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Archiv der Pharmazie - Chemistry in Life Science

Pyrazolobenzotriazinones Derivatives as COX Inhibitors: Synthesis Biological Activity and Molecular Modeling Studies

r Fo Journal:

Manuscript ID:

Wiley - Manuscript type:

Complete List of Authors:

ardp.200900317.R1 Full Paper 17-Feb-2010

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Date Submitted by the Author:

Archiv der Pharmazie

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Raffa, Demetrio; Università di Palermo, Dipartimento di Chimica e Tecnologie Farmaceutiche Migliara, Onofrio; Università di Palermo, Dipartimento di Chimica e Tecnologie Farmaceutiche Maggio, Benedetta; Università di Palermo, Dipartimento di Chimica e Tecnologie Farmaceutiche Plescia, Fabiana; Università di Palermo, Dipartimento di Chimica e Tecnologie Farmaceutiche Cascioferro, Stella; Università di Palermo, Dipartimento di Chimica e Tecnologie Farmaceutiche Cusimano, Maria; Università di Palermo, Dipartimento di Chimica e Tecnologie Farmaceutiche Giuseppe, Tringali; Università Cattolica del Sacro Cuore, Dipartimento di Farmacologia Cannizzaro, Carla; Università di Palermo, Dipartimento di Scienze Farmacologiche "Pietro Benigno" Plescia, Fulvio; Università di Palermo, Dipartimento di Scienze Farmacologiche "Pietro Benigno"

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vi

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Manuscript Keywords :

Anti-inflammatory activity, Synthesis, Pyrazole, benzotriazinones

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Pyrazolobenzotriazinones Derivatives as COX Inhibitors: Synthesis Biological Activity and Molecular Modeling Studies Demetrio Raffa

1*

, Onofrio Migliara 1 , Benedetta Maggio 1 , Fabiana Plescia 1 , Stella

Cascioferro 1 , Maria Grazia Cusimano 1 , Giuseppe Tringale 2 , Carla Cannizzaro 3 , and Fulvio Plescia 3 . 1

Department of Pharmaceutical Chemistry and Technology, Universit y of Palermo, Via Archirafi, 32, I-90123-Palermo, Ital y 2 3

Institut of Pharmacology, Catholic Universit y of S. Heart, Rome, Ital y

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Department of Pharmacological Sciences “Pietro Benigno�, Universit y of Palermo, Via del Vespro, 129, I-90127-Palermo, Ital y.

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Running title: P yrazolobenzotriazinones Derivatives as COX Inhibitors

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Correspondence: Prof. Demetrio Raffa, Dipartimento di Chimica e Tecnologie Farmaceutiche, Via Archirafi, 32, I-90123-Palermo, Ital y. Phone: + 39 91 6236117 e-mail: demraffa@unipa.it

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Archiv der Pharmazie - Chemistry in Life Science

Keywords: 2-(1H-pyrazol-1-yl)pyridines, 4(3H)-Benzotriazinones, docking, COX2 inhibitors.

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Archiv der Pharmazie - Chemistry in Life Science

Abstract Pyrazol ylbenzotriazinones are endowed with structural analogy with the COX-2 selective inhibitor celecoxib. Considering that our research group has long been interested in the 3-pyrazol yl-substituted benzotriazinones as anti-inflammator y agents, six new pyrazol ylbenzotriazinone derivatives 16a-c and 18a-c have been prepared by reacting the opportune eth yl 5-(2-aminobenzamido)-1-(p yridin-2-yl)1H-pyrazole-4-carboxylate

or

5-(2-aminobenzamido)-1-(p yridin-2-yl)-1H-

pyrazole-4-carbox yic acid with sodium nitrite in glacial acetic acid. The biological studies

revealed

a

pyrazol ylbenzotriazinones

good and,

pharmacological in

Fo

the

case

of

profile the

eth yl

for

some

5-(4-oxo-1,2,3-

benzotriazin-3(4H)-yl)-1-pyridin-2-yl-1H-p yrazole-4-carbox ylate, a good COX1/COX-2

selectivity.

biological results.

Molecular

modeling

studies

confirmed

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the

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INTRODUCTION Gastrolesivit y in all those therapies associated to an inflammatory process calls for the availabilit y of new antiinflammatory agents in order to overcome this heav y collateral effect that, for long periods of administration, can provoke ulcers with serious consequences. NSAIDS inhibit prostaglandin s ynthesis b y blocking the cyclooxygenation of arachidonic acid to prostaglandin H2, the common biosynthetic precursor to numerous prostaglandins including both the proinflammatory ones and those that provide protection for the gastric mucosa. The gastric damage is therefore strictly linked to the gastro protective prostaglandins s ynthesis inhibition.

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Cycloox ygenase exists in two isoforms: COX-1, constitutively expressed in most tissues and responsible for producing the prostaglandins involved in the normal physiological functions, and COX-2, that is present mainl y in inflammatory states

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induced b y proinflammatory stimuli such as mitogens, hormones, cytokines and growth factors [1,2]. Traditional NSAIDS, such as aspirin, ibuprofen and naproxen

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inhibit both isoenz ymes, and this lack of selectivit y explains the ulcerogenic side effects experienced with a chronic use [3]. The specific inhibition of COX-2 would

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be yields NSAIDS endowed of anti-inflammatory and analgesic activities without the gastric toxicit y associated with classical NSAIDS [4,5]. Celecoxib, Rofecoxib, Etoricoxib and Valdecoxib are examples of COX-2 selective NSAIDS endowed

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with similar efficacy to that of nonselective NSAIDS but with lesser gastrotoxicit y [6,7].

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Archiv der Pharmazie - Chemistry in Life Science

Our research group has long been interested in the 3-pyrazol yl–substituted benzotriazinones in order to evaluate the influence that the heterocyclic nuclei on the 3-position may have on anti-inflammatory, analgesic, ulcerogenic activities and acute toxicit y [8-10]. Taking into account the very low gastrolesivit y associated to our benzotriazinone derivatives and their structural analogy with the COX-2 selective inhibitor celecoxib 1 (figure 1) in this work new p yrazol ylbenzotriazinones have been s ynthesized, characterized and tested for their COX-1 and COX-2 inhibitory activities as well as for their effects on the production of inflammatory mediators by the CNS. In particular, the abilit y of compounds to inhibit the conversion of arachidonic acid to prostaglandin H 2 (PGH 2 ) and, prostaglandin E2 (PGE2) released from

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Archiv der Pharmazie - Chemistry in Life Science

hypothalamic explants, either under basal conditions or after stimulation b y th e proinflammatory cyt okine interleukin-lβ (IL-1β ), were taken as an index of COX2/COX-1 selectivit y and cycloox ygenase (COX) activit y respectivel y.

CHEMISTRY The s ynthesis of the novel benzotriazinones 16a-c and 18a-c was carried out following the s ynthetic pathway showed in scheme 1. In particular, eth yl 5-amino1-pyridin-2-yl-1H-p yrazole-4-carboxylate 12 was obtained by refluxing eth yl-2cyano-3-ethoxycarboxylate 10 with 2-h ydrazin ylpyridine 11. Appropriate eth yl 5(2-nitrobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carbox ylate 14a-c prepared by

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reaction of 2-nitrobenzoyl chloride derivatives 13a-c and ethyl 5-amino-1-p yridin2-yl-1H-pyrazole-4-carbox ylate 12, were used as starting materials. By reduction of 14a-c with stannous chloride in hydrochloric acid the corresponding eth yl 5-(2-

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aminobenzamido)-1-(p yridin-2-yl)-1H-pyrazole-4-carbox ylate 15a-c were easily obtained which, in turn, when treated with sodium nitrite in glacial acetic acid,

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afforded the ethyl 5-(4-oxobenzo[d][1,2,3]triazin-3-(4H)-yl)-1-(pyridin-2-yl)-1Hpyrazole-3-carbox ylate 16a-c. Moreover, 5-(4-oxobenzo[d][1,2,3]triazin-3-(4H)-

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yl)-1-(p yridin-2-yl)-1H-p yrazole-3-carbox ylate 18a-c were obtained b y h ydrol ysis of eth yl 5-(2-aminobenzamido)-1-(p yridin-2-yl)-1H-p yrazole-4-carbox ylate 15a-c followed b y diazotation with sodium nitrite in glacial acetic acid of the h ydrol ysis products

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5-(2-aminobenzamido)-1-(pyridin-2-yl)-1H-p yrazole-4-carbox yic

acid

17a-c. The structure of all the s ynthesized compounds was established on the basis of

1

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H NMR and IR spectral anal ysis. The results of elemental anal ysis of the

s ynthesized compounds were in all cases within ±0.4% of the theoretical values.

MOLECULAR MODELLING METHODS Molecular docking studies of compounds 16a-c and 18a-c were performed in order to rationalize the obtained biological results. Besides, molecular docking studies helped in understanding the difference of potency in vitro of our compounds respect to the reference drugs. Crystal structures (6COX [11] and 1Q4G [12]) from the Brookhaven Protein Data Bank provided Cartesian coordinates for COX-2 in complex with SC558 19 and ovine COX-1 in complex with α-methyl-4-biphenylacetic acid 20 respectivel y.

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Insigth II program 2005 (Accelrys Software Inc., San Diego, USA) was used to check the 6COX and 1Q4G structures for missing atoms, bounds and contacts. Cerius 2 4.10 program (Accelrys Software Inc., San Diego, USA) was used to add hydrogens to the enz yme structures, to manuall y delete ligands and water molecules. Discovery Studio Visualizer 2.5 (Accelrys Software Inc., San Diego, USA) was used to visualize and anal yze docking data. The structures of SC588 19 and α-meth yl -4-biphen ylacetic acid 20 (figure 2) were obtained with Cerius 2 4.10 by selecting and cutting the ligands from the proteinligand complex. The 3D-structures of N-[2-cyclohex ylox y)-4-nitrophenyl]methane sulphonamide 21

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(figure 2) and the new derivatives 16a-c and 18a-c were generated by using Build/3d-sketcher module of Cerius 2 4.10; their geometry were optimized using the Dreiding 2.21 force field and the charges calculated with the Gasteiger

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method. Ligand fit module in Cerius 2 4.10 was emplo yed for the docking study and binding site search using the default setting in LigandFit.

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The active sites of 6COX and 1Q4G were defined b y the “docked ligand” procedure in the Ligand fit module in Cerius 2 4.10 according to the shape of the

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ligands SC588 19 and α-methyl-4-biphen ylacetic acid 20 respectivel y. The site was thus obtained as the collection of all grid points occupied b y the ligand and unoccupied b y the proteins, setting the grid resolution to 0.5 Å and the radium of

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ligand heav y atoms and ligand hydrogens at 2.4 Å and 2.0 Å respectivel y. To verify if the docking program will be successful in reproducing the experimental

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Archiv der Pharmazie - Chemistry in Life Science

complex, the rigid fitting option in the Ligand fit module in Cerius 2 4.10 was selected to dock SC588 19 and α-meth yl-4-biphen ylacetic acid 20 into the active sites of 6COX and 1Q4G respectivel y. The ligand fit predicted conformations with their x-ray crystallographic structures show a very high overlap with a RMS of 0.37Å for 19 and 0.18 Å for 20. All inhibitors were docked into the active sites of 6COX and 1Q4G to generate the docked conformations emplo ying the flexible fitting option in the Ligand fit module in Cerius 2 4.10 setting the number of MC trials to 10000 and the max number of poses saved to 10. For all the docked structures obtained we computed the scores using the scoring functions as implemented in Cerius 2 4.10 including Ligscore1, Ligscore2, PLP1, PLP2, and PMF. A consensus score approach was

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Archiv der Pharmazie - Chemistry in Life Science

used, taking the conformer with best consensus score as the best docked conformation.

BIOLOGY The abilit y of compounds 16a-c and 18a-c to inhibit the conversion of arachidonic acid to prostaglandin H 2 (PGH 2 ) was determined using a chemioluminescence method suitable for fast screening as reported in literature [13] using N-[2(cyclohexilox y)-4-nitrophenyl]-methane sulphonamide (NS398 21) as reference compound. The results are shown in table 1. The ethyl ester 16a, showing the best COX-2 selectivit y was further tested for the

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anti-inflammatory effect of graded doses (1, 10, 100 µg/ml) on basal and IL-1β stimulated PGE2 release from h ypothalamic explants. The results are shown in figure 3.

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DISCUSSION

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An evaluation of data reported in Table 1 revealed that, to whom concern the COX-1 inhibition, compounds 16 and 18 resulted 2-6 times less potent than the

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reference compound NS398 21. Comparison of the different docking results of compounds 16, 18 and α-methyl-4-biphen ylacetic acid 20 shows that, in principle, they adopt the same binding mode in the COX-1 active site.

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However, in agreement with the literature, the ligand α-meth yl-4-biphen ylacetic acid 20 makes hydrogen bonds with Arg-120 and Thy-355 [14] and a cation Pi

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interaction with the same aminoacid (figure 4A). It is reported that ionic interactions or h ydrogen bonds of NSADS with the Arg-120 residue of COX-1 are essential for the potent inhibitory effect of such drugs while these are not universall y required for inhibition of COX-2 activit y[15]. Compounds 16a-c and 18b make hydrogen bonds with Ser-530 while compounds 18a,c make h ydrogen bonds with Ser-530 and Met-522. Moreover, compounds 16a and 18a-c showed onl y cation Pi interactions and no h ydrogen bonds with Arg-120 (figure 4B and 4C). The lack of the hydrogen bonds with Arg-120 in our compounds could explain their lower potency. Finall y, reference compound NS398 21, hydrogen bond Ser530 and Ala-527 with their nitro and sulphonamido groups respectivel y. In the case of COX-2 inhibition, compounds 16a-c and 18a-c are endowed with inhibitory activit y against the COX-2 ranging from 20.0-490.0 µM. Among these,

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compounds 16a, 18b and 18c showed the best activit y even if they resulted 70-300 times less potent than the reference compound NS398 21 (0.26 µM) with IC50 values of 20.0, 85.5 and 65.2 µM respectivel y. However, among the s ynthesized pyrazolobenzotriazinones, compound 16a showed a good COX-1/COX-2 selectivit y (14.1). Anal ysis of the docked structures of the s ynthesized pyrazolobenzotriazinones 16a-c and 18a-c in the active sites of 6COX (COX-2), reveals that this compounds are capable to take up the COX-2 binding site and to occupy the same space of known higher active inhibitors such as SC588 19. The cycloox ygenase binding site in both isoenz ymes is a long, narrow

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hydrophobic channel extending from the membrane-binding region of the protein [15]. A second cavit y branched off from the main channel that lead to the cyclooxygenase active site is observed in the COX-2. Compounds 16a-c and 18a-c

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are oriented so that their pyrazole N-phen yl ring overlapped with the 5bromophenyl ring of SC558 19. Furthermore, the benzotriazinone nucleus,

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overlapped with the phenylsulphonammide moiet y of SC558 19 and both take up the second cavit y branched off from the main channel.

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To rationalize the different inhibitory activit y of s ynthesized compounds, a comparison of 16a-c, 18a-c and reference compound NS398 21 was made. The sulphonamide oxygen atoms of NS398 21 makes three h ydrogen bonds with Arg-

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120 and an hydrogen bond with Tyr-355; a cation Pi interaction between the nitro group of NS398 21 and Tyr-355 is also formed. As regards our compounds,

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Archiv der Pharmazie - Chemistry in Life Science

different kinds of interaction were seen (figure 5D).

Most active compounds hydrogen bound with Arg-120 and form two Pi interactions between their pyrazole nucleus and Arg-120 (figure 5A) while, less active compounds hydrogen bound with Arg-120 but form onl y a Pi interaction between their p yrazole nucleus and Arg-120 (figure 5B). The worst inhibitor 16c, forms onl y two Pi interactions between their p yrazole nucleus and Arg-120 but no hydrogen bonds (Figure 5C). However, the lack of the second h ydrogen bond with Tyr-355 and the lower number of hydrogen bounds with Arg-120, explain wh y compounds 16 and 18 resulted less potent than the reference compound NS398 21. Compound 16a, showing the best COX-2 selectivit y was further studied in the prostaglandin E2 (PGE2) released from h ypothalamic explants, either under basal conditions, or after stimulation by the proinflammatory cytokine interleukin-lβ

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Archiv der Pharmazie - Chemistry in Life Science

(IL-1β ). In particular 16a was able to decrease basal prostanoid release, and to completel y reverse the increase in PGE2 release induced b y IL-lβ (10 ng/ml), at the concentration of 100 µg/ml (Figure 4A and 4B).

CONCLUSION The present findings show that 16a, possess a profile of in vitro anti-inflammatory activities, as shown by its common capabilit y to reduce prostanoid release under basal conditions and, following IL-lb stimulation. Moreover, the pharmacological screening of COX-1 and COX-2 inhibitory activit y showed that this last is also endowed with a good COX-2 selectivit y. These findings suggest that this

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compound may possess anti-inflammatory action in the brain and that, so far, this effect may be related to the inhibition of prostaglandin production. Further both

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molecular and clinical evidence are however necessary to ascertain the potential therapeutic properties of this compound.

EXPERIMENTAL

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Chemistry

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Reaction progress was monitored b y TLC on silica gel plates (Merck 60, F 2 5 4 , 0.2 mm). Organic solutions were dried over Na 2 SO 4 . Evaporation refers to removal of

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solvent on a rotary evaporator under reduced pressure. All melting points were determined on a Büchi 530 capillary melting point apparatus and are uncorrected.

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IR spectra were recorded with a Perkin Elmer Spectrum RXI FT-IR S ystem spectrophotometer as solid in Nujiol disc or nujol mull supported on NaCl disks. 1

H NMR spectra were obtained using a Bruker AC-E 250-MHz spectrometer

(tetrameth ylsilane as an internal standard): chemical shifts are expressed in δ values (ppm). Microanal yses (C, H, N) performed in the laboratories of the Dipartimento di Scienze Farmaceutiche, Università degli Studi di Catania, are within ±0.4% of the theoretical values. Yields refer to purified products and are not optimized. Synthesis of ethyl

5-amino-1-(p yridin-2-yl)-1H-pyrazole-4-carbox ylate 12. A

solution of ethyl 2-ciano3-ethox yacrylate 10 (10 mmol) and 2-h ydrazin ylp yridine 11 (10mmol) in ethanol (20 ml) was refluxed for 3 h. The solvent was then

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evaporated under reduced pressure and the residue poured into crushed ice, collected and recrystallized. Ethyl 5-amino-1-(pyridin-2-yl)-1H-pyrazole-4-carbox ylate 12: yield 65 %; mp 8990 °C (ethanol); I.R. (Nujiol) cm - 1 3419, 3294 (NH2), 1676 (CO); 1 H NMR (DMSO) δ 1.37 (t, 3H, CH 3 ); 4.29 (q, 2H, CH 2 ); 7.11-8.36 (a set of signals, 7H, NH 2 and aromatic protons). Anal. (C 1 1 H 1 2 N 4 O 2 ) C,H,N. Synthesis of ethyl 5-(5-R 1 -4-R 2 -2-nitrobenzamido)-1-(pyridin-2-yl)-1H-p yrazole4-carbox ylate

14a-c.

A

solution

of

5-amino-1-(pyridin-2-yl)-1H-p yrazole-4-

carbox ylate 12 (4 mmol), the appropriate 2-nitrobenzo yl chloride 13a-c (4 mmol)

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and trieth ylamine (4 mmol) in acetonitrile (20 ml) was refluxed for 20h. The solvent was evaporated under reduced pressure and the residue taken up with water, filtered and recrystallized.

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Ethyl 5-(2-nitrobenzamido)-1-(pyridin-2-yl)-1H-p yrazole-4-carbox ylate 14a: yield 70 %; mp 174-175 °C (ethanol); I.R. (Nujiol) cm - 1 3167 (NH), 1722, 1686 (CO); 1

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H NMR (DMSO) δ 1.30 (t, 3H, CH 3 ); 4.30 (q, 2H, CH 2 ); 7.40-8.56 (a set of

signals,

9H,

aromatic

(C 1 8 H 1 5 N 5 O 5 ) C,H,N. Ethyl

protons);

11.20

(s,

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1H,

NH,

exchangeable).

Anal.

5-(5-chloro-2-nitrobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carbox ylate

14b: yield 65 %; mp 192-193 °C (ethanol); I.R. (Nujiol) cm - 1 3193 (NH), 1712,

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1696 (CO); 1 H NMR (DMSO) δ 1.28 (t, 3H, CH 3 ); 4.27 (q, 2H, CH 2 ); 7.23-8.55 (a set of signals, 8H, aromatic protons); 11.32 (s, 1H, NH, exchangeable). Anal.

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(C 1 8 H 1 4 ClN 5 O 5 ) C,H,N. Ethyl

5-(4-chloro-2-nitrobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carbox ylate

14c: yield 60 %; mp 196-197 °C (ethanol); I.R. (Nujiol) cm - 1 3195 (NH), 1710, 1686 (CO); 1 H NMR (DMSO) δ 1.29 (t, 3H, CH 3 ); 4.28 (q, 2H, CH 2 ); 7.49-8.52 (a set of signals, 8H, aromatic protons); 11.35 (s, 1H, NH, exchangeable). Anal. (C 1 8 H 1 4 ClN 5 O 5 ) C,H,N. Synthesis

of

ethyl

5-(2-amino-5-R 1 -4-R 2 -benzamido)-1-(pyridin-2-yl)-1H-

pyrazole-4-carbox ylate 15a-c. Ethyl 5-(2-(5-R 1 -4-R 2 -2-nitrophen yl)-2-oxoeth yl)-1(pyridin-2-yl)- 1H-pyrazole-4-carbox ylate 14 a-c (3 mmol) were added to a magneticall y stirred suspension of finel y powdered stannous chloride (9 mmol) in hydrochloric acid (36%, 3 ml) at such a rate so that the temperature of the slurry Wiley - VCH


Archiv der Pharmazie - Chemistry in Life Science

was maintained below 5° C. After the complete addition, the mixture was allowed to stir for 24 hours. The white slurry thus obtained was diluted with cold water and, aqueous sodium hydroxide (40%) was added till the salts of tin were dissolved. The solution was extracted with eth yl acetate (2x80 ml), the extracts were dried (sodium sulphate) and evaporated under reduced pressure to give a residue which was recrystallized. eth yl

5-(2-aminobenzamido)-1-(p yridin-2-yl)-1H-p yrazole-4-carbox ylate

15a:

yield 70 %; mp 174-175 °C (ethanol); I.R. (Nujiol) cm - 1 3461-3342 (NH and NH 2 ), 1693-1670 (CO); 1 H NMR (DMSO) δ 1.29 (t, 3H, CH 3 ); 4.31 (q, 2H, CH 2 ); 6.73 (s, 2H, NH 2 , exchangeable); 6.65-8.37 (a set of signals, 9H, aromatic protons); 11.35

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(s, 1H, NH, exchangeable). Anal. (C 1 8 H 1 7 N 5 O 3 ) C,H,N. eth yl

5-(2-amino-5-chlorobenzamido)-1-(pyridin-2-yl)-1H-p yrazole-4-carbox ylate

15b: yield 75 %; mp 155-156 °C (ethanol); I.R. (Nujiol) cm - 1 3468-3355 (NH and

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NH 2 ), 1684 (CO); 1 H NMR (DMSO) δ 1.21 (t, 3H, CH 3 ); 4.22 (q, 2H, CH 2 ); 6.60 (s, 2H, NH 2 , exchangeable); 7.49-8.52 (a set of signals, 8H, aromatic protons);

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10.80 (s, 1H, NH, exchangeable). Anal. (C 1 8 H 1 6 ClN 5 O 3 ) C,H,N. eth yl

5-(2-amino-4-chlorobenzamido)-1-(pyridin-2-yl)-1H-p yrazole-4-carbox ylate

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15c: yield 60 %; mp 208-209 °C (ethanol); I.R. (Nujiol) cm - 1 3479-3368 (NH and NH 2 ), 1711-1670 (CO); 1 H NMR (DMSO) δ 1.22 (t, 3H, CH 3 ); 4.22 (q, 2H, CH 2 ); 6.50 (s, 2H, NH 2 , exchangeable); 7.78-8.47 (a set of signals, 8H, aromatic

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protons); 10.70 (s, 1H, NH, exchangeable). Anal. (C 1 8 H 1 6 ClN 5 O 3 ) C,H,N.

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Synthesis of Ethyl 5-(6-R 1 -7-R 2 -4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2yl-1H-pyrazole-4-carboxylate 16 a-c. To a magneticall y stirred cold solution (icebath,

0-5°C)

of

ethyl

5-(2-amino-5-R 1 -4-R 2 -benzamido)-1-(p yridin-2-yl)-1H-

pyrazole-4-carbox ylate 15a-c (3 mmol) in acetic acid (60 ml), an aqueous solution of potassium nitrite (7,0 mmol) was added dropwise. Stirring was continued for 2 h at 0-5°C and then the solution was warmed to room temperature. The solid which separated out by addition of cold water (100 ml) was collected and recrystallized. Ethyl

5-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2-yl-1H-p yrazole-4-

carbox ylate 16a: yield 75 %; mp 140-141 °C (ethanol); I.R. (Nujiol) cm - 1 17181700 (CO); 1 H NMR (DMSO) δ 1.09 (t, 3H, CH 3 ); 4.20 (q, 2H, CH 2 ); 7.10-8.43 (a set of signals, 9H, aromatic protons). Anal. (C 1 8 H 1 4 N 6 O 3 ) C,H,N.

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Ethyl 5-(6-chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-p yridin-2-yl-1H-p yrazole4-carbox ylate 16b: yield 60 %; mp 161-162 °C (ethanol); I.R. (Nujiol) cm - 1 1710 (CO); 1 H NMR (DMSO) δ 1.03 (t, 3H, CH 3 ); 4.14 (q, 2H, CH 2 ); 7.36-8.58 (a set of signals, 8H, aromatic protons). Anal. (C 1 8 H 1 3 ClN 6 O 3 ) C,H,N. Ethyl 5-(7-chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-p yridin-2-yl-1H-p yrazole4-carbox ylate 16c: yield 65 %; mp 105-106 °C (ethanol); I.R. (Nujiol) cm - 1 1715 (CO); 1 H NMR (DMSO) δ 1.03 (t, 3H, CH 3 ); 4.15 (q, 2H, CH 2 ); 7.36-8.54 (a set of signals, 8H, aromatic protons). Anal. (C 1 8 H 1 3 ClN 6 O 3 ) C,H,N. Synthesis of 5-(6-R 1 -7-R 2 -4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2-yl-1H-

Fo

pyrazole-4-carbox ylic acid 18a-c. Ethyl

5-(2-amino-5-R 1 -4-R 2 -benzamido)-1-(pyridin-2-yl)-1H-p yrazole-4-

carbox ylate 15a-c (3 mmol) dissolved in ethanol (20 ml),were treated with

rP

an

aqueous solution of sodium h ydroxide 4% (30 ml) and refluxed for 15 minutes. The mixture was then reduced to a small volume, cooled and acidified at pH 5.0

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with aqueous HCl. The product precipitated was collected, dissolved in acetic acid (60 ml) and, the obtained mixture was cooled (ice-bath, 0-5°C) and added, under

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magnetic stirrer, of an aqueous solution of potassium nitrite (7,0 mmol).After the complete addition, the solution was left on a magnetic stirrer for 45 minutes, then poured in a cold water. The solid which separated out was collected, and recrystallized from ethanol.

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5-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2-yl-1H-p yrazole-4-carbox ylic

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Archiv der Pharmazie - Chemistry in Life Science

acid 18a: yield 55 %; mp 205-206 °C (ethanol); I.R. (Nujiol) cm - 1 , 1714 (CO); 1 H NMR (DMSO) δ 7.31-8.46 (a set of signals, 9H, aromatic protons); 13.15 (s, 1H, COOH, exchangeable). Anal. (C 1 6 H 1 0 N 6 O 3 ) C,H,N.

5-(6-chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-p yridin-2-yl-1H-pyrazole-4carbox ylic acid 18b: yield 50 %; mp 215-216 °C (ethanol); I.R. (Nujiol) cm - 1 1708 (CO); 1 H NMR (DMSO) δ 7.33-8.56 (a set of signals, 9H, aromatic protons); 13.24 (s, 1H, COOH, exchangeable). Anal. (C 1 6 H 9 ClN 6 O 3 ) C,H,N. 5-(7-chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-p yridin-2-yl-1H-pyrazole-4carbox ylic acid 18c: yield 58 %; mp 230-231 °C (ethanol); I.R. (Nujiol) cm - 1 1710,1684 (CO); 1 H NMR (DMSO) δ 7.35-8.45 (a set of signals, 9H, aromatic); 13.39 (s, 1H, COOH, exchangeable). Anal. (C 1 6 H 9 ClN 6 O 3 ) C,H,N.

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Archiv der Pharmazie - Chemistry in Life Science

Biology The reference compounds Indomethacin and NS398 were purchased from Cayman Chemical, Ann Arbor, M I (cat. N° 70270 and 70590 respectivel y). Male Wistar rats, weighting 200-300 g, were used. They were kept four per cage and maintained at a temperature of 23 ± 1,5 °C, with a relative humidit y of 65 ± 2 %. The animals were exposed to 12 h of light (06.00 am – 06.00 pm) followed by 12 h of dark, and they had free accessed to food and water. Compounds 16a, 18a and 4 were dissolved in Dymethyl Sulfoxide (DMSO; Sigma-Aldrich S.r.l., Milano) and diluted to working concentrations in incubation media. IL-1β was obtained from Boeringher (Mannheim, German y). The cytokine was dissolved in phosphate-

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buffered saline containing 0.1% BSA and further diluted in incubation medium. All drugs tested did not interfere with the PGE 2 assay. COX1/COX2 inhibition

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Ovine COX1(cat. n° 60100), COX2 (cat. n° 60120), arachidonic acid (cat. n°

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90010) as well as the reference inhibitors NS398 (cat. n° 70590) and Indomethacin (cat. n° 70270) were purchased from Cayman Chemical, Ann Arbor, M I. Porcine

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hematin (cat. n° H3281), luminol (cat. n° A4685) and gelatine (cat. n° G7765) were from Sigma Aldrich srl.

Chemiluminescence measurements were performed in 96-well white pol yst yrene

ev

microtiter plates using a microtiter plate luminometer. Briefl y, COX1 or COX2 (50U/ml in 0.1M PBS, pH6.5, with 1 mg/ml gelatine),

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hematine (6µM in 0.1 M PBS, pH 6.5) and luminol (500 µM in 0.1M TR IS Buffer, pH 6.5) were added to the microtiter plate wells to obtain a concentration of 1U for COX, 350 µM for luminol and 1 µM for hematine, in a 150 µM final volume. Then, were added 10 µl of serial dilutions of test compounds in DMSO/H 2 O 1:10 (v/v), and the plate was incubated for 20min at 37°C. The enz ymatic reaction was started by adding to each well 50 µl of 80 µM arachidonic acid solution in water (final concentration 20 µM) and the CL emission was measured at 1-s intervals up to 60s after initiation of the reaction with substrate. The COX activit y was calculated b y interpolating the integrated CL signal (which is proportional to the amount of product formed) on a curve obtained by measuring in the same assay samples containing different amount of COX.

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Determination was performed in duplicate for two independent times and the results were expressed as IC 5 0 (µM). Hypothalamic incubation On day of the experiment the animals were decapitated between 09.00 and 10.00 a.m. to avoid circadian variation. The brains were rapidly removed and the hypothalami were dissected within the following limits: the posterior border of the optic chiasm, the anterior border of the mamillary bodies and the lateral hypothalamic sulci, with a depth of approximatel y 2 mm. Hypothalamic blocks were then divided longitudinall y in two halves to aid diffusion of medium. Total

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dissection time was less than 2 min from decapitation. The hypothalami were then incubated in a 24-well plate (one h ypothalamus/well), at 37°C in a humidified atmosphere consisting of 5% CO 2 and 95% O 2 in 300 µl incubation medium,

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Minimum Essential Medium (MEM) with Earle’s salts and with stable glutamine (Biocrhrom AG, Berlin, D), supplemented with 0.2% bovine serum albumin (BSA),

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0,005% ascorbic acid and 20 IU/ml aprotinin, pH 7.4. In this experimental model, hypothalamic tissues remain viable and functional, as assessed at the end of the

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experiments by the measurement of Lactic Dehydrogenase (LDH) activit y, taken as a marker of cytotoxicit y [16].

After a 60-min pre-incubation period (during which the medium was changed every

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20 min), the medium was replaced with fresh medium alone (control), or medium containing the test substance at appropriate concentrations. At the end of

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Archiv der Pharmazie - Chemistry in Life Science

experiments, the incubation media were collected and stored at – 35°C until measurement of PGE 2 immunoreactivit y. PGE 2 radioimmunoassay (RIA) PGE2 was measured b y radioimmunoassay (RIA) as previousl y described [17]. Incubation mixtures of 1,5 ml were prepared in disposable plastic tube. Two hundred

and

fift y

microliters

of

incubation

medium

were

assayed

from

hypothalamic explant. The remainder 1,25 ml was prepared b y adding together 2500-3000 cpm of [ 3 H]PGE 2 and antiserum at a final dilution of 1:115,000. A duplicate standard curve (ranging from 1 to 400 pg/tube) was run with each assay. The detection limit of the assay was 1 pg/tube and the EC 5 0 of 28 pg/tube. The intra-and interassay variabilit y was 5% and 10%, respectively. Separation of free

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Archiv der Pharmazie - Chemistry in Life Science

from antibody-bound PGE 2 was obtained with charcoal, which absorbs 95-98% of free PGE 2 . After centrifugation for 10 min. at 4 째C, supernatants were decanted directl y into 10 ml of liquid scintillation fluid. Radioactivity was measured by liquid scintillation counting.

Statistical anal ysis Data were anal yzed b y one-way ANOVA, followed b y post-hoc

Dunnet or

Newman-Keuls test for multiple comparisons among group means, using a Prism T M computer program (GraphPad, San Diego, CA, USA) and differences were considered statistically if P<0,05.

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ACNOWLEDGEMENT

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Financial support from MIUR (fund 60%-2006) is gratefull y acknowledged.

REFERENCES

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[1] R.G. Kurumbail, J.R.Kiefer, L.J Marnett, Curr. Opin.Struc. Biol. 2001,11 752-760.

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[2] D.L. Simmons, R.M. Botting, T. Hla, Pharmacol. Rev. 2004, 56, 387-437. [3] M. Gupta, G.M. Glenn, Current Gastroenterology Reports 2009, 32, 25-32. [4] B.M. Peskar, J. Physiol. 2001, 3-9.

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[5] M. Lazzaroni, A. Battocchia, G. Bianchi Porro, Digestive and Liver Disease

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2007, 589-596.

[6] M.D. Loren Laine, Seminars in Arthritis and Reumatism 2002, 32 25-32. [7] C. Blandizzi, M. Tuccori, R. Colucci, M. Fornai, L. Antonioli, N. Ghisu, M. del Tacca, Pharmacol. Res. 2009, 59, 90-100. [8] S. Plescia, D. Raffa, G. Daidone, B. Maggio, Il Farmaco 1994, 49, 505-507. [9] G. Daidone, S. Plescia, D. Raffa, M.L. Bajardi, M. Matera, A. Caruso, M.G. Leone, Il Farmaco 1990, 45, 391-398. [10] G. Daidone, B. Maggio, S. Plescia, D. Raffa, D. Schillaci, O. Migliara, A. Caruso, V.M.C. Cutuli, M. Amico-Roxas, Il Farmaco 1998, 53, 350-356. [11] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak, D. Gildehaus, J.M. Mi yashiro, T.D. Penning, K. Seibert, P.C. Isakson, W.C. Stallings, Nature 1996, 384 644-648.

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[12] K.Gupta, B.S. Selinsk y, C.J. Kaub, A.K. Katz, P.J. Loll, J. Mol. Biol. 2004, 335, 503-518. [13] A. Andreani, M. Granaioloa, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, A. Roda, M. Guardigli, S. Traniello, S. Spisani, Eur. J. Med. Chem. 2004, 39, 785-791. [14] J.A. Mancini, D. Riendeau, J.P. Falgueyret, P.J. Vickers, G.P. Oâ&#x20AC;&#x2122;Neill, J. Biol. Chem. 1995, 270, 29327-29377. [15] M.L. Plount Price, L. Jorgensen, J. Am. Chem. Soc. 2000, 122, 9455-9466. [16] G. Pozzoli, G. Tringali, C. Dello Russo, M. Vairano, P. Preziosi, P. Navarra, J Neuroimmunology 2001, 118, 268-276.

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[17] G. Tringali, G. Pozzoli, M. Vairano, N. Mores, P. Preziosi, P. Navarra, J Neuroimmunology 2005, 160, 61-67.

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Archiv der Pharmazie - Chemistry in Life Science

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Archiv der Pharmazie - Chemistry in Life Science

FIGURE LEGENDS Figure 1. Structural analogy of the 3-pyrazol ylbenzotriazinones with the COX-2 selective inhibitor celecoxib 1. Figure 2. Structures of ligands: SC588 19 and α-methyl-4-biphenylacetic acid 20 and, NS398 21. Figure 3. Effect of 16a at 1, 10, 100 µg/ml concentrations on basal (panel A) and on 10 ng/ml IL-1β stimulated (panel B) PGE2 release from rat h ypothalamic explans. Results are from two independent experiments performed in quadruplicate. Data are expressed as pg/ml of PGE2, means ± SEM of eiht replicates per group. *p<0.05 vs. Control; °p<0.05 vs. IL-1β .

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Figure 4. A. Reference compound α-meth yl-4-biphen ylacetic acid 20 bounded in the active site of COX-1. B. Compound 16a bounded in the active site of COX-1. C. Compound 18a bounded in the active site of COX-1.

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Figure 5. A. Reference compound 16a bounded in the active site of COX-2. B. Compound 18a bounded in the active site of COX-2. C. Compound 16c bounded in the active site of COX-2. D. Reference compound NS398 21 bounded in the active site of COX-2.

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Scheme 1. S ynthetic pathway of compounds 16a-c and 18a-c.

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Table 1. IC50 values (µM) for compounds 16a-c and 18a-c in COX-1 and COX-2 inhibition assay.

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Page 16 of 24

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r Fo

152x63mm (106 x 106 DPI)

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vi

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Archiv der Pharmazie - Chemistry in Life Science

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Archiv der Pharmazie - Chemistry in Life Science

r Fo Figure 1. Structural analogy of the 3-pyrazolylbenzotriazinones with the COX-2 selective inhibitor celecoxib 1. 90x43mm (500 x 500 DPI)

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Scheme 1. Synthetic pathway of compounds 16a-c and 18a-c. 189x139mm (500 x 500 DPI)

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Archiv der Pharmazie - Chemistry in Life Science

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Archiv der Pharmazie - Chemistry in Life Science

r Fo er

Pe Figure 2. Structures of ligands: SC588 19 and Îą-methyl-4-biphenylacetic acid 20 and, NS398 21. 90x55mm (500 x 500 DPI)

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Table 1. IC50 values (ÂľM) for compounds 16a-c and 18a-c in COX-1 and COX-2 inhibition assay. Comp. COX-1

COX-2 COX-1/COX-2 selectivity 20.0 14.1

16a

282.0

16b

115.9

123.2

0.94

16c

219.8

490.0

0.45

18a

225.0

204.5

1.10

18b

167.1

85.5

1.95

18c

409.3

65.2

6.2

NS398

61.3

0.26

236.9

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Archiv der Pharmazie - Chemistry in Life Science

Figure 3. Effect of 16a at 1, 10, 100 µg/ml concentrations on basal (panel A) and on 10 ng/ml IL-1β stimulated (panel B) PGE2 release from rat hypothalamic explans. Results are from two independent experiments performed in quadruplicate. Data are expressed as pg/ml of PGE2, means ± SEM of eiht replicates per group. *p<0.05 vs. Control; *p<0.05 vs. IL-1β. 140x45mm (500 x 500 DPI)

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Archiv der Pharmazie - Chemistry in Life Science

Figure 4. A. Reference compound Îą-methyl-4-biphenylacetic acid 20 bounded in the active site of COX-1. B. Compound 16a bounded in the active site of COX-1. C. Compound 18a bounded in the active site of COX-1. 90x230mm (500 x 500 DPI)

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Archiv der Pharmazie - Chemistry in Life Science

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Figure 5. A. Reference compound 16a bounded in the active site of COX-2. B. Compound 18a bounded in the active site of COX-2. C. Compound 16c bounded in the active site of COX-2. D. Reference compound NS398 21 bounded in the active site of COX-2. 140x149mm (500 x 500 DPI)

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Pyrazolobenzotriazinones Derivatives as COX Inhibitors  

Pyrazolobenzotriazinones Derivatives as COX Inhibitors - 2010

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