Antonio Scilimati

De Novo Synthesis of Cyclooxygenase-1 Counteracts the Suppression of Platelet Thromboxane Biosynthesis by Aspirin

Aspirin affords cardioprotection through the acetylation of serine529 in human cyclooxygenase-1 (COX-1) of anucleated platelets, inducing a permanent defect in thromboxane A2 (TXA2)–dependent platelet function. However, heterogeneity of COX-1 suppression by aspirin has been detected in cardiovascular disease and may contribute to failure to prevent clinical events. The recent recognized capacity of platelets to make proteins de novo paves the way to identify new mechanisms involved in the variable response to aspirin. We found that in washed human platelets, the complete suppression of TXA2 biosynthesis by aspirin, in vitro, recovered in response to thrombin and fibrinogen in a time-dependent fashion (at 0.5 and 24 hours, TXB2 averaged 0.1±0.03 and 3±0.8 ng/mL; in the presence of arachidonic acid [10 &mgr;mol/L], it was 2±0.7 and 25±7 ng/mL, respectively), and it was blocked by translational inhibitors, by rapamycin, and by inhibitors of phosphatidylinositol 3-kinase. The results that COX-1 mRNA was readily detected in resting platelets and that [35S]-methionine was incorporated into COX-1 protein after stimulation strongly support the occurrence of de novo COX-1 synthesis in platelets. This process may interfere with the complete and persistent suppression of TXA2 biosynthesis by aspirin necessary for cardioprotection.

Selective COX-1 inhibition: A therapeutic target to be reconsidered.

Since cyclooxygenase (COX) isozymes discovery, many papers and reviews have been published to describe the structural bases of COX inhibition, and to debate on the therapeutic and adverse effects of worldwide clinically used nonsteroidal anti-inflammatory drugs (NSAIDs), included COX-2 selective inhibitors (well known as Coxibs). COX-2 inhibition has been widely investigated, whereas the role of COX-1 in human pathophysiology is mostly not yet well ascertained. As time goes on, the cliché that the constitutively expressed isoform COX-1 is only involved in normal physiological functions, such as platelet aggregation, gastric mucosa protection and renal electrolyte homeostasis is going to be shattered. Low-dose aspirin, behaving as a preferential inhibitor of platelet COX-1, allowed to enlighten the role exerted by this isoenzyme in many mammalian cell types. This review would elucidate the most recent findings on selective COX-1 inhibition and their relevance to human pathology such as cancer, neuro-inflammation, cardioprotection, fever and pain. It would also focus on the design and development of new highly selective COX-1 inhibitors, useful tools in pharmacological studies aimed at gaining a deeper insight of the role of COX-1 in human health and disease. Among the traditional NSAIDs, other then aspirin and indomethacin, only few examples of selective COX-1 inhibitors (SC-560, FR122047, mofezolac, P6 and TFAP) have been so far identified. This review has also the scope to stimulate the development of novel drugs, which activity is COX-1 mediated.

Synthesis, Pharmacological Characterization, and Docking Analysis of a Novel Family of Diarylisoxazoles as Highly Selective Cyclooxygenase-1 (COX-1) Inhibitors

3-(5-Chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6), a known selective cyclooxygenase-1 (COX-1) inhibitor, was used to design a new series of 3,4-diarylisoxazoles in order to improve its biochemical COX-1 selectivity and antiplatelet efficacy. Structure-activity relationships were studied using human whole blood assays for COX-1 and COX-2 inhibition in vitro, and results showed that the simultaneous presence of 5-methyl (or -CF3), 4-phenyl, and 5-chloro(-bromo or -methyl)furan-2-yl groups on the isoxazole core was essential for their selectivity toward COX-1. 3g, 3s, 3d were potent and selective COX-1 inhibitors that affected platelet aggregation in vitro through the inhibition of COX-1-dependent thromboxane (TX) A2. Moreover, we characterized their kinetics of COX-1 inhibition. 3g, 3s, and 3d were more potent inhibitors of platelet COX-1 and aggregation than P6 (named 6) for their tighter binding to the enzyme. The pharmacological results were supported by docking simulations. The oral administration of 3d to mice translated into preferential inhibition of platelet-derived TXA2 over protective vascular-derived prostacyclin (PGI2).

Novel selective COX-1 inhibitors suppress neuroinflammatory mediators in LPS-stimulated N13 microglial cells

COX-1 plays a previously unrecognized part in the neuroinflammation. Genetic ablation or pharmacological inhibition of COX-1 activity attenuates the inflammatory response and neuronal loss. In this context, the effects of selective COX-1 inhibitors (P6, P10, SC-560, aspirin) and coxibs (celecoxib and etoricoxib) on LPS-stimulated microglial cell function (a worldwide accepted neuroinflammation model) were investigated, and the effects on COX-1/COX-2, cPGES mRNA and iNOS expression, PGE(2) and NO production and NF-κB activation by IκBα phosphorylation were evaluated. The total suppression of the expression of both COX-1 and COX-2 by their respective selective inhibitors occurred. NF-κB remained almost completely inactive in the presence of coxibs, as expected, and totally inactive in the presence of P6. P6 also markedly counteracted LPS enhancing cPGES mRNA expression and PGE(2) production. Since COX-1 is predominantly localized in microglia, its high selective inhibition rather than COX-2 (by coxibs) is more likely to reduce neuroinflammation and has been further investigated as a potential therapeutic approach and prevention in neurodegenerative diseases with a marked inflammatory component.

Diarylheterocycle core ring features effect in selective COX-1 inhibition.

The COX‐1 isoenzyme plays a significant role in a variety of diseases, as it catalyzes the bioprocesses behind many health problems. Among the diarylheterocycle class of COX inhibitors, the isoxazole ring has been widely used as a central heterocycle for the preparation of potent and selective COX‐1 inhibitors such as P6 [3‐(5‐chlorofuran‐2‐yl)‐5‐methyl‐4‐phenylisoxazole]. The role of the isoxazole nucleus in COX‐1 inhibitor selectivity has been clarified by preparing a set of new diarylheterocycles with various heterocycle cores. Replacement of isoxazole with isothiazole or pyrazole gave a drastic decrease in COX‐1 inhibitory activity, whereas the introduction of an electron‐donating group (EDG) on the N‐aryl pyrazole allowed recovery of COX‐1 inhibitory activity and selectivity. The EDG‐equipped 5‐(furan‐2‐yl)‐1‐(4‐methoxyphenyl)‐3‐(trifluoromethyl)‐1H‐pyrazole (17) selectively inhibits COX‐1 activity (IC50=3.4 μM; 28 % COX‐2 inhibition at 50 μM), in contrast to its inactive analogue, 3‐(furan‐2‐yl)‐1‐phenyl‐5‐(trifluoromethyl)‐1H‐pyrazole, which does not bear the methoxy EDG. Molecular docking studies of compound 17 into the binding site of COX‐1 shed light on its binding mode.

Constrained analogues of tocainide as potent skeletal muscle sodium channel blockers towards the development of antimyotonic agents

1-Benzyl-N-(2,6-dimethylphenyl)piperidine-3-carboxamide and 4-benzyl-N-(2,6-dimethylphenyl)piperazine-2-carboxamide, two conformationally restricted analogues of tocainide, were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. They showed, with respect to tocainide, a marked increase in both potency and use-dependent block.

Optically active sulfoxides by enantiospecific reactions of bromovinyl aryl sulfoxides with grignard reagents

Abstract Optically active bromovinyl aryl sulfoxides were subjected to reactions with phenyl- or alkyl-magnesium compounds, with the production of optically active diaryl or aryl alkyl sulfoxides, with inversion of configuration. The enantiospecificity of the process was found to be 98–100%.

Chemical and chemoenzymatic routes to 1-(benzothiazol-2-ylsulfanyl)-3-chloropropan-2-ol, a precursor of drugs with potential β-blocker activity

Abstract Several methods have been developed to prepare 1-(benzothiazol-2-ylsulfanyl)-3-chloropropan-2-ol 2 with good to high enantiomeric excess: 70 and 93% ees have in fact been obtained by bakers yeast-induced asymmetric reduction of the ketone precursor 1 and by kinetic resolution performed in the presence of lipase from Pseudomonas sp. (E=38), respectively. Compounds (R)-(+)-2 and (S)-(−)-2 have also been prepared by a chemical method in 90% yield and with enantiomeric excesses of 98 and 96.4%, respectively. HPLC on Chiralcel OD column separation of enantiomers (separability factor α=1.64) has also been successfully performed. Compound 2 could, in turn, be used for the synthesis in an optically active form of various molecules, including β-aminoalcohols 6, drugs with potential β-blocker activity.

Baker's yeast-induced asymmetric reduction of α-ketosulfides: synthesis of optically active 1-(benzothiazol-2-ylsulfanyl)-2-alkanols, 2-alkanols, and thiiranes

Abstract 1-(Benzothiazol-2-ylsulfanyl)-2-alkanols 3 were prepared in very high enantiomeric excess by bakers yeast-induced asymmetric reduction of 1-(benzothiazol-2-ylsulfanyl)-2-alkanones 1 . Conversion of 3 into optically active simple 2-alkanols 4 and thiiranes 2 by reductive desulfurization and base treatment, respectively, is also described. The absolute configuration of the new compounds synthesized has been established by chemical correlation and specific rotation comparison.

Decomposition of arylazides by thf/n-butyllithium-II-isolation of 1-aryl-4,5-dihydro-5-hydroxy-1h-1,2,3-triazoles

Abstract By reaction of arylazides having no electron-withdrawing groups with the enolate ion of the acetaldehyde (formed by cycloreversion of THF in the presence of n -butyllithium), 1-aryl-4,5-dihydro-5-hydroxy-1H-1,2,3-triazoles could be isolated and then characterized. Further reaction of such hydroxytriazolines with the same enolate ion afforded substantial amounts of the corresponding N-formylanilines, confirming their intermediacy in the previously reported decomposition of arylazides by THF/ n -butyllithium. On the other hand, using alkoxides as bases, the preferred scheme of conversion of the hydroxytriazolines was found to be the known dehydration to 1-aryl-1H-1, 2,3-triazoles.