Synthesis and SAR of potent and selective androgen receptor antagonists: 5,6-Dichloro-benzimidazole derivatives

The synthesis and in vivo SAR of 5,6-dichloro-benzimidazole derivatives as novel selective androgen receptor antagonists are described. During screening of 2-alkyl benzimidazoles, it was found that a trifluoromethyl group greatly enhances antagonist activity in the prostate. Benzimidazole 1 is a potent AR antagonist in the rat prostate (ID50 = 0.15 mg/day).     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

Insight from molecular modeling into different conformation and SAR of natural steroids and unnatural 7-oxa-steroids

Replacement of the 7-CH2 group of natural steroid with an oxygen atom led to identification of unnatural 7-oxa-steroids as potent and selective progesterone receptor antagonists. The unnatural 7-oxa-steroids exhibited a different structure-activity relationship (SAR) from natural steroids. Molecular modeling demonstrated that the switch of carbon to oxygen in the B-ring results in a subtle conformational change of the tetracyclic skeleton and induces a remarkable spatial shift at the terminal end of the side chain of the D-ring. This shift causes the phenyl ring on the D-ring to form a perfect parallel-displaced form of pi-pi interaction with the phenyl ring of Phe794. The unnatural 7-oxa-steroids were orally active in a rat complement C3 assay and showed comparable pharmacokinetic and metabolic profiles to those of the natural steroid, mifepristone.     

Dual binding site inhibitors of B-RAF kinase

Computer aided modeling guided the design of a series of diarylimidazole compounds (11–22) intended to interact with both the ATP and adjacent allosteric binding domains of B-RAF kinase. Their ability to inhibit the function of B-RAF kinase and intracellular ERK1/2 phosphorylation were evaluated.     

Novel 5-aryl-1,3-dihydro-indole-2-thiones. potent,orally active progesterone receptor agonists

During the course of our studies on 3,3-disubstituted-5-aryloxindoles derived progesterone receptor (PR) antagonists we discovered that changing the amide funtionality to a thio-amide resulted in compounds displaying potent PR agonist activity. In this communication, the synthesis, structure activity relationships (SAR) and in vivo activity of various 5-arylthio-oxindoles will be discussed.     

Selective estrogen receptor modulators protect hippocampal neurons from kainic acid excitotoxicity: differences with the effect of estradiol

Neuroprotective effects of estradiol are well characterized in animal experimental models. However, in humans, the outcome of estrogen treatment for cognitive function and neurological diseases is very controversial. Selective estrogen receptor modulators (SERMs) may represent an alternative to estrogen for the treatment or the prevention of neurodegenerative disorders. SERMs interact with the estrogen receptors and have tissue-specific effects distinct from those of estradiol, acting as estrogen agonists in some tissues and as antagonists in others. In this study we have assessed the effect of tamoxifen, raloxifene, lasofoxifene (CP-336,156), bazedoxifene (TSE-424), and 17beta-estradiol on the hippocampus of adult ovariectomized rats, after the administration of the excitotoxin kainic acid. Administration of kainic acid induced the expression of vimentin in reactive astroglia and a significant neuronal loss in the hilus. SERMs did not affect vimentin immunoreactivity in the hilus, while 17beta-estradiol significantly reduced the surface density of vimentin immunoreactive profiles. Estradiol, tamoxifen (0.4-2 mg/kg), raloxifene (0.4-2 mg/kg), and bazedoxifene (2 mg/kg) prevented neuronal loss in the hilus after the administration of kainic acid. Lasofoxifene (0.4-2 mg/kg) was not neuroprotective. These findings indicate that SERMs present different dose-dependent neuroprotective effects. Furthermore, the mechanisms of neuroprotection by SERMs and estradiol are not identical, because SERMs do not significantly affect reactive gliosis while neuroprotection by estradiol is associated with a strong down-regulation of reactive astroglia.     

The Transmembrane Helices of Beef Heart Cytochrome Oxidase

The locations of the transmembrane helices in the 12 subunits of beef heart cytochrome oxidase were predicted with a modified form of the von Heijne-Blomberg hydrophobicity scale. Based on ~20 residues per transmembrane helix, about 480 of the estimated 660 helical residues (36.8% of 1,793 total residues) are expected to be in transmembrane helices that have their axes tilted by a small angle α from the normal to the plane of the membrane. This angle is calculated to be ~30°, based on the observed overall tilt angle θ of 39° obtained from circular dichroism (CD) measurements on multilamellar films, or about 25°, based on the observed tilt angle θ of 36° obtained from the infrared linear dichroism of films. For 21 residues per transmembrane helix, the calculated values of α become 32° and 28°, respectively, depending upon the value of θ used. Thus, a transmembrane helical tilt angle of ~30° accounts for the predicted transmembrane stretches in cytochrome oxidase if 20-21 residues are sufficient to span the membrane. Additional helical residues in the lipid head region may deviate by a larger angle from the normal to the plane of the membrane in cytochrome oxidase.     

Evolutionary rates for tuf genes in endosymbionts of aphids

The gene encoding elongation factor Tu (tuf) in aphid endosymbionts (genus Buchnera) evolves at rates of 1.3 x 10(-10) to 2.5 x 10(-10) nonsynonymous substitutions and 3.9 x 10(-9) to 8.0 x 10(-9) synonymous substitutions per position per year. These rates, which are at present among the most reliable substitution rates for protein-coding genes of bacteria, have been obtained by calibrating the nodes in the phylogenetic tree produced from the Buchnera EF-Tu sequences using divergence times for the corresponding ancestral aphid hosts. We also present data suggesting that the rates of nonsynonymous substitutions are significantly higher in the endosymbiont lineages than in the closely related free-living bacteria Escherichia coli and Salmonella typhimurium. Synonymous substitution rates for Buchnera approximate estimated mutation rates for E. coli and S. typhimurium, as expected if synonymous changes act as neutral mutations in Buchnera. We relate the observed differences in substitution frequencies to the absence of selective codon preferences in Buchnera and to the influence of Muller's ratchet on small asexual populations.      

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000