European Journal of Medicinal Chemistry 50 (2012) 81e89

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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

Received 11 August 2011 (4ae4j) and pyrano[4,3-c]pyrazoles (6ae6e and 6g) were synthesized starting from 3-acetyl-4-chloro-
Received in revised form 6-methyl-2H-pyran-2-one (2). Estimation of pharmacotherapeutic potential, possible molecular mech-
18 January 2012
anism of action, toxic/side effects and interaction with drug-metabolizing enzymes were made for the
Accepted 19 January 2012
Available online 1 February 2012
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,
Keywords:
Dehydroacetic acid
6e, 6g exhibited good anti-inflammatory activity, whereas compounds 4b, 4c, 4h, 4i, 4j, 6b, 6e, 6g
Hydrazine showed excellent analgesic activity compared with standard drug Diclofenac sodium.
Pyranopyrazole Ó 2012 Elsevier Masson SAS. All rights reserved.
Docking study
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 The present study illustrates a new preparative strategy for
of interest due to their wide range of biological activities [1e6]. The synthesis of pyranopyrazoles by overcoming all the above
incorporation of another heterocyclic moiety in pyrones either in mentioned limitations.
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- 2. Results and discussion
diabetic, anti-tumour, antipyretic, anti-inflammatory, anti-hyper-
tensive and antidepressant agents [7e12]. Considering the 2.1. Chemistry
importance of pyrone and pyrazole derivatives, it was thought
worthwhile to combine both pharmacophoric groups (pyran-2- Our research work started with the preparation of 3-acetyl-4-
one þ pyrazole) in one molecular frame [13e20]. chloro-6-methyl-2H-pyran-2-one (Cl-DHA, 2) to be used as
These observations contemplated us to synthesize some new a substitute for 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one
pyranopyrazole derivatives of type 6 with a view to explore their (dehydroacetic acid, DHA, 1) and 4-chloro-3-(1-chlorovinyl)-6-
potency as good analgesic and anti-inflammatory agents. methyl-2H-pyran-2-one (dehydroacetchlorid, 8 ) for the synthesis
Earlier, the synthesis of pyranopyrazoles has been reported from of pyrano[4,3-c]pyrazoles, which was prepared following the
dehydroacetic acid (2-pyrone) following two different routes [21] literature method [22] (Scheme 1).
but the reported methods suffer from some serious drawbacks 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
* Corresponding author. Tel.: þ91 1744 239617 (0); fax: þ91 1744 238277/035/
NMR and 13C NMR) and elemental analysis data of the product
628. indicated the formation of new hydrazino pyrone 4a rather than
E-mail address: dromprakash50@rediffmail.com (O. Prakash). the expected hydrazone 5a. The scope of the reaction was studied

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

OH O Cl O treatment of dehydroacetic acid with hydrazines to form

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

NHNHR
COCH3

H3C O O
Cl O
4
CH3 EtOH
+ RNHNH2
H3C O O stir, 15min
Cl NNHR
2 3
CH3

H3C O O

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 83

NHNHR
COCH3
AcOH, reflux
H3C O O Method A R
N N
4 CH3

Cl O H3C O O
RNHNH2 6
CH3 AcOH, reflux
H3C O O Method B

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’ We have checked for the analgesic and anti-inflammatory
relationship analysis of PASS prediction results for the set of activity for the compounds which have Pa nearly equal to 0.5 and
studied compounds. Analgesic activity was predicted for all experimentally they have shown good results which means they
compounds with Pa value between 0.592 and 0.794 and anti- are NCE.
inflammatory activity with Pa between 0.421 and 0.592. Besides, Thus, our study showed that prediction of biological activity
all compounds were predicted as cyclooxygenase inhibitors with spectra for synthesized compounds by PASS and its analysis made
Pa > 0.3. For example PASS prediction results of one of the by PharmaExpert may estimate pharmacotherapeutic potential,
compound 6g are summarized here. 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 2.2.2. Analgesic activity

38 Substructure descriptors; 1 new.
The analgesic activity of the above mentioned derivatives was
104 Possible activities at Pa > Pi
Pa Pi Activity evaluated by following tail immersion methods [31] and acetic acid
0.702 0.011 Analgesic, non-opioid induced writhing assay [32,33] using diclofenac as a standard.
0.681 0.005 5 Hydroxytryptamine release inhibitor Results were expressed as mean  SEM differences between
0.677 0.025 Antineoplastic (ovarian cancer) control and treatment group and were tested using one-way
0.621 0.022 Analgesic
0.606 0.021 Antiarthritic
ANOVA followed by the least significant differences (LSD).
0.597 0.020 Cognition disorders treatment According to Tables 3A and 3B, compounds 4h, 4j, 6a, 6d, 6g, 6j
0.592 0.039 Anti-inflammatory exhibited equipotent analgesic effect or slightly less than that of
0.613 0.067 Antiviral (Arbovirus) standard after 1 h and 2 h post administration. While compounds
0.572 0.050 Complement factor D inhibitor
4a, 4b, 4h, 4i, 4j, 6a exhibited the analgesic effect after 1 h of
0.532 0.033 Immunomodulator
0.454 0.044 Immunosuppressant administration only. After 2 h compounds 4g, 4j, 6b also have
0.400 0.004 Cyclooxygenase inhibitor shown analgesic effect. Thus, it can be concluded that, compounds
0.431 0.050 HCV IRES inhibitor 4a, 4b, 4g, 4h, 4i, 6a, 6b, 6d, 6g have significant analgesic activity
0.411 0.036 Endothelial growth factor antagonist according to tail immersion method. The results from acetic acid
0.432 0.068 Histamine release inhibitor
0.466 0.122 Antianaemic
Induced writhing assay revealed that all tested compounds
0.352 0.016 Follicle-stimulating hormone agonist exhibited significant activity. Compounds 4b, 4c, 4f, 4h, 4i, 6b, 6e,
0.390 0.068 Mediator release inhibitor 6g have nearly the same activity as the reference drug.
0.302 0.016 Antithrombocytopenic
0.287 0.004 Cyclooxygenase 2 inhibitor
2.2.3. Anti-inflammatory activity
0.327 0.050 GABA receptor agonist
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 4a 267e268 89 C6H5 6a 206e207 209e210 76
4-MeC6H4 4b 209e210 87 4-MeC6H4 6b 170e172 172e173 82
4-ClC6H4 4c 207e208 96 4-ClC6H4 6c 180e182 179e182 85
4-BrC6H4 4d 185e186 95 4-BrC6H4 6d 289e290 e 87
4-MeOC6H4 4e 225e226 89 4-MeOC6H4 6e 153e154 e 79
4-O2NC6H4 4f 285e286 94 4-O2NC6H4 4f 285e286 e 86
4-Phenyl-2-thiazolyl 4g >300 93 4-Phenyl-2-thiazolyl 6g 248e249 e 80
2-Benzothiazolyl 4h 297e299 95 2-Benzothiazolyl 4h 297e299 e 93
4,6-Dimethyl-2-pyrimidinyl 4i 129e131 90 4,6-Dimethyl-2-pyrimidinyl 4i 129e131 e 90
4-Methyl-2-quinolinyl 4j 222e223 87 4-Methyl-2-quinolinyl 4j 222e223 e 87
84 A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89

OH O

CH3

H3C O O

RNHNH2 POCl3
MeOH, reflux reflux, 4.5 h

OH NNHR Cl Cl

CH3 CH2

H3C O O H3C O O
8
7

RNHNH2
DME, reflux, 49 h
or DME, reflux, 54 h

Xylene, reflux, 12 h

R R
O CH3
N N N N
CH3 CH3
+ N
H3C O N
H3C O O R H3C O O

9 6
10
+

H3C
H3C
N
N N N
R
R HO

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 Dose No. of Change in no. Percentage
30 min 60 min 90 min 120 min treatment (mg/kg) animals of wriths inhibition
1 Control 1.010  0.07 1.388  0.03 1.730  0.10 1.714  0.09 1 Control e 5 49.00  2.16** e
2 Standard 5.576  0.17** 7.420  0.36** 8.934  0.18** 8.464  0.24** 2 Standard 50 5 11.00  0.70** 77.55
3 4a 3.100  0.36 6.462  0.43** 3.656  0.22 2.744  0.25 3 4a 5 24.60  2.90** 49.79
4 4b 2.390  0.21 3.502  0.14** 2.540  0.24* 2.520  0.36 4 4b 5 14.60  2.61** 70.20
5 4c 3.262  0.43* 3.552  0.53 3.896  0.41 3.776  0.35 5 4c 5 12.20  2.05** 75.10
6 4d 3.496  0.85* 3.534  0.56 3.230  0.27 2.630  0.34 6 4d 5 23.00  3.03** 53.06
7 4e 2.924  0.30 3.174  0.19 3.006  0.20 2.840  0.81 7 4e 5 20.00  2.40** 59.18
8 4f 3.512  0.67* 4.276  0.79* 3.536  0.28 3.544  0.61 8 4f 5 25.80  2.31** 47.34
9 4g 3.094  0.41 4.114  0.58 5.234  1.07** 4.796  1.06** 9 4g 5 23.80  3.59** 51.42
10 4h 3.918  0.32** 4.582  0.30** 3.930  0.58 3.854  0.31 10 4h 5 17.20  2.91** 65.30
11 4i 4.136  0.61** 4.640  0.45** 4.562  0.56* 4.186  0.65* 11 4i 5 17.20  2.63** 64.89
12 4j 4.896  0.52** 4.970  1.06** 6.380  1.16** 4.594  0.42** 12 4j 5 12.40  1.07** 75.00
13 6a 3.664  0.35** 4.802  0.78** 5.516  1.02** 3.492  0.14 13 6a 5 24.40  2.37** 50.20
14 6b 4.134  0.84* 4.300  0.03* 3.680  0.97 3.376  0.91 14 6b 5 15.00  1.67** 69.38
15 6c 3.596  0.49* 3.972  0.85* 2.968  0.57 2.892  0.43 15 6c 5 17.00  2.56** 65.30
16 6d 3.690  0.52** 3.770  0.50 3.728  0.37 3.690  0.28 16 6d 5 27.00  4.82** 44.89
17 6e 3.578  0.19* 3.976  0.28* 5.078  1.13** 3.832  1.12 17 6e 5 11.80  2.22** 75.91
18 6g 4.086  0.29** 4.034  0.27* 3.812  0.27 3.512  0.23 18 6g 5 14.60  3.29** 70.20

N ¼ 5, values are expressed as mean  SEM and analyzed by ANOVA. 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. **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 of the ligands to give a number of conformations from which the
carrageenan. After the administration of carrageenan inflammation best mode could be achieved. In the analysis of docking results we
was developed in mice, the effect was measured in the interval of tried to find a correlation between the biological results and docking
1 h, 2 h, 3 h, and 4 h respectively. The percentage inhibition of studies.
oedema was calculated as a regard to saline control group, as From Table 5 and Figs. 1e5 the following results can be drawn:
depicted in Table 4. SC-558 (the original ligand) reveals docking score of 153.93 and
Most of the tested compounds have shown good results in forms five interactions shown as green dotted lines (Fig. 1),
comparison with standard drug. Amongst all the compounds, showing two hydrogen bonds between O of SO2NH2 moiety with
compounds 4h, 4j, 6e, 6g, have shown potent anti-inflammatory His90 and Arg513 of distances 2.70  A and 3.53 A respectively and
activity. 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-
2.3. Docking studies tively. Compound 4a exhibits relatively weak binding affinity with
docking score of 81.86 but forms two hydrogen bonds between
2.3.1. Aim of work C]O of pyrone with Ser530 and Tyr385. Further an increase in
To pre-asses the anti-inflammatory behaviour of hydrazino activity was observed in case of compound 4b, when eCH3 group
derivatives and pyrano[4,3-c]pyrazoles on a structural basis, auto- was introduced at para position. Compound 4b showed a docking
mated docking studies were carried out using Molegro Virtual score of 87.90 and forms two hydrogen bonds between C]O of
Docker, the scoring functions and hydrogen bonds formed with the pyrone with Ser530 and Tyr385 where interspatial distance
surrounding amino acids are used to predict their binding modes, between these is about 2.97  A and 3.19 
A respectively. Similar types
their binding affinities and orientation of these compounds at the of interactions were observed in case of compounds 4c, 4d when
active site of cyclooxygenase-II enzyme [35]. electron withdrawing groups (Cl and Br) were inserted at para
position. However, their cyclised analogues were found to be more
2.3.2. Binding affinities of the synthesized compounds into COX-II active as clear from their docking scores and compound 6g has the
The crystal structure was downloaded (PDB code: 1CX2) and our best docking score (123.978) being able to interact by its ring-N
compounds were docked into the active sites using Molegro Virtual and pyran-O with Ser353, Gln192, His90 and Asp515, respectively.
Docker 2008.3.2. software [35]. Compounds were ranked after Also, some interesting results were obtained in case of compounds
docking according to their docking scores and were visualised inside 4f, 4g, 4h, 4i, 4j as clear from their docking score which may be due
the pocket to view their fitting and closure to main residues. Molec- to insertion of heteroaryl moieties at the place of phenyl ring.
ular docking studies were revealed further insight into the nature of Hence, it can be concluded that relative COX-II inhibitory
interactions between the compounds and the active site amino acids potency order for this group follows the trend phenylthiazolyl >
to rationalize the obtained biological results. The next stage of protein benzothiazolyl > quinolinyl > pyrimidinyl > OCH3 > Br > CH3 > H.
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 3. Conclusion
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 The present study offers:(i) Synthesis of new hydrazino deriv-
well and we can see the possible interactions (Figs. 1e5). atives of dehydroacetic acid (4ae4j). (ii) Superior approach for the
synthesis of pyrano[4,3-c]pyrazoles (6ae6e and 6g). (iii) Prelimi-
2.3.3. Results and discussion nary prediction of biological activity spectra for title compounds by
Molegro Virtual Docker allows the flexible docking of ligands PASS computer program and further confirmation by experimental
into its site of action. It has the ability to use all the rotatable bonds evaluation and validation via docking studies. (iv) Identification of

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

1h 2h 3h 4h
Control 0.158  0.050 0.178  0.031 0.198  0.043 0.204  0.057
Standard 0.046  0.022(70.88)c 0.058  0.008**(67.41) 0.060  0.020**(69.70) 0.068  0.022*(66.67)
4a 0.064  0.008(59.49) 0.076  0.010*(57.30) 0.088  0.020*(55.56) 0.098  0.014(51.96)
4b 0.063  0.014(60.12) 0.083  0.017(53.37) 0.080  0.024(59.59) 0.102  0.015(50.00)
4c 0.072  0.023(54.43) 0.076  0.016*(57.30) 0.082  0.010*(58.59) 0.146  0.014(28.43)
4d 0.084  0.012(46.84) 0.094  0.015(47.19) 0.118  0.014(40.40) 0.166  0.020(18.63)
4e 0.079  0.015(50.00) 0.090  0.016(49.43) 0.110  0.018(44.44) 0.154  0.019(24.50)
4f 0.074  0.015(53.16) 0.080  0.025*(55.05) 0.108  0.023(45.45) 0.114  0.034(44.12)
4g 0.076  0.019(51.90) 0.092  0.024(48.31) 0.108  0.024(45.45) 0.156  0.021(23.53)
4h 0.076  0.023(64.56) 0.074  0.019*(58.42) 0.090  0.026*(54.55) 0.104  0.013(49.02)
4i 0.064  0.042(59.49) 0.068  0.046*(61.79) 0.088  0.042(55.56) 0.118  0.075(42.16)
4j 0.076  0.015(51.90) 0.080  0.026*(55.05) 0.088  0.026*(55.56) 0.100  0.008(50.98)
6a 0.068  0.019(56.96) 0.080  0.012*(55.05) 0.094  0.016*(52.53) 0.116  0.028(43.14)
6b 0.058  0.015(63.29) 0.074  0.014(58.42) 0.091  0.015(54.04) 0.119  0.021(41.66)
6c 0.066  0.044(58.23) 0.072  0.014*(59.55) 0.086  0.008*(56.57) 0.126  0.024(38.24)
6d 0.064  0.025(59.49) 0.088  0.012(50.56) 0.100  0.022(49.49) 0.126  0.022(38.24)
6e 0.056  0.038(64.56) 0.082  0.024(53.93) 0.084  0.024*(57.58) 0.118  0.020(42.16)
6g 0.052  0.016(67.09) 0.072  0.024*(59.55) 0.074  0.020*(62.63) 0.096  0.025(52.94)

**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 No. of H-bond Amino acid Interaction

scores hydrogen distance (A0) involved with compound
bonds or structural
feature
SC-558 153.933 5 2.70 His90 SO2NH2-O
3.53 Arg513 SO2NH2-O
3.18 Gln192 SO2NH2-N
3.09 Leu352 SO2NH2-N
2.98 Ser353 SO2NH2-N
4a 81.8629 2 3.13 Ser530 C]O
3.09 Tyr385 C]O
4b 87.9093 2 2.97 Ser530 C]O
3.19 Tyr385 C]O
4c 87.8557 2 2.97 Ser530 C]O
3.19 Tyr385 C]O
Fig. 1. Binding mode of compound SC-558 into cyclooxygenase II pocket. It has Mol-
4d 87.8676 2 2.97 Ser530 C]O
dock score 153.93 and forms 5 hydrogen bonds shown as green dotted lines (Table 5),
3.19 Tyr385 C]O
showing two hydrogen bonds between O of SO2NH2 moiety with His90, and Arg513 of
4e 86.4865 3 3.41 Ser530 Acetyl C]O
distances 2.70 A and 3.53 
A respectively and three other hydrogen bonds between N of
2.84 Arg120 OCH3-O
SO2NH2 moiety with Gln192, Leu352, and Ser353 of distances 3.18  A, 3.09 
A, and 2.98 
A
2.48 Tyr355 C]O
respectively. (For interpretation of the references to colour in this figure legend, the
4f 83.3152 8 2.94 Arg513 NO2-O
reader is referred to the web version of this article.)
2.60 Tyr355 NO2-O
3.27 His90 NO2-N
2.63 His90 NO2-O
3.05 Tyr355 NO2-N
2.92 Ser530 C]O electrical apparatus Labindia visual melting range apparatus and
2.82 Tyr385 C]O are uncorrected. IR spectra were recorded on a PerkineElmer 1800
3.56 Tyr385 Pyran-O
4g 108.078 4 2.47 Ser353 Thiaz-N
FT-IR spectrophotometer. The 1H NMR and 13C NMR spectra were
3.06 Thr94 Acetyl-O recorded in CDCl3 on Bruker Nuclear Magnetic Resonance (NMR)
3.43 His90 NHNH-2 spectrometer at 300 and 75 MHz respectively, using tetrame-
3.42 Gln192 NHNH-1 thylsilane (TMS) as an internal standard. Chemical shifts are
4h 108.721 3 3.01 His90 Pyran-O
expressed in d ppm. Mass spectra were recorded on 2500 eV (ESI
3.01 Tyr355 Acetyl C]O
2.67 Tyr355 C]O source) using a Water’s Q-TOF micro instrument.
4i 101.868 3 2.72 Tyr355 Acetyl-O
2.93 Tyr355 C]O 4.1.1. Synthesis of substituted hydrazino derivatives of
3.10 His90 Pyran-O dehydroacetic acid (4ae4j)
4j 106.178 3 3.20 Ser530 Pyran-O
4.1.1.1. Procedure. 3-Acetyl-4-chloro-6-methyl-2H-pyran-2-one (2,
3.46 Gly526 Acetyl-O
3.36 Ala527 Acetyl-O 1.86 g, 10 mmol) was treated with phenylhydrazine (3a, 1.08 ml,
6a 96.5692 2 3.43 Ser530 C]O 10 mmol) using ethanol (20 ml) as a solvent and stirred for
3.14 Tyr385 C]O 15e20 min at room temperature to give hydrazino derivative 4a in
6b 99.0714 2 3.05 Ser530 C]O
fair to good yields.
2.80 Tyr385 C]O
6c 99.0692 2 2.80 Ser530 C]O
Similar procedure was adopted for synthesis of other derivatives
3.05 Tyr385 C]O (4be4j).
6c 87.6278 2 2.99 Ser530 Pyran-O
2.94 Tyr385 Pyran-O
6d 99.787 2 3.26 Ser530 C]O
3.31 Tyr385 C]O
6e 103.644 3 3.07 Ser530 C]O
3.00 Tyr385 C]O
3.32 Arg120 OCH3-O
6g 123.978 4 2.77 Ser353 Thiaz-N
3.07 Gln192 Ring-N
3.50 His90 N-Attach
3.30 Asp515 Pyran-O

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 108.721 and forms 3 hydrogen bonds shown as green
4.1. Materials and methods dotted lines (Table 5), showing one hydrogen bond between eOe of pyran moiety with
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 106.178 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.
Gly526 and Ala527 of distances 3.46  A and 3.36 A respectively. (For interpretation of It has Moldock score 123.978 and forms 4 hydrogen bonds shown as green dotted lines
the references to colour in this figure legend, the reader is referred to the web version (Table 5), showing one hydrogen bond between eNe of thiazole moiety with Ser353 of
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
4.1.1.2. Characterization data of 4-(2-substitutedhydrazinyl)-3- distance 3.30 
A respectively. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
acetyl-6-methyl-2H-pyran-2-ones (4ae4j)
4.1.1.2.1. 4-(2-Phenylhydrazinyl)-3-acetyl-6-methyl-2H-pyran-2-
one (4a). IR (nmax, cm1, 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 1H), 6.55 (s, 1H, NH), 6.8e7.34 (m, 4H), 15.5 (s, 1H, NH). Anal. Calcd.
(s, 1H, NH), 6.8e7.3 (m, 5H), 15.6 (s, 1H, NH); MS (ESI) m/z ¼ 259.17 for C14H13ClN2O3: C, 57.44; H, 4.48; N, 9.57. Found: C, 57.39; H, 4.41;
(M þ Hþ). Anal. Calcd. for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85. N, 9.53.
Found: C, 65.03; H, 5.41; N, 10.79. 4.1.1.2.4. 4-[2-(4-Bromophenylhydrazinyl)]-3-acetyl-6-methyl-
4.1.1.2.2. 4-[2-(4-Methylphenylhydrazinyl)]-3-acetyl-6-methyl- 2H-pyran-2-one (4d). IR (nmax, cm1, KBr): 3448, 3356, 1713, 1651,
2H-pyran-2-one (4b). IR (nmax, cm1, KBr): 3413, 3358, 1705, 1649, 1612, 1543, 1450, 1389; 1H NMR (CDCl3): 2.2 (s, 3H), 2.71 (s, 3H),
1551, 1450, 1350; 1H NMR (CDCl3): 2.21 (s, 3H), 2.30 (s, 3H), 2.72 (s, 5.84 (s, 1H), 6.65 (s, 1H, NH), 6.75e7.42 (m, 4H), 15.6 (s, 1H, NH); MS
3H), 5.86 (s, 1H), 6.54 (s, 1H, NH), 6.7e7.4 (m, 4H), 15.5 (s, 1H, NH). (ESI) m/z ¼ 337.11 (M þ Hþ). Anal. Calcd. for C14H13BrN2O3: C, 49.87;
Anal. Calcd. for C15H16N2O3: C, 66.16; H, 5.92; N, 10.29. Found: C, H, 3.89; N, 8.31. Found: C, 49.83; H, 3.81; N, 8.29.
66.13; H, 5.87; N, 10.22. 4.1.1.2.5. 4-[2-(4-Methoxyphenylhydrazinyl)]-3-acetyl-6-methyl-
4.1.1.2.3. 4-[2-(4-Chlorophenylhydrazinyl)]-3-acetyl-6-methyl- 2H-pyran-2-one (4e). IR (nmax, cm1, KBr): 3418, 3364, 1697, 1651,
2H-pyran-2-one (4c). IR (nmax, cm1, KBr): 3434, 3368, 1713, 1643, 1620, 1558, 1474, 1366; 1H NMR (CDCl3): 2.18 (s, 3H), 2.57 (s, 3H),
1551, 1443, 1373; 1H NMR (CDCl3): 2.21 (s, 3H), 2.758 (s, 3H), 5.82 (s, 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.
4.1.1.2.6. 4-[2-(4-Nitrophenylhydrazinyl)]-3-acetyl-6-methyl-2H-
pyran-2-one (4f). IR (nmax, cm1, 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, cm1, 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-
Fig. 4. Binding mode of compound (6e) into the binding site of cyclooxygenase II pyran-2-one (4h). IR (nmax, cm1, KBr): 3433, 3402, 1703, 1620,
pocket. It has Moldock score 103.644 and forms 3 hydrogen bonds shown as green 1566, 1466, 1396,1358; 1H NMR (CDCl3): 2.29 (s, 3H), 2.69 (s, 3H),
dotted lines (Table 5), showing two hydrogen bonds between eOe of 2-pyran moiety 5.96 (s, 1H), 7.82e8.04 (m, 4H); 13C NMR 16.60, 19.88, 96.11, 104.95,
(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
106.65, 112.01, 113.8, 121.61, 127.41, 130.05, 135.46, 148.19, 162.89,
interpretation of the references to colour in this figure legend, the reader is referred to 179.23. Anal. Calcd. for C15H13N3O3S: C, 57.13; H, 4.16; N, 13.33.
the web version of this article.) Found: C, 57.03; H, 4.09; N, 13.29.
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:
methyl-2H-pyran-2-one (4i). IR (nmax, cm1, KBr): 3456, 3425, 1713, C, 63.03; H, 4.00; N, 12.89.
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, 4.2. Pharmacological assay
NH). Anal. Calcd. for C14H16N4O3: C, 58.32; H, 5.59; N, 19.43. Found:
C, 58.30; H, 5.47; N, 19.39. 4.2.1. Animals used
4.1.1.2.10. 4-(4-Methylquinolinyllhydrazinyl)-3-acetyl-6-methyl- Adult Swiss albino mice (20e25 g) were used in the experi-
2H-pyran-2-one (4j). IR (nmax, cm1, KBr): 3441, 3402,1713,1628,1543, ments. Animals were housed under standardized conditions for
1443, 1420, 1381; 1H NMR (CDCl3): 2.16 (s, 3H), 2.5 (s, 3H), 2.6 (s, 3H), light and temperature. Animals were randomly assigned to
5.99 (s,1H), 7.2 (s, IH), 7.1e7.6 (m, 4H), 7.3 (s, IH, NH),10.5 (s,1H, NH); 13C different experimental groups, each kept in a separate cage. Study
NMR (CDCl3) 16.08, 19.59, 19.77, 106.58, 113.96, 115.89, 121, 121.42, protocol was approved by the Institutional Animal Ethics
125.11, 125.90, 126.00, 129.93, 132.22, 136.39, 145.88, 149.49, 159.16, Committee before experiment.
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. 4.2.2. Analgesic activity screening
Acetic acid induced writhing model was used to evaluate anal-
4.1.2. Synthesis of 3,6-dimethyl-2-(substituted)pyrano[4,3-c] gesic activity of the synthesized compounds. Groups of five Swiss
pyrazoles 6 albino mice, each 20e25 g body weight were used and 0.6% acetic
4.1.2.1. Procedure. Method A: A solution of 4-(2-phenylhydrazinyl)-3- acid (10 ml/kg) was injected intra-peritoneally. The numbers of
acetyl-6-methyl-2H-pyran-2-one (4a, 0.258 g, 1mmol) in acetic acid wriths were counted for 10 min, immediately after 5 min of injec-
was refluxed for 6e7 h, cooled to room temperature and then poured tion of acetic acid in each mice. This reading was taken as control.
into an ice cold solution. The solid product thus obtained was filtered, Next day, same group of mice were used for evaluation. Each group
recrystallised using ethanol to gave pyrano[4,3-c]pyrazole 6a. was administered orally with the synthesized compounds. The dose
Method B: 3-Acetyl-4-chloro-6-methyl-2H-pyran-2-one (2, of 50 mg/kg of animal was given 30 min before injection of acetic
1.86 g, 10 mmol) was refluxed with phenylhydrazine (3a, 1.08 ml, acid. After 5 min of acetic acid injection, mice were observed for the
10 mmol) in acetic acid for 6e7 h, cooled to room temperature and number of writhing for the duration of 10 min. The mean value for
then poured into an ice cold solution. The solid product thus ob- each group was calculated and compared with control. Diclofenac
tained was filtered, recrystallised using ethanol to gave pyrano[4,3- sodium was used as a standard drug for comparison of analgesic
c]pyrazole 6a. activity. Percent protection was calculated using the formula:
Similar procedure was adopted for the synthesis of other
derivatives. ð1  Vc =Vt Þ  100

4.1.2.2. Characterization data of 3,6-dimethyl-2-(substituted)pyrano where Vt ¼ mean number of writhing in test animals; Vc ¼ mean
[4,3-c]pyrazoles number of writhing in control.
4.1.2.2.1. 3,6-Dimethyl-2-phenylpyrano[4,3-c]pyrazol-4(2H)-one Statistical significance was analyzed using one-way ANOVA
(6a). IR (nmax, cm1, KBr) 3070, 1715; 1H NMR (CDCl3) 2.2 (3H, s), followed by TurkeyeKrammer multiple comparison test and
2.6 (3H, s), 6.25 (1H, s), 7.45 (5H, s); 13C NMR (CDCl3) 11.8, 19.9, 96.7, p < 0.01 was considered significant.
105.8, 125.3, 129.3, 138.6, 143.2, 151.0, 157.4, 160.1
4.1.2.2.2. 3,6-Dimethyl-2-(4-methylphenyl)pyrano[4,3-c]pyrazol- 4.2.3. Anti-inflammatory activity screening
4(2H)-one (6b). IR (nmax, cm1, KBr) 3100, 1725 cm1; 1H NMR All the synthesized compounds were tested for their anti-
(CDCl3) 2.3 (3H, s), 2.4 (3H, s), 2.6 (3H, s), 6.3 (1H, s), 7.3 (4H, s); 13C inflammatory activity using carrageenan-induced rat hind paw
NMR (CDCl3) 11.8, 19.8, 21.0, 96.7, 106.0, 125.1, 129.8, 138.1, 139.1, oedema method of Winter et al. [34] the oedema hind paw was
143.1, 150.8, 157.2, 160.0 induced by injection of 0.1 ml of 1% carrageenan solution into
4.1.2.2.3. 3,6-Dimethyl-2-(4-chlorophenyl)pyrano[4,3-c]pyrazol- subplanter region of right hand paw. The volume of paw was
4(2H)-one (6c). IR (nmax, cm1, KBr) 3100, 1760 cm1; 1H NMR measured plethysmographically immediately and 1 h, 2 h, 3 h, 4 h
(CDCl3) 2.4 (3H, s), 2.7 (3H, s), 6.7 (1H, s), 7.3, 7.4, 7.6, 7.7 (4H, AB after the injection of irritant. The difference in volume gave the
system); 13C NMR (CDCl3) 11.3, 19.6, 95.2, 105.3, 127.4, 130.7, 133.1, amount of oedema developed. Percent inhibition of the oedema
138.5, 147.8, 150.2, 161.6, 163.0 between the control group and the compound treated group was
4.1.2.2.4. 3,6-Dimethyl-2-(4-bromophenyl)pyrano[4,3-c]pyrazol- calculated and compared with the group receiving standard drug at
4(2H)-one (6d). IR (nmax, cm1, KBr) 3100, 1760 cm1; 1H NMR 50 mg/kg b.w. The results are tabulated in Table 5.
(CDCl3) 2.32 (3H, s), 2.5 (3H, s), 6.636 (1H, s), 7.37e7.73 (m, 4H); 13C
NMR (CDCl3) 15.5, 19.78, 96.79, 106.58, 121.05, 121.45, 121.93, 4.2.4. Statistical analysis
129.19, 129.93, 149.72, 159.19, 162.12; MS (ESI) m/z ¼ 319.11 In analgesic and anti-inflammatory study, data are expressed as
(M þ Hþ). Anal. Calcd. for C14H11BrN2O2: C, 52.69; H, 3.47; N, 8.78. mean  SEM. Differences between vehicle control and treatment
Found: C, 52.57; H, 3.50; N, 8.73. groups were tested using one-way ANOVA followed by
4.1.2.2.5. 3,6-Dimethyl-2-(4-methoxyphenyl)pyrano[4,3-c]pyr- TurkeyeKrammer Multiple comparison test. A probability value
azol-4(2H)-one (6e). IR (nmax, cm1, KBr) 3086, 1713, 1620,1551, less than 0.01 was considered as significant.
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, 4.3. Computational methodology
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, 4.3.1. Ligand preparation
147.8, 150.2, 161.6, 163.0. Anal. Calcd. for C15H14N2O3: C, 66.66; H, The molecules were built using Maestro 8.5.207 or converted to
5.22; N, 10.36. Found: C, 66.59; H, 5.21; N, 10.26. 3D structure from the 2D structure using LigPrep.Version 2.3.
4.1.2.2.6. 3,6-Dimethyl-2-(4-phenylthiazol-2-yl)pyrano[4,3-c]pyr- Chemdraw 3D structures were energetically minimized by using
azol-4(2H)-one (6g). IR (nmax, cm1, KBr) 1636, 1543, 1404, 1319; 1H Schrodinger Macromodel Module and saved as MDL MolFile
NMR (CDCl3): 2.29 (s, 3H), 2.41 (s, 3H), 5.96 (s, 1H), 7.52e8.3 (m, (*.mol2).
 A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89 89

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