ABT-866, a novel alpha(1A)-adrenoceptor agonist with antagonist properties at the alpha(1B)- and alpha(1D)-adrenoceptor subtypes

N-[3-(1H-Imidazol-4-ylmethyl)phenyl]ethanesulfonamide, maleate (ABT-866) is a novel alpha(1)-adrenoceptor agent with mixed pharmacological properties in vitro. Compared to phenylephrine, ABT-866 demonstrates intrinsic activity at the alpha(1A)-adrenoceptor subtype present in the rabbit urethra (pD(2) = 6.22, with 80% of the phenylephrine response), reduced intrinsic activity at the alpha(1B)-adrenoceptor subtype in the rat spleen (pD(2)= 6.16, with 11% of the phenylephrine response), and no intrinsic activity at the rat aorta alpha(1D)-adrenoceptor subtype. ABT-866 also demonstrated antagonism at the rat spleen alpha(1B)-adrenoceptor (pA(2) = 5.39 +/- 0.08, slope = 1.20 +/- 0.12), and the rat aorta alpha(1D)-adrenoceptor (pA(2)= 6.18 +/- 0.09, slope = 0.96 +/- 0.13). This is in contrast to the weak non-selective activity seen with the alpha(1)-adrenoceptor agonist, phenylpropanolamine (2-amino-1-phenyl-1-propanol hydrochloride), and the alpha(1A/D)-adrenoceptor selective agonist 1-(2',5'-dimethoxyphenyl)-2-aminoethanol hydrochloride (ST-1059), the active metabolite of midodrine, that has been used clinically for the treatment of stress urinary incontinence. This study identifies a unique agent that may prove to be a valuable in vivo tool in testing the hypothesis that the alpha(1A)-adrenoceptor can be stimulated to contract the smooth muscle present in the urethra without evoking blood pressure elevations presumably caused by alpha(1B)- and alpha(1D)-adrenoceptor subtype involvements in the vasculature.     

8-NH2-boldine, an antagonist of alpha1A and alpha1B adrenoceptors without affinity for the alpha1D subtype: structural requirements for aporphines at alpha1-adrenoceptor subtypes

Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.     

New pyrimido[5,4-b]indoles as ligands for alpha(1)-adrenoceptor subtypes

A new series of compounds were designed as structural analogues of the alpha(1)-AR ligand RN5 (4), characterized by a tricyclic 5H-pyrimido[5,4-b]indole-(1H,3H)2,4-dione system connected through an alkyl chain to a phenylpiperazine (PP) moiety. These compounds were synthesized and tested in binding assays on human alpha(1A)-AR, alpha(1B)-AR, and alpha(1D)-AR subtypes expressed in HEK293 cells. Several structural modifications were performed on the PP moiety, the tricyclic system, and the connecting alkyl chain. Many of the new molecules showed a preferential affinity for the alpha(1D)-AR subtype. Some compounds, including 39 and 40, displayed substantial alpha(1D)-AR selectivity with respect to alpha(1A)-AR, alpha(1B)-AR, serotonergic 5-HT(1A), 5-HT(1B), 5-HT(2A), and dopaminergic D(1) and D(2) receptors. Two conformationally rigid analogues of 4, useful for studying the architecture of the receptor/ligand complex, were also prepared and tested. A subset of the new compounds was then used to evolve a preliminary pharmacophore model for alpha(1D)-AR antagonists, based on a more generalized model we had developed for alpha(1)-AR antagonists. This new model rationalized the relationships between structural properties and biological data of the pyrimido[5,4-b]indole compounds, as well as other compounds.     

Vascular activity of (-)-anonaine, (-)-roemerine and (-)-pukateine, three natural 6a(R)-1,2-methylenedioxyaporphines with different affinities for alpha1-adrenoceptor subtypes

We have studied the mechanism of action of three 6a( R)-1,2-methylenedioxyaporphines as vasorelaxant compounds. The alkaloids assayed showed different affinities for the three human cloned alpha (1)-adrenoceptor (AR) subtypes stably expressed in rat-1 fibroblasts, showing lower affinity for alpha(1B)-AR with regard to the alpha(1A)- or alpha(1D)-subtypes. These three natural compounds are more potent inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. As all these alkaloids inhibited noradrenaline (NA)-induced [ (3)H]-inositol phosphate formation in cerebral cortex and rat tail artery, they may be safely viewed as alpha (1)-AR antagonists, as is demonstrated by the vasorelaxant responses observed in isolated rat tail artery and/or aorta precontracted with NA. The alkaloids also inhibited the contractile response evoked by KCl (80 mM) but with a lower potency than that shown against NA-induced contraction. We have also examined their ability to inhibit the different forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aortic smooth muscle and endothelial cells, with negative results. We conclude that N-methylation favours the interaction of (R)-aporphines with all alpha (1)-AR subtypes, and that the topography of the binding site recognizing the basic or protonated nitrogen atom is similar in all three alpha (1)-AR subtypes. The presence of a hydroxy group at C-11 has different effects on the affinity for each alpha (1)-AR subtype but decreases the affinity for Ca (2+) channels. These results confirm and extend the view that subtle changes in the hydroxylation patterns on the aromatic ring of the aporphine structure affect the interactions of these compounds with the three alpha (1)-AR subtypes in different ways, suggesting that the binding site recognizing the aporphine skeleton is different in each of the three subtypes.     

Alpha(1A)-adrenoceptors mediate contractions to phenylephrine in rabbit penile arteries

BACKGROUND AND PURPOSE: Maintained penile erection depends on the absence of alpha-adrenoceptor (alpha-AR) activation and so can be facilitated by alpha-blockers. This study seeks the alpha(1)-AR subtypes involved in order to inform the pro-erectile consequences of subtype selective blockade. EXPERIMENTAL APPROACH: Wire myography was used with dorsal (nutritional supply) and cavernous (erectile inflow) penile arteries; standard alpha-AR-selective agonists and antagonists were employed to classify responses. KEY RESULTS: In both penile arteries noradrenaline (NA) and phenylephrine (PE, alpha(1)-AR agonist) caused concentration-dependent contractions. Sensitivity to NA was increased by NA uptake blockers, cocaine (3 microM) and corticosterone (30 microM). PE responses were antagonised by phentolamine (non-selective alpha-AR: dorsal pK(B) 8.00, cavernous 8.33), prazosin (non-subtype-selective alpha(1)-AR: dorsal 8.60, cavernous 8.41) and RS100329 (alpha(1A)-AR selective: dorsal 9.03, cavernous 8.80) but not by BMY7378 (alpha(1D)-AR selective: no effect at 1-100 nM) or Rec15/2615 (alpha(1B)-AR selective: no effect at 1-100 nM). Schild analysis was straightforward in cavernous artery, indicating that PE activates only alpha(1A)-AR. In dorsal artery Schild slopes were low, though alpha(1A)-AR was still indicated. Analysis using UK 14,304 and rauwolscine indicated an alpha(2)-AR component in dorsal artery that may account for low slopes to alpha(1)-AR antagonists. CONCLUSIONS AND IMPLICATIONS: Penile arteries have a predominant, functional alpha(1A)-AR population with little evidence of other alpha(1)-AR subtypes. Dorsal arteries (nutritional supply) also have alpha(2)-ARs. Thus, alpha-AR blockers with affinity for alpha(1A)-AR or alpha(2)-AR would potentially have pro-erectile properties; the combination of these perhaps being most effective. This should inform the design of drugs to assist/avoid penile erection.     

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.     

Design, synthesis, and structure-activity relationships of phthalimide-phenylpiperazines: a novel series of potent and selective alpha(1)(a)-adrenergic receptor antagonists

Beginning from the screening hit and literature alpha(1)-adrenergic compounds, a hybridized basic skeleton A was proposed as the pharmacophore for potent and selective alpha(1a)-AR antagonists. Introduction of a hydroxy group to increase the flexibility afforded B which served as the screening model and resulted in the identification of the second-generation lead 1. Using the Topliss approach, a number of potent and selective alpha(1a)-AR antagonists were discovered. In all cases, binding affinity and selectivity at the alpha(1a)-AR of S-hydroxy enantiomers were higher than those of the R-hydroxy enantiomers. As compared to the des-hydroxy analogues, the S-hydroxy enantiomers had slightly lower binding affinity at alpha(1a)-AR but gained more than 2-fold selectivity for alpha(1a)-AR over alpha(1b)-AR, and 2- to 6-fold selectivity for alpha(1a)-AR over alpha(1d)-AR. They also had less cross activities against a panel of 25-35 peripheral and CNS receptors. The S-hydroxy enantiomers 23 and 24 (K(i) = 0.29 nM, 0.33 nM; alpha(1b)/alpha(1a) >5690, >6060; alpha(1d)/alpha(1a) = 186, 158, respectively) were slightly less potent but much more selective at alpha(1a)-AR than tamsulosin (K(i) = 0.13 nM, alpha(1b)/alpha(1a) = 14.8, alpha(1d)/alpha(1a) = 1.4). In the functional assay, the S-hydroxy enantiomers 20, 23, and 24 were less potent than tamsulosin in inhibiting contractions of rat prostate tissue but more selective in the inhibition of tissue contractions of rat prostate versus rat aorta. Compound 24 was selected as the development candidate for the treatment of BPH.     

Synthesis, biological evaluation, and pharmacophore generation of new pyridazinone derivatives with affinity toward alpha(1)- and alpha(2)-adrenoceptors

A series of new pyridazin-3(2H)-one derivatives (3 and 4) were evaluated for their in vitro affinity toward both alpha(1)- and alpha(2)-adrenoceptors by radioligand receptor binding assays. All target compounds showed good affinities for the alpha(1)-adrenoceptor, with K(i) values in the low nanomolar range. The polymethylene chain constituting the spacer between the furoylpiperazinyl pyridazinone and the arylpiperazine moiety was shown to influence the affinity and selectivity of these compounds. Particularly, a gradual increase in affinity was observed by lengthening the polymethylene chain up to a maximum of seven carbon atoms. In addition, compound 3k, characterized by a very interesting alpha(1)-AR affinity (1.9 nM), was also shown to be a highly selective alpha(1)-AR antagonist, the affinity ratio for alpha(2)- and alpha(1)-adrenoceptors being 274. To gain insight into the structural features required for alpha(1) antagonist activity, the pyridazinone derivatives were submitted to a pharmacophore generation procedure using the program Catalyst. The resulting pharmacophore model showed high correlation and predictive power. It also rationalized the relationships between structural properties and biological data of, and external to, the pyridazinone class.     

Prazosin-related compounds. Effect of transforming the piperazinylquinazoline moiety into an aminomethyltetrahydroacridine system on the affinity for alpha1-adrenoreceptors

In a search for structurally new alpha(1)-adrenoreceptor (alpha(1)-AR) antagonists, prazosin (1)-related compounds 2-11 were synthesized and their affinity profiles were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO cells expressing human cloned alpha(1)-AR subtypes. Transformation of the piperazinylquinazoline moiety of 1 into an aminomethyltetrahydroacridine system afforded compound 2, endowed with reduced affinity, in particular for the alpha(1A)-AR subtype. Then, to investigate the optimal features of the tricyclic moiety, the aliphatic ring of 2 was modified by synthesizing the lower and higher homologues 3 and 4. An analysis of the pharmacological profile, together with a molecular modeling study, indicated the tetrahydroacridine moiety as the most promising skeleton for alpha(1)-antagonism. Compounds 5-8, where the replacement of the furoyl group of 2 with a benzoyl moiety afforded the possibility to evaluate the effect of the substituent trifluoromethyl on receptor binding, resulted, except for 7, in a rather surprising selectivity toward alpha(1B)-AR, in particular vs the alpha(1A) subtype. Also the insertion of the 2,6-dimethoxyphenoxyethyl function of WB 4101 on the tetrahydroacridine skeleton of 2, and/or the replacement of the aromatic amino function with a hydroxy group, affording derivatives 9-11, resulted in alpha(1B)-AR selectivity also vs the alpha(1D) subtype. On the basis of these results, the tetrahydroacridine moiety emerged as a promising tool for the characterization of the alpha(1)-AR, owing to the receptor subtype selectivity achieved by an appropriate modification of the lateral substituents.     

1-(2,6-二甲氧基)-2-(3,4-二甲基苯乙氨基)丙烷盐酸盐(DDPH)拮抗α1肾上腺素受体的特性

目的　研究DDPH对α₁-肾上腺素受体(α₁-AR)及其亚型的拮抗作用。方法　放射配体结合实验和离体血管收缩功能实验。结果　DDPH对¹²⁵I-BE2254与大鼠脑皮质和脾脏α₁-AR结合呈竞争性拮抗作用。pK_I值在两者间无显著性差别, Hill系数均接近于1.0。在分别稳定表达α_1A,α_1B或α_1D-AR的克隆HEK293细胞中,其拮抗的pK_I值α_1A和α_1D比α_1B-AR高约2倍,Hill系数均接近于1.0。并拮抗去甲肾上腺素(NE)介导大鼠主动脉,肾动脉和脾脏收缩的pA₂值,在三者间无显著差别,斜率接近1.0。结论　DDPH对α₁-AR有竞争性拮抗作用,但其作用对α₁-AR亚型无选择性。     

Failure of AH11110A to functionally discriminate between alpha(1)-adrenoceptor subtypes A, B and D or between alpha(1)- and alpha(2)-adrenoceptors

The potency of the putatively alpha(1B)-adrenoceptor selective drug, 1-[biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol (AH11110A), to antagonize contraction upon stimulation of alpha(1A)-adrenoceptors in rat vas deferens and rat perfused kidney, alpha(1B)-adrenoceptors in guinea-pig spleen, mouse spleen and rabbit aorta, and alpha(1D)-adrenoceptors in rat aorta and pulmonary artery was evaluated and compared to that of a number of subtype-discriminating antagonists. N-[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl-4H-1-benzopyran-8-carboxamide (Rec 15/2739) and (+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) were confirmed as selective for alpha(1A)-adrenoceptors, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378), 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro[4.5]decane-7,9-dione (MDL 73005EF), and cystazosin were found to be selective for alpha(1D)-adrenoceptors, whereas spiperone was weakly selective for alpha(1B)-over alpha(1A)-adrenoceptors. However, from the functional affinity profile obtained for AH11110A at alpha(1A)-adrenoceptors (pA(2)=6.41 in rat vas deferens), alpha(1B)-adrenoceptors (pA(2)=5.40-6.54) and alpha(1D)-adrenoceptors (pA(2)=5.47-5.48), the affinity and presumed selectivity previously obtained for AH11110A in radioligand binding studies at native alpha(1B)- and cloned alpha(1b)-adrenoceptors (pK(i)=7.10-7.73) could not be confirmed. Additionally, AH11110A enhanced the general contractility of rat vas deferens, produced a bell-shaped dose-response curve of vasodilation in perfused rat kidney, and its antagonism in most other tissues was not simply competitive. The affinity of AH11110A for prejunctional alpha(2)-adrenoceptors in rabbit vas deferens (pA(2)=5.44) was not much lower than that displayed for alpha(1)-adrenoceptor subtypes, revealing that AH11110A, besides alpha(1)-adrenoceptors, also interacts with alpha(2)-adrenoceptors, and thus may be unsuitable for alpha-adrenoceptor subtype characterization, at least in smooth muscle containing functional studies.     

Regulation of the cellular localization and signaling properties of the alpha(1B)- and alpha(1D)-adrenoceptors by agonists and inverse agonists

The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.     

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A-315456: a selective alpha(1D)-adrenoceptor antagonist with minimal dopamine D(2) and 5-HT(1A) receptor affinity.

In functional assays, A-315456, N-[3-(cyclohexylidene-(1H-imidazol-4-ylmethyl))phenyl]ethanesulfonamide, behaved as an alpha(1D)-adrenoceptor subtype selective antagonist (pA(2)=8.34) in the rat aorta. It was 83-fold less potent at the alpha(1B)-adrenoceptor subtype expressed in the rat spleen, and was inactive at the alpha(1A)-adrenoceptor subtype expressed in the rat vas deferens. Radioligand binding assays also revealed high affinity (pK(i)=8.71) for the alpha(1D)-adrenoceptor subtype and weaker affinities at the alpha(1A)-adrenoceptor (pK(i)=6.23) and alpha(1B)-adrenoceptor (pK(i)=7.86). In comparison to its potent affinity at the alpha(1D)-adrenoceptor subtype, A-315456 was 3020-, 794- and 38-fold weaker at the dopamine D(2)-, 5-HT(1A)-, and alpha(2a)-adrenoceptors, respectively. These studies indicate that A-315456 is a potent and selective alpha(1D)-antagonist that may serve as a useful pharmacological ligand to probe the physiological role of the alpha(1D)-adrenoceptor subtype in normal and disease states.     

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).     

Computer--based drug design, synthesis and biological evaluation of new pyrimidinone derivatives linked to arylpiperazine and 2'-carbethoxy-biphenylylmethyl moeities as alpha1-adrenoceptor antagonists and angiotensin II AT1 receptor antagonists

Two new series of pyrimidinone derivatives linked to arylpiperazine moieties and 2'-carbethoxy-biphenylmethyl moieties were designed, synthesized and biologically evaluated for their in vivo hypotensive activities. The design of arylpiperazine analogues (IIa-f, IIIa-c, VIIa.b, IX) was based upon structural modification of the newly discovered selective alpha1-AR antagonist drug; Urapidil. Compare/fit studies of these molecules with the previously generated and validated alpha1-AR antagonist hypothesis showed that these molecules have comparable affinities for the alpha1-AR antagonist hypothesis while compound IIIc had the highest fitting value. The in vivo biological evaluation of these compounds for their effects on blood pressure of normotensive cats in comparison to the lead compound prazosin, was consistent with the results of molecular modeling fit values. As expected, compound IIIc exhibited the highest hypotensive activity among the test set compounds. Meanwhile, the design of 2'-carbethoxy-biphenylylmethyl analogues (XIa,b) was based upon the molecular modeling simulation fitting of their carboxylic acid bioprecursors with the previously generated and validated Ang II receptor antagonist hypothesis. Such compare/fit studies predicted that the designed compounds (XIa,b) showed comparable fitting affinities between their de-esterified analogues and the Ang II antagonist pharmacophore. In vivo biological evaluation of these compounds for their effects on blood pressure of normotensive cats showed that compound Xla exhibited hypotensive activity more or less similar to losartan.     

Receptor subtype involved in α1-adrenergic receptor-mediated Ca^2＋ signaling in cardiomyocytes

Aim： The enhancement of intracellular Ca^＋signaling in response to α1-adrenergic receptor （α1-AR） stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role（s） of the α1-AR subtype（s） in mediating this response. Methods： We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca^2＋ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results： After being cultured for 48 h, the myocytes exhibited spontaneous local Ca^2＋ release, sparks, and global Ca^2＋ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca^2＋ transients with an EC5o value of 2.3 larnol/L. Blocking the α1A-AR subtype with 5-methylurapidil （5-Mu） inhi- bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca^2＋ spark numbers were also increased by the activation of the α1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency （EC50 value =6.9 nmol/L） in the potentiation of Ca^2＋ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion： These results indicate that α1-adrenergic stimulation of intracellular Ca^2＋ activity is mediated selectively by the α1-AR.     

trans-4-[4-(Methoxyphenyl)cyclohexyl]-1-arylpiperazines: a new class of potent and selective 5-HT(1A) receptor ligands as conformationally constrained analogues of 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1-arylpiperazines.

The present paper concerns the influence of conformational parameters on the recognition by rat 5-HT1A receptors of derivatives 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1-(2-pyridinyl)piperazine (1a) and 3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-N-[2-(2-pyridyloxy)ethyl]propanamine (3b), two highly potent and selective 5-HT1A receptor ligands. Fifteen corresponding flexible and rigid analogues were prepared following several synthetic routes and were tested in binding assays with radioligands at 5-HT1A, D2, and alpha1 receptors from rat brain membranes. Among the new derivatives emerged trans-4-[4-(3-methoxyphenyl)cyclohexyl]-1-(2-pyridinyl)piperazine (trans-8a) and trans-N-[4-(3-methoxyphenyl)cyclohexyl]-2-(2-pyridyloxy)ethylamine (trans-8b). These compounds can be considered as conformationally constrained analogues of compounds 1a and 3a, respectively. In fact, compounds trans-8a and trans-8b showed a marked enhancement in 5-HT1A receptor affinity when compared to the corresponding cis isomers. Because compound trans-8a was a potent and selective 5-HT1A ligand (K(i), nM: 5-HT1A = 0.028, D2 = 2194, alpha1 = 767), it was chosen as a lead to prepare other analogues that were tested at 5-HT1A, D2, and alpha1 receptors from rat brain membranes, showing high affinity at the 5-HT1A and selectivity vs D2 and alpha1 receptors. Selected compounds were tested for their affinity at the human cloned 5-HT1A, alpha1a, alpha1b, alpha1d receptor subtypes. They were also submitted to the [35S]GTPgammaS binding assay stimulating the 5-HT1A receptor-mediated G-protein activation, therefore behaving as full or as partial agonists. Finally, the ability of iv administration of trans-8a to induce fore-paw treading in rats was evaluated in comparison with 8-OH-DPAT. Although the affinity (K(i)) and in vitro activity (pD'2) of trans-8a at the 5-HT1A receptor were higher than those of 8-OH-DPAT, the compound was less potent than the reference standard in inducing the symptom.