The synthesis and pharmacological activity of a new series of 1-arylpyrazoles as potent σ(1) receptor (σ(1)R) antagonists are reported. The new compounds were evaluated in vitro in human σ(1)R and guinea pig σ(2) receptor (σ(2)R) binding assays. The nature of the pyrazole substituents was crucial for activity, and a basic amine was shown to be necessary, in accordance with known receptor pharmacophores. A wide variety of amines and spacer lengths between the amino and pyrazole groups were tolerated, but only the ethylenoxy spacer and small cyclic amines provided compounds with sufficient selectivity for σ(1)R vs σ(2)R. The most selective compounds were further profiled, and compound 28, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28 to be selected as clinical candidate.

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor's response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in alpha-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.

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A capillary electrophoresis method was developed for the enantioseparation of three novel cyclooxygenase-2 (COX-2) inhibitor drugs (E-6259, E-6036 and E-6087) with anti-inflammatory and analgesic activities using sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) as a chiral selector. The use of 50 mM sodium tetraborate at pH 9.2 with 30% v/v methanol, containing 7.1 mM SBE-beta-CD, as a background electrolyte (BGE) allowed the complete enantioseparation of the three neutral racemic mixtures (resolution = 2.4, 3.0 and 8.7, respectively) and their corresponding metabolites (oxidation products) in a single run. Migration times were shortened with some loss of enantioresolution by adding 1.75 mM dimethyl-beta-cyclodextrin (DM-beta-CD) to the previous BGE (dual CD system). The reversal of the migration order of E-6259 enantiomers in the dual CD system was also studied. Furthermore, the addition of DM-beta-CD to the BGE introduced a new chemoselectivity in the system that allowed E-6259 to be separated from the structurally similar compound E-6036.

The synthesis of a new series of 4-acylaminopyrazolo[3,4-]pyrimidines active on the sigma-1 receptor (σR) is reported. Compounds were efficiently prepared using a two to three step process starting from commercially available 1-pyrazolo[3,4-]pyrimidin-4-amine. A SAR study shows that the σR requires the presence of relatively highly lipophilic substituents at opposite sides of the central scaffold, while selectivity the σR can be improved by shortening the distance of the basic nitrogen to it. Compound was among the most active and selective derivatives and exhibited potent antinociceptive properties in several pain models in mice, indicating its antagonistic behaviour.

E-6087 is the most advanced compound among the cyclooxygenase-2 (COX-2) inhibitor drugs developed in our company. Its activity is mainly associated with the S(-)-enantiomer (E-6232), whereas the R(-)-enantiomer (E-6231) becomes an impurity whose content should be determined. Five main impurities and degradation products of E-6232 have been found (E-6144, E-6024, E-6072, E-6397 and E-6132), and some of them co-elute with the distomer when using a chiral high-performance liquid chromatography (HPLC) method. Consequently, we have optimized the separation of all the impurities from the two enantiomers of E-6087 by capillary electrophoresis (CE), in order to use the method for the enantiomeric purity determination of E-6232. The effect of the methanol (MeOH) content in the background electrolyte (BGE), the sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) and heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) concentration, and the capillary temperature have been studied. Separation of all compounds could be achieved in different systems, either in a single CD-system (with SBE-beta-CD) or in a dual CD-system (with DM-beta-CD as a neutral CD). By using the dual CD system a limit of detection (LOD) and a limit of quantitation (LOQ) of 0.03% and 0.1% of distomer, respectively, were achieved*.

The synthesis of a set of 1-aryl-2-aryl(3-pyridyl)ethanones 1-5 and the corresponding ketoximes 6-9 is reported. Structural studies of oximes 6, 7 and 9 were performed in solution using 1H-NMR and in the solid state by X-ray crystallography, providing evidence of H-bonding networks. The crystal packing was controlled by homomeric intermolecular oxime···oxime H-bond interactions for 6 and cooperative oxime···N(pyridyl) and CH/π interactions for 7 and 9.

A series of new N1-, N2-and N3-substituted 1,2,3-triazole derivatives has been synthesized by cycloaddition of butyltin azide with substituted alkynes followed by a N-alkylation reaction. The regioisomers have been isolated and characterized using NMR techniques. GIAO/B3LYP calculations of the absolute shieldings have been performed to verify the assignments and so the structures have been unequivocally identified. The proportion in which the three isomers are obtained corresponds with the relative order of stability indicated by the energy values calculated at the B3LYP level. CB1 cannabinoid receptor binding assays have been performed but none of the compounds showed significant activity.