COX-1/COX-2 inhibition assays and histopathological study of the new designed anti-inflammatory agent with a pyrazolopyrimidine core

Four pyrazolopyrimidine series were prepared with a substitution at position- 4 by Schiff base, triazole, oxadiazole and pyrazole moieties (7a-f, 8a,b, 9a-f, 10a,b and 13a,b), respectively. All the synthesized compounds were evaluated in vitro against COX-2 and in vivo against carrageenan-induced rat paw edema as anti-inflammatory agents. Regarding the anti-inflammatory activity (AI) compounds 7c, 7f, 8a, and 9a showed higher activity with respect to celecoxib. Compounds 9a, 7d, and 7f were closely selective to celecoxib. Also, 7c and 7d were safer than indomethacin and similar to celecoxib as resulted from the histopathological study. In addition, the docking study that showed the binding mode of prominent pyrazolopyrimidine compounds inside the COX-2 receptor. Formation of unexpected pyrazole 13a and 13b was briefly discussed using 2D NMR.     

甲基苯丙胺中毒大鼠纹状体COX-2、PGE2、EP2 受体及小胶质细胞 表达的研究

目的:通过研究COX-2、PGE2、EP2受体及小胶质细胞在甲基苯丙胺中毒大鼠纹状体内的表达变化探讨甲基苯丙胺中毒大鼠纹状体中COX-2/PGE2系统与小胶质细胞活化之间的关系。方法:将40只健康成年雄性SD大鼠,随机分成对照组10只和实验组30只(实验组分成三个亚组,分为末次给药后1天组、2天组和3天组,n=10)。实验组给予10mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射两次,注射时间为8:00、20:00,连续注射4天。分别于末次给药后的第1天、第2天、第3天处杀。用免疫组化技术对中毒大鼠纹状体(CPU)中COX-2、EP2受体及Iba1(钙离子接头蛋白,小胶质细胞内一种特异性标记物)的表达进行检测,并进行图像分析。另外,取大鼠的纹状体运用酶联免疫法检测PGE2的含量。结果:COX-2、PGE2、EP2受体及小胶质细胞在各组均有表达。与对照组相比,实验组中:COX-2、PGE2、EP2受体的1天组表达均不同程度下降;2天组中COX-2表达水平大幅度上升,PGE2、EP2受体表达仍低于正常水平;3天组COX-2表达水平继续升高,而PGE2、EP2受体表达趋于正常组水平。而小胶质细胞表达水平则是三个实验组均高于正常组,且3天组高于2天组,2天组高于1天组。对照组与实验组有显著性差异(P<0.05)。结论:COX-2/PGE2系统与甲基苯丙胺中毒大鼠纹状体内小胶质细胞活化无明显相关性;COX-2与甲基苯丙胺的神经毒性有关。     

Synthesis of new pyrazoles and pyrozolo [3,4-b] pyridines as anti-inflammatory agents by inhibition of COX-2 enzyme

New pyrazoles and pyrazolo[3,4-b] pyridines were synthesized and their structure was confirmed by elemental analyses as well as IR, ¹H NMR, ¹³C NMR, and mass spectral data. All the newly synthesized derivatives were evaluated in vitro for inhibitory activity against COX-1 and COX-2 enzymes and their IC₅₀ values were calculated, most of the derivatives showed good inhibitory activity with derivatives IVb, IVh and IVJ showing inhibitory activity better than celecoxib. Moreover, the eight most potent derivatives IVa, IVb, IVc, IVd, IVe, IVh, IVJ, and IVL were selected for in vivo assay to measure their effect on paw edema in rates and their ulcerogenic effect. Compounds IVa, IVb and IVc were found to be the most active and selective as COX-2 inhibitors and most effective in protection from edema, they were also found to have lowest ulcerogenic effect among all derivatives.     

Structural basis for selective inhibition of COX-2 by nimesulide

Nimesulide 1 is a novel nonsteroidal antiinflammatory drug which inhibits the enzyme cyclooxygenase 2 (COX-2) more selectively than cyclooxygenase 1 (COX-1). Molecular modelling studies have been carried out on complexes of 1 with COX-1 and with mutants of COX-1 simulating COX-2. These indicate that the mutations I523V and S516A largely contribute to the selectivity. A comparative study with SC-558 2 has also been performed.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

Synthesis and evaluation of 1,5-diaryl-substituted tetrazoles as novel selective cyclooxygenase-2 (COX-2) inhibitors

A series of 1,5-diaryl-substituted tetrazole derivatives was synthesized via conversion of readily available diaryl amides into corresponding imidoylchlorides followed by reaction with sodium azide. All compounds were evaluated by cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Tetrazoles 3a-e showed IC₅₀ values ranging from 0.42 to 8.1 mM for COX-1 and 2.0 to 200 μM for COX-2. Most potent compound 3c (IC₅₀ (COX-2) = 2.0 μM) was further used in molecular modeling docking studies.     

COX-1/COX-2 inhibition activities and molecular docking study of newly designed and synthesized pyrrolo[3,4-c]pyrrole Mannich bases

In the present paper we describe the biological activity of newly designed and synthesized series of pyrrolo[3,4-c]pyrrole Mannich bases (7a-n). The Mannich bases were obtained in good yields by one-pot, three-component condensation of pyrrolo[3,4–c]pyrrole scaffold (6a-c) with secondary amines and an excess of formaldehyde solution in C₂H₅OH. The chemical structures of the compounds were characterized by ¹H NMR, ¹³C NMR, FT-IR, and elemental analysis. Moreover, single crystal X-ray diffraction has been recorded for compound 7l. All synthesized derivatives were investigated for their potencies to inhibit COX-1 and COX-2 enzymes by colorimetric inhibitor screening assay. In order to analyse the intermolecular interactions between the ligands and cyclooxygenase, experimental data were supported with the results of molecular docking simulations. According to the results, all of the tested compounds inhibited the activity of COX-1 and COX-2.     

Molecular design,synthesis and biological evaluation of cyclic imides bearing benzenesulfonamide fragment as potential COX-2 inhibitors. Part 2

A group of cyclic imides (1–10) was designed for evaluation as a selective COX-2 inhibitors and investigated in vivo for their anti-inflammatory activity. Compounds 6a, 6b, 8a, 8b, 9a, 9b, 10a and 10b were proved to be potent COX-2 inhibitors with IC₅₀ range of 0.1–4.0 μM. In vitro COX-1/COX-2 inhibition structure–activity studies identified compound 8a as a highly potent (IC₅₀ = 0.1 μM), and an extremely selective [COX-2 (SI) > 1000] comparable to celecoxib [COX-2 (SI) > 384], COX-2 inhibitor that showed superior anti-inflammatory activity (ED₅₀ = 72.4 mg/kg) relative to diclofenac (ED₅₀ = 114 mg/kg). Molecular modeling was carried out through docking the designed compounds into the COX-2 binding site to predict if these compounds have analogous binding mode to the COX-2 inhibitors. The study showed that the homosulfonamide fragment of 8a inserted deep inside the 2°-pocket of the COX-2 active site, where the SO₂NH₂ group underwent H-bonding interaction with Gln¹⁹²(2.95 Å), Phe⁵¹⁸(2.82 Å) and Arg⁵¹³(2.63 and 2.73 Å). Docking study of the synthesized compound 8a into the active site of COX-2 revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

Synthesis and evaluations of selective COX-2 inhibitory effects: Benzo[d]thiazol analogs

A series of benzo[d]thiazole analogs were synthesized and evaluated for their anti-inflammatory and analgesic effects. Using an ear edema model, except for compounds 2k, 2m-2q and 3a, other compounds showed the anti-inflammatory effects. Among them, compounds 2c, 2d, and 2g showed the best anti-inflammatory activity (inhibition rate: 86.8%, 90.7% and 82.9%, respectively). By the acetic acid-induced abdominal writhing test, except for compounds 2e, 2l, 2m, 2o, 2p and 3a, other compounds showed the analgesic effects with inhibition rate values of 51.9–100% (2a-2r) and 68.6–100% (3a-3g). Next, compounds 2c, 2d, 2g, 3d, 3f, 3g that displayed the excellent anti-inflammatory and analgesic activities were evaluated for their inhibitory effect against ovine COX-1 and COX-2. Compounds 2c, 2d, 2g, 3d, 3f, 3g were weak inhibitors of the COX-1 isozyme but exhibited the moderate COX-2 isozyme inhibitory effects IC₅₀ from 0.28 to 0.77 μM and COX-2 selectivity indexes (SI: 18.6 to 7.2). This benzo[d]thiazole moiety will be proved to be of great significance for developing more potent COX-2 inhibitors.     

Toward the understanding of the molecular basis for the inhibition of COX-1 and COX-2 by phenolic compounds present in Uruguayan propolis and grape pomace

Propolis and grape pomace have significant amounts of phenols which can take part in anti-inflammatory mechanisms. As the cyclooxygenases 1 and 2 (COX-1 and COX-2) are involved in said mechanisms, the possibility for a selective inhibition of COX-2 was analyzed in vitro and in silico. Propolis and grape pomace from Uruguayan species were collected, extracted in hydroalcoholic mixture and analyzed. Based on phenols previously identified, and taking as reference the crystallographic structures of COX-1 and COX-2 in complex with the commercial drug Celecoxib, a molecular docking procedure was devised to adjust 123 phenolic molecular models at the enzyme-binding sites. The most important results of this work are that the extracts have an overall inhibition activity very similar in COX-1 and COX-2, i.e. they do not possess selective inhibition activity for COX-2. Nevertheless, 10 compounds of the phenolic database turned out to be more selective and 94 phenols resulted with similar selectivity than Celecoxib, an outcome that accounts for the overall experimental inhibition measures. Binding site environment observations showed increased polarity in COX-2 as compared with COX-1, suggesting that polarity is the key for selectivity. Accordingly, the screening of molecular contacts pointed to the residues: Arg106, Gln178, Leu338, Ser339, Tyr341, Tyr371, Arg499, Ala502, Val509, and Ser516, which would explain, at the atomic level, the anti-inflammatory effect of the phenolic compounds. Among them, Gln178 and Arg499 appear to be essential for the selective inhibition of COX-2.     

Utility of novel 2-furanones in synthesis of other heterocyclic compounds having anti-inflammatory activity with dual COX2/LOX inhibition

Inflammation is associated with the development of several diseases comprising cancer and cardiovascular disease. Agents that suppress cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, besides chemokines have been suggested to minimise inflammation. Here, a variety of novel heterocyclic and non-heterocyclic compounds were prepared from novel three furanone derivatives. The structures of all synthesised compounds were confirmed by elemental and spectral analysis including mass, IR, and ¹H-NMR spectroscopy. Anti-inflammatory activities of these synthesised compounds were examined in vitro against COX enzymes, 15-LOX, and tumour necrosis factor-α (TNF-α), using inhibition screening assays. The majority of these derivatives showed significant to high activities, with three pyridazinone derivatives (5b, 8b, and 8c) being the most promising anti-inflammatory agents with dual COX-2/15-LOX inhibition activities along with high TNF-α inhibition activity.     

Mucosal damage induced by preferential COX-1 and COX-2 inhibitors: role of prostaglandins and inflammatory response

Nonsteroidal anti-inflammatory drugs (NSAID) are well known to induce gastric mucosal damage including bleeding, ulceration and perforation in humans and animals too. These effects are related with the inhibition of the enzyme cyclooxygenase, which is the main established mechanism of action for these drugs. Fasted rats were given piroxicam, preferential COX-1 inhibitor (10-20 mg/kg) or meloxicam, preferential COX-2 inhibitor (7.5-15 mg/kg) orally. Six or nine hours (h) later, respectively, the stomach was excised, the severity of the damage assessed and myeloperoxidase (MPO) activity measured, as well as prostaglandin PGE(2) content. Furthermore, in order to assess the effects of these oxicams over previously damaged gastric mucosa, 1 ml of 0.6 N HCl was administered p.o. followed, 1 h after, of the correspondent dose of each NSAID, and the same parameters were determined. Oral administration of both drugs dose-dependently caused acute gastric haemorrhage erosions. Myeloperoxidase activity was significantly increased by piroxicam administration. In addition, PGE(2) content was significantly reduced. The association between the administration of the acid and NSAID caused a worsening of the damage and, while myeloperoxidase activity did not modify by both piroxicam and meloxicam, PGE(2) levels were reduced. These results suggest that the PG derived from both COX-1 and COX-2 pathway plays a beneficial role in the gastroprotection, and thus caution should be exercise in the clinical use of preferential COX-2 inhibitors.     

Novel chalcone/aryl carboximidamide hybrids as potent anti-inflammatory via inhibition of prostaglandin E2 and inducible NO synthase activities: design,synthesis, molecular docking studies and ADMET prediction

Two series of chalcone/aryl carboximidamide hybrids 4a–f and 6a–f were synthesised and evaluated for their inhibitory activity against iNOS and PGE2. The most potent derivatives were further checked for their in vivo anti-inflammatory activity utilising carrageenan-induced rat paw oedema model. Compounds 4c, 4d, 6c and 6d were proved to be the most effective inhibitors of PGE2, LPS-induced NO production, iNOS activity. Moreover, 4c, 4d, 6c and 6d showed significant oedema inhibition ranging from 62.21% to 78.51%, compared to indomethacin (56.27 ± 2.14%) and celecoxib (12.32%). Additionally, 4c, 6a and 6e displayed good COX2 inhibitory activity while 4c, 6a and 6c exhibited the highest 5LOX inhibitory activity. Compounds 4c, 4d, 6c and 6d fit nicely into the pocket of iNOS protein (PDB ID: 1r35) via the important amino acid residues. Prediction of physicochemical parameters exhibited that 4c, 4d, 6c and 6d had acceptable physicochemical parameters and drug-likeness. The results indicated that chalcone/aryl carboximidamides 4c, 4d, 6c and 6d, in particular 4d and 6d, could be used as promising lead candidates as potent anti-inflammatory agents.     

Synthesis of novel hybrids of pyrazole and coumarin as dual inhibitors of COX-2 and 5-LOX

In our previous study, we designed a series of pyrazole derivatives as novel COX-2 inhibitors. In order to obtain novel dual inhibitors of COX-2 and 5-LOX, herein we designed and synthesized 20 compounds by hybridizing pyrazole with substituted coumarin who was reported to exhibit 5-LOX inhibition to select potent compounds using adequate biological trials sequentially including selective inhibition of COX-2 and 5-LOX, anti-proliferation in vitro, cells apoptosis and cell cycle. Among them, the most potent compound 11g (IC₅₀ = 0.23 ± 0.16 μM for COX-2, IC₅₀ = 0.87 ± 0.07 μM for 5-LOX, IC₅₀ = 4.48 ± 0.57 μM against A549) showed preliminary superiority compared with the positive controls Celecoxib (IC₅₀ = 0.41 ± 0.28 μM for COX-2, IC₅₀ = 7.68 ± 0.55 μM against A549) and Zileuton (IC₅₀ = 1.35 ± 0.24 μM for 5-LOX). Further investigation confirmed that 11g could induce human non-small cell lung cancer A549 cells apoptosis and arrest the cell cycle at G2 phase in a dose-dependent manner. Our study might contribute to COX-2, 5-LOX dual inhibitors thus exploit promising novel cancer prevention agents.     

Synthesis,anti-inflammatory,analgesic, COX-1/2 inhibition activities and molecular docking study of pyrazoline derivatives

Design, synthesis and pharmacological activities of a group of 1,3,5-trisubstituted pyrazolines were reported. The chemical structures of the synthesized compounds have been assigned on the basis of IR, MS, ¹H NMR, and ¹³C NMR spectral analyses. The synthesized 1,3,5-trisubstituted pyrazoline derivatives were evaluated in vivo for anti-inflammatory, analgesic activities and in vitro for COX-1/2 inhibition assay. Among the tested compounds, derivatives 4h, 6e, 7a, 7e, and 9 showed more potent anti-inflammatory and analgesic activities than the reference drug celecoxib. On the basis of their higher activities in the in vivo studies compared with celecoxib, the five compounds 4h, 6e, 7a, 7e and 9 were selected to test their inhibitory activities against ovine COX-1/2 using an in vitro cyclooxygenase inhibition assay. Docking study of compounds 7a, 7e and 9 into the COX-2 binding site revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

Novel aryl carboximidamide and 3-aryl-1,2,4-oxadiazole analogues of naproxen as dual selective COX-2/15-LOX inhibitors: Design,synthesis and docking studies

A series of novel naproxen analogues containing 3-aryl-1,2,4-oxadiazoles moiety (4b-g) and their reaction intermediates aryl carboximidamides moiety (3b-g) was synthesized and evaluated in vitro as dual COXs/15-LOX inhibitors. Compounds 3b-g exhibited superior inhibitory activity than celecoxib as COX-2 inhibitors. Compounds 3b-d and 3g were the most potent COX-2 inhibitors with IC₅₀ range of 6.4 – 8.13 nM and higher selectivity indexes (3b, SI = 26.19; 3c, SI = 13.73; 3d, SI = 29.27; 3g, SI = 18.00) comparing to celecoxib (IC₅₀ = 42.60 nM, SI = 8.05). Regarding 15-LOX inhibitory activity, compounds belonging to aryl carboximidamide backbone 3b-e and 3g were the most potent with IC₅₀ range of 1.77–4.91 nM comparing to meclofenamate sodium (IC₅₀ = 5.64 µM). Data revealed that The levels of NO released by aryl carboximidamides 3b-g were more higher than 3-aryl-1,2,4-oxadiazole derivatives 4b-g, which correlated well with their COX-2 inhibitory activities.     

Synthesis,biological evaluation and molecular modeling study of pyrazole derivatives as selective COX-2 inhibitors and anti-inflammatory agents

A novel series of pyrazole derivatives were synthesized and evaluated in vivo for their anti-inflammatory activity in carrageenan-induced rat paw edema model. Among all compounds, 5a, and 5b showed comparable anti-inflammatory activity to Nimesulide, the standard drug taken for the studies. In silico (docking) studies were carried out to investigate the theoretical binding mode of the compounds to target the cyclooxygenase (COX-2) using Autodock 4.2.