Structure-activity relationship studies of chalcones as SRS-A receptor antagonists

A simple, reliable and highly sensitive bioassay with sensitized longitudinal strips of guinea pig ileum was used for screening the receptor antagonists of slow reacting substance of anaphylaxis (SRS-A). The SRS-A receptor antagonistic activities of 17 chalcones were studied. Most compounds in these chalcones were found to have SRS-A receptor antagonistic action at the concentration of 10(-4) mol.L-1. Among them, compounds 5, 13 and 17 were highly effective with IC50s of 7.5 x 10(-6), 7.5 x 10(-6) and 6.8 x 10(-5) mol.L-1, respectively. Under the same conditions, the IC50 of FPL 55712, a known leukotriene D4 receptor antagonist, was shown to be 3 x 10(-4) mol.L-1. It would appear that compounds 5, 13 and 17 were 40, 40 and 4.4 times more potent, respectively, than FPL 55712. From analysis of structure-activity relationship of chalcones, these results suggest that the following factors may be important for an active antagonist of SRS-A receptors: (a) There is a system of pi, pi conjugation in the molecule; (b) The ester group in the B ring of chalcones is more favorable than the carboxyl group; (c) Antagonism for meta- or para-substituted derivatives of carboxyl or ester group in the B ring are more potent than ortho-substituted compounds; (d) The length of carbon chain of alkyl group in the A ring of chalcones is more effective for 1, 4 or 6 carbon atoms than for 10 or 14 carbon atoms.     

Effects of adrenalectomy and glucocorticoid receptor antagonist, RU38486, on pituitary growth hormone-releasing hormone receptor gene expression in rats

We examined the effects of adrenalectomy and a glucocorticoid receptor antagonist, RU38486, on pituitary GH-releasing hormone (GRH) receptor gene expression in rats. GRH receptor mRNA levels were significantly decreased in adrenalectomized rats and replacement of dexamethasone reversed the decrease to normal. GH secretion was inhibited by adrenalectomy, whereas dexamethasone replacement failed to restore the impaired GH secretion. A high dose of RU38486 had an agonistic effect on GRH receptor mRNA levels. These results suggest that endogenous glucocorticoid is necessary for normal expression of pituitary GRH receptor mRNA in rats.     

Novel nonpeptide CCK-B antagonists: design and development of quinazolinone derivatives as potent, selective, and orally active CCK-B antagonists

We have designed a novel series of CCK-B receptor antagonists by combining key pharmacophores, an arylurea moiety of 1 and a quinazolinone ring of 3, from two known series. Our earlier studies showed that compounds with methylene linkers in our "target" produced moderate binding affinity and selectivity for CCK-B receptors, whereas its higher and lower homologues resulted in loss of affinity. Introduction of -NH- as a linker dramatically enhanced binding affinity and selectivity for CCK-B receptors, thus providing several compounds with single-digit nanomolar binding affinity and excellent selectivity. Analogous to the earlier studies of the series of quinazolinone derivatives 3, we also found 3-isopropoxyphenyl as a preferred substitution on the N-3 quinazolinone. Electron-withdrawing substitutions on the urea terminal phenyl ring enhanced the CCK-B potency. Representative compounds of this series were tested in the functional assay and showed pure antagonist profiles. Compounds 51 and 61 were orally active in the elevated rat X-maze test. These compounds were also evaluated for their pharmacokinetic profile. The absolute oral bioavailability of compound 61 was 22% in rats.     

Novel sodium binding properties of some cyclopentapeptide endothelin A selective receptor antagonists: electrospray and fast-atom-bombardment mass spectrometric studies

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.     

NGB 2904 and NGB 2849: two highly selective dopamine D3 receptor antagonists

N-(4-[4-?2, 3-dichlorophenyl?-1-piperazinyl]butyl)-3-fluorenylcarboxamide and N-(4-[4-?2, 3-dichlorophenyl?-1-piperazinyl]butyl)-2-biphenylenylcarboxamide were prepared in several steps from 2,3-dichloroaniline. These compounds were identified as highly selective dopamine D3 receptor antagonists.     

Distinct ontogeny of glucocorticoid and mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase types I and II mRNAs in the fetal rat brain suggest a complex control of glucocorticoid actions

Glucocorticoids (GCs) act via intracellular mineralocorticoid (MR) and glucocorticoid receptors (GR). However, it has recently been recognized that GC access to receptors is determined by the presence of tissue-specific 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) that catalyze the interconversion of active corticosterone and inert 11-dehydrocorticosterone. 11beta-HSD type 1 (11beta-HSD1) is a bidirectional enzyme in vitro that acts predominantly as a reductase (regenerating corticosterone) in intact neurons. In contrast, 11beta-HSD type 2 (11beta-HSD2) is a higher affinity exclusive dehydrogenase that excludes GCs from MR in the kidney, producing aldosterone-selectivity in vivo. We have examined the ontogeny of 11beta-HSD mRNAs and enzyme activity during prenatal brain development and correlated this with GR and MR mRNA development. These data reveal that (1) 11beta-HSD2 mRNA is highly expressed in all CNS regions during midgestation, but expression is dramatically reduced during the third trimester except in the thalamus and cerebellum; (2) 11beta-HSD2-like activity parallels closely the pattern of mRNA expression; (3) 11beta-HSD1 mRNA is absent from the CNS until the the third trimester, and activity is low or undectectable; and (4) GR mRNA is highly expressed throughout the brain from midgestation, but MR gene expression is absent until the last few days of gestation. High 11beta-HSD2 at midgestation may protect the developing brain from activation of GR by GCs. Late in gestation, repression of 11beta-HSD2 gene expression may allow increasing GC activation of GR and MR, permitting key GC-dependent neuronal and glial maturational events.     

Effects of the glucocorticoid agonist, RU28362, and the antagonist RU486 on lung phosphatidylcholine and antioxidant enzyme development in the genetically obese Zucker rat

The biochemical maturation of the lung in late gestation and in the young animal is regulated by glucocorticoids. The present study was aimed at dissociating the different glucocorticoid receptor sites involved in these regulatory functions. The obese Zucker rat was selected as a model for this study as it exhibits hypersensitivity to glucocorticoid hormone action by virtue of its elevated receptor numbers and activity. Two synthetic steroid analogues were administered to obese animals; RU28362, a specific type II receptor agonist, and the type II antagonist RU486. RU28362 promoted a strong catabolic effect, which was associated with reduced food intake and the abolition of growth in the rats. The agonist, RU28362, attenuated developmental increases in antioxidant enzyme activities, and altered the growth of the tissue. At the age studied, development of the lung phosphatidylcholine (PC) system was almost complete, but RU28362 increased disaturated PC 16:0/16:0 concentrations by almost 2-fold, and altered the molecular composition of total pulmonary PC. RU486 attenuated the growth of the rats and reduced their food intake. Treatment with the type II antagonist attenuated lung growth and increased the activities of pulmonary copper zinc (Cu/Zn) and manganese (Mn) superoxide dismutases. RU486 had no effect on lung PC concentrations and molecular composition. The data suggest a role for type I glucocorticoid receptors in the regulation of the antioxidant enzyme system in the lung, as type II antagonism will channel endogenous glucocorticoid binding to the type I site. Type II receptor binding would appear to play a role in regulating the lung PC content.     

Nonpeptide endothelin receptor antagonists. XI. Pharmacological characterization of SB 234551, a high-affinity and selective nonpeptide ETA receptor antagonist

The present study describes the pharmacological profile of ((E)-alpha-[[1-butyl-5-[2-[(2-carboxyphenyl)methoxy]-4-methoxy-phenyl ]-1H-pyrazol-4-yl]methlene]-6-methoxy-1,3-benzodioxole-5-propanoic acid) (SB 234551), a high-affinity, nonpeptide endothelin type A (ETA)-selective receptor antagonist. In human cloned ETA and endothelin type B (ETB) receptors, SB 234551 produced a concentration-dependent displacement of [125I]-endothelin-1 with Ki values of 0.13 and 500 nM, respectively. SB 234551 elicited concentration-dependent, rightward competitive shifts in the endothelin-1 concentration-response curves in isolated rat aorta and isolated human pulmonary artery (ETA receptor-mediated vascular contraction) with Kb values of 1.9 and 1.0 nM, respectively. SB 234551 antagonized ETB receptor-mediated vasoconstriction in the isolated rabbit pulmonary artery, as demonstrated by concentration-dependent, rightward shifts in the sarafotoxin S6c concentration-response curves (Kb = 555 nM). SB 234551 produced weak functional inhibition of sarafotoxin S6c-mediated endothelium-dependent relaxation (IC50 = 7 microM). SB 234551 (10 microM) had no significant effect against contraction produced by several other vasoactive agents and did not significantly influence radioligand binding to a number of diverse receptors. SB 234551 (0. 1-1.0 mg/kg i.v.) dose-dependently inhibited the pressor response to exogenous endothelin-1 in conscious rats. In vivo pharmacokinetic analysis in the rat demonstrated that SB 234551 was rapidly absorbed from the GI tract with a bioavailability of 30%. SB 234551 had a plasma half-life of 125 min and a systemic clearance of 25.0 ml/min/kg. The present study demonstrates that SB 234551 is an antagonist with high affinity for the ETA receptor, while sparing the ETB receptor. SB 234551 is a new pharmacological tool that should assist in the elucidation of the role of endothelin in pathophysiology.     

The ontogeny of 11 beta-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor gene expression reveal intricate control of glucocorticoid action in development

Glucocorticoids play important roles in development and 'fetal programming'. Fetal exposure to excess glucocorticoids reduces birth weight and causes later hypertension. To investigate these processes further we have determined the detailed category of 11 beta-hydroxysteroid dehydrogenase type2 (11 beta-HSD2, which potently inactivates glucocorticoids) and the mineralocorticoid receptor (MR) by in situ hybridisation from embryonic day 9.5 (E9.5, term = E19) until after birth in the mouse. Widespread abundant 11 beta-HSD2 mRNA expression from E9.5-E12.5 changes dramatically at approximately E13 to a limited tissue-specific pattern (kidney, hindgut, testis/bile ducts, lung and a few brain regions (later seen in cerebellum, thalamus, roof of midbrain, neuroepithelial regions in pons and near the subicular hippocampus)). Placenta (labyrinthine zone) and extra-embryonic membranes express abundant 11 beta-HSD2 mRNA until E15.5 but this ceases = E16.5. It is unclear to what extent rodent term placental 11 beta-HSD activity is due to persisting 11 beta-HSD2 protein. Convincing MR mRNA expression is seen from E13.5 and includes pituitary, heart, muscle and meninges with expression later in gut, kidney, thymus, discrete areas of lung and several brain regions (including hippocampus, rhinencephalon and hypothalamus). 11 beta-HSD2 and MR clearly co-localise = E18.5 in kidney and colon and might do so in discrete areas of lung (E14-15) and neuroepithelia near the subicular hippocampus. Probably elsewhere MR are non-selective and 11 beta-HSD2 is involved in protecting glucocorticoid receptors in fetal fetal tissues. Comparison with previous enzymology studies suggest the changing pattern of 11 beta-HSD2 mRNA is likely to be translated into enzyme activity and have significant parallels in human development.     

NMR and CD conformational studies of bradykinin and its agonists and antagonists: application to receptor binding

Most physiological processes are regulated by peptides that perform their functions by interacting with specific receptors on cells. Specific conformations of the peptides are required for correct interactions to take place, and a knowledge of the biologically important conformation is vital for the understanding of biological function. Over the last few years extensive studies using nuclear magnetic resonance and circular dichroism have been carried out on bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and its antagonists with the objective of developing new drugs to combat severe pathologies associated with its production. In the present review, these techniques for the determination of peptide conformation are reviewed and applied to the study of bradykinin and its antagonists. Modeling of these conformational data in the presence of the B2 receptor or an antibody allows the biologically active conformations to be deduced and these are presented in this review.     

Synthesis, biological activity, and molecular modeling of selective 5-HT(2C/2B) receptor antagonists

The synthesis and biological activity are reported for a series of analogues of the previously published indole urea 2 (SB-206553), designed to probe the 5-HT(2C) receptor binding site. Small molecule modeling studies have been used to define a region in space which is allowed at the 5-HT(2C) receptor but disallowed at the 5-HT(2A) receptor. In a complementary approach, docking of 2 into our model of the 5-HT(2C) receptor has allowed us to propose a novel primary binding interaction for this series of diaryl ureas, involving a potential double hydrogen-bonding interaction between the urea carbonyl oxygen of the ligand and two serine residues in the receptor. The difference of two valine residues in the 5-HT(2C) receptor for leucine residues in the 5-HT(2A) receptor is believed to account for the observed 5-HT(2C)/5-HT(2A) selectivity with 2.     

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.     

Protection against hypoxic-ischemic damage with corticosterone and dexamethasone: inhibition of effect by a glucocorticoid antagonist RU38486

We investigated whether the neuroprotection provided by dexamethasone against neonatal hypoxic-ischemic damage can be inhibited by a glucocorticoid antagonist and whether corticosterone, the endogenous glucocorticoid in the rat, also provides protection. Rats (6 days old) were treated with either vehicle (0.1 ml/10 g), corticosterone (3.5-80 mg/kg, s.c.) or dexamethasone alone or in combination with RU38486 (20-80 mg/kg, s.c.) 15 min prior to dexamethasone (0.1 mg/kg, i.p.). At 7 days of age, cerebral hypoxia-ischemia was produced by right carotid artery ligation under anesthesia and subsequent exposure to 2 h of hypoxia. Damage was quantified from brains perfusion-fixed and processed 2 days later. The reduction in somatic growth, thymus weight and the relatively elevated blood glucose levels at the end of hypoxia-ischemia were inhibited by RU38486. The protective effect of dexamethasone was also prevented by RU38486 (P < 0.001). Similar to pre-treatment with dexamethasone, administration of corticosterone (40-80 mg/kg) markedly reduced the extent of infarction compared to vehicle-treated controls (P < 0.0001). Thus, the endogenous glucocorticoid in the rat also provides protection against hypoxic-ischemic damage. RU38486 inhibits the beneficial effects of dexamethasone demonstrating that the neuroprotection observed with dexamethasone is a glucocorticoid receptor-mediated effect.     

Structure-activity relationships of 4-(phenylethynyl)-6-phenyl-1,4-dihydropyridines as highly selective A3 adenosine receptor antagonists

4-(Phenylethynyl)-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine) in the submicromolar range. In this study, structure-activity relationships at various positions of the dihydropyridine ring (the 3- and 5-acyl substituents, the 4-aryl substituent, and 1-methyl group) were probed synthetically. Using the combined protection of the 1-ethoxymethyl and the 5-[2-(trimethylsilyl)ethyl] ester groups, a free carboxylic acid was formed at the 5-position allowing various substitutions. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity analysis at adenosine receptors indicated that pyridyl, furyl, benzofuryl, and thienyl groups at the 4-position resulted in, at most, only moderate selectivity for A3 adenosine receptors. Ring substitution (e.g., 4-nitro) of the 4-phenylethylnyl group did not provide enhanced selectivity, as it did for the 4-styryl-substituted dihydropyridines. At the 3-position of the dihydropyridine ring, esters were much more selective for A3 receptors than closely related thioester, amide, and ketone derivatives. A cyclic 3-keto derivative was 5-fold more potent at A3 receptors than a related open-ring analogue. At the 5-position, a homologous series of phenylalkyl esters and a series of substituted benzyl esters were prepared and tested. (Trifluoromethyl)-, nitro-, and other benzyl esters substituted with electron-withdrawing groups were specific for A3 receptors with nanomolar Ki values and selectivity as high as 37000-fold. A functionalized congener bearing an [(aminoethyl)amino]carbonyl group was also prepared as an intermediate in the synthesis of biologically active conjugates.     

Synthesis and biological evaluation of new tetrahydronaphthalene derivatives as thromboxane receptor antagonists

New polysubstituted tetrahydronaphthalene derivatives were prepared as thromboxane receptor (TP-receptor) antagonists. Within this series of compounds S 18886 has been identified as an orally active, highly potent antagonist with a very long duration of action in different species.     

The discovery and structure-activity relationships of nonpeptide, low molecular weight antagonists selective for the endothelin ET(B) receptor

The systematic modification of the ETA selective N-(5-isoxazolyl)benzene-sulfonamide endothelin antagonists to give ETB selective antagonists is reported. The reversal in selectivity was brought about by substitution of the 4-position with aryl and substituted aryl groups. Of all the aromatic substituents studied, the para-tolyl group gave rise to the most active and selective ETB antagonist. Larger substituents caused a decrease in both ETB activity and selectivity. A similar trend was observed by substitution at the 5-position of the N-(5-isoxazolyl)-2-thiophenesulfonamide ETA receptor antagonists. The para-tolyl group was again found to be optimal for the ETB activity and selectivity. The structural features that were found to be favorable for binding to the ETB receptor, that is, the presence of a linear, conjugated pi-system of definite shape and size, have been successfully incorporated into the design of ETB selective polycyclic aromatic sulfonamides antagonists.