.57 (s, 3H),
3.817 (s, 3H, OCH3), 5.78 (s, 1H), 6.5 (s, 1H, NH), 6.82e7.27 (m, 4H),
15.45(s, 1H, NH). Anal. Calcd. for C15H16N2O4: C, 62.49; H, 5.59; N,
9.72. Found: C, 62.33; H, 5.56; N, 9.68.
4.1.1.2.3. 4-[2-(4-Chlorophenylhydrazinyl)]-3-acetyl-6-methyl-
2H-pyran-2-one (4c). IR (nmax, cmꢂ1, KBr): 3434, 3368, 1713, 1643,
1551, 1443, 1373; 1H NMR (CDCl3): 2.21 (s, 3H), 2.758 (s, 3H), 5.82 (s,
4.1.1.2.6. 4-[2-(4-Nitrophenylhydrazinyl)]-3-acetyl-6-methyl-2H-
pyran-2-one (4f). IR (nmax, cmꢂ1, KBr): 3430, 3369, 1720, 1643, 1612,
1551, 1450, 1373; 1H NMR (CDCl3): 2.2 (s, 3H), 2.76 (s, 3H), 5.83 (s,
1H), 6.52 (s, 1H, NH), 6.86e7.34 (m, 4H), 15.5 (s, 1H, NH). Anal.
Calcd. for C14H13N3O5: C, 55.45; H, 4.32; N, 13.86. Found: C, 55.43;
H, 4.27; N, 13.79.
4.1.1.2.7. 4-[2-(4-Phenylthiazol-2-ylhydrazinyl)]-3-acetyl-6-
methyl-2H-pyran-2-one (4g). IR (nmax, cmꢂ1, KBr): 3448, 3402,
1690, 1636, 1574, 1473, 1396, 1358; 1H NMR (DMSO): 2.2 (s, 3H),
2.35 (s, 3H), 6.16 (s, 1H), 7.4e7.9 (m, 6H), 8.26 (s, 1H, NH), 10.21 (s,
1H, NH); MS (ESI) m/z ¼ 342.10 (M þ Hþ). Anal. Calcd. for
C17H15N3O3S: C, 59.81; H, 4.43; N, 12.31. Found: C, 59.77; H, 4.37; N,
12.28.
4.1.1.2.8. 4-(2-Benzothiazolylhydrazinyl)-3-acetyl-6-methyl-2H-
pyran-2-one (4h). IR (nmax, cmꢂ1, KBr): 3433, 3402, 1703, 1620,
1566, 1466, 1396,1358; 1H NMR (CDCl3): 2.29 (s, 3H), 2.69 (s, 3H),
5.96 (s, 1H), 7.82e8.04 (m, 4H); 13C NMR 16.60, 19.88, 96.11, 104.95,
106.65, 112.01, 113.8, 121.61, 127.41, 130.05, 135.46, 148.19, 162.89,
179.23. Anal. Calcd. for C15H13N3O3S: C, 57.13; H, 4.16; N, 13.33.
Found: C, 57.03; H, 4.09; N, 13.29.
Fig. 4. Binding mode of compound (6e) into the binding site of cyclooxygenase II
pocket. It has Moldock score ꢂ103.644 and forms 3 hydrogen bonds shown as green
dotted lines (Table 5), showing two hydrogen bonds between eOe of 2-pyran moiety
ꢀ
ꢀ
(C]O) with Ser530, and Tyr385 of distances 3.07 A and 3.00 A and one hydrogen bond
ꢀ
between eOe of OCH3 moiety with Arg120 of distance 3.32 A respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

88
A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89
4.1.1.2.9. 4-(3,5-Dimethylpyrimidinyllhydrazinyl)-3-acetyl-6-
6H). Anal. Calcd. for C17H13N2O2S: C, 63.14; H, 4.05; N, 12.99. Found:
C, 63.03; H, 4.00; N, 12.89.
methyl-2H-pyran-2-one (4i). IR (nmax, cmꢂ1, KBr): 3456, 3425, 1713,
1620, 1551, 1435, 1373, 1342; 1H NMR (CDCl3): 2.29 (s, 3H), 2.43 (s,
3H), 2.66 (s, 3H), 2.69 (s, 3H), 5.90 (s, 1H), 5.96 (s, 1H), 6.62 (s, 1H,
NH). Anal. Calcd. for C14H16N4O3: C, 58.32; H, 5.59; N, 19.43. Found:
C, 58.30; H, 5.47; N, 19.39.
4.2. Pharmacological assay
4.2.1. Animals used
4.1.1.2.10. 4-(4-Methylquinolinyllhydrazinyl)-3-acetyl-6-methyl-
2H-pyran-2-one (4j). IR (nmax, cmꢂ1, KBr): 3441, 3402,1713,1628,1543,
1443, 1420, 1381; 1H NMR (CDCl3): 2.16 (s, 3H), 2.5 (s, 3H), 2.6 (s, 3H),
5.99 (s,1H), 7.2 (s, IH), 7.1e7.6 (m, 4H), 7.3 (s, IH, NH),10.5 (s,1H, NH); 13C
NMR (CDCl3) 16.08, 19.59, 19.77, 106.58, 113.96, 115.89, 121, 121.42,
125.11, 125.90, 126.00, 129.93, 132.22, 136.39, 145.88, 149.49, 159.16,
162.13; MS (ESI) m/z ¼ 324.20 (M þ Hþ). Anal. Calcd. for C18H17N3O3: C,
66.86; H, 5.30; N, 13.00. Found: C, 66.80; H, 5.21; N, 12.96.
Adult Swiss albino mice (20e25 g) were used in the experi-
ments. Animals were housed under standardized conditions for
light and temperature. Animals were randomly assigned to
different experimental groups, each kept in a separate cage. Study
protocol was approved by the Institutional Animal Ethics
Committee before experiment.
4.2.2. Analgesic activity screening
Acetic acid induced writhing model was used to evaluate anal-
gesic activity of the synthesized compounds. Groups of five Swiss
albino mice, each 20e25 g body weight were used and 0.6% acetic
acid (10 ml/kg) was injected intra-peritoneally. The numbers of
wriths were counted for 10 min, immediately after 5 min of injec-
tion of acetic acid in each mice. This reading was taken as control.
Next day, same group of mice were used for evaluation. Each group
was administered orally with the synthesized compounds. The dose
of 50 mg/kg of animal was given 30 min before injection of acetic
acid. After 5 min of acetic acid injection, mice were observed for the
number of writhing for the duration of 10 min. The mean value for
each group was calculated and compared with control. Diclofenac
sodium was used as a standard drug for comparison of analgesic
activity. Percent protection was calculated using the formula:
4.1.2. Synthesis of 3,6-dimethyl-2-(substituted)pyrano[4,3-c]
pyrazoles 6
4.1.2.1. Procedure. Method A: A solution of 4-(2-phenylhydrazinyl)-3-
acetyl-6-methyl-2H-pyran-2-one (4a, 0.258 g, 1mmol) in acetic acid
was refluxed for 6e7 h, cooled to room temperature and then poured
into an ice cold solution. The solid product thus obtained was filtered,
recrystallised using ethanol to gave pyrano[4,3-c]pyrazole 6a.
Method B: 3-Acetyl-4-chloro-6-methyl-2H-pyran-2-one (2,
1.86 g, 10 mmol) was refluxed with phenylhydrazine (3a, 1.08 ml,
10 mmol) in acetic acid for 6e7 h, cooled to room temperature and
then poured into an ice cold solution. The solid product thus ob-
tained was filtered, recrystallised using ethanol to gave pyrano[4,3-
c]pyrazole 6a.
Similar procedure was adopted for the synthesis of other
derivatives.
ð1 ꢂ Vc=VtÞ ꢃ 100
where Vt ¼ mean number of writhing in test animals; Vc ¼ mean
4.1.2.2. Characterization data of 3,6-dimethyl-2-(substituted)pyrano
[4,3-c]pyrazoles
4.1.2.2.1. 3,6-Dimethyl-2-phenylpyrano[4,3-c]pyrazol-4(2H)-one
(6a). IR (nmax, cmꢂ1, KBr) 3070, 1715; 1H NMR (CDCl3) 2.2 (3H, s),
2.6 (3H, s), 6.25 (1H, s), 7.45 (5H, s); 13C NMR (CDCl3) 11.8, 19.9, 96.7,
105.8, 125.3, 129.3, 138.6, 143.2, 151.0, 157.4, 160.1
number of writhing in control.
Statistical significance was analyzed using one-way ANOVA
followed by TurkeyeKrammer multiple comparison test and
p < 0.01 was considered significant.
4.2.3. Anti-inflammatory activity screening
4.1.2.2.2. 3,6-Dimethyl-2-(4-methylphenyl)pyrano[4,3-c]pyrazol-
4(2H)-one (6b). IR (nmax, cmꢂ1, KBr) 3100, 1725 cmꢂ1 1H NMR
;
All the synthesized compounds were tested for their anti-
inflammatory activity using carrageenan-induced rat hind paw
oedema method of Winter et al. [34] the oedema hind paw was
induced by injection of 0.1 ml of 1% carrageenan solution into
subplanter region of right hand paw. The volume of paw was
measured plethysmographically immediately and 1 h, 2 h, 3 h, 4 h
after the injection of irritant. The difference in volume gave the
amount of oedema developed. Percent inhibition of the oedema
between the control group and the compound treated group was
calculated and compared with the group receiving standard drug at
50 mg/kg b.w. The results are tabulated in Table 5.
(CDCl3) 2.3 (3H, s), 2.4 (3H, s), 2.6 (3H, s), 6.3 (1H, s), 7.3 (4H, s); 13
C
NMR (CDCl3) 11.8, 19.8, 21.0, 96.7, 106.0, 125.1, 129.8, 138.1, 139.1,
143.1, 150.8, 157.2, 160.0
4.1.2.2.3. 3,6-Dimethyl-2-(4-chlorophenyl)pyrano[4,3-c]pyrazol-
4(2H)-one (6c). IR (nmax, cmꢂ1, KBr) 3100, 1760 cmꢂ1 1H NMR
;
(CDCl3) 2.4 (3H, s), 2.7 (3H, s), 6.7 (1H, s), 7.3, 7.4, 7.6, 7.7 (4H, AB
system); 13C NMR (CDCl3) 11.3, 19.6, 95.2, 105.3, 127.4, 130.7, 133.1,
138.5, 147.8, 150.2, 161.6, 163.0
4.1.2.2.4. 3,6-Dimethyl-2-(4-bromophenyl)pyrano[4,3-c]pyrazol-
4(2H)-one (6d). IR (nmax, cmꢂ1, KBr) 3100, 1760 cmꢂ1 1H NMR
;
(CDCl3) 2.32 (3H, s), 2.5 (3H, s), 6.636 (1H, s), 7.37e7.73 (m, 4H); 13
C
4.2.4. Statistical analysis
NMR (CDCl3) 15.5, 19.78, 96.79, 106.58, 121.05, 121.45, 121.93,
129.19, 129.93, 149.72, 159.19, 162.12; MS (ESI) m/z ¼ 319.11
(M þ Hþ). Anal. Calcd. for C14H11BrN2O2: C, 52.69; H, 3.47; N, 8.78.
Found: C, 52.57; H, 3.50; N, 8.73.
4.1.2.2.5. 3,6-Dimethyl-2-(4-methoxyphenyl)pyrano[4,3-c]pyr-
azol-4(2H)-one (6e). IR (nmax, cmꢂ1, KBr) 3086, 1713, 1620,1551,
1497, 1435, 1373; 1H NMR (CDCl3) 2.12 (3H, s), 2.3 (3H, s), 3.82 (s,
3H, OCH3), 6.246 (1H, s), 7.37e7.68 (m, 4H); 13C NMR (CDCl3) 14.37,
18.42, 58.44, 109.78, 115.26, 126.12, 128.49, 131.23, 133.79, 152.91,
153.87, 159.86, 163, 11.3, 19.6, 95.2, 105.3, 127.4, 130.7, 133.1, 138.5,
147.8, 150.2, 161.6, 163.0. Anal. Calcd. for C15H14N2O3: C, 66.66; H,
5.22; N, 10.36. Found: C, 66.59; H, 5.21; N, 10.26.
In analgesic and anti-inflammatory study, data are expressed as
mean ꢀ SEM. Differences between vehicle control and treatment
groups were tested using one-way ANOVA followed by
TurkeyeKrammer Multiple comparison test. A probability value
less than 0.01 was considered as significant.
4.3. Computational methodology
4.3.1. Ligand preparation
The molecules were built using Maestro 8.5.207 or converted to
3D structure from the 2D structure using LigPrep.Version 2.3.
Chemdraw 3D structures were energetically minimized by using
Schrodinger Macromodel Module and saved as MDL MolFile
(*.mol2).
4.1.2.2.6. 3,6-Dimethyl-2-(4-phenylthiazol-2-yl)pyrano[4,3-c]pyr-
azol-4(2H)-one (6g). IR (nmax, cmꢂ1, KBr) 1636, 1543, 1404, 1319; 1H
NMR (CDCl3): 2.29 (s, 3H), 2.41 (s, 3H), 5.96 (s, 1H), 7.52e8.3 (m,

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