Pyrazolo[3,4-d]pyrimidines as sigma-1 receptor ligands for the treatment of pain. Part 1: 4-acylamino derivatives.

The synthesis of a new series of 4-acylaminopyrazolo[3,4-d]pyrimidines active on the sigma-1 receptor (σ1R) is reported. Compounds were efficiently prepared using a two to three step process starting from commercially available 1H-pyrazolo[3,4-d]pyrimidin-4-amine. A SAR study shows that the σ1R requires the presence of relatively highly lipophilic substituents at opposite sides of the central scaffold, while selectivity versus the σ2R can be improved by shortening the distance of the basic nitrogen to it. Compound 9a was among the most active and selective in vitro derivatives and exhibited potent antinociceptive properties in several pain models in mice, indicating its antagonistic behaviour.

Synthesis and biological evaluation of the 1-arylpyrazole class of σ(1) receptor antagonists: identification of 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862).

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-independent inhibitory effects on T cell activation of novel 4,5-dihydro-3 trifluoromethyl pyrazole cyclooxygenase-2 inhibitors.

Anti-inflammatory efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) has been related to their properties as inhibitors of cyclooxygenase (COX)-mediated prostaglandin (PG) synthesis. However, recent studies have suggested that variations of the in vivo anti-inflammatory actions among different NSAIDs could not be solely explained by COX inhibition. Here, we have analyzed the effects on T cell activation of novel 4,5-dihydro-3 trifluoromethyl pyrazole anti-inflammatory drugs with different potencies as COX-2 inhibitors, namely E-6087, E-6232, E-6231, E-6036 and E-6259 as well as the chemically related COX-2 inhibitor Celecoxib. These drugs inhibited mitogen-mediated T cell proliferation as well as Interleukin (IL)-2, tumor necrosis factor (TNF)-α and Interferon (IFN)-γ synthesis by activated T cells, independently of their ability to inhibit COX-2 enzymatic activity. Immunosuppressive effects of these drugs seem to be due to their interference on transcription factor activation as induced transcription from Nuclear Factor (NF)-κB and Nuclear Factor of Activated T cells (NFAT)-dependent enhancers was inhibited in a dose-dependent manner, being the latter effect the most sensitive to the action of those compounds. Both NFAT dephosphorylation, required for its nuclear translocation, as well as transcriptional activity of a GAL4-NFAT chimera were diminished in the presence of these compounds. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory actions of NSAIDs, which may have important implications in anti-inflammatory therapy, through inhibition of NFAT.

Factors influencing histological response after neoadjuvant chemoradiation therapy for rectal carcinoma.

Neoadjuvant chemoradiation therapy is one of the standard therapeutic regimens for rectal carcinoma. Nevertheless, chemoradiation therapy is not completely devoid of adverse effects, and it would be interesting to try to predict which patient will respond to neoadjuvancy. This study aimed at analyzing factors influencing pathological response after therapy. We reviewed the clinical and morphological data of 39 patients after neoadjuvant chemoradiation therapy. We performed immunohistochemistry for p53, cyclin D1, MIB-1 (Ki67), and bcl-2 protein in paraffin-embedded tissue. In our series, 12 patients did not respond to neoadjuvant therapy, 12 showed a complete response, and 15 a partial response. There was a statistically significant association between response and cardiomyopathy (p=0.02) and tenesmus (p=0.02) and a trend towards significance for age (p=0.08), preoperative TNM (p=0.08), peritumoral inflammatory response (p=0.07), and preoperative CEA (p=0.08). As for immunohistochemistry, we only found a trend towards significance for cyclin D1 (p=0.08). In our series of patients with rectal carcinoma receiving preoperative chemoradiation therapy, few factors were predictive of a histological response. The histological response seems to improve survival and reduce relapses.

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature.

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.

1,2-Diaryl(3-pyridyl)ethanone oximes. Intermolecular hydrogen bonding networks revealed by X-ray diffraction.

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 (1)H-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/pi interactions for 7 and 9.

Determination of enantiomeric purity of a novel COX-2 anti-inflammatory drug by capillary electrophoresis using single and dual cyclodextrin systems.

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*.

Enantioseparation of novel COX-2 anti-inflammatory drugs by capillary electrophoresis using single and dual cyclodextrin systems.

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.