TRPV1 antagonists that cause hypothermia, instead of hyperthermia, in rodents: Compounds' pharmacological profiles, in vivo targets, thermoeffectors recruited and implications for drug development.

AIM: Thermoregulatory side effects hinder the development of transient receptor potential vanilloid-1 (TRPV1) antagonists as new painkillers. While many antagonists cause hyperthermia, a well-studied effect, some cause hypothermia. The mechanisms of this hypothermia are unknown and were studied herein. METHODS: Two hypothermia-inducing TRPV1 antagonists, the newly synthesized A-1165901 and the known AMG7905, were used in physiological experiments in rats and mice. Their pharmacological profiles against rat TRPV1 were studied in vitro. RESULTS: Administered peripherally, A-1165901 caused hypothermia in rats by either triggering tail-skin vasodilation (at thermoneutrality) or inhibiting thermogenesis (in the cold). A-1165901-induced hypothermia did not occur in rats with desensitized (by an intraperitoneal dose of the TRPV1 agonist resiniferatoxin) sensory abdominal nerves. The hypothermic responses to A-1165901 and AMG7905 (administered intragastrically or intraperitoneally) were absent in Trpv1-/- mice, even though both compounds evoked pronounced hypothermia in Trpv1+/+ mice. In vitro, both A-1165901 and AMG7905 potently potentiated TRPV1 activation by protons, while potently blocking channel activation by capsaicin. CONCLUSION: TRPV1 antagonists cause hypothermia by an on-target action: on TRPV1 channels on abdominal sensory nerves. These channels are tonically activated by protons and drive the reflectory inhibition of thermogenesis and tail-skin vasoconstriction. Those TRPV1 antagonists that cause hypothermia further inhibit these cold defences, thus decreasing body temperature. SIGNIFICANCE: TRPV1 antagonists (of capsaicin activation) are highly unusual in that they can cause both hyper- and hypothermia by modulating the same mechanism. For drug development, this means that both side effects can be dealt with simultaneously, by minimizing these compounds' interference with TRPV1 activation by protons.

Synthesis and characterization of non-steroidal ligands for the glucocorticoid receptor: selective quinoline derivatives with prednisolone-equivalent functional activity.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

Bisphenolic compounds that enhance cell cation transport are found in commercial phenol red.

We have isolated two bisphenolic compounds (4 and 5) that have a marked effect on K+ and Na+ concentrations in human cells from commercial preparations of the pH indicator dye phenol red (phenolsulfonphthalein). We used a bioassay to identify active chromatographic fractions from the lipophilic impurities present in phenol red, and we determined the structure of two active components (4 and 5) by 1H and 13C NMR and mass spectrometry. When added to human fibroblasts in serum-free medium, the bisphenol fluorene derivative 9,9-bis(4'-hydroxyphenyl)-3-hydroxyfluorene (5) produced a rapid loss of K+ and a gain of Na+, at low concentrations, with an EC50 between 30 and 60 ng/mL (80-160 nM). The 2- and 4-hydroxy isomers of the fluorene 5 (i.e., compounds 6 and 7), prepared by synthesis, had similar activity, although compound 6 was somewhat less potent. The bisphenol xanthene derivative 9,9-bis(4'-hydroxyphenyl)xanthene (4) elicited a similar biological response but was less potent than 5-7; it also had a strong effect on cell adhesion, causing release of cells from the plastic substrate at concentrations as low as 2-5 microg/mL (5.5-14 microM). The structures of xanthene (4) and fluorene (5) bisphenols have been confirmed by synthesis from xanthone and hydroxyfluorenone, respectively, by Friedel-Crafts alkylation with phenol. In the latter case, the desired 3-hydroxyfluorene isomer was formed in situ by rearrangement of the 1-hydroxy isomer.

Benz[a]anthracene diols: predicted carcinogenicity and structure-estrogen receptor binding affinity relationships.

Benz[a]anthracenes are ubiquitous environmental carcinogens that exert estrogenic and antiestrogenic effects directly or via hydroxylated metabolites. In this paper, the structure-estrogen receptor binding relationships of four 3,9-benz[a]anthracene diols are described: unsubstituted, 7-methyl, 12-methyl, and 7,12-dimethyl. Compounds unsubstituted at the 12-position have flat molecular topology, whereas methyl substitution at the 12-position in the bay region induces twisting of the molecular framework. The oxygen-oxygen distances (11.94-11.98 A) are similar to diethylstilbestrol (12.1 A). The binding affinities range from 0.43% to 26% that of estradiol. Methyl substitution at the 7-position enhances affinity; 12-methyl substitution decreases it. These results are contrary to many estrogen receptor (ER) ligand systems, in which the compounds with the flatter molecular geometries typically have lower binding affinity. Molecular graphics were used to analyze the fit of the four compounds with a receptor excluded volume model for the ER. These studies suggest that these compounds bind to the ER in a manner in which the anthracene fragment acts as the steroid AB-ring mimic (i.e, the benz[a]anthracene 9-position corresponds to the estradiol 3-position). Molecular orbital (AM1) calculations were used to calculate the charges of selected atoms. The 7-methyl compound was found to have greater charge similarity to estradiol than the other three compounds. The high affinity of the 7-methyl compound is ascribed to its charge similarity to estradiol, hydrophobic interactions in the receptor region that would accommodate a substituent in the planar 6-position of a delta 6,7-steroid, and favorable dispersive interactions with the receptor secondary to its extended planar system. Molecular orbital calculations also suggest that some of the benz[a]anthracene monophenols and diphenols have sufficiently low ionization potentials to act as carcinogens by a radical cation process.

Evaluation of a highly efficient aryl azide photoaffinity labeling reagent for the progesterone receptor.

16 alpha,17 alpha-[(R)-1'-(4-Azidophenyl)ethylidenedioxy]pregn-4-ene- 3,20-dione (7) was prepared in high specific activity tritium-labeled form (20 Ci/mmol) and shown to bind to the progesterone receptor with an affinity (Kd = 0.80 nM) that is 47% of that of [3H]-R 5020 (Kd = 0.38 nM). [3H]Progestin aryl azide 7 exhibits high photoattachment efficiency (60% at 1 h) compared to the commonly used progesterone receptor photoaffinity labeling reagent [3H]-R 5020 (2.2% at 1 h) and is the most efficient progesterone receptor photoaffinity labeling reagent prepared to date. The photoattachment observed with 7 proceeds in a time-dependent fashion, with most of the attachment occurring within the first 10 min of photolysis. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of two adducts of molecular weight 108,500 +/- 800 and 87,000 +/- 1,500 (n = 3), the same species as labeled by [3H]-R 5020. The ratio of progesterone receptor subunits A:B was determined to be 3.3:1 with both [3H]progestin azide 7 and [3H]-R 5020. Information on the specific amino acid(s) that attach to the ligand during photolysis awaits further analysis of the covalently bound ligand-protein adduct.

The effect of acceptor group variation on the solvatochromism of donor-acceptor fluorophores.

The absorption and emission characteristics of five hydroxytetrahydrochrysenes substituted with acceptor groups (nitro, cyano, methylketone, 1 degree amide and methyl ester) (THC-NO2, THC-CN, THC-COCH3, THC-CONH2 and THC-CO2CH3, respectively) were investigated in an extensive set of solvents. The order of absorption and fluorescence bathochromicity are: THC-NO2 > THC-COCH3 > THC-CN > or = THC-CO2CH3 > THC-CONH2 and THC-NO2 >> THC-COCH3 > THC-CO2CH3 > THC-CN > THC-CONH2, respectively. The emission spectra of these compounds are sensitive to the solvent polarity (ET[30] scale) in the order: THC-NO2 > THC-COCH3 > THC-CO2CH3 > THC-CONH2 > THC-CN. The response of the emission maxima of these compounds to the solvent polarity and hydrogen-bond donor/acceptor properties (pi */alpha/beta and acity/basity scales) was also determined. The emission energies of THC-NO2 were most sensitive to pi *, beta, acity, and basity of the solvent; those of the amide were least sensitive to the solvent pi *, beta, and basity. The ground- and excited-state dipole moments were determined by semiempirical molecular orbital calculations and the absorption/fluorescence solvent-shift method, respectively. THC-NO2 had the largest ground- and excited-state moments. The ester and amide had the smallest ground- and excited-state moments, respectively. In general, unsatisfactory results were obtained for correlations of the emission and absorption energies, fluorescence solvatochromism and the ground- and excited-state dipole moments with the Hammett substituent constants of the five acceptor groups. Acceptable correlations were obtained for the absorption and emission energies and the fluorescence solvatochromism with the substituent constants if the cyano compound was excluded.

Molecular structures, conformational analysis, and preferential modes of binding of 3-aroyl-2-arylbenzo[b]thiophene estrogen receptor ligands: LY117018 and aryl azide photoaffinity labeling analogs.

Structural and computational modeling studies were performed on the antiestrogen LY117018 (3) and two photoaffinity labeling analogs, in which an azide replaces the basic ether side chain (methyl ether tetrafluoro azide 7 and its protio analog 8). These studies were undertaken in order to determine the conformational preferences of these compounds and to propose favorable orientational modes for their binding to the estrogen receptor. In the crystallographic studies, we found that, unlike tetrafluoro azide 7, which adopts a face-to-face stacking of the p-hydroxyphenyl and benzoyl groups in the solid state, the pendant rings in the corresponding protio analog 8 are found in a predominantly offset pi-stacked array. In LY117018, which has an ether on the benzoyl ring, stacking of the pendant rings does not occur in the crystal structure; it assumes a T-shape, with the benzoyl group oriented perpendicular to the benzo[b]thiophene nucleus. In modeling studies, analogs of LY117018, 7, and 8 were subjected to a conformational grid search by molecular mechanics, and for each compound, three low-energy conformers (and their atropisomers) were obtained. These conformers were further geometry optimized by semiempirical molecular orbital calculations. For each compound, one of the three minimum-energy conformers is quite similar to the solid-state geometry. The computational structure of the tetrafluoro azide showed the greatest stacking between the benzoyl group and the p-methoxyphenyl ring, but less stacking than was observed in the crystallographic structure. The orientational preferences of these benzo[b]thiophene ligands with the estrogen receptor were analyzed with the receptor volume mapping technique, a method based on the correspondence of the hydroxyl groups and the volume that the benzo[b]thiophene compound shares with a composite molecular volume of high-affinity estradiol-type ligands (the receptor excluded volume, RExV). If the benzo[b]thiophene nucleus is overlapped with the steroid AB rings, the best overlap with the RExV is achieved, but there is poor correspondence of the hydroxyl groups. An orientation and conformation in which the benzoyl group of the 3-benzoyl-2-aryl-benzo[b]thiophenes occupies a 7 alpha-like position relative to the steroid produces both ample volume overlap with the RExV and close approximation of the hydroxyl groups and is presented as the putative bioactive conformation.

Fluorine-18-labeled progestin ketals: synthesis and target tissue uptake selectivity of potential imaging agents for receptor-positive breast tumors.

We have studied two new fluorine-substituted progestins as potential imaging agents for progesterone-receptor-positive human breast tumors. The steroids are 16 alpha, 17 alpha-fluoroacetophenone ketals of 16 alpha, 17 alpha-dihydroxyprogesterone and 16 alpha, 17 alpha, 21-trihydroxy-19-norprogesterone. Synthesis of the latter compound in seven steps from 19-norandrost-4-ene-3,17-dione is reported. Both compounds demonstrate high affinity for the progesterone receptor (PgR) (52.5 and 240%, respectively, relative to R5020 = 100). The syntheses were adapted to 18F-labeling with 4'-[18F]-fluoroacetophenone, prepared from 4'-nitroacetophenone by nucleophilic substitution with K18F/Kryptofix. Considerable adjustment of reaction conditions was required to effect ketalization using tracer quantities of the ketone. In tissue distribution studies in estrogen-primed immature female rats, both ketals showed selective uterine uptake, which was blocked by coinjection of a saturating dose of the unlabeled progestin ORG 2058. Additionally, metabolic stability of the radiolabel was indicated by the low radioactivity levels seen in bone. Both compounds showed relatively high uptake in fat, in accord with their relative lipophilicities demonstrated by HPLC-derived octanol-water partition coefficients. The selective uterine uptake and metabolic stability of these compounds suggests that this class of PgR ligands might be promising for the selective imaging of receptor-positive tumors if derivatives of reduced lipophilicity can be prepared.

Progestin 16 alpha, 17 alpha-dioxolane ketals as molecular probes for the progesterone receptor: synthesis, binding affinity, and photochemical evaluation.

Chemical probes for steroid receptors have proven useful in providing molecular details about important hormone-receptor interactions. A series of progestin 16 alpha, 17 alpha-dioxolane ketals of acetophenone or substituted acetophenones that bind to the progesterone receptor (PgR) with comparable or higher affinities than the natural ligand, progesterone, have been prepared and evaluated as potential in vitro and in vivo probes for the progesterone receptor. p-Azidoacetophenone ketal 6, the tetrafluoro analog 8, and the p-(benzoyl)acetophenone ketal 9 demonstrate the required combination of high relative binding affinity (RBA) (6 = 15%, 8 = 14%, 9 = 6.6%, progesterone = 13%, R5020 = 100%) and photoinactivation efficiency (6 = 80%, 8 = 77%, 9 = 29% at 30 min) required for potential photoaffinity labeling reagents for the PgR. The synthesis of azide 6 has been modified to accommodate a palladium-catalyzed tritium gas hydrogenolysis of an iodoaryl precursor in the final stage of the synthetic sequence; this procedure has been verified by hydrogenation. In addition, the progestin p-fluoroacetophenone ketal 10 was selected for preparation in fluorine-18-labeled form, on the basis of its high affinity for the PgR (RBA = 53%). Fluorine-18-labeled progestins may be evaluated as potential diagnostic imaging agents for PgR-positive breast tumors. The radiochemical syntheses and further biochemical results with the fluorine-18-labeled ketal 10 and the tritium-labeled aryl azide 6 will be presented in an accompanying paper and elsewhere.