N-Substituted [phenyl-pyrazolo]-oxazin-2 - thiones as COX-LOX inhibitors: influence of the replacement of the oxo -group with thioxo-group on the COX inhibition activity of N-substituted pyrazolo-oxazin-2-ones

Targeting to the synthesis of potent dual acting COX/LOX inhibitors as future anti-inflammatory drugs, we attempted a modification of the compounds based on docking analysis results. A substitution of the oxygen of the oxo-group of the oxazin-2-one ring by sulphur resulted in a four to over ten fold improvement of COX and LOX inhibitory action. N-phenyl derivatives exhibited the best biological properties with the 4-methoxy-phenyl derivative showing the best COX-1 and LOX inhibitory action and the 4-Br-phenyl derivative exhibiting the best COX-2 inhibitory action combined with good COX-1 and LOX inhibitory capacity.


Introduction
Inflammation is a multifactorial process.It reflects the response of organism to various stimuli and is related to many disorders such as arthritis, asthma, and psoriasis, which require prolonged or repeated treatment.Release of arachidonic acid via the action of phospholipase A2 on membrane phospholipids is one of the first events of inflammation.Cyclooxygenase (COX) and lipoxygenase (LOX) produce two groups of arachidonic acid (AA) metabolites, prostaglandins (COX products), leucotrienes and lipoxins (LOX products), that play a key role in inflammation.Traditional NSAIDs which preferentially inhibit COX-1 are associated with gastrointestinal irritation 1 and mild bleeding diathesis.NSAIDs of new generation which selectively inhibit COX-2, lack these side effects.However, some of them were associated with cardiovascular thrombotic events. 2 Since COX inhibition blocks one pathway of arachidonic acid metabolism, administration of COX inhibitors leads to induction of the second pathway resulting to overproduction of leucotrienes (LTs) 3 and lipoxines (LXs) via the action of LOX.LTs were found to have pro-inflammatory action.They are associated with leucocyte activation and adhesion to vascular endothelium. 4Cysteinyl LTs are associated with edema formation, damage of gastric mucosa and are involved in the pathogenesis of bronchial asthma.So, LOX products have their share in undesired inflammation events and LOX pathway has to be blocked for better results.Because leucotrienes have their role in blood coagulation and gastric tract irritation, LOX inhibition seems to be able to ameliorate GI tract irritation resulting from COX-1 inhibition as well as the prothrombotic tendency resulting from COX-2 inhibition.Thus, many researchers focused on the development of dual acting COX/LOX inhibitors. 1,2,5It has been stated that a balanced modulation of several targets can provide superior therapeutic effects and a favorable side effect profile compared to the action of a selective ligand, particularly in cases of complex diseases. 6icofelone, a COX-2/5-LOX inhibitor, has been evaluated in a phase III clinical trial. 7Among compounds found to be potent dual inhibitors are derivatives of pyrazole, thiophene, indomethacin, dit-butyl-phenol, hydrazone, pyrrole, fenamic acid etc. 1 Most recently Knaus et al. 8 tested a number of 1,3-diarylprop-2-yn-1-one as well as rofecoxib derivatives for COX-1, COX-2, and 5, 15 LOX inhibitory activity.Ziakas et al. 9 tested a number of tolfenamic and BHT derivatives and Geronikaki et al. tested novel thiazolidinone derivatives for COX-1, COX-2 and LOX inhibition. 5xazinones and pyrazole derivatives attracted organic chemists due to their biological and chemotherapeutic importance.1][12][13][14][15][16][17][18][19] On the other hand, pyrazole and their derivatives are associated with various pharmaceutical activities. 20,21e reported previously the synthesis and biological activity evaluation of a new series of Nsubstituted pyrazolo-oxazin-2-one compounds on the basis of PASS prediction results.Positive COX-1 inhibition activity predicted by PASS was confirmed by experimental evaluation 22 although the compounds exhibited only mild inhibitory action.Targeting to the synthesis of potent dual acting COX/LOX inhibitors as future anti-inflammatory drugs, we attempted a modification of the compounds based on docking results.Preliminary docking analysis revealed that substitution of the oxygen of the oxo-group of the oxazin-2-one ring by sulphur resulted in formation of a lower energy, more stable complex, in many cases.
Thione derivatives have been designed and tested by many investigators and a number of compounds with good antitumor, 23,24 antimicrobial 25,26 and anti-inflammatory 26,27 activity have been emerged.][30] In the present study, seven novel thione derivatives were synthesised.All novel compounds and their oxo-analogues 22 were tested for COX-1 as well as COX-2 and LOX inhibitory action.

Chemistry
The synthesis of the title compounds (Figure 1) was straightforward as illustrated in Scheme 1.
Figure1.Structure of the synthesized compounds.

Scheme 1. Synthesis of the designed compounds.
The preparation of initial pyrazolyl-enaminone 3a-g started with reaction of commercially available dehydroacetic acid 1 with phenylhydrazine to give, in two steps, the 1,3-dicarbonyl compound 2 according to the literature procedure 31 .Reaction of derivatives 2 with aliphatic and aromatic primary amines lead to pyrazolyl-enaminones 3. 32 As it was shown previously, 22 in the presence of phosgene, these intermediates lead to N-substituted pyrazolo-oxazin-2-one compounds.In similar conditions to already described procedure, 22 pyrazolyl-enaminones 3 react with thiophosgene in presence of triethylamine to give N-substituted pyrazolo-oxazin-2thione compounds 4 in 69-86 % yields (Table 1 The structures of compounds 4a-g were confirmed by elemental analysis as well as spectroscopically (IR, 1 H and 13 C NMR and MS electrospray mass spectrometry).
The signal at around 177-179 ppm in 13 C NMR denoted the presence of the carbamate thiocarbonyl group C=S.The signals at around 158-159 and 164-165 ppm were attributed to the quaternary carbon of junction and carbonyl of pyrazolic nucleus respectively.The lack of OH signal in 1 H-NMR spectra is also in accordance to the assigned structures.All the compounds exhibit a strong thiocarbonyl (C=S) absorption in the region of 1227-1276 cm -1 of the IR spectra according to the reported value for this group 33 and no absorption of the OH band.This is in agreement with the ring closing and the proposed structures.
The reaction mechanism could be rationalized as shown in Scheme 2.
Scheme 2. Mechanism of synthetic root.

Evaluation of biological activity
Evaluation of COX-1 inhibitory action.All tested thioxo analogues exhibited COX-1 inhibitory activity, with the exception of the ethyl-derivative 4a and n-propyl-derivative 4b with IC50>200 M (Table 2).Insertion of the bulky hydrophobic phenyl group in compounds 4c-4g strongly improved inhibitory action (IC50: 6.3 -25.0 M).The p-methoxy-phenyl-derivative 4f showed the best inhibitory activity (IC50=6.3M).The absence of the p-methoxy-group in the phenyl-derivative, 4d, resulted in diminished activity (IC50=17.8M) while the presence of a pmethyl-substituent as well as a methylene-bridge between the phenyl and the oxazinone rings in compounds 4e and 4c further reduced inhibitory action (IC50 : 21.4 M and 25.0 M respectively).Interestingly, the p-Br-phenyl derivative, 4g, exhibited better inhibitory activity (IC50=11.2M) than the phenyl derivative, 4d (IC50=17.8M).Six of the seven tested compounds present better inhibitory action than naproxen (IC50=125.8M).Comparison of the results with that previously obtained for the 1,3-oxazin-2-one analogues showed a four to over thirty fold improvement of the inhibitory activity in the 1,3-oxazin-2-thione derivatives 4c-4g.Evaluation of COX-2 inhibitory action.Most compounds exhibit no COX-2 inhibitory action (IC50>200 M) (Table 2).However, both thioxo-and oxo-4-Br-phenyl-derivatives, 4g, exhibited COX-2 inhibitory action with IC50 values 62 and 78 M respectively.Thioxo derivative 4g was slightly more active than the oxo-analogue, 4g(O), in this case, as well.Evaluation of LOX inhibitory action.Thioxo derivatives 4d-g showed significant LOX inhibitory action, four to ten fold higher (IC50 3.6 -5.4 M) than the activity of their oxoanalogues (IC50 27.8 -52.0 M) (Table 2).Among the thioxo-analogues, the 4-methoxy-phenyl derivative, 4f, exhibited the best activity (IC50=3.6 M), followed by 4-Br-phenyl-, 4g, phenyl-, 4d, and 4-methyl-phenyl, 4e, derivatives with IC50 values of 4.2 M, 5.3 M, 5.4 M respectively.In accordance to the COX inhibitory action, existence of the bulky phenyl moiety seams to be mandatory for LOX inhibitory activity, as well.The ethyl-, 4a, and n-propyl-, 4b, derivatives showed no inhibition.Among the compounds sharing the phenyl substituent, derivative 4c having a methylene-bridge between the phenyl and the oxazinone rings exhibited no activity (IC50 >200 M) showing that the short distance between oxazinone ring and the bulky phenyl substituent is of great importance.The better activity of 4-methoxy 4f, and 4-Br-phenyl-, 4g, derivatives shows that existence of an electronegative atom at the 4 position of the phenyl ring favors inhibitory activity.On the contrary, the existence of a methyl group in derivative 4e reduced effectiveness of the inhibitor.Among, the oxo-analogues In conclusion, 4-methoxy-phenyl thione derivative, 4f is the most effective COX-1 and LOX inhibitor but lacks COX-2 inhibitory action.4-Br-phenyl thioxo-derivative, 4g, effectively inhibits all three enzymes, being the second most active COX-1 and LOX inhibitor.

Docking results
Docking of the oxo-and thioxo-derivatives of compound 4f into the COX-1 active site are presented as an example of the influence of the oxygen to sulfur replacement in complex stabilization.3), the thioxo-analogue of compound 4f(O), revealed that the phenyl group of pyrazole ring is also placed at the opening of the cleft of COX-1 binding site in proximity to the amino acids Leu93 and Tyr355, where it undergoes van der Waals interaction with Tyr355 (distance < 5 Å) while the sulfur atom of oxazine ring undergoes hydrophobic interaction with Val349 of the active site.The 4-methoxy phenyl group is oriented within the hydrophobic pocket consisting of Gly526, Val349, Tyr348, Tyr385, Trp387 and Leu384.Because of a slight change in orientation of the thioxo analogue, compared to the oxo derivative, the oxygen of the 4-methoxy group participates in hydrogen bond interaction with the hydrogen of the OH group of Tyr385 (distance = 2.79 Å).Moreover, the slightly different position of the keto-group of the pyrazole ring in the active site, enables the formation of two hydrogen bonds (HB).One HB between the oxygen atom of the keto-group of the pyrazole ring and the hydrogen of the NH group of Arg120 (distance= 3.02 Å) and a second one between the oxygen atom of the same keto-group and OH of Tyr355 (distance = 2.31 Å) flanking the entrance of the active site.
This difference in the number of hydrogen bonds, 3 instead of 1, may result in increased complex stability and improved inhibitory activity of the thioxo-analogue.
The ability to form hydrogen bonds with Tyr355 or Arg120 of COX-1 characterises many COX inhibitors.For example, S-Ibuprofen, the active isomer of the racemate used, forms three hydrogen bonds with COX-1 aminoacid remnants, one with Arg120 and two with Tyr 355. 34yr385, placed at the distant end of the cleft of the catalytic centre, is rarely reached by COX-1 inhibitors.Tyr385 is involved in catalytic action, since the tyrosyl radical formed is thought to be responsible for abstracting a hydrogen atom from the substrate.The tyrosyl radical produced removes hydrogen from the substrate. 35In the absence of substrate or inhibitor, Tyr385 forms a hydrogen bond with a water molecule which connects Tyr385 with Ser530 thus stabilising the molecule.The position of Tyr385 within an extremely hydrophobic neighbourhood makes any electrostatic interaction or hydrogen bond formation of great importance. 36Tyr-385/Ser-530 chelation of electron-rich centers was found to be critical for acetylation of COX-2 by aspirin and was mainly associated with time-dependent, non-covalent inhibition of COX-2 by inhibitors like diclofenac. 37

Conclusions
Among the seven previously synthesized oxo analogues, only two compounds, 4d(O) and 4e(O) had been found to exhibit COX-1 inhibitory action with IC50 values lower than 200 M.Evaluation of the oxo-derivatives for COX-2 and LOX inhibitory action, revealed that one of the compounds, 4g(O), exhibited mild COX-2 inhibitory action (IC50: 78.0 M), while four of the compounds, 4d(O), 4e(O), 4f(O) and 4g(O), exhibited LOX inhibitory action (IC50: 27.8 M -52.0 M).In conclusion, three of the oxo-derivatives were found to present both COX and LOX inhibitory action.In general, the presence of a pure bulky hydrophobic group such as 4-methoxyphenyl or phenyl-group at the 3-position of the oxazinone ring (4d(O), 4e(O)), favored COX-1 inhibitory action.Although, replacement of the 4-methyl substituent with CH3O-or Brsubstituent resulted in reduced COX-1 inhibitory action, 4-Br-substitution favored COX-2 as well as LOX inhibition.
Replacement of oxo-with thioxo-group strongly improved inhibitory activity of the compounds in all three enzymes.Five out of the seven tested compounds showed much better COX-1 inhibitory action (four to over thirty fold improvement), four out of seven exhibited increased LOX activity (seven to ten fold improvement) and one showed slightly improved COX-2 inhibition compared to their oxo-analogues.
The most active compounds have good inhibitory action compared to reference substances under experimental conditions and four out of the seven combine COX and LOX inhibitory action, making compounds of this series promising agents as novel dual action COX/LOX inhibitors.

Experimental Section
General.Melting points were obtained on a Bibby stuart scientific SMP1 apparatus and are uncorrected.Elemental analyses were obtained on an acceptable range (±/0.4%) in a Perkin-/Elmer 240B CHN analyzer (The Perkin-/Elmer Corporation Ltd., Lane Beaconsfield, Bucks, UK).IR spectra were recorded on a Perkin Elmer FTIR paragon 100PC spectrometer in KBr disks. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker Avance II spectrometer using tetramethylsilane as an internal standard,.Mass spectra were obtained by electrospray ionisation technique (ESI) on a Perkin Elmer SCIEX API 300 spectrometer.

Biological evaluation
Evaluation of LOX inhibitory action.Lipoxygenase inhibition was evaluated as reported previously. 38It has been shown that inhibition of plant LOX activity by NSAIDs is qualitatively similar to the inhibition of the rat mast cell LOX and can be used as a simple screen for such activity. 38The tested compounds, dissolved in DMSO, were pre-incubated for 4 min at 28 °C with soybean lipoxygenase at a concentration of 7 × 10 -7 w/v.Enzyme reaction was initiated by the addition of sodium linolate at a final concentration of 0.1 mM.The conversion of sodium linolate to 13-hydroperoxylinoleic acid was measured at 234 nm.Nordihydroguaretic acid, an appropriate standard inhibitor, was used as positive control (inhibition) 94.4% at 0.1 mM).
COX inhibitor screening assay.The COX-1 and COX-2 activity of the compounds was measured using ovine COX-1 and human recombinant COX-2 enzymes included in the "COX Inhibitor Screening Assay" kit provided by Cayman (Cayman Chemical Co., Ann Arbor, MI, USA).The assay directly measures PGF2a produced by SnCl2 reduction of COX-derived PGH2.The prostanoid product is quantified via enzyme immunoassay using a broadly specific antibody that binds to all the major prostaglandin compounds. 5,9The inhibitory activity of the compounds was tested at arachidonic acid concentration of 1 μΜ.The compounds were added at the reaction mixture at a final concentration of 200 μΜ for a first screening of inhibitory action.IC50 values were calculated for the most active compounds.Naproxen was used as a positive control.

Docking analysis
The Genetic Optimization for Ligand Docking (GOLD) version 2.2 on windows based PC, was used for docking studies 39 .The reported crystal structure of COX-1 (PDB code 1CQE.pdb) with co-crystal of flurbiprofen from the Brookhaven Protein Data Bank (PDB) was downloaded for the present docking study.The docked poses were scored using scoring functions: Goldscore (GS) and ChemScore (CS) to find the required docking pose.Initially, the protein was considered without ligand and water molecules for the purpose of docking studies.The proteinligand complexes of COX-1 (PDB code 1CQE.pdb) were minimized up to a gradient of 0.01 kcal/(mol Å) and hydrogens were added using the CHARMm force field, available in the software discovery studio 2.0 .The energy minimized structure was used for further docking analysis.In the GOLD program, the default parameters: population size (100); selection-pressure (1.1); number of operations (10,000); number of islands (1); niche size (2); and operator weights for migrate(0), mutate(100) and crossover(100) were applied.The active site was defined within 15 Å and the ligand binding sites of COX-1 were analyzed.The docking of the COX-1 X-ray structure with the docking settings of flurbiprofen (reference ligand) showed hydrogen bonding between the carboxylate of the flurbiprofen with the phenolic OH group of Tyr355(distance=2.646Å) and amino group of Arg120(distance=2.835Å).The best docked conformation in each case was taken to unravel the disparities in binding among the series of molecules.

Table 2 .
22X-1/2 and LOX inhibitory action of synthesized compounds and their oxo-analogues22 a Values are means of three determinations and deviation from the mean is < 10% of the mean value.