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

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 b c

Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136 119, India Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India 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: Received 11 August 2011 Received in revised form 18 January 2012 Accepted 19 January 2012 Available online 1 February 2012

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-chloro6-methyl-2H-pyran-2-one (2). Estimation of pharmacotherapeutic potential, possible molecular mechanism 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. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Dehydroacetic acid Hydrazine Pyranopyrazole Docking study Analgesic activity Anti-inflammatory activity

1. Introduction 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 antidiabetic, anti-tumour, antipyretic, anti-inflammatory, anti-hypertensive and antidepressant agents [7e12]. Considering the importance of pyrone and pyrazole derivatives, it was thought worthwhile to combine both pharmacophoric groups (pyran-2one þ pyrazole) in one molecular frame [13e20]. 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. 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: [email protected] (O. Prakash). 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2012.01.042

such as harsh reaction conditions, formation of by-products, unsatisfactorily yields, and long reaction time. 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-4chloro-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 (dehydroacetic acid, DHA, 1) and 4-chloro-3-(1-chlorovinyl)-6methyl-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

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A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89

OH O

Cl

O

O

O

PCl5 O

O

ground, 6hrs

1

2

Scheme 1. Synthesis of chloro derivative of dehydroacetic acid (2) from dehydroacetic acid (1).

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). 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 evidenced 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 hydrazines (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). It is noteworthy to mention here that the synthesis of pyrano [4,3-c]pyrazoles is reported in literature from DHA and its derivatives following two different routes (Scheme 4). First route involves

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 drawbacks 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. 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 conversion 2 / 6 proceeds via the intermediacy of hydrazine pyrones 4. 2.2. Biological evaluation 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 PharmaExpert: http://www.ibmc.msk.ru/PASS/ [23e30]. A biological activity spectrum for a substance is a list of biological 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 experimental 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).

NHNHR COCH3 H3C Cl

O

O 2

O

4 CH3

H3C

O

EtOH + RNHNH2

O

stir, 15min Cl

3

NNHR CH3

H3C

O

O

5

where R = C6H5 (a); 4-MeC6H4 (b); 4-ClC6H4 (c); 4-BrC6H4 (d); 4-MeOC6H4 (e); 4-O2NC6H4 (f); 4phenyl-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 COCH3 H3C

O

O

AcOH, reflux R N N

Method A

4 Cl

CH3 H3C

O RNHNH2 CH3

H3C

O

83

O

O

O

6

AcOH, reflux 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 antiinflammatory 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.

Activity prediction 38 Substructure descriptors; 1 new. 104 Possible activities at Pa > Pi Pa Pi 0.702 0.011 0.681 0.005 0.677 0.025 0.621 0.022 0.606 0.021 0.597 0.020 0.592 0.039 0.613 0.067 0.572 0.050 0.532 0.033 0.454 0.044 0.400 0.004 0.431 0.050 0.411 0.036 0.432 0.068 0.466 0.122 0.352 0.016 0.390 0.068 0.302 0.016 0.287 0.004 0.327 0.050

Activity 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

Table 1 Physical data of the synthesized compounds 4ae4j.

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. 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. 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 2 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 4j

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 4-Methyl-2-quinolinyl

6a 6b 6c 6d 6e 4f 6g 4h 4i 4j

206e207 170e172 180e182 289e290 153e154 285e286 248e249 297e299 129e131 222e223

209e210 172e173 179e182 e e e e e e e

76 82 85 87 79 86 80 93 90 87

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A. Kumar et al. / European Journal of Medicinal Chemistry 50 (2012) 81e89

OH

O CH3

H3C

O

O

1 RNHNH2

POCl3 reflux, 4.5 h

MeOH, reflux OH

Cl

NNHR

Cl CH2

CH3 H3C

O

H3C

O

O

O

8

7

RNHNH2

DME, reflux, 49 h

DME, reflux, 54 h

or Xylene, reflux, 12 h R

O

N N CH3

H3C

O

CH3

N

+ H3C

O

R N N

CH3

O

9

N R

H3C

O 6

10

+

O

H3C

H3C

N N R HO

N N

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 Analgesic activity of compounds 4ae4j and 6ae6e, 6g by tail immersion method. S. no Compound Time

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Control Standard 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 6a 6b 6c 6d 6e 6g

30 min

60 min

90 min

120 min

1.010  0.07 5.576  0.17** 3.100  0.36 2.390  0.21 3.262  0.43* 3.496  0.85* 2.924  0.30 3.512  0.67* 3.094  0.41 3.918  0.32** 4.136  0.61** 4.896  0.52** 3.664  0.35** 4.134  0.84* 3.596  0.49* 3.690  0.52** 3.578  0.19* 4.086  0.29**

1.388  0.03 7.420  0.36** 6.462  0.43** 3.502  0.14** 3.552  0.53 3.534  0.56 3.174  0.19 4.276  0.79* 4.114  0.58 4.582  0.30** 4.640  0.45** 4.970  1.06** 4.802  0.78** 4.300  0.03* 3.972  0.85* 3.770  0.50 3.976  0.28* 4.034  0.27*

1.730  0.10 8.934  0.18** 3.656  0.22 2.540  0.24* 3.896  0.41 3.230  0.27 3.006  0.20 3.536  0.28 5.234  1.07** 3.930  0.58 4.562  0.56* 6.380  1.16** 5.516  1.02** 3.680  0.97 2.968  0.57 3.728  0.37 5.078  1.13** 3.812  0.27

1.714  0.09 8.464  0.24** 2.744  0.25 2.520  0.36 3.776  0.35 2.630  0.34 2.840  0.81 3.544  0.61 4.796  1.06** 3.854  0.31 4.186  0.65* 4.594  0.42** 3.492  0.14 3.376  0.91 2.892  0.43 3.690  0.28 3.832  1.12 3.512  0.23

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.

Table 3B Analgesic activity of compounds 6ae6g and 4ae4j by writhing method. S. no

Drug treatment

Dose (mg/kg)

No. of animals

Change in no. of wriths

Percentage inhibition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Control Standard 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 6a 6b 6c 6d 6e 6g

e 50

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 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

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

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. 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, automated 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]. 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. Molecular 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). 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

85

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 153.93 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 respectively. Compound 4a exhibits relatively weak binding affinity with docking score of 81.86 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 87.90 and forms two hydrogen bonds between C]O of pyrone with Ser530 and Tyr385 where interspatial distance 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 (123.978) 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.

3. Conclusion The present study offers:(i) Synthesis of new hydrazino derivatives of dehydroacetic acid (4ae4j). (ii) Superior approach for the synthesis of pyrano[4,3-c]pyrazoles (6ae6e and 6g). (iii) Preliminary 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

Table 4 Anti-inflammatory activity of compounds 4ae4j and 6ae6e, 6g against carrageenan-induced rat paw oedema over 4 h. Compounda

Control Standard 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 6a 6b 6c 6d 6e 6g

Volume of oedema (ml)b 1h

2h

3h

4h

0.158  0.050 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)

0.178  0.031 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)

0.198  0.043 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)

0.204  0.057 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)

**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%).

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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

SC-558

153.933

Amino acid Interaction No. of H-bond with compound hydrogen distance (A0) involved or structural bonds feature 5

4a

81.8629 2

4b

87.9093 2

4c

87.8557 2

4d

87.8676 2

4e

86.4865 3

4f

83.3152 8

4g

108.078

4

4h

108.721

3

4i

101.868

3

4j

106.178

3

6a

96.5692 2

6b

99.0714 2

6c

99.0692 2

6c

87.6278 2

6d

99.787

2

6e

103.644

3

6g

123.978

4

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 Arg513 Gln192 Leu352 Ser353 Ser530 Tyr385 Ser530 Tyr385 Ser530 Tyr385 Ser530 Tyr385 Ser530 Arg120 Tyr355 Arg513 Tyr355 His90 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 Asp515

SO2NH2-O SO2NH2-O SO2NH2-N SO2NH2-N SO2NH2-N C]O C]O C]O C]O C]O C]O C]O C]O Acetyl C]O OCH3-O C]O NO2-O NO2-O NO2-N NO2-O NO2-N C]O C]O Pyran-O Thiaz-N Acetyl-O NHNH-2 NHNH-1 Pyran-O Acetyl C]O C]O Acetyl-O C]O Pyran-O Pyran-O Acetyl-O Acetyl-O C]O C]O C]O C]O C]O C]O Pyran-O Pyran-O C]O C]O C]O C]O OCH3-O Thiaz-N Ring-N N-Attach Pyran-O

Fig. 1. Binding mode of compound SC-558 into cyclooxygenase II pocket. It has Moldock score 153.93 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 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.)

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 tetramethylsilane (TMS) as an internal standard. Chemical shifts are expressed in d ppm. Mass spectra were recorded on 2500 eV (ESI source) using a Water’s Q-TOF micro instrument. 4.1.1. Synthesis of substituted hydrazino derivatives of dehydroacetic acid (4ae4j) 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. Similar procedure was adopted for synthesis of other derivatives (4be4j).

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 4.1. Materials and methods All reagents were purchased from commercial sources and were used without purification. Melting points were taken on slides in an

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 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 with Tyr355 of distance 2.67  A respectively. (For interpretation of the references to 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

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 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.)

4.1.1.2. Characterization data of 4-(2-substitutedhydrazinyl)-3acetyl-6-methyl-2H-pyran-2-ones (4ae4j) 4.1.1.2.1. 4-(2-Phenylhydrazinyl)-3-acetyl-6-methyl-2H-pyran-2one (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 (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-methyl2H-pyran-2-one (4b). IR (nmax, cm1, 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. 4.1.1.2.3. 4-[2-(4-Chlorophenylhydrazinyl)]-3-acetyl-6-methyl2H-pyran-2-one (4c). IR (nmax, cm1, KBr): 3434, 3368, 1713, 1643, 1551, 1443, 1373; 1H NMR (CDCl3): 2.21 (s, 3H), 2.758 (s, 3H), 5.82 (s,

Fig. 4. Binding mode of compound (6e) into the binding site of cyclooxygenase II pocket. It has Moldock score 103.644 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 A respectively. (For between eOe of OCH3 moiety with Arg120 of distance 3.32  interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

87

Fig. 5. Binding mode of compound (6g) into the binding site of cyclooxygenase II pocket. It has Moldock score 123.978 and forms 4 hydrogen bonds shown as green dotted lines (Table 5), showing one hydrogen bond between eNe of thiazole moiety with Ser353 of 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 legend, the reader is referred to the web version of this article.)

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-methyl2H-pyran-2-one (4d). IR (nmax, cm1, 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-methyl2H-pyran-2-one (4e). IR (nmax, cm1, 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. 4.1.1.2.6. 4-[2-(4-Nitrophenylhydrazinyl)]-3-acetyl-6-methyl-2Hpyran-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-6methyl-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-2Hpyran-2-one (4h). IR (nmax, cm1, 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.

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4.1.1.2.9. 4-(3,5-Dimethylpyrimidinyllhydrazinyl)-3-acetyl-6methyl-2H-pyran-2-one (4i). IR (nmax, cm1, 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. 4.1.1.2.10. 4-(4-Methylquinolinyllhydrazinyl)-3-acetyl-6-methyl2H-pyran-2-one (4j). IR (nmax, cm1, 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. 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)-3acetyl-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 obtained was filtered, recrystallised using ethanol to gave pyrano[4,3c]pyrazole 6a. Similar procedure was adopted for the synthesis of other derivatives. 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, cm1, 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 4.1.2.2.2. 3,6-Dimethyl-2-(4-methylphenyl)pyrano[4,3-c]pyrazol4(2H)-one (6b). IR (nmax, cm1, KBr) 3100, 1725 cm1; 1H NMR (CDCl3) 2.3 (3H, s), 2.4 (3H, s), 2.6 (3H, s), 6.3 (1H, s), 7.3 (4H, s); 13C 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]pyrazol4(2H)-one (6c). IR (nmax, cm1, KBr) 3100, 1760 cm1; 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]pyrazol4(2H)-one (6d). IR (nmax, cm1, KBr) 3100, 1760 cm1; 1H NMR (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, 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]pyrazol-4(2H)-one (6e). IR (nmax, cm1, 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. 4.1.2.2.6. 3,6-Dimethyl-2-(4-phenylthiazol-2-yl)pyrano[4,3-c]pyrazol-4(2H)-one (6g). IR (nmax, cm1, KBr) 1636, 1543, 1404, 1319; 1H NMR (CDCl3): 2.29 (s, 3H), 2.41 (s, 3H), 5.96 (s, 1H), 7.52e8.3 (m,

6H). Anal. Calcd. for C17H13N2O2S: C, 63.14; H, 4.05; N, 12.99. Found: C, 63.03; H, 4.00; N, 12.89. 4.2. Pharmacological assay 4.2.1. Animals used Adult Swiss albino mice (20e25 g) were used in the experiments. 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 analgesic 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 injection 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:

ð1  Vc =Vt Þ  100 where Vt ¼ mean number of writhing in test animals; Vc ¼ mean 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 All the synthesized compounds were tested for their antiinflammatory 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. 4.2.4. Statistical analysis 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).

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